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169

IX. On the Impregnation of the Ovum in the Amphibia. (First Series.)
By GEORGE NEWPORT, F.R.S., F.L.S. fyc.

Received June 20,— Read June 20, 1850.

THE communication which I have now the honour to present to the Royal Society
is a portion of a series of investigations on the Development of the Embryo on which
I have been for some years engaged, and which was commenced in a paper on the
Development of the Myriapoda, that was honoured with a place in the Philosophical
Transactions for 1841. I now propose to give the results of rny observations on the
Amphibia, reserving to a future early occasion the continuation of those on the Inver-
tebrata commenced in the paper alluded to.

The Amphibia, of all the vertebrated animals, afford to us the readiest means of
investigating the difficult subject of Impregnation by actual experiment, and it is
only, perhaps, by combining experiment with careful observations on the physical
conditions that affect the development of the germ, and comparing these with the
facts of the natural history and instincts of the species, that we may hope, ulti-
mately, to obtain some further insight into this one of Nature's most hidden secrets.

I shall endeavour, therefore, in this communication, to show the condition of the
ovum in the Amphibia through its earliest changes, and also before and immediately
after impregnation, and to detail experiments made with a view to learn by what
means its fecundation is effected ; — and in a future communication I propose to trace
the development of the embryo from the time of fecundation to that of its liberation
from the ovum, in the two chief divisions of the class, — the tailless and the tailed
Amphibia. The subjects thus naturally form two series — Impregnation and De-
velopment.

IMPREGNATION OF THE OVUM.

The history of what we can now prove to be the agent of impregnation, the sper-
matozoon, deserves to be especially noticed. Although great attention has been paid
by physiologists during the last thirty years to almost every point of inquiry connected
with the production and physical composition of the seminal fluid of animals, and its
relation to the fecundation of the ovum, we have remained to the present time without
any acknowledged proof either of the part which the different constituents of this
fluid take in impregnation, or of the mode in which it effects impregnation. This
perhaps is little to be wondered at when we remember how many years elapsed before
the great discovery of HAM and LEEWENHOEK of the existence of moving bodies in
the fluid, as part of its normal composition, was admitted. It is now one hundred
and eighty-three years since LEEWENHOEK communicated the important discovery

MDCCCLI. Z

596544

»*.•** v/lr/i: :

170 '" ,,. MR. NEWPORT ON THE IMPREGNATION OF

"of "these bodies to the Royal Society*, and regarded them as in some way essential to
the fecundation of the ova ; although his conjecture that they became the future
embryos was erroneous. Eighty-five years afterwards, Dr. PARSONS, Foreign Secre-
tary of the Royal Society, believed it to be " extreme nonsense to imagine that the
insignificant animals called spermatic animals can contribute anything towards pro-
pagation," &c.-f-, and it was not until the publication of the observations of LEDER-
MULLER^;, a few years after that, that the production of spermatozoa, as part of the
fluid, began to be admitted. Dr. HILL, in the notes to his English translation of
SWAMMERDAM'S Biblia Naturse^, two years later, mentioned them as abundant in the
Frog at the season of pairing, but that it was then the fashion to doubt even their
existence. YetNEEDHAM, ten years after this, while acknowledging that these bodies
are found in the fluid of all animals, adopting the views of BUFFON and DAUBENTON,
stated that they do not exist until after the fluid is removed from the vessels, and
decomposition has commenced \\. And later still, even in our own time, their existence
has been denied in the most positive manner by Sir EVERARD HOME^[. SPALLANZANI,
however, was so well acquainted with them, as found in the Frog and Toad, that he
has recorded his great surprise at not observing them in the latter on two occasions**.
BoNNET-j-f- and GLEICHEN^, also, well knew them to abound in the males of animals
of distinct species at the season of impregnation, but discovered that they are usually
absent in hybrids, a fact that has since been confirmed by PREVOST and DUMAS §§.
These two observers, regarding the spermatic bodies, with LEEWENHOEK, as essential
elements of the semen, believed that they actually penetrate bodily into the ovum, and
become by metamorphosis part of the future embryo. Still more recently it has been
stated byDr. BARRY || | to this Society that he has actually seen the spermatozoon within
the ovum, a statement which my own observations do not enable me to confirm.

Before any satisfactory conclusion could be arrived at respecting the importance
of the spermatic bodies in impregnation, it was necessary to ascertain their nature,
to trace their mode of development and production, to establish the periods of their
occurrence in different classes of animals, and to learn something of their chemical

* Philosophical Transactions, 1667, vol. xii. p. 1040.

f Philosophical Observations on the Analogy between the Propagation of Animals and that of Vegetables.
8vo. 1752 (note), p. 44.

J Physikalische Beobachtungen der Samenthierchen. Nuremb. 1756. And also, "Beytrage zu denen
Beobachtungen deerer Saamenthiergen und Kleiste Aale gehorig. 12mo. Frankfurt und Leipzig, 1759."

§ Book of Nature, folio, part 2 (note), p. 105, 1758.

|| Notes des Nouvelles Recherches sur les De'couvertes Microscopiques de 1'Abbe SPALLANZANI par M.
NEEDHAM. Lond. 1769, torn. i. p. 196.

1J Lectures on Comparative Anatomy, 4to, vol. v. pp. 332 and 337. 1828.

** Dissertations relative to the Natural History of Animals and Vegetables. Lond. 1789, p. 151.

ft " Contemplations de la Nature ; " and " OZuvres d'Histoire Naturelle," 4to, torn. iii. p. 454, &c. 1779.

JJ Abhandlung iiber der Samen- und Infusionsthierchen. Nuremb. 1788.

§§ Annales des Sciences Naturelles (Prem. Se'rie), torn. i. p. 182, 1824.

1111 Proceedings of the Royal Society, vol. iv. p. 432. Phil. Trans, part 1, 1843, p. 33.

THE OVUM IN THE AMPHIBIA. 171

composition, as well also as of the fluid portion of the semen in which they move.
Most of these inquiries have been well followed out by WAGNER, SIEBOLD, MULLER,
and more especially KOLLIKER, and more recently by WAGNER and LEUCKARDT, from
whose labours we have now some positive information which enables us to deduce
a fair conclusion respecting their function, although a direct proof of its correctness
is still to be supplied. Most observers now believe with KOLLIKER that the sperma-
tozoa (still so called) are not independent living organisms, but are merely elemen-
tary constituent parts of the male body, an opinion in which my own investigations
lead me fully to coincide. This opinion, indeed, is not entirely new, as a like view
was held by some observers at the beginning of the last century, when it was still
questioned whether the spermatozoa are normal constituents of the semen. Dr-
DRAKE*, in his "New System of Anatomy," while acknowledging that he had seen
the seminal animalcules, and combating on the one hand the theory of LEEWEN-
HOEK respecting them, and on the other the view that had previously been held with
regard to the ovum, doubted their separate organization, and suggested that they
" may be nothing more than some large particles of mixed fluid, whose motions and
different figure the microscope discovers to our eyes," &c. G. TREVIRANUS-J- more
recently held a similar opinion, that they are not independent animals, but are analo-
gous in their structure and properties to particles in the pollen of plants, and that
their motion is of the kind discovered by ROBERT BROWN in vegetables. KOLLIKER]:,
however, first distinctly referred them to a class of known organic constituents of the
living body, the vibratile cilia, a view which had previously been discussed and in-
clined to by MULLER^.

But however much our knowledge has become settled in regard to the nature of
the spermatic bodies themselves, and their mode of development, their relation to
the fluid portion of the semen in which they are contained is still a matter of doubt.
H. GOODSIR|) regards certain albuminous flakes in the fluid portions of the semen of
Crustacea as the debris of dissolved cells, and as the source of nourishment and deve-
lopment of the spermatozoa ; while a more recent observer, Dr. KIRKES^[, regards the
spermatozoa as the elaborators of the fluid, and the conveyers of it to the ovum at
the time of impregnation. This latter supposition was originally advocated by WAG-
NER, VALENTINE arid BISCHOFP. But two of these observers have recently changed
their views**, and now regard the fluid portion as only of secondary importance in
impregnation, and the spermatic bodies as of essential. This view, as WAGNER
states-f-f-, is founded chiefly on the fact that in some of the invertebrata the whole mass

* New System of Anatomy, by JAMES DRAKE, M.D., F.R.S. vol. i. p. 352, 1707.
f TIEDEMANN, Zeitschrift, vol. v. part 2, 1835.

I Beitriige zur Kenntniss der Geschlechtsverhiiltnisse und der Samen-flussigkeit wirbelloserThiere. Berlin, 1 84 1 .
§ Elements of Physiology (Eng. ed.), part 6, 1841, p. 1478.

|| Anatomical and Pathological Researches, 1844, p. 40. ^f Handbook of Physiology, 1848, p. 610.

** BISCHOFF in MULLER'S Archiv, 1847. WAGNER in Article " Semen," Cyclopaedia of Anatomy and Physi-
ology, part xxxvi. January 1849. ft Loc. cit. p. 507.

z 2

172 MR. NEWPORT ON THE IMPREGNATION OF

of the semen appears to be constituted almost or entirely of spermatozoa, while
scarcely any liquor seminis can be detected ; — and further, on the great improbability,
perhaps impossibility, of the liquor seminis of those animals which expel their ova
into water before impregnation being brought into contact with the ovum. But the
same author justly remarks, that " even up to the present day this hypothesis of the
influence of the liquor seminis has not met with any direct refutation." To this I
may add, that however strong the presumption may be in favour of the agency of
the spermatozoa in those instances in which a liquor seminis has not been observed,
it affords no sufficient reason for disbelieving that the spermatozoa are not resolved
into fluid at the moment of fecundation ; or that in those animals in which the liquor
seminis occurs in abundance it is not that which impregnates the ovum.

The question then, so far as proof is concerned, both of the direct agency of the
spermatozoa, and of the non-efficiency of the liquor seminis in impregnation, remains
open, as well also as that which involves the knowledge as to how impregnation is
effected.

It is to these questions that this communication which I have now the honour of
laying before the Royal Society is chiefly directed. I propose^r*# to show the time
and mode of disappearance of the germinal vesicle, and the condition of the ovum in
the Frog and Newt, immediately before and after impregnation, and to endeavour to
supply proof from actual experiments that the spermatozoa alone, in all cases of com-
munion of the sexes, are the sole agents in impregnating the ovum ; and further, that
impregnation cannot be effected by the liquor seminis ; and next to examine in what
way the agency of the spermatozoa is influenced, impeded, or exerted.

1. CHANGES IN THE OVUM WITHIN THE BODY.

The ovum of the Amphibia has so frequently been the subject of examination by
the best observers that a further detailed account of its development may at first ap-
pear to be useless, after what we already know of its changes through the labours of

SWAMMERDAM, LEEWENHOEK, RoESEL, SPALLANZANI, PflEVOST and DuMAS, RuSCONI,

BAER, REICHERT, VOGT, BELL and others. But apart from the fact already men-
tioned, that the ovum of the Amphibia affords us the best means of actual experiment
on impregnation, there are questions which relate to its earlier conditions on which
the observers named are not agreed, but which are of importance with regard to the
physiology of reproduction in the whole of the vertebrata.

I shall state, therefore, what I have myself observed with regard to these questions
from the time when the ovarian ovum is approaching to maturity to that of its expul-
sion from the body, before entering on the subject of its impregnation.

As our means of comparing and testing the accuracy of all observations in natural
history, and of experimental results in physiology, depend mainly on the correct
identification of the objects examined, I may here state at once that the objects of
the following details have been the Frog, Rana temporaria, and the Toad, Bufo vul-

I

THE OVUM IN THE AMPHIBIA. 1/3

garis, among the'dnoura ; and the Triton palustris, Lissotriton punctatm and L. pal-
mipes, among the Water Newts, the Urodela. Neither SPALLANZANI, PREVOST and
DUMAS, nor RUSCONI, to whose observations I shall have frequent occasion to refer,
mention the species they have examined. SPALLANZANI has given only the popular
names of his animals, but RUSCONI has given a figure of his, which appears to have
been Rana esculenta, and as the description he has given of the ovum of this species
agrees with the description given by PREVOST and DUMAS, it is probable that the
species they employed was the same.

The Ovarlum Ovum. — When the frog, Rana femporaria, is examined in the autumn,
after it has ceased to feed, and is preparing to retire to its winter quarters, the ova
within it have already attained to more than two-thirds of their ultimate dimensions
before leaving the ovaries, and have begun to distend the abdomen. They remain in
this state through the greater part of the winter, while the frog is hybernating, as
Professor BELL remarks*, in the mud at the bottom of ponds and stagnant waters.
But as the spring approaches, and the animal is aroused from its lethargy by increased
temperature, the ova then rapidly acquire their full development. A female frog, taken
at the end of September, with her body enlarged with ova, was confined in water in
a cold room, undisturbed through the winter, excepting only at intervals of examina-
tion. The weather being mild, and the temperature of the room during October
and November being sometimes at or but little below 50° FAHR., the frog remained
active, and came frequently to the surface to respire. In December the tempera-
ture sunk to below 40° FAHR., when the frog became lethargic, and scarcely changed
its place at the bottom of the water during nearly a fortnight, while the temperature
was almost stationary, and ranged only from 35° FAHR. to 37° FAHR. On the 10th of
January, when it had again risen to 40° FAHR., and that of the water to 37°' 5 FAHR.,
the animal was still submerged and motionless ; but on sudden exposure to the light
of a candle it crept languidly, and almost imperceptibly, to a distance of about two
inches, and again became quiet. On the 26th of January the temperature of the
room had risen gradually and continuously to 52° FAHR., and that of the water to 48°
FAHR., but the frog remained submerged and perfectly quiet with its eyes partially
closed. Although it was not cognizant of any object by sight, the irritability of its
body was now much increased, as it moved instantly when touched ever so lightly,
but quickly relapsed into its previous state of rest. On the morning of the 1st of
February, the temperature being then 48° FAHR., it was still submerged and motionless,
but in the evening, when the warmth was increased to 52° FAHR., I found it with its
nostrils only above the surface of the water, and evidently beginning to respire freely,
but its eyes were completely closed. When the light of a candle was suddenly cast
upon it, the eyes were slowly opened, but its body remained immoveable. On the fol-
lowing day, February 2, it was evident that its hybernation had been brought to a
close, as it was then active, with its head out of the water, and its eyes widely open,

* History of British Reptiles, 1832, p. 89.

174 MR. NEWPORT ON THE IMPREGNATION OF

and perfectly alive to external objects, as when the hand was slowly approached it
withdrew beneath the water. The temperature at this time was 53° FAHR. During
this period of hybernation the frog became slightly emaciated, and its abdomen,
instead of being more enlarged, was somewhat diminished in bulk, — a good proof
that during the inactivity of the respiratory and circulatory functions, the secretory
also are lessened, and the development of the ova is arrested. But although no food
was supplied to the frog at this time, and none probably is taken by the creature in its
natural haunts, as at the time it comes forth but few of the objects on which it feeds
are abroad, its body soon became enlarged, showing that the ova were then rapidly
attaining their full development. Between the 2nd and 22nd of February the tem-
perature of the room was occasionally as low as 42° FAHR., yet the creature remained
active beneath the water, without relapsing into its previous state of hybernation.
It only continued longer beneath the surface without rising to respire. At this period,
having found that some frogs in their natural haunts had already come forth, I
removed the subject of these observations also from the water to a damp locality, and
on the following day found it greatly changed in appearance. While confined in the
water it was of a dull dirty brown colour, but some hours after its removal it cast
its tegument, and changed to a bright yellow, with the usual brown markings, and
had increased in size, both in its body and limbs.

The conclusion to which these circumstances seemed to lead was, that quickly after
the frog leaves its hybernaculum, it casts its tegument as the insect escapes from its
puparium, and acquires new vigour, while the ova are attaining their full growth.
The Toad undergoes a similar change. About a fortnight later in the season than
the Frog, I have seen many toads in a shallow ditch of slow moving water in the act
of casting their dark brown tegument, and acquiring one of a greenish yellow.

On examining several frogs taken from their natural haunts, I found them in, as
nearly as posible, the same state of development with regard to the ova, judging from
external appearance, as the specimen I had watched through the winter. A few had
just paired, but the majority were still single. On opening the bodies of the latter,
I found the ovaries greatly enlarged, and the ova apparently ripe, but still contained
in the ovisacs.

It is at this period, therefore, immediately after hybernation, and before the ova
have left the ovarfes, that the condition of the ovum is a matter of great interest with
reference to the structure and contents of the germinal vesicle, the period at which
the vesicle is changed or disappears, and the circumstances under which the ovum
escapes from the ovary, and is received into the oviduct.

The Germinal Vesicle. — The fate of the germinal vesicle in the matured ovum is
still a matter of doubt. Previous to the embryological researches of Dr. MARTIN
BARRY, it was usually believed that the vesicle entirely disappears before or at the time
of fecundation of the ovum. But this view was combated by the author named, who,
quoting the opinions of previous inquirers, contended that the germinal vesicle in

THE OVUM IN THE AMPHIBIA. 175

Mammalia does not disappear, as believed by PURKINJE*, in Birds and Amphibia, by
bursting during the generative act, and pouring its contents into the germinal layer
of the fecundated ovum. Neither, as supposed by BAER-{~, by being urged forwards
and burst between the vitellus and its membrane before fecundation. Nor, as stated
by RATHKE|, with reference to the Crustacea, by disappearing while still within the
ovary. Or, as supposed by WHARTON JONES §, in Birds and Amphibia, by approaching
the surface of the yelk, and by the giving way of the coats of the vesicle, and the
effusion of its contents on the surrounding surface of the yelk. Or, further, as
believed by BISCHOFF ||, by disappearing at the time of exit of the ovum from the ovary.
On the contrary, Dr. BARRY has stated that the germinal vesicle returns to the centre
of the ovum, and the germinal spot to the centre of the vesicle, before the ovum leaves
the ovary, and that these do not become dissolved, but only are changed in character
by a process of cell development within them, which ends in the production of two
cells in the centre of the yelk, which are the foundation of the body of the future
embryo.

Perhaps what I am about to mention may assist us to reconcile or correct the views
of this embryologist, as well as those of the authorities he has quoted. It is well
known that if the ovum of the Frog is examined before the yelk has attained to one-
half of its ultimate dimensions, the germinal vesicle is distinctly visible as a large cir-
cular body near the centre of the yelk, apparently granular in its interior, and more
opake than the yelk itself. In the centre of the vesicle its nucleus, the germinal
spot, is then equally distinct. If the ovum is examined when it has nearly acquired
its full dimensions, the vesicle is still found to exist, but, as compared with the size
of the entire ovum, is relatively smaller than at earlier periods, and is recognized
with more difficulty, owing chiefly to the yelk cells having both increased in number
around it, and also acquired a darker colour. It is equally well known that all
appearance of the vesicle is lost in the Amphibia, as in Birds, before the ovum is pre-
pared for fecundation ; but as to the way in which it disappears, observers are not
agreed, or even as to the time.

Structure of the Germinal Vesicle. — Having collected a number of frogs that had
recently left their hybernacula, and had not yet paired, I placed some in spirit for
examination. On dissecting them afterwards, I found that in some the ova had
nearly reached maturity, but had not left the ovary (Plate XIV. fig. ^ p). The yelks /<3.
being rendered firm by the spirit, I was able, by gentle pressure, to break open some
of these beneath the microscope, without diffluence of the contents. The aggregated
yelk cells were then seen to consist of two kinds ; the one dark-coloured, which form

* Symbols ad Ovi Avium Historiam ante Incubationem, 1825 ; and Article "Ei," Encyclop. Worterbuch,
Bandx. p. 112, 1834.

t " Lettre sur la Formation de 1'CEuf," in Breschet. Repertorium, 1829.

J Untersuchungen ueber die Bildung und Entwickelung des Fluss Krebses, fol. Leipzig, 1829.

§ Philosophical Transactions, 1837, part 2. || R. WAGNER'S Lehrbuch, &c., 1839.

176 MR. NEWPORT ON THE IMPREGNATION OF

the upper portion of the yelk, and which diminish in intensity of colour from brown
or black at the circumference to a leaden or grey in the centre ; the other, which
forms the inferior portion, consisting of cells that become lighter-coloured as they
approach the surface. The whole form a mass of nucleated cells of nearly uniform
size, closely aggregated together. In the interior of the yelk, amidst the dark-
coloured cells, and much more near the surface of the dark than that of the white por-
tion of the ovum, I was surprised to find the germinal vesicle still entire (fig. 6), and
of a somewhat oval, lenticular form, although irregularly compressed by the contrac-
tion of the whole egg in the spirit. It was of a dense white colour, and opake, from
the action of the spirit, and was in striking contrast to the dark cells of the yelk,
which adhered to its surface, and amidst which it lay imbedded like the kernel in a
peach or apricot. On the surface of the black portion of the yelk was a minute
orifice, already noticed by PREVOST and DUMAS*, the outlet of a canal that passes
through this portion of the yelk to the germinal vesicle in its interior (fig. 6). This
I believe to be the result of the yelk cells having only imperfectly closed around
the germinal vesicle. It is however of some consequence in the future development
of the ovum, as it is in this canal that the cleavage of the yelk is commenced. The
vitelline membrane was already formed, but I could not discover any orifice or per-
foration in it, either corresponding to the canal in the yelk, or to any other part of
the surface. It is not possible to mistake the germinal vesicle for any portion of the
lighter substance of the yelk, — first, from the fact that the vesicle at this period is of
an intense, opake, white colour, very different from that of the yelk substance, —
next, from its being completely isolated from the lighter, and imbedded in the dark
substance, — and lastly, from its being still invested with a distinct envelope. On re-
moving the vesicle, and examining it separately, first, as taken from the yelk, without
crushing it, and next by gentle compression, and with the highest powers of the
microscope, the interior was seen to be filled with secondary cells. Each of these,
formed by a distinct envelope, appeared to contain other, or tertiary cells, and strongly
reminded me of the developmental cells of the spermatozoa in the male organs, since
these again seemed to contain granules, or quaternary cells. In the midst of the
secondary cells I was able to distinguish, in the centre of the germinal vesicle, in some
specimens, one or two cells of larger size than the rest, and which I regarded as the
remains of the germinal spot, or central nucleus. In those ova which, from their size
and general appearance, seemed to be the most mature, the peripheral series of cells
within the germinal vesicle were of smaller diameter than those nearer to the centre,
as if the earlier developed secondary cells had disappeared and liberated their con-
tents. The cells of the dark portion of the yelk that adhered to the germinal vesicle
were also nucleated, but were of much smaller size than the peripheral cells of this
body. I must remark, however, that saving the fact noticed, of having seen in some
vesicles one or two cells near the centre of larger size than the rest, I have not been

* Loc. cit, torn. ii. p. 104.

THE OVUM IN THE AMPHIBIA. 177

able to distinguish any separate nucleus, or germinal spot, which seems, at this time,
to have disappeared as a distinct body. The germinal vesicle thus near its matuiity
is a mother-cell that contains a multitude of daughter or secondary cells, each, appa-
rently, including its own progeny.

It is thus certain that the germinal vesicle exists in the ovum of the Frog until after
the period of hybernation. But in individuals that have been several days abroad, and
in which the ova are so far matured as greatly to distend the body, and be nearly
ready to leave the ovisacs, I have not always been able to detect the vesicle. The
yelk has then a greater proportion of white substance in its interior, intermingled with
the dark, and this, I suspect, is the result of the disappearance of the vesicle.

In the ovum of the Toad, when as nearly mature as that of the Frog before leaving
the ovisacs, I have been unable to detect any trace of the germinal vesicle. The
dark portion of the yelk is then quite distinct from the light, and forms a cortical
stratum of intensely black pigment, scarcely more than one-sixth of the diameter of
the yelk in thickness. The light portion is of a yellowish white, and from which the
germinal vesicle, when present, is not readily distinguished. But at a little earlier
period, although only a few days before the pairing of the Toad, I have found the
vesicle fully as large and as distinct as before its disappearance in the egg of the
Frog, but situated more distant from the centre, and nearer to the black, the future
dorsal surface of the egg.

In the great Water Newt, Triton palustris, the yelk of the matured ovarian ovum is of
a dull pea-green colour, which in spirit is changed to a greenish yellow. On breaking
open the yelk of this species, after it has been hardened in spirit, I have always found
the germinal vesicle of the same opake, intense white colour, of precisely the same
structure, and situated in the same part of the yelk as in the Frog. It has also a
canal passing from the middle of the surface of the darker-coloured portion of the
yelk, through its substance to the vesicle, as in the Frog.

In the lesser Newts, Lissotriton punctatus and L. palmipes, the yelk is of a brown
or liver colour on the future dorsal surface, with a white central spot, and of a white
or pale straw colour on the ventral. In ripe ovarian ova of these species also I have
found the germinal vesicle of the same white colour, and having the same structure
as in the larger Triton and the Frog.

In each of these instances, not merely when the ovum is immature, but even when
nearly ready to leave the ovisac, the germinal vesicle is situated in the interior of the
yelk (fig. 6), and not immediately at the surface, and thus far the fact is in accord-
ance with the observation of Dr. BARRY, that in the Mammalia the germinal vesicle
is in the interior of the ovarian ovum, and does not disappear on the surface. But,
nevertheless, it is not in the centre of the yelk, its place, as I have stated, is excentric.

Time of disappearance of the Vesicle. — It is well known that in frogs and toads
the ovum is never impregnated until after it has left the body, and consequently not
until long after it has left the ovary, and the germinal vesicle has entirely disappeared.

MDCCCLI. 2 A

178 MR. NEWPORT ON THE IMPREGNATION OF

This disappearance does not, I think, take place at the instant the ovum is about to
leave the ovisac, but a short time before ; as I have found the ova of the Frog in one
instance more matured than in that already described, still contained in the ovisacs,
and yet most of them without any remains of the vesicle which I could identify as such,
and with the yelks containing a larger proportion of white substance, as in the Toad.
In a very few, however, the vesicle was still present, and exhibited the structure I have
described. Certainly, then, the vesicle is not burst at the moment the egg escapes
from the ovisac. There seems to be a short period of time between the disappearance
of the vesicle and the full maturity of the ovum, during which the yelk itself undergoes
some further change, and acquires the appearance noticed in the matured eggs of the
Toad, and in the most advanced of those of the Frog. No trace of the germinal vesi-
cle can be detected in any ova that have left the ovary and are contained in the cavity
of the abdomen, before entering the oviducts. I have examined many ova, both of
the Frog and Newt, from the abdominal cavity, but in every instance the vesicle has
entirely disappeared. In some specimens, which seemed to have been in the act of
escaping from the ovary when the animal was killed by immersion in spirit, I have
found in the place occupied by the vesicle an aggregation of white nucleated cells,
which, examined by the microscope, exhibited a close resemblance to those seen in
the interior of the vesicle. In the midst of these there has occasionally been one or
two of larger size than the rest, and which I have imagined to be the remains of the
germinal spot, and possibly the origin of the future embryo vesicle of the impregnated
ovum, an opinion, however, which I have not had the means of verifying, and I must
further state that I have failed to recognize these larger cells in ova that were free in
the cavity of the abdomen. Each of the three species of Newt, as well as the Frog,
have presented similar appearances in the germinal vesicle and ovum under similar
circumstances.

Thus it is quite certain that the germinal vesicle disappears in the Amphibia before
the ovum enters the oviduct. I believe it does so in the interior of the yelk, not in
the centre, but nearer to the dorsal than to the future ventral or white surface ; and
not, as has been supposed, on the dorsal or dark surface, between the vitellus and the
vitelline membrane. This view is supported by the fact, that that portion of the yelk
which incloses the vesicle in an advanced stage of the ovum in the Frog is of a more
or less intense black colour, while the vesicle is perfectly white ; and that at a further
advanced stage, after the vesicle has disappeared, and its place is occupied by a col-
lection of white cells, the dark portion of the yelk still preserves its intense black
colour, except at the point that corresponds to the central canal, which then has a
leaden hue. PREVOST and DUMAS*, and also RUSCONI-J-, have mentioned that there
is a yellow spot at a corresponding part of the dark surface of the egg of the species
they have examined after impregnation, Rana esculenta ?, but these appearances must

* Annales des Sciences Naturelles, torn. ii. p. 104.

f De'veloppement de la Grenouille Commune, 4to. Milan, 1826. p. 9.

THE OVUM IN THE AMPHIBIA. 179

not be mistaken for the germinal vesicle arrived at the surface. There is a similar
spot, and that too of an elevated form, on the egg of each of the Lissotritons. But
independent of the fact that the germinal vesicle has entirely disappeared from the
interior of the egg before it escapes from the ovary, this spot is shown not to be the
vesicle, both in the fact that in the egg of Rana temporaria the dark portion of the
yelk is unchanged, while in each case the spot is perforated, and leads into the canal
that passed originally to the vesicle. I regard the spot as simply a protrusion outwards
of the edges of the canal while closing, after the vesicle has disappeared. I shall
presently show that a similar white spot is formed on the under surface of the egg of
the Frog soon after deposition, and which might equally well be mistaken for the ger-
minal vesicle.

Mode of disappearance of the Vesicle. — The mode in which the vesicle disappears
may be inferred from the facts of its structure. Being filled with a progeny of cells
which we may regard as of different periods of growth, and these again containing
others, it is fair to conclude that this process of cell formation is that by which the
parent vesicle is ultimately destroyed. At the time when the germinal vesicle has
nearly attained its full size, the peripheral cells are smaller than those nearer to its
centre, while the yelk cells that surround the vesicle are still smaller than either, and
are of a dark colour. When, therefore, the vesicle has acquired its full size, by the
simple vegetative endogenous growth of the contained cells, we may fairly presume
that the death of the parent mother-cell, or germinal vesicle, takes place as the result
of their enlargement, by the diffluence of its investing membrane ; and the enclosed
daughter cells, thus gradually set free in the midst of those of the yelk, as in the ideal
(fig. 7), form one mass with the latter, and the moment of the actual disappearance of
the vesicle thus escapes direct observation, its previous existence being indicated only
by the unbroken outline of the investing membrane.

These views lead me to agree with WAGNER and BARRY in regard to the structure
and mode of growth of the germinal vesicle, but not as to that by which it disappears.
Dr. BARRY indeed believes that the vesicle only becomes changed by its mode of de-
velopment, and does not cease to exist. But most certainly it does disappear in the
Amphibia, and, as I believe, through the growth of the young cells in its interior. I
cannot therefore agree with Dr. BARRY that the changes in the vesicle end in the pro-
duction of two cells in the centre of the yelk, that give immediate origin to the em-
bryo ; but rather believe that it is from one of the central cells of the germinal vesicle
that the future embryo vesicle takes its origin, while the remainder of the liberated
cells are distributed with this through the substance of the yelk, when the segmenta-
tion of this body takes place. This opinion is in accordance with that of VOGT*, who
found that in the ova of Alytes obstetricans the germinal spots increase in number,
and that a few hours after fecundation small vesicles, similar to these spots, are scat-
tered through the yelk. I have myself found similar vesicles in the fecundated egg

* Untersuchungen uber die Entwicklungsgeschichte der Geburtshelfer-Kraete (Alytes obstetricans), 4to, 1842.

2 A 2

180 MR. NEWPORT ON THE IMPREGNATION OF

of the Frog about three hours after impregnation (fig. 10 a), but have not traced
them to their origin. With regard to the disappearance of the spot in the germinal
vesicle, the facts observed in the ova of the Frog agree with those noticed by KOL-
LIKER* in the ova of intestinal worms, that all appearance of the spot is lost before
that of the vesicle. This circumstance, however, may be owing either to the spot
having given origin to cells in the vesicle which quickly attain to similar dimensions,
and from which it is not otherwise distinguished ; or to its becoming entirely obscured
by their multiplication. Although no observations have been made on the origin of
the embryo vesicle that appears in the yelk after the disappearance of the germinal,
I am still inclined to regard this as being in some way derived from the lost germinal
spot, notwithstanding that KOLLIKER found a certain period of time elapse between the
disappearance of the vesicle and that at which he was able to recognize this body.

Transit of the Ovum. — Thus, then, when the ovum escapes from the ovisac and
ovary into the cavity of the abdomen, the germinal vesicle and spot have entirely
disappeared, and it consists only of the yelk enclosed in an exceedingly delicate, struc-
tureless, vitelline membrane. This is its condition in the Frog, Toad and Newts. It
is then extremely delicate and easily lacerated. The mode in which the ovum passes
into the oviduct has been the subject of much inquiry. I am quite certain, as SWAM-
MERDAM long ago showed, that the ova, when mature, pass from the ovaries into the
cavity of the abdomen, and from thence into the oviducts, in the Frogs, Toads and
Newts, quite independent of any intercourse with the male, as I have myself recently
had an opportunity of proving. The frog I have already mentioned (p. 173.) as
having watched through its season of hybernation, was kept apart from all others
until the 2nd of April, at which time she had not deposited any ova. But from the
altered form of her body it was evident that the ova had passed, or were at that time
in the act of passing, into the oviducts. I then placed her in a vessel with others,
some of which were males, but not one of these joined with her. Nevertheless, on
the 6th of April she cast her ova, without having paired, and died on the following
day. On examination after death, I found that the whole of the ova had left the
ovaries, all of which, excepting only two still free in the cavity of the abdomen, had
passed through the oviducts without any intercourse with the male. These ova of
course were sterile, but it was worthy of note that their envelopes did not expand to
so great an extent as those of the eggs of paired individuals.

Passage into the Oviduct. — In what way the ova pass from the cavity of the abdomen
into the mouth of the duct has never been satisfactorily explained. SWAMMERDAM
examined the question with much care-f-, but was unable to form any decided opinion
respecting it, as he correctly states that the mouths of the ducts are at a distance
from the ovaries, and are not free to grasp the ova like the fimbriated extremities of
the Fallopian tubes in Mammalia, but are confined in the peritonaeum, which is con-
tinuous with that which passes over the pericardium and heart. PREVOST and DUMAS

* MULLER'S Archiv, 1843. t Book of Nature, part 2, pp. 108, 109.

THE OVUM IN THE AMPHIBIA. 181

have since stated* that the ova after leaving the ovaries are seized by the tubes ("sont
saisis par des trompes "), but they do not show in what way this seizure is effected.
They have omitted to describe the structure of the parts concerned in the act, and
have not mentioned the way in which the ova are conveyed to the tubes from the ova-
ries, or whether the mouths of the tubes approach the ovaries, as in Mammalia.

The entrance to the tubes in the Frog, as SWAMMERDAM has correctly shown, is in
the peritonaeum (PI. XIV. fig. 1 b) at each side of the heart (a), and I have found it in
nearly the same place in the Newts. The apex of the pericardium in the Frog is at-
tached to the cartilage of the sternum by two layers of peritonaeum, which together
form the mediastinum, and enclose between them the trunk of the median abdominal
vein, a branch of the vena cava. Tracing one of these layers of peritonaeum upwards
and over the pericardium, we find in it an orifice (fig. 1. and 2 b), at the part where
it is reflected on itself to form the lateral portion of the suspensory ligament (e) of
the liver (c). This orifice (b) is elongated, oval and funnel-shaped, and, when dilated,
forms a kind of pouch at the anterior boundary of the space or cavity between the
liver (c) and the heart (a), and laterally it is in free communication beneath the sus-
pensory ligament with the common cavity of the abdomen. The oviduct (g) com-
mences in this dilated orifice as a narrow tube with thick muscular parietes and with
a thick mucous lining. It passes at first upwards and forwards, confined to the peri-
tonaeum, and then outwards above the base of the lung (/), gradually increasing in
its dimensions. Immediately after it has passed the lung it becomes more enlarged,
and as it passes backwards to the side of the spine forms many convolutions, which
end in the dilated oviduct (fig. ]. and 4 h) or common receptacle for the eggs ready
to be deposited.

The commencement of the oviduct in the Newts is very similar to that of the Frog.
In the Triton palustris it differs only in the entrance being larger, and situated more to
the side of the body, and above the lung, to the base of which, as well as to the peri-
tonaeal investment of the heart, it is confined, as in the Frog ; but it has a more free
communication laterally with the common cavity of the abdomen than in that animal.

The ova escape from the ovaries into the cavity of the abdomen among the viscera
both in the Frogs and Newts, and are carried forwards to the spaces between the liver
and heart on each side to the dilated mouth of the oviduct. They certainly are not
seized by the tubes as they escape from the ovaries, as they are constantly found free
in the abdominal cavity, while the mouths of the tubes being confined in the perito-
naeum, and having no appendages, cannot be extended to reach them. Their transfer
seems to be effected in the Frog in part by the action of the abdominal muscles forcing
them onwards in the spaces between the viscera, aided perhaps by the peristaltic action
of the stomach and intestines ; and their entrance into the tubes, when arrived in the
vicinity, seems to be induced by an ingurgitory or suction action at the mouth, occa-
sioned by the alternating and pulsatory motion of the heart, with which the tube is

* Loc. cit. torn. ii. p. 105.

182 MR. NEWPORT ON THE IMPREGNATION OF

connected by means of the peritonaeum. The tube itself is formed of strong longitu-
dinal and transverse fibres, which are continued into the peritonaeum, and the former
especially into the suspensory ligament, the free external margin of which bounds the
outer side of the orifice. The transverse fibres are strongly marked at the commence-
ment of the orifice, where there is a slight pouch ; so that when the eggs are entering,
these fibres doubtless prevent their return and transfer them onwards. This, I believe,
is the way in which the eggs enter the oviducts. It is quite certain from the anatomy
of the parts that they cannot be grasped by the oviducts until they are conveyed to
them. I have not actually witnessed the passing of the eggs from the abdomen into
the ducts in the Frog, but I have seen the eggs moved onwards in the smaller Newt,
Lissotritonpalmipes. Having deprived a female of this species of sensation and power
of motion by division of the spinal cord through the medulla oblongata, I proceeded
to open the abdomen to obtain ova from the oviducts for experiments on artificial
impregnation. I then found that a number of ova were free in the abdominal cavity,
and that some had very recently entered the ducts, while others were in the immediate
vicinity of the mouths. The heart was still pulsating vigorously and with great regu-
larity, and I then saw that at each pulsatory action the ova passed slowly forward
between the liver and lung, towards the mouth of the oviduct, which still contained
two or three ova that appeared to have entered at the moment of the operation. I
did not witness the actual entrance of an ovum, but saw that the action of the heart
certainly had the effect of inducing the advance of it to the mouth of the tube, and
quite sufficient to lead me to regard this as one of the chief means of its entrance into
the duct.

It is not until the ovum has become clothed in the oviduct with its gelatinous en-
velope that it is susceptible of impregnation. This remark applies equally to the
Frogs, Toads and Newts. The ova of the Frog and Newt at large in the abdominal
cavity are always entirely without this envelope, and consist simply of the yelk mass
enclosed in an extremely delicate vitelline membrane. They are so easily injured that
it is only with great difficulty that they can be removed from the abdomen for exami-
nation unbroken. Those of the Newt, when taken up ever so carefully by means of
a hair pencil, often burst the membrane simply by their own weight. But immediately
after they have entered the oviduct and begin to acquire their envelopes, the yelk
appears to undergo some change, as it becomes much firmer and is less easily injured.
Shortly after the egg has entered the duct, it gains the first layer of an investment,
which, from the great similarity it bears to the gelatinous layer gained by the ovum
of the Rabbit in the Fallopian tube, and regarded by its discoverer in that animal,
Mr. WHARTON JONES*, as the origin of the chorion, I am disposed, with him, to look
upon as the analogue of that layer. This covering adheres very closely to the vitelline
membrane, and is scarcely to be distinguished from it, except at certain periods of
change. It is acquired before the egg has arrived at the first convolutions of the ovi-

* Phil. Trans, part 2, 1837.

THE OVUM IN THE AMPHIBIA. 183

duct. During the remainder of its passage the egg gains two other distinct layers of
similar investment, which, together, we afterwards recognize as the gelatinous enve-
lope of the Frog and Toad, and the capsule of the Newts. These envelopes are not
merely simple means of protection to the egg during the production of the embryo,
as has been supposed, but, as I shall presently show, are essential to it at the period
of fecundation, and without which the egg is not susceptible of impregnation. The
layer of envelope which I regard as the foundation of the chorion, is a dense, but
very transparent thin covering, in immediate contact with the vitelline membrane,
and is formed of cells so closely aggregated together as to have coalesced into a
fibrous structure. The two layers external to this are also formed of cells, which,
with their nuclei, are distinctly visible in the envelope of Triton palustris, in which
they alternate in regular series. Although these layers, which constitute the jelly in
the egg of the Frog, become detached in that of the Newts quickly after oviposition,
and, expanding as in the Frog, they leave the egg at liberty in a chamber in their
interior, they are nevertheless essential to the impregnation of the ovum, which takes
place before or at the time of leaving the body, as in Frogs and Toads. RUSCONI*
removed the envelopes of the egg of frogs, and found that the embryo still became
developed, and thence concluded that these coverings serve only mechanical purposes
during the changes ; but it will presently be seen that they have a more important
function at a much earlier period. During the time they are in course of formation
around the egg the yelk undergoes some further change. The light portion becomes
of a whiter, and the dark portion of a deeper colour. Internally the cells vary more
in size, the lighter-coloured being the largest. I have not succeeded in recognizing
any embryo or central vesicle up to this period.

2. CHANGES AFTER SPAWNING AND IMPREGNATION.

First period of development. — It has been long known that a division or cleavage of
the yelk of the Frog's egg is one of the earliest and apparently invariable results of
fecundation. The primary division was first seen by SWAMMERDAM, and was figured-}-
and mentioned, but was not understood by him. SPALLANZANI long afterwards re-
cognized it in the egg of the Toad (Alytes obstetricansty, which he says becomes about
a day after fecundation marked " with two furrows which meet to form an angle," —
that the furrows afterwards become deeper, and that "two small tumours arise on
each side of the furrows," — changes which have since been more accurately and com-
pletely described by VOGT§. SPALLANZANI also says that the egg of the common Toad
is " marked with four furrows which intersect each other at right angles nearly like
the husk of a chestnut half-opened," but he seems to have thought this was the usual
condition of the ovum. To PREVOST and DUMAS ||, however, we owe the important

* Developpement de la Grenouille commune. Milan, 1826.

•f Loc. cit. tab. xlviii. figs. 5, 8. J Loc. cit. vol. ii. p. 159.

§ Untersuchungen iiber die Entwicklungsgeschichte der Geburtshelfer-Krsete (Alytes obstetricans), 1 842.

H Annales des Sc. Nat. torn. ii. p. 110, 1824.

184 MR. NEWPORT ON THE IMPREGNATION OF

discovery of the cleavage of the yelk as a process of the fecundated egg ; to Rus-
CONI*, BAER-f-and others, its full exemplification in the Amphibia ; and to BARRY J and
BISCHOFF^ its detection and elucidation in the Mammalia.

The agent immediately concerned in these changes is believed to be the embryo
vesicle and its progeny, produced after the disappearance of the germinal vesicle.
But it is yet uncertain what is the origin of the embryo vesicle, or whether it exists
in the unfecundated ovum. As cleavage of the yelk certainly is not the result of the
disappearance of the germinal vesicle, whicli disappears from all ova of the Amphibia,
whether they are afterwards impregnated or not, I was desirous, at the commencement
of my experiments on impregnation, to learn/rom direct observation whether the un-
fecundated ovum ever passes through any stage of cleavage ; since the ascertainment
of the fact in the negative would be an important test in the experiments I was about
to make. For this purpose it was necessary to collect many pairs of frogs at the
proper season, and when from symptoms which are soon recognized, it was found
they were about to cast their ova, to wait patiently, perhaps for many hours, for the
result, in order that the exact condition of the ovum, impregnated naturally, should
be first ascertained. SWAMMERDAM long ago remarked that the spawning of the frog
takes place very rapidly "by a single effort ||." It is often completed, as I have
found in the English species, in a few seconds, and usually in less than a minute,
during which the male impregnates them, so that if the animals are not closely
watched the opportunity of observing the earliest appearances of the ovum is lost.
Having noted the condition of the impregnated ova of several pairs of frogs within
the first few minutes after spawning, I found those of different individuals vary much
with respect to the white or inferior surface, and exhibit appearances that may readily
be mistaken for the breaking up of a vesicle on the surface. This appearance is due to
a more or less complete state of maturity of the eggs of different broods, and according
as their spawning has been retarded or hastened. The peculiarities are the most
marked in the least matured, the white surface of the egg being the last completed
part, and forming the base of the egg in the ovisac (fig. 5). Having noticed the
appearances of the eggs when impregnated naturally, I was enabled to compare them
with others impregnated artificially, and these with some of the same brood not
impregnated.

Immediately after the frog has spawned the ova form a close rounded mass, which
at first is scarcely so large as a walnut. They then seem to consist almost entirely
of dark-coloured yelks with thin gelatinous envelopes. The form of the egg is then
somewhat oval, with the white portion a little more conical than the dark and
differing slightly in different ova. In some there is a dark spot in the centre of the
white, that looks like a depression or cavity, or perhaps a vesicle. I am not certain

* Loc. cit. f MULLER'S Archiv, 1834.

t Philosophical Transactions, 1839, 1840.

§ Entwickelungsgeschichte des Kaninchen-eies, 1842. || Loc. cit. part 2. p 111.

THE OVUM IN THE AMPHIBIA. 185

that this appearance is in reality a vesicle, and therefore am content to describe it as
a spot, although it conveys the idea of being a vesicle. In some ova there are two,
four or six of these spots imbedded each in a small portion of white substance. When
only a single spot exists, the white surface of the egg for some space around it is more
defined than afterwards, and exhibits faint indications of a crucial division of the
yelk on this surface immediately around the spot. This is the condition of a few ova
immediately after spawning ; but the majority have then advanced farther in their
changes, and show four or six rounded dark-coloured spots at a little distance in the
place of the single central one. When four spots occur they are usually arranged in
a quadrangle, and are less than their own diameter apart. They convey the idea
of being derived from the central one ; but I have never seen any division of this,
and if such division takes place, I think it must occur before or at the very moment
the ova are expelled. In a further advanced stage of the ovum the four dark spots
have become larger, and are each imbedded in a distinct portion of the white surface.

One minute after deposition the spots are more widely separated, and are then each
encircled by a separate patch of white substance. Two minutes after spawning six
dark spots have made their appearance, one of which is situated nearly in the centre,
and the remaining five are so arranged around this that the white patches in which
they were imbedded seem to have coalesced. In three minutes the spots are further
enlarged, and appear joined by a dark line of colour extending from each, so that the
whole form, as it were, a knotted ring that includes a patch of the white surface of
the yelk with one of the dark spots near the centre. In four minutes the ring
around the included white substance is more distinct, and the white surface of the
egg has increased in extent. In a further advanced stage at this period the white
portion included in the ring exhibits the appearance of a white, very opake patch, the
dark spot in the centre having disappeared. Around this opake white patch is the
dark-coloured knotted ring, now become more uniform, and resembling a ragged
chink or slight circular furrow or division in the white surface. The centre of this
hemisphere of the egg thus comes to be occupied by a white patch instead of the
dark spot. At Jive minutes this central white patch, — which, as before stated, and
from what afterwards occurs, may readily be mistaken, on casual inspection, for the
germinal vesicle, altered in its appearance and arrived at the surface, — were it not
that we now know that this has long before entirely disappeared, — becomes more
defined, and the dark circle around it is more uniform and distinct. At ten minutes
the central patch is a little reduced in size, and the circle that incloses it begins to
take the appearance of a diffused halo. Atjifteen minutes the central white patch is
more reduced, and the halo is spread wider, while the whole of this hemisphere of the
egg has acquired a whiter appearance, and become more distinct from the dark
colour of the sides and future dorsal hemisphere. At twenty minutes the central
patch has become still smaller and rounder, and the dark halo much broader.

At this period an interesting circumstance occurs which may hereafter be found

MDCCCLI. 2 B

186 MR. NEWPORT ON THE IMPREGNATION OF

to have some reference to changes in the interior of the yelk, possibly to some rapid
evolution of the so-called central or embryo vesicle in the locality originally occupied
by the germinal vesicle and spot, which, as we have seen, is nearest to the dark surface
— it is the partial rotation of the entire yelk. Up to about this period the ova re-
main undisturbed in the water in a mass as they are expelled, and lie indiscriminately,
some with the dark and some with the white portion of the yelk uppermost, or hori-
zontal. But during the time that has passed since the ova have been in contact with
water, the envelopes have imbibed fluid and expanded until these investments of the
yelk have acquired a thickness equal to about two-thirds of the diameter of the yelk
itself. The yelks that have remained to this time with their white surface uppermost
now change their position spontaneously by a partial rotation of the whole mass of
each on its axis, within the vitelline membrane, until the dark surface of the whole
is placed uppermost. Whether this change of position is merely the result of an ex-
pansion of the vitelline membrane at this period, when the ovum is rapidly ceasing
to be susceptible of impregnation, as I shall presently show is the case after this lapse
of time in the water, or whether it be also connected, as we may fairly believe, with
changes going on in the interior of the yelk, I am not prepared to decide. It is
important, however, to note that the change takes place at about the time at which I
have found a great abundance of bright clear rounded vesicles distributed throughout
the yelk, but chiefly in the place originally occupied by the germinal vesicle. In some
of these vesicles, which I regard as the progeny of the germinal vesicle, I have seen
irregular-shaped nuclei that appeared to be formed of a multitude of nucleoli. These
vesicles convey to me the same idea as those seen by BISCHOFF in mammalian ova,
excepting only that in the egg of the Frog they contain compound nuclei.

At thirty minutes the central patch on the white surface of the egg has almost dis-
appeared, and the halo around it is still more diffused. At forty-five minutes it has
entirely disappeared in most specimens, and its place is occupied by a broad dark
area which includes the boundary of the previous halo, and which appears to be
occasioned by a slight depression in the centre of this surface of the yelk. One hour
after spawning this depression is somewhat deeper. The white surface has become
still more denned, and the dark has acquired a more intensely black colour. The
egg remains in this state without further perceptible change during the succeeding
second and third hour, excepting only that the depression in the white surface becomes
a little deeper, but it has almost disappeared at the end of the fourth hour, when seg-
mentation or cleavage of the yelk is about to take place. But this is not invariably
the case. When it does remain it is always of an oval form, and the primary cleavage
of the yelk, as it proceeds on either side from above downwards, meets hi its centre
and in variably passes through it transversely to its long diameter. These are the first
perceptible changes in ova that are impregnated by the natural union of the sexes,
and when spawning has not been retarded. But in some broods of eggs that have
been retained longer than usual in the oviducts, the whole of these changes have

THE OVUM IN THE AMPHIBIA. 187

already taken place, in so far as regards those of the yelk, the white surface of which
then exhibits an uniform appearance.

Changes immediately before segmentation or cleavage of the yelk. — Segmentation
usually commences in from four to five hours. At about one hour and a half after
spawning, the peripheral layer of cells on the middle of the dark or uppermost por-
tion of the yelk of the impregnated ovum becomes separated from the inner surface
of the vitelline membrane, and this separation goes on until a broad free space
is left between this envelope and the superior layer of yelk-cells. This space, which
we may designate the respiratory chamber, is at first but a small area above
the middle of the dark surface of the yelk, and is commenced above the central
canal. It seems to be occasioned by a recedence towards the interior, or a shrinking,
at this period, of the yelk-cells of the dark hemisphere of the egg, commencing in the
centre of this part and extending gradually, but in a less degree, to the circumfe-
rence. This recedence goes on until the space left between the vitelline membrane
and the yelk is equal to about one-sixth of the diameter of the whole mass, when the
space appears to be occupied by a very transparent fluid, interposed between the now
depressed surface of the yelk and the vitelline membrane. In the centre of the black
surface is the minute orifice noticed by PREVOST and DUMAS*, and BAER!, which leads
into the central canal that communicated with the germinal vesicle in the ovarian
ovum. It is in the margins of this canal that segmentation is commenced. While
the space or chamber between the black portion of the yelk and the vitelline mem-
brane is being formed, and from fifteen to thirty minutes before there is any sign of
cleavage, the yelk becomes extended horizontally in a direction transverse to that in
which the first cleft afterwards takes place, and assumes a transitory obtuse oval
form, which it retains until the yelk begins to divide. The division, as correctly
shown by BAER^, commences in the extension in opposite directions of at first a faint
indentation in the margin of the central canal, which quickly becomes deeper, and is
carried across the surface of the yelk, and gradually more and more deepening and
widening as it proceeds, is carried round the sides, and meeting in the middle of the
depression on the under surface, or of the remains of the white patch when this has
not already disappeared, is completed by passing through the middle of the yelk ;
which is thus divided into two portions. This first division occupies from twenty to
thirty minutes before it is finished, and it is not until then that a second fissure is
commenced. I have not had any opportunity of proving whether this division is the
direct result of subdivision of the central vesicle, and the attraction of the yelk-cells
in equal proportions around each division of that body, as believed by KOLLIKER§,
and, as it seems fair to infer, is the case ; but in addition to the observation by Prof.
SHARPEY||, that the contraction of the entire yelk at the commencement of these
changes, and the movements he has observed among its granules as they proceed, are
in favour of this opinion — I may remark, that the extension of the yelk of the Frog's

* Loc. cit. torn. ii. p. 104, 1824. f MULLEE'S Archiv, 1834. } Loc. tit.

§ MULLER'S Archiv, 1843. || QUAIN'S Anat., Fifth Edition, 1848.

2 B 2

188 MR. NEWPORT ON THE IMPREGNATION OF

egg in a direction transverse to the first cleavage, may also be advanced as conform-
able to the same view. It is further supported by the fact which I have seen in the
egg both of the Frog and Newts, that before the second or crucial cleavage is com-
menced the yelk becomes contracted, so that the first cleft for a time is almost imper-
ceptible while it is extended in the transverse direction, or line of axis of the first
division, after which the second or crucial cleft is commenced. To this I may add
another fact which appears to be equally significant, and which occurs in the unim-
pregnated eggs both of the Frog and Newts, but more especially in the latter.
Although no recedence of the yelk from the vitelline membrane takes place in the
unimpregnated egg of the Frog, it becomes, nevertheless, slightly oval after the first
few hours, but returns to its original shape some time afterwards. But the unim-
pregnated egg* of the Newts is not only separated from the vitelline membrane, but
also is depressed, and has a distinct pit in the centre of its upper surface, and also
assumes an obtuse oval form, both which it retains, when the egg is preserved in
water, until decomposition has commenced.

Changes in the impregnated and unimpregnated Ovum compared. — Having traced
the egg impregnated by natural union of the sexes through its first period of deve-
lopment, I was able to compare the phases it exhibits with those of the artificially
impregnated, and these with the appearances in unimpregnated eggs of the same
brood, placed under precisely similar circumstances with reference to light, heat, air,
water, and locality. The ova experimented on were all procured from the same
female, and the seminal fluid from the male with which she was paired, and at the
time the female was about to spawn.

SPALLANZANI obtained unimpregnated eggs of the Frog for his experiments by open-
ing the body of the female and removing them from the distended oviducts. This
mode is exposed to the objection, that in the removal of the ova they are liable to be
brought into contact with the blood of the animal from the cut vessels, and that the
ova thus obtained may not be the most mature, and fitted for experiment, it seemed
desirable, therefore, to obtain them by another mode, — the total withdrawal of the
influence of sensation, and power of tension in the muscles by division of the spinal
cord through the medulla oblongata. The attempt was made with a female frog
that had been paired for several days, and, from appearances, would have deposited
her ova naturally in the course of a few hours. The spinal cord was divided as
quickly as possible with a strong pair of scissors immediately behind the brain, and
this organ was also destroyed, so that all consciousness was annihilated. The attempt
was successful. The muscles deprived of voluntary power instantly became relaxed
and allowed of the ova being passed, by gentle compression of the body, through the
natural passage without contact with the blood of the animal, in greater or smaller
number at pleasure, and thus afforded easy means of experiment.

I may once for all state that it was in this way that the ova were always obtained
in the following investigations.

* Or, possibly, partially impregnated.

THE OVUM IN THE AMPHIBIA. 189

Recourse was had to a similar expedient to procure the seminal fluid from the
male. SPALLANZANI had obtained the fluid, by vivisection, from the seminal vesicles
themselves. But there seemed more objection to the adoption of this mode with the
male than the female, the certainty of much of the fluid being lost, independent of
the severity of the operation. Indeed SPALLANZANI states, that he was never able to
procure more than from two to three grains from a single individual. I therefore
availed myself of a habit in the male frog, and which SPALLANZANI had previously
noticed and taken similar advantage of in the Newt, to obtain the fluid in greater
quantities than by the mode constantly adopted by that physiologist. When the male
frog, like the Newt, is taken in the hand, or slightly compressed, at the season of pairing,
a quantity of fluid is immediately passed. This consists chiefly of seminal fluid mixed
with water expelled from the effect of the compression, or during the efforts to escape,
as water is passed by other animals at the moment of capture. It abounds with
spermatozoa in their most active state, and thus is fitted for experiment. It required
therefore only to secure the limbs of the animal and compress it slightly, to obtain
the fluid without severe injury. This ready mode was adopted on all occasions
when the fluid was required, and the precaution taken always to examine a portion
with the microscope, to be assured of its nature before employing it. Spermatozoa
have never, during the season of pairing, been absent from it. At the end of the
season they have been less abundant, and spermatozoal cells in greater proportion
than at an earlier period. But in these cases I had reason to think that the chief
part of the fluid consisted of water. It is probable that this was the case in the two
instances of apparent absence of spermatozoa in the Toad, mentioned by SPALLANZANI*,
and that the fluid did really contain spermatozoa, although few in number, and con-
sequently easily overlooked, and that the ova were impregnated by these, and not by
the fluid portion of the semen, as he appears to have supposed.

On comparing the white surface of the yelk of the unimpregnated with that of the
impregnated egg, whether the egg had been fecundated naturally or artificially, I was
not able to detect any difference during the first twelve minutes. The changes went
on in both, and appeared to be almost identical in each. But after the time speci-
fied no further progress was perceptible in the unimpregnated ovum, which con-
tinued to exhibit the same appearance for several hours. But the white surface of
the impregnated egg became more and more changed, up to the time of cleavage of
the yelk, when it was almost an uniform surface.

These observations were afterwards repeated with similar results, and the conclu-
sion to which they led was, that changes take place in the yelk from the period when
the germinal vesicle disappears and the ovum leaves the ovary to the moment of its
expulsion from the body, and which changes may proceed for some time afterwards
quite independent of impregnation ; and that these have some reference to the evolu-
tion of the central or embryo vesicle: possibly also that they do not cease imme-

* Dissertations relative to the Natural History of Animals and Vegetables, 1789, vol. ii. p. 151.

190 MR. NEWPORT ON THE IMPREGNATION OF

diately, but subside gradually when the stimulus imparted by impregnation is not
supplied.

The experiments made to ascertain whether the unimpregnated ovum passes
through any stage of cleavage, consisted of four sets, placed in four vessels of equal
size, containing each about two ounces of water. Ova were passed from the same
female as quickly as possible at the same time into each of these vessels. To one of
these marked A, a considerable quantity of a mixture of seminal fluid, one part to
three parts of water, was immediately added ; to a second, B, only a single drop of
this mixture ; while the third and fourth, C and D, contained only water with the
unimpregnated ova, and the four vessels were then placed in every other respect
under precisely similar conditions.

A few minutes after the impregnating fluid had been added to A, I examined
some of the ova beneath the microscope, and found a vast abundance of spermatozoa
adhering to every part of the surface of their gelatinous envelopes. On other ova
from the set B, there were also many spermatozoa attached, but in much smaller
number than on the ova of set A.

In Jive hours andjifteen minutes, the temperature of the room during the interval
having ranged only from 53° FAHR. to 54° FAHR., segmentation had commenced vigor-
ously, and was strongly marked in the whole of set A. But it had not commenced in
set B. It did not occur in these until Jive hours and twenty-two minutes, when it
began in these also. Thus there were seven minutes' difference in the commence-
ment of the changes in these two sets of ova, a circumstance which led to the belief
that this difference might have some reference to the relative quantities of the impreg-
nating fluid employed, — an opinion which I had long before been led to by observa-
tions on the impregnation of the common Earwig, Forficula, in which it had appeared
to me that deficiency in the quantity of the impregnating fluid is unfavourable to
fecundation.

No segmentation or cleavage of the yelk took place in the sets of ova marked C
and D, which, except in becoming a little oval, as already mentioned of unimpreg-
nated eggs, remained, in so far as the appearance of the yelk surface was concerned,
in the same state as at a few minutes after spawning, and they continued in exactly
the same condition at the end of twenty-two hours. This I have since found con-
stantly to be the case with unimpregnated ova, whether they happen to be exposed to
a high or low temperature of the surrounding medium. The yelk of the impregnated
egg gradually acquires a more intense black colour, which strikingly contrasts with
the dull colour of the unimpregnated.

At the end of six days the majority of the ova in A and B were producing embryos,
while those in C and D were fast decomposing.

These trials afforded the positive test I required from direct observation, as a fixed
point in the experiments about to be commenced, — that segmentation certainly does
not take place in the unimpregnated ovum.

THE OVUM IN THE AMPHIBIA. 191

3. SUSCEPTIBILITY OF THE OVUM.

I was not aware, at the time of commencing my experiments in March 1849, nor
indeed until very recently, of the extent to which the original investigations of SPAL-
LANZANI, and of PREVOST and DUMAS had long ago been carried*, and it has only been
since my experiments were completed, and during the preparation of this paper for
presentation to the Royal Society, I have learned by careful reference to their first
memoirs, that they have anticipated me in part of this inquiry — that of endeavouring
to separate the spermatozoa by filtration from the more fluid portion of semen, and
testing the effect of these two constituents in artificial impregnation. To them
therefore be all honour for the result ; although even they, as they honourably men-
tion, had themselves been anticipated in this by SPALLANZANI, and that too with
similar success. The extraordinary results obtained by SPALLANZANI-|- in artificial im-
pregnation, and the imperfect knowledge which we possess of the nature of the means
by which it is effected, has induced me to endeavour to repeat and vary his experiment,
and to conceive others, which, so far as I am aware, have not yet been attempted. I
have been the more urged to this from the circumstance mentioned by SPALLANZANI,
and already alluded to (p. 189), the occasional supposed absence of spermatozoa from
fluid that is capable of fecundating ; and also from a belief formed long ago with
regard to the Articulata, that the spermatozoa, nevertheless, certainly are the efficient
agents in impregnation, although full proof of the fact has been wanted. I have
been desirous therefore of learning how far this belief can bear the test of direct
experiment, or the fact be capable of demonstration by artificial means in the Am-
phibia. As, however, the experiments I have myself made vary from those of the
authors mentioned, — have not been influenced by the result they had previously
arrived at, — have been somewhat more extended, and, as I believe, will now tend to
place the fact of the direct agency of the spermatozoa in impregnating the ovum
beyond doubt, — it has seemed desirable still to give them in detail, as assisting to
establish an important point of knowledge by facilitating a comparison of the results
of independent investigations.

Duration of susceptibility . — The length of time during which the ovum, after it has
been passed, remains susceptible of fecundation, is affected by several circumstances.
I had reason to believe at the commencement of my experiment that this time is very
short. SPALLANZANI found that when the egg of the Toad was expelled into water it
was not susceptible of fecundation after a lapse of fifteen minutes^. This was at a
raised temperature of the atmosphere, 81°'5 FAHR. On the other hand, he also found
that, at this temperature, ova retained fourteen hours within the body of the female
after death, and of course not in contact with air or water, might still be fecundated ;
and that when preserved in an ice-house fecundation might be effected at two days
after the death of the parent^. But PREVOST and DUMAS arrived at the conclusion||

* Annales des Sciences Naturelles, torn. i. et ii. 1824. t Dissertations, &c. vol. ii.

} Loc. cit. vol. ii. p. 178. § Loc. cit. vol. ii. pp. 176 and 177. II An. des Sc. Nat. vol. ii. p. 135.

192 MR. NEWPORT ON THE IMPREGNATION OF

that the time is much more extended in the Frog ; — that fecundation may take place
when the temperature ranges from 53° FAHR. to 59° FAHR. at the expiration of one
hour after immersion of the eggs ; — that some eggs are fecundated at two, and a
very few even at the end of three hours, after which no fecundation takes place.
The results obtained by myself both on the Frog and Toad have been most in ac-
cordance with those by SPALLANZANI. This is the more worthy of notice from the
circumstance that a slight difference between his and mine is readily accounted for
by a corresponding slight difference of temperature, which SPALLANZANI, and PRK-
VOST and DUMAS, have remarked, and since them also Mr. BELL*, has great in-
fluence on the changes of the eggs and young. The temperature at which my test
experiments were made, was a little lower even than that at which PREVOST and
DUMAS made theirs ; and yet I was not able to find any ova susceptible of fecunda-
tion after they had remained from thirty to forty minutes in water. On careful
examination of PREVOST and DUMAS' experiments, I think the difference may perhaps
be due to a circumstance which seems equally to affect some of SPALLANZANI'S
results, namely, the mode in which the impregnating fluid employed was obtained.
These authors state that the fluid they employed was expressed from the testicles of
the frogs, so that from what we now know of the mode of origin of the sperma-
tozoa, this fluid in all probability contained a large proportion of developmental cells
that included spermatozoa not fully matured, but which might become liberated in
the water at a longer or shorter period. Or, possibly, the fluid added to ova that
had been long in the water, had been very recently obtained ; in which case the
vigorous spermatozoa might effect the impregnation of ova that had become almost
insusceptible through the imbibition of water by their envelopes. I am led to this
view by the fact that the jelly-like envelope of the Frog's egg begins to imbibe and
expand the instant it is brought into contact with fluid ; and from having ascertained
that there is a close relation between the degree of expansion and imbibition of this
envelope and the susceptibility of the ovum to become impregnated, and that these
conditions are also greatly affected by temperature. The act of expansion of the
envelope is an act of endosmose, and possibly this is one of the means by which the
impregnating agent is made to exert its influence on the yelk. The yelk is not a
passive recipient during the endosrnic action of its coverings, but seems to partici-
pate in that action, as I have seen portions of its surface heave and contract within
the vitelline membrane during the first hour the egg has remained in water. It may
thence be inferred, that if the impregnating stimulus be not supplied quickly, the
fitness of the ovum to become impregnated is diminished in proportion as its enve-
lopes are expanded. If then it be proved that the spermatozoon is the agent in impreg-
nation, but, so far as can be discovered, does not penetrate bodily into the ovum or
its envelopes, and yet, as may be shown, must always come into contact with their
surface, the more rapidly and to the greater extent this expansion takes place, and
removes the efficient body from that which it is in some way destined to affect, the

* British Reptiles, p. 92.

THE OVUM IN THE AMPHIBIA. 193

less will be the chance of its impregnating the ovum, and the less will the ovum
become susceptible of impregnation even by the most healthy and vibratile sperma^
tozoa.

The extent and rate of expansion of the envelope of the Frog's egg, during the first
half-hour it remains in water, very nearly coincide with the diminution of the fitness
of the ovum to become fecundated. This is shown by observing the rate of expansion
of the envelope during the first fifteen minutes of submersion, and then testing the
fitness of the ovum, by experiment, during a similar period.

At the moment when the ovum is expelled from the body, the envelope is merely a
thin gelatinous layer, its entire diameter being equal only to about one-sixth of the
diameter of the yelk. After it has been one minute in water, and begun to imbibe
and expand, it is then equal to about one-fourth of the diameter of the yelk. At the
end of two minutes it is enlarged to one-third, and in three minutes to one-half the
diameter of this body. In four minutes it exceeds three-fifths, and in six minutes
two-thirds, and it continues to imbibe fluid and expand at the same rate, until, at from
ten to fifteen minutes, it very nearly equals in thickness the whole diameter of the
yelk ; and at half an hour (fig. 9) it is one-fourth greater than this. PREVOST and
DUMAS* noticed the expansion of the envelope during the first six hours, but entirely
overlooked the rate of expansion during the most important period, the first hour,
and noticed only the general fact that the diameter of the envelope, at the end of the
first hour and a half, was as 5 to 2-5 at the time of spawning, and that it had nearly
acquired its full size at the end of three hours. My own observations agree with this
latter statement. The expansion of the envelope is greatly retarded at the end of
the third or fourth hour, until after cleavage of the yelk has taken place, when it
again proceeds, but much more slowly than at first. If then we bear in mind the
rate of expansion of the envelope during the first half-hour, the following experiments
will give some idea of the degree of susceptibility of the ovum to become impreg-
nated during that period.

Set E. April 6, 1850. — The temperature of the room, at the commencement of this
set of experiments, being 60° FAHR., ova were obtained from a female frog and seminal
fluid from a male, by the mode already mentioned ; the latter being mixed with an
equal quantity of water.

I may here remark, that the ova in each of this set of experiments were placed
in nearly similar quantities of water, and that as it had been shown in the experi-
ments A, B, C and D (p. 190), that segmentation of the yelk proves the ovum to
have been impregnated, although, as we shall hereafter find, not always sufficiently
so as to produce the embryo, I adopted this as a fair test of the susceptibility of
the ovum. I may here also mention, that although the date of making the several
experiments detailed in this paper is recorded, it has been necessary, for reasons
that will be obvious, to disregard the order of time at which the several sets were

* Loc. cit., vol. ii. p. 108.
MDCCCLI. 2 C

194 MR. NEWPORT ON THE IMPREGNATION OF

made, and to detail them when considering the subject to which they more especially
refer.

No. 1 . P.M. 2U 52m. — Eighty-three ova were passed into water, and the impregnating
fluid added to them at the expiration of one minute. This was forty-three minutes
after the fluid had been obtained and mixed with water; but on examination with
the microscope at this period, the spermatozoa contained in it were still very active.
This long period was determined on with a view to the more effectually testing the
susceptibility of the ovum, as it will be shown that the spermatozoa are less and less
efficient in proportion to the length of time they have been mixed with water. Seg-
mentation commenced in a very large proportion of the ova at the end of three hours
andjifty-jive minutes. On the 14th of April, the eighth day, thirty-two embryos had
been formed.

No. 2. P.M. 21' 51m. — Ninety-two ova were passed into water, and at the expiration
of two minutes impregnating fluid was added to the same, forty-three minutes after it
had been obtained. Segmentation commenced in these also at three hours and Jifty -five
minutes, and took place in almost every ovum. On the eighth day there were forty-
five embryos.

No. 3. P.M. 2h 24m. — One hundred and twenty-seven ova were immersed in water
for three minutes, and then exposed and bathed with impregnating fluid during
twenty seconds, water being immediately afterwards added to them. No segmentation
had taken place at the end of four hours and three minutes, but it took place in manv
of the ova at a later period, the exact time having escaped my notice. The fluid
employed had been mixed with water only seventeen minutes. On the eighth day
there were thirty-three embryos.

No. 4. P.M. 2h 15m. — Eighty-one ova were exposed to the air on a dry surface for
three minutes without having been in contact with water, and were then bathed with
impregnating fluid during Jive seconds and water immediately afterwards added to
them. The fluid in this experiment had been obtained only eight minutes. Segmenta-
tion commenced in several ova at four hours and Jive minutes, and on the eighth day
there werefifty-three embryos.

No. 5. P.M. 2h 25m. — One hundred and thirty-six ova were passed into water for Jive
minutes, and were then exposed and bathed with impregnating fluid for several seconds,
and water immediately afterwards added to them. The fluid had been obtained
twenty minutes. Segmentation occurred in one ovum at four hours and eight minutes,
and in others quickly after. On the eighth day only ten embryos had been formed.

No. 6. P.M. 2h 17m. — One hundred and thirty-nine ova were exposed to the air, on
a dry surface, for Jive minutes, and were then touched freely with fluid during Jive
seconds, applied with a hair-pencil, and water was then quickly added to them. The
fluid employed had been obtained twelve minutes. Segmentation took place in four
hours and eleven minutes. On the eighth day there were thirty-seven embryos.

No. 7. P.M. 2h 21m. — Two hundred and Jive ova were retained in water for fifteen

THE OVUM IN THE AMPHIBIA.

195

minutes, were then exposed, well-bathed with impregnating fluid, and water imme-
diately added to them. The fluid employed had been obtained twenty-six minutes.
Segmentation commenced in four hours and fourteen minutes, but was more general
in four hours and seventeen minutes. On the eighth day there were forty-Jive em-
bryos.

No. 8. P.M. 2h 24™. — About one hundred ova were submerged for half an hour, and
impregnating fluid obtained forty four minutes before was then supplied to them, but
not more than six or eight ova became segmented, and only two embryos were formed.

The following summary will more immediately indicate the results: —

TABLE I.— Set E.

Experiment.

Ova.

Time.

Medium.

Fluid obtained.

Segmentation.

Embryos.

Per-centage.

a

//

/ //

No. 1

83

]

Water.

43

3 55

32

•38

No. 2

92

2

Water.

43

3 55

45

•49

No. 3

127

3

Water.

17

4 3

33

•26

No. 4

81

3

Air.

8

4 5

53

•65-5

No. 5

136

5

Water.

20

4 8

10

•07

No. 6

139

5

Air.

12

4 11

37

•26

No. 7

205

15

Water.

26

4 14

45

•22

No. 8

100

30

Water.

44

a

•02

Thus then at a temperature of 60° FAHR. the susceptibility of the ovum to become
impregnated is greatest at the time it is passed into water, and for two or three
minutes afterwards, and segmentation then takes place more quickly, even when the
seminal fluid has been for nearly three quarters of an hour mixed with water, than
after longer immersion. The fitness of the ovum to become impregnated is gradually
diminished, and segmentation takes place more tardily, according to the length of
time which the ovum has remained in water, as is seen by comparing the results of
Nos. 1 and 2 with 7 and 8. On the other hand, while the desiccating effect of expo-
sure to air more arrests the fecundation of the ovum and the occurrence of segmenta-
tion of the yelk than a continuance for a corresponding length of time in water, it
seems to be less prejudicial to the fecundity of the ovum than immersion in that fluid,
as appears to be shown by comparison of Nos. 4 and 6 with 3 and 5, the difference
in the number of ova produced being too great to lead us to attribute this to differ-
ence in the length of time the impregnating fluid had been obtained.

In the foregoing set of experiments, the quantity of impregnating fluid supplied to
the ova was but little attended to, it being added very freely in each case. In the
following set I was desirous of knowing what difference would result from the fluid
being applied more sparingly, or but for a very short space of time. SPALLANZANI
had made experiments with a similar view, but his appeared to be open to some ob-
jections, as he had not noted some important circumstances which greatly affect
the result, as the temperature of the medium, the length of time the fluid employed
had been obtained, &c. In the experiments now made, these circumstances were

2 c 2

196 MR. NEWPORT ON THE IMPREGNATION OP

attended to, and I noticed a curious fact which I first remarked in experiments in
1849. It is what I may designate partial impregnation, and is indicated by a por-
tion only of the yelk becoming segmented. This frequently happens with ova that
have been brought into contact with only very small quantities of seminal fluid, and
but for short spaces of time, as in some of the following experiments. These ova,
so far as I have observed, never produce embryos. Segmentation is arrested in some
at the very commencement (Plate XIV. fig. 11), in others it goes on to the second or
crucial fissure, and in a very few cases may proceed somewhat further (fig. 12), but is
never completed to granulation of the yelk. This, I think, is a fact which deserves
some consideration with reference to the formation of the embryo.

Set F. March 22, 1850. — Temperature of the room at the time of the experiment
was 48°'5 FAHR., and that of the water employed 46°-5 FAHR. As the proof of impreg-
nation is the segmentation of the yelk, and as my object now was to observe the effect
of small quantities of impregnating fluid applied only for very short periods of time
at a low temperature of the surrounding medium, it is not of consequence that this set
was not watched to the full development of the embryo. To show the degree of sus-
ceptibility of the ovum under the combined influence of these circumstances, it was
sufficient to attempt the impregnation at a low temperature, and after the lapse of an
interval to remove the ova to a room of the same, or nearly the same temperature as
in the set E. This was done at the end of one hour and a half.

No. 1. A.M. 12h 12m. — Fifty-one ova, passed on a dry surface, were each touched
lightly and quickly, once only, with a small hair-pencil dipped in impregnating fluid
mixed with water, at eleven minutes after it was obtained, and water was then added
to them. At the expiration of one hour and a half they were removed to a room of
the temperature of 59° FAHR. Segmentation did not occur until the expiration of six
hours and a half, and at the end of eight days only four embryos had appeared.

No. 2. A.M. 12U 20m. — Forty-two ova were immersed in water for Jive minutes, and
then exposed, and touched for an instant only as above, and again placed in water.
The impregnating fluid had now been obtained nineteen minutes. Segmentation com-
menced in some of these at six hours and three quarters, but nearly all of them
were only partially impregnated, and not a single specimen produced an embryo.

No. 3. A.M. 12h 19m. — Fifty-eight ova immersed in water for Jive minutes, were ex-
posed, touched for an instant as above, and again immersed for one minute, after
which, they were well rubbed in the water with a clean pencil, and fresh water then
supplied to them. The fluid employed had been obtained twenty-three minutes.

Not a single egg gave any signs of having been impregnated, either perfectly or
partially, nor did a single ovum produce an embryo.

No. 4. A.M. 12h 23m.— Forty-two ova were passed into a solution of carmine (the
pigment employed by water-colour painters) for Jive minutes, and were then washed
with water, touched for an instant, as above, with impregnating fluid at twenty-seven
minutes after it was obtained, and water then supplied to them. Not a single egg

THE OVUM IN THE AMPHIBIA. 197

'became fully impregnated or afterwards produced an embryo. In two or three ova
there were slight indications of partial impregnation.

No. 5. P.M. 12h 37m. — Sixty-Jive ova having remained^/z/ifeew minutes in water, were
exposed, and thoroughly bathed with impregnating fluid applied with a hair-pencil,
fifty-one minutes after it had been obtained, and water was then added to them. Seg-
mentation did not take place in these ova.

No. 6. P.M. 12h 38m. — Seventy-seven ova, passed into a solution of carmine iov fifteen
minutes, were exposed, and thoroughly bathed with impregnating fluid as in No. 5,
and water was then added ; but not a single egg gave any sign of impregnation.

Influence of Temperature. — From these experiments, it seemed evident that the
susceptibility of the ovum to become impregnated is diminished in proportion to the
degree of expansion of its envelope and its imbibition of fluid, conditions which are
greatly affected by the temperature of the medium in which the ovum is placed during
the first hour; and there seems reason to suppose that this diminution may be due to
the extent to which the envelope becomes influenced by temperature, rather than to
any insusceptibility at that time in the yelk itself. The following experiments, made
a few hours after the above, tend to support this view.

Set G. March 22, 1850. Atmosphere 48° FAHR. Water 47°.

No. 1. P.M. 4h 30m. — Fifty-eight ova were passed from the female from which the
ova used in the preceding experiments were obtained, four hours and a half after
division of the spinal cord ; and seminal fluid mixed with water, and obtained^/^mz
minutes before, was immediately added to the water in which they were immersed.
The ova were removed at the end of twenty-five minutes to a room in which the
temperature was then 62° FAHR. Segmentation took place in almost every ovum a
few minutes within the sixth hour, at which time the temperature of the air was 64°
FAHR., and that of the water 62°. At the eighth day, Jiffy-three out of fifty-eight ova
had produced embryos.

No. 2. P.M. 5h. — Sixty-three ova from the same female were well bathed with im-
pregnating fluid, and water was then added to them. The fluid in this case had been
obtained three quarters of an hour. Segmentation took place in the majority of these
at the end of Jive hours and a half.

No. 3. March 23. P.M. 12h 45m. — One hundred and twenty-four ova from the same
female, twenty-four hours after section of the spinal cord, were passed into water,
and impregnating fluid soon after it was obtained supplied to them. The ova were
placed in a temperature of about 60°, and nearly the whole produced embryos.

These facts proved that the ova employed were still fitted to become impregnated
when the fluid was supplied to them in sufficient abundance, and for a sufficient length
of time, and within the period during which the envelope continues to expand and im-
bibe most rapidly. This condition is always promoted by an early removal from a
low to a comparatively high temperature during the period of expansion, as in SetG,
in which segmentation took place in ftomjive hours and a half to six hours, and when

198 MR. NEWPORT ON THE IMPREGNATION OF

the removal from low to high temperature was within the first half hour ; while it did
not occur, in the only experiment in which it happened in Set F, No. 1, until the end
of sLr hours and a-half, when the removal from a similar low to a like high tempera-
ture was not made until one hour and a half after impregnation.

The influence of temperature is thus as marked in its effects on the impregnation
of the ovum as it can be proved to be on the future development of the embryo.
Impregnation is accelerated, and also is more certain in its occurrence in a high
than in a low temperature. In the latter it becomes retarded and is less determined.
This applies equally to the susceptibility of the ovum, and to the fitness of the im-
pregnating fluid to effect impregnation. But in proportion as this fitness is exalted
by increase of temperature, so is the duration of the capability to receive in the one,
and the efficiency to communicate in the other diminished. SPALLANZANI found that
the ova of toads placed in an ice-house could be impregnated at the end of forty-one
hours*. PREVOST and DUMAS -J- also mention that they were successful, and that too
to a great extent, with ova that had been twenty-four hours in water, the temperature
during the period ranging from 18° Cent. (64°'4 FAHR.) to 22° Cent. (71°'6FAHR.), and
with some eggs that had not been immersed even at thirty- six hours;}:, the temperature
being then from 12° Cent, to 15° Cent. (53°'6 to 59° FAHR.). The results obtained by
myself have been much less successful. Out of one hundred and forty ova obtained
from a female frog, killed twenty-four hours before and preserved at or below the tem-
perature of 550< 5 FAHR., at which the experiment was made, only a very few became
partially segmented, but not one produced an embryo ; although an abundance of im-
pregnating fluid, abounding with spermatozoa, and obtained only a few minutes before
it was employed, had been supplied to them. It is evident therefore that this failure
was due chiefly to the ova, and not to inefficiency of the impregnating fluid. On the
other hand, I have been equally unsuccessful with ova from a frog that had been
killed only two hours and a half when the impregnating fluid employed had been
more than four hours and a half mixed with water. In this case the failure appeared
to have been due chiefly to the spermatozoa, nearly the whole of which, on inspection
by the microscope, were found to be motionless and appeared to have lost their vita-
lity. At the same time it must be mentioned that the female from which the ova
employed in No. 3 of the last set of experiments were obtained still existed, in so far
as the vitality of the muscular system was concerned, and therefore can hardly be
mentioned in comparison with MM. PREVOST and DUMAS' observation. But while the
numerical results obtained by myself have been less favourable than those of SPAL-
LANZANI or the physiologists now mentioned, the general facts, so far as they are open
to comparison, are in full accordance with them. The difference in the details of
our respective observations appears to have been due in chief part to the influence of
temperature at the time of the impregnation of the ova, or within the first two or
three hours after the impregnating fluid has been supplied. Thus, if the temperature
* Dissertations, &c., vol. ii. p. 177. f Loc. cif., vol. ii. p. 140. } Id., p. 134.

THE OVUM IN THE AMPHIBIA. 199

has been gradually rising at the time of impregnation, the fecundation of the ovum,
as I have stated, has more certainly taken place than when the temperature was sub-
siding, the condition of the ova and of the impregnating fluid employed being equally
fit in each case. SPALLANZANI has shown that in his experiments ova did not become
impregnated after they had remained fifteen minutes in water. In the experiments
by myself I could rarely obtain fecundation after thirty minutes' immersion. The
difference of time between these results may fairly be attributed to difference in the
temperature at which the experiments were made, and in great measure to the in-
fluence of this on the endosmosis and expansion of the envelopes. But it was possible
that some other agent might be concerned in these results, and that light, as well as
heat and immersion in water, might greatly influence them. To put this to the test,
and to learn whether the difference depends entirely or chiefly on the amount of tempe-
rature, I have made two sets of experiments at precisely the same time, performed in
the same way, with ova from the same female and impregnating fluid from the same
male, the only difference being that within a very few minutes after the impregnating
fluid was supplied, one set was removed to a higher and slightly rising temperature,
from which all light was excluded ; while the other was allowed to remain freely ex-
posed to light, but in a room of ten or twelve degrees lower temperature, and which
was becoming still further reduced.

The influence of light and heat on the development of the embryo has already
been referred to by SPALLANZANI, PREVOST and DUMAS, RUSCONI, Dr. W. EDWARDS,
and Mr. BELL. RUSCONI expressly states that light has no influence on the develop-
ment of the germ*, but his observations, as well as those before made by SPALLANZANI,
show that heat has a very marked influence, and this has been fully confirmed by
Dr. W. EDWARDS, and Professor BELL. Very recently also the subject has been
referred to by Mr. HIGGINBOTTOM-J-, and I have great pleasure in stating that my
own observations on the influence of heat, and the little effect of light on the develop-
ment of the tadpole, are in accordance with the observations made by him. But
the object I have had most in view has been, as above stated, to mark the effect of
heat, without light, on the changes of the ovum, more especially during the period
of fecundation, the first three or four hours after the egg is laid ; and onwards to the
termination of what I shall hereafter propose to consider, when 'describing the deve-
lopment of the embryo, — as the end of the third period — the closure of the lamina**
dorsales and the establishment of ciliary aeration on the surface of the body.

Set H, March 20, 1850. Atmosphere 59° FAHR. Water 57° FAHR.

No. 1. P.M. lh 14m. — Eighteen ova, as they passed from the body of a frog, were
touched lightly once with a hair-pencil that had been dipped in impregnating fluid
obtained two minutes before, and mixed with about three parts of water. After
these ova had remained ten minutes in water, this was removed and fresh supplied.

These ova assumed the ovoid form at the expiration of three hours and thirty-six

* Loc. cit., p. 20. t Proceedings of the Royal Society, May 16, 1850; and Phil. Trans. Part II., 1850.

200 MR. NEWPORT ON THE IMPREGNATION OF

minutes, and segmentation commenced at three hours and fifty -six minutes, and was
general in two minutes longer, at which time the temperature of the dark cupboard
in which the ova of this set of experiments were placed had been raised to 64° FAHR.,
and that of the water they were contained in to 60° FAHR.

In four days and a half, thirteen of these ova had produced embryos that were then
at the end of the third period of development, and on the eighth day the whole of
them had advanced to the period when they leave the ovum and attach themselves
to the exterior of the envelopes, — the end of the fourth period of development. The
mean temperature of the locality in which these ova and the embryos produced from
them were placed, was about 60° FAHR. for the entire period of eight days.

No. 2. P.M. lh 55m. — Forty-eight ova were touched once, as they passed from the
body of the frog, with a hair-pencil that had been dipped in a small quantity of
residual fluid retained with spermatozoa on a filter, in separating these from the fluid
portion of frog's semen, obtained and mixed with water forty minutes previous.

The temperature of the cupboard having been raised as in No. 1, segmentation
commenced in three hours and Jifty-Jive minutes, bat was more general in four
hours. Many of these ova were only partially impregnated, and of consequence did
not produce embryos. Others passed through their changes, as in No. 1, and in
nearly similar periods of time. On the eighth day ten embryos had been produced.

No. 3. P.M. 2h 3m. — Fifty-seven ova were well bathed as they passed from the frog
with the fluid portion of semen that had passed through two filter papers and been
separated from most of the spermatozoa, and which when examined with the micro-
scope was found to contain only a very few of these bodies.

No segmentation had taken place in any of these ova at the end of four hours and
jive minutes, but several had become ovoid. At four hours and thirty-seven minutes
segmentation had taken place in one ovum, and this alone produced an embryo.

Set I, March 20, 1850. Atmosphere 48° FAHR. Water 47° FAHR.— This set was
the counterpart of the preceding, Set H.

No. 1. P.M. lh 15™. — Nineteen ova were treated in exactly the same way as in
No. 1 H, and at the expiration of ten minutes were removed to fresh water, and placed
where they were most exposed to light.

No segmentation occurred in any of these ova until the expiration of seven hours
and forty-five minutes. The temperature of the room had then sunk to 47° FAHR., and
that of the water with the ova to 46° FAHR. All the changes in these ova were so
exceedingly slow, that at the end of the eighteenth hour, the temperature during
the interval becoming slightly further reduced, the segmentation of the yelk had not
advanced further than to the formation of the first equatorial and secondary median
furrows. On the fifth day the development of the germ had not proceeded further
than to the commencement of the formation of the area germinativa, the end of the
second period, the mean temperature during the interval having been 45°'49 FAHR. ;
while the ova in No. 1 H had reached the end of the third period, the mean tern-

THE OVUM IN THE AMPHIBIA. 201

perature in which they had been retained being 590<5 FAHR. On the eighth day, when,
as already shown, the thirteen embryos of No. 1 H had left the egg, only three had
been developed in this set of observations, No. 1 I, and these had reached only to the
commencement of the formation of the laminse dorsales, the mean temperature of
the room during the entire period being then advanced to 45°72 FAHR.; and the
completion of their third phase of development did not take place until the tenth or
eleventh day.

Thus while three embryos only were produced in this experiment, No. 1 I, during
exposure to light and at a mean temperature now raised to 47°'38 FAHR., thirteen
in No. 1 H were developed in about one-half the space of time from a similar number
of eggs removed from the light, and at a mean temperature of 59°'5 FAHR. ; so that
we seem here to have good reason to believe that a low temperature of the medium
not only retards the development of the embryo, even when exposed to light, but
injuriously affects the fecundation of the ovum.

The result of the next experiment coincides with the above.

No. 2. P.M. Ih56m. — Fifty-one ova were touched in the same way with spermatozoa
from the filter paper, as in No. 2 H, and were retained in the same temperature as
the preceding.

A few of the yelks became ovoid in about six hours, but segmentation did not com-
mence until seven hours and two minutes, and then only in a very few. Two embryos
only were produced from this set.

No. 3. P.M. 2h 5™. — Fifty-jive ova were bathed with filtered fluid in exactly the
same way as in No. 3 H ; but not a single ovum became segmented. Not one pro-
duced an embryo.

Thus while segmentation took place in No. 1 H, in three hours and fifty-six minutes,
when the temperature was rising from 59° FAHR. to 64° FAHH., it did not occur in
No. 1 I, until seven hours and forty-five minutes, when the temperature during the
interval was sinking from 48° FAHR. to 47° FAHR. This sufficiently marks the great
influence of temperature during the earliest periods of change in the ovum ; and this
injurious effect of reduction of temperature at that period is further shown in the
relative number of embryos in these comparative experiments. That the injurious
effect of reduced temperature at the time of impregnation is mainly the cause of this
result, and not the diminution of temperature after the period of impregnation, seems
to be shown in the circumstance, that while at the end of the eighteenth hour the ova
in the set H had already passed through all the stages of segmentation, and the
surface of the yelk had become granulated, and the blastoderma had begun to be
formed even although the temperature in that case subsided a little after segmen-
tation had commenced, — from 59° to 57°, — the corresponding set of ova, No. 1 I, had
advanced only to the octuple division of the yelk. A similar difference in the rate
of development we have seen takes place in the growth of the embryo. At the end
of three days the embryos of set H were advanced to the stage at which the laminse
MDCCCLI. 2 D

202 ME. NEWPORT ON THE IMPREGNATION OP

dorsales are proceeding rapidly to meet, and form the median dorsal sulcus of the
growing body. But the corresponding ova in set I had not been carried further than
to the earliest perceptible indications of the area germinativa.

These facts sufficiently prove the great influence of temperature on the develop-
ment of the embryo in its earliest stages, as the comparative numerical results do
also its effects on the impregnation of the ovum. Out of eighteen ova placed in
the higher and increasing temperature, thirteen produced embryos at nearly similar
stages of growth ; while of nineteen ova maintained in a low and diminishing tempe-
rature only eight became segmented, and but three of them arrived at the tadpole
state.

A somewhat similar but more marked result took place with the ova of No. 2 in
the two sets of experiments. The impregnating means employed in these trials had
already been forty minutes mixed with water on the filter. Out of forty-eight ova
employed in set H, twenty-five became segmented, and ten of these produced young.
But in set I fifty-one ova gave birth to only two embryos.

In the third experiment of each set the difference is as strongly marked. As the
filtered fluid employed in both was the same, and the very few spermatozoa con-
tained in it were by the same means brought into contact with the ova in each, it
might have been expected that each would have produced embryos. But while the
production of a single tadpole in the one case, at a high temperature, may be looked
upon as leading to the inference that these bodies are the efficient agents in impreg-
nation, the entire absence of all appearance of impregnation in the ova of the other
set, to which the same fluid had been equally applied, seems to point to the cause of
failure in this case as depending on the prejudicial effect of a low temperature of the
surrounding medium on their agency.

The temperature of the surrounding medium ought, therefore, always to be borne
in mind when we are attempting to deduce conclusions from experiment on impreg-
nation and development. The presence of light appears to be only of secondary con-
sideration as compared with heat ; since in set H, from which light was carefully ex-
cluded, not only did impregnation take place more certainly and rapidly than in set I
which were exposed to light, but the embryos also were produced in greater number,
and acquired maturity in less than one-half the space of time than in the latter ; the
only difference of circumstance between the two sets being degree of temperature.

Influence of Aeration. — Next in importance to heat is a free aeration of the ovum.
This is of less consequence with reference to impregnation than to the subsequent
production of the embryo. In every set of experiments there are always some ova
more advanced than others. These are ova which have been nearest to the sur-
face of the water, and which, consequently, have been more completely aerated as
well as exposed to a slightly higher temperature than others at a greater depth. It
is from this cause chiefly that the results of experiments on artificial impregnation,
and even of observations on naturally impregnated ova, are always less complete and

THE OVUM IN THE AMPHIBIA. 203

successful than what takes place with regard to the ova in the natural haunts of the
species. The ova in a state of nature are usually deposited in well-aerated places,
clear, slow-moving water, or shallow and but slightly turbid water. It is almost
impossible to afford to ova that are the subjects of experiment, either in broad flat
dishes, or in glass vessels in one's study, the amount of aeration required to ensure
complete success. By too frequently changing the water in the vessels the embryos
often become injured ; while if the water be not changed, development is arrested,
and decomposition commences, and the experiment entirely fails. Even when these
difficulties are obviated by a gentle withdrawal of the water, and a renewal of it with
equal care, the perfect stillness of the fluid in the interval of our observations does not
allow of that extent of aeration to the embryo which it gains in a perfectly natural
state, either in slow-moving waters, where I have usually found the eggs deposited,
or in pools of still water, the surface of which is agitated by currents of air, and
affected by diurnal changes of temperature.

Thus then we may conclude that the procreative force of the germ, and of the im-
pregnating fluid, is augmented by increase of heat, but the duration of the force is
lessened. It becomes less and less energetic in proportion as the temperature is dimi-
nished, but the period during which it is capable of being exerted is extended. In
each of these conditions aeration is of essential consequence, and becomes more and
more necessary in proportion to the increase of heat.

4. THE AGENCY OF THE SPERMATOZOA IN IMPREGNATION.

It is evident from the last-mentioned experiments, that however great may be the
influence of temperature in accelerating or retarding impregnation and develop-
ment, and however much the operation of this influence may be interfered with by
want of proper aeration of the ovum, the impregnating force does not equally pertain
to all parts of the seminal fluid, but is to be found in some only of its constituents.
Experiments made before those now detailed, — and to which I had been led by a
conviction that the opinion formerly entertained, that impregnation is effected
through means of the fluid portion only of the semen, was not in accordance with
facts I had very long been acquainted with in the Articulata, — convinced me that
the spermatozoa themselves, and not the other constituents of the semen, are the
efficient agents of impregnation. LEEWENHOEK, and, as I have since found, PREVOST
and DUMAS, not only believed this to be the fact, but also held the opinion that the
spermatozoa penetrate bodily into the ovum ; and this view has been more recently
insisted on by Dr. MARTIN BARRY, with the additional belief that a perforation or fissure
exists in the envelopes of the ovum, through which the spermatozoon enters. On care-
ful examination of the envelopes of the ovum of the Frog, I have not been able to
detect any fissure or orifice. The question of the agency of the spermatozoa, never-
theless, appears to be capable of solution, however difficult it may be to ascertain the
mode and particular nature of such agency. The separation, as far as possible, of

2 D2

204 MR. NEWPORT ON THE IMPREGNATION OF

the spermatozoa, by filtration from the fluid in which they move, and testing the ova
with these and the fluid separately, afford good proof of the agency of these bodies ;
while immersion of the ova in coloured fluids, at the moment of their passage from
the body of the frog, seems equally fitted to ascertain the believed existence of a
fissure or perforation through the envelopes during their expansion. The experiment
of filtration was originally performed by PREVOST and DUMAS with well-marked re-
sults, and it has since been repeated by the first of these observers * by a different mode,
— endosmose through the operation of galvanic currents. The mode pursued by my-
self was that originally adopted by these observers : — careful mechanical filtration, by
simply passing the fluid portion of diluted semen through folds of filtering-paper. The
paper I have employed, and which alone was fitted for the purpose, was the best
Swedish filtering-paper employed by chemists in their most delicate analyses. A large
proportion of the spermatozoa were always retained, even on a single filter, although
a few usually passed through ; but this, as the results show, was not in reality a dis-
advantage, when a few only were present in the filtered portion. When three or four
folds of filtering-paper were employed, the whole of the spermatozoa were removed.

Filtration of Seminal Fluid.— Fluid obtained from a male frog, immediately after
removal from the female, was mixed with about twice its quantity of water and placed
on the filter. Portions of this fluid as they passed through were repeatedly examined
with the microscope. Some of these filtered specimens contained a very few sperma-
tozoa, usually not more than three or four in the drop 'examined, but sufficient
occasionally, as the results proved, to effect impregnation.

Filtration Experiments. — Set K. March 14, 1849. Atmosphere 55°- 5 FAHR. Water
55° FAHR.

No. 1. A single drop of the Jittered fluid was added to one ounce of water, in which
forty-six ova were immersed. Not a single egg became segmented or produced an
embryo.

No. 2. A single drop of the diluted fluid, not Jiltered, but two hours after it had
been obtained, was added to one ounce of water with ninety ova. Not a single egg
was segmented or produced an embryo.

No. 3. Two drops of Jiltered fluid were added to one ounce of water with sixty ova,
but not one egg became impregnated.

No. 4. Three drops ofjilteredfluid were added to one ounce of water with one hun-
dred and jive ova. Two of these were partially impregnated, as shown by their be-
coming imperfectly segmented (Plate XIV. fig. 11 and 12), but neither of them pro-
duced an embryo.

No. 5. Three drops of diluted fluid, not flltered, but two hours after being mixed
with water, were added to one ounce of water with seventy-six ova. Several of these
became segmented, but more tardily than in the following experiment, No. 7* At the
end of seventeen daysjifteen embryos had been produced from these ova.

* Journal de 1'Institut, 1840, No. 362, p. 908.

THE OVUM IN THE AMPHIBIA. 205

No. 6. Thirty drops of Jittered fluid were added to one ounce of water with two
hundred and ten ova. At the expiration of five hours two ova had become segmented,
and two embryos were afterwards produced.

No. 7. Thirty drops of diluted fluid, notjiltered, were added to water with two hun-
dred andjifty ova. At four hours and forty-two minutes segmentation had commenced
in two or three of these, and in Jive hours had occurred in almost every ovum. Nearly
the whole of these produced embryos.

No. 8. About thirty drops of the same diluted fluid, notjiltered, were added to
water that contained about two hundred ova, passed from the body of a frog killed
twenty hours before. A few of these ova became imperfectly segmented, but not
one produced an embryo.

From these first experiments with filtered fluid, it seemed that the portion of semen
which passes through the filter has not the power of impregnating, unless there are
spermatozoa present in it ; while similar quantities of diluted semen that has not been
filtered, are efficient and impregnate, as in Nos. 4, 5, 6 and 7- Further, it is shown,
from No. 8, if we may judge from one experiment, that ova which have remained in
the body of a frog twenty hours after actual death and cessation of the organic func-
tions, and in a temperature of 55° FAHR., may be affected by the stimulus of the im-
pregnating fluid, but not sufficiently so perhaps as to result in fruitful impregnation.

During the time these ova were under observation, in March 1849, an opportunity
occurred of observing the effect of reduced temperature on the rate of development
of the embryo when its formation has been somewhat advanced. On the seventh,
eighth and ninth days after impregnation of the ovum, and when the temperature
had already been considerably reduced, the season became severe, and in order to
test the effects of cold, the eggs were removed to the open air and exposed to a keen
wind. The temperature of the atmosphere was then 38° FAHR. During the night of
the tenth day, the water in which the ova were contained was frozen to a mass of ice.
Yet many of these ova, as above shown, produced embryos. SPALLANZANI had already
remarked, that the eggs of the Frog may be inclosed in ice, and yet afterwards pro-
duce embryos, if the envelope does not become frozen*.

The experiments made to ascertain whether cleavage of the yelk may be taken as
a test of impregnation (p. 190), seemed also to show that, within certain limits, a
large or small quantity of seminal fluid has some influence on the more or less early
occurrence of this phenomenon. It occurred to me, therefore, that in making the
experiments now given, two questions might be examined : one, as to whether the ex-
tremely minute quantities of seminal fluid disseminated in water, as mentioned by
SPALLANZANI, are as efficient to produce the embryo at the low temperature of the
season at which the Frog spawns in this country, as in the warmer region of
Italy; and the other, whether the presumed efficiency of such minute quantities de-
pended on the presence of the spermatozoa ; and it seemed possible to put these

* Dissertations, &c., vol. ii. p. 49.

206 MR. NEWPORT ON THE IMPREGNATION OF

questions to the test in one set of experiments. But, in attempting to do so, it was a
matter of importance, first, to obtain some approximative knowledge of the actual
quantity of spermatic fluid employed by SPALLANZANI, in his more remarkable expe-
riments, to enable me to compare the results of my observations with those of his.
SPALLANZANI states, that by his mode of obtaining the seminal fluid, by vivisection, he
could only procure from "two to three grains*" from each individual. Three grains
weight of fluid are equal, by measure, to nearly three minims of our medicinal standard.
But it may be presumed that in so delicate an operation as that of removing the fluid
from the vesiculse seminales, from which SPALLANZANI states he usually obtained it,
he could rarely be very precise in his determination of the quantity. I assume, there-
fore, for the sake of comparison, that the quantity he speaks of as "a grain" was
about equal to a minim of our medicinal measure. Three grains of fluid (? minims),
he says, were mixed with a pint and a half of water, and one drop of this mixture
(the seminal fluid in which was equal to 3 ^ pth part of the whole at 16 oz. per
pint) applied directly to an ovum on the point of a needle, was " frequently " sufficient
to render it fruitful-^-. The drop spoken of in this experiment was a much less quantity
than the grain or minim ; indeed, SPALLANZANI states that it did not exceed the
"fiftieth of aline" (? fifth) in diameter. The quantity of fluid obtained by myself
from a frog was usually about six minims, and this I mixed with twice its quantity
of water, thus making eighteen minims. One drop of this mixed fluid measured, as
I found, one-third of a minim, and consequently contained one-third part of seminal
fluid, or one-ninth of a minim of the seminal fluid. Yet the result of the employ-
ment of one drop of this mixed fluid added to one ounce of water, in which the pro-
portion of seminal fluid was then made to be T^Tjth Par' °f tne whole, did not lead
to the same result as the experiment by SPALLANZANI. The difference arose, perhaps,
from the operation of two or more causes : — first, the much lower temperature of
the atmosphere at the time of making my experiment, than at that of SPALLANZANI'S ;
next, the diminished efficiency of the seminal fluid, owing to the length of time (two
hours) it had been removed from the body. Both these circumstances, but especially
the first, as already shown, operate unfavourably in experiments on impregnation,
more eggs being fertilized at a high temperature, when the changes go on rapidly,
and especially when the seminal fluid has been most recently obtained, than under
the opposite conditions. The general results of my experiments, however, may be
regarded as quite confirmatory of SPALLANZANI'S more remarkable one, as they prove,
like that, that only an exceedingly small proportion of seminal fluid is necessary to
fertilize the ovum.

* Loc. cit., vol. ii. p. 189.

t Loc. cit., vol. ii. p. 192. There is some confusion of statement in the translation of SPALLANZANI'S work,
now referred to, respecting the quantities mentioned by the author, as " pint " is the word used in some pass-
ages and "pound" in others (p. 191), apparently synonymously, while the latter is further spoken of as
" twelve ounces." — G. N.

THE OVUM IN THE AMPHIBIA. 207

But however satisfactory these experiments were with regard to that fact, they still
left the question of the immediate agency of the spermatozoa in impregnation in
doubt. I therefore repeated them with greater precision, and with that object only
in view, and took especial care to obtain as perfect a filtration and separation of the
spermatozoa from the fluid as possible. The results were far more interesting and
instructive than I had anticipated, as I found by repeated examination by the micro-
scope that the filtered fluid was almost completely deprived of spermatozoa, one or
two only being occasionally detected in it, with a very few nuclei and spermatozoal
cells. One circumstance that tended to increase the value of this set of experiments,
and to prove the influence of the spermatozoa, was, that the ova employed were not
fully matured, and hence I had less expectation of a favourable result. The quantity
of seminal fluid obtained was larger than usual, and this was mixed with twice its
quantity of water. This mixed fluid was divided, as before, into two portions, one of
which was filtered, and the other not. The experiments were commenced in the
early part of the day, and the temperature of the atmosphere of the room, and that of
the water employed, was nearly the same, 51° FAHR., and the whole of the experi-
ments were placed as nearly as possible under similar circumstances.

Set L. March 18, 1849. Atmosphere 51° FAHR. Water 51° FAHR.

No. 1. Ten minims of the mixed fluid were added to two ounces of water, into
which one hundred and Jifty ova were immediately passed. One hour afterwards I
found a great abundance of spermatozoa adhering to the surface of the envelopes.
Segmentation of the yelk commenced in Jive hours and forty minutes in a few ova;
and took place in others at a later period. A few only of these ova produced em-
bryos.

No. 2. Ten minims of the filtered portion of the mixed fluid were added to two
ounces of water, and about one hundred and Jifty ova were passed into it. When
these ova were examined at the expiration of an hour, not a single spermatozoon was
detected on any of them ; and when repeatedly examined at the end of five and six
hours, not one showed any signs of cleavage. This change did not take place in any
of them even at a later period, and not one produced an embryo.

As the ova in these two experiments were the first that passed from the body of the
Frog, it was fair to regard them as being the most matured; segmentation ought,
therefore, if it occurred at all, to have taken place at an earlier period in these than
in others afterwards obtained from the same female.

No. 3. The Jilter paper employed in separating the fluid used in the last experi-
ment, No. 2, and which retained the separated spermatozoa in a minute quantity of
fluid that had not passed through, was placed in two ounces of water, and one hun-
dred and thirty ova from the same female were shed upon it. When some of these ova
were examined at the 'end of an hour and a half, spermatozoa in vast abundance
were found adhering to every part of their surface, but the whole were then motion-
less, and apparently dead and partially coiled on themselves (Plate XIV. fig. 8 c). In

208 MR. NEWPORT ON THE IMPREGNATION OF

some of these the body appeared to be slightly enlarged. Segmentation commenced
in very many of these ova in five hours and ten minutes, and was almost universal in
them a few minutes later. Nearly the whole of these ova produced embryos, there
being only nine out of the one hundred and thirty that were abortive. I have some-
times found a much larger proportional number of unproductive ova in some broods
in the natural haunts of the Frog.

There was one exceedingly interesting fact in this experiment, — it was that the
smaller and apparently less matured ova were as fully impregnated as the larger and
more perfect. The whole set of observations was the more interesting from this cir-
cumstance. These ova were smaller than usual and had not the white surface so com-
plete, but were very like the ova in which I have described the changes in the light-
coloured surface (p. 185). When first placed in water with the filter paper and
spermatozoa, the surface of the yelks became more contracted and irregular, within
the vitelline membrane, than in other ova I have employed. Hence I had much
doubt, at first, whether any satisfactory evidence would be obtained from this set.
But the contrary has been the case, as it is evident that some ova may be impreg-
nated at a little earlier period than usual ; and that when a great abundance of sper-
matozoa is supplied, ova less matured than others may be equally well impregnated.
The greater efficiency of a larger as compared with a smaller number of spermatozoa,
with reference to the earlier or later segmentation of the yelk, has already been
shown ; and the difference is very marked in the first and third of this set of experi-
ments, both with reference to the occurrence of segmentation and to the relative
fecundity of the ova. On the other hand, the experiment No. 2 proved that the
liquor seminis is not the fecundating portion of the seminal fluid.

Circumstances having prevented me from making known the result of these expe-
riments at the time they were obtained, I determined, during the past spring, to
repeat and vary them, to obtain, if possible, still more conclusive proofs that the
spermatozoa alone effect impregnation. Accordingly, in March and April of the pre-
sent year (1850) I repeated them, with the following precautions : — first, that the frogs
in each case had been some days paired, and at the time of the experiment were
nearly ready to spawn ; next, that the seminal fluid used was obtained from the male
paired with the female from which the eggs were taken ; further, that the condition
of the specimen of fluid used was correctly ascertained ; and lastly, that the ova
were placed in flat dishes, under precisely similar circumstances, with similar quan-
tities of water, repeatedly changed. Two sets of experiments were made at the same
time.

Set M. April 4, 1850. Atmosphere 60° FAHR.

The seminal fluid employed, mixed with an equal quantity of water, was placed on a
single, and caught on a double filter paper, and the clear fluid that passed was then ex-
amined with the microscope. The fluid that had traversed the three filters was almost
completely deprived of spermatozoa ; as, after many very careful examinations, both

THE OVUM IN THE AMPHIBIA. 209

of drops as they passed the last filter, and of portions from the capsule it was caught
in, there was only one instance of the presence of spermatozoa. In this I saw two, but
perfectly motionless. The fluid that passed through the first or top filter, I had
reason to suspect was contaminated by a small quantity having travelled over the
sides of the filter. I knew also, that a few spermatozoa always penetrate through a
single filter, or rather perhaps are carried through by the fluid. Accordingly, I de-
tected several spermatozoa in this portion. The fluid that remained in the interior of
this filter abounded with very active ones. It was not employed until fifty-three
minutes after it had been procured from the Frog.

No. 1. Seventy ova were passed on a dry surface, and a portion of the fluid which
had traversed three filters was poured carefully over them, and water was then added.

In four hours and twenty-five minutes I found one ovum partially impregnated, and
the yelks of others were somewhat shrunk, and a little irregular, an appearance fre-
quently seen in ova that have not been impregnated. Not a single ovum, however,
was fruitful.

No. 2. One hundred and twenty-seven ova were shed into water on the second of
the three filters, which still retained a portion of the fluid from the first, and in
which I had found a few spermatozoa. At four hours and fifty minutes several of the
ova had begun to be divided, and out of this set of eggs sixteen embryos were pro-
duced.

No. 3. One hundred and sixty-three ova were passed into water on the topmost of
the three filters, and four hours and fifty minutes afterwards a very large proportion
of them had become segmented, as was expected, although from the circumstance of
the seminal fluid having been more than one hour mixed with water before it was
employed, the number was not so great as usual. But it was not entirely due to
this cause. Many of the ova had been injured before impregnation, and these became
irregular and pear-shaped. At the end of twelve days forty-nine well-developed em-
bryos had been formed.

SetN. April 4, 1850.

Four filters were now used instead of three, the seminal fluid being obtained and
mixed as before. The fluid which had passed through the whole of these filters, con-
tained not a trace of spermatozoa or of the nuclei of cells. That which had passed
through two only still gave an occasional perfectly motionless spermatozoon, but
not a single one in motion ; the fluid from the uppermost or first filter, as in Set M,
still swarmed with myriads of active spermatozoa. It was thus proved that the filtra-
tion was complete.

No. 1 . One hundred and thirty-one ova were passed on a dry surface, and a portion
of the fluid which had traversed the four filters was poured over them, and water was
then added. But not a single ovum gave any sign of having been impregnated, not
one became segmented, nor was a single embryo produced. The ova became slightly
irregular, shrunken, and depressed at parts of the yelk, a condition which, as I had

MDCCCLI. 2 E

210 MR. NEWPORT ON THE IMPREGNATION OF

noticed this in other ova, I was inclined to attribute to some normal change in the
yelk itself, perhaps of an imbibing or endosmic character.

No. 2. One hundred and ninety-seven ova were shed into water mixed with the
remaining portion of fluid which had passed through the four filters, but not a single
egg gave even a trace of segmentation. Many of the yelks had the same peculiarly
irregular outline as in No. 1. These two experiments I regarded as a satisfactory
proof that it is not the fluid portion of the semen which impregnates.

No. 3. Two hundred and four ova were passed into water upon the third filter,
already immersed in it, and the fluid on which showed an occasional spermatozoon.
I was unable to detect any impregnated ova in this experiment, but, as the result
showed, a few had been affected, as four embryos were produced.

No. 4. Three hundred and seventy-one ova were passed into water upon the first
or topmost of the four filters, and which had already been placed in the water. At
four hours and thirty minutes, almost every yelk had become segmented. The change
had occurred some length of time before this, as the second or crucial segmentation
was commenced. This experiment seemed to be a most direct and conclusive proof
of the agency of the spermatozoa. At the end of four days almost the whole of the
eggs were producing embryos, many of which were advanced to the fourth period of
development. One hundred and twenty-seven became fully formed and vigorous,
besides nearly as many more which did not. complete their changes, from an accidental
cause.

Before these concluding experiments were made, I had already, in March last,
repeated the preceding; but, as the filtration was less perfect, have thought it un-
necessary to give them in detail ; they agreed however in the results. The whole
have confirmed in the fullest manner the experiments first made, and have proved,
as I trust, satisfactorily that the spermatozoa alone are those parts of the semen
which effect the impregnation of the ovum. Having repeated the filtration in five
separate sets of experiments, on different occasions, and with exactly the same gene-
ral results, I can no longer entertain any doubt of the direct agency of the sperma-
tozoa. The conclusion, I think, is rendered certain by facts now shown, which escaped
the notice of SPALLANZANI, and of PREVOST and DUMAS. Segmentation of the yelk
takes place earlier when impregnation is effected by a large, than when occasioned by
a very small number of spermatozoa, the temperature of the surrounding medium, and
all other circumstances, being alike in the two cases, as in the experiments Set A as
compared with Set B (p. 190). This fact is supported by another, equally significant.
When only a very small number of spermatozoa exist in the fluid, then the remarkable
result of partial impregnation often takes place, and the ova are unproductive. On
the other hand, when spermatozoa are supplied in full abundance to the ova, not
only does segmentation of the yelks take place more rapidly, but also more exten-
sively, and almost every ovum produces an embryo.

With regard to the liquor seminis, it seems equally decisive that this portion of

THE OVUM IN THE AMPHIBIA. 211

the seminal fluid does not effect impregnation. WAGNER and LEUCKARDT* have justly
remarked, that it is almost impossible that the liquor seminis can have any action on
ova which are expelled into water before the semen is ejected by the male, as in the
case of frogs and fishes ; and it is worthy of note, that in the experiments now detailed
not a single ovum was either completely or partially impregnated when immersed in
water mixed only with the liquor seminis, obtained through filtration; nor even when
the ova were carefully bathed, as they passed from the body of the Frog, and before
they had been brought into contact with water, with the filtered fluid from which the
spermatozoa had been completely separated, as in Set N, No. 1, or even when the fluid
still contained a very few dead and perfectly motionless spermatozoa. Yet this fluid
can hardly be regarded as entirely without use, although it now appears to be of
very secondary consequence. When the ova were placed in water with which only
the liquor seminis had been mixed, the yelks became contracted exactly as is often
the case in the unimpregnated ovum. Whatever may be the nature of this fluid, it
does not appear to be essential to the conveyance of the structural peculiarities of
the male parent to the offspring. These appear to be communicated by the sperma-
tozoa alone, as not only did the ova that were impregnated by spermatozoa from the
filter paper, as in Set N, No. 4, become segmented quickly, but the embryos pro-
duced from them came forth with all the usual characters of tadpoles, and have
passed, or are now passing (June 20) through their stages of growth as perfectly and
as quickly as others which have been produced in the natural haunts of tne species
through the mutual concurrence of both sexes. Thus the liquor seminis does not
even hasten the course of development of the young. Neither does it accelerate
that of fecundation, either through direct imbibition or from becoming a solvent to
the bodies of the spermatozoa ; as we have seen that segmentation of the yelk takes
place most quickly in proportion to the number of spermatozoa, within certain limits,
in contact with the ovum. And such also is the case in a state of nature.

These facts appear to give that direct negative and refutation to the hypothesis of
the immediate agency of the liquor seminis in impregnation which WAGNER and
LEUcKARDT'f- remark it has not hitherto met with ; and they lead to the supposition
that one of the chief uses of the fluid is merely that of a vehicle through which the
spermatozoa are more readily brought into contact with the ova. Possibly it may
bear that relation to the spermatozoa in the viviparous vertebrata, in which it chiefly
occurs, which the fluid medium into which the ova of Amphibia and Fishes are ex-
pelled, bears to the spermatozoa in those classes. This view may derive some sup-
port from the fact that the liquor seminis has recently been shown by chemical
analysis to consist chiefly of a thin solution of mucus, with small quantities of chlo-
ride of sodium and phosphates and sulphates of the alkalies;}:.

* Cyclopaedia of Anatomy and Physiology, vol. iv. " Semen," part xxxiv. January 1849, p. 507.
t Loc. cit., p. 507. t Id.

2 E 2

212 MR. NEWPORT ON THE IMPREGNATION OP

5. NATURE OF THE AGENCY OF THE SPERMATOZOA.

Having obtained full evidence by direct experiment that impregnation is effected
through means of the spermatozoa, we have now to inquire as to the manner in
which it is induced by them, and as to the nature of the agency they exert. Sperma-
tozoa have been found adhering to the surface of the impregnated ovum by BARRY,
BISCHOFF, POUCHET and others, in the Mammalia; as they were long ago seen by
PREVOST and DUMAS on the ovum of the Amphibia ; and since by SIEBOLD, KOLLIKER,
myself and others in the Invertebrata. They have been constantly present in those
experiments on artificial impregnation which I have now detailed, in which the yelks
became segmented after the egg had been in contact with seminal fluid, or with the
filter paper used to remove them from the liquor sanguinis ; but they have not been
detected on ova which did not undergo the cleavage of the yelk, or which had been
immersed only in fluid separated during nitration. Whenever present in fluid in which
ova have become impregnated, the spermatozoa have always been found in motion,
until after they have become attached to the surface of the ovum, when their motion
has soon ceased. In the Lissotritons, in the few experiments I have made on this divi-
sion of the Amphibia, I have seen the motion continue for a long time after they have
been in contact with ova. This leads to the supposition that a vibratile condition or
power of motion is in some way essential to their power to effect impregnation ; not-
withstanding that, as WAGNER and LEUCKARDT have remarked*, no movements have
as yet been perceived in the spermatozoa of the Isopoda and Amphipoda. If this vibra-
tile condition be essential to their function, then the length of time which it is con-
tinued may be of importance.

Duration of Motive Power in Spermatozoa. — SPALLANZANI found that water mixed
with but a small quantity of seminal fluid of the Frog retained the property of im-
pregnating ova longer than pure semen-f-. When inclosed in a glass tube, the semen
of the Toad was not impaired at the end of six hours, but was useless at the end of
nine;}; ; while a small quantity from the Frog, mixed with water and preserved at a
temperature of about 40° FAHR.§, was still efficient at the end of thirty-five hours. Re-
sults obtained by myself have fallen short of this extended period, even when the in-
fluence of temperature has been attended to. The difference of result may to some
extent be accounted for in the condition of the fluid, and in the way in which it has
been obtained. SPALLANZANI says he obtained it both from the vesiculae seminales
and the testes after opening the body. It is probable therefore that a large part of
what he procured had not arrived at maturity, and instead of consisting almost en-
tirely of active spermatozoa, as when it is obtained by compression of the body, the

* Loc. cit., p. 503. f Dissertations, vol. ii. p. 193. I Id. p. 168.

§ It may be well here to mention, that for the purpose of more easily comparing the observations recorded
by SPALLANZANI, and PREVOST and DUMAS, with the results obtained by myself, I have, throughout this paper,
reduced the data given by them to the scale of FAHEENHEIT, employed by myself; the scale used by SPALLAN-
ZANI being that of REAUMUR, and by PREVOST and DUMAS the Centigrade.

THE OVUM IN THE AMPHIBIA. 213

mode adopted in my experiments, it included a large proportion of developmental cells
from which the spermatozoa escaped at longer or shorter periods after the fluid had
been mixed with water, or had been retained for some time out of the body. I have
very rarely found the seminal fluid of Rana temporaria obtained in the way stated,
and at a temperature of about 50° FAHR., retain any impregnating influence for more
than four or five hours. Thus, after mixing it with an equal proportion of water, very
many of the spermatozoa have soon become motionless, and in less than two hours a
moderate proportion only have continued active. At three hours there have been
still fewer moving; while at four hours the great majority of them have exhibited
most unequivocal signs of lost vitality, being either extended at length or coiled
on themselves (Plate XIV. fig. 8 c), as they usually appear when motionless, arid adhe-
rent to the surface of ova. If any, at this length of time, have been still moving in
the fluid, they have been few in number, and their motions exceedingly feeble. Occa-
sionally I have detected others, at this lapse of time, in the act of escaping from the
cells (fig. 8 a and b), and these have always been the most energetic in their move-
ments immediately after their liberation. Further, I have noticed that in those
specimens of fluid which have contained most developmental cells, the spermatozoa
have been longest in a state of activity.

The following have been the results of observations on spermatozoa attached to the
surface of ova, or contained in the water in which ova were immersed. At three-quar-
ters of an hour after mixing recently obtained seminal fluid with the water and ova,
vibratile spermatozoa have continued to be exceedingly abundant and in a state of
great activity. At one hour and a quarter there were still an abundance in motion,
but many were now perfectly motionless, and apparently dead. At one hour and u
half I was not able to detect any movement in even a single spermatozoon out of a
vast abundance which adhered to the surface of the gelatinous coverings of the ova,
although I sought for this very carefully. Neither could I detect even the slightest
indication of the spermatozoa having penetrated into these coverings, either near
the surface or in the vicinity of the thicker envelope, which I regard as the chorion,
and which immediately covers the vitelline membrane. After a lapse of some time
all the appearance of spermatozoa on the exterior of the envelopes ceased. The
longest period, after contact with spermatic fluid in water, at which I have hitherto
been able to recognize these bodies on the surface of the frog's egg has been six
hours and one or two minutes, and about half an hour after segmentation of the yelk
had commenced. This was on ova impregnated artificially, on the 14th of March, at
a temperature of the atmosphere of the room of 54°'5 FAHR. and 530>5 of the water
employed. A few motionless spermatozoa were then still found on the surface, but
most of them appeared to be becoming disintegrated. The surface of the egg-envelope
was then covered at-places with numerous small granules, possibly the remains of
spermatozoa which had disappeared.

A somewhat similar result has ensued when spermatozoa have been two hours mixed

214 MR. NEWPORT ON THE IMPREGNATION OF

with water before ova were passed into it. hi Jive minutes after immersion of these
ova, I have found large quantities of spermatozoa already adhering to the surface of
their then expanding envelopes. But many of them have already been coiled on them-
selves, and were perfectly motionless. The water still contained very many dissemi-
nated through every part of it, but most of them, with few exceptions, have appeared
to be rigid, and to have become enlarged in diameter, but not increased in length.
This change has appeared to be due to the hygroscopic nature of these bodies, as
formerly pointed out by SIEBOLD*. Possibly this nature may have some reference to
impregnation. Repeated observations lead me to believe that, in whatever way the
spermatozoa are concerned in impregnation, they do not penetrate bodily into the
ovum, but merely adhere to the surface.

PREVOST and DUMAS concluded-)- from their investigations, that the spermatozoa of
the Triton and Frog do penetrate into the envelope of the egg ; and they state that
they had fecundated ova taken from the ducts of the Triton, and after the lapse of
three hours, having first carefully washed them to remove all that were merely
adhering to the surface, have made sections of the envelopes of the egg, and, with the
aid of the microscope, have found living spermatozoa still struggling within. Their
words are — " Une grande quantite" d'animalcules encore mouvans, et qui semblaient
se debattre dans cette espece de ge!6e ou ils se trouvaient emprisonne's. On en voyait
partout meme au contact des membranes de 1'oeuf." Further, that they had repeated
this experiment on the ova of the Frog, and found the envelope penetrated in like
manner with spermatozoa, still moving, but not changing place. I regret much that
my investigations do not enable me to confirm these observations, which seem to me
to be due to the circumstance of these physiologists having regarded the objects on the
surface as being in the interior. I have many times sought for spermatozoa within
the substance of the egg-envelope of the Frog, at different periods between that of
first contact with impregnating fluid and the time when cleavage of the yelk has com-
menced, and have constantly found them on the surface, but have never, even in a
single instance, observed any appearance of them in the substance of the envelope,
nor anything which induced me to suspect that they penetrate bodily into it. They
have been present on the surface, and adherent to it, even from within a few seconds
after contact, to so late as the sixth hour, but have usually been motionless ; and most
of them have had the caudal portion folded back on the body. I was led to make
these observations on the egg of the Frog, — before I was aware of MM. PREVOST and
DUMAS' views, — from the circumstance of Dr. MARTIN BARRY having mentioned;}; that
an orifice or fissure exists in the thick investing membrane of the ovum of the Rabbit,
through which, at the time of impregnation, he believed the spermatozoon to enter.
All the observations I have been able to make on the ovum of the Frog, both micro-
scopically and experimentally, are opposed to the belief in the existence of any perfo-

* MULLER'S Archiv, 1836. f Loc. cit., vol. ii. p. 133.

+ Philosophical Transactions, 1840, p. 535.

THE OVUM IN THE AMPHIBIA. 215

ration either before or at the time of impregnation. With regard to the ovum of the
Tritons, I have recently made the following observations, since becoming acquainted
with the views of the authors named.

A female Lissotriton punctatus, obtained on the 17th of May, produced several ova
on the morning of the 19th. Cleavage of the yelk (which, I may remark, was entirely
overlooked by RUSCONI* in his account of the Newts) commenced in two of these ova
at the expiration of eight hours, the temperature during the period having ranged from
56° FAHR. to 62° FAHR. On examining these ova very carefully about an hour before
the cleavage commenced, there were what I regarded as portions of the bodies of
spermatozoa on the surface, but certainly no traces of any in the interior. While
engaged in this examination the same female produced another egg, which she
inclosed as usual in a folded leaf. On this specimen, examined at the end of half an
hour, I could not detect any spermatozoa on the surface, which led me to imagine
that it had not been impregnated, a supposition which ultimately proved to be correct,
as no segmentation of the yelk took place in it. Some time after this, the tempera-
ture of the atmosphere being 62° FAHR., I saw the same newt enclosing another egg in
a leaf. This I immediately removed for examination, and thinking that this, like the
previously deposited egg, had not been impregnated, no traces of spermatozoa being
found on its surface, I placed it for about a minute in a small capsule filled with
water, into which a quantity of fluid had just been expressed from a male that had
been kept separate from the female. The fluid on examination was found to be com-
posed almost entirely of very active spermatozoa. The egg was examined three
minutes after immersion, and scarcely five minutes after it had been laid, and multi-
tudes of spermatozoa were then seen adhering to its surface. Most of them were still
vibrating rapidly, while others were motionless. But although I was able to distin-
guish every part of these bodies, I could not detect any in the act of penetrating, or
which had already penetrated into the substance of the envelope, and most certainly
not one was imbedded in the interior. Neither were there any in contact with the
yelk-membrane, or in the yelk-chamber of the envelope.

The egg of the Newt is peculiarly fitted for an examination of this kind, from the
fact of the existence of this yelk-chamber, or space in the interior of the envelope.
This is formed by the gelatinous covering which the egg gains in the oviduct imbi-
bing fluid by endosmose and becoming expanded immediately it comes into contact
with water, when the inner layer separates from the vitellary membrane, with which it
has been in contact within the duct ; and as the outer layers more and more expand,
the yelk, covered only by the vitellary membrane, is left free in a large cavity in the
interior, surrounded by a thin fluid. The spermatozoa of the Newts, as is well known,
are of large size, and are easily recognized ; so that in the event of their having pene-
trated the egg-covering before it leaves the duct, or at the moment of its expansion,
or after the chamber has been formed in it, they can hardly escape observation.

* Amours des Salamandres Aquatiques, 4to. Milan, 1821.

216 MR. NEWPORT ON THE IMPREGNATION OF

I preserved this egg confined to one spot in a minute glass capsule, and in exactly
the same position beneath the microscope, for forty-eight hours. During the first
three quarters of an hour many of the spermatozoa on its surface exhibited as vivid
motions as at first, but still adhered to the same parts, and had not, so far as I could
perceive, changed their posture or their place in the least, or had penetrated in the
slightest degree into the envelope. At the end of twelve hours I found that the yelk
had undergone the usual process of cleavage, which, at that time, had already been
advanced to the stage of coarse granulation of the surface, a fact which proved most
distinctly that this egg had been impregnated. Spermatozoa were still distinctly
recognized over the whole surface of the envelope, but their motions had now ceased.
On the following morning, May 20th, the changes were found to have proceeded un-
interruptedly, as the yelk was then finely granulated over its whole surface. At a
little later period, the end of twenty -four hours, a few spermatozoa were still adhering
to the surface of the envelope, but the whole were perfectly motionless, and many
had evidently disappeared. Still, not one could be detected in the substance or in
the chamber of the envelope. At twenty-eight hours the granulation of the yelk was
nearly completed, and its surface was becoming smooth, and there were still a very
few motionless spermatozoa on the exterior of the envelope. At the end of forty-eight
hours, May 21st, I was still able to recognize the bodies of several spermatozoa which
had not yet disappeared, but which had become very indistinct, as if in a state of
diffluence. At the end of sixty hours I could no longer detect any trace of them.
The egg that was the subject of these observations proceeded regularly in its changes,
and ultimately produced the embryo ; all the stages of which I have traced and
delineated for future communication to the Royal Society.

Subsequently to these observations I saw ova passed by another female, Lissotriton
palmipes, and on submitting these to the same close examination as the above, within
five minutes after their production, I again found what I regarded as the remains of
spermatozoa on the surface of the egg-covering, but not a trace of any in the in-
terior or in the vicinity of the yelk.

Endosmosis of the envelopes during impregnation. — But although the facts now
mentioned are so opposed to the view that the spermatozoa penetrate bodily into the
ovum, it is due to the distinguished observers to whom I have referred to enter some-
what more fully into the questions which their observations involve ; and while I am
free to admit the possibility of mistake or oversight on my part, to mention the
details of some experiments made expressly with the view to ascertain whether the
envelopes of the ovum of the Frog are permeable in any part to solid particles of
matter ; or whether there exists any orifice in them by which such particles can enter.
It is well known that during the transit of the ovum through the oviduct the vitellary
membrane becomes invested with a thick gelatinous covering, the first thick layer of
which may be regarded as the rudimentary chorion, and perhaps may be analogous
in its function, in some respects, as it seems to be in its place and mode of origin, to

THE OVUM IN THE AMPHIBIA. 217

the albuminous investment of the ovum of the Rabbit. I cannot, with RUSCONI*,
regard this envelope of the Frog's egg as being merely a mechanical protection
during the process of development. It is formed of cells with distinct nuclei, and
from what I shall presently mention, seems to be essential to the ovum at the com-
mencement of the changes, and to be intimately connected with the act of impregna-
tion. RUSCONI deprived the ova of the green aquatic frog, ? Rana esculenta, of their
gelatinous envelopes at a period subsequent to impregnation, and found that they
passed through their changes as well as when covered by them ; and he thence con-
cluded that the envelope is of no use further than to protect the egg from the injury
it might receive through mechanical disturbance, "des petits chocs qui pourroient
nuire a son developpement-{~." Certainly it affords this protection to the germ, but
to conclude that this is its sole office appears to be somewhat premature. I have
found that it is almost impossible to remove this envelope from the Frog's egg at
the moment of deposition, or even during the first few minutes after submersion,
and before it has become expanded by imbibition of fluid ; although it may be removed
without much difficulty from the egg of the Newt, the yelk of which, in the vitelline
membrane, lies free and unattached in its interior. But some time after the expan-
sion has taken place I have myself found that the frog's egg may be deprived of
a large portion of this covering, and yet produce an embryo equally well as if it
had remained protected. On the other hand, one most important function of this
investment seems to be indicated in the following facts. SPALLANZANI found that
ova of the Frog deprived of their envelopes before contact with the male influence,
were not impregnated ; and further, that ova taken directly from the ovaria, are not
susceptible of impregnation^.

A remarkable fact which I noticed, at a time when I was not fully aware of its
interest and importance, enables me to confirm this observation. I captured a pair
of frogs, the female of which, a short time after they were in my possession, had a
large hernia formed by a protrusion of part of the great oviduct through an acci-
dental wound in the posterior part of the right side of the body, and in consequence
of which she was unable to deposit her ova. This wound had been received before
the union of the sexes, but the hernia was formed afterwards, during the passing of
the ova from the ovaria. The result of this was, that when some of the ova had
passed into that part of the duct which protruded through the wound, the sac formed
by it was constricted, and became so enlarged by the expansion of the egg-envelopes,
that the remainder of the ova were prevented from entering it. On opening the ab-
domen of the frog after death, I found that a very large proportion of the ova which
had left the ovariurn on that side of the body, were lying in the cavity of the perito-
naeum, among the viscera, being entirely prevented from entering the duct, which
was filled throughout its whole extent, to its very orifice, with eggs which had already
entered, and were prevented from passing on. On the left side of the body the ova had
* Loc. cit., p. 8. f Loc. tit., p. 9. + Dissertations, &c., ii. 152, 3.

MDCCCLI. 2 F

218 MR. NEWPORT ON THE IMPREGNATION OP

also quitted the ovarium, but the whole on that side had passed into the oviduct in the
usual way. The eggs found in the cavity of the body consisted only of yelk-masses
in their vitelline membranes. I immediately placed some of these eggs in water, with
seminal fluid obtained from the male with which this female had been paired ; but
not a single egg became impregnated, or gave afterwards any sign of formation of
the embryo. I had some hesitation in regarding this experiment as quite conclusive, —
that impregnation cannot take place before the egg has gained its gelatinous envelope,
and consequently while it is still within, or has but just escaped from the ovary, —
from the possibility that these eggs might have been for some time in the cavity of the
body, and that some change might have been induced in them through long deten-
tion. In so far, however, as that this was in accordance with SPALLANZANI'S experi-
ment, it seemed to point to the nature and importance of the covering which the
egg gains in the oviduct.

Since my attention has been more particularly directed to this point of investi-
gation, I have repeated the experiment on the ova of the Newt, Lissotriton palmipes,
with precisely similar results. I opened the body of a female with great care (after
dividing the spinal cord through the medulla oblongata), for the purpose of ob-
taining ova from the oviducts, for artificial impregnation, and immediately saw that
a number of ova were free in the cavity of the abdomen, and were in the course of
being transferred to the entrance of the tubes, as stated in the first part of this paper.
These ova, like those which had recently escaped from the ovarium in the Frog, were
without any other covering than their vitelline membranes ; most certainly I was
unable to detect any other, and they were so delicate that it was with difficulty they
were removed into water to which fluid from the male had been added. But
although uninjured in the removal, and in every way carefully treated, not one gave
any sign of cleavage of the yelk, which, as I have before stated, I have constantly
found take place in the impregnated ova of newts as well as of frogs, although the
fact of its occurrence was overlooked by RUSCONI ; not one egg afterwards produced
the embryo. Thus then it seems fair to conclude that the egg in the Amphibia is
not fitted for impregnation until after it has entered the oviduct and acquired its
gelatinous covering.

I have already shown that there is a remarkable coincidence between the rate of
expansion of the gelatinous covering, immediately after the egg is placed in water,
and the susceptibility of the egg to become impregnated ; and that in proportion as
the covering becomes enlarged and distended by imbibition of water, the susceptibi-
lity of the egg becomes diminished ; until at the end of about half an hour it is
almost completely lost, at which time the rate of expansion of the envelope is also
greatly lessened, and the envelope itself has attained to more than two-thirds its future
diameter. Now SPALLANZANI found that the susceptibility of the ovum, when im-
mersed in water, had ceased at the end of fifteen minutes, at which time the envelope
is considerably enlarged. PREVOST and DUMAS also observed that the expansion of

THE OVUM IN THE AMPHIBIA. 219

the envelope is greatest during the first three hours, and rightly regarded the occur-
rence as connected with impregnation, and with this opinion made experiments to
test their views. They placed ova taken from the oviducts in ink, and found the en-
velopes blackened with the imbibed fluid* ; but they remark — " bientot cette imbi-
bition s'est arretee a cause de la reaction chimique de 1'encre qui coagulait la matiere
nmqueuse." Afterwards they employed the blood of the Frog mixed with water, and
found the envelope deeply reddened when immersed in it ; and thence concluded
that the envelope in its normal condition admits of the entrance into it of solid par-
ticles of matter held in suspension in the fluid. But each of these experiments
appears to be open to a different explanation. It is probable that the chemical action
of the ink, by altering the condition of the envelope, allowed of the admission into it
of solid particles only in proportion to the change in the tissue ; and that the colour
given to the envelope by frog's blood was due to particles of colouring matter which
adhered to the surface, rather than to the admission of these into the substance of the
tissue. This conclusion is founded on the following trials.

The imbibition of water by the covering of the egg being so distinctly marked, I
had intended, like the authors mentioned, to endeavour to ascertain whether coloured
water could be as readily absorbed as pure water. Through accident, however, I
omitted to put this question to the test until late in the season of last year, and after
the whole of my frogs had spawned. But having placed several ova in rectified spirit
for future examination, at the moment of passing them from the body of a frog, I
determined to test the result of the immersion of these in coloured fluid, although
well aware of the correct objection that would be made, that the experiment must of
necessity be inconclusive. I put some of these into a solution of carmine in water,
and watched the result. The envelope, which, while the egg was in spirit, was white,
opaque, and adhered closely around the yelk membrane, began to imbibe and expand
the instant it was placed in the solution ; and at the expiration of an hour there
seemed reason to believe that the trial had succeeded. The envelope was much en-
larged, and the fluid had penetrated into it, carrying with it some colouring matter ;
as on carefully washing the surface repeatedly in clean water, to remove the deposit
on the exterior, the substance of the interior was seen to be coloured, and it was evi-
dent that the colouring matter had penetrated as fay as the deepest or thick layer of
the envelope. This result appeared to favour the view that the spermatozoon enters
the ovum. When the ova were again examined at the end of the third day's immer-
sion, they were of a deep red; the deepest stratum of colour being then between the
vitelline membrane and the thick or innermost layer of the envelope. The entire egg
presented the appearance of a globule or bead of red glass, with a dark red centre,
surrounded by a lighter-coloured halo. The eggs were then thoroughly washed and
placed in clean water. At the end of six hours part of the colouring matter had
again been removed from the interior, and the eggs were of a less deep hue, and the

* Loc. tit., ii. p. 132.

2 F2

220 MR. NEWPORT ON THE IMPREGNATION OF

water they had been immersed in was coloured. I then again removed them to clear
water. At the end of sixteen hours they had parted with more colour, but were still
red, more especially between the inner portion of the envelope and the vitelline mem-
brane, and the water had again acquired a red hue, thus showing that both endos-
rnose and exosmose must have taken place.

As it might fairly be objected that these ova, changed by immersion in spirit, were
unfitted for experiment, I made trial with others which had not been impregnated,
and being infertile, had remained in water many days without giving signs of decay.
When these were placed in the carmine solution, their envelopes became as deeply
and thoroughly imbued with colouring matter throughout their whole substance as
in the former ; and when placed in clear water they parted as readily with a portion
of it, so that it was evident that whenever the density of the fluid in which these
dead and infertile ova were immersed, was altered, a change by endosmose or exosmose
immediately took place in the fluid retained mechanically in their tissues. To this
cause, perhaps, may be ascribed the colouring of the ova in PREVOST and DUMAS'S
first experiment with ink, while other experiments, which I shall mention, made on
living and impregnated ova, lead me to regard the colour in the experiment with
frog's blood as merely the result of adhesion of colouring matter to the surface.

The immediate objects I had now in view were, to learn whether impregnation is
effected by any direct and palpable infiltration of seminal matter through the enve-
lopes of the ovum ; — whether the admixture of other matters with the seminal fluid
will prevent or arrest impregnation; — and whether the spermatozoa collected on a
filter paper, and then placed with this in a fluid of great density, are as efficient as in
clear water.

With these views, I prepared a very dense solution of carmine pigment in water,
and added parts of this to small quantities of water with ova, either before the
seminal fluid was mixed with the water, or immediately afterwards, and I expected
the results to show whether any solid particles, held in suspension in the fluid, passed
through the envelopes. The previous trials had shown that solid particles do pass
through the dead tissue, but it was doubtful whether the like result would occur in
the living.

Carmine Experiments. — Set O. March 13, 1850. Atmosphere 53° FAHR.

No. 1. Eleven unimpregnated ova were passed into water mixed with carmine.

The envelopes became as fully expanded, and imbibed fluid as freely as in the im-
pregnated ova, and acquired a red tint ; but much of the colour was due to the deposi-
tion of granules of matter on the surface, while I was unable to detect any similar
granules within their texture. On the contrary, on removing part of the surface of
the envelopes, the interior, although slightly reddened, exhibited an uniform appear-
ance.

No. 2. Thirty ova were passed into water that had been mixed with seminal fluid,
and immediately afterwards a solution of carmine was also added.

THE OVUM IN THE AMPHIBIA. 221

Twenty-six of these ova became impregnated and produced embryos ; thus showing
that impregnation takes place very quickly, and is not prevented by the addition of
a dense colouring fluid, added after contact with the impregnating fluid.

No. 3. Forty-one ova were passed into a solution of carmine in water which had
been mixed with seminal fluid immediately before the passing of the ova.

Thirty-three of these ova also produced embryos. It was evident, therefore, that
when seminal fluid is freely mixed with a dense medium that holds solid particles of
matter in suspension, the spermatozoa are not necessarily prevented from effecting
impregnation of the ova. Thus the ova of the Frog, although usually deposited in
slow-running or clear still water, may be deposited even in slightly turbid water
without impediment to the natural process of impregnation, as the water and sper-
matozoa may be brought into contact with the ova at the same instant.

No. 4. Thirty ova were passed into water mixed with fluid that had been almost
completely deprived of spermatozoa by filtration.

Only one ovum exhibited any signs of impregnation, but not a single embryo was
produced.

No. 5. About two hundred and twelve ova were passed into a dense solution of car-
mine and water in which the filter paper with spermatozoa, separated from the fluid
employed in No. 4, had already been placed, and the water and ova were then freely
agitated together.

The result of this experiment was very marked. Only a few of these ova became
segmented, and the change proceeded much slower in them than in the ova of expe-
riments Nos. 2 and 3. At the end of twelve days only Jive embryos had been pro-
duced. Thus a dense solution of carmine, applied to the spermatozoa before they are
brought into contact with ova, may have the effect of preventing impregnation, appa-
rently by operating as a mechanical impediment. These ova, excepting only a few
removed for the following experiment, No. 6, which were taken from the mass as
stated, were allowed to remain in the carmine for twenty-four hours before they were
placed in clear water.

No. 6. Forty ova taken from the last experiment were^rernoved to clear water at
the end of one hour and a quarter, having first been thoroughly washed. The result
was as decided as in No. 5. Only two embryos were formed; so that there was
further reason to believe that impregnation takes place very quickly, and that the
result in No. 5 was not entirely due to long continuance in the solution, but to some
impediment at the time of contact.

No. 7- A thick solution of carmine was mixed with seminal fluid and water, and
three minutes afterwards a mass of ova were passed into it.

This experiment was similar to No. 3, excepting only that the solution of carmine
was much more dense, and the ova were not passed until three minutes after the
fluids had been mixed. There was a marked difference in the result. Only a few of
these eggs became segmented, and only eight out of a large mass produced embryos.

222 MR. NEWPORT ON THE IMPREGNATION OF

But there were several eggs that appeared to have been partially impregnated, the
whole of which were abortive. Partial impregnation, as before stated, is shown in a
very imperfect cleavage of the yelk, sometimes on one surface only, and sometimes
complete as regards one half of the yelk, but imperfect or irregular in the other.

No. 8. Forty-two ova from the last experiment, No. 7, were removed from the car-
mine at the end of thirty seconds, and were immediately well washed to get rid of the
adhering spermatozoa and granules of colouring matter. Not one of these ova pro-
duced an embryo, but several had become partially impregnated.

These experiments were made in the middle of March, when the season was un-
usually cold, and the mean lowest temperature of the room during twelve days
was only 43° FAHR., and the mean highest 47° FAHR. ; at a higher temperature the
results, I have little doubt, would have been more favourable. The eggs had been
impregnated however when the temperature, during the first twelve hours, was 55°
FAHR., so that the question respecting the infiltration of solid matter with the water
absorbed by the envelopes of the eggs was not affected.

About four hundred and twenty eggs were employed in this set of experiments ; yet I
could not detect any granules of the colouring matter of carmine held in suspension,
and of dimensions equal to those of the spermatozoa, which had passed into the tissue
of the envelopes of the eggs, although they had become tinged by the colouring matter
in combination with the water. Abundance of granules of colouring matter of most
minute size, and not more than one-third the diameter of the spermatozoa of the
Frog seen beside them, adhered to the surface of the envelopes, and it was to these
chiefly that the red colour of the whole was due. Every part of the envelope exhi-
bited the same uniform appearance, the granules being pretty equally distributed over
the surface, and the suffusion of colour was uniform in the interior. These facts ap-
peared to be conclusive with reference to the question of the presumed existence of a
fissure or perforation through the coverings of the egg of the Frog before, or at the
moment of fecundation, as is supposed to exist in the ovum of the Rabbit. I have
not been able to detect any appearance of orifice or fissure in the egg of the Frog-
envelopes, and the course of which, if such really exists, would no doubt be indicated
by some deposition of the colouring matter of the carmine, to a greater or less ex-
tent, in its tract. The result of these experiments was thus most unfavourable to the
belief that the spermatozoa penetrate bodily through the membranes of the ovum ;
and to that of the supposed existence of a special opening in these membranes for
their admission.

I ought now, however, to mention one experiment that seemed to favour the opi-
nion that the spermatozoon enters the ovum. I had taken several ova, together with
the oviducts into which they had passed, from the body of a Lissotritonpalmipes, and
others from that of Triton palustris, for the purpose of artificial impregnation. Some
of these I pressed from the oviducts into a very clear solution of carmine, taken from
a solution which had remained undisturbed for nearly a fortnight, so that the granules

THE OVUM IN THE AMPHIBIA. 223

in suspension had subsided, and only the colouring matter actually combined with
the water gave it its red hue. At the end of half an hour I removed the ova from the
solution to clear water for examination, and then found that the interior of the enve-
lope was coloured by the water which had entered, but that the greater portion of the
colouring matter had been arrested and separated at its entrance and adhered to the
surface. One ovum of Lissotriton palmipes, however, to my great surprise, had a little
dense mass of colour deposited at one point only of the dark surface of the ovum, not
merely within the envelope or its chamber, but actually beneath the vitelline mem-
brane, between it and the yelk, as was distinctly proved by turning the egg on one
side and viewing it in profile. Not one of the other eggs, placed in the solution, either
of the Triton or Lissotriton, showed any appearance like this ; so that while I am
debarred from expressing a decided opinion that the spermatozoon does not enter the
ovum, I can only regard the appearance mentioned as entirely accidental, and not as
a normal occurrence ; but as resulting, perhaps, from some minute puncture or other
accident during the removal of the eggs from the body or the oviduct.

But in order, if possible, to remove another source of doubt, it seemed necessary to
make some trial with the colouring material employed by PREVOST and DUMAS in their
experiments ; and some further examination of that used in my own ; and to ascertain
whether any solid particles or granules of matter, held in suspension in ink or in car-
mine, and equal in size to the spermatozoa of the Frog or the Newt, can be passed
through the filter, or can be separated from the fluid portion by filtration, like the
spermatozoa, when precisely the same mode is followed, and the same means and same
description and number of filter-papers are employed, as in the filtration of the seminal
fluid. The solution of these questions it was evident must tend to confirm or to
unsettle the previous conclusions. I first tried ink, and used a part of the identical
filtering-paper employed to separate the spermatozoa. The ink passed quickly and
freely through three filters without losing any of its intense black colour, and carried
with it only a very few extremely minute granules, much smaller in size than the
spermatozoa of the Frog ; so that it seemed fair to conclude that the colour imbibed
by the ova from ink, in MM. PREVOST and DUMAS' experiment, was due to the admis-
sion of the chemically combined colours of the fluid, and not to an admission into
the texture of the egg-envelopes of solid particles held merely in suspension in the
fluid. Consequently this experiment seemed to negative the supposition that, from
the fact of the interior of the egg-covering becoming blackened, solid particles of
matter, equal in size to the spermatozoa, must have penetrated into the envelope
during its expansion ; and there seemed less reason to believe that the spermatozoa, —
bodies very much larger than the ink-granules, — could enter it. Carmine was then
tried. A solution of this colour could scarcely be made to pass through even a single
filter. This seemed to be due chiefly to the fact that the greater proportion of the
colouring matter of the carmine used (the water colour pigment of artists) was com-
bined with gum and an earthy base, and consequently most of the colour was in

224 MR. NEWPORT ON THE IMPREGNATION OF

suspension rather than in chemical combination. When placed on a single filter, the
solution passed through with extreme difficulty and slowness. When a microscopic
drop of the fluid so passed was examined with a power of three hundred diameters,
it was found to contain a large quantity of granules suspended in clear fluid. When
made to pass, but with still greater difficulty, through a second filter, it still contained
a quantity of minute granules, but each less than one-half' the diameter of the sperma-
tozoon. It is possible, therefore, that some extremely minute granules may penetrate
into the texture of the envelope, formed as it is of aggregations of cells ; but it seems
to be very improbable that any of the larger-sized objects, such as the spermatozoa,
can enter : and it is even much more improbable, that if the chief colour of the ova in
my experiments was due, as I believe, to granules of carmine on the surface, and not
in the interior of the ova, that in MM. PREVOST and DUMAS' experiments with frog's
blood, the ova should have become reddened by the admission of particles of this
into their interior, since it need scarcely be mentioned that the colour of the blood is
due only to the particles suspended in it ; and MM. PREVOST and DUMAS remark,
that they were not able to detect any blood-globules on the surface. To what else,
then, than to these, or to their broken-down particles, could the reddened colour
of the ova in their experiments be due ?

The conclusion, then, to which I am led by these experiments is, that although the
envelopes of the egg imbibe coloured fluid, they do so less easily than when the fluid
is not coloured, unless it is in chemical combination ; and although atoms of solid
matter, very much smaller than the spermatozoa, may possibly be carried by infiltra-
tion into the texture of the egg-envelope by the act of endosmose during its expansion,
it appears to be extremely unlikely that the large bodies of the spermatozoa are so
carried in ; an improbability which is raised almost to a certainty by the fact that the
spermatozoa are not seen attached to the egg with a centripetal direction of the axis
of their bodies, but are constantly applied laterally to, or are entangled amongst the
loose tissue of the surface, extended at length or partially folded on themselves.

6. AGENCY OF SPERMATOZOA AS AFFECTED BY CHEMICAL MEDIA.

The experiments with carmine having led to an unexpected result in the impedi-
ment which this medium offers to the impregnation of the ovum when immersed in it
before contact with the spermatozoa, I was desirous of ascertaining what effect would
be produced on the ovurn by the destruction of the spermatozoa by chemical means,
immediately after they had been applied to it. Mr. GULLIVER* long ago showed that
the spermatozoa of different animals are variously affected by different chemical tests ;
and Dr. FRERICHS-^, more recently, has found that a solution of caustic potass has
the property of entirely dissolving and destroying them. This material, therefore,
seemed to be peculiarly fitted for the object in view. But before any experiment, in

* Proceedings of the Zoological Society, part 10. p. 101. July 26, 1842.

t In Cyclopaedia of Anatomy and Physiology, Article " Semen," p. 506, January 1849.

THE OVUM IN THE AMPHIBIA. 225

which this was employed, could be relied on, it was necessary to confirm the facts
ascertained by chemical investigation, by observing the mode of action of solutions of
caustic potass, and other chemical agents on the spermatozoa, by means of the micro-
scope. As my observations on the effect of chemical agents on the spermatozoa have
been confined for the present to those of the Frog, I shall state the results of these
observations with the microscope before mentioning the experiments.

All the observations were made immediately after the spermatozoa employed had
been obtained, by the course already mentioned, and not by vivisection from the
vesiculse seminales or the testes, sources which are objectionable from the facts shown
in Dr. FRERICHS' analyses, that a large quantity of albumen is always found in the
immature cells in the testes, with which the spermatozoa, obtained from that source,
are constantly mixed, while there is no trace of albumen in the mature spermatozoa.

1. Solution of Caustic Potass. — The solution employed was in the proportion of
twenty grains of caustic potass (Potassa fusa) to one ounce of water. This was the
solution employed on most occasions in the following experiments, and which quickly
and entirely dissolves the spermatozoa. When a drop of semen, in which the sper-
matozoa are active and abundant, covered by a pellicle of talc on a plate of glass, is
attentively examined, while a very small quantity of the potass solution is applied to
the edge of the talc, the act of dissolution is easily witnessed. As the solution spreads
beneath the talc the spermatozoa first brought into contact with it are instantly de-
stroyed, while the motions of those at a distance become slower and slower, until, when
the fluid has nearly approached, they entirely cease. The instant the fluid comes into
contact with the spermatozoa, they roll up on a sudden into a spiral form, the change
commencing at the apex of the caudal extremity,, and each becomes a rounded mass,
which quickly dissolves and disappears in the homogeneous fluid. The action of the
potass in this destruction of the spermatozoa, as seen by the microscope, is very similar
to, in appearance, and strongly reminds one of the action of fire on the barbs of a feather,
which become frizzled in an instant, leaving only a scoria that soon disappears.

2. Nitrate of Potass. — This, as in the preceding case, was in solution in the pro-
portion of twenty grains to an ounce of water. It destroys the spermatozoa much
less quickly than the caustic potass. When applied, as above, to the edge of the talc,
the spermatozoa first become on a sudden motionless, and are in general elongated,
and afterwards are very slowly dissolved.

3. Diluted Acetic Acid. — When this is applied to the spermatozoa in the same way
as the solution of potass, it quickly destroys all signs of vitality. The movements
immediately become slower and very soon entirely cease, and the spermatozoa are
extended at full length, and are but rarely folded on themselves, as they usually are
in natural death. I could not satisfy myself that the acid has any other effect on
them chemically than that of contracting and rendering them smaller. It did not
appear to dissolve them. Mr. GULLIVER* has mentioned that the spermatozoa of the

* Loc. cit.
MDCCCLI. 2 G

226 MR. NEWPORT ON THE IMPREGNATION OF

snake (Natrix torquata) are not affected by acetic acid, but he makes no reference to
its action on those of the Frog.

4. Gum-Arabic. — A thick solution of gum appears to act on the spermatozoa me-
chanically only, and almost immediately deprives them of motion by the obstruction it
opposes to them. When a minute drop of spermatic fluid is placed in the midst of
one of gum solution, and covered with talc, those spermatozoa which have become
mixed with the gum cease to move instantly, and remain with the tail and body coiled
in various directions ; while others at the edges and in the midst of the fluid, where
they are less mixed with gum, still move feebly for a few seconds, but become motion-
less as the gum collects around them.

These circumstances will better enable us to understand the following experiments,
the object of which was to endeavour to learn how far the influence of the sperma-
tozoa, and the act of impregnation of the ovum partake of a chemical or of a mecha-
nical nature ; and also will help to determine the length of period of contact requisite
for impregnation.

The following experiments bear on these inquiries : —

Potass experiments. — SetP. March 25,1850. Atmosphere 48° FAHR. Water46° FAHR.

No. 1. P.M. lh 40m. — Fifty-four ova were passed from the Frog on a dry surface,
and were instantly bathed with recently-obtained impregnating fluid mixed with
water ; and at the lapse ofjifteen seconds were washed by means of a hair-pencil
loaded with the solution of caustic potass before mentioned ; after which the eggs
were again washed freely with water.

No. 2. P.M. lh 45™. — Thirty-nine ova were treated in precisely the same way, except
that the interval between the application of impregnating fluid and the solution of
potass was only Jive seconds.

The ova were removed, after the first day, to a room in which the average tempera-
ture was about 60° FAHR., and at the end of the eighth day twenty-one embryos, ad-
vanced to near the end of the fourth period of development, had been produced in No. 1,
and two embryos, at a similar stage, in No. 2. This, at first thought, appeared to be an
extraordinary fact, seeing that the solution of potass so quickly decomposes the sper-
matozoa, and even renders the ovum sterile, as was afterwards found ; but, on exami-
nation of the details, the experiments admit of explanation -.—Jirst, the seminal fluid
was employed immediately it was obtained, and before the application of the potass,
which was not used, in the first case, until after a lapse ofjifteen seconds, and in the
second case, of Jive seconds ; next, that in both instances the solution of potass was, as
quickly as possible after its application, diluted and removed by repeated washing of
the ova with water. Nevertheless, these experiments prove that the ovurn becomes
impregnated very quickly after the application of spermatozoa, and, in these cases,
even within the short interval oijlfteen seconds in the one, and Jive seconds in the
other, the difference in the number of embryos produced in the two apparently in-
dicating the extent in each of the deleterious effect of the solution.

THE OVUM IN THE AMPHIBIA. 227

No. 3. P.M. lh 50m. — Forty-seven ova were passed on a dry surface, and spermatic
fluid was instantly applied to them, and within ten seconds afterwards the ova were
washed with the solution of potass, which was allowed to remain, and water was then
added. At the end of the eighth day not a single embryo had been produced. The
difference between this experiment and the above was the non-removal of the potass,
and the more free bathing of the ova.

No. 4. P.M. lh 53m. — Forty-jive ova were passed on a dry surface, and werejirst
bathed with solution of potass, and then with seminal fluid in water, and afterwards
they were removed with No. 1 and 2, to higher temperature.

At the expiration of the eighth day three embryos had been formed. This result at
first appeared to be more difficult of explanation than the former. But, when the
circumstances are considered, it seems to admit of quite as easy an interpretation.
The bathing of the ova freely with seminal fluid mixed with water,^e seconds after
the application of the solution of potass, diluted this solution too much to allow of
its effect on the whole of the spermatozoa applied to the ova, while this very dilution
enabled the impregnating influence of these bodies to take effect in some of the ova.
The fact, however, leads to an inference of some importance with reference to the
action of the potass on the envelopes of the ovum, and seems to show that this
action is less immediate on the envelopes than on the spermatozoa.

No. 5. Fifty-nine ova were bathed with seminal fluid and water, and Jive seconds
afterwards with a solution of nitrate of potass (in the proportion of twenty grains of
the salt to one ounce of water), and water was then added to them.

No. 6. Seventy-two ova were treated in precisely the same way.

Not one embryo was produced in either of these experiments.

No. 7- Forty-four ova were washed with diluted acetic acid immediately after they
had been shed from the female, and^we seconds afterwards, seminal fluid with water
was added to them.

No. 8. Seventy-six ova were washed with diluted acid, and treated in every way as
in No. 7-

Not a single embryo was produced in either of these trials.

The action of acetic acid is almost instantaneous on the envelope of the ovum,
which it quickly contracts, and renders slightly opaque.

Besides those media which act chemically on the spermatozoa and the ova, I
made trial, in this and the two following sets of experiments, with a solution of gum-
arabic, the effect of which appears to be entirely mechanical ; and as the results are
curious and seemingly important with reference to the nature of the agency of the
spermatozoa in impregnation, I defer the mentioning of them until I have to show
the effect of media which operate mechanically on the ovum or the spermatozoon in
impregnation.

The result of the preceding experiments was so remarkable, that it seemed necessary
that they should be repeated with greater precision, with reference to exact periods

2 G 2

228 MR. NEWPORT ON THE IMPREGNATION OF

of time, than can always be done when alone and unassisted. It was evident that a
proper understanding of the nature of the act of impregnation, if ever this becomes
known, will be led to chiefly by attention to the periods of time in which it is effected.
I obtained therefore the assistance of a friend, to note the spaces of time which
elapsed in each stage of the following experiments, so that these might be performed
with the quickest dispatch, and the attention of the experimenter be not withdrawn
from each until it was completed.

Set Q. March 30, 1850. Atmosphere 49° FAHR.

The seminal fluid employed in this set of experiments was not obtained from the
usual source. From some cause or other it could not be so procured. I therefore
killed a male frog, by dividing the spinal cord in the medulla oblongata, and pressed out
the fluid from the testes, which were gorged with spermatozoa, as found by examina-
tion with the microscope. From the presence of a great number of spermatozoal
cells, and from the water with which I mixed the fluid becoming slightly turbid and
albuminous, it was seen that it was not fully matured, a circumstance to be borne in
mind with reference to the experiments, which were commenced some minutes after
the fluid had been thus obtained. It was doubtful also whether the eggs were quite
mature.

It is necessary further to mention, that in these experiments two solutions of potass,
with different proportions of the salt, were employed ; one having twenty grains in one
ounce of water, and which, to avoid repetitions, I shall designate " strong solution ; "
and the other having only eight grains of the salt in one ounce of the fluid, and
which I shall refer to as the " weak solution"

No. 1. P.M. 4h 45m. — Forty-five ova, passed on a dry surface, were bathed with
seminal fluid and water, and^ve seconds afterwards with the weak solution of potass,
and were then washed, and placed in clean water. The whole time occupied in the
experiment did not exceed thirty-five seconds.

On the following morning, segmentation was found to have taken place in twelve
ova. This as well as the following sets of ova were then removed to a higher tem-
perature, in which they were allowed to remain ; but no embryos were produced.

No. 2. P.M. 4h58m. — Fifty-three ova were treated in exactly the same way, but with
the strong solution of potass ; the interval between the application of the impreg-
nating fluid and the solution being only two seconds, and the whole time occupied
forty -five seconds. v '

Segmentation took place in three or four ova, but not completely. Several ova
also were altered in form ; but not a single embryo was produced.

No. 3. P.M. 4h 52™. — One hundred and twenty-two ova, passed on a dry surface,
were immediately well bathed with seminal fluid and water, and two seconds after-
wards with weak solution of potass, which was allowed to remain with the ova, and
water was then added. The whole time occupied was forty-five seconds.

Segmentation took place in a great number of these ova, certainly from fifty to

THE OVUM IN THE AMPHIBIA. 229

sixty, and there were nine also which became shrivelled and decayed. Notwithstand-
ing the great proportion of ova segmented, not one produced an embryo.

No. 4. P.M. 5h. — Sixty-six ova, passed on a dry surface, were bathed as in No. 2,
with impregnating fluid, and one second afterwards were washed quickly with strong
solution of potass, and water was then added ; the period occupied being sixty-two
seconds.

Only two ova became segmented, while, excepting only eight or nine ova, the whole
of the remainder were shrivelled, irregular, or compressed in form, and distinctly
spoiled, apparently by the action of the potass, thus showing that endosmose and
exosmose through the envelopes had taken place.

No. 5. P.M. 4h 55m. — Fifty -four ova were passed, and washed with the weak solution
of potass, and afterwards, at an interval of two seconds, with impregnating fluid and
water, and water was then added and allowed to remain. The whole time occupied
was Jifly seconds.

One ovum only became segmented ; but several others had slightly altered their
form to the obtuse oval, as if about to become divided : no embryo was produced.

No. 6. P.M. 51' 2m. — Seventy-nine ova were washed with strong solution of potass,
and one second afterwards with impregnating fluid, and water was then added.

Not one ovum became segmented, nor did even one yelk retain its proper shape.
The whole were irregular and spoiled. Five ova had the envelope clouded and opaque,
and the surface of others was translucent with refracted light, like crystallized car-
bonate of lime. In one egg only had there been any attempt at segmentation. This
experiment, like No. 4, seemed to show that the act of expansion of the chorion is
an act of endosmose.

No. 7. P.M. 5h 15m. — Nitrate of Potass. Seventy-seven ova were passed on a dry
surface, and were well bathed with impregnating fluid in water, and one second after-
wards with a weak solution of nitrate of potass, and water was then added to them ;
the whole time occupied being thirty seconds.

Segmentation took place in forty-three of these ova, and the whole retained their
natural form and size. The effect of the nitrate of potass, as compared with the
caustic solution, was thus very marked, as showing that the momentary application
of the nitrate does not prevent or arrest impregnation in weak solution (eight grains
to one ounce of water), even when applied after the impregnating influence. The
experiment was also interesting in another respect. It proved that the ova were sus-
ceptible of being impregnated, and that the fluid from the testes was efficient to
induce the first evidences of impregnation. But none of these ova, or of the ova in
the preceding experiments of this set, produced embryos. Subsequent observations
will show that this failure was not due to the nitrate of potass, but perhaps was at-
tributable to the conjoint causes of low temperature at the time of impregnation, and
of some imperfection both in the seminal fluid and the ova.

No. 8. P.M. 5h 9m. — Seventy-six ova were washed with diluted acetic acid as in Set P,

230 MR. NEWPORT ON THE IMPREGNATION OF

No. 7? and one second afterwards with impregnating fluid, and water was then added
to them. The time occupied was forty -Jive seconds.

The result was more decided than in the experiment referred to. The envelopes
of the ova immediately became clouded, and no segmentation took place in any of
the yelks, some of which became shrivelled and changed in form.

The result of the preceding experiments being doubtful as to the cause of the non-
production of embryos, especially with reference to the first three, and the seventh
experiments, in which many ova became segmented, I obtained some additional pairs
of frogs from their native haunts, and within twenty-four hours afterwards, before
they had in any way become debilitated by confinement, repeated the experiments at
a higher temperature.

Set R. April 3, 1850. Atmosphere 60° FAHR.

No. 1 . P.M. 3h 5m. — Eighty-two ova, passed on a dry surface, were touched for an
instant only with a pencil dipped in impregnating fluid and water, and one second
afterwards were washed with strong solution of potass, and then with water, and
water was then added. The whole time occupied was only fifteen seconds.

No. 2. Twenty-Jive ova were treated in precisely the same way with the same solu-
tion (which also was employed in the following experiments) ; the interval being
two seconds, and the whole time twenty seconds.

Segmentation took place, but only very partially, in about twelve ova of the first,
but completely in one only of the second experiment. The whole of the remaining
ova were shrivelled and decayed ; their envelopes exhibiting the same clouded and
refractive property noticed in the last set. At the end of five days two embryos had
been produced in the first experiment, and one in the second. It was thus far con-
firmatory of the experiments with potass in Set P, that if this salt be applied to the
envelope several seconds after the application of the impregnating fluid, and be again
quickly removed or diluted with water, impregnation may already have taken place,
and the action of the caustic will not in that case affect the production of the em-
bryo ; especially if the experiment be made when the temperature of the surrounding
medium is becoming increased. But if the solution be applied before the application
of the seminal fluid, then the spermatozoa will in most cases be decomposed, and no
impregnation follow. In either case, however, the undiluted solution acts also on
the ovum itself within a very short period, and destroys or renders it sterile. This
was further proved in the succeeding experiments.

No. 3. P.M. 3h 10m. — Fifty-eight ova were passed on a moistened surface, and were
immediately afterwards washed with the solution, and at the expiration of one second
were bathed with seminal fluid and water ; the time occupied being only fifteen
seconds, as in No. 1 .

No. 4. Sixty-nine ova were treated in exactly the same way, the interval being one
second ; and the whole time occupied only twelve.

Partial segmentation had taken place in one ovum of No. 3 ; but the whole of the

THE OVUM IN THE AMPHIBIA. 231

remaining ova, both in No. 3 and 4, were destroyed. Many of the yelks had begun
to change form within the first hour, and the envelopes exhibited the same refractive
appearance as in the previous experiments.

Anticipating from the former experiments what probably might be the ultimate
result in these, I now determined to put beyond the possibility of doubt, both the
fitness of the seminal fluid employed to effect impregnation and the healthiness of
the ova, and their susceptibility to become impregnated ; and to show from these
facts that a non-production of the embryo in this set of experiments must be due to
the action of the potass solution, and not to any unfitness in the spermatozoa or the
ova. Accordingly, —

No. 5. P.M. 3h 17™. — Sixty-two ova, from the same female employed in the pre-
ceding experiments, were bathed with a portion of the seminal fluid and water which
had been employed in No. 1 and 2, and were then placed side by side with these, in
a separate dish.

At the expiration of four hours and thirteen minutes, the temperature being 60°
FAHR., from thirty to forty of these ova had become segmented. Some of the ova
had been injured mechanically, but nearly the whole that had not been injured were
impregnated. On the seventh day there were twenty-three embryos, thirteen of which
had already left the egg-envelopes ; others were somewhat less advanced, thus proving
the fitness of the seminal fluid to impregnate, and the ova to produce. The number
of embryos too was fully as great as could have been expected, seeing that many of
the ova had been slightly injured, and that the seminal fluid had already been one
hour and twenty-six minutes mixed with water.

The result of this experiment was borne out by the following.

No. 6. P.M. 3h 31m. — Nitrate of Potass. Seventy-four ova were well bathed with
impregnating fluid and water on a previously dry surface, and one second afterwards
with a strong solution of nitrate of potass (twenty grains to one ounce of water), and
water was then added to them ; the whole time of the experiment being twenty
seconds.

No. 7- Fifty-nine ova were treated in exactly the same way, save that the interval
between the application of the impregnating fluid and the solution of potass was
three seconds, and the whole period twenty-five seconds.

Segmentation commenced in each of these sets \nfour hours and fourteen minutes,
when from twelve to fifteen ova were undergoing this change in No. 6, and thirteen
in No. 7- At a later hour there were many more in each experiment only very par-
tially segmented, and which proved to be unproductive. At the end of the seventh
day there were twelve embryos in No. 6 advanced to the same stage as in the simply
artificial impregnation No. 5, and ten embryos in No. 7, but at a little less early stage
of growth, a circumstance which I attributed at the time to imperfect aeration.

No. 8. P.M. 3h 37m. — Seventy-nine ova were bathed with the same solution of nitrate
of potass as above, and three seconds afterwards with impregnating fluid in water;
the whole time occupied being twenty seconds.

232 MR. NEWPORT ON THE IMPREGNATION OP

Segmentation took place at a little later period in this than in the preceding expe-
riments. It commenced at four hours and twenty minutes, when twenty-five ova were
undergoing the change. This was a full proportion of impregnation as compared
with No. 5, seeing that the impregnating fluid had already been mixed with water one
hour and forty-six minutes. Twenty-Jive embryos were the result of this experiment.

The results thus support the explanation already given, with reference to the effect
produced on the envelopes of the ovum being less immediate than on the sperma-
tozoa ; since, in this case, twice as many ova became segmented, and ultimately
produced embryos, as in those experiments in which the solution was applied after
the seminal fluid, and while endosmosis of the egg was most rapid, and when the
solution remained undiluted.

The general results of this set of experiments, compared with those of the last Set,
Q, appear also to show that the non-production of embryos in the whole of that set, —
after segmentation had taken place in several of the experiments, as in Nos. 1, 2, and
especially No. 3, with solutions of the caustic potass ; — and still further, No. 7 with
the nitrate, — may fairly be attributed to some defect in the seminal fluid or in the ova ;
since, if such were not the cause, and the failure had been due either to the chemical
effect of the media on the ova, or to the moderate temperature of the atmosphere
(49° FAHR.) at the time of experiment, — segmentation of the yelk would hardly have
taken place. This supposition appears to be the more likely, when we recollect that
in the Set Q, and in that set only, the impregnating fluid was obtained from the testes,
compressed and broken down in water, — that the eggs were of doubtful maturity, —
and that this was the only set of experiments in which no embryos were ultimately
produced ; although, I may now mention, that greater care was taken to ensure a
favourable result than in most of these investigations, — the ova being removed at the
end of twenty hours to an average temperature of 60° FAHR., — were retained in flat
shallow dishes, — and had the water changed daily. Both sets, however, Q and R,
seem to prove that the act of impregnation, as evidenced in the fact of the yelk be-
coming segmented, must take place, or be commenced very rapidly ; and, apparently,
almost at the instant of contact of the spermatozoon with the coverings of the ovum ;
as seems to be shown in the fact, that segmentation took place in many of the ova
when the space of time between the application of the spermatozoon, and that of the
solution, — which previous observation (p. 225) showed was sufficient to decompose it
immediately, — was scarcely more than one or two seconds. Thus in Q 4, and R 1, i$
must have commenced in the interval of one second, even when the strong solution
was used ; and in Q 2 and R 2 with the same solution in two seconds. When the
weaker solution was used, a greater number of ova became affected in similar spaces
of time, as in Q 2 and 3. These experiments seem to show that the act of impreg-
nation had already been commenced before the application of the solution ; as, in the
experiments which are the converse of those now mentioned, in regard to the time
when the spermatozoa and the solution were applied, a different result ensued. Thus
when the solution was applied to the ovum Jirst as in Q 6 and R 3 and 4, and one

THE OVUM IN THE AMPHIBIA. 233

second afterwards the impregnating fluid with spermatozoa, no impregnation, or but
a very partial one, was effected. The ova in these three experiments amounted to two
hundred and six, and yet only one ovum became very partially affected. A like result
ensued even when the weaker solution was employed at an interval of two seconds, as
in Q 5, when out ofjifty-four ova segmentation occurred but in one.

When the interval between the application of the impregnating fluid, in the first
instance, and that of the solution subsequently, was extended to Jive seconds, then a
greater proportion of ova became segmented, as in Q 1, with the weak solution, when
out of forty-Jive ova twelve became changed.

These were the results when the experiments were made at different temperatures,
as at 49° FAHR. with the Set Q, and 60° FAHR. with the Set R. They cannot, therefore,
be attributable to inertness of the fecundating agent, or of the object to be fecundated,
occasioned by an unfavourable temperature of the surrounding medium. The fact
of the occurrence of segmentation in some ova, but not in the majority of the ova of
different experiments, as in Q 1, 2, 3 and 4, seems further to show that the influence
of the momentary application of the potash solution was produced chiefly, and in the
first instance, on the spermatozoa, or impregnating bodies, and not so immediately
on the ova ; since if the ova had been first, or most affected, none of them, probably,
would have become impregnated.

Further, I may perhaps be allowed to remark, that the arrest of impregnation was
due mainly to the nature of the chemical agent employed; and the extent of inter-
ference with the fecundatory process was in proportion to the more or less immediate
action of this agent on the spermatozoon. Thus we have seen that but few ova were
impregnated when the solutions of caustic potass were employed; but when the
nitrate of potass was used as in Q 7, forty-three out of seventy -seven ova were seg-
mented; while in R 6, 7 and 8, in which the total number of ova was two hundred
and twelve, there werejifty-three segmented, and these produced forty-seven embryos.

The object of these sets of experiments, therefore, — that of endeavouring to ascer-
tain within what period of time after the contact of the spermatozoon with the ovum
its fecundatory function is exerted, — appears to have been somewhat fulfilled ; — in so
far as that in these experiments on the Amphibia the commencement of the act of
impregnation appears to have been almost instantaneous. Yet there seems reason to be-
lieve that momentary contact of the impregnating body, even in the ovum of these
animals, is not in itself sufficient to complete the fecundation, although it may tend to
induce that condition of the yelk, segmentation, which we now are assured is always
indicatory of its having been influenced by the fecundatory agent. If momentary
contact were sufficient for the completion of the function, then partial impregnation,
which so frequently takes place when spermatozoa are few in number, or in contact
only for very brief periods, could hardly happen ; while every ovum in which the
process of cleavage is begun ought to pass through all its changes to the production
of the embryo, circumstances being favourable to its development. But this we have

MDCCCLI. 2 H

234 MR. NEWPORT ON THE IMPREGNATION OF

seen in the foregoing experiments is not the case. On the contrary, duration of at
least some seconds of contact, varying no doubt in different tribes of animals, and,
apparently also, quantity of spermatozoa, seem to be essential to fruitful and healthy
impregnation, as appears to be shown in the Jilt rat ion experiments, Set L 3, as com-
pared with L 1 (p. 207). Possibly momentary contact may suffice to occasion seg-
mentation, but certainly with duration of contact the ovum is fecundated.

This leads us further to inquire whether any endosmos-is of the material substance of
the spermatozoon is imbibed by the ovum during any period of impregnation, before
or during segmentation of the yelk? — and whether those media which do not act
chemically on the spermatozoon or the ovum can arrest the agency of the former ?
BISCHOFF has already shown that spermatozoa are in contact with the ovum in some
Mammalia, the Rabbit* and Dog-f-, from quickly after the entrance of the ovum into
the Fallopian tube until segmentation is nearly completed, and the yelk has acquired
a tuberculated or mulberry-like surface. In the ovum of the Frog I have shown that
the spermatozoa are in like manner seen on the envelopes from immediately after
immersion in impregnating fluid until segmentation has commenced. In the Newt
we have seen that when impregnation is effected artificially, they may be recognized on
the surface for a much longer period, — from the time of contact with fluid, until the
surface of the yelk has reacquired its original smoothness, a period, in my observa-
tions, of from thirty-six to forty-eight hours. The persistence of these bodies to a
period after the first evident changes in the yelk have commenced, seems to favour a
supposition that their function is not completed in momentary contact. Although
we are at present unable to trace their influence beyond what is now stated, I think
it can be shown that their function can be arrested by media which affect them me-
chanically, when submitted to such media at the moment of contact with the ovum.

7. AGENCY OF THE SPERMATOZOA AS AFFECTED BY MECHANICAL MEDIA.

The object of the next experiments was to learn whether the interposition of a dense
fluid medium, which does not act chemically on the spermatozoa, would be as effectual
in preventing the influence of these bodies on the ovum as in the carmine experiments,
the effect of which seemed to be mechanical.

As the experiments were made at different periods, it will be seen, that although
on two of these occasions the temperature of the atmosphere differed, the general
results were similar.

Gum and Starch Experiments. — Set S. March 25, 1850. Atmosphere 48°.

(a.) Gum. No. 1. P.M. 2h 22m. — Seventy-sir ova were passed on a dry surface and
were immediately bathed with a thick solution of gum-arabic, and Jif 'teen seconds
afterwards with seminal fluid and water, and fresh water was then added to them.
The whole time of the experiment was sixty seconds.

* Entwickelungsgeschichte des Kaninchen-eies. 4to. 1842, tab. 2, Z and 4, fig. 17 to 28.
f Entwickelungsgeschichte des Hunde-eies. 4to. 1845, tab. 1 and 2, figs. 10 to 16.

THE OVUM IN THE AMPHIBIA. 235

No. 2. Fifty-eight ova were treated in exactly the same way, the interval being
about Jifteen seconds, and the whole time sixty.

The seminal fluid employed was obtained from two males,the fluid used toNo.2 being
from a male which had paired four days before. Out of the whole number of eggs in
the two sets, amounting to one hundred and thirty-four, not one produced an embryo.

No. 3. March 30, 1850. P.M. 5h 5m. Atmosphere 49° FAHR.

One hundred and eight ova were passed on a moist surface, and were immediately
bathed with a thick solution of gum as above, and one second afterwards with seminal
fluid in water; the whole time occupied being sixty seconds.

Segmentation took place in two, or at most only three of these ova, and even in
them very imperfectly, and much slower than in the corresponding ova of the set to
which they belonged, Set Q (p. 228-9), in which the fluid employed was obtained
from the testes of the Frog, and regarded as immature.

No. 4. P.M. 5h 18m. — Fifty-eight ova passed on a moistened surface were immediately
bathed with solution of gum, and one second afterwards with seminal fluid from the
same male as No. 3. The whole time occupied was forty-five seconds.

The result of these two experiments, as compared with others of the set to which
they belonged, Set Q, was exceedingly curious. In the experiments with the nitrate
of potass as in Q 7, segmentation was carried to some extent, and the divisions of
the yelk were multiplied; while onlyfourova out of the fifty-eight, in this with gum,
gave any evidence of segmentation, and the process was not advanced further, either
in this or in the preceding experiment, No. 3, than to the completion of the primary
division of the yelk into two hemispheres. Thus not only was the process entirely
prevented in the great majority of the ova, but it was also very much retarded in
those in which it did take place, and this simply, as it appeared, by the mechanical
hindrance of the gum. Could it be that the effect was produced on the endosmic
action of the yelk ? These trials certainly appeared to show that the obstruction
was a mechanical one. I need scarcely remark, that no embryo was produced in
either of these experiments.

No. 5. April 3, 1850. P.M. 3h 25m. Atmosphere 60° FAHR.

Sixty-one ova were passed on a moistened surface and were immediately bathed with
impregnating fluid, and two seconds afterwards with a thick solution of gum-arabic,
and water was then added ; the whole time occupied being only twenty seconds.

This experiment, when compared either with the four preceding ones, made at a
temperature of the atmosphere eleven degrees lower, or with that which follows,
No. 6, seems to point to the exact nature of the operation of the gum. At four hours
and Jive minutes from Jifteen to twenty ova had become segmented, and others were
in the act of becoming so. At seven hours and a half more than one-half of the
whole number had changed, and were perfectly healthy. Thus, in this case, in which
the gum was applied after the seminal fluid, impregnation occurred earlier than in
corresponding experiments of the same set, R 6, 7 and 8, with nitrate of potass, when
it happened in from four hours, and fourteen to twenty minutes. It was even as

2 H 2

236 MR. NEWPORT ON THE IMPREGNATION OF

rapid as in the artificial impregnation, R 5 (p. 23 1 ), in which it took place in four hours
and thirteen minutes. On the eighth day twelve embryos had been produced.

These facts seemed to show, precisely as in experiments with solutions of potass,
that impregnation is commenced very quickly; and further, that it was not arrested
by the gum when applied only two seconds after the spermatozoa, but that the change
proceeds almost as uninterruptedly as in a perfectly natural impregnation, since
the number of embryos was almost as great as in No. 5 R, seeing that the fluid
employed had been obtained and mixed with water one hour and thirty-four minutes.

No. 6. P.M. 3h 22m. — Seventy ova, passed on a moistened surface, were bathed with
a thick solution of gum, and two seconds afterwards with some of the impregnating
fluid employed in the last experiment, and water was then added ; the whole time
occupied, as above, being only twenty seconds.

This experiment was the converse of the preceding. At four hours and twenty-eight
minutes only one egg out of the whole had become segmented ; but others gave signs
of being about to change, and some hours later a few had done so, but there were not
at most more than ten. At the end of the seventh day two embryos had been produced.

Thus while a comparison of these two experiments seems to show that the gum acts
simply as a mechanical obstruction to the process of fecundation, this experiment,
No.6, when compared with Nos. 1 to 4, made at eleven degrees lower temperature, shows
also the influence of a higher degree of temperature in accelerating fecundation.

The two following experiments were made with a view to test the efficiency of the
fluid and ova employed, now at one hour and fifty minutes after the fluid had been
obtained : when examined at this time with the microscope, there were still an abun-
dance of active spermatozoa.

No. 7- P-M. 3h 41m. — Eighty-Jive ova were accordingly placed in water with some
of the impregnating fluid and allowed to remain to test its efficacy.

At four hours and twenty-four minutes nearly the whole of the ova had become seg-
mented, and on the seventh day forty-two embryos had been produced.

No. 8. P.M. 5h 50m. — One hundred and thirty-two ova were now passed into the re-
mainder of the impregnating fluid, which had at this time been four hours mixed with
water.

When examined at the end of five hours and ten minutes, not a single specimen
had become impregnated. This was proved by the result, that at the end of seven
days not a single embryo had been formed. The fluid had thus lost its fecundating
property at the end of four hours in a temperature of 60° FAHR.

No. 9. April 6, 1850. Atmosphere 60° FAHR.

P.M. lh 50m. — One more experiment was now made with the gum solution, for the
purpose of comparing it with the following experiments with starch.

One hundred and twenty-two ova were passed on a dry surface and covered with a
thick solution of gum, and three seconds afterwards with impregnating fluid that had
been mixed with water only thirty minutes. The time occupied was not noted, but
fresh water was added to the ova at the end of fourteen minutes.

THE OVUM IN THE AMPHIBIA. 237

Segmentation took place only, in two or three ova, at/owr hours and twenty-four
minutes, and at the end of ten days only three embryos had been produced.

(b.) Starch. No. 10. P.M. lh 40™. — One hundred and Jifty-eight ova were passed
into a solution of starch in water, and at the end of ten seconds one-half of the whole
quantity of seminal fluid, obtained from a single frog, and previously mixed with
water, was added to them.

After the ova had remained in the solution and been gently agitated during twenty
minutes, they were carefully washed and removed to clear water.

At four hours and twenty-six minutes only a very few of these ova, not more than
eight or ten, had become segmented, notwithstanding the large quantity of recent
impregnating fluid that had been added to them. At the end of ten days only Jive
embryos had been produced.

It was remarkable that very few of the ova in this experiment cohered together, as
the frog's ova almost invariably do when placed in fluid. On the contrary, most of
them remained separate and isolated, although their envelopes had imbibed water and
expanded to their usual extent in a similar space of time.

No. 11. P.M. lh 47m. — Seventy-one ova were passed on a perfectly dry surface, and
immediately afterwards were covered, by means of a hair-pencil, with a thick solu-
tion of starch, and at the expiration of ten seconds with impregnating fluid, and water
was quickly added.

The water was changed at the end of fifteen minutes. At four hours and twenty-
Jive minutes, only two or three ova had become segmented. At the end of ten days
three embryos had been produced.

The concluding experiment with starch was the counterpart of No. 5, with gum.

No. 12. P.M. lh 44m. — One hundred and nineteen ova were passed into water, with
which one-eighth part only of the seminal fluid, obtained from the Frog, had already
been mixed. Two seconds afterwards a solution of starch was added to these ova,
and at the end of sixteen minutes they were removed to clear water.

In four hours and twenty-six minutes segmentation had commenced in many of these
ova. The exact number I omitted to ascertain. But the change had taken place in
a much shorter space of time, and was more general, although scarcely one-fourth
part of the quantity of seminal fluid that had been employed in No. 10 was used in
this case. Nevertheless, in ten days twelve embryos had been formed.

This experiment, therefore, was quite confirmatory of the conclusions drawn from
its counterpart, No. 5, with gum, and Nos. 10 and 11 as fully bore out those deduced
from Nos. 1 to 5, with the same ; while the entire set seem to be in full accordance
with the already arrived at conclusion, that fecundation is commenced almost imme-
diately the fecundating body is in contact with the ovum. Thus, then, with regard
to the nature of impregnation, we seem to have obtained sufficient proof that the act
is effected through the agency of the spermatozoon, and not through that of the liquor
seminis, as was formerly supposed.

238

MR. NEWPORT ON THE IMPREGNATION OF

TABLE II. — Experiments with Media that act chemically or me-

No. of ex-
periments

Date and hour of
experiment.

Tempera-
ture at the
time of the
experiment

Mean daily temperature
after the experiment.

Segmentation
comenced in
hours.

No. of eggs

Embryos at

Nature of the
impregnating fluid.

Time, pro-
cured and
mixed with
water.

!

I

I

1

"H.

1

!

v>

End of
third
stage.

In days

and hours.

1850.

Set P. 1
2

3

4
5
6
7

8.

SetS. 1.
2.

Setq. 1.
2.

3.

4.
5.
6.
7.
8.

SetS. 3.

4.

Set K. 1.
2.
3.
4.

5.

6.

7.

8.

5e< S. 5.
6.

7.
8.

&< S. 9.

10.

11.

12.

March 25. P.M. 1 40
P.M. 1 45

P.M. 1 50
P.M. 1 53
P.M. 2 21
P.M. 2 26
P.M. 2 16

P.M.

P.M. 2 22

P.M.

30. P.M. 4 45
P.M. 4 58

P.M. 4 52

P.M. 5

P.M. 4 55
P.M. 5 2
P.M. 5 15
P.M. 5 9

P.M. 5 5

P.M. 5 18
April 3. P.M. 3 5

48
48

48
48
48
48
48
48

48
48

49
49

49
49
49
49
49
49

49

49

60
60
60
60

60

60
60
60

60
60

60
60

60

60

60
60

46
46

46
46
46
46
46
46

46
46

48
48

48
48
48
48
48
48

48
48

61
61

61
61
61
61
61
61

61
61

61-5
61-5

61-5
61-5
61-5
61-5
61-5
61-5

54
39

47
45
59
72
44
76

76

58

45
53

122

66
54

79
77
76

108

58

82
25
58
69

62

74
59
79

61

70

85
132

122

158

71
119

20
2

Not one.
3
Not one.
Not one.
Not one.
Not one.

Not one.
Not one.

Not one.
Not one.

Not. one.
Not one.
Not one.
Not one.
Not one.
Not one.

Not one.

Not one.

2
1

Not one.
Not one.

23

12
10
25

11
2

42

3

5

3
12

4" 12h
4<i 12h

Not one.
5"

Semen and water.
Semen and water.

Semen and water.
Semen and water.
Semen and water.
Semen and water.
Semen and water.
Semen and water.

Semen and water.
Semen add water.

Semen from testes.
Semen from testes.

Semen from testes.
Semen from testes.
Semen from testes.
Semen from testes.
Semen from testes.
Semen from testes.

Semen from testes.

Semen from testes.

Semen and water.
Semen and water.
Semen and water.
Semen and water.

Semen and water.

Semen and water.
Semen and water.
Semen and water.

Semen and water.
Semen and water.

Semen and water.

10™
15m

20m
23m
Slm
56'"
46m
10m

52"

12™

10ra
23°

17"'
25m
20°
27m
40'"

34m

30°

43°
lh 12™

1M7"
lh

jh 24"
l' 38°

12
4

50 to 60
2
1

43

riSto 20M
< at temp. )•
[ 49 to 48° J

18 to 20h

41

6to7d
6to7d

5d

5"
5d
5d

5d

7d

6d

7"

7"

7"
54d

3

4

12 partial
1

. partially

P.M. 3 10

...

4i»

P.M. 3 17
P.M. 3 31

...

4h 13»

4h I4m

4h 14°

4h 2<r

4h 5m

4h 30™
41. 24°

30 to 40

12 to 15
13
25

33
10

74

Not one.

3

8 or 10
3

P.M. 3 37

P.M. 3 25
P.M. 3 22

P.M. 3 41
P.M. 5 50

6. PM. 1 50

P.M. 1 40

P.M. 1 47
P.M. 1 44

1" 46"

lh 34"
lh 31m

Jh 50-n

4 hoars

30°

20m

27"

24ra

...

...

41, 24m
4h 20°
4h 25°
4" 18»

Semen and water.

Semen and water.

Semen and water.
Semen and water.

Total number

2634

369

176

f in Sets P, 1

14*. as./

RECAPITULATION AND CONCLUSIONS.

It may now be well to recapitulate briefly some of the facts and views derived
from the foregoing observations and experiments. First, then, the germinal vesicle
disappears in the Amphibia before impregnation ; and before, or at the time of the
bursting of the ovisac, and extrusion of the egg from the ovary into the cavity of the

THE OVUM IN THE AMPHIBIA.
chanically on the Spermatozoon or the Ovum in Impregnation.

239

Material employed.

Period of experiment.

Remarks.

Operating
mechanically.

Operating
cbemically.

Quantity per
ounce of
water.

Interval
before
fluid.

Interval
after
fluid.

Whole
period.

Solut. caust. pot.
Solut. caust. pot.

Solut. caust. pot.
Solut. caust. pot.
Solut. nitr. pot.
Solut. nitr. pot.
Acetic acid.
Acetic acid.

9j to jj.
9j to 3J.

9j to sj.
9j to sj.
9j to 5J.

15 sec.
5 sec.

10 sec.

jm
40 sec.

30 sec.
30 sec.
30 sec.
30 sec.
20 sec.
20 sec.

jm
jm

35 sec.
45 sec.

45 sec.
62 sec.
50 sec.
45 sec.
30 sec.
45 sec.

lm

45 sec.

15 sec.
20 sec.
15 sec.
12 sec.

iNo. 1 to 8 of this set removed after fifty minutes to tempe-
rature 61° FAHR. These ova were passed on a dry surface,
and fluid applied freely with a pencil, and afterwards the
potass washed off freely with water.
Potass allowed to remain.
Ditto, well bathed with fluid after potass.
f Well bathed with solut. nitr. potass and water then added,
\ the solution allowed to remain,
f Impregnating fluid applied quickly and profusely, and water
\ then added.

("Water was added quickly after the gum, hut not sufficient
\ to remove it ; one or two ova segmented.

I" No. 1 to 8 of this set also removed at end of nineteen hours
•< to high temperature, 62° FAHR., No. 1 and 2 being
thoroughly bathed with seminal fluid.
Water added and solut. of potass not washed off.
Nearly the whole at twenty hours spoiled.

All spoiled in a few hours.
Ova passed on a moist surface and well bathed with fluid.

(" This was a thick solution of gum and applied with difficulty,
but freely and quickly ; the eggs were then well bathed
with fluid and allowed to remain in a low, and slightly
|_ diminishing temperature.

iOva touched lightly for an instant only with the fecun-
dating fluid, and water afterwards added.
Time of applying the potass was from two to three seconds ;
the eggs were spoiling within twenty minutes.
(Made with fecundating fluid without solution of potass to
test the above.
These ova were passed on a dry surface and were then
thoroughly bathed with seminal fluid and water.
The fluid was applied after the solution.

Impregnation must thus have occurred very quickly.

/ Made to test the fecundatory fluid at two hours after mixture
\ with water.
Not one egg segmented or one embryo found.

I" Gum applied thickly and the ova then bathed with fecun-
\ dating fluid.
f The ova passed into the starch, and a large quantity of fluid
\ then added.
Ova passed on a dry surface, starch applied with a pencil.
Changed to fresh water after sixteen minutes.

5 sec.

6 sec.
5 sec.

5J t° 3J-
SJtoSJ-

5 sec.
5 sec.

15 sec.
15 sec.

Solut. gum-arab.
Solut. gum-arab.

Solut. caust. pot.
Solut. caust. pot.

Solut. caust. pot.
Solut. caust. pot.
Solut. caust. pot.
Solut. caust. pot.
Solut. nitr. pot.
Acetic acid dilut.

gr.viij to $j.
dj to &

Weak.
Strong.
Weak.
Strong,
gr. viij to 5J.

5 sec.
2 sec.

2 sec.
1 sec.

1 sec.
1 sec.

2 sec.
1 sec.

Solut. gum-arab.
Solut. gum-arab.

Strong.
Strong.
Strong.
Strong.

1 sec.
1 sec.

i'sec.
1 sec.

Solut. caust. pot.
Solut. caust. pot.
Solut. caust. pot.
Solut. caust. pot.

Isec.
2 sec.

Solut. nitr. pot.
Solut. nitr. pot.
Solut. nitr. pot.

Strong.
Strong.
Strong.

3 sec.

1 sec.
3 sec.

2 sec.

20 sec.
25 sec.
20 sec.

20 sec.
20 sec.

Solut. gum-arab.
Solut. gum-arab.

2 sec.

Solut. gum-arab.

Solut. of starch.

Solut. of starch.
Solut. of starch.

3 sec.

10 sec.
10 sec.

......

2 sec.

16™

abdomen. It does not return to the centre of the yelk, nor escape to the surface,
but is lost much nearer to the latter than to the former position ; and its disappear-
ance is the result of the endogenous development of cells in its interior. The egg is
cast loose into the abdomen, and then consists only of the yelk mass in its vitelline
membrane, and it is transferred to the mouth of the oviduct by the joint action of

240 MR. NEWPORT ON THE IMPREGNATION OF

the abdominal muscles and the motions of the viscera, and not necessarily through
the aid of the male during copulation. Second, changes are going on in the consti-
tuents of the egg, both before and after oviposition as well in the unimpregnated as
in the impregnated condition ; but they soon cease in the former, and do not pro-
ceed to the cleaving or segmentation of the yelk. Third, that the egg is not sus-
ceptible of impregnation until after it has acquired the envelopes which it gains in
the oviduct. Fourth, that endosmosis of the entire egg takes place through these
envelopes, and is most rapid during the few minutes the egg is most susceptible of
impregnation. Further, that this endosmosis is augmented and hastened by an in-
crease, and is lessened and retarded by a diminution of temperature ; and that the sus-
ceptibility of the egg to become impregnated, and produce, is in exactly the same con-
dition with regard to heat ; whether the egg be exposed to, or whether it be excluded
from light. Fifth, that only extremely minute granules of solid matter can by any
possibility pass into the tissue of the envelopes during endosmosis ; and that there
is no evidence whatever of the existence of a fissure or orifice, in the envelopes of the
egg of the Amphibia, at the time of, or before impregnation, capable of admitting
the spermatozoon to the interior of the yelk-membrane or its contents. Sixth, that
it is the spermatozoon alone which effects impregnation ; and that this does not take
place until the spermatozoon is brought into immediate contact with the external
envelopes of the ovum. Seventh, that the liquor seminis, when entirely separated
from spermatozoa, certainly does not effect impregnation. Eighth, that although
direct contact of the spermatozoa with the ovum is indispensable to effect impreg-
nation, I have never been able to detect any traces of these bodies in contact with
the yelk-membrane, or even within the substance of the external envelope. Ninth,
that impregnation is commenced the instant the spermatozoa are brought into contact
with the egg, but a certain duration of contact is essential to its completion. Tenth,
that impregnation is not effected when the whole or the majority of the spermatozoa
in contact with the envelopes have previously become motionless and, apparently,
have lost vitality, as they are found to have done after the lapse of a longer or shorter
period. Eleventh, that although an exceedingly minute quantity of spermatozoa suffice
to impregnate the ovum, the phenomenon of impregnation takes place more tardily,
even with duration of contact when the number is extremely limited, than when it is
in full abundance, without excess ; while when the quantity is deficient, or the dura-
tion of contact too limited, then the phenomenon is incomplete, and partial impreg-
nation only is effected. Twelfth, partial impregnation is shown in imperfect segmen-
tation of the yelk ; and is due chiefly to the spermatozoa being insufficient in quantity,
or in duration of contact, or inefficient through diminished vitality ; and it may also
result from diminished susceptibility in the ovum. Thirteenth, partial impregnation
of the ovum is of frequent occurrence, as I found in my first experiments with fluid
that had passed through filtering-paper, but which still contained a very few sperma-
tozoa, either motionless or exceedingly feeble ; and further, partial impregnation is of

^o woiTAtfoaiWKi a "-IT xo Tfl.O'iwavf .HM . '.»*.-

THE OVUM IN THE AMPHIBIA. 241

>•»" ?v-i« lo Jr-tl> altkn'"--'' •; ' ' .-:•?'••:!•'. .^u; /*.> si""* N"> syqoJsvaa aril d.tr<w nooxo JjRf.n~.'K((B

much the most frequent occurrence when the ova are placed in dense fluid before
contact with the spermatozoa, as in the experiments with carmine. Lastly, when the
ova are only partially impregnated they are usually, and perhaps always unpro-
ductive.

These facts lead us to inquire, whether impregnation takes place through any cata-
lytic influence of the spermatozoa as suggested by BISCHOFF*, while in a state of
activity, and at the instant they are brought into contact with the ovum, or whether
impregnation results from a diffluence of the spermatozoa thus brought into contact
with the surface, the substance into which they may be dissolved being carried by
endosmosis with the water imbibed through the tissues ; or whether it is the result
of the conjoint influence of both these conditions ; — the first action induced being
instantaneous and catalytic, and possibly dependent on the persistence of organic
vitality in the spermatozoa, while the completion of the impregnation may depend on
the imbibition of some material influence or substance derived from the impregnating
body ; — a view which the gradual disappearance of the bodies of the spermatozoa
from the surface of the ovum, both in the Frog and Newt, seems to favour ; as we
have already seen that endosmosis is an active and important function of the enve-
lopes of the ovum at the very period when impregnation is effected.

All the experiments now detailed seem to show that in those vertebrata which
expel their ova into water before impregnation, as in the tail-less Amphibia, and in
which — from the nature of the medium into which the ova are passed — we may
infer that the function takes place most quickly, impregnation is commenced at
the very instant of contact of the spermatozoon with the ovum, and even may be
completed within very short spaces of time — but duration of at least some seconds
of actual contact, — even in these animals' ova, is essential to the perfection of the
function ; — but this period, we may fairly conclude, may differ in different classes of
animals, and possibly may have some relation to the greater or less facility with which
the spermatozoa are brought into contact with the ova.

When the experiments last detailed are compared, — the effects produced by the ap-
plication of media which influence the spermatozoon and the ovum chemically, — with
those of which the effect is merely mechanical, we seem to have made some advance
towards a future knowledge of the nature of the impregnating power. Although we
are as yet entirely without proof that any material influence or substance is actually
transmitted from the spermatozoon on the surface of the ovum to the yelk in the in-
terior, we have evidence that fluids are imbibed by the ovum by endosmosis through
its tissues ; and although not a trace of the spermatozoon is detected in the interior
of the ovum, we have seen that it remains for a long time on the surface, and gradu-
ally disappears, apparently by diffluence ; so that it may be fair to conclude, that the
agency of this body is material in its operation. On the other hand, the effect which
we find is produced on the yelk by the direct and even momentary contact of the

• *'"<' Uf*T» A. MM ^M^ --*•• » *•

* MULLER'S Archives, 1847.
MDCCCLI. 2 H*

242 MR. NEWPORT ON THE IMPREGNATION OF

spermatozoon with the envelopes of the ovum, seems closely to resemble that of the so-
called catalytic power of certain known bodies, in so far as that contact, during only
very short spaces of time, with the surface of the ovum, appears to be sufficient to in-
duce certain changes in the interior. These changes, too, as known of catalysis, are
carried only to a certain extent when the exciting agents, — in this instance the sper-
matozoa,— are feeble in action or but very few in number ; and then, as we have seen,
the yelk may become only more or less partially segmented ; or the changes in it,
having proceeded to a certain extent, may then become arrested, apparently from de-
ficiency of the originally exciting cause. Then, again, we find that although segmen-
tation of the yelk may take place, embryos are not produced unless there has been
some continuance or duration of contact of the impregnating with the impregnated
body; and that the number produced seems to have reference to the duration and to
the full sufficiency of the exciting cause. But neither what we at present know of
the so-called catalytic power or of endosmosis, appears alone to be sufficient to ac-
count for the whole of the phenomena of impregnation. Simple contact of the sper-
matozoon does not appear to be sufficient to determine the transmission of more or
less of the material structural characters of the male parent to the offspring ; while
diffluence and endosmosis of the substance of the spermatozoon can hardly be ima-
gined to occur in a brief second or two of time sufficiently to effect the full impreg-
nation of the yelk, and induce its invariable consequence, segmentation. Possibly,
we may hereafter find that the first changes induced by contact of the impregnating
body are completed by its diffluence, and by the material constituents into which it
is dissolved, being transferred to the yelk by endosmosis.

DESCRIPTION OF THE PLATE.

PLATE XIV.

Fig. 1. The female Frog, Rana temporaria, dissected to show the situation of the
entrance to the oviducts (a) at each side of the heart (b). The liver (c) is
drawn back and removed a little from its natural position to show the
spaces (d) along which the ova pass from the cavity of the abdomen to the
mouths of the oviducts (g), to be received into the dilated or uterine por-
tion of the ducts (h). (i.) The stomach. (&.) Intestine. (I.) Colon and
rectum, (m.) The bladder.

Fig. 2. A portion of the commencement of the oviduct magnified, partially concealed
by the root of the lung.

a. The entrance to the duct between the heart and liver, (e.) The suspen-
sory ligament of the liver. (/.) The base of the lung around which
the oviduct (g) passes.

Fig. 3. The female Frog, exhibiting the viscera in situ before the ova have left the
ovaries (/?) and with the oviducts (g) enlarged with secretion, for the for-

THE OVUM IN THE AMPHIBIA. 242*

mation of the envelopes of the ova as they pass through to the uterine or
dilated portions of the ducts (A).

Fig. 4. The female Frog after oviposition, with the organs of digestion and the liver
removed to show the condition of the ovaries (p) with their fatty append-
ages (q), the hyoid and thyroid muscles (n) (o) (p), the lungs (f) and the
contracted state of the oviducts (g), and their uterine enlargement (h) with
the rectum (/), and the bladder (m) then beginning again to be enlarged.

Fig. 5. Structure of the ovarian ovum.

a. The ovum while still attached to the inner surface of the ovary and pro-
jecting into the cavity, exhibiting the dark surface within the ovisac,
which is traversed by minute vessels (b).

Fig. 6. Vertical section of the ovum, showing the situation of the germinal vesicle
and the canal in the yelk, which corresponds to the centre of the dark
surface of the yelk.

Fig. 7- The presumed mode of disappearance of the vesicle.

Fig. 8. Spermatozoa of the Frog, a and b escaping from the vesicle of development,
c, as seen on the egg after contact.

Fig. 9. An ovum with spermatozoa half an hour after impregnation.

Fig. 10. A small portion of surface of the yelk at the commencement of segmentation,
highly magnified, (a.) Yelk-cells at the same period, (b.) The smaller
cells of the last, more highly magnified.

Figs. 11 and 12. Examples of partial impregnation at twenty-eight hours after con-
tact with the spermatozoa.

MDCCCLI. 2 t

Phil Trans MD C C CLI . PlattZN p

J

IV

I

Fig 7

1-185.

PijM.

Kg *.

[ 233 ]

X. On the Impregnation of the Ovum in the Amphibia. (Second Series, Revised.)
And on the Direct Agency of the Spermatozoon. By GEORGE NEWPORT, F.R.S.,
F.L.S. Sgc.

Received May 10,— Read June 17, 1852.

HAVING shown in a former series of investigations, which has been honoured by a
place in the Philosophical Transactions for 1851, that the sole agent of impreg-
nation of the ovum, in all cases of communion of the sexes, is the spermatozoon, —
and having then supplied both direct and negative evidence that impregnation is not
effected by the liquor seminis, — 1 endeavoured, in a subsequent communication to
the Royal Society, in June 1851*, to arrive at some .knowledge of the manner in
which impregnation is effected, and of the nature of the impregnating influence.

Up to that period, and indeed, until very recently, I had never been able to detect
any evidence of the existence of spermatozoa within the envelopes of the fecundated
egg, but had constantly found them in great abundance, and easily recognized, in
contact with the exterior surface. Experiment also, made by immersion of the egg
in coloured fluids, showed that the substance of the envelopes, although permeable by
fluids, is uniform in its structure; but no evidence was afforded by it of any natural
canal, fissure, or perforation through the envelopes of the egg of the Frog, capable of
admitting the spermatozoon to the interior, as has been supposed to exist in the egg
of the Mammalia-}-.

Hence the conclusion which seemed to be fairly led to, was, that some influence
was transmitted from the spermatozoon on the surface of the envelopes, through
their substance, to the yelk which they inclose, — as the commencement of impregna-
tion. But it was especially pointed out in my First Series^, that "simple contact of
the spermatozoon does not appear to be sufficient to determine the transmission of
more or less of the material structural characters of the male parent to the offspring ;"
and that, "possibly, we may hereafter find that the first changes induced by contact
of the impregnating body are completed by its diffluence, and by the material con-
stituents into which it is dissolved, being transferred to the yelk by endosmosis ;"
Further, in my second communication, I remarked, — " I am not yet prepared to
assent to the view, that simple contact alone, even of the spermatozoon, is sufficient
to complete the changes which result in the formation of the embryo $." Again, in

* Proceedings of Royal Society, vol. vi. p. 82.

f Philosophical Transactions, 1840, p. 533, Plate XXII. figs. 165, 167.
J Philosophical Transactions, 1851, p. 242.

§ See " Impregnation of the Ovum (Second Series)," June 19th, 1851, MS. No. 762, p. 49, in the Archives
of the Royal Society.

234 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

a subsequent part of the same communication, these considerations are further
referred to, as being those alone on which the transmission of structural peculiarities
seemed likely to be explained*.

Recent observation has now supplied to me a fact which renders these considera-
tions, which heretofore were regarded as hypothetical, much more probable; but, at
the same time, it necessitates a revision of the view that an impregnating influence is
transmitted from the surface to the interior of the egg; and also some correction of
the deductions from experiment by which this view seemed to be supported.

The fact referred to, is, that the spermatozoon of the Frog penetrates bodily into
the substance of the thick envelopes of the egg, and comes into direct communication
with, at least, the membrane which incloses the yelk in the interior.

It is my duty, therefore, to Science, and to the Royal Society, knowing this to be
the case, to revise and to extend tny views, and to submit them again to the Society
in their amended form ; making, in self-correction, the certainty of fact now take the
place of negative observation.

In doing this, I shall endeavour to show, that, although still regarding the sperma-
tozoon, for reasons to be adduced, as the organ of a special condition of force, or
vitality in the male body, — its influence on the egg in fecundation is direct and
immediate, and not operative, as heretofore supposed, merely through the envelopes;
but, nevertheless, as I have before shown, that it is exerted only so long as the
spermatozoon continues to give evidence of its vitality or force in its power of
motion.

Before proceeding to show that the spermatozoon penetrates into the envelopes of
the egg it is necessary that I should point out some of the conditions which affect the
fulfilment of its fecundatory function, both in regard to the spermatozoon itself and
to the ovum. These are, on the one hand, the persistence of vital power in the
spermatozoon as influenced by the temperature of the surrounding medium ; and on
the other, the endosmic property of the gelatinous coverings of the egg, and the
susceptibility of the yelk which they inclose, to become impregnated.

1. THE VITALITY OF THE SPERMATOZOON AND OVUM COMPARED.

1. Of the Spermatozoon. — It has been elsewhere shown -f- that when the mature
spermatic fluid of the Frog has been more than four hours removed from the living
body, and mixed with water, at a temperature of 50° FAHR. or but a few degrees
higher, it usually has already ceased to have any power to fecundate the egg; and
its efficiency has been found to have been diminished in proportion to the length of
time it has been obtained and so mixed. Nearly the whole of the observations I have
since made have coincided with these results. Yet there have been two marked
instances in which fecundation was effected by fluid, which had been obtained, and

* Loc. cit. p. 56. f Philosophical Transactions, 1851, p. 213.

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 235

mixed with a small quantity of water, twenty -four hours before it was employed. In
the first case, the fluid was obtained when the temperature of the atmosphere was
51° FAHR., and afterwards sunk to 49° FAHR., but had again risen to 51° FAHR. at the
time of the experiment. When examined with the microscope, immediately before it
was used, the fluid still contained a quantity of very active vibratile spermatozoa, and
also an abundance of spermatozoal cells, still in course of development. This fact
seemed to afford an explanation of the cause of the efficiency of the fluid for so great
a length of time, spermatozoa being liberated from the cells during the whole period.
Thus the fact of the prolonged fecundity of this fluid seems to confirm, instead of
oppose the conclusion arrived at, — that, the more mature the fluid is before its
removal from the body, the more efficacious it is, but only for a given extent of time,
since it is then composed almost entirely of very active spermatozoa, with but very
few cells of development. SPALLANZANI, as formerly mentioned*, found that the fluid
of the foetid terrestrial Toad (Bufo calamita ?) at a high temperature of the season,
70° FAHR. to 73° FAHR., at which this species spawns in Italy, had lost its fecundatory
influence at the end of six hours-f- ; but that in the temperature of an ice-house,
40° FAHR. it retained its efficacy for twenty-five hours;};. Further, that when the fluid
was preserved in the testes of the dead animal, at the temperature of the season
mentioned, it became effete in nine or ten hours; but that it continued to be effica-
cious, when retained in those organs in the temperature of an ice-house for thirty-
four hours. Again, he found that the fluid of the green aquatic Frog (Rana esculenta),
when mixed with a large quantity of water, and placed in an ice-house, the tempera-
ture of which he gives at 3| REAUM. (39'87° FAHR.) retained its fecundatory property
for thirty-five hours §. But it must be borne in rnind, with regard to these observa-
tions, that the species of Toad (Bufo), pair later in the season, and at a higher
temperature than those of the Frog ; and also that the fluid employed by SPALLANZANI
in all his experiments, was obtained by vivisection, either from the testes or the
vesicles; and no doubt contained, as in my experiment just mentioned, many unde-
veloped cells; and, consequently, not being fully matured, retained its influence
longer than it would have done, under the same circumstances, in its perfect con-
dition.

But PREVOST and DUMAS ||, in some experiments made to ascertain the length of
time during which the fluid of the species they experimented on, — the Rana esculenta,
— retains its fecundatory property, observed that it was efficient at twenty-four hours,
although removed from the body and preserved during that time, in a temperature
that varied from 18 to 22 cents., or from 64' to 71° FAHR.^[ This result, differing so
much, in regard to temperature, from the results formerly arrived at by SPALLANZANI,
in regard to the Frog, and from the majority of those since obtained by myself, may,
I think, in great part be accounted for in the fact that, like the Toads, the Rana

* Loc. cit. p. 212. f Dissertations, &c., vol. ii. p. 169. J Loc. dt. p. 169. § Loc. cit. p. 194.
|| Annales des Sc. Naturelles, torn. ii. 1824. f Loc. cit. p. 140.

MDCCCLIII. 2 I

236 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

esculenta spawns at a higher temperature, and later in the season than the R. tempo-
raria, the subject of my observations, and that the fluid employed by PREVOST and
DUMAS, was always obtained, as they state, by vivisection, and was expressed from
the testes, and necessarily must have been in great part immature, and contained
many spermatozoal cells, from which the fecundatory agents were liberated at dif-
ferent periods during the observations, and thus appeared to show that the fluid
continues to be efficacious, at a high temperature of the atmosphere, for a much
longer period than is really the case. In my own experiments the fluid employed
being obtained, without vivisection, by simple compression of the body from the
seminal ducts and vesicles, has usually contained only perfectly mature and very
active spermatozoa, with but few cells of development. In this condition, as I have
stated, it has retained its fecundatory property only during the first three or four
hours. Yet, in the exception above mentioned, and also in the second one alluded to,
which occurred more recently, the fluid was efficacious at the end of twenty-four
hours in the first, and twenty-six hours in the second instance, the temperature being
similar on the two occasions. In both instances the fluid contained a very large
quantity of developmental cells, and had been procured from individuals which were
not fully prepared for the exercise of their fecundatory function. Further, I may
add, that in each of these instances, after the fluid had been supplied to some eggs
to test its effect, I found many undeveloped spermatozoal cells adhering to the
surface of the eggs, and detected some spermatic bodies in the act of being liberated
from them. These facts, then, are confirmatory of the suggestion respecting the
results obtained by PREVOST and DUMAS.

The presence of undeveloped cells in the fluid obtained from the living Frog is
explicable in two or three ways: first, in that of the animal having but recently
been taken from its natural haunts, either early in the season, or at a later period,
after a continuance of unusually cold weather and easterly wind, or after the sexes
have but recently united ; in either of which cases, as also in certain others, in which
the individual itself has been late in the development of its reproductive organism,
developmental cells are thrown off from the testes together with already liberated
spermatozoa, and are usually abundant in the efferential ducts and vesicles. It may
thus be seen that in judging of the length of time during which the fluid preserves
its fecundatory influence, and consequently, the spermatozoon its vitality, after removal
from the body, it is necessary to take into consideration, not only the temperature
of the surrounding medium, but, also the precise condition of the fluid itself. In
perfectly natural impregnation by the Frog there is reason to believe that the fluid
contains but very few undeveloped cells, and that it is not employed until it has
acquired its full maturity, and that, — as the time of its actual employment is exceed-
ingly brief, — the oviposition of Rana temporaria occupying scarcely a single minute,
the retention of the impregnating influence and vital force of the spermatozoon may
be even for a much shorter space of time than I have mentioned — from three to four

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 237

hours. But this time may differ in different species of the Anoura, and of the higher
vertebrata, and may have some relation to the particular economy of each animal.

The general conclusion which seems to be deducible from a comparison of the
observations of SPALLANZANI and of PREVOST and DUAIAS, with those by myself, in
regard to the tail-less Amphibia, is, that — making allowance for the difference of
species and habit of the animals experimented on, and for the degree of maturity of
the fluid, which there seerns reason to think does not attain its perfect condition
until it has passed into the efferential ducts and vesicles — the vitality of the sperma-
tozoon, and the duration of its fecundatory power, are in a ratio inverse to that of an
increase of temperature in the surrounding medium.

2. EVOLUTION OF VITALITY IN THE SPERMATOZOON AND OVUM.

WAGNER and LEUOKARDT have already pointed out* that the power of motion
possessed by the spermatozoon is closely connected with the completion of its struc-
ture and composition, and is gradually evolved as the development of this body
proceeds. This power of motion I shall presently endeavour to show I regard as the
visible exponent of its fecundatory force, or form of vitality, and that a similar power
is evolved concurrently with this in the spermatozoon in the contents of the egg, the
evolution in both being more or less influenced by temperature, according to the
species. These facts are well shown both in the Frog and Toad.

Several pairs of Frogs were collected on the 2nd of March, when the temperature
of the season was low, and strong easterly winds prevailed, during which Frogs
seldom spawn, except in very sheltered places. As one female had passed some eggs,
and believing that the others were equally advanced, I selected a pair, which
appeared to be the most mature, for experiment. The fluid from the male was
obtained with the greatest ease, in full quantity of a white and somewhat opake
colour. When examined with the microscope it was found to consist in chief part of
developmental cells, containing each its motionless immature spermatozoon. Besides
these cells it also included many others less far advanced, and in which I was unable
to distinguish this body. In addition to these there were some also from which the
spermatozoa were in the act of being liberated, besides many very active spermatozoa
already set free. These constituted nearly one third of the whole mass. It was
evident from these circumstances that the fluid was immature, and not fitted for
experiment. Five days afterwards, March 9th, I again examined fluid from this
male, which had been kept since the previous examination, in a separate vessel, so
that there was no mistake in its identity. The mean temperature of the room during
the interval was 42'°5 FAHR., and the water in which the Frogs were preserved
40° FAHR., but none of the Frogs had spawned. The fluid was then less dense and
of a less white colour, and was composed almost entirely of very active and fully
developed spermatozoa, with but very few cells from which these bodies had not

* Article " Semen " in Cyclopaedia of Anatomy and Physiology, part xxxvi. (January, 1849) vol. iv. p. 504.

2 I 2

238 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

been liberated. There were, however, some of which the contents appeared to be
perfectly granular, and these I regarded as spermatozoal cells in the earlier stage of
development.

The female with which this male had been paired was killed on the 4th, at the
time the experiments were to have been commenced. It was then found that the
eggs, like the fluid from the male, were immature. The whole of the eggs had escaped
from the ovaries into the cavity of the peritoneum among the viscera, and were
crowded together in a mass on each side, at the anterior part of the abdomen, and
were without any gelatinous covering. A few only had begun to enter the oviduct
on the right side of the body, and acquire their envelopes. In most of these eggs the
germinal vesicle had already disappeared.

The fluid and eggs of the common Toad (Bufo vulgaris) are developed in precisely
the same way as in the Frog. On the 30th of March 1851, I examined the bodies of
a male and female Toad for the purpose of experiment. The great efferential ducts
and vesicles in the male were then entirely empty, no fluid having yet descended
into them. But the testes contained a vast quantity of spermatozoal cells, some of
which were distended by the spermatozoon, as if in the act of being liberated. But
not a single spermatozoon was free, or showed any sign of motion. The whole were
still immature, the caudal portion being as yet short and imperfect.

In the female the ovaries were distended with eggs and occupied the whole of the
visceral cavity between the intestines. The eggs were of a sooty black colour, with
but a slight trace of the grey surface, and appeared to be nearly mature, but had not
yet left the ovary. The germinal vesicle of a large size, circular in its outline, and
somewhat flattened, still existed in nearly the whole of them.

It is thus evident that the pairing of the sexes, both of Frogs and Toads, takes
place before the semen is fully matured, or the eggs have descended into the oviducts,
and that there is a near concurrence in the time of maturity in both.

These facts seem to lead to the conclusion, that when, as in some of the fore-men-
tioned experiments, the male fluid contains a very large proportion of developmental
cells, which occasion its white appearance, it is not fully matured for its function ;
that the first portions of fluid which descend into the efferential vessels are usually
immature ; and that it is not absolutely necessary that the spermatozoa should be fully
formed before the cells escape from the testes. It seems fair then to infer from these
facts that the completion of the development of the spermatozoal bodies takes place
in the deferential vessels, or in the vesicles, perhaps in both : further, that the
quantity of fluid produced, when too great to be contained in the ducts, may become
accumulated in the vesicles, to be furnished at the instant it is required ; and that
there is a near concurrence in the time of maturity in the fluid in the male, and of
the eggs in the female, the former being only slightly in advance, in regard to time,
of the latter, at the natural period of their encounter ; a condition which perhaps
may be necessary to the full and healthy fecundation of the whole brood.

'•• AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 239

3. OF THE OVUM.

The vitality of the eggs of the Frog appears to be of longer duration than that of
the fertilizing agent, the spermatozoon. The egg, as formerly shown*, leaves the
ovary quickly after, or at the time when its germinal vesicle disappears ; but, as
experiment has proved, it is not then entirely fitted for its function. It has yet to be
enveloped in a thick covering, which it gains in its transit through the oviduct,
before it is susceptible of impregnation. Its condition when escaping from the
ovary to the oviduct seems to be analogous to that of the spermatozoon when it
leaves the testis to enter the efferential duct. Neither the one nor the other
has yet attained its full maturity. When the eggs are collected in the dilated or
uterine portions of the oviducts they seem to be in a condition parallel to that of the
spermatozoa in the lower portion of the efferential ducts and vesicles. When nearly
the whole of the eggs are collected in the uterine cavities their maturity is then
almost completed ; but a period of retention, even in these structures, seems to be
necessary to ensure the fertilization of the whole, as some of them, — as I have
found by the persistence of the germinal vesicle for a short time, even after the egg
has escaped from the ovary into the cavity of the abdomen, as the undeveloped
spermatozoal cell passes from the testis into the efferential duct, — are later in their
development than others. Further, it may be remembered -}-, that so far from the
contents of the yelk being in a dormant condition at the time of oviposition, as some
inquirers have supposed;}:, there are changes still going on within it, and which are
perceptible to the eye, in the condition of the white surface, for ten or twelve minutes
after oviposition ; after which they become less and less marked, and soon entirely
cease if the egg be not fecundated.

Thus then from a comparison of the state of the fecundatory agent with the body
to be fecundated, we might have expected to have found some close coincidence in
the retention of their vitality, and this indeed, in a state of nature, seems to be the fact.
Spawning seldom or never takes place by the act of the species until each sex has
attained its full maturity of function. The eggs are sometimes voluntarily retained
by the female Frog, for several hours or days within the uteri, although arrived at
maturity, if the proper evolution of spermatozoa in the male be not completed. I
have had evidence of this in the fact, that when the union of the sexes has been of
long continuance, the female has sometimes passed a very few ova, and then has con-
tinued without further oviposition for many hours, sometimes for a day or two,
before the mass was expelled, and the function of the sexes consummated. These first
extruded eggs have almost always been found to be unimpregnated.

When the eggs have been deposited, the circumstances which affect their fecunda-
tion are precisely similar to those which affect the spermatozoa, — the temperature
of the surrounding medium, and the length of time during which they remain im-

* Philosophical Transactions, 1851, p. 180. f Loc. cit. p. 185. J WAGNER and LEUCKARDT, loc. cit.

240 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

mersed in water after expulsion from the body. The length of time during which
they are susceptible of impregnation has relation to each of these conditions ; and
these have now acquired additional interest in the establishment of the fact that the
spermatozoon penetrates into the envelopes of the eggs, and that, consequently, the
rate of expansion of the envelopes has a further direct relation to its function.

The following experiments will illustrate the foregoing observations: —

It may here be remarked, that although, as in my former paper, the date of the
experiment is recorded, it is so only for the convenience of reference, and not with
any regard to priority of period at which the experiments were made ; the details
being given only in illustration of the general statements enunciated.

April 2, 1852. Atmosphere 55° FAHR. The first three of the following experiments
were made with the eggs and fluid from a pair of Frogs which had been obtained
from their natural haunts only three hours before the female was killed, by division
of the spinal cord, for the purpose of the experiments.

No. 1 . Eighty-six eggs passed from the Frog at one hour and a half after death,
were supplied with fluid from the male with which this female had been paired. — the
fluid having been obtained and mixed with water one hour and a half before it was
employed.

On the seventh day Jifty-two embryos had been produced from these eggs, and
some of them were then escaping from their envelopes.

No. 2. Thirty-seven eggs, obtained from the same female, were supplied with a
portion of the same fluid as the above, after it had been mixed with water about two
hours and three-quarters.

Twenty-eight embryos were produced from these eggs at the same time as the
above.

No. 3. Sixty-eight eggs from the same Frog at six hours and three-quarters after
death were immersed in the same portion of mixed fluid and water in which the eggs
of No. 2 had been fecundated. In this experiment the fluid had been six hours and
three-quarters mixed with water.

At the end of three days the eggs still had a healthy appearance, but on the seventh
day not one had given any evidence of having been impregnated, nor was any
embryo ultimately produced. Thus the fluid had lost its fecundatory property, at a
temperature of 55° FAHR. between the end of the third and of the seventh hours.

As it was possible that the failure in this experiment was due as much to the eggs
as to the fluid employed, it having already been shown that impregnation is some-
times effected with fluid which has been twenty-four hours removed from the body, —
the following experiments were then made : —

No. 4. Sixty-nine eggs obtained from the same female at one hour and twenty
minutes after death were bathed with fluid procured from another male, and which had
been mixed with water thirty hours previous. These eggs preserved their regularity
of outline for nearly six hours, at the end of which time one egg showed traces of

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 241

partial impregnation, while the yelks of the others were changing form and becoming
irregular. But not a single embryo was produced.

No. 5. Eighty-one eggs were obtained from another Frog, a few minutes after it
had been killed by division of the spinal cord, and a portion of fluid which had been
mixed with water twenty-four hours and a half, and kept in a temperature which had
sunk from 54° FAHR. to 51°FAHR., but at the time the fluid was employed had again
risen to 55° FAHR. — was supplied to them.

This fluid on examination by the microscope was found to contain an abundance
of spermatozoa, the whole of which were perfectly motionless, so that I was not able
to detect even one which gave any sign of vitality. Yet the eggs in this experiment
retained their natural healthy appearance for several hours ; but not one was im-
pregnated, nor was even a single embryo produced. On the contrary, after thirteen
hours, the yelks of some of them assumed a pyriform shape, while those of others
became shrivelled and withered.

No. 6. Thirty-six eggs were passed from a Frog, which had been killed about
three-quarters of an hour, and some fluid which had been obtained and mixed with
water forty -four hours before was shed over them, and pure water was then added.

The envelopes of these eggs became expanded as under perfectly healthy and
favourable conditions ; but within two hours of the contact of the eggs with the fluid
the yelks of the whole became very irregular, shrivelled, and contracted, and assumed
an appearance very similar to that which first results from the application of a strong
solution of potass. Not one egg retained its spherical shape, but the whole entirely
perished ; and it may be needless to add, not an embryo was produced.

These facts then support the statement already made, that only while the sperma-
tozoon continues to give evidence of vitality in its power of motion does it exert any
fecundatory influence on the egg. They seem, too, to show that the period during
which this vital force is retained by the mature spermatozoon after it is passed from
the body of the Frog is perhaps shorter even than what I have stated, and that the
limit, in regard to time, depends much on physical influences. In addition to this,
they seem to show that after all ocular evidence of power in the spermatozoon is
lost, this body is inert, until decomposition has commenced, when its material con-
stituents become injurious and destructive.

The following experiments have more direct reference to the vitality of the egg,
and appear to indicate that this is much longer retained than that of the spermato-
zoon, and is less quickly affected by external causes.

April 2, 1851. Atmosphere 55°.

No. 1. Thirty-eight eggs were passed from a Frog killed twenty-four hours and a
half before, and some fluid which had been obtained and mixed with water about
one hour and a quarter was then added to them.

Segmentation commenced in some of these eggs in about four hours and a half,
and on the seventh day eight embryos had been produced.

242 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

No. 2. Seventy-three eggs were taken from a Frog which had been killed about
thirty hours, and were bathed with fluid which had been one hour and twenty minutes
mixed with water.

At the end of six hours several of these eggs had become irregular, but one had
certainly been fecundated, as one embryo afterwards came to maturity.

No. 3. One hundred and sixty eggs horn the Frog which supplied those employed in
the experiment No. 2, were bathed with fluid from another male, immediately after
it had been obtained, but not a single egg was impregnated.

No. 4. Thirty-seven eggs were passed from a Frog which had been killed forty-four
hours before, the temperature of the atmosphere during this period having risen from
50° FAHR. to 55° FAHR. These eggs were bathed with fluid which had been obtained
about three quarters of an hour. At the end of six hours I found, to my surprise, that
one egg had become segmented, but that the yelks of the others were flattened,
shrivelled, and irregular, as if they had been affected by the solution of potass, or by
decomposing seminal fluid. On the seventh day the egg which had been segmented
had produced an embryo.

From these results, then, it appears that while the spermatozoon at a mean tem-
perature of the atmosphere of 55° FAHR. usually loses its vitality, and is inert in less
than four hours, and but rarely is efficient at a longer period after immersion in
water, the egg retains its reproductive property for a very much longer time, especially
if not removed from the body of the dead animal, only losing it at twenty-four hours
after the death of the parent, and occasionally retaining it for forty hours.

The experiment No. 4, in which the latter fact is shown, is interesting, from the
circumstance that the same results are produced by the living vibratile spermatozoon
on the yelks of the dead eggs, as the majority of these were, as that which is occa-
sioned by the application of dead and decomposing spermatozoa to the envelopes of
the living one, as shown in No. 6 of the preceding set ; and further, that these results
so closely resemble in appearance the first effects produced on the yelks of living eggs
by the application of strong solution of potass ; thereby showing not only that powerful
endosmic action takes place rapidly through the coverings of the egg, but also that
the influence of the spermatozoon on the yelk, whatever may be its precise nature,
is direct and immediate.

4. ENDOSMOSIS OF THE EGG IN RELATION TO ITS VITALITY.

It has before been shown that the endosmic action of the envelopes of the egg, on
immersion in water, is closely connected with the act of fecundation ; and we have
now further proof of this in the fact that the vitality of the egg may be preserved for
many hours if the envelopes be not brought into contact with water; so that the
insusceptibility of the egg to become impregnated, after a lengthened period of
immersion, is due to a diminution of the expansive property of the tissue of the

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 243

envelopes, and the distension of the tissue with fluid, rather than to the loss of vitality
in the contents of the yelk.

This view has led me to endeavour to ascertain more precisely than heretofore, —
fast, at what length of time, after immersion in water, the egg usually becomes
insusceptible of impregnation ; and next, to what extent the act of fecundation is
accelerated by a given increase of temperature, when the experiments are made with
portions of fluid from the same male, and with eggs from the same female, at, as
nearly as possible, the same time, all the conditions being similar except in regard to
the temperature of the surrounding medium.

With this object, I have recently made two sets of observations consisting each of
nine experiments. One set was conducted in a room, the temperature of which was
raised artificially to 61° FAHR., and the water in which the eggs were immersed to
60° FAHR. ; while the other was carried on in an adjoining room where the tempera-
ture was only 55° FAHR., and the water employed 53° FAHR. In order to place the
whole of the experiments under as similar conditions as possible, it was necessary
that the corresponding experiments in each set should not only be made at about the
same time, but should also be supplied with fluid in exactly the same state, and the
eggs be immersed in like quantities of water, for the time specified in each experi-
ment, before the addition of the fluid. When the time of immersion had elapsed the
water was quickly withdrawn, and the fluid poured over the eggs, and fresh water
was immediately supplied to them. As much time was unavoidably occupied in
passing the eggs into their respective vessels, and noting the period of immersion, it
became necessary to commence the experiments with those which had been longest
in water, in order that the fluid supplied to the whole should be as nearly as possible
in the same condition, in each experiment, at the moment of its application, and be
furnished to the eggs in similar quantities. To ensure this, the fluid was mixed, as
soon as it was obtained, with fifteen times its quantity of water, and was employed
at about thirty-Jive minutes afterwards. The quantity of this mixed fluid poured over
the eggs of each experiment was ten minims by measure, excepting only to the first
and second experiments of each set, to which twenty minims were added; but even
with this advantage, as was seen, with no more favourable change in the result.
After the end of the first hour, the eggs which had remained in the lower temperature
were removed to the higher, and the two sets of experiments were then placed side
by side to watch the time of commencement of segmentation, as indicatory of the
more or less accelerated impregnation. The number of embryos subsequently pro-
duced showed the ratio in which the eggs had been fecundated after different periods
of immersion*. Each set consisted of nine separate experiments, the time of immer-
sion of the eggs being forty-five, thirty-five, twenty-five, fifteen, ten, seven, five, three,
and one or two minutes in each.

At the highest temperature, 61° FAHR. atmosphere, and 60° FAHR. water, a tem-
* For the details of the experiments, see MS. paper in the Archives of the Society.

MUCCCLIII. 2 K

244 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

perature which is always most favourable to fecundation, there were no signs that any
change had even been commenced in those eggs which had been immersed for a longer
period than thirty-five minutes. But it must be borne in mind that the fluid employed
had been thirty-five minutes mixed with water, and consequently had begun to lose
some of its efficacy. The results of the corresponding experiments at the lower tem-
perature— Atmosphere 55°, Water 53° — were similar, but less marked.

On comparing the two sets of investigations, it was found that no evidence of
fecundation occurred in either of them, when the eggs had been immersed during
forty-five minutes before the impregnating fluid was supplied to them ; and that only
the very earliest symptoms of any influence having been communicated to the eggs,
after thirty-five minutes' immersion, occurred in those which were submitted to the
higher temperature, in the irregular contraction of the yelks ; while in each set, both
in the higher and lower temperature, some of the eggs, after twenty-five minutes'
immersion, showed unequivocal signs of partial fecundation in the formation of the
respiratory chamber, the result of the contraction and depression of the upper portion
of the yelk previous to segmentation. But in both sets there were some eggs which,
after fifteen minutes' immersion, had become fecundated, and produced embryos.
The relative number of embryos produced after immersion for this and shorter spaces
of time was certainly greatest in the higher temperature ; since, when the total
number of eggs employed in the last six experiments in each of the two sets, in
which only any embryos were produced, were compared with the number of embryos,
it was found that in the higher temperature there were severity-eight embryos from
five hundred and thirty-six eggs; while in the lower temperature, from six hundred
and two eggs, there were only seventy-six embryos.

The period at which segmentation occurred in the fecundated eggs of the two sets
corresponded also with the above results. In most of the eggs fecundated in the higher
temperature segmentation commenced about ten minutes earlier than in those which
had been fecundated, and remained during the first hour in the lower temperature.

These results seem to show, that while the eggs are more rapidly affected, they are
also more certainly impregnated, and the embryos produced in greater numbers at
a higher than at a lower temperature. But it must be borne in mind that the number
of eggs fecundated in these experiments can only be regarded as comparative, and
as mere approximations to what takes place in a state of nature, the experiments
having been made with fluid which had been removed from the body, and con-
siderably diluted with water, more than half an hour before it was employed, and
consequently when its efficacy had been considerably diminished.

These facts, then, go to confirm the previous conclusions, that the organic force or
vitality of the spermatozoon is of shorter duration than that of the egg ; since, if the
results of these experiments be compared with those obtained from former ones, it
will be seen that the insusceptibility of the egg depends very much more on the
endosmic action of its envelopes after immersion in water, than on any loss of vitality :

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 245

further, that the non-fecundation of the egg may depend much more on diminished
vitality and inefficiency of the spermatozoon, than on the insusceptibility of the egg
itself.

5. QUANTITY OF SPERMATOZOA ESSENTIAL TO FECUNDATION.
On a former occasion* I endeavoured to arrive at some conclusion as to the mini-
mum quantity of spermatozoa necessary to effect impregnation, and to draw some
comparison between the results obtained by myself and certain of the more remark-
able ones by SPALLANZANI -f-. It then seemed to rne, that although the general results
obtained were in accordance with SPALLANZANI'S, yet that a larger proportion of fluid,
and consequently a greater quantity of spermatozoa, was required than was supposed
by him. I have, therefore, made further investigation on this subject, and have
sought to obtain some knowledge of the quantity really necessary to fecundate the
egg. PREVOST and DUMAS formerly made a similar attempt;}:, and M. QUATREFAGES
has recently done the sarnefy. The results of the observations by PREVOST and DUMAS,
QuATREFAGEs,and myself, although differing greatly in detail, coincide with the general
results obtained by SPALLANZANI, — that only an exceedingly small quantity of seminal
influence, and consequently only a very limited number of vibratile spermatozoa is
necessary to effect impregnation. They agree, too, in the fact that the number of
eggs impregnated is always much fewer than that of the spermatozoa supplied to
them ; and, consequently, that there is reason to think that fecundation is not the
result of a single, isolated spermatozoon, although it has not been ascertained what
is the number actually required. The course I have pursued in putting this curious
question to the test, has been different from that of either of the distinguished
naturalists mentioned. It has not been that of diluting the impregnating fluid in a
very large quantity of water, and then applying a minute drop of the water to the
Frog's egg, as done by SPALLANZANI ; — nor has it been that of reckoning the number
of spermatozoa on a given surface of a micrometer plate, and then immersing the
plate in water with a quantity of eggs, and afterwards observing how many of them
became fecundated, — the course followed by PREVOST and DUMAS ; — nor has it been
by estimating the number of spermatozoa in a given bulk of fluid, and then ascertain-
ing the number of eggs fecundated by them, as done by QUATREFAGES with those of
the Hermella and Teredo; but it has been by direct application of the spermatozoa
on the point of a pin to the surface of a single egg in a glass cell ; and then observing
beneath the microscope the number of spermatozoa deposited from the point of the
pin, applied in a similar way as to the egg, on a plate of glass. Yet only approxi-
mative results can be arrived at by this, or by the other modes of proceeding,
although it is satisfactory to find that the general results obtained in this way are
in agreement with those obtained by the observers mentioned ; and further, that the
mode adopted has led to some additional results, which seem to be of value.

* Philosophical Transactions, 1851, p. 206. t Dissertations, &c., vol. ii. p. 189.

\ Annales des Sc. Naturelles, torn. ii. 1824. § Annales des Sc. Nat. 3me aerie, torn. xiii. p. 129.

2x9

246 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

6. TESTS OF FECUNDATION.

I have given some account in my former paper* of a space, or chamber, which is
developed between the vitellary envelope of the egg and the upper surface of the yelk,
within the first hour and a half, subsequent to the encounter of the spermatozoon with
the egg ; and I have referred to it in the details of these experiments, as occurring in
some of the eggs which had been immersed during twenty-five minutes before the
fecundatory agent was supplied to them, and as marking their having been affected
by its influence. The formation of this chamber is one of the earliest and most cer-
tain indications that the egg has been more or less impregnated. The earliest
symptom of impregnation occurs somewhat sooner than the formation of the chamber,
or within the first half-hour, and consists in a marked increase in the degree of undu-
latory heaving and sinking of portions of the surface of the yelk, motions which take
place slightly in the unirnpregnated egg, but are increased in the impregnated;
although, even in that, they are not sufficiently permanent to be easily recognized as
proofs of fecundation. The irregular contraction and shrivelled appearance of the
yelks, noticed in the preceding observations, may, perhaps, be due in part to this
cause. The formation of the chamber is the result of the contraction and depression
of the upper surface of the yelk, occasioned, as there is reason to think, by changes
which are going on within the substance of the upper hemisphere of the yelk, around
the embryo vesicle, or its progeny, and which changes appear to be due to the opera-
tion of the spermatozoon ; as I have never observed the chamber in any egg which
has not been more or less fecundated. The fact of the existence of a chamber, therefore,
may always be regarded as a. proof of fecundation. It is commenced sooner or later
according to the temperature of the surrounding medium, and to the more or less
complete fecundation of the egg. It is usually perceptible in about one hour, or one
hour and a quarter after the impregnating fluid has been supplied to the egg, and is
distinctly marked in less than one hour and a half, when the eggs are preserved in a
temperature of from 50° to 55° FAHR., and its size is increased during the first three
hours. It is commenced in a slight depression in the centre of the upper surface of
the yelk, at a point which corresponds to the entrance to the central canal, and it
takes place much sooner in eggs which have been completely fecundated, even at
moderate temperatures, than in others which have been more sparingly affected. I
have seen it begun in a fully impregnated egg, in a temperature of 52° FAHR., at fifty-
three minutes after encounter with very mature spermatozoa. When it has attained its
greatest superficial extent it appears like a clear watch-glass cavity, filled with a per-
fectly transparent, limpid fluid above the dark upper surface of the egg. After the
third hour it becomes slightly further enlarged in its axis from above downwards by
the formation of a shallow, funnel-shaped depression on the surface of the yelk, the
centre of which is the minute orifice seen by PREVOST and DUMAS, and BAKU, and this
is continuous with the canal above alluded to. I have never found this chamber

* Philosophical Transactions, 1851, p. 187.

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 247

absent from any egg which has produced an embryo. Its formation is preliminary to
the subsequent cleavage of the yelk. It thus affords, within a very, short space of
time, a certain test of fecundation, and as such is exceedingly valuable in all experi-
ments by artificial impregnation.

7. PARTIAL, OR INCOMPLETE FECUNDATION.

But although the formation of the chamber gives certain proof that the egg has
been influenced by the spermatozoon, it is only the actual segmentation of the yelk,
carried through the first four stages, which shows that its fecundation has been com-
pleted ; since the egg may be only partially influenced, and consequently may not
produce an embryo, although the respiratory chamber may be formed, and the early
stages of segmentation of the yelk be commenced. When this is the case the change
seldom proceeds beyond the second or third stage of cleavage, and even then there is
usually some irregularity in its occurrence. Sometimes only the chamber itself is
developed, and the process then stops ; at other times the chamber is completed and
a little white spherical body makes its appearance, and is but just perceptible within
the entrance to the central canal ; but more frequently a white spherical body, not
more than one-fourth the size of the original germinal vesicle, appears on the surface
of the yelk within the chamber ; at other times there are two of these white bodies,
each of which is scarcely more than one-half the size of the single one, and sometimes
the bodies are of a dark or grey colour. In other cases the yelk begins to be divided
in the margins of the canal, but does not proceed with its change ; or it may go on,
as just stated, to the several stages. In other cases the yelk divides very unequally.
These instances of partial impregnation or fecundation may occasionally be produced
by application of exceedingly minute quantities of seminal influence, as some of the
following experiments will show.

8. DEFICIENCY OF FECUNDATORY INFLUENCE.

(a.) Pin-point application of the fluid. — These experiments were commenced in the
season of last year (1851), but have been repeated at different periods during the
season of the present year.

March 30, 1851. Atmosphere 56°FAHR. — A single egg was passed into each of
four glass cells, with a small quantity of water, sufficient only to preserve the eggs
moist and promote the expansion of their envelopes. Within two minutes afterwards
two of the eggs were each touched once with the point of a common-sized pin, which
had been so lightly dipped, into a mixture of one part of fecundatory fluid, obtained
about one hour and five minutes before, and four parts of water, — as to cover a por-
tion of its surface equal to about one thirtieth of an inch in extent. The two remain-
ing eggs were touched, the first once only, and the second twice, with the point of a
pin slightly curved, and which thus was made to retain a much greater number of
spermatozoa in the fluid which adhered to it than the pin with the straight point.

248 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

Each cell was then filled with water, so that the egg which it contained was completely
immersed. Within the second minute after the eggs had been touched with the
loaded pin, there arose from each, at the point of contact, a microscopic bubble of
air, the egg which had been twice touched having two bubbles. These were found
by subsequent observations to be due to the circumstance of the eggs having been
exposed to the air, for a short time before they were touched, and their surface
become slightly dried, so that when the fluid was applied to it a small bubble of air
became involved and imprisoned in it, before the eggs were immersed in water.
This circumstance and its explanation are mentioned simply to show that the occur-
rence had no relation to the act of fecundation, as at first I had supposed was pro-
bable.

I endeavoured to obtain some idea of the number of spermatozoa deposited from
the pin point, during its momentary contact with the egg, in these experiments. But
the result seemed to give but little hope that either of the experiments would be suc-
cessful, or that any fecundation could be effected by the application of so minute a
quantity of influence, as it was found that the number of spermatozoa deposited on
a plate of glass, beneath the microscope, when the pin point was applied to the glass
in the same way as to the egg, did not exceed from six to ten or twelve of these
bodies. Yet the results of the experiments were watched. Within the first half-hour
the eggs were removed from the temperature of 56°FAHR. to one of 65°FAHR. But
at the end of ten hours it was found that only in one instance had any fecundation
been effected. The grey central spot on the dark surface of the yelk was still pre-
sent in three of the eggs, and no respiratory chamber had been formed in either of
them. But partial fecundation had been effected in the fourth egg, which had been
twice touched with the loaded pin point. In this instance the dark surface of the
yelk was depressed, and the respiratory chamber had been formed between it and
the envelope, and two spherical opake white bodies appeared within it, lying side by
side on the middle of the surface of the yelk ; but no segmentation of this had taken
place, or even had been commenced.

One cause of failure, in these first experiments, appears to have been the diminished
efficacy of the fluid, which had been obtained and mixed more than one hour before ;
as it will presently be seen that even a less proportion of the fluid sometimes is
effectual when employed immediately it is procured.

As it was late in the season of last year when these experiments with minimum
quantities were commenced, the spawning of the frogs being then nearly at an end,
I was fortunate in procuring, on the second of April, a single unspawned pair of
frogs from their natural haunts, and thus was enabled to repeat the experiments with
the fewest disadvantages. This was done within four hours after the frogs were
captured. The fluid in this instance was very mature, was obtained in good abun-
dance, and was mixed with an equal quantity of water, and employed in the experi-
ments a few minutes afterwards.

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 249

April 2, 185 1 . Atmosphere 55°.— Ten eggs were passed into a large glass cell, and
each egg was touched once only with the pin point dipped lightly into the fluid for each
egg. This experiment was perfectly successful, as the chamber was formed above
the yelk in four or five of these eggs, and at the end of five days three of them had
produced embryos. In these instances the pin had been allowed to remain in contact
with the egg, and the fluid to drain off it for one or two seconds.

Six eggs were also placed in another cell and each was touched with the loaded
pin point when the fluid employed had been obtained about a quarter of an hour.
Two of these eggs became partially fecundated, as shown in the formation of the
respiratory chamber. In each of these the yelk became slightly elongated, a symptom
which always precedes the first cleavage ; yet they did not undergo further alteration,
but with the remaining four were abortive.

Twenty-three eggs were included in a large cell as a further experiment, and each
egg was touched once with the pin-point loaded each time, when the fluid employed
had been one hour and three quarters mixed with water. Four of these also had the
chamber formed, and segmentation commenced in them, at the end of four hours
and a half; but the majority were only partially fecundated, as only one of the
twenty-three eggs produced an embryo. The fact of this production, however, shows,
that an exceedingly small amount of influence, even at a long period after the fecun-
datory fluid has been removed from the body in which it is generated, is sometimes
sufficient to occasion the development of an animated being ; and that although a
plurality of spermatozoa, supplied to the egg, appears always to be necessary to
ensure fecundation, yet that the result seems to depend less upon the abolute given
number, or numerical relation of these bodies, to the egg, than on the measure of
vitality possessed by those which are brought into actual encounter with it, as a very
small number appears to be more efficient when employed immediately after their
removal from the body, than a much larger number after a prolonged interval.

(b.) Pin-head application of fluid. — The preceding experiments having been made
with the smallest quantity of fluid, and consequently with the fewest spermatozoa
that could be applied directly to the egg, it was desirable to increase the number of
these bodies without applying them in excess, and for this purpose the head of a very
small pin, the smallest size used by insect-collectors, was employed, instead of the
point of a larger sized pin. An endeavour was also made, as in the previous trials,
to ascertain the number of spermatozoa which the head of this sized pin, dipped
lightly into fluid, was likely to be the means of conveying to the egg. It was then
found that the loaded pin-head deposited on a plate of glass, in the minute quantity
of fluid which adhered to it, from at least fifty to one hundred and fifty spermatozoa ;
a number which was not only fully sufficient to fecundate each egg to which the pin-
head was applied, but also other eggs which might happen to come into contact with
these in the same cell and water in which the experiment was made.

Six eggs, placed in a single cell, were each touched once only with the pin-head,

250 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

which had been dipped into fluid only once for the whole, the time occupied in
touching them being about five seconds. Each of these eggs became fertilized, and
segmentation commenced in two of them in exactly Jive hours, and in the remaining
four in from three to four minutes later. The period at which segmentation com-
menced having been very carefully watched, and the whole of the eggs having been
submitted, during the interval, to the same physical conditions of heat, light, quantity
of water and degree of aeration, the result, in regard to the difference of time at
which they began to change, seemed to be accounted for only on the assumption
that it was due to a greater quantity of influence applied to the first two than to the
others ; and this opinion has since been more fully borne out by other experiments.
The fluid at the time it was employed had been thirty-five minutes mixed with
water. On ihejifth day each of these eggs was producing an embryo.

Three eggs were placed in separate cells, and each egg was touched once with the
pin-head loaded only once for the three, the fluid employed having been twenty-three
minutes mixed with water. After four hours and forty minutes two of these eggs were
undergoing segmentation, and on the fifth day both were producing embryos. The
third egg was not impregnated.

Twenty-two eggs, in a large cell, were each touched once with the pin-head loaded
for each egg when the fluid had been one hour and forty-seven minutes mixed with
water. Some of the eggs in this experiment were touched on the white surface,
others on the dark, and others at the side, or about midway between the white and
dark surfaces.

In the same cell with these eggs, but at a little distance from them, were placed
eighteen other eggs, which were not touched with the pin-head, but were simply
allowed to remain in the same cells with the twenty-two eggs experimented on, and
the cell was then filled with pure water. At the end of four Jiours and Jifty minutes
the chamber had been formed, and segmentation had commenced in each of the
twenty-two eggs experimented on. But in addition to these there were also two of
the eighteen g which had not been experimented on undergoing segmentation.
These, during the expansion of their envelopes, had come into contact with some of
the fecundated eggs, and had also become fecundated by some of the spermatozoa
which had been supplied to them. Five days afterwards the whole of these twenty-
four eggs were producing embryos ! so that not only had the quantity of spermatozoa
employed been fully sufficient to fecundate the eggs actually touched, but also others
present in the same water with them.

In another experiment, made before the preceding, but with some of the same brood
of eggs and same sample of fluid, I placed five eggs in a cell, and touched each once,
very lightly, with the pin's head loaded once only for the whole. This was when the
fluid had been only eighteen minutes mixed with water. The object of this experi-
ment was to learn whether there is any difference in the result when the eggs are
touched at different parts of their surface. Accordingly the trial was made by touch-

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 251

ing some eggs on the dark surface, others on the under or white surface, and others
at the sides between the two. At the end of Jive hours and seven minutes the chamber
had been formed and segmentation was taking place in each egg, and on \hejifth day
each had produced an embryo, thus appearing to show that it is of little consequence,
with reference to fecundation, as to which part of the egg the fecundatory agent is
applied to. But this conclusion is subject to certain conditions, as I shall proceed to
explain.

9. PORTION OF THE YELK MOST SUSCEPTIBLE OF FECUNDATION.

On attentively examining the preceding experiments, it seemed difficult to under-
stand how it happened that when similar quantities of the same fluid were applied by
the pin-head to different eggs of the same brood, at the same time, and the whole of
them placed in exactly similar conditions in regard to heat, light and aeration, that
some should become fecundated and others not, if, what appeared to be shown in the
last detailed experiment, — that the egg can be fecundated equally well by the appli-
cation of the fecundatory agent to any part of its surface, be strictly correct. There
at first seemed to be no other way of accounting for the differences in the results,
except on the hypothesis, either that it was due to some insusceptibility in the entire
egg itself, — which thus presupposed great imperfection in each brood of eggs, a con-
clusion at variance with observation, — or that it resulted from the mode in which
fecundation was attempted.

During the past season (1852) I have put the question, thus raised to a more rigid
test, in order to learn whether the egg really is as susceptible of fecundation in one
part of its structure as in another.

In order to obtain some decisive results I repeated my experiments on three sepa-
rate occasions, with eggs and fluid from three pairs of frogs, at nearly similar tempe-
ratures of the atmosphere. On the first occasion, April 5th, the temperature was
54°FAHR., on the second, the following day, it was 55°FAHR., and on the last occa-
sion, April 9th, it was 53° FAHR.

In all the preceding experiments the eggs had been allowed to remain in exactly
the same position, at the time of applying the fluid, as that in which they had passed
from the body of the frog ; so that sometimes the dark, and sometimes the white
surface was uppermost, and at other times inclined more or less to either side, and
occasionally the two were perfectly horizontal. When the fluid was applied by means
of the pin's-head to the part desired, some portion of it necessarily flowed over other
parts, if the surface which received it happened to be inclined to one side or the
other.

But in my more recent investigations care was taken to place every egg in a per-
fectly vertical position, before attempting to fecundate it, so that in one set of eggs
the central part of the dark surface of the yelks was uppermost, and in another the
centre of the white surface. These precautions led to very definite results. In the

MDCCCLIII. 2 L

252 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

first experiment, made on the 5th of April, with fluid which had been mixed with
water about one hour and Jifty minutes before, twelve eggs contained in a single cell
were each twice or thrice freely touched with the loaded pin's-head over the centre
of the white surface. Three of these eggs became partially impregnated, as shown
in the formation of the chamber in each, and segmentation commenced in one of
them, but no embryo was produced from either. Fourteen eggs in another cell were
touched at the same time, and in the same way as the preceding, with the loaded
pin's-head, over the centre of the black surface of the yelk. The whole of these eggs
produced healthy and well-formed embryos.

These experiments were repeated on the following day on the eggs of another frog,
with fluid which had been obtained only about twenty minutes before it was employed.
Fourteen eggs were each touched once only on the centre of the white surface, the
pin-head being loaded for each egg. But not one of these eggs became fecundated,
nor was even one of them partially impregnated. In another cell, at the same time
with these, thirteen eggs were touched on the centre of the dark surface, and four of
them became partially fecundated, but only two embryos were produced. The failure
in this experiment certainly appeared to be due to the eggs, rather than to the mode
of attempting their fecundation.

At the time of making this experiment I placed two eggs, each in a separate cell,
with their white surface uppermost, and then filled the cells, the one with nearly pure
fluid, and the other with equal parts of fluid and water. Each of these eggs became
fecundated, and underwent segmentation, although more slowly than usual, but the
development of their embryos was not completed, as it became arrested at a parti-
cular period, a circumstance which is presently to be explained. Yet these experi-
ments showed that when the egg is completely immersed in fluid its position at the
moment has but little reference to the act of fecundation, provided the fecundatory
agents are present in good abundance. A further trial was made on the 9th of April,
when six eggs, placed in one cell, were each twice touched on their white surface, the
pin's-head being loaded each time it was employed. But neither of these eggs pro-
duced an embryo. Six eggs, placed in another cell, were also twice touched, like the
preceding, on their dark surface. The whole of these eggs became fecundated, but
only Jive embryos were sufficiently matured as to leave their envelopes. The fluid
employed in these two experiments had been mixed with water about Jifty-Jive minutes
before it was applied to the eggs, and consequently was becoming slightly deterio-
rated.

The conclusion which seems to be deducible from a comparison of these experi-
ments is, — that when the egg of the frog is completely immersed in water charged
with the fecundatory agents, or to which these are supplied quickly after the immer-
sion of the egg, as in the natural fecundation of the species, the actual position of the
egg at the moment of its encounter with the spermatozoon, has but little reference
to the act of fecundation, every part of its surface having then an equal chance of

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 253

becoming affected. In like manner when the fluid is applied directly to any given
part of the surface artificially, when the egg is exposed in an empty cell, and the
fluid is allowed to gravitate, before the egg is immersed in water, it is of little con-
sequence what part is first touched with it, since it may spread over the surface to
the most susceptible portion ; there being in reality one part of the egg which is
more susceptible than another. The extremes of susceptibility and insusceptibility
appear to be, on the one hand the centre of the dark, or future upper surface of the
yelk, and on the other that of the white or under surface*.

That these conclusions, deduced from experiment, are correct, seems to be shown
in the fact, formerly pointed out-f-, that the germinal vesicle originally, and its pro-
geny subsequently, at the time of fecundation, occupies the centre, not of the entire
egg itself, but of the upper or dark hemisphere of the yelk ; and this possibly may be
the structure to be fecundated.

These facts deserve consideration in connexion with that of the first changes in the
yelk taking place in this hemisphere; first, in the shrinking of the yelk at this part;
the formation of the chamber above it, subsequent to fecundation; the existence of a
canal in its centre, in the margin of which the cleavage of the yelk commences ; and,
lastly, in this being the portion of the yelk in which the first lineaments of the embryo
become apparent.

In connexion with these remarks, it is but just to mention that a former observer,
Dr. MARTIN BARRY, has distinctly referred to the changed germinal vesicle as being
the structure in which the future embryo, in the Mammalia, originates!.

10. EXCESS OF FECUNDATORY INFLUENCE.

At the time of commencing the foregoing experiments with small quantities of
fluid, I made others of a directly opposite character with an excess of the same.
Four eggs were placed each in a separate cell, and within one minute afterwards the
cells were filled with a mixture composed of equal parts of seminal fluid and water.
This was half an hour after the fluid had been obtained. I had expected, from the
vast quantity of spermatozoa present in the fluid, that segmentation of the yelk in
these eggs would have taken place very quickly; but to my great surprise, although
the eggs were preserved in a temperature of 60° FAHR. no segmentation whatever had
commenced in them at the expiration of twelve hours ! at which time an abundance
of spermatozoa adhered to every part of the surface of their envelopes. But no
chamber was formed in either of these eggs, as neither of them had been fecundated.

* During the present year I have most fully confirmed these views by numerous experiments, and have proved
to my full satisfaction that the white surface of the egg is least, and perhaps not at all, susceptible ; while of
the dark surface, that the centre, which is occupied by the canal, and its contents, is the fertilizable part. — G. N.,
April 18, 1853.

t Philosophical Transactions, 1851, p. 176. J Ibid. 1840.

2 L 2

254 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

This result appeared to be far more inexplicable than any I had previously noticed.
A somewhat similar one had occurred once before, but I had attributed that to some
imperfection in the eggs employed, an explanation which did not apply in this case,
as eggs from the same female had all been fecundated in other experiments.

Four eggs were then passed from another frog, immediately after death, each into
a separate cell, which was filled with a mixture of two parts of water and one of
seminal fluid, obtained only a few minutes before it was employed. Two of the eggs
were allowed to remain in this mixture in their cells, but the others, after a few
minutes' immersion, were well-washed, and supplied with pure water, and the whole
were then placed in a temperature of 60° FAHR. At the end of four hours I found
the surface of the first two eggs covered with an abundance of spermatozoa ; and a
chamber had been commenced, and the upper surface of the yelk was depressed, in
each, thus showing that some degree of fecundation had been effected, but no segment-
ation had taken place in either of these eggs at the end of Jive hours and a half',
although the temperature of the room had been raised during the interval from
60° FAHR. to 67° FAHR. The sixth hour was almost completed before either of these
yelks gave any indication of the probability of this change. Segmentation then com-
menced, but in a very irregular manner. In one egg the division was not in the
centre, but at the side of the yelk, and proceeded no further than to about one-half
the extent across the surface. In the other the surface of the yelk within the
chamber merely became sulcated twice in the same direction. Neither of these eggs
proceeded further with their changes, and of course no embryos were produced.
The two remaining eggs did not undergo any change whatever, and consequently
had not been impregnated.

Although these failures were still attributed to some immaturity of the eggs, I
began to suspect that they might be occasioned by the immersion, or smothering of
the eggs in fluid of too great a density ; yet this suspicion appeared to be so hypo-
thetical as scarcely to merit consideration, when it was remembered that on a former
occasion when I had covered eggs with a solution of gum* and afterwards applied
the impregnating fluid to them, some of them became fecundated. I was well aware
that SPALLANZANI had found in his experiments that the greatest number of frogs'
eggs became fecundated when the fluid employed was very much diluted with water-f- ;
but I did not then know that M. QUATREFAGES had made an observation :}: which
completely coincided with the result obtained by myself. This acute naturalist found
that when he employed the pure semen of the sea-worms, the Hermella and the
Teredo, not a single egg was fecundated, and that when he mixed it with only a small
quantity of water, so as to obtain " un liquide tres opalin," that then only a very small
number of eggs showed any traces of fecundation. He ascribed these results simply

* Philosophical Transactions, 1851, p. 236. t Dissertations, vol. ii. p. 190.

t Annales des Sc. Naturelles, 3me Serie, torn. xiii. p. 129.

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 255

to the circumstance of the fluid being too thick ; but as we shall presently find, this,
perhaps, may admit of a different interpretation.

My first object having been to learn how small a number of spermatozoa is suffi-
cient to fecundate an egg, it may well be supposed that the facts just mentioned
were exceedingly puzzling, and they were rendered still more so by the circumstance
that a different result occurred with three other eggs from the same frog, employed
at the same time as those in the preceding experiments. Two of these eggs, placed
like the others, each in a separate cell, in a mixture of one part of seminal fluid to
six parts of water, two hours and twenty minutes after the fluid had been obtained,
became fecundated. In each of these the chamber was formed above the yelk,
segmentation took place, and proceeded regularly, and each egg subsequently pro-
duced an embryo. In the third egg the chamber was formed, but the yelk became
irregular in outline, and did not undergo segmentation. It had merely three parallel
depressions on its upper surface, after which it became strangely altered in form, and
was abortive. The cause of this failure seemed to be the same as in the preceding
experiments, — an excess of spermatozoa in encounter with the egg, as subsequent ex-
periments tended to show.

Seven eggs were passed into one large cell, and one egg into each of two separate
cells, and the cells were then filled with a mixture of fluid and water, which was so
dense as to appear turbid and opaline. This appearance was in part due to the fluid
containing a large proportion of developmental cells. The result in each instance
was very marked. The fluid at the time it was employed had been mixed with water
about one hour and jive minutes, and the temperature at the time the experiment was
made was 56° FAHR., but the eggs were soon afterwards removed to a temperature of
62° FAHR. At the end of Jive hours the yelks of two of the eggs had become partially
segmented, but the changes were continued only in one. The remaining eggs had
been more injuriously affected. Their yelks had become shrivelled and contracted,
and resembled withered apples. In this experiment it was evident that the effect had
been occasioned by an excess of impregnating influence, but in what way it was not
then easy to understand. The yelks of these eggs looked very like those of eggs
which had been in contact with solution of potass, or of those to which stale and de-
composing fluid had been supplied.

But it was possible that this change of form might be due to some injury sustained
by the eggs in their removal from the body of the frog, or to some accidental cause
in their removal to the cells, before fecundation. In order, therefore, to remove all
doubt in this respect, another trial was made with two other eggs, which were so
carefully passed into cells as to avoid all suspicion of injury to them, and the cells
were then filled with mixed fluid like the preceding. The yelks of each of these eggs
also changed their form, and one of them became irregularly contracted and pear-
shaped. Of the whole of the eggs employed in these experiments only one underwent
segmentation, and produced an embryo. The others were not fecundated. It was

256 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

thus evident that an excess of fecundatory fluid is as unfavourable to the production
of the embryo as a deficiency.

11. DEFICIENCY AND EXCESS OF FECUNDATORY INFLUENCE COMPARED.

The results of these experiments with the fecundatory agent in excess require to
be further explained, and also to be compared with those obtained with deficiency of
the agent, in order that the nature of both may be properly understood. If this be
done it will be seen that fecundation of the frog's egg depends very much on the
physical influence of temperature ; and that this has a close relation with the intensity
of the force, or the degree of vitality evolved in the spermatozoon, at the time of its
application to the egg ; and probably also with the influence which the spermatozoon
supplies through a greater or less amount of material substance to the body to be
fecundated, at the time of its encounter, as in the following experiments made in
March 1852.

The experiments by pin-point application of fluid show that only a very small
amount of influence is actually required to set up those changes in the yelk which
result in the formation of the embryo ; and other observations have convinced me
that the result is the more certain at a slight increase than at ever so slight a dimi-
nution of temperature. The effect of the application of minimum quantities is most
certain when the influence has only very recently been obtained, and consequently
while still endowed with its greatest degree of vitality. But even at that time a
given amount is required, and if such be not supplied the result is incomplete, and
partial fecundation only is effected. First then in regard to minimum quantities.

Six eggs were placed each in a separate cell, and three of these were touched once
only with the pin-head loaded with fluid obtained about twenty-five minutes before,
while the other three were each touched once with the fine pin-point loaded in like man-
ner. Yet although there was only an interval of one minute between the application
of fluid to the two sets of eggs, two of the former by pin- head application had the
chamber developed, and segmentation commenced at the end of three hours and forty
minutes ; while one egg only of the three supplied by pin-point application began to be
segmented in three hours and forty-eight minutes, although the whole were placed under
precisely similar conditions in regard to light, heat, quantity of water and degree of
aeration. The third egg of the first set was not impregnated, neither were the two
remaining ones of the second set. Thus there was a difference between the two sets of
eggs, not only in the extent to which they were fecundated, but also in the rate of the
accomplishment of fecundation to the extent of eight minutes, in about three hours and
three-quarters. There was also a perceptible difference of time in the formation of the
respiratory chamber of the two, after the first hour, as was remarked by a distinguished
physiologist who was present with me when these comparative trials were made. The
two eggs of the first set, and the single egg of the second, which underwent seg-
mentation, afterwards produced embryos ; the remaining eggs were abortive.

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 257

A second comparative trial was made on the 15th of March. Three eggs in sepa-
rate cells were each touched once with the fine pin-point loaded with fluid which
had been obtained only twenty minutes before. The temperature at the time of the
experiment was 56° FAHR., but the eggs were removed within a few minutes after-
wards to a higher temperature, which was gradually increased to 64°-5 FAHR. Seg-
mentation took place in one of these eggs in three hours and thirty -nine minutes, and
this egg afterwards produced an embryo. But the remaining 'two were not impreg-
nated. Three other eggs in separate cells, were, at the same time as the preceding,
each once touched with the loaded pin-head, and two out of the three underwent
segmentation at the end of three hours and thirty-three minutes. In a further
experiment, made at the same time, sixteen eggs included in one cell were touched
with the loaded pin's-head when the fluid had been obtained about half an hour
before. Four of these eggs underwent segmentation in three hours and Jifty '-three
minutes, and afterwards produced embryos, the remaining eggs being unproductive.
Another experiment was made with nineteen eggs in a single cell, by pin-head
application of the fluid, and eleven of these were fecundated and began to be seg-
mented in three hours and forty-two minutes, and ultimately produced embryos, the
remaining eight being sterile. Some of these failures were probably due to the cir-
cumstance that the fecundatory fluid was not applied to the most susceptible part of
the egg, yet the general results sufficiently show not only that fecundation is more
surely and quickly effected at a moderately high than at a low temperature, but also
that it is so, more or less certainly and quickly, in proportion to the amount of
influence supplied.

These facts lead us to some further consideration of the results of the application
of definite quantities of the fecundatory agent to the egg, as affecting the develop-
ment of the embryo, and possibly also as influencing both the evolution of its physical
structure and psychical condition. I have little hesitation in believing that whatever
be the precise nature of the influence communicated by the fecundatory agent to the
egg, that it is only after full and complete impregnation that perfectly normal and
healthy embryos are formed, and ultimately attain to the maturity of the species.
And yet we have already seen that when there is great excess of the agent, either
that no embryo is produced, or that its development is not completed.

A few days after making the last-mentioned experiments I repeated them with
another object in view, and then obtained a result which had occurred on previous
occasions, but being less distinctly marked had been almost overlooked.

About twenty eggs had been placed in separate cells, and fecundation attempted
by pin-head application of fluid which had been obtained from a partially exhausted
and debilitated male, at the end of the season, and which had been employed from
want of a more healthy individual. The fluid, when examined by the microscope, was
found to contain very many perfectly motionless spermatozoa, besides a large quantity
of cells. The inefficiency of this male was inferred from the circumstance that the

258 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

female was prepared to spawn, as was proved by her having passed a few eggs ; but
these remained unimpregnated, and she still retained the remainder within her for
nearly a day, at which time the male was removed for the experiment. The employ-
ment of this male was thus accidental, it being the only one I then possessed.

The fluid was applied to the eggs within a quarter of an hour of its being obtained,
and the eggs were at once placed in a temperature of 61° FAHR. I remarked that
development appeared to go on more slowly than usual, although most of the eggs
imderwent segmentation, and^/^lfeewof them produced embryos, which were placed
together in one vessel. A few days afterwards I was surprised to find that two of
these embryos were greatly malformed. One had a short falcated tail narrowed at
its base, where it was not broader than at its extremity, and the tail of the other
was scarcely more than a short narrow stump, so that the embryo had much diffi-
culty of locomotion. A few days later three other of the embryos became distorted,
and one of them died much altered in form at the period of absorption of the external
branchiae.

These circumstances are recorded merely as simple occurrences which point to the
necessity for further experiment on this curious and important subject, and not as
results from which any very positive conclusions can be deduced. Nevertheless, I
may add, that I think I have before observed that embryos which have been the
result of fecundation with very small quantities of fluid, are usually smaller than
others which are produced from full and natural impregnation.

The effect produced by immersion of the egg in pure fluid is as curious as the
results above mentioned obtained from minimum quantities, and at first it is less easily
to be understood. I can find no more expressive term by which to indicate it than
that of smothering, and this, perhaps, may be found to be a more literally correct ex
pression of the fact than at first is apparent. It has already been shown that free
aeration is most essential to the development of the embryo, and I believe it is equally
so, in a minor degree, to fecundation. Thus while the egg may be fecundated in a
dense fluid, composed of equal parts of seminal fluid and water, its changes usually
take place much more slowly than when the proportion of water is considerably
increased; while if it be immersed in pure fluid, and be allowed to remain in that for
some hours, it may still be fecundated, and its changes be commenced, but these will
not proceed to the full development of the embryo. If the egg when immersed in
pure fluid be retained in a moderately low temperature, then the development of tne
embryo may go on slowly for a time, but with less rapidity than when the egg is
freely aerated in pure water. But if, on the other hand, it be retained in seminal
fluid, at a high temperature, then it usually happens that although the respiratory
chamber be formed, as the result of fecundation, segmentation will proceed slowly, or
irregularly, and the embryo, which is begun to be formed, perish. That want of
proper aeration is in part the cause of this infertility seems to be shown in the fol-
lowing experiments.

- AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 259

April 5, 1852. Atmosphere 54°. — Two eggs were placed in separate cells, which
were immediately filled with nearly pure fluid. In about one hour afterwards the
respiratory chamber was beginning to be formed in both these eggs, but its progress
was slow. One egg was then washed as completely as possible with a powerful jet
of water, thrown upon it by a syringe, and the cell was then filled with pure water,
while the cell with the other egg was replenished with more of the fecundatory
mixture. After the addition of fresh water to the first egg its envelopes began to
expand, and it was then seen that the substance of these coverings was Jilled with
spermatozoa, lying in every direction and so crowded around the envelope which
immediately invests the yelk, as to render the whole semi-opaTte. The envelopes of the
second egg were much more opake than those of the first. Yet the respiratory
chamber was developed, after a lengthened interval, in both, but earlier in the egg
which had been washed and placed in water than in that which remained in the fluid.
At the expiration of twelve hours the second egg was washed and placed in pure
water like the first. Both eggs were preserved, from within an hour or two after
their first encounter with seminal fluid, for four days in a temperature which ranged
from 59° FAHR. to 64° FAHR. At the end of three days and a quarter, or about seventy-
eight hours, in this temperature the development of the embryo had advanced so far
in the first as to the union of the laminae dorsales, and the narrowing and elongation
of the body, a stage of development at which the function of aeration is becoming
more energetic in the evolution of ciliary action. At this stage of formation the
development of the embryo became arrested, although the egg was properly supplied
with water, and from this period it decayed. The second egg, which had remained
twelve hours in the mixed fluid before it was supplied with fresh water, perished much
earlier, and before there were any distinct evidences of the formation of an embryo.

On other occasions I have found that the egg does not become fertilized at all,
but the yelk contracts irregularly, and resembles the yelk of eggs affected by solution
of potass.

These are the usual results of immersion in undiluted fluid, which, contrary to
what occurs with minimum quantities, appears to be most prejudicial to the egg, in
its pure state, when the temperature of the surrounding medium is much increased;
so that the cause of failure in these cases of excess may reasonably be attributed, in
great part, to a smothering of the egg, through impeded aeration ; accelerated,
perhaps, by some chemical change, through increased temperature, in the remains of
the spermatozoa, with which the envelopes of the egg are crowded in their interior.

12. THE MOTION OF THE SPERMATOZOON IN RELATION TO ITS FUNCTION.

The possibility of the fecundatory function being in some way connected with the
motion exhibited by the contents of the pollen in plants, and with that of the
spermatozoa in animals, has not escaped the consideration of the best observers.
Mr. Brown, the most distinguished of botanists, many years ago, made this the

MDCCCLIII. 2 M

260 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

subject of his particular investigation *; and M. BRONGNIART-|-, SCHLEIDEN;}:, NAGELI^,
GRIFFITH ||, and more recently SUMINSKI^[ and HENFREY** have done the same.
But although neither of these able inquirers succeeded by direct experiment in
proving that the motion of the particles of plants is essential to the act of impregna-
tion, M. HoFMEisTER-{~f-, and very recently also Mr. HENFREY^, have noticed facts
in regard to that of the spermatozoid filaments discovered by NAGELI and SUMINSKI,
in the Cryptogamia, which seem to show that, in plants, it is of great importance to
the function of these bodies. The motion of the spermatozoon in animals has equally
attracted the attention of zoologists. PREVOST and DUMAS§§, as already stated,

SlEBOLD, MuLLER, WAGNER, KoLLIKER, BlSCHOFF, QuATREFAGES, and especially

WAGNER and LEUCKARDT, have studied it attentively ; but so intricate is the inquiry
concerning its nature and import, that the last two authors dismiss the consideration
of the question without arriving at any conclusion, and state that they do not venture
to decide || ||. Heretofore I regarded impregnation as being commenced by transmission
from the spermatozoon on the surface of the egg, to the contents in the interior, of
some influence characterized by motion. But I have regarded this motion as being
only the visible indication of a. peculiar force, or form of vitality, in the impregnating
agent, the spermatozoon, by which it is destined to arrive at, and is to expend on the
object to be fecundated, and the effect of which is to strengthen, to augment, and
possibly also to modify the nature of the formative changes, which are going on in
the yet unimpregnated egg, per se ; but which will subside, and soon entirely cease,
if not reinforced through the agency of the spermatozoon. Nevertheless, I have not
been prepared to assent to the view that simple contact of the spermatozoon, even with
the vitelline membrane, is sufficient to complete the changes which result in the forma-
tion of the embryo (see p. 233). The powerful endosmic action of the envelopes of the
ovum, at the time of oviposition, is opposed to this conclusion ; since, if simple contact

* A brief account of microscopical observations, made in the months of June, July and August, 1827, on
the particles contained in the pollen of plants, and on the general existence of active molecules in organic and
inorganic bodies, by ROBERT BKOWN, F.R.S., etc., 8vo. July, 1828 ; also additional remarks on active molecules.
(Id.) July, 1829.

t Recherches sur la generation et le developpement de 1'embryon dans les Vegetaux Phanerogames (Notes).
Annales des Sciences Nat. torn. ix. 1828.

J Grundziige der wissenschaftliche Botanik.

§ SCHLEIDEN und NAGELI'S Zeitschr. fur Wiss.-Botanik ; Heft i. 168; Zurich, 1844.

|| Transactions of the Linnean Society of London, vol. xx.

5f Zur Entwickelungsgeschichte der Farrnkriiuter, 4to. Berlin, 1848.

** On the development of the ovule in Orchis mono, Transactions of the Linnean Society of London,
vol. xxi., and Proceedings, vol. ii. p. 27, April 8, 1849.

ft Untersuchung des Vorganges bei der Befruchtung der OZnothereen, Botanische Zeitung, v. 785, 1847.

\\ Annals and Magazine of Natural History, vol. ix. June, 1852 ; Transactions of Linnean Society, vol. xxi.
Part II. ; also Proceedings, June 17, 1852, vol. ii.

§§ Annales des Sciences Naturelles, torn. ii. 1824.

Illl Article " Semen," Cyclopaedia of Anatomy and Physiology, vol. iv. par. xxxiv. p. 508.

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 261

or encounter of the spermatozoon with the ovum were sufficient, it might be expected,
as has been well remarked*, that the influence of the spermatozoon of any animal of
the same class would be competent to effect the impregnation of any species.

Yet all the phenomena connected with the origin and death of the spermatozoon
seem to be in accordance with the view, that its motion is essential to its function.
Whatever be the relation of this motion to its peculiar faculty, it is evident that
motion is intimately associated with, and dependent on, its material composition, and
structural development. In the Frog, the spermatozoa are usually completed very
early in the season, when the animals begin to emerge from their hybernacula, in the
beginning or middle of February ; but, as we have seen, they only begin to pass into
the efferential ducts from the testicle, at the time when the pairing of the sexes is
commenced ; from which time, to that of spawning, a period of from ten days to a
fortnight or three weeks, according to the temperature of the season, they are more
fully matured, and acquire greater vibratory power, and are collected in the vesiculse
seminales for expulsion at the instant after oviposition. In the Toad the spermatozoa
are developed at a later period of the season, but at a relatively corresponding period
in the life of the animal. The male Toad, like that of the Frog, usually emerges
from its hiding-place a few days, or a week or two earlier than the female. I have
taken the males in the middle and at the end of March, at which time I have not
been able to detect any seminal fluid in their vesiculae seminales ; yet they are then
exceedingly salacious and disposed to pair, and I have sometimes found the contents
of the reproductive organs in a similar state, even after the sexes have been for two
or three days in union. The testicles, nevertheless, are then filled with an abundance
of spermatozoal cells in the course of development, and also with a great quantity of
spermatozoa, each still included in its vesicle of development ; but as yet immature,
motionless, and with only a very short caudal extension from its thick cylindrical
body. Besides these there are usually a few spermatozoa, more matured than the
rest, which exhibit movements while still retained within their cells, which are enlarged,
and from which they are soon to be liberated.

Thus motive power in the spermatozoon is coincident with the completion of its
structure and composition, and as such, may fairly be regarded as essential to its
function. In the Frog the motion of the spermatozoon is most intense and persistent
at the full period of connubiality. On the other hand, 1 have constantly noticed in
all my experiments on artificial impregnation, that where impregnation has not been
effected, all the conditions being favourable to it, or when I have found by trial that
the male fluid has ceased to be efficient, that then nearly the whole, or perhaps all of the
spermatozoa, have been perfectly motionless, and apparently dead. This is also the
condition in which I have usually found the few spermatozoa which are retained in
the reproductive organs a week or two after pairing, when the male Frog and Toad
may be regarded as in the state of aged individuals, the season of reproduction

* WAGNEB and LEUCKARDT, loc. tit. p. 508.
2 M 2

262 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

having passed, and the function being fulfilled. Thus too, I have noticed that, when
from accident, but more especially when from reduction of the temperature in the
surrounding medium, the season of spawning has been greatly retarded, the impreg-
nating power of the male is much diminished, and perhaps is almost exhausted,
through constant shedding of the spermatic fluid, which, I have found, often takes
place when the oviposition of the female is delayed, and the individuals are disturbed
or interfered with. The female is then forsaken by her partner, and when this occurs
it rarely happens that the connubial intercourse of these two individuals is recom-
menced. When this separation has taken place, there is usually but a small quantity
of fluid remaining in the male organs, and even in that, the number of spermatozoa
is considerably diminished, and their power of motion is exceedingly feeble ; while the
quantity of molecules and cells is increased. When several days, or a week or two
have elapsed, there are not only fewer spermatozoa, but those which remain are
much more feeble in action. This is exactly what occurs also in the Toad. On the
sixth of June I found that the testes and efferential ducts in a male Toad, which had
been kept from pairing during the whole season, were still filled with spermatozoa,
together with a very small quantity of liquor seminis with active molecules moving
in it ; but that, though the spermatozoa were in full abundance, nearly the whole of
them were entirely motionless, while the motions of the few which still gave evidences
of vitality, were exceedingly feeble, whether the spermatozoa were examined simply
in the fluid portion of the semen, or whether they were mixed with water, in which,
as is well known, the motions are always at first greatly increased.

A similar reduction in the number of the spermatozoa and diminution of their
motive power, appears to exist in animals which have become exhausted through long
confinement or want of food ; at least, if we may so judge, from a few observations
on the Tritons. A Triton palustris, which had been captured on the seventh of May,
and accidentally confined without food till the sixth of June, was examined immediately
after death. The efferential ducts were well-filled with spermatozoa contained in a
distinctly perceptible quantity of liquor seminis. When the spermatozoa were ex-
amined, without the addition of water, their motions were regular, but apparently
very much slower than usual, being uniform and undulating, without that peculiar
rapid ciliary action of the spirally twisted tail, which is so constantly referred to as
characteristic of the spermatozoa of the Tritons and of some other Amphibia. When
water was added, the motions were immediately accelerated, and the tail, which
before was merely flexed, and almost longitudinally extended from the body, became
folded and entwined around it, and its rapid ciliary movements were commenced.
But these gradually subsided within a very few minutes. In another specimen, which
had been captured at the same time as the preceding, and confined under similar
circumstances, but which, at the time of examination, had been already dead for
more than twenty-four hours, scarcely any spermatozoa remained in the testes, or in
the efferential ducts. There was a great quantity of cells, with granular nuclei, in

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 263

the body of each testis, but scarcely a single spermatozoon. The few spermatozoa
which remained were most of them dead, and decomposition appeared to have com-
menced in them, and there was a large proportion of active molecules. Amongst these
were one or two spermatozoa still in action, and the. molecules were accumulated
around them by the attraction of the current in the fluid, induced by the ciliary action
of the tail, and were propelled onwards, frequently in a spiral direction, from the base
of the tail to the anterior extremity of the body ; thus showing that the motions
around the spermatozoon are due to the action of the filamentous tail, flexed upon,
and twisted spirally round the body ; in accordance with the observation of WAGNER
and LEUCKARDT.

But it has been remarked, in opposition to the view, that power of motion in the
spermatozoon is essential to its function, that motion is not detected, or but very
faintly, in many instances in which the spermatozoa are united into simple and
uniform cords, as they often are in the deferential vessels of insects ; and that then
only a slight waving, or trembling of the mass, the consequence of hygroscopic con-
ditions induced by the fluid around, is, under such circumstances, observed. An
aggregation of the spermatozoa into cords takes place in the Tritons as well as in
insects. When the semen is expelled by the Triton at the time of conjugation, it is
composed almost entirely of white cord-like, flocculent masses of spermatozoa, which,
at that moment, exhibit only the faint undulatory motion noticed in them while still
within the deferential vessels. Yet, immediately after these masses are ejected from
the vessels, and are in contact with water, at the moment of being received into the
cloaca of the female, as I shall hereafter have occasion to show, — the motions of the
spermatozoa are not only greatly increased, but that peculiar vibratory ciliary action
of the tail, which, while the spermatozoa are still within the seminal ducts is very in-
distinct, and perhaps can scarcely be said to occur — is immediately set up, and the
motion of the body of the spermatozoon is changed from the undulatory action before
observed, to the jerking or watch-spring movement pointed out by observers, and
thought to be the motion peculiar to the spermatozoon of this class of animals.

Thus then, the fact of only a slight wavy motion of the spermatozoon being per-
ceived in some cases, ceases to be of importance, as an argument against the view
that motion is closely connected with the function, since it is evident that the true
spermatic force is not set up until the spermatozoa have become more or less separated
from each other, through their residence in some fluid vehicle. The movements of
the spermatozoon in insects are very similar to those of the spermatozoon of the
Triton, and the circumstances which affect both are in full accordance with the
fact of the existence of, and the necessity for, a distinct liquor seminis, as part of
the normal composition of the seminal fluid, in the Mammalia; while this portion of
the fluid is not needed, and consequently is almost entirely absent in the Amphibia,
Fishes and most Invertebrata. A comparison of the facts in the two divisions of
animals seems to point to the true nature and use of the liquor seminis, as being that

264 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

of a mere vehicle for the ready transmission of the spermatozoon to the ova, and as
allowing greater freedom of motion to these bodies.

It is true, as WAGNER and LEUCKARDT have stated, that but little motion can be
observed in the spermatozoa of Insects when they are united into cords ; yet this
seems to be due rather to their union into these masses, and to have reference to
their mode of ejection from the body of the male, as in the Triton, and is known to
take place during their collection in the deferential vessels, than to any real absence
of a power of motion in them. This is not their condition when brought into contact
with the egg at the time of impregnation. When the fluid of the male insect, during
pairing, is passed into the sperrnatheca of the female, where it may be destined to
remain for an indefinite time, the spermatozoa compose the chief portion of it, as in
the Tritons; but, during their residence in the spermatheca, the spermatozoa are
mixed with a fluid, which is supplied by a gland attached to that organ, and which
becomes to them a vehicle like the liquor seminis, and allows of their separation and
independent motion, and thus appears to answer the purpose of the true liquor
seminis in the Mammalia. At the period when the ova are descending from the
ovaries, the movements of the spermatozoa become more distinct ; and when these
bodies are brought into contact with the egg, as it passes the outlet of the sperma-
theca, they become more isolated, and their movements more intense, as I have seen
in the case of the Orthoptera. From these circumstances I am led to believe that
some degree of motion will ultimately be observed to mark the perfect condition ot
the impregnating agent in all animals. The facts already mentioned coincide with
this view, and point to the probability that the degree of impregnating force in each
individual may perhaps be indicated relatively by the degree or intensity of motion
in the spermatozoon, and the duration of this force by the length of time which the
spermatozoon continues in motion.

The established fact, that a difference in the structural conformation of a body is
the invariable result of a difference in the relations, proportions, and composition of
its material constituents, has always afforded reason for presuming that some material
influence may be transferred from the substance of the spermatozoon to the contents
of the ovum, at the time of impregnation. The function of impregnation appears to
be one of definite relations and proportions. Thus we have seen, when only a few
spermatozoa were applied to the ovum on the point of a pin, that full impregnation
was but rarely effected. In most instances of such limited application, the yelk
underwent only partial segmentation, and its changes were then gradually arrested,
and no embryo was produced. If, however, the pin point which had been charged
with spermatozoa, instead of being applied to the ovum for an instant only, was
allowed to remain in contact with the egg for a second or two, and thus by capillary
attraction became drained of the spermatozoa which adhered to it, and, as a conse-
quence, thus made to deposit a greater number of these bodies, which were not
afterwards attempted to be removed or destroyed, then impregnation was sometimes

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 265

completed, and an embryo became developed. But if, instead of the spermatozoa
being thus supplied in a minimum quantity from the point of a pin, they were sup-
plied in greater quantity from the head, then impregnation was almost always effected ;
and this result was rendered the more certain by allowing the head to be drained of
the adhering bodies for a moment or two, as from the point, by which a much larger
quantity of the impregnating agents was deposited. In these instances it was rare
that impregnation was not effected. These results appear to show, that, whatever may
be the precise quantity necessary to effect healthful impregnation, it has some definite
relation to the effect to be produced in the contents of the ovum — that a definite
quantity of spermatozoa, or spermatic influence is required to fecundate, and that
the perfection of fecundation has relation to the degree of impregnating influence.
They seem to show, also, that fecundation is not the simple result of the pene-
tration into the egg of a single isolated spermatozoon, but probably of some definite
number of these bodies, or of a definite amount of influence supplied through their
encounter. We are thus led to perceive that the same law of relation between
cause and effect, — between a definite amount of influence expended, and definite
results, — which has long constituted the basis of our knowledge respecting chemical
affinities, and which is now being demonstrated, as that also of the other forces of
inorganic nature, may equally pervade and control these material combinations
among the organic affinities. Further, the observations, now mentioned, seem to
put to rest the last remaining question respecting the independent animality of the
spermatic bodies, and to show that these, like the cilia, are mere elementary parts
of the adult male organization, — as many physiologists believe*, — as the contents of
the ova are of that of the female.

All my experiments on the egg of the Amphibia serve to show, that even with a
definite quantity or number of spermatozoa, no impregnation is effected, if, before
the spermatozoa are brought into contact with the egg, they have all ceased to exhibit
that motion which constitutes their marked characteristic. Thus then, it seems that,
independent of the important question as to whether these bodies yield any material
substance for combination with the constituents of the egg, through the usual chemical
affinities of matter, — the quantity of influence to be supplied to produce the healthful
result is definite, and that, whatever be its nature or essence, it is always charac-
terized by a definite degree of motion in the spermatozoon. Further, all the observa-
tions I have made on the spermatozoa tend to show that their motion is rendered
more vivid and intense by an increase of heat, but that, in proportion to such increase,
it is so much the sooner exhausted ; as, on the other hand, it has long been known
that it is diminished by reduction of temperature, but increased in duration. Many
of the observations lead to the view, that the power, in a given quantity or number
of the spermatozoa, to effect impregnation, is more in proportion to the amount,
quantity, or intensity of the motion exhibited by these bodies than to their actual

* KOLLIKER, SlEBOLD, MlJLLER, WAGNER, &C.

266 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

numbers. Thus the spermatozoa, like the ova, are more efficient at a given tempera-
ture, within the first two or three minutes after they are passed from the body, than
at a later period. At that time there is not only a greater number of spermatozoa in
a given quantity of fluid in a state of activity ; but the whole of them exhibit a
greater intensity of motion than after they have been for some time mixed with water.
In the Toad, which, in this country, as already stated, pairs at a few weeks later in
the season than the Frog, and which seems not only to require a higher temperature
of the surrounding medium, but also a relatively higher temperature for the develop-
ment of the ova, the spermatozoa are most active, and most fitted to impregnate at
the instant of expulsion ; as is shown in the mode of intercourse of the sexes in that
animal, and in the fact that, if the fecundatory fluid be not immediately applied to
the strings of ova as these are passed, the unsprinkled ova are infertile. The oviposi-
tion of the Toad is an exceedingly slow process, and usually lasts, as SPALLANZANI
observed, from ten to fifteen hours : in one instance I found it continued during
seventeen hours, but it was completed in others in eight or ten. The act of impreg-
nation, therefore, is necessarily also prolonged. But the length of time during which
the spermatozoa continue efficient to impregnate after removal from the body, is
shorter than in the Frog. SPALLANZANI found that, at a temperature of 81° FAHR.,
it does not exceed fifteen minutes. I also have noticed that the motions of the
spermatozoa cease much earlier than in the spermatozoa of the Frog.

Thus the conclusions to be drawn from the facts of natural impregnation in the
Toad, fully agree with those deduced from artificial impregnation in the Frog, and
seem to establish the view, that while an increase of temperature is required for the
fulfilment of the reproductive function in that animal, and to maintain the efficiency
of the spermatozoa, the fecundatory force of the agent is of shorter duration, and
corresponds to its more early cessation of motion.

13. PENETRATION BY THE SPERMATOZOON IN EFFECTING FECUNDATION.

The penetration of the spermatozoon into the substance of the egg, or even into
its envelopes, has been so much disputed, and so repeatedly denied, that it is only on
what may perhaps be regarded as indisputable evidence, that any one ought to re-
assert it. Up to a very recent period I could not, myself, admit it as probable ;
because, throughout my investigations, I had not been able to detect any appearance
in the fecundated egg or its envelopes, either of the Frog or Newt, which rendered it
likely that the spermatozoon penetrates into or through them. Yet the fact of pene-
tration was stated by the older naturalists, not so much, perhaps, from any decided
proof of its occurrence, as from a confidence that it was only in this way that the
function of the spermatozoon could then be understood. LEEUWENHOEK, and those of
his day, not only believed that the spermatozoon penetrates bodily into the substance
of the egg, but also that it becomes the future embryo. In later times, M. PREVOST
having, with M. DUMAS, seen the spermatozoon within the gelatinous envelope of the

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 267

egg of the Frog, as they show, conceived that this body becomes, — not the embryo itself,
but the foundation of the nervous system of the embryo. Since then, Dr. MARTIN
BARRY announced to the Royal Society that he had seen spermatozoa within the
substance of the egg of the Rabbit ; through the envelopes of which, he has stated, that
there is a natural perforation or cleft at the time of fecundation* ; a view which has
met with much opposition, as no physiologist has hitherto (1852) verified his observa-
tion with regard to the existence of a perforation or cleft in the egg-envelopes ; or
has announced that he has seen the spermatozoon within the ovurn of that animal.
Indeed so general has been the opinion that there is no penetration by the sperma-
tozoon, even into the envelopes of the egg, notwithstanding WAGNER'S-^- announce-
ment that he had seen spermatozoa within the envelopes in the eggs of Fishes, that
one of the most recent investigators, M. QUATREFAGES, in alluding to MM. PREVOST
and DUMAS' views, observes, that " it is useless to allude to the question of penetra-
tion by the spermatozoon, as he believes that the only living author of the theory has
himself renounced it|." But very recently it has been announced, in a paper on
the Ascaris Mystax, by Dr. NELSON, communicated to the Royal Society §, that the
spermatozoon in that animal does penetrate into the substance of the yelk ; and a
somewhat similar account of that of the Earth-worm was formerly given by Dr. ARTHUR
FARRE||. Yet, with all due respect for the observations of these able investigators, I
must still have hesitated to admit the fact of any penetration, even into the envelopes
of the egg, had I not more recently been convinced of this fact by direct observa-
tion, in correction of my former opinions, in so far as relates to the penetration of
the spermatozoon into the envelopes of the eggs of the Frog, and its arrival at the
vitelline membrane. To them, then, be all honour on this subject, while I subjoin
my testimony to a fact which I had heretofore failed to observe.

It was during the month of March last (1852), while making experiments on the
Frog's egg by artificial impregnation, before some scientific friends, that certain
appearances were noticed beneath the microscope within the expanded jelly of the
egg, which led to a suspicion of the probability of penetration by the spermatozoon.
Heretofore I had employed, in my observations, either a deep glass cell, in which the
egg was completely immersed in water, or the common object-glass of the microscope.
In the experiments now referred to, a shallow glass cell was employed, which was
capable of containing only a single egg. This, while it admitted light freely around
the egg, was too shallow for its complete immersion, and the egg was therefore
covered by a drop of water, into which the object-glass of the microscope was passed,

* Philosophical Transactions, Part II. 1840, p. 533, Plate XXII. figs. 164, 165 and 167. Ibid. Part I.
1843, p. 33.

f Elements of Physiology (English Edit, by Dr. WILLIS), note, p. 74, 1841.

I Annales des Sciences Nat. 3me Se"rie, torn. xiii. 1850.

§ Proceedings, June 19, 1851, vol. vi. p. 86. Philosophical Transactions, 1852.

|| See Dr. CARPENTER'S Principles of Human Physiology, 1st edit. 1842, p. 617.
MDCCCLIII. 2 N

268 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

while viewing the egg, instead of the observation being made through the double
medium of air and water. This was the method of examination in all the subsequent
experiments.

The appearance which first led me to suspect that spermatozoa do penetrate into
the envelope, was produced by an acicular body, which seemed to have its narrowed
extremity in near proximity to the vitelline membrane. The direction of the longi-
tudinal axis of this body was in a line with the centre of the yelk. But what appeared
to be its larger end was farthest removed from the yelk ; and this circumstance
seemed to show that the appearance could hardly be due to the presence of a sper-
matozoon, which, if the observation were correct, must have penetrated in a direc-
tion the reverse of that of its usual motion.

It was necessary, therefore, that the suspicion raised by this observation should be
settled. I had again and again found, as before shown, that the egg of the frog may
be impregnated, under certain conditions, by the direct application of spermatozoa
to almost any part of its surface, and this enabled me to put the question of penetra-
tion to the test. In the first trial, an egg was placed in a single cell, and immersed
in water for one minute — the water was then removed, and the egg touched on one
point only of its surface with the head of a pin, loaded with the fecundating fluid,
which had been obtained and mixed with water about two hours before. I had ex-
pected that on watching the egg beneath the microscope, from the instant of contact
with the pin's-head and the re-filling of the cell with water, to have been able to
detect the spermatozoon during its passage through the envelope. But this I failed
to do, in the present instance, — no spermatozoa were detected in the interior of the
envelope, although many were easily observed on the surface at the point to which
they had been applied by the pin. Yet, this egg, placed in a temperature of 66° FAHR.,
underwent segmentation in three hours and thirty-two minutes, and ultimately pro-
duced an embryo. A similar trial was made, at the same time, with an isolated egg
after immersion for two minutes in water, which was then withdrawn, and the fecun-
dating fluid applied as before. In this instance, several spermatozoa had penetrated
for a short distance into the envelope, but had not reached the covering which imme-
diately invests the yelk. No respiratory chamber was formed above the yelk in this
egg, nor was any embryo afterwards produced. In a third instance, with an egg,
which had been immersed {or Jive minutes, the experiment was equally unsuccessful.
On the 24th of March a further trial was made with an isolated egg, after one minute's
immersion. The fecundating fluid employed had been obtained only sixteen minutes
before it was used, and was applied by the head of a pin, once only, to one point of the
egg. In this case, at the expiration of half an hour, I distinctly saw a single sperma-
tozoon sticking by its larger extremity into the vitelline membrane, and a few minutes
later there was evidence of the respiratory chamber being about to be formed. This
egg underwent segmentation, and afterwards produced a good embryo. In a second
egg immersed for three minutes, and in a third for Jive minutes, there were no appear-

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 269

ances of spermatozoa in the interior of the jelly, although they were in abundance
at the point on the surface, to which they had been applied by the pin. Neither
of these eggs underwent any change. But in a fourth trial, with an egg immersed
for ten minutes, and to which the spermatozoa were applied in the same way as in the
preceding, I saw, at the expiration of fifty minutes, four spermatozoa sticking in the
vitellary membrane ; soon after which the chamber was commenced, and at a later
period segmentation of the yelk took place, and an embryo was produced. This was
with fluid which had been obtained nearly an hour before it was employed. In a
further trial, with an isolated egg, to which a full quantity of fluid was added, by
three or four applications of the loaded pin's-head, after the egg had been one minute
in the water, I detected several spermatozoa, within half an hour afterwards, sticking
like the preceding into the vitelline membrane, and this egg also produced a good
embryo.

It was thus evident, that in some cases, spermatozoa certainly penetrated through
the envelope ; but as there was also one instance, in which the egg had become
fecundated, and afterwards produced an embryo, in which I had not seen sperma-
tozoa within the envelopes, it was necessary to pursue the investigation further, to be
quite assured of the fact of penetration as connected with the act of fecundation.

On the following day an opportunity occurred to me of examining the egg after
impregnation by the natural union of the sexes. I had several pairs of frogs in
a basin of water covered by a glass bell jar. when, having my attention directed for
a few minutes to some other object, one pair of frogs spawned suddenly, as I knew
by the sound of a plunge or splash in the water, which always occurs after the act,
at the moment of separation of the sexes. Nearly the whole of the eggs were depo-
sited in a mass, but there were a few which were detached from the rest, and were
lying at the bottom of the water. This seemed a good opportunity to examine these
eggs, which had been expelled naturally, singly, in search of the spermatozoa within
the envelopes. Fifteen of these detached eggs were placed, each in separate cells,
and carefully examined, during the first hour and a half after their expulsion. In
the first eight or nine of these eggs, I was not able to detect even a single spermato-
zoon within their substance, and I began to look upon the previous observations,
made by artificial impregnation, rather as the result of accident. In the tenth egg,
however, I most distinctly saw spermatozoa in contact with the vitellary membrane,
but in one or two eggs examined after this no spermatozoa were to be seen. Out of
the fifteen eggs examined, there were only two in which I could detect spermatozoa.
As each egg had been carefully marked, and a note made, as to whether or not
any spermatic bodies had been detected within it ; and as previous investigation had
shown, that when any fecundation has been effected the yelk of the fecundated egg
becomes depressed, and a chamber begins to be formed, in about one hour, or little
more, after the spermatozoon is supplied to the egg, between its upper surface and
the investing membrane, — there were ready means of learning, within a short time,

2 N 2

270 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

whether any, and which of these eggs had been fecundated. At the expiration of one
hour and twenty minutes, the temperature being 54° FAHR., the chamber was beginning
to be formed in the two eggs in which I had seen spermatozoa sticking in the vitel-
lary membrane, and these bodies were still readily detected in them, and continued
to be so for two or three hours afterwards. These two eggs ultimately produced
embryos. But no change took place in any of the thirteen eggs, in which I could
not detect spermatozoa, — no respiratory chamber was formed within, nor was any
embryo produced. It was evident, therefore, that these had not been fecundated,
and it is probable that the whole of the detached eggs were those which had last
been ejected from the oviducts, possibly after the act of fecundation by the male
had been completed. Having found spermatozoa in two only of these detached eggs,
I then examined several of those from the mass. In each of them I found numerous
spermatozoa sticking around the yelk membrane, and very many, in the clear space
between the membrane and the granular middle portion of the envelope, which had
not arrived at the membrane, and in every instance the eggs had been fecundated, as
the chamber was being formed at the time of examination, about an hour and a
quarter after the eggs had been deposited, so that the act of penetration by the sper-
matozoon through the envelopes as far as the vitellary membrane, seemed thus to be
clearly established as connected with the act of fecundation. This was the case
with all the eggs taken from the upper and middle portion of the mass. But in a
very few eggs taken from the sides of the mass, I was not able to detect any sperma-
tozoa, within the envelopes, or found only solitary instances of them. In these cases
it was remarkable that no chamber had yet been formed above the yelk, although in
some, in which spermatozoa were detected, it commenced at a later period, so that
these appeared to confirm the deduction from experiment with reference to quantity
of influence, or number of spermatozoa required to effect fecundation ; while the
penetration of the spermatozoa, as far as the vitellary membrane, and the subsequent
development of the chamber above the yelk, appeared in the relation of cause and
consequence, direct or indirect. I could not then observe, in the eggs thus examined,
any penetration by the spermatozoa completely through the vitellary membrane into
the substance of the yelk ; although numerous spermatozoa were attached, by their
larger ends, to every portion of the membrane, sticking out from it at right angles,
with what, at first, appeared to be a knob or knot at the distal end, or rather as if that
part of the spermatozoon had been shrivelled up or scorched. This appearance, as
I subsequently found, was due to a loop, or distortion of the tail of the spermatozoon,
consequent, apparently, on the death of this body. It was this looping of the tail
which gave the appearance of a thick end to this part of the spermatic body first
detected within the envelopes in a previous observation. All the spermatozoa seen
in connexion with the vitellary membrane were perfectly motionless ; so that when
the looping of the tail has taken place the fecundatory influence of these bodies may
be held to have been already exercised and exhausted.

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 271

At the expiration of three hours from the commencement of these observations,
although the number of spermatozoa which had first been noticed within the coverings
of the eggs submitted to examination appeared to have been diminished, there was still
an abundance attached to the vitellary membrane. These appeared to afford a good
opportunity for endeavouring to ascertain whether any part of the body of the sper-
matozoon is passed through the membrane into the cavity of the yelk. With this
object in view, I placed a glass cell, which contained an egg, on one side, and waited
for the rotation, or rather the gravitation, of the yelk within its envelopes. By this
change of position of the yelk within its coverings — and which always takes place, if
fecundation, or any change in the position of the entire egg, has been effected, — the
flattened surface of the yelk, and the chamber above it, were brought beneath that
part of the vitellary membrane into which spermatozoa appeared to have sunk
deepest, — so that if any of these bodies had passed, or were in the act of passing, or
had been partially protruded through the membrane into its cavity, which contained
the yelk, it was fair to expect that they might thus have been detected. But not the
slightest trace of penetration, even by a single spermatozoon, could then be observed*.

I may here remark, in anticipation of a future communication, that, previous to
segmentation, the mass of the yelk does not appear to be invested by any distinct
envelope within its so-called vitellary membrane, but the whole seems to be kept
together by the natural coherence of the granules or cells of which it is composed ; so
that when the position of the egg is changed the yelk mass rotates within the vitel-
lary envelope as a consequence of this change, owing probably to the excentric posi-
tion, within its substance, of the progeny of the germinal vesicle-f-, contained in the

* Since this paper was communicated to the Society, I have succeeded, through the adoption of a different
mode of examination, in detecting spermatozoa within the vitelline cavity in direct communication with, and
penetrating into the yelk. They were first seen by myself, in company with a friend, on the 25th of March of
the present year (1853) within the clear chamber above the yelk, at about forty minutes after fecundation,
when the chamber begins to be formed. I have since repeatedly observed them within the chamber, and in
some instances still in motion, in which state I have had opportunities of showing them to my friend Professor
ELLIS of University College, and to two other medical friends, so that the presence of active spermatozoa within
the vitelline cavity in the fecundated egg of the Frog may now be regarded as indisputable. The details of my
investigation I reserve for a future communication, and will merely now add, that the spermatozoa do not reach
the yelk of the Frog's egg by any special orifice or canal in the envelopes, but actually pierce the substance of
the envelopes at any part with which they may happen to come into contact ; as I have constantly observed while
watching their entrance : sometime after they have entered the yelk chamber they become disintegrated, and
are resolved into elementary granules. The importance of this fact of actual penetration by the spermatozoon
into the yelk is indicated by WACNEK and LEUCKARDT, in their late Article, " Semen" (loc. cit. p. 507), in the
following remark: — "The truth is, the 'how' of the fecundation is as far from our knowledge to-day as it
was thousands of years ago ; this process is still enveloped in what we feel inclined to consider ' its sacred
mystery.' It would be different if we could prove that the spermatozoa really yielded the material foundation for
the body of the embryo ; that they penetrated into the ovum, and were developed into the animal (which was the
assumption of LEEUWENHCEK, ANURY, GUATIER), or else, that they became metamorphosed into the central parts
of the nervous system." — G. N., April 18, 1853.

f Philosophical Transactions, Part I. 1851, p. 176.

272 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

middle of the dark hemisphere of the yelk, and which, possibly, may be of less speci-
fic gravity than the contents of the light-coloured hemisphere.

The facility with which the yelk can thus be made to rotate, and its flattened
surface, and the clear space, or chamber above it, — commenced within one hour after
the first encounter of spermatozoa with the egg, — can be brought beneath any portion
of the vitellary membrane into which spermatozoa have penetrated, — thus enabled
me to observe, within a comparatively short period after fecundation, whether any of
those bodies are at that time in the act of passing through it. But although no
spermatozoa could then be detected within the vitellary cavity, it must be borne in mind
that these observations were made after the commencement of the formation of the
clear space, or respiratory chamber, the consequence of a shrinking and depression of
the surface of the dark hemisphere of the yelk as the result of impregnation, so that
these negative observations did not afford any proof that penetration by the sperma-
tozoon had not previously taken place. Indeed, all circumstances considered, there
seems reason to believe that impregnation is effected very quickly after the
encounter of the spermatozoon with the egg, as appeared to be shown in my former
experiments with solutions of potass*. It is probable that it is commenced, as then
suggested, within a few seconds, or at most a very few minutes, after such encounter ;
as in one instance of artificial impregnation in some observations made subsequently
to those above detailed, I detected a spermatozoon in contact with the vitellary
membrane, within one minute after the impregnating bodies had been supplied to an
egg beneath the microscope. Certainly I believe that it is commenced, and probably
completed, within the first half -hour ; because, after that lapse of time, I have not
yet been able to detect any change of place or position of the spermatozoa which
have passed into the envelopes of the egg ; whether they may have already arrived at,
and become partially imbedded in the vitellary membrane, or whether they are still
contained in the clear substance of the outer coverings, their force of penetration
being exhausted before arrival at the membrane. After the lapse of half an hour,
and frequently of a much shorter time, the caudal portion of the body of a sperma-
tozoon which has penetrated into the envelopes becomes looped on itself, and this is
a certain indication of the death of the object.

As no spermatozoa were seen, in these observations-f-, within the vitellary cavity at
the early period above stated, it was not to be expected that they were likely to be
observed at a later ; although the eggs which had been the subject of these investiga-
tions, and which had been fecundated naturally, were continued to be watched until
segmentation of the yelks commenced. This took place in the two eggs, in which
alone, out of the fifteen first examined, spermatozoa were detected, — at the expira-
tion of jive hours and twenty minutes. But this was much earlier than in the great
mass of eggs, of which these were a part; and was due to their having been exposed
to a higher temperature, owing to heat radiated from my own body during their

* Philosophical Transactions, 1851. t See the preceding note.

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 273

constant observation beneath the microscope, — rather than to that to which the
undisturbed mass of eggs was exposed. The temperature of the atmosphere of the
room at the time the eggs were deposited was 54° FAHR., but it gradually sunk to
51°'5 FAHR., and the water in which the eggs were contained to 50° FAHR. at the time
when segmentation commenced, at the end of six hours and thirty Jive minutes. In
the most submerged it did not take place until six hours and forty-Jive minutes had
elapsed.

In nearly the whole of the eggs of this mass which had been fecundated by the
natural union of the sexes, I was struck with the fact that the quantity of sperma-
tozoa which had penetrated into every part of their envelopes was very considerable.
Very many of these had arrived at, and were sticking by their larger end into the
vitellary membrane, from which they projected like spines from the head of a thistle ;
while there were many others which had not arrived at this part, their power of
penetration being exhausted, and their progress inwards being arrested before they
had passed more than half-way through the outer gelatinous coverings. Others,
again, had penetrated to scarcely more than their own length into them, while a still
greater number were simply in contact with, and adhering to the external surface.

When segmentation took place in these fully impregnated eggs, their yelks seemed
to contract more powerfully, and the clear space, or respiratory chamber, formed in
each became much larger than in others from the same mass in which but a few
spermatozoa were detected ; so that the inference deducible from the fact seemed to
be, that a plurality of spermatozoa is necessary for the full impregnation of the egg,
and the production of the robust and healthy embryo — although simple fecundation
may result from the influence of only a very few spermatozoa, especially when such
influence is aided by a considerable increase in the temperature of the surrounding
medium.

Mode of Penetration by the Spermatozoon. — The preceding observations on natu-
rally fecundated eggs were so decisive of the fact of penetration by the spermatozoa
into at least the envelopes of the egg, and of the arrival at, and partial imbedment of
these bodies in the vitellary membrane, that it seemed desirable to make further
observations, by the artificial method, with a view to ascertain more directly the
mode and circumstances of their entry ; and these it was hoped might be learned
through the facility with which the egg may be impregnated by direct application
of spermatozoa to almost any part of its surface. Accordingly, on the following
day, I placed an egg in a glass cell, beneath the microscope, and quickly afterwards
applied to one side of it, by means of a pin-head, a quantity of spermatic fluid, ob-
tained from the male only a few minutes before, and immediately filled the cell with
water, and commenced the observation. The fluid was applied four times to the
same part of the egg, the pin-head being loaded for each application, in order that a
full sufficiency of spermatozoa might be furnished, before the water was added. The
temperature of the room at the time of the experiment had been intentionally raised

274 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

to 65° FAHR., to afford a greater chance of success ; as all my previous experiments
had shown that more eggs became fecundated at a moderately elevated, than, under
otherwise similar circumstances, at a low temperature. The instant the object-glass
(the half-inch of Ross's microscope) was brought into focus, a vast quantity of sperma-
tozoa were seen adhering to the surface of the egg at the part to which the pin's head
had been applied. Many of them, attached laterally to the surface, had already
ceased to move ; others, only partially attached by their larger end, still vibrated the
caudal or ciliated extremity rapidly, but did not appear to penetrate ; while others,
more centripetally attached by their body portion, vibrated the free extremity rapidly,
and were seen in the act of gradually penetrating into the substance of the envelopes.
I distinctly recognised one of these bodies which had just entered the envelope to a
depth equal to about twice its own length, and when first seen had not reached so
far as the middle or granulous layer of the envelope. Its motion was then slightly
serpentine, and its course from without inwards was in a perfectly centripetal direc-
tion, with its thicker or body portion extended forwards, and in a line with the
centre of the yelk, its progress inwards, as seen beneath the microscope, being as
continued and as direct as that of an arrow. I kept this object in focus for several
seconds, and watched it through the granulous layer, but ultimately lost it in the
more dense and, as yet, unexpanded portions of the inner layers of the envelope,
after it had been distinctly seen by a friend, who was with me at the time of the ob-
servation. A few seconds afterwards, as the envelopes became more expanded, very
many of these spermatic bodies were seen to have already arrived at, and be in the
act of passing through the inner or laminated portion of the envelopes, — the portion
which, when the envelopes have acquired their full distension, by the imbibition of
water, is seen to be that which immediately covers the vitellary membrane. A few
seconds later a great abundance of them were seen in contact with the vitellary
membrane itself; and some were even partially imbedded by their thicker extremity
in its substance, and some of these showed an appearance as if they were actually
penetrating through it. But in no one instance could I then satisfy myself that they
did really pass through ; since by alternately elevating and depressing the lens, and
carefully noticing when the margin of the vitellary membrane was most distinctly
defined, the appearances of perforation which some of them showed, seemed to be
due to the spermatic bodies being imbedded in the membrane at some inclination to
the plane of observation and to that of their direction being not quite centripetal.
Yet there were other circumstances which seemed to show a likelihood that some
spermatozoa do actually pass through the membrane. Thus, within the first few
minutes after the impregnating fluid had been supplied to the egg under observation,
and at the time when the spermatozoa, which had penetrated its envelopes, were first
seen to have arrived at, and begun to enter the laminated portion, or zona pellucida,
some of them were noticed to pass gradually onwards for a time, and then suddenly
to disappear in an instant ; as if, having passed into this tissue, they had also escaped

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 275

through the vitelline membrane which it covers. This mode of disappearance
certainly is that by which the spermatozoa might be supposed to penetrate to the
yelk, if in reality they do so*. Thus, presuming the body, or thicker portion of the
spermatozoon, to perforate and pass through the vitelline membrane, the more
slender or tail portion would follow quickly. But if this occurs with some, then it
would be fair to expect that it would happen to the whole which arrive at and become
partially imbedded in the membrane ; especially to those which have sunk into it to
a depth equal to one-half the length of the thicker or body portion ; or, further, that
the progress of others might be arrested before they had completely passed into the
vitelline cavity ; and, consequently, that some would occasionally be seen protruding
into the interior. But I was not able, in the instance of the observations now detailed,
nor in others afterwards made, to prove either of these suggested conditions.

In the observation now referred to, as well as in others since made, there were
many spermatozoa which remained distinctly visible for several hours, in the same
place, and in almost precisely the same position, sticking into the vitelline membrane,
and retaining, at the end of a lengthened period, the same appearance as at first,
excepting only that they seemed to have become smaller in diameter and to have
their caudal portion more looped. In the present instance, at the high temperature
of 65° FAHR. to 66° FAHR., not only were they distinctly seen at the commence-
ment of segmentation of the yelk, which happened at the end of three hours and
twenty-two minutes, but many of them were present until after the yelk had under-
gone several of its subsequent divisions. This was the case not only with those
which had arrived at the vitelline membrane, but also with others which had never
reached it, and had not penetrated further than to about the middle, or granulous
portion of the envelopes ; as happened with many spermatozoa, both in the eggs
which were fecundated naturally, as well as in those which were the subjects of ex-
periment, and were artificially affected. It was those which remained in the sub-
stance of the envelopes which usually disappeared earliest, becoming at first gradually
fainter, and then more undefined in outline. This change was supposed to be due to
a gradual diffluence of the substance of the spermatozoon, through the influence of
the water imbibed by the envelopes ; but whether this happened as part of the fecun-
datory process, or whether it was simply the natural process of decay, as other cir-
cumstances to be mentioned seemed to intimate, there was no distinct proof. It
was remarkable, however, with reference to the act of fecundation, that in almost
every instance, even of those spermatozoa which never arrived at the vitelline mem-
brane, the body portion was always directed towards the yelk, usually peripherally,
but sometimes inclined at slight angles to one side or the other; thus showing that
it is invariably the body portion which penetrates.

* That this is really the fact, and that the actual penetration by the spermatozoon into the yelk chamber
was observed on this occasion, is now rendered almost certain by my recent observations stated in the preceding
note, p. 271.— G. N., April 18, 1853.

MDCCCLIII. 2 O

276 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

It may be matter of surprise, that, considering the immense quantity of spermatozoa
which exist even in a microscopic drop of fluid, and considering' also the abundance
which come in contact with the surface of the egg, even when but a small quantity
of fluid is employed, as in artificial impregnation, a much greater number do not
penetrate jthan are usually observed to do so. There are conditions and circum-
stances which affect this result. Thus I have found, in repeated observations, that
only those spermatozoa which, at the moment of first contact with the egg-enve-
lopes, are in rapid action, and have their body portion directed, either perfectly
centripetally towards the yelk, or at angles but slightly inclined to it, do by any
possibility enter ; while those which happen to be directed horizontally to the
surface of the egg at the instant of contact, always adhere to it laterally, and lose
their power of motion, but do not penetrate ; and the like also is the case with
those which become attached by their caudal end, and even with many which
adhere by their thicker end, when they come into contact with the egg at very
acute angles. Further, I have noticed, that a relatively much greater number of
spermatozoa penetrate the envelopes when supplied to the egg immediately after
this has been removed from the female into water ; — especially when the spermatic
fluid also has been recently passed from the male ; — and more decidedly so when
passed from a male in full season, at which time the movements of the spermatozoa
are most energetic. Thus the chances of penetration through the envelopes, and
consequently of fecundation of the egg by the spermatozoon, are in direct relation to
these circumstances; and inversely to those of an opposite character, being less in
proportion to the length of time the egg has been removed from the female, or the
fluid from the male ; the healthfulness of the parent, and consequent power of motion
in the spermatozoon ; — the temperature of the season, and the quantity supplied to
the egg.

These were the conclusions deduced from the previous observations, and they have
been fully borne out by subsequent experiments, some of which have been made in
the presence of my friends Professors BELL, BOWMAN, CARPENTER, and Mr. BUSK,
Fellows of the Royal Society, and Professor ELLIS, who permit me to mention the
circumstance.

Narcotization of the Spermatozoon. — I may now mention some experiments which
were made with the view to test the fecundatory influence of recently obtained sper-
matozoa when narcotized by chloroform. These experiments were suggested by a
communication made to me by Mr. BUSK, F.R.S., who, after witnessing my mode of
procuring the eggs of the Frog, conceived that a similar result might be attained by
narcotizing the gravid animal without killing it, as is necessarily done in my experi-
ment ; and on putting this opinion to the test he found that it may be accomplished
with ease and success. It then occurred to him to try the effect of exposing the
spermatozoa to the vapour of chloroform, by simply covering the spermatic fluid
contained in a watch-glass covered with blotting-paper wetted with the liquid,

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 277

and afterwards to apply the narcotized bodies to some eggs in the way done by
myself.

The facility with which the spermatozoa can be narcotized by the mode now men-
tioned has enabled me to test the relation of their power of motion to that of their
fecundatory property, and although my results differ somewhat from those obtained
by Mr. BUSK, it may be well, perhaps, to relate them.

MM. PREVOST and DUMAS found, in addition to their many other excellent results,
that spermatozoa are rendered motionless by an electric shock, and that then they
do not impregnate the egg. They also found that opium and strychnine have a
similar paralysing effect on these bodies. But it has since been suggested that the
latter agents act on these bodies only in so far as they affect the chemical composi-
tion of their substance, and that the operation of electricity on them also is similar.
But it yet remains to be shown whether any chemical change is produced in the
substance of the spermatozoon, when simply narcotized by the vapour of chloroform,
and not mixed with it in the fluid state; and when, although the power of motion is
arrested, the vitality of the body is not destroyed.

Three eggs were placed in separate cells, and the spermatic fluid, immediately it
had been obtained, was applied, once only to each, by means of the pin's head. These
trials with the fluid in its natural state, mixed only with a small quantity of water,
were made for the purpose of comparing their results, with those of others to be
made with portions of the same fluid after it had been narcotized, and applied to
eggs in this state at different periods. One minute after the application of the fluid I
found an abundance of spermatozoa on the surface of each egg at the part touched,
and some spermatozoa had not only already penetrated into the envelopes, but had
arrived at, and were in contact with the vitelline membrane, or rather the zona pel-
lucida. The respiratory chamber was afterwards formed above the yelk in each of
these eggs, and two of these subsequently formed embryos ; the third was only par-
tially fecundated.

The spermatic fluid was then exposed to the influence of chloroform, in the way
mentioned, about fifteen minutes after it had been obtained ; and after seven minutes'
exposure to it, and when the majority of the spermatozoa it contained had become
narcotized, was employed in experiments. The signs of full narcotization are the
entire cessation of all motion in the spermatozoon, which lies with its body extended
at length, and not looped on itself. In the latter condition it is usually dead.

Three eggs placed in separate cells were then supplied with the narcotized sperma-
tozoa applied to each egg three times by means of the pin's head. On examining the
eggs six minutes afterwards, I was unable to detect even a single spermatozoon within
the envelopes of either of them, either in contact with the vitelline membrane or in
any part of the substance of their envelopes ; although there was a great abundance
of perfectly motionless spermatozoa on the surface. No chamber was formed above
the yelk in either of them, nor did either of them produce an embryo.

2 o2

278 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

Twenty minutes after narcotization, and during which time not a single sperma-
tozoon had been observed in motion, a portion of them were applied in the same way as
above to three other eggs, but not one of these became fecundated, no chamber being
formed within them, and no embryo was produced. Many of the spermatozoa at this
time were dead, as was shown by the looping of their bodies, but there was still a
quantity which had been simply narcotized and lay extended at length.

One hour after narcotization another portion was applied to two eggs, one of which
became partially fecundated. The chamber appeared in it above the yelk, the yelk
underwent segmentation, and afterwards an embryo was begun to be formed, but its
development was not completed, and the egg then perished. The second was not
fecundated. The circumstance of this egg having been fertilized at so long a period
after the fluid had been narcotized, leads to the conclusion that impregnation had
been effected by revived spermatozoa.

On the following day I repeated these experiments with very mature fluid imme-
diately after it was obtained, and in which the spermatozoa were exceedingly vigorous.
One minute and a half after it had been exposed to chloroform, I found that the same
effect had been produced on the spermatozoa as that which is produced in them by
their admixture with very weak solution of potass ; their power of motion was
greatly increased, and the whole were in a state of intense action. After a few
minutes' longer exposure their motion was perceptibly diminished, and at the end of
about ten minutes it had almost entirely ceased, as only a few of them were then
observed to move.

Three eggs were supplied, by means of the pin's head, with these narcotized sper-
matozoa. The respiratory chamber was formed in two of these eggs, and one of
them underwent segmentation, and at the end of the fifth day an embryo had begun
to be produced, but its development did not proceed, and this egg like the other
two perished.

Six eggs were then supplied copiously by means of the loaded pin's head, three
times applied to each, nearly the whole of the spermatozoa employed being then mo-
tionless, and having already remained so for about ten minutes, only an occasional
one being observed in feeble action. Two of these eggs became partially impreg-
nated, the usual chamber being formed in them, but neither of them underwent seg-
mentation, nor was any embryo formed either in these or the others.

Three eggs were then supplied as above after the spermatozoa had been motionless
during twenty minutes, not one being observed with the slightest action at the time
of the experiment. No chamber was formed in either of these, but the whole
remained unimpregnated.

Three eggs were the subject of further trial, when the spermatozoa employed had
been motionless during half an hour. At this time one or two spermatozoa were
again observed to be in feeble action, but no impregnation was effected.

Thirteen eggs were employed when the spermatozoa had remained motionless

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 279

during one hour, and when there was reason to believe that the majority of them had
perished, but when there were still a very few among- them in motion, having either
revived, or but recently escaped from spermatozoal cells contained in the fluid. No
impregnation took place in either of these instances.

Thus the results of these experiments appear to show that the spermatozoon does
not impregnate when entirely deprived of its power of motion by narcotization, and
disenabled to penetrate into the envelopes of the egg; and, consequently, that its
fecundatory power has a close relation with its motion, or force of vitality. In this,
then, they coincide with those of the previously detailed experiments, and go with
them to show that the act of fecundation of the egg of the Frog, and probably also of
all the Vertebrata, is the result of a power in the spermatozoon, which, in its operative
condition, is characterized by motion ; and that this power is totally independent of
everything approaching to volitional influence, in the impregnating body, but seems
to be in direct relation to physical causes.

MM. PREVOST and DUMAS appear to have thought that the spermatozoa which they
observed within the egg-envelopes had entered through means of the infiltration, or
imbibition of water by the envelopes ; and the gist of their experiments was to show
that particles of solid matter do enter during such infiltration, or, as it has since been
designated, endosmosis. But some experiments, elsewhere detailed*, have led me to
believe, that only such solid particles as are very much smaller in diameter than the
spermatozoon can so enter with the water by endosmosis ; while the circumstances
now detailed fully prove that the entrance of the spermatozoon is not the result of
simple infiltration, but is that of the operation of direct mechanical or physical power
in that body. Thus, if it were mainly dependent on endosmosis, the perfectly motion-
less spermatozoon would enter the tissues and fecundate the egg equally well with
the active, but this, as we have seen, is not the case, and this was shown by the
physiologists now referred to. On the other hand, the circumstance that the spermato-
zoon invariably enters the tissues with its thicker or body portion directed forwards,
and sometimes even at an angle slightly inclined to the centre of the yelk, and always
impelled by an oscillatory or vibratile motion of its caudal portion, seems to show
that its power of penetration is not only a necessary condition of its function, but is
inherent as such in its organic composition. It may yet be true, nevertheless, that
the passing of the spermatozoon through the substance of the envelopes to the vitel-
lary membrane may be indirectly aided by the endosmic action of the envelopes.
But this aid does not consist of any imbibing property in the tissues. It appears to
be simply of a negative character, and to be the result of a decrease of density in the
tissues, which, through their imbibition of water, and consequent expansion, are
caused to offer less and less resistance to the propulsive force of the spermatozoon.
This I believe is the proper explanation of the fact of penetration by this body.
But although penetration be not caused by the endosmic action of the envelopes,

* Philosophical Transactions, 1851, p. 224.

280 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

it may yet be thought to be induced by some attractive power in the substance of
the yelk itself; and that, therefore, the entrance of the spermatozoon may be as much
due to the egg as to any power of motion in the penetrating body. An accident has
enabled me to test the validity of this surmise.

I had promised to show the fact of penetration to a friend, but circumstances pre-
vented me from doing so until the season had nearly passed, and the whole of my
frogs had spawned. I determined therefore, as a last resource, to endeavour to ob-
tain some fecnndatory fluid from a male which had already paired two or three days
previously, and to employ it with the only eggs I had then left, which remained in
the body of a frog that had been killed twenty-six hours before, and which, as former
experiments had shown, it was probable had lost their vitality. The fluid required was
obtained with ease, but mixed with a large quantity of spermatozoal cells. This was
supplied to some eggs from the dead frog, since, although I did not expect that the eggs
would be fecundated, I hoped for an opportunity of again witnessing the penetration
by the spermatozpon. The spermatozoa in the fluid obtained were very active, and
fully efficient, and were supplied in abundance to several eggs in separate cells under
the microscope. The envelopes of the eggs expanded as usual, and endosmosis went
on in a perfectly natural way, and an abundance of spermatozoa adhered to their
surface. At the expiration of from fourteen to twenty minutes I found that several
spermatozoa had penetrated the envelopes and were adhering in the usual way to
the vitelline membrane. In one egg there were six, in another five, and in a third
four, distinctly visible in the plane of observation that could be brought at once
within view with the microscope, besides others recognisable on changing the focus.
It was thus evident that spermatozoa, even of the previously paired Frog, still retained
their power of penetrating into dead eggs, as these ultimately proved to be, after
careful preservation to the sixth day in a favourable temperature. The spermatozoa
were distinctly visible within the envelopes, without change of position, for several
hours, but no fecundation was effected by them ; no chamber was formed in either
of the eggs, no segmentation took place, nor was any embryo produced. These
circumstances seem to show that the eggs were already dead, as was supposed, before
contact of the spermatozoon ; consequently that the entrance of the spermatozoon
into the envelope is due to a power inherent in the penetrating body, and not simply
to an attraction on the part of the yelk; although from the fact that the spermatozoa
usually enter in a centripetal direction, it is probable that some influence may be
exerted by the yelk or its vesicle, although penetration is mainly the result of force
in the spermatozoon.

The facts now stated of penetration by the spermatozoon seem to lead us better
to understand the nature of some experiments with solutions of caustic potass, which
are detailed in my former paper. I have repeated these experiments, during the past
season, in the presence of several friends, Professors SHARPEY, ELLIS, and BELL, and
Messrs. BUSK, TOMES and WATERHOUSE, with results precisely similar to those which

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 281

i

are detailed in that paper; viz. that when diluted spermatic fluid, recently obtained,
is applied to a set of eggs, and — as soon afterwards as the experiment can be made,
a solution of potass, — of such a strength as is known by previous microscopic observa-
tion to have the property of instantly decomposing the spermatic body, — the solution
being washed away quickly after its application, by repeated quantities of water, —
to prevent its affecting the egg itself — that then — in some instances — even when the
interval of time between the application of the spermatic fluid and the subsequent ap-
plication of the potass does not exceed a few seconds, — impregnation of the egg is
effected; as is proved by the formation of the chamber, the segmentation of the
yelk, and perhaps the formation of an embryo. Further, that, all circumstances
being similar, excepting only that the interval of time between the application of the
spermatic fluid, and, subsequently, that of the solution of potass be prolonged, — the
production of an embryo is not only more certain to take place, but the number of
embryos produced is increased. The now ascertained fact of almost instantaneous
penetration by the spermatozoon, which, as before shown, sometimes arrives at the
vitelline membrane in less than one minute after its application to the egg, confirms
the principal conclusion deduced from the potass experiments at the time they were
made, viz. that impregnation is commenced at the instant the spermatozoon is in con-
tact with the egg ; while it also seems to afford the true explanation of the nature of
those experiments, in which it may now be presumed that some spermatozoa had
actually penetrated into the substance of the envelopes before the application of the
solution of potass, and thus had already passed out of the reach of its destructive
influence, the effect of which on the egg itself was obviated by speedy dilution and
ablution with water.

The potass experiments may thus be regarded as confirming by anticipation the
results now obtained by direct observation with the microscope, with respect to the
rapidity of operation by the spermatozoon; and they seem also to support the view
of the essentiality of the motor power of this body to its functional action. A similar
view may be taken, as indeed was held at the time, of the nature of the Carmine, the
Gum, and the Starch experiments, that the operation of these substances in preventing
the fecundation of the egg is entirely mechanical, and that they do so simply by offering
a mechanical impediment to the spermatozoon, a conclusion which seems to be fully
supported in the present inquiry.

14. NATURE OF THE INFLUENCE OF THE SPERMATOZOON.

Having seen in the preceding experiments and observations that fecundation of
the egg is effected by the spermatozoon only while this body retains and continues to
give evidence of its vitality in its power of motion, and that its vitality either may be
destroyed, or its operation be for a time entirely arrested by electricity and by chlo-
roform,— the question naturally arises — in what way, then, is its fecundatory influence
to be explained ? Is it simply by diffluence of the substance of the spermatozoon, and
the chemical fusion or combination of this with the contents of the egg, after the sper-

282 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

matozoon has penetrated through the envelopes and arrived at the vitelline membrane,
or the yelk i Or is it by its expenditure on the yelk, of aforce or power of vitality, which
is inherent in the body of the spermatozoon ? Or is it by the joint cooperation of both
these conditions — the expenditure of aforce with diffluence of material substance ? I
have endeavoured to put the first of these questions to the test of experiment ; —
although, it must be remarked, that the experiments made seem to be open to some
objections, which at present cannot be fully answered. Yet knowing what we do of
the disposition and great readiness of all matter to enter into new combinations, and
affect, or entirely change the condition of the whole body operated upon, — as, — not
to mention the effect of the poison of serpents, or the introduction of dead matter into
the living, in dissection, — can be shown in the effect, on the yelk of the Frog's egg,
of extremely minute quantities of chemical compounds diffused in water, — there has
seemed fair reason to think that the question thus started may be examined by
experiment. The rapid endosmic action of the envelopes of the egg at the instant of
their contact with any aqueous fluid, and the rapidity with which the yelk itself then
becomes affected, seemed to favour the idea that if the bodies of the newly obtained
spermatozoa could be quickly reduced to a state of diffluence simply by mechanical
means, without the addition of any menstruum, saving only a very small quantity of
water, and be applied in this state of diffluence to the egg at the instant after it has
left the body of the female, — that then an experiment thus made would be a fair test
of the question. Some imbibition of the substance of the spermatozoon with the
water might thus be expected to take place, and some changes in the egg to follow,
if impregnation be the result of simple chemical combination of the substance of the
male with that of the female. The presence in the experiment of a portion of water
holding the diffluent spermatic substance in suspension, appeared at first sight to
be an objection, but this seemed to be met by the fact that water must always be pre-
sent to ensure the natural fecundation of the Frog's egg ; and that it not only perme-
ates the envelopes, and is the means of facilitating the entrance of the spermatozoon,
but that it passes even to the substance of the yelk itself, as shown in the experiments
with large quantities of potass before alluded to, and also in others made by immer-
sion of eggs in water with given proportions of potass in solution. As the details of
these latter experiments are contained in a paper which is now in the Archives of
the Royal Society*, I need give but little more than the results of these experiments,
in the present communication, before proceeding to relate those now referred to with
the diffluent spermatozoa.

Immersion of Ova in Solutions of Potass and Soda. — These experiments were
commenced in April 1850, before the publication of some which were made with
solutions of caustic potass, by M. QUATREFAGES, on the spermatozoa of one of the
marine worms, Hermella, and read to the Institute on the 24th of June 1850-f-.

* MS., No. 762. p. 13 to 26; also Proceedings, vol. vi. p. 83.

f Comptes Rendus, June 24, 1850, and Annales des Sciences Naturelles, 3m' s6rie, torn. xiii. in Nos.
marked " February " and " March 1850."

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 283

The object which I had in view was to endeavour to learn what proportion of the
carbonates of potass and soda may be present in a given quantity of water without
destroying the fertility of the egg. The first trials with potass were made with eggs
which had already been fecundated, and were undergoing segmentation. It was
then found that when fecundated eggs, arrived at the second stage of segmentation
of the yelk, were immersed in a solution, composed of twenty grains of the fused
carbonate of potass to one ounce of water, the yelks became shrivelled, and decom-
position was commenced within three minutes after immersion ; thus proving that
even when the envelopes of the egg are almost fully expanded, and, consequently,
when their endosmic action may be expected to have become greatly diminished,
that endosmosis may still be going on with much energy. It is right to mention,
however, that in this and the following experiments, the endosmic action may have
been, and probably was, increased by the removal of the eggs — which already retained
water in their tissues — to the more dense fluid solution ; but as the experiments are
comparative, the circumstance is not of much importance.

On the contrary, when some eggs were immersed in a solution of one-fourth of a
grain of the salt to one ounce of water, and the potass in admixture with the water
was thus reduced to ywotn part of the whole, development went on more quickly
than in other eggs of the same brood, impregnated at the same time, but which were
placed in pure water apparently through the potass occasioning a slight increase of
temperature.

These results appear to be explicable only on the assumption that the weak potass
solution acts as a chemical stimulus, — perhaps both to the egg and to the sperma-
tozoon,— at the time of fecundation, a view which derives some support, in so far as
refers to the spermatozoon, from direct observation with the microscope. Thus,
when a drop of the two-grain solution of potass is applied to spermatozoa on a plate
of glass, covered with talc, beneath the microscope, the movements of these bodies
are not instantly arrested, as by the stronger solutions, but gradually become slower
and slower, until they entirely cease, apparently, in proportion as their substance
becomes affected by the potass. The one-grain solution very much accelerates the
movements of these bodies during the first few seconds ; but in a few seconds longer
these also become diminished like the preceding. But the effect of the half-grain
solution, although exciting the spermatic bodies to activity as soon as it comes into
contact with them, continues its effect for a much longer time without acting
injuriously upon their substance; and it is not until after the lapse of many minutes
that the intensity of the motion excited by it is observed to be diminished, and the
movement to slacken and afterwards gradually cease.

With regard to the carbonates of soda, I may state that the general effect of these
on the impregnated and on the unimpregnated egg are very similar to those of
potass, but are far less prejudicial, or marked, so that it is unnecessary to detail the
experiments.

MDCCCLHI. 2 P

284 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

The general conclusions deducible from these observations, with reference to the
nature of the influence of the spermatozoon in impregnation, seemed to be, that
as water which holds in suspension or solution any chemical substance, conveys some
portion of that substance at all times, with great rapidity, to the immediate vicinity
of the yelk itself, which becomes affected by it, and especially so at the time of its
first coming into contact with the envelopes of the newly deposited egg, — the sub-
stance of the spermatozoon also, if diffused in water, might be so conveyed, in the
experiments proposed. In addition to this conclusion, there are others, which,
although less directly connected with the object in view, are not less important.
Thus it appears that an alkaline fluid, in large quantity, is as injurious to the vitality
and fecundatory influence of the spermatozoon as to the fertility of the egg.

Trituration Experiments. — The endeavour in these experiments was to put to the
test the question proposed in the preceding observations, — whether, if the sperma-
tozoon be reduced to a state of diffluence in water quickly after it has left the body
of the Frog, and before it can reasonably be supposed that any change in the chemical
constituents of its body has taken place, the egg can be fecundated through simple
imbibition of the substance of the spermatozoon, conveyed to the vicinity of the yelk
with the water during endosmosis, at the time when endosmosis of the envelopes of
the egg is most energetic ?

Although it had been constantly found, both by the authorities before cited, and
since by myself, that when the spermatozoon has ceased to move, and is believed
to be dead, no impregnation results from the contact of its motionless body with
the egg; yet there still appeared to remain some doubt as to what this want of
operation is then due ; whether it is to be attributed to the organic death and
incipient decomposition of the body, or whether to the loss or suspension of some
power which is characterized by motion, and through which its function is exercised?

The following experiments, with reference to this question, were commenced before
I was aware of the fact that the spermatozoon penetrates into the envelopes of the
egg ; a fact which, when known, seemed further to necessitate the inquiry, as probably
tending to show whether impregnation is merely the result of a union of the sub-
stance of the spermatozoon with that of the yelk or its vesicle ; or whether it be not
primarily due to the transmission of some dynamic influence, force, or peculiar
vitalizing power from the spermatozoon to the egg at the time of, or previous to any
fusion with it of its material substance.

The first experiments were made by breaking down the bodies of the spermatozoa
mixed with water, and, after filtration, applying the filtered liquid directly to the
egg ; the following being the method pursued : —

The bottom and sides of a very small glass mortar, and the glass pestle employed
with it were ground together with fine sand and water, for the purpose of slightly-
roughening their surfaces, and of ensuring their contact at every part, and thus
to render the crushing of the spermatozoa more certain.

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 285

The spermatic fluid first employed was procured from a Frog early in the season,
and consequently contained a large quantity of spermatozoal cells from which the
spermatic bodies had not been liberated. A difficulty immediately occurred which
had not been anticipated. The fluid was divided into two parts, one of which was
triturated gently with the pestle and mortar for two minutes, and was then examined
with the microscope. It still contained an abundance of active spermatozoa, and
many cells. The trituration was continued for three or four minutes longer, without
any marked result, as active spermatozoa were still abundant in the specimens of
fluid examined. It was then repeated for several minutes with increased rapidity
and force, when the fluid suddenly became partially coagulated, and separated into
opake white flocculi, composed almost entirely of cells and granules, and a trans-
parent fluid in which there were scarcely any granules. It was then found that nearly
the whole of the spermatozoa had been destroyed, as I was not able to detect even one
in motion. The thinner portion only of this fluid, when placed on the filter, passed
through, and it did so with great slowness and difficulty.

These circumstances are stated to show the mode of proceeding, and the accidents
to be guarded against. The conclusions deduced from these trials were, that the
spermatic fluid was not fully matured, and probably contained albumen, which, it is
stated*, does not exist in mature spermatozoa ; also that the substance of the sperma-
tozoa had undergone some chemical decomposition, perhaps from excess of heat
evolved in the act of trituration, through too much force being employed ; so that
the results of these experiments could not be depended on. A further trial was
therefore made a few days afterwards. The fluid obtained from a mature Frog was
divided into two-parts, one of which was reserved for simple artificial impregnation ;
while the other was triturated slowly and carefully in the mortar with but little more
exertion than was necessary to keep the pestle in constant motion for about fifteen
minutes ; no water being added to the fluid. When placed on the filter it became
necessary to add to it about an equal quantity of water to facilitate its filtration.
Portions of the fluid which then passed through, holding the substance of the broken-
down spermatozoa in solution, were caught in separate glass cells.

Ten eggs were passed into a cell about half filled with the filtered fluid, and the
cell was afterwards filled up with water. No other change than a slight enlargement,
after the lapse of some hours, occurred in these eggs ; no chamber was formed, nor
did any segmentation take place in either of them, consequently no embryos were
produced.

Fifteen eggs were placed in a cell which contained about half the quantity of filtered
fluid employed in the preceding ; but no fecundation took place in either of these
eggs.

Eleven eggs in a third cell with filtered fluid were equally unfruitful.

Ten eggs in a fourth cell gave similar results.

* Article " Semen," Cyclop. Anatomy and Physiology, vol. iv. p. 506.

2 P2

286 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

One hundred and forty -seven eggs were then placed on the topmost of the filter
papers, which had been employed in separating the fluid used in the preceding trials,
and on which were the remains of the broken-down spermatozoa, with, probably,
some which had not been injured. This was about four hours and a half after
the spermatic fluid had been obtained, so that on this ground but little success
could be expected. Not a single embryo was produced.

The portion of reserved fluid which had not been triturated was then applied to
four eggs in separate cells, by means of the pin's head, when each of these eggs
became fecundated, and not only underwent segmentation, but each afterwards pro-
duced an embryo. As this experiment was made for the purpose of comparison with
the preceding, it is clear that the failure in the case of the mass of eggs on the filter
paper was not due to the length of time the fluid had been obtained, but to the effect
of trituration ; and it seems equally clear that the substance of the broken-down
spermatozoa dissolved in water and passed through the filter is as inefficient as the
liquor seminis has heretofore been proved to be*.

These experiments were afterwards repeated, in the presence of Professors SHARPEY
and ELLIS, with similar results. Usually there are still some spermatozoa which
escape being crushed during trituration, and, being uninjured, remain on the filter
paper capable of effecting impregnation when eggs are passed upon it in water, as
was the case on the following occasion.

Seventy eggs were first added to some filtered fluid, which resulted from the
trituration of spermatic fluid, and had passed through four filter papers, but not one
egg became affected, not one embryo was afterwards produced from them ; while of
sixty-two eggs placed on the topmost filter paper in water, amidst the remains of
broken-down spermatozoa, with many which were still active and uninjured, more
than one-third of the eggs became fertilized, and twenty-three afterwards produced
embryos.

It was thus evident that no impregnation results from an immersion of the egg in
fluid which is derived from the broken-down body of the spermatozoon, mixed with
water, and separated from the more solid parts by filtration. But as some objections
may fairly be offered as to the validity of this experiment, it seemed desirable that
trial should be made without having recourse to filtration, and without removing
any portion of the destroyed spermatozoon; but, on the contrary, by applying the
whole of the triturated fluid immediately to the egg before it is immersed in water;
and at the same time to compare the result obtained with that of an experiment by
simple artificial impregnation, with a sample of the same spermatic fluid which had
not been triturated; the experiments in both cases being made at the same time,
and with eggs from the same female, and placed under precisely similar conditions
in regard to light, heat, &c. A comparison of the relative number of embryos which
might result from these experiments, it was thought, would show, not merely whether

* Philosophical Transactions, 1851.

AND ON THE DIRECT AGENCY OF THE SPERMATOZOON. 287

impregnation can or cannot be effected by the direct application of the diffluent sub-
stance of the spermatozoon to the egg, but also whether the influence of the
spermatic body is essentially dynamic in its character.

The quantity of spermatic fluid obtained for these experiments, mixed with a
small proportion of water, measured about fifteen minims ; and to this a further
quantity of water was added, making the whole thirty minims. This was divided
into three equal portions. The first portion (a) was triturated slowly and con-
tinuously for fifteen minutes, after which it was slightly turbid, and on examination
with the microscope was found to contain a large quantity of granules, each of which
did not exceed in size one half of the diameter of a spermatozoon. It also still con-
tained a good abundance of active spermatozoa, although the quantity of these was
greatly reduced, and the movements of those which remained were impeded by the
quantity of granules. The second portion of fluid (b) was triturated with the addition
to it of about one grain weight of well-washed sand, and after three or four minutes
presented a turbid appearance. At the end of seven minutes, when examined by the
microscope, it was found to contain a great abundance of organic granules, inter-
mixed with the sand, and the number of spermatozoa had been greatly reduced ; and
three or four minutes later nearly the whole of the spermatozoa were destroyed.
The following experiments were then made with eggs obtained from the female with
which the male that supplied the fluid had been paired. The temperature of the
atmosphere at the commencement of the experiments was 57° FAHR., and that of the
water employed 55° FAHR. At the end of the experiment, the commencement of
segmentation of the yelks, the atmosphere was 61° FAHR., and the water 59° FAHR.

One hundred and seventy-nine eggs were passed into a glass dish, and the triturated
fluid (a) was immediately poured over them, and the dish quickly filled with water.
The yelks of many of the eggs soon became irregular and contracted, and at the end
of four hours and thirty-one minutes segmentation had commenced in some of them,
and ultimately, sixty-seven embryos were produced; so that one hundred and twelve
eggs were not fecundated.

One hundred and sixty-seven eggs were passed into a second dish, and the fluid (b)
which had been triturated with sand was immediately added to them, and afterwards
the dish filled with water as in the preceding. But not a single egg became fecun-
dated, nor was a single embryo produced. It must be stated, however, that much of
this result was afterwards found to have been due to the admixture of sand with the
fluid acting mechanically, as I shall presently show.

Two hundred and thirty-nine eggs were also passed into a dish of the same dimen-
sions as the preceding, and the third portion of fluid (c) reserved for simple artificial
impregnation, and which had not been triturated, was supplied to them at, as nearly as
possible, the same time as the triturated fluid to the preceding. This was at thirty-
five minutes after it was obtained. Segmentation commenced in nearly the whole of
these eggs in four hours and fourteen minutes, and at the end of the fifth day two

288 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA

hundred and six embryos were produced, so that only thirty-three eggs failed; thus
leading to the inference that the failure in the experiments (a) and (b) was owing
to the spermatozoa being destroyed ; and, consequently, that the application of the
substance of the body of the spermatozoon to the egg is not alone sufficient to effect
fecundation ; so that fecundation cannot be regarded as the result of simple chemical
combination of the substance of the spermatozoon with that of the egg, but, essentially,
may be due to some dynamical influence in that body.

At the time of making these experiments I made also two others with a view to this
hypothesis ; and for the sake of correct comparison employed eggs from the same
female, and placed them in a precisely similar condition with regard to light, heat,
and the quantity of water employed. The fecundatory fluid, however, was not from
a male in full season, as above, but was what may properly be regarded as senile,
it being purposely obtained from some frogs which had been kept separate from the
females, having already paired and spawned from five to ten days previous. It was
slightly translucent, and contained, relatively, but few living spermatozoa, the
majority of which were languid in their movements ; but there were many which
appeared perfectly dead and motionless, and there was a good proportion of sperma-
tozoal cells. In the first experiment one hundred and sixty-seven eggs were supplied
with a portion of this fluid, and segmentation commenced in some of them in four
hours and forty-two minutes, or not so soon as in the experiment (c) by twenty-eight
minutes, and afterwards only forty-three embryos were produced. In the second trial,
made at the same time with one hundred and seventy -three eggs, segmentation took
place in four hours and forty-one minutes, and one hundred and twenty-six embryos
were produced, forty-seven eggs being unirnpregnated. If the difference in the
length of time which elapsed between the encounter of the spermatozoon with the
egg and the commencement of segmentation in these two experiments, as compared
with the preceding one, be not fairly referable to a less degree of vitalizing power in
the spermatozoon in the latter, it seems difficult to understand to what other cause
it can be assigned ; seeing that the eggs employed were from the same female in each
experiment, and that all the conditions were similar. It seems, as it were, inversely,
to show, that the most important condition of the impregnating agent is its possession
of some dynamical quality, the degree or intensity of which is expressed in that of
its power of motion, and which, possibly, it may transmit from itself to the egg, during
the act of fecundation.

These results sufficiently mark the injurious effect produced on the fecundatory
fluid by trituration, whether with or without admixture of foreign substances ; and
while these seem to prove that attrition of the spermatic bodies with particles of solid
matter, by lacerating and mechanically destroying them, is fatal to their function,
other experiments showed that even their simple admixture with any finely com-
minuted solid materials greatly interferes with their operation, by the mechanical
impediment which such materials oppose to their free motion, and to their penetra-

AND O.N THE DIRECT AGENCY OF THE SPERMATOZOON. '289

tion into the envelopes of the eggs, whether such admixture be made with the bodies
themselves, or with the water in which the eggs are .immersed before they are sup-
plied to it.

The question proposed in these experiments by trituration we may now look upon
as to some extent, if not entirely, answered ; as it seems that although the spermatozoon
be broken down mechanically before any chemical change can be supposed to have
taken place in it, — indeed, at the very time it is giving evidence of its vitality, — and
its organic substance so broken down be quickly afterwards brought into contact with
the egg in water, at the moment when the egg is most susceptible of its influence, yet
that no fecundation is then effected by it. It may be urged, it is true, that there was
no direct proof that the spermatic substance was actually conveyed to the yelk by,
or with, the water in these trials ; but there was one circumstance which gave fair
reason to believe that it really was so conveyed. The yelks of some of the eggs
became much contracted, and, as it were, shrivelled, as when affected by potass solu-
tion ; of the penetration of which to the yelk there is absolute proof in the decomposi-
tion of the egg which quickly succeeds to its introduction. It has also been shown
in a preceding experiment (p. 241) that the yelk becomes similarly affected when
decomposing spermatic fluid is applied to it.

The conclusion then which seems to me to be deducible from these investigations
is, that fecundation is not simply the result of a mere fusion or chemical admixture of
the substance of the spermatozoon with that of the egg; although such fusion,
probably, is necessary to the production of the organized body of the embryo, in
determining its structural and psychical peculiarities, and its definite species ; and
more or less of which may, possibly, help to determine the sex, and the extent to
which the structural and psychical peculiarities of the male parent are transmitted
to the offspring. That this fusion of some portion of the spermatic substance with
the egg does actually take place, either when the spermatozoon has arrived at, and
become imbedded in the vitelline membrane, as in the Frog; or, as stated by a
recent observer*, to occur in the Ascaris Mystax, when it is in immediate contact
with the yelk itself, is probable, from the considerations now adduced. These views
are countenanced by the now established fact that the spermatozoon of the Frog, and
probably also of other Vertebrata, does not fertilize the egg either when it is perfectly
motionless, — whether from actual death, or from suspension of vitality by narcotiza-
tion; nor even, as we now find, though living, while simply in contact with the
surface ; nor until after it' has actually passed into the envelopes, and arrived at the
immediate vicinity of the yelk, — facts which seem, I think, inferentially, to show a
probability that, whatever conjugation of materials may be effected, some vitalizing
dynamic influence is also expended by the spermatozoon on the contents of the yelk in
the production of its changes, — phenomena which have not been found to take place

* Dr. NELSON, " On the Reproduction of the Ascaris Mystax," Proceedings of the Royal Society, vol. vi.
p. 86. Philosophical Transactions, 1852.

290 MR. NEWPORT ON THE IMPREGNATION OF THE OVUM IN THE AMPHIBIA.

on the simple application to the egg of the diffluent spermatic substance. In the
absence, then, of all proof that simple fusion only of the substance of the spermato-
zoon with the contents of the egg is the chief condition of fecundation, we have, in
the facts now referred to, much reason to regard fecundation as primarily dependent
on the existence of a force or power in the spermatozoon, by which this body is
enabled to arrive at the object to be fertilized, and which, visibly expressed in that
of its power of motion, is lost quickly after it has penetrated into the yelk-membrane
or yelk : — this, probably, is the instant of fecundation. May not the spermatozoon,,
then, be viewed as the organ of a special form of force in the male body for the pro-
duction of these results, in the same way as the nervous structure is regarded as
that of the nervous, and the muscular as that of muscular power? We have already
seen that its function is exercised more or less readily and perfectly in relation to
two conditions : first, that of its full maturity, in which its power of motion is most
intense ; and next, in relation to the influence of external physical agencies, which
cause this power to be evolved through its organic composition, in a greater or less
degree, whether the external influence be that of heat, or the operation of a chemical
power, as in the instance of potass in the preceding experiments.

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