Mortality in pike-perch eggs in hatcheries

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DEPARTMENT OF COMMERCE

J.”. BUREAU OF FISHERIES
HUGH M. SMITH, Commissioner

MORTALITY IN PIKE-PERCH EGGS
IN HATCHERIES

By
FRANZ SCHRADER
Formerly Fish PatHologist, U.S. Bureau of Fisheries
and

SALLY HUGHES SCHRADER
Temporary Assistant, U. S. Bureau of Fisheries

APPENDIX V TO THE REPORT OF THE U. S. COMMISSIONER
OF FISHERIES FOR 1922

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MORTALITY IN PIKE-PERCH EGGS IN HATCHERIES.’

By Franz ScHraver, formerly lish Pathologist, U. S. Bureau of Fisheries,
and
SALLY HuGHeEs ScurADER, Temporary Assistant, U. S. Bureau of Fisheries.

The remarkable losses in the hatching of pike-perch (Stizostedion
vitreum) eggs have frequently made this phase of fish culture a
ground for ‘Investigation. It is, however, not unjust to say that very
little detailed study has been made and that no definite conclusion
as to the causes of the high death rate has ever been reached.

The methods used in the handling of parent fish and eges are, in
the main, very much alike at the various stations that hatch pike
perch. The fishes are caught in nets that are usually pulled once a
day, weather permitting. “Those that are ripe are stripped immedi-
ately, either in the field or at the hatchery, while the rest are retained
in pens or live boxes. ‘They are examined in turn daily until found to
be ripe. In some years the sexes are found to be disproportionate in
number. If males are scarce, the same individual may be used to
obtain milt on several -suecessive days. Fertilization is by the dry
method, no water, or very little water, being used in the process. Milt
and eges are stripped into a bowl in more or less regular alternation,
and the whole is gently stirred at frequent intervals to insure the con-
tact of the eggs with the sperm. When the bowl is sufficiently full—
generally after 10 to 15 minutes—the contents are diluted with water
which after a varying period is poured off and renewed until the eggs
are contained in clear water. Cohesion of the eggs, which at this time
are extremely sticky, is prevented by active stirring or by adding
silt or starch to the water in addition to such mechanical agitation.
Finally, after several hours, the eggs are put into the hatching jars
through which a gentle current of water is kept flowing.

As “already indicated, the losses are very great. Nevin (1887)
considers a hatch of 50 per cent’ a very fair success, and this would
be agreed to by most fish-culturists. The cause of this great mor-
tality i is, In general, ascribed to failure of the eggs to be fertilized
or else to injuries incurred while the eggs are being handled, espe-
cially the active stirring and the addition of foreign materials to
prevent cohesion. It seems almost certain that these last-named
crude procedures—which so far are unavoidable—are very apt to be

1 Appendix V to the Report of the U. S. Commissioner of Fisheries for 1922. B. F.
Doc. No. 926.

91376°—22 al

2 U. S. BUREAU OF FISHERIES.

harmful, and a certain amount of loss is probably due to them.
Reighard (1890) found that by very careful handling of the eggs at
and immediately after fertilization the percentage of eggs that
started segmentation soon after fertilization could be considerably
increased. Describing the loss met with in the ordinary course of
the routine methods, he states that in 252 samples examined 11 per
cent had died due to lack of impregnation and 33 per cent due to
injury. Unfortunately, although he designates the day and hour
at which these observations were made, the age of the eggs is not
specifically given. From the context it would appear as 29 hours.
It seems that an egg was designated as dead when it showed an
opaque white color, a criterion which was adopted also in the present
investigation.

The figures of the losses in ordinary handling of pike-perch eggs
given by Reighard are somewhat at variance with those given by
L. H. Almy in some unpublished notes on the pike perch. His find-
ings and those of the authors follow:

Almy. | Schrader and Schrader
= |
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Age of eggs. eee | Age of eggs. cea

S hourse = eases 8 2e5y hI hoursty Ayer eh ee bee 0.5
27 to 29 hours......-- 5.4 || 4 hours 30 minutes. - .. 1.0
2 daysustss FIST eas 8.1 | 8 hours 15 minutes... 2.4
BOF BSE eee Se eeeire Lean mente lacs 12;16',||/Qhourss?*: cates seeeeeecee eee 4,3
A URYSE Seis ace ope ee ee ane ae tLe SPT 29 OUTS. Fe Sa uke on esos ee te ee 5.0
DGRYSLE ate pated. Byte eb Re ae Bo.'0 I (Zidays-2-e22. See: £5 See eee 8.3
MUGS HEAL sean lira at ote ie gede geste ieee tan fe BOSONS OAVSs 2. ook ees Soe ee Lee ee 13.3
Oidaysa he Leute VERE) VE Waa RE RENE) SBbaN LA days vlh We Mee eee Oe ee 33. 2
5: AB Y See on vicelak sc1-5 dates e nee eee eS 37.1

It will be seen that in contrast to the 33 per cent of white eggs
given by Reighard, Almy observed only 5.4 per cent at 27 hours,
while our own observations are lower still. Almy’s and our figures
agree fairly well, the latter beng lower up to two days and a trifle
higher at four days. It is not quite clear to what such a discrepancy
could be due, although Reighard’s hypothesis of injury as a cause
of mortality would, of course, itself allow for large differences on
account of the varying skill and care bestowed on the eggs. (Tem-
perature conditions were in all cases apparently the same, the water
being in the neighborhood of 45° F.) Reighard describes the in-
jury as taking place most easily over the oil globule, and there is
no reason to dispute the observation. However, the following ex-
planation which he advances to account for this phenomenon does
not seem to rest on a very firm physical basis (Reighard, 1890, pp.
33, 84):

In the natural position the yolk sphere lies with its lower half against the
egg membranes. These membranes, therefore, support this half of the yolk,
surrounding it as if it were resting at the bottom of a cup. The upper half
of the yolk is, on the contrary, not of the same form as the investing membrane;
its spherical surface is interrupted by the protruding oil globule.

The result of this arrangement is that when any pressure is brought to bear
on the egg membranes, so that the space within which the yolk lies is reduced,
the yolk is able to resis. this pressure by fitting itself against the egg mem-
brane at every part of its surface except over the oil globule. The strain,

MORTALITY IN PIKE-PERCH EGGS IN HATCHERIES. 3

therefore, comes on that part of the protoplasmic investment of the yoke which
covers the oil globule and here it bursts. In almost every case the white spot
which indicates the rupture of the yoke investment makes its appearance at
the oil globule, usually at the equator.

Almy’s as well as our own observations show that the death rate
increases rapidly and steadily to the fourth day and then advances
more slowly. To begin with, it must be noticed that a small per-
centage of dead eggs is found practically as soon as the fishes are

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Fig. 1.—Graph showing the variation in numbers of abnormal and unsegmented pike-
perch eggs during development.

stripped. It is surmised that these may have been injured in the
process of stripping, or that they may have died through some de-
velopmental irregularity while still in the fish.

Coming now to the hypothesis that failure of fertilization. is re-
sponsible for a greater part of the mortality of hatching eggs, it is
generally assumed that lack of impregnation and failure to segment
are closely correlated. A detailed examination of the material does
not bear this out. We found that in eggs 4 hours 30 minutes old there
was a considerable percentage which showed no trace of cleavage.
This was true also at 5 hours 30 minutes, 6 hours 45 minutes, and

4 U. S. BUREAU OF FISHERIES.

even 8 hours 15 minutes. Most of the unsegmented eggs at the
latter stages on being examined cytologically appeared to be normal.
They were therefore merely lagging behind. The proportion of
eggs which has failed to cleave becomes progressively less with age,
which in itself supports the idea that we are dealing here rather with
a delay in cleavage than with a lack of impregnation. The curve
in figure 1 (p. 3) shows graphically the numerical conditions en-
countered. These data are emphasized here merely to show that it is
manifestly impossible even after eight hours to designate eggs as un-
fertilized when the absence of segmentation is taken as a criterion.

Figs. 2 to 10.—Surface views of pike-perch eggs. Magnification, approximately x 100.

Fic. 2.—Normal 6-hour 4-celled blastoderm. Fic. 7.—Abnormal 19-hour blastoderm.
Fic. 3.—Normal 19-hour blastoderm, Fie. 8.—Abnormal 19-hour blastoderm,
Fic. 4.—Abnormal 8-hour blastoderm. Fic. 9.—Abnormal 29-hour blastoderm.
Fic. 5.—Abnormal 10-hour blastoderm. Fic. 10.—Abnormal 29-hour blastoderm.

Fic. 6.—Abnormal 10-hour blastoderm.

In addition to all this it must be considered that in the artificial
insemination of the pike perch the eggs are immersed in milt which
is diluted very little. The chance of a normal ripe egg remaining
unfertilized must therefore be extremely small, and, as a matter of
fact, it is surprising that polyspermy is not more often encountered.

In addition to the eggs which are found to be dead almost imme-
diately, and to those which are slow to cleave, there is a third class
which has been designated as “abnormal.” In explanation it must
be stated that minor irregularities in cleavage are not necessarily an
indication of pathological conditions (H. V. Wilson, 1891), and only
such extreme cases as are shown in figures 4 to 10 were rated as

. MORTALITY IN PIKE-PERCH EGGS IN HATCHERIES. 5

abnormal. Eggs showing normal cleavage are shown in figures 2 and
3. Extreme variations, “such as are shown in figures 4 to 10, in
size of cleavage cells were found in nearly all cases to be correlated
with internal conditions which presaged embryonic death sooner or

Fies. 11 to 17.—Sections of pike-perch eggs. Magnification, approximately x 200.

Fig. 11.—Horizontal section of a normal egg of 64 or more cells.

Fic. 12.—From a 29-hour egg showing cytasters and abnormal spindles.

Fic. 13.—From a 29-hour egg showing size variation in eytasters.

Fic. 14—From an 8-hour 15-minute egg, showing elongated nucleus.

Fig. 15.—From a 29-hour 15-minute egg, showing partial segmentation.

Fic. 16—From a 29-hour 15-minute egg showing ‘degeneration in chromatin and multi-
plication of chromosomes.

Fig. 17.—From an 8-hour 15-minute egg showing monaster.

later. The number of.such abnormal eggs increased steadily with
age, ranging from 1 per cent at 4 hours 30 minutes to 21 per cent
at 19 hours. As the curve in figure 1 shows, this increase runs
parallel with a decrease in the number of unseemented eges, which
suggests the possibility that such abnormal cases are derived chiefly

6 U. §. BUREAU OF FISHERIES.

from eggs which are slow to cleave. It may be remarked that it is
difficult to draw a strict line at times between the abnormal and the

22

Fias. 18 to 22.—Sections of pike-perch eggs. Magnification, approximately X 1620.

Fic. 18a, 18b, 18¢e.—Side views of three sections of a plate showing 30 chromosomes,
the normal number.

Fig. 19.—Side view of a plate in another cell in the same blastoderm as figure 11,
showing 15 chromosomes, the haploid number.

Fig. 20.—Polar view of normal plate.

Fig. 21.—Abnormal multiplication of chromosomes.

Fic, 22.—Abnormal elongated nucleus—abortive division.

unsegmented types in the first few hours. At that time one or a few
minute excrescences are occasionally budded from the germinal disk,

————

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MORTALITY IN PIKE-PERCH EGGS IN HATCHERIES. rf

‘and it becomes a question whether these should be regarded as cells
-or not.” On the other hand, at the 29-hour stage, abnormal eggs
are often segmented into such small cells in such a way that, exter-
nally, it is very difficult to tell them from normal eggs, although in-
ternally they may be shown to be very irregular in behavior. The
intermediate stages are therefore the best material for this phase of
the investigation.

Those cytological features in the development of normal eggs
which bear on the work in hand are as follows: Up to the 16-cell
stage cell walls are sometimes partially or completely absent, but
casters and spindles are normal in size and occupy the same position
that they would if there were a distinct separation into cells. Fol-
lowing the 16-cell stage, the cleavage, which becomes externally com-
plete at least in the surface layer of cells, gives evidence of this fact
internally by the presence of very distinct cell walls. Mitosis is at
first synchronous in all the cells, but this regularity is soon lost, so
that certain cells of an egg may be in the resting condition while
neighboring cells may be undergoing mitotic division.

As already indicated, cytological examination of uncleaved eggs at
4 to 8 hours showed the majority to be normal (fig. 11, p. 5). The
few exceptions were found to have anomalous mitotic figures, and
their number was increased in the 8-hour stage. At 29 hours every
uncleaved egg showed anomalous internal features. The exceptional
‘8-hour eggs often show a very large monaster (fig. 17, p. 5). Other
egos may show several cytasters and an occasional spindle (figs. 12
to 16, p. 5). At 29 hours no such large monasters are found in eggs
of this type or in those called abnormal and generally there is only
‘an increase of cytasters in the former.

The abnormal eggs often present a curious mixture of spindles
and asters of varying sizes, drawn-out nuclei, chromosomal irregu-
Jarities, and partially formed cell walls (figs. 12 to 17, 21, and 22).
Frequently an egg is found in which a part has undergone regular
cleavage while the rest is filled with cytasters and shows no indica-
tion of cell walls (fig. 15). As it was expected that such irregu-
larities would be reflected in the distribution of the chromosomes in
division, evidence of such chromosomal abnormalities was sought.
But, as in other teleosts, the chromosomes are usually so clumped that
‘an exact’ analysis of them is very difficult. In at least one case, how-
ever, the metaphase plate in one cell showed close to 30 chromosomes
(which seems to be the diploid number as obtained from counts in
normal eggs, fig. 20), while the adjoining cell contained only about
15 (figs. 18 and 19). This might be explained as a case of partial
fertilization, in which the sperm has instigated a division of the
egg nucleus and later has fused with one of the nuclei resulting from
this first division of the egg nucleus. The fusion nucleus would then
be diploid and the purely maternal nucleus haploid.

Trregularities in cleavage and mitotic figures practically identical
with those here described have been obtained experimentally by a
number of investigators. It will be noted that in every experiment
of this nature the effect is to induce development with one of the

2Reighard (1890a) mentions excrescences as occurring in correlation with the flow of
protoplasm in the formation of the protoplasmic cap. Since the number of eggs showing
the exerescences mentioned above increases long after the formation pf the cap, in our
-case, the phenomenon described by Reighard is probably not related to it.

8 U. S. BUREAU OF FISHERIES.

parent nuclei absent or in a weakened condition, with both in a
weakened condition, or with the pronuclei incompatible through
hybridization. A limited survey of the extensive literature on this.
subject will suffice to show the trend of the work.

E. B. Wilson (1901) found that in artificial parthenogenesis, where,
of course, only one of the parent nuclei is present, there occur such
abnormalities as the formation of cytasters, the multiplication of
chromosomes without accompanying cell division, multipolar mitoses,,
and delay in cleavage. The chromosome number in the eggs which
seem to show normal development is haploid.

Dungay (1913) weakened or injured sperms of several species of
invertebrates by means of chemical treatment, heat, or staling, and
development in eggs fertilized by such sperms resulted in delayed
cleavage, abnormally sized cells, multipolar figures, and similar
defects.

O. Hertwig (1911) and G. Hertwig (1912), among others, have
described the effect of fertilizing eggs with sperms treated with
radium. Both authors remarked especially a budding phenomenon
correlated with delayed development. They also describe drawn-out.
nuclei, multiplication of chromosomes in a nucleus, giant nuclei, and
cytasters, all almost identical with phenomena which we have de-
scribed in the abnormal pike-perch eggs.

C. Packard (1914) found that sperms which had been treated with
radium may stimulate the eggs to cleave but fail to take part them-
selves in the subsequent development. When the eggs are “radi-
ated,” they show various irregularities, such as abnormal divisions
or the failure of pronuclei to unite.

G. and P. Hertwig (1914) produced similar effects to those already
mentioned by weakening sperms with methlyn blue among other
reagents. Still more striking are the phenomena produced by fer-
tilizing the eggs of teleosts with sperms of another species of teleost.
The whole list of abnormalities given above was reproduced in such
development.

It is not within the province of apphed biology to go into a
theoretical consideration of these phenomena. Suffice it to say that
the weakening or injury of either sperms or unfertilized eggs will
produce the same defects in the development of all animals so far
investigated. Physiological and cytological phenomena identical in
appearance with those produced experimentally in this way have been
observed also in abnormally developing pike-perch eggs, and it sug-
gests itself that the cause of such irregularities is of similar nature.
In other words, there is a weakening of either sperms or eggs before
fertilization.

Jt is highly improbable that natural conditions should induce a
state that would cause such a large mortality in the embryos, and it.
becomes almost certain that the injury is incurred during the period
of the captivity of the fishes.

As has been said in the introduction, most of the fishes are found
not to be ready for stripping when first caught. They are therefore
retained in pens or crates until the reproductive products can be
obtained from them by stripping; in other words, until they are
“ripe.” The penning of fishes prior to spawning is a practice of

~

MORTALITY IN PIKE-PERCH EGGS IN HATCHERIES. 9

long standing in the handling of pike perch as well as many other
species. Some of these species are known to stand such confinement
fairly well, but many show various ill effects, such as hardening of
the ovaries, wateriness of milt, and low percentage of hatched fry.
Whatever the cause and physiological process involved, be it abnor-
mal hydrogen ion concentration due to the crowding of the parent
fishes, or more directly circulatory and nervous relations, the result
is a degeneration of eggs and sperms. In the case of the pike perch
especially the consequent mortality may. of course, fluctuate from
year to year due to such causes as sudden changes of temperature
(a sudden change of temperature is known to materially retard the
ripening of the fishes in the pens) and weather conditions which
may prevent pulling the nets and therefore postpone examination of
the caught fishes. Some specimens do not lay eggs even when ripe
under such conditions.

It may be of interest in this connection to give the opinions of men
who have the supervision of pike-perch hatcheries which are located
at Constantia, N. Y., Swanton, Vt., Put in Bay, Ohio, and Duluth,
Minn. Their opinions, given in response to a letter of inquiry, are

Fig. 25.—Eggs of Stenotomus prior to maturity. a, Normal egg; b, egg from a parent
kept in a tank for two weeks.

not based on numerical data but are the results of practical obser-
vation. All of these four superintendents and a fifth, who was
formerly connected with pike-perch work, agree that the mortality
af eggs is proportional to the time that the adult pike perch are
retained in pens and, conversely, that the percentage of hatched eggs
from fish stripped when taken from the net is much greater than that
of eggs from penned fish. Four of the men believe that both male
and female are affected by penning, but that the female is more
susceptible, while the fifth does not commit himself on this point
but cautions against using the males more than once, i. e., on several
days.

Bearing more directly on the problem are some experiments made
by the senior author in connection with some other work. Females
of the common scuppaug (Stenotomus chrysops) were netted shortly
before the spawning period and retained in a tank supplied with a
continual flow of fresh sea water. Specimen of these impenned fishes
were dissected at intervals of a few days and the ovaries examined
histologically. A progressive deterioration of the nearly ripe ova
was observed, which at the end of two weeks had reached such a stage
as shown in figure 28.

10 U. S. BUREAU OF FISHERIES.

The generalization that the high death rate in pike-perch eggs is
due to lack of impregnation thus seems to be unwarranted. That the
present methods of preventing cohesion of the eggs are responsible
for a certain percentage of the mortality is probable, but they do not
account for all the loss. On the other hand, it has been shown that
about 25 to 30 per cent of representative samples of 29-hour eggs show
abnormalities that must lead to either malformation or death. Lf an
average loss is then considered as 50 to 60 per cent (and that is a fair
estimate), about half of this is due to the agency which manifests
itself in abnormal development. This cause is in all probability to
be found in the practice of retaining captured fishes in pens for the
purpose of permitting eggs and sperms to mature.

BIBLIOGRAPHY.

DunGay, NEIr S.

1913. A study of the effects of injury upon the fertilizing power of sperm.
Biological Bulletin, Marine Biological Laboratory, Woods Hole,
Mass., Vol. XXV, No. 4, p. 213-260, Pls. I-II. Press of New Era
Printing Co., Lancaster, Pa.

HeErtTWIG, GUNTHER.
1912. Das Schicksal des mit Radium bestrahlten Spermachromatins im
Seeigelei. Eine experimentell-cytologische Untersuchung. Archiv
fiir mikroskopische Anatomie und Entwicklungsgeschichte, Bd. 79,
Abt. 2, p. 201-241, 3 Taf. Bonn.
, und PAULA HERTWIG. :

1913. Beeinfiussung der minnlichen Keimzellen durch chemische Stoffe.
Archiv fiir mikroskopische Anatomie und Entwicklungsgeschichte,
Bd. 83, Abt. 2, p. 267-806, 2 Taf. Bonn.

1914. Kreuzungsversuche an Knochenfischen. Jbid, Bd. 84, Abt. 2, p.
49-88, 1 Taf.

HERTWIG, OSCAR.

1911. Die Radiumkrankheit tierischen Keimzellen. Ein Beitrag zur ex-
perimentellen Zeugungs- und Vererbungslehre. en ue EOS
skopische Anatomie und Entwicklungsgeschichte, Bd. PN Oe 20) Os
1-95+ 97-164, 4 Tuf.+2 Taf. Bonn.

NEVIN, JAMES.

1887. Hatching the wall-eyed pike. Transactions, American Fisheries So-

ciety, Seventeenth Annual Meeting, p. 14-16. New York.
PACKARD, CHARLES.

1914. The effect of radium radiations on the fertilization of Nereis. The
Journal of Experimental Zoology, Vol. 16, No. 1, p. 85-129, 3 pls.
Philadelphia.

REIGHARD, JACOB.

1890. Experiments in the impregnation of pike-perch eggs.? Transactions,
American Fisheries Society, Nineteenth Annual Meeting, p. 30-36.
New York.

1890a. The development of the wall-eyved pike, Stizostedion vitreuwm Raf.
A popular introduction to the development of bony fishes. Ap-
pendix, Ninth Biennial Report, Michigan State Board of Fish Com-
missioners, Dec. 1, 1888, to Oct. 1, 1890, p. 98-158, Pls. I-X. Lan-
sing.

1898. The ripe eggs and the spermatozoa of the wall-eyed pike and their
history until segmentation begins. Jbid., Tenth Biennial atts
Oct. 1, 1890, to Dec. 1, 1892, p. 89-166, Pls. I-\V.

Witson, E. B.

1901. Experimental studies in cytology. I. A cytological study of artificial
parthenogenesis in sea-urchin eggs. Archiv fiir Entwicklungs-
mechanik der Organismen, Bd. 12, p. 529-596, 7, Taf., 12 figs.
Leipzig.

1901a. Experimental studies in cytology. II. Some phenomena of fertili-
zation and cell division in etherized eggs. III. The effect on cleay-
age of artificial obliteration of the first cleavage furrow. Jbid.,
Bd. 18, p. 353-395, mit Taf.

WILSON, HENRY V.

1891. The embryology of the sea-bass (Serranus atrarius). Bulletin, U.S.
Fish Commission, Vol. IX, for 1899, p. 209-277, Pls. LXXXVITI-
CVII, 12 text figs. Washington.

3“ Presented at the meeting by Herschel Whitaker and erroneously attributed to him by
the editor of the Transactions.’ (Quotation from Dean’s Bibliography of Fishes, Vol. Il,
p. 829, published by the American Museum of Natural History, New York, 1917.)

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