Special scientific report

WATER SOLUBLE VITAMIN
REQUIREMENTS OF SILVER SALMON

Marine Biological Laboratory

FEB !■) ~iy;)9
WOODS HOLE, MASS.

SPECIAL SCIENTIFIC REPORT- FISHERIES No. 281

UNITED STATES DEPARTMENT OF THE INTERIOR
FISH AND WILDLIFE SERVICE

EXPLANATORY NOTE

The series embodies results of Investigations, usually of restricted
scope, Intended to aid or direct management or utilization practices and as
guides for administrative or legislative action. It Is Issued In limited quantities
for official use of Federal, State or cooperating agencies and In processed form
for economy and to avoid delay In publication .

United States Department of the Interior, Fred A. Seaton, Secretary
Fish and Wildlife Service, Arnie J. Suomela, Commissioner

WATER-SOLUBLE VITAMIN REQUIREMENTS
OF SILVER SALMON

By

John A. Coates* and John E. Halver
Western Fish Nutrition Laboratory
Cook, Wash.
Bureau of Sport Fisheries and Wildlife

*Present address: Ralston Purina Co., Checkerboard Square, St. Louis, Mo.

Special Scientific Report- -Fisheries No. 281

Washington, D. C.
November 1958

The Library of Congress has cataloged this publication
as follows:

Coales, John A

Wiiti'i-sohilile vitamin ivcjuireineiits of silver sahnon, by
John A. CoiUes and John E. Ilalver. AVashinjrton. U. S.
Di'pt. of tlie Interior. Fisli and AVildlife Service. 1058.

t) I). tliiiKis., tables. 27 ciu. (Special scientific report— fisheries,
110.281)

Bibliography : p. 9.

1. Salniuii. 2, Vitamins. 3. Fishes — Foixl. i. Halver, Joliu Emil,
1922- joint author, u. Title. (Series: U. S. Fish and Wild-
life Service. Special scientific report: fisheries, no. 281)

SH11.A335 110.281 (i;]9.375 59-60279

Library of Congress

The Fish and Wildlife Service series, Special Scientific
Report — Fisheries, is cataloged as follows:

U. S. Fish and Wildlife Service.

Special scientific report : fisheries, no. 1-
[WashingtoU] 1949-

no. illiis., maps, diagrs. 27 cm.
Supersedes in part the Service's Special scientific report.

1. Fisheries — Research.
SH11.A335 639.2072 59-60217

Library of Congress

CONTENTS

Page

Experimental 1

Results and discussion 3

Summary 8

Literature cited 9

ABSTRACT

Qualitative vitamin requirements of silver salmon (Oncorhynchus
kisutch) were determined by feeding groups of fingerlings a complete vitamin
test diet (casein 54, gelatin 15, com oil 7, cod liver oil 2, dextrin 8, miner-
als 4, methionine 1.0, tryptophan 0.5, celluflour plus crystalline vitamins
8.5) as the control diet; and deleting one water-soluble vitamin at a some from
the ration for the respective deficient lots. A 16 -week feeding period resulted
in vitamin deficiency syndromes appearing in thiamine, pyridoxine, folic acid,
biotin, pantothenic acid, inositol and choline deficient groups. Nicotinic acid,
riboflavin and cyanocobalamine deficient lots gave inconclusive results, and
under experimental conditions used no deficiency syndromes were observed
for fish without ascorbic acid in the diet.

WATER-SOLUBLE VITAMIN REQUIREMENTS
OF SILVER SALMON

Good fish husbandry requires the use of
diets which provide the nutrients essential for
normal growth of the fish. A lot of "practical"
information has been accumulated by fish cultur-
ists on food for silver salmon (Oncorhynchus
kisutch), but there is little published data on
specific nutritional requirements for this species.
Nutritional investigations with other fish, rain-
bow trout (Salmo gairdneri), chinook salmon (O.
tshawytscha) and sunfishes (Lepomis), have
yielded more concrete data on qualitative and
quantitative needs for growth and metabolism .
Vitamin requirements (Phillips et al. 1945, 1947,
1949, 1950; McLaren etal. 1947; Wolf 1951;
Halver 1957a), general protein requirements
(Tunison et al. 1942, 1943; Gerking 1952; De
Long, Halver and Mertz 1957) and amino acid
requirements (Halver 1957b; Halver, DeLong
and Mertz 1957, 1958) have been described
which can serve as a basis for formulating test
diets and experimental rations for determining
the basic nutritional requirements of silver sal-
mon. If the general requirements for all
nutrients is in the same general range as that
found for trout and chinook salmon, and if the
same experimental techniques can be applied to
studies with silver salmon, then it should be
possible to use existing test diets to develop
specific nutritional deficiency syndromes in sil-
ver salmon and determine the spectrum of the
water-soluble vitamin requirements for this
species .

As a preliminary logical step, silver
salmon were tested with the same vitamin -test
diet used for qualitative vitamin requirement
studies with chinook salmon (Halver 1957a) and
which also maintained rainbow trout for at least
one reproductive cycle (Halver and Coates 1957).
Since silver salmon fingerlings grew when fed
this diet as sole ration, it was then possible to
delete one vitamin at a time from the complete
vitamin mixture in the diet, to feed these specif-
ic water-soluble vitamin deficient diets to
various individual lots of fingerlings, to describe
the specific vitamin-deficiency syndromes as
they occurred in each respective lot of fish, and
to determine which water-soluble vitamins were

required for growth and fresh water survival of
silver salmon.

EXPERIMENTAL

The complete vitamin-test diet was
formulated from the materials listed in table 1.
The procedure used was the same as that de-
scribed in detail for the preparation of diets
used previously to induce vitamin deficiency
syndromes in chinook salmon (Halver 1957a).
Prior to mixing the diet, the crystalline vitamin
supplement, the amino acid supplement and the
alpha -cellulose flour were mixed for two hours
in a ball mill and then stored at 5° to 10° C,
until used. To ensure more accurate and re-
producible weights, sufficient alpha -cellulose
flour, amino acids, and vitamins for at least 4 kg
of diet were mixed at one time. The mineral
mixture was also ground for two hours in a ball
mill and stored in the cold in a tight container
until needed.

To prepare 400 gm of mixed diet contain-
ing 25 percent solids and 75 percent water, 15 gm
of purified gelatin were added to 300 ml of water
and heated on a hot plate until the temperature
rose to 60° to 70° G. After the gelatin had lique-
fied, the container was removed from the hot
plate, placed in a mechanical mixer and stirred
at medium speed with a dough hook until the tem-
perature dropped to 40° to 50° C. Then 54 gm
vitamin test casein, 7 gm purified corn oil, 8 gin
white dextrin and 4 gm mineral mixture were
added and thoroughly blended. Finally, the cod
liver oil and the alpha -cellulose flour containing
the vitamin mixture and the amino acid supplement
were added and stirred until a homogeneous mass
was obtained (30° to 35° C.). For convenience in
feeding, the mixture was poured into ice cube con-
tainers, hardened in a refrigerator at 10° C., and
stored in screw-top glass jars at 5° to 10° C.
until used.

Approximately 7,000 silver salmon from
the 1956 brood were fed two weeks on a complete
vitamin test diet containing one-fourth the normal
amount of vitamin mix at the Washington State

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Department of Fisheries Issaquah Hatchery.
This vitamin level was fed in order to deplete
any large storage of water-soluble vitamins by
the fish . At the end of two weeks the fish were
randomly divided into 13 groups and placed in
standard screen-covered hatchery shallow
troughs. Each group was fed a diet deficient in
only one of the 11 water-soluble vitamins and
the growth was compared with two control lots
fed the complete vitamin-test diet. The fish
were fed three times daily, six days weekly with
no ration offered on Sundays . The food was
presented to the fish by grating small cubes of
the ration over the water in such a manner that
the food presented was eaten in approximately
one minute .

The fish were weighed biweekly accord-
ing to a standardized experimental technique
(Burrows, Robinson and Palmer, 1951; Halver
1957a) and a random sample of five fish was
taken for subsequent histopathological analysis.

Each group was under close examination
for the appearance of vitamin deficiency syn-
dromes listed for fish (Halver 1957a; McLaren
et al. 1947; Wolf 1951). Dead and moribund fish
were removed, recorded daily and were exam-
ined microscopically for any indication of the
presence of fish pathogens.

When the growth pattern of any of the test
diets differed significantly from the control or
when more than 20 percent of the population had
died, the remaining population of the test group
was divided, one -half fed the complete diet and
the other half continued on the vitamin -deficient
diet until the end of the experiment or the total
loss of the population. If that portion of the pop-
ulation fed the complete diet recovered from the
symptoms, it was then felt that the malady was
directly due to the lack of the vitamin in question
and that silver salmon required this vitamin.

The regular hatchery water supply from
Issaquah Creek was used. The water to the in-
dividual trough was screened through a 64-mesh
screened box to eliminate as much as possible
the natural food that might come in through the
water supply. Finer mesh screen was tried but
this had a tendency to plug and overflow subse-
quently cutting off the water supply to the
experimental fish.

Throughout the experiment many pathojren-
ic diseases were apparent. The two encountered
most often were "cold water disease" and "Octo-
mitus" . The effect of the pathogen certainly
influenced the results, and it was impossible to
evaluate growth and mortalities critically.
Growth curves of the deficient groups were plotted
however, and can be generally compared with the
growth and mortality rates of the two control lots
fed the complete vitamin-test diet (figs.l ana 2).

RESULTS AND DISCUSSION

The two control groups gained weight con-
sistently. There was little if any significant
difference between the two. This group suffered
approximately 50 percent loss over the 16 -week
period but all mortalities were heavily infected
with Hexamita salmonis and/or myxobacteria.
The greatest mortality occurred during the fourth
week. A summary of the specific vitamin de-
ficiency syndromes observed in the various lots
of fish was tabulated in table 2.

The diet used as the control ration for
this study contained approximately 70 percent
protein. Subsequent work has indicated that
about 50 percent protein might have been a more
desirable level for rapid growth with fish living
in this temperature range (DeLong, Halver,
Mertz 1957). At the time the experiment started,
however, this information had not yet been con-
clusively demonstrated and it was decided to use
the complete vitamin-test diet which did produce
near normal growth in chinook salmon for a time
period sufficient for the development of water-
soluble vitamin deficiency syndromes. For a
similar reason, the protein component of the
diet was supplemented with methionine and
tryptophan even though some evidence had been
accumulated that these amino acid supplements
to this diet might not be needed for satisfactory
growth. High levels of tryptophan would, in
addition, probably interfere with the development
of severe nicotinic acid deficiencies but since the
tryptophan requirements of fish were not known,
and this diet had grown chinook salmon, trypto-
phan was added.

The thiamine deficiency group compared
favorably with the control groups for the first
12 weeks of the experiment. After 12 weeks

Figure 1 and 2: -

-Growth and mortality of vitamin -deficient silver salmon.
Upper curves show growth of specific deficient lots and
the control lots. Lower curves show biweekly mortality
percentage of survivors . The junction points in growth
and mortality curves represent division of the deficient
groups into two sublets after the deficiency syndromes be-
came apparent in a large portion of the population.

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feeding gradually ceased and many emaciated
fish or "pin heads" with a characteristic severe
concave or "pinched" abdomen occurred. Some
loss of ability to control the dorsal and pectoral
fins was noted in moribund specimens. Loco-
motion consisted of a slow, rolling action and
a general loss of equilibrium . The coloration
of affected fish changed from a dark hue at the
onset of the clinical manifestations of the de-
ficiency to a more transparent condition when
moribund or dead.

The pyridoxine deficiency syndrome
manifested itself by loss of equilibrium and by
rapid flashing and erratically skipping action
along the surface of the water just prior to death .
Upon death, these fish assumed a lateral cres-
cent shape and were light in color. The char-
acteristic blue color previously described in
Chinook salmon (Halver 1957a) was not noted.
During the early stages of the malady, the ap-
petite was fair but changed to a complete lack
of feeding during the late stages of the experi-
ment. Severely affected fish exhibited indiffer-
ence to strong lig^t in contrast to the controls'
definite negative phototropism .

The folic acid group exhibited no clinic-
al syndrome and periodic blood counts did not
indicate any gross anemia. However, when this
group was divided, the section receiving the
complete diet indicated a definite benefit from
the addition of folic acid by a rapid growth
response and a significant increase in weight
over the deficient lot.

The biotin deficiency syndrome was
characterized by loss of appetite, emaciated
bodies and the caudal fins were contracted to
form a triangular point. Although the feeding
habits of the deficient fish were retarded, the
individuals seemed fairly active throughout the
experiment. The recovery group regained
normal appetite and weight quickly, and within
2 weeks these fish appeared normal. Mortalities,
fairly consistent in other groups, were virtually
non-existent in the biotin recovery gfroup after
replacement of the missing vitamin in the diet.

Pantothenic acid depleted fish had the
characteristic clubbed gills previously noted by
many investigators. As the syndrome started
to appear, these fish gradually lost their appetite

until they ceased feeding entirely near the end
of the experiment. The recovery group exhibited
a rapid positive response in appetite and growth
as soon as the gill damage was repaired, and
near the end of the experiment, was gaining
rapidly .

The appetite and growth of the inositol
deficient group was reduced, although a con-
sistent gain in weight was noted. Upon replace-
ment of the missing vitamin in the diet the
recovery group showed a significant growth
response and a corresponding reduction in mor-
tality from that of the deficient lot.

The choline deficient fish fed actively
throughout the experiment but failed to gain
weight, resulting in emaciated fish in the entire
population. When one -half the group was fed the
complete diet, they immediately started to gain
whereas the group continued on the choline de-
ficiency continued to lose weight . The growth
curve of the choUne deficient group was not
graphed because a general straight line relation
existed from the start to the end of the experi-
ment, and the mortality rate was consistently
low throughout.

Disease decimated the niacin deficient
group before any specific syndromes became ap-
parent. This group was split at the end of 12
weeks but at the 16-week period there were no
significant differences between the deficient and
recovery groups. Since these fish were held in-
doors, the common "sunburn" symptom described
by DeLong, Yasutake and Halver (1958) in Chin-
ook salmon and rainbow trout did not appear .
Exposure of these fish to ultraviolet light in- a
more carefully controlled environment might have
resulted in the appearance of a specific deficiency
syndrome.

Similarly, the riboflavin deficient group
did not exhibit any gross clinical symptoms.
Growth continued parallel to the controlled lot
throughout the course of the experiment but a
slight clouding of the lenses of the eyes appeared
in some of the moribund fish late in the experi-
ment. Some food was probably introduced through
the screens in the creek water supply and may
have been sufficient to partially supplement the
diet to an extent preventing severe clinical man-
ifestations of riboflavin deficiency.

In the vitamin 8^2''^^^^'^^^°^ ^°^> °° ^^'
ficiency syndromes were noted, even when this
group was divided after 12 weeks on the depleted
diet. Those fish receiving the complete test
diet exhibited little difference from those fed
the diet without vitamin 8^2"

In the ascorbic acid deficient group,
growth comparable with the controls was ob-
served throughout the course of the experiment.
The mortality rate paralleled closely that of the
control groups at least for the first 14 weeks of
feeding. The group as a whole seemed healthy
and showed less disease incidence than any other
group, except the controls, until the last 2 weeks
of the experiment. During the last 2 weeks of
feeding, most fish examined in all lots showed a
hig^ incidence of Hexamita, myxobacteria, or
both.

The entire course of the experimental
feeding period was beset with problems of
Hexamita infestation, myxobacterial infection
and natural-food problems. The water temper-
ature gradually increased from a low of 54° F.
to a hig^ of 68° F . during the middle of the feed-
ing period. Unfortunately, the temperature
remained high (in the low 60 's) until the 16 -week
feeding period was terminated. In the mornings
the temperature was in the middle 50 's, in-
creasing from 7° to 10° F. during each day. Be-
cause of the severe disease incidence, repeated
prophylactic treatments with pyridal mercuric
acetate were given for one hour every two weeks
starting at the sixth week of feeding. Undoubt-
edly, some adverse physiological effects were
experienced by the fish which may have tended
to obscure the appearance of specific vitamin
deficiency syndromes.

These experiments show again the neces-
sity of having a fish disease -free water supply
for conducting critical nutrition experiments in
order to prevent the disease symptoms from
masking the appearance of the deficiency syn-
dromes. The desirability of a more stable or
controlled water temperature also manifested
itself with the extreme variation in even daily
temperatures reflecting severely on the feeding
habits of the various groups of fish and on the
calculation of the correct dietary intake for each
lot. Perhaps more descriptive information of
subclinical manifestations of the deficiency

syndromes will be obtained upon completion of
the histopathology investigations of the tissues
of fish collected during the course of the experi-
ment.

SUMMARY

A complete vitamin-test diet successfully
used to induce specific water-soluble vitamin de-
ficiencies in Chinook salmon was fed to silver
salmon for a 16 -week feeding period. Deleting
the water-soluble vitamins one at a time from
this complete diet caused deficiency syndromes
to appear. Under the experimental conditions
used, some deficiency syndromes for thiamine,
pyridoxine, folic acid, biotin, pantothenic acid,
inositol, and choline were induced in silver sal-
mon. Inconclusive results were obtained with
niacin, riboflavin and vitamin 8^2 deficient diets.
No deficiency syndromes were observed for the
ascorbic acid deficient lot and since p -amino
benzoic acid was not included in the vitamin mix-
ture, it also was probably not required.

Appreciation is expressed to G. Duane
Gahimer of the U.S. Fish and Wildlife Service
and Daniel C. Coyle, Washington State Depart-
ment of Fisheries, for their help in the prelimin-
ary hatchery operation, diet preparation, care
and feeding of fish. W. C. Ashcraft, the
Issaquah Hatchery superintendent, was extremetly
helpful, and Brian W . Earp, Washington State
Department of Fisheries, generously investigated
and checked the fish pathology. The facilities
and the fish were supplied by the Washington
State Department of Fisheries, and the diet in-
gredients and laboratory equipment were furnished
by the Western Fish Nutrition Laboratory.

LITERATURE CITED

Burrows, R.E., L.A. Robinson, and D.D.
Palmer
1951. Tests of hatchery foods for

blueback salmon 1944-48. U.S.
Dept. of the Int., Fish & Wildl.
Serv., Spec. Sci. Report 59: 2-3.

DeLong, D.C., J.E. Halver, andE.T.Mertz
1957. Protein requirements of chinook

salmon at two water temperatures .
Fed. Proc. 16: 1644.

1947. The nutrition of rainbow trout.

I. Studies of vitamin requirements .
Arch. Biochem. J^ 169-178.

Phillips, A.M., A.V. Tunison, H.B. Shaffer,
G.K.White, M.W.SuUivan, C.
Vincent, D.R. Brockway, and CM.
McCay
1945. The nutrition of trout. Cortland

Hatchery Report 14. N.Y. State
Cons. Dept., 31 pp., illus.

1958.

, , andW.T. Yasutake

A possible cause of "sunburn"
in fish. Prog. Fish -Cult. 20:111-
113.

D.

Gerking, S .

1952. The protein metabolism of sun-

fishes of different ages. Physiol.
Zoology 25: 358-372.

Halver, J.
1957a.

1957b.

Nutrition of salmonoid fishes.

III. Water-soluble vitamin require-
ments of Chinook salmon.

Jour. Nutri. 62: 225-243.

Nutrition of salmonoid fishes.

IV. An amino acid test diet for
Chinook salmon. Jour. Nutri. 62:
245-254.

1947.

, D.R. Brockway, E.O. Rodger s,
H.L. Robertson, H. Goodell, J. A.
Thompson, and H. Willoughby

The nutrition of trout. Cortland
Hatchery Report 16. N.Y. State
Cons. Dept., 35 pp., illus.

, , , M . Bryant,

H. Goodell, C. Walker, P. Frank,
and H . Newman
1949. The nutrition of trout . Cortland

Hatchery Report 17. N.Y. State
Cons. Dept., 31pp., illus.

, , A.J.J. Kolb, J.M.

Maxwell, W. Curry, M. Hoaglund,
C. Morefield, and O. Cox.
1950. The nutrition of trout. Cortland

Hatchery Report 19. N.Y. State
Cons. Dept., 24 pp., illus.

1957.

1957.

1958.

and J. A. Coates

A vitamin test diet for long-term
feeding studies. Prog. Fish-Cult.
19: 112-118.

, D.C. DeLong, and E.T. Mertz
Nutrition of salmonoid fishes .
V. Classification of essential
amino acids for chinook salmon.
Jour. Nutri. 63: 95-105.

Threonine and lysine require-
ments of chinook salmon. Fed.
Proc. 17: 1873.

McLaren, B.A., E. Keller, D.J. O'Donnell,
andC.A. Elvehjem.

Tunison, A.V., D.R. Brockway, J.M. Maxwell,
A.L, Dorr, and CM. McCay.
1942. The nutrition of trout . Cortland

Hatchery Report 11. N.Y. State
Cons. Dept., 52 pp., Illus.

_, H.B. Shaffer, J. M.
CM. McCay, G.E. Palm,

1943.

Wolf, L.
1951.

Maxwell

and D.A. Webster

The nutrition of trout . Cortland
Hatchery Report 12. N.Y. State
Cons. Dept., 26 pp., illus.

Diet exp)eriments with trout .
I. A synthetic formula for dietary
studies. Prog. Fish -Cult. 13: 17-24.

INT.-DUP SEC. . WASH.

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