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A study of the viability of salmon eggs and sperm after varying periods of storage Barrett, Izadore 1949

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(1*1 ft* A S T U D Y O F T H E V I A B I L I T Y O F S A L M O N E G G S A N D S P E R M A F T E R V A R Y I N G P E R I O D S O F S T O R A G E I Z A D O R E B A R R E T T A thesi8 submitted in partial fulfilment of the requirements for the degree of M A S T E R O F A R T S in the Department of Z O O L O G Y T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A April, 1949. ABSTRACT Experiments on the--viability of salmonoid eggs and sperm under conditions o f - - a r t i f i c i a l storage were eonducxed at L l o y d ' s creek, Kelson "creek and Cultus lake", B.C.,- from May to November» 1948. In May and June, preliminary experi-ments on the holding of Salmo g a i r d n e r i i kamloops eggs at Lloyd-1-s creek- indicated the I'easibilty of the storage, espec-i a l l y " at lev; temperatures. In October, further experiments at Nelson creek on Oneorhynchus keta eggs and sperm resulted i n the development of a storage technique which was subsequently employed at Cultus lake "in November. At Cultus lake, "mature 0. keta;eggs were held i n sealed, s t e r i l e 500 ce. Mason j a r s , one t h i r d f u l l of eggs, f o r multiples -of 12 hours up to 192 hours (8 days) at temperatures averaging 3.5°C* The 0> keta sperm were held i n 2 ounce screw-cap j a r s , approximately bee. of m i l t i n each j a r , under conditions si m i l a r to those f o r the eggs. Controls were run i n a l l eases* • The stored eggs and sperm were f e r t i l i z e d with, or used to f e r t i l i z e , f r e s h sperm and eggs. The f e r t i l i z e d eggs were incubated f o r 24 hours. Tfce eggs were then examined under the dissecting microscope for,evidences of cleavage* Two hundred egg l o t s for each time peri, od were examined and the percent i n f e r t i l i t y determined-The re s u l t s showed that 0. keta eggs may be s t o r e d f o r 108 hours, under these conditions, with l e s s than 20$ i n f e r t i l i V . The Q. keta sperm may be held f o r 36 hours w i t h less than 10$ i n f e r t i l i t y . Beyond 36 hours, unde r the conditions of t h i s experiment, the sperm res u l t s were e r r a t i c . The use of these r e s u l t s f o r f i s h c u l t u r a l practices and for the salvage of mature eggs and sperm i s suggested. TABLE OP CONTENTS Page L i s t of I l lustrat ions • i i Acknowledgments i l l Introduction • v Review of Literature 1 Materials and Methods 10 Results 25 Discussion * 28 Summary and Conclusions • 42 Literature Cited 43 Appendix A (Lloyd's creek data) ••• 45 Appendix B (Nelson creek data) 48 Appendix C (Cultus lake data) 52 LIST OP ILLUSTRATIONS Page P l a t e I Pig« 1. Diagram o f salmonoid egg 11 P i g . 2. Diagram o f salmonoid sperm 11 P l a t e I I P i g . 3.. Pour c e l l stage 24 P i g . 4. Germinal d i s c 24 P i g . 5. Graph d e p i c t i n g egg d a t a 26 P i g . 6. "Graph d e p i c t i n g sperm data 27 i i ACKNOWLEDGMENTS The writer wishes to express appreciation to Dr. W.A. Clemens f o r suggesting the problem and for h i s sus-ta i n i n g i n t e r e s t . The writer i s also much indebted to Dr. W.S. Hoar fo r h e l p f u l advice, valuable c r i t i c i s m s and constant encour-agement offered him during the course of the study. Grateful acknowledgments are also made to: B.C. Packers, Ltd., f o r f i n a n c i a l assistance; The B.C. Game Commission, f o r affording hatchery f a c i l i t i e s both at Lloyd's creek and Cultus lake; The Dominion Department of Fi s h e r i e s , f o r permitting the use of spawning chum salmon from Nelson and Sweltzer creeks; The International P a o i f i c Salmon Fisheries Commission for the use of the salmon traps on Sweltzer creek; The w r i t e r s fellow students, whose advice, c r i t i -cisms and ai d contributed m a t e r i a l l y to t h i s study; Mr. T. Step, Superintendent of the salmon traps at Cultus lake, for much valuable assistance i n procuring the chum salmon specimens; i i i Mr. J.O. Lake, Dominion Fisheries Inspector, f o r h i s most welcome a i d with many of the problems at Nelson Creek; and Mr. F. P e l l s , Superintendent of the Lloyd*s creek and Cultus lake hatcheries, f o r h i s constant cooperation and s c i e n t i f i c i n t e r e s t . I t i s also a pleasure to acknowledge the support and encouragement of the writer by his parents. i v A STUDY OP THE VIABILITY OP SALMON EGGS AND SPERM AFTER VARYING PERIODS OF STORAGE INTRODUCTION One of the d i f f i c u l t i e s commonly experienced i n spawn-taking operations i s the shortage of either male or female f i s h at some time during the spawning run. In such cases as these, the storing of eggs and sperm f o r some length of time with a low m o r t a l i t y , rather than holding the f i s h In pens, would appear to be advantageous. This storage would be e s p e c i a l l y valuable i n the case of female salmon where I t has been shown that a delay i n spawning, leading to an over-ripe condition of the eggs, r e s u l t s In an increase i n the number of deaths i n the o f f s p r i n g , i n the number of mal-formed young and i n the number of males produced (Mrsic,1923). Another hatchery d i f f i c u l t y , In some areas of B r i t i s h Columbia, occurs where spawn c o l l e c t i o n s are made at points distant from the hatcheries, and the f e r t i l i z e d eggs have to be transported long distances. In cer t a i n cases t h i s procedure i s r e l a t i v e l y i n e f f i c i e n t , since i t r e s u l t s i n high m o r t a l i t i e s due to the greater f r a g i l i t y of the f e r t i l i z e d v -egg- as compared with the u n f e r t i l i z e d egg (Gray, 1920) and since i t poses such problems as temperature and oxygen control, the prevention of agitation and the physical problem of trans-portation. The p o s s i b i l i t y that these d i f f i c u l t i e s might be lessened by transporting the eggs and sperm i n an appropriate manner to the hatchery, and then completing the f e r t i l i z a t i o n , i s indicated by t h i s study of the v i a b i l i t y of the eggs and sperm. Trout eggs are at present c o l l e c t e d at various spawning streams i n B r i t i s h Columbia, are allowed to develop at l o c a l hatcheries u n t i l "eyed", and are then shipped, with a low mortality, to the central hatchery where they are hatched and reared. Holding the eggs at the l o c a l hatchery u n t i l they are "eyed" involves the maintenance of hatchery crews, f o r an extra six to eight week period. The advantage of transporting the eggs and sperm, as soon as they are taken, to the central hatcheries, rather than holding the eggs u n t i l they are "eyed", i s indicated. There i s a considerable waste of mil t and roe i n connection with the Indian f i s h e r y i n B r i t i s h Columbia where, i n many cases, mature salmon are taken as food. I f t h i s wastage could be halted (or at least reduced) by the salvage of the eggs and sperm, the m i l t and roe thus saved would be of not inconsiderable importance. Also, knowing the v i a b i l i t y , under cert a i n standard conditions, of mature salmon eggs and v i sperm, i t might be possible to work back, on the basis of •this knowledge, to a determination of the v i a b i l i t y of immature eggs and sperm, with further salvage p o s s i b i l i t i e s . Prom the biological point of view, the knowledge of the v i a b i l i t y of livin g cells under a r t i f i c i a l conditions, and their mortality rates, is of value. In this study an attempt has been made to develop a f i e l d technique for holding eggs and sperm of salmon. v l i REVIEW OP LITERATURE The v i a b i l i t y of the eggs and sperm of f i s h e s , p a r t i c u l a r l y salmonoid f i s h e s , has aroused some attention from f i s h c u l t u r i s t s since the middle of the 19th century. Vrasski, a Russian land-owner who performed a r t i f i c i a l f e r t i l -i z a t i o n experiments i n 1854 (Soudakevicz, 1874) and who devised the'Russian 1 or 'dry' method of f e r t i l i z a t i o n , was among the f i r s t to determine the v i a b i l i t y of f i s h sperm i n the absence of water. The report by Soudakevicz (1874) on Vrasski'a experiment states that "on (Acerlna vulgaris C.) m i l t that had not yet reached i t s mature state, he (Vrasski) found that some drops of m i l t kept i n a pe r f e c t l y dry and w e l l -closed cylinder had not l o s t t h e i r o r i g i n a l q u a l i t i e s , even a f t e r s i x days i n the ordinary temperature of a room." Vogt, a Swiss b i o l o g i s t , whose observations i n the 1850»s on the v i a b i l i t y of whitefish sperm are noted by Milner (1874), states that: "at low temperatures, t h i s power (the f e r t i l i z i n g power of the spermatozoa) i s retained for hours and even days i f the m i l t remains i n the organs by which i t i s secreted. In the Lake of Neufchatel (Switzerland) the palee (Coregonus palea), a f i s h of the trout family, i s taken, during the winter months, by night or at sunset. I have often received these f i s h s t i f f frozen and succeeded p e r f e c t l y i n im-pregnating spawn with the m i l t taken from the genitals of the male the day af t e r . " - 1 -- 2 -Atkins (1874), working i n New England on the A t l a n t i c salmon, Salmo s a l a r , attempted the preservation of the eggs and sperm of t h i s species. One l o t of eggs was kept i n the hatchery i n a pan covered over, hut not secluded from a i r , with samples of the stored eggs being f e r t i l i z e d at 0, 12, 30, 48 and 96 hours. The m o r t a l i t i e s were respectively 0, 10, 12^, 25 and 87£#. F e r t i l i z i n g eggs from females that had been dead for two hours, and for f i f t e e n hours, resulted i n m o r t a l i t i e s of 41-| and 100$. With m i l t kept i n an open dish f o r ten minutes, "several hours", and four days, and then used to f e r t i l i z e f r e s h l y spawned eggs, Atkins found respective m o r t a l i t i e s of 7-|, 0, and 100$. From his experimental work on the sperm of an un-named species of French trout, Henneguy (1877) made the following observations: 'Du sperms est conserve pendant quatre jours (du 7 au 11 novembre) dans un flacon bouche, a une temperature de 10 a" 15 degres, l e s spermatozoides au contact de l*eau pure, sal6e ou a l c a l i n l s e e , ont des mouvements aussi v i f s et aussi prolonges qu'a l ' e t a t f r a l s . Sur quarante oeufs feconde's avec ce sperme, hult seulement ne se sont pas developpes. Des oeufs fecondes avec du sperme datant de deux jours, t r a i t e s par l'eau faiblement a l c a l i n l s e e par l a potasse, se sont tous bien developpes dans l e mdme temps qui des oeufs fecondes normalement.1 Rutter (1902a) working i n C a l i f o r n i a on the quinnat salmon, Oncorhynchus tshawytscha, attempted the preservation, tinder varying conditions, of the eggs and sperm of this species. Storing milt from several f i sh for 24 and 48 hours in open, large-mouthed jars , Rutter found mortal i t ies of 36 and 100$ in the f e r t i l i z e d eggs. Bggs f e r t i l i z e d with sperm that had been stored for 24 hours i n a sealed v i a l showed a mortality of 100$. With ova exposed to a i r but in ovarian f l u i d , for 12, 30 and 50 minutes at 2 5 ° C , and then f e r t i l i z e d normally, mortali t ies of 0, 1, and 34/6 respective-l y were noted. In a subsequent experiment with the eggs l e f t in the bodies of two dead females for varying periods of time, Rutter found less than 8% mortality after 6 hours storage, and 100$ mortality after 24 hours storage. He concludes that eggs stored in this manner for less than 5 hours may safely be used for f i sh cul tural purposes. Several experiments are reported by Calderwood (1910) on the transportation of mil t from one area to another for f i sh cul tural purposes. Male salmon (presumably S. salar) were stripped and the mi l t placed in sealed bot t les . These bottles were then packed in moss. The mil t was in transit for 21 hours and was then used to f e r t i l i z e normal eggs. Of the 22,000 eggs mixed with this transported m i l t , 10,510 (48$) were unfer t i l i zed . In another t r i a l , the milt was in t ransi t 40 hours and, of the 700 eggs used, 411 died in 7 days. For reasons not stated, this experiment was considered to be a - 4 -f a i l u r e . A t h i r d t r i a l involved the transportation of the m i l t fo r 63|- hours, with only 2000 of 4000 eggs being f e r t i l i z e d . Later, a l l the eggs died. These r e s u l t s are of value only as an ind i c a t i o n of the p o s s i b i l i t i e s of t h i s method, since i n no case i n these experiments were any controls run, were the temperatures recorded or were the techniques s i m i l a r i n a l l d e t a i l s . Gray (1919) states that •milt obtained from a ripe male uncontaminated by excreta or water, retains i t s power of f e r t i l i z i n g eggs for at least 24 hours. 1 but does not state the conditions of storage or the percent mortality of the eggs f e r t i l i z e d with t h i s stored sperm. Hakano and Nozawa (1925), i n preservation experi-ments on the eggs and sperm of 0. masou, express t h e i r observ-ations i n terms of the " v i t a l i t y " of the eggs or sperm. Thus, eggs stored i n a s t e r i l i z e d vessel at a temperature range of 5.3 to 13.0"C.= "maintained v i t a l i t y " f o r 97 hours. Sperm, s i m i l a r l y stored, "maintained v i t a l i t y " for 97 hours. Eggs and sperm stored i n the dead parents and then f e r t i l i z e d , "maintained t h e i r v i t a l i t y " f o r f i v e hours, with the eggs having a greater " f e r t i l i z i n g power" than the sperm. E l l i s and Jones (1939), i n experiments on the a c t i v i t y of the sperm of S. s a l a r , observed that sperm stored i n ovarian f l u i d at s l i g h t l y above 0*0. remained a l i v e up to - 5 -7 days. No f e r t i l i z a t i o n tests were made, the only c r i t e r ion of v i a b i l i t y being the ac t iv i ty of the sperm in water, when examined under the microscope. Working in the State of Washington on both the steelhead (Salmo galrdneri l galrdneri i) and the spring salmon (Oneorhynchus tshawytscha), Smith and Quistorff (1943) preser-ved only the milt from each species, using the subsequent hatching percentages as a basis of comparison. The mortal i t ies of the eggs which had been f e r t i l i z e d with steelhead sperm that had been stored in f u l l , sealed v ia l s for 0, 2%, and 5 hours at 6,7 to 7.0°C. were, respectively, 4.2, 96.8 and 99$. The mortali t ies of eggs f e r t i l i z e d with spring salmon sperm which had been s imi lar ly stored were 0, 9.5, and 45.9$. Spring salmon sperm stored in dead males for 0, 2-§-, and 5 hours at 6.7 to 7.0"C. produced mortali t ies of 6.1, 5.5, and 7.1$ when used to f e r t i l i z e freshly stripped eggs. Kleerekoper (1946) used for his studies on the v i a b i l i t y of f i sh mil t and roe, the Braz i l i an Atherinid, Odonthestes b ras i l l ens l s . This i s a f i sh which dies immediate-l y on capture and which, as a resul t , cannot be retained al ive for future use in case of a shortage of male or female speci-mens during spawn-taking operations. He was able to store the sperm in Salisbury's yolk-ci t ra te mixture in proportions of 1:3 and 1:4 at le^C. for 24 and 48 hours with mortal i t ies of 40 and 60$, respectively. The eggs,which were le f t in the ovary and suspended i n a thermos b o t t l e at room temperature, showed a m o r t a l i t y of 40% a f t e r 24 hours s t o r a g e . Kleerekoper (1949) a l s o s t a t e s t h a t he has found an optimum p r e s e r v a t i o n temperature of 5-6"C. f o r the eggs and sperm o f the s p e c i e s used. No new e f f e c t s o f any importance were found w i t h the use o f v a r i o u s pH's and b u f f e r m i x t u r e s . The r e s u l t s of egg and sperm storage by v a r i o u s workers are presented t a b u l a r l y i n Tables I (egg storage) and I I (sperm s t o r a g e ) . TAB IE I RESULTS CF EGG STORAGE BY VARIOUS • WORKERS TIME PERCENT HELD WORKER SPECIES CONDITIONS TEMPERATURE MORTALITY 0 hours Atkins (1874) Salmo salar clotlxjovered pan room 0 12 minuted Rutter (1902a) 0. tshawytscha in coelomic fluid exposed to a i r 25°C 0 30 minutes Rutter (1902a) 0, tshawytscha in ooelomic fluid exposed to air 25°C 1 50 minutes Rutter (1902a) 0. tshawytscha in coelomic fluid exposed to air 25°C 34 2 hours Atkins (1874) Salmo salar in dead female not given 41.5 5 hours Nakano and Nozawa (1925) 0. jHasou in dead female not given "maintained v i t a l i t y " 6 hours Rutter (1902a) 0, tshawytscha in dead female • not given 8 12 hours Atkins (1874) S. salar cloth-covered pan room 10 15 hours Atkins (1874) S. salar in dead female not given ' 100 24 hours Rutter (1902a) 0, tshawytscha in dead female not given 100 ELeerekoper (1946) Odonthestes brasiliensis ovary suspended in thermos room 40 30 hours Atkins (1874) S, salar cloth-covered pan room 12.5 48 hours Atkins (1874) S. salar cloth-oovered pan room 25 Nakano & Nozawa (1925) 0. mas-ou ^"sterilized vessel" 5.3 - 13.0°C "maintained v i t a l i t y " 96 hours Atkins (1874) S, salar cloth-covered pan room 87.5 I -3 TffBLE II . RESULTS OF SPERM STORAGE BY VARIOUS WORKERS TIME HELD WORKER SPECIES CONDITIONS TEMPERATURE PERCENT MORTALITY 0 hours Smith and Ouistorff (1943) S. gairdnerii in f u l l , sealed vials 6.7 - 7.0°C 4.2 ti n 0. tshawytscha in f u l l , sealed vials 6.7 - 7.0°C 0 tt n 0. tshawytscha in dead males not given 6.1 10 minutes Atkins (1874) S. salar in open dish ro am 7.5 2.5 hours Smith and &uistorff (19*3) s. gairdnerii in f u l l , sealed vials 6.7 - 7.0°C 96.8 « « 0. tshawytscha in f u l l , sealed vials 6.7 - 7.0°C 9.5 tt it 0. tshawytscha in dead males not given 5.5 5 hours Smith and Ctuistorff (1943) s. gairdnerii in f u l l , sealed vials 6.7 - 7.0°C 99 , ti tt 0. tshawytscha in f u l l , sealed vials 6.7 - 7.0°C 45.9 CD II » o. tshawytscha in dead males not given 7.1 • Nakano and Nozawa (1925) o. masou in Sead males not given "maintained v i t a l i t y " "several hours" Atkins (1874) s. salar in open dish room 0 24 hours Vogt (Milner, 1874) Coregonus palea in dead males o;°c "perfect impregnation" Rutter (1902a) 0. tshawytscha in sealed vials room 100 Rutter (1902a) 0. tshawytsoha in open jars room 36 Gray (1919) S t salar not given not given "retains fe r t i l i z i n g power" ELeerekoper (1946) Odonthestes brasiliensis in Salisbury's yolk-citrate, 1:3 and 1:4 16°C 40 48 hours Henneguy (1877) Salmo sp. in stoppered bottle 10 - 15°C 0 Rutter (1902a) 0. tshawytscha in open jar room 100 ELeerekoper (1946) Odonthestes brasiliensis in Salisbury's yolk-citrate, 1:3 and 1:4 16°C 60 TABLE II RESULTS OF S PERM STORAGE BY VARIOUS WORKERS (CONTINUED) TIME HELD 96 hours 97 hours 144 hours 168 hours WORKER Henneguy (1877) Atkins (1874) Nakano and Nozawa (1925) E l l i s and JQnes (1939) SPECIES Salmo sp«  Salmo salar 0. masou Vrasski (Soudakevioz, .1874) Aoerina vulgaris S . salar CONDITIONS TEMPERATURE PERCENT MORTALITY in stopped bottle 10 - 15°C 20 in open dish room 100 in steriie v i a l , kept dark 5,3 — 13*C "maintained v i t a l i t y " tightly corked v i a l not stated "perserves its fer t i l i z i n g power" in coelomic fluid 0 °C "remained alive" I I MATERIALS AND METHODS The b a s i c p l a n f o l l o w e d i n t h i s s e r i e s of e x p e r i -ments was the s t r i p p i n g of mature roe and m i l t i n t o s t e r i l e j a r s , s t o r i n g these j a r s at a low temperature, and then f e r t i -l i z i n g t h i s s t o r e d spawn i n order to determine the r e s u l t a n t m o r t a l i t y . C o n t r o l s were run i n a l l c a s e s . The s p e c i e s of salmonoids employed were Oncorhynchus  k e t a (the chum salmon) and Salmo g a i r d n e r i i kamloops ( the Kamloops t r o u t ) . These s p e c i e s were used because of t h e i r a v a i l a b i l i t y , because of t h e i r r e p r e s e n t a t i o n of two c l o s e l y r e l a t e d genera i n the one f a m i l y , and because of the conven-i e n t d i f f e r e n c e i n t h e i r spawning times. The egg o f the salmonoid f i s h i s diagrammed i n f i g u r e 1. The eggs are produced i n p a i r e d o v a r i e s i n the salmon and, when mature, break away from the ovary i n t o the abdominal c a v i t y . There are o n l y v e s t i g i a l o v i d u c t s i n the salmon, so t h a t the eggs are extruded i n t o the water d i r e c t l y from the abdominal c a v i t y through the g e n i t a l p a p i l l a . The egg, when extruded, v a r i e s i n s i z e from l e s s than 3 mm. i n diameter (the t r o u t ) t o approximately 7 mm. i n diameter (the chum salmon). They are s o f t , f l a c c i d b o d i e s when extruded. Immediately on c o n t a c t w i t h water, the eggs b e g i n to absorb i t and from 15 to 60 minutes l a t e r (depending on the temperat-ure) the c h o r i o n i s t u r g i d and h i g h l y e l a s t i c . The c h o r i o n - 10 -PLATE I t e r m i n a l d i s c F i g u r e I. 'Diagram o f Salmonoid Egg, x 45. (From Gray, 1920) F i g u r e 2. Diagram o f Salmonoid Sperm, x 900. (From R u t t e r , 1902a) - 12 -of the egg i s p e r f o r a t e d by t i n y h o l e s , approximately 1 micron i n diameter. The micr o p y l e appears to be a funnel-shaped opening through which the sperm gains entrance t o the egg. During l i f e , the eggs are almost completely t r a n s p a r e n t and of a salmon p i n k c o l o u r , w h i l e dead eggs r a p i d l y become opaque and m i l k white i n c o l o u r . Sperm are diagrammed i n f i g u r e 2. When mature, the sperm, known as m i l t , are r e l e a s e d Into the water through the g e n i t a l p a p i l l a i n a milky-white stream. The f l u i d v a r i e s i n c o n s i s t e n c y from a watery m i l k i n e s s t o the t h i c k n e s s o f cream. The c o l o u r i s dead white, w i t h sometimes a shade o f ye l l o w * Peart (1920) has estimated the number of sperm c e l l s i n a m i l l i g r a m o f t r o u t m i l t t o be 48,500,000, the m i l t i t s e l f having an average d e n s i t y o f 1.021. The sperm are m o t i l e o n l y a very s h o r t time (up to one minute) i n f r e s h w a t e r , ( L i n d r o t h , 1947). The experimental storage of the eggs and sperm was c a r r i e d out a t three p l a c e s i n B r i t i s h Columbia. The p r e l i m -i n a r y i n v e s t i g a t i o n s i n t o the f e a s i b i l i t y o f the problem were made at L l o y d ' s creek Hatchery, Paul l a k e , B.C.; f u r t h e r In-v e s t i g a t i o n s were made a t Nelson creek In West Vancouver; and the f i n a l work was done at the P r o v i n c i a l T r out Hatchery a t Cu l t u s l a k e , B.C. Lloyd'8 creek The p r e l i m i n a r y work on the Kamloops t r o u t was done - 13 -at the Provincial trout hatchery at Lloyd's creek, B . C . , from June 9 to June 30, 1948. Only the eggs of the Kamloops trout were used experimentally, since the males were in short supply. The general procedure involved was the taking of;the f i sh from the traps, the removal of eggs by the incis ion method, and the f e r t i l i z a t i o n of the eggs after various periods of storage. After 10 days of incubation in the hatch-ing troughs, both the l i v e and dead eggs (on the basis of opacity) were counted, the percentage of dead eggs being an taken to constitute the percent f e r t i l i z e d . Nelson creek The next experiments were performed from October 15 to November 17, 1948, at Nelson creek, a small stream which empties Into Burrard Inlet on i t s north shore, near Eagle Harbour. This stream normally receives a large run of spawning chum salmon and i s only about ten miles from the university. The spawning f i sh were taken from the stream with a large dip net and held in two storing pens, one for each sex, u n t i l they were required. However, great d i f f i c u l t y was encountered i n capturing f i sh that had not already spawned. Evidently, the salmon were fu l ly mature when they entered the creek and spawned almost immediately. No immature f i sh were taken during the 5 week period. Of approximately 60 female f i sh netted, only 15 s t i l l retained their eggs. The situation was different with the males since there usually was, for - 14 -the purposes of the experiment, enough m i l t l e f t i n the male, even a f t e r he had spawned. However s u f f i c i e n t f i s h were c a p t u r -ed so t h a t 16 samples of eggs and 10 samples o f m i l t were s t o r e d . A t y p i c a l experiment ran as f o l l o w s . The mature female whose eggs were to be used was taken from the pen and stunned w i t h a blow on the head. The body was then d r i e d w i t h t o w e l l i n g , and the f i s h b l e d by c u t t i n g the d o r s a l a o r t a behind the head. The c l o a c a l aperture was extended to approx-i m a t e l y 3 cms. and the eggs were allowed to flow g e n t l y i n t o a p e r f e c t l y d r y , l a r g e s t a c k i n g d i s h , open to the a i r . Approx-i m a t e l y 85% of these eggs were poured i n t o two dry, s t e r i l e , b l a c k - p a i n t e d 500 c c . Mason j a r s , each j a r b e i n g about one-t h i r d f u l l and c o n t a i n i n g approximately 800 eggs. These j a r s were then t i g h t l y s e a l e d , l a b e l l e d , and p l a c e d i n the icebox.. The time of storage was noted. The remaining 15$ o f the eggs was d i v i d e d i n t o two l o t s and p l a c e d i n two d r y s t a c k i n g d i s h e s . A male was then taken from the h o l d i n g pen, stunned and d r i e d but not b l e d . The m i l t was s t r i p p e d , by the u s u a l method, i n t o two dry, s t e r i l e , b l a c k - p a i n t e d 2 ounce j a r s , and i n t o one of the two s t a c k i n g d i s h e s of eggs. The two s m a l l j a r s were s e a l e d , l a b e l l e d and s t o r e d i n the icebox. In some cases the m i l t was not s t r i p p e d i n t o the two j a r s t o be s t o r e d , but was merely used to f e r t i l i z e the c o n t r o l l o t of eggs. Water was then added to b o t h l o t s of eggs i n the s t a c k i n g - 15 -d i s h e s . T h i s , of course, completed the f e r t i l i z a t i o n p r o c e s s i n the d i s h to which the m i l t had been added. The two l o t s o f eggs were then p l a c e d i n l a b e l l e d baskets i n a h a t c h i n g trough which had been b u i l t i n the stream. The eggs were allowed to incubate f o r 24 hours and then taken from the basket and p r e s e r v e d i n Davidson's f l u i d . The f e r t i l i z e d eggs served as the c o n t r o l s (Tables IV & V) and the u n f e r t i l i z e d eggs served as a p o i n t of r e f e r e n c e f o r any u n f e r t i l i z e d eggs i n the experimental l o t s . A f t e r the a p p r o p r i a t e storage time, the experimental eggs and sperm were then used to f e r t i l i z e or were f e r t i l i z e d w i t h f r e s h spawn, Incubated i n the trough f o r 24 hours and p r e s e r v e d i n Davidson's f l u i d . No c o n t r o l s were run f o r i the f r e s h spawn. The icebox temperatures are g i v e n i n Table V i , while the data f o r the experimental eggs and sperm are g i v e n i n T a bles IV & V. , Davidson's f l u i d Is prepared as f o l l o w s : Formalin 20% G l y c e r i n e , 10$ A l c o h o l (95$) 30% G l a c i a l a c e t i c a c i d 10$ D i s t i l l e d water 30% The eggs remain In t h i s s o l u t i o n f o r 24 hours and are then t r a n s f e r r e d t o the same s o l u t i o n minus the a c e t i c a c i d . - 16 -C u l t u s l a k e Experiments were run at C u l t u s l a k e , B.C., from November 19 to November 28, 1948." Here, a g a i n , the chum salmon was used as a source o f eggs and sperm. The f i s h were " r e a d i l y o b t a i n e d from the salmon t r a p s l o c a t e d on Sweltzer creek, the o u t l e t stream of C u l t u s l a k e . The t r a p s are operated by the I n t e r n a t i o n a l P a c i f i c Salmon F i s h e r i e s Commission and to the Commission and i t s r e p r e s e n t a t i v e a t C u l t u s l a k e , Mr. T. Step, the w r i t e r g i v e s thanks f o r the c o u r t e s i e s and a i d extended. When dipped from the t r a p s , the f i s h were s o r t e d as to mature males and females, and each c l a s s was p l a c e d i n a separate h o l d i n g pen. The f i s h were dipped from the t r a p s every morning and every a f t e r n o o n and, s i n c e there was an adequate spawning run, no specimens were h e l d i n the pens l o n g e r than 24 hours. T h i s minimized the p o s s i b l e e f f e c t s of the o v e r - r i p e n e s s o f the g e n i t a l p r o d u c t s . Pour iceboxes of the o r d i n a r y household type were used i n which to s t o r e the j a r s of eggs and sperm. They were i n s u l a t e d wooden boxes measuring on the i n s i d e 63 cms. i n l e n g t h , 43 cms. i n width and 40 cms. i n depth. The l i d s were hinged i n the middle so t h a t the contents o f h a l f the box c o u l d be i n s p e c t e d a t one time. The i c e was p l a c e d i n one h a l f of the box and the j a r s c o u l d be s t o r e d i n the o t h e r h a l f . A s h e l f d i v i d e d the storage p o r t i o n so t h a t two l a y e r s o f j a r s c o u l d be s t o r e d ; one l a y e r on the f l o o r on a r a c k and the o t h e r 17 -l a y e r on the s h e l f r a c k . Ice was added to the boxes from time t o time, to m a i n t a i n the low temperature. Temperatures, taken d a i l y for- each o f the f o u r boxes, are r e c o r d e d i n Table V I I . They ranged from 2.0 C. t o 5.8" C , the average b e i n g 3.5 0.8°C. The f o u r iceboxes themselves were kept i n a s m a l l b u i l d i n g a t the h a t c h e r y . T h i s b u i l d i n g was about 60 yards from the t r a p s . The hatchery troughs used were i n the main h a t c h e r y b u i l d i n g , which was perhaps 60 yards from the b u i l d i n g i n which the Iceboxes were kept and about 120 yards from the t r a p s . The troughs themselves were standard s i z e d wooden hatch-er y troughs 22 cms. deep, 27 cms. wide and ran the l e n g t h o f the b u i l d i n g . The baskets used were made o f wire mesh and were 4 cms. deep, 31 cms. wide and 39 cms. l o n g . The water temperature i n the troughs v a r i e d from 3.5 aC. to 7 . 7 ° C , the average b e i n g 7.1 " C , from November 19 to November 27. The eggs and sperm were s t o r e d f o r m u l t i p l e s of 12 hours up t o 192 hours (8 days) i n j a r s i n the iceboxes, and were then employed as p r e v i o u s l y d e s c r i b e d i n the a r t i f i c i a l f e r t i l i z a t i o n p r o c e s s . Four f i s h , two males and two females, were used f o r each time p e r i o d , the eggs or sperm from each f i s h b e i n g d i v i d e d Into two storage l o t s , making a t o t a l o f e i g h t samples f o r each time p e r i o d . The eggs were h e l d i n d r y , s t e r i l e , wide mouth 500 c c . Mason j a r s , which c o u l d be t i g h t l y s e a l e d ; the sperm, i n d r y , s t e r i l e , 2 ounce, 30 c c . screw-cap -\18 -jars . The exact procedure was as follows. The desired female salmon was dipped from the holding pen and examined for maturity. I f eggs flowed freely from the genital pap i l l a when pressure was exerted on the abdomen, the female was considered to be fu l ly mature. At the traps, the f i sh was stunned with a blow on the head, the body dried with towelling, and the dorsal aorta cut just behind the head so that the f i sh bled freely. The f i sh was then suspended by the head from a hook, so that the genital opening was approximately 120 cms. above the ground. The cloacal aperture was then extended to 3 cms. by a scissor cut, so that the eggs flowed freely from the abdominal cavity into the storage ja r . This, the incis ion method, was used rather than the expression method of taking the eggs so that the pos s ib i l i t i e s of obtain-ing broken or immature eggs were reduced. The f i r s t few eggs were allowed to drop to the ground in case there was any contamination by excreta or blood. This la t ter poss ib i l i ty , was, however, mostly negated by bleeding the animal. The eggs remaining in the body cavity were then collected in a s t e r i l e 500 cc. ja r . Enough eggs were usually collected from one female to f i l l the jar (approximately 2000 eggs). Care was taken that no water got into the jars . With the eggs came some coelomic f l u i d , so that the eggs were quite moist at a l l times. The mouth of the jar , except for the space allowed - 19 -f o r t he e n t r a n c e o f t h e e g g s , was k e p t c o v e r e d b y t h e l i d . As soon as t h e sample was t a k e n , two l o t s o f a p p r o x -i m a t e l y 200 eggs e a c h were p o u r e d i n t o two l a r g e s t a c k i n g d i s h e s . The sample j a r was t h e n t i g h t l y c l o s e d . To one o f t he s m a l l samples was added m i l t f rom a male w h i c h had been t a k e n f rom the pen and s t u n n e d b y a b l o w on t h e h e a d . The m i l t r e m a i n i n g i n t h i s male was s t r i p p e d i n t o two s t e r i l e , 2 ounce j a r s f o r s t o r a g e , (about 5 c c . i n e a c h j a r ) and t h e l i d s were s c r ewed t i g h t . F o l l o w i n g t h i s , t h e two s t a c k i n g d i s h e s o f e g g s , t h e s e a l e r o f eggs and t h e two s m a l l j a r s o f sperm were c a r r i e d f rom t h e t r a p s t o the b u i l d i n g where t h e i c e b o x e s were s t o r e d . H e r e , h a l f o f t he eggs i n t h e 500 c c . j a r were p o u r e d g e n t l y I n t o a second s t e r i l e 500 c c . j a r so t h a t e a c h j a r c o n t a i n e d about 800 eggs and was a p p r o x i m a t e l y o n e - t h i r d f u l l . The two j a r s o f eggs were t h e n s e a l e d t i g h t l y , l a b e l l e d and p l a c e d i n the I c e b o x . The two j a r s o f m i l t were s i m i l a r l y s t o r e d . The two s t a c k i n g d i s h e s c o n t a i n i n g the eggs were t h e n t a k e n t o the m a i n h a t c h e r y b u i l d i n g where w a t e r was added t o b o t h d i s h e s j t h a t i s , t o t h e eggs p l u s t h e . m i l t , and t o the eggs a l o n e . The d i s h e s were t h e n a l l o w e d t o s t a n d f o r one m i n u t e , i n o r d e r t o c o m p l e t e t h e f e r t i l i z a t i o n p r o c e s s i n one d i s h . A f t e r t h i s t he two l o t s o f eggs were e a c h t r a n s f e r -r e d t o t a g g e d h a t c h e r y t r a y s , and a l l o w e d t o r e m a i n I n t h e t r o u g h f o r 24 h o u r s . A f t e r 24 h o u r s i n c u b a t i o n , t he eggs were removed - 20 -and stored i n Davidson's f l u i d . The f e r t i l i z e d l o t of eggs was used as a control on both the experimental eggs and sperm and the u n f e r t i l i z e d l o t was used for comparison. I t was r e a l i s e d that, should any great mortality occur i n the con t r o l , either the eggs or sperm for that p a r t i c u l a r time period could be at f a u l t , so that both the egg and sperm experimental r e s u l t s would have to be discarded. However, while the run was comparatively good, the f i s h were not so p l e n t i f u l that d i f f e r e n t males and females could be used as controls. As may be seen from Table V I I I , the maximum mortality f o r the control l o t s i n any case was 8%, with the average mortality being 1.5 ± 2.6%. To determine the f e r t i l i z i n g power of the stored sperm, a mature female salmon was stripped of her eggs by the i n c i s i o n method and the eggs divided into f i v e l o t s , one i n each of f i v e dry, l a b e l l e d stacking dishes. Into each of four of these f i v e stacking dishes of eggs was added one of the four samples of sperm stored for the p a r t i c u l a r time period under inve s t i g a t i o n . The four dishes of normal eggs plus experimental m i l t were carried to the hatchery b u i l d i n g from the icebox b u i l d i n g , and there the f e r t i l i z a t i o n was completed by the addition of .water. The eggs were transferred to l a b e l l e d baskets i n the troughs, allowed to incubate for 24 hours, and then removed to Davidson's f l u i d . The four stacking dishes were then washed and - 21 -thoroughly d r i e d , i n order that they might be used f o r the f o l l o w i n g procedure. Into each of the four s t a c k i n g dishes was poured one of the four egg samples stored f o r t h a t par-t i c u l a r p e r i o d . M i l t from a f r e s h l y taken male, brought from the traps to the storage b u i l d i n g , was added to the f o u r ex-perimental egg samples and to the s t a c k i n g d i s h of eggs r e -maining from the eggs used to check the s u r v i v a l of the stored sperm. This l a s t sample served as a c o n t r o l on the f r e s h eggs and m i l t which were used to determine the s u r v i v a l of the experimental spawn. These c o n t r o l data are given i n Table IX, the average percent i n f e r t i l i t y being 1.1 1 1*8%, The f i v e l o t s of eggs were then t r a n s f e r r e d to the hatchery, f e r t i l i z e d , incubated f o r 24 hours and then removed to Davidson's f l u i d f o r l a t e r examination. I t should be noted t h a t the stored m i l t tended to s e t t l e out. This phenomenon, which produced a t h i n , watery-o v e r l a y on a t h i c k , white suspension, was n o t i c e d i n most of the samples. The f e r t i l i z a t i o n procedure i n these cases was as f o l l o w s . The watery f l u i d was poured over the eggs to be f e r t i l i z e d , and the eggs were placed d i r e c t l y i n t o the j a r which s t i l l had the t h i c k m i l t suspension In i t . These eggs were s w i r l e d around i n the storage j a r and then were poured out, b r i n g i n g the t h i c k suspension w i t h them. This method e f f e c t i v e l y cleaned the storage j a r of a l l the m i l t . The m i l t was then mixed w i t h the eggs i n the s t a c k i n g d i shes - 22 by a gentle rotary motion of the fingers and the dish trans-ferred to the hatchery b u i l d i n g , where the usual f e r t i l i z i n g procedure was followed. Thirty-two males and 32 females were used as the source of the experimental eggs and sperm, and 128 samples i n a l l were held i n the iceboxes, over a period of 192 hours. To check the f e r t i l i t y of the stored samples, another male and female salmon were employed f o r each time period, so that i n a l l , 96 f i s h were taken. Although death i s evidenced i n salmonoid eggs by an opacity of the egg due to a p r e c i p i t a t i o n of the globulins (Gray, 1919), t h i s opacity does not give any i n d i c a t i o n as to whether the egg was f e r t i l i z e d or not. Conversely, some eggs may continue to be transparent and have a l l the appearances of normally f e r t i l i z e d eggs f o r some period of time, although these eggs have not been f e r t i l i z e d . Thus, obviously, the count of opaque and transparent eggs w i l l not give an even reasonably accurate i n d i c a t i o n of the f e r t i l i t y of the sample. For t h i s reason, each egg was i n d i v i d u a l l y examined. Whether the eggs were u n f e r t i l i z e d or not was deter-mined by inspection, under the dis s e c t i n g microscope, of the germinal d i s c , which had been exposed by the removal of the chorion. I f the egg had been f e r t i l i z e d , the 1st to 4th cleavages (depending on the temperature of the water and the length of Incubation) were p l a i n l y v i s i b l e on the germinal - 23 d isc , ( f i g , 3) . I f f e r t i l i z a t i o n had not taken place, there were, of course, no evidences of cleavage on the germinal d isc , which then appeared as a white, unmarked button, 1 mm, diameter in the chum egg ( f i g , 4), The number of unfer t i l ized eggs present in a 200 egg lo t of each experimental sample was determined. For the control samples, only 50 egg lots in each sample were counted. In some cases, s l igh t ly less than 200 eggs in the experimental samples were examined, this being due to the smaller number of the eggs preserved in Davidson's f l u i d . Some 35,000 eggs were individual ly examined during the course of the experi-ment • The data concerning the Cultus lake experimental samples are given in Tables X and X I , PLATE I I F i g u r e 4. Diagram of Germinal D i s c o n . U n f e r t i l i z e d Egg, x 55. RESULTS P r e l i m i n a r y Experiments (Kamloops T r o u t ) The experiments at L l o y d ' s creek i n d i c a t e d t h a t the storage of eggs and sperm was f e a s i b l e and t h a t t h i s storage was b e s t done at low temperatures. The data are p r e s e n t e d i n Appendix A. , ., Nelson creek Experiments (Chum Salmon) The data for. the s t o r e d eggs and sperm, and the icebox temperatures are t a b u l a t e d i n Appendix B. While the Nelson creek data are not r e p r e s e n t e d i n the c a l c u l a t i o n s o f the t r e n d l i n e s i n f i g s . 5 and 6, they are i n d i c a t e d on the graphs f o r the purposes of comparison. C u l t u s l a k e Experiments (Chum Salmon) The data f o r the s t o r e d eggs and sperm, and the i c e b o x temperatures are t a b u l a t e d i n Appendix C. P i g . 5 r e p r e s e n t s the data f o r the s t o r e d eggsj f i g . 6, the s t o r e d sperm. In f i g s . 5 and 6, the percentages of u n f e r t i l i z e d eggs have been p l o t t e d a g a i n s t the times the samples were s t o r e d . Each p o i n t r e p r e s e n t s one sample a t t h a t p a r t i c u l a r time p e r i o d . The numbers adjacent to some of t h e ^ p o i n t s i n d i c -ate the number of times t h a t p a r t i c u l a r percentage i s r e p r e -sented on the graphs a t t h a t time p e r i o d . A t r e n d l i n e f o r each graph was p l o t t e d by a moving average o f t h r e e s . - 25 -v-Figure 5. E f f e c t o f Storage on the F e r t i l i z a b i l i t y o f Mature 0, k e t a Eggs Legend,: Percentages f o r eggs s t o r e d a t C u l t u s l a k e , . . . X Percentages f o r eggs s t o r e d a t Nelson creek .. N Moving averages f o r C u l t u s l a k e d a t a . . . . . . . . . . 6 100 - X X X X X i { x 3 * V 90 N N X / X Q 80 fsl • X Ul H X PERCENT UNF o o X X X X X f X N 4Q X X X X N N 30 X 2 X X 20 N N X X X X N 10 0 X 0 i _ J X 3 i # * X • • x2 1 _JL X —1 1— I I 1 X i i 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 HOURS STORED .Figure 6# Effect of Storage on the ^Fert i l iz ing A b i l i t y of 0. keta Sperm Legend: Percentages for sperm stored at Cultus lake X Percentages for sperm stored at Kelson creek N Moving averages for Cultus lake data 0 DISCUSSION When the egg i s detached from the ovary, i t b e g i n s t o l e a d an e x i s t e n c e o f i t s own. F o l l o w i n g i t s detachment, the egg i s i n a f a i r l y q u i e s c e n t s t a t e , ready to develop but r e q u i r i n g some a d d i t i o n a l impulse to i n i t i a t e the p r o c e s s . T h i s impulse i s n o r m a l l y s u p p l i e d by f e r t i l i z a t i o n . A c c o r d i n g to Weiss (1939) the egg can p e r s i s t i n the u n f e r t i l i z e d s t a t e d u r i n g which c e r t a i n m e tabolic f u n c t i o n s (e.g. r e s p i r a t i o n ) are c a r r i e d on, f o r a c e r t a i n l e n g t h o f time, but not i n d e f -i n i t e l y . T h i s l a s t statement i s e s p e c i a l l y t r u e i n t h e case of the salmonoid f i s h e s where, i n many cases, the female d e p o s i t s eggs over a p e r i o d o f s e v e r a l days and must, as i t were, s t o r e them w i t h i n h e r s e l f f o r v a r y i n g l e n g t h s o f time.. In t h i s s e r i e s o f experiments, the eggs have been s t o r e d i n a manner comparable to a degree w i t h t h a t found i n n a t u r e . * In the n a t u r a l h o l d i n g o f the eggs i n the abdominal c a v i t y , i t must be repeated, the eggs l e a d an e x i s t e n c e sep-a r a t e from t h a t of the female. However, the parent does supply c e r t a i n r e q u i s i t e s t o the egg, without which the chances o f s u r v i v a l of the egg would be g r e a t l y reduced. F i r s t l y , the n a t u r a l storage a f f o r d s p r o t e c t i o n from harmful i n f l u e n c e s . The chances o f mechanical i n j u r y t o the egg p r i o r t o e x t r n s i o n are at a minimum and the p o s s i b i l i t i e s o f harmful b a c t e r i a l - 28 -29 -i n f e c t i o n of the egg are r e l a t i v e l y few. Secondly, storage w i t h i n the body o f the parent probably i n s u r e s the n e c e s s a r y supply of oxygen f o r the eggs. The oxygen, i n t h i s case,, comes not from a d i r e c t b l o o d supply to the egg, but by d i f -f u s i o n through the coelomic f l u i d . A t h i r d p o i n t i s t h a t the f o o d supply o f the egg w i t h i n the abdominal c a v i t y i s not s u p p l i e d by the p a r e n t . As a matter of f a c t , i f the eggs are h e l d s u f f i c i e n t l y l o n g w i t h i n the body c a v i t y , they b e g i n t o form a f o o d supply f o r the parent ( M r s i c , 1923). L a s t l y , storage w i t h i n the body of the parent p r o v i d e s a proper p h y s i -c a l and chemical environment f o r the q u i e s c e n t egg. The eggs are bathed i n an i s o t o n i c coelomic f l u i d which i s produced w i t h i n the body c a v i t y of the female at the time of spawning (Greene, 1904). There are b u f f e r systems p r e s e n t , and mechan-isms f o r the removal of wastes, so t h a t a pH proper f o r the eggs i s maintained. The temperature o f n a t u r a l storage i s , of course, r e g u l a t e d by the temperature of the p a r e n t ' s environment. The above are some of the c o n d i t i o n s o f n a t u r a l s t o r a g e . How do the c o n d i t i o n s of experimental storage compare w i t h them? Under the experimental c o n d i t i o n s , p r o t e c t i o n from harmful i n f l u e n c e s was a f f o r d e d t o the eggs. They were s t o r e d under near a s e p t i c c o n d i t i o n s , w h i l e the only danger of p h y s i c a l i n j u r y d u r i n g the storage procedure was i n the - 30 -s t r i p p i n g o f the eggs,which was done as g e n t l y as p o s s i b l e . The oxygen supply t o the s t o r e d eggs may or may not have been as good as t h a t a v a i l a b l e from the p a r e n t . The oxygen a v a i l a b l e was t h a t s e a l e d i n the c o n t a i n e r and t h a t supply was not r e -newed. I t was pr o b a b l y s u f f i c i e n t , though, f o r the s m a l l number of eggs (approximately 800) In the j a r . In a d d i t i o n , the m e t a b o l i c r a t e of the c e l l was probably lowered t o some extent by storage at a low temperature and t h i s a l s o would have tended to reduce the amount o f oxygen r e q u i r e d f o r the r e s p i r a t o r y f u n c t i o n s o f the c e l l . There may, however, have been l i t t l e or no c i r c u l a t i o n of a i r around the eggs so t h a t some of the eggs may have obtained l e s s oxygen than the o t h e r s . The food supply may, on the whole, be n e g l e c t e d except to s t a t e t h a t the eggs were not s u b j e c t to the e f f e c t s of r e -s o r p t i o n as they would have been, s t o r e d i n the parent body. The p h y s i c a l and chemical c o n d i t i o n s o f the experimental storage were d i f f e r e n t t o a degree from those i n the p a r e n t . There was no o p p o r t u n i t y under the experimental c o n d i t i o n s f o r the removal of any waste m a t e r i a l s , e s p e c i a l l y the carbon-d i o x i d e g i v e n o f f d u r i n g r e s p i r a t i o n . Thus, the pH o f the surrounding medium may have been lowered, although t h i s con-t i n g e n c y was probably handled by the b u f f e r systems p r e s e n t In the coelomic f l u i d i n which the eggs were s t o r e d . The storage temperature i n t h i s case was lower than t h a t of the parent body; a f a c t o r which may e i t h e r p r o l o n g the l i f e o f the - 31 -c e l l by r e d u c i n g the r a t e o f metabolism or hasten the death of the c e l l by c o o l i n g i t beyond a minimum p o i n t . The longer the eggs were h e l d i n experimental s t o r -age, the g r e a t e r was the number o f i n f e r t i l e eggs r e s u l t i n g , a l though the r e l a t i o n s h i p was not a d i r e c t one. The i n a b i l i t y of the eggs to be f e r t i l i z e d c o u l d be due to one or a combin-a t i o n o f s e v e r a l f a c t o r s , namely: 1. the death of the c e l l , 2. the l e n g t h of time the egg had a l r e a d y been h e l d i n the abdominal c a v i t y o f the female, 3. the i n a b i l i t y o f the l i v i n g c e l l t o b e f e r t i l i z e d 4. impotent m i l t and/or poor f e r t i l i z a t i o n t e c h n ique. C o n s i d e r i n g the l a s t p o i n t f i r s t , i t can be s t a t e d t h a t t h i s f a c t o r was of n e g l i g i b l e importance i n t h i s s e r i e s of experiments. C o n t r o l s were run on a l l experimental samples and, i n a d d i t i o n to' t h i s , the more e f f i c i e n t "dry" method of f e r t i l i z a t i o n was employed throughout. The death of the c e l l s , which may have been due to a number of causes, i s of more importance. The f a c t o r o f i n j u r y was e v e r - p r e s e n t , but was kept t o a minimum by a g e n t l e h a n d l i n g of the eggs a t a l l times. The l a c k o f n u t r i e n t s f o r the c e l l s may have been a f a c t o r c o n t r i b u t i n g to t h e i r death. Of more e f f e c t i n regard to the death of the eggs was the oxygen supply, or l a c k of i t . T h i s l a c k would tend t o have a cumulative e f f e c t , which may be r e p r e s e n t e d i n the t r e n d l i n e - 32 -shown In f i g u r e 5. An accumulation o f waste products may a l s o have had a c o r r e s p o n d i n g l y i n c r e a s i n g e f f e c t . The f a c t o r of p r e v i o u s storage w i t h i n the parent body b e f o r e the eggs were used e x p e r i m e n t a l l y , w h i l e an im-. p o r t a n t one, i s one t h a t cannot be e v a l u a t e d r e a d i l y . Nakano and Nozawa (1925) s t a t e t h a t "the e f f e c t of f e r t i l i z a t i o n o f the preserved eggs i s v a r i a b l e a c c o r d i n g to the c o n d i t i o n o f the parent f i s h " but do not put forward any experimental p r o o f . M r s i c (1923) has shown f o r the European rainbow t r o u t t h a t the h o l d i n g o f eggs In the abdominal c a v i t y o f the female does i n c r e a s e the p e r c e n t m o r t a l i t y of the eggs. For t h i s s e r i e s of experiments, the e f f e c t s o f p r e v i o u s storage i n the p a r e n t ' s abdominal c a v i t y may be regarded as having had a comparable e f f e c t on a l l samples, alt h o u g h the somewhat h i g h e r m o r t a l i t i e s a f t e r 84, 120 and 180 hours storage might be p a r t i a l l y e x p l a i n e d on t h i s b a s i s . The i n a b i l i t y o f the l i v i n g egg t o be f e r t i l i z e d may have been due to one o f a t l e a s t two f a c t o r s . The f i r s t was the p o s s i b l e c l o s u r e of the m l c r o p y l e so t h a t the sperm was unable to g a i n entrance t o the egg. T h i s c l o s u r e may have been due to a s w e l l i n g o f . t h e c h o r i o n i n the presence o f water ( P e a r t , 1920; L i n d r o t h , 1947) or to, the p l u g g i n g o f the m i c r o p y l e by the y o l k m a t e r i a l of broken eggs ( C a r l , 1941). I t i s u n l i k e l y t h a t e i t h e r o f these two f a c t o r s were o p e r a t i v e d u r i n g t h i s experiment. The second, and p o s s i b l y major, 33 -reason t h a t the eggs were i n f e r t i l e was a p p a r e n t l y the ageing o f the c o l l o i d a l system o f the protoplasm t o a p o i n t beyond which i t c o u l d not be r e a c t i v a t e d , even by f e r t i l i z a t i o n . While the egg, a f t e r b e i n g detached from the ovary and b e f o r e b e i n g f e r t i l i z e d , appears to be i n a q u i e s c e n t s t a t e , " i t i s a c t u a l l y undergoing changes which make i t s i n k from an optimum developmental c a p a c i t y t o a c o n d i t i o n from which development cannot o c c u r " (Weiss, 1939, p. 194). Weiss f u r t h e r s t a t e s t h a t a s i g n i f i c a n t c o r r e l a t e o f the egg's developmental c a p a c i t y i s the c o l l o i d a l c o n d i t i o n o f the egg. That i s , the changes which occur i n the egg as i t ages a r e , i n a sense, comparable t o the"ageing" o f a c o l l o i d . C o l l o i d s are heterogenous systems made up o f two phases; the d i s p e r s e d phase ( c o n s i s t i n g o f d i s p e r s e d par-t i c l e s ) and the continuous phase (the d i s p e r s i o n medium). The i n d i v i d u a l suspended p a r t i c l e s are termed u l t r a m i c r o n s . The c o l l o i d a l system o f a mature egg i s h i g h l y d i s p e r s e d and, as such, i s r e l a t i v e l y u n s t a b l e . The system tends t o decrease i t s d i s p e r s i t y and thereby i n c r e a s e i t s s t a b i l i t y . The u l t r a -microns tend to aggregate w i t h time i n t o l a r g e u n i t s . T h i s coming out of d i s p e r s i o n proceeds at a slow r a t e , along w i t h concomitant chemical and p h y s i c a l changes. E v e n t u a l l y , the c o l l o i d a l system tends t o separate out i t s two phases; the s o l i d d i s p e r s o i d assuming g r e a t e r d e n s i t y w h ile the l i q u i d d i s p e r s o i d becomes f r e e from the i n t e r f a c e s t o which i t was 34 -formerly bound. It i s Weiss' suggestion that the changes i n a c e l l due to ageing correspond to the l i f e history of a c o l l o i d -a l system, with i t s decrease in dispersi ty, hydration, elec-t r i c a l conductivity, e l a s t i c i t y and s o l u b i l i t y , and i t s i n -crease in ultramicronic s ize , s t a b i l i t y and e l ec t r i ca l res i s t -ance. Thus by changes progressing towards s t a b i l i t y i n the co l lo ida l system of the egg, the mature c e l l becomes less and less susceptible to f e r t i l i z a t i o n , with the passage of time. To recapitulate. The i n f e r t i l i t y of the egg may be due to the death of the egg, because of oxygen lack and waste accumulation, along with the lesser factors of food lack and -physical injury. Or, i t may be due to the i n f e r t i l i t y of the l i v i n g ce l l s caused by closure of the micropyle, or, mainly, by the progressive ageing of the co l lo ida l system of the egg beyond a point from which i t cannot be reactivated by f e r t i l -i za t ion . It i s with this la t ter factor that Weiss tends to correlate the decrease with age of the developmental capacity of the egg. A study of f i g . 5 shows a definite relationship between the length of storage and the f e r t i l i t y of the stored eggs. As the trend l ine indicates, however, this relationship i s not a precise one. No attempt has been made to determine the exact mathematical nature of this relationship of the two since (a) the data are insufficient for this purpose and (b) the problem at hand does not concern a mathematical treatment - 35 -of m o r t a l i t y r a t e s . Prom f i g . 5 i t i s ev i d e n t t h a t chum eggs may be s t o r e d f o r 72 hours under the c o n d i t i o n s of t h i s experiment w i t h l e s s than 10$ i n f e r t i l i t y . The c o n t r o l s f o r these eggs e x h i b i t e d an i n f e r t i l i t y o f s l i g h t l y l e s s than 1%. The o n l y e x c e p t i o n , In these d a t a , was a t 48 hours where one o f the e i g h t d e t e r m i n a t i o n s showed an i n f e r t i l i t y o f 16%, Storage o f the eggs f o r 108 hours r e s u l t e d i n l e s s than 20$ i n f e r t i l i t y i n 32 out of 36 cases, the f o u r e xceptions b e i n g a f t e r 84 hours s t o r a g e , where between 20 and 30$ i n f e r t i l e eggs r e s u l -t e d . The average percentage of i n f e r t i l e eggs i n the 108 hour p e r i o d was 9.0* 7.8$. The average percentage of i n f e r t i l e eggs i n the c o n t r o l s f o r t h i s p e r i o d was l e s s than 1$. A f t e r 108 hours s t o r a g e , the percentage of i n f e r t i l e , eggs r a p i d l y r ose t o a maximum of 85$. The average percentage of i n f e r t i l i t i e s a f t e r storage from 108 t o 192 hours was 52.6-19.4$; the c o n t r o l s , l e s s than 3$. The Nelson creek d a t a , added f o r comparison, show a g e n e r a l f o l l o w i n g o f the t r e n d l i n e , a circumstance which tends t o enhance-the a p p l i c a b i l i t y o f f i g . 5 i n f i s h c u l t u r a l p r a c t i c e s . The sperm c e l l s o f the salmon are not r e l e a s e d i n t o the body c a v i t y of the p a r e n t , as are the eggs o f the female. - 36 -The sperm are produced i n the t e s t e s , s t o r e d t h e r e , and then, at the time of spawning, pass t o the o u t s i d e through the vasa d e f e r e n t i a . The c e l l s are non-motile i n the t e s t e s , moving onl y when they come i n c o n t a c t w i t h water, a f t e r which t h e i r l i f e i s very s h o r t , ( L i n d r o t h , 1 9 4 7 ) . The sperm are produced over a longer p e r i o d of time than are the eggs. The h o l d i n g o f the sperm i n the t e s t e s r a t h e r than i n the abdominal c a v i t y g i v e s a h i g h s u r v i v a l v alue to the c e l l s . The sperm c e l l i s g i v e n a h i g h degree o f p h y s i c a l p r o t e c t i o n w hile w i t h i n the t e s t e s , as w e l l as p r o t e c t i o n from b a c t e r i a l i n j u r y . While i n the t e s t e s , the spermatozoa r e c e i v e t h e i r n u t r i e n t s from the b l o o d supply o f the t e s t e s . The oxygen supply to the c e l l s i s a l s o gained from the b l o o d system of the pare n t , i n d i r e c t l y . Carbon d i o x i d e and m e t a b o l i c waste removal i s normal. The pH o f the f l u i d surrounding the sperm i s maintained a t a constant l e v e l by b u f f e r systems, the f l u i d i s i s o t o n i c w i t h the c e l l s and temperature changes are not r a p i d . The c o n d i t i o n s of experimental storage v a r i e d t o some degree from those found In n a t u r e . The sperm, under ex-p e r i m e n t a l c o n d i t i o n s , were p r o t e c t e d from p h y s i c a l i n j u r y and from b a c t e r i a l i n f e c t i o n . The food supply under the a r t i -f i c i a l c o n d i t i o n s was n o n - e x i s t e n t . The oxygen supply t o the c e l l s waa onl y t h a t l e f t i n the v e s s e l s when they were s t o r e d . M e t a b o l i c wastes and carbon d i o x i d e were not removed. The pH - 37 -was probably maintained f o r a s h o r t w h i l e , due t o the b u f f e r -i n g systems present i n the m i l t . Temperature was maintained constant over a range from 2.0 to 5.8*0. The m i l t was not kept i n motion, so t h a t s e t t l i n g out d i d o c c u r . Comparatively, then, the experimental c o n d i t i o n s were on l y able to supply to the sperm p r o t e c t i o n , some s l i g h t pH c o n t r o l and a constant temperature, somewhat lower than normal. S e v e r a l normal environmental f a c t o r s were absent i n the experimental samples. These were fo o d , oxygen, waste removal, pH c o n t r o l , and slow v a r i a t i o n s i n temperature. The above n a t u r a l f a c t o r s , which c o u l d not be s u p p l i e d under the c o n d i t i o n s of the experiment, are necessary to some degree f o r the s u r v i v a l of the sperm c e l l s . K leerekoper (1949) over-came some of these d i f f i c u l t i e s by p r e s e r v i n g sperm i n S a l i s b u r y ' s y o l k - c i t r a t e mixture, thereby r e d u c i n g the concen-t r a t i o n of the sperm c e l l s , p r o v i d i n g n u t r i e n t s and m a i n t a i n -i n g a b u f f e r e d s t a t e . These f a c t o r s should produce a m o r t a l i t y curve f o l l o w i n g the p a t t e r n shown f o r the eggs i n f i g . 5. However, the data p l o t t e d In f i g . 6 show a wide range of m o r t a l i t i e s i n the s t o r e d m i l t samples. Up to 36 hours storage, 10 o f 12 samples s t o r e d had l e s s than &% i n f e r t i l i t y . The remaining two samples i n t h i s time p e r i o d showed I n f e r t i l i t i e s o f 43 and 55$. From 0 to 96 hours storage, 24 of 32 samples had l e s s than 50$ i n f e r t i l i t i e s , the average b e i n g 8.9 * 14.5$. The - 38 -remaining samples i n the time period had an average i n f e r t i l i t y of 73 i19$, while the average i n f e r t i l i t y of the entire 32 samples was 23 t 31$. From 96 to 192 hours, 24 of the 32 m i l t samples showed i n f e r t i l i t i e s of above 50$, the average being 92 - 11$. The remaining 8 samples had an average i n f e r t i l i t y ot 23 - 15$, while the average for the 32 samples was 75 "= 35$. The data are obviously e r r a t i c with, for example, an i n f e r t i l i t y of 99$ r e s u l t i n g i n one case a f t e r only 48 hours storage while a 192 hour storage of another sample resulted i n no i n f e r t i l i t i e s . Several factors may be held to account f o r t h i s d i v e r s i t y , a l l or none of which may have been operative. One of the factors which may have had some ef f e c t was the possible i n i t i a l difference i n the sperm samples, when f i r s t taken from the f i s h . This i s doubtful, however, sinoe controls were run i n a l l cases, with no I n f e r t i l i t i e s higher than 8$. Some of the greatest differences In f e r t i l i z i n g power were between two samples from the same f i s h (Table X I ) . S l i g h t , un-noticed variations from time to time i n the technique of storage may have been of some effe c t i n pro-ducing the wide range of i n f e r t i l i t i e s . Of those samples stored up to 96 hours, the eight samples with i n f e r t i l i t i e s above the 50$ l e v e l might possibly be dismissed as being due to some negative v a r i a t i o n i n technique, which tended to k i l l o f f these p a r t i c u l a r samples. However, I f technique were a fa c t o r , i t seems improbable that more errors i n technique were made - 39 -with the samples which were held longer than 96 hours. Also, what were the positive variations i n technique, I f any, which allowed 8 stored samples to show low i n f e r t i l i t i e s up to 8 days? Some major d i f ferent ia l factor was probably at hand, having a greater effect with time. It i s quite possible that this major factor was the matter of moisture in some form coming into contact with the sperm. I f this were the case, the death of the sperm would follow the short period of mot i l -i t y . There are at least three pos s ib i l i t i e s in this connection. One, which could not be controlled, was the entrance of urine with the sperm sample. The second was the presence of water i n the storage jars , due to the method of s t e r i l i z i n g the ja rs . This i s rather improbable, although quite possible. Thirdly, a factor to which some of the: low f e r t i l i t i e s : may" be at t r ibu-ted was the entrance of moisture to the sperm while they were in storage. As noted above, the jars were of the screw-cap type and did not have rubber seals, as did the egg storage jars . I t seems possible that in a humid atmosphere* such as that of the icebox, moisture may have gained entrance to some of the jars , producing the wide range of resul ts . Another factor possibly leading to these errat ic mortali t ies was the variations in the depth of the mil t i n the jars . No attempt was made to keep the depth constant. I t i s quite possible that, In jars where the level of the stored 40 -m i l t was low, the d i f f u s i o n of gases to and from the c e l l s was more r e a d i l y accomplished than i n those j a r s where the m i l t l e v e l was h i g h . In other words, to those c e l l s i n the " l o w - l e v e l " j a r s , b o t h the oxygen supply and the carbon d i o x i d e removal (and the maintainence of a constant pH l e v e l ) were p o s s i b l y b e t t e r than to the c e l l s i n the h i g h - l e v e l j a r s . The s h o r t e r l i f e t i m e f o r the sperm as compared to the eggs may have been due t o the g r e a t e r number o f sperm c e l l s s t o r e d . For the approximately 800 eggs h e l d , there were about one q u a r t e r b i l l i o n sperm s t o r e d . The oxygen consumption and the carbon d i o x i d e and m e t a b o l i t e p r o d u c t i o n of the sperm were undoubtedly many times g r e a t e r than those of the eggs. Again, t h i s d i v e r s i t y o f r e s u l t s may be e x p l a i n e d by the p o s t u l a t e t h a t the l e n g t h and c o n d i t i o n s of storage employ-ed i n these experiments are not the major determinants i n the v i a b i l i t y o f the salmon sperm. There does appear to be, however, some s l i g h t r e l a t i o n s h i p between the time o f storage and the v i a b i l i t y o f the sperm. Some of the i n f e r t i l i t i e s a t 120, 168 and 192 hours (Table XI) do i n d i c a t e t h a t sperm may remain v i a b l e f o r some l e n g t h of time, depending on the c o r r e c t mode o f s t o r a g e . Work along the l i n e s o f a more l i m i t e d temper-ature range, an absolute s t a n d a r d i z a t i o n of storage techniques, the use o f b u f f e r e d n u t r i e n t mediums and the t e s t i n g of a g r e a t e r number o f samples i s i n d i c a t e d . The Nelson creek d a t a , added t o f i g . 6 f o r compari-son, show the same wide range o f i n f e r t i l i t i e s . - 41 -Prom the p r a c t i c a l p o i n t o f view, sperm may he s t o r e d w i t h a low m o r t a l i t y f o r a t l e a s t 36 hours. Beyond t h i s time, i f sperm were to be s t o r e d under the c o n d i t i o n s s p e c i f i e d , i t would probably be wi s e r to s t o r e s e v e r a l sam-p l e s inthe:hope t h a t one or two of these samples would show a h i g h f e r t i l i t y . F u r t h e r i n v e s t i g a t i o n o f the problem w i l l un-doubtedly remove t h i s aspect o f u n c e r t a i n i t y as regards m i l t storage f o r p e r i o d s l o n g e r than 36 hours. P o s s i b i l i t i e s f o r the salvage o f mature salmon eggs and sperm are i n d i c a t e d . I f the c e l l s are taken from the mature f i s h upon capture and h e l d i n the manner d e s c r i b e d , much In the way o f r e d u c i n g the l o s s e s o f mature eggs and sperm can be accomplished. The methods of storage are simple and inexpensive; the p o s s i b l e a p p l i c a t i o n s o f the r e s u l t s are complex and v a l u a b l e . SUMMARY AND CONCLUSIONS 1. A technique has been developed f o r s t o r i n g , under f i e l d c o n d i t i o n s , salmon eggs and sperm f o r v a r y i n g p e r i o d s p r i o r t o f e r t i l i z a t i o n . 2. The eggs o f 0. ket a may be s t o r e d i n s t e r i l e j a r s a t a low temperature f o r 108 hours w i t h an average i n f e r t i l i t y o f l e s s than 20$. 3. The sperm o f 0. k e t a may be s t o r e d up t o 36 hours i n s t e r i l e j a r s In the absence o f moisture and a t a low temperature w i t h an average of l e s s than 10$ i n f e r t i l i t y . 4. The sperm o f 0. k e t a are l e s s s u c c e s s f u l l y s t o r e d than the eggs and show a wide v a r i a t i o n In t h e i r r e a c t i o n t o sto r a g e . 5. The l i m i t e d data f o r Kamloops t r o u t eggs I n d i c a t e t h a t , w i t h f u r t h e r i n v e s t i g a t i o n , r e s u l t s s i m i l a r t o those f o r 0. k e t a eggs would be ob t a i n e d . - 42 -LITERATURE CITED A t k i n s , C.G., 1874. On the salmon o f e a s t e r n North America and i t s a r t i f i c i a l c u l t u r e . Rept. o f Comm. o f U.S. P i s h and F i s h e r i e s , 2: 226-337. Calderwood, W.L., 1909. Note on c r o s s i n g salmon by t r a n s -p o r t i n g r i p e m i l t . 28th Aran. Rept. F i s h . Bd. S c o t l a n d , p a r t 2: 61-62. C a r l , 6.C., 1941. Beware the broken egg. Prog. F i s h C u l t . , 53: 30-31. E l l i s , W.G. and Jones, J.W., 1939. The a c t i v i t y o f the spermatozoa o f Salmo s a l a r i n r e l a t i o n t o osmotic p r e s s u r e . J o u r . Exp. B i o l . , 16: 530-534. Gray, J . , 1919. Some notes on the p h y s i o l o g y o f the t r o u t egg. The Salmon and Trout Mag., London, #19: 13-19. 1920. The r e l a t i o n of animal c e l l s t o e l e c t r o l y t e s . I . A p h y s i o l o g i c a l study o f the egg o f the t r o u t . J o u r . P h y s i o l . , 53: 308-319. Greene, C.W., 1904. P h y s i o l o g y of the Chinook salmon. B u l l . U.S. Bur. F i s h . , 24: 431-456. Henneguy, L.F., 1877. Recherches sur l a v i t a l i t e des sperm-a t o z o i d e s dans l a t r u i t e . C.-R. Acad. S c i . , P a r i s , 84: 1333-1335. Kleerekoper, H . , 1946. A p r e l i m i n a r y note on the p r e s e r v a t i o n of the eggs and sperm o f " p e i x e r e i " (Odonthestes  b r a s i l l e n s l s V a l . ) , an A t h e r i n i d of southern B r a z i l , Uruguay, and A r g e n t i n a . B o l e t i n Agronomico ( B r a z i l ) 119-120: 291-293. 1949. P r i v a t e communication. L i n d r o t h , A., 1947. Time o f a c t i v i t y o f freshwater f i s h spermatozoa i n r e l a t i o n t o temperature. Z o o l . B i d r a g . , Uppsala, 25: 165-168. M i l n e r , J.W., 1874. The prog r e s s of f i s h c u l t u r e i n the Unit e d S t a t e s . Rept. of Comm. of U.S. F i s h and F i s h e r i e s , 2: 523-566. - 43 -- 44 -M r s i c , W . , 1923, Die Spatbefruchtung und deren B I n f l u s s auf Entwicklung und G e s c h l e c h t s b i l d u n g e x p e r i m e n t e l l e nachgepruft an der Regenbogenforelle. Arch, MIkr. Anat. u. Entwmech., 98: 129-209. Nakano S. and Nosawa, A., 1925. On the v i t a l i t y of the eggs and sperm o f Oncorhynchus masou ( l a n d l o c k e d ) . 3"our. Imp. P i s h . Inst.., Tokyo, 21: 17. P e a r t , A.B., 1920. The mechanics o f f e r t i l i z a t i o n i n t r o u t . The Salmon and Trout Mag., London, #21: 33-43. R u t t e r , C , 1902a. The n a t u r a l h i s t o r y o f the quinnat salmon. B u l l . U.S. Bur. P i s h . , 22: 65-141. 1902b. S t u d i e s i n the n a t u r a l h i s t o r y of the Sacramento salmon. Pop. S c i . Mon., 61: 195-211. Smith, R.T. and Q u i s t o r f f , B., 1943. Experiments w i t h the spermatozoa o f the s t e e l h e a d t r o u t , Salmo g a i r d n e r i i and the Chinook salmon, Oncorhynchus tshawytscha. Copeia, 1943: 164-167. Soudakevicz, T., 1874. Report on the p r o g r e s s of p i s c i c u l t u r e i n R u s s i a . Rept. of Comm. of U.S. P i s h and F i s h e r i e s , 2: 493-513. Weiss, P., 1939. P r i n c i p l e s of development. New York, Henry H o l t and Company. Appendix A: Lloyd's creek data - 46 -T A B L E I I I DATA FOR EGGS STORED AT LLOYD'S CREEK Hours held % mortality Temperature Conditions 0.0 0.8 17.5°C 4 oz. jar 0.5 2.2 t i 1.0 1.4 i i 1.5 8.5 i t 2.0 0.0 i i 2.0 18.0 17.0 stacking dish 2.5 8.3 17.5 4 oz. jar 4 24 17.0 stacking dish 6 30 • • 8 26 • • 10 56 • • 12 56 • • 15 14 • • 24 76 TABLE I I I (cont'd) Hours held % mortality Temperature Conditions 48 90 17.0 stacking dish 67 61 4.0 i n dead f i s h 67 - 54 4.0 stacking d i s h 72 67 17.0 t Appendix B: Nelson creek d a t a . 49 -T A B L E I V D A T A F O R E G G S S T O R E D A T N E L S O N C R E E K Percent Hours Sample Eggs Eggs Percent u n f e r t i l i z e d Stored: Number :Examined : U n f e r t i l i z e d : U n f e r t i l i z e d : i n controls 24 S13A 40 4 10 S13B 74 4 5 0 48 32A 110 0 0 S2B 110 0 0 0 72 S12A 100 2 2 S12B 92 4 4 3 96 S3A 110 0 0 S3B 110 3 3 0 120 S7A 31 15 48 S7B 20 6 30 0 144 S5A 100 42 42 S5B 111 37 34 0 S8A 130 43 33 S8B 130 39 30 0 168 S9A 100 41 41 4 S9B 100 56 56 - 50 TABLE V DATA FOR SPERM STORED AT NELSON CREEK Percent Hours Sample Eggs Eggs Percent U n f e r t i l i z e d Stored:Number:Examined: U n f e r t I l i z e d : U n f e r t i l i z e d : i n controls 48 M2A 121 23 19 0 M2B 52 16 31 72 M3A 110 76 69 0 M3B 110 100 91 72 M7A 130 115 88 4 M7B 120 24 20 168 M4A 115 48 42 6 M4B 115 47 41 192 M5A 130 65 50 0 M5B 120 22 18 - 51 -TABLE VI TEMPERATURE DATA FOR ICEBOX AT NELSON CREEK Date Temperature Oct 27 4. O X 28 2.75 29 1.5 31 2.5 Nov 1 3.5 3 2.5 5 3.5 6 3.5 7 2.0 8 1.5 9 2.0 10 1.5 11 2.0 12 2.0 Appendix C: C u l t u s l a k e d a t a 5 3 -TABLE VII TEMPERATURES OP THE POUR ICEBOXES USED FOR THE STORAGE OF Qt KETA EGGS AND SPERM AT CULTUS LAKE, B.C. Date Icebox 1 Ieebox 2 Icebox 3 Icebox 4 Nov 19 3.OX 3.5 5.0 4.5 20 3.5 4.0 3.3 4.8 21 4.0 4.0 3.0 3.0 22 2.0 4.3 4.0 5.8 23 2.5 4.0 3.0 2.5 24 3.3 3.0 3.0 2.8 25 3.5 2.8 3.0 4.1 26 3.8 2.5 3.5 4.0 ' 27 4.0 4.3 3.0 2.5 - 54 -TABLE VIII DATA FOR CONTROL SAMPLES OF EGGS AND SPERM STORED AT CULTUS LAKE Sample Number Eggs : Examined Eggs U n f e r t i l i z e d Percent : U n f e r t i l i z e d X1Y1 50 4 8 X2Y2 50 3 6 X3Y3 50 0 0 X4Y4 50 0 0 X5Y5 50 0 0 X6Y6 50 0 0 X7Y7 50 4 8 X8Y8 50 0 0 X9Y9 50 0 0 XIOYIO 50 0 0 X11Y11 50 4 8 X12Y12 50 0 0 X13Y13 50 0 0 - 55 -TABLE VIII (cont'd) Sample Number Eggs : Examined • Eggs U n f e r t i l i z e d Percent : U n f e r t i l i z e d X14Y14 50 0 0 X15Y15 50 0 0 X16Y16 50 2 4 X17T17 50 0 0 X18Y18 50 1 2 X19Y19 50 0 0 X20Y20 50 2 4 X21Y21 50 0 0 X22Y22 50 1 1 X23Y23 50 0 0 X24Y24 50 1 2 X25Y25 50 0 0 X26Y26 50 0 0 X27Y27 50 0 0 - 56 -TABLE V I I I (cont'd) Sample Number : Eggs Examined Eggs : u n f e r t i l i z e d P ercent : U n f e r t i l i z e d X28Y28 50 0 0 X29Y29 50 0 0 X30Y30 50 0 0 X31Y31 50 2 4 X32Y32 50 0 0 - 57 -TABLE IX CONTROL DATA OP MALES AND FEMALES USED TO TEST THE FERTILITY OF THE STORED EGGS AND SPERM Sample Number Eggs : Examined Eggs : U n f e r t i l i z e d : Percent U n f e r t i l i z e d 1 50 0 0 2 50 0 0 5 50 1 2 4 50 0 0 5 50 0 0 6 50 0 0 7 50 2 4 8 50 0 0 9 50 0 0 10 50 1 2 11 50 3 6 12 50 1 2 13 50 0 0~ 14 50 0 0 TABLE IX (cont'd) Sample Number Eggs : Examined Eggs Unfert i l ized • • Percent Unfert i l ized 15 50 0 0 16 50 0 0 17 50 3 6 18 50 0 0 19 50 0 0 20 50 0 0 21 50 1 2 22 50 0 0 23 50 0 0 24 50 2 4 25 50 0 0 26 50 2 4 27 - 50 0 0 28 50 0 0 29 50 1 2 - 59 -TABLE IX (cont'd) Sample Number Eggs : Examined Eggs : u n f e r t i l i z e d P ercent : U n f e r t i l i z e d 30 50 0 0 31 50 0 0 32 50 1 2 - 60 -TABLE X DATA FOR EGGS STORED AT CULTUS LAKE Hours Sample Eggs Eggs Percent Stored : Number : Examined t U n f e r t i l i z e d : U n f e r t i l i z e d 12 X31A 200 13 6.5 X31B 200 13 6.5 X32A 200 1 0.5 X32B 200 3 1.5 24 X27A 200 7 3.5 X27B 200 3 1.5 X28A 165 6 4.0 X28B 158 13 8.0 36 X29A 200 12 6.0 X29B 200 13 6.5 X30A 200 13 6.5 X30B 200 10 5.0 48 X21A 200 17 8.5 X21B 200 12 6.0 X22A 200 33 16.5 X22B 181 29 16.0 48 X23A 200 5 2.5 X23B 200 2 1.0 X24A 200 4 2.0 X24B 200 7 3.5 72 X19A 200 5 2.5 X19B 200 4 2.0 X20A 200 18 9.0 X20B 200 11 5.5 84 X25A 157 43 27.0 X25B 157 46 28.0 X26A 168 41 24.5 X26B 176 41 23.5 - 61 -TABLE X (cont'd) Hours Sample Eggs Eggs Percent Stored i : Number : Examined : U n f e r t i l i z e d : U n f e r t l l i z e i 96 X13A 167 5 3.0 X13B 176 24 14.0 X14A 181 6 3.5 X14B 173 34 20.0 108 X17A 200 12 6.0 X17B 200 18 9.0 "X18A 200 30 15.0 X18B 200 41 20.5 120 X7A 200 119 59.5 X7B 200 106 53.0 X8A 200 97 48.5 X8B 200 95 47.5 132 X15A 200 67 33.5 X15B 200 39 19.5 X16A 200 30 15.0 X16B 200 66 33.0 144 X5A 200 37 18.5 X5B 200 23 11.5 X6A 200 120 60.0 X6B 200 122 61.0 156 X11A 250 162 65.0 X11B 200 99 49.5 X12A 183 116 63.5 X12B 190 107 56.0 168 X3A 200 128 64.0 X3B 200 138 69.0 X4A 200 99 49.5 X4B 200 104 52.0 - 62 -TABLE X (cont'd) Hours Sample Eggs Eggs Percent Stored : Number : Examined : TJnf.ertilized : u n f e r t i l i z e d 180 X9A 200 130 65.0 X9B 200 129 64.5 X10A 250 212 85.0 X10B 200 168 84.0 192 X1A 200 102 51.0 X1B 200 103 51.5 X2A 200 149 74.5 X2B 200 150 75.0 - 63 -TABLE XI DATA FOR SPERM STORED AT CULTUS LAKE Hours Sample Eggs Eggs Percent Stored : Number : : Examined : U n f e r t i l i z e d : U n f e r t i l i z e d 12 Y31A 200 2 1.0 Y31B 200 0 0.0 Y32A 200 1 0.5 Y32B 200 5 2.5 24 Y27A 200 7 3.5 • Y27B 200 85 42.5 Y28A 200 2 1.0 Y28B 200 2 1.0 36 Y29A 200 0 0.0 Y29B 200 1 0.5 Y30A 181 0 0.0 Y30B 140 77 55.0 48 Y21A 200 62 31.0 Y21B 200 5 2.5 Y22A 200 198 99.0 Y22B 200 122 61.0 48 Y23A 200 0 0.0 Y23B 200 4 2.0 Y24A 200 86 43.0 Y24B 200 1 0.5 72 Y19A 200 26 13.0 Y19B 200 195 97.5 Y20A 200 73 36.5 Y20B 200 5 2.5 84 Y25A 50 0 0.0 Y25B 50 0 0.0 Y26A 50 3 6.0 Y26B 50 27 54.0 64 -TABLE XI (cont'd) Hours Sample Eggs Eggs Percent Stored : Number : Examined : U n f e r t i l i z e d \ : U n f e r t i l i z e d 96 Y13A 200 97 48.5 Y13B 200 117 ' 58.5 Y14A 200 37 18.5 Y14B 200 200 100.0 108 Y17A 200 34 17.0 Y17B 200 94 47.0 Y18A 200 80 40.0 Y18B 200 197 98.5 120 Y7A 161 45 28.0 Y7B 150 133 89.0 Y8A 138 39 28.0 Y8B 120 68 57.0 132 Y15A 200 7 3.5 Y15B 200 198 99.0 Y16A 200 36 18.0 Y16B 173 173 100.0 144 Y5A 175 175 100.0 Y5B 200 197 98.5 Y6A 150 150 100.0 Y6B 162 162 100.0 156 Y11A 166 166 100.0 Y11B 188 188 100.0 Y12A 175 129 74.0 Y12B 185 183 99.0 168 Y3A 128 128 100.0 Y3B 150 150 100.0 Y4A 135 36 27.0 Y4B 140 140 100.0 65 -TABLE XI (cont'd) Hours Sample Eggs Eggs Percent Stored : Number : Examined : U n f e r t i l i z e d : U n f e r t i l i z e d 180 Y9A 227 157 69 .0 Y9B 232 231 100.0 Y10A 223 223 100.0 Y10B 200 200 100.0 192 Y1A 170 170 100.0 Y1B 200 1 0 .5 Y2A 200 198 99 .0 Y2B 166 158 95 .0 

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