A Preliminary Study of Induced Ovulation in Coho Salmon (Oncorhynchus kisutch) at Robertson Creek Salnlon Hatchery

Transcription

1 A Preliminary Study of Induced Ovulation in Coho Salmon (Oncorhynchus kisutch) at Robertson Creek Salnlon Hatchery George A. Hunter, Edward M. Donaldson, Helen M. Dye and Karl Petersen Department of Fisheries and Oceans Resource Services Branch 4160 Marine Drive West Vancouver, British Columbia V7V 1 N6 August 1979 Fisheries and Marine Service Technical Report No. 899

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3 Fisheries and Marine Service Technical Report 899 August 1979 A PRELIMINARY STUDY OF INDUCED OVULATION IN COHO SALMON (ONCORHYNCHUS KISUTCH) AT ROBERTSON CREEK SALMON HATCHERY by George A. Hunter, Edward M. Donaldson, and Helen M. Dye and 1 Karl Petersen Resource Services Branch Department of Fisheries and Oceans 4160 Marine Drive \.Jest Vancouver, British Columbia V7V ln6 1 Enhancement Services Branch Department of Fisheries and Oceans Rob2rtson Creek Salillon Hatchery Por~ Aljerni, B~itish Columbia V9Y 7L9

4 - ii - (c) Minister of Supply and Services Canada 1979 Cat. no. Fs97-6/899 ISSN

5 - iii - ABSTRACT Hunter, George A., Edward M. Donaldson, Helen M. Dye and Karl Petersen A preliminary study of induced ovulation in coho salmon (Oncorhynchus kisutch) at Robertson Creek salmon hatchery. Fish. Mar. Servo Tech. Rep. 899: 15 p. Salmon pituitary extracts and an acetone powder of salmon pituitaries were used to induce the early maturation and ovulation of coho salmon at the Robertson Creek Salmon Hatchery. On Oct 30, seventy-five adult female coho salmon were divided into four groups of 30, 19, 18 and 18 fish. A group of 20 control fish were injected with saline on Day 0 (Oct 30) and Day 3 (Nov 2). Two groups of 19 and 18 fish received a 10 mg/kg injection of salmon pituitary extract (SPE) on Day 0 followed by a 50 mg/kg injection of the same solution on Day 3. The pituitary extract (SPE-WV) given the former of the two groups was prepared from chinook pituitaries collected in The latter group (SPE-SD) received an extract prepared from chum pituitaries collected in The fourth group consisted of 18 fish which were injected with an acetone powder of chum pituitaries collected in 1978 (AP-SD). The dosages given these fish were 1.3 mg/kg on Day 0 and 6.5 mg/kg on Day 3. The mean days to ovulation for the SPE-WV, SPE-SD, AP-SD and control groups were 9.45 ~ 2.9, 11.8 ~ 2.7, 10.0 ~ 3.9 and Analysis of time to ovulation data indicated that while no significant differences occurred between the three hormone treated test groups, all three groups ovulated earlier than the controls. The cumulative percent ovulations for the SPE-WV, SPE-SD, AP-SD and control groups were 68.8%, 100.0%, 86.7% and 57.9%, respectively. Two fish in the SPE-WV group were stripped prematurely. The remaining fish in each group died prior to spawning. The greater number of fish ovulating in the treated groups resulted in larger egg takes for these groups. The percent survivals to the eyed stage for eggs from the SPE-WV, SPE-SD, AP-SD and control groups were 86.4%, 93.6%, 83.7% and 89.4%. Pooling of egg lots prevented statistical analysis of egg survival to the eyed stage. It is apparent from the results of this preliminary experiment that the technique of induced ovulation with salmon pituitary extracts shows considerable promise for the aleviation of prespawning mortality in coho salmon.

6 - iv -, / RESUME Hunter. George A Edward M. Donaldson. Helen M. Dye and Karl Petersen A preliminary study of induced ovulation in coho salmon (Oncorhynchus Kisutch) at Robertson Creek salmon hatchery. Fish. Mar. Servo Tech. Rep. 899: 15 p. Des extraits d'hypophyse de saumon et une poudre d'hypophyse de pr~par~e par pr~cipitation ~ l'ac~tone ont servi a acc~l~rer la maturation et l'ovulation du saumon coho dans la piscifacture de Robertson Creek. Le 30 octobre, on a s~par~ 75 femelles adultes de saumon,.. coho en quatre groupes de 20, 19 et deux fois 18 poissons. On a injecte a un groupe de 20 poissons t~moins une solution saline le jour 0 (30 octobre) et le jour 3 (2 novembre). Deux groupes de 19 et 18 poissons ont re~u une injection de 10 mg/kg d'extrait pituitaire de saumon (SPE) le jour 0, puis une injection de 50 mg/kg de la meme solution le jour 3. L'extrait pituitaire (SPE-WV), ", "" administre au premier des deux groupes a ete prepare a partir d'hypophyses de saumon quinnat pr~lev~es en Le deuxieme groupe (SPE-SD) a re~u un,;,., " extrait prepare a partlr d'hypophyses de saumon keta prelevees en Le quatrieme Qroupe consistait en 18 poissons ayant recu une ~,, " > injection de poudre, preparee par precipitation a l'acetone, d'hylophyses de saumon k~ta pr~lev~es en 1978 (AP-SO). Les dosages administres aces poissons ~taient de 1.3 mg/kg au jour 0 et 6.5 mg/kg au jour 3.,. '. La duree moyenne, en Jours. de la perlode allant jusqu'a l'ovulation pour les groupes SPE-WV, SPE-SD, AP-SD et le groupe t~moin ont ~t~ de , , et 21, L'analyse des donn~es concernant le d~l~i jusqu'a-l'ovulation a-indiqu~ que, s'il n'y a pas eu de diff~rences significatives dans les trois groupes trait~s aux hormones. ces trois groupes ont pr~sent~ une ovulation anticip~e par rapport au groupe temoin. Le pourcenta~e cum~latif des ovualtions dans les groupes SPE-WV. SPE-SO et le groupe temoin etait de 68.8%, 100%. 86.7% et 57.9% res~ectivement. Pour deux poissons du groupe SPE-WV, l'extraction des oeufs a eu lieu de,, facon prematuree. }.. de se reprodulre. Dans chaque groupe, les poissons restants sont morts avant La fr~quencellus ~lev~e de l'ovulation chez les poissons des groupes trait~s a permis de prelever un plus grand nombre d'oeufs dans ces groupes. Le pourcentage de survie jusqu'au stade embryonn~, pour les oeufs provenant des groupes SPE-WV. SPE-SD, AP-SD et le groupe t~oin. ~taient de 86.4%, 93.6%, 83.7%, et 89.4%. Le group age des lots d'oeufs a empech~ que soit pratiquee, une analyse statistique de la survie des oeufs jusqu'au stade embryonne. Les resultats, d e cette experlence '.' preliminaire mont rent que 1 a technique de l'ovulation provoqu~e a l'aide d'extraits d'hypophyse de saumon ouvre des perspectives int~ressantes pour la r~duction de la mortalit~ ant~rieure a la fraye chez le saumon coho.

7 INTRODUCTION 1 1 recent years presp<lwning mort;}1 i ty has become evident in some of the s,llmon hatcheries in ti\(' P;Jcific Region. During the holding period, prior to spawning, the adults are confined in concrete holding ponds often at high density. The occurrence of prespawning mortality is probably linked to stress associated with confinement together with the increased opportunity for disease transmission. While it is clear that the ideal solution would be the elimination of the factors producing stress a more realistic immediate solution would be the use of induced ovulation techniques ta accelerate oocyte maturation and ovulation. This approach is base.g. on the hypothesis that spawning the fish earlier would increase egg take if the treatment itself did not increase the mortality rate. The procedure has the added advantage of permitting the synchronization of the spawning of large numbers of fish once the optimal dosage and time to ovulation have been determined. The consequent reduction in fish handling would minimize the exposure of the fish to prespawning stress and also reduce the manpower needs for the spawning operation. To date, our investigations of techniques for the induced ovulation of coho have involved the use of pituitary extracts, partially purified salmon gonadotropin and various synthetic hormones (Jalabert et al., 1978; Donaldson et al., 1978a, 1979). These studies were conducted exclusively at the laboratory level. The occurrence of prespawning mortality has, however, been successfully aleviated in an experiment on chinook salmon at the Capilano Salmon Hatchery (Hunter et al., 1978). In this study the time to ovulation was significantly reduced in fish treated with either partially purified salmon gonadotropin (SG-GlOO) or salmon pituitary extract (SPE) without increasing the rate at which mortality occurred. This resulted in a corresponding increase in the numbers of eyed eggs in the treatment groups (Hunter et al., 1978). Since the completion of the Capilano study, collaborative work between the Department of Fisheries and Oceans and Syndel Laboratories Ltd., has resulted in the availability of commercially produced frozen salmon pituitary glands and an acetone powder of salmon pituitary glands. The Robertson Creek Salmon Hatchery presents an example of prespawning mortality in coho salmon. After the withdrawal of the certification of malachite green as a treatment for fungal infections in adult salmon there has been an increase in the prevalence of Saprolegnia in adult coho being held at this hatchery. While stressors such as crowding and high water temperatures during the fall accelerate the development of the disease (Neish, 1977), the problem is not yet serious enough to affect the number of eggs taken for hatchery use. It has, however, had an effect on the numbers of excess fish available for sale. The purpose of this study has been to investigate the use of induced ovulation techniques on coho salmon at Robertson Creek Salmon Hatchery using the newly available commercially produced salmon pituitary preparations.

8 -2- MATERIALS AND METHODS This study was conducted in the fall of 1978 at the Robertson Creek Ilatchery. Robertson Creek is part of the Somass River - Alberni Inlet system on th~ west coast of Vancuuver Island. Seventy-five adult 3-yr old female coho salmon (Oncorhynchus kisutch) from the 1978 broodstock, which had been selected as having little or no Saprolegnia infection, were used as test animals. The normal peak spawning period of coho salmon at Robertson Creek is between Nov 7 and Nov 21. HOLDING FACILITIES The test animals were maintained in 2 pens, 2.29 m long, 1.83 m wide and 1.68 m high. During the injection procedure one of the pens was divided into 2 smaller pens for separating injected from uninjected fish. The pens were situated along one wall of the hatchery raceway which held the coho broodstock. Thewalls of the pens were constructed of 2.5 cm x 5 cm galvanized wire mesh which allowed unimpeded water flow between the pens and the holding pond. The water depth of both the pond and the pens was 0.90 m. The water temperature ranged from 11.9 C on Oct 30 to 7.0 C on Dec 3. HANDLING PROCEDURE The procedure used for anaesthetization and tagging of the fish was identical to that used by Hunter et al., (1978). On the first day (Day 0) test fish were removed from the holding enclosures and transferred to a fiberglas anaesthetic bath containing 0.5 ml/l of 2-phenoxyethanol (Sehdev et al., 1963). After the fish had been anaesthetized, they were weighed to the nearest 10 g and tagged with a numbered Floy spaghetti anchor tag (Floy Tag and Manufacturing Inc., Seattle, Wash.), inserted in the musculature below the dorsal fin. An egg sample was taken by forcing eggs out of the urogenital vent by abdominal massage. This was followed by an injection of the appropriate test solution. The time required for the above procedure did not exceed 2 min. The procedures followed on the fourth day (Day 3) differed from Day 0 in that it was not necessary to weigh and tag the fish. The fish were checked by the Robertson Creek Hatchery staff at 3 day intervals following the second injection. Egg samples used for determining egg maturity were taken on the 2 injection days (Day 0 and Day 3). INJECTION PROC EDURE Separate 3 ml syringes fitted with 2lG 1 1/4 needles (Becton, Dickinson and Co., Rutherford, N.J.), were used for each group. The needle was introduced posterior to the right pelvic fin and inserted anteriorly at a shallow angle into the peritoneal cavity between the pelvic girdle and the ovary. All injection volumes were based on 0.4 ml/kg body weight. TEST SOLlITIONS Saline (0.65%) was used as the vehicle for all preparations tested.

9 -3- Frozen chinook salmon ~ tshawytscha) pituitaries collected in 1973 from the Spring Creek National Fish Hatchery, Washington State, U.S.A., were used in the preparation of a salmon pituitary extract SPE-WV. The pituitaries were weighed and homogenized in saline for 2 min in a Sorvall Omni mixer. The homogenate was centrifuged (9770 g) for 5 min at 2 C. The supernatant was decanted and brought up to the required volume with saline (Hunter et al., 1978). Using the same procedure a second pituitary extract SPE-SD was prepared from pituitaries collected between Oct 11-13, 1978 from maturing chum salmon ~. keta) caught at sea in the commercial fishery during their anadromous migration. The third test solution, AP-SD was made by dissolving an acetone dried pituitary powder at the required concentration in saline. These pituitaries were collected on Oct 5, 1978, also from maturing chum salmon caught at sea. The frozen pituitaries used in the SPE-SD preparation and the acetone dried pituitary powder used in the AP-SD preparation were supplied by Syndel Laboratories Ltd., Vancouver, British Columbia. Determination of egg stage and ovulatory response Egg samples were taken by abdominal massage, on each of the two injection days. The stage of maturity was determined for each sample according to the criteria of Jalabert (1976). The ovulatory response of each fish was judged by the hatchery staff according to standard hatchery procedure. The fish were considered to be ovulated (ripe) on the basis of physical and morphological characteristics such as softness of the abdominal region, body colour, degree of hypertrophy and colour of the urogenital papilla, and ease of egg discharge. SPAWNING Ripe females were stripped according to standard hatchery procedures. The eggs were incubated in Heath incubators (Heath Tecna Corp., Seattle, Wash.), supplied with river vater which rarig~d in temperature from 8 C in mid-nov to 3 C in mid-jan. EXPERIMENTAL PROTOCOL On Oct 30 the 75 fish were divided randomly into 4 groups of 20, 19, 18 and 18 fish. The first group (N = 20, mean wt kg) received an injection of the saline vehicle on both Day 0 (Oct 30) and Day 3 (Nov 2). The second group (N = 19, mean wt 3.04 ~ 0.62 kg) received 10 mg/kg and 50 mg/kg of SPE-WV on Day 0 and Day 3, respectively.

10 - 4 - The third group (N = 18, mean wt 3.11 ~ 0.8 kg) was given 10 mg/kg and 50 mg/kg injections of SPE-SD on Day 0 and Day 3, respectively. The fourth group (N = 18, mean wt kg) received a 1.3 mg/kg injection of AP-SD on Day 0 followed by a 6.5 mg/kg injection of AP-SD on Day 3. RESULTS The cumulative percent ovulations for each experimental group are presented in Fig. 1. Mean days to ovulation for those fish which ovulated within each group are presented in Table 1. Statistical analysis (Analysis of variance) of time to ovulation data indicated that while no significant differences occurred between test groups, all test groups ovulated significantly earlier (P < 0.001) than the control group. Analysis of the cumulative ovulation data (chi square with Yate's correction and the normal approximation to Fisher's exact test) indicated that a significantly greater number of fish ovulated in the SPE-SD groups than in the control group (chi p = , Z P = ) or the SPE-WV group (chi p = 0.036, Z P = 0.038). Comparison of the numbers of fish ovulated in the AP-SD and control groups resulted in p values of and for the chi square and Fishers exact test, respectively. The cumulative percent prespawning mortalities for the control, SPE-WV and AP-SD groups are presented in Fig. 2. The tabulation of ovulated fish did not include 11 fish which had lost tags prior to ovulation or 2 fish (2 SPE-WV) which were stripped prior to the completion of ovulation (Table 1). Two fish from the control group and one from each of the SPE-SD and AP-SD groups were found to be dead but 100% ovulated at the time of sampling. These fish were included in the tabulation of ovulated fish, but no attempt was made to fertilize and rear their eggs. The rate of egg maturation was increased in all test groups following the first injection. Approximately 50% of fish in these groups had eggs undergoing GVBD on Day 3, as compared with 5% in the control group (Table 2). Statistical analysis (Analysis of variance) indicated no significant differences between the mean weights of fish contributing eggs in each group. The number of eggs surviving to the eyed stage for each experimental group are given in Table 3. The percentage survival ranged from 83.7 in the AP-SD group to 93.6 in the SPE-SD group (Table 3). Eggs from individual fish in each group which spawned on a particular day were pooled. This precluded statistical analysis of the percentage survival. The hatchery staff noted that the amount of ovarian fluid present was much greater than normal in a few test fish. The excess ovarian fluid was held responsible by the hatchery staff for the premature stripping of the 2 SPE~~V fish mentioned previously. The eggs were fertilized without draining off this excess fluid. It must be noted that the percentage survivals given in Table 3 represent the total eggs per group surviving to the eyed stage. The percentage survi~al for egg lots taken on individual days were either very high ( > 95%) or relatively low «80-85%). This variation in survival rates occurred in

11 - 5 - both control and test groups with the exception of the SPE-SD group (Table 4). DISCUSSION While the absence of both non-injected and production control groups prevents direct comparisons between the experimental groups and normal production fish the successful results of this first hatchery study on the induced ovulation of coho salmon indicate that this technique shows considerable promise for the aleviation of prespawning mortality in this species. In all treated groups, the mean time to ovulation was significantly shorter than in the control group. As a consequence, there was a higher incidence of prespawning mortality in the control group compared to the three treatment groups. Examin~tion of the cumulative prespawning mortalities in the AP-SD, SPE-WV and control groups (Fig. 2) indicates that seven (37% of control group) of the eight mortalities in the control group occurred during the period of time in which the fish in the three test groups were ovulating (Day 5-16, Fig. 1). It is interesting that mortalities did not occur in the SPE-WV and AP-SD groups until the end of the above period (Days 12-19, Fig. 2). This would indicate that while increased handling stress due to experimental protocol may have increased the rate of mortality in the controls relative to the production stock the rate was not increased relative to the three test groups. This agrees well with the results obtained by Hunter et al., (1978) with chinook salmon. It is notable that these differences were found even though only the healthiest fish, based on severity of saprolegniasis, were selected from the raceway. Furthermore, this acceleration of ovulation was possible even though 33-35% of the fish in each group had ova at the germinal vesicle migration stage at the time of the first injection. The induction of ovulation in treated fish resulted in a larger egg take for those groups. As there were no overall differences in the percentage survival to the eyed stage, there was a resultant increase in the numbers of eyed eggs in all the treated groups. The smallest increase over controls was in the SPE-WV group where two females were stripped early in the experiment and prior to the completion of the maturation and ovulatory process. There is no clear cut explant ion for the differences in survival to the eyed stage among fish in the various groups spawned on different days. Only in the SPE-SD group were there no instances of survival below 80%. This suggests that the hormone treatments per se were not responsible for the variation. Survival of coho eggs to the eyed stage at Robertson Creek Salmon Hatchery normally varies between '94 and 97%. At the time of stripping, hatchery personnel noted that there was an abnormal accumulation of ovarian fluid in several of the females. As the ova from these fish were not incubated separately, it is not possible to tell whether there was a relationship between ovarian fluid accumulation and low survival to the eyed stage. Excess ovarian fluid has been observed in chinook salmon (Hunter et al., 1978) and coho salmon (Donaldson et al., 1978, unpublished) treated with salmon pituitary extracts. However, the phenomenon has

12 - 6 - also been observed in control females. Information from unpublished laboratory tests indicates that the accumulation of ovarian fluid may be related to the use of pituitary extracts as opposed to purified gonadotropin preparations. Recent work on coho salmon indicates that eggs taken from females treated with salmon pituitary extracts had survival percentages of 97% and 98% (Donaldson et al., 1977 and 1978, unpublished). In the above experiment~ excess ovarian fluid was drained prior to fertilization. The dosage chosen for both the SPE-WV and SPE-SD treatments was equivalent to the dose found effective for inducing ovulation in chinook salmon (Hunter et al., 1978). The dosage for the acetone powder preparation (AP-SD) was arrived at by calculating the yield of acetone powder from frozen pituitary glands. No allowance was made for any losses of biological activity during processing. As indicated above, the mean time to ovulation was not significantly different between the hormone treated groups but all were different flom the contlol gloup. The measule of mean days to ovulation however, only takes into account those fish which ovulated. The statistical analysis of the number of fish which ovulated or died in each group indicated that the SPE-SD, and to a lesser degree the AP-SD groups,had significantly higher cumulative ovulations than the control group. Differences between the number of fish ovulating in the SPE-WV and SPE-SD groups were also determined. If the two SPE-WV fish which were stripped prior to ovulation had been allowed to ovulate the differences between the three test groups would have been negated. There are several possible variables which could give rise to differences in the success of the three treatment groups. The superior performance of the salmon pituitary extract could be a result of a slight loss in activity during the processing of the acetone powder. The salmon pituitaries used for the SPE-WV had been collected from chinook salmon in 1973 and held frozen since that time, while the SPE-SD pituitaries had been collected from chum salmon in 1978, just prior to the experiment. While pituitary glands or salmon gonadotropin can be held in frozen storage for long periods, it is possible that some loss of activity may occur (Donaldson et al., 1978b). Apart from this, it is possible that there may h ave been a s pecies difference in gonadotropin content. The SPE-WV preparation has induced 100% ovulation in maturing coho in laboratory tests at West Vancouver (Donaldson et al., 1978, unpublished), suggesting that it was the premature stripping of the two females early in the experiment that resulted in the lower cumulative percentage ovulation in this group. Based on the results presented, future research on the induced ovulation of coho in production facilities will be oriented towards the use of single injection procedures to minimize the handling of the fish, and the use of promising synthetic inducing agents. Also, further work is required to determine the optimum date on which to treat the fish at a particular facility.

13 - 7 - ACKNOWLEDCMENTS We wish to thank the staff of the Spring Creek National Fish Hatchery for assistance in the collection of chinook salmon pituitary glands and Syndel Laboratories Ltd. for the provision of churn salmon pituitary glands in the frozen state and prepared as an acetone powder. We also acknowledge the assistance provided by the staff of the Robertson Creek Hatchery and by Dr. Maria Alice Ramos, a visiting scientist from Servi~o de Inspec~a da Ca~a E Pesca, Lisboa, Portugal. REFERENCES Donaldson, E.M., G. Hunter, and H.M. Dye Induced ovulation in the coho salmon (Oncorhynchus ~isutch) using salmon pituitary preparations, gonadotropin releasing hormones and an antiestrogen. Western Reg. Conf. on Gen. Compo Endocrinol., Univ. California, Santa Cruz. March Abstr. 24. Donaldson, E.M., H.M. Dye, and B.F. Wright The effect of storage conditions on the biological activity of salmon gonadotropin. Ann. BioI. animo Bioch. Biophys. 18(4) Donaldson, E.M., G.A. Hunter, and H.M. Dye Relative potency of gonadotropin releasing hormone and gonadotropin releasing hormone analogues for induced ovulation in the coho salmon Oncorhynchus kisutch. West. Reg. Conf. Compo Endocrinol., Eugene, Oregon, March Abstr. Hunter, G.A., E.M. Donaldson, E.T. Stone, and H.M. Dye Induced ovulation of female chinook salmon Oncorhynchus tshawytscha at a production hatchery. Aquaculture. 15: Jalabert, B., C. Bry, B. Breton, and C. Campbell Action de la l7a-20b dihydroprogest~rone et de la progest~rone sur la maturation et l'ovulation in vivo et sur Ie niveau d'hormone gonadotrope plasmatique, t-gth chez la Truite Arc-en-ciel Salmo gairdneri. C.R. Acad. Sci. Paris, Ser. D. 283: Jalabert, B., F.W. Goetz, B. Breton, A. Fostier, and E.M. Donaldson Precocious induction of oocyte maturation and ovulation in coho salmon Oncorhynchus kisutch. J. Fish. Res. Board Can. 35: Neish, G.A Observations on saprolegniasis of adult sockeye salmon, Oncorhynchus nerka (Walbaum). J. Fish BioI. 10: Sehdev, H.S., J.R. McBride, and U.H.M. Fagerlund phenoxyethanol as a general anaesthetic for sockeye salmon. J. Fish. Res. Board Can. 20:

14 Table 1. Fate, cumulative percent ovulation and mean days to ovulation of induced and control female coho salmon. No. of females Cumulative ovulation No. of females Treatment retaining Prespawning stripped prior Mean days to group original tags mortality to ovulation number percent ovulation Control SPE-WV SPE-SD co AP-SD

15 - 9 - Table 2. Effect of treatment on the rate of egg maturation as indicated by germinal vesicle migration (GYM) and germinal vesicle breakdown (GVBD). Stage of egg maturation Day 0 Day 3 Treatment group % GYM % GVBD 0/0 GYM % GVBD Control SPE-WV SPE-SD AP-SD

16 Table 3. Number and mean weight of females contributing eggs and the number and percent survival of eggs at the eyed stage. No. of eggs Treatment No. of females Mean weight surviving to Percent survival group contributing eggs of females eyed stage to eyed stage Control SPE-W SPE-SD AP-SD

17 Table 4. Number of eggs surviving and percent survival to the eyed stage of individual egg lots. Control SPE-WV SPE-SD AP-SD No. eggs No. eggs No. eggs No. eggs Date of at eyed Percent at eyed Percent at eyed Percent at eyed Percent egg take stage survival stage survival stage survival stage survival Nov r-' r-' Dec

18

19 SPE-SD 90 AP-SD Z ~ ~...I ::) ~ 50 ~ 40 Z w U 0:: 30 w 0.. SALINE DAYS 0 DATE Oct Nov 9 20 Nov 19 ~ + Fig. 1 - Induced ovulation of coho salmon at Robertson Creek. The number of fish in the SPE-WV, SPE-SD, AP-SD and control 30 Nov 29 groups which ovulated are expressed as a cumulative percentage of the respective groups. Arrows indicate the days on which the two injections were administered.

20

21 > t I 50 ~ 0=: o ~ 40 _ ~SiAALI NE t Z w 30 u ~ W Q.. 20 SPE-WV 10 DAYS 0 DATE Oct Nov 9 20 Nov 19 Fig. 2 - The cumulative percent pres pawning mortalities for the AP-SD, SPE-WV and control group are presented. No mortalities occurred in the SPE-SD group. Arrows indicate dates on which in'ections were administered. 30 Nov 29