1+1. Canadian Technical Report of Fisheries and Aquatic Sciences , ~r i~i~:-.:" ~.

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1 Scientific Excellence Resource Protection & Conservation Benefits for Canadians Excellence scientifique Protection et conservation des ressources Benefices aux Canadians The Effect of Hormone Dose, Water Temperature and Salinity on Growth, Smolting and Histology of Underyearling Coho Salmon (Oncorhynchus kisutch) Sterilized by Treatment with 17 a-methyltestosterone J. E. Shelboum, W. C. Clarke, J. R. McBride, D. A. Higgs and E. M. Donaldson, ~r i~i~:-.:" ~. Department of Fisheries and Oceans /. ~ tj ' Biological Sciences Branch <:t Pacific Biological Station { Nanaimo, British Columbia V9R 5K Canadian Technical Report of Fisheries and Aquatic Sciences Fisheries Peches and Oceans et Oceans Canada

2 Canadian Technical Report of Fisheries and Aquatic Sciences Technical reports contain scientific and technical information that contributes to existing knowledge but which is not normally appropriate for primary literature. Technical reports are directed primarily toward a worldwide audience and have an international distribution. No restriction is placed on subject matter and the series reflects the broad interests and policies of the Department of Fisheries and Oceans, namely, fisheries and aquatic sciences. Technical reports may be cited as full publications. The correct citation appears above the abstract of each report. Each report is abstracted in Aquatic Sciences and Fisheries Abstracts and indexed in the Department's annual index to scientific and technical publications. Numbers in this series were issued as Technical Reports of the Fisheries Research Board of Canada. Numbers were issued as Department of the Environment, Fisheries and Marine Service, Research and Development Directorate Technical Reports. Numbers were issued as Department of Fisheries and the Environment, Fisheries and Marine Service Technical Reports. The current series name was changed with report number 925. Technical reports are produced regionally but are numbered nationally. Requests for individual reports will be filled by the issuing establishment listed on the front cover and title page. Out-of-stock reports will be supplied for a fee by commercial agents. Rapport technique canadien des sciences halieutiques et aquatiques Les rapports techniques contiennent des renseignements scientifiques et techniques qui constituent une contribution aux connaissances actuelles, mais qui ne sont pas normalement appropries pour la publication dans un journal scientifique. Les rapports techniques sont destines essentiellement a un public international et ils sont distribues a cet echelon. 11 n'y a aucune restriction quant au sujet; de fait, la serie reflete la vaste gamme des interets et des politiques du ministere des Peches et des Oceans, c'est-a-dire les sciences halieutiques et aquatiques. Les rapports techniques peuvent etre cites comme des publications completes. Le titre exact parait au-dessus du résumé de chaque rapport. Les rapports techniques sont résumés dans la revue Résumés des sciences aquatiques et halieutiques, et ils sont classes dans l'index annual des publications scientifiques et techniques du Ministere. Les numeros 1 a 456 de cette serie ont ete publies a titre de rapports techniques de ]'Office des recherches sur les pecheries du Canada. Les numeros 457 a 714 sont parus titre de rapports techniques de la Direction generale de la recherche et du developpement, Service des peches et de la mer, ministere de l'environnement. Les numeros 715 a 924 ont ete publies a titre de rapports techniques du Service des peches et de la mer, ministere des Peches et de l'environnement. Le nom actuel de la serie a ete etabli lors de la parution du numero 925. Les rapports techniques sont produits a ]'echelon regional, mais numerotes ]'echelon national. Les demandes de rapports seront satisfaites par l'etablissement auteur dont le nom figure sur la couverture et la page du titre. Les rapports epuises seront fournis contre retribution par des agents commerciaux.

3 Canadian Technical Report of Fisheries and Aquatic Sciences THE EFFECT OF HORMONE DOSE, WATER TEMPERATURE AND SALINITY ON GROWTH, SMOLTING AND HISTOLOGY OF UNDERYEARLING COHO SALMON (ONCORHYNCHUS KISUTCH) STERILIZED BY TREATMENT WITH 17u-METHYLTESTOSTERONE. by J. E. Shelbourn, w. c. Clarke, J. R. McBride 1, D. A. Higgs 1 and E. M. Donaldson 1 Department of Fisheries and Oceans Biological Sciences Branch Pacific Biological Station Nanaimo, British Columbia V9R 5K6 1 West Vancouver Laboratory 4160 Marine Drive West Vancouver, British Columbia V7V 1N6

4 - ii - (c) Minister of Supply and Services Canada 1992 Cat. No. Fs 97-6/1869E ISSN Correct citation for this publication: Shelbourn, J. E., w. C. Clarke, J. R. McBride, D. A. Higgs and E. M. Donaldson The effect of hormone dose, water temperature and salinity on growth, smolting and histology of underyearling coho salmon (Oncorhynchus kisutch) sterilized by treatment with 17u-methyltestosterone. Can. Tech. Rep. Fish. Aquat. Sci. 1869: 15 p.

5 - iii - ABSTRACT Shelbourn, J. E., W. C. Clarke, J. R. McBride, D. A. Higgs and E. M. Donaldson The effect of hormone dose, water temperature and salinity on growth, smolting and histology of underyearling coho salmon (Oncorhynchus kisutch) sterilized by treatment with 17a-methyltestosterone. Can. Tech. Rep. Fish. Aquat. Sci. 1869: 15 p. Sterilization of coho alevins was initiated by immersion in a 200 ~/1 solution of 17a-methyltestosterone (MT) at emergence and 1 week later. Subsequently, MT was provided in the diet at 12.5 or 25 mg/kg for 8 or 12 weeks to complete sterilization. Fry were reared in fresh water at 8, 10 and 12 C for 16 weeks, then transferred to 30 sea water at 14 C. Control fish had outgrown steriles at 8 and 12 C by the time of seawater entry and they had surpassed all sterilized fish, regardless of treatment, after a further 10 weeks in sea water. Blood sodium levels were below 170 rnrnol/1 at the time of seawater entry, indicating that the fish had smolted. There were no signs of gonadal development at seawater entry. However, incomplete sterilization was noticed at the terminal examination in a few male fish. There was no gonadal development in females. Epidermal thickening was noted in fish given hormone. Skin thickness was normal at termination. Key words: coho, sterilization, growth, smolting, histology

6 - iv - RESUME ' ' Shelbourn, J. E., w. C. Clarke, J. R. McBride, D. A. Higgs and E. M. Donaldson The effect of hormone dose, water temperature and salinity on growth, smelting and histology of underyearling coho salmon (Oncorhynchus kisutch) sterilized by treatment with 17u-methyltestosterone. Can. Tech. Rep. Fish. Aquat. Sci. 1869: 15 p. La sterilisation des larves du saumon coho a ete initiee au moment de!'emergence puis une semaine plus tard, en les immergeant dans une solution de 17u-methyltestosterone (MT) a La MT a ensuite ete ajoutee a!'alimentation a une 200~gjl. concentration de 12.5 ou 25 mgjkg pendant huit a douze semaines afin de completer la sterilisation. Des alevins ont ete eleves dans l'eau douce a 8, 10 et l2 C pendant 16 semaines puis transferee dans l'eau de mer a 30 salinite et a 14 C. Lors de leur entree dans l'eau de mer, les poissons temoins ont une taille superieure a celle des poissons sterilises eleves a 8 et 12 C et ont depasse tous les autres poissons sterilises, independamment du traitement, apres une periode prolongee de dix semaines. Les niveaux de sodium du sang etaient inferieurs a 170 mmol/1 au moment de!'entree dans l'eau de mer, ceci indiquant que les poissons sont devenus des saumoneaux. Aucun s1gne de developpement L de gonades n'a ete ' ' observe ' lors de leur entree dans l'eau de mer. Neanmoins, une sterilisation incomplete a ete notee chez quelques poissons males lors du dernier examen. Aucun developpement des gonades n'est apparu chez les femelles. Un epaississement de l'epiderme a ete observe chez les poissons ayant re~u des hormones. L'epaisseur de l'epiderme est devenu normale a la fin de!'experience. Mots cles: histologie coho, sterilisation, croissance, saumoneau,

7 INTRODUCTION Hunter et al. (1982) demonstrated that yearling coho salmon were sterilized by immersion of eggs and larvae in solutions of 17a-methyltestosterone (MT) followed by inclusion of MT in the diet for 12 weeks. These fish grew for at least 2 years beyond the age of normal sexual maturation (and death in most Pacific salmon). Recently, Shelbourn et al. (1992) tested the same technique of immersion and oral MT treatment for 12 weeks on zero age coho. However, in contrast to the response of yearlings, zero-age coho grew poorly relative to controls both in fresh water after the withdrawal of the hormone and after subsequent transfer to sea water. Losses of 3% were reported. In the current study, coho salmon from Big Qualicum River stock were raised using different combinations of MT dose, duration of treatment and rearing temperature. The object was to determine the minimal effective dose and duration of treatment for producing sterile zero age coho smolts, fully seawater tolerant and having growth performance comparable with normal intact coho. MATERIALS AND METHODS Several returning adult coho to the Big Qualicum River {Vancouver Island) were stripped at the Big Qualicum hatchery on 28 November, and the pooled gametes were brought to the Pacific Biological Station where the eggs were fertilized. The eggs were incubated in screened hatchery trays, and hatched on 11 January. Incubation temperatures were maintained at approximately 10.5 C. Eggs designated for sterilization were given a 2-hour bath of 17a-methyltestosterone (MT) at a concentration of 200~g/l on 14 January and again on 21 January (Goetz et al. 1979). On 9 February 1987, the alevins were ponded into stock tanks at l0 C, and on 26 February they were distributed randomly, putting 100 fish into each of 18 tanks in two rooms. The daylength was fixed at 9.5 hours. At this time there was a residual showing of yolk, although the fish were fully buttoned up. All the fish in each tank were lightly anaesthetized {2-phenoxyethanol, 0.5 ml/1) and weighed on 26 February, and the temperatures were adjusted to the 8 and l2 C levels on the next day: this was deemed the start of the experiment. The fish were weighed subsequently at three week intervals. Data were subjected to analysis of variance (ANOVA) using the SAS GLM

8 - 2 - procedure -- a linear regression model for unbalanced data (SAS, 1985). Temperatures were recorded daily. The ranges of means of the recorded temperatures were , and C respectively in fresh water, and C in sea water. Photoperiod was held at 9.5 h/d until 10 April, when the on/off timers were set to the current 14.5 h/d natural daylength, and were reset daily thereafter to follow the natural cycle. Samples of ten fish per tank were removed for the seawater challenge test (Blackburn and Clarke, 1987) on 22 June and the tank water supply was then switched to local sea water (30 ), adjusted to 14 C by 2 /d steps and maintained at that level for the duration of the experiment. The fish were weighed seven days after transfer and three times more before termination of the study on 1 September. On 10 April, five fish per tank were removed for histological examination, and again on 8 May and 18 June. Twenty-five fish per tank were taken at the termination of the experiment on 1 September. The fish were fed the P-10 diet (Higgs et al., 1985) with either 0 (control), 12.5 or 25 mg of supplemental MT/kg, depending on temperature level (see Table 1). Feeding with MT started 13 February and continued for 8 or 12 weeks from this date, i.e., to 10 April or 8 May respectively. The 12.5 mg/kg dose level in P-10 was higher than the dosage of MT incorporated into the Oregon Moist (OMP) diet at 10 mgjkg in the previous study (Shelbourn et al., 1992). This higher dosage was necessary in order to ensure that the fish in the current study received a similar absolute intake of hormone to the previous study. The energy density of OMP is lower than that of P-10 on an as-fed basis and hence more OMP is consumed by the fish than P-10 to meet their daily energy needs. After 8 weeks treatment, the fish on the shorter duration were fed control P-10, and after 12 weeks all groups were changed to White Crest dry feed, a local commercial diet. The initial stocks were reduced by approximately 10% in the first month by culling pinheads, by escapement or from physical damage during tank cleaning.

9 - 3 - RESULTS GROWTH PERFORMANCE Initial weight means for the 18 groups ranged from 0.37 to 0.40 g, with an overall mean of 0.38 ±O.Ol(s.d.). The means were not significantly different (p=0.47). Mean weights and s.e. of the replicates at time of entry to sea water are shown in Figure la, and similarly at termination in sea water in Figure lb. Analysis of variance showed that the effects of temperature on weight were highly significant regardless of hormone dose or duration (p<0.01). In subsequent evaluations the data were analyzed as three separate temperatures. Analysis of the data for Figure 1 showed no significant effect of duration of hormone treatment on weight during the experiment (p=0.27), hence treatment duration was pooled during subsequent statistical analyses. However, the effect of dosage (MT level) on weight was significant throughout the experiment (p<0.05), except for the l0 C groups in fresh water (p=0.44). Hormone-treated fish weighed less than the controls in all other cases (p<o.ol), while the two MT levels tested at l0 C did not result in significant differences in weight (Tukey's HSD test, p>0.05). The marked discrepancy in growth in sea water between the two replicates raised initially at l0 C, and fed 25 mg MT/kg for 8 weeks (Fig. lb) is due to the unexplained failure of one of the two replicates to perform in sea water and the success of the other. It is apparent from Figure 1 that, although variability in weight had increased by the end of the experiment, the trend was to greater discrepancy between control fish and those previously treated with MT during the sea water phase than was noted during the time of hormone administration in fresh water. PLASMA SODIUM LEVEL The results of plasma sodium level determinations are shown in Table 1. The number of individual fish whose sodium values fall below the 170 rnrnol/1 level indicates the percentage accepted as seawater tolerant according to the method of Blackburn and Clarke (1987). The average plasma sodium level was below 170 mmol/1 in every case, indicating that most fish had smolted. There were no significant differences in plasma sodium levels due to temperature, dose, or duration of treatment (p>0.05).

10 - 4 - HISTOLOGY In histological samples, no pathology was noted in the liver or kidney of any of the groups - in all cases the structure of these organs was as expected for normal, actively feeding juvenile salmonids. Some fish developed darkened skin on the caudal region ("blacktail'') after two weeks from the start (Table 2). This was found in all groups, although more frequently in hormone-fed fish than in controls. Samples examined by the Fish Health Unit at the Pacific Biological Station, Nanaimo, B.C. showed that no infectious agents were present. The darkening disappeared upon anaesthesia or death. Histological sections of skin from those fish with marked darkening of the tail region showed no differentiation from those with tails of normal pigmentation. Skin thickening occurred predictably in the MT treated fish (McBride and Fagerlund, 1973; Yamazaki, 1972). The epidermis had reduced to near normal thickness at the time of seawater entry (Table 3). Skin thickness at both week 8 and at week 12 (end of the two dosing times) was not significantly affected by temperature (p>o.os), but the difference was highly significantly in respect to hormone dose (p<o.ool, Table 3). Some partially sterile fish were found in the terminal sample (males only). This suggests that the marked atrophy of the germ cells in the earlier samples might have been masking the recognition of incomplete sterilization (Fig. 2). It is estimated that this area of active differentiation was less than 5% of the area of the testis in a comparable control fish. One incompletely sterilized fish of the 247 sampled had been given MT for 12 weeks, 11 others had received hormone for 8 weeks. However, only these few males were not fully sterile: all the females appeared to be completely sterile. ROOM (PHOTOPERIOD) EFFECTS Photoperiod was not designed to be a variable in this study, but the accidental exposure of fish in one room to 24 h light for a possible maximum of 3 weeks after the start of the experiment allowed an examination of the effect of daylength on performance. It was noted that fish in the groups exposed to extra daylength tended to grow less rapidly than the others, but there was no statistical significance to room effect (p>0.05).

11 - 5 - PERCENT MORTALITY There was high mortality in both control and experimental groups at 10 C and 12 C in fresh water, which decreased later when the fish were in sea water. Early in the freshwater stage of the experiment, infestations of gill and tail fungus (Saprolegnia sp.) were found in several tanks of fish, including controls. Treatment with malachite green on two occasions helped to reduce the outbreak, with the loss of some fish during the second treatment. Survivors resumed a normal appetite and growth. Percent mortality data are shown in Table 2. DISCUSSION GROWTH Specific growth rates for control and treated fish in fresh water are comparable with those reported elsewhere (e.g., 3.3 and 3.2%/d for control and treated fish at 10 C here, compared with 3.2%/d at 10 C for 12 weeks initial growth reported by Clarke et al., 1981). Fagerlund and McBride (1975) examined the response of intact coho fry to dietary MT from a size of 0.8 g for a period of 72 weeks, with the last 14 weeks in sea water. They reported that a dose level of 1 mgjkg diet promoted growth to a greater degree than food without supplemental hormone throughout the experiment. A level of 10 mg/kg had a greater effect, but the growth of hormone-treated coho in sea water decreased to the level of controls. The weights reported by Fagerlund and McBride are comparable with those here (until the change to 14 C sea water). The fish used in the present study were accelerated underyearling coho. Some of the coho were transferred from fresh water at 10 C, and after 10 weeks in 14 C sea water they had mean body weights of 21.1 g and 32.8 g for sterile and control fish respectively. From the previous study of accelerated underyearling coho (Shelbourn et al., 1992, Table 5.), we note means of 21.8 g and 32.7 g for sterile and control underyearling coho reared at l0 C in fresh water and in 10 -l3 C sea water for 10 weeks. Clearly, the mean body weight of coho in the previous study was lower in sterile fish than in controls to the same degree as in the current one (ca. 35%). This finding emphasizes

12 - 6 - the lower growth performance of MT-sterilized coho in sea water in both instances. Reference to Figure 1 shows that this is also apparent for the current coho underyearlings reared in fresh water at 8 and 12 C. Other workers have reported that sterilized fish have grown for several months after the sexual maturation of their non-sterile cohorts. For example, length data reported by Hunter et al. (1976) shows an increase from 43 to 48 ern in sterile coho after one year's growth in sea water. Fagerlund and McBride (1975) reported good survival with reduced growth in coho given MT at 10 rngjkg and transferred to sea water, and to a lesser extent these trends were seen in a group previously treated with MT at 1 mgjkg. For this last group, the mortalities were less than the 4% recorded for controls. Hunter et al. (1982) reported 6% mortality in MT-sterilized coho when the dose of hormone was 10 rngjkg diet. Mortality was low after the fish were transferred to sea water in the present study, and given no occurrence of disease it would appear to be possible to rear MT-sterilized coho for an extended time in sea water, albeit at a slower rate of growth than in normal fish. Similarly, it should be possible to extend the life of sterile coho in sea water beyond the time of normal maturation. It is noteworthy that complete sterilization of juvenile coho via the oral route required 12 weeks of treatment with MT at a dosage of 12.5 rngfkg using a dry pelleted feed. The growth performance of the sterile coho underyearling srnolts was below established levels for similar, non-sterilized coho. STERILIZATION Sterilization was successful, with minor exceptions. Other authors have reported various degrees of sterilization in salmonids following androgen treatment by immersion and/or by inclusion in the diet. For example, Miwa and Inui (1986) reported spermatogenesis in some of the juvenile amago salmon (Oncorhynchus rhodurus) sterilized by immersion in MT followed by oral administration of MT at 10 mgjkg diet for 60 days. Baker et al. (1988) obtained sex-reversal but not sterilization in chinook salmon fry given single immersions in MT, without application of hormone in the diet. Goetz et al. (1979) reported full sterilization of coho salmon when the supplemental feed level of MT was 20 mg/kg.

13 - 7 - SMOLTING The present stock was raised under a short (9.5 h) daylength for two months from first feeding, which has been shown to accelerate smolt development in coho fry (Clarke and Shelbourn 1986). Our data suggest that most fish had smolted according to criteria of the seawater tolerance test of Blackburn and Clarke (1987). Although MT treatment has been shown to decrease smolting in salmonids (e.g. Miwa and Inui 1986), our fish would likely have recovered from the effects of hormone treatment as indicated by the pervasive low plasma sodium values (regardless of dose and duration), and the reversal of the skin-thickening phenomenon after hormone withdrawal (McBride and Fagerlund 1973). CONCLUDING REMARKS Goetz et al. (1979) reared coho underyearlings under natural daylength and local water temperature (ca. 5 C). They reported complete sterilization in coho fry weighing 2.2 g, 13 weeks after swim-up. The fish were given MT orally at 20 mgjkg diet. Coho underyearlings reared in our previous experiment, under a short daylength (growth-promoting) in 10 C water, weighed 5.8 g after 12 weeks of feeding a moist diet with MT at 10 mg/kg. They were fully sterile (Shelbourn et al. 1992). In the present study, the 8 week MT treatment at 12.5 mg/kg did not provide complete sterilization, whereas 12 weeks of treatment was successful (with one exception). Our records show that 8 weeks after ponding, the MT-fed coho held at 10 C had a mean weight of 2.5 g, and after 12 weeks they weighed 4.7 g. It is suggested that a weight of 5 grams may be the threshold size for coho to complete sterilization at a dietary level of 12.5 mg/kg with the P-10 diet, presented for at least 8 weeks but less than 12 weeks. A higher dietary level of MT (25 mgjkg), although not shown to be toxic, was not seen to be advantageous for sterilization, seawater tolerance or growth performance of accelerated coho smolts in sea water. Thus, MT treatment of newly-hatched coho is not at present a recommended strategy for production of sterile, accelerated, zero age coho smolts, because of the poor growth performance obtained. To date, coho fry sterilized by MT treatment, regardless of hormone dose and duration or rearing temperature, have not exhibited growth performance comparable to their intact counterparts. This situation may be a consequence of

14 - 8 - suboptimal titres of anabolic hormones (e.g. steroids and thyroid hormones) in the sterilized fish. Further investigation should elucidate the solution to the problem. ACKNOWLEDGEMENTS The study would not have been possible without the particular assistance of Ian Baker, John Blackburn, Bill Damon, Ulf Fagerlund, Dominique Hervio, Karen Mullen and Igor Solar: all of whom we thank for their effort on our behalf. REFERENCES Baker, I. J., I. I. Solar, and E. M. Donaldson Masculinization of chinook salmon (Oncorhynchus tschawytscha) by immersion using 17«-methyltestosterone around the time of hatching. Aquaculture 17: Blackburn, J. and w. C. Clarke Revised procedure for the 24 hour seawater challenge test to measure seawater adaptability of juvenile salmonids. Can. Tech. Rep. Fish. Aquat. Sci. 1515: 35 p. Clarke, w.c. and J. E. Shelbourn Delayed photoperiod produces more uniform growth and greater seawater adaptability in underyearling coho salmon (Oncorhynchus kisutch). Aquaculture 56: Clarke, W. C., J. E. Shelbourn, and J. R. Brett Effect of artificial photoperiod cycles, temperature, and salinity on growth and smelting in underyearling coho (Oncorhynchus kisutch), chinook (0. tshawytscha), and sockeye (0. nerka) salmon. Aquaculture 22: Fagerlund, U. H. M. and J. R. McBride Growth increments and some flesh and gonad characteristics of juvenile salmon receiving diets supplemented with 17a-methyltestosterone. J. Fish Biol., 7: Goetz, F. w., E. M. Donaldson, G. A. Hunter, and H. M. Dye Effects of estradiol-17~ and 17a-methyltestosterone on gonadal differentiation in the coho salmon, Oncorhynchus kisutch. Aquaculture 17:

15 - 9 - Higgs, D. A., J. R. Markert, M. D. Plotnikoff, J. R. McBride, and B. s. Dosanjh Development of nutritional and environmental strategies for maximizing the growth and survival of juvenile pink salmon (Oncorhynchus gorbuscha). Aquaculture 47: Hunter, G. A., E. M. Donaldson, F. M. Goetz, and Production of all-female and sterile experimental evidence of male heterogamety. Fish. Soc. 111: P. R. Edgell. coho salmon, and Trans. Amer. McBride, J. R. and U. H. M. Fagerlund The use of 17"-methyltestosterone for promoting weight increases in juvenile Pacific salmon. J. Fish. Res. Board Can. 30: Miwa, S. andy. Inui Inhibitory effects of 17«-methyltestosterone and estradiol-17~ on smoltification of sterilized amago salmon (Oncorhynchus rhodurus). Aquaculture, 53: SAS SAS User's Guide: Statistics, Version 5 Edition. SAS Institute Inc., Cary, NC, USA. 56 pp. Shelbourn, J. E., W. c. Clarke, J. R. McBride, u. H. M. Fagerlund, and E. M. Donaldson The use of 17" methyltestosterone and 3,5,3'-triiodo-L-thyronine for sterilizing and accelerating the growth of zero-age coho salmon smelts (Oncorhynchus kisutch). Aquaculture 103: Yamazaki, F Effects of methyltestosterone on the skin and gonads of salmonids. Gen. Comp. Endocrinol. Supp. 3:

16 Table 1. Mean plasma sodium levels (mmol/1 ±s.e.) of fish in all groups sampled prior to entry into sea water with percentage of fish having plasma sodium values below 170 mmol/1, indicated by square brackets. (Sample size in parenthesis). For further details, see text. Hormone dose (ppm) MT dose duration: 8 weeks 12 weeks 8 c series Control ±1.6 [90%] ±2.1 [85%) ( 20) (20) ±1.2 [95%) (19) 10 c series Control ±1.4 [77%] ±1. 6 [85%] ±1.7 [90%) (20) (20) ±0.9 [97%] (20) (20) ±1.0 [92%] ( 2 0) l2 C series Control ±1. 3 [90%] ±1. 8 (100%) (10) ( 5 )

17 Table 2. Percent mortality for the replicate groups (percent of number at start of each period shown), and the recorded incidence of blacktail 1 Dose Natural mortality level I time Fresh water 14 c sw Blacktail recorded on 22L4, mglkg weeks 26L2-10L4 1oL4-22L6 22L6-1L9 % of stock 8 c Series c 2.0% 1. 3% 0% 7% c Series c C Series c see text for description of blacktail.

18 Table 3. Mean epidermal thickness (~m ± s.e.) of fish reared in fresh water at three temperatures and fed P-10 with MT at one of three levels of concentration for 8 and 12 weeks, and of all fish at week two weeks prior to seawater entry. 8 c series Week 8 Week 12 Week 16 Hormone level n n n Control 10 3~.0 ± ± ±1. 3a 12.5 ppm ± ± ±3. 4b 10 c series Control ± ± ±1. 2a 12.5 ppm ± ± ±3. 8b 25.0 ppm ± ± ±3. 1 b 12 c series Control ± ±1. 2a 12.5 ppm ± ±4. 4a Note: Means at week 16 with the same superscript at any one temperature are not significantly different (Tukey's HSD test, p>0.05).

19 MT laval (ppm) C Duration (whks) 8 12 Temperatura (OC) 8 c c Fig. la. Final mean body weights of coho in fresh water, 16 wk after pending, arranged by level and duration of MT treatment and temperature. Bars are replicate pairs (except 12 C group), T-bars are one standard error above the mean (s.e.) :g 20 l:! 0 Gi ;= MT laval (ppm) Duration (waaks) Temperatura (OC) c c c Fig. lb. Final mean body weights of coho after 10 wk in sea water at 14 C. Bars show prior treatment (Fig. la., above).

20 Figure caption. Figure 2. Sections of gonads of juvenile coho salmon depicting responses to administration of MT. All sections stained with H and E. Samples were collected at termination when fish were held in sea water at 14 C. A, ovary of control coho originally reared in fresh water at 8 C. Note oocytes ~ in diameter in perinucleolar stage of development, x 40. B, testis of control coho (orig. 8 C, fresh water). Note nests of spermatogonia and very thin tunica (enclosing membrane), x 40. C, testis of control coho (B, above), showing nests of spermatogonia, x 160. D, testis of treated coho (10 C f.w., 25 mg/kg MT for 12 wk): presumptive sterile. Note absence of spermatogonia and thick tunica (cf. B), x 64. E, testis of treated coho (10 C f.w., 12.5 mg/kg MT for 8 wk): incomplete sterilization. Note spermatogonia (reduced numbers cf. B, above), x 64. F, testis of treated coho (E, above). Note spermatogonia in centre of testis and thickened tunica, x 160.

21 15