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Feminization of coho salmon (Oncorhynchus kisutch) and chinook salmon (Oncorhynchus tshawytscha) by immersion of alevins in a solution of estradiol-17β
Aquaculture, 53 (1986) 295-302 Elsevier Science,Publishers B.V., Amsterdam - Printed in The Netherlands
295
FEMINIZATION OF COHO SALMON (ONCORHYNCHUS KISUTCH) AND CHINOOK SALMON (ONCORHYNCHUS TSHA W YTSCHA) BY IMMERSION OF ALEVINS IN A SOLUTION OF ESTRADIOL-170
G.A. HUNTER, II. SOLAR’,
I.J. BAKER and E.M. DONALDSON
West Vancouver Laboratory, Fisheries Research Branch, Department of Fisheries and Oceans, 4160 Marine Dn’ue, West Vancouver, B.C., V7V IN6 (Canada) (Accepted
31 January 1986)
ABSTRACT Hunter, G.A., Solar, II., Baker, I.J. and Donaldson, E.M., 1986. Feminization of coho salmon (Oncorhynchus kisutch) and chinook salmon (Oncorhynchus tshawytscha) by immersion of alevins in a solution of estradiol-17p. Aquaculture, 53: 295-302. Immersion treatment of newly-hatched coho and chinook salmon alevins in water containing estradiol-17p in concentrations ranging from 200 to 1600 pg/l, for two 2-h periods, 1 week apart, produced 86-96% females and 66-91% females in coho and chinook, respectively. Gonadal morphology of the females produced resembled that of the control females when sampled at 100 and 190 days post-hatch. Variable proportions of male and intersex fish were also observed. There was no evidence of dose-related mortality or differential growth, nor was there a direct relationship between dose level and percent of female fish produced. Compared with effective feminization treatments previously described for these two salmonid species, which include several immersions starting at the eyed-egg stage plus dietary treatment for up to 10 weeks, the treatment described in this report is easy to apply and highly efficient in enhancing the proportion of females for the commercial culture of Pacific salmon.
INTRODUCTION
Among the hormonal techniques to control sex in salmonids, the production of all-female stocks is of considerable interest because of its potential as a management tool in different situations for resource enhancement or for aquaculture. Detailed objectives of the techniques for direct and indirect production of all-female stocks have been reviewed by several authors (Yamamoto, 1969; S&reck, 1974; Donaldson and Hunter, 1982; Hunter and Donaldson, 1983; Yamazaki, 19.83). The indirect technique requires the production (and segregation) of phenotypic males of female genotype (XX) by androgen treatment. The isolated homogametic males will produce a monosex (100% female) progeny when crossed with normal females (Hunter et al., 1982). This technique ’ To whom reprint requests should be sent. 0044-8486/86/$03.50
o 1986 Elsevier Science Publishers B.V.
296
produces fish which are both phenotypically and genotypically female. The main drawback is the lead time required since the desired all-female stock is produced in the second generation. The direct approach involving the use of estradiol-170 (EJ, the major naturally-occurring estrogen in teleosts and higher vertebrates including mammals, is considered a practical alternative because the desired phenotypic females are produced in the immediate generation. The estrogen treatment is fully effective, the induced females are phenotypically indistinguishable from normal females and there is no treatment-related mortality at the appropriate dosage. The main drawback of this method is that the feminized fish may not be used as broodstock. One-half of the phenotypic females produced are genotypic males. They would therefore produce a 3:l male:female ratio when mated with normal males [25% female (XX), 50% male (XY), 25% male (YY)] (Hunter et al., 1982). Earlier attempts to directly feminize Oncorhynchus species using two to 13 immersions of eyed-eggs and alevins followed by dietary treatment for 3-10 weeks have been successful in inducing 60-100% females in coho salmon (0. hisutch) (Goetz et al., 1979; Hunter et al., 1982) and 96100% females in chinook salmon (0. tshawytscha) (G.A. Hunter and E.M. Donaldson, unpublished data, 1978). These results and further tests conducted at the West Vancouver Laboratory show that treatment prior to first feeding is essential and that a high percentage of female coho or chinook salmon may be obtained by treatment at the alevin stage alone. In this report we describe experiments conducted in 1983-1984 at the West Vancouver Laboratory on the direct feminization of coho and chinook salmon by administration of immersion treatments to newlyhatched alevins in water containing the estrogen estradiol-170. MATERIALS
AND METHODS
Experimental fish The chinook salmon gametes were obtained from the Big Qualicum Hatchery (Vancouver Island, B.C.). Fertilization and incubation were carried out at the West Vancouver Laboratory (West Vancouver, British Columbia). For the experiments with coho salmon, eyed-eggs which had been incubated for 94 days at ambient water temperatures ranging from 2.5 to 75°C (398°C days) were obtained from the Capilano Hatchery (North Vancouver, British Columbia). At West Vancouver, the eggs of both species were incubated in Heath trays supplied with 9 l/min well water at 9.5-10.5”C. Prior to hatching the eyed-eggs of each species were divided into five groups of approximately 100 eggs and placed in plexiglass chambers with 1 mm plastic mesh top and bottom which were inserted in the Heath trays (Goetz et al., 1979). Mean (50%) hatch took place on Day 53 post-fertilization (513°C days)
297
for chinook coho salmon.
salmon
and
on Day
96 post-fertilization
(418°C
days)
for
Hormonal treatments On Days 2 and 9 post-hatch the alevins were immersed in estrogen for 2 h (estradiol 17~, Sigma Co. St. Louis, MO) at concentrations of 200, 400, 800 and 1600 pg/l. ,‘l’he immersion treatments were conducted as described by Goetz et al. (1979). Following treatments the Plexiglass chambers containing the alevins were returned to the incubators in trays located below those containing the control groups which were left untreated. On the 5th day after the first immersion treatment, a breakdown in the well water supply to the incubator holding the chinook alevins caused interruption of the normal water flow. The alevins were subsequently maintained for 4 days in static water which was replaced daily by 1.5-3-h flushes with a mix of hot and cold untreated municipal water adjusted to approximately 10°C. Three days after the second treatment, heavy mortalities were observed in these groups including untreated controls. At this time continuous water flow was re-established using the cold municipal water supply. As a result, development of the chinook alevins took place (after treatment) at variable temperatures ranging from 3.5”~~34°C until swim-up, at which time they were transferred to the Rosewall Creek Experimental Hatchery, Vancouver Island. The final incubation, hormone treatments, and post-treatment culture of the coho eggs and alevins were carried out at a different time, hence they were not affected by the accidental failure of the support system described above. Rearing, sampling, and histological procedures At swim-up the alevins were transferred to 50-l tanks supplied with aerated well water at 9-10°C. The fry were fed regular (untreated) Oregon moist pellets (OMP). Mortalities were recorded daily. At 100 and 190 days post-hatch (coho and chinook, respectively), random samples of 4050 fish were taken from each group, weighed (to 0.01 g) and measured (fork length to 0.1 cm). Cross-sections were cut at a point just caudal to the pectoral fins, fixed in Bouin’s, sectioned, and stained with hematoxylin/ eosin for histological sex identification. At Day 146, the remaining coho fry in each experimental group were dissected for visual examination of the gonads. Statistical analysis Means and standard deviations were calculated for length (L, cm), weight (W, g), and condition factor (K). The condition factor was calculated using the formula K=W/L3e2’ X 1000 (Vanstone and Markert, 1968).
298
One-way analysis of variance (ANOVA) was applied to W and K data to examine independently the effect of dosage levels on the size of the experimental fish at the end of the treatment, compared with each other and the control groups (Table 2). Analysis for sex ratio was performed with the Chi-squared test. Male and intersex were combined into one category. Differences (in absolute numbers) from the expected 1:l ratio were considered significant when
P < 0.001. RESULTS
Coho saimon The estrogen (E2) immersion treatment at the preswim-up stage significantly altered the normal sex ratios in all the treated groups compared with the untreated control, which had close to a 50:50 male:female ratio (Table 1). Close to 97% of the fish in the group submitted to two 4OO+g/l estradiol immersions had gonads containing oocytes in the perinucleolar stage, with normal appearance similar to the control ovaries. In the treated groups, the lowest proportion of females (87%) and highest proportion of males (10%) were produced with 200 pg/l Ez. From 2 to 9% of the TABLE 1 Effect of two 2-h estradiol-170 immersion treatments (Days 2 and 9 post-hatch) on sex differentiation of coho (Oncorhynchus kisutch) and chinook (Oncorhynchus tshowytscha) salmon (N = number sexed) Gonadal morphology %
Treatment dose (fig/I)
Survival %
N
Coho salmon* * 200 400 800 1600 0
84.9 86.1 59.8 55.1 98.3
68 59 51 63 32
10.3 3.4 7.8 3.2 56.0
2.9 0.0 2.0 9.5 0.0
86.ga 96.6a 9o.2a 87.38 44.0
-
36 44 44 45 44
16.7 6.8 15.9 0.0 45.5
16.6 11.4 18.2 9.1 0.0
66.7b 81Ba 65.gb 91.18 54.5
Chinook salmon* * * 200 400 800 1600 0
Male
Intersex
Female*
*Significance of difference from 1:l ratio a = P < 0.001; b = P < 0.025 (Chi-squared test). **Age at sampling: 100 days (histological) and 146 days (visual), post-hatch. ***Age at sampling: 190 days post-hatch.
299
TABLE 2 Weight, length and condition factor (K) of coho (Oncorhynchus kisutch) and chinook (Oncorhynchus tshawytscha) salmon treated with two 2-h estradiol-17@ immersions on Days 2 and 9 post-hatch (N = number sampled) N
Length (cm) mean (SD)
Weight (g) K* Mean (SD) mean (SD)
Coho salmon** 200 400 800 1600 0
44 42 42 40 44
5.8 5.9 5.6 5.9 5.9
(0.7) (0.7) (0.8) (0.7) (0.6)
2.29 2.33 2.16 2.37 2.32
(0.9) (0.8) (1.0) (0.7) (0.8)
7.2 7.2 7.6 7.5 7.2
(0.6) (0.8) (0.9)+ (1.2) (0.8)
Chinook salmon*** 200 400 800 1600 0
40 52 50 50 50
9.0 9.1 9.2 8.9 8.9
(0.5) (0.7) (0.6) (0.5) (0.5)
8.31 8.65 8.73 7.78 7.73
(1.5) (2.1) (1.8) (1.7) (1.7)
6.4 6.5 6.4 6.4 6.3
(0.4) (0.4) (0.3) (0.3) (0.6)
Treatment dose (at/l)
*Condition factor K = ( W/L3-25) x 1000. **Age at sampling: 100 days post-hatch. ***Age at sampling : 190 days post-hatch. +Significantly different from control groups (P < 0.05).
estrogen-treated fish in different groups had gonads containing both oocytes, usually in small numbers, and localized areas of undeveloped germinal cells resembling spermatogonia. They were classified as intersex. Survival was higher in the groups receiving the lower doses. The differential mortalities were produced by a fungus-like infection in the groups that received treatments at higher doses (800 and 1600 pg E2). Statistical analysis of weight and condition factor did not indicate significant differences in either parameter, with the exception of the group treated with 800 pg/l where the condition factor was significantly higher (P < 0.05) than those of the control and the other treated groups (Table 2). Chinook salmon The sex ratio was also substantially altered in the experimental chinook salmon groups compared with the controls which showed a normal 1:l ratio. Proportions of female fish with normal ovarian morphology ranged from 66% to 91%, while proportions of fish with gonads containing germinal elements similar to the control males ranged from 0 to 16% (Table 1). The largest number of males was found in the group that received the lowest dose while no males were present in the group treated with the highest dose of the estrogen. The intersex condition, where underdeveloped germinal tissue (spermatogonia) and reduced numbers of oocytes
300
coexisted in the same gonad, was observed in all the experimental groups with a frequency ranging from 9% to 18%. Growth did not seem affected by the dose levels of the immersion treatments. Although the group that received the highest dose had a lower mean weight than the other treated groups, this weight was equivalent to that of the control means and not significantly different from the other treated groups (Table 2). Mortalities in all the chinook groups during the first few days immediately following treatments were negligible. However, as a consequence of a breakdown in the water supply system all the groups suffered similar losses. The mortality was attributed largely to the temporary sub-optimal culture conditions and therefore was not computed. DISCUSSION
These results demonstrate the feasibility of significantly altering the normal sex ratio of coho and chinook salmon towards an enhanced proportion of females by immersion of alevins for two 2-h periods (2 and 9 days post-hatch) in water containing the natural piscine estrogen estradiol-178. This treatment is of much shorter duration than those previously described by Goetz et al. (1979), Hunter et al. (1982), and Donaldson and Hunter (1982) for coho salmon, and by Nakamura (1981) for masu salmon (Oncorhynchus masou). The earlier studies with coho salmon mentioned above included several immersions of eyed-eggs and yolk-sac alevins plus the administration of estrogen in the diet at a dose of 5-10 mg/kg for up to 90 days from first feeding. Nakamura (1981) obtained 100% female masu salmon by 18-day continuous immersion in static water containing 0.5-5 pg/l Ez starting 5 days post-hatch. In this study we did not produce complete (100%) sex reversal of the male portion of the experimental groups: however, the proportions of females produced confirm that the process of sex differentiation in salmon is remarkably labile and highly responsive to the influence of exogenous sex steroids even for very short periods. Close to 97% female coho were produced in this experiment by two 2-h immersions in 400 pg/l estradiol. The rather lower level of success achieved in chinook salmon could be due to the influence of the lower temperature of the rearing water during the critical period of sex differentiation at the time of and subsequent to the treatments. Several authors have discussed the potency of sex steroids in connection with the route of administration (for reviews see Yamamoto, 1969; Hunter and Donaldson, 1983). Estradiol has been described as the most potent naturally-occurring estrogen when administered either in the diet or in the rearing water. Typically, effective treatments for sex reversal in Pacific salmon have included exposure to the sex steroid at a very early stage, including eyed-egg and alevin immersion plus dietary treatment for several
301
weeks. This study shows that the effective treatment for feminization of chinook and coho salmon could be reduced to short-time immersions of newly-hatched alevins in water containing a relatively high concentration of estradiol. A possible explanation for the success of such an unusually short treatment period has been discussed by Hunter and Donaldson (1983). It is suggested that the period during which the differentiating gonads are exposed to the effect of the steroid is in effect much longer than the treatment period itself. This exposure period would be influenced, among several possible factors, by the dose and the rate of uptake and turnover of the steroid. The results obtained by Nakamura (1981) with masu salmon (0. masou) using longer exposure at lower doses (0.5-5 pg/l for 18 days) seem to reinforce the possibility of an inverse relationship between dose and duration of treatment. Continuous immersion at concentrations above 5 pg/l (ranging from 10 to 200 pg/l) resulted in very high mortality (Nakamm-a, 1981). The mortality in the coho alevins administered the high doses of E, (800 and 1600 pg/l) was higher than in those groups treated with lower doses and the controls. However, the major apparent reason for mortality was a fungus-like infection on the skin and fins which may not be related to the dose levels. In fact, no similar infection occurred in the chinook groups where most mortalities were caused by sub-optimal culture conditions as previously described. The percentages of female fish of both species produced by the immersion treatments do not seem to be directly influenced by the increase of the dose of the steroid within the range used in this experiment. On this basis, we conclude that the dosage levels in this protocol could already be above the required effective limits and that a lower dose in conjunction with adjustment of other factors such as duration and/or timing and frequency could contribute to maximize the success of the feminization treatment. Additionally, further investigation of the rates of uptake and retention of the steroid in relation to its concentration in the immersion solution could provide the information required to establish the minimum effective dosage to feminize coho and chinook salmon. The main advantage of this treatment is its overall simplicity in relation to its effectiveness. Although complete feminization was not achieved in this study, we believe that enhancing the sex ratio of a salmonid stock towards 80-95s females should be of significant economic benefit to the commercial aquaculture of coho and chinook salmon. ACKNOWLEDGEMENTS
The assistance of the staff of the aquarium facility of the West Vancouver Laboratory, Capilano Hatchery and Rosewall Creek Experimental Hatchery is appreciated. We wish to thank Dr. N.E. Down for valuable suggestions on the statistical analysis of the data, Ms. H.M. Dye for comments to improve the manuscript and Mrs. M. Booth for typing the manuscript.
302 REFERENCES Donaldson, E.M. and Hunter, G.A., 1982. Sex control in fish with particular reference to salmonids. Can. J. Fish. Aquat. Sci., 39: 99-110. Goetz, F.W., Donaldson, E.M., Hunter, G.A. and Dye, H.M., 1979. Effects of estradiol17~ and 17e-methyltestosterone on gonadal differentiation in the coho salmon, Oncorhynchus kisutch. Aquaculture, 17 : 267-278. Hunter, G.A. and Donaldson, E.M., 1983. Hormonal sex control and its application to fish culture. In: W.S. Hoar, D.J. Randall and E.M. Donaldson (Editors), Fish Physiology, Vol. IX, Part B, Behaviour and Fertility Control. Academic Press, New York, NY, Chap. 5, pp. 223-303. Hunter, G.A., Donaldson, E.M., Goetz, F.W. and Edgell, P.R., 1982. Production of all-female and sterile groups of coho salmon (Oncorhynchus kisutch) and experimental evidence for male heterogamety. Trans. Am. Fish. Sot., 111: 367-372. Nakamura, M., 1981. Feminization of masu salmon, Oncorhynchus ma.sou, by administration of estradioL17p. Bull. Jpn. Sot. Sci. Fish., 47(11): 1529. Schreck, C.B., 1974. Hormonal treatment and sex manipulation in fishes. In: C.B. Schreck (Editor), Control Of Sex In Fishes. VPI-SG-74-01, Extension Division, Virginia Polytechnical Institute and State University, Blacksburg, VA, pp. 84-106. Vanstone, W.E. and Markert, J.R., 1968. Some morphological and biochemical changes in coho salmon, Oncorhynchus kisutch, during parrsmolt tranformation. J. Fish. Res. Board Can., 25: 2403-2418. Yamamoto, T., 1969. Sex differentiation. In: W.S. Hoar and D.J. Randall (Editors), Fish Physiology, Vol. III. Academic Press, New York, NY, pp. 117-175. Yamazaki, F., 1983. Sex control and manipulation in fish. Aquaculture, 33: 329-354.
295
FEMINIZATION OF COHO SALMON (ONCORHYNCHUS KISUTCH) AND CHINOOK SALMON (ONCORHYNCHUS TSHA W YTSCHA) BY IMMERSION OF ALEVINS IN A SOLUTION OF ESTRADIOL-170
G.A. HUNTER, II. SOLAR’,
I.J. BAKER and E.M. DONALDSON
West Vancouver Laboratory, Fisheries Research Branch, Department of Fisheries and Oceans, 4160 Marine Dn’ue, West Vancouver, B.C., V7V IN6 (Canada) (Accepted
31 January 1986)
ABSTRACT Hunter, G.A., Solar, II., Baker, I.J. and Donaldson, E.M., 1986. Feminization of coho salmon (Oncorhynchus kisutch) and chinook salmon (Oncorhynchus tshawytscha) by immersion of alevins in a solution of estradiol-17p. Aquaculture, 53: 295-302. Immersion treatment of newly-hatched coho and chinook salmon alevins in water containing estradiol-17p in concentrations ranging from 200 to 1600 pg/l, for two 2-h periods, 1 week apart, produced 86-96% females and 66-91% females in coho and chinook, respectively. Gonadal morphology of the females produced resembled that of the control females when sampled at 100 and 190 days post-hatch. Variable proportions of male and intersex fish were also observed. There was no evidence of dose-related mortality or differential growth, nor was there a direct relationship between dose level and percent of female fish produced. Compared with effective feminization treatments previously described for these two salmonid species, which include several immersions starting at the eyed-egg stage plus dietary treatment for up to 10 weeks, the treatment described in this report is easy to apply and highly efficient in enhancing the proportion of females for the commercial culture of Pacific salmon.
INTRODUCTION
Among the hormonal techniques to control sex in salmonids, the production of all-female stocks is of considerable interest because of its potential as a management tool in different situations for resource enhancement or for aquaculture. Detailed objectives of the techniques for direct and indirect production of all-female stocks have been reviewed by several authors (Yamamoto, 1969; S&reck, 1974; Donaldson and Hunter, 1982; Hunter and Donaldson, 1983; Yamazaki, 19.83). The indirect technique requires the production (and segregation) of phenotypic males of female genotype (XX) by androgen treatment. The isolated homogametic males will produce a monosex (100% female) progeny when crossed with normal females (Hunter et al., 1982). This technique ’ To whom reprint requests should be sent. 0044-8486/86/$03.50
o 1986 Elsevier Science Publishers B.V.
296
produces fish which are both phenotypically and genotypically female. The main drawback is the lead time required since the desired all-female stock is produced in the second generation. The direct approach involving the use of estradiol-170 (EJ, the major naturally-occurring estrogen in teleosts and higher vertebrates including mammals, is considered a practical alternative because the desired phenotypic females are produced in the immediate generation. The estrogen treatment is fully effective, the induced females are phenotypically indistinguishable from normal females and there is no treatment-related mortality at the appropriate dosage. The main drawback of this method is that the feminized fish may not be used as broodstock. One-half of the phenotypic females produced are genotypic males. They would therefore produce a 3:l male:female ratio when mated with normal males [25% female (XX), 50% male (XY), 25% male (YY)] (Hunter et al., 1982). Earlier attempts to directly feminize Oncorhynchus species using two to 13 immersions of eyed-eggs and alevins followed by dietary treatment for 3-10 weeks have been successful in inducing 60-100% females in coho salmon (0. hisutch) (Goetz et al., 1979; Hunter et al., 1982) and 96100% females in chinook salmon (0. tshawytscha) (G.A. Hunter and E.M. Donaldson, unpublished data, 1978). These results and further tests conducted at the West Vancouver Laboratory show that treatment prior to first feeding is essential and that a high percentage of female coho or chinook salmon may be obtained by treatment at the alevin stage alone. In this report we describe experiments conducted in 1983-1984 at the West Vancouver Laboratory on the direct feminization of coho and chinook salmon by administration of immersion treatments to newlyhatched alevins in water containing the estrogen estradiol-170. MATERIALS
AND METHODS
Experimental fish The chinook salmon gametes were obtained from the Big Qualicum Hatchery (Vancouver Island, B.C.). Fertilization and incubation were carried out at the West Vancouver Laboratory (West Vancouver, British Columbia). For the experiments with coho salmon, eyed-eggs which had been incubated for 94 days at ambient water temperatures ranging from 2.5 to 75°C (398°C days) were obtained from the Capilano Hatchery (North Vancouver, British Columbia). At West Vancouver, the eggs of both species were incubated in Heath trays supplied with 9 l/min well water at 9.5-10.5”C. Prior to hatching the eyed-eggs of each species were divided into five groups of approximately 100 eggs and placed in plexiglass chambers with 1 mm plastic mesh top and bottom which were inserted in the Heath trays (Goetz et al., 1979). Mean (50%) hatch took place on Day 53 post-fertilization (513°C days)
297
for chinook coho salmon.
salmon
and
on Day
96 post-fertilization
(418°C
days)
for
Hormonal treatments On Days 2 and 9 post-hatch the alevins were immersed in estrogen for 2 h (estradiol 17~, Sigma Co. St. Louis, MO) at concentrations of 200, 400, 800 and 1600 pg/l. ,‘l’he immersion treatments were conducted as described by Goetz et al. (1979). Following treatments the Plexiglass chambers containing the alevins were returned to the incubators in trays located below those containing the control groups which were left untreated. On the 5th day after the first immersion treatment, a breakdown in the well water supply to the incubator holding the chinook alevins caused interruption of the normal water flow. The alevins were subsequently maintained for 4 days in static water which was replaced daily by 1.5-3-h flushes with a mix of hot and cold untreated municipal water adjusted to approximately 10°C. Three days after the second treatment, heavy mortalities were observed in these groups including untreated controls. At this time continuous water flow was re-established using the cold municipal water supply. As a result, development of the chinook alevins took place (after treatment) at variable temperatures ranging from 3.5”~~34°C until swim-up, at which time they were transferred to the Rosewall Creek Experimental Hatchery, Vancouver Island. The final incubation, hormone treatments, and post-treatment culture of the coho eggs and alevins were carried out at a different time, hence they were not affected by the accidental failure of the support system described above. Rearing, sampling, and histological procedures At swim-up the alevins were transferred to 50-l tanks supplied with aerated well water at 9-10°C. The fry were fed regular (untreated) Oregon moist pellets (OMP). Mortalities were recorded daily. At 100 and 190 days post-hatch (coho and chinook, respectively), random samples of 4050 fish were taken from each group, weighed (to 0.01 g) and measured (fork length to 0.1 cm). Cross-sections were cut at a point just caudal to the pectoral fins, fixed in Bouin’s, sectioned, and stained with hematoxylin/ eosin for histological sex identification. At Day 146, the remaining coho fry in each experimental group were dissected for visual examination of the gonads. Statistical analysis Means and standard deviations were calculated for length (L, cm), weight (W, g), and condition factor (K). The condition factor was calculated using the formula K=W/L3e2’ X 1000 (Vanstone and Markert, 1968).
298
One-way analysis of variance (ANOVA) was applied to W and K data to examine independently the effect of dosage levels on the size of the experimental fish at the end of the treatment, compared with each other and the control groups (Table 2). Analysis for sex ratio was performed with the Chi-squared test. Male and intersex were combined into one category. Differences (in absolute numbers) from the expected 1:l ratio were considered significant when
P < 0.001. RESULTS
Coho saimon The estrogen (E2) immersion treatment at the preswim-up stage significantly altered the normal sex ratios in all the treated groups compared with the untreated control, which had close to a 50:50 male:female ratio (Table 1). Close to 97% of the fish in the group submitted to two 4OO+g/l estradiol immersions had gonads containing oocytes in the perinucleolar stage, with normal appearance similar to the control ovaries. In the treated groups, the lowest proportion of females (87%) and highest proportion of males (10%) were produced with 200 pg/l Ez. From 2 to 9% of the TABLE 1 Effect of two 2-h estradiol-170 immersion treatments (Days 2 and 9 post-hatch) on sex differentiation of coho (Oncorhynchus kisutch) and chinook (Oncorhynchus tshowytscha) salmon (N = number sexed) Gonadal morphology %
Treatment dose (fig/I)
Survival %
N
Coho salmon* * 200 400 800 1600 0
84.9 86.1 59.8 55.1 98.3
68 59 51 63 32
10.3 3.4 7.8 3.2 56.0
2.9 0.0 2.0 9.5 0.0
86.ga 96.6a 9o.2a 87.38 44.0
-
36 44 44 45 44
16.7 6.8 15.9 0.0 45.5
16.6 11.4 18.2 9.1 0.0
66.7b 81Ba 65.gb 91.18 54.5
Chinook salmon* * * 200 400 800 1600 0
Male
Intersex
Female*
*Significance of difference from 1:l ratio a = P < 0.001; b = P < 0.025 (Chi-squared test). **Age at sampling: 100 days (histological) and 146 days (visual), post-hatch. ***Age at sampling: 190 days post-hatch.
299
TABLE 2 Weight, length and condition factor (K) of coho (Oncorhynchus kisutch) and chinook (Oncorhynchus tshawytscha) salmon treated with two 2-h estradiol-17@ immersions on Days 2 and 9 post-hatch (N = number sampled) N
Length (cm) mean (SD)
Weight (g) K* Mean (SD) mean (SD)
Coho salmon** 200 400 800 1600 0
44 42 42 40 44
5.8 5.9 5.6 5.9 5.9
(0.7) (0.7) (0.8) (0.7) (0.6)
2.29 2.33 2.16 2.37 2.32
(0.9) (0.8) (1.0) (0.7) (0.8)
7.2 7.2 7.6 7.5 7.2
(0.6) (0.8) (0.9)+ (1.2) (0.8)
Chinook salmon*** 200 400 800 1600 0
40 52 50 50 50
9.0 9.1 9.2 8.9 8.9
(0.5) (0.7) (0.6) (0.5) (0.5)
8.31 8.65 8.73 7.78 7.73
(1.5) (2.1) (1.8) (1.7) (1.7)
6.4 6.5 6.4 6.4 6.3
(0.4) (0.4) (0.3) (0.3) (0.6)
Treatment dose (at/l)
*Condition factor K = ( W/L3-25) x 1000. **Age at sampling: 100 days post-hatch. ***Age at sampling : 190 days post-hatch. +Significantly different from control groups (P < 0.05).
estrogen-treated fish in different groups had gonads containing both oocytes, usually in small numbers, and localized areas of undeveloped germinal cells resembling spermatogonia. They were classified as intersex. Survival was higher in the groups receiving the lower doses. The differential mortalities were produced by a fungus-like infection in the groups that received treatments at higher doses (800 and 1600 pg E2). Statistical analysis of weight and condition factor did not indicate significant differences in either parameter, with the exception of the group treated with 800 pg/l where the condition factor was significantly higher (P < 0.05) than those of the control and the other treated groups (Table 2). Chinook salmon The sex ratio was also substantially altered in the experimental chinook salmon groups compared with the controls which showed a normal 1:l ratio. Proportions of female fish with normal ovarian morphology ranged from 66% to 91%, while proportions of fish with gonads containing germinal elements similar to the control males ranged from 0 to 16% (Table 1). The largest number of males was found in the group that received the lowest dose while no males were present in the group treated with the highest dose of the estrogen. The intersex condition, where underdeveloped germinal tissue (spermatogonia) and reduced numbers of oocytes
300
coexisted in the same gonad, was observed in all the experimental groups with a frequency ranging from 9% to 18%. Growth did not seem affected by the dose levels of the immersion treatments. Although the group that received the highest dose had a lower mean weight than the other treated groups, this weight was equivalent to that of the control means and not significantly different from the other treated groups (Table 2). Mortalities in all the chinook groups during the first few days immediately following treatments were negligible. However, as a consequence of a breakdown in the water supply system all the groups suffered similar losses. The mortality was attributed largely to the temporary sub-optimal culture conditions and therefore was not computed. DISCUSSION
These results demonstrate the feasibility of significantly altering the normal sex ratio of coho and chinook salmon towards an enhanced proportion of females by immersion of alevins for two 2-h periods (2 and 9 days post-hatch) in water containing the natural piscine estrogen estradiol-178. This treatment is of much shorter duration than those previously described by Goetz et al. (1979), Hunter et al. (1982), and Donaldson and Hunter (1982) for coho salmon, and by Nakamura (1981) for masu salmon (Oncorhynchus masou). The earlier studies with coho salmon mentioned above included several immersions of eyed-eggs and yolk-sac alevins plus the administration of estrogen in the diet at a dose of 5-10 mg/kg for up to 90 days from first feeding. Nakamura (1981) obtained 100% female masu salmon by 18-day continuous immersion in static water containing 0.5-5 pg/l Ez starting 5 days post-hatch. In this study we did not produce complete (100%) sex reversal of the male portion of the experimental groups: however, the proportions of females produced confirm that the process of sex differentiation in salmon is remarkably labile and highly responsive to the influence of exogenous sex steroids even for very short periods. Close to 97% female coho were produced in this experiment by two 2-h immersions in 400 pg/l estradiol. The rather lower level of success achieved in chinook salmon could be due to the influence of the lower temperature of the rearing water during the critical period of sex differentiation at the time of and subsequent to the treatments. Several authors have discussed the potency of sex steroids in connection with the route of administration (for reviews see Yamamoto, 1969; Hunter and Donaldson, 1983). Estradiol has been described as the most potent naturally-occurring estrogen when administered either in the diet or in the rearing water. Typically, effective treatments for sex reversal in Pacific salmon have included exposure to the sex steroid at a very early stage, including eyed-egg and alevin immersion plus dietary treatment for several
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weeks. This study shows that the effective treatment for feminization of chinook and coho salmon could be reduced to short-time immersions of newly-hatched alevins in water containing a relatively high concentration of estradiol. A possible explanation for the success of such an unusually short treatment period has been discussed by Hunter and Donaldson (1983). It is suggested that the period during which the differentiating gonads are exposed to the effect of the steroid is in effect much longer than the treatment period itself. This exposure period would be influenced, among several possible factors, by the dose and the rate of uptake and turnover of the steroid. The results obtained by Nakamura (1981) with masu salmon (0. masou) using longer exposure at lower doses (0.5-5 pg/l for 18 days) seem to reinforce the possibility of an inverse relationship between dose and duration of treatment. Continuous immersion at concentrations above 5 pg/l (ranging from 10 to 200 pg/l) resulted in very high mortality (Nakamm-a, 1981). The mortality in the coho alevins administered the high doses of E, (800 and 1600 pg/l) was higher than in those groups treated with lower doses and the controls. However, the major apparent reason for mortality was a fungus-like infection on the skin and fins which may not be related to the dose levels. In fact, no similar infection occurred in the chinook groups where most mortalities were caused by sub-optimal culture conditions as previously described. The percentages of female fish of both species produced by the immersion treatments do not seem to be directly influenced by the increase of the dose of the steroid within the range used in this experiment. On this basis, we conclude that the dosage levels in this protocol could already be above the required effective limits and that a lower dose in conjunction with adjustment of other factors such as duration and/or timing and frequency could contribute to maximize the success of the feminization treatment. Additionally, further investigation of the rates of uptake and retention of the steroid in relation to its concentration in the immersion solution could provide the information required to establish the minimum effective dosage to feminize coho and chinook salmon. The main advantage of this treatment is its overall simplicity in relation to its effectiveness. Although complete feminization was not achieved in this study, we believe that enhancing the sex ratio of a salmonid stock towards 80-95s females should be of significant economic benefit to the commercial aquaculture of coho and chinook salmon. ACKNOWLEDGEMENTS
The assistance of the staff of the aquarium facility of the West Vancouver Laboratory, Capilano Hatchery and Rosewall Creek Experimental Hatchery is appreciated. We wish to thank Dr. N.E. Down for valuable suggestions on the statistical analysis of the data, Ms. H.M. Dye for comments to improve the manuscript and Mrs. M. Booth for typing the manuscript.
302 REFERENCES Donaldson, E.M. and Hunter, G.A., 1982. Sex control in fish with particular reference to salmonids. Can. J. Fish. Aquat. Sci., 39: 99-110. Goetz, F.W., Donaldson, E.M., Hunter, G.A. and Dye, H.M., 1979. Effects of estradiol17~ and 17e-methyltestosterone on gonadal differentiation in the coho salmon, Oncorhynchus kisutch. Aquaculture, 17 : 267-278. Hunter, G.A. and Donaldson, E.M., 1983. Hormonal sex control and its application to fish culture. In: W.S. Hoar, D.J. Randall and E.M. Donaldson (Editors), Fish Physiology, Vol. IX, Part B, Behaviour and Fertility Control. Academic Press, New York, NY, Chap. 5, pp. 223-303. Hunter, G.A., Donaldson, E.M., Goetz, F.W. and Edgell, P.R., 1982. Production of all-female and sterile groups of coho salmon (Oncorhynchus kisutch) and experimental evidence for male heterogamety. Trans. Am. Fish. Sot., 111: 367-372. Nakamura, M., 1981. Feminization of masu salmon, Oncorhynchus ma.sou, by administration of estradioL17p. Bull. Jpn. Sot. Sci. Fish., 47(11): 1529. Schreck, C.B., 1974. Hormonal treatment and sex manipulation in fishes. In: C.B. Schreck (Editor), Control Of Sex In Fishes. VPI-SG-74-01, Extension Division, Virginia Polytechnical Institute and State University, Blacksburg, VA, pp. 84-106. Vanstone, W.E. and Markert, J.R., 1968. Some morphological and biochemical changes in coho salmon, Oncorhynchus kisutch, during parrsmolt tranformation. J. Fish. Res. Board Can., 25: 2403-2418. Yamamoto, T., 1969. Sex differentiation. In: W.S. Hoar and D.J. Randall (Editors), Fish Physiology, Vol. III. Academic Press, New York, NY, pp. 117-175. Yamazaki, F., 1983. Sex control and manipulation in fish. Aquaculture, 33: 329-354.