- Email: [email protected]
Production of all-female triploid rainbow trout
Aquaculture, 30 (1983) 375-380 Elsevier Scientific Publishing Company, Amsterdam -Printed
in The Netherlands
375
Short Communication PRODUCTION
OF ALL-FEMALE
TRIPLOID RAINBOW TROUT
R.F. LINCOLN and A.P. SCOTT Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory, Lowestoft, Suffolk NR33 OHT (Great Britain) (Accepted
22 February 1982)
ABSTRACT Lincoln, R.F. and Scott, A.P., 1983. Production of all-female triploid rainbow trout. Aquaculture, 30: 375-380. Triploid rainbow trout were produced by heat shock treatment of eggs soon after fertilization with either normal sperm or sperm from masculinised females. The proportion of triploid fry, as judged from red blood cell nuclear volume, varied between 75% and 100% in three experiments using different batches of eggs from an autumn-spawning strain of trout while a single batch of eggs from a winter-spawning strain yielded 50% triploids. A microscopic examination of the gonads was made on 5-month-old fish weighing between 1 and 3 g. In female controls the ovaries were packed with oocytes while those from female triploids, although showing the typical lamellar structure of an ovary, contained no oocytes, thus indicating that female triploids are sterile. The testes from triploid males appeared to be developing normally. The use of masculinized females combined with heat shock treatment of eggs to produce triploids, allowed the production of sterile all-female triploids. This should have considerable potential for aquaculture.
INTRODUCTION
A major problem in salmonid fish cultivation is the detrimental effect of sexual maturation on the growth, survival and marketability of fish. These effects are most acute in male fish and research over the past decade has led to the commercial production of al&female fish using masculinized females (Johnstone et al., 1979; Bye and Lincoln, 1981). The production of sterile fish on a commercial scale has so far proved difficult. One possible method which has aroused considerable interest in recent years, however, is the production of triploid fish by subjecting newly-fertilized eggs to a temperature shock (Purdom, 1972). Extensive early research on amphibians has shown that triploidy is associated with impaired gonad development. Similar observations have also been made in fish (Swarup, 1957; Purdom, 1972; Purdom and Lincoln, 1973). One aspect of the work in fish, not fully explored, is the difference in 0044S486/83/0000-0000/$03.00
0 1983 Elsevier Scientific Publishing Company
316
gonad development between male and female triploids. In amphibians, for example, while ovaries of triploids are usually markedly underdeveloped, the testes can develop to the same size as those of diploid males (Fankhauser, 1945). A similar development has been reported in triploid male plaice (Hew-one&es plutessa) and in its hybrid with the flounder (Platichthys flesus) (Lincoln, 1981a) - little or no reduction in testis growth occurred compared with diploid controls, and viable sperm were produced which could fertilize eggs. Mature triploid hybrid females, on the other hand, had ovaries which were less than 5% of the weight of those in control diploid fish (Lincoln, 1981b). Thorgaard and Gall (1979) have also noted in naturallyoccurring triploid rainbow trout that females show almost no gonad development, while male triploids are morphologically indistinguishable from diploid males. Clearly, there is no commercial advantage in producing male triploid fish if testis growth is unaffected by chromosome number. On the other hand, the production of female-only triploids could be a significant advantage. Early attempts to induce triploidy in salmonids have been unsuccessful (Svardson, 1945; Lincoln et al., 1974; Lemoine and Smith, 1980) but recent work by Chourrout (1980) has shown that high levels of triploidy can be achieved in rainbow trout using a mild heat shock treatment soon after fertilization. The use of this technique combined with the production of allfemale rainbow trout should enable the commercial production of sterile triploid rainbow trout to be achieved. MATERIALS
AND METHODS
Trout of two strains were used: Mount Lassen (winter spawner) and Caribou (autumn spawner). Eggs were collected from 3-year-old fish. These were held at a farm remote from the laboratory which necessitated a period of some 6 h or more between stripping and fertilization. Eggs were stripped into polythene tubs (450 ml), flushed with oxygen, and placed in insulated containers containing crushed ice. The sperm used to produce all-female ova came from a second generation of all female fish that were masculinized during the fry stage (Bye and Lincoln, 1981). Sperm collected from these homogametic XX males and from normal males were similarly stored under oxygen in 25 ml polythene vials. Dry fertilization was carried out at the laboratory, and the eggs were activated and held in water at a constant temperature of 10°C prior to the thermal shock. The heat shock was applied for 10 or 15 min at 40 min after fertilization by transferring eggs to water at 27°C or 28°C (T 0.2%); in one experiment the shock was started over a range of time intervals after fertilization. Determination of ploidy was made on measurements of nuclear volume of red blood cells (RBC) taken from the severed caudal fin of 5-month-old fish. Dried blood films were fixed and stained in Giemsa and magnified 2000 times
377
on to a ground-glass screen using a Zeiss Photomicroscope II. Nuclear outlines (ten per fish) were traced on to transparent film and the volumes were calculated from the measurements of the major and minor axes according to the formula: a2.b
1.91’ where a = minor axis and b = major axis (Becak et al., 1967). RESULTS
Table I lists the results of four experiments. No difficulty was experienced in identifying triploids and typical mean nuclear volume ratios and S.E.‘s for experiments 3 and 4 gave values of 1.63 (+ 0.028) and 1.56 (+ 0.027) respectively. These two experiments utilized sperm from masculinized females, which was confirmed when a microscopic examination of the gonads revealed only the presence of ovaries in control and treated fish from both experiments. Experiment 2 comprised a mixed group of fry derived from four separate batches of eggs heat shocked at different times after fertilization with normal milt (0-15,15-30,30-45,45+0 min). The batches were combined after hatching due to insufficient facilities, as it was considered more important to investigate the development of the all-female triploid broods. All fish tested in this combined group were triploid however, suggesting that the timing of the heat shock may be irrelevant over the first hour after fertilization. In Experiments l-3 on the autumn-spawning Caribou strain, the yield of triploids was very high, whereas in Experiment 4 with the winter-spawning Mount Lassen strain, the yield was lower. TABLE I DetaiIs of heat-shock treatment and percentage triploid production. Numbers in brackets are number of fish scored Experiment
1 2
Trout strain
Caribou Caribou Caribou Mount Lassen
Percent triploid
Heat shock Duration (min)
Time after fertilization (min)
10 15 10 10
40 0-45s 40 40
74 100 100 50
aResults combined from heat-shocking eggs at 0,15, 30 and 45 min. bEggs fertilized with a masculinized female - progeny all-female.
( 24) ( 14) ( 10) (149)
378
Egg mortality up to hatching was recorded only in Experiment 1, which gave survivals of 59% and 30% for control and treated groups respectively. Survival of alevins up to first feeding and beyond was not recorded but, by observation, treated fish showed a higher mortality relative to controls until feeding was fully established after which mortalities ceased. Gonad development was assessed microscopically at 5 months of age on the same fish used for ploidy determinations. At this stage the fish weighed between 1.5 and 3 g. The ovaries of female controls together with those from diploid females identified in the treated groups, were packed with developing oocytes. Ovaries from triploid fish, although showing the distinct lamellar structure of an ovary, contained no oocytes (Fig. 1). In every case a direct correlation existed between sterile ovary growth and triploidy as revealed from the RBC nuclear volume ratio. Sterility alone could be a useful criterion in identifying triploids in all female broods of rainbow trout. Testes development in male fish at this stage had not proceeded beyond the spermatogonial stage and no obvious differences could be observed between diploid and triploid fish. DISCUSSION
The high levels of triploidy achieved in the experiments reported here confirm the effectiveness of heat shock treatment in suppressing Meiosis II in rainbow trout eggs as reported by Chourrout (1980). The most important observation is the apparent sterility of triploid female fish. The complete suppression of oocyte development in the ovaries of immature triploid female trout parallels similar events in the ovaries of immature female triploid flatfish, first reported by Purdom (1972). Although sexually-mature triploid flatfish are able to produce a sizeable ovary (Lincoln, 1981b) this may not be the case in rainbow trout, since Thorgaard and Gall (1979) reported thread-like gonads in mature female triploid rainbow trout at 2 years of age. The use of masculinized female trout, which are now readily available, combined with heat shock treatment after fertilization to induce triploidy, would enable the production of sterile female rainbow trout to be carried out. Although quantitative data on egg mortality was recorded for only one experiment, where triploid survival was half that of controls up to hatching, subjective assessments in other cases indicate survival of triploids can equal that of the controls. This does not appear to be the case during the alevin stage up to the period of established feeding, however, where again subjective assessment indicates a greater mortality occurs in triploid fish. Further research will concentrate on both the need to improve survival up to first feeding and the identification of particular strains of rainbow trout which might give consistently high yields of triploids. However, even where triploidization is not 100%~ diploids will be female, so the problems associated with male maturity (Bye and Lincoln, 1981) will still be avoided.
Fig. 1. Photographs of wet mount preparations of ovaries from (a) diploid and (b) triploid rainbow trout at 5 months of age (x87).
380 REFERENCES Becak, W., Becak, M.L., Lavalle, D. and Schreiber, G., 1967. Further studies on polyploid amphibians (Ceratophrydidae). II. DNA content and nuclear volume. Chromosoma (Berl.), 23: 14-23. Bye, V.J. and Lincoln, R.F., 1981. Get rid of the males and let the females prosper. Fish Farmer, 4: 22-24. Chourrout, D., 1980. Thermal induction of diploid gynogenesis and triploidy in the eggs of the rainbow trout (Saimo gairdneri Richardson). Reprod. Nutr. DBv., 20 (3A): 727-733. Fankhauser, G., 1945. The effects of changes in chromosome number on amphibian development. Q. Rev. Biol., 20: 20-78. Johnstone, R., Simpson, T.H., Youngson, A.F. and Whitehead, C., 1979. Sex reversal in salmonid culture. Part II. The progeny of sex-reversed rainbow trout. Aquaculture, 18: 13-19. Lemoine, H.L. and Smith, L.T., 1980. Polyploidy induced in brook trout by cold shock. Trans. Am. Fish. Sot., 109: 626-631. Lincoln, R.F., 1981a. Sexual maturation in triploid male plaice (Pleuronectes platessa) and plaice X founder (Platichthys flesus) hybrids. J. Fish Biol., 19: 415-426. Lincoln, R.F., 1981b. Sexual maturation in female triploid plaice (Pleuronectes platessa) and plaice X flounder (Platichthys flesus) hybrids. J. Fish Biol., 19: 499-507. Lincoln, R.F., Au&ad, D. and Grammeltveat, A., 1974. Attempted triploid induction in Atlantic salmon (Salmo salar) using cold shocks. Aquaculture, 4: 287-297. Purdom, C.E., 1972. Induced polyploidy in plaice (Pleuronectes platessa) and its hybrid with the flounder (Platichthys flesus). Heredity, 29: 11-23. Purdom, C.E. and Lincoln, R.F., 1973. Chromosome manipulation in fish. In: J.H. Schrijder (Editor), Genetics and Mutagenesis of Fish. Springer-Verlag, Berlin, pp. 83-89. Sviirdson, G., 1945. Chromosome studies on Salmonidae. Medd. Statens Unders. FSrsAnst. SGtvattFisk., 23: l-151. Swarup, H., 1957. The production and effects of triploidy in the 3-spined stickleback Gasterosteus aculeatus (L.). PhD Thesis, The University, Oxford, mimeographed. Thorgaard, G.H. and Gall, G.A.E., 1979. Adult triploids in a rainbow trout family. Genetics, 93: 961-973.
in The Netherlands
375
Short Communication PRODUCTION
OF ALL-FEMALE
TRIPLOID RAINBOW TROUT
R.F. LINCOLN and A.P. SCOTT Ministry of Agriculture, Fisheries and Food, Directorate of Fisheries Research, Fisheries Laboratory, Lowestoft, Suffolk NR33 OHT (Great Britain) (Accepted
22 February 1982)
ABSTRACT Lincoln, R.F. and Scott, A.P., 1983. Production of all-female triploid rainbow trout. Aquaculture, 30: 375-380. Triploid rainbow trout were produced by heat shock treatment of eggs soon after fertilization with either normal sperm or sperm from masculinised females. The proportion of triploid fry, as judged from red blood cell nuclear volume, varied between 75% and 100% in three experiments using different batches of eggs from an autumn-spawning strain of trout while a single batch of eggs from a winter-spawning strain yielded 50% triploids. A microscopic examination of the gonads was made on 5-month-old fish weighing between 1 and 3 g. In female controls the ovaries were packed with oocytes while those from female triploids, although showing the typical lamellar structure of an ovary, contained no oocytes, thus indicating that female triploids are sterile. The testes from triploid males appeared to be developing normally. The use of masculinized females combined with heat shock treatment of eggs to produce triploids, allowed the production of sterile all-female triploids. This should have considerable potential for aquaculture.
INTRODUCTION
A major problem in salmonid fish cultivation is the detrimental effect of sexual maturation on the growth, survival and marketability of fish. These effects are most acute in male fish and research over the past decade has led to the commercial production of al&female fish using masculinized females (Johnstone et al., 1979; Bye and Lincoln, 1981). The production of sterile fish on a commercial scale has so far proved difficult. One possible method which has aroused considerable interest in recent years, however, is the production of triploid fish by subjecting newly-fertilized eggs to a temperature shock (Purdom, 1972). Extensive early research on amphibians has shown that triploidy is associated with impaired gonad development. Similar observations have also been made in fish (Swarup, 1957; Purdom, 1972; Purdom and Lincoln, 1973). One aspect of the work in fish, not fully explored, is the difference in 0044S486/83/0000-0000/$03.00
0 1983 Elsevier Scientific Publishing Company
316
gonad development between male and female triploids. In amphibians, for example, while ovaries of triploids are usually markedly underdeveloped, the testes can develop to the same size as those of diploid males (Fankhauser, 1945). A similar development has been reported in triploid male plaice (Hew-one&es plutessa) and in its hybrid with the flounder (Platichthys flesus) (Lincoln, 1981a) - little or no reduction in testis growth occurred compared with diploid controls, and viable sperm were produced which could fertilize eggs. Mature triploid hybrid females, on the other hand, had ovaries which were less than 5% of the weight of those in control diploid fish (Lincoln, 1981b). Thorgaard and Gall (1979) have also noted in naturallyoccurring triploid rainbow trout that females show almost no gonad development, while male triploids are morphologically indistinguishable from diploid males. Clearly, there is no commercial advantage in producing male triploid fish if testis growth is unaffected by chromosome number. On the other hand, the production of female-only triploids could be a significant advantage. Early attempts to induce triploidy in salmonids have been unsuccessful (Svardson, 1945; Lincoln et al., 1974; Lemoine and Smith, 1980) but recent work by Chourrout (1980) has shown that high levels of triploidy can be achieved in rainbow trout using a mild heat shock treatment soon after fertilization. The use of this technique combined with the production of allfemale rainbow trout should enable the commercial production of sterile triploid rainbow trout to be achieved. MATERIALS
AND METHODS
Trout of two strains were used: Mount Lassen (winter spawner) and Caribou (autumn spawner). Eggs were collected from 3-year-old fish. These were held at a farm remote from the laboratory which necessitated a period of some 6 h or more between stripping and fertilization. Eggs were stripped into polythene tubs (450 ml), flushed with oxygen, and placed in insulated containers containing crushed ice. The sperm used to produce all-female ova came from a second generation of all female fish that were masculinized during the fry stage (Bye and Lincoln, 1981). Sperm collected from these homogametic XX males and from normal males were similarly stored under oxygen in 25 ml polythene vials. Dry fertilization was carried out at the laboratory, and the eggs were activated and held in water at a constant temperature of 10°C prior to the thermal shock. The heat shock was applied for 10 or 15 min at 40 min after fertilization by transferring eggs to water at 27°C or 28°C (T 0.2%); in one experiment the shock was started over a range of time intervals after fertilization. Determination of ploidy was made on measurements of nuclear volume of red blood cells (RBC) taken from the severed caudal fin of 5-month-old fish. Dried blood films were fixed and stained in Giemsa and magnified 2000 times
377
on to a ground-glass screen using a Zeiss Photomicroscope II. Nuclear outlines (ten per fish) were traced on to transparent film and the volumes were calculated from the measurements of the major and minor axes according to the formula: a2.b
1.91’ where a = minor axis and b = major axis (Becak et al., 1967). RESULTS
Table I lists the results of four experiments. No difficulty was experienced in identifying triploids and typical mean nuclear volume ratios and S.E.‘s for experiments 3 and 4 gave values of 1.63 (+ 0.028) and 1.56 (+ 0.027) respectively. These two experiments utilized sperm from masculinized females, which was confirmed when a microscopic examination of the gonads revealed only the presence of ovaries in control and treated fish from both experiments. Experiment 2 comprised a mixed group of fry derived from four separate batches of eggs heat shocked at different times after fertilization with normal milt (0-15,15-30,30-45,45+0 min). The batches were combined after hatching due to insufficient facilities, as it was considered more important to investigate the development of the all-female triploid broods. All fish tested in this combined group were triploid however, suggesting that the timing of the heat shock may be irrelevant over the first hour after fertilization. In Experiments l-3 on the autumn-spawning Caribou strain, the yield of triploids was very high, whereas in Experiment 4 with the winter-spawning Mount Lassen strain, the yield was lower. TABLE I DetaiIs of heat-shock treatment and percentage triploid production. Numbers in brackets are number of fish scored Experiment
1 2
Trout strain
Caribou Caribou Caribou Mount Lassen
Percent triploid
Heat shock Duration (min)
Time after fertilization (min)
10 15 10 10
40 0-45s 40 40
74 100 100 50
aResults combined from heat-shocking eggs at 0,15, 30 and 45 min. bEggs fertilized with a masculinized female - progeny all-female.
( 24) ( 14) ( 10) (149)
378
Egg mortality up to hatching was recorded only in Experiment 1, which gave survivals of 59% and 30% for control and treated groups respectively. Survival of alevins up to first feeding and beyond was not recorded but, by observation, treated fish showed a higher mortality relative to controls until feeding was fully established after which mortalities ceased. Gonad development was assessed microscopically at 5 months of age on the same fish used for ploidy determinations. At this stage the fish weighed between 1.5 and 3 g. The ovaries of female controls together with those from diploid females identified in the treated groups, were packed with developing oocytes. Ovaries from triploid fish, although showing the distinct lamellar structure of an ovary, contained no oocytes (Fig. 1). In every case a direct correlation existed between sterile ovary growth and triploidy as revealed from the RBC nuclear volume ratio. Sterility alone could be a useful criterion in identifying triploids in all female broods of rainbow trout. Testes development in male fish at this stage had not proceeded beyond the spermatogonial stage and no obvious differences could be observed between diploid and triploid fish. DISCUSSION
The high levels of triploidy achieved in the experiments reported here confirm the effectiveness of heat shock treatment in suppressing Meiosis II in rainbow trout eggs as reported by Chourrout (1980). The most important observation is the apparent sterility of triploid female fish. The complete suppression of oocyte development in the ovaries of immature triploid female trout parallels similar events in the ovaries of immature female triploid flatfish, first reported by Purdom (1972). Although sexually-mature triploid flatfish are able to produce a sizeable ovary (Lincoln, 1981b) this may not be the case in rainbow trout, since Thorgaard and Gall (1979) reported thread-like gonads in mature female triploid rainbow trout at 2 years of age. The use of masculinized female trout, which are now readily available, combined with heat shock treatment after fertilization to induce triploidy, would enable the production of sterile female rainbow trout to be carried out. Although quantitative data on egg mortality was recorded for only one experiment, where triploid survival was half that of controls up to hatching, subjective assessments in other cases indicate survival of triploids can equal that of the controls. This does not appear to be the case during the alevin stage up to the period of established feeding, however, where again subjective assessment indicates a greater mortality occurs in triploid fish. Further research will concentrate on both the need to improve survival up to first feeding and the identification of particular strains of rainbow trout which might give consistently high yields of triploids. However, even where triploidization is not 100%~ diploids will be female, so the problems associated with male maturity (Bye and Lincoln, 1981) will still be avoided.
Fig. 1. Photographs of wet mount preparations of ovaries from (a) diploid and (b) triploid rainbow trout at 5 months of age (x87).
380 REFERENCES Becak, W., Becak, M.L., Lavalle, D. and Schreiber, G., 1967. Further studies on polyploid amphibians (Ceratophrydidae). II. DNA content and nuclear volume. Chromosoma (Berl.), 23: 14-23. Bye, V.J. and Lincoln, R.F., 1981. Get rid of the males and let the females prosper. Fish Farmer, 4: 22-24. Chourrout, D., 1980. Thermal induction of diploid gynogenesis and triploidy in the eggs of the rainbow trout (Saimo gairdneri Richardson). Reprod. Nutr. DBv., 20 (3A): 727-733. Fankhauser, G., 1945. The effects of changes in chromosome number on amphibian development. Q. Rev. Biol., 20: 20-78. Johnstone, R., Simpson, T.H., Youngson, A.F. and Whitehead, C., 1979. Sex reversal in salmonid culture. Part II. The progeny of sex-reversed rainbow trout. Aquaculture, 18: 13-19. Lemoine, H.L. and Smith, L.T., 1980. Polyploidy induced in brook trout by cold shock. Trans. Am. Fish. Sot., 109: 626-631. Lincoln, R.F., 1981a. Sexual maturation in triploid male plaice (Pleuronectes platessa) and plaice X founder (Platichthys flesus) hybrids. J. Fish Biol., 19: 415-426. Lincoln, R.F., 1981b. Sexual maturation in female triploid plaice (Pleuronectes platessa) and plaice X flounder (Platichthys flesus) hybrids. J. Fish Biol., 19: 499-507. Lincoln, R.F., Au&ad, D. and Grammeltveat, A., 1974. Attempted triploid induction in Atlantic salmon (Salmo salar) using cold shocks. Aquaculture, 4: 287-297. Purdom, C.E., 1972. Induced polyploidy in plaice (Pleuronectes platessa) and its hybrid with the flounder (Platichthys flesus). Heredity, 29: 11-23. Purdom, C.E. and Lincoln, R.F., 1973. Chromosome manipulation in fish. In: J.H. Schrijder (Editor), Genetics and Mutagenesis of Fish. Springer-Verlag, Berlin, pp. 83-89. Sviirdson, G., 1945. Chromosome studies on Salmonidae. Medd. Statens Unders. FSrsAnst. SGtvattFisk., 23: l-151. Swarup, H., 1957. The production and effects of triploidy in the 3-spined stickleback Gasterosteus aculeatus (L.). PhD Thesis, The University, Oxford, mimeographed. Thorgaard, G.H. and Gall, G.A.E., 1979. Adult triploids in a rainbow trout family. Genetics, 93: 961-973.