ELSEVIER Animal Reproduction Science 42 ( 19%) 38 1-392

REPSON SCIENCE

Manipulation of reproduction in farmed fish

Edward M. Donaldson West Vancouuer Laboratory. Biological Sciences Branch, Department of Fisheries and Oceans, West

Vancouver, B.C. V7V lN6, Canada

Abstract

The production of fish by aquaculture bas increased rapidly in recent years. An important factor in this growth has been the application of controlled reproduction biotechnologies. Recent progress in the development of induced ovulation and spermiation technologies has facilitated the reproduction of many commercial species. The use of a variety of gonadotropin-releasing hormone analogues with or without dopamine antagonists is replacing the use of pituitary preparations. Current research is focused on optimal administration techniques utilizing intramuscular, intraperi- toneal and oral routes. Sex control technologies have also seen rapid development and application in salmonids and tilapias, as a means of increasing both production efficiency and reproductive containment. Gender control wil1 be applied in the near future to other farmed species including flatfsh and seabass. The most effective techniques involve the production of monosex gametes by either endocrine manipulation dming early development or a combination of endocrine and genetic technologies.

1. Introduction

The total harvest of the world Capture fishery reached a maximum leve1 in 1989 (New, 1991) and has recently entered a period of decline (Garcia and Newton, 1!994), brought about by multiple factors including increased demand, development of wide ranging fishing and processing vessels (Larkin, 19911, environmental degradation and climate change. Aquaculture is increasingly recognized as a sustainable means of producing high quality aquatic foods for human consumption. Production bas increased rapidly during the last decade and especially over the last half decade to the extent that aquaculture now accounts for over 20% of the world production of aquatic foods of marine and fresh water origin.

0378-4320/%/$15.00 8 1996 Elsevier Science B.V. Al1 rights reserved. PI1 SO378-4320(96)01555-2

382 E.M. Donaldson /Animal Reproduction Science 42 (1996) 381-392

In aquaculture as in agriculture there are areas where science plays an important role. These include reproduction, early development, nutrition, health and genetics (Donald- son, 1988). While agricultural production focuses on a limited number of mammalian and avian species, aquaculture production involves a large number of different species, each with their own distinct biological characteristics. Individual species are adapted to specific aquatic environmental conditions which vary according to temperature, salinity, oxygen concentration, pH, turbidity, flow rate and photoperiod. Thus biological tech- nologies developed for a particular species are not directly transferable to other species.

The ability to produce viable offspring from captive brood stock is an almost ubiquitous characteristic of the aquaculture of economically important fn fsh. Repro- duction in captivity has been the key which has opened the door to successful early rearing, metamorphosis and grow out to market size. Above all, reproduction in captivity permits domestication and genetic improvement to proceed.

Another aspect of controlled reproduction of fin fish which is of increasing impor- tante is the regulation of sex. Almost half a century ago Huxley (1938) stated that It thus becomes of great interest to discover the mechanism by which sex is determined, and to find whether by any means we can bring it under our control.

While progress in mammalian and avian species has been slow, there has been rapid progress in the development and application of sex control technologies in fin fish aquaculture. Gender control is of importante for maximizing the economie efficiency of production systems. In many species one sex either grows faster, matures later or has a higher market value than the other sex. Thus in tilapia, males are preferred, whereas in flatfish and salmonids, females are normally preferred for culture. Furthermore in some species the production of sterile fish is a viable option. Sex control is of importante both from a production standpoint, and as a means of reproductive containment. The culture of monosex or sterile populations can reduce or eliminate reproductive interaction between escaped farmed fish and wild conspecifics. It can also prevent cultured exotic species from forming self sustaining feral populations. In the future sex control wil1 als0 be used for the reproductive containment of genetically modified fish (e.g. transgenics) (Devlin and Donaldson, 1992).

In this paper 1 review recent developments in both the regulation of brood stock maturation and the development of monosex and sterile stocks.

2. Induced ovulation and spermiation

Current technologies for the induction of flnal maturation, ovulation and spermiation in cultured fishes have evolved from first generation techniques developed in the 1930s which involved the administration of pituitary glands from mature donors homogenized in physiological saline (Donaldson and Hunter, 1983). Second generation technologies involve the use of acetone dried homologous or heterologous fish pituitary glands, partially purified fish gonadotropins and human chorionic gonadotropin (hCG). Third generation technologies utilize gonadotropin releasing hormone analogs (GnRHAs) with or without dopamine antagonists such as domperidone, pimozide, sulpiride and metoclo- pramide or utilize antiestrogens such as clomiphene citrate to black feed back inhibition

E.M. Donaldson /Animal Reproduction Science 42 (1996) 381-392 383

1 234 5 6 18 9 10

MAMMAL ~GLU-HIS-TRP-SER-TIR-OLY-LEU-ARO-PR~~LY-~

SALMON pGLU-HIS-TRP-SER-TYR-GLY-TRP-LEU-PRO-GLY-NH2

SEA BREAM pGLU-HIS-TRP-SER-TYR-GLY-LEU-SER-PRO-GLYWIZ

CATFISH pGLU-HIS-TRP-SER-lil.%GLY-LEU-ASN-PRO-GLY-NH2

CHICKBN-II pGLU-HIS-~-SER-HIS-GLY-T~-~-PRO-GLY-~2

Fig. 1. Amino acid sequences of the GnRH peptides found to date in boney fishes. Amino acids that differ from those in the mammalian peptide are shown in bold.

by estradiol 17p at the hypothalamic leve]. It is the third generation techniques that have attracted significant interest in recent years especially: 1. the discovery of new natura1 forms of GnRH, 2. the development and testing of potent GnRH analogs, 3. the development of novel means of GnRH administration including oral and con-

trolled release intra muscular (i.m.1 implants. The first novel form of GnRH to be identified in fsh was the salmon Oncorhynchus

sp. GnRH (sGnRH) (Sherwood et al., 1983). Since then novel forms have been identified in the sea bream Sparus aurata (Powell et al., 1994), and the Thai catfish Clurius macrocephalus (Ngamvongchon et al., 1992a). The arnino acid sequences of GnRH forms found to date in boney fishes are shown in Fig. 1. Novel forms have also been identifed in an elasmobranch, the spiny dogfish (Squalus ucunfhius) (Lovejoy et al., 1992), and an agnathan, the lamprey (Pefromyzon marinus) (2 forrns) (Sherwood et al., 1986). The presence of two or three distinct forms of GnRH has been demonstrated in several fish species. Thus in the sea bream, the presence of sea bream GnRH, salmon GnRH and chicken GnRH 11 (cGnRH 11) has been demonstrated (Powell et al., 1994), while in the catfish both cGnRH 11 and catfsh GnRH (cfGnRH1 are present (Ngam- vongchon et al., 1992a).

The evolution of GnRHs throughout the vertebrates has been reviewed by Sherwood et al. (1993). The recent discovery of two farms of GnRH in the tunicate Chelyosoma productum has extended the evolutionary line of this peptide family back into the protochordates (Sherwood, 1995). Two of the most frequently seen GnRH forms in the teleosts are sGnRH and cGnRH 11. Comparisons of the potency of natura1 forms of GnRH in stimulating gonadotropin (GtH) release in vitro or in vivo or in inducing ovulation have shown that cGnRH 11 is generally more potent than natural mGnRH or sGnRH. Thus in the Thai carp Puntius gonionutus, cGnRH 11 was more potent than mGnRH or sGnRH in increasing plasma GtH in vivo and in inducing ovulation (Sukurnasavin et al., 1993) while in the Thai catfish, cGnRH 11 and dogfish GnRH (dfGnRH) were capable of inducing ovulation in 71 and 52% of mature females, respectively, at 300 pg kg-. However cfGnRH, sGnRH and mGnRH were ineffective (Ngamvongchon et al., 1992b). Of the 10 amino acids present in the above 5 natura1 GnRHs, the only variations are seen in positions 5, 7 or 8 (Fig. 1). Several studies have shown that the potency of the various native GnRHs in different species is the result of

384 E.M. Donaldron / Animal Reproduction Science 42 (19%) 381-392

the combination of amino acids in positions 5, 7 and 8 (Habibi et al., 1992; Ngamvong- chon et al., 1992b; Schulz et al., 1993).

The development of potent GnRH analogues by modification of mammalian, avian and piscine GnRHs has had a significant impact on the development of practica1 means for the induction of final maturation, ovulation and spermiation in farmed teleosts. Substitution of the glycine in position 6 with an appropriate D amino acid such as PAla, D-Arg, D-Trp, D-Lys, D-Nal, D-Ser has been particularly effective in increasing potency and to a lesser extent the deletion of the terminal glycine in position 10 and its rcplacement with an ethylamide group. The GnRH analogue with the longest history of use in aquaculture is [u-Ala6,des-Gly ] mGnRH ethylamide (Donaldson and Hunter, 1983). It stimulates gonadotropin secretion (Van Der Kraak et al., 1983) steroidogenesis (Van Der Kraak et al., 1984) and ovulation (Van Der Kraak et al., 1986; Taranger et al., 1992; Haraldsson and Sveinsson, 1993) in salmon and in many other species. Another GnRH analogue that has been widely used in salmonids (Wei1 et al., 1991) and in carps (Peter et al., 1988) is [D-Arg6,des-Gly] sGnRH ethylamide. Other less widely used analogues include [~-Lys~] sGnRH in the Indian catfish Heteropneusres fossilis (Alok et al., 1993) and [D-Arg6,des-Glyo] cGnRH 11 ethylamide, [D-Na16,des-Gly] cGnRH 11 ethylamide and [o-Arg6,des-Gly] dfGnRH ethylamide in the Thai catfsh (Ngam- vongchon et al., 1992a; Ngamvongchon et al., 1992b). Key factors in the determination of the potency of individual GnRH analogues in vivo include affnity for GnRH receptors (Habibi et al., 1989; Schulz et al., 1993) and resistance to degradation by peptidase (Zohar et al., 1990).

While most successful protocols for induction of ovulation involve i.m. or i.p. injection of an aqueous solution of GnRH analogue, other forms of administration have been tested. Initial controlled release devices consisted of cholesterol or cholesterol/cel- lulose mixtures compressed into pellets (Sherwood et al., 1988). Polyanhydride micro- spheres have been utilized to induce spawning in the sea bream Spurus aurutu (Myolonas et al., 1995). Ovulation in salmon (e.g. Oncorhynchz4s tshfwytscha) is usually induced with two aqueous injections of GnRHA spaced three days apart and we have recently shown that a single polymer implant is more effective than a single aqueous injection owing to the sustained release of GnRHA from the implant (Fig. 2) (Solar et al., 1995).

Recent studies have demonstrated that GnRH can be administered orally to fish (McLean et al., 1991) and this technique has been utilized to induce ovulation in both warm water (spotted seatrout, Cynoscion nebulosus, Thomas and Boyd, 1989) and cold water marine species (sablefish, AnopZopoma~mbriu, Solar et al., 1990). We have also demonstrated tbe oral induction of ovulation in the Thai catfish when GnRHA is coadministered with domperidone (Sukumasavin et al., 1992).

3. Sex control

Studies on the endocrine control of sex differentiation were initially conducted on omamental species such as the medaka (Oryzius hfipes) in the, 1950s and 1960s (Yamamoto, 1969). Since then, studies on a range of economically significant species

E.M. Donalakon/Animal Reproduction Science 42 (1996) 381-392 385

+ CONTROL + D-ALA 6 INJ -+ D-AU 6 IYPLANl

Fig. 2. Influence of a single dose of 250 pgfish- [txAla6,des-Glyo] mGnRH, administered either in aqueous solution or as a slow release implaut (Reproboost) on ovulation in mature chinook salmon. Modified from Solar et al. (1995).

has led to the utilization of sex control in commercial aquaculture (Hunter and Donaldson, 1983). Sex control is of particular importante in the culture of tilapias (McAndrew, 1993), Pacific salmonids Oncorhynchus sp. (Piferrer and Donaldson, 1993), and Atlantic salmon, Sulmo dar (Johnstone, 1993) and its utilization is being examined in other cultured fish such as carps (Cyprinidae), flatfish (Pleuronectiformes) and seabass (Serrunidae).

There are two distinct means of endocrine sex control. The direct technique involves masculinization or feminization by androgen or estrogen treatment dwing early develop- ment of production fish. The indirect technique utilizes endocrine, genetic or environ- mental manipulation in the previous generation to produce monosex gametes (usually sperm). In current practice, monosex male tilapia are produced by direct masculinization (McAndrew, 19931, while monosex female salmonids are produced by indirect feminiza- tion (Fig. 3) (Solar and Donaldson, 1991; Donaldson and Devlin, 1996) and sterile (monosex female triploid) salmonids are produced by indirect feminization plus the use of pressure or temperature shock to induce triploidy (Fig. 3) (Benfey and Sutterlin, 1984; Guoxiong et al., 1989; Jungalwalla, 1991; Johnstone et al., 1991; Johnstone, 1993).

Significant progress has recently been achieved in several aspects of sex control in cultured fsh including: characterization of the labile period, determination of the minimal effective dosage of steroid and selection of the optimal natura1 or synthetic androgen or estrogen. Furthermore, the first Y specific DNA probes for fish have been developed and progress has been reported in the use of a number of chromosome manipulation techniques for sex control in aquaculture. The labile period is the time prior to morphological sex differentiation when a fish is most susceptible to endocrine intervention. In salmonids this occurs in the late pre batch to early post batch period (Piferrer and Donaldson, 1993) and its characterization has facilitated the use of single immersion treatments as short as two hours in duration to induce phenotypic feminiza-

386 E.M. Donaldion/ Animal Reproducrion Science 42 (19%) 381-392

BROODSTOCK PRODUCTION

1

t LOW

Androgen

Female

+ 1

Phenotypic Ovulation Male

+ Normal

+ Female

. Grow-out

as monosex females

Pressure shock 9500 psi for 5 min

30 min after fertiliz.

Ova\ Pm Fertilization

1 Genotypic Female Fertilized Eggs

I I

I

Grow out as monosex female

Market at any time

Fig. 3. Integrated production systems for the production of monosex female and monosex female triploid salmonids. Modifed from Donaldson and Benfey (1987).

tion (Piferrer and Donaldson, 1992) or masculinization (Piferrer et al., 1993) in chinook salmon shortly after hatching. In Atlantic salmon the labile period for masculinization appears to occur somewhat later (Johnstone and MacLachlan, 1994; 1.1. Solar and E.M. Donaldson, unpublished data, 1994). While single low doses of androgen can induce masculinization in salmonid alevins, high doses of aromatizable androgens can result in paradoxical feminization (Piferrer and Donaldson, 1991; Piferrer et al., 1993) and high dosages especially over longer periods can result in sterilization (Piferrer et al., 1994). Comparison between the potency of natura1 and synthetic androgens has shown that testosterone is ineffective in inducing masculinization while the male specific androgen 11 ketotestosterone is effective. Furthermore the non-aromatizable androgen 17cu meth- yldihydrotestosterone is capable of inducing masculinization without paradoxical femi- nization (Piferrer and Donaldson, 1991; Piferrer et al., 1993). Studies on the uptake and clearance of isotopically labeled 17/3 estradiol and testosterone have shown that in the salmonid egg, uptake and clearance are slow while in the alevin, uptake is rapid and clearance is slow and in the fry, uptake and clearance are both rapid. Thus for a given immersion dose, the alevin is exposed to a greater dose than either the egg or the fry (Piferrer and Donaldson, 1994). When initiating the production of a new stram of monosex female salmonids by the indirect technique, the identification of masculinized genetic females has required a variety of approaches. Initially this was accomplished by either a two generation masculinization process which involved progeny testing (Hunter et al., 1983) or by masculinization via dietary treatment after sex differentiation had been initiated which can result in genetic females developing testes which have no sperm ducts and which must be removed surgically when mature (Bye and Lincoln,

EM. Donaldson / Animal Reproduction Science 42 (1996) 381-392 387

1986). There have recently been two advances which facilitate the production of monosex female salmonid sperm. First, the production of masculinized gynogenetic salmonids has resulted in the generation of monosex female coho and Atlantic salmon (1.1. Solar and E.M. Donaldson, unpublished data). Second, the development of the frst piscine Y specific DNA probes has enabled the sorting of masculinized mixed sex stocks into genotypic males which are discarded and genotypic females which produce monosex female sperm at maturity. The first sex probe is specific to chinook salmon (Devlin et al., 1991) and has been used commercially to generate monosex female stocks (Devlin et al., 1994a). The second sex probe is derived from a growth hormone pseudogene which is located on the Y chromosome in four species of salmon, the chinook, coho, pink (Oncorhynchus gorbuscha) and chum (Orcorhynchus kets) (Du et al., 1993).

In tilapia, the production of al1 male populations is usually achieved by direct masculinization (Guerrero, 1975; McAndrew, 1993), however there has been consider- able progress recently in the development of indirect techniques for the production of monosex male tilapia. Thus in Oreochromis niloticus and 0. mossambicus sperm from YY supermales obtained by gynogenesis from XY females has been used to generate al1 male offspring (Scott et al., 1989; Pandian and Varadaraj, 1989). In Oreochromis aureus 17a ethynylestradiol has been used to produce phenotypic females having a homozy- gous ZZ male genotype. When these females were crossed with normal males, 100% male progenies were obtained (Melard, 1995).

In salmonids chromosome set manipulation techniques other than gynogenesis (see above) have been used for sex control. Thus monosex female triploid salmonids are generally sterile although limited egg development has been reported in older individu- als (Johnstone, 1993). Triploids have been reported to be less tolerant of extreme environmental conditions such as temperature (Ojolock et al., 1995) and in some studies they have been reported to grow at a slower rate than diploids probably as a result of the absente of endogenous anabolic steroids. On the other hand triploids can reach a larger size if they are grown past the time when diploids reach sexual maturity, e.g. brook trout Salvelinus fontinalis (Boulanger, 1991).

4. Future directions for induced ovulation and spermiation

We can expect increasing utilization of gonadotropin releasing hormone analogues to induce ovulation and spermiation in farmed fish. Various methods of administration for rapid and controlled release GnRHA preparations wil1 be utilized including injection, implantation and dietary treatment depending, among other factors, upon the species, the degree of maturity and whether the species is a multiple spawner. Choice of GnRHA wil1 be based upon in vivo biological activity in relation to peptide tost per effective dose. It is expected that attention wil1 be directed to the development of long term GnRHA administration protocols which wil1 accelerate reproductive development in species that have not yet matured and reproduced in captivity e.g. the giant cattsh of the Mekong River (pla buk) (Pangasius gigas). Also we can expect further research on the induction of gonadotropin release by manipulation of feed back inhibition through the use of antiestrogens and other mechanisms.

388 E.M. Donaldson / Animal Reproduction Science 42 (1996) 381-392

5. Future directions for sex control

Sex control technologies wil1 be utilized to produce fast growing al1 female stocks of the European sea bass (Dicentrurchus lubrar) and flatfish such as the turbot (Scophrhalmus marimus> and tbe olive flounder (Purulichthys olivuceous). They wil1 also be used to achieve reproductive containment of farmed strains, exotic species and genetically modifed fish. In exotic species such as Atlantic salmon grown in Pacific rim countries, reproductive containment could be achieved by farming monosex female stocks, as escaping fish would have no wild conspecific males with which to breed. (Donaldson et al., 1993). Monosex technology could also provide for the reproductive containment of genetically modified fish providing that they were exotic to the location of culture and could not hybridize with indigenous species. In situations where these conditions are not met it would be essential to sterilize genetically modified fish which are to be utilized outside the brood stock quarantine facility. The development of fast growing transgenic salmon based upon the first al1 fish DNA construct (Du et al., 1992; Devlin et al., 1995) and the first al1 salmon DNA construct (Devlin et al., 1994b) has led to countries such as Canada developing draft policy and guide lines for transgenic aquatic organisms which include reference to the need for both physical and biological i.e. reproductive, containment (Canada Dept. of Fisheries and Oceans, 1994). As fish are mobile organisms that cannot be recovered once released and furthermore can cross intemational boundaries there is a need to consider the development and harmonization of global guide lines for the containment of genetically modifed aquatic organisms.

Acknowledgements

1 thank Margaret Mattson for typing the manuscript and Helen M. Dye and Igor 1. Solar for preparation of diagrams.

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