Edward M. Donaldson

9 – Hormonal Enhancement of Growth

This chapter reviews the comparative endocrinology of the growth regulating hormones in fish and describes the research aimed at the application of the knowledge regarding fish culture. Three types of hormones have been shown to increase growth rates in fish both alone and in combination. These are the pituitary growth hormones, the anabolic steroid hormones, and the thyroid hormones. In addition to these, the insulins are a fourth group of hormones, which play a significant metabolic role and may be capable of growth promotion alone or in combination with other hormones. While pituitary materials have been collected from most classes of fish, they have only been tested for growth promoting activity in the teleosts, amphibia, or mammalia. Most of these fish pituitary preparations have been tested in the mammalia, specifically in the rat tibial plate assay. Of the various classes of fish, only the teleostei have been used as bioassay recipients for growth hormone preparations from other vertebrates. It is clear from the range of donor and recipient response relationships shown in the chapter that the growth hormone of teleosts is distinct from that of other vertebrates including the other classes of fish.

5 Hormonal Sex Control and its Application to Fish Culture

Publisher Summary A discussion of hormonal sex control inherently involves consideration of the assumptions on which the action of the hormone is based, ultimately the mechanisms of sex determination and sex differentiation. Although pursued intensively during the 19OOs, a unifying model of sex determination and sex differentiation has proven illusive. This is, perhaps, not surprising in light of the bewildering diversity of sexual expression found within the vertebrates, particularly the largest group the Pisces. The chapter focuses on the theoretical context within which hormonal sex control studies are conducted. The chapter examines the various components of hormonal sex control studies with particular reference to those factors that influence treatment success. Studies that have involved several economically important species are discussed in detail. It is notable that the majority of studies on hormonal sex control have involved gonochorist species. Therefore, these species are also discussed in the chapter.

7 Induced Final Maturation, Ovulation, and Spermiation in Cultured Fish

Publisher Summary Major progress has occurred in recent years toward the development of reliable and economic procedures for the induction of ovulation and spermiation in economically important teleosts, and that “second generation” techniques are being applied for these purposes. This chapter discusses induced final maturation, ovulation, and spermiation in cultured fish. The application of induced-maturation techniques in fish falls into two basic categories. The first involves the induction of maturation and spawning in fish, which would not otherwise reproduce in captivity; the second involves the manipulation of spawning time in fish, which do normally reproduce in captivity. The search for synthetic alternatives to gonadotropin in the 1970s has led to the development of second generation techniques for the induction of final maturation, ovulation, and speriniation in fish. These techniques either operate at a higher level in the axis by stimulating the production and/or release of gonadotropin in the pituitary gland or they operate at a lower level of the axis by supplying ovarian hormones that would normally be stimulated by endogenous or exogenous gonadotropin.

Preparation of gonadotropin from salmon (Oncorhynchus tshawytscha) pituitary glands

Abstract A procedure is described for the purification of pituitary gonadotropin from the salmon ( Oncorhynchus tshawytscha ) by means of ethanol extraction, gel filtration on Sephadex G-100 (preparation SG-G100) and ion exchange chromatography on DEAE-cellulose; (preparations SG-DEAE-1 to 7). Bioassays were carried out using either the goldfish spermiation response or the augmentation of radiophosphate uptake by chick testis. In the goldfish assay, 1 mg SG-G100 and SG-DEAE-2 were equivalent to approximately 2150 IU HCG and 10,000 IU HCG, respectively. In the chick assay, SG-G100 had a potency of 0.10 × NIH-LH-S16. The sedimentation coefficients s 20 w for SG-DEAE-3 and SG-G100 were determined by ultracentrifugation to be 2.57 and 2.65, respectively. Molecular weights (M) calculated using the Svedberg equation for SG-DEAE-3 and SG-G100 were 28,524 and 29,411, respectively. At pH 1.4, s 20 w for SG-G100 was 1.38 and M = 12,805. Direct determination of M by gel filtration using nonglycoproteins as standards gave an M value for SG-G100 of 40,000. Stokes radius for SG-G100 was determined by gel filtration to be 26.5 A and a diffusion coefficient of 8.9 × 10 −7 cm 2 sec −1 was calculated.

Gonadal differentiation in coho salmon, Oncorhynchus kisutch, after a single treatment with androgen or estrogen at different stages during ontogenesis

Abstract The labile period for hormonal treatment for purposes of controlling sexual differentiation in coho salmon was deternined by administration of estradiol-17β (E2) and 17α-methyltestosterone (MT) through immersion for 2 h at a concentration of 400 μg/l, to groups of coho salmon sampled weekly from the late eyed-egg stage until the third week of feeding. Histological samples of the gonadal region were prepared from a control group at each weekly treatment interval using a plastic embedding process. It was found that a single estrogen treatment significantly increased the proportion of phenotypic females when administered between 8 days pre-hatch and 13 days post-hatch, while a single androgen treatment significantly increased the proportion of phenotypic males when administered between 6 and 13 days post-hatch. The maximum response for E2-treated groups (84% females) occurred as a result of treatment 1 day before 50% hatch while the maximum response for MT-treated groups (73.1% males) occurred as a result of treatment 1 week later. Thus, the labile period during which the fish were able to respond to a single administration of exogenous steroid was determined to last 3 weeks, and coincided with the time of hatching and the first days of the alevin stage. Sexual differentiation was first observed at 27 days post-hatching, 21–28 days after the time of maximum steroid sensitivity and coinciding approximately with the time of first feeding.

Induction of triploidy in rainbow trout (Salmo gairdneri Richardson) by heat shock, and investigation of early growth

Abstract Heat shocks were applied to wild rainbow trout eggs for 10 min, starting at 1 and 40 min after fertilization and at temperatures ranging from 24 to 30°C. Analysis of blood samples by flow cytometry revealed triploid induction ranging from 10 to 100% depending on the temperature and timing of the treatment. Survival from fertilization to 60 days and growth of the treated groups were lower than the control. The best temperature shock treatment with regard to percentage of triploids and percentage survival relative to controls was 26°C starting 1 min after fertilization. Histological analysis of the gonads revealed sterility in triploid female fish.

Effects of LH-RH and des-gly10[D-Ala6]LH-RH-ethylamide on plasma sex steroid profiles in adult female coho salmon (Oncorhynchus kisutch)

17 beta-Estradiol, testosterone, and 17 alpha, 20 beta dihydroxy-4-pregnen-3-one (17 alpha 20 beta P) levels were measured in plasma samples obtained from coho salmon (Oncorhynchus kisutch) during the preovulatory period and following the injection of mammalian gonadotropin releasing hormones. Spontaneous reproductive activity was characterized by a rapid decline in plasma 17 beta-estradiol 10 days prior to ovulation and a large increase in plasma 17 alpha 20 beta P 6 days before ovulation. Testosterone levels remained high (greater than 125 ng/ml) throughout the preovulatory period, with a small peak evident 6 days prior to ovulation. Oocyte development was not accelerated in fish injected with mammalian LH-RH, whereas des-Gly10[D-Ala6]LH-RH-ethylamide (LH-RHA DAla6) promoted germinal vesicle breakdown (GVBD) in 10 out of 14 fish within 96 hr. Only LH-RHA DAla6-injected fish which completed GVBD displayed the characteristic steroid changes observed during spontaneous reproductive activity. In LH-RH-injected fish, there was a transient increase in plasma 17 alpha 20 beta P levels which persisted for less than 24 hr. LH-RHA DAla6-injected fish which failed to complete GVBD maintained high 17 alpha 20 beta P levels, but the peak concentrations were lower than those in fish which completed GVBD. These fish also maintained high plasma 17 beta-estradiol levels when compared to fish which completed GVBD. The appearance of high plasma 17 alpha 20 beta P levels during spontaneous and LH-RHA DAla6-induced reproductive activity was coincident with the time of GVBD. This finding was consistent with the view that 17 alpha 20 beta P functions as the maturation-inducing steroid in salmonids. The induction of GVBD using gonadotropin-releasing hormones was related to the elevation of plasma gonadotropin levels for greater than 24 hr [G. Van Der Kraak, H. R. Lin, E. M. Donaldson, H. M. Dye, and G. A. Hunter (1983) Gen. Comp. Endocrinol. 49, 470-476] and a decrease in 17 beta-estradiol production.

TRANSMISSION AND PHENOTYPIC EFFECTS OF AN ANTIFREEZE/GH GENE CONSTRUCT IN COHO SALMON (ONCORHYNCHUS KISUTCH)

Abstract Transmission of the opAFPGHc gene construct from parental transgenic coho salmon to F 1 progeny has been observed. Just prior to first feeding, these offspring were found to fall into two distinct phenotypic classes on the basis of morphology and external colouration. One group possessed the normal brown colouration typical of coho salmon alevins, whereas the other had a distinct green colouration and showed signs of cranial deformities and opercular overgrowth. Polymerase chain reaction analysis revealed that green phenotype was correlated with the presence of the opAFPGHc gene construct, and thus colouration could be used to identify transgenic progeny. On this basis, frequencies of transgene transmission to F 1 progeny from four individuals ranged from 2.2 to 18.9%, while a fifth male produced no transgenic progeny. Prior to first feeding, the transgenic progeny were found to be 21.2% heavier and 11.9% longer than their non-transgenic siblings, suggesting that the expression of GH in early development can influence the rate or efficiency of conversion of yolk energy reserves. After 1 year of development of F 1 progeny, the atypical phenotype associated with overgrowth of cartilage in the cranial and opercular regions became progressively more severe and resulted in reduced viability.

Effects of estradiol-17β and 17α-methyltestosterone on gonadal differentiation in the coho salmon, Oncorhynchus kisutch

Abstract Methods are described for controlling sex differentiation in the coho salmon, Oncorhynchus kisutch . Eyed eggs and alevins were immersed in solutions of estradiol-17β and 17α-methyltestosterone and fed these steroids in the diet for a period of 10 weeks postswim-up. In seven out of 10 groups that received estradiol all fish resembled normal females when sampled at 4 months posthatch. In fish treated with methyltestosterone at all except the lowest dose (25 and 50 μg/l) the gonads resembled neither normal males nor normal females; these gonads were composed largely of connective tissue with only occasional germ cells. Various proportions of male, female and intersex gonads were observed in groups which received androgen or estrogen only in the diet. The implications of the work for salmon culture include the production of sterile fish and increasing the proportion of female salmon in hatchery populations.

Effects of LH-RH and des-Gly10[d-Ala6]LH-RH-ethylamide on plasma gonadotropin levels and oocyte maturation in adult female coho salmon (Oncorhynchus kisutch)

Plasma gonadotropin (GtH) levels and state of oocyte development were determined in adult female coho salmon following single intraperitoneal injections of LH-RH or its superactive analog des-Gly10[D-Ala6]LH-RH-ethylamide (LH-RHA DAla6). The peptides injected at dosages of 1.0 or 0.2 mg LH-RH and 0.2 or 0.02 mg LH-RHA DAla6/kg bw elevated plasma GtH by 1.5 hr postinjection. The response to the analog was of longer duration. Plasma GtH levels returned to basal levels 24 hr following LH-RH injection while elevated plasma GtH levels were maintained for at least 96 hr in response to the analog. Only those fish injected with LH-RHA DAla6 showed an accelerated rate of germinal vesicle breakdown (GVBD). Based on their relative effects on GVBD, LH-RHA DAla6 has at least 50 times the biological potency of LH-RH in adult female coho salmon.