Intra‐annual changes in reproductive indices of male and female Himalayan snow trout,Schizothorax richardsonii (Gray, 1832)

Periodic changes in reproductive hormone levels, gonadal histology and gonadosomatic index (GSI) of snow trout, Schizothorax richardsonii, were examined to ascertain annual cycle of gonadal development and reproductive status in their natural habitat. In females, there were coherent changes in plasma 17β‐oestradiol and vitellogenin along with GSI, oocyte maturation and vitellogenic progression, collectively indicating two distinct maturation peaks during the months of September and February. Coinciding with this, in males, plasma 11‐keto testosterone was also noticeably higher during September and February, with highest GSI values in September. However, plasma 17α, 20β‐dihydroxyprogesterone levels in males were found to be persistently high from September to February. This observation suggests the potential presence of matured oozing males over a longer period, unlike in females. Overall, the close association between reproductive hormone levels, GSI and gonadal maturation stages in males and females (particularly, the presence of postovulatory follicle complexes) with apparent natural synchronization clearly indicates that S. richardsonii breeds twice in a year, possibly during late September to early November and late February to early April in the coldwater riverine habitats of the Indian Himalayan region.     

Studies on the mode of action of neuropeptide Y (NPY) on maturational gonadotropin (GtH) secretion from perifused rainbow trout pituitary glands

The action of neuropeptide Y (NPY) and gonadotropin releasing hormone (s-GnRH) have been compared on the release of gonadotropin (GtH) by perifused rainbow trout pituitary glands sampled from freshly ovulated female rainbow trout. We have already demonstrated that NPY stimulated the secretion of GtH in vitro. The pituitary responses to s-GnRH and NPY were similar either after repeated 10 min infusions or a one hour prolonged application. In both cases, after the first application, the pituitary did not responded to subsequent secretagogues stimulations, and appeared to be desensitized. The stimulatory action of s-GnRH was partly inhibited (60%) by LH-RH antagonist (DpGlu¹, DPhe², DTrp^3–6) LH-RH, which completely inhibited the response to NPY in perifused pituitary glands sampled from freshly ovulated females, but did not modify the response of pituitaries taken from vitellogenic animals in which NPY induced a transient inhibition of the GtH secretion. These results may indicate that the mode of action of NPY would differ between vitellogenic and matured animals. NPY also stimulated the GtH secretion from perifused pituitary dispersed cells prepared from pituitaries taken from freshly ovulated rainbow trout, indicating that NPY may act directly on the pituitary gonadotropic cells to stimulate GtH secretion.     

Neuropeptide Y (NPY) modulatesin vitro gonadotropin in release from rainbow trout pituitary glands

This work investigated the action of neuropeptide Y (NPY) on thein vitro pituitary release of the maturing gonadotropic hormone (GtH) of the rainbow trout using a perifusion system employing trout balanced salt solution (pH 7.5) at 15°C and a 12.5 ml/h flow rate. In vitellogenic females a 20 minutes NPY application (10⁻⁷ M) induced a 20–30% decrease in GtH secretion. Removal of NPY was followed by a rebound in GTH secretion. On the contrary, in ovulated females, NPY (15 minutes, 10⁻⁷ M) directly stimulated GTH secretion. The greatest stimulation was obtained the day of ovulation where the stimulatory effect of NPY was similar to those induced by s.GnRH in the same conditions, reaching 400% of the basal GTH level. In vitellogenic females treated with 1-4-6 androstadien 3–7 dione, an inhibitor of aromatase activity, the pituitary response to NPY was similar to that obtained in ovulated females. Thus thein vitro action of NPY might depend on thein vivo steroidogenic environment.     

Preliminary report on hybridization experiments in trout — Growth and survival of F1 hybrids

Hybridization experiments were carried out by crossing females of common rainbow trout with the males of albino rainbow trout. Survival rates in egg, hatchling, fry and yearling stages, and the annual growth rate were higher than those of either parent.Reciprocal crosses with females of albino rainbow trout and males of common rainbow trout showed poor survival rate. Gonadial development of these hybrids was normal.     

Regulation of LH synthesis and release by GnRH and gonadal steroids in masu salmon

The roles of salmon GnRH (sGnRH) and gonadal steroid hormones in regulation of LH synthesis and release were examined in primary pituitary cell cultures of masu salmon (Oncorhynchus masou). Pituitaries were taken from fish at four reproductive stages: in March (initiation of sexual maturation); May (early maturation); July (pre-spawning); and September (spawning period). Amounts of LHβ subunit mRNA in the pituitary cells were determined by real-time PCR, and LH levels in the medium were determined by RIA. sGnRH and gonadal steroids including estradiol-17β (E2), testosterone (T) and 11-ketotestosterone (11-KT) were added to the cultures to examine their direct effects on LH response. sGnRH had no significant effect on LHβ mRNA levels at any stages, although a stimulatory trend was noted in March. In contrast, E2 and T considerably increased LHβ subunit mRNA levels in March and May during initial stages of maturation, and the effects were less pronounced in July and September. On the other hand, sGnRH stimulated LH release at all stages in the males and the effects were most prominent in July and September. E2 and T also stimulated LH release in July and September, but their effects were weaker than that of sGnRH. The present results indicate that sGnRH and gonadal steroids directly regulate LH synthesis and release in masu salmon pituitary cells: sGnRH mainly stimulates LH release in the late stage of sexual maturation; whereas, E2 and T are effective in stimulating LH synthesis at earlier stages of maturation.     

Sex differences in circulating steroid hormone levels in the red drum, Sciaenops ocellatus L.

Steroid profiles of cultured and captive red drum (Sciaenops ocellatus L.) were investigated to evaluate the potential use of circulating sex steroid levels as a tool for gender identification in this species. Cultured 18‐month‐old fish were maintained on a 120‐day shortened photothermal cycle to induce precocious maturation. Additionally, wild‐caught fish were maintained in captivity under simulated natural photothermal conditions from late spring to early fall. Circulating 11‐ketotestosterone (11‐KT) levels were significantly higher in males compared with females during the early stages of gonadal growth in both cultured and captive fish. Plasma testosterone (T) levels showed a similar trend; however, the differences were significant only when males were already producing sperm. 17β‐estradiol (E₂) concentrations were low in males and females before gonadal recrudescence but increased significantly with the progression of vitellogenesis in females. These results show that a test using a minimum concentration of circulating 11‐KT could be developed to differentiate between sexes in the early stages of gonadal maturation in red drum. Moreover, plasma E₂ concentrations could be used to identify vitellogenic females. The two steroids considered together could help avoid possible error in gender identification due to unusually high levels of certain steroids encountered in some individuals.     

The effects of gonadal development and sex steroids on growth hormone secretion in the male tilapia hybrid (Oreochromis niloticus × O. aureus)

Profiles of plasma growth hormone (GH) in male tilapia hybrid (Oreochromis niloticus x O. aureus) were measured and compared at different times of the year. The profiles did not appear to be repetitive, however, differences in their nature were observed at the different seasons; the most erratic profiles were seen in the height of the reproductive season (July), while the peaks were more subdued in the spring and disappeared in the autumn. Peaks in male fish were more prominent than in the females when measured in July. Perifused pituitary fragments from fish with a high GSI responded to salmon gonadotropin-releasing hormone (sGnRH) analog (10 nM-1 M), while those from fish with a low GSI barely responded to even the highest dose. Exposure of perifused pituitary fragments from sexually-regressed fish to carp growth hormone-releasing hormone (cGHRH; 0.1 M) or sGnRH (I M) stimulated GH release only after injection of the fish with methyl testosterone (MT; 3 injections of 0.4 mg kg ¹). The same MT pretreatment did not alter the response to dopamine (DA; 1 or 10 M). GH pituitary content in MT-treated fish was lower than in control fish, which may be explained by the higher circulating GH levels in these fish, but does not account for the increased response to the releasing hormones. Castration abolished the response of cultured pituitary cells to sGnRH (I fM-100 nM) without altering either their basal rate of secretion or circulating GH levels. Addition of steroids to the culture medium (MT or estradiol at 10 nM for 2 days) enabled a GH response to sGnRH stimulation in cells from sexually regressed fish. Pituitary cells which had not been exposed to steroids failed to respond to sGnRH, although their response to forskolin or TPA was similar to that of steroid-exposed cells. It would appear, therefore, that at least one of the effects of the sex steroids on the response to GnRH is exerted proximally to the formation of cAMP, or PKC, presumably at the level of the receptor. An increase in the number of receptors to the GH-releasing hormones, following steroid exposure, would explain also the changing nature of the GH secretory profile in different stages of the reproductive season.     

A releaser pheromone that attracts methyltestosterone- treated immature fish in the urine of ovulated female rainbow trout

SUMMARY: Behavioral experiments concerning a releaser pheromone in the urine of female rainbow trout were performed using immature fish administered orally with 17α-methyltestosterone (MT) during the non-spawning season. The urine was collected by catheter. The frequency of entries of test fish was recorded in each channel scented by test and control solutions in a Y-maze trough. The behavior of both MT-treated and control fish demonstrated that they could not discriminate the differences between distilled and environmental water as control solutions. There was also no difference between MT-treated and control fish when distilled and environmental water were introduced. The MT-treated immature fish were attracted to the channel scented by ovulated female urine. Neither coelomic fluid nor the immature female urine had any effect on the behavioral responses of MT-treated fish, while immature control fish had no preference for the urine of ovulated females. These results suggest in rainbow trout that ovulated female urine contains a releaser pheromone to attract mature males, and that androgens are involved in the sensory mechanisms detecting the releaser pheromone in fish.     

Sex reversal in salmonid culture: Part III. The production and performance of all-female populations of brook trout

Oral administration of 17β-oestradiol at 20 mg/kg diet for a period from 0 to 60 days following “swim-up” resulted in the sex reversal of male brook trout and the production of essentially all-female populations. The gonads of these animals were indistinguishable from the ovaries of untreated fish.Oral administration of this hormone at the same concentration but for the period from 0 to 40 days resulted in a population which comprised 67% females, 21% males and 12% bisexual animals.Within the control group, the growth rate of males was significantly greater than that of females. Growth in the all-female stocks produced by dietary oestradiol administration (0–60 days) was significantly lower than that of the untreated population taken as a whole but was identical to that of the control females.The growth of brook trout during the first year was closely comparable to that of rainbow trout reared under identical conditions. During the second year, the growth of the rainbow trout exceeded that of the brook trout. At 19 months of age, all the animals in the control and oestradiol treated brook trout populations were fully mature; maturity in the rainbow trout was substantially confined to males.     

Hormonal profile of growing male and female diploids and triploids of the blue tilapia,Oreochromis aureus,reared in intensive culture

Triploidy as a result of thermal shock exposure of fertilized eggs decreases the growth rate ofOreochromis aureus as compared to their diploid controls, but this is due to the higher female ratio present in triploids (86%) and the lower growth rate of females. When females and males are considered separately, the growth rate is not significantly different in diploids and triploids. Since triploidy results in a malfunctioning steroidogenesis in females (mainly testosterone (T) and 17β-estradiol (E₂)), but does not affect the growth rate, it is concluded that female gonadal steroids do not influence growth unless in pharmacological concentrations. These low levels of gonadal steroids are generally accompanied by higher levels of gonadotropin (GtH), but the difference is not always significant. Despite their lower growth rate diploid females have higher plasma concentrations of growth hormone (GH) during several months compared to the triploid females and diploid males. 3,5,3′-triiodo-L-thyronine (T₃) levels, however, are comparable between diploid and triploid females (except for 1 month), but higher in diploid males in 4 of the 5 months studied. 11-ketotestosterone (11kT) is always higher in males. These results indicate that the higher growth rate of males may be related to the high circulating levels of T₃ and 11kT.     

The development of a radioimmunoassay for carp,Cyprinus carpio,vitellogenin

The development of an easily — performed and robust radioimmunoassay (RIA) to carp, Cyprinus carpio, vitellogenin (c-VTG) is described. Purified c-VTG was iodinated using Iodogen. The resulting c-VTG label was useful for up to 60 days. High titre antibodies were raised in rabbits to the purified c-VTG. The practical operating range of the c-VTG RIA was between 2 and 200 ng. ml^-1. VTG was detected in plasma from all female mirror carp investigated, and all plasmas diluted parallel to the standard. The plasma VTG level in female carp increased concomitantly with the GSI; levels increasing from the ng. ml^-1 level in juveniles to a maximum of 1 mg. ml^-1 in fully mature females. VTG level was a far more sensitive index of the degree of sexual development than was GSI. In males, blood levels of VTG were always undetectable. Vitellogenic plasma from all subspecies of C. carpio (e.g. mirror, common, Koi) diluted parallel to the standard, as did blood from most other female cyprinids, such as the roach (Rutilis rutilis), bream (Abramis brama), and dace (Leuciscus leuciscus), but not all. These results suggest that the structure of VTG is highly conserved within this family. However, plasma from vitellogenic salmonids did not cross-react in the RIA. The relationship between plasma VTG and calcium levels was studied in both carp and rainbow trout. In rainbow trout it was found that plasma calcium levels do not rise above basal levels until the VTG level exceeds about 1 mg. ml^-1, and therefore in this species it is only useful as an indicator of the degree of ovarian development in the later stages of the reproductive cycle. In the carp, however, and probably other cyprinids, blood VTG levels do not appear to naturally exceed about 1 mg.ml^-1, and plasma calcium levels are not suitable as an indirect measure of the VTG level.     

Effects of partial or total fish meal replacement by agricultural by-product diets on gonad maturation, sex steroids and vitellogenin dynamics of African catfish (Clarias gariepinus)

The establishment of the first sexual maturation was characterized in African catfish (Clarias gariepinus) in order to study the efficiency of replacement of fish meal (FM) by diets composed of local vegetable ingredients. Four diets were formulated containing decreasing levels of FM (50-0% for diet 1 to diet 4) and increasing proportions of vegetable ingredients (50-100%). Gonadosomatic index (GSI), diameter and percentages of developmental stages of oocytes, plasma sex steroids and vitellogenin dynamics were investigated from February to June using one-year-old fish. Fish were individually tagged, and 12 individuals from each diet were investigated monthly. Replacement of FM with plant ingredients did not affect the GSI neither in males, nor in females. All males were spermiating, and no abnormal gonads were found. In females, GSI and percentages of advanced stages of oocytes decreased during the dry season, indicating seasonal changes in gonad development. Moreover, oocytes were in late exogenous vitellogenesis, but no final maturation stages were observed, whatever the diet. Higher plasma levels of E2 in females and of androgens (T and 11-KT) in both sexes were observed in fish fed diet 4 than in those receiving diet 1 depending on the season. Levels of plasma E2 and ALP (indicator for vitellogenin) in males did not differ among treatments and seasons suggesting no phytoestrogenic activity. The results showed that total replacement of FM by vegetable diets composed of groundnut oilcakes, bean and sunflower meals has no deleterious effect on the onset of sexual maturation in African catfish but, may stimulate the sex steroid production and in turns may potentially exert some positive actions on reproductive success.     

Seasonal changes in circulating growth hormone (GH), hepatic GH-binding and plasma insulin-like growth factor-I immunoreactivity in a marine fish,gilthead sea bream,Sparus aurata

We have studied the seasonal relationship between growth and circulating growth hormone (GH), hepatic GH-binding and plasma insulin-like growth factor-I immunoreactivity in gilthead sea bream,Sparus aurata. The seasonal increase in plasma GH levels preceded by several weeks the summer increase in growth rates. In contrast, a marked increase in hepatic GH-binding with a high degree of endogenous GH occupancy was found during the period of maximum growth which suggests an enhanced sensitivity of liver to GH action. Thus, circulating levels of immunoreactive IGF-I, probably derived from the liver in response to GH action, were positively correlated with growth throughout the experimental period although a consistent relationship between growth and circulating GH was not found. In spite of this, we consider that, in gilthead sea bream, as in several other teleosts, the availability of endogenous GH can limit growth. Thus, under environmental conditions of suboptimal growth, a single intraperitoneal injection of recombinant rainbow trout GH (rtGH) induced over the dose range tested (0.75, 1.5, 3 μg g BW⁻¹) an increase in plasma IGF-I-like immunoreactivity comparable to that seen during the period of maximum growth.     

Sex-Reversal of Oreochromis aureus by Immersion in Mibolerone,a Synthetic Steroid1

Mibolerone (MI), a synthetic steroid, was used to sex-reverse undifferentiated Oreochromis aureus fry. Fry were exposed to 0.0, 0.3, 0.6, or 1.0 ppm MI in static-water solutions for five weeks (immersion treatments) or were fed a diet containing 1.0 ppm MI for four weeks in a flow-through system. Following hormone treatment, the fish were grown to over 60 mm total length prior to sex determination using gonadal squash examination. Fish immersed in 1.0, 0.6, or 0.3 ppm MI had average tissue MI concentrations of 14.4, 5.6, and 3.3 ppm, respectively. Immersion in either 1.0 or 0.6 ppm MI for five weeks resulted in an average of 82% males and 18% ovo-testicular fish (inter-sex fish) with no gonadal females being produced. Exposure to 0.3 ppm MI resulted in 78.7% males, 20.7% ovo-testicular fish, and 0.7% gonadal females. Feeding a diet containing 1.0 ppm MI resulted in 85% males, 11% ovo-testicular fish, and 4% gonadal females. Fry growth and survival were negatively correlated with the MI concentration of the immersion treatments. Exposing tilapia fry to static-water solutions of 0.6 ppm MI for five weeks appears to be a feasible method of eliminating the production of functional females. Immersion solutions should be changed at least weekly to maintain an effective hormone concentration.     

Sex-Reversal of Oreochromis aureus by Immersion in Mibolerone,a Synthetic Steroid 1

Mibolerone (MI), a synthetic steroid, was used to sex-reverse undifferentiated Oreochromis aureus fry. Fry were exposed to 0.0, 0.3, 0.6, or 1.0 ppm MI in static-water solutions for five weeks (immersion treatments) or were fed a diet containing 1.0 ppm MI for four weeks in a flow-through system. Following hormone treatment, the fish were grown to over 60 mm total length prior to sex determination using gonadal squash examination. Fish immersed in 1.0, 0.6, or 0.3 ppm MI had average tissue MI concentrations of 14.4, 5.6, and 3.3 ppm, respectively. Immersion in either 1.0 or 0.6 ppm MI for five weeks resulted in an average of 82% males and 18% ovo-testicular fish (inter-sex fish) with no gonadal females being produced. Exposure to 0.3 ppm MI resulted in 78.7% males, 20.7% ovo-testicular fish, and 0.7% gonadal females. Feeding a diet containing 1.0 ppm MI resulted in 85% males, 11% ovo-testicular fish, and 4% gonadal females. Fry growth and survival were negatively correlated with the MI concentration of the immersion treatments. Exposing tilapia fry to static-water solutions of 0.6 ppm MI for five weeks appears to be a feasible method of eliminating the production of functional females. Immersion solutions should be changed at least weekly to maintain an effective hormone concentration.     

Reproduction related immunoglobulin changes in rainbow trout

Annual changes in plasma immunoglobulin (IgM) levels were investigated in three strains of rainbow trout Oncorhynchus mykiss which have different spawning periods, i.e., September–October, November–December, and January, reared under constant water temperature and natural day length. Plasma IgM levels decreased during the spawning season in all strains tested. The IgM changes became reversed in response to significant increases in plasma testosterone (T) and estradiol-17 in females and T and 11-ketotestosterone in males. Though the IgM decline showed a connection with suppressed immunocompetence, since many mature fish caught fungal diseases, no clear differences were observed in the plasma IgM levels between infected and noninfected fish during the spawning season. Incidentally, plasma IgM levels in infection prone fish were higher than in noninfection prone fish prior to the spawning season, whereas coincident differences in the plasma steroid levels were observed. Immature fish reared under lower water temperatures showed lower IgM levels. The effect of water temperature may have to be considered when analyzing the defense mechanism during the spawning season in rainbow trout.     

Changes in levels of GnRH in the brain and pituitary and GTH in the pituitary in male masu salmon,Oncorhynchus masou,from hatching to maturation

Levels of two types of gonadotropin-releasing hormone (salmon GnRH and chicken GnRH-II) in the brain and pituitary, and content of gonadotropin (GTHIβ and IIβ) in the pituitary were measured in male masu salmon from hatching to gonadal maturation for three years in order to clarify the involvement of GnRHs in precocious maturation. Underyearling precocious males were distinguishable in summer of year 1 and were marked by an increased GSI. Spermiation was observed among these individuals thereafter every autumn. Pituitary GTHIβ content in both precocious and immature males, and GTHIIβ content in precocious males showed seasonal fluctuations — high in autumn and low in winter. Pituitary GTHIIβ content was low in immature males. Pituitary sGnRH content in precocious males increased from spring to autumn during the three-year period. sGnRH concentrations in discrete brain areas showed seasonal changes — high during autumn to winter and low in summer. Concentrations in the olfactory bulbs and hypothalamus increased significantly in association with testicular maturation during year 3. sGnRH concentrations in the hypothalamus were significantly higher in precocious males than in immature males; this was possibly due to positive feedback of steroid hormones. cGnRH-II was undetectable in the pituitary and no distinct changes were observed in its concentration in the brain in relation to maturation. The phenomenon of underyearling precocious maturation is considered to be triggered before the onset of early summer. It is suggested that males which mature precociously are larger in size and contain much sGnRH in the pituitary before the outward signs of precocity appear; sGnRH may stimulate GTH II synthesis and induce precocious maturation.     

The growth and reproductive endocrinology of adult triploid Pacific salmonids

This paper describes the effect of triploidy on growth and reproductive endocrinology in the months leading up to and including spawning in rainbow trout,Salmo gairdneri, and pink salmon,Oncorhynchus gorbuscha. Growth rates were the same for diploid and triploid rainbow trout, but triploid female pink salmon were smaller than maturing diploid females and diploid and triploid males of the same age. Triploid males of both species developed typical secondary sexual characteristics and had normal endocrine profiles, although their cycle appeared to be delayed by about one month. Triploid females remained silvery in appearance and showed no endocrine signs of maturation, even at the level of the pituitary. Thus, although triploids of both sexes are genetically sterile, only the females do not undergo physiological maturation.Reported in part at the Third International Symposium on Reproductive Physiology of Fish, St. John's, Newfoundland, August 2–7, 1987.     

Stocking density and cohort sampling effects on endocrine interactions in rainbow trout

Studies were conducted to explore the effect of cohort sampling and stocking density on the interactions between plasma growth hormone (GH), thyroid hormone and cortisol concentrations in rainbow trout. Depending on the experimental design, plasma GH concentrations were either suppressed, or elevated by sequential removal of cohorts from the holding aquarium. Since plasma cortisol concentrations consistently increased during cohort sampling, regardless of experimental design, it would appear that the apparent correlation (direct or inverse) is the result of concomitant changes, i.e. not necessarily a cause-effect relationship. Plasma GH concentrations of rainbow trout were not correlated with eviscerated body weight.Trout stocked at 150 kg m^–3 exhibited a significantly lower mean growth rate, hepatosomatic index, hepatic lipid reserve, plasma triiodo-l-thyronine (T₃) concentration andin vitro hepatic T₃ production than trout stocked at 60 kg m^–3. These observations are consistent with the former group being food deprived or ration restricted. Stocking density appeared to have no effect on plasma GH or cortisol concentrations, or on the pituitary-interrenal axis response to stressor challenge, or the thyroid tissue response to exogenous TSH challenge.     

Molecular cloning and dimorphic expression of growth hormone (gh) in female and male spotted scat Scatophagus argus

Spotted scat Scatophagus argus exhibits a typical sexual growth dimorphism in which the females grow faster than the males. Growth hormone (GH) is best known as an anterior pituitary hormone fundamental in regulating growth. To clarify the roles in sexual growth dimorphism in the spotted scat, gh cDNA was isolated from the pituitary. A phylogenetic tree was constructed based on the GH amino acid sequences of spotted scat and other vertebrates, and the resulting topology clearly reflects the taxonomic relationship of the Perciformes species selected. Alignment of GH amino acid sequences displayed very high similarity between the spotted scat and the other Perciformes species. The qRT-PCR analysis demonstrated that females exhibited higher pituitary gh mRNA levels than males at 6, 18, and 30 months (P < 0.05), which was 1.84, 4.61 and 6.34 times greater, respectively. In addition, immature males (6 months) exhibited higher pituitary gh mRNA levels than mature males (18 and 30 months). These results imply that the sexual growth dimorphism may be ascribable to the gh levels in pituitary in spotted scat.