Effects of dietary soy isoflavones on estrogenic activity,cortisol level,health and growth in rainbow trout,Oncorhynchus mykiss

Soy isoflavones (the phytoestrogens genistein, daidzein and glycitein) may act as estrogen receptor agonists or antagonists. The aim of this study was to examine the effect of dietary isoflavones on growth, reproduction and health in rainbow trout (Oncorhynchus mykiss). Rainbow trout fed three experimental diets containing different concentrations of isoflavones (0, 500 and 1,500 ppm). Growth, estrogenic activity, plasma estradiol levels, gonadal development, state of stress and histological changes in selected tissues were evaluated at the end of 70 days. Neither growth performance nor the relative mRNA levels of Insulin Growth Factor I (igf‐I) in the liver were influenced by different levels of dietary isoflavones. Plasma and liver vitellogenin (VTG) protein levels and plasma 17‐β‐estradiol (E₂) were unaffected by treatments, although the correlation between plasma levels of E₂ and VTG densitometry values was significant (p < .05). The fish gonadosomatic index (GSI) did not significantly differ among the three experimental groups but correlated with plasma VTG densitometry values (p < .05). Plasma, muscle and fin cortisol concentrations fell within the normal welfare range and were not correlated with isoflavone levels. Histologically, the distal intestine showed a normal morphology with well‐differentiated enterocytes and in the liver hepatocytes were also normal. A supranuclear accumulation of lipid droplets in enterocytes and some lipid droplets in hepatocytes were observed in all tested groups, suggesting an impact of basal dietary lipid on transport/metabolism of fat in the fish. Overall, the present results suggest that the doses of isoflavones tested do not compromise rainbow trout reproduction, growth and health.     

Comparison of in vitro vitellogenin syntheses by three phenols in primary cultures of Chinese minnow, Rhynchocypris oxycephalus hepatocytes

The in vitro effects of bisphenol-A (BPA), nonylphenol (NP) and 4-tert-octylphenol (OP) on vitellogenin (VTG) synthesis were examined using primary cultures of Chinese minnow, Rhynchocypris oxycephalus hepatocytes. In vitro VTG syntheses were induced by exposure to 10^?5 M of BPA and 10^?4 M of both NP and OP, suggesting that BPA had the highest estrogenic potential among the three phenols.     

Seasonal changes in hepatosomatic index,gonadosomatic index and plasma estradiol‐17β level in captively reared female rabbit fish (Siganus guttatus)

The seasonal changes in hepatosomatic index (HSI), gonadosomatic index (GSI) and plasma estradiol‐17β (E₂) level in female rabbit fish (Siganus guttatus) were investigated. The relationship between plasma E₂ levels with these indices and ovarian growth was also evaluated. Each month, at least 10 female broodfish were sacrificed to collect liver, ovary and blood for HSI, GSI and plasma E₂, respectively. GSI and HSI were calculated as percentage (%) of relative weight of gonad and liver to total body weight, respectively. Plasma E₂ level was measured using enzyme‐linked immunosorbent assay method (ELISA). Ovaries were cut and stained for histological observation. The results included seasonal changes in plasma E₂ levels, stages of ovarian development, GSI and HSI. The highest level of E₂ was observed in June (1,445.62 pg/ml) and during vitellogenesis (2,305 pg/ml). GSI and HSI values significant fluctuated monthly. The highest HSI and GSI were 1.72% in May and 3.58% in June, respectively. The pattern of plasma E₂ levels showed a relationship with GSI and different stages of ovarian development. HSI was associated with ovarian stages. During vitellogenesis, the highest value (1.9%) of HSI was observed. Histological sections showed that rabbit fish is a multiple spawner. These results contribute to further understanding of female rabbit fish reproductive biology in captivity. Important reproductive parameters such as HSI, GSI and E₂ can be used to indicate maturation status of this fish species.     

Effects of low dietary protein level on serum oestradiol,testosterone and sex reversal in rice field eel,Monopterus albus (Zuiew)

We investigated the effects of low dietary protein in isocaloric diets on sex reversal of Monopterus albus by evaluating the oestradiol (E₂) and testosterone (T) concentrations, gonadosomatic index (GSI), sex ratio and gonad structure at the histological level. Fish (9.50 ± 1.50 g average initial weight; n=3 per group) were fed with five practical diets containing 100, 150, 200, 250 or 400 g kg^?1 crude protein to apparent satiation for 15 months. Serum E₂ and T concentrations were determined by radioimmunoassays. E₂ concentrations and GSI significantly increased while T concentrations decreased as the dietary protein level was raised. Fish fed 400 g kg^?1 of dietary protein had significantly higher E₂ concentrations and GSI than those fed with lower dietary protein levels. The T concentrations of fish fed 100 g kg^?1 of dietary protein was significantly higher than that of fish fed higher dietary protein levels. The shift of sex ratio towards more male and intersex fish was observed with decreasing dietary protein levels. Therefore, low dietary protein level may promote sex change from female to male in M. albus. This study provides important information for successful reproductive management and may be exploited for aquaculture of this species.     

Structural and functional effects of early exposure to estradiol-17β and 17α-ethynylestradiol on the gonads of the gonochoristic teleost Dicentrarchus labrax

This study investigated the effects of estrogens on sexual differentiation in sea bass (Dicentrarchus labrax L.), a gonochoristic marine teleost that under culture conditions has a histologically sexual undifferentiated period that covers most of the first year of life, after which most individuals develop as males. Sea bass that had no noticeable histological sign of sex differentiation were fed estrogens at two doses (5 or 10 mg kg^-1 food) and for different periods ranging from 48 to 426 days post fertilization (DPF). Exposure to the synthetic estrogen 17-ethynylestradiol (EE₂) at 10 mg kg^-1 food from 60 to 260 DPF, including the sensitive period to equivalent doses of synthetic androgens previously determined for this species (126-226 DPF), significantly (p < 0.05) more than doubled the number of juvenile females to 80%, compared to the control value of 33%, and completely suppressed gonadal development in the remaining 20% of the population. This suggests that the period during which sea bass gonads exhibit high sensitivity to androgens is also very sensitive to estrogens. A comparable exposure to the natural estrogen estradiol-17 (E₂) resulted in 13% of the fish having suppressed gonadal development, but induced 57% of the fish to develop gonads with germinal tissue of both sexes, suggesting a pivotal role for E₂ during this sensitive period. Earlier exposure to EE₂ at 10 mg kg^-1 food from 48-88 DPF, significantly (p < 0.05) increased the number of females to 62% from 36% in the control group, allowing for the normal testicular development in the remaining fish. In contrast, a later chronic exposure (226-426 DPF) to E₂, at either 5 or 10 mg kg^-1 food, starting when the gonads showed no sign of sexual differentiation but past the critical sensitive period, had no effect on the resulting overall sex ratios, indicating that after this period responsiveness of the gonads to estrogens decreases as gonadal sexual differentiation progresses. However, the consequences of this apparently innocuous exposure were later manifested in adults, exemplified by a significant dose-dependent reduction in the number of mature males at 626 DPF, coinciding with the second reproductive season, the time when males normally reach sexual maturation in cultured sea bass. This suggests that chronic exposure to E₂ past the critical sensitive period may not affect the sex ratio, but could result in alterations in the male reproductive organs. This was later verified by histological analysis which revealed a significant (p < 0.05) dose-dependent reduction of the surface of the testicular lobules in the remaining males that did not mature. Together, these experiments illustrate both readily observable and subtle effects of estrogens on sex proportions, gonadal morphology and maturation rates, providing evidence that estrogen exposure can have delayed action in a teleost in a manner similar to the effects described for mammalian species. The possible existence of effects of this latter type in adult fish could be considered when evaluating the consequences of deliberate or accidental exposure to estrogens or putative estrogenic chemicals, particularly if such exposure had taken place during sex differentiation.     

Relationship between metabolic and reproductive hormones in salmonid fish

Circulating concentrations of estradiol (E₂), vitellogenin (VTG), thyroxine (T₄), triiodothyronine (T₃) and insulin were measured in reproductively maturing four and five year-old Atlantic salmon. Blood samples were collected from the fish in seawater for one year prior to their spawning in November in fresh water. In females, E₂ and VTG were low but detectable from December to July, and then increased to peak levels in September and October. Plasma levels of T₄ and T₃ were relatively constant in winter and spring, and decreased in July. Plasma concentration of T₄ increased in November when the fish returned to fresh water. Plasma T₃ levels remained low during the autumn. Both T₄ and T₃ levels tended to be higher in males than in females during September through November. Plasma insulin concentrations increased during the spring to peak values in May, and then decreased in June and July in fish of both sexes. There was a significant elevation of plasma insulin in males during October, and the levels in males tended to be higher than those found in females during final maturation.     

The reproductive physiology of three age classes of adult female diploid and triploid brook trout (Salvelinus fontinalis)

Triploid female fish show impaired gametogenesis and are unable to produce viable offspring. The reproductive physiology of artificially-induced triploid female salmonids has been well described up until the time of first sexual maturation in diploids, but few reports exist for older triploids. This study reports the influence of triploidy on growth, ovarian development and reproductive endocrinology among three age classes of female brook trout (Salvelinus fontinalis) in comparison to sibling diploids. Triploids were larger than diploids for most of the study period, but the difference was statistically significant only during maturation and spawning of 2⁺ diploids. Plasma estradiol-17 (E₂), testosterone (T) and vitellogenin (VTG) levels in triploids were generally lower than in diploids, and VTG was the only parameter to show seasonal fluctuations resembling those of diploids. Triploids showed significantly lower GSI and total oocyte number than diploids of similar age, and only half of all triploids sacrificed during the study (n=56) had developing oocytes in their ovaries. At age 3⁺, 13 of 19 triploid females had oocytes at various stages of development, including perinucleolar, yolk vesicle and yolk globule stages. In addition, three of these fish had collectively produced 72 mature stage oocytes. Thus, whereas diploid brook trout can produce mature oocytes as two-year-olds, triploids cannot do so until four years of age, with the number of mature oocytes being greatly reduced.     

Distinct expression of three estrogen receptors in response to bisphenol A and nonylphenol in male Nile tilapias (Oreochromis niloticus)

Environmental estrogens, such as bisphenol A (BisA) and nonylphenol (NP), have been shown to affect the estrogen receptor (ER) expression and induce male reproductive abnormalities. To elucidate molecular mechanisms of action of xenoestrogenic chemicals on the expression of estrogen receptors in the testes of Nile tilapia (Oreochromis niloticus), three full-length cDNAs respectively encoding ntERα, ntERβ1 and ntERβ2 were cloned from testes. The amino acid sequences of ntERα, ntERβ1 and ntERβ2 showed a high degree of similarity to the relevant fish species. Tissue-specific expression study showed that three receptors were highly expressed in pituitary, liver, testis, kidney and intestine tissues. The ntERα, ntERβ1 and ntERβ2 mRNA expressions were significantly higher at the sexual early recrudescing stage than at other recrudesced stages. After being exposed to xenoestrogens from weeks 2 to 4, the ntERα mRNA levels were increased significantly in testes after NP treatment at all sampling times or after 4 weeks of exposure to BPA. The ntERβ1 mRNA levels remained unchanged, while a significant decrease of the ntERβ2 mRNA level was observed in testes after exposure to NP and BPA. The present study demonstrates that the regulation of all three ntER subtypes in testes may act via different molecular mechanisms of exposure to NP and BPA.     

Relative potencies of natural estrogens on vitellogenin and choriogenin levels in the Indian freshwater spotted snakehead, <Emphasis Type="Italic">Channa punctata</Emphasis>: in vivo and in vitro studies

The relative efficacies of three natural estrogens viz., estrone (E₁), estradiol-17β (E₂) and estriol (E₃) to induce synthesis of vitellogenin (Vg) and choriogenin (Chg) were assessed in primary hepatocyte cultures of the Indian freshwater spotted snakehead, Channa punctata. Hepatocytes were isolated from the spotted snakehead liver by a non-enzymatic protocol. Optimum culture conditions were standardized for ensuring their viability and functioning. Isolated hepatocytes were cultured for 48 h for monolayer formation and then exposed to various concentrations (0.001–10 μM) of the three estrogens. Competitive homologous ELISAs, developed and validated for spotted snakehead Vg and Chg were employed to determine the amounts of these two proteins secreted into the culture medium after 48 h of incubation. The results reveal that although all the three estrogens were effective in inducing the production of Vg and Chg in a dose-dependent manner, there were differences in their relative potencies. Of three estrogens, E₁ was the least potent and could induce synthesis of Vg and Chg only at a minimum concentration of 0.5 μM; whereas significant levels of both the proteins were quantified in culture medium by exposing the hepatocytes to E₂ or E₃ even at a concentration of 0.001 μM. All three estrogens were effective in inducing synthesis of Vg and Chg in vivo also. These results suggest the possibility of employing the above in vitro experimental design to monitor the presence of estrogens/estrogen-like chemicals in natural waters, which could interfere with the estrogen receptor system of fish. This study further points to the possibility of using Chg, in addition to Vg, as a parameter for screening various chemicals for their estrogenic activity.     

Effects of 17β-estradiol and 4-nonylphenol on smoltification and vitellogenesis in Atlantic salmon (Salmo salar)

The impact of 17-estradiol (E₂) and the putative estrogenic compound, 4-nonylphenol (4-NP), on smoltification and vitellogenesis in Atlantic salmon (Salmo salar) was investigated during a 30 day period starting late April. Three groups of fresh water (FW) fish (1 year old, mixed sexes, average weight 23 g) were injected once a week with 50 µg (0.18 µmol) 17-estradiol, 3 mg (13.6 µmol) 4-nonylphenol dissolved in peanut oil, or peanut oil alone as control. Every ten days, subgroups were challenged with 28 ppt seawater (SW) for 24h, and sampled together with subgroups of FW fish. Treatment effects were examined on vitellogenic and osmoregulatory parameters. E₂ and 4-NP treatment increased the total calcium and protein level in plasma and the hepatosomatic index of FW fish, both indicating an activated vitellogenesis in the liver. The presence of vitellogenin in the plasma of 4-NP- and E₂-treated groups was further indicated by the appearance of a high molecular weight vitellogenin band (550 kDa) in electropherograms produced by native gel electrophoresis. This band appeared in exactly the same position in both the E₂- and the 4-NP-treated groups but could not be detected in controls. During the 30 day treatment period, control fish approached the peak of smoltification, as indicated by a distinct silvery appearance, decreasing condition factor, increasing levels of gill Na⁺,K⁺-ATPase and improved hypoosmoregulatory performance in the SW-challenge test. Both E₂ and 4-NP treatments significantly inhibited the progress of smoltification, as judged by a significant reduction of gill Na⁺,K⁺-ATPase activity, relative -subunit Na⁺,K⁺-ATPase mRNA expression, gill chloride cell density and a poorer hypoosmoregulatory performance of treated fish. The impaired SW-tolerance of E₂- and 4-NP-treated fish was strongly correlated with a decreased gill Na⁺,K⁺-ATPase activity. Despite a difference in relative potency, the present study shows that 4-nonylphenol and 17-estradiol may have qualitatively similar inhibitory effects on smoltification and hypoosmoregulatory physiology of Atlantic salmon. Both 4-NP and E₂ activated the vitellogenic system, and the study supports the hypothesis that sexual maturation and smoltification are antagonistic, developmental phenomena in salmon. It is suggested that the presence of estrogenic compounds in the environment may negatively influence smoltification and migration in wild stocks of salmon.     

Involvement of Gonadotropin-Releasing Hormone in Thyroxine Release in Three different forms of Teleost Fish: Barfin Founder, Masu Salmon and Goldfish

Effects of gonadotropin-releasing hormone (GnRH) on thyroxine (T₄) release in vivo and in vitro were studied in barfin flounder Verasper moseri, masu salmon Oncorhynchus masou and goldfish Carassius auratus. Seabream GnRH (sbGnRH) at a dose of 200 ng/50 g body weight (BW) significantly increased plasma T₄ levels 1 h after the in vivo injection in the barfin flounder, but thereafter the levels normalized. Salmon GnRH (sGnRH) significantly increased plasma T₄ levels l h after the injection with a significant return to initial levels in male masu salmon and male goldfish. In contrast, sGnRH and cGnRH-II in barfin flounder, and cGnRH-II in male masu salmon and male goldfish were not effective in stimulating T₄ release. To clarify direct involvement of GnRH in T₄ release, dissected lower jaw including scattered thyroid follicles was incubated with sbGnRH (1 μg/well) in barfin flounder, and with two doses (0.1 and 1 μg/well) of sGnRH in masu salmon and goldfish in vitro. T₄ concentrations of control were stable during 24 h. Incubation of lower jaw with high dose (1 μg/well) of GnRH significantly (P<0.05) increased T₄ concentrations of incubation medium at 1 h in all experimental fishes. These results indicate that direct stimulation of T₄ secretion by GnRH occurs widely in teleost fish.     

Photoperiodic manipulations of the reproductive cycle of sea bass (Dicentrarchus labrax) and their effects on gonadal development, and plasma 17ß-estradiol and vitellogenin levels

The annual profile of plasma vitellogenin (VTG) and 17ß-estradiol (E₂) levels, as well as gonadal development and spawning characteristics were investigated in captive female sea bass (Dicentrarchus labrax). Endocrine and gonadal changes were studied in fish reared under natural conditions or exposed to manipulated photothermal cycles. In natural conditions of photoperiod and temperature, sea bass spawned from February through March (East coast of Spain, 40°N 0°E). One or two months of constant long-days (15L/9D) in a constant short-day photoperiod regime (9L/15D) all-year-round, given early in the year (March and March–April), advanced spawning by 3 months. The same treatment applied later in the year (September–October) delayed spawning by 1 month, compared to controls.In all groups, changes in plasma VTG levels were correlated with E₂ levels, oocyte growth and spawning time. In control females, VTG was low (<100 ng ml^-1) during the summer, until its first surge in plasma 4 months before the beginning of spawning. The VTG (3.1 ± 0.3 mg ml^-1) and E₂ (4.1 ± 0.5 ng ml^-1) levels showed a single annual peak during late vitellogenesis, the time of the highest proportion of vitellogenic oocytes in the ovary. Constant high levels of VTG (1–1.4 mg ml^-1) and E₂ (1.6–1.9ng ml^-1) were maintained during the entire spawning time, together with the presence of vitellogenic oocytes, suggesting the existence of several waves of oocyte growth in the ovary and thus, several spawns per female. Endocrine profiles and oocyte development in fish exposed to constant photoperiods were similar to controls, but were shifted in time in relation to the displacement of the spawning time. In the fish showing advanced spawns, the duration of the gametogenic proces was compressed when compared to controls. The differences observed in the evolution of the reproductive-related factors in the advanced groups, which were exposed to a reduction in temperature to 15°C, suggest an influence of the temperature in the early stages of the reproductive cycle in sea bass.     

Disparate effects of gonadal steroid hormones on plasma and liver mRNA levels of insulin-like growth factor-I and vitellogenin in the tilapia, Oreochromis mossambicus

The tilapia, Oreochromis mossambicus, exhibits a sexually dimorphic pattern of growth, males growing larger than females. We examined the effects of E₂ and DHT on the GH/IGF-I axis and on VTG production in the tilapia. Sexually mature tilapia were injected with 5 μg g body weight of E₂ (males) or DHT (females) every 5 days for a total of 3 injections. Female tilapia had significantly higher plasma GH levels than males. However, plasma and liver mRNA levels of IGF-I were significantly lower in females than in males, whereas VTG levels in both the plasma and liver mRNA were significantly higher in females than in males. Although significant amounts of VTG were detected in control males (8 ± 0.3 μg ml), the levels in control females (3000 ± 500 μg ml) were about 400 times higher than in males. Males treated with E₂ exhibited a female-like GH/IGF-I profile. That is, they had significantly elevated levels of plasma GH with lower plasma IGF-I and liver IGF-I mRNA levels. Estradiol treatment significantly elevated both plasma and liver mRNA VTG levels. Dihydrotestosterone treatment in females induced a male-like GH/IGF-I profile: plasma GH levels were significantly reduced, whereas plasma and liver IGF-I mRNA levels were significantly elevated. Both plasma and liver mRNA levels of VTG were not altered by DHT treatment. Pituitary GH mRNA levels were similar in all treatment groups. These results clearly indicate that estrogens and androgens feminize and masculinize the GH/IGF-I axis, respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hormonal changes accompanying sexual maturation in captive milkfish (Chanos chanos Forsskal)

Steroid hormone profiles accompanying sexual maturation in captive milkfish are described. There were no significant differences in levels of serum estradiol 17- (E₂) and testosterone (T) between immature male and female fish. Mean E₂ levels rose from 0.54±0.11 ng/ml in immature females (Stage 1) to 4.53±1.16 ng/ml in vitellogenic females (Stage 5), while T levels increased from 2.06±0.28 ng/ml to 38.4±9.26 ng/ml. E₂ and T levels were positively correlated to GSI and oocyte diameter. In males, serum T levels increased from 2.5±0.40 ng/ml in immature males to 27.73±5.02 ng/ml in spermiating males. A significantly higher T level was found in males with thick and scantly milt (spermiation index, SPI, 2) compared to males with scanty milt (SPI, 1) or males with copious, fluid milt (SPI, 3).Serum levels of E₂ and T, and the GSI in females rose significantly during the breeding season (April–June 1983). The levels of both steroids dropped below 1 ng/ml in spent females sampled in succeeding months. In immature males, T levels ranged from 1.11 ng/ml to 2.78 ng/ml and rose significantly to 21.52±8.38 ng/ml during the breeding season when GSI peaked. Serum T levels dropped to around 10 ng/ml in the succeeding months when only spent or regressed males were sampled.     

Comparative responses in rare minnow exposed to 17β-estradiol during different life stages

Present in the excrement of humans and animals, 17β-estradiol (E₂) has been detected in the aquatic environment in a range from several nanograms to several hundred nanograms per liter. In this study, the sensitivities of rare minnows during different life stages to E₂ at environmentally relevant (5, 25, and 100 ng l⁻¹) and high (1000 ng l⁻¹) concentrations were compared using vitellogenin (VTG) and gonad development as biomarkers under semistatic conditions. After 21 days of exposure, VTG concentrations in whole-body homogenates were analyzed; the results indicated that the lowest observed effective concentration for VTG induction was 25 ng l⁻¹ E₂ in the adult stage, but 100 ng l⁻¹ E₂ in the larval and juvenile stages. After exposure in the early life stage, the larval and juvenile fish were transferred to clean water until gonad maturation. No significant difference in VTG induction was found between the exposure and control groups in the adults. However, a markedly increased proportion of females and appearance of hermaphrodism were observed in the juvenile-stage group exposed to 25 ng l⁻¹ E₂. These results showed that VTG induction in the adult stage is more sensitive than in larval and juvenile stages following exposure to E₂. The juvenile stage may be the critical period of gonad development. Sex ratio could be a sensitive biomarker indicating exposure to xenoestrogens in early-life-stage subchronic exposure tests. The results of this study provide useful information for selecting sensitive biomarkers properly in aquatic toxicology testing.     

Effect of in vitro xenoestrogens on steroidogenesis in mature female fish, Chasmichthys dolichognathus

In this study, fully vitellogenic oocytes of the longchin goby (Chasmichthys dolichognathus) were exposed to in vitro xenoestrogens such as diethylstilbestrol (DES), bisphenol A (BPA) and nonylphenol (NP) at concentrations of 100 ng/ml in the presence of [³H]17α-hydroxyprogesterone as precursor. The major metabolites produced in vitro are progestogens [17α-hydroxy,20α-dihydroprogesterone (17α20αOHP) and 17α-hydroxy,20β-dihydroprogesterone (17α20βOHP)], androgens [androstenedione (A4) and testosterone (T)] and estrogens [estrone (E1) and estradiol-17β (E2)]. Comparative activities of these chemicals in oocyte steroid biosynthesis showed as follows: NP treatment resulted in clear stimulation of estrogen production, while the opposite occurred in DES treated incubation. Treatment with DES resulted in a higher synthesis of 17α20αOHP. BPA treatment increased the androgen, while estrogen synthesis was slightly inhibited. These results suggest that NP exhibited clearly estrogenic activity on the three chemicals.     

Purification,characterization and immunoassay of striped bass (Morone saxatilis) vitellogenin

The egg yolk precursor, vitellogenin (VTG), was purified from blood plasma of striped bass by chromatography on hydroxylapatite or DEAE-agarose. The fish were first implanted with estradiol-17β (E₂), which induced vitellogenesis. A rabbit antiserum (a-FSPP) raised against plasma from mature female striped bass, and then adsorbed with mature male plasma, was used to detect female-specific plasma protein (FSPP) in the chromatography fractions. Striped bass VTG (s-VTG) was collected from the peak fraction that was induced by E₂, reacted with a-FSPP, and contained all detectable phosphoprotein. It appeared as a single band (M_r ≂ 170,000) in SDS-PAGE or Western blots using a-FSPP, and as a pair of closely-spaced phospholipoprotein bands in native gradient-PAGE, suggesting that there is more than one circulating form of s-VTG. The relationship of s-VTG to the yolk proteins was verified using a-FSPP. The antiserum reacted with the main peak from gel filtration of saline ovary extracts, and it specifically immunostained the two main bands in Western blots of the extracts and the yolk granules of mature oocytes. The amino acid composition of s-VTG was similar to that of VTG from other fish and Xenopus. A radial immunodiffusion assay for s-VTG was developed using a-FSPP and purified s-VTG as standard. The s-VTG was not detected in blood plasma of males, immature females, or regressed adult females, but plasma s-VTG levels were highly correlated with plasma E₂ and testosterone levels, and oocyte growth, in maturing females. The results indicate that the maturational status of female striped bass can be identified by s-VTG immunoassay.     

Effect of aromatase inhibitors on the ovarian development of stunted yearlings of rohu (Labeo rohita): a preliminary study

In India, many of the fish farmers stock 1-year-old stunted fishes (stunted yearlings) of Indian major carps (IMC) for enhancing fish production through compensatory growth, but many of them observed problems of early maturation in these fishes. Application of aromatase inhibitors for deceleration of ovarian maturation is one of the probable solutions to mitigate this issue. In the present study, a synthetic aromatase inhibitor letrozole [25 (L₂₅) and 50 (L₅₀) mg kg^?1 feed] and a plant-derived aromatase inhibitor, grape seed extract [100 (G₁₀₀) and 200 (G₂₀₀) mg kg^?1 feed], were fed to stunted yearlings of rohu (Labeo rohita) for 45 days well before the onset of breeding season. Maturation indices such as gonadosomatic index (GSI) and serum oestradiol (E) levels indicated a dose-dependent inhibition of ovarian development in the aromatase-inhibitor-treated fish. Higher dose of letrozole (GSI, 15.12 ± 0.18; E, 3.19 ± 0.42) and grape seed extract (GSI, 16.90 ± 0.40; E, 3.60 ± 0.75) were found to be more effective since control fish showed further advancement in maturation (GSI, 21.20 ± 1.10; E, 7.33 ± 0.74) during the peak breeding season (15th June). Histological observations also confirmed the results revealing a delayed initiation of ovarian development in the case of higher doses of letrozole and grape seed extracts. These results indicate the possible use of aromatase inhibitors in arresting the early maturation process in IMC.     

Control of reproduction in Nile tilapia Oreochromis niloticus (L.) by photoperiod manipulation

A major problem in tilapia aquaculture is the frequent reproduction of female fish, leading to increased competition for supplemented food and stunted somatic growth. The feasibility of using photoperiod manipulation to arrest the reproductive performance of tilapia Oreochromis niloticus was therefore examined. Newly hatched O. niloticus were reared in the laboratory under 12L:12D photoperiod at 28 °C. Fish (230–340 g) were maintained under 6L:6D, 12L:12D and 14L:10D photoperiod at 28 °C during the course of this study. Effect of photoperiod manipulation on reproductive parameters of fecundity, gamete quality, offspring viability and overall reproductive success were evaluated. Steroid levels (estradiol-17β, E₂; testosterone, T) during the spawning cycles of fish were analyzed on days 1 and 3 postspawning and at 3-day intervals thereafter. A total of 72 female fish from each photoperiod treatment were investigated the changing pattern of E₂, T and GSI with fish that have spawned once, twice and three times. Fish exposed to 12L:12D and 14L:10D photoperiod spawned successfully throughout the study. Whereas the spawning of fish exposed to 6L:6D photoperiod was arrested after three to four spawning cycles. The arrestment of spawning in fish exposed to 6L:6D photoperiod was paralleled by a significant decrease in plasma levels of E₂ (P<0.05). By contrast, there was no major difference in T levels among the treatments. These findings suggest that photoperiod manipulation can be used to arrest the spawning in tilapia O. niloticus. The utility of this in controlling the problem of overcrowding due to excess offspring in tilapia aquaculture is applicable.     

Characterization,specificity and sensibility of produced anti-<Emphasis Type="Italic">Rhamdia quelen</Emphasis> vitellogenin in Brazilian fish species

Endocrine-disrupting chemicals (EDCs) are widespread used and can interfere on hormone regulation with adverse consequences for both biota and human. Vitellogenin (vtg) is a yolk precursor protein synthesized by the liver in response to estrogen. In order to characterize the vtg of tropical fish Rhamdia quelen and establish a molecular biomarker, adult male individuals were exposed to 17-β-estradiol (E₂) for vtg induction and anti-R. quelen vtg polyclonal antibodies production. Vitellogenic female fish were used as positive control group. E₂-induced vtg was characterized as a glycolipophosphoprotein of high molecular mass with peptide mass fingerprint very similar in E₂-exposed male and vitellogenic female fish. A polyclonal serum containing anti-R. quelen vtg antibodies was produced and showed high specificity and sensibility to detect the vtg of three fish species: R. quelen, Piaractus mesopotamicus and Prochilodus lineatus. Wildlife and laboratory studies reported that EDCs released into the environment may alter the levels of plasma vtg in male fish, making this protein a valuable biomarker of xenoestrogens exposure. Then, we propose the use of anti-R. quelen vtg as a tool for biomonitoring studies and water quality assessment in Brazil and South American countries where the three fish species occur.