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

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.     

Effect of hormone injection frequency on the lipid content and fatty acid compositions in gonad,muscle and liver of Anguilla japonica during artificial maturation

Two groups of Anguilla japonica were treated with hormone, one on a weekly basis, the other on a biweekly basis. Intramuscular injection was applied at a dose of 0.75 mg/kg BW carp pituitary extract (CPE) plus 150 IU/kg BW human chorionic gonadotropin (hCG) for female fish, while males received half of this dose. The average total lipid content of the gonads from the weekly treated group, i.e. 20.6 ± 1.2 % for females and 18.0 ± 2.3 % for males, was significantly higher than the biweekly treated group, 16.8 ± 0.7 and 15.5 ± 1.3 %, respectively (p < 0.05). For both muscle and liver, the readings were not significantly different. The gonads from the weekly treated fish had more fatty acids, particularly saturated fatty acids, polyunsaturated fatty acids, highly unsaturated fatty acids, eicosapentaenoic acid (20:5n-3, EPA), docosahexaenoic acid (22:6n-3, DHA) and arachidonic acid (20:4n-6, ARA). Histological examination showed that the ovaries of both the weekly and the biweekly treated fish were mainly at stage IV. However, the weekly treated females had bigger oocyte diameter (722.0 ± 60.9 μm) than the biweekly females (611.6 ± 22.6 μm). These results suggest that CPE and hCG promoted the maturation process for both scheduled induction and that the frequency of hormone injection influenced the biochemical composition of gonads, especially their lipids. Our study describes for the first time the effect of hormone injection frequency on the lipid content and fatty acid composition in the gonads of A. japonica during artificial maturation.     

Dietary carotenoids do not improve motility or antioxidant capacity in cichlid fish sperm

Carotenoids may act as antioxidants under many circumstances. We examined the importance of carotenoids as antioxidants in the gonads of male convict cichlids (Amatitlania nigrofasciata), a species in which males lack the carotenoid-based breeding coloration that characterizes females. Male fish were fed one of four diets that included different combinations of xanthophyll and carotene carotenoids, and then we measured carotenoid concentration of the gonads, gonadosomatic index (GSI), sperm motility, and the antioxidant capacity of the gonads. Significant differences were found in gonadal carotenoid content among treatment groups, suggesting that dietary carotenoids were indeed sequestered in the gonads. There were no differences among diet groups, however, in GSI, sperm motility, or gonadal antioxidant capacity. These findings suggest that carotenoids are required only in small amounts in the testes of male convict cichlids or that they play a limited role in protecting sperm from oxidative damage.     

Study of sex determination system in ship sturgeon, Acipenser nudiventris using meiotic gynogenesis

The sex ratios and sex determination mechanism of gynogenetic diploids of ship sturgeon, Acipenser nudiventris, have been investigated to verify the possibilities of sex control by chromosome manipulation in this species. Meiotic gynogenesis was induced in a female ship sturgeon using cold shock after egg activation with UV-irradiated sperm of a male Siberian sturgeon. Microsatellite DNA analysis was applied for verification of uniparental inheritance in the gynogenetic diploid group of fish. All the analyzed gynogenetic diploids possessed only maternal genotype in the examined experimental group of fish. In this study, a minimum of two distinctly selected diagnostic loci in the offspring was used to confirm exclusively maternal contribution. Also, these fish were analyzed for sex diagnostic. Histological analysis of gonads from gynogenetic diploids, obtained from one family, showed 73.3 % of females and 27.7 % of males. The observed sex ratio has suggested that the ship sturgeon have a female heterogametic sex determination system. Gynogenesis in this species with female heterogametic sex determination system will have important role in breeding program and reclamation of its natural population to produce both female and male progeny, while this species has been introduced in the red list of IUCN (International Union for Conservation of Nature and Natural Resources).     

Expression of dmrt1 and sox9 during gonadal development in the Siberian sturgeon (Acipenser baerii)

The sex differentiation period of the Siberian sturgeon was investigated through expression profiling of two testicular markers (dmrt1 and sox9). At the molecular level, a clear sexual dimorphism of dmrt1 and sox9 was observed in 3-year-old fish with immature gonads, in which males showed higher expression of these genes. Among 16-month-old sturgeons cultured in Uruguay, gonad morphology analyses showed one group of fish with undifferentiated gonads and a second group which had started their histological differentiation into ovaries or testes. dmrt1 showed a significantly higher expression in testes of recently differentiated fish, but this was not the case for sox9. In undifferentiated fish, we observed two clearly different groups in terms of expression: one group of fish over-expressing male markers (dmrt1, sox9) and another group of fish showing very low expression of these genes. This suggests that fish undergoing male differentiation can be identified by their profiles of gene expression before they undergo morphological differentiation.     

Induced sex reversal of dusky grouper, Epinephelus marginatus (Lowe)

The present paper describes sex reversal in dusky grouper, Epinephelus marginatus (Lowe). Over a 4-month feeding period, 5 mg kg^?1 of the male hormone 17-α methyltestosterone was combined with frozen fish as feed. After 2.5 months of treatment, the gonads of all six fish treated in the experiment presented tissues with oocytes and spermatogonia, and after 3 months, there were only a few remnants of oogonial tissue left in the gonads. All fish in the experiment gave sperm of varying quality after 4 months of the trial. The quantity of the hormone administered to the fish over these 4 months was 89-113 mg kg^?1. Spermatozoid motility varied from 95% to 40%, and lasted for about one hour in sea water. Using the combined sperm of three males, fertilization ranged from 50% to 95%. Fertilized eggs and hatched larvae continued to develop normally.     

Reproductive cycle of yellow croaker <Emphasis Type="Italic">Larimichthys polyactis</Emphasis> in southern waters off Korea

The reproductive cycle and batch fecundity of yellow croaker Larimichthys polyactis were examined in 981 specimens (286 males, 695 females) caught from waters around Mokpo, Busan, and Jeju-do (Korea) between February 2008 and January 2009. Males and females with ripe-stage gonads were collected between April and June, at which time the mean gonadosomatic indices were also high, indicating that the spawning season of yellow croaker around the waters off southern Korea occurs from April to June. Degenerating postovulatory follicles as well as oocytes in the migratory-nucleus stage or hydrated stage were found in ripe-stage gonads, suggesting that this species can spawn repeatedly in a season. Batch fecundity in 45 females with migratory-nucleus or hydrated oocytes ranged from 19,396 to 106,311 eggs in fish varying from 20.0 to 26.7 cm in total length and was found to be positively related to total length and body weight. Our findings should assist in implementing a proper stock assessment of yellow croaker around the waters off Korea.     

Comparison of aspects of the reproductive biology of Pomadasys,Otolithes and Pampus spp. in Kuwaiti waters

Aspects of the reproductive biology of Pomadasys argenteus, Otolithes argenteus and Pampus argenteus are described. Maturation of the gonads is divided into 8 arbitrary stages, and the distribution of the stages in an annual cycle is reported. In Pomadasys argenteus there is a major spawning period from February to April and another minor one in October. In Otolithes argenteus there is one distinct spawning period from January to April. The spawning season of Pampus argenteus extends from March to August. The histology of the gonads and the distribution of oocytes of different diameters in the ovaries of the 3 fish species are described. The distribution of oocyte diameters in the ovary of Pampus argenteus is polymodal and characteristic of serial spawners, whereas the distribution of oocyte diameters in Pomadasys argenteus and Otolithes argenteus corroborate the finding that these fish are single spawners. The estimated potential fecundity for Pomadasys argenteus ranged from 6 25 848 to 2 424 846, for Otolithes argenteus from 53 676 to 7 163 954 and for Pampus argenteus from 765 675 to 2 537 774.     

Cytogenetic and histological studies of the brook trout, Salvelinus fontinalis (Mitchill), and the Arctic char, S. alpinus (L.) hybrids

Although brook trout and the Arctic char hybrids are able to reproduce, individuals with decreased fertility or even fish that are unable to produce any gametes have been also described. Abnormal gonadal development and disturbances in the gamete production in the char hybrid offspring may be triggered by the odd chromosome number and disturbances in their pairing during meiosis. To verify this hypothesis, cytogenetic examination and the gonadal histology analysis of the brook trout x Arctic char hybrids were carried out. Diploid chromosome number in the studied char (F₁) hybrids varied from 82 to 84 (FN = 99–102). Among 28 hybrids, 12 males, three females, nine intersex individuals and two sterile specimens were described. In the case of two individuals, gonads were not found. Diploid chromosome numbers in the males and intersex individuals varied from 82 to 84. Chromosome numbers in the females were 82 and 83 chromosomes. Two sterile fish exhibited karyotypes composed of 82 and 84 chromosomes. Predominance of the ovarian component in the intersex gonads and gonadal sex ratio distortion towards the males suggested hybrid females had problems with gonadal differentiation. However, the lack of the clear relationship between chromosome number and gonadal development in the studied hybrids did not support our hypothesis that odd chromosome number may be responsible for such reproductive disturbances in the hybrid individuals. We have presumed that sterility and intersexual development of the gonads may be caused by interactions between brook trout and Arctic char genes on the sex chromosomes and autosomes rather than unpairing of the parental chromosomes.     

Effects of Natural EstradioI-17β and Synthetic 17α- Ethynylestradiol on Direct Feminization of European Sea Bass Dicentrarchus labrax

To produce a monosex female population of European sea bass Dicentrarchus labrax, fry were fed dry diets containing dosages of 12.5, 25, and 50 mg/kg food of either the natural estrogen estradiol-170β(E₂) or the synthetic estrogen 17α-ethynylestradiol (EE₂) for 60 d starting at 88 d post-hatch (dph). A complete feminization (100%) was achieved in all E₂-treated groups at the age of 11 mo (330 dph). All affected fish had ovaries similar in size and histological structure to those of control females. In the E₂-treated groups, feminized fish were heavier and longer than untreated controls (males and females combined). In control groups females exhibited significantly higher body weight and total length than males. Untreated females from control groups and females from the group treated with E₂ at 12.5 mg/kg food had similar body weight, suggesting that in sea bass growth is related to phenotypic sex. In the Entreated groups, survival rates were similar to those of the control fish. A relatively high percentage of females was obtained in the EE₂-treated groups (from 38.6 to 96.5%). However, the gonadal development in these fish was significantly suppressed and a dose-dependent reduction of gonadal sizes was evident. Treatments with the EE₂ (12-5, 25, and 50 mg/kg food) resulted in many fish having abnormal (2.9-5.4-39.8%, respectively) and sterile (0.6-6.0-21.6%, respectively) gonads. Effects also included significantly lower weight and shorter length when compared with controls. Furthermore, fish fed with EE₂ at the dosage of 50 mg/kg food had high mortality rate. A simple protocol was developed for the complete feminization in sea bass in which the fry (80-100 dph) were fed to satiation two times daily with a diet containing 12.5 me of E₂/ks food for a period of 60 d.     

Age and growth of the yellow gurnard (Trigla lucerna L. 1758) from the Thermaikos Gulf (Greece) with some comments on its biology

Age, growth and reproduction of yellow gurnard in the Thermaikos Gulf were studied. Otoliths from 250 yellow gurnards (138 males and 112 females) were read for age-determination. The sexes differ in growth rate, and the body length—otolith radius relationship is linear (male, L = 2.65 + 5.58 R; female, L = ?43. 9 + 7.04 R; where L = fish length in mm, R = otolith radius 18 ×). The life-span of females seems to be longer than that of males. Weight increased in females and males as the 3.110026 and 3.146985 power of the length, respectively. The growth of yellow gurnard of the Thermaikos Gulf lies between that of yellow gurnard of Morocco and that of those in the Catalane Sea. Reproduction lasts from January to May, and occasionally also from October to July. Males begin to mature after completion of the third annual ring, females after the fourth. Migration of bigger fish to greater depths is evident. The male to female ratio is usually 1 : 1.     

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.     

Ultrasound evaluation of the gonadal structure in sex-reversed rainbow trout females

Ultrasound approach has been used to assess morphology of the gonadal structure in the sex-reversed rainbow trout females (neomales) provided in the course of 11β-hydroxyandrostenedione treatment applied within gonadal differentiation period. Eighteen matured individuals (in range 38.0–48.6 cm of body length and 802–1644 g of body weight) were scanned using digital ultrasound apparatus (DP-6900 model) Mindray Ltd., during the spawning season. After screening, fishes were killed to validate the morphological configuration and position of the gonads in the body cavity. The favorable place for cross-sectional imaging of gonadal lobes was half of the distance between the pectoral and pelvic fins below lateral line of neomales. Most of the examined specimens (61 %) had properly shaped, paired testis. Moreover, presence of individuals with asymmetrical gonads (33 %) and one bisexual fish was confirmed. There were no differences between total volume of sampled semen and sperm motility, but statistically significantly different in sperm concentration within selected (with symmetrical and asymmetrical testis) groups of neomales. It was confirmed that ultrasonic imaging is an efficient and accurate method to determine the state of gonads of mature sex-reversed rainbow trout females during spawning season and offers the opportunity for non-invasive detection of any morphological anomalies in their gonads.     

Helminth parasitism of Galaxias maculatus (Jenyns 1842) in southwestern Australia

Abstract –  One cestode, Ligula sp. [possibly Ligula intestinalis (L.)], one trematode, Diplostomum sp., and two nematode parasitic worms, Eustrongylides sp. [possibly Eustrongylides gadopsis (Royal Society of South Australia, 64, 340)] and Contracaecum sp. are reported from Galaxias maculatus inhabiting a permanent freshwater lake and two intermittently flowing, saline rivers in southwestern Australia. Worms infecting fish are all larval; the definitive hosts are piscivorous waterfowl. Ligula sp. infected 12% of fish in the lake. Effects of infection include reduced lifespan, significant weight reduction of gonads of males and females and body weight of females. Infection reduces the proportion of males that attain spawning gonad stage more severely than it does in females. The prevalence and intensity of Ligula sp. infection were much less in the rivers. The infection of Pseudogobius olorum (Sauvage 1880) by this cestode is reported for the first time in Western Australia. Trematodes were much more benign in their effect on G. maculatus .     

Effect of temperature on the efficiency of feminization of medaka (Oryzias latipes) by hormonal (estradiol) manipulation

During the critical time of sex determination, fish gonads are liable to chemicals and environmental factors that can disrupt the normal course of sex differentiation. This study was conducted to examine the possibility of a combined effect of water temperature and an exogenous steroid hormone (estradiol-17β, E₂) on the process of sex differentiation of Japanese medaka (Oryzias latipes) of the d-rR strain. Sex reversal experiments conducted at different temperatures (14, 18, 22, 26, 30, and 34 °C) and E₂ concentrations (0, 1 and 10 ppb) showed that the percentage of feminization of genotypic males into phenotypic females decreased with increasing temperature for both E₂ concentrations, suggesting a marked synergism between the two factors.     

Aspects of the reproductive biology of the sharpsnout seabream Diplodus puntazzo (Cetti, 1777). I. Gametogenesis and gonadal cycle in captivity during the third year of life

A histological study was made of the reproductive cycle of captive sharpsnout seabream Diplodus puntazzo during the 3rd year of life. All specimens contained both ovarian and testicular tissue. Ovarian tissue prevailed in 64 out of 120 fish sampled throughout one year. The ovary underwent a long resting phase from October to August, during which time it contained oogonia, primary growth phase (PGP) and occasional yolk vesicle stage oocytes. Intense ovarian development took place in September leading to the formation of advanced vitellogenic oocytes. Final maturation and spawning were not achieved in any of the fish. The testicular area in the predominantly female gonads contained only spermatogonia and was found to be histologically normal in 76% of individuals, but severely regressed in the remaining fish. The testicular portion was found to be dominant in 56 out of 120 fish sampled throughout one year. These underwent a resting phase from November to July, a maturing/pre-spawning phase in August, and a spawning phase with spontaneous sperm release in September to October, as in the natural environment. The ovarian portion in the functional male gonads contained only oogonia and oocytes which did not develop beyond the perinucleolus stage at any point during the reproductive cycle. Apart from the pre-spawning/spawning phase, during which the gonadosomatic index (GSI) was comparable in the two groups, the GSI values were approximately ten-fold higher in those fish in which the ovary predominated than in those in which the testis predominated.     

Cloning and expression of prostaglandin E2 receptor subtype 1 (ep 1 ) in Bostrichthys sinensis

Our previous studies suggested that prostaglandin E₂ (PGE₂) is a putative sex pheromone in Chinese black sleeper Bostrichthys sinensis, a fish species that inhabits intertidal zones and mates and spawns inside a muddy burrow. We found immunoreactivities of PGE₂ receptor subtypes (Ep_1–3) expressed in the olfactory sac, but only Ep₁ presented higher density of immunoreactivity in mature fish than that in immature fish in both sexes. To gain a better understanding of the underlying molecular mechanism for the detection of PGE₂ in the olfactory system, we cloned an ep ₁ cDNA from the adult olfactory sac. The open-reading frame of the ep ₁ consisted of 1,134-bp nucleotides that encoded a 378-amino acid-long protein with a seven-transmembrane domain, typical for the G protein-coupled receptors superfamily. Expression of ep ₁ mRNA was observed in all tissues examined, with higher levels obtained in the olfactory sacs and testes. The expression of ep ₁ mRNA in the olfactory sacs and gonads was significantly higher in both sexes of mature fish than in those of immature ones. Taken together, our results suggested that Ep₁, which is highly expressed in the olfactory sacs and gonads of mature fish, is important for the control of reproduction and may be involved in PGE₂-initiated spawning behavior in B. sinensis.