Seasonal fluctuations in androgen levels in females of the hermaphroditic gag, Mycteroperca microlepis, with an emphasis on juvenile animals

Androgens are known to play many roles in the reproductive physiology of teleosts, but less information exists on the role that they play in the development of larval and juvenile fish. This study examines an observed seasonal cycle of 11-ketotestosterone (11KT) in females of the hermaphroditic gag grouper (Mycteroperca microlepis). Otoliths, gonads, and plasma samples from gag were collected quarterly (spring, summer, fall, and winter), with complete data (age, sex, and androgen levels) obtained from a total of 225 individuals. Ages ranged from zero to 11 years, and all individuals were female. Testosterone (T) peaked in the spring, coincident with spawning, and was low throughout the remainder of the year. The androgen 11KT peaked in summer and declined through the following spring. 11KT levels were negatively correlated with fish size in both the summer and winter, while T was negatively correlated in the summer and positively correlated in the winter. T levels showed little seasonal variation in juveniles (0-1 and 2-3 age groups), but showed a seasonal increase from fall through spring in older fish (4-5 and 6+ age groups). Age 0-1 fish had significantly higher levels of 11KT than the age 4-5 group during the summer and both the 4-5 and 6+ age groups in the winter. The gag is a protogynous hermaphrodite that goes through several ontogenetic shifts during its life, and this seasonal fluctuation in plasma levels of 11KT may play a role in the growth and development, behavior, or control of sex change of gag.     

Induction of female-to-male sex change in the honeycomb grouper (Epinephelus merra) by 11-ketotestosterone treatments

The honeycomb grouper, Epinephelus merra, is a protogynous hermaphrodite fish. Sex steroid hormones play key roles in sex change of this species. A significant drop in endogenous estradiol-17beta (E2) levels alone triggers female-to-male sex change, and the subsequent elevation of 11-ketotestosterone (11KT) levels correlates with the progression of spermatogenesis. To elucidate the role of an androgen in sex change, we attempted to induce female-to-male sex change by exogenous 11KT treatments. The 75-day 11KT treatment caused 100% masculinization of pre-spawning females. Ovaries of the control (vehicle-treated) fish had oocytes at various stages of oogenesis, while the gonads of the 11KT-treated fish had transformed into testes; these contained spermatogenic germ cells at various stages, including an accumulation of spermatozoa in the sperm duct. In the sex-changed fish, plasma levels of E2 were significantly low, while both testosterone (T) and 11KT were significantly increased. Our results suggest that 11KT plays an important role in sex change in the honeycomb grouper. Whether the mechanism of 11KT-induced female-to-male sex change acts through direct stimulation of spermatogenesis in the ovary or via the inhibition of estrogen synthesis remains to be clarified.     

Variation in the reproductive potential of Schistocephalus infected male sticklebacks is associated with 11-ketotestosterone titre

Parasites can impact host reproduction by interfering with host endocrine systems, but the adaptive nature of such effects is disputed. Schistocephalus solidus plerocercoids are parasites of three-spined sticklebacks Gasterosteus aculeatus that are often associated with impaired host reproduction. Here, we relate reproductive behavior and physiology to levels of the androgen 11-ketotestosterone (11KT) in naturally infected and non-infected male sticklebacks from two UK populations. In one population infected males harbored heavy infections and showed uniformly reduced 11KT titres and kidney spiggin (nesting glue protein) content compared to non-infected fish. However in a second population infection levels were more variable and males with smaller infections recorded 11KT and spiggin titres that overlapped those of non-infected fish; among infected males from this population 11KT and kidney spiggin also both correlated negatively with infection severity. Male reproductive behavior correlated closely with 11KT titre in both populations, and infected males with high 11KT levels exhibited normal reproductive behavior. Our results suggest that Schistocephalus infection per se does not block reproductive development in male sticklebacks, and that some male fish may have the ability to breed whilst infected. Our results are not consistent with the hypothesis that Schistocephalus adaptively castrates male hosts via endocrine disruption; rather they support the hypothesis that reproductive disruption is a side effect of the energetic costs of infection.     

Determination of sex and maturity stage of great sturgeon (Huso huso) using steroid sex hormones profiles,histology and laparoscopic images of gonads

We studied sex and maturity stage of great sturgeon with different reproductive stages using sex steroid hormones concentrations in blood plasma, gonad histology and laparoscopic technique. One hundred sixty farmed great sturgeons at ages between 1 and +12 years and weight ranged from 0.45 to 49.65 kg (80 males and 80 females, 20 fish of each maturity stage) were selected. Mean sex steroid levels showed that testosterone (T) and 11ketotestosterone (11KT) in plasma varied significantly by sex and maturity stage. But the mean concentrations of 17B‐estradiol (E2) varied significantly only between the maturity stages I and III. All fish were sex determined by laparoscopy and histology examinations confirmed it. Our results showed, however laparoscopy was effective method for sex determination at four maturity stages, but young fish at maturity stage I required gonadal histology examinations to confirm sex. The present study results showed that some steroid sex hormones (T and 11KT) can be used to determine sex and reproductive stage in great sturgeon, especially in later maturity stages. Although the laparoscopy may be stressful and cause small scars on the body of the fish, but it is a fast, effective, and cheap method for sex and maturity stage assessment (30 to 40 s for each fish), especially at later maturity stages. Histology of gonads is a very accurate, but it is an invasive method and not a suitable technique for commercial purposes. Histology is more suited for scientific studies than commercial application. The ability to use minimally invasive methods for sex determination will assist broodstock management and conservation in sturgeons.     

Hormonal and body size correlates of electrocommunication behavior during dyadic interactions in a weakly electric fish, Apteronotus leptorhynchus

Brown ghost knife fish, Apteronotus leptorhynchus, produce sexually dimorphic, androgen-sensitive electrocommunication signals termed chirps. The androgen regulation of chirping has been studied previously by administering exogenous androgens to females and measuring the chirping response to artificial electrical signals. The present study examined the production of chirps during dyadic interactions of fish and correlated chirp rate with endogenous levels of one particular androgen, 11-ketotestosterone (11KT). Eight males and four females were exposed to short-term (5-min) interactions in both same-sex and opposite-sex dyads. Twenty-four hours after all behavioral tests, fish were bled for determination of plasma 11KT levels. Males and females differed in both their production of chirps and their ability to elicit chirps from other fish: males chirped about 20-30 times more often than females and elicited 2-4 times as many chirps as females. Among males, chirp rate was correlated positively with plasma 11KT, electric organ discharge frequency, and body size. Combined with results from experimental manipulation of androgen levels, these results support the hypothesis that endogenous 11KT levels influence electrocommunication behavior during interactions between two male fish.     

Regulation of protogynous sex change by competition between corticosteroids and androgens: an experimental test using sandperch, Parapercis cylindrica

Cortisol, the dominant corticosteroid in fish, and 11-ketotestosterone (11KT), the most potent androgen in fish, are both synthesized and (or) deactivated by the same two enzymes, 11beta-hydroxylase and 11beta-hydroxysteroid dehydrogenase. Cortisol is synthesized in response to stress (such as that caused by interaction with a dominant conspecific), whereas 11KT is synthesized during protogynous sex change. It has been hypothesized that corticosteroids (such as cortisol) inhibit 11KT synthesis via substrate competition, thereby providing a mechanism for the regulation of socially mediated, protogynous sex change. We tested this hypothesis by administering cortisol (50 microg g(-1) body weight) to female sandperch (Parapercis cylindrica) under social conditions that were permissive to sex change (i.e. in the absence of suppressive male dominance). Twenty-one days later, mean physiological cortisol concentration in cortisol-treated fish was 4.2-fold greater than that in 'socially stressed' female fish maintained in a semi-natural system. Although the dosage of cortisol was therefore considered to be favorable for engendering competitive inhibition of 11KT synthesis, all cortisol-treated fish changed sex, as did all sham-treated and control fish (n=7 fish per treatment). In addition, there was no effect of cortisol treatment on the rate of sex change or on the pattern of steroidogenesis. Thus, our results refute the hypothesis that protogynous sex change is regulated by substrate competition between corticosteroids and androgens.     

Differential Responses of Brain,Gonad and Muscle Steroid Levels to Changes in Social Status and Sex in a Sequential and Bidirectional Hermaphroditic Fish

Sex steroids can both modulate and be modulated by behavior, and their actions are mediated by complex interactions among multiple hormone sources and targets. While gonadal steroids delivered via circulation can affect behavior, changes in local brain steroid synthesis also can modulate behavior. The relative steroid load across different tissues and the association of these levels with rates of behavior have not been well studied. The bluebanded goby (Lythrypnus dalli) is a sex changing fish in which social status determines sexual phenotype. We examined changes in steroid levels in brain, gonad and body muscle at either 24 hours or 6 days after social induction of protogynous sex change, and from individuals in stable social groups not undergoing sex change. For each tissue, we measured levels of estradiol (E₂), testosterone (T) and 11-ketotestosterone (KT). Females had more T than males in the gonads, and more E₂ in all tissues but there was no sex difference in KT. For both sexes, E₂ was higher in the gonad than in other tissues while androgens were higher in the brain. During sex change, brain T levels dropped while brain KT increased, and brain E₂ levels did not change. We found a positive relationship between androgens and aggression in the most dominant females but only when the male was removed from the social group. The results demonstrate that steroid levels are responsive to changes in the social environment, and that their concentrations vary in different tissues. Also, we suggest that rapid changes in brain androgen levels might be important in inducing behavioral and/or morphological changes associated with protogynous sex change.     

A mechanism for rapid neurosteroidal regulation of parenting behaviour

While systemic steroid hormones are known to regulate reproductive behaviour, the actual mechanisms of steroidal regulation remain largely unknown. Steroidogenic enzyme activity can rapidly modulate social behaviour by influencing neurosteroid production. In fish, the enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) synthesizes 11-ketotestosterone (KT, a potent teleost androgen) and deactivates cortisol (the primary teleost glucocorticoid), and both of these steroid hormones can regulate behaviour. Here, we investigated the role of neurosteroidogenesis in regulating parenting in a haremic bidirectionally hermaphroditic fish, Lythrypnus dalli, where males provide all requisite parental care. Using an in vitro assay, we found that an 11β-HSD inhibitor, carbenoxolone (CBX), reduced brain and testicular KT synthesis by 90% or more. We modulated neurosteroid levels in parenting males via intracerebroventricular injection of CBX. Within only 20 min, CBX transiently eliminated parenting behaviour, but not other social behaviour, suggesting an enzymatic mechanism for rapid neurosteroidal regulation of parenting. Consistent with our proposed mechanism, elevating KT levels rescued parenting when paired with CBX, while cortisol alone did not affect parenting. Females paired with the experimental males opportunistically consumed unattended eggs, which reduced male reproductive success by 15%, but some females also exhibited parenting behaviour and these females had elevated brain KT. Brain KT levels appear to regulate the expression of parenting behaviour as a result of changes in neural 11β-HSD activity.     

Further refinement of the non‐invasive procedure for measuring steroid production in the male three‐spined stickleback Gasterosteus aculeatus

Measurement of steroids that are released into the water via the gills has previously been shown to be an effective way of studying the reproductive endocrinology of the male three‐spined stickleback Gasterosteus aculeatus without having to kill the fish. In the present paper, a previous observation on the existence of a compound other than 11‐ketotestosterone (11‐KT) in water, which cross‐reacted in the 11‐KT radioimmunoassay was repeated. The amounts of this compound, however, were not sufficient to warrant a separation step prior to carrying out assay. The lack of association between androstenedione levels in water and those in plasma was also confirmed. For the first time, the amounts of testosterone released into the water were shown to be positively correlated with the amounts in plasma, the sampling procedure (placing the fish for 30 min in 50 ml water) had no effect on the rate of release of cortisol but caused a rapid drop in the rate of release of 11‐KT (which means that the fish should not be sampled twice in short succession), physical interaction between two nesting males (which was accompanied by aggression) significantly increased the rate of release of 11‐KT, androstenedione and testosterone (but not of cortisol) and the rate of release of 11‐KT was at its maximum between 2 and 4 h after exposure.     

Sex steroid dynamics during embryogenesis and sexual differentiation in Eurasian perch, Perca fluviatilis

It is widely accepted that sex steroid hormones play an important and a specific role during the process of sex differentiation in fish. In order to describe the role of the three main sex steroid hormones (testosterone--T, 17beta-estradiol--E2 and 11keto-testosterone--11KT) during embryogenesis and sex differentiation in Eurasian perch, Perca fluviatilis, eggs, larvae and juveniles originating from two mixed-sex and two all-female progenies were regularly sampled from fertilization to hatching (D0) and from hatching to day 70 post-hatching (D70). Just after spawning, a significant amount of sex steroids [T (1634.2pgg(-1)), E2 (554.4pgg(-1)) and 11KT (1513.2pgg(-1))] was measured in non-fertilised eggs suggesting a maternal transmission of these steroids. From D2 to D70 post-hatching, E2 levels were significantly higher in mixed-sex progenies (median: 725.7pgg(-1)) than in all-female progenies (156.2pgg(-1)) and significantly increased after the onset of the histological differentiation of the gonad in both progenies (D35). Levels of 11KT were significantly higher in mixed-sex (median: 431.5pgg(-1)) than in all-female progenies (below the limit of assay detection) and significantly increased at D35 in all-female progenies (median value: 343.2pgg(-1)). Mean 11KT to E2 ratio was six-fold higher in mixed-sex progenies (1.35) than in all-female progenies (0.24). The data suggest that the 11-oxygenated androgen (11KT) plays a major role in the male differentiation process, and that sex differentiation in Eurasian perch is probably determined by the 11KT to E2 ratio.     

Seasonal changes of serum sex steroids concentration and aromatase activity of gonad and brain in red-spotted grouper (Epinephelus akaara)

Levels of serum sex steroids (estradiol-17beta, E2; testosterone, T; 11-ketotestosterone, 11-KT) in male, female and natural sex-reversing red-spotted grouper (Epinephelus akaara), and aromatase activity of gonad and brain in both male and female were investigated throughout an annually reproductive cycle. In females, serum E2 and T peaked during vitellogenesis, but in males and natural sex-reversing fish, 11-KT, T and E2 reached peak during spermatogenesis. In addition, in females, serum 11-KT levels (monthly means: 0.32 +/- 0.03 ng/ml) which were very low did not significantly fluctuate during the annual reproductive cycle. In breeding season, females displayed higher E2 levels than males and sex-reversing fish, while males and sex-reversing fish showed higher 11-KT levels and, to a lesser extent, higher T levels than females. Furthermore, the changing pattern of sex steroids in males was similar to that in natural sex-reversing fish, and a second peak of serum androgens 11-KT and T appeared in December both in male and natural sex-reversing fish; significantly higher serum 11-KT levels were observed in natural sex-reversing fish than that in females from December to April. In females, but not in males, aromatase activity of brain and gonad demonstrated significantly seasonal changes (exhibiting a peak in breeding season); moreover, aromatase activity in females was higher than that in males. Furthermore, significantly lower aromatase activity in testis was observed in breeding season, in contrast to that in ovary. Taken together, the present findings indicated that changes of serum sex steroids levels and aromatase activity in red-spotted grouper were closely associated with sex inversion. In addition, the present results also suggested that sex inversion in red-spotted grouper peaked mainly from December to March.     

Natural sex change in the temperate protogynous Ballan wrasse Labrus bergylta

Wild Ballan wrasse Labrus bergylta were sampled monthly over 2 years in western Norway to identify the natural process of sex change in this species. Light microscopy of standard histological‐stained and immunohistochemistry‐treated gonad tissue showed that spermatogonial germ cells tended to proliferate around the periphery of the lamellae before filling into the slowly receding, apoptotic central areas of the lamellae. Sex change occurred following the breeding season. From July to September, fish were most often in an early state of gonadal transition (ET), characterized by degenerating previtellogenic oocytes and pockets of proliferating spermatogonia in the germinative epithelia. The majority of fish with late transitional gonads, that were typically dominated by spermatogenic cells, developing efferent ducts and the beginning of lobule formation, were found between October and November. Sex steroid profiles of fish representing the different sexual phases showed that breeding females had the highest concentrations of 17β oestradiol (E₂) and the lowest concentration of 11 ketotestosterone (11KT). Concentrations of E₂ decreased greatly in ET fish at the beginning of sex change and remained low in all subsequent phases. The opposite trend was demonstrated in 11KT profiles. Initial‐phase female fish had minimal concentrations of 11KT, but these increased during subsequent transitions. Sex change occurred most often in fish 34–41 cm total length (L_T) and the median of fish in the size‐frequency overlap of female and male fish was 36 cm L_T.     

Comparison of antimicrobial activity in the epidermal mucus extracts of fish

The mucus layer on the surface of fish consists of several antimicrobial agents that provide a first line of defense against invading pathogens. To date, little is known about the antimicrobial properties of the mucus of Arctic char (Salvelinus alpinus), brook trout (S. fontinalis), koi carp (Cyprinus carpio sub sp. koi), striped bass (Morone saxatilis), haddock (Melanogrammus aeglefinus) and hagfish (Myxine glutinosa). The epidermal mucus samples from these fish were extracted with acidic, organic and aqueous solvents to identify potential antimicrobial agents including basic peptides, secondary metabolites, aqueous and acid soluble compounds. Initial screening of the mucus extracts against a susceptible strain of Salmonella enterica C610, showed a significant variation in antimicrobial activity among the fish species examined. The acidic mucus extracts of brook trout, haddock and hagfish exhibited bactericidal activity. The organic mucus extracts of brook trout, striped bass and koi carp showed bacteriostatic activity. There was no detectable activity in the aqueous mucus extracts. Further investigations of the activity of the acidic mucus extracts of brook trout, haddock and hagfish showed that these fish species had specific activity for fish and human pathogens, demonstrating the role of fish mucus in antimicrobial protection. In comparison to brook trout and haddock, the minimum bactericidal concentrations of hagfish acidic mucus extracts were found to be approximately 1.5 to 3.0 times lower against fish pathogens and approximately 1.6 to 6.6 folds lower for human pathogens. This preliminary information suggests that the mucus from these fish species may be a source of novel antimicrobial agents for fish and human health related applications.     

Comparative study of the reproductive characteristics of XY male and hormonally sex-reversed XX male Eurasian perch, Perca fluviatilis

In order to compare the reproductive capacity of XY male versus XX male (neomales) Eurasian perch (Perca fluviatilis), we determined the sperm quality (sperm concentration and motility) and reproductive characteristics such as gonadosomatic index (GSI), fertilization rate and sex steroid levels (testosterone, T; 17beta-estradiol, E2 and 11-ketotestosterone, 11KT) during the reproductive season. Median GSI was not significantly different between XY males (7.9%) and XX males (7.5%). Fertilization rates ranged between 30.0 and 98.0%. Sperm concentration ranged between 27.9 x 10(9) and 42.0 x 10(9) spermatozoa ml(-1). Median level of T, 11KT and E2 levels increased in the middle of the reproductive season (2136.0, 2409.0 and 3252.0 pg ml(-1), respectively) and decreased at the end (1657.0, 2006.6 and 431.0 pg ml(-1), respectively). Sperm motility was assessed by CASA and expressed by the curvilinear velocity (VCL), straight line velocity (VSL), average path velocity (VAP), linearity (LIN), percentage of motile sperm (% MOT) and motile concentration (MOC). Overall, there were not any significant differences between XY and XX males. In conclusion, no differences of reproductive capacities were observed between XY males and XX males suggesting that the last can be crossed with females to improve the productivity of Eurasian perch by producing all-female stock.     

Androgens and Immune Function in Male Alternative Reproductive Morphotypes of the Peacock Blenny Salaria pavo

Species with alternative reproductive tactics offer the opportunity to analyse how behavioural and morphological traits are tuned to produce successfully competing phenotypes within one sex. In the teleost fish Salaria pavo , alternative reproductive tactics are sequential. The older ornamented males compete for access to females by guarding a cavity to which they attract females to spawn. Ornamented males that are found without a nest are called 'floaters'. Younger mature males which are too small to compete with nest-holders attempt to 'sneak' as female mimics into successful nests and release their sperm to fertilize freshly spawned eggs. The alternative tactics in S. pavo are associated with different levels of the androgens testosterone and 11-ketotestosterone, which have been found to suppress immune function in several teleost fish. A field study was carried out to analyse the relationship between these reproductive tactics, androgens and blood levels of lymphocytes as a monitoring method of immune function. We expected highest investment in the immune system in sneakers because these have the lowest androgen levels and functionally because investing in self-maintenance increases their future prospect to switch tactic and to reproduce as a nest-holder. Indeed, the relative count of lymphocytes correlated negatively with body size and thus was highest in sneakers and lowest in nest-holders. In concordance, 11-ketotestosterone and testosterone levels were found to be higher in floaters and nest-holders than in sneakers. However, no correlation was found between individual levels of testosterone or 11-ketotestosterone and lymphocytes. Thus, a trade-off between reproductive traits associated with high androgen levels and immunocompetence might exist at the level of alternative tactics but this might not be explained by acute immunosuppressive effects of circulating androgens.     

Contextual modulation of androgen effects on agonistic interactions

Seasonal changes in steroid hormones are known to have a major impact on social behavior, but often are quite sensitive to environmental context. In the bi-directionally sex changing fish, Lythrypnus dalli, stable haremic groups exhibit baseline levels of interaction. Status instability follows immediately after male removal, causing transiently elevated agonistic interactions and increase in brain and systemic levels of a potent fish androgen, 11-ketotestosterone (KT). Coupling KT implants with a socially inhibitory environment for protogynous sex change induces rapid transition to male morphology, but no significant change in social behavior and status, which could result from systemically administered steroids not effectively penetrating into brain or other tissues. Here, we first determined the degree to which exogenously administered steroids affect the steroid load within tissues. Second, we examined whether coupling a social environment permissive to sex change would influence KT effects on agonistic behavior. We implanted cholesterol (Chol, control) or KT in the dominant individual (alpha) undergoing sex change (on d0) and determined the effects on behavior and the degree to which administered steroids altered the steroid load within tissues. During the period of social instability, there were rapid (within 2 h), but transient effects of KT on agonistic behavior in alphas, and secondary effects on betas. On d3 and d5, all KT, but no Chol, treated females had male typical genital papillae. Despite elevated brain and systemic KT 5 days after implant, overall rates of aggressive behavior remained unaffected. These data highlight the importance of social context in mediating complex hormone–behavior relationships.     

Circadian changes in serum concentrations of steroids in Japanese char Salvelinus leucomaenis at the stage of final maturation

Circadian changes in serum concentrations of testosterone (T), 11-ketotestosterone (11KT), estradiol-17beta (E2), 17alpha,20beta-dihydroxy-4-pregnen-3-one (DHP), 17alpha-hydroxyprogesterone (OHP), cortisol (F) and progesterone (P) were investigated in the spermiated/ovulated Japanese char Salvelinus leucomaenis for over three days using newly developed time-resolved fluoroimmunoassays. Testosterone and DHP in both sex and 11KT in male showed significantly (P<0.05) higher serum levels just before/after onset of darkness (15:00 or 18:00), and the levels during night and daytime were significantly (P<0.05) lower than those of the peak levels. Serum F levels in both sex during dark phase were significantly (P<0.05) higher than those levels during daytime. A surge of serum OHP concentrations in both sexes was observed at the time of twilight (03:00). The peak time of serum T, 11KT and DHP levels were approximately 6 hours prior to those of serum F and OHP levels. Serum E2 in female and P in both sex fluctuated intensely during sampling period, and did not show remarkable changes. These results strongly suggest the existence of circadian-like diel changes in serum T, DHP, F and OHP levels in both sex and 11KT in male, and no variations in serum E2 in female and P in both sex in spermiated/ovulated Japanese char under the stage of final maturation. Furthermore, relationship between circadian rhythms of steroid hormones and spawning behaviors are discussed in the present study.     

Elevated 11-ketotestosterone during paternal behavior in the Bluebanded goby (Lythrypnus dalli)

The relationship between androgens and paternal behavior is not straightforward, potentially because of the diversity of tasks a male must undertake to maximize reproductive success, notably alternating between courtship, aggression, and offspring care. In some species, these events are separated in time, but in others they are coincident. The endocrine profiles of species that simultaneously court, parent, and defend a nest, such as male bluebanded gobies (Lythrypnus dalli), are not well understood. We sampled a potent fish androgen, 11-ketotestosterone (KT), at different life history stages (experienced parenting males, experienced males not actively parenting, inexperienced males with their first clutch, and females), to examine this relationship. We found that experienced parenting L. dalli males have the highest KT levels of any group, while none of the other groups differed significantly. Males showed elevated KT levels when they had eggs compared to when they did not. Our data suggest that KT facilitates at least some aspects of parental care in L. dalli.     

Plasma concentrations of sex steroids and fighting in male Tilapia zillii

In laboratory-held Tilapia zillii the gonadosomatic index ( I _G) is an accurate predictor of dominance in pairwise fights between males. Immediately after fighting no differences were detected between winners, losers or controls, in plasma concentrations of testosterone (T) or 11-ketotestosterone (11KT). 11KT was correlated negatively with I _G and positively with T. 11KT appeared to be the major androgen in this species, but there was no evidence that simple differences in levels of these sex steroids mediated aggressive behaviour.     

Milt characteristics and plasma levels of gonadal steroids in greenback flounder Rhombosolea tapirina following treatment with exogenous hormones

Fish were treated with exogenous hormones, and milt and blood samples were collected for up to 96 h post‐treatment. Blood plasma samples were assayed for the gonadal steroids testosterone (T), 11‐ketotestosterone (11KT) and 17,20ß‐dihydroxy‐4‐pregnen‐3‐one (17.20ßP). Milt volume, spermatocrit and sperm motility were measured from milt samples. Non‐spermiated fish showed increased plasma T and 11KT in response to human chorionic gonadotropin (hCG) but not luteinising hormone releasing hormone analogue (LHRHa). Fish did not become spermiated in response to treatment with hCG, LHRHa, 11KT, 17‐hydroxyprogesterone (17P) or 17,20ßP. Spermiated fish showed an increase in milt volume in response to hCG and LHRHa but not exogenous steroids. Sperm motility declined to zero over 120 s and was not affected by hormone treatment or sampling time. Increased milt volume was accompanied by increased plasma T and 11KT, but not 17.20ßP levels. In a separate experiment, LHRHa delivered by injection or pellet was equally effective at increasing milt volume but had no effect on plasma steroid levels. Spermatocrit declined with stripping but was not affected by hormone treatment, nor was sperm motility. Co‐treatment of fish with 17P plus LHRHa had no additive effect on plasma steroid concentrations or milt volume. The results suggest that as in other teleosts, gonadotropin mediates milt production in greenback flounder.