Effect of exogenous hormones on ovulation and gonadal steroid plasma levels in starry flounder, <Emphasis Type="Italic">Platichthys stellatus</Emphasis>

The present study was designed to examine the potential for inducing ovulation in starry flounder (Platichthys stellatus) using gonadotropin-releasing hormone analog (GnRHa) and human chorionic gonadotropin (hCG) to assess whether starry flounder are differentially responsive to GnRHa and hCG. Female starry flounder were injected or implanted with different doses of hCG or GnRHa pellets to examine their ovulation-inducing potential and effects on plasma levels of testosterone (T), 17β-estradiol (E2), and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP). Blood samples were collected for up to 10 or 25 days post-injection or post-implantation in two separate experiments designed to mimic the early and middle stages of spawning, respectively. Fish treated with the GnRHa pellets (100 µg) showed a significant increase in the total number of stripped eggs relative to the controls. GnRHa administration had no effect on the floating rate or fertilization rate of ovulated eggs in the both experiments, whereas hCG injection affected both of these rates. Plasma T levels were not significantly different between the exogenous hormone-treated and control fish. In contrast, the plasma E2 level was elevated in those fish treated with GnRHa, regardless of injection or implantation, and was accompanied by increased numbers of stripped eggs in both experiments. Treatment with GnRHa resulted in higher 17,20βP levels compared to the controls, and there was a positive relationship between elevated plasma 17,20βP and an increase in ovulated eggs in response to GnRHa treatment. The implantation of starry flounder with GnRHa-containing pellets was effective at inducing sustained ovulation compared to hCG treatment.     

Effects of slow release gonadotropin releasing hormone analog on milt characteristics and plasma levels of gonadal steroids in greenback flounder, Rhombosolea tapirina

Spermiated male greenback flounder (Rhombosolea tapirina) were implanted with gonadotropin releasing hormone analog (GnRHa) pellets at different dosages to examine their effects on milt characteristics and plasma levels of testosterone (T), 11-ketotestosterone (11KT) and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP). Milt and blood samples were collected for up to 28 or 42 days post-implantation (p.i.) in two separate experiments using fish that were slightly or moderately spermiated, respectively. In both experiments, fish treated with GnRHa pellets showed a consistent and significant increase in milt volume relative to controls, and the increase of milt volume was more rapid than the increase in numbers of spermatozoa. Spermatocrit (Sct) was significantly lower in GnRHa-treated fish than in controls. Plasma levels of T and 11KT were elevated at 7 and 14 days p.i. in slightly spermiated fish treated with GnRHa and elevated plasma T and 11KT levels were accompanied by increased milt volume early in the spermiation period. In contrast, there was no significant difference in plasma T levels between GnRHa-treated and control fish in fish that were moderately spermiated at the time of implant. Treatment with GnRHa tended to result in lower plasma levels of 11KT and higher levels of 17,20βP relative to controls, and there was a positive relationship between the elevation of plasma 17,20βP and the increase in milt volume in response to implantation of GnRHa pellet. Slow release GnRHa administration had no effect on sperm activity or pH and osmolality of seminal plasma in either experiment.     

The combined effects of temperature and GnRHa treatment on the final stages of sexual maturation in Atlantic salmon (Salmo salar L.) females

Atlantic salmon (Salmo salar L.) females (2 SW), maturing for the first time, were reared under one of three temperature regimes (high: 14.3 ± 0.5°C; natural: 10.6 ± 1.0°C; and cold: 6.9 ± 1.0°C) in combination with one of two experimental treatments; an injection of GnRH analogue (GnRHa) contained in biodegradable microspheres, or a sham injection (microspheres only). The six experimental groups were then reared under simulated natural photoperiod for 4 weeks. Blood samples were drawn for analysis of plasma steroid levels and the fish were inspected for ovulation weekly. Batches of stripped eggs were incubated in triplicate incubators in raceways until the eyed stage. Treatment with GnRHa resulted in a substantial advancement and synchronization of ovulation at all temperatures, while exposure to cold water also appeared to advance ovulation slightly. While 75% (warm and cold) to 90% (natural) of GnRHa fish ovulated during the 4-week trial, only 30% of sham-treated females exposed to cold water, and none of the sham-treated fish held at higher temperatures, ovulated during this period. Survival rates of embryos to the eyed-stage were significantly higher for broodstock exposed to cold water. Plasma levels of testosterone (T), 17β-oestradiol (E₂), and 17α,20β-dihydroxy-4-pregnen-3-one (17,20βP) were all significantly affected by treatment with GnRHa and, to a lesser extent, temperature. The efficiency of GnRHa in counteracting the negative effects of high temperature on ovulation and the associated changes in circulating sex steroids suggest that temperature inhibition operates at least in part at the brain or pituitary.     

Effects of temperature on the final stages of sexual maturation in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.)

Maturing male and female Atlantic salmon (Salmo salar L.) were held under three temperature regimes for 10 weeks between September and December: warm (constant 14–16 °C), ambient (decreasing from 11 to 5 °C), and cold (decreasing from 7 to 3 °C). Blood samples were analyzed for plasma steroid levels, and the fish were inspected for the presence of expressible milt (total volume and spermatocrit) and ovulation weekly. Samples of eggs were dry-fertilized with milt stripped from three males held at the same temperatures and incubated until the eyed stage. In females, levels of plasma testosterone (T) and 17β-oestradiol (E₂) dropped as ovulation approached, concurrent with a rapid increase in levels of plasma 17α,20β-dihydroxy-4-pregnen-3-one (17,20β-P). In males, levels of T and 11-ketotestosterone (11-KT) peaked 2–3 weeks after the first appearance of expressible milt, while levels of 17,20β-P increased steadily and did not exhibit a definite peak. Exposure of females to cold water amplified and advanced the profiles of all three steroids compared with the ambient group, and increased the survival rates to the eyed egg stage. Cold water had no immediate effect on the male steroid profiles, but later, higher levels of 17,20β-P were evident compared with both the ambient controls and the warm water group, while the effects on 11-KT and T were more variable. Exposure to warm water completely inhibited both milt production and ovulation. Moreover, warm water modulated the steroid profiles of the males with lower 11-KT levels compared with ambient controls and lower 17,20β-P level compared with cold-water-treated males. In females, warm water resulted in total inhibition of the peri-ovulatory peak in 17,20β-P and prevented the normal decline of T and E₂ levels associated with ovulation. The findings of the present study are highly relevant for broodstock management in aquaculture, as well in understanding the impact of climate change/temperature variability on wild salmon spawning.     

Hormonal and pheromonal control of spawning behavior in the goldfish

Species that employ sexual reproduction must synchronize gamete maturity with behavior within and between genders. Teleost fishes solve this challenge by using reproductive hormones both as endogenous signals to synchronize sexual behavior with gamete maturation, and as exogenous signals (pheromones) to synchronize spawning interactions between fish. This dual role of hormonal products is best understood in the goldfish, an external fertilizer with a promiscuous mating system. Female gonadal growth and vitellogenesis is stimulated by 17β-estradiol (E2) which also evokes release of a recrudescent pheromone. At the completion of vitellogenesis, ovarian E2 production drops and plasma testosterone increases, sensitizing the female gonadotropin II (luteinizing hormone; LH) system to environmental cues (temperature, spawning substrate, pheromones). These cues eventually trigger a LH surge that alters steroidogenesic pathways to favor the production of progestins including 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P). Plasma 17,20β-P stimulates oocyte maturation but is also released to the water along with sulfated 17,20β-P and androstenedione to serve as a preovulatory pheromone. This pheromone stimulates male behavior, LH release, and sperm production. At the time of ovulation, females become sexually active in response to prostaglandin F2α (PGF2α) synthesized in the oviduct. PGF2α and its metabolites are released as a postovulatory pheromone that induces male spawning behavior which further increases male LH and sperm production. Androgenic hormones are required for male behavior and LH release. Although goldfish are gonochorists, hormone treatments can induce heterotypical functions in adults. Similar findings in other fish demonstrate that a sexually bipotential brain is not restricted to hermaphroditic fishes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Fertility and motility of sperm from Atlantic halibut (Hippoglossus hippoglossus) in relation to dose and timing of gonadotrophin-releasing hormone agonist implant

In broodstocks of Atlantic halibut, Hippoglossus hippoglossus, male and female gamete production often becomes unsynchronised towards the end of the spawning season—milt becomes very viscous and difficult to express while the females are still producing batches of good quality eggs. Gonadotrophin-releasing hormone agonist (GnRHa) has been shown to stimulate spermiation in a number of fish species. Therefore, we conducted two experiments where male halibut were implanted intramuscularly with pellets containing GnRHa. The effect of the pellets was tested at three periods: before, at the height of and at the end of spermiation. In the middle period, GnRHa was tested at two doses (5 and 25 μg/kg bodyweight). Measurements were made of milt hydration, sperm motility and fertilisation rate. Implanted males began spermiation at least 4 weeks before control males. Both doses of GnRHa increased the fluidity of the milt. This effect lasted for at least 20 days in the low dose group and for 40 days in the high dose group. When applied at the end of the season, GnRHa reversed the normal trend for the milt to become more viscous. GnRHa treatments did not affect fertilisation rates obtained with the sperm. However, towards the end of the spawning season, sperm motility was enhanced in males treated with the high dose of GnRHa (25 μg/kg) compared to controls. As described previously, plasma concentrations of the gonadal steroids, 5β-pregnane-3β,17,20β-triol 20-sulphate and 17,20-dihydroxy-4-pregnen-3-one, were significantly enhanced by GnRHa treatment. Concentrations of testosterone on the other hand decreased when spermiating males were treated with GnRHa. Our data suggest that 17,20β-dihydroxy-4-pregnen-3-one or its metabolites are involved in milt hydration, possibly through affecting ion transport.     

Exposure to 17α,20β-dihydroxy-4-pregnen-3-one changes seminal characteristics in Nile tilapia, Oreochromis niloticus

This experiment was conducted to evaluate the seminal characteristics of Nile tilapia males exposed to water‐borne 17α,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20βP). Male Nile tilapia (Oreochromis niloticus L.) were exposed to the steroidal pre‐ovulatory pheromone 17,20βP, added to water at a concentration of 5×10^?9 M. The pheromone‐exposed males had higher sperm volume and concentration. In addition, the spermatozoa contained in the sperm had higher motility and the motility duration was longer than ethanol‐exposed males (control group). The percentage of live spermatozoa was not affected by the treatments. Our results suggest that this pheromone can improve sperm quality characteristics and could become a non‐invasive method for enhancing spawning in Nile tilapia.     

Production, release and olfactory detection of sex steroids by the tench (Tinca tinca L.)

The present study is concerned with pheromone communication in tench (Tinca tinca L.), establishing firstly whether males have a high olfactory sensitivity to some typical teleost sex steroids and prostaglandins; and secondly whether males and females might be able to synthesise and release some of these steroids into the water. The C₂₁ steroid, 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) was found to give large electro-olfactogram responses with an estimated threshold of detection of 10⁻¹² M. The male tench were equally sensitive to glucuronidated 17,20β-P (10^−11.6 M) but 100 times less sensitive to sulphated 17,20β-P (11^−9.7 M). Preliminary data from cross-adaptation studies suggest that both the free and conjugated forms are detected by the same olfactory receptor(s). Male tench also had high olfactory sensitivity to prostaglandin F_2α (PGF_2α) and 15-keto PGF_2α (11^−11.5 and 10^−11.4 M). They were relatively insensitive, however, to testosterone (T), androstenedione (AD), 11-ketotestosterone (11-KT), 17β-oestradiol (E₂), 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P) and 17,20α-dihydroxy-4-pregnen-3-one (17,20α-P). Radioimmunoassays were used to measure the steroids in plasma and water and all samples were processed for the measurement of free, sulphated and glucuronidated fractions. In females, free 17,20β-P, 17,20α-P, free and glucuronidated T, and AD in plasma showed the largest increases in response to injection with mammalian gonadotropin-releasing hormone analogue (GnRHa) or Ovaprim (a mixture of GnRHa and a dopamine inhibitor). Free 17,20β-P was released into the water at the greatest rate. Plasma concentrations of the two conjugated forms of 17,20β-P were also elevated 18 h after the administration of GnRHa, but not by as much as the free steroid. In males, AD and 11-KT showed the greatest increase in response to GnRHa and were moreover released into the water at a higher rate in the treated group than in the control. The data support a possible pheromonal role for free and glucuronidated 17,20β-P. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hormone Profiles of Captive Striped Bass Morone saxatilis During Spermiation, and Long-Term Enhancement of Milt Production

Abstract— Plasma profiles of reproductive and thyroid hormones were studied in captive striped bass Morone saxatilis during an 11-wk period encompassing the spawning season, and the effect of a sustained-release gonadotropin-releasing hormone agonist (GnRHa)-delivery system (GnRHa-implant) on milt production was evaluated. The highest percentage of spermiating fish was observed between mid-April and mid-May, and mean total expressible milt ranged from 3.5 to 6.0 mL/kg. Plasma gonadotropin II (GtH II) increased significantly, though inconsistently, during the spermiation period, whereas testosterone and 11-ketotestosterone levels declined continually. Plasma 17,20β-dihydroxy-4-pregnen-3-one and 17,20β,21-dihydroxy-4-pregnen-3-one remained low and unchanged during the peak of the spermiation period, while thyroid hormones were high and fluctuated without exhibiting a trend consistent with spermiation. The observed endocrine profiles suggest that captivity can diminish plasma GtH II and triiodothyronine levels in striped bass. Transfer of spermiating males from large holding tanks to small spawning tanks reduced total expressible milt after 14 d, but treatment with a GnRHa-implant restored milt volume, presumably due to the prolonged elevation of plasma GnRHa and GtH II induced by the GnRHa-implant. Also, treatment with the GnRHa-implant induced a two- to four-fold elevation of expressible milt for at least 20 d compared to control fish, while resulting in only a 5 to 15% decrease in sperm density. It appears that captivity and hatchery operations can diminish milt production in striped bass, and that GnRHa-delivery systems, via sustained elevation of plasma GtH II, can induce long-term enhancement in milt volume without affecting sperm density greatly.     

Seasonal changes in plasma gonadal steroid concentrations and gonadal morphology of male and female tench (Tinca tinca, L.)

In order to gain a better understanding of the reproductive cycles of male and female tench (Tinca tinca), gonadosomatic index, gonad histology and plasma concentrations of estradiol‐17β (E₂), testosterone, an drostenedione, 11‐ketotestosterone (11‐KT), 17,20β, 21‐trihydroxy‐4‐pregnen‐3‐one (17,20β,21‐P), 17,20β‐dihydroxy‐4‐pregnen‐3‐one (17,20β‐P) and 17,20α‐dihydroxy‐4‐pregnen‐3‐one (17,20α‐P) were measured at the four seasons of the year, plus a further sampling coincident with the peak of spawning in early July. As expected, in both males and females, the plasma concentrations of androgens (excluding 11‐KT in females – undetectable) and C₂₁ steroids were significantly more elevated in the spring and summer (when most gonadal development took place) than in the autumn and winter. The only unexpected finding was that 17,20β‐P and 17,20β,21‐P, the steroids that are normally associated with oocyte final maturation in females and spermiation in males, were found in substantial amounts in both pre‐vitellogenic, pre‐spermatogenic and post‐spawning fish. This suggests that these steroids may have other as yet unidentified roles in this species.     

Changes of plasma steroid concentrations associated with spontaneous or induced ovulation in yellow perch Perca flavescens

This study examined the changes in plasma steroids during natural (Experiment 1) and induced (Experiment 2) final maturation in yellow perch Perca flavescens. In experiment 1, ovulating yellow perch were stripped of eggs and blood samples collected to determine the concentrations of testosterone (T), estradiol-17β (E2), and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP). Eggs from individual females were weighed and fertilized. Fertilization rate was determined at the embryo eyed stage. In experiment 2, females were randomly assigned to one of the following treatment groups: (1) saline (0.7% NaCl), (2) des-Gly¹⁰[D-Ala⁶] LHRH-ethylamide (100 μg LHRHa/kg), and (3) LHRHa plus 17,20βP (100 μg LHRHa/kg + 2 mg 17,20βP/kg). Fish were injected intraperitoneally with two doses at a two-day interval. Blood was collected prior to injections and at the time of ovulation/spawning and concentrations of T, E2, and 17,20βP (free and conjugated) were determined. In experiment 1, low concentrations of 17,20βP were recorded at spawning. In experiment 2, all surviving fish injected with LHRHa (5 of 5) released their eggs spontaneously during the week following injections. None of the surviving control fish (0 of 5) ovulated during this period, whereas only 1 of 3 surviving fish injected with LHRHa + 17,20βP released eggs. In the control group, concentrations of E2 and 17,20βP did not show significant differences over the experimental period, whereas plasma T concentrations increased significantly. In fish injected with LHRHa, the concentrations of T and 17,20βP increased significantly after the first injection but then declined at ovulation/spawning. It also appears that 17,20βP was conjugated to its sulfated form. Mortality reached 62.5% in the group injected with LHRHa + 17,20βP indicating that this treatment was severe. Thus, LHRHa alone appears highly effective in inducing ovulation in yellow perch. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gonadotropin releasing hormone-analogue treatment increases sperm motility, seminal plasma pH and sperm production in yellowtail flounder Pleuronectes ferrugineus

Compared to control fish, gonadotropin releasing hormone-analogue (GnRHa) treatment delivered either by microspheres or cholesterol pellets successfully increased sperm production (cells kg–1) and milt volume (ml kg–1) in mature yellowtail flounder Pleuronectes ferrugineus during the spawning season. Spermatocrit decreased in both treated and control groups between 12 and 29 days post-implantation, indicating a seasonal decrease in sperm concentration, rather than an effect of the GnRHa treatments.Plasma levels of testosterone, 11-ketotestoterone and 17,20dihydroxy-4-pregnen-3-one (1720P) showed no clear pattern either across treatments or over days, however this does not exclude the possiblity that GnRHa had its effect on milt volumes via the stimulation of steroid production since the sampling protocol did not allow for the rapid clearance of steroids from the plasma. GnRHa treatment did not have a negative effect on sperm fertilizing ability, percentage hatch or larval morphology. Sperm motility and seminal plasma pH were increased by GnRHa treatment.     

Induced Ovulation of Southern Flounder Paralichthys lethostigma Using Gonadotropin Releasing Hormone Analogue Implants

Implanted pellets that provide a sustained release of [D-Ala⁶ Des-Gly¹⁰] LHRH-ethylamide (GnRHa) were used to induce maturation and ovulation of Southern flounder Paralichthys lethostigma . Of the 12 females whose ovaries contained follicles with a maximum diameter ≥500 μm, 11 ovulated for the first time within 90 h of hormone implantation. Only 1 fish with a maximum follicle diameter less than 500 μm ovulated within 2 wk after implantation. Ovulated eggs were manually stripped from the females and mixed with sperm from several males. Most females were spawned 1 to 3 times on consecutive days with variable fertility. One female was spawned 11 times producing 668,000 eggs. Fertility was evaluated by examining the incubated eggs for early stages of embryonic cleavage. The percentage of fertile eggs in subsamples of incubated eggs ranged from 7–95%. The results indicate that GnRHa implants can be used to induce repeated ovulation in this species. The variability in fertility is discussed in relation to egg quality.     

Application of controlled-release,GnRHa-delivery systems in commercial production of white bass X striped bass hybrids (sunshine bass), using captive broodstocks

Hatchery-produced white bass (Morone chrysops) and striped bass (M. saxatilis) reared to maturity in a commercial aquaculture facility, were successfully spawned using controlled-release delivery systems containing the gonadotropin-releasing hormone analog DAla⁶, Pro⁹[NEt]-GnRH (GnRHa). Two-year-old white bass females (mean weight, 0.81 kg) were implanted with different polymer-based, GnRHa delivery systems at doses ranging from 40 to 89 μg GnRHa kg⁻¹ body weight. GnRHa treatment on 20 February 1994, when females contained oocytes up to 720 μm in diameter, induced ovulation of all fish between 35 to 82 h after treatment. The white bass eggs produced were fertilized with sperm from striped bass for the production of sunshine bass. An average of 294500 eggs kg⁻¹ were produced, with a mean fertility of 81.2%, 24 h survival of 46.5%, and overall hatching success of 45%. Survival from hatch to 30 days post-hatch was 78% and the fry weighed between 0.07 and 0.1 g. Overripening of eggs began within 1 h from ovulation and maximum fertilization (60%) was observed when eggs were stripped 0.5 h after ovulation. Fertilization success decreased thereafter to 31% and 10% by 1 h and 3 h after ovulation, respectively. Control fish not treated with GnRHa did not show any signs of final oocyte maturation during the period of the study. GnRHa administration via controlled-release delivery systems appears to be a very effective method for inducing high fecundity ovulation of captive white bass broodstocks, and producing eggs of high fertility and hatching success.     

Effect of Oxygen Saturation in Water on Reproductive Performances of Pacu Piaractus brachypomus

Broodstock pacu Piaractus brachypomus as well as their eggs during their embryonic development were exposed to either normoxia (5.5–7.5 mg O₂/L) or hypoxia (2.0–4.5 mg O²/L) conditions. The plasma concentrations of 11-ketotestosterone in males and estradiol-17β in females, as well as that of their precursor testosterone (T) were significantly ( P < 0.01) higher in fish maintained under normoxic conditions than in fish exposed to hypoxia. After ovulation and spermiation induced by hormonal treatments, the plasma concentrations of T and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) significantly ( P 0.05) increased in both sexes under both normoxic and hypoxic conditions. The plasma levels of T and 17,20βP achieved under normoxic conditions were higher than the ones recorded under hypoxia, except for those of 17,20βP in males. Males responded positively to the hormonal treatments, and the concentration of spermatozoa was 10.5 ± 0.8 10⁹/mL under both oxygen conditions. Hypoxia resulted in significantly lower survival of embryos (17.3 ± 28%) in comparison to normoxic conditions (68.5 ± 25%). Moreover, larval deformities were found when exposed to hypoxia (91.6 ± 6%). During embryonic development of this species 4 mg/L of oxygen is tolerated at 26–27 C without negative impact. We conclude that despite the highly adaptable nature of adult pacu to environmental hypoxia, oxygen concentrations below 4 mg/L severely impacted survival of embryos.     

The relationship between plasma and ovarian levels of gonadal steroids in the repeat spawning marine fishesPagrus auratus (Sparidae) andChromis dispilus (Pomacentridae)

The relationship between plasma and ovarian levels of gonadal steroids was examined in two New Zealand fish species with multiple spawning cycles of differing length. Snapper (Pagrus auratus) have a daily cycle of oocyte development, ovulation and spawning, whereas demoiselles (Chromis dispilus) spawn over 2–3 days during a repeat spawning cycle of 7–9 days. Ovarian and plasma levels of the gonadal steroids 17β-estradiol (E₂), testosterone (T), 17-hydroxyprogesterone (17P) and 17,20β-dihydroxy-4-pregnen-3-one (17,20βP) were measured in reproductively active fish captured from the wild. Ovarian levels of E₂, T and 17P changed in relation to spawning cycle and gonad stage in both snapper and demoiselles. E₂ and T levels were detectable at all times, but highest during vitellogenesis in both species. Cyclic changes of 17P occurred in both species, and levels appeared to depend on the rate of conversion of 17P to other hormones. No changes in ovarian levels of 17,20βP were detected in relation to stage of the spawning cycle in snapper; however, ovarian levels of 17,20βP were highest in demoiselles before spawning when fish undergoing final oocyte maturation predominated. Plasma levels of E₂ and T were strongly correlated with ovarian concentrations (r=0.850 and r=0.819 for E₂ and T respectively) in demoiselles but there was poor correlation between ovarian and plasma levels of 17P and 17,20βP (r=0.004 and 0.273 respectively), or between ovarian and plasma levels of E₂, T, 17P or 17,20βP of snapper (r=0.135, 0.277, 0.131 and 0.279). The poor correlation between plasma and ovarian levels of some steroid hormones suggests that plasma concentrations of steroids may not adequately reflect the reproductive status of the fish during short-term cyclic ovarian changes. It is suggested that this disparity is likely to be most marked in species with ovulatory periodicity of short duration.     

Sperm production and quality in brill Scophthalmus rhombus L.: relation to circulating sex steroid levels

The aims of the present study were to characterize sperm quality and to quantify seasonal changes in sexual hormone (testosterone [T], 11-ketotestosterone [11-KT] and 17,20β-dihydroxypregn-4-en-3-one [17,20β-P]) levels in male brill (Scophthalmus rhombus) plasma, as well as to test a more intensive sampling strategy to establish relationships between sex steroid levels and sperm production parameters. Sperm concentration ranged from 0.5 to 3.1 × 10⁹ spermatozoa mL^?1, and changes in sperm quality parameters depending on sampling date were observed. Plasma sexual steroid levels remained high and changed in parallel during the spawning season and afterwards decreased to very low levels in summer. The analysis of annual changes of 11-KT and T ratios suggests that 11-KT can be the main circulating androgen for stimulating spermatogenesis in S. rhombus and that T could be involved in the beginning of spermatogenesis through the positive feedback on brain-pituitary-gonad axis. Finally, daily 11-KT and T levels showed similar patterns of variation in males sampled, whereas 17,20β-P amounts showed somewhat opposite trends. These differences could be related with the different role of androgens and progestin during the spermatogenesis.     

Involvement of sex steroids in final stages of oogenesis in Eurasian perch, Perca fluviatilis

Significant plasma 17,20β-dihydroxy-4-pregnen-3-one peaks were measured for the first time in female Eurasian perch, Perca fluviatilis, during the pre-ovulatory period, reaching 3.5 ng ml^?1, but was not synchronized with final maturation and ovulation stages.     

Steroid synthesis in follicular cells and induction of germinal vesicle breakdown in spotted wolffish (Anarhichas minor) oocytes

Incubation of follicular cells from postvitellogenic spotted wolffish ovaries with tritiated steroid precursors revealed that granulosa cells were able to convert 17-hydroxyprogesterone (17-P) to 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) and androstenedione. Theca cells had limited ability to synthesise additional steroids from 17-P but converted 17,20β-P to 17,20β-P sulphate. Neither of the two cell layers was able to synthesise 5β-pregnane-3α,17,20β-triol-20-sulphate (3α,17,20β-P-5β-S) which is found in high concentrations in plasma. 17,20β,21-trihydroxy-4-pregnen-3-one (17,20β,21-P), 17,20β-dihydroxy-5β-pregnan-3-one (17,20β-P-5β) and 17,20β-P were most potent in inducing germinal vesicle breakdown (GVBD). Sulphation of 17,20β-P resulted in loss of GVBD inducing activity.     

Gonadotropin releasing hormone‐analogue (GnRHa) treatment improves the milt production and sperm motility of endangered Caspian brown trout,Salmo trutta caspius,over the course of a spawning season

To determine the effect of gonadotropin releasing hormone‐analogue (GnRHa) treatment on the milt quality of endangered Caspian brown trout, Salmo trutta caspius, the sperm motility (percentage and duration of motility), sperm production (sperm density, spermatocrit and milt volume) and milt pH were measured for GnRHa‐treated (the treatment group) and untreated groups (the control group) during the spawning season. For untreated brooders, the values of the motility per cent, sperm density and spermatocrit decreased continuously during the spawning season while the milt volume, duration of motility and milt pH showed only a significant decrease at the end of the season. For GnRHa‐treated males, these parameters increased 14 days after GnRHa treatment (first milt collection) and then decreased continuously towards the end of the season. In addition, the values of milt and sperm density yielded per treated male were higher than that in the untreated group, although these were not statistically different. In any case, the total sum of yielded milt from the treatment group over the spawning season was higher than that in the untreated group. In this experiment, significant positive correlations were found between milt parameters as follows: sperm motility vs. milt pH; sperm density vs. spermatocrit; milt volume vs. spermatocrit; and milt volume vs. sperm density. The results show that the treatment of Caspian brown trout by GnRHa can improve the milt quality in terms of sperm motility and sperm production during a spawning season.