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.     

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.     

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.     

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.     

Gonadotrophin-releasing hormone agonist raises plasma concentrations of progestogens and enhances milt fluidity in male Atlantic halibut (Hippoglossus hippoglossus)

In two separate spawning seasons, spermiating male Atlantic halibut were implanted with pellets containing gonadotrophin-releasing hormone agonist (GnRHa). Males were bled repeatedly, and milt samples were collected. Blood samples were assayed for free and conjugated steroids: testosterone, 11-ketotestosterone, 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,20-dihydroxy-4-pregnen-3-one (17,20-P), 17,20,21-trihydroxy-4-pregnen-3-one and steroids with a 17,20 configuration. Towards the end of the first season, pellets were implanted into three wild-caught and three hatchery-reared males. No control fish were available. The major progestogen in plasma was identified as sulphated 5-pregnane-3,17,20-triol (3,17,20-P-5-S). Concentrations of this steroid were stimulated by the GnRHa. Sulphated 17,20-P was also identified in the plasma, but at 10-fold lower concentrations than 3,17,20-P-5-S. In the middle of the second season, pellets were implanted into five hatchery-reared males; five unimplanted males were used as controls. Levels of androgens fell following GnRHa treatment, levels of progestogens rose briefly, and there was a significant increase in the fluidity of the milt. Of all the measured steroids, free and sulphated 17,20-P showed the best correlation with milt fluidity.     

Effects of photoperiod, temperature and GnRHa treatment on the reproductive physiology of Atlantic salmon (Salmo salar L.) broodstock

The present study demonstrates that acceleratedphotoperiod advances ovulation in Atlantic salmon, and that exposure to cold water prior to spawning further advances and synchronizes this process while improving egg-survival. High water temperature inhibited both sperm release and ovulation, whereas a GnRHa treatment overrode this temperature effect in most individuals. A decrease in water temperature seemed to accelerate both ovulation and sperm release, and water temperature modulated the plasma 17,20βP profiles around ovulation and sperm release. The GnRHa treatment markedly increased the volume of strippable milt and the plasma 17,20βP levels in males.     

Comparative analysis of reproductive performance between spring and autumn forms of Persian sturgeon,Acipenser persicus,Bordin, 1897: with an emphasis on sex steroids and milt quality

To compare the reproductive performance of spring and autumn forms of Persian sturgeon males, Acipenser persicus, the relative abundance of spermiating and non‐spermiating males, milt quality, sex steroids and cortisol were investigated after pituitary preparation (PP) treatment and at the time of spermiation. Our results showed that higher numbers of spring males are spermiated after PP treatment than autumn individuals. The autumn spermiating males had higher and lower values of sperm density and duration of motility than spring males respectively. The milt volume was higher in spring males than in autumn individuals. The percentage of motility was statistically equal in both the groups examined. In both forms of brooders, the serum levels of testosterone (T), 11‐ketotestosterone (11‐KT), progesterone (P), 17α, 20β, 21‐trihydroxy‐4‐pregnen‐3‐one (20βS) and cortisol (C) increased after PP treatment, although such increases did not occur in non‐spermiating males. Our results showed the probable involvement of 20βs, P, T, 11‐KT and C in the final maturation of Persian sturgeon. In addition, it was demonstrated that the spring form of Persian sturgeon shows better reproductive performance than the autumn form in terms of a response to artificial induction and milt quality.     

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.     

Effects of hemi-castration on plasma steroid levels in two teleost fishes; the three-spined stickleback, Gasterosteus aculeatus, and the Atlantic salmon, Salmo salar

In order to study the possible homeostatic regulation of gonadal steroids in fishes, plasma steroid levels were measured in hemi-castrated and sham-operated nesting male three-spined sticklebacks, Gasterosteus aculeatus, and in mature 2-year old male Atlantic salmon, Salmo salar. Hemi-castration significantly suppressed androgen levels in both species. In sticklebacks, plasma levels of 11-ketotestosterone (11KT) were 56% and levels of testosterone (T) 55% of those found in sham-operated males. In hemi-castrated salmon the levels of 11KT were 63%, and the levels of T were 75% of the levels in sham-operated males. In contrast, levels of 17α,20β-dihydroxy-4-pregnen-3-one (17,20-P) in salmon (not measured in sticklebacks) were not different between hemi-castrated and sham-operated males. The results suggest that, although levels of the steroid 17,20-P were compensated in hemi-castrated salmon, the androgen levels in fish males in full spawning condition are not closely regulated by negative feedbacks. This revised version was published online in July 2006 with corrections to the Cover Date.     

Reproduction of striped bass, Morone saxatilis (Walbaum), broodstock: monitoring maturation and hormonal induction of spawning

Abstract Levels of gonadal steroid hormones were quantified in an adult striped bass, Morone saxatilis (Walbaum), broodstock during their gametogenic cycle. Blood plasma concentrations of Estradiol (E₂) and testosterone in females, or 11-ketotestosterone(11-KT) and testosterone (T) in males, were used as indicators of maturation. In both sexes, hormone levels were low in summer but increased significantly by late October to intermediate levels which were then maintained until late January. They then increased again rapidly to maximum pre-spawning values attained in late February or March, and subsequently decreased during the spawning period (April and May) with an increased incidence of spent fish with low hormone levels. The changes in blood hormone concentrations coincided with annual changes in photoperiod and water temperature that may be useful landmarks for maturation in captive broodstock. Mature females were implanted with pellets containing a dose of approximately 20 μg/kg body weight of [D-Ala⁶-Pro⁹-Net]-LHRH (GnRHa) in a matrix of cholesterol (CH) and cellulose. In April, they had not yet begun final oocyte maturation (FOM) and were too immature for conventional induction of spawning by injection with human chorionic gonadotropin (hCG). In early April, females given two 95% CH (slow hormone-release) GnRHa pellets (95/95) or females given one 80% CH (fast hormone-release) GnRHa pellet and one 95% CH GnRHa pellet (80/95) spawned within 13 days treatment (n= 4) with good egg fertility (76 ± 7% of total) and hatch rates (62 ± 15% of fertile). Females given dual fast-release GnRHa pellets (80/80) or control (Sham) pellets did not spawn or show evidence of increased oocyte diameter or development. In late April, four of six females given the 80/95 GnRHa pellet combination spawned within 9 days. Three fish produced fertile eggs (54 ± 18%). one spawned overripe eggs, and the remaining two increased oocyte diameter and maturation. Three corresponding controls did not spawn, and two of these showed clear signs of atresia within 11 days. In early May, some females were undergoing early FOM and were mature enough to be spawned by hCG injection. Three were given a single 80% CH GnRHa pellet and spawned within 6 days of treatment to produce fertile eggs (44 ± 6%). Of two other females given dual 80% CH GnRHa pellets, one spawned infertile eggs and the other failed to spawn within 9 days. GnRHa implants show promise as a technique for inducing spawning of captive striped bass broodstock although the optimum hormone delivery systems, dosages and release rates should be verified for fish at specific maturational stages.     

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.     

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.     

Sperm characteristics and androgens in Acipenser ruthenus after induction of spermiation by carp pituitary extract or GnRHa implants

Spermiation and changes in androgen (testosterone, T and 11-ketotestosterone, 11-KT) levels were studied in sterlet (Acipenser ruthenus) treated with GnRH agonist implants (dAla⁶-Pro⁹-LHRHa) at 25 and 75?μg?kg^?1 b.w. and compared with those males treated with 4?mg?kg^?1 b.w. of carp pituitary extract (CPE) and 3 pellets of Ovopel kg^?1 b.w., which contains dAla⁶-Pro⁹NEt-mGnRH and metoclopramide. Sperm quality (sperm mass, spermatozoa concentration and sperm motility and velocity) was evaluated 24, 48 and 72?h after hormonal treatments. Males did not release sperm in the control group injected with physiological solution, while sperm could not be collected 7?days after treatments in all hormonally treated groups. Spermiation rates were 100?% in the CPE and Ovopel groups and 25–50?% in the GnRHa-treated groups. Sperm production was significantly lower in the GnRHa-treated groups than in the CPE and Ovopel groups and decreased 72?h after hormonal treatment. Sperm motility and velocity were higher in the Ovopel and GnRHa (75?μg) groups compared to the CPE and GnRHa (25?μg) groups and decreased 72?h after hormonal treatment. Androgens were only affected in spermiating males and changed in the Ovopel and GnRHa (75?μg) after hormonal treatment. Significant correlations were observed between sperm production, sperm motility and sperm velocity, but not androgens. The present study suggests involvement of dopamine in sturgeon spawning. Additionally, better sperm quality observed in the Ovopel group and particularly sperm motility in the GnRHa (75?μg) suggests enhancement of sperm quality in sturgeon treated with GnRHa. Therefore, further study is needed to induce fully spermiation using GnRHa implants in combination with a dopamine inhibitor.     

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.     

Effects of aromatase inhibitors on sexual maturation in Atlantic salmon,Salmo salar,male parr

Atlantic salmon, Salmo salar, male parr were implanted with Silastic capsules filled with different aromatase inhibitors: 1,4,6-androstatriene-3,17-dione (ATD), 4-hydroxy-4-androstene-3,17-dione (4OH), and the non-steroidal CGS16949 A, 4-benzonitrile monohydrochloride (CGS). Aromatization in brain homogenates were lower in salmon implanted with CGS and ATD than in controls. This was not the case for 4OH, but administration of 4OH to brain homogenates reduced the aromatase activity. All three aromatase inhibitors had effected gonadal weights in fish sampled in the summer, but the effects were markedly different among inhibitors. Plasma levels of the androgen 11-ketotestosterone (11KT) and the progestin 17, 20-dihydroxy-4-pregnen-3-one (17,20P) were measured by means of radioimmunoassay. CGS and ATD, but not 4OH, significantly decreased the plasma 17,20P levels in the autumn. Plasma levels of 11 KT were not influenced by ATD or CGS treatment, but 4OH had a lowering effect in one autumn sampling. ATD and 4OH (CGS not tested) increased the proportion of maturing males.These findings suggest that aromatization is of physiological importance in different mechanisms controlling reproduction in salmon.     

Endocrine changes during the annual reproductive cycle of the red porgy, Pagrus pagrus (Teleostei, Sparidae)

Changes in 17-estradiol (E2), estrone (E1), testosterone (T), 11-ketotestosterone (11KT), and 17,20-dihydroxy-4-pregnen-3-one (17,20-P) levels were correlated to changes in gonadosomatic index (GSI), vitellogenin concentration (Vg), ovarian and testicular histology during the annual reproductive cycle of the red porgy, Pagrus pagrus. The production of E2, E1, T and 17,20-P was confirmed by analysis of the steroidogenic activity of ovaries. In females, the average concentration of E2 was lower than 2 ng ml^–1. E2 values first increased significantly at the stage of endogenous vitellogenesis and remained high during exogenous vitellogenesis. E1 levels were lower values than E2 (less than 300 pg ml^–1), but they increased at the beginning of exogenous vitellogenesis. Estrogens concentrations followed similar pattern to Vg and were significantly correlated. Mean levels of T were mostly lower than 1 ng ml^–1. They followed a pattern similar to that of E2 except for a further increase observed at the stage of final maturation. T and E2 levels were significantly correlated. The concentration of 11KT did not change significantly. The levels of 17,20-P ranged between 0.22 and 1.22 ng ml^–1 but changes were not related to gametogenesis. In males, the concentrations of T and 11KT fluctuated significantly during the sexual maturity stages, showing a similar pattern and were significantly correlated to GSI changes. T levels increased during spermiogenesis and spermiation stages to reach about 3 ng ml^–1. 11KT levels stayed about half those of T. The levels of estrogens showed no significant changes. Level of 17,20-P showed no significant variation related to male maturity. Results are discussed in relation to changes in plasma steroid levels during gametogenesis of other multiple spawner species.     

Effects of 11-ketotestosterone and gonadotropin-releasing hormone on follicle-stimulating hormone and luteinizing hormone gene expression in castrated and sham-operated male red seabream <Emphasis Type="Italic">Pagrus major</Emphasis>

ABSTRACT:   In order to clarify the roles of androgen and gonadotropin-releasing hormone (GnRH) on gonadotropin (GTH; luteinizing hormone [LH] and follicle stimulating hormone [FSH]) synthesis, effects of castration and implantation of GnRH analog (GnRHa) or 11-ketotestosterone (11-KT) on expression of GTH subunit, α-glycoprotein subunit (αGSU), FSHβ, and LHβ genes, during the early spermatogenic stage in male red seabream Pagrus major were examined. Male red seabream underwent castration or sham-operation and were subsequently implanted with cholesterol pellets containing GnRHa, silicone capsules filled with 11-KT, or blank capsules (control). FSHβ mRNA levels increased due to castration, and it was reversed by treatment with 11-KT. 11-ketotestosterone treatment also decreased FSHβ mRNA levels in sham-operated fish. These results suggest that 11-KT acts on the pituitary to suppress FSH synthesis in male red seabream. On the other hand, neither castration nor replacement of 11-KT in castrated fish had effects on LHβ mRNA levels, whereas 11-KT treatment had slightly but significantly decreased LHβ mRNA in sham-operated fish. αGSU mRNA levels were not changed by castration or 11-KT treatment in both sham-operated and castrated fish. Meanwhile, treatment with GnRHa significantly decreased FSHβ mRNA levels in sham-operated fish, but not in castrated fish. This suggests that GnRHa may down-regulate expression of FSHβ mRNA through the production of 11-KT in testis. LHβ and αGSU mRNA levels in sham-operated fish, but not in castrated fish, were significantly elevated by treatment with GnRHa.     

Artificial induction of maturation and fertilization in the Japanese eel, Anguilla japonica

Repeated injections of salmon pituitary extract (20 mg per fish per week) induced vitellogenesis in feminized, cultivated Japanese eels (Anguilla japonica). Oocytes were attained at the migratory nucleus stage after 11 or 12 injections. Addition of 17,20-dihydroxy-4-pregnen-3-one (DHP) into the incubation medium induced germinal vesicle breakdown (GVBD) in the oocytes at the migratory nucleus stage. An injection of DHP (2 µg g^-1 BW), given 24h after an injection of salmon pituitary extract (20 mg fish^-1), succeeded in inducing maturation and ovulation in females which contained occytes at the migratory nucleus stage. Most fish ovulated 15–18h following the DHP injection. Eggs that were ovulated within 15h after the DHP injection showed high fertility and hatchability, but eggs ovulated 18 or 21h after the DHP injection, showed considerably lower fertility and hatchability. A delay between ovulation and stripping of the eggs rapidly decreased both the fertility and hatchability within 6–9h after ovulation, indicating that artificial fertilization must be carried out immediately after ovulation. Repeated injections of human chorionic gonadotropin (hCG) at a concentration of 1 IU g^-1 BW week^-1 induced spermatogenesis, spermiation, and the acquisition of potential for sperm motility in cultivated males. Most males spermiated after the fifth or sixth injection of hCG, and the milt weight gradually increased and remained constant (1–2 g) from the 11th to 31th injection. Sperm motility peaked 24h after each weekly injection, and decreased from the 3^rd day after the injection. Potassium ions are an essential constituent for the maintenance of motility in the eel spermatozoa. Artificial seminal plasma containing 15.2 mM KCl is applicable as a milt diluent. Using these techniques developed for female and male eels, we have succeeded in obtaining many fertilized eggs from cultivated eels.     

Effects of experimental hypo- and hyperthyroidism on iodothyronine deiodinases in Nile tilapia, Oreochromis niloticus

In the present study, we examined the effects of experimentally-induced increases or decreases in plasma concentrations of thyroid hormones on iodothyronine deiodinases in tilapia, Oreochromis niloticus. To obtain hyperthyroid tilapia, fish were injected with porcine follicle stimulating hormone (pFSH) 36 hours before sampling or fed on demand for 11 days with tilapia pellets containing 12 ppm T3. Tilapias were made hypothyroid by providing them food containing 0.2% methimazole for 11 days. Plasma T4 and T3 and the in vitro deiodinase activity in liver, kidney, brain and gill were measured at the end of the treatment period. Injection with pFSH caused an increase in plasma T4 but had no influence on plasma T3 levels. A small increase in plasma T3 was observed in T3-fed fish. Plasma levels of both T4 and T3 were decreased by methimazole treatment. We observed no changes in kidney type I deiodinase (D1), whereas liver type II deiodinase (D2) was increased during hypothyroidism and decreased during hyperthyroidism. Hypothyroidism resulted in a significant decrease in brain, gill and liver type III deiodinase (D3). An pFSH-induced increase in T4 stimulated brain and gill D3 but not liver D3, whereas the opposite was true in T3-fed fish. We conclude that the regulation of D1 and D3 in tilapia is probably different compared to mammals.     

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.