Controlled reproduction of an important indigenous fish species, Spinibarbus denticulatus (Oshima, 1926), in Southeast Asia

Aquaculture of Spinibarbus denticulatus (Oshima, 1926), a fish species indigenous to North Vietnam and Eastern China, is constrained by lack of fingerlings for stocking ponds and cages. As these fish do not naturally breed in captivity, carp pituitary extract (CPE), luteinizing hormone-releasing hormone analogue (LHRHa) with domperidone (DOM) and human chorionic gonadotropin (HCG) were administered at various doses to induce ovulation. A first set of experiments evaluated the response to LHRHa (30, 40 and 50 μg kg⁻¹) with or without DOM (10 mg kg⁻¹), CPE (20, 30 and 40 mg kg⁻¹) and HCG (3000, 4000 and 5000 IU kg⁻¹). A second set of experiments evaluated the dose response to LHRHa (30, 35, 40 and 50 μg kg⁻¹) primed with 6 mg kg⁻¹ of CPE, and HCG (3000, 3500, 4000, 5000 IU kg⁻¹) primed with 6 mg kg⁻¹ of CPE. The treatment groups were compared with each other and the control (injected with 0.9% saline solution). Only 25% and 50% ovulation resulted when treated with LHRHa at 40 and 50 μg kg⁻¹, whereas 100% ovulation was achieved with an addition of DOM to LHRHa. Both 30 and 40 mg kg⁻¹ CPE induced 100% ovulation. However, HCG (4000 and 5000 IU kg⁻¹) induced ovulation in only 33% of females. When primed with CPE, the minimum dose of LHRHa required was 35 μg kg⁻¹ to achieve 70% ovulation. Priming HCG with CPE also resulted in 100% ovulation, and the minimum effective dose of HCG to induce ovulation was 3500 IU kg⁻¹ with 60% ovulation. Fertilization and hatch rates observed in this study with different hormonal stimulation were high (80–93%). The results indicate that while the use of combined hormone strategy has no apparent advantage over a single hormone strategy, LHRHa+DOM (40 μg kg⁻¹+10.0 mg kg⁻¹) and CPE (30 mg kg⁻¹) are most effective in consistently inducing ovulation and thus can be used for commercial hatchery production of S. denticulatus larvae.     

Quantity, motility and fertility of tench Tinca tinca (L.) sperm in relation to LHRH analogue and carp pituitary treatments

The spermiation of tench males was stimulated with Supergestran containing mammalian LHRHa lecireline at the following doses: 5, 10, 20 and 40 g kg⁻¹ b.w.; then with carp pituitary suspension (CPS) at a dose of 2 mg kg⁻¹ b.w. and with a control of saline physiological solution. The following days, meaning 24, 48 and 72 h after injection, sperm was collected to evaluate volume and the number of sperm per male per kg body weight (B.W.) The percentage of motile sperm and velocity of spermatozoa were measured 48 h after hormonal injection, and 72 h after hormonal injection the sperm was evaluated for fertilization and hatching ability. All 42 males in experimental groups were diploid. Live weight did not differ significantly among experimental groups. The strongest stimulation of spermiation was achieved with LHRHa in dosage of 20 and 40 g kg⁻¹ b.w. and CPS compared to males of the control group and lower dosage of LHRHa. Analysis of variance showed no significant influence of the treatment on the velocity and percentage of motile spermatozoa. The effect of different treatment on the fertilization capacity (the number of spermatozoa per egg was equilibrated) was significant. Significantly the highest quality of sperm collected 72 h after injection expressed by percentage of fertilization and hatching (62–65% fertilization and 61–64% hatching rates, respectively) was found for LHRHa in dosage of 20 and 40 g kg⁻¹ b.w. Significantly the lowest parameters of fertilization and hatching were found for the control group, on the 12% level.     

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.     

Spermiation time affect the milt quality indices of the Russian sturgeon,Acipenser gueldenstaedtii,Brandt & Ratzeburg, 1833

In this study, the effects of spermiation time are investigated on milt quality of Russian sturgeon over the course of the spawning season. The milt samples were collected from three broodstock batches at three time points including: the beginning, middle and at the end of the spawning season. According to the results, the milt quality parameters including pH, sperm density, spermatocrit, duration of sperm motility and percentage of sperm motility were significantly low in the beginning and end of season than middle of season. The values of milt quality parameters in the middle of season were as follows: (motility percentage: 69.6 ± 3.5, motility duration: 460.3 ± 37.2 s, sperm density: 8.7 ± 0.4 × 10⁹, milt volume: 86.3 ± 8.1 and milt pH: 8.3 ± 0.15). Significant positive correlations were also found between milt pH and sperm motility as well as between sperm density and spermatocrit. In conclusion, our study showed that the middle of season is the best time for collection of milt with appropriate quality in Russian sturgeon. Selection of milt with good quality is necessary aim to cryopreservation of spermatozoa in endangered fish species including Russian sturgeon.     

The effect of luteinizing hormone-releasing hormone analogue (LHRHa) on sperm count of Oreochromis niloticus (L.)

An experiment was conducted to compare the potency of different levels of luteinizing hormone-releasing hormone analogue (LHRHa) for increasing the sperm counts in sexually mature male Oreochromis niloticus (L.). One hundred males (106.49 ± 3.59g, 15.2 7 ± 0.20 cm) were randomly distributed in four treatment groups, each receiving intramuscular injections of: (i) control, 0.05 ml phosphate-buffered saline, (ii) 10 μg kg^-1 LHRHa, (iii) 20 μ kg^-1 LHRHa and (iv) 30 μ kg^-1 LHRHa. Sperm counts were determined prior to injection (day 0) and for four consecutive days (days 1 to 4) thereafter. Results showed a significant increase in sperm counts (P < 0.01) 1 day after injection among males injected with 10 to 20 μ kg^-1 LHRHa. The effect of injecting 30 μ kg^-1 LHRHa is similar to that in the control group. Across all treatments, sperm counts declined with time over the 4-day sampling period.     

Delayed gametogenesis and spawning of sea bass (Dicentrarchus labrax L.) kept under different photoperiod and temperature regimes

The present work investigates the importance of day length and temperature in the control of reproduction of sea bass, as well as the effectiveness of LHRHa and HCG in inducing spawning out of season in this species. A controlled regime was produced and seasonal cycles with high components of temperature and photoperiod were extended from the summer solstice for at least 6 months, followed by a short photoperiod regime for 3 months before a new increase in these components. Natural spawning in the control fish occurred more frequently in mid February, although it was also observed in January and early March. Temperature manipulation delayed the spawning one month with respect to the controls, although some of the animals entered into gonadal regression. Photoperiodic manipulation delayed maturation for three months with respect to controls but it was necessary to perform hormonal induction of spawning. Although LHRHa and HCG were both applied, only intraperitoneal injections of LHRHa were effective in inducing spawning of sea bass out of season when the temperatures were 17°C. Dephasing between the annual changes in photoperiod in relation to the coordination of the different events of the sexual cycle of sea bass is considered.     

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.     

Effect of different methods for the induction of spermiation on semen quality in European eel

Five hormonal treatments with human chorionic gonadotropin (hCG) were tested for the induction of maturation and spermiation in male farmed eels. The main aim was to optimize previously used hormonal treatments to achieve shorter induction treatments, longer spermiation periods and/or higher sperm quality. Fish treated for just 3 weeks (treatment E) or until the onset of spermiation (treatment C) showed the worst results, while the treatment consisting of weekly administration of 1.5 IU hCG g^?1 fish (treatment A) induced the highest percentage of spermiating males, the highest number of sperm samples and sperm volumes and densities similar to the rest of the treatments (B: half hormone dosage, or D: biweekly administration). Evaluation of the sperm quality was performed by computer‐assisted sperm analysis (CASA), considering the percentage of total motile spermatozoa, the percentage of fast and medium‐velocity spermatozoa, as well as different motility parameters. Sperm samples from A‐D groups showed between 44% and 54% motile spermatozoa, and between 10% and 15% fast spermatozoa, while samples from E‐treated males showed 0% motile cells. No significant differences were found in the spermatozoa straight line velocity (VSL), curvilinear velocity (VCL) or the angular velocity (VAP), neither spermatozoa beating cross frequency (BCF) between A–D groups.     

Stimulation of spermiation and induction of ovulation in pike (Esox lucius)

Mature northern pike were given various hormonal treatments in March or April in order to stimulate spermiation or to induce ovulation. In males the total amount of sperm collected after treatment increased, in comparison with saline-injected males, by 3–11 times with partially purified salmon gonadotropin (PPSG-activity: half of the highly purified s-GTH; injected at doses of between 5 and 100 μg/kg body weight); 3–6 times with crude carp pituitary extract (0.5–3 mg/kg body weight); and 3–7 times with fresh pike pituitaries (14 and 1.2 mg wet weight/kg body weight). The sperm obtained after hormonal treatment was of good quality. Intracardiac injection of superactive LRH analogue had no effect. In females, PPSG induced 90 and 100% ovulation at the doses of 50 and 25 μg/kg body weight. Dried salmon pituitaries (2.5 mg/kg, equivalent to 50 μg of PPSG) gave 25% ovulation; at 10 mg/kg, 25% complete ovulation was again recorded, but in addition 70% of the females showed oocyte maturation and partial ovulation. Similarly, dried carp pituitary (3 mg/kg) induced only oocyte maturation but no ovulation. The oocytes obtained after hormonal treatment were in general fertile. Intraperitoneal injection of LRH in an emulsified form induced neither oocyte maturation nor ovulation. The lack of effect of LRH analogue is discussed and shows that the use of this compound as a substitute for pituitary preparation is not very promising.     

Changes in Sperm Production,Sperm Motility,and Composition of Seminal Fluid in Caspian Brown Trout,Salmo trutta caspius,Over the Course of a Spawning Season

This study was carried out to evaluate milt quality in male Caspian brown trout (Salmo trutta caspius) over the course of the winter spawning season. Milt samples were collected biweekly during December and January. Chemical composition of seminal fluid, sperm production (milt volume, sperm density, spermatocrit,) and sperm motility characteristics (percentage and duration of motility) were measured. Milt volume, sperm density, osmolality, seminal minerals (Ca²⁺, Mg²⁺, K⁺, Na⁺, Cl^?), and total protein gradually decreased over the spawning season. Glucose and triglyceride content of milt did not show significant changes over the spawning season. Milt pH and the percentage and duration of motility were comparatively stable, declining only at the end of the season. Significant positive correlations were found between sperm density and seminal minerals, total protein and spermatocrit; percentage of motile spermatozoa and seminal minerals, total protein; and duration of motility and K⁺, Cl^?, total protein, and pH. Results show that season has a significant influence on milt quality in male Caspian brown trout, with the best milt being available at the beginning of spawning season.     

Effect of two commercial preparations containing different GnRH analogues with dopamine antagonists on barbel Barbus barbus (L.) sperm quantity and quality

The effectiveness of applying Ovaprim [(D-Arg⁶, Pro⁹NEt)-sGnRH + domperidone] and Ovopel [(D-Ala⁶, Pro⁹NEt)-mGnRH + metoclopramide] to male barbel Barbus barbus (L.) 6, 12 and 24 h after hormonal stimulation was analyzed. The control group (Control) during each time interval was stimulated with 0.9 % NaCl. Milt was collected from seven fish only once (n = 7) for Ovopel, Ovaprim and Control group in order to determine total volume of milt, volume of milt per kg of body weight, sperm concentration, total sperm production, seminal plasma osmotic pressure, pH of milt and pH of seminal plasma. Woynarovich’s solution (68 mM NaCl + 50 mM urea) with the addition of 0.5 % BSA (pH 7.7; 181 mOsm kg^?1) was used as the activating liquid. Selected parameters of sperm motility (MOT %) and progressively motile sperm (%), curvilinear velocity (VCL, μm s^?1), straight-line velocity (μm s^?1), movement linearity (%), wobbling index (%), amplitude of lateral head displacement (μm) and beat cross frequency (Hz) were determined using the Computer-assisted sperm analysis system. A time of 6 h proved to be too short to obtain milt from barbel following hormonal stimulation with Ovaprim and Ovopel. Extending the time to 12 h, however, resulted in 100 % spermiation in males, regardless of hormonal preparation used for stimulation. The stimulation of spermiation in barbel is best performed using Ovopel 12 h upon application. Extending the latency period to 24 h following the application of this preparation results in a significant decrease in the volume of milt obtained, sperm count and motility parameters, including MOT and VCL, which may influence sperm fertilization ability.     

Sperm density, seminal plasma composition and their physiological relationship in the endangered Caspian brown trout (Salmo trutta caspius)

Sperm density, mineral and organic composition of the seminal plasma and their physiological relationship were investigated in the Caspian brown trout (Salmo trutta caspius). To establish a rapid and accurate method for assessment of sperm density, three different techniques were used: sperm counting, spectrophotometry (at 480 nm) and determination of the spermatocrit. The seminal plasma contained 159.26±8.84 mM sodium (Na), 33.72±2.01 mM potassium (K), 133.04±5.96 mM chlorine (Cl), 1.68±0.2 mM calcium (Ca) and 0.988±0.13 mM magnesium (Mg). The following organic components were found: total protein 0.75±0.14 mg 100 mL⁻¹, cholesterol 2.86±0.58 mg L⁻¹ and glucose 3.81±1.04 mM L⁻¹. The mean sperm density was estimated to be 3.3 × 10⁹ spermatozoa mL⁻¹. The spermatocrit (%) ranged from 25 to 52 in sperm samples. Highly significant linear relationships were found between sperm density and spermatocrit (R²=0.703, P<0.001) and sperm density and optical density (R²=0.909, P<0.001), indicating that optical density can be used as a quick and accurate method of estimating sperm density. Significant relationships were also found between sperm density and Ca, Mg and total protein of seminal plasma. A significant correlation was also observed between the Ca and Mg concentrations (R²=0.774, P<0.01). The following correlations were observed between mineral and organic components: total protein and Ca (R²=0.462, P<0.05), total protein and Mg (R²=0.518, P<0.05) and glucose and Cl (R²=0.374, P<0.05). These parameters should be considered when developing procedures for either artificial fertilization or for cryopreservation of sperm.     

Spermatocrit and spermatozoa density in Atlantic cod (Gadus morhua): correlation and variation during the spawning season

The utility of spermatocrit (the proportion of solid packed material in semen after centrifugation) as an indicator of spermatozoa density and male spawning stage was tested in Atlantic cod (Gadus morhua). Semen was collected from captive male cod over three spawning seasons. Spermatocrit was positively and significantly correlated with spermatozoa density measured with a Coulter counter (Multisizer), but not with spermatozoa counts in a haemacytometer. Spermatocrit increased significantly as the spawning season progressed. However, day of season explained only 35% of the variation because spermatocrit varied among individual males. Spermatozoa size remained unchanged throughout the sampling period and was not correlated with spermatocrit, indicating that variation in spermatocrit was due to variation in spermatozoa number and not their size. Spermatocrit is a good estimator of sperm density but is not reliable as indicator of spawning stage because of the variation among individual males.     

Effects of hormonal manipulation on stress responses in male and female broodstocks of pikeperch Sander lucioperca

This study was designed to determine the effects of hormonal manipulation on stress responses in female and male pikeperch. Two-year-old cultured female and male broodstocks with an average weight of 337.4 ± 20.1 (mean ± SE; n = 16) and 318.7 ± 15.1 g (n = 16), respectively, were randomly allocated into four hormonal treatments each containing 4 fish. Two sexual groups of 16 fish for each gender were considered. Sexually mature male and female pikeperch were injected with either physiological saline solution (as control group), common carp pituitary extract (CPE), human chorionic gonadotropin (hCG) and or luteinizing hormone-releasing hormone analog (LHRHa₂). The blood samples were taken before hormonal injection and after ovulation and spermiation. Then the plasma levels of stress indices (cortisol, glucose, and lactate) were determined. The results showed that all CPE-, HCG-, and LHRHa_2- injected males produced sperm. In females treated with CPE and hCG, three of four ovulated, but none of LHRHa_2- and saline-injected fish spawned. Significant changes in cortisol, glucose, and lactate levels were observed among the females injected with different hormones. Plasma cortisol and glucose levels increased significantly in males injected with CPE and females injected with hCG, but no significant change was observed in lactate levels before and after hormonal induction. Comparison of two sexes revealed significant differences in glucose levels for females in some groups before injection, while CPE-injected sexes showed significant changes in cortisol and lactate concentrations. The results indicated that the induction of ovulation or spermiation stimulated stress responses especially in female pikeperch, and therefore, all the procedures should be made to minimize the disturbance during the artificial spawning.     

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.     

The Effect of Luteinizing Hormone Releasing Hormone Analog Regime and Stage of Oocyte Maturity for Induced Ovulation of Channel Catfish,Ictalurus punctatus

This study addresses rapid methods to identify mature channel catfish, Ictalurus punctatus, females for induced spawning with luteinizing hormone releasing hormone analog (LHRHa) and common carp pituitary extract (CPE) and the effects of stage of maturity, hormone dose, season, and cannulation before hormone injection. Hormonal intervention is the most effective method for inducing maturation and spawning in channel catfish × blue catfish, Ictalurus furcatus, hybrids. A total of 80 mature channel catfish were staged for maturity based on secondary sexual characteristics and 20–30 oocytes cannulated to ascertain their maturation stage based on the position of gonadal vesicle. Twenty females were randomly assigned to one of the four hormone regimes (priming + resolving dose): CPE 2 + 8 mg/kg (CPEC); LHRHa 20 + 40 µg/kg (LHRHaL); LHRHa 20 + 60 µg/kg (LHRHaM); and LHRHa 20 + 80 µg/kg (LHRHaH) and a fifth treatment consisted of 20 females selected based on apparent maturity as evidenced by external appearance were injected with CPE, 2 + 8 mg/kg without cannulation (CPEO). Two trials were conducted in early part of the spawning season and two trials in peak season. External methods to identify maturity correlated (r = 0.63) with that of “germinal vesicle position” method to identify the stage of maturity in females. Mean percent of females that ovulated, hatched, and fry produced per kg body weight did not differ (P > 0.05) among the five hormone treatments. The mean percent females that ovulated was higher (P < 0.05) for CPE‐induced females that were identified as “excellent” females based on external examination. The performance of cannulated females did not differ (P > 0.05) with that of non‐cannulated catfish. The mean egg quality of hormone‐induced females and percent of females ovulated in response to hormone treatment established a weak but significant linear relationship (Y = 3.85 + 0.008 × ovulation, r² = 0.39, P < 0.05).     

Estimation of sperm concentration of wild and reconditioned brown trout, Salmo trutta L.

Sperm concentrations were assessed for milt samples taken from three stocks of brown trout, Salmo trutta L., (wild resident trout, and reconditioned and seareared sea trout) using direct counts, spermatocrit and an optical density of 1:1000 dilutions. There were significant linear relationships between spermatocrit and absorbance, and both spermatocrit and absorbance with sperm concentration. The results gave sperm concentrations from 2.2 × 10⁹ mL^?1 to 26 × 10⁹ mL^?1. Mean concentrations differed significantly between stocks; the reconditioned males had a mean concentration of 9.1 × 10⁹ mL^?1, sea-reared males 18.0 × 10⁹ mL^?1 and 8.0 × 10⁹ mL^?1 in 1994 and 1995, respectively, and wild resident males 13.3 × 10⁹ mL^?1. Sperm concentrations from all three stocks of trout were negatively correlated with length, and therefore, age. It was indicated that sufficient milt was being produced to achieve good rates of fertilization, although some of the larger trout did have low sperm counts, suggesting that fertilizing ability may decrease with male size or age within any one stock.     

LHRHa‐induced ovulation of the endangered‐Caspian brown trout (Salmo trutta caspius) and its effect on egg quality and two sex steroids: testosterone and 17α‐hydroxyprogestrone

To induce synchronized ovulation, migrating wild Caspian brown trout (Salmo trutta caspius) females were treated with two interperitoneal injections of Des‐Gly¹⁰, d ‐Ala⁶ LHRH (LHRHa), given 3 days apart. Two injections of 100 μg kg^?1 body weight of this hormone effectively induced ovulation. Within 27 days from the second injection, all fish injected with 100 μg kg^?1 LHRHa had ovulated compared with 54.5% of the controls. The mean time to ovulation was reduced significantly (P<0.05) from 31.67±4.84 days in control fish and 28.83±7.31 days in sham‐treated fish to 16.36±1.61 days in fish injected with 100 μg kg^?1 LHRHa. The fertilization rate in 50 and 100 μg kg^?1 LHRHa‐injected fish was significantly lower than that in the control fish (P<0.05). In fish injected with 50 and 100 μg kg^?1 LHRHa, significant (P<0.05) changes in testosterone (T) and 17α‐hydroxyprogestrone (OHP) levels were observed. After the second LHRHa injection, the fish injected with 100 μg kg^?1 showed the highest serum levels of testosterone and OHP. These results demonstrate that the use of LHRHa can effectively reduce the mean time to ovulation and induce synchronized ovulation in Caspian brown trout.     

The use of luteinizing hormone releasing hormone analogue for ovulation induction in black sea bass (Centropristis striata)

Interest in commercial production of black sea bass has increased in recent years, but reliable spawning methods remain problematic. The objective of this study was to evaluate the effects of oocyte size and luteinizing hormone releasing hormone analogue (LHRHa) dosage and delivery systems on ovulatory success for in vitro fertilization. Vitellogenic females with maximum oocyte diameters of 400–625 μm were implanted with a 95% cholesterol–5% cellulose pellet containing 50 μg of LHRHa. Fish with maximum oocyte diameters < 450 μm failed to ovulate. In contrast, 90% of fish with 500 μm oocytes spawned within 36 h and 40% of this group ovulated a second time. All of the females containing oocytes > 550 μm ovulated. In a second experiment, females with uniformly vitellogenic oocytes (> 500 μm) and implanted with 50 μg LHRHa ovulated substantial numbers of eggs (45,000–192,000 eggs/kg body weight (BW), but fertility was consistently low (0–15%). In a third experiment, 19 of 39 females receiving implants containing 6.3–23.6 μg LHRHa/kg BW during the spawning season ovulated, but fecundity (17,000–339,000 eggs/kg) and fertilization (0–98%) were highly variable. When fish were grouped by developmental index, calculated as the number of oocytes with diameters > 400 μm/total number of oocytes measured, there were no statistical differences among groups with respect to the number of spawns, fecundity or fertilization success. In a fourth experiment, 11 of 13 females with a clutch of fully vitellogenic oocytes that were injected with 20 or 100 μg/kg BW LHRHa ovulated between 1 and 2 times on consecutive days. Five of seven given an implant containing 12.5 μg LHRHa ovulated one or more times. Fish implanted with shams or injected with vehicle alone did not ovulate in any of the experiments. No differences were found in the number of spawns, fecundity or fertilization success from fish receiving different doses of injected or implanted LHRHa. Incubation of pooled eggs produced 155,000 larvae (60% hatch) and 95,000 one-gram juveniles. These results demonstrate that injected or implanted LHRHa is effective for inducing ovulation in black sea bass with maximum oocyte diameters > 500 μm.     

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.