Protective role of antifreeze proteins on sterlet (<Emphasis Type="Italic">Acipenser ruthenus</Emphasis>) sperm during cryopreservation

The loss of sperm quality in sterlet (Acipenser ruthenus) due to freeze-thaw process in cryopreservation was investigated in the present study. Two antifreeze proteins (AFPI or AFPIII) were used at different concentrations of 0.1, 1, 10, and 100 μg/mL. We compared motility, curvilinear velocity, and plasma membrane integrity of fresh, cryopreserved sperm, and sperm cryopreserved in the presence of antifreeze proteins. Fresh sperm (control) had 85?±?4% motility and 160?±?2 μm/s curvilinear velocity, respectively. After cryopreservation, the motility of frozen-thawed sperm without addition of antifreeze proteins significantly decreased (44?±?9%), compared to the control. The highest motility of frozen-thawed sperm was obtained in cryopreserved sperm with addition of 1 μg/mL of AFPIII (58?±?14%). No significant differences were observed in curvilinear velocity between fresh sperm and cryopreserved sperm with/without addition of AFPI or AFPIII. The flow cytometry analysis revealed that fresh sperm contained 94.5?±?6% live cells, while the cryopreserved sperm only contained 26.6?±?14% live cells. Supplementation of antifreeze proteins has significantly improved the percentage of live cells in frozen-thawed sperm, except 0.1 μg/ml of AFPI group. No significant difference in percentage of live cells was detected in the sperm cryopreserved with 10 μg/mL of AFPI or AFPIII, compared to fresh sperm. Thus, addition of antifreeze proteins to cryopreservation medium could be considered to improve the post-thawed sperm quality of sterlet.     

Greenlip abalone (Haliotis laevigata Donovan, 1808) sperm cryopreservation using a programmable freezing technique and testing the addition of amino acid and vitamin

This study investigated factors key to the development of sperm cryopreservation in the greenlip abalone Haliotis laevigata using a programmable freezing technique, including (1) permeable cryoprotectant agent (CPA) selection; (2) cooling rate; (3) endpoint temperature; (4) thawing temperature; (5) sperm to egg ratio and (6) sugar, vitamin and amino acid supplementation, using sperm motility, fertilization rate, plasma membrane integrity, mitochondrial membrane potential or acrosome integrity as quality assessment indicators. Results showed that among the permeable CPAs evaluated, 10% dimethyl sulfoxide was the most suitable for greenlip abalone sperm cryopreservation. The highest post‐thaw sperm motility was achieved with the sperm being frozen at a cooling rate of ?5°C min^?1 to ?30°C from 0°C and thawed and recovered in 40°C and 18°C seawater baths respectively. The addition of sugars in 10% dimethyl sulfoxide did not significantly improve the post‐thaw sperm motility and fertilization rate. The addition of 0.6% glycine, 0.2% taurine or 0.02% L‐ascorbic acid, on the other hand, significantly improved the post‐thaw sperm motility. However, only the addition of 0.6% glycine improved the post‐thaw sperm fertilization rate, which was further confirmed by the improvement of the post‐thaw sperm mitochondrial membrane potential and acrosome integrity through flow cytometry analysis.     

Determination of spermatological properties of male Liza abu (Heckel, 1843) in Atatürk Dam Lake, Şanlıurfa

The aim of this study was to determine the spermatological characteristics in male L. abu during the spawning season. Semen was collected weekly by abdominal massage from 26 males in March. In collected semen, volume, motility, duration of motility, concentration and pH were determined. In the L. abu sperm, volume (μl), motility (%), duration of motility (s), concentration (×10⁹/ml), and pH values were found 45.76 ± 3.55, 54.25 ± 2.93, 330.15 ± 37.92, 4.27 ± 0.40 and 7.87 ± 0.05, respectively. A correlation was found between semen volume and semen pH. Semen volume and the duration of sperm motility were higher in the 2nd and 3rd sampling dates than in the 1st and 4th sampling dates (P < 0.05; P < 0.01, respectively). Neither sperm motility nor sperm concentration was affected by sampling dates. Major changes in semen pH were observed in the 4th sampling date (P < 0.001). The Pearson correlation test presented significant relationships with the duration of motility, semen volume, and motility. Semen pH values were significantly correlated with the sperm concentration and semen volume. Sperm concentration was inversely correlated with semen volume. Sperm motility and duration significantly correlated with total weight. Total length significantly correlated with the duration of motility and total weight. In conclusion, these characteristics represent a valuable baseline dataset for establishing a semen quality standard and provide background information that may be useful for assisted breeding programs in this species.     

Optimization of sperm irradiation protocol for induced gynogenesis in Siberian sturgeon,Acipenser baerii

The great diversity of optimal UV irradiation doses are used for DNA inactivation in fish sperm forcing authors to repeat optimization of irradiation treatment every time. Analysis of sperm UV irradiation protocol for induction of gynogenesis showed the importance of sperm UV light absorption estimations. The UV absorption investigation in Siberian sturgeon sperm showed average extinction coefficient 7.69 × 10^?8 ± 1.04 × 10^?8 cm². It is resulted in high heterogeneity of UV irradiation of undiluted sperm samples. Therefore, it is strongly suggested to specify doses only with defined concentration of spermatozoa; otherwise, the difference in absorbance level between samples can bring a significant error to optimal UV dose estimation. This was confirmed by UV-irradiated sperm motility investigation. Results of motility investigation of UV-irradiated sperm revealed high sensitivity of Siberian sturgeon spermatozoa motion mechanisms to UV irradiation, with complete loss of motility after homogeneous UV irradiation at doses above 2,000 J/m². Partial gynogenesis was conducted using diluted and undiluted sperm. Ploidy level of hatched larvae was estimated by flow cytometry. Percentage of haploid hatched larvae revealed sperm DNA inactivation efficiency. The highest percentage of haploid putative gynogenotes 19.67 ± 4.19 % was obtained at UV irradiation dose 200 J/m² with sperm diluted to 1:4.     

Effect of ascorbic acid supplementation on zootechnical performance,haematological parameters and sperm quality of lebranche mullet Mugil liza

The feed provided to breeding fish is one of the main factors influencing the quality of fish gametes. This study was carried out to evaluate the influence of ascorbic acid on growth, haematological parameters and sperm quality of Lebranche mullet males (Mugil liza). Six diets with different levels of ascorbic acid (0; 53; 107; 216; 482 and 708 mg/kg) were tested in triplicate for 75 days. During spermiation (first gonadal maturation), 144 individuals (205.7 ± 11.5 g and 25.7 ± 0.4 cm) were randomly distributed in 18 experimental tanks. Growth parameters were evaluated at the beginning and end of the experiment. Fish blood was collected to analyse glucose, total protein and erythrocyte count (EC) (n = 9). Fish (n = 12) from each treatment were euthanized to determine hepatosomatic index (HSI) and gonadosomatic index (GSI). Semen was collected to evaluate spermatic density, cell membrane integrity and sperm motility. No difference (p > .05) was found on growth parameters, GSI, HSI and total protein. However, EC was lower in fish fed without ascorbic acid (the control group). Ascorbic acid supplementation provided positive effects on sperm characteristics. Fish from treatments with 53 and 107 mg/kg presented the best results for motility time (133.30 ± 4.25 and 135.00 ± 2.77 respectively). Treatments with 107, 216 and 708 mg/kg provided the best results for motility rate (92.0 ± 2.9%, 93.0 ± 5.8% and 93.0 ± 5.8% respectively). Supplementation with 107 and 216 mg/kg provided the best results for plasma membrane integrity (70.12 ± 9.10% and 76.3 ± 3.1% respectively). Lower spermatic density was observed in fish without ascorbic acid supplementation, although no difference was found in sperm density among the treatments with ascorbic acid (p < .05). Considering these results, supplementation of dietary ascorbic acid between 107 and 216 mg/kg optimizes the spermatic quality in males of lebranche mullet.     

Comparative gonad histology and semen quality of normal (XY) and neo‐males (XX) of Atlantic salmon (Salmo salar)

With an overarching objective of improving the hatchery production of Atlantic salmon (Salmo salar) all‐female progeny, this study comparatively evaluated the reproductive parameters between normal (genotype XY) and neo‐males (genotype XX). Four normal (XY) and seven neo‐ (XX) males, from the same brood stock, distinguished by their ability to or lack of expressing semen, respectively, were comparatively evaluated. The left testicular lobe was used for histomorphometric analyses, while the right for semen collection and sperm quality analyses. Histomorphometric observations revealed that neo‐male testes are irregularly shaped, and have poorly formed seminiferous ducts, higher proportions of interstitial tissue and lower gonadosomatic index (p < .05). In addition, hypertrophied and cyst forming Sertoli cells were found in these individuals which collectively appear to form a physical barrier, precluding the semen collection by standard stripping techniques and reducing sperm quality. Particularly, semen motility (80.69 ± 2.4% and 57.2 ± 36.5% for XY and XX respectively) and duration of motility (99.31 ± 28.03 s and 66.84 ± 23.83 s for XY and XX respectively) of neo‐males were most compromised (p < .05). Interestingly, the TUNEL assay indicated no signs of apoptotic tissue suggesting that the histological differences may relate to delayed physiological/sexual maturity of neo‐males.     

Milt quality and spermatozoa morphology of captive Brycon siebenthalae (Eigenmann) broodstock

In order to develop artificial reproduction in freshwater fish for potential species to be developed in South American aquaculture, milt quality and sperm morphology were studied in yamú (Brycon siebenthalae) under captive conditions during the natural middle spermiation period. The volume of milt collected for each male was 1.8±1.2 mL and the sperm concentration was 13.9±4.0 × 10⁹ spermatozoa mL^?1. Spermatocrit (41.5±10.8%) was positively associated (r²=0.30) with sperm density calculated using a corpuscle counting chamber. Sperm motility was 88±9% and the average duration of forward motility was 41±7 s. Fertilization rate was 84±8% and there was no association between this trait and sperm motility (r²=0.009) or with sperm density (r²=0.073). These results suggest that captive B. siebenthalae broodstock can be reproduced successfully.     

Hybridization between Haliotis rufescens and Haliotis discus hannai: evaluation of fertilization,larval development,growth and thermal tolerance

Two introduced abalone species are currently produced in Chile, red abalone Haliotis rufescens and Japanese abalone Haliotis discus hannai. However, red abalone accounts for 99% of total production, while the Japanese abalone has not adapted well to Chilean coastal waters. This study reports the hatching, growth and thermal tolerance performance in interspecific hybrids produced between red (R) and Japanese (J) abalone. Our results show that egg age and sperm concentration were critical factors to produce hybrids. The cross R♀ × J♂ showed a fertilization rate of 55.3 ± 3.5% using 20‐min‐old eggs and sperm concentrations of 14 × 10⁶ cells mL^?1, while the reciprocal cross (J♀ × R♂) was not successful. Further, larval development stages were similar in RR, JJ and RJ hybrid abalones. Among the experimental trials, settlement rate varied from 12.3% to 18.6% and final survival from 20.1% to 31.7%, being the RJ hybrid rates intermediate between parental species. The final shell lengths were similar between RR and RJ hybrids, but significantly higher in JJ abalones. In addition, thermal tolerance was ascertained due its pivotal role for the abalone physiology. Thus, RJ hybrids showed the highest HSP70 gene expression and offers new possibilities to expand Chilean abalone production in warm waters zones.     

Production and storage of sperm from the black sea bass Centropristis striata L

Black sea bass Centropristis striata L. are protogynous hermaphrodites that develop and spawn as females before changing sex to male. Since all fish eventually become males, determining the relationship between sperm production, sperm quality and seasonal changes in plasma levels of testosterone (T) and 11‐ketotestosterone (11‐KT) could be useful for identifying appropriate males to maintain as broodstock. Milt and blood samples were collected three times during an 8‐week spawning season. Milt volume (3.5±0.76 mL kg^?1), sperm density (3.2 × 10⁸± 0.31 cells mL^?1), sperm production [11 × 10⁸±3.4 cells kg^?1 body weight (BW)] and sperm motility (80±0.6%) were at their highest during the first sampling interval and coincided with the highest 11‐KT levels (1.0± 0.11 ng mL^?1). All of the sperm indices decreased to their lowest levels during the final 3 weeks of the study. Sperm viability was highly correlated (adjusted R²=0.84) with sperm motility. Sperm cryopreserved in modified Mounib's extender (MME) had the highest post‐thaw motility compared with two other extenders. Post‐thaw motility of sperm cryopreserved in MME was not different from fresh after 90 days of storage. There was no difference in fertilization rates between fresh (69±2.4%) and post‐thaw (67±4.1%) sperm samples taken from the same male or among males. These results demonstrate that the quality of black sea bass spermatozoa is higher earlier in the spawning season and that acceptable post‐thaw fertilization rates can be obtained from cryopreserved sperm.     

Preservation of black sharkminnow,Labeo chrysophekadion (Bleeker, 1849) spermatozoa

The objective of this study was to investigate the effects of extenders and storage time on motility, viability and fertilization of preserved black sharkminnow, Labeo chrysophekadion spermatozoa. Sperm were diluted 1:3 in one of five extenders: modified Cortland solution (MC); Hanks' balanced salt solution (HBSS); 0.9% sodium chloride (NaCl); Kurokura solution (KU); and modified extender, and undiluted sperm samples were used as control and stored at 4°C for 5 days. Motility, viability and fertilization rates were evaluated every day. After a storage time of three days, the highest motility, viability and fertilization rates (61.27 ± 2.26%, 58.60 ± 2.29% and 40.58 ± 0.57, respectively) were achieved with sperm diluted with modified extender. Motility, viability and fertilization rates decreased significantly (P < 0.05) with increasing storage time in all treatments. In addition, this study found that motility, viability and fertilization had a positive significant correlation (P < 0.01). The results indicate that isotonic extender is suitable for the short‐term preservation of black sharkminnow spermatozoa.     

Cryopreservation of sperm in farmed blacklip abalone (Haliotis rubra Leach, 1814)

This study developed a technique of sperm cryopreservation using liquid nitrogen (LN) vapour in farmed blacklip abalone Haliotis rubra through evaluating the following five key factors: (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature; (3) thawing temperature; (4) sperm to egg ratio and (5) sugar addition, using sperm motility or fertilization rate as quality assessment indicators. The results demonstrated that 6% dimethyl sulfoxide (DMSO) was the best single CPA for sperm cryopreservation in this species. The highest post‐thaw sperm motility was achieved when sperm were exposed to LN vapour for 10 min at 5.2 cm above the LN surface and thawed at 60°C and recovered at 16°C in seawater baths. Post‐thaw sperm motility was found to be significantly higher when 6% DMSO was used in combination with 1% or 2% glucose than 6% DMSO alone. Further evaluation of fertilization rate between these CPAs showed that 6% DMSO+2% glucose achieved the highest fertilization rate of 70% at a sperm to egg ratio of 10 000:1.     

Effect of Hybrid Abalone,Haliotis discus hannai × Haliotis discus discus,Cultivation on the Carbon Cycle: Carbon Source/Sink

Respiration, calcification, and bio‐deposition of hybrid abalone, Haliotis discus hannai × Haliotis discus discus, fed on different foodstuffs have been measured to evaluate the effect of hybrid abalone culture on carbon source/sink in coastal areas. Fed with Laminaria japonica, Undaria pinnatifida, Gracilaria lemaneiformis, U. pinnatifida, and Ulva pertusa, alternated mutually, the carbon bio‐deposition rate of hybrid abalone was 24.29 ± 6.39, 65.40 ± 10.55, 21.48 ± 5.99, and 29.28 ± 6.47 µg/g/h, respectively. Hybrid abalone fed on U. pinnatifida had a higher carbon bio‐deposition rate compared to that fed on other foodstuff (P < 5%). Rate of CO₂ released by respiration of hybrid abalone fed on the experimental foodstuff was 24.53 ± 8.57, 32.73 ± 7.99, 29.31 ± 6.39, and 33.67 ± 12.37 µg/g/h, respectively. Results indicated that calcification presented less relationship with body weight type of the foodstuff. The rate of CO₂ released by calcification into seawater and atmosphere was 2.77 ± 1.89 and 6.53 ± 3.36 µg/g/h, respectively. The total rate of CO₂ released because of bio‐deposition, respiration, and calcification processes was 16.19 ± 4.67 µg/g/h, while the total rate of carbon sequestered in shells and tissues was 8.94 ± 2.07 µg/g/h. The study revealed that hybrid abalone culture is a source of CO₂.     

Transport and cryopreservation of sperm of the common snook, Centropomus undecimalis (Bloch)

Sperm were collected in Florida from wild common snook, Centropomus undecimalis (Bloch), and were shipped to Louisiana State University for analysis and cryopreservation. Threshold activation of sperm (10% motility) occurred at 370 mOsmol kg^?1, and complete activation occurred at 680 mOsmol kg^?1. These values were significantly different. Sperm samples stored at 1°C in Hanks' balanced salt solution (HBSS) or in 0.6% NaCl solution at 200 mOsmol kg^?1 retained motility for as long as 22 days. Mean motility remained above 50% for 9 days for sperm stored in HBSS and for 7 days for sperm stored in NaCl solution. Sperm exposed to 5% dimethyl acetamide (62±10%; mean±SD), 10% dimethyl sulphoxide (DMSO) (39±16%), 5% glycerol (26±5%) or 10% glycerol (6±2%) for 30 min had significantly lower motility than did unexposed sperm (89±9%). When used as a cryoprotectant, samples frozen with 5% or 10% DMSO or 5% methanol had significantly higher post‐thaw motility than did samples frozen with other cryoprotectants. Sperm cryopreserved with 10% DMSO (38±12%) had significantly higher post‐thaw motility than did sperm cryopreserved with 15% DMSO (19±10%) or 20% DMSO (4±4%). There were no significant differences in hatch rates of eggs fertilized with fresh sperm (54±29%) or cryopreserved sperm (41±35%). Survival to first feeding was not different between fish produced with fresh sperm (37±30%; range, 0–86%) or with thawed sperm (24±29%; 0–77%). Transport of sperm to a cryopreservation laboratory and back to a hatchery for thawing and use enabled collaboration between groups with specific expertise and provides a model for the application of cryopreservation by transport of fresh and frozen samples.     

High‐throughput Cryopreservation of Sperm from Sex‐reversed Southern Flounder,Paralichthys lethostigma

The Southern flounder, Paralichthys lethostigma, is a valuable aquaculture fish with established markets in the USA. All‐female production in this species is an important technology for aquaculture because the females usually have body sizes twice those of males at the same age, and sex‐reversed males (genotypic XX neomales) are used for all‐female production by crossing with genetically normal females. However, sperm volume from the neomales is usually small (<0.5 mL) and limits their application for all‐female fish production. Cryopreservation of sperm from these sex‐reversed neomales will provide access on demand with increased efficiency to extend the application of neomales. The goal of this study was to develop a protocol for cryopreservation of sperm from the Southern flounder by using an automated high‐throughput processing system. The objectives were to: (1) determine the effect of osmolality on activation of sperm motility; (2) evaluate the effect of extender solutions on sperm motility capacity; (3) evaluate the acute toxicity of cryoprotectants (dimethyl sulfoxide [DMSO], propylene glycol, and polyethylene glycol) on sperm motility, and (4) estimate the effect of cooling rate on sperm cryopreservation and post‐thaw fertilization. Sperm motility was activated when osmolality was 400 mOsmol/kg or higher. Of the three extender buffers tested, HEPES4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) at 300 mOsmol/kg resulted in better protection for sperm motility than did Hanks' balanced salt solution and Mounib solution at 300 mOsmol/kg during 7 d of refrigerated storage. After 30 min equilibration with the cryoprotectant of 15% DMSO, sperm motility was 24 ± 21% (fresh sperm motility without any cryoprotectants was 42%). After cooling at a rate of 20 C/min, post‐thaw sperm motility was 8 ± 5% and fertilization was 63 ± 40% evaluated at the 32–64 cell stage (5 × 10⁵ sperm per egg). Overall, a protocol was developed for sperm cryopreservation in the Southern flounder with high‐throughput processing, which provides a tool to preserve the valuable genetic resources from neomale flounders, and enables germplasm repository development for the Southern flounder.     

Effect of the addition of six antioxidants on sperm motility,membrane integrity and mitochondrial function in red seabream (<Emphasis Type="Italic">Pagrus major</Emphasis>) sperm cryopreservation

The present study was to evaluate the effects of six antioxidants on frozen-thawed sperm motility, viability, membrane integrity and mitochondrial function in red seabream (Pagrus major) by computer-assisted sperm analysis system and flow cytometry, respectively. All the parameters tested in this study were determined using one-way ANOVA and identified using the SNK test (P < 0.05). The results demonstrated that on the first day, the highest motility and longevity occurred in 100 mM trehalose (78.34 ± 3.41 %, 29 ± 4.00 days) and 50 mM taurine (77.46 ± 1.54 %, 29.33 ± 4.04 days), followed by 25 mM vitamin C (79.03 ± 5.37 %, 17 ± 1.00 days), 25 mM vitamin E (69.64 ± 1.64 %, 27.67 ± 1.53 days) and 25 mM vitamin A (78.89 ± 2.81 %, 9.33 ± 1.53 days), which were all higher than frozen-thawed sperm without antioxidant (control) (66.80 ± 5.55, 5.67 ± 1.15 days). Especially, the percentages of class A sperm with the addition of 100 mM trehalose (40.39 ± 5.20 %) and 50 mM taurine (37.78 ± 3.22 %) were significantly improved compared to the control (19.63 ± 5.44 %). The viability of all groups on the third and sixth day showed a similar trend. Moreover, during the 4 °C storage process, the decrease of frozen-thawed sperm motility was closely associated with the decrease in membrane integrity and mitochondrial function. In conclusion, the present study indicated that antioxidant (100 mM trehalose and 50 mM taurine) provided the most pronounced protective effect in improving frozen-thawed quality of red seabream sperm. The addition of antioxidant may be capable of scavenging the ROS generated during the cryopreservation process and 4 °C storage.     

Effect of storage time and cryoprotectant concentrations on the fertilization rate and hatching rate of cryopreserved sperm in red seabream (Pagrus major Temminck & Schlegel, 1843)

This study examined the effects of storage time and cryoprotectant concentrations on the post‐thaw sperm of red seabream, Pagrus major. Sperm treated with 12%, 15%, 18% and 21% DMSO were cryopreserved for 10, 30, 60 and 360 days, and fertilization and hatching rates were analysed. For all groups, there were no differences in the fertilization rates and hatching rates between sperm cryopreserved for <60 days and fresh sperm (98.8±0.8%, 96.4±1.3%). However, for sperm cryopreserved for 360 days, both fertilization rates (88.6±3.0% to 7.0±1.9%) and hatching rates (79.4±7.2% to 3.3±0.8%) decreased drastically. Furthermore, the cryoprotectant concentrations affected sperm quality significantly (P<0.05). When cryopreserved for 360 days, sperm treated with 15% DMSO obtained the best results compared with other concentrations. We suggest that 15% DMSO may be an effective cryoprotectant for long‐term sperm cryopreservation of red seabream.     

An investigation on the gamete quality of Black Sea trout (Salmo trutta labrax) broodstock fed with mealworm (Tenebrio molitor)

In this study, the effect of combined feeding Black Sea trout (Salmo trutta labrax) broodstock with commercial feed and mealworm larvae (Tenebrio molitor) for 50 days before the breeding period in order to evaluate the gamete quality. While the control group was fed with commercial feed, mealworm larvae were given as an additional protein source 2 days a week and 3 days a week to the experimental groups formed from female and male individuals. In addition to the growth parameters, the number of eggs (number/individual) and the egg diameter (mm), sperm volume (ml), density of spermatozoa (×10⁹ cell/ml), total motility (%), progressive motility (%) and average curvilinear velocity (VCL, µm/sec) values were determined. At the end of the study, the highest live weight gain was found as 46.2 g in the control group of male. The egg diameters were 4.3 ± 1.8 mm and 4.5 ± 1.4 mm in the worm treatment groups fed twice and three times with mealworm respectively. As a result, it was determined that feeding fish with mealworm larvae as an addition to the commercial diet in female individuals did not affect the amount of eggs (p > .05), but the egg diameters were significantly smaller in the control group than the mealworm groups (p < .05). In spermatological characteristics, only the amount of sperm was different between the groups (p < .05); all other parameters were found to be similar to each other with no statistical differences (p > .05).     

Fertilization by refrigerator stored sperm of the Indian major carp, Labeo calbasu (Hamilton, 1822)

This study reports on the spermatological properties, and on the development of a protocol for refrigerator storage (4°C) of Labeo calbasu (Hamilton, 1822) sperm for artificial breeding. Volume, motility, concentration and pH of the freshly collected sperm were 2.21 ± 0.53 (μL g^?1 of fish weight) (mean ± SD), 95 ± 1 (%), 1.93 ± 0.44 × 10⁹ (cells mL^?1) and 7.56 ± 0.17 respectively. Sperm activation was evaluated at different osmolalities of NaCl solution, and motility ceased completely when osmolality of the extender was ≥287 mOsmol kg^?1. Sperm retained motility for 24, 72 and 108 h, after refrigerator storage when sperm were undiluted, suspended in Alsever's solution and suspended in Alsever's solution containing 5% methanol respectively. Fertilization rate of fresh eggs with sperm stored at 4°C in Alsever's solution and Alsever's solution containing 5% methanol was 77% and 60% with a hatching rate of 60% and 43% respectively. The fertilization and hatching success of the stored sperm suggests potential to use refrigeration for transporting genetic material to hatcheries for artificial breeding of L. calbasu in Bangladesh.     

二倍体泥鳅与大鳞副泥鳅及杂交F1精子结构与活力

二倍体泥鳅与大鳞副泥鳅杂交能够顺利获得杂交后代,其正反交核型都为（2n=49）,但杂交后代精巢表现出明显的发育迟缓现象,杂交泥鳅自交与回交实验表明,其雄性不可育。为了解二倍体泥鳅和大鳞副泥鳅杂交F₁雄性不育的原因,实验通过计算机辅助精子活力分析系统（CASA）、光学显微镜、扫描电镜、透射电镜、流式细胞技术分别对二倍体泥鳅（DD）、大鳞副泥鳅（PP）、其正反杂交F₁（DP）和（PD） 4种泥鳅精子的形态结构与活力进行了分析。结果显示,激活30 s时,DD和PP的精子运动率分别为76.50%±0.70%和75.17%±8.60%,极显著高于DP （3.65%±1.75%）和PD （2.68%±0.63%）,且杂交泥鳅的精子的平均运动速度（VAP）、平均曲线速度（VCL）、平均直线运动速度（VSL）等运动参数也极显著低于DD和PP,说明杂交泥鳅精子的活力极其低下。应用流式细胞技术对4种泥鳅精巢内细胞进行倍性鉴定,发现DD和PP精巢内大多为1N精子,而DP和PD精巢内除了正常单倍体精子外,还有大量的2N和4N以及少量的8N细胞。通过扫描电镜和透射电镜...     

Refrigerated storage and cryopreservation of sperm of red drum, Sciaenops ocellatus L.

To aid in artificial spawning of sciaenid fishes, the present authors developed techniques to collect, handle and cryopreserve sperm from red drum, Sciaenops ocellatus L. Sperm were collected by removing and slicing the testis, and adding Hanks' balanced salt solution (HBSS) or NaCl solution (each at 200-400 mOsm kg^?1) as an extender. Sperm were activated with 800 mOsm kg^?1 artificial sea water (ASW) to characterize motility. Sperm reached maximum motility (highest percentage motility observed for that sample) within 8 ± 1 s (mean ± SD) and remained at maximum motility for 33 ± 4 s. Sperm were exposed to graded osmotic pressures of ASW (8-800 mOsm kg^?1) to determine the range of osmolalities that elicited motility. Threshold activation (defined as ～10% motility) occurred at 351 ± 4 mOsm kg^?1 and complete activation occurred at 539 ± 2 mOsm kg^?1. Sperm stored at 200 mOsm kg^?1 retained motility for up to 13 days. Dimethyl sulphoxide (DMSO) was used as a cryoprotectant at concentrations ranging from 7.5% to 15% (v:v) in HBSS (200 mOsm kg^?1). There were no significant differences among post-thaw motilities of sperm cryopreserved at any concentration of DMSO. Sperm thawed on the benchtop at 21°C had lower post-thaw motility than did sperm thawed at 10, 20, 30, 40, 50 or 60°C in a water bath.