Identification of calcium-binding proteins in fish seminal plasma

Calcium ions play an important role in the activation of fish sperm movement. The mechanism of their binding in semen is still unknown. The goal of this study was the development of a method for identifying calcium-binding proteins in fish seminal plasma. Two methods of calcium-binding proteins detection were tested with the use of Quin2 and Stains-all dyes. The first method was useful for the identification of calcium-binding proteins of fish seminal plasma. It consisted of proteins separation using SDS–PAGE, transfer on PVDF membrane, incubation with CaCl₂, staining with Quin2 and illumination with UV light to reveal calcium-binding protein bands. Using Quin2 allowed the detection of calcium-binding proteins with low and high molecular weight. Electrophoretic species-specific profiles of calcium-binding proteins were identified in the seminal plasma of carp, whitefish, roach, brook trout, brown trout and rainbow trout. Staining of calcium-binding proteins with Quin2 is a quick and safe method, allowing the identification of calcium-binding proteins in fish semen.     

Physiological and Biochemical Determination of Rainbow Trout,Oncorhynchus mykiss,Semen Quality for Cryopreservation

In rainbow trout, Oncorhynchus mykiss, parameters to determine semen fitness for cryopreservation and quality control of cryopreserved semen were investigated. The following parameters can be used to evaluate semen fitness for cryopreservation as they are statistically significant (P < 0.01) correlated to the post-thaw fertilization rate: motility rate of fresh semen (y = 4.996x - 0.0958x² + 0.0006x³ - 5 1.7363); sperm velocity of fresh semen (y = 6.741x - 0.036x² - 268.37); seminal plasma osmolality (y = 0.539x - 125.59); seminal plasma pH (y = -82.768x + 728.133); seminal plasma triglyceride levels (y = 0.069x + 29.863); seminal plasma ß-D-glucuronidase activity (y = -1.112x + 0.0058x² + 82.229); seminal [lasma lactate dehydrogenase activity (y = -0.096x + 0.00006x² + 583.80); spermatozoan acid phosphatase activity (y = -132.51x + 126.38x² + 66.48); spermatozoan adenylate kinase activity (y = 3.474x + 4.925). Quality of deep-frozen semen can be evaluated by motility parameters (P < 0.01): frozen/thawed semen motility rate and post-thaw fertilization rate: y = 1.943x + 28.002; sperm velocity and post-thaw fertilization rate: y = 0.8812x - 0.0059x² + 24.9686.     

Characterization of rainbow trout milt collected with a catheter: semen parameters and cryopreservation success

Collection of fish milt by stripping risks the danger of milt contamination by urine. This may seriously influence milt characteristics and quality, including usefulness for cryopreservation. Urine contamination of milt may be avoided by using a catheter for sperm collection. The objectives of this study were to provide basic characteristics of milt collected with a catheter, to test the usefulness of this milt for cryopreservation, and to correlate characteristics of fresh and cryopreserved semen with sperm fertility rates. Milt from 25 rainbow trout Oncorhynchus mykiss (Walbaum) males were used. All samples were cryopreserved using the pellet method within 1 h of collection, using 0.6 m sucrose and 10% dimethyl sulphoxide (DMSO) as an extender. Catheterization resulted in semen of very good motility (> 90% motile spermatozoa) and high fertilization rates after cryopreservation (mean fertilization rate 81.8 ± 13.3% of control, at a sperm/egg ratio of 2.4 ± 0.3 × 10⁶). Osmolality of seminal plasma and concentrations of sodium, potassium and magnesium ions had low variability, which suggests that they are important for creating a stable environment for sperm storage in the sperm duct. Higher variability of certain seminal plasma characteristics, such as protein concentration and antiproteinase activity, suggests that these characteristics are related to individual semen features of particular males. A strong correlation of seminal plasma zinc concentration with protein concentration may reflect an importance of zinc in semen biology. Cryopreservation caused a significant release of protein and acid phosphatase from spermatozoa. Our results did not reveal any single characteristic of semen collected by catheter that could be used as a powerful predictor of cryopreservation success, presumably because all samples were of high quality.     

Motility Parameters of Rainbow Trout (<Emphasis Type="Italic">Oncorhynchus mykiss</Emphasis>) Spermatozoa in Relation to Sequential Collection of Milt,Time of <Emphasis Type="Italic">Post-mortem</Emphasis> Storage and Anesthesia

The present study investigated the effects of sequential collection of milt, time of post-mortem storage and anesthesia on rainbow trout (Oncorhynchus mykiss) sperm motility parameters (using computer-assisted sperm analysis – CASA) as well as seminal plasma osmolality and sperm concentration. The post-mortem storage and time of anesthesia altered motility characteristics of rainbow trout sperm to different extents. The moderate impact of time of anesthesia was manifested in a shortened duration of sperm motility after 10 min exposure of fish to anesthetic. The prolonged post-mortem storage (≥40–60 min), in addition to lowering sperm motility duration, also significantly influenced sperm motility parameters, such as sperm velocities, percentage of motile sperm and sperm trajectory parameters. These results clearly demonstrate that when milt from sacrificed fish is used for sperm motility studies, the time of post-mortem storage significantly alters sperm motility characteristics. Since sperm motility rate and swimming velocity could predict fertilizing ability, detrimental effects of prolonged post-mortem storage may lead to reduced fertilization success. Sperm concentration and seminal plasma osmolality were lower in the first fractions and increased with successive collections of milt. It suggests the presence of urine contamination of the first milt fractions which were collected by stripping. Therefore, testing of sperm concentration and/or seminal plasma osmolality should be mandatory while handling stored milt.     

Quality of testicular semen of the African catfish Clarias gariepinus (Burchell, 1822) and its relationship with fertilization and hatching success

Quality differences of testicular semen of the African catfish, Clarias gariepinus, and their influence on fertilization and hatching success were investigated. In accordance with an earlier study, two semen types of the African catfish were distinguished according to testicular maturity stage. Semen type I derived from males with white mature testes whereas type II semen derived from males with grey, partly mature testes. Semen volume, sperm cell concentration and seminal plasma pH was significantly higher in type I semen than in type II semen, while sperm motility was similar. Similar fertilization percentages were obtained with semen type I and semen type II. However, the hatching percentage was higher and the percentage of deformed hatched larvae was lower for type I semen. There were significant (P<0.01) positive correlations between sperm motility and fertilization percentage, seminal plasma pH and hatching percentage and a negative correlation between seminal plasma pH and percentage of deformed larvae. Therefore seminal plasma pH and sperm motility are useful to predict semen quality of the African catfish.     

Variation in semen production of farmed Atlantic salmon and rainbow trout

The variation in semen production among farmed Atlantic salmon (Salmo salar) and rainbow trout (Salmo gairdneri) has been studied. Both species were stripped at weekly intervals, the Atlantic salmon four times and the rainbow trout three times.The individual variation in volume of semen was very high, particularly in rainbow trout. The total volume of semen obtained was 137 ml (20 ml/kg body weight) in Atlantic salmon and 23 ml (5 ml/kg body weight) in rainbow trout. The intraclass correlation for volume of semen was estimated at 0.73 in Atlantic salmon and at 0.59 in rainbow trout. The correlations between volume of semen and body size (weight and length) were all positive. They were all significant and medium in Atlantic salmon whereas in rainbow trout they were all low and significant only for volume of semen at first stripping.The number of males needed to supply the Norwegian fish farming industry with semen is discussed. It is concluded that the possibility of disseminating genetic improvement throughout the whole population of farmed Atlantic salmon and rainbow trout by transport of semen from selected males is considerable.     

Effects of season and dietary ascorbic acid on some biochemical characteristics of rainbow trout (Oncorhynchus mykiss) semen

During the reproductive season, rainbow trout spermatozoa are stored in the sperm ducts for several months. There is no sperm production at this time since spermatogenesis is completed before spawning. To leam more about characteristics of semen during such a long storage, we analyzed changes in protein concentrations, anti-proteinase activity in seminal plasma and sperm aspartate aminotransferase activity during an extended reproductive period during which fish were fed diets supplemented with various ascorbic acid concentrations. Seminal plasma protein concentration and anti-proteinase activity declined toward the end of the reproductive season. These phenomena may be related to oncoming proteolytic events leading to degradation of the sperm. Protein concentrations and anti-proteinase activities were strongly correlated within groups of different ascorbic acid supplementations and several sampling dates (r=0.6–0.9 in most cases, p<0.05). Ascorbic acid deficiency resulted in a decrease in both parameter levels as compared to levels in groups with vitamin C supplement (p<0.08). Deficiency also resulted in lower stimulation of aspartate aminotransferase by an exogenous pyridoxal 5-phosphate in comparison to fish fed vitamin C-supplemented diets (p<0.05). These results support earlier studies suggesting a protective role of ascorbic acid toward maintaining sperm quality.     

Some properties of bream Abramis brama L. sperm and its cryopreservation

Concentration and motility of spermatozoa, total protein content and its electrophoretic profile, glucose content, activity of aspartate aminotransferase (AspAT) and acid phosphatase (AcP) were assessed in 18 samples of semen from common bream Abramis brama L. males, which were hormonally stimulated to spermiation. Also, milt pooled from four donors was cryopreserved as pellets in vapours of liquid nitrogen (?80 °C) using four extenders (each with or without the addition of hen egg yolk). Mean spermatozoa concentration was 11.68 × 10⁹ mL^?1, and mean spermatozoa motility was about 60%. Protein content in seminal plasma was 2.08 mg mL^?1; both PAGE and SDS–PAGE showed considerable heterogeneity of protein fractions. Mean glucose content was over 11 mg%. AspAT and AcP activities were detected in both seminal plasma and spermatozoa extracts. As calculated to 1 × 10⁹ spermatozoa, AcP and AspAT activities were almost sixfold and 46-fold higher in spermatozoa than in seminal plasma respectively. In the best variant, cryopreservation attempts resulted in 66.6% of eyed embryos (compared with control fertilization) obtained after fertilization of eggs with cryopreserved semen.     

Fertility of teleost semen as affected by dilution and storage in a seminal plasma-mimicking medium

Fertility trials were run with fish semen diluted in Fish Extender #6 and stored for up to 24 h at 10°C. After 30 min of storage, rainbow trout (Salmo gairdneri) and northern pike (Esox lucius) semen diluted 1:255 maintained control values of fertility. Brown trout (Salmo trutta) and pink salmon (Oncorhynchus gorbuscha) semen diluted 1:511 also maintained control values of fertility after 30 min of storage. As storage times increased, the dilution ratios that maintained control values of fertility decreased. After 24 h of storage, rainbow trout semen diluted 1:31 and brown trout semen diluted 1:63 maintained control values of fertility. A field trial with rainbow trout semen showed that semen diluted 1:8 maintained control values of fertility after 30 min of storage. Non-motile sperm cells did not significantly (P>0.05) interfere with the fertilizing ability of motile sperm cells. Calculations from the dilution trials on the number of inseminated sperm cells showed that approximately 4000–169 000 motile sperm cells/egg were required to maintain control values of fertility.     

Chemical composition and osmolality of seminal fluid of Acipenser persicus; their physiological relationship with sperm motility

Determination of semen quality is necessary to understand the basic biochemical processes occurring during motility of sperm and during fertilization to evaluate the reproductive ability of different fish species and to create an optimal environment for storage of spermatozoa; in this regard less information is available for Acipenseridae compared with Cyprinidae and Salmonidae. The aim of the present study is to determine chemical composition and osmolality of seminal fluid and their relationship with sperm motility in Acipenser persicus. The results obtained show that sodium (Na⁺), chloride (Cl^?) and potassium (K⁺) were predominant ions in the seminal plasma and the average of osmolality of seminal plasma was 82.56 mOsm kg^?1. The higher chemical contents and osmolality compared with other sturgeon species reveal species‐specific characteristics and high secretory activity of spermatic duct in A. persicus. Significant positive correlations were observed between osmolality‐Cl^?, Na⁺‐osmolality and Na⁺–Cl^? (P<0.05, P<0.001 and P<0.05 respectively). But statistically significant correlation was not observed between seminal plasma parameters and sperm motility. Probably, the Na⁺ and Cl^? are the main electrolytes playing a major role in maintaining the osmolality of the seminal plasma and the viability of the spermatozoa in vivo.     

Proteins of seminal fluid and spermatozoa in the trout (Oncorhynchus mykiss): Partial characterization and variations

The protein composition of seminal fluid, blood serum, sperm plasma membrane and flagellum of rainbow trout were analysed by SDS-polyacrylamide gel electrophoresis. Immunological identity between proteins of the 2 fluids and sperm components was studied using crossed immunoelectrophoresis, rocket immunoelectrophoresis and immunoblotting. Results indicate that many seminal proteins are antigenically-related to serum proteins, proteins of sperm origin are present in seminal fluid in varying amounts, depending on the animals and sampling time, and several serum-like seminal proteins are bound to spermatozoa. Lipoproteins were isolated from seminal fluid (mean level: 33 μg/ml) and characterized. They were identified as being HDL-like lipoproteins. A possible physiological role is proposed for these seminal lipoproteins.     

Arginine effects on biochemical composition of sperm in rainbow trout,Oncorhynchus mykiss

The present study investigated the effect of arginine on seminal plasma composition in rainbow trout. Male rainbow trout broodstocks (2500 ± 200 g) were fed five practical diets (each consisting of three triplicates) supplemented with Arginine at 0.50%, 1.50% and 2.00%. The control group were fed without arginine. Broodstock feeding lasted for 90 days, and then fish semen was sampled. Results indicated no significant differences in LDH, ALP, Fe²⁺ and phosphorous content among the different treatments. The lowest levels of AST and ALT and the highest levels of Ca²⁺ and Mg²⁺ ions were observed in the treatment fed with 1.50% arginine, which showed significant differences from other treatments (P < 0.05). Moreover, the amount of Cl^?, Na⁺ and K⁺ ions was significantly increased in the seminal plasma in fish fed diets containing arginine in comparison with the control. As the amount of arginine was increased, the levels of uric acid became significantly greater in contrast to urea and glucose levels. The highest amounts of cholesterol, fructose and total protein were observed in treatments fed on 2.00%, 0.50% and 1.00% arginine, respectively, showing significant differences from other treatments (P < 0.05). The highest pH value was assayed in the 1.50% arginine treatment. Results indicated that arginine had a potential efficacy on semen quality in rainbow trout broodstocks.     

添加氯化钾和蛋黄对虹鳟精液冷冻保存的效果

为筛选出更有效的虹鳟(Oncorhynchus mykiss)精液冷冻保存液,在以10％ 甲醇和0.3 M葡萄糖作为主要成分的基础上,分别或同时添加0.9 g/L氯化钾和10％ 蛋黄形成四种冷冻保存液,用于虹鳟精液冷冻保存试验,分别形成4个处理组:Ⅰ#(不添加氯化钾和蛋黄)、Ⅱ#(添加氯化钾)、Ⅲ#(添加蛋黄)和Ⅳ#(...     

Preservation of trout sperm in liquid or frozen stateFlüssig- und gefrierkonservierung von regenbogenforellensperma

A series of experiments to study the preservation of rainbow trout semen in the liquid and frozen state was carried out.For the preservation of liquid semen both undiluted and diluted semen was stored in total darkness at 20° or 4°C under a number of different gasses (air, O₂, O₂ + N₂ (1:1), N₂, CO₂, N₂ + H₂ + CO₂ (8:1:1)). The best results were obtained when storing semen in an undiluted state at 4°C under O₂ (hatching rate after 15 days was 80.6%, controls were 98.2%). Good results were also obtained under air (after 9 days 94.7% vs 94.9% for the controls). Anaerobic conditions, dilution or an increase in temperature all rendered less favourable results.For the purpose of cryopreservation a diluent was developed on the basis of the composition of rainbow trout seminal plasma. The semen was mixed with the diluent immediately before freezing at a ratio of 1:1 or 1:3. The pellet-technique (Nagase, 1964) was found to be more convenient and successful than freezing in straws. The pellets were thawed in a 1% NaHCO₃-solution. The hatching results after storage periods of 1–4 months were very variable (2.6–80.3%).     

Ovarian fluid pH enhances motility parameters of rainbow trout (Oncorhynchus mykiss) spermatozoa

All evidence to date suggest that sperm motility is the primary determinant of fertilization success in externally fertilizing fish species. Ovarian fluid, which comprises 10–30% of the total egg volume in salmonids, enhances sperm motility with respect to swimming speed, trajectory and the duration of movement. It was recently demonstrated that there is individual variability in sperm motility enhancing potential of ovarian fluid of particular females. In the present study we examined the effect of particular ovarian fluids collected from 31 females on the sperm motility parameters of one male of rainbow trout (Oncorhynchus mykiss) using computer-assisted sperm analysis (CASA). During our experiment we also monitored the pH of ovarian fluid. We found that particular fluids differed in the ability to activate spermatozoa; sperm remained immotile in four fluids and exhibited 50–100% motility in 27 samples. The percentage of motile sperm, velocity and duration of movement positively correlated with ovarian fluid pH (r² = 0.34–0.62). These data strongly suggest that the pH of the ovarian fluid is the primary determinant of sperm motility in rainbow trout under natural conditions of fertilization.     

Cryopreservation and transplantation of sexually immature gonads of rainbow trout

The objective of this investigation was to cryopreserve sexually immature gonads of rainbow trout (RBT). Gonads from juvenile male or female RBT were cryopreserved and stored in liquid nitrogen. Individual testes were thawed and surgically transplanted to isogenic male trout; individual ovaries were autografted. Four weeks following transplantation, all male fish were injected with salmon pituitary extract for 8–10 weeks. The transplanted testis was present in 5 of 8 animals in replicate 1 and in 3 of 6 animals in replicate 2. Fertility of sperm obtained from individual testes in replicate 2 was evaluated by in vitro fertilization. Sperm from the intact control testes had a fertilization rate of 95±3% (mean ± SEM), and sperm from transplanted testes had a fertilization rate of 78±7%. Ovaries that were frozen in liquid nitrogen, thawed and autografted to an ectopic site were not present at five months following surgery.     

圆口铜鱼精浆的理化性质分析

对圆口铜鱼(Coreius guichenoti)精浆离子和氨基酸成分及精液生理特性进行了检测分析。结果显示,圆口铜鱼精液pH值为7.3,呈弱碱性;精液浓度为39.7%,精子密度为5.3×10~9个/mL;精浆离子以Na~+含量88.7 mmol/L最高,其次是K~+,之后为Ca~(2+)、Mg~(2+)、Zn~(2+)、Cu~(2+);精浆水解氨基酸总量为2 872.69μmol/100mL,其中以脯氨酸含量最高,蛋氨酸、酪氨酸和组氨酸含量最低。该结果填补了圆口铜鱼繁殖生物学的相关数据,为圆口铜鱼规模化人工繁育提供了基础资料。     

Indicators of reproductive performance in rainbow trout Oncorhynchus mykiss (Walbaum) selected for high and low responsiveness to stress

A number of indicators of reproductive performance were assessed in rainbow trout divergently selected on the basis of their plasma cortisol response to a standardized stressor. For both male and female rainbow trout Oncorhynchus mykiss (Walbaum), body weight was significantly related to stress responsiveness. High‐responding (HR) females were significantly heavier than low‐responding (LR) females overall, and HR males were significantly heavier than LR males overall. There was no significant difference in the specific growth rate (SGR) of HR and LR female fish during the experimental period, whereas in contrast the mean SGR of HR males was significantly greater than that of LR males. The divergent confinement‐induced levels of blood cortisol in HR and LR groups were stable for more than 1 year after selection. There was no significant difference in the extent to which confinement stress reduced blood oestradiol‐17β levels in HR and LR females, despite a large difference in relative blood cortisol levels. The onset and rate of ovulation was similar within the HR and LR groups. Differences in fecundity, relative fecundity and egg volume and weight were wholly attributable to the different mean body weights of the HR and LR fish. There was no difference between the sperm counts of HR and LR males. There was significantly higher mortality among eggs derived from HR parents overall between fertilization and hatch, but maximum mean mortality did not exceed 12%. Mean time to eyeing in the HR groups was 224 degree days, and in the LR groups was 244 degree days. Hatching commenced at 344 and 347 degree days in the HR and LR groups, respectively, and was complete within 412 and 416 degree days respectively. Overall, selection for stress responsiveness was associated with no significant benefits or costs in reproductive performance.     

二倍体和三倍体虹鳟精液低温保存寿命的研究

与二倍体（2n组）精原液做比较,以不同密度为组别,研究了4℃下两组（3n-HD和3n-LD）三倍体虹鳟（Oncorhynchus mykiss）精原液的保存时限。结果发现,随着保存时间的延长,精液中精子的活力和寿命逐渐降低。2n组、3n-HD组和3n-LD组虹鳟精原液中精子分别在保存5d、4.5d和12.5d后失去活力;在7d、15d和14.5 d后死亡,即三倍体虹鳟精原液4℃保存时间约为二倍体的2倍,说明低温短期保存三倍体虹鳟精原液可以取得较好效果。     

光照颜色对虹鳟行为反应、血浆皮质醇和生化指标的影响

光照是影响鱼类行为、生理以及生长的主要环境因子之一。为研究光照颜色对虹鳟行为反应、血浆皮质醇、生化指标的影响,实验根据鱼类行为学研究方法,分别将白、红、黄、蓝和绿5种光照颜色灯带铺设在室内试验水槽中央底部,将虹鳟放置在水槽同一侧,观察虹鳟在水槽中穿越灯带尾次数及行为反应;根据行为生理学研究方法,分析比较在不同光照颜色下虹鳟血浆皮质醇、总蛋白(TP)、白蛋白(ALB)、甘油三酯(TG)、总胆固醇(TC)、谷草转氨酶(AST)、谷丙转氨酶(ALT)的含量差异。结果显示:5种光照颜色下虹鳟穿越灯带尾次数均低于空白组,其中黄光、白光条件下穿越灯带尾次数均显著低于空白组和其余3种光照颜色,红光、蓝光、绿光和空白组之间无显著差异。黄光条件下血浆皮质醇浓度显著高于空白组和其余4种光照颜色,空白组和其余4种光照之间无显著差异。5种光照颜色下总蛋白(TP)、白蛋白(ALB)、甘油三酯(TG)、总胆固醇(TC)、谷草转氨酶(AST)、谷丙转氨酶(ALT)均具有显著差异。研究表明,虹鳟比较适应蓝光和绿光,对黄光和白光有一定的趋避反应,且黄光的趋避效果最强;虹鳟可通过自身生理调节以适应不同的光照颜色变化。