The antioxidant system of seminal fluid during in vitro storage of sterlet <Emphasis Type="Italic">Acipenser ruthenus</Emphasis> sperm

The role of the seminal fluid antioxidant system in protection against damage to spermatozoa during in vitro sperm storage is unclear. This study investigated the effect of in vitro storage of sterlet Acipenser ruthenus spermatozoa together with seminal fluid for 36 h at 4 °C on spermatozoon motility rate and curvilinear velocity, thiobarbituric acid reactive substance level, and components of enzyme and non-enzyme antioxidant system (superoxide dismutase and catalase activity and uric acid concentration) in seminal fluid. Spermatozoon motility parameters after sperm storage were significantly decreased, while the level of thiobarbituric acid reactive substances, activity of superoxide dismutase and catalase, and uric acid concentration did not change. Our findings suggest that the antioxidant system of sterlet seminal fluid is effective in preventing oxidative stress during short-term sperm storage and prompt future investigations of changes in spermatozoon homeostasis and in spermatozoon plasma membrane structure which are other possible reasons of spermatozoon motility deterioration upon sperm storage.     

Sex steroids in the seminal fluid and milt quality indices in the endangered Caspian brown trout,Salmo trutta caspius

In this study, the relationships between the seminal fluid sex steroids sperm quality parameters (i.e. sperm motility and sperm production were investigated in the endangered Caspian brown trout. According to results, the variation of sex steroid concentrations was comparatively high. The seminal fluid levels of 17α‐Hydroxyprogesterone (OHP) had positive correlation with percentage and duration of sperm motility. Similar correlation was found between the seminal fluid concentration of testosterone (T) and estradiol‐17β (E2) with sperm concentration. As well as, there was no significant correlation between progesterone (P) with sperm quality indices. The significant correlation of some steroids i.e. OHP, T and E2 and sperm quality parameters of Caspian brown trout confirms the key role of these steroids in acquisition of the potential of motility during final stage of the maturation.     

Ionic composition and osmolality of paddlefish (Polyodon spathula, Acipenseriformes) seminal fluid

Changes in ionic composition as Na⁺,K⁺, Ca²⁺ and Mg²⁺, osmolality inseminal fluid, percentage of motile spermatozoaand velocity were investigated in response toCPP and different dosage of LHRHa. The lowestvelocity of sperm was observed after use CPPtreatment. The velocity of spermatozoa,significant main effect of the treatment(P < 0.0001) and the time of sperm collection(P < 0.0104) were evaluated. The osmolality ofseminal fluid was different betweenexperimental groups of LHRHa (48.0–62.7mOsmol.kg^–1) and CPP (33.0–46.3mOsmol.kg^–1) treatments. The osmolalitywas significantly higher on the first day andone-half, then declined on day three, rangingfrom 33.0 to 62.7 mOsmol.kg^–1. Analysisof variance showed significant main effects ofthe treatment (P < 0.0001) and the time ofsperm collection (P < 0.0002) on the osmolalityof seminal fluid. The level of Na⁺ andK⁺ ion was different between experimentalgroups of LHRHa and CPP treatment. The highestconcentration of 11.11 mmol.l^–1 wasobserved at Na⁺ ion. Then theconcentrations declined on the level 1.56, 0.52and 0.36 mmol.l^–1 for K⁺, Ca²⁺and Mg²⁺ ions, respectively. There werehighly positive correlations between osmolalityof seminal fluid and dosage of LHRHa treatment(r = 0.84), velocity of spermatozoa andosmolality of seminal fluid (r = 0.57) andosmolality of seminal fluid and Na⁺concentration at seminal fluid (r = 0.70).Injection with LHRHa increased quality of spermas velocity of sperm, level of Na⁺,K⁺ and osmolality at seminal fluidcompared to CPP treatments.     

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.     

Sperm cryoconservation in Anarhichas sp., endangered cold-water aquaculture species with internal fertilization

This study contributes to the identification of an adequate cryopreservation medium for wolffishes sperm (Anarhichas minor and A. lupus). Our results also confirm the presence of antifreeze proteins in the seminal fluid of the Atlantic wolffish (Anarhichas lupus) that could facilitate the process of cryoconservation in association with increased post-thaw motility and fertilization rates. Cryo-Fish, a commercial diluent was the most efficient in our trials, with 80% motility post-thaw.     

Low seminal plasma buffering capacity corresponds to high pH sensitivity of sperm motility in salmonids

The buffering capacity of air-equilibrated seminal plasma from the chinook salmon Oncorhynchus tshawytscha and steelhead Oncorhynchus mykiss were determined. Buffering capacities were significantly less at pH values at and above 7.3 than over the range 6.0 to 7.0 and were particularly low between pH values of approximately 7.5 to 8.5. The potential of chinook salmon sperm to become motile upon addition to water was highly sensitive to pH. Only very low levels of motility were demonstrable when sperm had been maintained at pH values at or below 7.5 prior to water exposure while maximum motility was noted when sperm had been maintained at pH values of about 8.0 and above. Thus, the potential for motility appeared especially sensitive to that pH range over which seminal plasma exhibited its lowest buffering capacity. Buffering capacity, protein content and inorganic phosphate content of chinook salmon and steelhead seminal plasmas were less than those of corresponding blood plasma or ovarian fluid suggesting that seminal plasma is unique among salmonid body fluids in its composition and low buffering capacity. This low buffering capacity is likely to facilitate the role of the sperm duct epithelium in semen pH regulation and thus, in the sperm's acquisition of the potential for motility upon exposure to water. This revised version was published online in August 2006 with corrections to the Cover Date.     

Thyroxine-induced alterations in the testis and seminal vesicles of air-breathing catfish, Clarias gariepinus

Effect of experimentally induced thyroxine overdose on the testis and seminal vesicles was studied in the air-breathing catfish, Clarias gariepinus during the preparatory and the pre-spawning phase. The present study revealed a marked reduction in testosterone level in serum, testis and seminal vesicles (SV). Histological examination showed a considerable reduction in the number of spermatozoa/spermatids in the seminiferous tubular lumen as well as depletion of fluid in the loculi of SV. SDS-PAGE analysis of SV fluid proteins demonstrated a significant decrease in the level of a ~27 kDa protein in thyroxine treated fishes. Evidences are presented here to indicate that thyroid hormone plays a role in regulating testis and SV function in catfish. T.N. Jacob and J.P. Pandey contributed equally     

蟹类精荚贮存和裂解研究进展

甲壳动物在海洋中分布广泛,有多种精子传输方式,大部分产生精荚,粘于雌体腹部或通过交接器输送到纳精囊中。研究表明,在蟹类交配,精子转移,贮存和受精等过程中,存在如精子竞争和亲体保护,多次受精等众多复杂事件。这些过程主要是以蟹类纳精囊为核心异型,为此本文综述了纳精囊的结构和功能,精子活力的维持,精荚形态,精子竞争和转移等近年研究进展,旨为相关研究提供参考。     

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.     

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.     

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.     

金乌贼纳精囊组织结构及其精子的储存和利用

为揭示金乌贼精子进入纳精囊及产卵过程中的精子利用方式,丰富金乌贼繁殖生物学研究内容,本研究利用实验生态学和组织切片技术,检测了交配后不同时间段雌性口膜表面精子囊和纳精囊中精子数量的变化,观察分析了雌性金乌贼纳精囊的组织结构。结果显示,金乌贼纳精囊位于繁殖期雌性个体口膜腹面的突起处,共1对。纳精囊开口于口膜内表面,通过一根中央管连通整个纳精囊。中央管内壁含有大量褶皱和纤毛。在中央管两端,有12~20个储精小囊与之相连。储精小囊四周具有发达的环肌,其中储存有大量精子,并且大部分精子头部均朝向腔室内壁。完成一次交配后,雌性金乌贼对精子囊和纳精囊中精子的利用可以分为三个阶段,主要利用精子囊中的精子(交配后1~2 d);由利用精子囊中的精子向纳精囊中的精子过渡(交配后2~3 d);主要利用纳精囊中的精子(交配后3 d以上)。研究表明,从精子囊释放出的精子进入雌性口膜表面的褶皱中,通过自身运动到达纳精囊。进入纳精囊的精子通过自身运动及中央管内壁纤毛的摆动进入储精小囊,其中大部分精子头部朝向储精小囊内壁有规律地分布。在产卵过程中,雌性优先利用精子囊中的精子,而在精子囊中精子不足时,纳精囊通过肌肉收缩以及纤毛摆动将其中的精子逐渐释放出来,卵子在雌性口膜附近完成体外受精。     

Relationship between biochemical constituents of fish semen and fertility: the effect of short-term storage

Spermatozoa and seminal plasma obtained from rainbow trout and whitefish were analyzed in respect to their aspartate aminotransferase (AspAT) and alkaline phosphatase activities. In particular, the experiments characterized AspAT optimum pH, optimization of assay conditions and action of coenzyme, pyridoxal 5-phosphate (vitamin B₆). The effect of short-term semen storage at 0°C on biochemical indicators and fertilization rate was examined in both species. The concentrations of reduced and oxidized ascorbic acid in seminal plasma of both species were several folds higher than in spermatozoa and blood plasma of fish. Highly significant correlations were found for both species between AspAT activity (sperm or seminal plasma) and fertilization rate (% of eyed-stage or hatched embryos). For rainbow trout, highly significant correlations were found between sperm concentration, motility and fertilization rate. These results suggest that several biochemical indicators of seminal plasma can be used as measures of sperm quality of fish. Some common biochemical parameters for fish and mammal's semen provide evidence for using fish sperm as a model in biomedical research.     

Seasonal changes in the biochemistry of seminal plasma and sperm motility in the ocean pout,Macrozoarces americanus

Sperm motility, pH and osmolality of seminal plasma varied throughout the reproductive season spanning the period from June to September. Initially, sperm motility was low, peaked in July and August and then fell again towards the end of the spawning season. While the pH of seminal plasma increased from pH 7.4 to 7.9 during the period of spermiation, the average seasonal pH (7.78 ± 0.03) remained close to an experimentally determined optimum pH range for ocean pout sperm motility (pH 8–9). Likewise, although the values for seminal plasma osmolality fell during the reproductive season, from 416–339 mmol kg^-1, the average osmolality value 356 ± 3 was within the optimum for sperm motility (300–400 mmol kg^-1). In comparing fluctuations in sperm motility with the biochemical composition of ocean pout seminal plasma during the spawning season, this analysis showed that increased Mg⁺⁺ levels were correlated with the summer period of maximum sperm motility. A seasonal decline in Na⁺ and Cl⁻ ion levels was reflected in lower seminal plasma osmolality values.     

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.     

Sperm motility in the steelhead Oncorhynchus mykiss (Walbaum): influence of the composition of the incubation and activation media

This study examined the pH sensitivity of steelhead, Oncorhynchus mykiss (Walbaum), sperm motility relative to the composition of incubation and activation media. The percentage of sperm that initiated motility following incubation in a sperm immobilizing solution (SI) titrated to different pH values and subsequent activation by dilution in buffered swimming medium (SM) at pH 8.5 or 50% ovarian fluid (OF) showed little or no pH sensitivity; sperm diluted in de‐ionized water (DI) showed no motility after incubation at any pH. In contrast, motility of sperm diluted in tap water (TAP) was highly sensitive to the pH of the incubation medium. Sperm incubated with buffered seminal plasma at high, but not low pH demonstrated high percent motility when diluted with DI. Sperm incubated in low‐pH SI demonstrated high motility only when diluted into high‐pH SM. The effects of the composition of incubation and activation media on sperm motility were generally reflected in comparable effects on fertility. Therefore, these data indicate that the pH sensitivity of sperm motility and fertility depends on the composition of commonly used incubation as well as activation media.     

中华倒刺鲃、白甲鱼和岩原鲤精子的生理特性比较

对中华倒刺鲃(Spinibarbussinensis)、白甲鱼(Onychostomasimus)和岩原鲤(Procyprisrabaudi)3种鱼的鲜精在不同水溶液和不同浓度梯度的NaCl溶液中的活力以及精子形态、密度和精液pH值、浓度等进行了比较研究。结果表明:3种鱼的精液pH值都偏弱碱性,位于7·2~7·7之间;精液浓度依次为71·1%,76·1%和71%;精子头长依次为(3·89±0·53)μm,(2·98±0·08)μm和(6·42±0·59)μm,全长依次为(41·63±3·66)μm,(65·67±2·97)μm和(58·89±5·25)μm;精子密度依次为1·527×1010尾/mL,1·336×1010尾/mL和1·362×1010尾/mL。分析表明,3种鱼的精子大小与密度无相关性。在不同水溶液中,3种鱼的精子活力均以池塘水中最高;在不同浓度NaCl溶液中,3种鱼精子的最适浓度位于0·45%~0·55%之间,有效运动时间以中华倒刺鲃最高,为(85·23±12·02)s,其次是岩原鲤,为(50·45±6·89)s,白甲鱼最短,为(31·44±5·53)s。3种鱼的精子全长与精子活力存在负相关性,即精子越长,活力越低,精子越短,活力越高。     

First results on a relation between ovarian fluid and egg proteins of Salmo trutta and egg quality

By use of sodium dodecyl sulfate polyacrylamide gel electrophoresis, ovarian fluid proteins and main proteins of unfertilized eggs were qualitatively and quantitatively investigated in the brown trout, Salmo trutta, to see whether some of them were correlated with the rate of embryos reaching the eyed embryo stage. In the ovarian fluid, 12 types of proteins in the range of 39–166 kDa were detected whereby three proteins were lipoproteins and two were glycoproteins. Ovarian fluid proteins with a molecular weight of 85, 68, 62 and 39 kDa were negatively correlated with the percentage of eyed stage embryos. The statistical significance of the relations was low in simple and multiple regression models (R²≤0.534) indicating that the relations were influenced and superposed by other factors. Therefore, ovarian fluid proteins give only poor information about maturity and quality of eggs. In the eggs, nine major types of proteins in the range of 95–15 kDa were identified. The 95 kDa protein was a lipoprotein, the 85 and the 62 kDa protein were glycoproteins, and the 15 kDa protein was a phosphoprotein. The 95, 85, 77 and 39 kDa protein were positively correlated with embryo survival to the eyed embryo stage. The explanatory effect of the multiple regression model was very high (R²=0.961) indicating that distinct egg proteins are closely related with egg quality.     

Carbon dioxide reversibly inhibits sperm motility and fertilizing ability in steelhead (Oncorhynchus mykiss)

The effects of different carbon dioxide (CO₂) levels on the short-term storage of semen samples from hatchery-produced steelhead (Oncorhynchus mykiss) were evaluated. Sperm motility and fertilizing ability were significantly reduced following 4 h incubation under a relatively modest (0.9 kPa = 1%) amount of CO₂. The dose-dependent reductions, however, were not the result of cell death as sperm viability was unaltered even at the highest (5.2 kPa = 5.6%) CO₂ exposures. Reductions in sperm motility and fertilizing ability were reversible. Although previous work has indicated a direct relationship between salmonid sperm motility and sperm ATP content, the inhibitory effects of CO₂ on sperm motility were not the result of reduced sperm ATP levels. Decreasing the pH of the seminal fluid (to below 7.5) significantly reduced sperm motility. However, this effect was only observed after prolonged (4 h) exposure; short-term (1 min) exposure to this lowered pH did not alter sperm motility. Moreover, acetazolamide, a carbonic anhydrase inhibitor, attenuated the inhibitory effects of CO₂ on sperm motility. These results suggest that CO₂ inhibits steelhead sperm motility and therefore fertility in a dose-dependent manner, by reversibly lowering intracellular pH.