中华乌塘鳢精子的生物学特性及其超低温保存

对中华乌塘鳢精子激活的生理生态学进行了研究,探讨了中华乌塘鳢精子超低温保存方法,中华乌塘鳢精子激活的适宜盐度为５－１５;其精子的激活不仅与激活溶溶盐度有关,而且与激活溶液的离子成份有关,激活溶液中Ｋ＾＋的存在一定程度上对精子的活动有抑帛和;中华乌塘鳢精子对ＰＨ的适应性强,在ＰＨ５．５－９．５范围内,激活率均在７０９％以上,尤以中性及弱酸性条件下的激活率最高,在ＰＨ６．０时精子活力最好。使用筛选出的     

Research Progresses on Anti-oxidation in Male Reproductive Systems

It is believed that active metabolism in male reproductive tract is contributed to production of ROS (reactive oxygen species).Low physiological ROS plays a fundamental role in mammalian reproduction, but the excessive accumulation of potentially damaging ROS can affect the function of sperm.Thus, it is important to maintain normal reproductive ability by a protective mechanism against oxidative stress.Here, we review the progress of antioxidant enzymes and redox systems in male reproductive systems, in order to provide references for physiology and pathology researches in male animals.     

Identification of sperm motility markers in bovine transition protein genes

Transition proteins (TNPs) are essential in chromatin condensation during spermiogenesis, and hence, they are the candidate genes for identifying sperm motility markers. Coding and in silico predicted promoter regions of these genes were investigated in crossbred and purebred cattle, and also, their mRNA quantification was done to explore its use as a diagnostic tool of infertility. PCR‐SSCP analysis revealed two band patterns in fragment III of TNP1 and fragment II of TNP2 gene. Sequence analysis revealed a deletion of “G” nucleotide in 3′UTR region of TNP1 and C>T SNP in intronic region of TNP2 gene. Least square analysis of variance did not reveal any significant influence of nucleotide deletion on any sperm motility parameters in both crossbred and purebred cattle. However, C>T SNP had a significant effect on initial progressive motility (p < 0.05) in purebred cattle and post‐thaw motility in overall cattle population. RT‐qPCR analysis did not reveal any significant variation in TNP1 and TNP2 gene expression among poorly motile and good quality spermatozoa of Vrindavani bulls.     

牛精子全蛋白二维电泳等电聚焦程序优化及染色方法的比较

以牛精子全蛋白为试验材料进行二维电泳试验研究,优化和改进了一向等电聚焦参数,比较了不同的染色方法,并运用Image Master 6.0软件分析了二维电泳图谱。结果表明,采用24 cm,pH3～10线性IPG胶条进行牛精子全蛋白二维电泳,等电聚焦80000 Vh和结合硝酸银染色方法可得到较多蛋白点和较高分辨率的二维电泳图谱。     

Relation between respiratory activity and sperm parameters in boar spermatozoa cryopreserved with alpha‐tocopherol and selected by Sephadex

Our aim was to evaluate the effect of Sephadex filtration on respiratory activity of porcine spermatozoa and its relation with quality and functional sperm parameters. Samples were evaluated regarding oxygen uptake and sperm parameters: motility, plasma and acrosome membrane integrity, capacitation and acrosome reaction induction in vitro, plasma membrane functionality, determined by the hypo‐osmotic swelling test (HOST), and lipid peroxidation assessed by thiobarbituric acid assay. Sephadex filtration improved all routine quality parameters (motility, plasma and acrosome membrane integrity) and functional parameters (HOST, in vitro capacitation and true acrosome reaction levels) and produced a significant decrease in cryocapacitation and lipid peroxidation. Oxygen uptake increased in Sephadex samples (41 ± 7%) respect to single washing. Oxygen addition of carbonyl‐cyanide‐m‐chlorophenylhydrazone (CCCP) confirmed mitochondrial coupling in washed and Sephadex samples; showing an increase of 2.6 and 4.2 times for oxygen consumption in single washing and Sephadex ones, respectively. The increase in oxygen uptake with succinate addition with respect to basal oxygen uptake was significantly lower in Sephadex samples (63 ± 25%) than in the washed ones (183 ± 35%). Sephadex samples showed higher mitochondrial activity measured by oxygen consumption and improved quality and functional parameters. Our study recommends this protocol due to the fact that this filtration method removes dead or damaged spermatozoa allowing to obtain cryopreserved boar spermatozoa with optimized fertilizing capacity.     

Improved conception rates in sows inseminated with cryopreserved boar spermatozoa prepared with a more optimal combination of osmolality and glycerol in the freezing extender

Cryoprotectant agents (CPAs) are added in freezing extenders to prevent intracellular ice crystal formation. However, it has been reported that high dose of CPAs confer toxicity on spermatozoa. Recently, the reduction of intracellular water by a high osmolality solution has also resulted in the suppression of ice crystal formation in spermatozoa, suggesting that the optimal combination of glycerol concentration and freezing extender osmolality could contribute to the development of effective sperm cryopreservation techniques. In this study, we investigated the motility, membrane and acrosomal integrity of frozen-thawed boar spermatozoa treated with freezing extender (NSF) of varying osmolalities (300, 400, 500 mOsm/kg) and final concentrations of glycerol (0.5, 1, 2, 3%). The spermatozoa that were treated at 400 mOsm/kg and 2% glycerol showed significantly higher rates of motility and membrane integrity compared with those in other treatment groups. In addition, the conception and implantation rates of swine artificially inseminated with spermatozoa frozen by the novel freezing extender (conception; 79%, implantation; 57.5%) were significantly higher than those of frozen-thawed spermatozoa treated in the conventional NSF (300 mOsm/kg, 3% glycerol) (conception; 29%, implantation; 33.8%). From these results, we concluded that the novel hyperosmotic (400 mOsm/kg) and low-glycerol (final concentration 2%) freezing extender is beneficial for the cryopreservation of boar spermatozoa.     

哺乳动物性别控制的研究进展

目前哺乳动物的性别控制主要是通过XY精子分离和早期胚胎性别鉴定来实现的。其他常用的性别控制的方法主要有：受精时间控制法、阴道pH值调节法和营养调节法等。本文就性别决定的机理、XY精子分离以及早期胚胎性别鉴定的几种方法进行综述，并对性别控制存在的问题及发展前景进行讨论。     

三疣梭子蟹精子活力评价方法的研究

采用染色法和钙离子载体A23187诱导精子顶体反应法,对三疣梭子蟹精子的活力进行评价研究.结果表明,采用台盼蓝染色无法清晰辨别死、活精子.采用曙红B染色,精子分别呈现不同的染色特征:活精子无色,细胞边界清晰可辨,在光学显微镜下观察可见顶体中央突起的圆锥状结构和辐射臂;死精子细胞边界有稍许模糊,核杯和顶体均着色.最适的曙红B浓度和染色时间分别为2%和2 min.钙离子载体.A23187诱导精子顶体反应的结果显示:在诱导时间和A23187浓度分别为50 min和30μg·mL-1时,校正顶体反应率达到(92.73±2.43)%.对这两种活力检测方法的比较分析显示,曙红B染色法和钙离子载体A23187诱导精子顶体反应法的活力检测值与样品理论值呈显著正相关(P<0.01),他们二者之间亦呈显著正相关(P<0.01),说明这两种方法均可用于精子活力检测,其结果具有可比性.     

Effect of cooling rate and cryoprotectant on the cryosurvival of equine spermatozoa

Cryosurvival of cells is reduced if the cooling rate used is suboptimal. If cells cool too rapidly, intracellular water will freeze, causing intracellular ice crystals. However, if spermatozoa are cooled too slowly, excessive cellular dehydration occurs, causing irreversible damage to cellular compartments. In addition, cryoprotectants are added to the freezing diluent to protect cells from damage during cryopreservation. This study was conducted to determine the optimal cooling rate for stallion spermatozoa frozen in the presence of three different cryoprotectants. Spermatozoa were frozen in a skim milk, egg yolk diluent containing 4% glycerol, and ethylene glycol or dimethyl formamide at 10 different cooling rates ranging from 5°C/min to 50°C/min. The percentage of viable spermatozoa was higher for spermatozoa cooled at 10°C/min than at 50°C/min (P < .05). Spermatozoa frozen using glycerol as the cryoprotectant had higher percentages of motile and progressively motile spermatozoa compared with spermatozoa frozen using the other two cryoprotectants (P < .05). In conclusion, the cryosurvival of stallion spermatozoa is similar when cooling rates of 5°C/min to 45°C/min are used, and when 4% cryoprotectant is used, glycerol is a more effective cryoprotectant than ethylene glycol or dimethyl formamide.