低温和超低温保存对中国大鲵成熟精子的影响

摘要

Chinese giant salamander (4ndrias davidianus) is a rare and endangered endemic amphibian caudata in China.Chinese giant salamander sperm was cryopreserved in liquid nitrogen or stored at 0-4℃ on a short-term basis. The objective was to establish reliable technologies for the conservation of this endangered species and for artificial assisted reproduction in Chinese giant salamander farms. The studies showed that the spermatozoa remained motility only 3-5h at the room temperature but 6d at 4℃ and 9d at 0℃. Besides, the viability of spermatozoa precipitous declined after adding a dilute solution. An improved cryopreservation method for Chinese giant salamander sperm was established according to the mature technologies for cryopreservation of fish. Cryopreseved sperm was diluted in buffered extenders (pH between 7 and 7.5) containing intracellular (DMSO). The concentration of intracellular cryoprotectants ranged from 5％ to 25％. In this paper, ten to fifteen percents of the sperm were motile after thawing. The ultrastructures of Chinese giant salamander sperm before and after cryopreservation were observed by SEM and TEM. Many deformations were found in the cryopreserved sperm, such as swelling or breaking of the plasma membrane, disappearing of the nuclear envelope, fracture of the perforatorium and axoneme, degenerating of the undulating membrane and disappearing of the mitochondrial ridges. The results indicated that these damages might be the main reasons for the decline of the motility in the cryopreserved sperm. This study provided an important reference for the establishment of standardized procedures of the short-term storage and cryopresevation of Chinese giant salamander's sperm as well as the artificial breeding of Chinese giant salamander.%中国大鲵Andrias davidianus是中国特有的一种濒危有尾两栖类动物,为了保护这一珍稀物种并且为中国大鲵人工辅助繁育建立一套可靠的技术,对中国大鲵的精子在0-4℃下低温短期保存和在液氮中超低温长期保存进行了研究.研究表明中国大鲵精子原液在常温下一般仅存活3-5h,4℃下能存活6d,0℃下存活时间可达9d.精子原液中添加Ringer氏液或Holtferter氏液,精子存活率比原液保存显著降低.在改良的超低温冻存条件下,中国大鲵精子在液氮中保存两周后解冻,精子复苏率可达10%-15%.利用扫描和透射电镜观察冻融前后中国大鲵精子的超微结构,发现受冷冻损伤精子质膜出现膨胀、破裂;精子穿孔器和轴丝显著弯曲,甚至断裂;波动膜明显脱落或膜结构损坏;精子线粒体嵴变形.结果表明,超低温冻存导致部分中国大鲵精子超微结构发生变化而产生冻伤,进而导致精子活力、复苏率下降.研究为建立规范化的中国大鲵精液保存程序和中国大鲵规模化人工繁育提供重要参考.