不同渗透压的稀释液对猕猴精子低温冷冻保存的影响

Effects of Extenders Varying in Osmolality on Rhesus Monkey (Macaca mulatta) Spermatozoa Cryopreservation

以稀释液TTE(382mOsm/kg)为对照,研究了5种渗透压(688、389、329、166、43mOsm/kg)的TEST稀释液(TEST、mTEST1、mTEST2、mTEST3、mTEST4)在冷冻过程中对猕猴精子功能的影响。精液一步稀释于含甘油的防冻液中,甘油的终浓度为5%(v/v)。在冷冻前后分别检测精子的运动度和质膜完整性,后者用Hoechst33342和碘化丙锭双色标记流式细胞术分析。结果表明:冷冻之前,与鲜精相比,用TEST和mTEST4稀释的精子运动度和质膜完整性显著降低(P<0·001),其余组中除mTEST2稀释的精子质膜完整性显著降低(P<0·05)外,精子运动度无差异;冷冻复苏后,TTE、mTEST3和mTEST1冻存精子的运动度和质膜完整性最高,其次是mTEST2,TEST和mTEST4冷冻效果最差(P<0·05)。提示等渗、适当高渗或低渗的稀释液适合猕猴精子的冷冻保存;对精子产生高渗毒害作用是导致猕猴精子用TEST冷冻存活率低的主要原因。

Effects of five TEST extenders varying in osmolality on rhesus monkey sperm cryopreservation were studied, using TTE as the control. These five extenders were designated as TEST, mTEST1, mTEST2, mTEST3 and mTEST4, and the corresponding osmolalities were 688, 389, 329, 166 and 43 mOsm/kg, respectively. Semen were diluted in one step into freezing media containing glycerol, and the final concentration of glycerol was 5%(v/v). Sperm survival was determined by assessment of sperm motility and membrane integrity prior to and after cryopreservation. Membrane integrity was evaluated using Hoechst 33342/propidium iodide dual staining procedure and flow cytometry. The results showed that, before freezing, motility and membrane integrity of spermatozoa diluted in TEST and mTEST4 were drastically reduced (P<0.001) compared with fresh sperm. In remaining groups, sperm function remained unaffected after equilibration except that the membrane integrity of spermatozoa diluted in mTEST2 was significantly lower than that of fresh semen (P<0.05). The sperm cryopreservation efficiency for these extenders, evaluated by post-thaw sperm motility and membrane integrity, is: TTE, mTEST3 and mTEST1> mTEST2> TEST and mTEST4 (P<0.05). The results suggest that isosmotic, moderately hyperosmotic or hypoosmotic extenders are favorable for the cryopreservation of rhesus monkey spermatozoa and the hyperosmotic stress of TEST accounts for the lower survival of rhesus monkey spermatozoa frozen in this medium.