Rasul Z, Ahmad N, Anzar M
Animal Sciences Institute, National Agricultural Research Centre, Islamabad, Pakistan.
J Androl. 2001 Mar-Apr;22(2):278-83.
Motion characteristics, plasma membrane integrity, and acrosome morphology of buffalo spermatozoa after different stages of cryopreservation (ie, dilution, cooling to 4 degrees C, equilibration at 4 degrees C, and freezing and thawing) were examined. Semen ejaculates from 4 buffalo bulls were pooled (n = 5), diluted in tris-citric acid extender, cooled to 4 degrees C over 2 hours, equilibrated at 4 degrees C for 4 hours, dispensed into 0.5-mL straws, and frozen in a programmable cell freezer before plunging into liquid nitrogen. Frozen semen was thawed at 37 degrees C for 15 seconds. After completion of each stage, sperm motion characteristics, plasma membrane integrity, and acrosomal morphology were determined using computer-assisted semen analysis, hypo-osmotic swelling assay, and phase-contrast microscopy, respectively. Data were presented as mean +/- standard error of the mean. Visual and computerized motility did not differ due to dilution, cooling, or equilibration (77.3% +/- 2.3% and 90.5% +/- 1.2%, respectively), but was reduced (P < .05) after freezing and thawing (53.0% +/- 4.6% and 48.6% +/- 6.5%, respectively). Linear motility of spermatozoa was lower (P < .05) after dilution or equilibration (56.2% +/- 2.4%) than after cooling or freezing and thawing (79.6% +/- 1.4%). Sperm curvilinear velocity was reduced (P < .05) from 112.4 +/- 5.3 microm/sec after dilution to 96.0 +/- 5.8 microm/s after cooling, and from 87.6 +/- 4.1 microm/s after equilibration to 69.4 +/- 2.0 microm/s after freezing and thawing. Sperm lateral head displacement differed (P < .05) after each stage (ie, dilution, 3.9 +/- 0.2 microm; cooling, 2.3 +/- 0.2 microm; equilibration, 3.1 +/- 0.3 microm; and freezing and thawing, 1.7 +/- 0.2 microm). Spermatozoa with intact plasma membranes were 80.2% +/- 3.9% after dilution, reduced (P < .05) to 60.4% +/- 5.6% after equilibration, and then to 32.6% +/- 3.8% after freezing and thawing. The percentage of spermatozoa with normal acrosomes remained higher after dilution, cooling, or equilibration (73.2% +/- 2.4%) than after freezing and thawing (61.8% +/- 2.4%; P < .05). In conclusion, the maximal damage to the motility apparatus, plasma membrane, and acrosomal cap of buffalo spermatozoa occurs during freezing and thawing followed by equilibration.
研究了水牛精子在不同冷冻保存阶段（即稀释、冷却至4℃、在4℃平衡以及冷冻和解冻）后的运动特征、质膜完整性和顶体形态。将4头水牛公牛的射精精液混合（n = 5），用三羟甲基氨基甲烷 - 柠檬酸稀释液稀释，在2小时内冷却至4℃，在4℃平衡4小时，分装到0.5毫升细管中，然后在程序降温细胞冻存器中冷冻，再投入液氮。冷冻精液在37℃解冻15秒。在每个阶段完成后，分别使用计算机辅助精液分析、低渗肿胀试验和相差显微镜测定精子运动特征、质膜完整性和顶体形态。数据以平均值±平均标准误差表示。视觉和计算机化的活力在稀释、冷却或平衡后没有差异（分别为77.3%±2.3%和90.5%±1.2%），但在冷冻和解冻后降低（P < 0.05）（分别为53.0%±4.6%和48.6%±6.5%）。精子的直线运动在稀释或平衡后（56.2%±2.4%）低于冷却或冷冻和解冻后（79.6%±1.4%）（P < 0.05）。精子曲线速度从稀释后的112.4±5.3微米/秒降低（P < 0.05）至冷却后的96.0±5.8微米/秒，从平衡后的87.6±4.1微米/秒降低至冷冻和解冻后的69.4±2.0微米/秒。精子头部横向位移在每个阶段后均有差异（P < 0.05）（即稀释后为3.9±0.2微米；冷却后为2.3±0.2微米；平衡后为3.1±0.3微米；冷冻和解冻后为1.7±0.2微米）。质膜完整的精子在稀释后为80.2%±3.9%，平衡后降低（P < 0.05）至60.4%±5.6%，冷冻和解冻后降至32.6%±3.8%。顶体正常的精子百分比在稀释、冷却或平衡后（73.2%±2.4%）高于冷冻和解冻后（61.8%±2.4%；P < 0.05）。总之，水牛精子的运动装置、质膜和顶体帽在冷冻和解冻随后平衡的过程中受到的损伤最大。
