Correa J R, Rodriguez M C, Patterson D J, Zavos P M
Department of Animal Sciences, University of Kentucky, Lexington, KY 40546, USA.
Theriogenology. 1996 Aug;46(3):413-20. doi: 10.1016/0093-691x(96)00163-x.
The objective of this study was to evaluate the effects of thawing and processing temperatures on post-thaw sperm viability, occurrence of osmotic shock and sperm membrane functional status. The occurrence of osmotic shock, characterized by increased spermatozoa with coiled tails, eventually results in reduced sperm viability and sperm membrane integrity. The effects of different thawing temperatures were assessed by thawing frozen specimens at 37, 21 or 5 degrees C for 1 to 2-min, followed by processing at these temperatures. A subset of frozen specimens were thawed at 37 degrees C for 10 to 15-sec and transferred to a water bath at 21 or 5 degrees C for 1 to 2-min to complete thawing, followed by processing at these temperatures. Sperm processing (washing) consisted of dilution, centrifugation and resuspension to remove glycerol from the medium and to gradually return the spermatozoa to isotonic conditions. Post-thawed specimens (0.5 mL) were slowly diluted 1:1 (v/v) at a rate of 0.1 mL/min, centrifuged, and resuspended to 0.5 mL (37 degrees C). Diluted specimens were equilibrated for 1 to 2-min after dilution and for 5-min after resuspension. The specimens were then incubated for 2-h (37 degrees C) and assessed at 60-min intervals for the percentage of motility, for progressive motility (Grades 0 to 4), for the percentage of spermatozoa with coiled tails, and for the percentage of swollen spermatozoa. The percentage of swollen spermatozoa (measurement of sperm membrane integrity) was assessed by exposing spermatozoa to a modified hypoosmotic swelling (HOS) test. The results obtained seem to indicate that physiological thawing and processing temperatures (37 degrees C) are required to maintain sperm motility. However, thawing and processing at lower temperatures (< 37 degrees C) seems to prevent the occurrence of osmotic shock and to maintain sperm membrane functional integrity. In this study, thawing at 37 degrees C (10 to 15-sec) and transfer to a water bath at 21 degrees C (1-min) to complete thawing, followed by processing at 21 degrees C, yielded better results in terms of increased sperm viability, reduced occurrence of osmotic shock and higher reactivity to the HOS test.
本研究的目的是评估解冻温度和处理温度对解冻后精子活力、渗透休克的发生以及精子膜功能状态的影响。渗透休克的发生表现为尾部卷曲的精子数量增加，最终导致精子活力和精子膜完整性降低。通过在37℃、21℃或5℃解冻冷冻样本1至2分钟，然后在这些温度下进行处理，来评估不同解冻温度的影响。将一部分冷冻样本在37℃解冻10至15秒，然后转移到21℃或5℃的水浴中1至2分钟以完成解冻，随后在这些温度下进行处理。精子处理（洗涤）包括稀释、离心和重悬，以从培养基中去除甘油，并使精子逐渐恢复到等渗状态。解冻后的样本（0.5 mL）以0.1 mL/min的速度缓慢1:1（v/v）稀释，离心，并重悬至0.5 mL（37℃）。稀释后的样本在稀释后平衡1至2分钟，重悬后平衡5分钟。然后将样本在37℃孵育2小时，并每隔60分钟评估活力百分比、进行性活力（0至4级）、尾部卷曲精子的百分比以及肿胀精子的百分比。通过将精子暴露于改良的低渗肿胀（HOS）试验来评估肿胀精子的百分比（精子膜完整性的测量）。获得的结果似乎表明，需要生理解冻和处理温度（37℃）来维持精子活力。然而，在较低温度（<37℃）下解冻和处理似乎可以防止渗透休克的发生，并维持精子膜功能完整性。在本研究中，在37℃解冻（10至15秒）并转移到21℃的水浴中（1分钟）以完成解冻，随后在21℃进行处理，在提高精子活力、减少渗透休克的发生以及对HOS试验的更高反应性方面产生了更好的结果。
