Liu Z, Foote R H, Brockett C C
Department of Animal Science, Cornell University, Ithaca, New York, 14853, USA.
Cryobiology. 1998 Nov;37(3):219-30. doi: 10.1006/cryo.1998.2117.
The effects of freezing procedures, osmolarity, trehalose, and sucrose on survival of bull sperm in whole milk (WM) and egg yolk-Tris (EYT), semen extenders used worldwide, were studied. Sperm were added to extenders at 25 degreesC, cooled slowly to 5 degreesC, glycerolated, packaged in 0.5-ml straws, and frozen. Different freezing rates were accomplished in two steps. Straws were transferred from +5 degreesC to nitrogen vapor at temperatures ranging from -10 to -100 degreesC in the first step and to liquid nitrogen in the second step. Straws were thawed in water at 35 degreesC. A substantial decrease in sperm motility occurred between -10 and -20 degreesC, as abrupt nucleation occurred following supercooling to -13 degreesC. To study the interactions between osmolarity x cooling rate, WM and EYT extenders were prepared to yield media measuring 220 to 420 mOsm/L. The optimal first-step range of cooling in the two-step procedure was -30 to -70 degreesC, and the highest proportions of motile sperm after freezing and thawing were 61 to 62 in 260 to 300 mOsm/L WM and 63 to 64% in 300 to 340 mOsm/L EYT, equivalent to the results with the control procedure used commercially. As the cooling rate increased (first step to -100 degreesC) sperm motility was much higher in hypertonic than in hypotonic extenders (P < 0.05), indicating the importance of partial dehydration before rapid cooling. Replacing part of EYT and WM with equivalent solutions (same mOsm/L) of sucrose or trehalose had no appreciable effect. These results provide a basis for further investigating simple freezing systems that might be more effective in preserving bull sperm than those currently available.
研究了冷冻程序、渗透压、海藻糖和蔗糖对全球范围内使用的精液稀释剂全脂牛奶（WM）和蛋黄 - 三羟甲基氨基甲烷（EYT）中公牛精子存活的影响。精子在25℃下添加到稀释剂中，缓慢冷却至5℃，甘油化处理，包装在0.5毫升细管中并冷冻。通过两步实现不同的冷冻速率。第一步，细管从+5℃转移至温度范围为-10至-100℃的氮蒸气中，第二步转移至液氮中。细管在35℃的水中解冻。在-10至-20℃之间精子活力大幅下降，因为过冷至-13℃后会突然成核。为研究渗透压×冷却速率之间的相互作用，制备了WM和EYT稀释剂以产生渗透压为220至420 mOsm/L的培养基。两步冷冻程序中第一步的最佳冷却范围为-30至-70℃，冷冻和解冻后活力精子的最高比例在260至300 mOsm/L的WM中为61%至62%，在300至340 mOsm/L的EYT中为63%至64%，与商业使用的对照程序结果相当。随着冷却速率增加（第一步至-100℃），高渗稀释剂中的精子活力远高于低渗稀释剂（P < 0.05），表明快速冷却前部分脱水的重要性。用等渗的蔗糖或海藻糖溶液替代部分EYT和WM没有明显影响。这些结果为进一步研究比现有方法更有效地保存公牛精子的简单冷冻系统提供了基础。
