New methods for cryopreservation of chicken semen using Hiroshima solution (trehalose solution)

自Polge等人的初次报告以来。世界卫生组织发现甘油对冷冻至-79ﾟC的鸡精子具有保护作用，目前已有许多鸡精子冷冻保存的研究。然而，冷冻精液的受精率明显低于新鲜精液。本研究的目的是评价海藻糖对鸡精液冷冻保存的效果，并改进颗粒冷冻方法。用含有不同水平海藻糖和甘油的稀释剂稀释精液，并将其冷冻成颗粒。冷冻精液解冻后，测定其胞外培养液的SPEM活力和乳酸脱氢酶(LDH)活性。随着海藻糖水平的增加，冷冻精液中LDH活性降低，精子活力增加。冻融前后乳酸脱氢酶渗漏的最小差值为0.26M海藻糖。9%甘油解冻后精子活力恢复最好，LDH活性最低。研究了含海藻糖的稀释液的pH值和渗透压对冷冻解冻后精子活力和乳酸脱氢酶漏出的影响。冷冻精液的最适pH值为7.2 mOsm，渗透压为370 mOsm。此外，还研究了精子密度对精子活力和冻融后乳酸脱氢酶漏出的影响。冷冻时精子浓度越低，精子活力恢复越好，细胞外液中LDH活性最低。结果表明，用含9%甘油的0.26M海藻糖溶液(pH=7.2，渗透压370mOsm)稀释精液冷冻保存家禽精液是可行的。

Since an initial report by Polge et al. who found a protective action of glycerol in chicken spermatozoa deep-frozen to -79ﾟC, there have been many investigations of freeze-storage of chicken spermatozoa. However, fertility by freeze-thaw semen is markedly below that obtained with fresh semen. The purpose of the present study is to evaluate an effectiveness of trehalose and to improve pelleted methods for cryopreservation of chicken semen. The semen was diluted with an extender containing different levels of trehalose and glycerol, and was frozen in pellets. After thawing, the spem motility and lactate dehydrogenase(LDH)activity of extracellular medium of freeze-thaw semen were measured. Along with the increased levels of trehalose, the motility of postthaw sperm increased and LDH activity in freeze-thaw semen decreased. The minimum difference between LDH leakage before and after freeze-thawing was shown to be at 0.26 M trehalose. The 9% glycerol level gave the best postthaw recovery in the sperm motility and the lowest LDH activity. The effects of PH and osmotic pressure of the extenders containg trehalose on the sperm motility and LDH leakage after freeze-thawing were investigated. The optimum pH value and osmotic pressure for the extender used to freeze semen was 7.2 and 370 mOsm, respectively. Furthermore, the effects of sperm concentration on the sperm motility and LDH leakage after freeze-thawing were estimated. The lowest sperm concentration at freezing gave the best postthaw recovery in the sperm motility and the lowest LDH activity in the extracellular medium of freeze-thaw semen. From those results, it was concluded that cryopreservation of semen diluted with 0.26 M trehalose solution (PH=7.2 ; osmotic pressure, 370 mOsm) containing 9% glycerol was the method feasible for poultry breeding programs.