Bóveda P, Esteso M C, Castaño C, Toledano-Díaz A, López-Sebastián A, Muñiz A, Prieto P, Mejía O, Ungerfeld R, Santiago-Moreno J
Department of Animal Reproduction, INIA, Madrid, Spain.
D.T. Jaén, Consejería de Medio Ambiente y Ordenación del Territorio, Junta de Andalucía, Spain.
Anim Reprod Sci. 2018 May;192:193-199. doi: 10.1016/j.anireprosci.2018.03.010. Epub 2018 Mar 8.
This study examines the effectiveness of two methods for cryopreserving post-mortem epididymal sperm - conventional slow freezing employing a short equilibration time with glycerol, and ultra-rapid freezing - from the wild ruminant species Ovis musimon (mouflon) and Dama dama (fallow deer). A Tris-citric acid-glucose (TCG) + 12% egg yolk-based medium was used for the conventional slow freezing of the fallow deer sperm, whereas a Tes-Tris-glucose (TEST) + 6% egg yolk-based medium was used for the mouflon sperm. Glycerol was added to a final concentration of 5% to both media. The same diluents were used for ultra-rapid freezing but replacing the glycerol with 100 mM of sucrose. Sperm variables (motility, viability, acrosome integrity, membrane integrity, and morphological abnormalities) were analyzed before and after cryopreservation. Although values were generally better after the thawing of the conventionally cryopreserved sperm, total sperm motility (38.40 ± 4.44% in mouflon and 31.25 ± 3.37% in fallow deer) and total live sperm (47.19 ± 5.18% in mouflon and 43.13 ± 2.43% in fallow deer) were acceptable for the ultra-rapidly cooled sperm. Independent of the cryopreservation method, membrane integrity, acrosome integrity and the percentages of dead sperm and sperms with a damaged acrosome were better for the cryopreserved mouflon sperm than the fallow deer sperm (P < 0.05). Despite exerting a more harmful effect on sperm variables than conventional freezing, ultra-rapid freezing may be a useful alternative for the cryopreservation of these species' epididymal sperm in the field, as this simple technique does not require sophisticated equipment and expertise.
本研究考察了两种冷冻死后附睾精子的方法——采用甘油短时间平衡的传统慢速冷冻法和超快速冷冻法——对野生反刍动物摩弗伦羊（欧洲盘羊）和黇鹿精子的效果。采用基于柠檬酸三钠 - 葡萄糖（TCG）+ 12%蛋黄的培养基对黇鹿精子进行传统慢速冷冻，而采用基于三羟甲基氨基甲烷 - 三羟甲基氨基甲烷 - 葡萄糖（TEST）+ 6%蛋黄的培养基对摩弗伦羊精子进行冷冻。两种培养基中甘油的最终添加浓度均为5%。超快速冷冻采用相同的稀释液，但用100 mM蔗糖替代甘油。在冷冻保存前后对精子变量（活力、存活率、顶体完整性、膜完整性和形态异常）进行了分析。虽然传统冷冻保存的精子解冻后各项指标总体上更好，但超快速冷冻精子的总精子活力（摩弗伦羊为38.40±4.44%，黇鹿为31.25±3.37%）和总活精子率（摩弗伦羊为47.19±5.18%，黇鹿为43.13±2.43%）是可以接受的。无论采用何种冷冻保存方法，冷冻保存的摩弗伦羊精子在膜完整性、顶体完整性以及死精子和顶体受损精子的百分比方面均优于黇鹿精子（P < 0.05）。尽管超快速冷冻对精子变量的有害影响比传统冷冻更大，但它可能是在野外冷冻保存这些物种附睾精子的一种有用替代方法，因为这种简单技术不需要复杂的设备和专业知识。
