Van den Berghe Femke, Paris Monique Christina Johanna, Briggs Michael Brent, Farstad Wenche Kristin, Paris Damien Boyd Bertrand Paul
Discipline of Biomedical Science, College of Public Health, Medical and Veterinary Sciences, James Cook University, Solander Drive, Townsville, QLD 4811, Australia; Institute for Breeding Rare and Endangered African Mammals (IBREAM), 9 Ainslie Place, Edinburgh, EH3 6AT, Scotland, UK.
Discipline of Biomedical Science, College of Public Health, Medical and Veterinary Sciences, James Cook University, Solander Drive, Townsville, QLD 4811, Australia; Institute for Breeding Rare and Endangered African Mammals (IBREAM), 9 Ainslie Place, Edinburgh, EH3 6AT, Scotland, UK; Wageningen Livestock Research, De Elst 1, 6708, WD Wageningen, The Netherlands.
Cryobiology. 2018 Feb;80:18-25. doi: 10.1016/j.cryobiol.2017.12.095. Epub 2017 Dec 27.
Conservation management of endangered African wild dogs (AWD; Lycaon pictus) can benefit greatly from development of sperm freezing and artificial insemination. Previous freezing attempts yielded nearly 0% motile sperm within 2 h of thawing. In this study, two canine freezing protocols were tested: Protocol 1: a one-step dilution in TRIS-20% egg yolk containing 8% glycerol; and Protocol 2: a two-step dilution in TRIS-20% egg yolk containing a final extender concentration of 5% glycerol and 0.5% Equex STM, coupled with a TRIS-citrate-fructose thawing solution. Semen was collected by electroejaculation from n = 24 AWDs, of which eight ejaculates of sufficient quality (four good quality with initial sperm motility of 75.0 ± 4.4% and four poor quality; showing rapid decrease in sperm motility to 3.3 ± 3.3% prior to freezing) were frozen. For good quality samples, motility and sperm motility index persisted for up to 8 h for Protocol 2, and was higher between 2 and 6 h after thawing with a decrease from 4 h of incubation. Motility dropped to nearly 0% after 2 h incubation for Protocol 1. Viability was higher for Protocol 2 throughout the 8 h of incubation, with a decrease after 6 h, compared to 4 h for Protocol 1. Acrosome integrity was higher for Protocol 2 throughout post-thaw incubation, with a decrease after 2 h for both protocols. Protocols did not differ in normal sperm morphology or DNA integrity. Poor quality samples yielded similar results, except for acrosome integrity, which declined for Protocol 2. In conclusion, a two-step dilution in TRIS-egg yolk-glycerol extender containing Equex STM yields significantly improved post-thaw quality and longevity of AWD spermatozoa, making it suitable for sperm banking and artificial insemination initiatives.
濒危非洲野犬（AWD；非洲野犬属三色豺）的保护管理可从精子冷冻和人工授精技术的发展中大大受益。此前的冷冻尝试在解冻后2小时内可游动精子的比例几乎为0%。在本研究中，测试了两种犬类冷冻方案：方案1：在含有8%甘油的TRIS-20%蛋黄中进行一步稀释；方案2：在含有最终稀释剂浓度为5%甘油和0.5% Equex STM的TRIS-20%蛋黄中进行两步稀释，并使用TRIS-柠檬酸盐-果糖解冻溶液。通过电刺激法从n = 24只非洲野犬采集精液，其中8份质量足够的射精样本（4份质量良好，初始精子活力为75.0±4.4%，4份质量较差；冷冻前精子活力迅速下降至3.3±3.3%）被冷冻。对于质量良好的样本，方案2的精子活力和精子活力指数在长达8小时内持续存在，解冻后2至6小时更高，并在孵育4小时后开始下降。方案1在孵育2小时后精子活力降至几乎0%。在整个8小时的孵育过程中，方案2的精子存活率更高，在6小时后下降，而方案1在4小时后下降。在解冻后的整个孵育过程中，方案2的顶体完整性更高，两种方案在2小时后均有所下降。两种方案在正常精子形态或DNA完整性方面没有差异。质量较差的样本产生了类似的结果，但方案2的顶体完整性有所下降。总之，在含有Equex STM的TRIS-蛋黄-甘油稀释剂中进行两步稀释可显著提高非洲野犬精子解冻后的质量和寿命，使其适用于精子库保存和人工授精计划。
