Sandfoss Mark R, Cantrell Jessica, Roberts Beth M, Reichling Steve
Conservation and Research Department, Memphis Zoo, Memphis, TN 38112, USA.
Animals (Basel). 2022 Jul 17;12(14):1824. doi: 10.3390/ani12141824.
Cryopreservation of sperm to preserve the genetic diversity of declining populations is a promising technique to aid in the recovery of endangered species such as the Louisiana pinesnake (). However, this technique has been performed on only a handful of snake species and with limited success. Here, we tested a cryoprotective agent (CPA) mixture containing Lake's buffer with 10% ,-dimethyl formamide (DMF), 2% methanol, 5% clarified egg yolk, (/% final concentration) against 16 other CPA-treatment mixtures. These contained either Lake's buffer or TEST egg yolk buffer as the base diluent with a penetrating or non-penetrating CPA on the post-thaw recovery of sperm motility and viability. We also investigated the effect of post-thaw incubation treatment in TL HEPES supplemented with 10% fetal bovine serum (H10) alone or with caffeine on post-thaw motility parameters. Sperm from 16 Louisiana pinesnakes was cryopreserved, and the effectiveness of the CPA treatment mixtures and post-thaw treatments was determined based on measurements of sperm motility and viability. Sperm cryopreservation significantly reduced initial post-thaw sperm quality for all of the extender treatments. Viability of sperm was best maintained when cryopreserved in an CPA treatment mixture containing Lake's buffer with 10% DMF, 2% methanol, and 5% clarified egg yolk with the addition of 5 mg/mL bovine serum albumin (BSA). For several extender mixtures a similar percent of post-thaw motility was observed, but no forward motility returned in any post-thaw samples prior to incubation in dilution treatments. Following incubation in both post-thaw treatments, the percent of forward motility and the index of forward progressive movement improved significantly. Post-thaw dilution with H10 containing caffeine improved motility parameters over H10 alone, suggesting further investigation of post-thaw treatment in caffeine could be beneficial. Although, cryopreservation of sperm from the Louisiana pinesnake continues to present a challenge, post-thaw dilution and the addition of BSA to CPA mixtures provides areas for improving cryopreservation methods for this endangered species.
冷冻保存精子以保护数量不断减少的种群的遗传多样性，是一种很有前景的技术，有助于恢复濒危物种，如路易斯安那松蛇（此处原文括号内内容缺失）。然而，这项技术仅在少数蛇类物种上进行过，且成功率有限。在这里，我们测试了一种冷冻保护剂（CPA）混合物，它含有莱克氏缓冲液以及10%的二甲基甲酰胺（DMF）、2%的甲醇、5%的澄清蛋黄（最终浓度为/%），并与其他16种CPA处理混合物进行了对比。这些混合物要么以莱克氏缓冲液要么以TEST蛋黄缓冲液作为基础稀释剂，同时含有一种渗透性或非渗透性CPA，用于研究其对解冻后精子活力和生存能力的恢复情况。我们还研究了在仅添加10%胎牛血清（H10）或添加咖啡因的TL HEPES中进行解冻后孵育处理，对解冻后活力参数的影响。对16条路易斯安那松蛇的精子进行了冷冻保存，并根据精子活力和生存能力的测量结果，确定了CPA处理混合物和解冻后处理的有效性。对于所有的稀释剂处理，精子冷冻保存均显著降低了解冻后的初始精子质量。当精子在含有莱克氏缓冲液、10% DMF、2%甲醇、5%澄清蛋黄并添加5毫克/毫升牛血清白蛋白（BSA）的CPA处理混合物中冷冻保存时，精子的生存能力得到了最佳维持。对于几种稀释剂混合物，观察到解冻后活力的百分比相似，但在进行稀释处理孵育之前，任何解冻后样本中均未恢复向前运动能力。在两种解冻后处理中孵育后，向前运动能力的百分比和向前渐进运动指数均显著提高。与仅使用H10相比，在含咖啡因的H10中进行解冻后稀释可改善活力参数，这表明进一步研究咖啡因在解冻后处理中的作用可能是有益的。尽管如此，路易斯安那松蛇精子的冷冻保存仍然是一项挑战，但解冻后稀释以及在CPA混合物中添加BSA为改进这种濒危物种的冷冻保存方法提供了方向。
