Pradiee J, Esteso M C, Lopez-Sebastián A, Toledano-Díaz A, Castaño C, Carrizosa J A, Urrutia B, Santiago-Moreno J
Departamento de Reproducción Animal, SGIT-INIA, Madrid, Spain; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brasilia DF, Brazil.
Departamento de Reproducción Animal, SGIT-INIA, Madrid, Spain.
Theriogenology. 2015 Dec;84(9):1513-22. doi: 10.1016/j.theriogenology.2015.07.036. Epub 2015 Aug 3.
A method for cryopreserving wild ibex sperm at high cooling rates was developed. To design a freezing solution based on Tris, citric acid, and glucose (TCG), two preliminary experiments were performed using glycerol (GLY) and dimethyl sulfoxide (DMSO) at different concentrations (5%, 10%, 20%). The 10% GLY + 10% DMSO combination reduced (P < 0.05) frozen-thawed sperm motility, which reached a minimum when 20% GLY + 20% DMSO was used. In the second experiment, sperm tolerance to three sucrose concentrations was evaluated (100-mM sucrose, 300-mM sucrose, 500-mM sucrose). Frozen-thawed sperm motility and sperm viability decreased (P < 0.05) at concentrations above 300 mM. The ultrarapid cooling procedure finally used involved a TCG egg yolk (ey)-based extender with 100-mM sucrose, either alone or with 5% GLY with or without BSA. Two warming procedures (37 °C vs. 60 °C) were also evaluated. The TCG ey with 100-mM sucrose but without GLY/BSA returned the best sperm quality variables. Slow warming at 37 °C strongly affected (P < 0.05) sperm motility and viability in all groups. Sperm selection by density gradient centrifugation produced no motile sperm when slow warming was performed. In contrast, when fast warming was used, sperm selection increased (P < 0.05) percentage of motility, viability, and the percentage of sperms with intact acrosomes. Heterologous in vivo fertilization involving domestic goats was performed to evaluate the in vivo fertilization capacity of the ultrarapidly cooled cryopreserved sperm (in TCG-ey + 100 mM sucrose), with warming undertaken at 60 °C. Inseminations of domestic goats resulted in three pregnancies (3 of 16, 18.7% fertility). In conclusion, ibex spermatozoa are strongly sensitive to high concentrations of permeable cryoprotectants and sucrose. However, the combination of ultrarapid cooling, using TCG-ey + 100-mM sucrose, and fast warming at 60 °C, followed by sperm selection by density gradient centrifugation to collect the motile sperm, has a positive effect on sperm viability.
开发了一种以高冷却速率冷冻野生北山羊精子的方法。为了设计一种基于Tris、柠檬酸和葡萄糖（TCG）的冷冻溶液，进行了两项初步实验，使用不同浓度（5%、10%、20%）的甘油（GLY）和二甲基亚砜（DMSO）。10% GLY + 10% DMSO组合降低了（P < 0.05）冻融后精子活力，当使用20% GLY + 20% DMSO时活力降至最低。在第二项实验中，评估了精子对三种蔗糖浓度（100 mM蔗糖、300 mM蔗糖、500 mM蔗糖）的耐受性。浓度高于300 mM时，冻融后精子活力和精子存活率降低（P < 0.05）。最终使用的超快速冷却程序涉及一种基于TCG蛋黄（ey）的稀释液，含有100 mM蔗糖，单独使用或与5% GLY一起使用，有或没有牛血清白蛋白（BSA）。还评估了两种升温程序（37°C与60°C）。含有100 mM蔗糖但没有GLY/BSA的TCG ey产生了最佳的精子质量变量。在所有组中，37°C缓慢升温对精子活力和存活率有强烈影响（P < 0.05）。进行缓慢升温时，通过密度梯度离心法选择精子未产生活动精子。相比之下，当使用快速升温时，精子选择提高了（P < 0.05）活力百分比、存活率以及顶体完整精子的百分比。进行了涉及家山羊的异种体内受精，以评估超快速冷却冷冻保存精子（在TCG-ey + 100 mM蔗糖中）在60°C升温时的体内受精能力。对家山羊进行授精导致三次怀孕（16次中有3次，受孕率18.7%）。总之，北山羊精子对高浓度的渗透性冷冻保护剂和蔗糖非常敏感。然而，使用TCG-ey + 100 mM蔗糖进行超快速冷却，然后在60°C快速升温，接着通过密度梯度离心法选择活动精子来收集精子，对精子存活率有积极影响。
