Pradiee J, Esteso M C, Castaño C, Toledano-Díaz A, Lopez-Sebastián A, Guerra R, Santiago-Moreno J
Departamento de Reproducción Animal, INIA, Madrid, Spain.
Conselho Nacional de Desenvolvimento Cientifico e Tecnológico - Cnpq, Brasilia, Brasil.
Andrologia. 2017 Apr;49(3). doi: 10.1111/and.12629. Epub 2016 Jul 4.
This work examines the effectiveness of a TCG (Tris, citric acid, glucose, 6% egg yolk and 5% glycerol) and a TEST (TES, Tris, glucose, 6% egg yolk and 5% glycerol) sperm extender in the freezing of mouflon spermatozoa at slow cooling rates, using different pre-freezing equilibration times (2-3 hr). It also examines the tolerance of mouflon spermatozoa to different concentrations of cryoprotectants (5, 10, 20% glycerol; 5%, 10%, 20% dimethyl sulfoxide; 6% polyvinylpyrrolidone) and/or sucrose (100, 300, 500 mm). The highest quality (p < .01) thawed spermatozoa were obtained when using the TEST extender and an equilibration time of 3 hr. Sperm motility and membrane integrity were strongly reduced when using rapid freezing rates (60-85°C min ), independent of the concentration of cryoprotectants. The lowest sucrose concentration (100 mm) provided the highest (p < .05) percentage of motile spermatozoa and live spermatozoa with an intact acrosome. Vitrified-warmed sperm variables were at their best when the spermatozoa was diluted in TCG-6% egg yolk + 100 mm sucrose and warmed at 60°C. Slow warming at 37°C strongly reduced (p < .05) sperm motility and viability. However, sperm vitrification returned lower fertility, sperm motility and sperm viability values than conventional sperm freezing.
本研究考察了TCG（三羟甲基氨基甲烷、柠檬酸、葡萄糖、6%蛋黄和5%甘油）和TEST（TES、三羟甲基氨基甲烷、葡萄糖、6%蛋黄和5%甘油）两种精液稀释液在缓慢冷却速率下冷冻摩弗伦羊精子时的效果，采用了不同的预冻平衡时间（2 - 3小时）。研究还考察了摩弗伦羊精子对不同浓度冷冻保护剂（5%、10%、20%甘油；5%、10%、20%二甲亚砜；6%聚乙烯吡咯烷酮）和/或蔗糖（100、300、500 mM）的耐受性。使用TEST稀释液和3小时平衡时间时，解冻后精子质量最高（p < 0.01）。当采用快速冷冻速率（60 - 85°C/分钟）时，精子活力和膜完整性显著降低，与冷冻保护剂浓度无关。最低蔗糖浓度（100 mM）时，具有完整顶体的活动精子和活精子的百分比最高（p < 0.05）。当精子在TCG - 6%蛋黄 + 100 mM蔗糖中稀释并在60°C升温时，玻璃化复温后的精子各项指标最佳。在37°C缓慢升温会显著降低（p < 0.05）精子活力和存活率。然而，精子玻璃化冷冻后的生育力、精子活力和精子存活率值低于传统精子冷冻。
