Departamento de Reproducción Animal, INIA, 28040, Madrid, Spain; Facultad de Ciencias Agropecuarias, Universidad de Cuenca, EC010205, Cuenca, Ecuador.
Departamento de Reproducción Animal, INIA, 28040, Madrid, Spain.
Theriogenology. 2019 Jul 15;133:29-37. doi: 10.1016/j.theriogenology.2019.04.027. Epub 2019 Apr 25.
The poor fertility of ram semen stored chilled for long periods has encouraged the development of protocols designed to improve the kinetic vigour and cervical barrier-crossing capacity of sperm. The present work evaluated the effect of sperm selection with Sephadex filtration and the supplementation of 2% glycerol (GLY) to extenders based on ultra-heat-treated skimmed milk (UHT) or Tris-Tes-Glucose (TEST) on ram sperm kinetic parameters, plasma membrane integrity, acrosome integrity, mitochondrial function and fertilizing ability, over long chilling times. The results showed that for non-filtered semen, values for progressive sperm motility (%PSM), straight line velocity (VSL, μm/s) and the percentage of sperm with an intact plasma membrane/intact acrosome/a high mitochondrial function index (%IPIAHM) at all times up to 96 h of chilling were higher when the UHT extender (P < 0.01) was used compared to TEST extender irrespective of the presence of GLY. When semen was previously filtered with Sephadex, the addition of GLY to the UHT extender improved total motility (%TM), the %PSM and the VSL at 96 h compared to all other treatments (P < 0.01). The best results of all were obtained with non-filtered semen and UHT either with or without GLY. Heterologous IVF using zona-intact bovine oocytes was used to assess the fertilizing capacity of non-filtered fresh (FS0), chilled-for-24 h (CS24) or chilled-for-48 h (CS48) ram semen diluted in UHT extender (GLY-free). Heterologous IVF showed that ram sperm, either FS0, CS24 or CS48, were equally capable of penetrating zona pellucida intact bovine oocytes, leading to pronuclear formation and hybrid embryo cleavage (46.3 ± 3.2; 48.8 ± 3.2; and 43.3 ± 3.5, respectively). No differences were seen with respect to fresh sperm in terms of sperm binding, penetration, polyspermy, pronucleus formation or cleavage rates (P > 0.05). In conclusion, neither Sephadex filtration nor addition of glycerol provided extra benefits to ram sperm chilled up to 96 h. Chilled, non-filtered sperm extended with UHT without GLY showed better sperm functionality than did similar sperm extended with TEST extenders. Indeed, sperm diluted in UHT extender, maintained fertilizing ability up to 48 h.
长期冷藏会降低公羊精液的受精能力，因此人们开发了一些方法来提高精子的活力和穿透宫颈的能力。本研究评估了使用葡聚糖凝胶过滤法（Sephadex filtration）选择精子和在以超高温处理脱脂乳（UHT）或 Tris-Tes-Glucose（TEST）为基础的稀释液中添加 2%甘油（GLY）对精子动力学参数、质膜完整性、顶体完整性、线粒体功能和受精能力的影响，研究的最长冷藏时间为 96 小时。结果表明，在非过滤精液中，无论是否添加 GLY，UHT 稀释液（P＜0.01）在 96 小时内对直线速度（VSL，μm/s）、质膜完整/顶体完整/高线粒体功能指数（%IPIAHM）的百分率、总运动精子（%TM）和精子活力都有显著提高；而当精液经 Sephadex 过滤后，UHT 稀释液添加 GLY 能提高总运动精子（%TM）、直线速度（VSL）和 96 小时时的 %PSM，与其他处理相比有显著差异（P＜0.01）。未经过滤且未经过滤的精液与 UHT 稀释液结合，无论是否添加 GLY，效果最好。非过滤的新鲜精液（FS0）、冷藏 24 小时（CS24）或冷藏 48 小时（CS48）的公羊精子在 UHT 稀释液（无 GLY）中与牛卵母细胞体外受精（heterologous IVF），评估精子的受精能力。结果显示，FS0、CS24 和 CS48 精子都能穿透完整的牛卵透明带，形成原核和胚胎卵裂（分别为 46.3±3.2；48.8±3.2；43.3±3.5）。在精子结合、穿透、多精受精、原核形成或卵裂率方面，FS0 精子与新鲜精子没有差异（P＞0.05）。总之，Sephadex 过滤或添加甘油都不能提高公羊精子的活力，冷藏 96 小时内精子活力没有明显提高。非过滤的、未经处理的公羊精子用 UHT 稀释，不添加 GLY，其精子功能优于用 TEST 稀释液处理的精子。实际上，UHT 稀释液中稀释的精子在 48 小时内仍保持受精能力。
