Mao Jiude, Wu Guang-Ming, Prather Randy S, Smith Michael F, Cantley Tom, Rieke August, Didion Brad A, Day Billy N
Division of Biological Sciences, University of Missouri-Columbia, Columbia, MO 65211, USA.
Theriogenology. 2005 Dec;64(9):1913-27. doi: 10.1016/j.theriogenology.2005.03.024.
A series of experiments were carried out to develop a new method to reduce pig polyspermic fertilization and produce more normal embryos, in vitro. Experiment 1 determined the effect of methyl-beta-cyclodextrin (MCD) treatment during cryopreservation on sperm acrosome reaction and sperm fertilization. Compared to the non-MCD-treated control, MCD treatment increased the percentage of acrosome-reacted spermatozoa at thawing and 2h after incubation in fertilization medium (P<0.01). Treatment with MCD also increased (P<0.05) sperm-penetration rate, number of spermatozoa in oocytes, and fertilization efficiency in the caffeine-free fertilization medium. Experiment 2 was designed to examine the effect of withdrawal of caffeine (caffeine-free) from fertilization medium on fertilization parameters and early embryo development. Using MCD-treated spermatozoa, there was no difference in sperm-penetration rate, oocyte cleavage rate, and blastocyst formation rate between the caffeine-free and caffeine-supplemented groups. However, polyspermic fertilization rate was lower, and fertilization efficiency and blastocyst cell number were higher in the caffeine-free group compared to the caffeine-supplemented group (P<0.05). Experiment 3 studied the effect of caffeine and different concentrations of spermatozoa on fertilization parameters. Sperm-penetration rate did not differ between the caffeine-free and the caffeine-supplemented groups at different sperm concentrations. Caffeine and sperm concentration had an effect on the number of spermatozoa in oocytes and on the polyspermic fertilization rate (P<0.002). Caffeine also affected fertilization efficiency (P<0.05). In conclusion, treating spermatozoa with MCD and withdrawing caffeine from fertilization medium may provide a new method to produce a large number of normal embryos, in vitro.
进行了一系列实验，以开发一种新方法来减少猪的多精受精并在体外产生更多正常胚胎。实验1确定了冷冻保存期间甲基-β-环糊精（MCD）处理对精子顶体反应和精子受精的影响。与未用MCD处理的对照组相比，MCD处理增加了解冻时和在受精培养基中孵育2小时后顶体反应精子的百分比（P<0.01）。MCD处理还提高了（P<0.05）无咖啡因受精培养基中的精子穿透率、卵母细胞中的精子数量和受精效率。实验2旨在研究从受精培养基中去除咖啡因（无咖啡因）对受精参数和早期胚胎发育的影响。使用MCD处理的精子，无咖啡因组和添加咖啡因组之间的精子穿透率、卵母细胞裂解率和囊胚形成率没有差异。然而，与添加咖啡因组相比，无咖啡因组的多精受精率较低，受精效率和囊胚细胞数量较高（P<0.05）。实验3研究了咖啡因和不同浓度精子对受精参数的影响。在不同精子浓度下，无咖啡因组和添加咖啡因组的精子穿透率没有差异。咖啡因和精子浓度对卵母细胞中的精子数量和多精受精率有影响（P<0.002）。咖啡因也影响受精效率（P<0.05）。总之，用MCD处理精子并从受精培养基中去除咖啡因可能提供一种在体外产生大量正常胚胎的新方法。
