Development of a transient co-incubation IVF system in the pig

本研究项目是为了在猪体内开发一种瞬时共孵育体外受精系统，以获得有效的正常单精子穿透。(A)用腺苷预处理精子和瞬时配子与咖啡因共孵育的效果一种新的瞬时共孵育体外受精系统，将脱光的卵母细胞与精子在含咖啡因的培养基中共培养5 ~ 30min，然后在不含咖啡因的培养基中继续培养，可以减少多精穿透的发生率。在短暂共孵育IVF之前，用腺苷预孵育新鲜精子也可以提高单精子的穿透率。-巯基乙醇（bME）的存在即使在咖啡因的存在下也能阻止精子获能的诱导，而H_2O_2则能诱导精子获能和自发顶体反应。(C)短暂共育过程中配子氧化应激的调节在体外受精过程中补充bME对维持配子的功能、正常受精的发生以及体外受精胚胎的质量有有益的作用，除了在有咖啡因存在的配子的短暂共培养期间。(D)选择抗氧化剂提高猪低温保存延长期精液的功能谷胱甘肽和半胱氨酸能提高猪精子在液体保存期间的活力和功能状态，即使在10℃半胱氨酸存在下保存29 D，猪精子也能在体外穿透。(E)瞬时体外受精（transient IVF）系统的IVM系统的建立虽然经罗斯科维汀预处理48 h的猪卵母细胞的细胞质成熟程度与目前IVM系统中的对照卵母细胞不同，但储存的卵母细胞可以在体外成熟、受精并发育到囊胚期。

This research project was undertaken to develop a transient-co-incubation IVF system in the pig, in order to obtain an efficient normal monospermic sperm penetration. Obtained sights are as follows :(A)Effect of pretreatment of spermatozoa with adenosine and a transient gamete co-incubation with caffeineA new transient co-incubation IVF system, in which denuded oocytes are co-cultured with spermatozoa in medium containing caffeine for 5 to 30 min and then continuing the culture in caffeine-free medium, will reduce the incidence of polyspermic penetration. Preincubation of fresh spermatozoa with adenosine before the transient co-incubation IVF can also improve the monospermic penetration.(B)Effective regulation of sperm capacitation during transient IVF periodThe presence of beta-mercaptoethanol (bME) prevents induction of sperm capacitation even in the presence of caffeine, whereas H_2O_2 induces sperm capacitation and the spontaneous acrosome reaction.(C)Regulation of oxidative stress of gametes during transient co-incubationSupplementation with bME during IVF procedures, except during a transient co-culture period of gametes in the presence of caffeine, has a beneficial effect in maintaining the function of gametes, the incidence of normal fertilization and, consequently, the quality of IVF embryos.(D)Selection of antioxidants to improve the function of extended boar semen stored at a low temperatureGlutathione and cysteine can improve the viability and functional status of boar spermatozoa during liquid preservation and boar spermatozoa penetrated in vitro even after preservation in the presence of cysteine at 10℃ for 29 days.(E)Development of an IVM system for the transient IVF systemAlthough cytoplasmic maturation in porcine oocytes pretreated with roscovitine for 48 h did not equal that of control oocytes in the current IVM system, those stored oocytes can matured, fertilized and developed in vitro to the blastocyst stage.