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

本研究旨在开发猪的瞬时-共孵化体外受精系统，以获得有效的正常单精子穿透。研究结果如下：(1)用腺苷处理精子并与咖啡因瞬时共孵育配子的效果一种新的瞬时共孵育体外受精系统，即将去皮的卵母细胞与精子在含咖啡因的培养液中共培养5~30min，然后在不含咖啡因的培养液中继续培养，将减少多精子穿透的发生率。在短暂的体外受精过程中，用腺苷预先孵化新鲜精子也可以提高单精子的穿透率。(B)在短暂的体外受精期，有效地调节精子的获能。即使在咖啡因存在的情况下，β-巯基乙醇(BME)的存在也能阻止精子获能的诱导，而H_2O_2可以诱导精子获能和自发性顶体反应。(C)在短暂的共孵育过程中，配子的氧化应激的调节在试管受精过程中补充BME，除了在有咖啡因的短暂的配子共培养期间，在维持配子的功能、正常受精的发生率以及因此，在维持配子的功能、正常受精的发生率方面都是有益的。(D)选择抗氧化剂改善低温保存的猪延长精液的功能谷胱甘肽和半胱氨酸可以提高猪精子液体保存期间的存活率和功能状态，即使在10℃的半胱氨酸存在下保存29天后，猪精子也能在体外穿透。(E)建立了瞬时体外受精系统。尽管在目前的体外卵母细胞培养系统中，经Rosovvitine预处理48h的猪卵母细胞的细胞质成熟程度不如对照卵母细胞，但这些保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.