用荧光染料（Hoechst 33342）和激光处理精子对牛卵母细胞体外受精的影响

用流动细胞计数法分离牛的Ｘ，Ｙ精子，需要对精子进行活体荧光染色及激光照射处理。本研究进行２个实验，以探索荧光染料（Ｈｏｅｃｈｓｔ３３３４２）和激光对牛精子以及牛卵母细胞的体外受精和胚胎的体外培养的影响。实验１包括４个精子处理组：（１）０处理组（对照组）；（２）染色组，用９ｍｇ／Ｌ的Ｈｏｅｃｈｓｔ３３３４２对精子进行染色；（３）激光照射组，用强度为１５０ｍＷ的激光对精子进行照射；（４）染色及激光照射组，综合（２）和（３）的处理。实验２用不同浓度的Ｈｏｅｃｈｓｔ３３３４２处理精子，以研究染料浓度与体外受精效果的关系。实验结果表明，Ｈｏｅｃｈｓｔ３３３４２作用于精子之后，能显著降低牛卵母细胞体外受精后的分裂率、囊胚率、一级囊胚率、囊胚孵化率及胚胎发育速度，而且降低的幅度随着染料浓度的升高而增加（卵裂率除外）。精子经激光照射后对体外受精的效果没有显著影响。

EFFECTS OF HOECHST 33342 AND LASER ON BOVINE IN VITRO FERTILIZATION

Hoechst 33342 and laser are necessarily employed to treat sperm when flow cytometry is used to separate sperm into X-and Y-chromosome bearing populations. Two experiments were conducted to determine the effects of dye (Hoechst 33342) and laser on X and Y sperm and their subsequent in vitro fertilization (IVF) and culture (IVC) of bovine oocytes to blastocyst (BL) and hatched BL stages. Experiment 1, four treatment groups were involved: (1) untreated sperm as control;(2) sperm stained with Hoechst 33342 at a level of 9 mg/L only; (3) unstained sperm treated with laser at a level of 150 mW only; (4) sperm treated with both Hoechst 33342 and laser. IVF with sperm of the four treatments and IVC were carried out according to the method described by Lu et al (1988). Experiment 2, different concentrations of Hoechst 33342 were used to investigate the relationship between dye concentration and the results of IVF and IVC.It was found in this study that Hoechst 33342 significantly reduced rates of cleavage,BL,grade one BL,hatched BL and BL developmental speed. These effects, except the effect on cleavage rate, were greater when the concentration of Hoechst 33342 was higher. No effect of laser was found on the results of IVF.