Studies On Fertilization In Vitro Of The Poreine Ooeytes

This study has taken abandoned porcine ovarian from slaughterhouse as the original materials, obtained immature porcine oocytes via mechanical extraction methods and utilized them in IVF( In Vitro Fertilization) experiments after they were maturation in vitro culture. This thesis systematically compared influencing factors of porcine ovarian IVF, attempting to further improve the pig embryo in vitro production system and provide technical support for the further development of the embryo technique.Study contents included: 1) Compared influences of adding different concentration of caffeine in m TBM, different fertilization time and sperm flaoting time to porcine IVF. 2) Compared influences of different maturation time of porcine oocytes in vitro, existence situations of granulosa cells in fertilization process and hypoxia environment to porcine IVF 3) studied effects of adding different concentration of Ionomycin and Ionomycin to sperm capacitation liquid on porcine IVF.The study results indicate that:1) Adding caffeine significantly accelerated the cleavage and blastocyst of fertilized embryos, cleavage in group of adding 3mmol/L caffeine markedly higher than that of 5mmol/L group and none-adding caffeine group(P<0.05), and its blastocyst is the highest; cleavage and blastocyst in sperm floatation processing 1h group obviously higher than other groups(P<0.05); Time of incubation of oocytes and sperm doesn’t have significant effects(P >0.05), with the fertilizing time extended to 9h, blastocyst signally decreased. Through comparing all experimental groups it is showed that add 3mmol/L caffeine in fertilization liquid, float sperm for 1h and be fertilized for 6h can obtain preferable IVF results.2) The cleavage and blastocyst of fertilized embryos in IVM(in vitro maturation) 40-42 h group is dramatically higher than that of 36~38h and 46~48h group(P<0.05); groups of reserved 2-3 layers of granule cells and not taken off the granulosa cells not significantly different(P>0.05), while these teo groups are obviously higher than none- granule cells group(P<0.05). In terms of cleavage and blastocyst, 5% CO2 group is higher than 10% and 15% group. Comparing results showed that culture porcine oocytes in vitro for 40-42 h, reserve all the granule cells and keep 5% CO2 in air can obtain preferable IVF results.3) Adding 10μmol/L Ionomycin in capacitation fluid can markedly increased the cleavage and blastocyst of fertilized embryos P<0.05). processing sperms with Ionomycin for 30 min can obtain preferable IVF results.