Study On The Affect Factors Of IVM,IVF And Development Of Embryo In Sheep

The aim of this study was to establish an steady and efficient IVF system for sheep oocytes maturation in vitro in our lab.Experiments were carried out to analyze the diffetent factors which been reported influence the results of IVF and to optimize the technology of IVF in sheep and establish a preliminary IVF system.Two methords,surface-follicles cutting and aspiration,were used for harvesting usable cumulus-oocyte complex(COC) from surface-visable follicles of slaughtered ewe ovaries.The results showed that the number of COC recovered per ovary by surface cutting is significantly higher than that collected by aspiration( per ovary 6.77 vs per ovary 4.08) at the short time, and the quality of oocytes by surface-follicles cutting were better than that of latter.We also studied several factors which affected the results of IVM in sheep and got these conclusions as following. First, the maturation rate of oocytes had no different by congregate culture oocytes comparied with micro-dropset culture methord( p>0.05,82.5% vs 78.6). Second,the different concentration Fetal cattle serum (FCS) and culture time were compared to study the effects of IVM. The results suggested that the maturation rate of oocytes which cultured in non-FCS culture medium had significant different comparied with the maturation rate of oocytes added FCS in culture medium (p<0.01). Besides, the maturation rate of oocytes which added 5% FCS in culture medium had also significant different compared with those which added 10%, 15% and 20% FCS (p<0.01). However,the last three groups had no difference( p>0.05).But, the maturation rate of oocytes would trend to arise while the concentration of FCS were added gradually. In addition, the maturation rate would got up with culture time postpone from 18 hr to 26 hr.Third, the maturation rate of oocytes had significant difference comparied with or without 17β-E₂ and adding 10μg/m117β-E₂ were lower than with those just add 1,2,5μg/m117 β-E₂(p<0.01).Fourth, the maturation rate of oocytes while addition of FCS or EES had no significant different to addition of FCS or estrous sheep serum (EES) in the same concentration (p>0.05).The sheep semen were processed according to the following procedure treatments:by swim up or Percoll methord.As a results, Percoll methord obtained higher cleavage rate(77.6%),and development rate of morula or blastocyst(46.1%),whereas, swim up methord were cleavage rate 64.3%,and development rate of morula or blastocyst 34.4%.There have no significent difference between two methords (p>0.05).The different concentration(0,5%,10%,20%) EES were added to cultured medium of IVF due to many paper reported that EES could increase cleavage rate. The results showed that the cleavage rate and morula or blastocyst of free-serum were all significantly lower than that of added serum(5%,10%,20%). The cleavage rate and morula or blastocyst of added 10% and 20% serum had no difference( p>0.05) except of adding 5% serum.The fertilization eggs were incubated in medium TCM199,M16 and SOF in order to compare the...