An efficient method for the sanitary vitrification of bovine oocytes in straws

Background：At present,vitrification has been widely applied to humans,mice and farm animals.To improve the efficiency of vitrification in straw,bovine oocytes were used to test a new two-step vitrification method in this study.Results：When in vitro matured oocytes were exposed to 20%ethylene glycol（EG20） for 5 min and 40%ethylene glycol（EG40） for 30 s,followed by treatment with 30%glycerol（Gly30）,Gly40 or Gly50,a volume expansion was observed in Gly30 and Gly40 but not Gly50.This indicates that the intracellular osmotic pressure after a 30 s differs between EG40 and ranged between Gly40（approximately 5.6 mol/L） and Gly50（approximately 7.0 mol/L）.Since oocytes are in EG40 just for only a short period of time（30 s） and at a lower temperature（4℃）,we hypothesize that the main function of this step in to induce dehydration.Based on these results,we omitted the EG40 step,before oocytes were pretreated in EG20 for 5 min,exposed to pre-cooled（4℃） Gly50,for 30 s,and then dipped into liquid nitrogen.After warming,81.1%of the oocytes survived,and the surviving oocytes developed into cleavage stage embryos（63.5%） or blastocysts（20.0%） after parthenogenetic activation.Conclusions：These results demonstrate that in a two-step vitrification procedure,the permeability effect in the second step is not necessary.It is possible that the second step is only required to provide adequate osmotic pressure to condense the intracellular concentration of CPAs to a level required for successful vitrification.

Expression of α_(6) integrin subunit in bovine oocyte and its potential role during fertilization

Fertilization in mammals requires the successful completion of a sequence of steps, starting with the transport of gametes in the reproductive tract and ending with sperm-egg membrane fusion to produce a zygote. Although some integrin subunits are known to be associated with the plasma membrane of some mammalian oocytes and spermatozoa, the presence of α6 integrin on bovine oocytes with intact zona pellucida has not been reported. The present study was undertaken to evaluate the expression of α6 integrin subunit in bovine oocyte and to determine if in vitro binding to the zona pellucida and fertilization were affected by treating oocytes with α6 integrin subunit antibody. The α6 integrin subunit was identified on the bovine oocyte by immunocytochemistry. In vitro fertilization was significantly decreased when in vitro matured bovine oocytes were pre-incubated with α6 integrin subunit antibody at concentration 5 and 20 μg/mL, and spermoocyte binding increased. These studies demonstrated the presence of α6 integrin subunit on bovine oocyte, and its importance in fertilization and polyspermy.

Roles of trifluoperazine and verapamil in the oocyte maturation and cumulus expansion of bovine cumulus－oocyte complexes

Bovine cumulus-oocyte complexes were cultured in the maturation medium containing 4 different concentrations of verapamil and trifluoperazine to testify the necessity of extracellular Ca2+ and Ca2+-calmodulin complex for the resumption and completion of meiosis as well as cumulus expansion.Ultrastructure of the treated oocytes was also observed to investigate the cytoplasm maturation. The results showed that verapamil didn't influence the cumulus expansion,meiosis resumption and completion and cytoplasm maturation significantly.TFP inhibited cumulus expansion in a dose-dependent manner.25 μm trifluoperazine significantly inhibited the GVBD and maturation(p<0.01),wherease 1 μm TFP had no effect.Both oocytes and cumulus cells treated with 25 μm TFP severely degenerated.Our observations suggest that the resumption and completion of meiosis and cumulus expansion are Ca2+ -CaM dependent and blocking membrane Ca2+ channel does not influence oocyte germinal vesicle breakdown,nuclear and cytoplasm maturation significantly in cattle.