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.