Vitrification Of Porcine Oocytes At MⅡ Stage

In order to establish an efficient, economic, and stable protocol for the vitrification of porcine oocytes, we performed four experiments to explore the effects of some key factors including cryoprotectants (CPA), loading devices and pretreatments (chemical-delipation)on in vitro development of swine matured oocytes after vitrification and warming. (1) In Experiment 1, the effect of chemical delipation on the vitrification was examined. The results showed that:â‘ As for the survival rate, no significant difference was found among the delipation group, the direct vitrification group, and centrifugal refrigeration group (P>0.05).â‘¡Pretreatment by delipation notably increased the rate of cleavage compared to the direct vitrification group and the centrifugal refrigeration (P<0.05), while similar cleavage rate was obtained in groups of direct vitrification and centrifugal vitrification group (P>0.05).â‘¢the delipation group remarkably improved the blastocyst rate compared to the directc vitrification group (P<0.05), but no significant difference was observed between delipation group and the centrifugal refrigeration group (P>0.05). These results indicate that delipation before vitrification is helpful to the subsequent development of porcine matured oocytes.(2)The effect of OPS, Cryotop, and self-made carriers (Cryotip) on the vitrification of porcine MII oocytes was investigated. The results showed that similar survival rates, cleavage rates, and blastocyst rates were obtained from oocytes vitrified/warmed by using Cryotip, OPS(control group 1) or Cryotop(control group 2) as loading devices (P>0.05). Thusfore cryotip can replace expensive commercial carriers in doing porcine oocyte vitrification.(3)The effect of combinations of CPAs, such as ES1+VS1, ES2+VS2, ES2+VS3, and ES2+VS4 on the procine MII oocytes vitrification was explored. Results showed that:â‘ for the rates of survival, and cleavage, no significant difference was found among four groups (P>0.05).â‘¡obvious differences were observed among ES2+VS2, ES2+VS3 and ES1+VS1 (control group) regarding to the blastocyst rate (P<0.05). These results demostrate that when EG act as CPA at certain higher concentration, similar post vitrification/warming develomental ability can be obtained in procine matured oocytes.(4) The effect of different sugar CPAs on procine MII oocytes vitrification was evaluated. The results showed that:â‘ among trehalose, ficoll 400 and sucrose (control group), similar survival rate, cleavage rate or blastocyst rate was observed respectively (P>0.05).â‘¡vitrification by using trehalose and sucrose as protective agent, blastocysts were exclusively obtained, but not in the Ficollg roup. Results suggest that the use of trehalose in vitrification of procine MII oocytes is feasible.In summary, use Cryotop or cryotip as vitrification carriers and EG+DMSO+ sucrose or trehalose as CPAs, and pretreatment by delipation, an efficient, economical, simple and stable vitrification/warming protocol in porcine matured oocytes is sccessfully established.