| Oocyte cryopreservation is of the great importance,and this work will be helpful for biological basic researches and practicle applications,such as in vitro fertilization,nuclear transfer and transgenic technique.Porcine oocytes are sensitive to lower temperature due to rich lipid droplets in their ooplasm.The research can also provide a reference for such species. The present study aimed firstly to evaluate toxic effects of permeable cryoprotectants.Base on this,then,the effects of permeable cryoprotectants,cytochalasin B treatment time,oocytes loading devices and delipation methods on developmental potential of the in vitro matured porcine oocytes after vification were investigated.The porcine matured oocytes were treated with 15%EG,15%DMSO and 15%ED(7.5% EG+7.5%DMSO)for 2 or 5 min,and then treated with 30%EG,30%DMSO and 30%ED (15%EG+15%DMSO)for 30 s,respectively.After that,the permeated cryoprotectants were removed,and the oocytes were parthenogenetically actived.The control oocytes were actived without cryoprotectant treatment.Results showed that when treated with 15%EG for 2 min, porcine oocytes cleavage and blastocyst rates were 93.7%and 16.0%,respectively,while the rates of cleavage and blastocyst in control oocytes were 97.2%and 20.6%,respectively.No significant difference was found between them(P＞0.05).The cleavage rate in 15%ED for 2 min was 92.05%,and had no difference from the control,but the blastocyst rate was significantly lower than that in the control(15.1%vs 20.6%;P＜0.05).The cleavage rate and blastocyst rate in 15%ED treatment of 2 min and in all the groups with cryoprotectants treatment for 5 min were significant lower than those of the control(P＜0.05).The results indicated that 15%EG treatment of 2min minimized the toxicity before porcine oocytes vitrification;and that the toxicity to porcine oocytes increased when pretreatment time prolonged from 2 min to 5 min.The oocytes were pretreated with 15%EG or 15%ED for 2 min.The control oocytes were not treated with cryoprotectants or vitrified.As a result,the oocytes morphology integrity and survival rate at the group of EG(85.9%and 87.8%)and ED(65.9%and 63.2%)had no significant difference(P＞0.05),but was lower than that in the control(100%and 98.6%) (P＜0.05).The result indicated that the effects of two cryoprotectants on porcine oocyte morphology and survival are similar.The oocytes were treated with CB for 0 min,min,30 min and 45 min respectively before treated with EG.The result showed that the morphology integrity rate,viability and cleavage rates after parthenogenetic actived had no significant difference among them(P＞0.05).But the morphology integrity rate,viability and cleavage rates in CB treatment for 30 min(82.8%,62.7%and16.4%respectively)were slightly higher than those in other groups.The CB treatment for 45 min didn't enhance the cryopreservation effect.There was no significant difference in viability and cleavage rates between OPS and hemi-straw vitrification group(P＞0.05),but all higher than those in the straw group significantly(P＜0.05).The result indicated that OPS and hemi-straw as loading devices are more suitable to porcine MⅡoocytes cryopreservation.Oocytes cleavage rates had no significant difference among the group of lipid removal by centrifugation and micro-manipulation vitrification,without lipid removal group after parthenogenetic activation(P＞0.05),but were lower than the control(fresh oocytes without vitrification)significantly(P＜0.05).Blastocyst rate after lipid removal by micro-manipulation (11.4%)was significantly higher than those in lipid removal by centrifugation and without lipid removal group(0%)(P＜0.05),and there is,however,no significant difference from the control(P＞0.05).These results indicated that cryopreservation after delipation by micro-manipulation can improve the viability of porcine MII oocytes which could develop to blastocyst stage,whereas the effect of cryopreservation can not be enhanced by centrifugation only.Our study shows that in the experiments of porcine MII oocytes vitrification,OPS as loading device,CB treatment and lipid removal by micro-manipulation,15%EG pretreatment for 2 min,30%EG treatment for 30 s play together can result in better viability.
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