Vitrification Of Porcine Oocytes At Stage Germinal Vesicle

The cryopreserved mammalian oocytes have been increasingly used in embryo biotechnologies,such as in vitro fertilization,nuclear transfer and transgene.The cryopreservation of oocytes can facilitate the transportation and,therefore,the research work.Nowadays,it has been a widely used basic biology.Porcine oocyte cryoperservation is of the great importance in both husbandary and human clinics.On the other hand,porcine oocyte cryopreservation has got less progress than that of other mammalian oocytes.Porcine oocytes are highly sensitive to low temperature stress due to the rich lipid droplets in their ooplasm.It is necessary to carry out more researches in order to optimize porcine oocytes cryopreservation.The vitrification is a recently developed method for oocyte cryopreservation.It has quite a few advantages. For instance,it is simple,handy,fast and effective without expensive instruments.The present study was to investigate the vitrification of porcine oocytes at germinal vesicle (GV) stage,including the effects of equilibration time in vitrification solution before cryopreservation,and the effects of cumulus cell existence carriers of oocytes on the survival rate and further developmental potential of thawed oocytes.In order to choose the appropriate equilibration time of oocytes in vitrification solution before cryopreservation,fresh porcine GV oocytes were treated with 10% and then 20%ethylene glycol(EG) for 1 min and 1 min(totally 2 min) respectively, or 3 min and 2 min(totally 5 min) respectively.And then all the oocytes were treated with 30%EG solution for 30 sec.Afterwards,the treated oocytes were moved into the Hemi-straws,and were cryopreserved in liquid nitrogen directly.The survival rate and further developmental potential of the thawed oocytes were examined after thawing and then in vitro maturation for 44 h.The oocytes were treated with hyaluronidase (HE) to remove the cumulus cells around oocytes,and then were stained by trypan blue to evaluate the survival of the oocytes.The oocytes with first polar body(PB1) were regarded as mature oocytes.The results showed that the survival rates of thawed porcine oocytes equiliberated in 10%and 20%EG for 2 min and 5 min were 51.33% and 45.16%respectively,which had no significant difference(P＞0.05).The maturation rate of oocytes equilibrated in EG for 5 min was significantly higher than that for 2 min after in vitro maturation(10.48%vs 3.54%,P＜0.05).To test the effects of the number of cumulus cell layer around oocyte on the survival and maturation of frozen-thawed oocytes,the fresh GV oocytes with different layer of cumulus cells(multiple layers and two layers) were treated with 10%,20% and 30%EG for 3 min,2 min and 30s respectively followed by cryopreservation. Then their survival and maturation of the thawed oocytes were evaluated after staining and in vitro maturation.The fresh porcine GV oocytes were directly matured in vitro without cryoperservation as control group.The results showed that the oocytes enclosed with two layers of cumulus cells had a significantly higher survival rate that those enclossed with two layers(42.11%vs 18.18%,P＜0.01).In contrast,the maturation rates didn't differ significantly(9.77%vs 4.55%,P＞0.05).It indicated that the removal some cumulus cells around oocyte before vitrified cryopreservation may reduce the damage of vitrification.The survival rates in control group were 98.10% and 94.33%,respectively while the maturation rates in control group were 69.43% and 68.79%,respectively.Both the survival and maturation rates in control group were most significant higher than those of the couterparts(P＜0.01),which indicated the cryopreservation injury against porcine GV oocytes.The third experiment was undertaken to investigate the effects of cryopreserved oocyte carriers on the survival and further developmental potential of the thawed oocytes.Two kinds of carriers of porcine oocytes,namely,open pulled straw(OPS) and Hemi-straw,were used with the same procedures of equiliberation, cryopreservation,thawing and maturation culture.The results showed that the survival rate of the oocytes in OPS and Hemi-straw were 51.47%and 55.41%, respectively after in vitro maturation,and they had no significant difference (P＞0.05).The maturation rates didn't have NO statistical difference was found for maturation rate either(8.82%vs 10.81%,P＞0.05).It was considered that both of OPS and Hemi-straw be used as the carriers of vitrified cryopreservation of porcine oocyte at stage of GV and Hemi-straw be a little better than OPS.It was concluded that the procedure of cryopreservation of porcine GV oocytes be as follows:to remove part of cumulus cells around fresh oocytes,treat oocytes with 10%,20%and 30%EG for 3 min,2 min and 30 s respectively,use Hemi-straw as cryopreservation carrier and cryopreserve oocytes in liquid nitrogen.