Cryopreservation of immature and in vitro matured porcine oocytes by solid surface vitrification.
Cryopreservation of normal, lipid-containing porcine oocytes has had limited practical success. This study used solid surface vitrification (SSV) of immature germinal vesicle (GV) and mature meiosis II (MII) porcine oocytes and evaluated the effects of pretreatment with cytochalasin B, cryoprotectant type (dimethylsulfoxide (DMSO), ethylene glycol (EG), or both), and warming method (two-step versus single-step). Oocyte survival (post-thaw) was assessed by morphological appearance, staining (3',6'-diacetyl fluorescein), nuclear maturation, and developmental capacity (after in vitro fertilization). Both GV and MII oocytes were successfully vitrified; following cryopreservation in EG, more than 60% of GV and MII stage porcine oocytes remained intact (no significant improvement with cytochalasin B pretreatment). Oocytes (GV stage) vitrified in DMSO had lower (P<0.05) nuclear maturation rates (31%) than those vitrified in EG (51%) or EG+DMSO (53%). Survival was better with two-step versus single-step dilution. Despite high survival rates, rates of cleavage (20-26%) and blastocyst formation (3-9%) were significantly lower than for non-vitrified controls (60 and 20%). In conclusion, SSV was a very simple, rapid, procedure that allowed normal, lipid-containing, GV or MII porcine oocytes to be fertilized and develop to the blastocyst stage in vitro.
Gupta
Mukesh Kumar
MK
Department of Animal Biotechnology, Bio-Organ Research Center, Konkuk University, 1 Hwayang-dong, Gwangjin-Gu, Seoul 143 701, South Korea.
Uhm
Sang Jun
SJ
Lee
Hoon Taek
HT
D000818:Animals; D002470:Cell Survival; D002970:Cleavage Stage, Ovum; D015925:Cryopreservation / Q000379:methods / Q000662:veterinary*; D003451:Cryoprotective Agents / Q000494:pharmacology; D003470:Culture Media; D003571:Cytochalasin B / Q000494:pharmacology; D005260:Female; D005307:Fertilization in Vitro / Q000662:veterinary*; D006358:Hot Temperature; D008540:Meiosis; D009865:Oocytes / Q000166:cytology / Q000187:drug effects / Q000254:growth & development*; D013552:Swine / Q000502:physiology*
D003451:Cryoprotective Agents; D003470:Culture Media; D003571:Cytochalasin B