83 EFFECTS OF CYTOCHALASIN B AND VITRIFICATION PROCEDURE ON SURVIVAL AND IN VITRO MATURATION OF SWAMP BUFFALO OOCYTES CRYOPRESERVED AT THE IMMATURE STAGE

Abstract

Cryopreservation of oocytes from swamp buffaloes of high genetic and individual values is important for conservation of animal genetic resources. This study investigated the effect of cytochalasin B (CB) pretreatment and the efficiencies of solid surface vitrification (SSV) and cryotop (CT) vitrification methods to cryopreserve immature buffalo oocytes. Cumulus oocyte complexes harvested from abattoir-derived ovaries were randomly separated into 5 groups: 1) CT method with pretreatment by 7.5 μg mL–1 CB in base medium (BM, TCM-199 HEPES+20% FBS) for 15 min [CT(+)CB]; 2) CT method without CB pretreatment [CT(–)CB]; 3) SSV method with pretreatment by 7.5 μg mL–1 CB in BM for 15 min [SSV(+)CB]; 4) SSV method without CB pretreatment [SSV(–)CB]; and 5) fresh control. Immature oocytes were partially denuded, and those with 2 layers of cumulus cells were placed in 10% dimethylsulfoxide (DMSO) + 10% ethylene glycol (EG) for 1 min followed by exposure to 20% DMSO + 20% EG + 0.5 M sucrose in BM for 30 s. The oocytes were then vitrified either by SSV or CT method and stored in liquid nitrogen. Vitrified oocytes were thawed by placing them directly in warming solution (0.5 M sucrose in BM) for 5 min at 38.5°C and then washed in BM for 5 min. Oocyte viability was assessed by fluorescein diacetate staining. Surviving oocytes were in vitro matured (IVM) for 23 h (Parnpai et al. 1999 Buffalo J. 15, 371–384) and their meiotic progression was investigated by nuclear staining. Data were analysed by 1-way ANOVA. The survival rate of oocytes in CT groups [CT(+)CB, 82%; CT(–)CB, 82%] was significantly lower (P < 0.05) than that of the control group (100%), but significantly higher (P < 0.05) than those in the SSV groups [SSV(+)CB, 71%; SSV(–)CB, 72%]. After IVM, the proportions of viable oocytes were similar among vitrified groups [CT(+)CB, 84%; CT(–)CB, 86%; SSV(+)CB, 80%; SSV(–)CB, 79%], but significantly lower than those of the control group (96%). Pretreatment with CB did not increase oocyte survival. Among vitrified groups the highest maturation rate was obtained in CT(–)CB (32%), which was significantly higher than that of SSV(+)CB (13%). Vitrified oocytes in all groups reached metaphase-II at significantly lower rates compared with the control group (55%). In conclusion, CB did not improve the survival and maturation of vitrified oocytes. Higher survival and maturation rates in CT groups indicate the superiority of this method. This work was supported by Thailand Research Fund, Suranaree University of Technology and Japan Society for the Promotion of Science – National Research Council of Thailand (JSPS-NRCT) scientific cooperation program.