155 EFFECT OF VITRIFICATION OF MORULAE ON PREGNANCY RATE IN GOAT

Abstract

This work evaluated alternative methods for goat morulae cryopreservation by using the High Security Vitrification Kit (Cryobiosystem): vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). Vitrification was applied according to the method described by Vajta et al. (1998). Both treatments used a vitrification solution (VS) containing 20% ethylene glycol (EG), 20% dimethylsulfoxide (Me2SO), 0.5 mol L^–1 sucrose in DPBS with 10% BSA. In our experiment we used the Vit-Master™ (MTG, Germany). Super-cooled liquid nitrogen (LN) facilitates heat transmission between LN and the cryosolution interface suggested to be beneficial for bovine semen and blastocyst cryoconservation. By surgical flushing of 30 super-stimulated goats, 137 transferable morulae were harvested; 41 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 47) or SCURV (n = 49), respectively thawed, and transferred to recipients. Embryos were vitrified using the HSV Kit. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS followed by vitrification (group V). Accordingly, morula of SCURV group were exposed to 50% VS for 2 min and to 100% VS for 30 s at 37°C. Thereafter, embryos were transferred into the VIT-Master for freezing with liquid nitrogen using negative pressure. Thawing of vitrified embryos was accomplished by placing the vitrified embryos in solutions of 0.25 M sucrose for 2 min and 0.125 M sucrose for 3 min, respectively. After thawing only survived embryos were transferred. Statistical analyses were performed with Student's t-test. After transfer of fresh or frozen-thawed embryos of V and SCURV groups, 25, 17, and 19 kids were born. No statistical difference was found for the percentage of viability of thawed embryos after vitrification (36.2 ± 4.4%), and SCURV methods (38.7 ± 6.5%). The survival of fresh embryos, however, was significantly higher (60.9 ± 5.3%). Differences were statistically significant (P < 0.05). Importantly, our data suggest that the SCURV method can be used for cryopresevation of goat morulae. Nevertheless, further work regarding the developmental competence of embryos cryopreserved with the SCURV method is needed.