44 SUCCESSFUL KIDDING AFTER ULTRARAPID VITRIFICATION OF GOAT EMBRYOS

Abstract

This work evaluated methods for goat morulae cryopreservation by using cryoloop: vitrification (V) and super-cooling ultra-rapid vitrification (SCURV). The vitrification method was applied according to the method described by Vajta et al. (1998 Mol. Reprod. Dev. 51, 53–58). Both treatments used a vitrification solution [VS: 20% (3.6 mol L⁻¹), ethylene glycol (EG), 20% (2.4 mol L⁻¹) dimethylsulfoxide (Me₂SO)] 0.5 mol L⁻¹ sucrose in DPBS with 10% BSA in both methods. In our experiment, we used the Vit-Master™ (MTG, Bruckberg, Germany). The super-cooled LN facilitates heat transmission between LN and the cryosolution interface and this is efficient for bovine semen and blastocyst cryoconservation (Arav et al. 2002 Mol. Cell. Endocrinol. 187, 77–81). By surgical flushing 25 super stimulated goats, 127 transferable morulae were harvested; 39 morulae were transferred fresh to synchronized recipients (control) and the others were cryopreserved by V (n = 46) or SCURV (n = 42), respectively thawed or warmed, and transferred to recipients. Embryos were vitrified using the cryoloop. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by cryoloop, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the cryoloop into DPBS + 0.25 M sucrose for 5 min. Embryos were then transferred into DPBS + 0.125 M sucrose for 3 min and finally to DPBS until transfer. The SCURV morulae were then exposed to 50% and 100% VS at 37°C for 2 min and 30 s, respectively. Embryos after saturation in VS were transferred by cryoloop and using negative pressure of liquid nitrogen in the chamber for freezing with the VIT-Master. Thawing vitrify embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 M and 0.125 M with 2- and 3-min exposures accordingly. After thawing, embryos were transferred. Statistical analysis was done using Student’s test. The kidding rate following transfer of fresh, frozen-thawed vitrification, and SCURV methods were 22, 16, and 16 kids, respectively. No statistical difference was found for the percentage of does kidding following transfer thawed after vitrification (34.7 ± 4.5%^a), and SCURV methods (38.1 ± 5.9%^b). The survival rate following transfer of fresh embryos (56.4 ± 4.9^c) was higher and in line with previous findings using VS. Differences were statistically significant (^{ac, bc} P < 0.05). Importantly, our data suggested that the SCURV method can be used for cryopreservation of goat morulae and has similar success to the vitrification method. While further work on the developmental competence of embryos cryopreserved with the SCURV method is needed, we hypothesise that SCURV, with a faster freeze rate and potentially a lower level of cryoprotectants, may be able to minimize ice crystal formation; SCURV should be further evaluated as a routine mechanism for cryopreserving goat embryos.