120 SUCCESSFUL LAMBING AFTER SUPER-COOLING ULTRARAPID VITRIFICATION SHEEP MORULAE

Abstract

The aim of this work was to establish alternative methods for sheep morulae cryopreservation by using vitrification by open pulled straw (OPS) methods and super-cooling ultra-rapid vitrification (SCURV). Both treatments used a vitrification solution (VS) of 20% (3.6 mol L^-1) ethylene glycol (EG), 20% (2.4 mol L^-1) dimethylsulfoxide (DMSO), 0.5 mol L^-1 sucrose in DPBS with 10% BSA in both methods. In our experiment we used the Vit-Master™ (MTG, 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). By surgical flushing of 24 super stimulated ewes 121 transferrable morulae were harvested; 30 morulae were transferred fresh to synchronised recipients and the others were cryopreserved by OPS (n = 49) or SCURV (n = 42), respectively thawed or warmed, and transferred to recipients. Embryos were vitrified using the OPS method. They were first incubated in 50% VS for 2 min and then transferred for 30 s into 100% VS. Each embryo was loaded by touching a 1-μL drop with the straw, which was immediately submerged into and stored in liquid nitrogen. Warming was done by placing the narrow end of the straw into DPBS + 0.25M 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 VS have been transferred by on a surface of a nylon loop (volume 20 μL, diameter 0.5 mm) and using negative pressure temperature of liquid nitrogen in the chamber for freezing with the VIT-Master. Thawing vitrified embryos was accomplished by placing the vitrified embryos in solutions of sucrose 0.25 and 0.125 with expositions of 2 and 3 min, accordingly. After embryos were thawed, only good quality embryos were transferred. Importantly, our data suggest that by using the SCURV method, the toxic elements contained in the cryopreservation solution can be reduced while maintaining a similar ability to produce viable morulae for implantation as the OPS method. Although further work on the developmental competence of embryos cryopreserved with the SCURV method are needed, these data suggest that the faster freeze rate and lower levels of cryoprotectants of SCURV are able to minimize ice crystal formation and should be further evaluated as a routine mechanism for cryopreserving sheep morulae.

Table 1.  Effect vitrification and ultra-rapid super-cooling vitrification on the viability and lambing of sheep morulae