41 Vitrification of bovine in vitro-produced embryos: can it replace slow freezing in bovines?

Cryopreservation of embryos has allowed for the sharing of precious germplasm across the globe, significantly minimising the risk of disease transmission. The only challenge with this approach is that cryopreserved embryos should result in pregnancies. It has become evident from different studies that in vitro-produced (IVP) embryos are less cryotolerant than their in vivo counterparts (IVD). This led to the quest to find an alternate method of cryopreservation, particularly for in vitro embryos (Nedambale et al. 2004). Among available alternatives, vitrification has so far been explored by different research groups, but its effectiveness in bovines is still being debated. However, vitrification has now become the most preferred cryopreservation method in the human embryo industry. The present study was, therefore, undertaken to observe the effectiveness of vitrification of bovine embryos over slow freezing. Bovine embryos were produced from Holstein-Friesian cross-bred animals through ovum pickup (OPU) following the standard in vitro embryo production (OPU-IVEP) process. Only blastocysts and expanded blastocysts of grade I, as per IETS, were considered for the present study. The embryos produced through OPU-IVEP were subjected to different slow-freezing protocols (post-seeding cooling rates of 0.3°C min⁻¹ and 0.5°C min⁻¹) and vitrification. 30 embryos were considered in each group. Vitrification was performed using high security vitrification (HSV) straws, which is a closed method of vitrification. The embryos, cryopreserved through different methods, were then subjected to thawing and culture for observing re-expansion and hatching rate in in vitro culture media. Embryos were observed at 0 h, 2 h, 24 h, and 48 h for viability, re-expansion, and hatching. Blastocyst survival was defined according to the number of viable cells observed in each blastocyst after warming through morphological evaluation of OPU-IVEP embryos. The blastocyst was defined as “survived” if more than 50% of the cells were noted viable, i.e. grade I, as per IETS. A comparison between each group was performed using Fisher’s exact test for comparing 3 × 2 contingency tables. The groups were considered significantly different when P < 0.05 was used. The vitrified group had significantly higher (P < 0.05) viable blastocysts observed (93.3%) compared with slow freezing with a rate of 0.5°C min⁻¹ (79.2%) or 0.3°C min⁻¹ (77.3%). In accordance to the viability, significantly (P < 0.05) higher re-expansion was observed in the vitrified group (90.0%) compared with slow frozen at a rate of 0.5°C min⁻¹ (62.5%) or 0.3°C min⁻¹ (59.1%). Also, the hatching rate was higher in the vitrified group (63.3%) compared with slow frozen at a rate of 0.5°C min⁻¹ (41.7%) or 0.3°C min⁻¹ (36.4%), though the difference was not significant. Analysis of the results revealed that vitrification is less damaging to the bovine embryos and post-thaw viability is higher than slow-frozen embryos. Thus, vitrification could be a promising alternative for cryopreservation of IVEP bovine embryos.