Viability of ovarian tissue after cryopreservation by slow freezing or vitrification in sheep

Ovarian tissue cryopreservation is a valuable technique for preserving fertility, yet the relative efficacy of vitrification compared to slow freezing remains to be fully explored. This study evaluates the post-thawed viability of ovarian tissue cryopreserved by either method using ovarian samples from healthy ewes aged one year or younger. The ovarian tissue was sectioned into 5 mm × 5 mm × 1 mm slices, cryopreserved, and cultured in vitro for 5 days. Follicle morphology was evaluated via histology, while DNA integrity in primordial follicles and stromal cells was evaluated using TUNEL staining. Histological analysis revealed that the fresh tissue contained a higher proportion of morphologically normal primordial follicles and greater DNA integrity compared to both cryopreserved groups. On day 0, TUNEL staining indicated that vitrification better preserved primordial follicle DNA integrity than slow freezing (p<0.001). However, after 5 days of culture, slow freezing more effectively maintained stromal cell DNA integrity than vitrification (p<0.001). Although primordial follicles exhibited promising self-repair ability, stromal cells exhibited limited recovery. In general, vitrification produced results comparable to slow freezing in ovarian tissue cryopreservation. These findings highlight the potential of vitrification to match or even surpass slow freezing in certain aspects.