44 The effect of various cryoprotective agents and slow cooling rate on viability of goat ovarian tissue

Preservation of animal biodiversity of local farm animals is very important, because these animal breeds are highly adapted for breeding in local climatic conditions. The preservation of animal ovary tissue as a means of preserving domestic animals has become the focus of research. Therefore, the aim of this study was to compare the effectiveness of different cryoprotectors on morphology of cryopreserved goat ovarian tissue. Ovarian tissue from 10 local Kazakh goat breed was transported to the laboratory within 30 min at 32 to 34°C, divided into smaller pieces (2.0 × 1.2 × 1.0 mm), and randomly distributed into four groups: (1) control (fresh tissue), (2) pieces after freezing/thawing with 1.5 M ethylene glycol (EG); (3) pieces after freezing/thawing with 1.5 M propanediol (PROH); (4) pieces after freezing/thawing with 1.5 M dimethyl sulfoxide (DMSO). The ovarian pieces were placed in a cryovial and equilibrated sequentially in freezing medium containing 0.25, 0.75, and 1.5 M cryoprotectors with 0.5 M sucrose (5 min each), precooled at 4°C, and stored in a −80°C freezer for 24 h. Then, the cryovials containing the ovarian pieces were placed in liquid nitrogen and stored (for 2 months) until thawing. The ovarian pieces were cultured in vitro for 7 days in TCM-HEPES + 10% native goat serum (NOS). After thawing, the pieces of ovarian tissue were removed from the cryo tubes and placed in 5 mL of thawing solution (0.75 M sucrose solution in PBS with 10% NOS) for 15 min at room temperature. The tissue fragments were then placed in 0.5 M sucrose solution for 15 min, then in 0.25 M sucrose solution for 15 min, and finally transferred to PBS solution (with 10% NOS). After 7 days of culture, we evaluated the effects of different cryoprotectors on frozen-thawed ovarian tissue morphology by light microscopy after hematoxylin and eosin staining of tissue sections. The number of viable and damaged preantral follicles was counted. All morphologically normal follicles had healthy and intact oocytes, each containing a round nucleus and clearly visible nucleolus surrounded by well-organised granulosa cells without a pyknotic nucleus. Integrity rate of tissue after treatment was evaluated by Student’s t-test. In groups 1, 2, 3, and 4, the mean densities of follicles per 1 mm³ was 17.2 ± 4.5, 16.3 ± 7.1, 13.1 ± 5.2, and 11.9 ± 4.7, respectively (P > 0.05). For these groups, respectively, 69.3 ± 8.3%, 61.7 ± 8.5%, 52.5 ± 8.7%, and 48.6 ± 8.5% preantral follicles were morphologically normal (P > 0.05), and 30.7 ± 3.5%, 38.3 ± 3.8%, 47.5 ± 4.4%, and 51.4 ± 4.6% preantral follicles were morphologically damaged (P > 0.05), respectively. We did not find significant differences between groups. The analysis of comparative histology revealed no significant differences between all experimental groups. Thus, all cryoprotectants tested can be used for goat ovary tissue cryoconservation to preserve biodiversity in the goat.