42 THE EFFECT OF VARIOUS CRYOPROTECTIVE AGENTS AND SLOW COOLING RATE ON VIABILITY OF SHEEP OVARIAN TISSUE

Abstract

Priority objects of protection in agrobiocenoses are grades of cultural plants and local breeds of the cultivated animals. The most general criteria for preservation of local breeds are viability, adaptability, a state of health, reproductive abilities, and unique genetic polymorphism at the molecular and morphological levels. Therefore, the aim of this study was to compare the effectiveness of different cryoprotectors on morphology of ovine ovarian tissue cryopreserved by a passive cooling method. Ovarian tissue from 10 indigenous Chuyi 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 4 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 ovine serum (NOS) (Seisenbayeva et al. 2015 Reprod. Fertil. Dev. 28, 193–194). After 7 days of culture, we evaluated the effects of passive cooling methods with different cryoprotectors on ovarian tissue morphology by light microscopy after hematoxylin and eosin staining of tissue sections. The number of viable and damaged follicles was counted. All morphologically normal primordial, primary, and secondary 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 test. In groups 1, 2, 3, and 4, the mean densities of follicles per 1 mm³ was 17.0 ± 4.6^a, 16.2 ± 7.2^b, 13.0 ± 5.1^c, and 11.9 ± 4.8^d, respectively (^{ab, ac, ad} P > 0.05). For these groups, respectively, 69.2 ± 8.2%^a , 61.7 ± 8.6%^b , 52.4 ± 8.8%^c and 48.5 ± 8.8%^d preantral follicles were morphologically normal (^{ab, ac, ad} P > 0.05). We did not find significant differences between groups. The analysis of comparative histology shows that 1.5 M EG is more effective on viability of ovarian follicles than 1.5 M DMSO or 1.5 M PROH.