126 EFFECTIVE METHOD FOR IN VITRO CULTURE OF CRYOPRESERVED OVINE OVARIAN TISSUE

Abstract

Today, ovarian tissue cryopreservation is used for preserving the reproductive function of women, as well as the genetic material of rare and endangered species or domestic animals breeds. In the last 20 years genetic diversity of farm animals breeds suffered considerable losses in Kazakhstan; therefore, genetic preservation of valuable local breeds is desirable. The aim of this study was to compare the effectiveness of different in vitro culture media on morphology of ovine ovarian tissue cryopreserved by a slow-freezing protocol with 1.5 M dimethyl sulfoxide (DMSO). Ovaries were collected from indigenous Chuyi breed and immediately transported to the laboratory at 30°C within 1 h. Ovaries were rinsed several times in PBS supplemented with antibiotics (75 mg L^–1 of penicillin-G, 50 mg L^–1 of streptomycin sulfate). In Hepes-buffered medium 199, halved and the medulla removed with curved iris. Using a scalpel, the cortex was cut into 5- × 3- × 1-mm strips. Ovarian strips were equilibrated sequentially in freezing medium containing 0.25, 0.75, and 1.5 M DMSO with 0.5 M sucrose (5 min each). Then, ovarian strips were frozen in plastic straws using a programmable freezer Planer Kryo-360 3,3 (Planer, UK) and cooled as follows: stabilised at 20°C for 5 min, cooled from 20°C to –70°C at 5°C min^–1, seeded to the temperature –7°C, cooled again to –30°C at 0.3°C min^–1, cooled to –150°C at 35°C min^–1, and finally plunged into liquid nitrogen and stored for 10 days. The straws were thawed at room temperature for 1 min, and then immersed in a water bath at 37°C for 2 min, warmed at room temperature with Dulbecco’s PBS (DPBS), supplemented with 10% FCS and 0.75 M sucrose (15 min), then DPBS + 10% FCS (30 min), and finally placed in the culture media for 10 min. Fresh and frozen tissue pieces were randomly distributed into 12 groups for further culture: 1) TCM 199 + 10% FBS; 2) TCM 199 + 10% native ovine serum (NOS); 3) TCM-Hepes + 10% FBS; 4) TCM-Hepes + 10% NOS; 5) DMEM + 10% FBS; 6) DMEM + 10% NOS; with and without 7.5 mg mL^–1 of FSH. After 7 days of culture, the effects of different culture media on ovarian tissue morphology was evaluated by light microscopy after hematoxylin and eosin staining of tissue sections. The best result was observed when frozen ovarian tissue was cultured in the presence of FSH. The best result was observed in group 3 and 4 with FSH. The percentages of normal primordial, primary, and preantral follicles were: 1) TCM 199 + 10% FBS + FSH = 53.5 ± 3.1, 39.7 ± 3.8^a, 28.5 ± 3.2; 2) TCM 199 + 10% NOS + FSH = 49.4 ± 2.3^a, 36.7 ± 3.3^a, 25.3 ± 4.1; 3) TCM-Hepes + 10% FBS + FSH = 66.3 ± 2.5, 45.7 ± 3.9, 35.1 ± 3.8; 4) TCM-Hepes + 10% NOS + FSH = 86.5 ± 3.8^b, 75.4 ± 4.2^b, 45.7 ± 3.5; 5) DMEM + 10% FBS + FSH = 42.1 ± 3.5^a, 33.7 ± 2.9^a, 21.3 ± 4.9, 20.7 ± 3.9; 6) DMEM + 10% NOS + FSH = 41.3 ± 3.9^a, 32.9 ± 2.5^a; control group = 98.2 ± 1.1, 93.7 ± 1.7, 90.3 ± 1.9 (^ab, P < 0.01). The majority of follicles in groups without FSH were degenerated. In group 4) TCM-Hepes + 10% NOS without FSH, a damaged structure of primordial follicles was observed.