53 Cryopreservation of Sheep Ovarian Tissue Using Different Cryoprotectants

Abstract

Biological diversity is the key to maintaining life. The global trend in industrialization of agriculture bears in itself a set of risks. One of them is a reduction of a national genetic resource or gene pools of animals. Today, new methods of auxiliary reproductive technology permit using them for preserving not only the reproductive potential of the person, but also the biodiversity of wild and endangered species of farm animals. The art of cryobiology involves the addition of one or more cryoprotectants and change of time and temperature of exposition with tissue, and cooling speed. Therefore, the purpose of this work was to identify an optimum method of cryopreserving ovarian tissue of sheep using various cryoprotectants. Ovarian tissue from 8 indigenous Chuyi breed was transported to the laboratory at 36°C, divided into smaller pieces (5 × 3 × 1.0 mm) in the holding medium (Dulbecco’s PBS + 10% FCS), and randomly distributed into 8 groups. The pieces were equilibrated sequentially in freezing medium containing 0.25, 0.75 (5 min each), and 0.5 M sucrose with 1.5 M cryoprotectants (10 min) and placed in a cryo straw. The ovarian tissue was frozen in vapors of liquid nitrogen at a distance of 4 or 6 cm from the surface within 20 min with use of various cryoprotectants: 1.5 M dimethyl sulfoxide (DMSO), 1.5 M propylene glycol (PROH), 1.5 M ethylene glycol (EG), and 1.5 M glycerol (GL). After that, straws were plunged in liquid nitrogen. For thawing, straws were warmed in air at room temperature for 30 s and then immersed in a water bath at 37°C for 40 s. Then, ovarian pieces were transferred into the solution 0.75 M sucrose + 10% FCS + Dulbecco’s PBS (10 min, 37°C), then 0.3 M sucrose + Dulbecco’s PBS + 10% FCS (15 min, twice), and the holding medium (15 min). After thawing, the effects of cooling methods with different cryoprotectants on ovarian tissue morphology were evaluated by light microscopy after hematoxylin and eosin staining of tissue sections. The number of viable and damaged preantral follicles was counted. Data obtained in the experiments were analysed using Student’s t-test. Data are given as mean values ± standard errors. The level of significance was set at P < 0.05. In groups that were frozen at 6 cm using 1.5 M DMSO, 54.6 ± 4.8% of follicles were morphologically normal. In groups that were frozen at 4 cm using 1.5 M GL and 1.5 M DMSO, 69 ± 3.0% and 58.4 ± 3.7% of follicles, respectively, were morphologically normal. Using 1.5 M PROH and 1.5 M EG did not yield good results. Differences were statistically significant compared with fresh control (99 ± 2.4%). The analysis of comparative histology showed that use of 1.5 M DMSO for freezing at distance of 6 cm or 1.5 M GL at 4 cm were more effective in terms of viability of ovarian follicles compared with other cryoprotectants.