Optimization of medium for in vitro culture of sheep ovarian tissue

Abstract

Creation of genetic cryobanks is a way of preserving animal genetic materials. The gene bank can be used actively for increasing populations as well as minimization of interrelated crossing. One of the ways of preserving animal genetic material is ovarian tissue cryopreservation. After cryopreservation and storage, part of the ovarian tissue can be thawed and cultured in vitro in order to check a quality of follicles and stromal cells. The viability rate indicates whether or not it is possible to restore the reproductive function of the animal. Our study aimed to search and test a new medium for in vitro culture of sheep ovarian tissue. Ovarian pieces from 32 ewes (n = 320 and n = 180 in two series of experiments) were cultured during 16 days in three basic media: Tissue Culture Medium (TCM) 199, Buffered Tissue Culture Medium (TCM-HEPES), and Dulbecco Modified Eagle Medium (DMEM). These basic culture media used in different experimental groups (12 and 6) were supplemented with fetal calf serum (FCS), ewe estrous serum (EES) as well as follicles stimulating hormone and luteinizing hormone (FSH and LH, respectively). After the cultures of ovarian tissue and histological treatment, respectively, developmental rates of primordial, primary and secondary follicles as well as the secretion of hormones by tissues (progesterone and estradiol 17ß) were evaluated. Maximal developmental rate of follicles and hormonal activity were observed in the group with the culture medium TCM-HEPES + EES + FSH + LH. For in vitro culture of sheep ovarian tissue, the following medium is recommended: TCM-HEPES (supplemented with 100 μg/mL penicillin-streptomycin, 50 μg/mL gentamicin, and 2 mM l-Glutamine) +10% EES + 5 μg/mL FSH + 5 μg/mL LH.