Sheep ovarian tissue vitrification by two different dehydration protocols and needle immersing methods

The present research investigated the effects of two vitrification methods on sheep ovarian tissue. The base medium (BM) of the vitrification solutions contains 60% HEPES tissue culture medium (HTCM), 15% ethylene glycol (EG) and 15% dimethyl sulfoxide (DMSO). Ovarian cortex pieces were dehydrated by two different regimens, the 2-step which consisted of 50% BM and a 90% solution of 0.25 mol/L sucrose in BM for 10 minutes each at 4 degree C and the 4-step method which utilized: a) 25% BM, b) 50% BM, c) 75% BM and d) a 90% solution of 0.25 mol/L sucrose in BM for 5 minutes each at 4 degree C. After warming, the proportion of intact antral follicles in the control (non-vitrified) and 2-step vitrification groups was significantly higher than in the 4-step vitrification group. The number of apoptotic follicles in the ovarian pieces was significantly different between the control and 4-step vitrification groups. These results indicated that sheep ovarian tissue vitrification by the 2-step method was simpler and more effective than the 4-step method.