Ethylene Glycol and Dimethyl Sulfoxide Combination Reduces Cryoinjuries and Apoptotic Gene Expression in Vitrified Laying Hen Ovary.

Successful cryopreservation of avian gonads is important not only for avian breeding but is also crucial for preservation of species, especially of endangered birds. The aim of this study was to evaluate the effect of vitrification by several cryoprotectants on the ovarian tissues of laying hens. Ovarian tissues were randomly divided into six groups: control (nonvitrified: C), dehydrated using ethylene glycol (EG), dehydrated with propylene glycol (PROH), dehydrated using dimethyl sulfoxide (DMSO), and two combined groups, EG+DMSO and EG+PROH. The composition of vitrification solutions was as follows: EG group: V1 = 7.5% EG and V2 = 15% EG +0.5 M sucrose, DMSO group: V1 = 7.5% DMSO and V2 = 15% DMSO +0.5 M sucrose, PROH group: V1 = 7.5% PROH and V2 = 15% PROH +0.5 M sucrose, EG+DMSO group: V1 = 7.5% EG +7.5% DMSO and V2 = 15% EG +15% DMSO +0.5 M sucrose and EG+PROH group: V1 = 7.5% EG +7.5% PROH and V2 = 15% EG +15% PROH +0.5 M sucrose. Ovarian tissues of each group were dehydrated for 10 minutes with V1 solution and 2 minutes with V2. Among the vitrified groups, intact primordial and primary follicles showed significant increase in EG+DMSO, but follicular attrition had the highest rate in the PROH group (p < 0.05). Immunohistochemical analysis showed that the percentage of active caspase 3-positive cells was lower (p < 0.05) when using EG+DMSO versus PROH. Further gene expression of caspase 3, 8, and 9 was highest in the PROH group (p < 0.05). Vitrification of ovaries of laying hens using EG+DMSO can afford effective protection of primordial and primary follicles during preservation and may therefore be successfully used for storing avian gonadal tissues.