Successful cryopreservation of whole sheep ovary by using DMSO-free cryoprotectant

The study aims to assess the protective effects of dimethyl sulfoxide (DMSO)-free solution based on trehalose on the cryopreservation of a whole sheep ovary and evaluate its use as an efficient cryoprotectant. Twenty-one ovaries collected from 6- to 8-month-old non-pregnant female sheep were randomly distributed into three groups, namely, a fresh group, a DMSO-free group, and a DMSO group. The morphology, cell apoptosis (by hematoxylin and eosin (HE) staining and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay), and mRNA transcript of Bcl-2-associated X protein (BAX) and cold inducible RNA-binding protein (CIRP) (by real-time PCR) of the thawed sheep ovaries and fresh controls were tested to establish a criterion for appraising the results of the cryopreservation. (i) The histological assessment indicated that the structure of the DMSO-free ovaries remained largely intact and comparable to those of the fresh control groups; whereas, significant damage was observed in the ovaries of the DMSO group (P < 0.05). (ii) The TUNEL assay and mRNA transcript of the BAX assessment showed that the apoptosis parameter in the fresh group was the lowest among all the groups (P < 0.05), and the parameter in the DMSO-free group was significantly lower than that in the DMSO group (P < 0.05). (iii) The level of the CIRP transcripts increased the most in the DMSO-free group followed by the DMSO group and the fresh control group (P < 0.05). These results indicate that a DMSO-free cryoprotectant solution, especially a trehalose cryoprotectant, is an efficient cryoprotectant and has a beneficial effect on the cryopreservation of whole sheep ovaries.