Purpose 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. Method 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 deoxynucleotidyltransferasemediated 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.Results (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). Conclusions 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.Capsule These results indicate that a DMSO-free and trehalose-containing cryoprotectant solution has a beneficial effect on the cryopreservation of intact sheep ovaries.Tianqi Du and Lan Chao contributed equally to this work.

In this work, Chinese hamster ovary (CHO) cells were used to examine the protective effects of glycyrrhizic acid (GA) on the reproductive toxicity of heavy metal ions, including Cd 2+ and Cu 2+ . As a result, both metal ions induced significant toxicity in CHO cells after 48-h treatment, as revealed by a severe decrease in cell viability. GA and glutathione (GSH) largely reduced the toxicity caused by Cd 2+ and Cu 2+ , which could be due to their recovery of GSH levels and superoxide dismutase (SOD) activities in CHO cells. In addition, GA and GSH significantly up-regulated the gene expressions of glutathione S-transferase (gst), sod, and heme oxygenase (ho)-1, which could be another explanation for their protective effects. More importantly, GA exhibited comparable protective effects as GSH but at much lower concentrations (50-100 µM v.s. 500-1000 µM). Therefore, GA could be effective for the alleviation of reproductive toxicity of Cd 2+ /Cu 2+ , which needs further investigation in animal models.