Our objective was to evaluate the function of treahlose and erythritol in reducing ROS concentrations, which is associated with a general improvement in the quality of frozen-thawing miniature pig sperm. Semen was mixed in modified Modena B extender, added to cooling media and freezing media, followed by the supplement of 100 mM trehalose and/or 100 mM erythritol with spermatozoa (1000x 109cells/straw). The trehalose plus erythritol (TE) added group had less intracellular H2O2 than did control and trehalose (36.6±1.6 vs. 49.0±5.8 and 48.8±7.9; P<0.05). The percentage of viable acrosome-intact sperm (FITC-PNA-/PI-) was higher in erythritol and TE than controls (57.0±5.5% and 62.5±4.3% vs. 45.4±5.4%; P<0.05 and P<0.001). The percentage of sperm with high fragmented DNA was observed in control group when compared with erythritol and TE also trehalose (65.5±1.3% vs 59.3±0.7% and 59.0±0.3% vs 62.2± 0.8%; P<0.001). The percentage of sperm LPO was higher in control and trehalose than erythritol (4.4±0.5% and 5.0±0.5% vs. 3.5±0.2; P<0.01 and P<0.001), and was lowest in the TE (control and trehalose vs. TE: P<0.001, erythritol vs. TE: P<0.05). Also, we performed that surgical insemination based on above data to evaluate the function of new cryoprotectant such as trehalose plus erythritol in vivo. Finally, 1 pregnant gilt showed natural estrus was allowed to go to term and 8 live piglets were born. In conclusion, miniature pig sperm was successfully cryopreserved with trehalose plus erythritol provided the increasing the sperm quality and reducing the ROS.

Cryopreservation of miniature pig sperm is essential because of high demand of organ transplant in mass production. However, miniature pig sperm are vulnerable to oxidative stress more than other mammals. Erythritol is a naturally occurring sugar alcohol with powerful antioxidant property. Thus, the aim of our study is to verify if erythritol could reduce lipid peroxide and enhance viability of frozen thawed miniature pig sperm. Ejaculated semen samples were frozen with cryoprotectant subjected to erythritol treatment (0, 10, 100, 500 mM). After frozen thawed, spematozoa viability were examined using the computer assisted sperm analysis (CASA) system. The product of lipid peroxidation, malondialdehyde (MDA) were quantified using spectrophotometer with DPPH and ABTS assays as ROS scavenger markers. Our result showed that erythritol enhanced sperm viability (p<0.05), reduced lipid peroxides significantly (p<0.05), proving the concentration of 100 mM erythritol to be an effective for lowing oxidative damage. Data from our study suggest that erythritol exhibits significant lipid peroxidation scavenging characteristics which may prevent oxidative damage, enhance viability of frozen thawed sperm and thus could be a effective additive as cryoprotectant.