Quality Improvement of Frozen Sperm by Combined Usage of Trehalose and Fructose in Black-Bone Sheep
GUO Zhilin1*, YANG Yongmei1, YU Fuli2, WU Di3, REN Junguang3
The impact of trehalose and fructose on cryopreservation of Black-bone sheep sperm was researched. Tris-citric acid-egg yolk diluent containing 82.65 mmol/L fructose was supplemented with 0, 5, 10, 15, 25, 50 and 100 mmol/L trehalose, respectively. The assay was divided into T0, T5, T10, T15, T25, T50 and T100 groups. Twenty-seven semen samples from 12 Black-bone sheeps were cryopreserved by two-step dilution method. Before and after thawing, sperm motility was evaluated by sperm quality analyzer. The percentage of survival sperm was evaluated by eosin aniline black staining. Sperm plasma membrane status was evaluated by carboxyl fluorescein diacetate/propidium iodide double staining. Sperm acrosome status was evaluated by pea agglutinin staining complexed with fluorescein isothiocyanate. The hydrogen peroxide content and malondialdehyde content of thawed sperm in each experimental group were detected by hydrogen peroxide content determination kit and malondialdehyde content determination kit to evaluate the lipid peroxidation of sperm. The results showed that the sperm movement and survival rate of T5, T10 and T15 groups were significantly improved after freezing. The status of sperm plasma membrane of T5 group was significantly improved. Compared with the T0 group, adding trehalose could not improve sperm acrosome status. The addition of trehalose could not reduce the hydrogen peroxide content of thawed sperm, but could significantly reduce the malondialdehyde content of sperm. The results suggest that adding 5 mmol/L trehalose and 82.65 mmol/L fructose to Tris-citric acid-yolk dilution can improve the quality of frozen sperm of Black-bone sheep, increase the sperm movement, survival and plasma membrane integrity after thawing, and reduce the lipid peroxidation of sperm membrane after thawing.
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