Effects Of Three Kinds Of Antioxidants On Cryopreservation Of Boar Spermatozoa

In order to improve the effectiveness of pig raising, artificial insemination has been widely used. Whereas, semen could be injured by the low temperature during the process of cryopreservation. The motility of boar sperm would be unsatisfied after thawing, which could affect the reproductive rates. Therefore, it is necessary to find some practical cryoprotectants. The membrane of boar sperm contains many unsaturated fatty acids. They are easily oxidized during the processes of freeze thawing, thereby undermining the integrity of the sperm membrane. However, bamboo leaf flavonoid, sodium erythorbate and butyl hydroxyl anisd are efficient antioxidants which commonly served as food additives. There are many studies about their functions about food antioxidation, but their influence on boar semen cryopreservation has not been reported yet. This experiment added different concentrations of these three reagents into boar semen as cryoprotectants. And spermatozoa viability, plasma membrane integrity, mitochondrial membrane integrity and acrosomal integrity were tested by microscopic examination of the propidium iodide(PI), rhodamin 123(Rh 123) staining, eosin staining, hypoosmotic swelling test(HOST), and fluorescein isothiocyanate-peanut agglutinin(FITC-PNA) staining to evaluate the protection of these antioxidants to boar semen cryopreservation. Additionally, the malonaldehyde(MDA) levels, the antioxidase activities of superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) were evalueated by SOD, GSH-Px and MDA assay kits. The results were shown below:1. The spermatozoa motility, viability, mitochondrial membrane integrity, plasma membrane integrity and acrosomal integrity and of frozen-thawed boar semen were significantly improved with the addition of different concentrations of bamboo leaf flavonoid into boar semen freezing dilution solution(P<0.05). The boar sperm motility, viability, mitochondrial membrane integrity, plasma membrane integrity, and acrosomal integrity would be highest when bamboo leaf flavonoid was added with the concentration of 5 mg/mL.2. The spermatozoa motility, viability, mitochondrial membrane integrity, plasma membrane integrity and acrosomal integrity and of frozen-thawed boar semen were significantly improved with the addition of different concentrations of sodium erythorbate into boar semen freezing dilution solution(P<0.05). The highest motility, viability, mitochondrial integrity, plasma membrane integrity, and acrosomal integrity were achieved when sodium erythorbate was added with the concentration of 6 mg/mL.3. The spermatozoa motility, viability, mitochondrial membrane integrity, plasma membrane integrity and acrosomal integrity of frozen-thawed boar semen were significantly improved with the addition of different concentrations of butyl hydroxyl anisd into boar semen freezing dilution solution(P<0.05). The highest motility, viability, plasma membrane integrity, and acrosomal integrity were achieved when butyl hydroxyl anisd was added with the concentration of 0.2 mg/mL. Whereas, the highest mitochondrial integrity was found when added 0.15 mg/mL of butyl hydroxyl anisd.4.Compared with the control group, added bamboo leaf flavonoid of 2 mg/mL~6 mg/mL into boar semen freezing dilution solution could significantly lower the MDA level and improve the antioxidase activities of SOD & GSH-Px(P<0.05). With addition of bamboo leaf flavonoid, the MDA level decreased most at 4 mg/mL and 5 mg/mL,and the antioxidase activities of SOD showed best at 5 mg/mL and while the antioxidase activities of GSH-Px at 4 mg/mL. Freezing dilution solution supplemented with 6 mg/mL of sodium erythorbate decreased the MDA level at the most significant extent,and the antioxidase activities of SOD showed best at 8 mg/mL and while the antioxidase activities of GSH-Px was significantly different with other groups. The MDA level, the antioxidase activities of SOD and GSH-Px were significantly different with the addition of different concentrations of butyl hydroxyl anisd into freezing dilution solution(P<0.05), and the best concentration was 0.2 mg/mL.5. The joint action of two kinds of antioxidants can significantly increase the thawed boar sperm vitality, living rate and membrane integrity(P<0.05). Of 5 mg/mL bamboo leaves flavonoids and 6 mg/mL sodium ascorbate compatibility effect after the best results.