Studies Of Salvia Miltiorrhiza Polysaccharides And Alpha-lipoic Acid On Boar Spermatozoa Cryopreservation

In recent years the artificial insemination has been widely used in animal breeding and production. Spermatozoa cryopreservation has been a high quality genetic resource for artificial insemination. In modern cattle production, the frozen semen has been widely used all over the world. But the use of boar semen is limited in commercial swine production. Because compared to other domestic animals, boar spermatozoa is especially susceptible to cold shock. And the sperm motility, plasma membrane integrity, mitochondrial activity and acrosome integrity of frozen semen is lower than fresh semen. The conception rate and litter size of frozen semen is decreased after artificial insemination. It is necessary to add frozen cryoprotectants in the extenders for protect sperm from cold damage. In our study, Salvia miltiorrhiza polysaccharides and alpha-lipoic acid were added in the extenders, to investigate the effects Salvia miltiorrhiza polysaccharides and alpha-lipoic acid on sperm quality, antioxidant enzyme activity, expression of apoptosis gene and artificial insemination after freezing and thawing. And the results were shown following:1.The addition of Salvia miltiorrhiza polysaccharides to the extender results in a higher sperm quality: The optimal concentration of Salvia miltiorrhiza polysaccharides was determined to be 0.4 mg/mL. It lead the highest boar sperm motility, sperm viability, membrane integrity, acrosomal integrity and mitochondrial activity(P<0.05). The results were 51.45%, 48.17%, 51.44%, 48.61% and 54.14%, respectively. The addition of Salvia miltiorrhiza polysaccharides to the extender also increase antioxidant enzyme activity after freezing and thawing: Add 0.2 mg/mL, 0.4 mg/mL and 0.6 mg/mL Salvia miltiorrhiza polysaccharides in the extender have the highest SOD activity, but there are no significant difference between them(P<0.05); Add 0.4 mg/mL Salvia miltiorrhiza polysaccharides in the extender have the highest LDH, GOT and CAT activity(P<0.05).2.The addition of alpha-lipoic acid to the extender results in a higher sperm quality: Add 6.0 mg/mL alpha-lipoic acid lead the highest boar sperm motility, sperm viability, acrosomal integrity and mitochondrial activity(P<0.05). The results were 52.85%, 50.52%, 50.89% and 53.87%, respectively; Add 4.0 mg/mL and 6.0 mg/mL alpha-lipoic acid have the highest membrane integrity(P<0.05). The addition of alpha-lipoic acid to the extender also increase antioxidant enzyme activity after freezing and thawing: Add 4.0 mg/mL, 6.0 mg/mL alpha-lipoic acid in the extender have the highest SOD and GOT activity, but there are no significant difference between them(P<0.05); Add 6.0 mg/mL alpha-lipoic acid in the extender have the highest LDH and CAT activity(P<0.05).3.The addition of compatibility Salvia miltiorrhiza polysaccharides and alpha-lipoic acid could increase boar sperm quality after freezing and thawing. And boar sperm motility, sperm viability, membrane integrity, acrosomal integrity and mitochondrial activity were higher than add Salvia miltiorrhiza polysaccharides or alpha-lipoic acid individually, but there are no significant difference between compatibility groups(P<0.05). S1A3 group(0.3 mg/mL SMP +7.0 mg/mL ALA), S2A1 group(0.4 mg/mL SMP +5.0 mg/mL ALA), S2A2 group(0.4 mg/mL SMP+6.0 mg/mL ALA) and S2A3 group(0.4 mg/mL SMP+7.0 mg/mL ALA) lead the highest SOD activity, but there are no significant difference between them(P<0.05). S1A3 group, S2A1 group and S2A2 group have the highest LDH activity, but there are no significant difference between them(P<0.05). Compared with other groups, S2A1 group have the highest GOT activity(P<0.05). S2A1 group(0.4 mg/mL SMP +5.0 mg/mL ALA) and S2A2 group have the highest CAT activity, but there are no significant difference between them(P<0.05).4.The addition of 0.4 mg/mL SMP could decrease the expression of GADD45 BB and BAX gene, and increase the expression BCL-2 gene(P<0.05); The addition of 0.8 mg/mL SMP could decrease the expression of SURVIVIN gene(P<0.05). Add 4.0 mg/mL and 6.0 mg/mL ALA in the extender, the expression of GADD45 BB is lower than other groups, but there are no significant difference between them(P<0.05). Add 4.0 mg/mL ALA in the extender have the highest BCL-2 gene expression, and 6.0 mg/mL ALA have the lowest BAX gene expression(P<0.05). S1A3 group(0.3 mg/mL SMP +7.0 mg/mL ALA) and S2A1 group lead the lowest GADD45 BB gene expression but here are no significant difference between them(P<0.05). S3A1 group(0.5 mg/mL SMP +5.0 mg/mL ALA) have the lowest SURVIVIN gene expression. S2A1 group have the highest BCL-2 gene expression(P<0.05). S2A1 group and S3A2 group(0.5 mg/mL SMP +6.0 mg/mL ALA) lead the lowest BAX gene expression, but tere are no significant difference between them(P<0.05).5.The artificial insemination results demonstrated that the non-return rate, conception rate, farrowing rate and litter size are all decrease after sperm cryopreservation. For the frozen semen, 0.4 mg/mL SPM group, 6.0 mg/mL ALA group and compatibility of 0.4 mg/mL SPM and 5.0 mg/mL ALA group, have the same non-return rate and conception rate, there are70%. 0.4 mg/mL ALA group have 60% farrowing rate, lower than other frozen semen groups. The compatibility of 0.4 mg/mL SPM and 5.0 mg/mL ALA group have the highest litter size in frozen semen groups(P<0.05).Our results show that the supplementation of extender with SMP and ALA could provide improved boar sperm quality, antioxidant enzyme activity, inhibit sperm apoptosis and DNA damage, non-return rate, conception rate, farrowing rate and litter size. The optimal concentration is compatibility of 0.4 mg/mL SMP and 5.0 mg/mL ALA.