Study On Dimethylformamide And Two Polysaccharides For Crypreservation Of Boar Semen

The technology on cryopreservation of boar semen was gradually developed as the wide application of artificial insemination, but the utilization rate of frozen semen for swine artificial insemination was less than 1%. It was mainly because of the sensitivity of boar spermatozoa towards cooling, the more abnormal sperm, the low sperm viability after freezing-thawing and low fecundity after artificial insemination.In order to further improve the quality of frozen-thawed boar semen, the freezing extenders were improved in three aspects in this present study: using dimethylformamide (DMF) instead of the conventional cryoprotectant glycerol; using pigeon egg yolk instead of the traditional egg yolk and adding laminarin and Salvia polysaccharide to diluents. Sperm viability, sperm motility, mitochondrial activity, plasma membrane integrity, acrosome integrity and DNA integrity were evaluated by using computer-assisted sperm analysis (CASA) system, propidium iodide (PI), rhodamine 123, osmotic swelling test (HOST), isothiocyanate fluorescence-linked peanut agglutinin(FITC-PNA) and single cell gel electrophoresis (SCGE), respectively. The protective effects on frozen boar semen of DMF, two polysaccharides and pigeon egg yolk; and the effects of the cryoprotectants on the SOD activity, total antioxidant capacity (T-AOC) and the cholesterol content of sperm during freeze-thaw process were studied.The results in this paper were as follows:1. When DMF concentration was 5%, the equilibration time was 90 min in 4℃, the sperm motility, viability, mitochondrial activity, acrosomal integrity and plasma membrane integrity were significantly higher than 3% glycerol control group (P<0.05). These results showed that DMF could be used to replace glycerol on cryopreservation of boar semen, and could improve boar sperm quality during freeze-thaw process.2. Laminarin and Salvia polysaccharide were added to the conventional diluent (TCG), the optimum concentrations were 0.8mg/mL and 0.4mg/mL, respectively. The results showed that the sperm motility, viability, mitochondrial activity, acrosome integrity and plasma membrane integrity in 0.8mg/mL laminarin and 0.4mg/mL Salvia polysaccharide groups were significantly higher than control group after freezen and thawed(P<0.05). Meanwhile, two polysaccharides could be used as effective antioxidants.3. The results showed that the sperm viability, acrosome integrity, plasma membrane integrity and DNA integrity in the 15% pigeon egg yolk group were significantly higher than 20% chicken egg yolk group after freezen and thawed(P<0.05). The pigeon egg yolk could replace egg yolk on cryopreservation of boar semen, and could obtain a better protective effect.4. During cooling and freeze-thaw process, the SOD activity, total antioxidant capacity (T-AOC) and cholesterol content of boar sperm all decreased. When adding pigeon egg yolk, the sperm SOD activity, T-AOC and cholesterol content increased; they were significantly higher in 15% pigeon egg yolk than 20% chicken egg yolk (P<0.05); SOD activity and T-AOC significantly increased in 5%DMF group (P<0.05), but the DMF has little effect on sperm cholesterol content; when adding 0.6～0.8mg/mL laminarin and 0.4～0.6mg/mL Salvia polysaccharide, the sperm SOD activity, T-AOC and cholesterol content significantly increased (P<0.05).