| The present study is to improve boar semen cryopreservation by reforming cryoprotectants, cooling procedure and evaluation methods.The hand-held acquisition of boar semen, pretreatment of sperm, the seminal plasma is removed by centrifugation, diluted. Cooling and balancing of semen, packed by tubule, then freezing. The frozen pig sperm motility, acrosome integrity rate, membrane integrity, membrane integrity and nuclear DNA integrity deteced by different means, thus feedback cryopres ervati on.Comparie and correlation analysis of single cell gel electrophoresis assay method (SCGE) and acridine orange staining (AOT) of pig sperm DNA integrity, results shows there is highly significant correlation between SCGE and AOT(P<0.01), the correlation coefficient is0.992. the effect of five different cooling rates, namely, A, B, C, D and E (61.9℃/min,45.5℃/min,32.2℃/min,24.9℃/min and14.1℃/min) on the quality of boar sperm after it has been cryopreserved, cooling rate of Group D offered the best protection to sperm, the motility is44.63%, however, with the reduction of the cooling rate, the differences between motilities of boar sperm in Group D in Group E are not apparent (P>0.05) although the former is still higher than the latter. The membrane integrity of boar sperm frozen at a cooling rate of Group D is remarkably higher than that of the remaining four groups (P<0.05), hitting55.45%. However, with the reduction of the cooling rate, the acrosome integrity was gradually climbing, with acrosome integrity of boar sperm in Group D much higher than that in Group A, B and C (P<0.05), but lower than that of Group E, and the differences between the boar sperm in the two groups are not so palpable (P>0.05). Ratio of normal and mature sperm nuclei of the boar sperm frozen at a cooling rate of Group D is much higher than that of Group A, B and C (P<0.05), hitting79.14%, but not so significantly higher than that of Group E(P>0.05). The effect of different glycol concentrations on the boar sperm cryopreservation, the motility of boar sperm which added4%glycol is significantly higher than the other four group(P<0.05), but there is no significant difference between that of added5%glycol and6%glycol(P>0.05). The acrosome integrity is positively correlated to the glycol concentration, but there is no significant difference among4%glycol,5%glycol,6%glycol(P>0.05). The membrane integrity of boar sperm which added4%glycol is significantly higher than that of added2%glycol,3%glycol and6%glycol(P<0.05), higher than that of added5%glycol(P>0.05). Ratio of normal and mature sperm nuclei of the boar sperm which added4%is the highest, significantly higher than that of added2%glycol,3%glycol and6%glycol(P<0.05), but there is no significant difference between that of added5%glycol and4%glycol(P>0.05), there is no significant difference between that of added5%glycol(P>0.05). The effect of different permeable cryoprotectants on the boar sperm cryopreservation, the motility of boar sperm which added4%glycol is40.98%, significantly higher than that of added3%glycerol and5%DMA(P<0.05). Acrosome integrity of added4%glycol is significantly higher than that of added5%DMA(P<0.05) and higher than that of4%glycerol. The membrane integrity which added4%glycol is45.52%, significantly higher than3%glycerol and5%DMA(P<0.05). Ratio of normal and mature sperm nuclei of the boar sperm which added4%glycol is79.32%, significantly higher than3%glycerol and5%DMA(P<0.05), but there is no significant difference between3%glycerol and5%DMA(P>0.05). The effect of different poultry egg-yolk on boar sperm cryopreservation, the sperm motility of which added francolin egg yolk (FEY) is42.54%, significantly higher than the other four protectants(P<0.05). The boar sperm acrosome integrity of which added FEY is significantly higher than the other four protectants(P<0.05). The membrane integrity of which added FEY is significantly higher than which added duck egg-yolk(DEY), pigeon egg-yolk(PEY), quail egg-yolk(QEY)(P<0.05), but there is no significant difference between which added chicken egg-yolk(CEY) and FEY(P>0.05). Ratio of normal and mature sperm nuclei of the boar sperm which added FEY is highest, significantly higher than DEY, but ti is not significantly different with the other there protectants(P>0.05). The effect of different trehalose concentrations on the sperm cryopreservation, the sperm motility of in the group of1Og/L the trehalose complex is highest, reached45.44%, is significantly higher than in Og/L,5g/L,15g/L and20g/L groups(P<0.05), the acrosome integrity of lOg/L is significantly higher than in Og/L,5g/L,20g/L groups(P<0.05), but there is no significantly difference between lOg/L and15g/L groups(P>0.05).The ratio of nonnal and mature spenn nuclei of the boar sperm is positively correlated to trehalose concentration, ratio of normal and mature sperm nuclei of the boar sperm when added20g/L trehalose is79.1%, significantly higher than the other four groups(P<0.05), there is no significant difference between15g/L and20g/L groups(P>0.05), respectively reached77.84%and77.42%.SCGE and AOT in sperm DNA integrity rate detection, the results showed a highly significant correlation between SCGE, can replace the method of SCGE which is simple and cheap; Cryopreservation by self-made refrigerating device, the results show24.9℃/min freezing the speed of boar semen freezing damage minimum; Ethylene glycol storage effect of glycerol, better than DMA on porcine sperm cryopreservation, ethylene glycol toxicity is low, viscosity is small, more conducive to detect porcine frozen semen; Ethylene glycol, the optimum concentration was4%, effect of cryopreservation of4%is better than in2%groups,3%groups,5%group,6%groups.Chicken egg, duck’s egg, partridge eggs, quail egg, egg yolk four dove group, preservation of partridge eggs best, small, large particles in the yolk and easy to dissolve, can replace egg yolk; Trehalose has effect of cryopreservation of boar semen freezing, concentration is lOg/L, the frozen thawed sperm quality best, as the concentration increased to20g/L, the sperm motility drop. |
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