Studies On LDL And Polysaccharides On Boar Cryopreservation Sperm

Althougth the artificial insemination and sperm cryopreservation were extensive used in bovine industry, the fertility and litters of boar sperm were lower with the insemination of boar cryopreservation sperm which resulted the less application of boar freezing and thawing sperm in boar industry. Compared to the others domestic animals, boar sperm is featured with higher volume, smaller sperm density, higher lipid content on the head of boar sperm and more senstive to temperature during freezing and thawing, so boar sperm was damaged easily in the process of freezing and thawing. However, this damage could be decreased by cryopretectant added into the extenders. Although glycerol has been used in cryopreservation of different cell type, it con't be used widely in boar cryopreservation sperm because the motility of boar sperm after freezing and thawing was increaing with the glycerol concentration raising but the acrosome integrity was decreasing.Therefore, it is the one of the most important to look for another better cryopretectant. In present study, Gynostemma polysaccharide(GP), Radix Phodiolae Polyose(RPP), LBP and low density lipolipid (LDL) were added in the extenders to investegate the effects of polysaccharides and LDL on boar sperm parameters after freezing and thawing, and the results were shown following:1,The motility of boar cryopreservation sperm is up to 37.82% and 42.66%(P<0.05)with the addition of 3% GP and 4% RPP, respectively. Boar sperm plasma was better protected with higher contentration of GP(4%,5%,6%)and RPP(6%,8%,10%)(P<0.05).The concentration of 6%,8% and 10% RPP were also benificial for acrosome integrity(P<0.05). The capacitation-like variation of boar cryopreservation sperm was increased with the increasing of the concentration of RPP(P<0.05). The motility and plasma integrity of boar cryopreservation sperm were up to 42.25% and 49.33%, 60.26% and 62.68% with the additive of 8% and 9% LDL(P<0.05), respectively. LDL was also beneficial for acrosome of boar cryopreservation sperm, but it was not related to the concentration of LDL. Moreover, addition of LDL in boar sperm extender couldn't change the capacitation-like varation of boar sperm after freezing and thawing.2,Computer-Aided Analysis System was used for motile characteristics of boar sperm after freezing and thawing to well understand the effects of cryopreservation on boar sperm. In 0.25ml France straw, the total motile sperm (TMS,%) of boar cryopreservation sperm were increased at the concentrations of 8～10% LDL and the values were 42.25%,49.33% and 43.21%, respectively.The velocity of straight-line (VSL,um/s) were up to 40.35,44.18 and 36.71 um/s with addition of 8～10% LDL after freezing and thawing.The velocity of curvilinear (VCL,um/s) was 37.14 um/s at the concentration of 6% LDL in extender and it was the highest group(P<0.05). All the concentration of LDL couldn't change the ALH. However, TMS (%) and VSL(um/s) could be increased with the addition of 8~10% LDLin 0.5ml France straw.3,Apoptotic-like variation of boar cryopreservation sperm could be induced by DNA damage and decreasing of activity of anti-oxidition enzyme. In 0.25ml France straw, Comet rate (CR) of boar cryopreservation sperm were significantly decreased at the concentration of 8% and 9% LDL, tail DNA(TD) and tail moment(TM) were decreased with the addition of 8～10% LDL(P<0.05) in boar cryopreservation sperm. The activity of SOD in boar sperm was increased at all the concentrations of LDL, but the activity of CAT was just increased with the additive of 8～10% LDL(P<0.05)after freezing and thawing. All the concentrations of LDL were benificial for mitochondrial of boar cryopreservation sperm. In 0.5ml straw, Comet rate (CR) and tail DNA(TD) were decreased at the concentration of 8~10%LDL and only the activity of SOD was increased with the addition of 8% and 9% LDL. The activity of caspases were decreased at the the concentration of 10% LDL in the straw of 0.25ml and 0.5ml.4,Different methods of RNA extration from boar cryopreservation sperm were used to investigate the mollicular mechnism of freezing and thawing on boar sperm. Boar sperm was treated by trizol with three temperatures of room temperature, 45℃and 70℃to extract total RNA. The results were shown that we could get enough numbers of RNA for gene expression only used the temperature of 70℃. Therefore, RNA extraction from boar sperm was related to the temperature of Trizol.5,The relative genes expression were detected by quantitive real-time PCR. The results showed that low density lipolipid receptor (LDLR) could be detected in boar cryopreservation sperm but the expression of LDLR was not affected by LDL. GADD45B expression was down-regulated by the addition of 9%～10% LDL in boar extender(P<0.05).The expression of SURVIVIN was always low in all the groups and no difference was observed between the groups of treatment and control and among the groups of LDL. The expression of BCL-2 gene was up-regulated by the addition of 9% and 10% LDL and the expression of BAX was down-regulated by 9% and 10% LDL addition ,respectively. Both the expression of BCL-2 and BAX could not be changed by the addition of 6%～8% LDL in extender of boar sperm .According to all the results, the positive effects of LDL on most parameters of boar sperm after freezing and thawing were founded, expercially the DNA damage were decreased and the expression of apototic-related genes were varied with the addition of LDL. It demonstrated that LDL could be used for boar cryopreservation sperm.