Efefcts Of Supplementing Boar Semen Cryopreservation Media With Dimethylacetamiden, Trehalose And Low Denisity Lipoprotein On Post-thawed Spermatozoa Quality

Compared with the semen storaged at room temperature, the conception and litters rates arelower relatively with the artifical insemination of boar cryopreserved semen for sows, whichcaused the less application of boar cryopresevation semen in pig production industry. Compared tothe other domestic animals, pig’s ejaculation is featured with higher semen volume, lower spermconcentration, higher polyunsaturated fatty acids (PUFAs) content in pig sperm plasma membraneand more particularly sensitive to temperature changes during the freezing and thawing process, soboar sperm is damaged easily in the process of cryopreservation. However, the damage can bedecreased by cryoprotectant added into extenders. Glycerol (3%concentration) is the mostcommom use cryoprotective agents (CAs) in boar semen cryopreservation extender. However,some studies indicate that glycerol has chemical and penetration toxic effects on metabolism andplasma membrane of sperm cells, leading the plasma membrane split. Also, it has a contraceptiveeffect on spermatozoa. Therefore, it promotes the research of other CAs.In an attempt to improve boar cryopreserved semen fertility, these experiments weresupplemented trehalose, dimethylacetamide (DMA), low density lipoprotein (LDL) to thecryopreservation diluent of boar in order to increase the post-thawed sperm motility, viability,plasma membrane and acrosome integrity, mitochondrial activity and decrease lipid peroxidationlevel of sperm. Research results on the cryopreservation of boar spermatozoa were as follows:1. Using5%DMA (v/v) instead completely3%glycerol in the diluents to freeze boar semen,the effect of lactose, trehalose, DMA, glycerol combination on sperm cryopreservation wasinvestigated. The results showed that trehalose-DMA-egg yolk extender significantly increased thepost-thawed sperm viability (49.17±1.78%) and acrosome integrity (47.22%±0.74%)(P<0.05).2. With trehalose replaced lactose, we evaluated the effect of different concentration of DMAon the cryopreservation of boar semen. The results have showed that when DMA was added at4%concentration, the sperm motility, viability, acrosome integrity after thawing were (45.17±0.56%),(50.33±0.67%),(48.30±1.44%) respectively, and were superior to3%,6%DMA groups (P<0.05),sperm motility was significantly higher than5%DMA added group (P<0.05), but sperm viability,acrosome integrity between them were no significant difference (P>0.05). Therefore, we suggestedthat when the use of trehalose as the basis of dilution of cryopreservation, the volume fraction of4%DMA showed a better effect on the quality of boar cryopreservation semen.3. Boar spermatozoa were frozen at different cooling rates using set the straws at differenthigh in the freezing machine designed to minimize thermal variables involved in the cooling process, to see whether sperm cryosurvival may be improved by changing cooling rate.Computer-assisted analysis syntem was used to measure spermatozoa motile characteristics. Whenspermatozoa in straws were cryopreservation with placing at4cm distance from liquid nitrogensurface, the percentage of total motile sperm (TMS%)(58.17±0.87%) and rapid progressivelymotile sperm (RPM%)(38.75±1.84%) after thawed were significantly higher than other groups(P<0.05). There were no significant difference (P>0.05) about thawed sperm curvilinear velocity(VCL), straight linear velocity (VSL) and average path velocity (VAP) between the4cm groupand the3cm group, but these were higher than the other two groups (P<0.05). The post-thawedsperm viability (54.8±1.42%) and membrane integrity (48.30±1.36%) in4cm group weresignificant higher than other three groups (P<0.05). The post-thawed sperm acrosome integrity(50.37±1.15%) and mitochondrial activity (49.31±1.57%) were no significantly difference (P>0.05)between the4cm and the3cm group, they were significantly higher than5cm,6cm groups(P<0.05). In addition, the minimum of malondialdehyde production (1.35±0.26nmol/10~8spermatozoa) of sperm after post-thawed was4cm group. To set up, when spermatozoa in strawswere cryopreservation with placing at4cm distance from liquid nitrogen surface would produce abetter cryopreservation effect.4. In order to improve the post-thawed spermatozoa quality, boar semen was frozen usingLDL, which was replaced the chicken egg yolk, in the extenders. Different concentration of LDL(at concentration of7%,8%,9%and10%) was added to the semen cryopreserved extenders andits effect on the quality of cryopreservation was assessed. The results showed that: compared withother experimental groups,8%LDL supplementation group significantly improved thepost-thawed sperm motility (54.38±1.48%)(P<0.05); the post-thawed sperm viabiliy and plasmamembrane integrity between supplementation at8%and9%LDL groups were no significantdifference (P>0.05), they were (52.26±1.57%),(50.68±0.87%) and (54.46±1.78%),(53.62±1.26%)respectively, but significantly higher than other groups (P<0.05); compared with the control group(20%chicken egg yolk group), different concentrations of LDL were significantly increasedacrosome integrity (P<0.05), and supplementation8%LDL group was the highest acrosomeintegrity, up to (58.60±1.54%). Comprehensive the above results: the concentration of8%LDLindicated a better effect on the quality of boar cryopreservation semen.