Study on Semen Freezing Preservation of German Shepherd Dogs

In order to prolong semen preservation and improve pregnancy rate, semen freezing was studied with German shepherd dogs for experimental animals. The semen of four dogs was collected 40 times in four dilution frozen into two formulations, according to the sperm motility to compare the advantages and disadvantages. The result indicated that the sperm motility of the pellet frozen semen in dilute 2 was significantly higher than that in dilution 1, 3, and 4 （P0.01）. The sperm motility of dogs semen with fried smoked method was notablely higher than that of frozen semen of program method （P0.01）. The dilution which contained yolk-Tris mainly was the best; the pellet semen frozen with fried smoked method was superior to tuble semen frozen with program freezing method; sperm motility of pellet semen was higher than that of tuble semen in the same dilution. The conception rate and litter size of the natural matting were higher than AI.

Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing

Resveratrol(3,5,4’-trihydroxystilbene,RSV) has been widely used in mammalian cells,but whether it can be used during freezing boar semen is still unknown.The effects of RSV treatment during boar semen freezing on its anti-freezing ability,apoptosis,and possible apoptotic pathways were observed in this study.Sperm motility,mitochondrial membrane potential(ΔΨm),adenosine triphosphate(ATP) content,terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL)-positive apoptotic state,and messenger RNA(mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested.The results showed that:(1) Compared with fresh sperm,the motility,normal acrosome rate,and plasma membrane integrity rate of frozen boar sperm decreased significantly(P<0.05),and RSV did not significantly increase the sperm motility(0.44 vs.0.40,P>0.05),but it did significantly improve the normal acrosome rate(57.65% vs.47.00%,P<0.05) and plasma membrane integrity rate(46.67% vs.38.85%,P<0.05).(2) After freezing,most boar sperm showed low mitochondrial ΔΨm.RSV treatment could increase the rate of high mitochondrial ΔΨm of boar sperm.(3) RSV treatment significantly decreased reactive oxygen species(ROS) levels(58.65% vs.88.41%,P<0.05)and increased the ATP content(0.49 μmol/L vs.0.25 μmol/L,P<0.05) of boar sperm during freezing.(4) The apoptotic rate of the freezing group(80.41%) with TUNEL detection increased significantly compared to the fresh group(9.70%,P<0.05),and RSV treatment greatly decreased the apoptotic rate(68.32%,P<0.05).(5) Real-time polymerase chain reaction(RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway(tumor necrosis factor-α(TNF-α),Fas ligand(FasL),and Caspase-8),but also the genes from the mitochondria-mediated apoptotic pathway(manganese superoxide dismutase(MnSOD),B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),and Caspase-9) were both significantly changed after freezing.RSV treatment during freezing greatly changed their expression levels.Although RSV treatment during boar semen freezing did not significantly increase motility after thawing,it still played an efficient antioxidant role,which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor-and mitochondria-mediated apoptotic pathways.

Effect of taxifolin on cold-shock damages in spermatozoa in rabbits

Objective:To investigate the effect of taxifolin added to rabbit semen on freezing-induced cold-shock damages in spermatozoa.Methods:Semen was collected from six adult New Zealand rabbits once a week by artificial vagina.The collected semen was pooled at 38℃ and divided into four equal volumes.They were diluted with 0,50,100 and 200μM taxifolin-containing Tris+egg yolk extender at 38℃ and their temperatures were lowered to 4℃.Following equilibration,semen drawn into 0.25 mL straws were frozen in an automatic semen freezing device and stored in liquid nitrogen container at-196℃.Samples were thawed in 38℃ water for 25 s and the analyses of motility,kinematic parameters,morphological deformities,changes in membrane integrity,mitochondrial membrane potential,dead-live ratio,acrosomal damages and as well as oxidative stress analyses were performed in semen.Results:Addition of 50μM taxifolin significantly improved motility(total,progressive,rapid and static),high mitochondrial membrane potential and the ratios of spermatozoa with acrosomal damage compared to the control group.Compared to the control group,malondialdehyde(MDA)levels in the 50 and 100μM taxifolin groups were significantly lower,while the MDA level was high and viable spermatozoa ratio was low in the 200μM taxifolin group.There were no significant differences between the groups in terms of kinematic parameters,morphological deformities,membrane integrity and antioxidant levels.Conclusions:The low dose of taxifolin(50μM)has a positive effect and the high dose(200μM)has a negative effect.Therefore,it is concluded that the addition of low-dose(50μM)taxifolin to the extenders would be a useful additive in reducing cold-shock damage that occurs during freezing of rabbit semen.