| Artificial insemination technology(AI)can greatly improve the reproductive efficiency of pigs.One of the key links of AI is semen preservation in vitro.In liquid preservation method,the quality of sperm will significantly decrease within 72 hours,and its transportation tends to cause stress,these disadvantages of this method limits the application scopes of boar semen.Cryopreservation can break those limitations on time and space.However,porcine sperm is easy to be damaged by low temperatures and accumulation of reactive oxygen species(ROS)during the cryopreservation,which results in a significant decline in sperm quality after thawing.But the specific mechanism of damage is not still completely understood.Lecithin,the active component of yolk,can fuse with the plasma membrane of sperm to change the phase transition temperature of sperm,and finally reducing the sensitivity of sperm to low temperature,but this efficacy may be affected by the uneven structure of yolk.Chitosan is the deacetylated product of chitin,which is capable to reduce the oxidative damage of sperm by the means of binding hydroxyl radicals(a component of ROS)during liquid storage.As a natural antioxidant,honey has good effect on cryopreservation by protect the fertilization ability of sperm.Therefore,the sperm features,and indexes of oxidative damage and mitochondrial functions were detected here to screen the key damaged steps which cause the decline of sperm quality during the cryopreservation of boar semen.Homogenized yolk,chitosan and honey were added to semen dilution medium to optimize the protocol of cryopreservation.Finally,the fertilization of thawed semen was identified.An efficient method of cryopreservation of boar semen was aimed to be established to promote the industrial application and extension of frozen semen in pig.Part 1 Screening of key damaged steps in cryopreservation of boar semenIn order to screen out the key steps which cause the damage of pig semen during cryopreservation,the viability,motility,plasma membrane integrity and acrosome integrity,oxidative damage and mitochondrial function index of sperm after 25℃equilibrium,centrifugation,dilution,4℃ equilibrium,fumigation with liquid nitrogen and freezing storage in liquid nitrogen were detected,the fresh semen acted as control group.The results were as following:(1)With the progression of cryopreservation,the viability,motility,plasma membrane integrity and acrosome integrity of sperm showed a downward trend.Compared with the control group,values of these detected indexes in 4℃ treatment group,fumigation with liquid nitrogen treatment group and freezing storage in liquid nitrogen treatment group were significantly lower(P<0.01),which indicates that the quality of semen is beginning to decline after 4℃ equilibrium.(2)The content of ROS and the degree of MPTP opening of sperm in 4℃ treatment group,fumigation treatment with liquid nitrogen treatment group and freezing storage in liquid nitrogen treatment group were significantly increase than these in fresh sperm control group,respectively(P<0.01),and the content of SOD and mitochondrial membrane potential in these treatment groups were significantly decrease than these in fresh sperm control group,respectively(P<0.01),while the values of those detected indexes had not significantly difference among three treatment groups(P>0.05).Compared with 4℃ equilibrium treatment group,the viability(P<0.01),motility(P<0.01)and plasma membrane integrity(P<0.05)of sperm were significantly declined,and the degree of MPTP opening was significantly increased(P<0.01).Therefore,4℃ equilibrium,fumigation with liquid nitrogen and freezing storage in liquid nitrogen will result in the decline of sperm quality,especilaay,the 4℃equilibrium,fumigation with liquid nitrogen are the key damaged steps during the cryopreservation of boar semen.Part 2 Optimization of protocol on cryopreservation of boar semenBased on the key damaged steps during the cryopreservation of boar semen in part 1,the homogenized yolk,chitosan(0 mg/m L,0.05 mg/m L,0.1 mg/m L,0.15mg/m L,0.2 mg/m L or 0.25 mg/m L)and/or honey(0%,2.5%,5% or 10%)were added into semen dilution medium before the 4℃ equilibrium step,and the viability,motility,plasma membrane integrity and acrosome integrity,oxidative damage index and mitochondrial function index of sperm were detected to optimize the protocol on cryopreservation of boar semen,alleviate the damages caused by low temperature and oxidation,and improve the quality of cryopreserved sperm.The results showed that:(1)Compared with the unhomogenized yolk group,homogenized yolk could significantly improve the viability,motility,plasma membrane integrity and acrosome integrity of thawed sperm(P<0.01),and significantly increased the mitochondrial membrane potential(P<0.05),but there were no significant differences in the content of ROS,SOD and MDA,and the opening degree of MPTP(P>0.05)between these two groups.Therefore,the homogenized yolk can improve the stability of sperm membrane and thus improve the quality of frozen sperm.It was used in subsequent experiments for cryopreservation of boar sperm.(2)Compared with 0 mg/m L chitosan control group,0.1 mg/m L chitosan could significantly improve the viability(P<0.01),vitality(P<0.05)and plasma membrane integrity(P<0.05)of the thawed sperm,while significantly decreased the mitochondrial membrane potential(P<0.01),the content of ROS(P<0.05)and the opening degree of MPTP(P<0.05);In addition,the MDA content of thawed sperm in 0.15 mg/m L chitosan treatment group was significantly lower than that in 0.1 mg/m L chitosan treatment group(P<0.01).Therefore,supplement of 0.1 mg/m L or 0.15 mg/m L chitosan can improve the quality of thawed sperm.(3)Compared with 0% honey control group,supplement of 2.5% or 5% honey before cryopreservation could significantly increase the sperm viability,motility,plasma membrane integrity,acrosome integrity,SOD content and mitochondrial membrane potential,and significantly decreased the content of ROS and the opening degree of MPTP of sperm(P<0.01),but there was no significant difference in content of MDA between 0% honey control group and 2.5% or 5% treatment group(P>0.05).No significant difference in the plasma membrane integrity of sperm was observed between 2.5% honey IV treatment group and fresh sperm control group(P>0.05).Therefore,supplement of honey can effectively improve the quality of sperm,especially 5% honey I and 2.5% honey Ⅱ.(4)The viability,motility,plasma membrane integrity,acrosome integrity,mitochondrial membrane potential and the opening degree of MPTP of the thawed sperm in 0.1 mg/m L chitosan + 2.5% honey Ⅱ treatment group was significantly higher than these in 0.1 mg/m L chitosan treatment group(P<0.01),but there were not significant different in the contents of ROS,SOD and MDA between these two groups,respectively(P>0.05);Compared with 2.5%honey Ⅱ treatment group,the mitochondrial membrane potential was significantly increased(P<0.01),the opening degree of MPTP was significantly decreased(P<0.01)in 0.1 mg/m L chitosan + 2.5% honey Ⅱ treatment group.The viability,activity,plasma membrane integrity and acrosome integrity of the thawed sperm in 0.1 mg/m L chitosan+ 2.5% honey Ⅱ treatment group were higher than that in 2.5% honey Ⅱ treatment group,but no significant difference was observed between these two groups(P>0.05).Therefore,homogenized yolk,0.1mg/m L chitosan and 2.5% honey Ⅱ is suitable to be supplemented into semen dilution medium for the cryopreservation of boar semen.Part 3 Identification of in vitro fertilization with cryopreserved and thawed spermIn order to identify the in vitro fertilization ability of cryopreserved and thawed sperm in boar,porcine oocytes were collected and matured in vitro,the thawed sperms which were cryopreserved with the screened protocol in part 2 were used to fertilize the oocytes in vitro,and their fertilization ability was identified by detecting the cleavage rate and blastocyst rate.The results showed that the maturation rate of oocytes was 67.41%,and the cleavage rate and blastocyst rate after in vitro fertilization were 50.65% and 31.17%,respectively,which indicated that semen preserved with homogenized yolk,0.1 mg/m L chitosan and 2.5% honey Ⅱ had good fertilization ability in vitro.Conclusions: 4℃ equilibrium and fumigation with liquid nitrogen are the key steps which cause the decline of boar semen quality during the cryopreservation.Supplements of the homogenized yolk,chitosan or honey before 4℃ equilibrium can significantly increase the quality of frozen sperm.The combination of homogenized yolk,0.1 mg/m L chitosan and 2.5% honey Ⅱ is further conducive to improving the quality of cryopreserved and thawed serum,and this kind of boar serum show good ability of fertilization in vitro. |
