| The objective of this study is to screen related separate parameter of flow cytometry for boar sperm and evaluate the influences of sex-sorting by flow cytometry on the physiological functions of boar semen, including sperm motility, acrosome intactness and capacitation status of sperm using fluorescent staining techniques. And further more, we examined the effect of antioxidants AA-2G on sperm sex-sorting, which would be of great value in the exploration of the reasons for the decrease fertility of sex-sorted semen and improvement of this technique and other assisted reproductive technology (ART).The whole experiment includes four parts. First, by tuning the parameters of the flow cytometry, we found the optimum levels dye of Hoechst33342 and the pressures for sex-sorting. The results showed that added to 7.5-16.5μl of Hoechst33342 which concentrations is 8.89mM and performed the sorting pressures at 40.2-40.8psi would be the optimal condition for boar sperm sex-sorting.Second, the influences of antioxidants on the physiological functions of porcine semen were evaluated by examining sperm viability, acrosome intactness, integrity of plasma membranes using fluorescent staining techniques and Laser Scanning Confocal Microscope(LSCM). Then we selected the best suitable antioxidant which can protect the sperm from the damage of sorting. The results showed that there were significant differences(P<0.05) between the groups A(AA-2G) and (P+A)(10% seminal plasma+AA-2G) and others in sperm viability(0.87 and 0.85); And AA-2G could prolong the lifespan of the sex-sorting semen(more than 4.5h). Third, the influences of antioxidants AA-2G on the physiological functions of swine semen were evaluated by examining sperm viability, acrosome intactness, capacitation status, using fluorescent staining techniques and Laser Scanning Confocal Microscope(LSCM) and computer assisted sperm analysis(CASA). We obtained the optimal concentration of AA-2G for boar sex-sorted spermatozoa. The results showed significant differences(P<0.05) between the sex-sorting treated with AA-2G at the dosage of 200μM and the control without any antioxidant and others in boar sperm STR, VAP and ALH. Also, there were significant differences(P<0.05) between the AA-2G treatment and the control in 5h and 6h under 17℃in the boar sperm viability(0.45% vs 0.38%,0.38% vs 0.30%); It was showed no significant difference (P>0.05) between the AA-2G treatment and control semen in sperm acrosome intactness (82.4% vs 80.2%,75.45% vs 77.2%,59.0% vs 64.4%) within 3h at 17℃. However, the sperm acrosome intactness (60.4% vs 46.0%) and capacitation status (40.6% vs 47.2%) appeared significantly different (P<0.05) following in vitro incubation for 4-6h. Especially, the capacitation status (45.2% vs 58.8%) showed highly significantly different (P<0.01) at 6h. Fourth, the efficiency of antioxidant AA-2G on artificial insemination with boar sex-sorted spermatozoa was evaluated by examining the rate of pregnancy of sow. The experiment showed that the pregnancy of sow was 63.64% when insemination with fresh sex sorted boar spermatozoa processed in the presence of anti-oxidative substance of AA-2G. The accurate of X sperm was 95.35%(82/86). and Y sperm was 99.17%(120/121). When sorted spermatozoa were kept at 17℃for 2 h after sorting,61.02% of the animals became pregnant, whereas only 59.25% of the sows went to term when inseminations were performed 24 h after sorting. But the date was similar as the level of the insemination with sex sorted fresh boar spermatozoa without any antioxidant (59.26%) was gained.Conclusion:We conclude that added to 17.78-36.67μM of Hoechst33342 and performed the sorting pressures at 40.2-40.8psi for boar sperm sex-sorting, the antioxidant AA-2G play a crucial role in protecting the acrosome intactness, plasma membranes integrity and improving the sperm viability. We conclude that the optimal concentration of AA-2G for boar sex-sorted spermatozoa is 200μM with CASA. Meanwhile, it is helpful for increasing the boar sex-sorted spermatozoa viability, acrosome intactness and reducing the sperm capacitation status in vitro incubation at 17℃.Simultaneity, enhancing the pregnancy rates after artificial insemination with sex-sorting boar fresh spermatozoa after processing. This experiment established a theoretical foundation for the pig sex-sorting sperm commercial applications.
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