| Flow-cytometric sorting of X-and Y-sperm is a promising technology for accelerating the genetic improvement and altering the sex ratio of buffalo. Though the technology of buffalo sperm sorting had been established, however, the blastocyst development rate after IVF and the conception rate after AI with sorted sperm were still lower than that with unsorted sperm. There were potential damages inflicted on sperm during the flow sorting procedure involves high levels of dilution, nuclear staining, exposure to UV lasers, cooling and cryopreservation which affected on sperm quality such as motility, plasma membrane, acrosomal integrity, mitochondrial membrane potential, DNA integrity, apoptosis. The objectives of this study were to detect the sperm damage in the different stages of flow sorting process (fresh, stained, sorted and frozen), so as to optimize the sorting procedure, improve sperm quality and finally accelerate buffalo breed improvement. A series of experiments were carried out in this study as follows:1. Evaluation of sperm quality, such as motility, plasma membrane integrity, apoptosis and DNA integrity was studied during the flow sorting procedure including four different stages (fresh, stained, sorted and frozen). The results indicated that sperm motility of sorted and frozen semen samples was significantly lower than that of fresh and stained samples (54.72%,52.38%vs.98.07%,96.41%, P<0.05). The percentage of sperm with plasma membrane integrity was higher in fresh, stained and sorted samples compared to the frozen samples by fluorescence microscopy (72.12%,69.39%,77.26%vs.58.45%) and by flow cytometry64.74%,63.69%,66.25%vs.36.98%, P<0.05). The percentage of apoptosis was significantly increased in sorted and frozen samples compared to the fresh samples by fluorescence microscopy (13.23%,18.74%vs.8.11%) and by flow cytometry (12.86%,22.4%vs.7.56%, P<0.05). The percentage of DNA fragment was much lower in the sorted sample than those of in fresh, stained and frozen samples by sperm chromatin dispersion test (SCD,0.47%vs.2.23%,2.25%,2.26%) and by sperm chromatin structure assay (SCSA,0.51%vs.2.55%,2.64%,2.15%, P<0.05).2. Sperm motility, plasma membrane integrity, apoptosis and DNA integrity of sorted frozen and unsorted frozen sperm from three Nili-Ravi buffalo bulls were evaluated. The percentage of sperm motility was significantly lower in sorted frozen sperm than unsorted frozen sperm by computer assisted sperm analysis system (CASA,59.6%vs.38.9%;58.1%vs.35.8%;59.1%vs.34%, P<0.05. for bull No.1,2and3, respectively). The DNA fragment index (DFI) was much lower in the sorted group than that in the unsorted one by sperm chromatin structure assay (SCSA,1.86%vs.2.47%,2.05%vs.3.0%,3.45 vs.4.38%, P<0.05), however, bull3had higher DFI than the other two bulls regardless of sorted or unsorted sperm. The percentage of sperm with plasma membrane integrity from sorted group was higher than that from unsorted group by fluorescence microscopy (55.3%vs.42.4%,52.7%vs.41.7%,51.6%vs.43.3%, P<0.05). The percentage of apoptosis was significantly increased in the sorted sperm compared to that of unsorted sperm by fluorescence microscopy (28.3%vs.21.1%,31.5%vs.23.6%,32%vs.23%, P<0.05). The percentage of apoptosis from sorted sperm was significantly higher in bull3than the other two bulls.3. Effects on buffalo sperm motility, plasma membrane integrity, apoptosis, DNA integrity of different antioxidant substances (folic acid and GSH) supplemented in the semen stained, sorted and frozen semen extenders were evaluated. The results of the study indicated that sperm motility, membrane integrity and DNA fragment were no significant differences (P>0.05) either with or without folic acid or GSH supplemented in the semen extender (stained, sorted and frozen). The percentage of apoptosis was much lower in frozen semen supplemented with folic acid or GSH in the extender compared to that in the control (P<0.05).4. A detection system based on laser tweezers Raman spectroscopy of sorted buffalo sperm was established. The results of the study indicated that the band assigned to PO2diester symmetric stretching shifted from788cm-1to783,785cm-1in fresh group compared to that in sorted and frozen groups. The band assigned to C-O stretching at1010cm-1shifted to1004,1005and1005cm-1in fresh group compared to that in stained, sorted and frozen groups. The scatter plots of principal component analysis could be distinguished the buffalo sperm from fresh, stained, sorted and frozen stages after the normalization of original Raman spectrum.5. Effects of different antioxidant substances (folic acid and GSH) supplemented to the semen stained, sorted and frozen extenders on sperm Raman spectrum were evaluated. The results showed that:(1) The band assigned to C-O stretching at1010cm-1in the fresh group shifted to1004cm-1in the stained group. When supplemented with folic acid or GSH in stained extender, the band assigned to C-O stretching at1004cm-1in control group shifted to1008cm-1(folic acid) and1005cm-1(GSH) in treatment groups, while for the frozen semen the C-O stretching from1004cm-1in the control semen shifted to1008cm-1(folic acid) and1010cm-1(GSH). The band assigned to PO2diester symmetric stretching at788cm-1and the PO2-group symmetric stretching band at1094cm-1only had different Raman peak intensity in the stained, collected and frozen semen supplemented with folic acid or GSH.(2) There was a peak of880cm-1considered as characteristic frequency of H2O2which was only existed when differential Raman spectrum was subtracted between the control and the stained, collected and frozen semen supplemented folic acid or GSH.(3) The scatter plots of principal component analysis (PCA) could be indentified from different semen treatments after the normalization of original Raman spectrum. 6. Three experiments were conducted to evaluate in vitro embryonic development after IVF with (1) sorted and unsorted sperm from three Navi-Ravi buffalo bulls;(2) X-and Y-sperm and (3) sorted sperm treated with folic acid or GSH in the stained, sorted and frozen semen extender. The results showed that (1) the rates of cleavage and blastocyst after IVF were much lower (P<0.05) for sorted sperm than that for unsorted sperm (11.6%vs.21.8%,11.9%vs.22.6%,9.2%vs.16.4%, for the three Navi-Ravi buffalo bulls, respectively).(2) there were no significant differences (P>0.05) in cleavage and blastocyst rates and the percentage of blastocyst developed on Day6, Day7, Day8after insemination with X sperm and Y sperm.(3) there were no defferences (P>0.05) in cleavage and blastocyst rates between the control and the sperm treated with folic acid in the stained, sorted and frozen semen extender after IVF. However, the blastocyst rate was increased (P<0.05) when the sperm was treated with GSH in the stained, sorted and frozen semen extender after IVF compared to the control group.7. Pregnant rate was investigated after AI with X sperm treated with folic aicd or GSH in the stained, sorted and frozen semen extenders. The results showed that (1) There were no significant difference (P>0.05) in conception rate after AI with X sperm between the folic acid treatment and control groups, but the abortion rate for the treatment group was much lower (P<0.05) than that for the control.(2) A significant higher conception rate was found in X sperm treated with GSH than that in the control (P<0.05), but no differerence in abortion rate (P>0.05). |
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