Study Of Sex-Preselection In Buffalo

China is a habitat for more than twenty million swamp buffalo.Thus,if the procedures of sex-preselection by flow-cytometric sorting of X-and Y-chromosome bearing spermatozoa is established and applied in buffalo breeding,great improvement in terms of genetics and economics would be possible.Therefore,a series experiments assorted in 5 parts were carried out in this study and the issues relating to the sperm sexing and its subsequent use in IVF and AI were investigated.PartⅠ,the difference in DNA content characterizing the X-and Y-chromosome bearing sperm of buffalo,which indicates the feasibility of sex sorting,was to be identified in this study.Two symmetrical,separate but overlapping peaks presumed to be X-and Y-chromosome bearing sperm were detected in this study.The difference in fluorescence intensity,which related to the DNA content,between the X-and Y-sperm was 3.59±0.11%for Murrah buffalo and 3.55±0.14%for Nili-Ravi buffalo,respectively.Significant differences were observed among males within each breed,but there were no differences between the averages of the two breeds.The results indicate that flow cytometric sorting of X- and Y-sperm of buffalo is feasible.PartⅡ,viability,acrosome integrity and microbe contamination in sexed and unsexed buffalo semen was to be analyzed in this study,which would provide the needed information of semen quality to facilitate the sexing procedures and the AI and IVF protocols by using sexed sperm.The results revealed that there was a tight correlation among the sperm motility examined by flow cytometry,fluorescent microscopy and computer assisted sperm analysis(r＞0.85).Results of analysis for the semen quality revealed that the sperm sexing procedure appeared to be helpful in removing the dead sperm.An increase in sperm motility from 72.4%before sexing to 86.4% after sexing was observed.Moreover,the post-thawed motility of sexed sperm was higher than unsexed sperm(44.1%vs 33.9%,P＜0.05).And the motility of sexed and unsexed sperm dropped to 24.2%and 9.0%(P＜0.05)respectively after incubation in 38℃for 3h,and to 15.6%and 6.7%(P＜0.05)respectively for 6h.The sperm sexing procedure also appeared to be helpful in removing the sperm with damaged acrosome.The percentage of sperm with acrosome damaged decreased from 22.9%before sexing down to 3.9%after sexing.After frozen-thawed,percentage of sperm with damaged acrosome in sexed and unsexed semen was similar(30.0%vs 26.8,P＞0.05),which indicated a higher damaged effect of cryopreservation on the acrosome of sexed sperm than on unsexed sperm.Following incubation in 38℃for 3h and 6h,percentage of sexed sperm with damaged acrosome was 33.9%and 38.3%respectively,and 32.8% and 35.6%respectively for unsexed semen(P＞0.05).Analysis of microbe in the semen revealed that the average number of microbe clonies presented in 1.0 mL fresh and frozen buffalo semen was 10619 and 299 repectively,while 18935 clonies was found in 1.0 mL sexed frozen semen(P＜0.05).Classification of the microbes from the fresh buffalo semen revealed that Gram-positive cocci accounted for 48%,Gram-negtive bacilli accounted for 41%,Gram-positive bacilli accounted for 9%and fungi accounted for 2%.However,all the microbes from the sexed frozen semen were Gram-negtive bacilli,which were subsequently identified as Enterobacter cloacae and Klebsiella oxytoca.The results of antimicrobial susceptibility test indicated that,the microbes derived from sexed frozen semen appeared resistant to Tobramycin and Gentamicin,but showed high susceptibility to Amikacin and Ciprofloxacin.PartⅢ,the sex chromosome of Holstein was to be microdissected in this study,followed by preparation of the X- and Y-chromosome probes for identification of the purity of the sexed buffalo sperm by FISH.The metaphase karyotype of Holstein sex chromosome was clearly identified on the slide and successfully dissected by using micromanipulator,from which the probes for X- and Y-chromosome were prepared.The results of FISH using Y-chromosome probe suggested that the Y-sperm accounted for 47.7%in the unsexed buffalo semen samples.The X sperm accounted for 48.9%in the unsexed buffalo semen samples while using X-chromosome probe.Both of the results were in concord with the theoretical distribution of X-and Y-sperm(50% for each)in unsexed semen(P＞0.05),which indicated the feasibility of FISH procedures by using sex chromosome probes.Sexed Y-semen samples were used in the FISH for verification of the sexing purity.Y-sperm accounted for 82.2%while hybridized by Y-chromosome probe and X-sperm accounted for 9.6%while hybridized by X-chromosome probe.In average,the percentage of Y-sperm in the sexed Y-semen was 86.2%,which was similar to that of 87% previously identified by flow cytometer(P＞0.05).PartⅣ,sexed buffalo sperm was to be used in the IVEP in this study to establish a system for efficient production of sex-preselected embryos and offspring with superior genetic metrit.The results of the study indicated that leptin supplemented in the IVM and IVC medium could significantly boost the oocyte maturation and its subsequent embryo development following IVF with unsexed buffalo sperm.The percentage of polar extrusion and blastocyst developed from oocytes matured in 10ng/mL was 61.2%and 22.4%respectively,significantly higher than those matured in 0ng/mL(51.9%and 13.0%,respectively,P＜0.05).Blastocyst development rate was 26.1%while supplementing 10ng/mL leptin in the IVC medium,higher than that of the control group(17.5%,P＜0.05).Simultaneously supplementation of 10ng/mL leptin in IVM and IVC resulted in a further increase in blastcyst development comparing to control group(27.0%vs 19.7%, P＜0.05).However,when the oocyte was fertilized by sexed sperm, supplementation of 10ng/mL leptin in IVM and IVC resulted in little improvement over control group in terms of cleavage and blastocyst development rate(P＞0.05).Sexed buffalo sperm and OPU-derived oocytes were used in this study to produce sex-preselected embryos,which were subsequently transferred into recipients to produce calves.The results revealed that OPU-derived oocytes fertilized by sexed sperm had similar developmental competence to those fertilized by unsexed sperm in terms of cleavage rate(50.5 vs 50.9%,P＞0.05) and blastocyst development rate(15.3 vs 19.1%,P＞0.05).Of the embryos produced in OPU-IVF system,9 of 34 sexed fresh embryos(26.5%)and 5 of 43 sexed frozen embryos(11.6%)transferred to recipients established pregnancies, whereas 7 of 26 unsexed fresh embryos(26.9%)and 6 of 39 unsexed frozen embryos(15.4%)transferred to recipients established pregnancies.No significant difference in pregnant rate was found following transfer of fresh embryos produced by sexed and unsexed sperm,nor frozen embryos produced by sexed and unsexed sperm.While the data from sexed and unsexed embryos were combined,difference in pregnancy rate was observed between fresh and frozen embryos.Transfer of embryos produced by unsexed sperm resulted in birth of 10 buffalo calves(6 females and 4 males)and 3 abortions(3 males), whereas transfer of sex-preselected embryos resulted in birth of 10 buffalo calves(10 females and 1 male)and 3 abortions(2 females and 1 male). Deviation of sex ratio from 1:1(male:female)in calves produced by sexed sperm was significant(P＜0.05).This study provided the proof of concept for further research and wider field application of these technologies in buffalo.PartⅤ,sexed sperm from buffalo and Holstein was to be used in AI in this study and the factors playing on conception rate were investigated to refine the AI procedures for more efficient production of sexed calves.The results revealed that the conception in Holstein heifers and parous cows was 61.4%and 42.9%(P＜0.05)respectively while bred by sexed sperm,and 61.3%and 46.2%(P＜0.05)respectively while bred by unsexed sperm.More pregnancies were obtained in Holstein heifers than parous cows bred by either sexed or unsexed sperm(P＜0.05).But no significant difference was found between the conception rate following AI by sexed and unsexed sperm(P＞0.05).Similar conception rate after AI by using sexed and unsexed sperm was also observed in buffalo(69.7%vs 66.5%,P＞0.05).And low abortion rate was found in both pregnancies established by using sexed and unsexed sperm(3.3% vs 0.5%,P＞0.05).A slight increase in conception rate in heifer over parous buffaloes bred by sexed sperm was also observed(77.8%vs 67.6%),but not statistical different(P=0.699).The conception rate in cross breed(river type×swamp type,F1)bred by sexed sperm was slightly higher than native swamp buffalo(85.7%vs 62.1%,P=0.114).The results in this study also revealed that, whatever Holstein or buffalo,sexed sperm derived from different bulls significantly affected the conception rate,and different management conditions (housing,feeding,breeding et al.)played an important role in the efficiency of pregnancy establishment.A total of 306 Holsteins were bred by sexed sperm in this study and 179 of which got pregnant,accounting for 58.5%.Seven abortions were observed and 172 sex-preselected calves were successfully delivered,in which heifers accounted for 89.0%(153/172).A total of 65 buffaloes were bred by sexed sperm and 35 of which got pregnant,accounting for 53.8%.Two abortions were observed and 33 sex-preselected buffalo calves were successfully delivered,in which heifers accounted for 84.8%(28/33).Deviation of sex ratio from 1:1 (male:female)in calves produced by AI using sexed sperm was significant (P＜0.01).This study suggested the feasibility for a wider field application of AI by using sexed sperm to accelerate the improvement in genetics and economics in native buffalo.