摘要
中国拥有超过两千万头沼泽型水牛,但绝大多数只能作为役用,若能通过性别控制技术将其快速改良成乳用或乳肉兼用的杂交水牛,将能带来巨大的经济和社会效益。本论文利用流式细胞仪分离水牛X和Y精子法进行水牛性别控制研究,以期建立和完善水牛性别控制技术体系,并应用到水牛繁殖、育种的研究和生产实践,加速水牛品种改良工作进展。具体研究内容分为如下五个部分:
研究一,分析和鉴定水牛X精子和Y精子DNA含量的差异,为进行分离水牛XY精子性别控制研究提供理论基础。
研究结果显示,摩拉水牛的X精子和Y精子的DNA含量差别为3.59±0.11%,尼里—拉菲水牛X精子和Y精子的DNA含量差别为3.55±0.14%,两个品种之间没有显著差异(P＞0.05),但是品种内的牛个体之间有差异(P＜0.05)。本研究结果揭示了水牛XY精子DNA含量差异,同时表明了分离的可行性。
研究二,对水牛精子分离之后的体外存活能力、顶体完整情况及微生物污染等精液品质相关参数进行系统分析,以了解影响水牛分离精子活率和受精能力的因素,为改进水牛精子分离方法及提高其在IVF和AI应用效率提供参考。
研究结果表明,流式细胞仪法、荧光显微镜法与精子图像分析仪法检测所得精子活率的相关系数大于0.85,相关性非常显著,表明均能有效评定精子活率。对分离精子品质分析结果表明,新鲜水牛精子经过分离后,死亡精子被去除,活精子率从分离前的72.4%提高到了86.4%(P＜0.05)。经过冷冻解冻后,分离精子的活率高于未分离精子的活率(44.1%vs 33.9%,P＜0.05);体外孵育3h、6h后,分离精子活精子率分别为24.2%、15.6%,未分离精子活精子率分别为9.0%、6.7%,二者均下降明显(P＜0.05),而同一孵育时间的分离精子活率均高于未分离精子活率(P＜0.05)。在顶体反应方面,新鲜水牛精子经过分离后,顶体损坏的精子也被去除,发生顶体反应精子的比例从分离前的22.9%减少到分离后3.9%(P＜0.05),但是经过冷冻解冻后增加到30.0%,与未分离冷冻精子的26.8%没有显著差异(P＞0.05),表明分离精子受冷冻损伤更严重。解冻并体外孵育3h、6h后,分离精子顶体反应比例分别为33.9%、38.3%,未分离精子顶体反应比例分别为32.8%、35.6%,同一孵育时间的分离精子顶体反应率稍微高于未分离精子,但无统计差异(P＞0.05)。
精液微生物分析试验结果表明,新鲜采集的水牛精液平均菌落密度10619个/毫升,常规冻精平均菌落密度为293个/毫升,而分离冷冻精液平均菌落密度达到18935个/毫升,三者差异显著(P＜0.05)。新鲜精液检测到的菌落中,革兰氏阳性球菌占48%,阴性杆菌占41%,阳性杆菌占9%,霉菌占2%;而分离精子冷冻精液中的菌落100%为革兰氏阴性杆菌,经检测确定为阴沟肠杆菌和产酸克雷伯氏菌,对妥布霉素和庆大霉素呈耐药性,而对丁胺卡那霉素以及环丙氟哌酸药物敏感性较强。
研究三,通过制作奶牛性染色体涂染探针,检测水牛分离精子的纯度,以期建立一种独立评估水牛分离精子纯度的手段。
研究结果表明,运用显微操作仪能够有效分离荷斯坦奶牛的性染色体并制作探针。应用荷斯坦奶牛Y染色体探针杂交水牛正常精子显示,Y精子比例为47.7%;应用荷斯坦奶牛X染色体探针杂交水牛正常精子显示,X精子比例为48.9%,二者均接近50%理论值(P＞0.05),表明所制备染色体探针用于鉴定XY精子比例的有效性。在杂交水牛分离精子中,应用Y染色体探针杂交水牛分离Y精子样本表明,Y精子所占的比率为82.2%;应用X染色体探针杂交水牛分离Y精子样本表明,X精子所占比例为9.6%,也即Y精子所占的比率为90.4%。两种探针检测分离Y精子的综合纯度为86.2%,与流式细胞仪重分析87%的结果接近(P＞0.05)。
研究四,将水牛分离精子应用于活体采卵(OPU)和体外受精(IVF)技术体系,以提高水牛分离精子生产优良性控胚胎和后代的效率。
试验结果表明,在水牛卵母细胞体外成熟培养中添加瘦素不但能有效促进水牛卵母细胞成熟,而且提高其体外成熟的质量及受精后的胚胎发育潜能,其中以成熟培养液中添加10ng/mL的效果最为明显,卵母细胞成熟后极体排出率达到61.2%,IVF囊胚发育率达到22.4%,与不添加瘦素的对照组51.9%的极体排出率和13.0%的囊胚率均有显著差异(P＜0.05)。研究还发现,在胚胎培养液中添加瘦素有助于促进水牛IVF胚胎的发育,其中以10ng/mL的效果最为明显,囊胚发育率达到26.1%,与不添加瘦素的对照组17.5%的囊胚率有显著差异(P＜0.05)。此外,在成熟液和胚胎培养液中同时添加10ng/mL的瘦素,IVF囊胚发育达到27.0%,与对照组19.7%的囊胚率差异显著(P＜0.05)。但在分离精子IVF时,在成熟液和胚胎培养液中同时添加10ng/mL的瘦素,虽然IVF后卵母细胞的分裂率和囊胚率都有一定提高,但是与对照组没有统计差异(P＞0.05)。
在OPU-IVF研究中,OPU得到的水牛卵母细胞与分离精子和未分离精子IVF后的分裂率分别为50.5%和50.9%,二者没有显著差异(P＞0.05);而分离精子IVF后的囊胚率为15.3%,稍微低于未分离精子19.1%,但二者也没有显著差异(P＞0.05)。分离和未分离精子与OPU来源卵母细胞IVF生产的鲜胚移植受胎率分别为26.5%和26.9%,二者没有显著差异(P＞0.05);分离和未分离精子与OPU来源卵母细胞IVF生产的冻胚移植受胎率分别为11.6%和15.4%,二者也没有显著差异(P＞0.05)。将分离精子和未分离精子生产的胚胎移植数据合并分析显示,冻胚的总体受胎率(13.4%)显著低于鲜胚受胎率(26.7%,P＜0.05)。未分离常规精子与OPU来源卵母细胞生产的体外胚胎移植后,产下10例正常水牛犊(6母,4公),流产3例(3公);分离X精子与OPU卵母细胞生产的体外性控胚胎移植后,产下11例正常水牛犊(10母,1公),流产3例(2母,1公),雌性率为85.7%,远高于未分离精子46.1%(6/13)的雌性率(P＜0.05)。
研究五,通过将水牛分离精子应用于AI,并与荷斯坦牛分离精子AI相比较,探讨和分析水牛分离精子在AI中应用的问题,以提高其利用效率,为利用分离精子性别控制技术加快水牛品种改良奠定基础。
研究结果表明,在荷斯坦奶牛方面,分离和未分离的常规精子配种处女牛的受胎率分别为61.4%和61.3%(P＞0.05),配种经产母牛的受胎率分别为42.9%和46.2%(P＞0.05);无论是分离精子还是常规精子,配种经产母牛的受胎率均显著低于处女牛(P＜0.01)。在水牛方面,分离精子和常规精子配种水牛情期受胎率分别为69.7%和66.5%,二者无显著差异(P＞0.05);分离精子配种处女牛受胎率为77.8%,比配种经产母牛67.6%的受胎率高,但没有统计差异(P＞0.05);在流产率方面,水牛分离精子和常规精子配种妊娠后流产率都很低,二者没有显著差异(P＞0.05)。而分离精子配种杂交水牛受胎率(85.7%)高于本地沼泽型水牛(62.1%),但二者没有统计差异(P＞0.05)。
此外,本研究还表明,无论是荷斯坦奶牛还是水牛,不同公牛来源的分离精子配种后受胎率差异显著(P＜0.05);在不同管理条件下的母牛群进行分离精子配种的受胎率差异明显(P＜0.05)。
本研究中,利用分离精子总共配种荷斯坦奶牛306头,受胎179头,情期受胎率58.5%,流产7头,产犊172头,其中产母犊153头,产犊性别准确率89.0%;利用分离精子总共配种水牛65头,妊娠35头,情期受胎率53.8%,流产2头,经产下牛犊33头,其中母犊28头,产母犊性别准确率84.8%。奶牛和水牛分离精子母犊率与常规精子配种后50%的理论值差异显著(P＜0.01)。
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.
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