CN116158405A - 一种提高奶山羊后代母羔率的方法 - Google Patents
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Abstract
本发明提供了一种提高奶山羊后代母羔率的方法：首先确定奶山羊Y精子中特异性表达的蛋白DPY30，然后包装山羊DPY30基因干扰慢病毒，将山羊DPY30基因干扰慢病毒注射入种公羊睾丸，47d后采集种公羊精液鉴定精液品质及上游精子中X、Y精子的比例，使用注射DPY30基因干扰慢病毒的种公羊精液人工授精后母羔率达到58.20％，提高了22.08％，显著增加了出生母羔的数量。本发明采用干扰慢病毒调控编码奶山羊Y精子中特异性表达蛋白的基因DPY30在山羊睾丸中表达，高效地提高后代的母羔率，并不影响公羊精液品质及母羊的妊娠率、产羔率，获得了有效调控奶山羊后代性别的技术，具有较好的推广应用前景和经济价值。
Description
技术领域
本发明属于动物繁殖技术领域，具体涉及奶山羊后代性别比例调控的方法。
背景技术
奶山羊是我国发展特色奶制品的重要奶畜动物，而羊奶因其高营养价值备受关注。目前传统的奶山羊养殖模式不能满足产业发展的需求，为积极推动奶山羊养殖科学化、规模化、现代化发展，需要改进、优化现有的动物繁殖技术，干预动物的正常繁殖过程，选择性地提高母羊在养殖场中的占比，加速奶山羊的扩群及种质提升、提高乳制品的产量，这对于发展奶山羊养殖、增加市场羊乳产品供应具有重要意义。
奶山羊后代性别调控有多种方法，常见的方法主要有：流式细胞术分离奶山羊X、Y精子；TLR7/8受体激活分离奶山羊X、Y精子；SRY抗体分离奶山羊X、Y精子。这些不同的奶山羊后代性别调控方法的均可分离奶山羊X、Y精子以达到后代性别调控的目的，但都存在安全性差、难以推广的问题。
1.流式细胞术分离奶山羊X、Y精子
流式细胞术分离X、Y精子是目前主流的性别控制技术手段，基于该方法分离的牛X、Y逐渐市场化，商品化，在国内外获得了良好的效果。流式细胞术也可高效分离奶山羊X、Y精子，利用X、Y精子分离技术配合人工授精技术，母羊可生产出性别比符合率达到83％以上的性别控制后代。然而，与传统精液相比，这种方法的生产经济、时间成本更高(每小时仅7-12支)，性控冷冻精液的价格是普通冻精的3-5倍数。与此同时，母羊宫颈长、狭窄、曲折，且存在多个褶皱，严重阻碍输精枪进入的深度从而影响人工授精效率；为了达到较高的受胎率，只能靠提高输精剂量，但这样显著提高了奶山羊性别控制的成本，难以有效应用于生产。此外，流式细胞术分离后的精子受到明显损害，分离和冷冻过程严重影响精子受精能力和胚胎发育，这降低了流式细胞术分离X、Y精子的安全性。
2.TLR7/8受体激活分离奶山羊X、Y精子
利用奶山羊X、Y精子差异表达的蛋白TLR7/8受体，在稀释后的精液中添加TLR7/8配体(R848)影响X精子运动能力，使得Y精子上游，分离的精子用于体外受精，可以获得80.52％±6.75％的山羊雌性胚胎。该分离过程会不可逆的激活精子的糖酵解途径，使得精子由直线运动变为“之”字形运动。因此该方法只适用于体外受精，并不适用于人工授精，这严重限制了该技术在的生产中的应用。
3.SRY抗体分离奶山羊X、Y精子
使用制备SRY抗体可以特异性识别山羊Y精子，但SRY蛋白不属于精子膜蛋白。若要利用SRY抗体高效分离奶山羊X、Y精子，需要增加精子细胞的通透性，会无差别的对X、Y精子造成额外的损伤。
综上，尽管多年来有大量研究推动了奶山羊后代性别控制相关研究技术的改进与发展，但是现有的技术还是存在诸多弊端，不能有效应用于奶山羊产业。目前仍需要开发一种更安全、高效、经济、易于操作推广的奶山羊后代性别控制手段。
发明内容
本发明的目的在于提供一种奶山羊后代性别控制的方法。
为达到上述目的，本发明采用了以下技术方案：
1)测定奶山羊X、Y精子中DPY30的表达；
2)包装山羊DPY30基因干扰慢病毒，滴度达到6.25×106-7TU/ml以上；
3)将山羊DPY30基因干扰慢病毒注射入种公羊睾丸，总共注射3ml病毒；
4)47d后检测DPY30基因干扰慢病毒注射后种公羊精液品质；
5)采集处理后种公羊精液人工授精配种，记录配种后的母羊妊娠率、产羔率、母羔率。
所述步骤2)中，使用慢病毒浓缩试剂(Takara)浓缩包装后的慢病毒，离心的条件为：4℃条件下3000g离心30min。
所述步骤3)具体包括以下步骤：使用5号(25G)长针头(60mm)，采用由浅入深的多点注射方式，将DPY30基因干扰慢病毒注射入种公羊两侧睾丸。
所述步骤4)中，检测评价精液密度，精子活率、活力，及上游精子中X精子的比例。
本发明的有益效果体现在：
本发明主要采用在精子发生过程中调控奶山羊性别的方法；利用奶山羊Y精子特异性表达蛋白，在睾丸内下调DPY30的表达影响Y精子功能的特点(不影响X精子)，达到奶山羊后代性别控制的目的。与现有奶山羊后代性别控制方法相比，本研究具备成本低(＜100元/每只公羊)，简单易行(10min内完成注射)，安全性高(不影响公羊、母羊繁殖性能)，因此获得了有效调控奶山羊后代性别的技术效果，具有较高的推广应用前景和经济价值。
附图说明
图1为本发明所述奶山羊后代性别控制方法的流程图；
图2 Y精子特异性表达蛋白的鉴定；
图3山羊DPY30基因干扰慢病毒载体的包装与干扰效率检测；
图4山羊DPY30基因干扰慢病毒的注射；
图5公羊精液品质的分析；
图6母羊繁殖性能评定；
图7出生母羔比例分析。
具体实施方式
下面结合附图和实施例对本发明作详细说明。
(一)奶山羊后代性别控制实施例(参见图1)
1)选取分离后的X、Y精子，加入RIPA裂解液(1x蛋白酶和磷酸酶抑制剂，1mM PMSF)混匀后，将蛋白样品置于摇床冰浴裂解30min(120r/min)，随后12000r/min，4℃离心10min。吸取上清测定蛋白浓度后加入适量5×SDS-PAGE蛋白上样缓冲液并混匀，100℃沸水浴变性10min。
20μg的X、Y精子总蛋白利用SDS-PAGE电泳进行分离，浓缩胶使用80V，30min；分离胶使用120V，80min。随后应用半干转膜系统将目的蛋白恒压转移至PVDF生物膜，转膜后的生物膜置于5％脱脂奶粉封闭2h。随后将目的蛋白置于相应的一抗反应液，4℃过夜孵育。目的蛋白用TBST洗涤3次，用与一抗反应液对应种属的辣根过氧化物酶标记的二抗孵育目的条带。最后洗涤三次后，使用ECL化学发光剂与二抗偶联的辣根过氧化物酶发生化学反应，检测目的蛋白信号。使用ImageJ程序量化目的条带的信号，β-Actin作为内参评价目的蛋白的相对表达量。
2)根据NCBI在线的山羊DPY30转录本序列，设计两对山羊DPY30基因特异性干扰shRNA，使用退火体系合成双链。将shRNA序列连接到PLL3.7质粒以构建山羊DPY30基因干扰shRNA表达载体，分别命名为PLL3.7-shRNAC1与PLL3.7-shRNAC2。
在100mm皿中培养293T细胞，待其生长至80％-90％汇合度时，将PLL3.7、PLL3.7-shRNA M1、PLL3.7-shRNA M2分别与pMD2.G、psPAX2载体组合转染293T细胞，转染12h后细胞全换液，收集转染后48h及72h细胞培养基，离心去除细胞碎片。利用慢病毒滴度检测卡测定慢病毒滴度后，使用慢病毒浓缩试剂浓缩提高慢病毒滴度，随后再次测定慢病毒滴度。使用慢病毒侵染山羊精原干细胞，使用实时定量PCR技术测定山羊DPY30基因干扰慢病毒的干扰效率。
3)将山羊DPY30基因干扰慢病毒于4℃保存运输至养殖场，保定种公羊，依次使用75％酒精、碘酒对山羊睾丸进行消毒处理，使用5号(25G)长针头(60mm)，采用由浅入深的多点注射方式，将DPY30基因干扰慢病毒注射入种公羊两侧睾丸。
4)注射47d后采集山羊精液，使用精液稀释液1:3稀释精液，使用计算机辅助精液分析系统(CASA)(南宁松景天伦生物科技有限公司)分析采集后的公羊精液品质。取稀释后的奶山羊精液5μl，置于37℃预热台保温10min，随后使用CASA系统分析奶山羊精液品质(精液密度、精子活率、精子活力、精子直线运动速率)。使用精子上游法收集上游精子，Hoechst33342(28μM)染色后分析其中X、Y精子的比例。
5)使用公羊鉴定为发情的待配母羊，12h后第一次配种。使用注射病毒组公羊与未注射组公羊精液，将奶山羊输精枪置于母羊子宫颈口内1-2cm处输精，发情36h后第二次配种。记录奶山羊配种情况，待母羊生产后，分析奶山羊妊娠率、产羔率、母羔率。
(二)奶山羊后代性别控制结果分析
1.使用蛋白免疫印迹方法鉴定DPY30蛋白在X、Y精子中的表达。经过鉴定，DPY30的表达量在Y精子中显著高于X精子，X精子中检测不到DPY30的信号，参见图2。
2.将慢病毒载体组合共转染293T细胞，利用绿色荧光评价转染48h后的转染效率，经观察发现质粒转染效率较高，同时细胞内也激发表达较强的荧光强度。取收获的病毒上清，检测病毒滴度，收取的病毒滴度可达到T1级别(6.25×106-7TU/ml)以上，满足注射动物体内的要求。
使用包装后的奶山羊DPY30基因干扰慢病毒侵染奶山羊精原干细胞，PLL3.7-shRNA C1干扰DPY30的效率达到近70％，PLL3.7-shRNA C2在精原干细胞中干扰DPY30的效率达到50％。PLL3.7-shRNA C1的干扰效率更强，于是选择该shRNA序列包装得到的慢病毒，使用慢病毒浓缩试剂提高病毒滴度。参见图3。
3.使用5号(25G)长针头(60mm)，采用由浅入深的多点注射方式，将DPY30基因干扰慢病毒注射入种公羊两侧睾丸。注射方式参见图4。
4.奶山羊种公羊睾丸注射DPY30基因干扰慢病毒之后，假阴道法采集公羊精液，稀释后使用CASA系统分析奶山羊精液品质。评价精液密度、活率、活力、直线运动速率后，发现DPY30基因干扰慢病毒并不影响奶山羊精液品质。
Hoechst33342染色后分析上游精子中X、Y精子的比例。发现DPY30慢病毒处理后高活力的山羊精液中，X精子的比例更高。参见图5。
5.将包装的高滴度的慢病毒注射公羊睾丸后，使用山羊DPY30基因干扰慢病毒注射后种公羊精液进行人工授精，共配种119只母羊，其中83只母羊最终怀孕产出110只羔羊；使用对照组公羊精液共配种183只母羊，129只母羊妊娠后生出182只羔羊。经过统计分析，使用对照组和山羊DPY30基因干扰慢病毒注射的公羊精液人工授精后，母羊的妊娠率(70.27％±5.50％，70.29％±5.13％)、产羔率(142.23％±20.46％，132.63％±4.37％)无显著统计学差异。参见6图。
山羊DPY30基因干扰慢病毒注射组的母羔率显著高于对照组(35.40％±6.00％，58.20％±5.50％)。参见7图。
Claims (4)
1.一种提高奶山羊后代母羔率的方法，其特征在于：包括以下步骤：
1)测定奶山羊X、Y精子中DPY30的表达；
2)包装山羊DPY30基因干扰慢病毒，滴度达到6.25×106-7TU/ml以上；
3)将山羊DPY30基因干扰慢病毒注射入种公羊睾丸，总共注射3ml病毒；
4)47d后检测DPY30基因干扰慢病毒注射后种公羊精液品质；
5)采集处理后种公羊精液人工授精配种，记录配种后的母羊妊娠率、产羔率、母羔率。
2.根据权利要求1所述一种提高奶山羊后代母羔率的方法，其特征在于：所述步骤2)中，使用慢病毒浓缩试剂(Takara)浓缩包装后的慢病毒，离心的条件为：4℃条件下3000g离心30min。
3.根据权利要求1所述一种提高奶山羊后代母羔率的方法，其特征在于：所述步骤3)具体包括以下步骤：使用5号(25G)长针头(60mm)，采用由浅入深的多点注射方式，将DPY30基因干扰慢病毒注射入种公羊两侧睾丸。
4.根据权利要求1所述一种提高奶山羊后代母羔率的方法，其特征在于：所述步骤4)中，检测评价精液密度、精子活率、活力、及上游精子中X精子的比例。
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