CN110079495B - 一种牛冻精激活液 - Google Patents
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Abstract
本申请属于生物技术领域和畜牧业人工授精技术领域,具体地,本申请提供了一种牛冻精激活液,其中包含大豆蛋白或小麦低聚肽的非动物源性蛋白,pH为7.0‑9.0。
Description
技术领域
本申请属于生物技术领域和畜牧业人工授精技术领域,具体地,本申请提供了一种牛冻精激活液,其中包含大豆蛋白或小麦低聚肽的非动物源性蛋白,pH为7.0-9.0。
背景技术
精液冷冻保存是将精液用含特殊冷冻保护剂和营养成分等的稀释液稀释后,保存于液氮或干冰中,以达到在体外长期保存精子的目的。精液冷冻技术是现代畜牧业家畜品种保持/构建,特别是牛、羊品种保持/构建最重要的手段之一——人工授精中几乎必不可少的技术(鲜配受时间、地域、操作条件限制很大)。
精液冷冻技术最重要的指标是复苏/复温后精子的活力和完整度,目前细管牛羊精液解冻一般采用在30-50℃水浴中直接解冻5-40s的方法。实践中,这种方法虽然简单可靠,但由于样本质量、保存条件、操作失误(养殖厂环境中,操作大量样本时操作失误如解冻超时等常出现),经常出现复苏/复温后精子的活力和完整度,授精成功率不足/授精失败的问题,特别是对于较为珍贵/昂贵的,数量较少的精液样本(对于优质品种牛羊精液,经常由于保密、价格等原因经常无法获得足够试错用的精液样本),这种失败带来的经济和时间损失更为严重。
因此,本领域中有开发提高冻精复苏效果的方法/试剂的需求。
发明内容
申请人在冻精稀释液研究的基础上,进一步发现在冻精解冻后给予少量非动物源蛋白——大豆蛋白和小麦低聚肽,并且以较高pH环境处理可以有效提高解冻后精子活力和完整度并提高人工授精效果,进一步的研究表明,这样的处理还可以有效提高膜完整性和流动性(可能是人工授精效果提高的原因之一)。
一方面,本申请提供了一种牛冻精激活液,其中包含非动物源性蛋白、氯化钠和缓冲液成分。
进一步地,所述牛冻精激活液中的非动物源性蛋白为大豆蛋白或小麦低聚肽。
进一步地,所述牛冻精激活液中的非动物源性蛋白为小麦低聚肽。
进一步地,所述牛冻精激活液中缓冲液为Tris-HCl,pH为7.0-9.0。
进一步地,所述牛冻精激活液中缓冲液为0.2M的Tris-HCl,pH为8.5。
进一步地,所述牛冻精激活液组成为:每100mLpH为8.5的0.2M Tris-HCl缓冲液中,加入小麦低聚肽1g,NaCl 0.8g。
另一方面,本申请提供了上述牛冻精激活液在牛人工授精繁殖中的应用。
进一步地,所述应用中将解冻后的牛冻精和激活液混合,轻柔搅拌混合后静置,进行人工授精。
进一步地,所述应用中将解冻后的牛冻精按照牛冻精:激活液 3:1的比例混合,轻柔搅拌混合后静置10分钟。
本申请中的氯化钠、Tris、HCl、小麦低聚肽等成分可以选用符合相应标准的各生产商产品。Tris-HCl也可以选用市售成品。根据各生产商的大豆蛋白、小麦低聚肽产品性质的不同,配方可以做适当调整。
本申请的牛冻精激活液可用于各种自制和市售的各种牛冻精产品,只要其中所用冻精稀释液其中不包含与本申请的牛冻精激活液中成分有反应性的成分即可。
本申请的牛冻精激活液可用于各品种人工授精,包括但不限于安格斯牛、荷兰牛、神户肉牛、夏洛莱牛、西门塔尔牛、鲁西黄牛、大额牛等。经过本领域常规技术手段的改进和验证后也有望用于羊、马、猪等家畜。
具体实施方式
主要试剂和仪器
小麦低聚肽 食品级,河南兴源化工产品有限公司生产;
Tris、HCl、氯化钠由国药集团生产;
ANS、DPH标记物:北京百奥博莱科技有限公司;
冻精封灌设备,MRSP3,由法国卡苏公司生产;
细管冷冻仪,007209,由法国卡苏公司生产;
精子检测仪以及配套软件,WLJY9000,由北京新世纪科技发展有限公司生产;
荧光光度计(配偏振附件),F7000,由日本日立公司生产;
其他未专门标明的非关键仪器和试剂为常规国产种类/型号。
实施例1 牛冻精激活液的配制
按照以下配方配制各种冻精激活液(筛选配方时使用的部分效果不佳配方未列出):
配方1:
每100mLpH为8.5的0.2M Tris-HCl缓冲液中,加入小麦低聚肽1g,NaCl 0.8g;
配方2:
每100mLpH为8.5的0.2M Tris-HCl缓冲液中,加入大豆蛋白1g,NaCl 0.8g;
配方3:
每100mLpH为5.5的0.2M Tris-HCl缓冲液中,加入小麦低聚肽1g,NaCl 0.8g;
配方4:
每100mLpH为7.0的0.2M Tris-HCl缓冲液中,加入小麦低聚肽1g,NaCl 0.8g。
实施例2 牛冻精来源
筛选和对比试验使用申请人2017年保存的(至实验时保存1年2个月时间)同批次大额牛精液细管,该细管使用卡苏OPTIXcellTM冷冻稀释液以及卡苏的冷冻设备制备。将5份样品直接以37℃水浴解冻30s后测量复苏精子基本指标,结果表明该批次大额牛精液细管精液性能良好且较为均一(鲜精活力达到0.65以上,精子密度达到10亿/ml,复苏率和活力百分数相差不超过0.5%)。
实施例3 牛冻精激活实验
冻精解冻/激活方法:
对照组,不加激活剂,37℃水浴解冻30s;
配方1-5:将解冻后的牛冻精按照牛冻精:激活液 3:1的比例混合,轻柔搅拌混合后静置10min。
复苏精子基本指标检测:
使用前述的伟力精子分析仪以及配套软件分析总活精子数、精子复苏率、活力、平均路径速率(VAP)、曲线运动速率(VCL)、直线运动速率(VSL)、顶体完整率和畸形率(每个配方10份样本平均值,由于伟力精子分析仪原用途为人精子分析,用于牛时部分高密度或软件中出现问题样本可能出现数据混乱明显失真情况,对此类伟力精子分析仪分析结果明显有问题的样本,参考现有技术人工观察和统计)。同时取样本200微升检测复苏精子存活时间(人工监控,99%精子死亡/失活的时间)结果见下表:
激活液配方 | 精子复苏率(%) | 活力(%) | VAP(u/s) | VSL(u/s) | VCL(u/s) | 顶体完整率(%) | 畸形率(%) | 存活时间(h) |
对照 | 63.93±0.25 | 51.23±0.37 | 88.43±2.32 | 65.31±1.96 | 127.33±5.79 | 79.33±2.21 | 13.52±1.78 | 7.7±0.5 |
配方1 | 68.91±0.64 | 58.96±0.43 | 88.43±2.23 | 66.32±2.87 | 129.29±11.21 | 80.93±5.21 | 12.38±2.09 | 11.9±0.4 |
配方2 | 67.74±0.94 | 57.90±0.37 | 89.43±1.65 | 64.31±5.22 | 129.93±10.32 | 81.25±6.45 | 13.08±4.21 | 9.5±0.5 |
配方3 | 64.15±0.74 | 49.14±0.62 | 83.43±0.94 | 63.21±1.97 | 120.33±11.77 | 81.55±6.75 | 12.98±3.81 | 7.6±0.8 |
配方4 | 63.51±0.68 | 52.96±0.43 | 85.43±1.07 | 65.06±4.13 | 128.56±10.70 | 80.06±2.47 | 13.02±2.87 | 8.0±0.4 |
上述结果表明,配方1-2均实现了良好的精液保存效果,存活时间上明显超过直接解冻不做激活处理的对照,复苏率和活力上也有优势。特别是配方1存活时间超过其他配方50%以上,这对人工授精操作带来了很大的便利(统一母牛发清周期,成批操作人工授精时,由于一般养殖厂操作人数有限,往往需要很长时间完成,这期间经常会遇到精子存活时间/存活状态不佳的问题)。
配方1和配方3-4的比较则表明了pH对激活效果的重要意义:现有技术一般认为由于哺乳动物阴道中pH环境为酸性,精液的冷冻和活化也都应在弱酸性条件下进行(冷冻稀释液为中性或弱酸性,解冻时不专门调整pH),而我们发现,弱碱性的环境可能引起的新陈代谢速率变化以及细胞质膜流动性的变化(见下面实验)可能对延长精子存活时间有益。
为进一步验证pH对细胞质膜流动性的影响,进行以下精子膜荧光强度和偏振检测:
将复苏精子以pH同激活液体的0.1M Tris-HCl缓冲液稀释到2x107个精子/ml,加入ANS至终浓度3x10-5mol/L,室温孵育10min,以375nm激发波长,480nm发射波长检测荧光强度。
将复苏精子以pH同激活液体的0.1M Tris-HCl缓冲液(不使用激活液的样本,使用pH 6.0的Tris-HCl缓冲液)稀释到2x107个精子/ml,加入DPH标记液至1x10-6 mol/L,室温下孵育30min,离心去上清,Tris-HCl缓冲液重悬后相同条件处理一遍。再次重悬后,以362nm激发波长,432nm发射波长检测荧光偏振度。
结果如下表:
激活液配方 | 荧光强度 | 荧光相对偏振度 |
配方1 | 6350 | 0.549 |
配方3 | 5730 | 0.591 |
配方4 | 5862 | 0.604 |
上述结果证明了碱性pH对细胞质膜完整性和流动性有正面作用,明显优于弱酸性和中性激活液,这很可能是精子生存时间延长的原因。
Claims (4)
1.一种牛冻精激活液,其组成为:每100mL pH为8.5的0.2M Tris-HCl缓冲液中加入小麦低聚肽1g,NaCl 0.8g。
2.根据权利要求1的牛冻精激活液在牛人工授精繁殖中的应用。
3.根据权利要求2所述应用,其中将解冻后的牛冻精和激活液混合,轻柔搅拌混合后静置,进行人工授精。
4.根据权利要求3所述应用,其中将解冻后的牛冻精按照牛冻精:激活液 3:1的比例混合,轻柔搅拌混合后静置10分钟。
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CN101310729A (zh) * | 2007-05-23 | 2008-11-26 | 辽宁省辽宁绒山羊育种中心 | 牛羊无动物源冻精稀释液及牛羊冷冻精液的制作方法 |
CN104145944A (zh) * | 2014-08-25 | 2014-11-19 | 山东省海洋生物研究院 | 一种魁蚶精子超低温冷冻保存与激活方法 |
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CN104145944A (zh) * | 2014-08-25 | 2014-11-19 | 山东省海洋生物研究院 | 一种魁蚶精子超低温冷冻保存与激活方法 |
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