CN106511386B - 一种牛冷冻精液制剂及其制备方法 - Google Patents

一种牛冷冻精液制剂及其制备方法 Download PDF

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CN106511386B
CN106511386B CN201610910884.3A CN201610910884A CN106511386B CN 106511386 B CN106511386 B CN 106511386B CN 201610910884 A CN201610910884 A CN 201610910884A CN 106511386 B CN106511386 B CN 106511386B
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许晓玲
刘彦
白佳桦
路永强
冯涛
肖霖力
王天坤
宋玉清
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

本发明提供了一种冷冻精液制剂及其制备方法,所述精液制剂包括冷冻精液以及含有褪黑素和前列腺素的溶液。精液和所述溶液储存于细管中，两者之间用空气或惰性气体隔离。使用时，执细管一端甩管即可实现精液与溶液的混合。本发明通过奶牛实验发现，所述制剂能够提高奶牛冻精输精后的受胎率，可以在家畜养殖领域广泛推广应用，具有良好的市场前景和社会效应。

Description

一种牛冷冻精液制剂及其制备方法
技术领域
本发明涉及家畜人工授精的改良技术领域，具体涉及一种牛冷冻精液制剂及其制备方法。
背景技术
人工授精技术是家畜繁殖中一项效果非常好、非常成熟的专门技术，在推进品种改良、提高和改善畜产品产量和品质方面意义非常重大。奶牛的人工授精技术从20世纪80年代开始在中国广泛使用,随后冷冻精液逐渐取代了鲜精授精。近年来随着规模化、集约化的普及，奶牛的产奶量在逐年增加，但是奶牛的繁殖效率却在下降。如何提高奶牛输精后的受胎率进而提高奶牛的繁殖效率,成为繁殖学家研究的热点。
前列腺素(prostaglandin)最早发现存在于人的精液中，主要来自精囊。此后发现从动物体多种组织，如睾丸、子宫、胎盘、及卵巢等均可提取。其主要具有溶解黄体的功能以及促使平滑肌收缩，这对发情、排卵、分娩时子宫的蠕动、产后胎衣排出、精子在生殖道内运行，均有一定的影响。目前，前列腺F2α(PGF2α)及其类似物已有效地用于奶牛的同期发情，持久黄体、黄体囊肿等卵巢疾病的治疗。Abdussamad等(2015，Comparison of various semenextenders and addition of prostaglandin F2αon pregnancy rate in cows)研究表明，冷冻精液中添加1.5mg/ml PGF 2α,受胎率可达到56％。发明专利(一种提高母牛受胎率的方法，申请号，201510265568.0)在人工授精前1至2小时，用输精管吸入0.2mg前列腺素2ml，注射到子宫颈分叉处，可以提高奶牛的受胎率。
褪黑素(melatonin)是一种主要由松果体分泌的神经内分泌激素，化学结构为N-乙酰-5-甲氧色胺(N-acetyl-5-methoxytryptamine)，其在生物钟、生殖、免疫、抗氧化等方面有重要作用。同时，也有研究表明，褪黑素可以直接参与卵泡的发育与排卵,提高牛卵母细胞成熟率以及胚胎的发育率。发明专利(一种提高奶牛受胎率的制剂及其制备方法与应用，申请号，201510085043.9)10-3～10-1mol/L的褪黑素和浓度为10-6～10-4mol/L的维生素E可以有效提高奶牛受胎率。
提高奶牛受胎率的方法和制剂还有很多，但是奶牛受胎率的提升幅度仍然有很大的空间。并且，很多技术操作复杂，需要经过专业训练，在一定程度上影响了技术的推广应用。
发明内容
本发明的目的是提供一种牛冷冻精液制剂及其制备方法。
本发明的技术方案如下：
一种牛冷冻精液制剂，所述冷冻精液制剂包括冷冻精液以及含有褪黑素和前列腺素的溶液(下称为MP溶液)，精液和所述溶液储存于细管中，两者之间用空气或惰性气体隔离。
所述细管的材料可以是任何对精子活性不产生不良影响的材质，例如塑料细管、玻璃细管，优选为塑料细管。细管内径一般为0.5～5.0mm，例如可以是0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5或5.0mm，一种优选的例子是2.5mm。管径可以根据气体分隔两端液体的效果进行调整。
优选地，所述褪黑素与前列腺素的重量比为：1:6250-625000，更优选，所述褪黑素与前列腺素的重量比为：1:62500。
在所述MP溶液中，所述褪黑素的浓度为优选为4.8-480ng/ml，更优选为48ng/ml。所述前列腺素的浓度优选为2-4mg/ml，更优选为3mg/ml。
本领域技术人员应当理解，上述MP溶液的浓度可以根据商品的需要进行浓缩或稀释，在使用时MP溶液可以进行相应稀释后使用。另外，MP溶液的溶剂可以是本领域所能接受的任何可用于稀释精子的溶剂，例如常规精子稀释液。
本发明还提供一种制备所述牛冷冻精液制剂的方法，其包括如下步骤：1)在细管中装入一段待冷冻精液；2)装入一段空气或惰性气体；3)装入一段所述含有褪黑素和前列腺素的溶液；4)封闭细管；5)预冷冻后投入液氮保存。
细管的封口材料可以是棉栓，或者其他具有封闭功能、对精子活性不产生不良影响，并能够被针头穿刺的材料，例如硅胶。为了便于灌装，细管的至少一端的封口材料应当是透气材料，例如棉栓。细管的另一端也可以通过烧结或者烫结的方式封闭。细管的两端封口内侧，也可以分别置入一段封闭液，一端封闭液与待冷冻精液之间用空气或惰性气体隔离，另一端封闭液与含有褪黑素和前列腺素的溶液之间同样用空气或惰性气体隔离。封闭液可以是任何对精子不会产生不良影响的溶液，例如MP溶液。当然封闭液不是必须的。
冷冻精液的制备可以按照常规方法，例如将检验合格的精液加入冷冻保护液，预平衡直4℃即可。一般而言，细管中装入精液的量以150～250μl为宜，精液与MP溶液按体积比优选为1:0.5-1.5，更优选1:1。
预冷冻可以采用常规程序进行，例如在程序降温仪中从4℃开始，1℃/min，降到-6℃，再经液氮熏蒸15min后投入液氮保存。在使用时，从液氮中取出，于38℃水浴锅中解冻30秒，甩管使精液与MP溶液混合，上下颠倒使之混合均匀。
本发明通过奶牛实验发现，所述制剂能够提高奶牛冻精输精后的受胎率，该方法简单易操作，通过受胎率的提高，进而提高了奶牛的繁殖效率，降低了成本，增加了经济效益，因此具有良好的应用前景。
附图说明
图1是本发明牛冷冻精液制剂装管示意图，其中1封口，2精液，3空气，4MP溶液。
具体实施方式：
以下实施例用于进一步说明本发明，但不应理解为对本发明的限制。在不背离本发明精神和实质的前提下，对本发明所作的修饰或者替换，均属于本发明的范围。
地诺前列腺素购自辉瑞、精液稀释液都购自Minitueb(ref.13500/0260)，褪黑素购自Sigma(货号：M5250)。精液采自荷斯坦种公牛。
实施例1牛冷冻精液制剂的制备
1.1MP溶液的制备
1)称取8.16mg褪黑素，溶于1mL的无水乙醇中，用PBS稀释1000倍，成终浓度8.16ng/μL，备用。
2)在0.5mL EP管中，分别加入浓度为5mg/ml的地诺前列腺素102μL,精液稀释液67μL，褪黑素1μL，共计170μL MP溶液，混匀。
MP溶液中褪黑素的浓度为48ng/ml，地诺前列腺素的浓度为3mg/ml。所有操作均在暗室中完成。
1.2精液的制备
采集荷斯坦种公牛精液，迅速带回实验室。预热的精液稀释液稀释20倍后用精子图像分析系统检查精子密度以及活力。在保证输精有效精子数在1000万以上，加入相应量预热的精液冷冻液(果糖0.99w/v％，Tris 4.84w/v％，柠檬酸2.77w/v％，甘油7.00v/v％，卵黄20v/v％，链霉素800IU/ml，青霉素800IU/ml，SDS 0.15w/v％)，制成精液混合液。
1.3灌装与保存
取内径2.5mm的毛细管，一端用棉栓封闭，装入精液混合液170μL，装入0.5cm空气，装入170μL MP溶液，封闭。预平衡至4℃，在程序降温仪中从4℃开始，1℃/min，降到-6℃，再经液氮熏蒸15min后投入液氮保存。
实施例2配种实验
1)从液氮中取出细管冻精，置于38℃水浴锅中解冻30秒，甩管并上下颠倒，使精液与MP溶液混匀；
2)剪开细管封口，混合液中，褪黑素的终浓度为24ng/ml，地诺前列腺素的浓度为1.5mg/ml。另设对照组、褪黑素实验组和地诺前列腺素实验组，对照组为精液稀释液，褪黑素实验组和地诺前列腺素实验组分别仅含褪黑素和地诺前列腺素，制备方法同实施例1。各组别操作同。具体分组如下：
表1各组别精液混合液中褪黑素组和地诺前列腺素的含量
组别 褪黑素(ng/ml) 前列腺素(mg/ml) 总体积(μL)
对照组 0 0 340
褪黑素组 24 0 340
地诺前列腺素组 0 1.5 340
褪黑素组+地诺前列腺素组 24 1.5 340
将混合液装入输精枪，准备输精；
3)输精对象的选择
选择发情周期正常的泌乳奶牛，随机分成四组，每组平均胎次相近。
4)输精时间的确定
严格控制在排卵前的3-5小时输精，即奶牛接受爬跨后12-14小时进行直肠检查，卵泡有一触即破时为最佳输精时间。并结合外观观察，奶牛表现安静，外阴肿胀开始消失，阴门流出半透明的牵缕性黏液。
5)输精方法
严格消毒母牛外阴。采用子宫体深部输精，输精部位要求在排卵侧子宫角深处2-5厘米处，这样保证有足够精子到达与卵子授精部位。并且输精时做到“轻插、慢推、缓出”，以免对母牛的子宫造成损伤。
6)早期妊娠诊断
配种后28天，采集血液5ml，分离血清，利用爱德士可视早孕检测试剂盒，检查奶牛的受胎情况，并统计结果。
表2各组别精液混合液输精后的受胎率
处理 处理奶牛头数 妊娠头数 受胎率(％)
对照组 25 9 36
褪黑素组 24 9 37.5
地诺前列腺素组 22 10 45.5
褪黑素组+地诺前列腺素组 27 14 51.9
统计结果显示：该制剂可显著提高奶牛配种后的受胎率。
结论：由以上结果可知褪黑素和地诺前列腺素联合使用可以提高奶牛输精后的受胎率。因此，该组合试剂可应用于奶牛的体外输精。

Claims (5)

1.一种牛冷冻精液制剂，所述冷冻精液制剂包括冷冻精液以及含有褪黑素和前列腺素的溶液，精液和所述溶液储存于细管中，两者之间用空气或惰性气体隔离，其中褪黑素的浓度为48ng/ml，其中前列腺素的浓度为3mg/ml，所述精液与所述溶液体积相等。
2.根据权利要求1所述的冷冻精液制剂，其特征在于，所述细管为塑料细管。
3.根据权利要求1或2所述的冷冻精液制剂，其特征在于，所述细管内径为0.5～5.0mm。
4.根据权利要求3所述的冷冻精液制剂，其特征在于，所述细管内径为2.5mm。
5.一种制备权利要求1-4任一项所述牛冷冻精液制剂的方法，其包括如下步骤：1)在细管中装入一段待冷冻精液；2)装入一段空气或惰性气体；3)装入一段所述含有褪黑素和前列腺素的溶液；4)封闭细管；5)预冷冻后投入液氮保存。
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