CN105994251B - 一种杀灭动物精液病毒细菌的稀释保存液 - Google Patents
一种杀灭动物精液病毒细菌的稀释保存液 Download PDFInfo
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- CN105994251B CN105994251B CN201610439771.XA CN201610439771A CN105994251B CN 105994251 B CN105994251 B CN 105994251B CN 201610439771 A CN201610439771 A CN 201610439771A CN 105994251 B CN105994251 B CN 105994251B
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- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
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- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
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Abstract
本发明提供了一种杀灭动物精液病毒细菌的稀释保存液，按重量计，包括以下成分：葡萄糖25‑40份、柠檬酸2.5‑3份、柠檬酸三钠5‑8份、碳酸氢钠1.2‑1.3份、乙二胺四乙酸二钠1.2‑1.3份、氯化钾0.7‑0.9份、三羟甲基氨基甲烷2.8‑3份、半胱氨酸0.05‑0.08份、聚维酮碘0.025‑0.030份和抗菌肽0‑0.002份。用于经济动物精液的稀释、保存和杀灭细菌病毒。
Description
技术领域
本发明属于畜牧业动物人工授精领域，具体涉及一种杀灭动物精液病毒细菌的稀释保存液。
背景技术
人工授精技术的优势是能够通过人工采集动物优质精液，通过一定的技术手段使它能够长时间保存运输，为畜牧业品种改良和提高生产绩效带来巨大经济效益。但是人工授精技术也有它的天然缺陷，那就是将供体精液里面可能带有的病原微生物都传染给它的受体。以猪为例，公猪精液中通常含有104~105 CFU/ml细菌以及可能污染的未知病毒及其他病原微生物。这些细菌和病毒会影响公猪精液的保存时间及精子的活率，并且能够通过人工授精技术将公猪所可能携带病原微生物传染给母猪和小猪，导致母猪繁殖障碍、小猪疾病的流行和生产绩效的大幅度下降，造成严重的经济损失。
国内外学者的研究表明公猪精液中能检出的危害养猪生产的主要病毒有：口蹄疫病毒（FMDV）、猪瘟病毒（CSFV）、猪圆环病毒（PCV-2）、猪伪狂犬病毒（PRV）、猪繁殖与呼吸综合症病毒（PRRSV）、猪细小病毒（PPV）、日本乙型脑炎病毒（JEV）。其中已证实CSFV、PRRSV、PPV、PRV能通过人工受精感染母猪引起死胎、流产等繁殖障碍。种公猪精液带毒是猪群病毒病垂直传播的一个重要途径，也是制约人工受精技术发展和推广的一大难题。
动物精液稀释保存液在人工授精行业应用广泛，技术难度不高，配方简单容易生产，其主要功能是增加精液体积、防止冷应激、提供精子营养、作为高pH值的缓冲液，延长精子的存活时间。但针对公猪精液污染，现有的技术是通过在精液稀释保存液中加入抗生素（如青霉素、链霉素、庆大霉素、林可霉素等）来抑菌，但是抗生素仅能抑制精液中的部份细菌繁殖，而对病毒完全无效，并且有滥用抗生素的风险。因此，目前在经济动物人工授精领域还没有更好的办法来处理精液中的细菌和病毒污染问题。
如发明专利申请201510756292.6，公开了一种猪鲜精保存长效稀释粉及制备方法与应用。它是由重量份数的葡糖糖35-40，乙二酸四乙酸钠1.2-1.4，柠檬酸钠4-8，碳酸氢钠1.2-1.4，氯化钾0.7-0.8，聚乙烯醇0.4-0.6，半胱氨酸0.1-0.2，肌醇0.4-0.6，青霉素1.0-1.5，链霉素1.0-1.5等原料组成，存在滥用抗生素的问题。
发明专利申请201410164618.1，公开了一种猪抗病毒精液稀释液的配方组成。基于每1000毫升双蒸馏水，猪抗病毒精液稀释液含有：15.50份的葡萄糖；11.65份的柠檬酸三钠；2.35份的EDTA钠盐；1.75份的碳酸氢钠；1.00份的聚乙烯醇；5.50份的三羧甲基氨基甲烷；4.10份的柠檬酸；0.07份的半胱氨酸；1.00份的维生素C；0.10份的恩诺沙星；以及50ml抗病毒剂。配方中含有利巴韦林等抗病毒成分，利巴韦林虽然有一定抑制病毒生长的效果，但却是国家严禁畜牧兽医行业使用的抗病毒药物。
杀灭动物精液病毒细菌的精液稀释保存液的开发难度体现在：
a、安全性，首先要不损伤精子活率，不对受精卵、胚胎以及母猪的繁殖造成影响，对精子的遗传性能无影响。
b、有效性，对精子里面的有害病原，特别是病毒有效，是这项技术的关键。
c、能对精子提供有效保护，精子保存期可以达到7-10天。
d、安全性和有效性是一对矛盾，很难对立统一。
e、研究条件要求高，要求对药剂学、兽医细菌学、病毒学、病毒细胞培养、以及畜牧学等相关专业以及试验条件,单一企业或研究单位难以满足。
文献“一种精液净化保护剂对公猪精子活率的影响及对精液中5种病毒的杀灭作用研究”（中国畜牧杂志，繁殖生理，2015年第51卷第7期），公开了一种精液净化保护剂对公猪精子活率的影响，以及对存在于公猪精液中病毒的杀灭作用。该文献的精液净化保护剂不适用于含碘的消毒剂。
发明内容
为了解决上述技术问题，本发明提供了一种杀灭动物精液病毒细菌的稀释保存液。在公猪精液稀释保存液中加入低毒聚维酮碘和抗菌肽，用于经济动物精液的稀释、保存和杀灭细菌病毒。
本发明为了实现上述发明目的，采用如下技术方案：
一种杀灭动物精液病毒细菌的稀释保存液，按重量计，包括以下成分：葡萄糖25-40份、柠檬酸2.5-3份、柠檬酸三钠5-8份、碳酸氢钠1.2-1.3份、乙二胺四乙酸二钠1.2-1.3份、氯化钾0.7-0.9份、三羟甲基氨基甲烷2.8-3份、半胱氨酸0.05-0.08份、聚维酮碘0.025-0.030份和抗菌肽0-0.002份。
优选地，按重量计，包括以下成分：葡萄糖35-40份、柠檬酸2.8-3份、柠檬酸三钠5-6份、碳酸氢钠1.25-1.3份、乙二胺四乙酸二钠1.25-1.3份、氯化钾0.7-0.8份、三羟甲基氨基甲烷2.8-2.9份、半胱氨酸0.05-0.07份、聚维酮碘0.025-0.030份和抗菌肽0.001-0.002份。
本稀释保存液分为两部分，第一部分为基础稀释营养液部分：葡萄糖、柠檬酸、柠檬酸三钠、碳酸氢钠、乙二胺四乙酸二钠、氯化钾、三羟甲基氨基甲烷和半胱氨酸，其中的半胱氨酸为精子保护剂；第二部分为杀灭病毒细菌有效成分：聚维酮碘。本发明配方通过对三羟甲基氨基甲烷、乙二胺四乙酸二钠与氯化钾的加入比例的控制，使稀释保存液的pH值为6.20—6.30，渗透压为347mOsm。
本发明所述稀释保存液的pH为6.20—6.30，渗透压为347mOsm。针对精子生存的严格环境要求，在该环境中精子的保存稳定性最好。
本发明所述稀释保存液采用的溶剂为双蒸水。
水质对于精子活力和保存时间的影响很大，不同地区不同养殖企业水质都不同，其中的关键指标如pH、矿物质及微量元素含量、微生物含量、重金属含量等对精子质量影响很大。本发明采用稳定的双蒸水水质，克服了使用不便、效果不稳定的缺点。
聚维酮碘是目前畜禽养殖场常用的碘制剂之一，是将碘载于强碱性阴离子树脂上形成的一种接触型消毒剂，可杀死大部分细菌、真菌和病毒。但是，在对精子安全的浓度范围内，聚维酮碘对部份细菌和病毒有很好的效果，但不能完全杀灭精液里面常见五种病毒，特别是猪伪狂犬病毒和猪瘟病毒效果不佳。
抗菌肽是生物体内经诱导产生的一种具有生物活性的小分子多肽，分子量在2000～7000左右，由20～60个氨基酸残基组成，对细菌和病毒都有广谱杀灭效果，且对精子安全性非常好，特别是对精液常见病毒特别是猪瘟病毒、伪狂犬病毒、蓝耳病病毒有很好的杀灭作用，和聚维酮碘配合使用有强烈互补性。
进一步优选地，所述的抗菌肽为蟑螂内源性抗菌肽。
本发明选用蟑螂内源性抗菌肽，其氨基酸序列为SEQIDNO:1。这种抗菌肽具有良好稳定性以及广谱高效抗菌、抗病毒等特点，且生产工艺成熟和完善，其生产工艺是以蟑螂内源性抗菌肽的氨基酸序列为模版，以固相多肽合成方法将氨基酸序列重组后用工程菌表达发酵，得到重组广谱抗菌肽纯品。蟑螂内源性抗菌肽在适当的浓度下对精子安全性非常好，而且对精液常见病毒特别是猪瘟病毒、伪狂犬病毒、蓝耳病病毒有很好的杀灭作用，和低毒聚维酮碘配合使用有强烈互补性。
本发明的稀释保存液已经得到全国超过100家大中型养猪企业的生产验证，在使用本发明产品后生产成绩大幅改善的有超过80％，普遍反映母猪繁殖障碍疾病大幅降低，疾病发生率显著下降，妊娠率显著提高，窝产仔猪头数增加，生产绩效普遍提高，具有显著经济效益和社会效益。
本发明采用了优选的稀释保存液基础配方和聚维酮碘消毒剂，使稀释保存液配方之间的稳定性最好，再加上统一稳定的双蒸水水质，克服了使用不便、效果不稳定的缺点。
具体实施方式
下面结合具体实施方式对本发明的实质性内容作进一步详细的描述。
实施例1
一种杀灭动物精液病毒细菌的稀释保存液，按重量计，包括以下成分：葡萄糖25份、柠檬酸2.5份、柠檬酸三钠5份、碳酸氢钠1.2份、乙二胺四乙酸二钠1.2份、氯化钾0.7份、三羟甲基氨基甲烷2.8份、半胱氨酸0.05份和聚维酮碘0.025份。
实施例2
一种杀灭动物精液病毒细菌的稀释保存液，按重量计，包括以下成分：葡萄糖40份、柠檬酸3份、柠檬酸三钠8份、碳酸氢钠1.3份、乙二胺四乙酸二钠1.3份、氯化钾0.9份、三羟甲基氨基甲烷3份、半胱氨酸0.08份、聚维酮碘0.030份和抗菌肽0.002份。
该稀释保存液的pH值为6.2，渗透压为347mOsm。
所述稀释保存液的溶剂为双蒸水。
实施例3
一种杀灭动物精液病毒细菌的稀释保存液，按重量计，包括以下成分：葡萄糖30份、柠檬酸2.6份、柠檬酸三钠6份、碳酸氢钠1.22份、乙二胺四乙酸二钠1.22份、氯化钾0.8份、三羟甲基氨基甲烷2.9份、半胱氨酸0.06份和聚维酮碘0.026份。
该稀释保存液的pH值为6.3，渗透压为347mOsm。
所述稀释保存液的溶剂为双蒸水。
实施例4
一种杀灭动物精液病毒细菌的稀释保存液，按重量计，包括以下成分：葡萄糖35份、柠檬酸2.8份、柠檬酸三钠5份、碳酸氢钠1.25份、乙二胺四乙酸二钠1.25份、氯化钾0.7份、三羟甲基氨基甲烷2.8份、半胱氨酸0.05份、聚维酮碘0.025份和抗菌肽0.001份。
该稀释保存液的pH值为6.22，渗透压为347mOsm。
所述稀释保存液的溶剂为双蒸水。
实施例5
一种杀灭动物精液病毒细菌的稀释保存液，按重量计，包括以下成分：葡萄糖40份、柠檬酸3份、柠檬酸三钠6份、碳酸氢钠1.3份、乙二胺四乙酸二钠1.3份、氯化钾0.8份、三羟甲基氨基甲烷2.9份、半胱氨酸0.07份、聚维酮碘0.030份和蟑螂内源性抗菌肽0.002份。
该稀释保存液的pH值为6.25，渗透压为347mOsm。
所述稀释保存液的溶剂为双蒸水。
实施例6
一种杀灭动物精液病毒细菌的稀释保存液，按重量计，包括以下成分：葡萄糖37份、柠檬酸2.9份、柠檬酸三钠6份、碳酸氢钠1.25份、乙二胺四乙酸二钠1.25份、氯化钾0.75份、三羟甲基氨基甲烷2.8份、半胱氨酸0.07份、聚维酮碘0.026份和蟑螂内源性抗菌肽0.0015份。
该稀释保存液的pH值为6.26，渗透压为347mOsm。
所述稀释保存液的溶剂为双蒸水。
实施例7
一种杀灭动物精液病毒细菌的稀释保存液，按重量计，包括以下成分：葡萄糖37份、柠檬酸2.9份、柠檬酸三钠6份、碳酸氢钠1.25份、乙二胺四乙酸二钠1.25份、氯化钾0.75份、三羟甲基氨基甲烷2.8份、半胱氨酸0.08份、维生素C 1 份、聚维酮碘0.026份和蟑螂内源性抗菌肽0.0012份。
该稀释保存液的pH值为6.22，渗透压为347mOsm。
所述稀释保存液的溶剂为双蒸水。
毒理学试验
毒理学试验采用动物试验观察其急性毒性、亚急性毒性及刺激性毒性结果。
结果：本精液净化消毒剂对大鼠和小鼠急性经口LD50均大于5000 m份/k份·BW，其原液对兔眼、完整皮肤及破损皮肤无刺激性；给药剂量在1000 m份/k份·BW以下，与阴性对照组比较，对大鼠的体重、血常规及血液生物化学指标无显著影响，试验动物各脏器均未见明显病理改变。仔猪口服精液净化保护剂原液，每天两次，1ml／每次，连续两天给药，用药前后试验仔猪可食用组织中碘浓度均低于最高残留限量，聚维酮碘在猪使用时无休药期。精液净化保护剂在使用浓度下毒理学是安全的。
本发明精液净化消毒剂稳定性好，37℃恒温箱放置三个月后碘含量下降率低于5％，六个月碘含量下降率低于10％。
本发明用于猪精液常温稀释保存液稳定性好，密闭常温避光保存有效期可达1年。pH为6.20±0.05、渗透压为347mOsm。
制备方法：在无菌厂房内，37ºC 995.00ml灭菌双蒸水中依次加入配方量的葡萄糖、EDTA-Na2、柠檬酸三钠：、柠檬酸、碳酸氢钠、Tris、氯化钾和半胱氨酸，边加边低速搅拌，保持温度在37 ºC不变，搅拌均匀后待温度降至室温，加入配方量的聚维酮碘，低速搅拌均匀后无菌分装。
本发明的成品检验项目与操作方法
1、有效碘含量检测
按照《中华人民共和国兽药典》（2005版）有效碘检测方法，本品含聚维酮碘按有效碘( I )计算，应为标示量的0.85%～1.20% 。
【性状】本品为无色液体。
【鉴别】
(1)取本品1～5滴，加水10ml与淀粉指示液1滴，即显蓝紫色。
(2)取本品10ml，置50ml锥形瓶中(瓶内颈切勿沾污)，瓶口覆盖一张用淀粉指示液湿润的滤纸，放置80秒钟，不显蓝色。
【检查】pH值 应为6.20±0.05。
2、不同品牌精液稀释保存液以及精液稀释保存粉对精液常温保存时间的影响
市场购买四种不同品牌精液稀释保存液或精液稀释保存粉（1号、2号、3号和4号）稀释后待用。
表1 不同稀释液对猪精液常温保存的影响
由表1可知，本发明稀释保存液有效保存时间极显著高于1、2、3、4号液（Ｐ＜０．０１），生存指数也显著高于1、2、3、4号液（Ｐ＜０．０５）。
3、本发明稀释保存液常温保存精液配种成绩
用本发明稀释保存液稀释猪精液，在１７℃恒温箱中保存５ｄ，精子活力在０．５以上的稀释精液用于人工授精试验。共输精母猪１２０头，具体结果见表2: 可见，本发明稀释保存液常温保存５ｄ的猪精液情期受胎率、平均窝产仔数明显高于1、2、3、4号液。
表2 不同稀释液常温保存５ｄ的猪精液情期受胎率、平均窝产仔数
序列表
<110>成都美强兽医技术服务有限公司
<120>一种杀灭动物精液病毒细菌的稀释保存液
<160>1
<170>PatentInversion3.3
<210>1
<211>37
<212>PRT
<213>蟑螂内源性抗菌肽
<400>1
Lys Trp Lys Val Phe Lys Lys Ile glu Lys Met gly Arg Asn Ile Arg
1 5 10 15
Asn gly Ile Val Lys Ala gly Pro Ala Ile Ala Val Leu gly glu Ala
20 25 30
Lys Ala Lys Ala Leu
35
Claims (3)
1.一种杀灭猪精液病毒细菌的稀释保存液，其特征在于：按重量计，由以下成分组成：葡萄糖35-40份、柠檬酸2.8-3份、柠檬酸三钠5-6份、碳酸氢钠1.25-1.3份、乙二胺四乙酸二钠1.25-1.3份、氯化钾0.7-0.8份、三羟甲基氨基甲烷2.8-2.9份、半胱氨酸0.05-0.07份、聚维酮碘0.025-0.030份和抗菌肽0.001-0.002份；
该稀释保存液的pH值为6.2-6.3，渗透压为347mOsm。
2.根据权利要求1所述的一种杀灭猪精液病毒细菌的稀释保存液，其特征在于：所述稀释保存液的抗菌肽为蟑螂内源性抗菌肽。
3.根据权利要求1所述的一种杀灭猪精液病毒细菌的稀释保存液，其特征在于：所述稀释保存液的溶剂为双蒸水。
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| CN108588014A (zh) * | 2018-03-15 | 2018-09-28 | 广州伯瑞丁生物新技术开发有限公司 | 一种猪精子培养基及其制备方法 |
| CN109938008A (zh) * | 2019-03-20 | 2019-06-28 | 上海创宏生物科技有限公司 | 一种动物精液处理剂及其应用方法 |
| CN111869658A (zh) * | 2020-08-22 | 2020-11-03 | 南京健邦锦源医疗科技有限公司 | 一种灭活病毒保存液及其制备方法 |
| CN114794085A (zh) * | 2022-06-08 | 2022-07-29 | 杭州东源生物科技有限公司 | 一种猪精液低温保存用稀释剂及其制备方法 |
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| CN104304237A (zh) * | 2014-11-11 | 2015-01-28 | 成都美强兽医技术服务有限公司 | 一种安全高效的动物精液消毒剂及其制备方法 |
| CN105010304A (zh) * | 2014-04-23 | 2015-11-04 | 马乃祥 | 猪抗病毒精液稀释液的配方组成 |
| CN105325401A (zh) * | 2015-11-10 | 2016-02-17 | 天津市畜牧兽医研究所 | 一种猪鲜精保存长效稀释粉及制备方法与应用 |
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| CN101168564A (zh) * | 2007-10-22 | 2008-04-30 | 中国科学院昆明动物研究所 | 人源性抗菌肽及其衍生物的用途 |
| CN105010304A (zh) * | 2014-04-23 | 2015-11-04 | 马乃祥 | 猪抗病毒精液稀释液的配方组成 |
| CN104304237A (zh) * | 2014-11-11 | 2015-01-28 | 成都美强兽医技术服务有限公司 | 一种安全高效的动物精液消毒剂及其制备方法 |
| CN105325401A (zh) * | 2015-11-10 | 2016-02-17 | 天津市畜牧兽医研究所 | 一种猪鲜精保存长效稀释粉及制备方法与应用 |
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