CN105325401B - 一种猪鲜精保存长效稀释粉及制备方法与应用 - Google Patents
一种猪鲜精保存长效稀释粉及制备方法与应用 Download PDFInfo
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Classifications
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
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Abstract
本发明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,乙二胺四乙酸钠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。其使用方法是猪精液采精后,用该稀释粉配制的1 L稀释液进行稀释,稀释后的精液在17℃恒温冰箱中能够保存5‑7天,精液质量仍能满足输精要求,精子活率在60%以上。本发明内容是历时3年,经过200多次试验所取得的成果,在猪场应用证明该猪精液保存稀释粉质量较高,精子保存5天以上仍具有授精能力,效果稳定。
Description
技术领域
本发明属于家畜繁殖技术领域,涉及一种猪鲜精保存长效稀释粉及制备方法与应用。
背景技术
随着猪人工授精技术在集约化养猪场中的推广与应用,精液保存技术越来越受到重视,并日益显示出降低公猪饲养量和饲养成本、加快猪群品种改良、减少疾病传播等多方面优势。为此,近年来常温保存精液技术已广泛应用于猪的人工授精中,国内外研究者围绕猪精液常温保存稀释液开展了大量的研究工作,并研制出应用效果较好的稀释液配方。市场上的稀释粉一般分为短效(2-3天)、中效(3-5天)和长效(5-7天)等3种。在文献中能够检索到的稀释液种类中,BTS最为常见,为保存2-3天的短效稀释液。就目前而言,国内养猪企业使用的鲜精稀释粉大多从韩国、日本、西班牙、美国、比利时等等国家进口,价格较高。据市场调查,保存2-3天的短效稀释粉平均需5-7元/每袋,中效和长效的稀释液价格更高。我国大多数大、中型养猪企业人工输精所用精液来自当天采集,或来自存储2-3天的精液。如此段的精液存储时间必将给企业增加人力、物力和财力负担,降低企业经济效益。随着我国生猪市场集约化和规模化发展,猪精液高效、长期保存技术越来越重要。本项目组成员在结合多年猪精液鲜精保存技术研究的基础上,借鉴国内外猪精液保存方面最新研究成果,经过大量试验验证开发出一种新的猪精液保存长效稀释粉,而且成本较低,平均每袋长效稀释液成本在3元左右,能够更好服务于我国生猪企业的快速、健康发展。
就稀释液成分和功能分析,稀释液中的主要成分有:稀释剂(扩大精液容量)、营养剂(提供精子代谢所需的营养)、保护剂(缓冲不良环境对精子的影响)和抗菌剂。附睾中的精子存活时间很长(可长达17d之久),这与附睾中精子所处的理化环境密切相关,附睾中的精子处于酸性环境中,并且保持高渗脱水状态,精子的活动完全被抑制,同时附睾中精子带有负电荷可防止精子凝集。除此之外,稀释液中含有针对性的抗菌成分可防止精液中细菌的生长和生殖性疾病的传播。然而,稀释剂在延长保存精子活性的同时,对精子也有些负面影响,会导致精子活力逐渐丧失及膜损伤增加。如果稀释过度,尤其是使用单纯的电解质则对精子的损伤更大。可能是稀释剂保存过程中精子细胞失去了一些重要的成分,而且稀释剂中的保护剂可能也对精子的结构和活性造成负面影响,从而影响了精子质膜的功能。
鲜精的pH值一般在7.2-7.5之间,较低的pH值能抑制精子活力和新陈代谢。大多数猪精液稀释剂中有较高浓度的葡萄糖使精子内pH值维持在6.0以下,使猪精子的糖酵解代谢很弱。稀释剂中的离子强度对精子保存可能不起关键性作用。稀释液中非离子物质(如葡萄糖等)保证了渗透压浓度。稀释剂中碳酸氢钠和柠檬酸钠作为缓冲液。氯化钾以低浓度用于维持Na+-K+泵,防止细胞外K+耗竭而使细胞失去活力。EDTA能捕获二价的金属离子,尤其是Ca2+,可能抑制跨膜运动而阻止获能的启动和顶体反应。稀释剂中使用EDTA对推广猪精子液态保存是很重要的一步。此外,在精液保存过程中,精子呼吸代谢会产生大量的活性氧族(reactive Oxygen Species,ROS),其中的氧自由基(O2-、H2O2和-OH)能使精子细胞膜中不饱和脂肪酸发生过氧化反应,导致精子质膜损伤。利用抗氧化剂如含有硫氢基团的物质,可能对稳定精子质膜有重要作用,也可抑制获能,从而提高精子液态保存的成活率。基于对上述猪鲜精保存原理的认识,本项项目组经过长期大量试验后,在常规稀释粉BTS基础上,适当添加了①聚乙烯醇,其作用是增加稀释液渗透压而且能够有效降低精子聚头现象,增加单个精子游动性;②半胱氨酸,其增强精子在储存过程中的抗氧化损伤能力;③肌醇,其是细胞膜的稳定剂,更好维持精子在储存过程中质膜的稳定性,④提高青霉素含量,其作用是增强精液抗菌能力。在猪场和种公猪站的应用证明了该猪精液保存长效稀释粉质量的可靠性。
发明内容
为开发高效、低价猪鲜精稀释粉,打破我国养猪企业稀释粉长期依赖进口的局面,本科研团队经过不断的研究和创新,最终选择在BTS基础上适量添加聚乙烯醇、半胱氨酸、肌醇和青链霉素等物质,分别从增加精子游动性、抗氧化能力、增加精子质膜稳定性和提高精液抗菌能力等方面,综合增加猪精液体外保存质量,达到长期(5-7天)保存的目的。
本发明有效降低了猪场运行管理成本,提高猪场生产水平。本发明提供的猪鲜精保存长效稀释粉配方如下:
一种猪鲜精保存长效稀释粉,其特征在于它是由下述重量份数的原料组成:
葡萄糖 35-40
乙二胺四乙酸钠 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
青霉素 1.0-1.5
链霉素 1.0-1.5
聚乙烯醇 0.4-0.6
半胱氨酸 0.1-0.2
肌醇 0.4-0.6。
本发明进一步公开了猪鲜精保存长效稀释粉的使用方法,其特征在于按如下的步骤进行:
(1)按照上述重量份数准确称量原料;
(2)将上述准确称量的原料放置于1000 mL烧杯中,然后在烧杯中加入1000 mL双蒸水,最后在37 ℃水浴锅中加热溶解和平衡1 h以上待用;
(3)猪精液采集后,在室温下稍作放置,使精液温度降到35-37 ℃,采用步骤(2)的稀释液进行稀释,稀释后将精液分装于集精瓶中,每瓶80 mL,分装后,将集精瓶放在室温下缓慢降温30-60 min,最后放置在17 ℃恒温冰箱中储存,并每隔12 h在17 ℃恒温箱中翻动集精瓶一次,避免精子处于假死状态。
本发明更进一步公开了猪鲜精保存长效稀释粉在制备提高猪精液抗菌能力,特别是在提高猪精子存活率方面的应用。
本发明内容是历时3年,经过200多次试验所取得的成果,其特点是,在猪鲜精常规稀释液BTS中(葡萄糖37g,乙二胺四乙酸钠 1.25g,柠檬酸钠6g,碳酸氢钠1.25g,氯化钾0.75g,青霉素1g,链霉素1g),适当的添加了①聚乙烯醇,其作用是增加稀释液渗透压而且能够有效降低精子聚头现象,增加单个精子游动性;②半胱氨酸,其增强精子在储存过程中的抗氧化损伤能力;③肌醇,其是细胞膜的稳定剂,更好维持精子在储存过程中质膜的稳定性,④提高青霉素含量,其作用是增强精液抗菌能力。在猪场应用证明该猪精液保存稀释粉质量较高,精子保存5天以上仍具有授精能力,效果稳定。
本发明公开的猪鲜精保存长效稀释粉及使用方法与现有的技术相比所具有的积极效果在于:
(1)效果稳定。众所周知,不同品种、不同个体公猪精液本身质量差异较大,且易受到季节变化等环境的影响。市售的猪精液稀释粉效果大都不稳定,保存效果时好时坏。本项目组经过长期试验探索和有效物质的筛选,最终适量应用了抑菌剂、抗氧化剂和质膜稳定剂等药品,经过大量试验表明效果非常稳定,能够很好克服不同品种不同个体公猪精液本身差异,使保存前5天的精子活率均在0.6以上。
(2)价格低。市售的长效稀释粉(精子有效保存5-7天)价格大都在15元/袋以上,价格较贵。而本长效稀释粉从研发开始就瞄准低成本、高效果这一目标,从数目众多的低价药品中开展大量筛选试验,预计本长效稀释粉市售价格最多7元/袋。给养猪企业节约成本一倍以上。
(3)使用方便。市售大多长效稀释粉都含有两个小包装,使用前需将两个包装拆开、然后将两个包装的稀释粉混到一块,最后再稀释;而本研发的长效稀释粉只有一个包装,打开后直接稀释,使用起来较为方便。
具体实施方式
通过以下实施例对本发明作一步说明,但不作为本发明的限制。本发明的实施例中所述的重量份数原料指的是克,使用前用1000 mL双蒸水进行稀释,即配成稀释液。所用到的葡糖糖、乙二胺四乙酸钠、柠檬酸钠、碳酸氢钠、氯化钾、聚乙烯醇、半胱氨酸、肌醇、青霉素、链霉素均有市售。
实施例1:
稀释液配制:
按照上述重量份数准确称量如下药品:葡萄糖37g,乙二胺四乙酸钠1.25g,柠檬酸钠6g,碳酸氢钠1.25g,氯化钾0.75g,青霉素1.4g,链霉素1.4g,半胱氨酸0.1g,聚乙烯醇0.5g,肌醇0.5g。上述药品准确称量于1000 mL烧杯中,然后在烧杯中加入1000 mL双蒸水,最后在37 ℃水浴锅中加热溶解和平衡1 h以上待用。
实施例2:
猪精液稀释,分装和保存:
猪精液采集后,在室温下稍作放置,使精液温度降到37 ℃左右,根据原精密度用本发明的稀释液进行稀释,使稀释后的精液密度在0.5亿/mL左右。稀释后将精液分装于集精瓶中,每瓶80 mL。分装后,将集精瓶放在室温下缓慢降温30-60 min,最后放置在17 ℃恒温冰箱中储存,并每隔12 h在17 ℃恒温箱中翻动集精瓶一次,避免精子处于假死状态。
实施例3:
精子保存质量检测:
精子活率、活力等运动状态质量检测,将集精瓶中的精液充分摇动使瓶中的精子分布均匀,取10 uL精液于专用精子计数板中,并盖上盖玻片,置于37℃的检测仪载物台上,保温2分钟后随机取10个视野进行分析。每天检测一次精子质量,当精子活率低于0.6时放弃保存,从而得出精子有效保存天数。
精子质膜完整性检测,采用低渗肿胀原理进行检测。
低渗液配制:准确称量二水柠檬酸钠7.35g,果糖13.51g,完全溶解于1000mL双蒸水中即可。
精子质膜完整性的检测
取50µL精液放在1mL低渗液(37℃)中混匀。30 min后取出20µL滴在血细胞计数板上,400倍相差显微镜下观察,计算弯尾精子的百分率。因为质膜完整的精子在低渗液进入质膜后,会由于肿胀而导致精子尾部弯曲,而质膜损伤的精子由于膜的通透性增大,精子尾部不会弯曲。基于上述原理可以检测出精子质膜的完整性。
精子顶体损伤检测,采用金霉素(CTC)荧光染色法进行检测。
染色液配制:CTC 缓冲液: Tris 1.21g/500ml; Nacl 3.7986g, 用Hcl溶液调节pH值到7.8。CTC染液:CTC 0.0019g, Cystein 0.0033g, CTC缓冲液 5ml。该液最好现用现配,并避光低温储存。12.5%的多聚甲醛液:Tris6.05g/100ml(用盐酸调节PH=7.4);多聚甲醛12.5g/100ml,3%的PVP溶液:PVP(40,000)3g;PBS:100ml,增光剂:甘油:PBS(9:1)10ml;1,4-diazabicyclo[2,2,2]octane 0.2468g。
精子顶体完整性检测方法:取出50µL精液用2mL孵育液稀释后,在37℃下孵育20min。在1.5mL离心管中放入1mL3%的PVP溶液,把精液轻轻地铺在PVP溶液上,500×g离心6min,去上清。沉淀精子用50µL mBO液悬浮均匀。再取45µL精子悬液加入45µL的CTC染液,混匀。然后加入8µL12.5%的多聚甲醛溶液,混匀。取10µL精子悬浮液加入等量的增光剂混匀后盖上盖玻片,用无色指甲油封片,避光保存并尽快用荧光显微镜观察。CTC荧光染料能着色精子顶体,在激发波长400-440nm、辐射波长470nm的紫外光照射下精子的顶体发出黄绿色,以此区分顶体的完整性。每次检测,用血细胞计数器计数200个精子,计算顶体完整性。
实施例4
对比试验
(1)试验材料
试验组:选用实施例1所述的稀释液;
对照组:选用常规的BTS稀释液。
(2)试验方法
将采集下来的原精平均分在2个500 mL预热的烧杯中,分别标记为试验组和对照组。然后用相应的稀释液根据原精密度进行等倍稀释,使稀释后的试验组和对照组精子密度相近,大约在0.5 X 108/mL左右。然后集精瓶分装、并室温下放置30-60 min,最后把集精瓶放在17 ℃恒温箱中保存。精液稀释当天为第0天,以后每隔24 h检测精子质量,当精子活率低于0.6时终止保存,计算精子在17 ℃恒温箱中有效保存天数。
(3)试验结果
本试验用不同猪场的不同公猪,如大白、长白和杜洛克等公猪精液重复50次。试验结果表明,采用本课题研发的稀释粉配制的稀释液,使来自不同猪场、不同公猪的精液在17℃保存5天时,其精子活率均稳定在0.7以上,试验结果稳定、可靠。在精液保存5天以后,由于公猪个体和身体质量差异等原因可造成精子保存质量发生变化。但从实验的总体观察,当精液保存达7天时,仍有70%以上的公猪精子活率维持在0.6以上,仍能满足输精要求。而采用常规稀释液BTS进行稀释保存的对照组,在50次的对比试验中,只有7次的精液能保存到第3天仍能满足输精要求(活率在0.6以上),17次能有效保存2天,剩下的26次试验中的精液只能有效保存1.5天左右。在精子质膜完整性和顶体完整性方面,在50次的对比试验中观察到,在有效保存天数内(活率在0.6以上)精子的质膜完整性和顶体完整性都非常高,而本项目研发的鲜精稀释液对精子质膜和顶体的保护性更高。总之,本发明的猪鲜精长效稀释粉配方能够较长时间(5-7天)使精子处于质量较高状态,仍能满足输精要求。
Claims (2)
1.一种提高猪精液抗菌能力的猪鲜精保存长效稀释粉,其特征在于它是由下述重量份数的原料组成:
葡萄糖 35-40
乙二胺四乙酸钠 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
青霉素 1.0-1.5
链霉素 1.0-1.5
聚乙烯醇 0.4-0.6
半胱氨酸 0.1-0.2
肌醇 0.4-0.6。
2.权利要求1所述猪鲜精保存长效稀释粉的使用方法,其特征在于按如下的步骤进行:
(1)按照上述重量份数准确称量原料;
(2)将上述准确称量的原料放置于1000 mL烧杯中,然后在烧杯中加入1000 mL双蒸水,最后在37 ℃水浴锅中加热溶解和平衡1 h以上待用;
(3)猪精液采集后,在室温下稍作放置,使精液温度降到35-37 ℃,采用步骤(2)的稀释液进行稀释,稀释后将精液分装于集精瓶中,每瓶80 mL,分装后,将集精瓶放在室温下缓慢降温30-60 min,最后放置在17 ℃恒温冰箱中储存,并每隔12 h在17 ℃恒温箱中翻动集精瓶一次,避免精子处于假死状态。
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