WO2017139908A1 - 一种小反刍动物精液冷冻稀释液 - Google Patents

一种小反刍动物精液冷冻稀释液 Download PDF

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WO2017139908A1
WO2017139908A1 PCT/CN2016/000656 CN2016000656W WO2017139908A1 WO 2017139908 A1 WO2017139908 A1 WO 2017139908A1 CN 2016000656 W CN2016000656 W CN 2016000656W WO 2017139908 A1 WO2017139908 A1 WO 2017139908A1
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Prior art keywords
semen
frozen
resveratrol
small ruminant
sperm
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PCT/CN2016/000656
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English (en)
French (fr)
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权国波
洪琼花
邵庆勇
吴国权
吕春荣
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权国波
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Priority to US16/078,009 priority Critical patent/US20200060259A1/en
Publication of WO2017139908A1 publication Critical patent/WO2017139908A1/zh

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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
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • 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
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • 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
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0215—Disinfecting agents, e.g. antimicrobials for preserving living parts
    • 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
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients

Definitions

  • the invention relates to the technical field of animal reproductive physiology and reproduction, in particular to a small ruminant semen frozen dilution liquid.
  • the composition of the diluent can reduce the damage of the sperm to the sperm, mainly by adding a certain osmotic organic solvent and sugar, egg yolk, ice crystal inhibitor, etc. in the semen diluent, and the osmotic organic solvent, the sugar and the egg yolk can be improved.
  • the survival rate of sperm cells after freezing but does not inhibit the formation of ice crystals, while ice crystal inhibitors can inhibit the formation of ice crystals, but there are still many problems in practical applications.
  • the existing ice crystal inhibitors are best for antifreeze proteins, but the antifreeze protein technology has been in the hands of developed countries.
  • the antifreeze proteins that can be used at present are imported from abroad, expensive, and antifreeze. After the protein, although the amount of ice crystal formation is reduced, the formed ice crystals are needle-like, and the mechanical damage to the cells is sometimes greater.
  • the low temperature balance of the semen before freezing at 0-5 °C is critical for the survival of frozen sperm, usually this process requires 2-4 hours, but the metabolic activity of sperm at 0-5 °C did not completely stop, its metabolites may cause severe oxidative damage and apoptosis of sperm, and ultimately cause damage to cell structure. It is common practice in the art to add certain antioxidants (vitamin C, vitamin E, superoxide dismutase, catalase, glutathione peroxidase, etc.) to the semen freezing dilution to remove the freezing and thawing process.
  • vitamin C vitamin E
  • superoxide dismutase catalase
  • glutathione peroxidase etc.
  • an inositol compound, resveratrol is further added, and the concentration of the two is compatible with the cryopreservation effect of the semen of the small ruminant animal, thereby avoiding The defects in the use of ice crystal inhibitors alone have not been reported in the literature.
  • the small ruminant semen frozen diluent of the present invention comprises the following components per 100 ml:
  • the balance is ultrapure water.
  • the inositol compound is one of 1,3-cyclohexanediol, 1,4-cyclohexanediol, and 1,3,5-cyclohexanetriol.
  • the monosaccharide is glucose or fructose.
  • the osmoprotectant is glycerin or ethylene glycol.
  • fresh egg yolk is inactivated at 56 ° C for 30 min.
  • the preparation method of the small ruminant semen frozen dilution liquid of the invention comprises the steps of weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of monosaccharide, 5-20% of osmotic protective agent, penicillin.
  • the small ruminant semen cryopreservation of the present invention can be subjected to one-step cryopreservation of semen of small ruminants, including sheep and goats, by mixing sheep semen and the semen frozen dilution of the present invention in a ratio of 1:10. Uniform, then sub-packaged in 0.25ml plastic tubing, first slowly cooled to 5 ° C, and then quickly moved into the liquid nitrogen phase fumigation for 5min, and finally the thin tube was put into liquid nitrogen for cryopreservation.
  • the small ruminant semen frozen dilution of the present invention can also be subjected to two-step cryopreservation of semen of small ruminants, including sheep and goats, by the step of freezing the sheep semen and the osmotic protective agent-free frozen product of the present invention.
  • the diluent is uniformly mixed in a ratio of 1:4-1:10, and then dispensed into a 2 ml cryotube, slowly lowered to 5 ° C, and then the suspension and the lyophilized diluent containing the osmotic protective agent of the present invention.
  • the mixture was uniformly mixed in a ratio of 1:1, further equilibrated at 5 ° C for 1-3 hours, and pipetted with 0.2 ml of a drop to dry ice to be pre-frozen, and finally the frozen pellet was placed in liquid nitrogen for cryopreservation.
  • the two-step cryopreservation method of the present invention comprises the osmotic protective agent-free frozen diluent, which is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, and 1.0 g of monosaccharide, respectively.
  • Penicillium 100,000 IU, 100,000 IU of streptomycin, 50-600 mMol of inositol, 0.1-50 ⁇ Mol of resveratrol dissolved in ultrapure water stirred evenly, and fresh yolk 5 inactivated at 56 ° C for 30 min before use. 20 ml, mix well, dilute to 100 ml, centrifuge at 15000 rpm for 1 hour at 4 ° C, and take the supernatant for filtration with a 0.45 ⁇ m disposable filter.
  • the quality of the semen used in the cryopreservation of the present invention is: the vigor is greater than 75%, and the sperm density is higher than 1 x 10 9 /ml.
  • Resveratrol is a research hotspot in recent years. Resveratrol is widely believed to have anti-oxidation, anti-mutation, cardiovascular, anti-inflammatory and antibacterial effects. It has been successfully applied in the fields of food, cosmetics and pharmaceuticals.
  • the invention aims at the main problems existing in the research of cryopreservation of livestock semen at present. For the first time, the ice crystal inhibitor of chemical synthesis source and the natural plant extract, resveratrol, are used for the cryopreservation of semen of small ruminant livestock.
  • resveratrol reduces the mechanism of ice crystal damage to sperm cells
  • a large number of experiments have shown that the addition of resveratrol to semen cryopreservation can improve sperm motility, viability and acrosome integrity after semen freezing and thawing.
  • Quality parameters such as plasma membrane integrity, mitochondrial membrane potential normal rate, and non-return rate after artificial insemination, which may be due to the similar molecular structure of resveratrol and the inositol compound disclosed in the present invention, which can reduce extracellular formation.
  • Ice crystals damage mechanical structures such as plasma membrane and mitochondria; resveratrol is also an antioxidant that can effectively reduce oxidative damage of sperm at 0-5 °C low temperature balance; resveratrol can also prevent cell inflammation, this pair It is also of positive significance to protect cells and reduce damage when semen cells are frozen.
  • the present invention is the first to successfully apply resveratrol to the field of semen freezing of small ruminants.
  • the inositol compound and resveratrol used in the present invention are common chemical reagents, and are convenient in source, low in cost, and chemically stable.
  • the preparation method and the using method of the semen frozen diluent provided by the invention are simple and easy to operate.
  • the semi-fine wool sheep semen of Yunnan was collected by electric stimulation, and then the semen quality was tested.
  • the semen index for cryopreservation must meet: viability greater than 75%, sperm density above 1 ⁇ 10 9 /ml, and semen volume 1-2 ml.
  • the semen cryopreservation solution is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 5% of glycerol, 100,000 IU of penicillin, 100,000 IU of streptomycin, and 1,3, respectively.
  • the sheep semen and frozen dilutions that meet the freezing requirements are uniformly mixed in a ratio of 1:5, and then subdivided into 0.25 ml plastic thin tubes, and then slowly cooled to 5 ° C, and then quickly transferred to liquid nitrogen phase for fumigation for 5 min, and finally the liquid is poured. Nitrogen is stored frozen.
  • the method of cryopreserved livestock semen the sperm survival rate after thawing is 76.14 ⁇ 6.84%, the vigor is 49.47 ⁇ 5.19%, the acrosome integrity is 68.46 ⁇ 6.27%, the plasma membrane integrity is 47.55 ⁇ 6.93%, and the mitochondrial membrane potential
  • the normal rate is 73.41 ⁇ 5.39%, and the rate of non-return after artificial insemination reaches over 70%.
  • the false vaginal collection of Yunling goat semen was performed, followed by semen quality testing.
  • the goat semen index for cryopreservation must meet: viability greater than 75%, sperm density higher than 1 ⁇ 10 9 /ml, and semen volume 1-2 ml.
  • the semen frozen dilution solution is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of fructose, 5% of glycerol, 100,000 IU of penicillin, 100,000 IU of streptomycin, and 1,4 -cyclohexan Alcohol 50mMol, resveratrol 10 ⁇ Mol dissolved in ultrapure water, stirred evenly, add 20ml of fresh egg yolk inactivated at 56 ° C for 30min before use, mix well and dilute to 100ml, centrifuge at 15000 rev / min at 4 ° C After 1 hour, the supernatant was filtered through a 0.45 ⁇ m disposable filter for use.
  • the method of cryopreserved livestock semen the survival rate of sperm after thawing is 72.68 ⁇ 5.42%, the vigor is 48.58 ⁇ 3.27%, the acrosome integrity is 68.19 ⁇ 4.26%, the plasma membrane integrity is 45.79 ⁇ 8.16%, mitochondrial membrane potential The normal rate is 71.55 ⁇ 7.41%, and the rate of non-return after artificial insemination is above 70%.
  • the electrical stimulation was used to collect the Dorset ram semen, and the semen quality test was performed immediately.
  • the semen index for cryopreservation must meet: viability greater than 75%, sperm density above 1 ⁇ 10 9 /ml, and semen volume 1-2 ml.
  • a frozen diluent without a osmotic protective agent which is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of glucose, 100,000 IU of penicillin, and 100,000 IU of streptomycin, respectively.
  • 1,3,5-cyclohexanetriol 50mMol, resveratrol 8 ⁇ Mol dissolved in ultrapure water stir well, add 15ml of fresh egg yolk inactivated at 56 °C for 30min before use, mix well and dilute to 100ml, at 4 Centrifuge at 15000 rpm for 1 hour at ° C, and take the supernatant for filtration with a 0.45 ⁇ m disposable filter.
  • the semen cryopreservation solution is configured by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of fructose, 10% of glycerol, 100,000 IU of penicillin, 100,000 IU of streptomycin, and 1,3 5-cyclohexanetriol 50mMol, resveratrol 8 ⁇ Mol dissolved in ultrapure water, stir well, add 15ml of fresh egg yolk inactivated at 56 °C for 30min before use, mix well and dilute to 100ml, 15000 at 4 °C The rate of revolution/minute was centrifuged for 1 hour, and the supernatant was taken and filtered with a 0.45 ⁇ m disposable filter for use.
  • the semen which meets the freezing requirement and the frozen diluent which does not contain the osmotic protective agent are uniformly mixed in a ratio of 1:5, and then sub-packaged in a 2 ml cryotube, and then slowly lowered to 5 ° C, and then the above suspension and the present invention
  • the frozen liquid dilution of the livestock semen was uniformly mixed in a ratio of 1:1, and 0.2 ml of the drop was pipetted into dry ice to be pre-frozen, and finally the frozen particles were frozen and stored in liquid nitrogen.
  • cryopreserved sheep semen The method of cryopreserved sheep semen, the sperm survival rate after thawing is 74.58 ⁇ 7.35%, the vigor is 50.47 ⁇ 2.84%, the acrosome integrity is 71.32 ⁇ 5.43%, the plasma membrane integrity is 50.09 ⁇ 6.91%, mitochondrial membrane potential The normal rate is 75.16 ⁇ 6.38%, and the rate of non-return after artificial insemination is above 75%.

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Abstract

本发明涉及一种小反刍动物精液冷冻稀释液，每100ml包括如下组分：三羟甲基氨基甲烷2.71g，柠檬酸1.4g，单糖1.0g，新鲜蛋黄5-20ml，渗透性保护剂0-10ml，青霉素10万IU，链霉素10万IU，肌醇类化合物50-600mMol，白藜芦醇0.1-50μMol，余量为超纯水。本发明首次成功地将白藜芦醇应用于小反刍动物精液冷冻领域，提高了冷冻精液的质量，成本低廉、化学性质稳定，使用方法简单易操作。

Description

一种小反刍动物精液冷冻稀释液 技术领域
本发明涉及动物生殖生理与繁殖技术领域，具体涉及一种小反刍动物精液冷冻稀释液。
背景技术
尽管家畜的精液冷冻保存研究已经开展了50多年，但是解冻后的精液活力仅在30％-50％左右，人工授精后不返情率和产仔率均显著低于新鲜和低温冷藏精液。在动物的精液冷冻过程中，精子细胞受到的损伤主要是细胞膜内外形成的冰晶造成的物理损伤，因此，降低冰晶的产生一直是低温生物学领域研究的热点，已经有大量研究证明，通过改变精液稀释液的组成成分可以降低冷冻对精子的损伤，主要是在精液稀释液中添加一定的渗透性有机溶剂和糖类、蛋黄、冰晶抑制剂等，渗透性有机溶剂和糖类、蛋黄都能提高冷冻后精子细胞的活率，但是并不能抑制冰晶的形成，而冰晶抑制剂虽然能够抑制冰晶形成，但现实应用中还存在很多问题。
首先是现有的冰晶抑制剂以抗冻蛋白为最佳，但是抗冻蛋白技术一直掌握在发达国家手里，目前能使用的抗冻蛋白都是从国外进口的，价格昂贵，而且使用抗冻蛋白后，虽然冰晶形成量减少，但形成的冰晶为针状，有时对细胞的机械损伤反而更大。
其次，申请人在专利(专利号201210491289.2)中阐述了采用化工合成的冰晶抑制剂——肌醇类化合物来代替抗冻蛋白应用于哺乳动物精液的冷冻保存，由于肌醇类化合物特有的六角形结构，使得形成的冰晶也为六角形，从而降低冷冻过程中冰晶形成对精子结构和功能的损伤。然而，尽管肌醇类化合物的冰晶抑制剂可以缓解冷冻解冻过程冰晶对精子质膜和线粒体等结构的不利影响，但是冷冻对精子细胞结构的损伤仍然很严重。特别是由于精液冷冻保存程序的特殊性，冷冻前精液于0-5℃低温平衡对于冷冻精子的存活非常关键，通常这个过程需要 2-4小时，但是在0-5℃下精子的代谢活动并没有完全停止，其代谢产物可能对精子造成严重的氧化损伤和细胞凋亡，最终造成细胞结构损伤。本领域常规做法是在精液冷冻稀释液中加入一定的抗氧化剂(维生素C、维生素E、超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶等)清除冷冻解冻过程中产生的活性氧，但是由于0-5℃下低温平衡时精子细胞代谢机制还不清楚，造成的细胞损伤机制也不清楚，不同种动物的精子受损伤的情况也不一致，而且精子细胞微小脆弱，各种化学试剂的配伍及剂量稍有变化，产生的冷冻结果就大不相同，因此，即不能通过单纯添加抗氧化剂来减少0-5℃下低温平衡时精子细胞的损伤，也不能用一种配伍方法针对所有种类的动物。
对家畜育种业而言，优秀公畜都是通过若干轮选择才选出，为了最大化的利用优秀公畜扩群，业内常用的做法就是将优秀公畜的精液尽量多的给发情母畜进行授精，最科学和经济的方法是将采到的精液稀释后冷冻，到使用时解冻后给母畜实施人工授精，这样可以将优秀公畜的基因快速扩散，但是目前这一做法只在养牛业上普遍实施且已经形成体系，但在小反刍动物方面，包括山羊和绵羊上，由于物种特点，羊的射精量本来就比较少，且精子对冷冻损伤比较敏感，精液冷冻技术在养羊业上使用和推广都远不如养牛业，实践中很多地方还在使用活羊本交的方式就行授精，大大限制了优秀公羊的扩群，使得经过多年精心选择的优秀公羊基因不能快速扩散，羊群改良进展缓慢。
发明内容
本发明在前期采用化学来源的冰晶抑制剂——肌醇类化合物冷冻保存家畜精液的基础上，进一步添加白藜芦醇，观察二者浓度配伍对小反刍动物精液的冷冻保存效果，从而避免了单独使用冰晶抑制剂所存在的缺陷，相关的研究未见文献报道。
本发明的小反刍动物精液冷冻稀释液，每100ml包括如下组分：
Figure PCTCN2016000656-appb-000001
Figure PCTCN2016000656-appb-000002
余量为超纯水。
进一步，所述肌醇类化合物为1，3-环己二醇、1，4-环己二醇和1，3，5-环己三醇其中之一。
进一步，所述单糖为葡萄糖或果糖。
进一步，所述渗透性保护剂为甘油或乙二醇。
进一步，所述新鲜蛋黄经56℃、30min的灭活处理。
本发明的小反刍动物精液冷冻稀释液的制备方法，其步骤是，分别称取三羟甲基氨基甲烷2.71g、柠檬酸1.4g、单糖1.0g、渗透性保护剂5-20％、青霉素10万IU，链霉素10万IU，肌醇类化合物50-600mMol，白藜芦醇0.1-50μMol溶于超纯水中，搅拌均匀，用三羟甲基氨基甲烷调整pH值至6.8-7.2，使用前加入56℃30min灭活处理的新鲜蛋黄5-20ml，混合均匀后定容至100ml，于4℃下15000转/分的速率离心1小时，取上清液用0.45μm一次性滤器过滤备用。
本发明的小反刍动物精液冷冻稀释液可以对小反刍动物，包括绵羊和山羊的精液进行一步法冷冻保存，其步骤是，将羊精液和本发明的精液冷冻稀释液按照1∶10的比例混合均匀，而后分装于0.25ml塑料细管内，先缓慢降温至5℃，而后迅速移入液氮气相中熏蒸5min，最后将细管投入液氮冷冻保存。
本发明的小反刍动物精液冷冻稀释液还可以对小反刍动物，包括绵羊和山羊的精液进行两步法冷冻保存，其步骤是，将羊精液和本发明所述不含渗透性保护剂的冷冻稀释液按照1∶4-1∶10的比例混合均匀，而后分装于2ml冻存管内，先缓慢降温至5℃，而后将上述悬浮液和本发明所述含有渗透性保护剂的冷冻稀释液按照1∶1的比例混合均匀，于5℃下进一步平衡1-3小时，用移液器吸取0.2ml滴于干冰预冻，最后将冷冻颗粒投入液氮冷冻保存。
本发明的两步法冷冻保存方法中所述不含渗透性保护剂的冷冻稀释液，其制备方法为，分别称取三羟甲基氨基甲烷2.71g、柠檬酸1.4g、单糖1.0g、青霉 素10万IU、链霉素10万IU、肌醇类化合物50-600mMol，白藜芦醇0.1-50μMol溶于超纯水，搅拌均匀，使用前加入56℃30min灭活处理的新鲜蛋黄5-20ml，混合均匀后定容至100ml，于4℃下15000转/分的速率离心1小时，取上清液用0.45μm一次性滤器过滤备用。
对于山羊精液的冷冻保存而言，为了清除磷酸酯酶A与蛋黄相互作用对精子存活的不利影响，优选将精液和清洗液(按照1∶10的比例混合，600g离心15min，取上清，重复洗涤精子沉淀一次。所述清洗液制备方法为，分别称取三羟甲基氨基甲烷2.71g、柠檬酸1.4g、单糖1.0g、青霉素10万IU、链霉素10万IU、肌醇类化合物50-600mMol，白藜芦醇0.1-50μMol，用超纯水定容至100ml，用0.22μm一次性滤器过滤备用。
本发明中用来进行冷冻保存的家畜精液品质为：活力大于75％，精子密度高于1×109/ml。
本发明的有益效果如下：
1.将白藜芦醇应用于动物遗传物质冷冻领域：白藜芦醇是近年来的研究热点，普遍认为白藜芦醇具有抗氧化、抗突变、保护心血管、抗炎抗菌的作用，已经被成功的应用于食品、化妆品、药品领域。本发明针对目前家畜精液冷冻保存研究中存在的主要问题，首次将化学合成来源的冰晶抑制剂和天然植物提取物——白藜芦醇配伍应用于小反刍家畜精液的冷冻保存。虽然白藜芦醇减少冰晶对精子细胞损伤的机制尚不明确，但大量实验证明，精液冷冻稀释液中添加白藜芦醇的确可以提高精液冷冻解冻后的精子活率、活力、顶体完整性、质膜完整性、线粒体膜电位正常率、人工授精后不返情率等质量指标，这可能因为白藜芦醇与本发明公布的肌醇类化合物的分子结构类似，可以减少细胞外形成的冰晶对细胞质膜和线粒体等结构的机械损伤；同时白藜芦醇也是一种抗氧化剂，可以有效降低0-5℃低温平衡时精子的氧化损伤；白藜芦醇还能防止细胞发炎，这对精液细胞冷冻时保护细胞、减少损伤也具有积极的意义。本发明首次成功地将白藜芦醇应用于小反刍动物精液冷冻领域。
2.提高了冷冻精液的质量。大量实验表明，通过应用本发明的精液冷冻稀释液进行冷冻保存的小反刍动物精液，解冻后精子的活力在50％以上，顶体完整性在65％左右，质膜完整性在50％以上，线粒体膜电位正常率在70％以上，人工授 精后不返情率达到70％以上。
3.成本低廉、配制简单。本发明中使用的肌醇类化合物和白藜芦醇是常见的化学试剂，来源方便、成本低廉、化学性质稳定。本发明提供的精液冷冻稀释液的配制方法及使用方法简单易操作。
具体实施方式
以下通过具体实施例进一步说明本发明的技术方案，但是本发明的技术方案不以实施例为限。
实施例1：
采用电刺激法采集云南半细毛羊精液，而后进行精液品质检测。用于冷冻保存的绵羊精液指标必须满足：活力大于75％，精子密度高于1×109/ml，精液量为1-2ml。
配制精液冷冻稀释液，其步骤为，分别称取三羟甲基氨基甲烷2.71g、柠檬酸1.4g、葡萄糖1.0g、甘油5％、青霉素10万IU、链霉素10万IU、1，3-环己二醇100mMol，白藜芦醇1μMol，溶于超纯水，搅拌均匀，使用前加入56℃30min灭活处理的新鲜蛋黄10ml，混合均匀后定容至100ml，于4℃下15000转/分的速率离心1小时，取上清液用0.45μm一次性滤器过滤备用。
将符合冷冻要求的绵羊精液和冷冻稀释液按照1∶5的比例混合均匀，而后分装于0.25ml塑料细管内，先缓慢降温至5℃，而后迅速移入液氮气相中熏蒸5min，最后投入液氮冷冻保存。
该方法冷冻保存的家畜精液，解冻后精子的活率在76.14±6.84％，活力在49.47±5.19％，顶体完整性在68.46±6.27％，质膜完整性在47.55±6.93％，线粒体膜电位正常率在73.41±5.39％，人工授精后不返情率达到70％以上。
实施例2：
采用假阴道采集云岭山羊精液，而后进行精液品质检测。用于冷冻保存的山羊精液指标必须满足：活力大于75％，精子密度高于1×109/ml，精液量为1-2ml。
配制精液冷冻稀释液，其步骤为，分别称取三羟甲基氨基甲烷2.71g、柠檬酸1.4g、果糖1.0g、甘油5％、青霉素10万IU、链霉素10万IU、1，4-环己二 醇50mMol，白藜芦醇10μMol溶于超纯水，搅拌均匀，使用前加入56℃30min灭活处理的新鲜蛋黄20ml，混合均匀后定容至100ml，于4℃下15000转/分的速率离心1小时，取上清液用0.45μm一次性滤器过滤备用。
该方法冷冻保存的家畜精液，解冻后精子的活率在72.68±5.42％，活力在48.58±3.27％，顶体完整性在68.19±4.26％，质膜完整性在45.79±8.16％，线粒体膜电位正常率在71.55±7.41％，人工授精后不返情率在70％以上。
实施例3：
采用电刺激法采集陶赛特公羊精液，而后马上进行精液品质检测。用于冷冻保存的绵羊精液指标必须满足：活力大于75％，精子密度高于1×109/ml，精液量为1-2ml。
配制不含渗透性保护剂的冷冻稀释液，其制备方法为，分别称取三羟甲基氨基甲烷2.71g、柠檬酸1.4g、葡萄糖1.0g、青霉素10万IU、链霉素10万IU、1，3，5-环己三醇50mMol，白藜芦醇8μMol溶于超纯水，搅拌均匀，使用前加入56℃30min灭活处理的新鲜蛋黄15ml，混合均匀后定容至100ml，于4℃下15000转/分的速率离心1小时，取上清液用0.45μm一次性滤器过滤备用。
配置精液冷冻稀释液，其步骤为，分别称取三羟甲基氨基甲烷2.71g、柠檬酸1.4g、果糖1.0g、甘油10％、青霉素10万IU、链霉素10万IU、1，3，5-环己三醇50mMol，白藜芦醇8μMol溶于超纯水，搅拌均匀，使用前加入56℃30min灭活处理的新鲜蛋黄15ml，混合均匀后定容至100ml，于4℃下15000转/分的速率离心1小时，取上清液用0.45μm一次性滤器过滤备用。
将符合冷冻要求的精液和不含渗透性保护剂的冷冻稀释液按照1∶5的比例混合均匀，而后分装于2ml冻存管内，先缓慢降温至5℃，而后将上述悬浮液和本发明所述家畜精液冷冻稀释液按照1∶1的比例混合均匀，用移液器吸取0.2ml滴于干冰预冻，最后将冷冻颗粒投入液氮冷冻保存。
该方法冷冻保存的绵羊精液，解冻后精子的活率在74.58±7.35％，活力在50.47±2.84％，顶体完整性在71.32±5.43％，质膜完整性在50.09±6.91％，线粒体膜电位正常率在75.16±6.38％，人工授精后不返情率在75％以上。
上述实施例仅用于说明本发明，对本发明的保护范围不构成任何限制。
以上对本发明所提供的小反刍动物精液冷冻稀释液进行了详细介绍。本文通过具体实施方式对本发明的原理和实施方式进行了阐述，以上说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (5)

  1. 一种小反刍动物精液冷冻稀释液，其特征在于，每100ml包括如下组分：
    Figure PCTCN2016000656-appb-100001
  2. 根据权利要求1所述小反刍动物精液冷冻稀释液，其特征在于，所述肌醇类化合物为1，3-环己二醇、1，4-环己二醇和1，3，5-环己三醇其中之一。
  3. 根据权利要求1或2所述的小反刍动物精液冷冻稀释液，其特征在于，其中所述单糖为葡萄糖或果糖。
  4. 根据权利要求1或2所述的小反刍动物精液冷冻稀释液，其特征在于，其中所述渗透性保护剂为甘油或乙二醇。
  5. 根据权利要求1或2所述的小反刍动物精液冷冻稀释液，其特征在于，其中新鲜蛋黄经56℃、30min的灭活处理。
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CN108244095B (zh) * 2017-09-08 2018-11-02 东北农业大学 一种猪冻精解冻稀释液及其配制方法
CN107624752A (zh) * 2017-09-11 2018-01-26 吉林省农业科学院 一种猪冷冻精液稀释液的制备方法
CN109258625A (zh) * 2018-10-14 2019-01-25 青海大学 一种用于提高牦牛冷冻精液冻后品质的稀释液配方
CN109287621A (zh) * 2018-11-16 2019-02-01 翼城县富华养殖有限公司 一种提高奶牛受胎率的冻精稀释液
CN112369409B (zh) * 2020-11-23 2023-08-22 青岛农业大学 猪冷冻精液基础稀释液及其制备方法和应用
CN112674075A (zh) * 2020-12-25 2021-04-20 南京农业大学 一种绵羊精液冷冻保护剂
CN113016779B (zh) * 2021-02-07 2022-06-14 内蒙古大学 一种奶绵羊精液冷冻保存方法及其稀释液
CN113812396B (zh) * 2021-09-28 2022-09-16 广东海洋大学 一种鸭精液冷冻保存稀释液
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