WO2017139909A1 - Diluant de congélation pour sperme de bétail - Google Patents

Diluant de congélation pour sperme de bétail Download PDF

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Publication number
WO2017139909A1
WO2017139909A1 PCT/CN2016/000657 CN2016000657W WO2017139909A1 WO 2017139909 A1 WO2017139909 A1 WO 2017139909A1 CN 2016000657 W CN2016000657 W CN 2016000657W WO 2017139909 A1 WO2017139909 A1 WO 2017139909A1
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WO
WIPO (PCT)
Prior art keywords
semen
polyvinyl alcohol
livestock
sperm
cryopreservation
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PCT/CN2016/000657
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English (en)
Chinese (zh)
Inventor
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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Application filed by 权国波 filed Critical 权国波
Publication of WO2017139909A1 publication Critical patent/WO2017139909A1/fr

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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
    • 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 frozen dilution solution of livestock semen.
  • antifreeze protein reduces the mechanical damage of ice crystals to tissue cells by modifying ice crystal traits and inhibiting recrystallization. Some studies have shown that antifreeze proteins can improve the survival rate of human and some mammalian semen after cryopreservation. However, the application of antifreeze proteins also has certain limitations. First, as a heterologous protein, antifreeze proteins may cause certain immune responses in animals. Secondly, the main source of antifreeze proteins is extracted from the blood of polar marine fish, and these technologies and patents are mainly in Europe and America.
  • the antifreeze protein inhibits the formation of ice crystals by changing the shape of the ice crystals, which leads to the formation of some sharp needle-shaped ice crystals, thereby aggravating the mechanical damage of the sperm by freezing.
  • antifreeze protein can improve the quality of frozen sperm.
  • Polyvinyl alcohol is a high molecular organic compound, and pharmaceutical grade polyvinyl alcohol is non-toxic and has no side effects. It is widely used as an emulsifier and film former in the fields of medicine and cosmetics.
  • Chinese patent "a kind of pig semen diluent" patent number: 201310478237.6 announced the use of polyvinyl alcohol in the dilute preservation of pig semen at room temperature
  • the Chinese patent "pork semen cryoprotectant" patent number: 201210196733.8 announced the polyvinyl alcohol It is applied to the low temperature dilution of 0-4 °C of pig semen.
  • the present invention finds for the first time that the non-permeable high molecular polymer polyvinyl alcohol has the same effect as the antifreeze protein for inhibiting the re-formation of ice crystals, and can be used instead of the antifreeze protein for cryopreservation of livestock semen, thereby avoiding Defects related to the application of antifreeze proteins have not been reported in the literature.
  • the frozen liquid dilution of livestock semen of the present invention comprises the following components per 100 ml:
  • the balance is ultrapure water.
  • the polyvinyl alcohol has a molecular weight of 9 KD.
  • the monosaccharide is glucose, fructose, mannitol, sorbitol, galactose or xylitol.
  • the osmoprotectant is glycerin.
  • the method for preparing a frozen liquid dilution of livestock semen comprises the steps of weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of monosaccharide, 0-10 ml of osmotic protective agent, and 100,000 penicillin. IU, streptomycin 100,000 IU, polyvinyl alcohol 0.05-5 g, dissolved in ultrapure water, stir evenly, adjust the pH to 6.8-7.2, add 5-20ml of fresh egg yolk inactivated at 56 °C for 30 min before use. After mixing well, the volume was adjusted to 100 ml, and the mixture was centrifuged at 15,000 rpm for 1 hour at 4 ° C, and the supernatant was filtered through a 0.45 ⁇ m disposable filter for use.
  • the frozen liquid dilution of the livestock semen of the present invention can be subjected to one-step cryopreservation of the livestock semen by mixing the livestock semen and the frozen semen of the livestock semen of the present invention in a ratio of 1:4-1:10, and then sub-packaging.
  • a 0.25 ml plastic thin tube slowly cool down to 5 ° C, then quickly move into the liquid nitrogen phase for 5 min, and finally put the thin tube into liquid nitrogen for cryopreservation.
  • the livestock semen freezing dilution liquid of the invention can also perform the two-step cryopreservation of the livestock semen by mixing the livestock semen and the frozen diluent without the osmotic protective agent in a ratio of 1:4, and then sub-packaging.
  • a 2 ml cryotube slowly cool down to 5 ° C, then mix the above suspension and the frozen semen dilution of the livestock semen of the present invention in a ratio of 1:1, further equilibrate at 1-5 h for 5 h, using pipetting
  • the device was pipetted into a drop of 0.2 ml of dry ice and finally frozen pellets were 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.
  • the semen and washing solution were mixed at a ratio of 1:10, centrifuged at 600 g for 15 min, and the supernatant was taken. Repeat the washing of the sperm pellet once, and the rest of the steps are the same as the sheep.
  • the cleaning liquid for freezing the goat semen is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of monosaccharide, 100,000 IU of penicillin, and a chain. 100,000 IU of the peptide and 0.05-5 g of polyvinyl alcohol were made up to 100 ml with ultrapure water and filtered through a 0.22 ⁇ m disposable filter.
  • the quality of the semen used in the cryopreservation of the present invention is: the vigor is greater than 75%, the deformity rate is less than 10%, and the sperm density is higher than 1 x 10 9 /ml.
  • the trishydroxymethylaminomethane is a buffer substance
  • citric acid is a solute, and may also participate in glycolysis
  • the monosaccharide is a sperm metabolism substrate
  • the egg yolk is a low temperature shock plasma membrane.
  • the protective agent is a osmotic protective agent
  • the penicillin and streptomycin are antibiotics
  • the polyvinyl alcohol is an ice crystal inhibitor.
  • Our research shows that although the molecular structure is different from the antifreeze protein, polyvinyl alcohol has a significant inhibitory effect on the re-formation of ice crystals after thawing, thereby reducing the secondary damage of ice crystals to sperm cells.
  • polyvinyl alcohol inhibits ice crystal formation. The ability is higher than other high molecular polymers, such as dextran and polyvinylpyrrolidone.
  • the present invention is aimed at the main problems in the current research on the cryopreservation of livestock semen.
  • the common high molecular polymer, polyvinyl alcohol is used as ice crystal suppression.
  • the agent replaces the natural antifreeze protein for the cryopreservation study of livestock semen.
  • Polyvinyl alcohol can effectively inhibit the formation of ice crystals during the thawing process of frozen semen, reduce the mechanical damage of ice crystals to sperm, improve the quality of frozen semen in livestock, and improve the rate of artificial insemination.
  • the sperm semen stored frozen by the frozen semen of livestock semen of the present invention has a sperm activity of about 75% after thawing, an activity of more than 50%, acrosome integrity of more than 65%, and plasma membrane integrity. At about 50%, the rate of non-return after artificial insemination is over 70%.
  • the polyvinyl alcohol used in the present invention is a common chemical raw material with wide source, low cost, guaranteed purity, stable chemical properties, and ice crystal suppression using polyvinyl alcohol as a semen frozen diluent.
  • the agent has the advantages of no immune reaction, low toxicity, guaranteed purity, and low price.
  • 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 higher than 1 ⁇ 10 9 /ml, deformity rate less than 10%, semen volume 1-2ml.
  • 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 ml of glycerol, 100,000 IU of penicillin, 100,000 IU of streptomycin, and polyvinyl alcohol 0.1. g, soluble in ultrapure water, stir well, add 10ml of fresh egg yolk inactivated at 56 °C for 30min before use. After mixing well, the volume was adjusted to 100 ml, and the mixture was centrifuged at 15,000 rpm for 1 hour at 4 ° C, and the supernatant was filtered through a 0.45 ⁇ m disposable filter for use.
  • the method of cryopreservation of livestock semen the survival rate of sperm after thawing is about 75%, the vitality is above 50%, the acrosome integrity is about 65%, the plasma membrane integrity is about 50%, and the rate of return after artificial insemination is not More than 70%.
  • the Nubian sheep semen was collected using a fake vagina, and then the semen quality test was performed.
  • the semen index for cryopreservation must meet: viability greater than 75%, sperm density greater than 1 x 10 9 /ml, malformation rate less than 10%, semen volume 0.75-2 ml.
  • the semen cleaning solution is prepared by weighing 2.71 g of tris (hydroxymethyl) aminomethane, 1.4 g of citric acid, 1.0 g of fructose, 100,000 IU of penicillin, 100,000 IU of streptomycin, 0.2 g of polyvinyl alcohol, and dissolved in Ultra-pure water, mix well and dilute to 100 ml, and filter with 0.22 ⁇ m disposable filter for use.
  • 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, 7 ml of glycerol, 100,000 IU of penicillin, 100,000 IU of streptomycin, and 0.2 of polyvinyl alcohol. g, soluble in ultrapure water, stir well, add 15ml of fresh egg yolk inactivated at 56 ° C for 30 min before use, mix well and dilute to 100 ml, centrifuge at 15000 rpm for 1 hour at 4 ° C, take supernatant The solution was filtered through a 0.45 ⁇ m disposable filter for use.
  • the goat semen and washing solution meeting the freezing requirements were uniformly mixed at a ratio of 1:4, and centrifuged at 2500 rpm for 10 minutes at room temperature. After removing the supernatant, the sperm was washed once under the same centrifugation conditions. Add the cleaning solution and resuspend the sperm to the original volume. The washed sperm suspension and the frozen diluent are uniformly mixed in a ratio of 1:8, and then sub-packed in a 0.25 ml plastic thin tube, and then slowly cooled to 5 ° C, further equilibrated at this temperature for 2 hours, and then rapidly transferred into the liquid. Fumigation in the nitrogen phase for 5 min, and finally into liquid nitrogen for cryopreservation.
  • the method of cryopreserved livestock semen is about 75%, the vitality is above 50%, the acrosome integrity is about 60%, the plasma membrane integrity is about 50%, and the rate of return after artificial insemination is not Reach More than 70%.
  • Semen terracotta ram semen was collected using a false vaginal method, and semen quality testing 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.
  • 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 ml of glycerol, 100,000 IU of penicillin, 100,000 IU of streptomycin, and polyvinyl alcohol 1.0. g, soluble in ultrapure water, stir well, add 20ml of fresh egg yolk inactivated at 56 °C for 30min before use, mix well and dilute to 100ml, centrifuge at 15000 rpm for 1 hour at 4 °C, take supernatant The solution was filtered through 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 1:4, 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, further equilibrated at 5 ° C for 1 h, and a 0.2 ml drop was pipetted into dry ice to be pre-frozen, and finally the frozen pellet was placed in liquid nitrogen for cryopreservation.
  • the molecular weight of the polyvinyl alcohol used is 9KD. Since the polyvinyl alcohol is a high molecular polymer, if the molecular weight is too high, it may form an "ice mass" during the freezing process, causing more serious damage to the sperm cells. If the molecular weight is too low, it may not be effective in inhibiting the formation of ice crystals.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
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  • Engineering & Computer Science (AREA)
  • Environmental Sciences (AREA)
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  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
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Abstract

La présente invention concerne un diluant de congélation pour le sperme de bétail. Le diluant de congélation, pour 100 ml, comprend les composants suivants : 2,71 g de trihydroxyméthylaminométhane, 1,4 g d'acide citrique, 1,0 g de monosaccharide, 5 à 20 ml de jaune d'œuf frais, 0 à 10 ml d'un agent de protection perméable, 0,1 million d'UI de pénicilline, 0,1 million d'UI de streptomycine, 0,05 à 5 g d'alcool polyvinylique, et le reste étant de l'eau ultrapure. Selon la présente invention, pour la première fois, un alcool polyvinylique de polymère de haut poids moléculaire courant est utilisé en tant qu'inhibiteur des cristaux de glace au lieu d'une protéine antigel naturelle, et est appliquée à la recherche en cryoconservation de sperme de bétail. Le taux de survie du sperme décongelé est d'environ 75 %, son activité est de 50 % ou plus, l'intégrité de l'acrosome est de 65 % ou plus, l'intégrité de la membrane plasmique est d'environ 50 %, et le taux de non-retour après insémination artificielle est supérieur à 70 %
PCT/CN2016/000657 2016-02-16 2016-11-25 Diluant de congélation pour sperme de bétail WO2017139909A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610086324.0 2016-02-16
CN201610086324.0A CN105638643B (zh) 2016-02-16 2016-02-16 一种羊精液冷冻稀释液

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Cited By (2)

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CN114304139A (zh) * 2022-01-19 2022-04-12 山东畜牧兽医职业学院 一种提高精液保存品质的精液保存液及其制备方法
CN117158415A (zh) * 2023-11-02 2023-12-05 黑龙江八一农垦大学 一种含伊拉米肽的猪精子冷冻保存稀释剂及其应用

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CN105638643B (zh) * 2016-02-16 2018-01-12 云南省畜牧兽医科学院 一种羊精液冷冻稀释液
CN111066775B (zh) * 2016-06-29 2022-02-01 许红喜 一种冷藏保存动物精子的方法
CN106508886B (zh) * 2016-09-14 2019-11-12 西北农林科技大学 番茄红素与α-硫辛酸作为哺乳动物精液冷冻保存剂的应用
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
CN108103012B (zh) * 2017-12-14 2021-04-02 内蒙古赛科星家畜种业与繁育生物技术研究院有限公司 小鼠x/y精子分离精液的生产方法及应用
CN109221091A (zh) * 2018-11-01 2019-01-18 洛宁农本畜牧科技开发有限公司 一种动物鲜精低温稀释液及其制备方法与应用
CN110463687B (zh) * 2019-07-10 2021-07-27 安徽农业大学 一种羊精液的保存液以及保存方法
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
CN114009427B (zh) * 2021-12-11 2023-06-02 江苏省农业科学院 一种兔精液冷冻保存稀释液和制备方法、兔精液冷冻保存方法和应用

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CN114304139A (zh) * 2022-01-19 2022-04-12 山东畜牧兽医职业学院 一种提高精液保存品质的精液保存液及其制备方法
CN117158415A (zh) * 2023-11-02 2023-12-05 黑龙江八一农垦大学 一种含伊拉米肽的猪精子冷冻保存稀释剂及其应用
CN117158415B (zh) * 2023-11-02 2024-02-02 黑龙江八一农垦大学 一种含伊拉米肽的猪精子冷冻保存稀释剂及其应用

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