CN101622986B - Glutathione-contained boar semen cryopreservation liquid and cryopreservation method thereof - Google Patents
Glutathione-contained boar semen cryopreservation liquid and cryopreservation method thereof Download PDFInfo
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- CN101622986B CN101622986B CN2009101009657A CN200910100965A CN101622986B CN 101622986 B CN101622986 B CN 101622986B CN 2009101009657 A CN2009101009657 A CN 2009101009657A CN 200910100965 A CN200910100965 A CN 200910100965A CN 101622986 B CN101622986 B CN 101622986B
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Abstract
The invention provides glutathione-contained boar semen cryopreservation liquid and a cryopreservation method thereof. The boar semen cryopreservation liquid uses diluent which is generally suitable for boar semen cryopreservation as a substrate and also comprises 2-6 mg/ml of glutathione, 5-11 mg/ml of lactose, 10-30 percent (v/v) of yelk, 5-10 percent (v/v) of glycerin and 0.5-2 mg/ml of sodium lauryl sulfate. The invention applies the glutathione to the boar semen cryopreservation, can effectively remove redundant free radicals generated in a refrigeration process, solves the problem of low survival rate of the prior boar semen cryopreservation, has lower economic cost and is convenient to popularize and apply in production.
Description
(1) technical field
The present invention relates to a kind of Cryopreservation of Boar Semen liquid and freezing and storing method thereof that contains glutathione, belong to the livestock reproduction technical field.
(2) background technology
In China, pig is topmost stock raising, and pork output accounts for 70% of national meat total amount.Along with the raising of living standards of the people, the breeding direction of pig improved the local flavor of meat from mainly being conceived to the raising of growth rate and lean meat percentage, changing into improve pork quality in recent years.Traditional seed selection mode time generation interval is long; Can not meeting the market requirement, and adopt pig frozen semen technology, can make full use of the good species poultry; Shorten time fine-variety breeding generation interval, set up the boar and the market pig crossbreed system of new suitable market demands fast.Simultaneously, raising history in nearly thousand has formed hundreds of landrace that differ from one another in China; This is a very valuable property, and the competition of livestock breeding from now on quite partly is a genetic resources; The competition that excellent genes utilizes, but the introduction of external pig kind hybridization have in recent years made China that a considerable amount of landrace extinctions are arranged or have faced extinction; And adopt the Cryopreservation of Boar Semen technology can effectively preserve China's local pig breed matter resource, have immeasurable social effect and benefit.
Though the freezing preservation technology of herbvore livestock semen ultralow temperature (196 ℃) is changed herding production in modern times, germ plasm resource is being brought into play important effect in preserving, and has produced obvious economic and social benefit.But the pig frozen semen is difficult to apply on a large scale aborning always; Its reason has two; The one, C/P ratio significantly is lower than plant-eating animal in the pig spermoblast plasma membrane; The ion permeability of sperm membrane strengthens in refrigerating process, and lipid material runs off, and causes freezing that smart motility rate is low, conception rate is low.The 2nd, the pig sperm often receives the infringement of free radical and oxidative stress in refrigerating process, thereby influences motility rate and fertility behind the pig sperm freezing.Free radical is that eubolism produces in the cell, organism contain natural antioxidant (free radical scavenger) with in the toxic and side effect of free radical.Under the adjusting of these antioxidants, the generation of interior free yl and elimination are in a kind of dynamic equilibrium just.But the level of above-mentioned antioxidant is lower than level in the body in the boar semen dilution of routine, and in the dilution equilibrium process before sperm freezing; Be exposed to air inevitably; And airborne oxygen concentration is higher than oxygen concentration (20%VS.7%) in the genital tract, and the influence of metal ion in light and the dilution when adding manipulation in vitro makes free radical level increase in sperm when freezing; Break the poised state of oxidation antioxidation, caused the damage of sperm oxidative stress.Therefore, the new cryoprotector of screening has become the focus of boar semen research field to improve freezing back sperm motility rate.
Glutathione is the non-albumen tripeptides that contains sulfydryl that methionine, glycine and cysteine are formed, and has stronger antioxidation, can eliminate free radicals such as superoxide anion and hydrogen peroxide.It is a non-albumen sulfhydryl compound main in the histocyte, is the substrate of glutathione peroxidase and two kinds of enzymes of glutathione S-transferase, and is essential for these two kinds of enzyme decomposition of hydrogen peroxide institutes, can stablize the enzyme that contains sulfydryl.For the existing bibliographical information of the research of glutathione.Multiple patent applications such as CN100455592C, CN100503837C, CN1164737C, CN1325511C, CN1313597C, CN100360679C disclose the preparation method of glutathione.Multiple patent such as CN1185008C, CN100435793C discloses the purposes of glutathione as medicine.The relevant glutathione of Shang Weijian is used for the livestock reproduction technical field, especially for the report of Cryopreservation of Boar Semen but up to the present.
(3) summary of the invention
The purpose of this invention is to provide a kind of Cryopreservation of Boar Semen liquid and method for using thereof of containing glutathione that reduces freezing damage, improves cold smart motility rate to sperm.
The technical scheme that the present invention adopts is:
The application of glutathione in preparation Cryopreservation of Boar Semen liquid.Concrete; Described Cryopreservation of Boar Semen liquid is matrix with the dilution that is applicable to Cryopreservation of Boar Semen; The glutathione that also contains 2～6mg/mL in the said Cryopreservation of Boar Semen liquid, the lactose of 5～11mg/mL and 10～30% (v/v) yolk, the glycerine of 5～10% (v/v) and the sodium lauryl sulfate of 0.5～2mg/mL.A kind of Cryopreservation of Boar Semen liquid that contains glutathione; Described Cryopreservation of Boar Semen liquid is applicable to that with routine the dilution of Cryopreservation of Boar Semen is a matrix; The glutathione that also contains 2～6mg/mL in the said Cryopreservation of Boar Semen liquid, the lactose of 5～11mg/mL and 10～30% (v/v) yolk, the glycerine of 5～10% (v/v) and the sodium lauryl sulfate of 0.5～2mg/mL.
Said glutathione has multiple preparation method; Belong to already known processes and mature technology; And existing commodity selling, comparatively ripe manufacturer has U.S. Sigma-Aldrich company, Shaanxi to melt to rise bio tech ltd, Chongqing Yaoyou Pharmaceutical Co., Ltd., Huzhou is bright is Bioisystech Co., Ltd etc. deeply.Used glutathione is provided by U.S. Sigma-Aldrich company among the present invention, and purity is 98%.
Concrete, the every 1000mL of said Cryopreservation of Boar Semen liquid forms as follows:
Glutathione 2～6g
Lactose 50～110g
Yolk 100～300mL
Glycerine 50～100mL
Sodium lauryl sulfate 5～20g
The dilution that is applicable to Cryopreservation of Boar Semen complements to 1000mL.
The said dilution of Cryopreservation of Boar Semen that is applicable to is the conventional dilution that is used for Cryopreservation of Boar Semen, like the Seminark of BTS dilution, Korea S's product
TMDilution etc.Preferably; Saidly be applicable to that the dilution of Cryopreservation of Boar Semen is BTS dilution (Beltsville thawingsolution); Every 1000mL presses following formulated: glucose 37g, sodium citrate 6g, potassium chloride 0.75g, sodium bicarbonate 1.25g, EDTA 1.25g, penicillin 100000 units; Streptomycin 100mg, deionized water complements to 1000mL.
Preferably, the every 1000mL of said Cryopreservation of Boar Semen liquid forms as follows:
Glutathione 4g
Lactose 80g
Yolk 200mL
Glycerine 80mL
Sodium lauryl sulfate 5g
The BTS dilution complements to 1000mL.
The invention still further relates to a kind of method that the said Cryopreservation of Boar Semen liquid that contains glutathione carries out Cryopreservation of Boar Semen of using, said method comprises:
(1) with the fresh pig seminal fluid of taking with one times of the BTS diluted of isothermal;
(2) under 15 ℃ of conditions, left standstill (balance) 2 hours, 1000 left the heart 10 minutes;
(3) supernatant is removed in centrifugal back, and deposition is with the boar semen diluted (not containing glycerine and sodium lauryl sulfate) that contains glutathione, and making sperm concentration is 2 * 10
9Individual/mL, the seminal fluid after the dilution leaves standstill (balance) after 2 hours under 5 ℃ of conditions, and with one times of 5 ℃ of Cryopreservation of Boar Semen liquid dilution that contain glutathione of preserving down, the final densities that makes sperm is 1 * 10 again
9Individual/mL, the seminal fluid after obtaining diluting;
The every 1000mL of said boar semen dilution forms as follows:
Glutathione 2～6g
Lactose 50～110g
Yolk 100～300mL
The BTS dilution complements to 1000mL;
The every 1000mL of said Cryopreservation of Boar Semen liquid forms as follows:
Glutathione 2～6g
Lactose 50～110g
Yolk 100～300mL
Glycerine 50～100mL
Sodium lauryl sulfate 5～20g
The BTS dilution complements to 1000mL;
(4) with the conventional freezing pipe of packing into of the seminal fluid after the chill back, place on the liquid nitrogen surface 3 centimetres,
Fumigate after 5 minutes, directly drop in the liquid nitrogen, freezing preservation is subsequent use.
Cryopreservation of Boar Semen liquid of the present invention is according to needs anti-oxidant in the Cryopreservation of Boar Semen process, Green Tea Extract, develops in conjunction with the biological function of glutathione.Glutathione has stronger antioxidation, can eliminate free radicals such as superoxide anion and hydrogen peroxide.It is a non-albumen sulfhydryl compound main in the histocyte, is the substrate of glutathione peroxidase and two kinds of enzymes of glutathione S-transferase, and is essential for these two kinds of enzyme decomposition of hydrogen peroxide institutes, can stablize the enzyme that contains sulfydryl.Therefore glutathione is applied to Cryopreservation of Boar Semen, can remove the unnecessary free radical that produces in the refrigerating process effectively, has solved the low problem of present Cryopreservation of Boar Semen survival rate.Its Financial cost is lower, is convenient to apply aborning.
(4) embodiment
Below in conjunction with specific embodiment the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: preparation contains the Cryopreservation of Boar Semen liquid and the freeze-extender of glutathione
Contain the Cryopreservation of Boar Semen liquid preparation of glutathione:
Accurately take by weighing glutathione 400mg; (every 1000mL presses following formulated to first BTS dilution with 50mL: glucose 37g, sodium citrate 6g, potassium chloride 0.75g, sodium bicarbonate 1.25g, EDTA 1.25g, penicillin 100000 units; Streptomycin 100mg; Deionized water complements to 1000mL) dissolving; In dilution, add the sodium lauryl sulfate of yolk (from egg, extract and obtain), 8mL glycerine and the 0.5g of 8g lactose, 20mL then, add the BTS dilution again to 100mL, once more stirring and evenly mixing.After the micro-filtration membrane suction filtration sterilization with 0.22 μ M, store down at 5 ℃, subsequent use.
Contain the boar semen dilution preparation of glutathione:
Accurately take by weighing glutathione 400mg,, in dilution, add the yolk of 8g lactose and 20mL then, add the BTS dilution again to 100mL, once more stirring and evenly mixing earlier with the BTS dilution dissolving of 50mL.After the micro-filtration membrane suction filtration sterilization with 0.22 μ M, store down at 5 ℃, subsequent use.
Embodiment 2: the Cryopreservation of Boar Semen liquid that contains glutathione is used for big York Cryopreservation of Boar Semen
Select fertility height, athletic big York stock boar, adopt the hand grip semen collection.Collect sperm and enrich part, through 4 layers of sterile gauze filter remove colloid after, survey sperm volume immediately, and the inspection sperm motility rate, the collection sperm motility rate is at the about 50mL of the seminal fluid more than 75%, with one times of the BTS diluted of isothermal.Under 15 ℃ of conditions, leave standstill balance 2 hours, 1000 left the heart 10 minutes then.
Seminal plasma is removed in the centrifugal back of seminal fluid; The sperm deposition is with boar semen dilution (embodiment 1) dilution that contains glutathione; This dilution is the freezing preservation liquid of the present invention that does not contain glycerine and sodium lauryl sulfate; Because under the glycerine cryogenic conditions to the sperm toxic side effect, add behind the glycerine sperm want at once freezing can not stand at low temperature.Making sperm concentration is 2 * 10
9Individual/mL.Seminal fluid after the dilution left standstill balance 2 hours under 5 ℃ of conditions.Use one times of Cryopreservation of Boar Semen liquid (embodiment 1) dilution again, the final densities that makes sperm is 1 * 10
9Individual/mL.The control group seminal fluid is with freezing preservation liquid (BTS dilution) dilution that does not contain glutathione, then experimental group and control group seminal fluid packed in the 0.25mL conventional freezing pipe, places on the liquid nitrogen surface 3 centimetres, and is stifling after 5 minutes, directly drops into the medium-term and long-term preservation of liquid nitrogen.
Embodiment 3: big yorker is frozen essence and thaws
In order to detect the implementation result of patent of the present invention; Big yorker seminal fluid will be equipped with the experimental group and the control group cryovial that freeze essence respectively and place 37 ℃ of water-baths to thaw in 10 seconds after handling according to embodiment 1, embodiment 2 methods successively, and microscopically detects the energy motility rate; Wherein control group freeze essence thaw the back motility rate be 30.2%; Freezing the essence back sperm motility rate of thawing in the experimental group cryovial is 36.7%, improves 6.5% than control group, both significant differences of statistical results show.The result confirms, the Cryopreservation of Boar Semen liquid cooling that contains glutathione is frozen and preserved big yorker seminal fluid and can effectively improve the survival rate after cold essence is thawed.
Claims (4)
1. Cryopreservation of Boar Semen liquid that contains glutathione; It is characterized in that described Cryopreservation of Boar Semen liquid is matrix with the dilution that is applicable to Cryopreservation of Boar Semen; The glutathione that also contains 2～6mg/mL in the said Cryopreservation of Boar Semen liquid, the lactose of 5～11mg/mL and 10～30% yolk, 5～10% glycerine and the sodium lauryl sulfate of 0.5～2mg/mL.
2. Cryopreservation of Boar Semen liquid as claimed in claim 1 is characterized in that the every 1000mL of said Cryopreservation of Boar Semen liquid forms as follows:
The dilution that is applicable to Cryopreservation of Boar Semen complements to 1000mL.
3. according to claim 1 or claim 2 Cryopreservation of Boar Semen liquid is characterized in that saidly being applicable to that the dilution of Cryopreservation of Boar Semen is the BTS dilution.
4. Cryopreservation of Boar Semen liquid as claimed in claim 1 is characterized in that the every 1000mL of said Cryopreservation of Boar Semen liquid forms as follows:
The BTS dilution complements to 1000mL.
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| CN2012101003455A Division CN102630665A (en) | 2009-08-10 | 2009-08-10 | Method for freezing and preserving pig semen |
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| CN101622986B true CN101622986B (en) | 2012-11-14 |
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| CN102258007A (en) * | 2011-09-02 | 2011-11-30 | 大连海洋大学 | Refrigerant and method for storing Pacific codfish sperms |
| CN103651330A (en) * | 2012-09-21 | 2014-03-26 | 临沂思科生物科技有限公司 | Animal semen preservative with biological activity and preparation method thereof |
| CN103314948B (en) * | 2013-05-31 | 2014-07-23 | 中国科学院海洋研究所 | Superfreeze preservation method of starry flounder sperms |
| KR20210142751A (en) * | 2019-04-09 | 2021-11-25 | 페킹 유니버시티 서드 호스피탈 | Bionic ice control material and cryopreservation solution containing same |
| CN113331178B (en) * | 2021-06-01 | 2023-04-21 | 西北农林科技大学 | Application of methionine in preparing normal-temperature preservation diluent for boar semen |
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Non-Patent Citations (4)
| Title |
|---|
| 杜立银等.抗氧化剂在猪精液冷冻保存技术中的应用.《中国畜牧杂志》.2008,(第01期), * |
| 王争光等.圆筒形贮存管对猪精液冷冻保存效果的影响.《浙江农业科学》.2009,(第02期), * |
| 王前.关于公猪精液采集的有关问题.《畜禽业》.2005,(第10期), * |
| 郭志儒.我国将首次把猪精液送至太空.《中国兽医学报》.2005,(第05期), * |
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