CN102578074B - Method for preparing livestock sex control frozen sperm by adding antioxidants of CAT (Catalase) and VE (Vitamin E) - Google Patents
Method for preparing livestock sex control frozen sperm by adding antioxidants of CAT (Catalase) and VE (Vitamin E) Download PDFInfo
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- CN102578074B CN102578074B CN 201110433773 CN201110433773A CN102578074B CN 102578074 B CN102578074 B CN 102578074B CN 201110433773 CN201110433773 CN 201110433773 CN 201110433773 A CN201110433773 A CN 201110433773A CN 102578074 B CN102578074 B CN 102578074B
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Abstract
The invention relates to a method for preparing livestock sex control frozen sperm by adding antioxidants of CAT (Catalase) and VE (Vitamin E), belonging to the technical field of animal propagation. The method comprises the following steps: adding CAT and VE in the semen separation and frozen processes by mainly using the characteristics that the antioxidants of CAT and VE can resist ROS (Reactive Oxygen Species) carrying free radicals so as to reduce peroxidative damage to the semen caused by the ROS, effectively protect the semen physiological function in the livestock sperm separation and frozen processes, improve the unfrozen semen viability and prolong the semen survival time, therefore, the livestock sex control frozen sperm capable of fertilizing and conceiving are prepared.
Description
Technical field
The invention belongs to the animal reproduction technical field, mainly provide by adding catalase (CAT) and vitamin E (V
E), protect effectively that spermatozoa separates, sperm physiological function in refrigerating process, improve sperm thaw rear vigor with extend the sperm time-to-live, thereby the method for the domestic animal property control frozen semen that preparation can normal fertilization be impregnated.
Background technology
Spermatozoa separation-property control techniques is since the industrialization in 2000, and more than ten countries apply in the whole world at present.But, in actual production, compare with common domestic animal frozen semen, due to the property control freeze essence will be through centrifugal, dyeing, separation process, cause the damage to property control sperm, vigor and the time-to-live after essence is thawed frozen in the control of impact property, and the low problem of smart conception rate is frozen in the control of causing property.Freeze the quality of essence for the control of raising property, researched and developed this property control and frozen process for producting concentrated.
Summary of the invention
The invention provides a kind of interpolation antioxidant CAT and V
EThe method for preparing domestic animal property control frozen semen, this know-why are to utilize antioxidant CAT and V
ECan resist active chalcogen (ROS) characteristics that carry free radical, separate at sperm, add CAT and V in freezing process
E, reduce the effect of the peroxide injury that active chalcogen causes sperm, to thaw rear sperm viability and extend the sperm time-to-live thereby improve, the control of raising property is frozen smart product quality and is reached the purpose of being impregnated.
The objective of the invention is to be achieved through the following technical solutions: a kind of interpolation antioxidant CAT and V
EPrepare the method for domestic animal property control frozen semen, it comprises the following steps:
(1), CAT liquid and V
EThe preparation of liquid:
The CAT that takes 4.2mg under room temperature adds in the distilled water of 1ml and dissolves, and keeps in Dark Place at-20 ℃, and the pot-life is a week; Take the V of 4mg
EAdd in the DMSO of 1ml and dissolve, keep in Dark Place at 4 ℃, the pot-life is a week;
(2), the antibiotic of the former essence of domestic animal is added:
According to the former essence of 1ml domestic animal add 6 μ l woodss can-ratio of miramycin, 6 μ Taylor l mycins, the 8 large mycins in μ l east adds antibiotic in the former essence of domestic animal;
(3), sperm separates:
1) will add the former essence of antibiotic domestic animal and be filtered in new centrifuge tube with 50 μ m seminal fluid filters, remove the impurity in former seminal fluid, and use Density Measuring Instrument to detect former smart actual concentrations after filtering, and put in 18-20 ℃ of refrigerator and preserve;
2) calculate per hour separated sperm sum 1,200,000,000/former smart actual concentrations of the former smart volume number that six seperators are used for dyeing=six seperator;
3) according to step 2) the volume number of the former essence that is used for dyeing that calculates, with the 15ml centrifuge tube from step 1) the former essence of taking-up, add Staining Talp to 3ml, 360g centrifuge washing, 5 minutes;
4) enter 75 μ l Hoechst33342 fluorescent dyes, add the ratio of 6ml Staining Talp according to 1,200,000,000 former finishings in another new centrifuge tube, Hoechst33342 fluorescent dye and StainingTalp are mixed, and vibrated 3 seconds with the suspension oscillator is slight, the concentration of Hoechst33342 fluorescent dye is 5mg/ml;
5) step 3) after centrifugal stopping, inhaling and to abandon the 2ml supernatant, retention volume 1ml transfers them to above-mentioned steps 4) the 15ml centrifuge tube in, 34 ℃ of dyeing added the 4%eggyolk of 6ml after 45 minutes;
6) step 5) the former essence processed of dyeing is by the former capable packing that progresses greatly of each separator tube 200,000,000, and the former smart sample after packing is dyeed is well put into the sample stage of seperator, separates former essence;
7) sperm according to every the seperator of read-record on seperator separates sum, and the sperm that calculates six seperators separates sum=every instrument separation number * 6 (unit: ten million);
8) in the buffer buffer solution that takes out, add 5 μ l CAT liquid, 1 μ l V according to 1ml from-20 ℃ of refrigerators
EThe ratio of liquid is added antioxidant CAT liquid and V
ELiquid, room temperature preheating half an hour, as the recovery liquid of sperm after separating, said buffer buffer solution contains 84% the freezing equilibrium liquid of TrisA and 16% ultra-pure water; Under separated sperm, after machine, balance is 90 minutes under 4 ℃ of conditions, and 850g abandons supernatant after centrifugal 20 minutes, with the sperm precipitation, slightly shakes with the suspension oscillator, and the sperm after after concussion, all being separated is all poured in an empty centrifuge tube;
(4), add the preparation that essence is frozen in the antioxidant control:
1) add antioxidant in Tris-A, Tris-B, adding proportion is: 1ml Tris-A adds respectively 51 μ l CAT liquid, 1 μ l V
ELiquid, 1ml Tris-B add respectively 5 μ l CAT liquid, 1 μ l V
ELiquid after having added antioxidant, mixes Tris-A, Tris-B respectively, is placed under 4 ℃ of conditions balance 90 minutes;
2) the total number of sperm that separates according to lower machine calculates by step 1) consumption of the Tris-A that balance is good, M represents the gross mass of Tris-A and sperm precipitation, total number of sperm ÷ 10 * 80% * 1.03 ÷ 2 that M (g)=lower machine separates, at first the sum that machine under sperm is separated is converted into ten million by 1,000,000, multiply by 80% the rate of recovery is exactly the sperm volume, multiply by averag density 1.03g/ml, adding exactly the gross mass of Tris-A and Tris-B, is exactly to add by step 1 divided by 2) quality of the Tris-A that balance is good;
3) with the step 8 in step (3)) all be housed separate after centrifuge tubes of sperm be placed on electronic balance, add by step 1 in centrifuge tube) Tris-A that balance is good, until reading is step 2) M (g) that calculates;
4) glycerine: get with M (g) equivalent by step 1) Tris-B that balance is good, 15 minutes equivalent of minute 2 minor tick adds step 3 in above-mentioned steps (4)) centrifuge tube in, put upside down gently after each the interpolation and shake up;
5) tubulature and freezing preservation: after separated sperm shakes up, the 0.25 milliliter of semen freezing pipe of packing under 4 ℃ of conditions;
6) every 300 are spread in a single layer on freezing washboard for standard, adjusting liquid nitrogen container gas-bearing formation height is 12.6in on liquid nitrogen surface, with dust catcher aspirated liquid blanket of nitrogen after-125 ± 1 ℃, product is placed on the gas-bearing formation position of 8.5-9.0cm and carried out freezing processing 20 minutes, then, every 25 tubules are contained in one to be moulded in the essence cylinder, with label, date, batch and number, freeze the information flags such as smart people on white label, during transferred product, can not surpass 3 seconds in air, drop at last-180 ℃ of liquid nitrogen and preserve;
Said Staining Talp:PH=7.4, it consists of:
Said 4%eggyolk:PH=5.5, it consists of:
Consisting of of said buffer buffer solution:
Tris-A 84％
Pure water 16%
Said domestic animal is ox, sheep, deer, pig.
Advantage of the present invention and beneficial effect are: utilize antioxidant CAT (catalase) and V
E(vitamin E) can resist active chalcogen (ROS) characteristics that carry free radical, separates at sperm, adds CAT and V in freezing process
E, reduce the effect of the peroxide injury that active chalcogen causes sperm, protect effectively that spermatozoa separates, sperm physiological function in refrigerating process, thaw rear sperm viability and extend the sperm time-to-live thereby improve, reach the purpose of being impregnated.
Embodiment:
Embodiment: 2,000,000/pipe milk cow X control frozen semen is made:
1, CAT liquid and V
EThe preparation of liquid:
The CAT that takes 4.2mg under room temperature adds in the distilled water of 1ml and dissolves, and keeps in Dark Place at-20 ℃, and the pot-life is a week; Take the V of 4mg
EAdd in the DMSO of 1ml and dissolve, keep in Dark Place at 4 ℃, the pot-life is a week;
2, the antibiotic of the former essence of milk cow is added:
Gather fresh cattle bull semen 6ml, add 36 μ l woodss can-miramycin, 36 μ Taylor l mycins, the 48 large mycins in μ l east;
3, sperm separates:
(1) with the above-mentioned former essence of antibiotic milk cow of having added, after removing impurity in former essence with 50 μ m seminal fluid filters, be filtered in new centrifuge tube, using Density Measuring Instrument to detect former smart actual concentrations is 15.8 hundred million/ml, puts in 18-20 ℃ of refrigerator and preserves;
(2) select SX-MoFlo sperm seperator, every sperm seperator per hour separates 200,000,000 sperms, the required former smart volume number that is used for dyeing of 6 seperators=2 * 6/15.8=0.759ml;
(3) take out former smart 0.759ml with the 15ml centrifuge tube from (1), add Staining Talp to 3ml, 360g is centrifugal, 5 minutes;
(4) add 75 μ l Hoechst33342 fluorescent dyes, 6mlStaining Talp to mix in another new centrifuge tube, and vibrated 3 seconds with the suspension oscillator is slight, the concentration of Hoechst33342 fluorescent dye is 5mg/ml;
(5) after centrifugal the stopping of step (3), inhale and to abandon the 2ml supernatant, retention volume 1ml transfers them in the 15ml centrifuge tube that step (4) is equipped with fluorescent dye and Staining Talp, and 34 ℃ of dyeing 45 minutes, add the 4%eggyolk of 6ml;
(6) with the former essence of separator tube packing step (5), the former essence of each separator tube packing 200,000,000 is put into the sample stage of seperator, and with the speed separated sperm of 5000 of per seconds, dead smart rate is in 20% left and right;
(7) the sperm separation according to 6 seperators of read-record on seperator adds up to 558,000 ten thousand;
(8) take out the buffer buffer solution 4ml of the ultra-pure water contain 84% TrisA and 16% from-20 ℃ of refrigerators, add CAT liquid 20 μ l and V
ELiquid 4 μ l, room temperature preheating half an hour, as the recovery liquid of sperm after separating, under separated sperm, after machine, balance is 90 minutes under 4 ℃ of conditions, 850g is centrifugal, 20 minutes, abandon supernatant, sperm is precipitated, slightly shake with the suspension oscillator, the sperm after after concussion, all being separated is all poured in an empty centrifuge tube;
4, add the preparation of antioxidant frozen sexed X-semen of dairy cow AI:
(1) add respectively 5 μ l CAT liquid, 1 μ l V by 1ml Tris-A
ELiquid, 1ml Tris-B add respectively 5 μ l CAT liquid, 1 μ l V
ELiquid adds antioxidant CAT liquid 75 μ l and V in 15ml Tris-A
ELiquid 15 μ l, add antioxidant 75 μ l CAT liquid and V in 15ml Tris-B
ELiquid 15 μ l mix Tris-A, Tris-B respectively, are placed under 4 ℃ of conditions balance 90 minutes;
(2) calculate the amount of adding by the good Tris-A of step (1) balance, M (g)=558 ÷ 10 * 80% * 1.03 ÷ 2=22.99g according to the total number of sperm after lower machine separation;
(3) all centrifuge tubes that separate rear sperm will be housed and be placed on electronic balance, add by the good Tris-A of step (1) balance in centrifuge tube, until reading is 22.99g;
(4) glycerine: get 22.99g by the good Tris-B of step (1) balance, minutes 2 times, interval 15 minutes adds in the centrifuge tube of step (3), puts upside down gently after each the interpolation to shake up;
(5) tubulature and freezing preservation: the separated sperm tubulature after step (4) is shaken up, be standard by 300, be spread in a single layer on freezing washboard, adjusting liquid nitrogen container gas-bearing formation height is 12.6in on liquid nitrogen surface, with dust catcher (Haier ZB800-1 vacuum cleaner) aspirated liquid blanket of nitrogen after-125 ± 1 ℃, product is placed on the gas-bearing formation position of 8.5-9.0cm and carried out freezing 20 minutes, every 25 tubules are contained in one to be moulded in the essence cylinder, with label, date, batch and number, freezing smart people waits information flag on white label, during transferred product, can not be over 3 seconds in air, dropping into-180 ℃ of liquid nitrogen preserves,
5, smart every quality index detection is frozen in the control of milk obstinacy:
Get 1 and add according to the method described above antioxidant CAT and V
EThe milk cow X control frozen semen of producing, ox number: 15503832, date of manufacture: 2011.1.25 carries out every quality index and detects after thawing; Meanwhile, get same ox and number do not add antioxidant CAT and V according to conventional method
EThe milk cow X control of producing is freezing to be contrasted as quality index; The property control freeze the essence product specification be 0.25ml; Microscopy: each quality inspection index calculating method is as follows:
(1) the separated sperm vigor detects: get the control of a property and freeze smart product, detect after thawing in 35 ℃ of tepidariums, in advance slide and cover glass were placed on 37 ℃ of heating plates preheating 10 minutes, get 15 μ l sample drops on slide with the pipettor centrifuge tube, compressing tablet is placed on the microscopically microscopy, select the uniform sample area of compressing tablet, a compressing tablet is got 5 visuals field, and middle 1, surrounding is respectively got 1, count respectively live number of sperm and Necrospermia number
After observing 5 visuals field, the number of sperm alive in each visual field and Necrospermia number statistics are as follows:
The calculating vigor: vigor=work number of sperm/work number of sperm and Necrospermia number and,
Add CAT and V
EThe freezing smart vigor of milk cow X control of producing=268/ (268+181) * 100%=59.7%;
Do not add CAT and V
EThe freezing smart vigor of milk cow X control of producing=209/ (209+202) * 100%=50.9%;
(2) the separated sperm time-to-live: get the control of a property and freeze essence, defreezing method is the same, sample after thawing keeps in Dark Place under 37 ℃ of environment, after 4 hours with seminal fluid transposition in tubule in the 1.5ml centrifuge tube, get 15 μ l sample drops with pipettor on ready slide from centrifuge tube, light is observed the sperm survival condition under microscope natural, and method of counting is the same;
(3) separated sperm sum:
Mix take 1 specification as 0.25ml and freeze essence as example, thaw according to above method, mixed semen after getting 50ul and thawing, after 20 times of dilutions of formalin solution with 950 μ l, count with the blood counting chamber point sample, see the sperm sum of looking at microscopically, the total number of sperm of counting under natural daylight: add CAT and V
ECombination: 88,74,58,100,96, mean is 83.2, namely the total sperm concentration of this batch products is 83.2*20*10000=1664 ten thousand/ml, total number of sperm 3,320,000/agent; Do not add CAT and V
ECombination: 82,84,81,80,89, mean is 83.2, namely the total sperm concentration of this batch products is 83.2*20*10000=1664 ten thousand/ml, total tens thousand of 3,320,000/agent of sperm.
Claims (1)
1. one kind is added antioxidant CAT and V
EPrepare the method for milk obstinacy control frozen semen, it comprises the following steps:
(1), CAT liquid and V
EThe preparation of liquid:
The CAT that takes 4.2mg under room temperature adds in the distilled water of 1ml and dissolves, and keeps in Dark Place at-20 ℃, and the pot-life is a week; Take the V of 4mg
EAdd in the DMSO of 1ml and dissolve, keep in Dark Place at 4 ℃, the pot-life is a week;
(2), the antibiotic of the former essence of milk cow is added:
According to the former essence of 1ml milk cow add 6 μ l woodss can-ratio of miramycin, 6 μ Taylor l mycins, the 8 large mycins in μ l east adds antibiotic in the former essence of milk cow;
(3), sperm separates:
1) will add the former essence of antibiotic milk cow and be filtered in new centrifuge tube with 50 μ m seminal fluid filters, remove the impurity in former seminal fluid, and use Density Measuring Instrument to detect former smart actual concentrations after filtering, and put in 18-20 ℃ of refrigerator and preserve;
2) calculate per hour separated sperm sum 1,200,000,000/former smart actual concentrations of the former smart volume number that six seperators are used for dyeing=six seperator;
3) according to step 2) the former smart volume number that is used for dyeing that calculates, with the 15ml centrifuge tube from step 1) the former essence of taking-up, add Staining Talp to 3ml, 360g centrifuge washing, 5 minutes;
4) enter 75 μ l Hoechst33342 fluorescent dyes, add the ratio of 6ml Staining Talp according to 1,200,000,000 former finishings in another new centrifuge tube, Hoechst33342 fluorescent dye and Staining Talp are mixed, and vibrated 3 seconds with the suspension oscillator is slight, wherein, the concentration of Hoechst33342 fluorescent dye is 5mg/ml;
5) step 3) is centrifugal stop after, inhale and to abandon the 2ml supernatant, retention volume 1ml transfers them to above-mentioned steps 4) the 15ml centrifuge tube in, 34 ℃ of dyeing added 4% eggyolk of 6ml after 45 minutes;
6) step 5) the former essence processed of dyeing is by the former capable packing that progresses greatly of each separator tube 200,000,000, and the former smart sample after packing is dyeed is well put into the sample stage of seperator, separates former essence;
7) sperm according to every the seperator of read-record on seperator separates sum, and the sperm that calculates six seperators separates sum=every instrument separation number * 6, unit: ten million;
8) in the buffer buffer solution that takes out, add 5 μ l CAT liquid, 1 μ l V according to 1ml from-20 ℃ of refrigerators
EThe ratio of liquid is added antioxidant CAT liquid and V
ELiquid, room temperature preheating half an hour, as the recovery liquid of sperm after separating, said buffer buffer solution contains 84% the freezing equilibrium liquid of TrisA and 16% ultra-pure water; Under separated sperm, after machine, balance is 90 minutes under 4 ℃ of conditions, and 850g abandons supernatant after centrifugal 20 minutes, with the sperm precipitation, slightly shakes with the suspension oscillator, and the sperm after after concussion, all being separated is all poured in an empty centrifuge tube;
(4), add the preparation that essence is frozen in the antioxidant control:
1) add antioxidant in Tris-A, Tris-B, adding proportion is: 1ml Tris-A adds respectively 5 μ lCAT liquid, 1 μ l V
ELiquid, 1ml Tris-B add respectively 5 μ l CAT liquid, 1 μ l V
ELiquid after having added antioxidant, mixes Tris-A, Tris-B respectively, is placed under 4 ℃ of conditions balance 90 minutes;
2) the total number of sperm that separates according to lower machine calculates the consumption by the good Tris-A of step 1) balance, M represents the gross mass of Tris-A and sperm precipitation, total number of sperm ÷ 10 * 80% * 1.03 ÷ 2 that M (g)=lower machine separates, at first the sum that machine under sperm is separated is converted into ten million by 1,000,000, multiply by 80% the rate of recovery is exactly the sperm volume, multiply by averag density 1.03g/ml, adding exactly the gross mass of Tris-A and Tris-B, is exactly the quality of adding by the good Tris-A of step 1) balance divided by 2;
3) with the step 8 in step (3)) all be housed separate after centrifuge tubes of sperm be placed on electronic balance, add by the good Tris-A of step 1) balance in centrifuge tube, until reading is step 2) M (g) that calculates;
4) glycerine: get with M (g) equivalent by the good Tris-B of step 1) balance, 15 minutes equivalent of minute 2 minor tick adds step 3 in above-mentioned steps (4)) centrifuge tube in, put upside down gently after each the interpolation and shake up;
5) tubulature and freezing preservation: after separated sperm shakes up, the 0.25 milliliter of semen freezing pipe of packing under 4 ℃ of conditions;
6) every 300 are spread in a single layer on freezing washboard for standard, adjusting liquid nitrogen container gas-bearing formation height is 12.6in on liquid nitrogen surface, with dust catcher aspirated liquid blanket of nitrogen after-125 ± 1 ℃, product is placed on the gas-bearing formation position of 8.5-9.0cm and carried out freezing processing 20 minutes, then, every 25 tubules are contained in one to be moulded in the essence cylinder, with label, date, batch and number, freeze smart people's information flag on white label, during transferred product, can not surpass 3 seconds in air, drop at last-180 ℃ of liquid nitrogen and preserve;
Said Staining Talp:pH=7.4, it consists of:
Said 4% eggyolk:pH=5.5, it consists of:
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| CN103710303A (en) * | 2013-12-30 | 2014-04-09 | 内蒙古赛科星繁育生物技术股份有限公司 | Method for lowering teratosperm ratio and dead sperm ratio in cattle and sheep sperm |
| CN107251892A (en) * | 2017-05-25 | 2017-10-17 | 中国热带农业科学院热带作物品种资源研究所 | It is a kind of suitable for the solution for dilution of cock semen of torrid areas, preparation and its application process |
| CN111053080A (en) * | 2019-11-01 | 2020-04-24 | 中国农业大学 | Semen diluent for improving semen low-temperature preservation quality and application method thereof |
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| CN101554152B (en) * | 2009-05-21 | 2012-05-09 | 西北农林科技大学 | Frozen semen diluent and preparation method thereof |
| CN101843238A (en) * | 2010-06-12 | 2010-09-29 | 昆明亚灵生物科技有限公司 | Ultralow temperature cryopreservation method of macaca primate semen |
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