CN101613677A - A kind of method of separating pig X sperm and y sperm - Google Patents
A kind of method of separating pig X sperm and y sperm Download PDFInfo
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Abstract
The present invention relates to a kind of method of separating pig X sperm and y sperm, the main operation of this method is: (1) is gathered boar semen and is temporarily preserved under 30 ℃ ± 2 ℃ temperature; (2) dilute former seminal fluid with semen diluent by suitable proportion, add fluorescent dye black out hatching dyeing in water-bath of proper concn then, add food pigment dyeing after-filtration again; (3) under 20 ℃～25 ℃, utilize flow cytometer to separate X, y sperm; (4), after centrifugal concentrating, can be used for again preserving or inseminating with X, y sperm after the receiving tube collection separation that sperm reception liquid is housed.Adopt the inventive method to separate resolving power that the pig sperm can improve X, y sperm and separates accuracy rate, the separation accuracy rate is more than 90%; And effectively keep the high quality of sperm and the high-level efficiency that separated sperm reclaims, and the inventive method is combined with breeding technologys such as artificial insemination and in vitro fertilizationes, can improve efficiency of farming industry effectively.
Description
Technical field
The present invention relates to a kind of method of separating pig X sperm and y sperm.
Background technology
In aquaculture is produced, breed other livestock and poultry offspring of a large amount of foreseeabilities selectively, can improve the efficient of production rate and production widely.Use X or y sperm to inseminate, can produce the offspring of many " precognition sexes ", change offspring's sex ratio significantly, bring tangible economic and social benefit according to our needs to dam.For example, in Swine Production, the breeding potential of general bacon hogs is low, lean ratio is high, and breeding potential height, the lean ratio of native breed pig (as Taihu Lake pig, Luchuan pig) are low, join local sow as the boar of using bacon hogs aborning, not only can improve litter size but also can improve offspring's lean ratio, raising raiser's economic benefit and the people's standard of living all are beneficial to; In addition, in breeding work, we need a large amount of paternal boar and maternal sow, so that the kind of cultivating as early as possible, and enlarge the quantity of planting swinery.Under the natural propagation state, the ratio of male and female is bordering on 50%, obviously can't achieve the goal, if use X, y sperm isolation technique, makes offspring 90% used for us, then can significantly improve the economic benefit of production, improves the speed and the efficient of breeding work.
Pig X, y sperm isolation technique are controlled other effective ways.If can in advance X, y sperm be separated,,, just can access the offspring of certain sex far above 50% ratio according to the karyomit(e) theory of mammal sex determination (being that XX is that female, XY is male) through artificial insemination or technology in vitro fertilization.Separated since nineteen twenty-five Lush reported first since the sperm of rabbit, scientists was being done many work aspect the sperm separation, comprise that characteristics such as density according to sperm, vigor, size, electric charge, surface antigen attempt sperm is separated, the result is a lot of for report, but inefficiency in general, and poor reliability, repeated low, be not enough to instruct and produce.Up to now, carry out science that sperm separates has become that people generally acknowledge, sperm separation method reliably and efficiently by flow cytometer.The flow cytometer separated sperm is a kind of principle higher than Y according to the dna content of Mammals X sperm, by fluorescent dye, use equipment such as flow cytometer then and,, can control offspring's sex reliably if in conjunction with effectively insemination measure with the method that X, y sperm in the seminal fluid separate.
At present, flow cytometer separates that pig sperm technology exists also that the sperm separation efficiency is lower, separation purity is not high enough, cost is than problems such as height, and because the structure problem and the separation condition problem of pig sperm, the pig sperm separates the back vigor through flow cytometer and result of use can be affected, up to the present, domesticly do not see sophisticated pig sperm separation method report.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of sperm separation method that is applicable to the pig sperm, and when guaranteeing separated sperm purity, the vigor and the result of use of improve isolating efficient, separating the back sperm are prepared for commercially producing.
The present invention solves the problems of the technologies described above with following technical scheme:
(1) boar semen of gathering is slowly cooled to 30 ℃ ± 2 ℃ temporary transient preservations, and dilute as early as possible and separating treatment.
(2) to dilute former seminal fluid to sperm concentration by proper proportion be 8～15 * 10 with not containing semen diluent that yolk, 30 ℃ of balances crosses
7/ ml adds the fluorescent dye of proper concn then, black out hatching dyeing in the water-bath of proper temperature; Fluorescent dye adds an amount of food pigment after finishing, at 20 ℃～25 ℃ 2min～5min after-filtration that dye down;
Wherein the composition of semen diluent and content are:
| Composition | Content (g/1000ml) |
| Glucose | ??26.0 |
| ??EDTANa 4 | ??2.4 |
| Trisodium Citrate | ??8.0 |
| Sodium bicarbonate | ??1.2 |
| Hepes acid | ??8.5～9.5 |
| ??BSA | ??2.0～3.0 |
| Gentamicin | ??0.3 |
Add deionized water during preparation to 1000ml, and transfer PH to 6.9～7.2.
(3) utilize flow cytometer to separate required X, y sperm under 20 ℃～25 ℃ temperature, the composition of used sheath fluid and content are when wherein separating X, y sperm:
| Composition | Content (g/1000ml) | Composition | Content (g/1000ml) |
| Sodium-chlor | ??8 | Magnesium chloride | ??0.1 |
| Repone K | ??0.2 | Glucose | ??1.0 |
| Sodium phosphate dibasic | ??1.15 | ??BSA | ??0.8～1.2 |
| Potassium primary phosphate | ??0.2 | ??EDTANa 4 | ??0.0～1.5 |
| Sodium.alpha.-ketopropionate | ??0.036 | Penicillin | ??0.12 |
| Calcium chloride | ??0.1 | Streptomycin sulphate | ??0.1 |
Add deionized water to 1000ml;
After earlier being dissolution with solvents calcium chloride with the 200ml deionized water during preparation, joining with the deionized water is to go in the solution of other compositions of dissolution with solvents again, is mixed with 1000ml solution.
(4) receiving tube of using the sperm that is equipped with 20 ℃～25 ℃ in advance to receive liquid is collected X, the y sperm after separating, separate the seminal fluid that receives and after centrifugal concentrating, obtain X, y sperm, can be used for preserving or insemination after process is suitably handled, wherein the basal liquid composition and the content of sperm reception liquid are as follows:
| Composition | Content (g/1000ml) |
| ??TES | ??11.0～13.0 |
| ??TRIS | ??1.8～2.2 |
| Glucose | ??30.0～34.0 |
| Penicillin | ??0.627 |
| Streptomycin sulphate | ??1.0 |
Add deionized water to 1000ml;
Before the use, according to volume percent basal liquid and fresh yolk 1%～5% and seminal plasma 1%～10% are mixed with sperm and receive liquid.
In (2) step, the volume ratio of diluent and former seminal fluid is: 1～2: 1; The fluorescent dye that adds is Hoechst33342, and its add-on is: the fluorescent dye that adds 25～125 μ g in rare seminal fluid of every 1ml; Food pigment is FD﹠amp; C#40, its add-on is: add 12.5～37.5 μ g in the rare seminal fluid of the fluorescent dye of every 1ml; The suitable temperature of fluorescent dye is 32 ℃～37 ℃; The fluorescent dye time is 30min～60min;
In (4) step, centrifugal spissated condition is: 20 ℃～25 ℃ of temperature, 2000～3000 revolutions per seconds of speed of rotation, time 10min～20min.
The present invention is when detecting the isolating accuracy rate of sperm, and take a morsel (about 100～1,000 ten thousand) are separated the X or the y sperm sample that obtain and docked by pulse ultrasonic wave, and adding Hoechst33342 redyes and (adds staining agent concentration at 25 μ g/ml～100 μ g/ml; Dyeing time can shorten to 20min～45min; 32 ℃～37 ℃ water-bath dyeing of black out), then by flow cytometer weight analysis sperm DNA under identical condition, utilize the matching degree of DNA histogram and Gaussian distribution figure to determine the isolating accuracy rate of sperm.
Use the inventive method to separate the pig sperm and have following advantage:
(1) adopts the inventive method to separate resolving power that the pig sperm can improve X, y sperm and separate accuracy rate.
With individuality, the sperm concentration adjustment of pig, generally density is 8～15 * 10 after dilution aspect the concentration of using staining agent and dyeing time in the present invention
7In the seminal fluid of/ml, add fluorescent dye Hoechst33342 (being formulated as the liquid of concentration 5mg/ml) 5～25 μ l/ml, behind the mixing in 32 ℃～37 ℃ water-bath water-bath black out dyeing 30min～60min, and then dye with food pigment.
Use diluent of the present invention and dyeing procedure, per dilution and dyeing of carrying out one batch of sperm in 2 hours separates after dyeing finishes as early as possible, and vigor that can fine preservation sperm, and the resolving power height of sperm separate accuracy rate generally all more than 90%.
(2) adopt the inventive method to separate the pig sperm and can effectively keep the high quality of sperm and the high-level efficiency that separated sperm reclaims.
Present method adopts diluent that former seminal fluid is diluted 1～2 times at most, makes sperm concentration 8～15 * 10
7/ ml, the protective substance concentration dilution in the pig seminal fluid is little, still can play enough effects of protection sperm.Through repetition test, dyeing temperature, time and the staining agent concentration used when present method dyes water-bath are all adjusted to suitable scope, and vigor, the quality of protecting sperm all had very big benefit.After water-bath dyeing, re-use food pigment dyeing and filtration, the sperm alive that a cell membrane is complete separates.In receiving liquid, added yolk and seminal plasma (seminal fluid removes the later part of sperm), can the sperm after separating have been played a very good protection.Concentrated research after sperm received optimized the centrifugal condition when concentrated, the sperm after efficient recovery is separated and to guarantee that sperm quality has suitable.
(3) the inventive method whole sperm sepn process with separate after concentration process in, adopt 20 ℃～25 ℃ constant temperatures; Semen diluent, sheath fluid, reception liquid all pass through temperature equilibrium, and this vigor to protection pig sperm is very important.It is very big that the pig sperm quality is influenced by variation of temperature, particularly is lower than 16 ℃ and when being higher than 37 ℃.No matter be sample introduction, separation, acceptance, concentration process, use this temperature can both avoid high temperature the cold strike of the influence in motility of sperm, life-span and low temperature to sperm.Can also reduce microbial reproduction speed, help the protection of separated sperm.
(4) will use the isolating pig sperm of the inventive method to combine, can improve efficiency of farming industry effectively with breeding technologys such as artificial insemination or in vitro fertilizationes.
Description of drawings
Fig. 1 is that embodiment one uses the detected XY sperm DNA of flow cytometer to contain spirogram.
Fig. 2 is that embodiment one separates X sperm sample by the detected dna content figure of weight analysis method.
Fig. 3 is that embodiment one separates the y sperm sample by the detected dna content figure of weight analysis method.
Fig. 4 is that embodiment two uses the detected XY sperm DNA of flow cytometer to contain spirogram.
Fig. 5 is that embodiment two separates X sperm sample by the detected dna content figure of weight analysis method.
Fig. 6 is that embodiment two separates the y sperm sample by the detected dna content figure of weight analysis method.
Embodiment
Below be that the inventive method is further described:
Embodiment one:
1, the good boar semen of fresh collection is transported to split site rapidly under 30 ℃ ± 2 ℃ and temporarily preserves, and dilute as early as possible and separating treatment.
2, the semen diluent of use balance excess temperature (30 ℃) is 1: 1 dilution proportion pig seminal fluid by volume, and the density of dilution back sperm is 1.15 * 10
8/ ml.
3, in the pig seminal fluid after dilution, add Hoechst33342 (being mixed with the liquid that concentration is 5mg/ml) 15 μ l/ml (promptly adding fluorescent dye to concentration is 75 μ g/ml), behind the mixing in 34 ℃ water-bath water-bath black out dyeing 50min.Water-bath adds food pigment (FD﹠amp after finishing in the rare seminal fluid of fluorescent dye; C#40) to 25 μ g/ml.
Dyeing 5min after-filtration under 25 ℃ room temperature, inferior quality sperms such as elimination conglomeration, death carry out separating treatment at once.
Wherein the composition of semen diluent and content are:
| Composition | Content (g/1000ml) |
| Glucose | ??26.0 |
| ??EDTANa 4 | ??2.4 |
| Trisodium Citrate | ??8.0 |
| Sodium bicarbonate | ??1.2 |
| Hepes acid | ??8.5 |
| ??BSA | ??3.0 |
| Gentamicin | ??0.3 |
During preparation, add deionized water and be mixed with 1000ml solution as solvent; And transfer about PH to 7.0; The full name of Hepes acid is N2 hydroxyethyl piperazine N ' 2 ethyl sulfonic acids; BSA is the calf serum albumin.
4, installing temperature is 22 ℃ sheath fluid, adjust the fluidic cell instrument parameter, the plastic centrifuge tube that uses 50ml is as receiving tube, and the sperm of pre-installing 20 ℃～25 ℃ in the receiving tube receives liquid 1.5ml, and soak along tube wall and to drop down one time, under 20 ℃～25 ℃ conditions, separate.
The composition and the content that wherein separate the used sheath fluid of pig sperm are as follows:
| Composition | Content (g/1000ml) | Composition | Content (g/1000ml) |
| Sodium-chlor | ??8 | Magnesium chloride | ??0.1 |
| Repone K | ??0.2 | Glucose | ??1.0 |
| Sodium phosphate dibasic | ??1.15 | ??BSA | ??0.8 |
| Potassium primary phosphate | ??0.2 | ??EDTANa 4 | ??0.9 |
| Sodium.alpha.-ketopropionate | ??0.036 | Penicillin | ??0.12 |
| Calcium chloride | ??0.1 | Streptomycin sulphate | ??0.1 |
During preparation, earlier be dissolution with solvents calcium chloride with the 200ml deionized water after, joining with the deionized water is to go in the solution of other compositions of dissolution with solvents again, is mixed with 1000ml solution; BSA is the calf serum albumin; EDTA-Na
4Be sodium ethylene diamine tetracetate.
The basal liquid composition and the content of sperm reception liquid are as follows:
| Composition | Content (g/1000ml) |
| ??TES | ??11.0 |
| ??Tris | ??2.2 |
| Glucose | ??30.0 |
| Penicillin | ??0.627 |
| Streptomycin sulphate | ??1.0 |
During preparation, add deionized water as solvent preparation 1000ml solution; Before use, the sperm that is mixed with by the fresh yolk of 95% basal liquid+3%+2% seminal plasma receives the receiving tube of packing into after liquid (in volume percent) mixes.TES is N three (methylol) methyl 2 taurines; Tris is a Tutofusin tris.
5, the sperm that separated and collected is obtained, under 22 ℃ condition,, stay the small amount of liquid that is rich in sperm after removing supernatant liquor with the centrifugal concentrated 20min of 2200 revolutions per seconds speed, semen diluent with the front suitably dilutes again, takes a sample then and detect sperm motility rate under the sperm Image analytical system.Through 8 replications, sperm motility rate result is 62 ± 2.7%.
6, take a morsel (about 8,000,000) separate X or the y sperm sample obtain and dock by pulse ultrasonic wave, add Hoechst33342 and redye that (the Hoechst33342 concentration of adding is reduced to 50 μ g/ml; Dyeing time shortens to 30min; 34 ℃ of temperature), by flow cytometer weight analysis sperm DNA under identical condition, its result as shown in Figure 1 then.Utilize the gaussian curve approximation figure to determine the isolating accuracy rate of sperm, the X sperm that obtains in six repeated experiments separates accuracy rate 94% (as shown in Figure 2), and y sperm separates accuracy rate 91% (as shown in Figure 3).
7, the y sperm of getting after the separation carried out artificial insemination to a sow, in down a brood of 6 male piglets of daily output in June 18 in 2009; The X sperm of getting after the separation carried out artificial insemination to a sow, in down a brood of 4 female piglets of daily output in June 20 in 2009.This batch separates pig sperm sex control farrowing sex rate of accuracy reached to 100%.
Embodiment two
1, the good boar semen of fresh collection is transported to split site rapidly under 30 ℃ ± 2 ℃ and temporarily preserves, and dilute as early as possible and separating treatment.
2, the semen diluent of use balance excess temperature (30 ℃) is 2: 1 dilution proportion pig seminal fluid by volume, and the density of dilution back sperm is 0.95 * 10
8/ ml.
3, in the pig seminal fluid after dilution, add Hoechst33342 (being mixed with the liquid that concentration is 5mg/ml) 12.5 μ l/ml (promptly adding fluorescent dye to its concentration is 62.5 μ g/ml), behind the mixing in 37 ℃ water-bath water-bath black out dyeing 30min.Water-bath adds food pigment (FD﹠amp in the rare seminal fluid of fluorescent dye after finishing; C#40) to 20 μ g/ml.
Dyeing 2min after-filtration under 25 ℃ room temperature, inferior quality sperms such as elimination conglomeration, death carry out separating treatment at once.
Wherein the composition of semen diluent and content are:
| Composition | Content (g/1000ml) |
| Glucose | ??26.0 |
| ??EDTANa 4 | ??2.4 |
| Trisodium Citrate | ??8.0 |
| Sodium bicarbonate | ??1.2 |
| Hepes acid | ??9.5 |
| BSA (calf serum albumin) | ??2.0 |
| Gentamicin | ??0.3 |
During preparation, add deionized water and be mixed with 1000ml solution as solvent; And transfer about PH to 7.0; The full name of Hepes acid is N2 hydroxyethyl piperazine N ' 2 ethyl sulfonic acids.
4, installing temperature is 23 ℃ sheath fluid, adjust the fluidic cell instrument parameter, the plastic centrifuge tube that uses 50ml is as receiving tube, and the sperm of pre-installing 20 ℃～25 ℃ in the receiving tube receives liquid 2.0ml, and soak along tube wall and to drop down one time, under 20 ℃～25 ℃ conditions, separate.
The composition and the content that wherein separate the used sheath fluid of pig sperm are as follows:
| Composition | Content (g/1000ml) | Composition | Content (g/1000ml) |
| Sodium-chlor | ??8 | Magnesium chloride | ??0.1 |
| Repone K | ??0.2 | Glucose | ??1.0 |
| Sodium phosphate dibasic | ??1.15 | ??BSA | ??1.2 |
| Potassium primary phosphate | ??0.2 | ??EDTANa 4 | ??0.75 |
| Sodium.alpha.-ketopropionate | ??0.036 | Penicillin | ??0.12 |
| Calcium chloride | ??0.1 | Streptomycin sulphate | ??0.1 |
During preparation, earlier be dissolution with solvents calcium chloride with the 200ml deionized water after, joining with the deionized water is to go in the solution of other compositions of dissolution with solvents again, is mixed with 1000ml solution; BSA is the calf serum albumin; EDTANa
4Be sodium ethylene diamine tetracetate.
The basal liquid composition and the content of sperm reception liquid are as follows:
| Composition | Content (g/1000ml) |
| ??TES | ??12.5 |
| ??Tris | ??1.8 |
| Glucose | ??32.0 |
| Penicillin | ??0.627 |
| Streptomycin sulphate | ??1.0 |
During preparation, add deionized water as solvent preparation 1000ml solution; Before use, the sperm that is mixed with by the fresh yolk of 92% basal liquid+2%+6% seminal plasma receives the receiving tube of packing into after liquid (in volume percent) mixes.TES is N three (methylol) methyl 2 taurines; Tris is a Tutofusin tris.
5, the sperm that separated and collected is obtained, under 22 ℃ condition,, stay the liquid that is rich in sperm on a small quantity after removing supernatant liquor with the centrifugal concentrated 15min of 2400 revolutions per seconds speed, semen diluent with the front suitably dilutes again, takes a sample then and detect sperm motility rate under the sperm Image analytical system.Through 6 replications, sperm motility rate result is 61 ± 3.2%.
6, take a morsel (about 6,000,000) separate X or the y sperm sample obtain and dock by pulse ultrasonic wave, add Hoechst33342 and redye that (the Hoechst33342 concentration of adding is reduced to 37.5 μ g/ml; Dyeing time shortens to 30m; 32 ℃ of temperature), by flow cytometer weight analysis sperm DNA under identical condition, its result as shown in Figure 4 then.Utilize the gaussian curve approximation figure to determine the isolating accuracy rate of sperm, the X sperm that obtains in six repeated experiments separates accuracy rate 93% (as shown in Figure 5), and y sperm separates accuracy rate 92% (as shown in Figure 6).
7, the X sperm of getting after the separation carried out artificial insemination to a sow, in down a brood of 5 female piglets of daily output in July 16 in 2009.This batch separates pig sperm sex control farrowing sex rate of accuracy reached to 100%.
Claims (3)
1. a method of separating pig X sperm and y sperm according to the principle that the dna content of pig X sperm is Duoed than y sperm, is separated by using flow cytometer behind the fluorescent dyeing, it is characterized in that:
(1) boar semen of gathering is temporarily preserved under 30 ℃ ± 2 ℃ temperature, and diluted as early as possible and separating treatment;
(2) to dilute former seminal fluid to sperm concentration by suitable proportion be 8～15 * 10 with not containing semen diluent that yolk, 30 ℃ of balances crosses
7/ ml adds the fluorescent dye of proper concn then, black out hatching dyeing in the water-bath of proper temperature; Fluorescent dye adds an amount of food pigment after finishing again, the 2min～5min after-filtration that under 20 ℃～25 ℃ temperature, dyes, and wherein the composition of semen diluent and content are:
Add deionized water to 1000ml;
(3) utilize flow cytometer to separate required X, y sperm under 20 ℃～25 ℃ temperature, sheath fluid composition and content used when wherein separating X, y sperm are:
Add deionized water to 1000ml;
(4) receiving tube of using the sperm that is equipped with 20 ℃～25 ℃ in advance to receive liquid is collected X, the y sperm after separating, separate the seminal fluid that receives and after centrifugal concentrating, obtain X, y sperm, can be used for preserving or insemination after process is suitably handled, wherein the basal liquid composition and the content of sperm reception liquid are as follows:
Add deionized water to 1000ml;
Before the use, according to volume percent basal liquid and fresh yolk 1%～5% and seminal plasma 1%～10% are mixed with sperm and receive liquid.
2. the method for separation pig X sperm according to claim 1 and y sperm, it is characterized in that: in (2) step, the volume ratio of diluent and former seminal fluid is: 1～2: 1; The fluorescent dye that adds is Hoechst33342, and its add-on is: the fluorescent dye that adds 25～125 μ g in rare seminal fluid of every 1ml; Food pigment is FD﹠amp; C#40, its add-on is: add 12.5～37.5 μ g in the rare seminal fluid of the fluorescent dye of every 1ml; The suitable temperature of fluorescent dye is 32 ℃～37 ℃; The fluorescent dye time is 30min～60min.
3. the method for separation pig X sperm according to claim 1 and y sperm, it is characterized in that: in (4) step, centrifugal spissated condition is: 20 ℃～25 ℃ of temperature, 2000～3000 revolutions per seconds of speed of rotation, time 10～20min.
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| CN103299987A (en) * | 2013-05-29 | 2013-09-18 | 西北农林科技大学 | Diluent for normal temperature preservation of boar semens and preparation method thereof |
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| CN103525760A (en) * | 2013-09-29 | 2014-01-22 | 大连金弘基种畜有限公司 | Separation method of sexed semen |
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| CN106417256B (en) * | 2016-10-12 | 2020-01-31 | 中国水产科学研究院黑龙江水产研究所 | method for improving sperm utilization rate of rainbow trout pseudo-male fish |
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| CN114085809A (en) * | 2021-11-30 | 2022-02-25 | 吉林大学 | Method for separating X sperm and Y sperm |
| CN114085809B (en) * | 2021-11-30 | 2023-08-25 | 吉林大学 | Method for separating X sperm and Y sperm |
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