CN103525760B - The separation method of sexing semen - Google Patents
The separation method of sexing semen Download PDFInfo
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- CN103525760B CN103525760B CN201310456121.2A CN201310456121A CN103525760B CN 103525760 B CN103525760 B CN 103525760B CN 201310456121 A CN201310456121 A CN 201310456121A CN 103525760 B CN103525760 B CN 103525760B
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Abstract
The present invention relates to animal reproduction field, particularly to the separation method of sexing semen, comprise the following steps: be proficient in the filtration for the first time of 40 60 micron filters by former, and obtained seminal fluid after the filter of removal spermatium and foreign material；The vigor of seminal fluid after detection filter, the vigor of obtaining be 0.6 0.85 filter to be dyed after seminal fluid；It is that seminal fluid dyes after the filter to be dyed of 0.6 0.85 by vigor, obtains the seminal fluid that dyes；Dyeing seminal fluid is used flow cytometric sorting, obtains the seminal fluid containing X chromosome and the seminal fluid containing Y chromosome.The separation method of sexing semen that the present invention provides, it is possible to ensure uniformity and the stability of seminal fluid dyeing after filter to be dyed, and then be beneficial to separate the seminal fluid containing X chromosome and the seminal fluid containing Y chromosome efficiently.Additionally, seminal fluid is difficult to the phenomenon nozzle of blocking flow cytometer occur after Lv, and then ensure that the smooth and easy of whole separation process is carried out.
Description
Technical field
The present invention relates to animal reproduction field, in particular to the separation method of a kind of sexing semen.
Background technology
Sex controll (sex control) technology of animal is by artificially doing the normal reproductive process of animal
In advance, Adult female animal output is made it is intended that a biotechnology of sex offspring；Concrete, this technology utilizes animal
Containing the difference of DNA content in X chromosome and the seminal fluid containing Y chromosome, dye by specific stain agent, then according to sending out
The fluorescence intensity gone out, by computer discriminant sperm classification, then by seminal fluid drop additional charge, according to electrostatic principle, will
Seminal fluid containing X chromosome separates with the seminal fluid of Y chromosome, and is optionally applied in animal reproduction.
In the related, in the method separate sexing semen, common operation is directly by after former essence detection vigor
Dyeing, dyeing separates after terminating；But, owing to former essence typically can contain bigger spermatium and some foreign material, these essences
Son group and foreign material often can cause former essence dyeing uneven, in turn result in the seminal fluid of the seminal fluid containing X chromosome and Y chromosome
Separating effect is undesirable.
Summary of the invention
It is an object of the invention to provide the separation method of a kind of sexing semen, to solve above-mentioned problem.
The separation method of the sexing semen provided in embodiments of the invention, including: it was proficient in 40-60 Mm filter by former
Device filters for the first time, obtains seminal fluid after the filter of removal spermatium and foreign material；The vigor of seminal fluid after detection filter, the vigor of obtaining is 0.6-
Seminal fluid after the filter to be dyed of 0.85；It is that seminal fluid dyes after the filter described to be dyed of 0.6-0.85 by vigor, obtains dyeing essence
Liquid；Described dyeing seminal fluid is used flow cytometric sorting, obtains the seminal fluid containing X chromosome and the seminal fluid containing Y chromosome.
The separation method of the sexing semen that embodiments of the invention provide, during separating seminal fluid, was proficient in former
40 micron of-60 micron filter filters, and obtains removing seminal fluid after the filter of spermatium and foreign material, then Activity determination, dyeing and
Obtain the seminal fluid containing X chromosome and the seminal fluid containing Y chromosome after separation, and then realize the whole mistake former essence separated
Journey.Owing to former essence is filtered by 40 micron of-60 micron filter before testing, the filter of 40 microns-60 microns can be by
The spermatium and the filtering foreign that contain in former essence fall, and then during follow-up dyeing, it is possible to ensure essence after filter to be dyed
The uniformity of liquid dyeing and stability, and then be beneficial to divide the seminal fluid containing X chromosome and the seminal fluid containing Y chromosome efficiently
From.
Additionally, the separation method of this sexing semen of the present embodiment, it was proficient in 40 micron of-60 micron filter by former
Once filtering, obtain removing seminal fluid after spermatium and foreign material filter, therefore during later separation, after filter, seminal fluid is difficult to appearance
The phenomenon of the nozzle of blocking flow cytometer, and then ensure that the smooth and easy of whole separation process is carried out.
Accompanying drawing explanation
Fig. 1 shows the flow chart of the separation method of the sexing semen that the embodiment of the present invention one provides；
Fig. 2 shows the flow chart of the separation method of the sexing semen that the embodiment of the present invention two provides.
Detailed description of the invention
Below by specific embodiment and combine accompanying drawing the present invention is described in further detail.
The separation method of sexing semen provided in embodiments of the invention, comprises the following steps, and refer to Fig. 1:
Step 101: be proficient in 40-60 micron filter carry out filtering for the first time by former, and obtained removing spermatium and foreign material
Filter after seminal fluid；
In a step 101, can be realized the spermatium in former essence and filtering foreign by the filtration of 40-60 micron filter
Effect, and then ensure the patency in subsequent separation process, it is preferred that the effect that 50 micron filters realize filtering can be chosen
Really.
Step 102: the vigor of seminal fluid after detection filter, the vigor of obtaining be 0.6-0.85 filter to be dyed after seminal fluid；
After filter, seminal fluid is by after viability examination, selection vigor be 0.6-0.85 filter after seminal fluid, i.e. obtain to be dyed
Seminal fluid after filter.
Step 103: be that seminal fluid dyes after the filter to be dyed of 0.6-0.85 by vigor, obtain the seminal fluid that dyes；
Step 104: dyeing seminal fluid is used flow cytometric sorting, obtains the seminal fluid containing X chromosome and dyes containing Y
The seminal fluid of body.
Concrete, the temperature of dyeing controls between 32 DEG C-36 DEG C, when after filter to be dyed seminal fluid be in 32 DEG C-36 DEG C it
Between time, its activity is higher, and therefore, carrying out dyeing the temperature ranges of 32 DEG C-36 DEG C can ensure that the effect of dyeing.
The separation method of the sexing semen that embodiments of the invention provide, during separating seminal fluid, was proficient in former
40-60 micron filter filters, and obtains removing seminal fluid after the filter of spermatium and foreign material, then Activity determination, dye and separate
Obtain the seminal fluid containing X chromosome and the seminal fluid containing Y chromosome afterwards, and then realize the whole process former essence separated；By
Yu Yuanjing is filtered by 40-60 micron filter before testing, the essence that the filter of 40-60 micron can will contain in former essence
Son group and filtering foreign fall, and then during follow-up dyeing, it is possible to the uniformity that after ensureing filter to be dyed, seminal fluid dyes
And stability, and then it is beneficial to separate the seminal fluid containing X chromosome and the seminal fluid containing Y chromosome efficiently.
Additionally, the separation method of this sexing semen of the present embodiment, it was proficient in 40-60 micron filter for the first time by former
Filtering, obtain removing seminal fluid after the filter of spermatium and foreign material, therefore during later separation, after filter, seminal fluid is difficult to occur stifled
The phenomenon of plug separator, and then ensure that the smooth and easy of whole separation process is carried out.
So that the method for floating of the embodiment of the present invention one is preferably applied, more efficient it is applied to sexing semen
Separating technology in, the present invention also provides embodiment two on the basis of above-described embodiment one, embodiment two be implement
That makes on the basis of example one further limits and increases, and is now described in detail and explains, refer to Fig. 2:
Embodiment two
Step 201: be proficient in the filtration for the first time of 40-60 micron filter by former, and obtained removing spermatium and the filter of foreign material
Rear seminal fluid；
Step 202: after drawing the filter of 15-25 microlitre, seminal fluid dropping keeps on the microscope slide through 32 DEG C-36 DEG C preheatings
Under 200-400 power microscope, the vigor of seminal fluid after filtering is observed after 0.5-1.5 minute；
Concrete, during detection vigor, can be carried out by the operation of step 202；Additionally, live in concrete survey
During, the seminal fluid reaching separation standard can be with vigor as 0.6-0.85, density >=1,000,000,000/ml, for standard, for density 6-
Seminal fluid after filter between 800000000/ml, can make density carry out dying operation after reaching the standard separated after being centrifuged.
Step 203: by pre-to Staining Talp liquid and 4%Egg yolk Talp liquid difference at a temperature of 32 DEG C-36 DEG C
Hot 10-20 minute, the Staining Talp liquid after being preheated and the 4%Egg yolk Talp liquid after preheating；
Wherein, every 1000ml Staining Talp liquid is by HEPES(40.00mM) 9.520g, Magnesi μm
Chloride6-Hydrate crystal(0.40mM) 0.080g, Sodi μm Chloride(95.00mM) 5.518g,
Potassi μm Chloride(3.00mM) 0.224g, Sodi μm Bicarbonate Dibasic/Anhydrous(0.30mM)
0.040g, Sodi μm Bicarbonate(10.00mM) 0.840g, Pyruvic Acid(2.00mM) 0.220g, Glucose
(5.00mM) 0.900g, Lactic Acid, 60%Syrup25.00 (mM) 3.610ml, Bovine Ser μm Alb μ
Min3.000g configuration forms；
Concrete collocation method includes: is sequentially placed into by reagent needed for above-mentioned data precise and fills this dosing total amount 3/
The ultra-pure water beaker of 4 fully dissolves；By NaOH solution, pH value is adjusted to 7.40 after dissolving；Solution is moved fully into volume relative
In the volumetric flask answered, repeatedly rinse the immigration volumetric flask filling of beaker inwall with ultra-pure water and fully mix to scale；Configure complete
Filtration sterilization is carried out afterwards with leaf filter after sterilizing；After filter, the every 1000 milliliters of liquid of liquid add gentamicin injection 2.5ml.
Every 1000ml4%Egg yolk Talp liquid is by Staining Talp liquid 957.5ml, 5%FD(ml) 2.6ml, Egg-
Yolk40ml forms；Concrete collocation method includes；The Staining Talp liquid of respective volume is moved into corresponding volumetric flask
In, then with pipet, 5%FD is added volumetric flask；Fill to the scale of volumetric flask the most mixed with Staining Talp liquid
Even；Remove remaining Ovum Gallus domesticus album with aseptic filtration paper after Fresh Egg reject Ovum Gallus domesticus album after disinfecting in alcohol, rack film by egg yolk
Clamp-on at the scale that graduated cylinder is the most volume required；Fully mix after the graduated cylinder mouth sealed with sealed membrane.Solution is poured into beaker again use
Magnetic stirrer 30 minutes, stands overnight (12 hours) at 5 DEG C；Solution after standing sucks graduated cylinder upper strata 5-10 milliliter liquid
Discarded, the most slowly tilt graduated cylinder and supernatant is poured into beaker (preventing from pouring precipitate into)；With HCL solution, pH is adjusted to
5.50, suck beaker upper strata grease after standing 30 minutes.Residue supernatant is distributed into centrifuge tube, balances two-by-two.Put into from
In scheming centrifugal (5 DEG C, 10000-12000 rev/min, 30 minutes), after centrifugal end, suck each centrifuge tube upper strata grease,
Use, in centrifugal rear solution is collected reagent bottle, gone out bacterium leaf filter (filter membrane of upper strata 1.2 μm, the mistake of lower floor 0.22 μm
Filter membrane) filtration sterilization, after filter, the every 1000 milliliters of liquid of liquid add gentamicin injection 2.6ml.
Step 204: at a temperature of 32 DEG C-36 DEG C, adds fluorescent dye after filter to be dyed, obtains after mixing in seminal fluid
Just contaminate seminal fluid；
In step 204, concrete, can be, after filter to be dyed, seminal fluid adds fluorescence after addition antibiotic solution
Dyestuff, obtains after mixing just contaminating seminal fluid；
Antibacterial contained in seminal fluid after filter to be dyed can be played the effect that suppression is even killed, in order to realize by antibiotic
Preferably effect, the most in the present embodiment, antibiotic solution is combined with antibiotic, the most mould including: the mould cellulose solution of Taylor, celebrating
Cellulose solution and the Lincoln-grand solution being made into by cillimycin and spectinomycin；And preferably, the mould cellulose solution of Taylor, celebrating are greatly
The volume ratio of mould cellulose solution and Lincoln-grand solution is 3:3:4；
Wherein, the collocation method of the mould cellulose solution of Taylor includes: accurately weigh Taylor's rhzomorph (tylosin) 0.19g the most molten
Solution is in the sodium citrate of the 2.9% of 10ml, and making ultimate density is 19.0mg/ml.
The collocation method of gentamycin solution includes: accurately weighs gentamycin (gentamicin) 1.0g and is dissolved completely in
The sodium citrate of the 2.9% of 10.1ml, making ultimate density is 99.01mg/ml.
The collocation method of Lincoln-grand solution includes: every milliliter of ultra-pure water dissolves 0.05g cillimycin (linco
And 0.1g spectinomycin (spectino mycin) mycin).Dosing amount can be increased according to above-mentioned data according to producing needs, and
It is dispensed in 2 milliliters of centrifuge tubes.
The tylosin solution configured, Lincoln-grand solution, gentamycin solution are added on one in 3:3:4 ratio
Playing mixing, the solution mixed is combined with antibiotic.
Further, in this step, concrete, after seminal fluid adding 20 microlitre antibiotic solutions after every milliliter of filter to be dyed
Obtain after adding fluorescent dye mixing just contaminating seminal fluid；
20 microlitre combined with antibiotic, so its bacterial species that can suppress are joined many with seminal fluid after every milliliter of filter to be dyed,
And inhibition is obvious.
In step 204, fluorescent dye includes: Hoechst33342；Hoechst33342 is that one can be with penetration cell
The blue fluorescent dyes of film, relatively low to the toxicity of cell.Therefore, in the present embodiment as the fluorescent dye of seminal fluid after filter.
Step 205: mix after the Staining Talp liquid after adding preheating in just dye seminal fluid, hatch 40 minutes-50 points
Clock, the seminal fluid after being hatched；
Concrete, in step 205, mixed after being specially the Staining Talp liquid after adding preheating in just dye seminal fluid
Even, in the water-bath of 32 DEG C-36 DEG C, darkroom is hatched 40 minutes-50 minutes, the seminal fluid after being hatched；Preferably, hatching
During choose the operation utilizing water-bath darkroom to hatch, water-bath be easy to control incubation temperature.
Step 206: pass through 40 micron-60 after adding the 4%Egg yolk Talp liquid after preheating in seminal fluid after incubation
Micron filter carries out second time and filters, and obtains the seminal fluid that dyes；
Owing to seminal fluid after incubation adds 4%Egg yolk Talp, therefore, follow-up separation process,
Prevent the phenomenon that the nozzle of blocking flow cytometer occurs in separation process, it is preferred that the seminal fluid after hatching can be carried out
Second time filters.In concrete operation, preferably 50 micron filters carry out second time and filter, and filter it in second time
Before, need to shake up the seminal fluid adding 4%Egg yolk Talp.
Step 207: dyeing seminal fluid is used flow cytometric sorting, obtains the seminal fluid containing X chromosome and dyes containing Y
The seminal fluid of body.
Just can be realized the process separated by dyeing seminal fluid by flow cytometer, and then obtain the essence containing X chromosome
Liquid and the seminal fluid containing Y chromosome, and be applied in the controlled other reproductive process of braking physical property.
Additionally, in the Lincoln-grand solution of above-described embodiment, it is preferred that the concentration ratio of cillimycin and spectinomycin
For 1:2.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, that is made any repaiies
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (7)
1. the separation method of sexing semen, it is characterised in that comprise the following steps:
It is proficient in the filtration for the first time of 40-60 micron filter by former, obtains seminal fluid after the filter of removal spermatium and foreign material；
Detect the vigor of seminal fluid after described filter, the vigor of obtaining be 0.6-0.85 filter to be dyed after seminal fluid；
Staining Talp liquid and 4%Egg yolk Talp liquid are preheated at a temperature of 32 DEG C-36 DEG C 10-20 respectively divide
4%Egg yolk Talp liquid after clock, the Staining Talp liquid after being preheated and preheating；
At a temperature of 32 DEG C-36 DEG C, after seminal fluid adding antibiotic solution after described filter to be dyed, add fluorescent dye,
Obtain after mixing just contaminating seminal fluid；
Mix after Staining Talp liquid after adding described preheating in described just dye seminal fluid, hatch 40 minutes-50 minutes,
Seminal fluid after being hatched；
Seminal fluid after described hatching adds the 4%Egg yolk Talp liquid after described preheating, after shaking up, obtains dyeing essence
Liquid；
Described dyeing seminal fluid is used flow cytometric sorting, obtains the seminal fluid containing X chromosome and the essence containing Y chromosome
Liquid；
Wherein, described antibiotic solution includes the mould cellulose solution of Taylor, gentamycin solution and is joined by cillimycin and spectinomycin
Lincoln-grand the solution become；And the described mould cellulose solution of Taylor, described gentamycin solution and the body of described Lincoln-grand solution
Long-pending ratio is 3:3:4；
Wherein, every 1000ml Staining Talp liquid is by hydroxyethyl piperazine ethanesulfonic acid 9.520g, magnesium chloride hydrate crystal
0.080g, sodium chloride 5.518g, potassium chloride 0.224g, anhydrous sodium bicarbonate 0.040g, sodium bicarbonate 0.840g, acetone acid
0.220 gram, glucose 0.900 gram, Lactic Acid, 60%Syrup 3.610ml, bovine serum albumin 3.000g configuration forms.
The separation method of sexing semen the most according to claim 1, it is characterised in that seminal fluid after the described filter of described detection
Vigor, the vigor of obtaining be 0.6-0.85 filter to be dyed after seminal fluid step in, the vigor of seminal fluid after the described filter of described detection,
Specifically include:
Draw seminal fluid dropping holding 0.5-1.5 minute on the microscope slide through 32 DEG C-36 DEG C preheatings after the filter of 15-25 microlitre, so
After under 200-400 power microscope, observe the vigor of seminal fluid after described filter.
The separation method of sexing semen the most according to claim 2, it is characterised in that after described filter to be dyed in seminal fluid
Add fluorescent dye after adding antibiotic solution, obtain after mixing, in the first step contaminating seminal fluid, specifically including:
Fluorescent dye mixing is added after seminal fluid adding antibiotic solution described in 20 microlitres after filter to be dyed every milliliter described
After obtain described just contaminating seminal fluid.
The separation method of sexing semen the most according to claim 3, it is characterised in that after described filter to be dyed in seminal fluid
Add fluorescent dye after adding antibiotic solution, obtain after mixing in the first step contaminating seminal fluid；
Described fluorescent dye includes: Hoechst 33342.
The separation method of sexing semen the most according to claim 3, it is characterised in that add institute in described just dye seminal fluid
Mix after stating the Staining Talp liquid after preheating, hatch 40 minutes-50 minutes, the step of the seminal fluid after being hatched, specifically
Including:
Mix, at the water-bath of 32 DEG C-36 DEG C after Staining Talp liquid after adding described preheating in described just dye seminal fluid
In, darkroom is hatched 40 minutes-50 minutes, obtain described in hatch after seminal fluid.
The separation method of sexing semen the most according to claim 3, it is characterised in that add in the seminal fluid after described hatching
Enter the 4%Egg yolk Talp liquid after described preheating, obtain the step of described dyeing seminal fluid after shaking up, specifically include:
40 microns-60 microns are passed through after seminal fluid after described hatching adds the 4%Egg yolk Talp liquid after described preheating
Filter carries out second time and filters, and obtains described dyeing seminal fluid.
The separation method of sexing semen the most according to claim 5, it is characterised in that by cillimycin and spectinomycin
In the described Lincoln being made into-grand solution:
The concentration of described cillimycin and described spectinomycin is than for 1:2.
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| CN108575989A (en) * | 2018-07-05 | 2018-09-28 | 河南省鼎元种牛育种有限公司 | A kind obstinacy control sperm Cryopreservation |
| CN108865982A (en) * | 2018-07-05 | 2018-11-23 | 河南省鼎元种牛育种有限公司 | A kind of frozen cattle semens X, y sperm separation method |
| CN114459857B (en) * | 2022-01-29 | 2023-09-05 | 上海益诺思生物技术股份有限公司 | Pretreatment method for maintaining animal sperm motility and animal sperm apoptosis determination |
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| CN101613677A (en) * | 2009-07-30 | 2009-12-30 | 广西大学 | A kind of method of separating pig X sperm and y sperm |
| US9140688B2 (en) * | 2011-09-30 | 2015-09-22 | Inguran, Llc | Sperm staining and sorting methods |
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