CN102095675A - Method for identifying and detecting mixed frozen sexed semen product in dairy cow - Google Patents
Method for identifying and detecting mixed frozen sexed semen product in dairy cow Download PDFInfo
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- CN102095675A CN102095675A CN 201010554665 CN201010554665A CN102095675A CN 102095675 A CN102095675 A CN 102095675A CN 201010554665 CN201010554665 CN 201010554665 CN 201010554665 A CN201010554665 A CN 201010554665A CN 102095675 A CN102095675 A CN 102095675A
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Abstract
The invention relates to a method for identifying and detecting a mixed frozen sexed semen product in dairy cow, which belongs to the animal reproductive biotechnology. The method comprises the following steps: thawing the frozen sexed semen of dairy cow, examining with a microscope in 30 minutes after thawing to observe the sorted sperm emitting fluorescence and the undyed mixed sperms of other animals to determine that the sample is mixed frozen sexed semen of dairy cow; identifying the type of the mixed sperms except the sorted sperm of the dairy cow according to the DNA specific difference of X and Y sex chromosomes determining the sex of animals; and detecting the motility and the total number of the sorted sperms, and the total number of the mixed sperms in the mixed frozen sexed semen product. The method is convenient in identifying the mixed frozen sexed semen product, and can objectively detect the various quality indexes of the sorted sperms in the mixed frozen sexed semen.
Description
Technical field
The invention belongs to animal reproduction bioengineering new technology, mainly provide the control of a kind of mixed milk obstinacy to freeze smart product and identify and detection method.
Background technology
Dairy cow sperms separation-property control techniques since the industrialization in 2000, at present surplus the whole world ten country apply.But the subject matter that this technology exists is that production efficiency is low, cost of products is higher, is the key factor that limits this technology large-scale promotion application.At present, developed based on the milk cow low dosage control of heterogenous animal mixing sperm and frozen smart production patent new technology, patent name: a kind of low dosage livestock semen and production method and application, the patent No.: CN 2006100149168 increases substantially this technology production efficiency.Freeze smart product for discriminating mixed milk obstinacy control on market, detect every quality index that separated sperm in the essence is frozen in the Combination control more objectively simultaneously, researched and developed this technology.
Summary of the invention
The invention provides the control of a kind of mixed milk obstinacy and freeze smart product evaluation and detection method, this know-why is to utilize the characteristics of milk cow separated sperm fluorescent dye, thaw and observe sperm at fluorescence and lamp microscopically respectively in back 30 minutes, show according to the sperm head color, determine whether this product is that essence is frozen in the control of mixed milk obstinacy, adopt animal sperm specific antibody staining technique simultaneously, can identify milk cow separated sperm mixing animal sperm kind in addition; And measure this Combination control by fluorescence and natural light microscope inspection and freeze smart milk cow separated sperm vigor, milk cow separated sperm sum, mix the every quality index of sperm sum.
The objective of the invention is to be achieved through the following technical solutions: smart product evaluation and detection method are frozen in the control of a kind of mixed milk obstinacy, it is characterized in that: it comprises that the control of mixed milk obstinacy freezes the authentication method of smart product and the detection method that smart product is frozen in the control of mixed milk obstinacy, wherein:
(2) authentication method of smart product is frozen in the control of mixed milk obstinacy, and it may further comprise the steps:
1) milk obstinacy control is frozen essence and thawed: getting 1 specification is that smart product is frozen in the milk obstinacy control that the 0.25ml tubule is preserved in liquid nitrogen, put into 37 ℃ of tepidariums rapidly, seminal fluid is fetched into the time of putting into water from liquid nitrogen container must not be above 10 seconds, treat seminal fluid all melt after with seminal fluid transposition in the tubule in the 1.5ml centrifuge tube, put into 37 ℃ of water-bath temperature baths 5 minutes;
2) control of mixed milk obstinacy is frozen smart product evaluation and is confirmed: open microscope fluorescence and natural light simultaneously, regulate light intensity and focus, reach the state that can observe the dyeing sperm and the sperm that is unstained by microscope color filter control respectively, in advance microslide and cover glass be placed on 37 ℃ of heating plates preheating 10 minutes with
(2) evaluation and the affirmation of smart product frozen in the control of mixed milk obstinacy: open microscope fluorescence and natural light simultaneously, regulate light intensity and focus, reach the state that to observe the dyeing sperm and the sperm that is unstained by the control of microscope color filter respectively, in advance microslide and cover glass were placed on 37 ℃ of heating plates preheating more than 10 minutes, drop on the ready microslide with getting 15 μ l products in the centrifuge tube of pipettor from (1), compressing tablet is placed on microscopy under the fluorescent microscope, in this visual field, the dyeing sperm head presents blue-fluorescence, the sperm that is unstained shows grey, thawed back 30 minutes in, if can observe the sperm that is unstained, confirm that this product is that essence is frozen in the control of mixed milk obstinacy, as shown in Figure 1, what show blue-fluorescence is to separate Niu Jingzi, and what show the Dark grey natural light is the sheep sperm;
(3) animal beyond the milk cow separated sperm mixes the evaluation of sperm kind:
With the goat sperm is example, and the milk obstinacy control of mixing goat sperm is frozen smart authentication method and is:
Select Y chromosome specificity DNA probing needle G-SRY (Berta et al.Nature, 1990,348:448-450, Koopman et al.Nature, 1991,351:117-121) as evaluation of foundation, get the control of potion tubule and freeze essence, put 37 ℃ of water-baths and thaw, push in the 1.5ml test tube, after ultrasound wave 10s removes sperm tail, use PBS phosphate buffer eccentric cleaning 3 times; Getting 10ul sperm head sample is added on the microslide, with the fixing 10min that handles of sperm immobile liquid 0.05%Formalin, use goat sperm specificity DNA probing needle G-SRY probe then, 37 ℃ down and sperm head hybridize, shading treatment 4 hours, after the hybridization shading treatment, handle 20min with the one-level fluorescent antibody staining successively, microslide after the processing washes the back gently with clear water and drips mounting oil, after the covered, under fluorescent microscope, detect and add up sperm count with yellow hybridization point, if find that sperm beyond the separated sperm of UV blue-fluorescence has about 50% sperm yellow hybridization point is arranged, illustrate that it is goat sperm that the sperm kind of mixing in the smart product is frozen in this obstinacy control, if there is not the sperm of yellow hybridization point, can select the specificity DNA probing needle of other animal, adopt and identify with quadrat method, till finding yellow hybridization point, the animal varieties of determining to mix sperm.
Embodiment 2: present embodiment is to be that 0.25ml mixes that to freeze essence be example with 1 international specification, and every dose of mixing is frozen in the essence 0.2ml and mixed and freeze essence, and the tally capacity is 0.1 μ l; It is freezing equilibrium liquid A of Tris alkali and B that smart refrigerating fulid is frozen in the property control, and the ratio of freezing equilibrium liquid A of Tris alkali and B is 1: 1,
The freezing equilibrium liquid A:PH=6.8 of Tris-, it consists of:
TRIS trishydroxymethylaminomethane 291.7mM
Citric acid 95.1mM
Fructose 70.2mM
Yolk 20%
Ultrapure water;
The freezing equilibrium liquid B:PH=6.8 of Tris-, it consists of:
TRIS trishydroxymethylaminomethane 291.7mM
Citric acid 95.1mM
On, to get product with pipettor from centrifuge tube and drop on the ready microslide, compressing tablet is placed on microscopy under the fluorescent microscope, and in this visual field, the dyeing sperm head presents blue-fluorescence, and the sperm that is unstained shows grey; Thawed back 30 minutes in, and if can observe the sperm that is unstained, confirm that this product is that essence is frozen in the control of mixed milk obstinacy;
3) the mixing sperm kind beyond the milk cow separated sperm is identified: the mixing sperm kind beyond the milk cow separated sperm is identified, its method is: the sperm that may be used for beyond the preparation milk cow mixes specific DNA molecular probe (the list of references 1:Hassanane M etc. that use animal varieties, Molecular Reproductionand Development, 1999,53:407-412; List of references 2:Hamano K, Li X etc., Journalof MammOvaRes, 2001,18:81-88), carry out molecular hyridization with the sperm that freezes smart product and handle, have or not sperm demonstration specificity fluorescent hybridization point to differentiate the kind of mixing animal sperm in the sperm by detecting;
(2) control of mixed milk obstinacy is frozen milk cow separated sperm vigor in the essence, milk cow separated sperm sum, is mixed sperm sum detection method, and it may further comprise the steps:
1) milk cow separated sperm vigor detects, and it may further comprise the steps:
Freeze essence with the mixed milk obstinacy control that pipettor is got after thawing from centrifuge tube, drop on ready 37 ℃ of heating microslides, compressing tablet is placed under the UV light source fluorescent microscope, select the uniform sample area of compressing tablet to observe, counting shows the sperm count alives and the Necrospermia number of blue-fluorescence respectively, calculate separated sperm vigor=works separated sperm number/works separated sperm number and extremely the separated sperm number with;
2) milk cow separated sperm sum and mixing sperm sum detect, and it may further comprise the steps:
Get the control of partially thawed mixed milk obstinacy and freeze smart sample, with dropping on the blood counting chamber behind the formalin solution fixed dilution, counting has the separated sperm sum of fluorescence under the UV fluorescent microscope, and calculates every dose and freeze separated sperm sum in the essence; Again microscope is transferred under the natural light, the total sperm count of counting under natural light, and calculate every dose and freeze in the essence total sperm sum.
The invention has the beneficial effects as follows: can differentiate easily that 1, smart product is frozen in the control of mixed milk obstinacy on the market, detect every quality index that separated sperm in the essence is frozen in the Combination control simultaneously more objectively.2, the present invention also can identify milk cow separated sperm mixing sperm kind in addition simultaneously, comprises ox, sheep, pig, deer, horse and donkey.3, because the smart new product that is is frozen in the control of mixed milk obstinacy, also there is not conventional process at present at this product detection and evaluation, this invention is detection and the authentication method according to the XY sex chromosome DNA specificity difference design that causes the animal gender differences, does not also have the comparability with additive method at present.
Description of drawings
Fig. 1 is that smart sem photograph is frozen in the mixing of thawing in 30 minutes.
Embodiment
Embodiment 1: the authentication method of smart product is frozen in the control of a kind of mixed milk obstinacy, and it may further comprise the steps:
(1) milk obstinacy control is frozen essence and thawed: getting 1 specification is that smart product is frozen in the milk obstinacy control that the 0.25ml tubule is preserved in liquid nitrogen, put into 37 ℃ of tepidariums rapidly, seminal fluid is fetched into the time of putting into water from liquid nitrogen container must not be above 10 seconds, after treating that seminal fluid all melts, seminal fluid transposition in the tubule in the 1.5ml centrifuge tube, is put into 37 ℃ of water-bath temperature and bathed 5 minutes;
Fructose 70.2mM
Yolk 20%
Glycerine 12%
Ultrapure water;
The detection method that the control of a kind of mixed milk obstinacy is frozen separated sperm vigor in the smart product, separated sperm sum and mixed the sperm sum, wherein:
(1) the separated sperm vigor detects, it comprises the steps: in advance microslide and cover glass were placed on 37 ℃ of heating plates preheating more than 10 minutes, from embodiment 1, thaw after confirm as the control of mixed milk obstinacy and freeze and get 15 μ l sample drops in the centrifuge tube of smart product on ready microslide with pipettor, compressing tablet is placed on microscopy under the fluorescent microscope, open fluorescent microscope, select the UV light source, select the uniform sample area of compressing tablet to observe simultaneously, a compressing tablet is got 5 visuals field, middle 1, respectively get 1 all around, counting shows the sperm count alives and the Necrospermia number of blue-fluorescence respectively, calculate separated sperm vigor=works separated sperm number/works separated sperm number and extremely the separated sperm number with;
With 1 specification is that 0.25ml mixes that to freeze essence be example, after thawing, and microscopy, each quality inspection index calculating method is as follows: after observing 5 visuals field, each visual field sperm count (unit: individual):
| Separated sperm number alive | 26 | 25 | 17 | 15 | 25 | 108 |
| Dead separated sperm number | 18 | 13 | 10 | 16 | 12 | 69 |
This batch separated sperm vigor=108/ (108+69) * 100%=61%;
(2) milk cow separated sperm sum and mixing sperm sum detect, it may further comprise the steps: freeze essence according to the thaw mixing of 0.25ml specification of the method among the embodiment 1, get the 50ul mixed semen, drop on the blood counting chamber after 20 times of dilutions of formalin solution with 950ul, placing and counting total sperm count under the microscope natural light is (unit: individual): 106,100,98,96,109, average is 101.8, promptly the total sperm of this batch products is: 101.8*20*10000=2036 ten thousand/ml, and the mixing sperm of every product adds up to 2,036,/5=,407 ten thousand/agent; Under microscope UV light, add up to (unit: individual) again: 25,28,27,28,27 with blood counting chamber counting fluorescence separated sperm, average is 27, promptly this batch products separated sperm density is: 27*,20*,100,00=,540 ten thousand/ml, and the milk cow separated sperm of every product adds up to: 540,/5=,108 ten thousand/agent;
Because the smart new product that is is frozen in the control of mixed milk obstinacy, also there is not conventional process at present at this product detection and evaluation, this invention is detection and the authentication method according to the XY sex chromosome DNA specificity difference design that causes the animal gender differences, does not also have the comparability with additive method at present.The present invention can identify the mixing sperm kind beyond the milk cow separated sperm simultaneously, comprises ox, sheep, pig, deer, horse and donkey.
Claims (1)
1. mixed milk obstinacy control is frozen smart product and identified and detection method, it is characterized in that: it comprises that the control of mixed milk obstinacy freezes the authentication method of smart product and the detection method that smart product is frozen in the control of mixed milk obstinacy, wherein:
(1) authentication method of smart product is frozen in the control of mixed milk obstinacy, and it may further comprise the steps:
1) milk obstinacy control is frozen essence and thawed: getting 1 specification is that smart product is frozen in the milk obstinacy control that the 0.25ml tubule is preserved in liquid nitrogen, put into 37 ℃ of tepidariums rapidly, seminal fluid is fetched into the time of putting into water from liquid nitrogen container must not be above 10 seconds, treat seminal fluid all melt after with seminal fluid transposition in the tubule in the 1.5ml centrifuge tube, put into 37 ℃ of water-bath temperature baths 5 minutes;
2) control of mixed milk obstinacy is frozen smart product evaluation and is confirmed: open microscope fluorescence and natural light simultaneously, regulate light intensity and focus, reach the state that to observe the dyeing sperm and the sperm that is unstained by the control of microscope color filter respectively, in advance microslide and cover glass were placed on 37 ℃ of heating plates preheating more than 10 minutes, getting product with pipettor from centrifuge tube drops on the ready microslide, compressing tablet is placed on microscopy under the fluorescent microscope, in this visual field, the dyeing sperm head presents blue-fluorescence, and the sperm that is unstained shows grey; Thawed back 30 minutes in, and if can observe the sperm that is unstained, confirm that this product is that essence is frozen in the control of mixed milk obstinacy;
3) the mixing sperm kind beyond the milk cow separated sperm is identified: the mixing sperm kind beyond the milk cow separated sperm is identified, its method is: the sperm that may be used for beyond the preparation milk cow mixes the specific DNA molecular probe that uses animal varieties, carry out molecular hyridization with the sperm that freezes smart product and handle, have or not sperm demonstration specificity fluorescent hybridization point to differentiate the kind of mixing animal sperm in the sperm by detecting;
(2) control of mixed milk obstinacy is frozen milk cow separated sperm vigor in the essence, milk cow separated sperm sum, is mixed sperm sum detection method, and it may further comprise the steps:
1) milk cow separated sperm vigor detects, and it may further comprise the steps:
Freeze essence with the mixed milk obstinacy control that pipettor is got after thawing from centrifuge tube, drop on ready 37 ℃ of heating microslides, compressing tablet is placed under the UV light source fluorescent microscope, select the uniform sample area of compressing tablet to observe, counting shows the sperm count alives and the Necrospermia number of blue-fluorescence respectively, calculate separated sperm vigor=works separated sperm number/works separated sperm number and extremely the separated sperm number with;
2) milk cow separated sperm sum and mixing sperm sum detect, and it may further comprise the steps:
Get the control of partially thawed mixed milk obstinacy and freeze smart sample, with dropping on the blood counting chamber behind the formalin solution fixed dilution, counting has the separated sperm sum of fluorescence under the UV fluorescent microscope, and calculates every dose and freeze separated sperm sum in the essence; Again microscope is transferred under the natural light, the total sperm count of counting under natural light, and calculate every dose and freeze in the essence total sperm sum.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103822816A (en) * | 2013-12-16 | 2014-05-28 | 内蒙古赛科星繁育生物技术股份有限公司 | Method for identifying mixed sperms by using fluorescent dyes FITC (fluorescein isothiocyanate) and MITO (Mitotracker) |
| CN111879721A (en) * | 2020-06-19 | 2020-11-03 | 西安交通大学 | Method for identifying fine spot species based on Fourier transform infrared spectroscopy and chemometrics |
Citations (3)
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|---|---|---|---|---|
| WO2004087177A1 (en) * | 2003-03-28 | 2004-10-14 | Monsanto Technology Llc | Process for the staining of sperm |
| CN101411722A (en) * | 2008-10-22 | 2009-04-22 | 李喜和 | Freezing mixed sperm for controlling X/Y gender of milk cattle and preparation method thereof |
| CN100551382C (en) * | 2006-07-26 | 2009-10-21 | 李喜和 | A kind of low dosage livestock semen and production method and application |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004087177A1 (en) * | 2003-03-28 | 2004-10-14 | Monsanto Technology Llc | Process for the staining of sperm |
| CN100551382C (en) * | 2006-07-26 | 2009-10-21 | 李喜和 | A kind of low dosage livestock semen and production method and application |
| CN101411722A (en) * | 2008-10-22 | 2009-04-22 | 李喜和 | Freezing mixed sperm for controlling X/Y gender of milk cattle and preparation method thereof |
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| 《中国牛业科学》 20090131 赵国琳等 奶牛性控冻精应用效果研究 17-18 1 第35卷, 第1期 2 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103822816A (en) * | 2013-12-16 | 2014-05-28 | 内蒙古赛科星繁育生物技术股份有限公司 | Method for identifying mixed sperms by using fluorescent dyes FITC (fluorescein isothiocyanate) and MITO (Mitotracker) |
| CN103822816B (en) * | 2013-12-16 | 2015-12-30 | 内蒙古赛科星繁育生物技术（集团）股份有限公司 | Use the method for fluorescent dye FITC and MITO qualification mixing sperm |
| CN111879721A (en) * | 2020-06-19 | 2020-11-03 | 西安交通大学 | Method for identifying fine spot species based on Fourier transform infrared spectroscopy and chemometrics |
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Address after: 011517 the Inner Mongolia Autonomous Region Helingeer County of Hohhot city Shengle Economic Zone Development Avenue North Building 6 floor of Mengniu Applicant after: Saikexing Reproductive Biotechnology CO., Ltd. Address before: 011517 the Inner Mongolia Autonomous Region Helingeer County of Hohhot city Shengle Economic Zone Development Avenue North Building 6 floor of Mengniu Applicant before: Mengniu Reproductive Biltechnology Co., Ltd. |
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Address after: 011517 the Inner Mongolia Autonomous Region Helingeer County of Hohhot city Shengle economic Park gathering service area management committee building 4 New South Building Room 4422 Patentee after: Inner Mongolia saikexing reproductive biotechnology (Group) Limited by Share Ltd Address before: 011517 the Inner Mongolia Autonomous Region Helingeer County of Hohhot city Shengle Economic Zone Development Avenue North Building 6 floor of Mengniu Patentee before: Saikexing Reproductive Biotechnology CO., Ltd. |