CN117147548A - Novel method for sperm survival rate - Google Patents
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- CN117147548A CN117147548A CN202311119420.7A CN202311119420A CN117147548A CN 117147548 A CN117147548 A CN 117147548A CN 202311119420 A CN202311119420 A CN 202311119420A CN 117147548 A CN117147548 A CN 117147548A
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- 230000004083 survival effect Effects 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title claims abstract description 26
- 238000001514 detection method Methods 0.000 claims abstract description 39
- 210000000582 semen Anatomy 0.000 claims abstract description 37
- 239000000243 solution Substances 0.000 claims abstract description 36
- 239000000975 dye Substances 0.000 claims abstract description 32
- SEACYXSIPDVVMV-UHFFFAOYSA-L eosin Y Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C([O-])=C(Br)C=C21 SEACYXSIPDVVMV-UHFFFAOYSA-L 0.000 claims abstract description 29
- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000001044 red dye Substances 0.000 claims abstract description 25
- 241001465754 Metazoa Species 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000007853 buffer solution Substances 0.000 claims abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 7
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 238000010790 dilution Methods 0.000 claims abstract description 4
- 239000012895 dilution Substances 0.000 claims abstract description 4
- 239000011521 glass Substances 0.000 claims description 22
- 230000035899 viability Effects 0.000 claims description 19
- 210000000805 cytoplasm Anatomy 0.000 claims description 7
- 239000000872 buffer Substances 0.000 claims description 3
- 238000004364 calculation method Methods 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 238000010186 staining Methods 0.000 claims description 3
- 238000007619 statistical method Methods 0.000 claims description 3
- 230000000007 visual effect Effects 0.000 claims description 3
- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 0.000 claims description 3
- 230000003833 cell viability Effects 0.000 claims 2
- 238000002156 mixing Methods 0.000 claims 1
- 230000019100 sperm motility Effects 0.000 claims 1
- 238000012545 processing Methods 0.000 abstract description 2
- 241000283690 Bos taurus Species 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 230000003287 optical effect Effects 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 3
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- FWLHAQYOFMQTHQ-UHFFFAOYSA-N 2-N-[8-[[8-(4-aminoanilino)-10-phenylphenazin-10-ium-2-yl]amino]-10-phenylphenazin-10-ium-2-yl]-8-N,10-diphenylphenazin-10-ium-2,8-diamine hydroxy-oxido-dioxochromium Chemical compound O[Cr]([O-])(=O)=O.O[Cr]([O-])(=O)=O.O[Cr]([O-])(=O)=O.Nc1ccc(Nc2ccc3nc4ccc(Nc5ccc6nc7ccc(Nc8ccc9nc%10ccc(Nc%11ccccc%11)cc%10[n+](-c%10ccccc%10)c9c8)cc7[n+](-c7ccccc7)c6c5)cc4[n+](-c4ccccc4)c3c2)cc1 FWLHAQYOFMQTHQ-UHFFFAOYSA-N 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 229920000767 polyaniline Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000010835 comparative analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 210000003313 haploid nucleated cell Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- 210000003794 male germ cell Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000033458 reproduction Effects 0.000 description 1
- 230000014639 sexual reproduction Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
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Abstract
The application relates to the technical field of sperm detection, and discloses a novel method for sperm survival rate, which comprises the following detection steps of S1: preparing a reagent, namely preparing an eosin Y dye solution and a neutral red dye solution according to the dosage, and then preparing a buffer solution for later use after the eosin Y dye solution and the neutral red dye solution are prepared; s2: semen collection treatment, namely collecting semen from cultured animals, placing the collected semen in a constant-temperature water bath tank for heat preservation treatment, then carrying out high-power dilution on the semen after heat preservation treatment, and estimating the semen to obtain an estimated score. The sperm survival rate of the cultured animals is detected by combining the estimated score of the semen during collection and processing and the detection data of various sperm survival rates, so that the detection precision is high, the detection is convenient, the detection of the sperm survival rate of the cultured animals by the animal husbandry culture institution is convenient, and the popularization and use value are high.
Description
Technical Field
The application relates to the technical field of sperm detection, in particular to a novel method for sperm survival rate.
Background
Sperm, transfer sperm and plant sperm, male germ cells in sexual reproduction of animals, germ cells of male animals, male gametes in heteroleptic reproduction, haploid germ cells produced by sperm organs, while more sperm in life refers to mature germ cells of men, formed in spermary, semen is an organic matter, contains fructose and proteins, and some enzymes, inorganic salts and organic salts.
In the process of animal husbandry cultivation, the breeding efficiency of the cultivated animals is an important factor affecting the profit of the animal husbandry cultivation institutions, so that the sperm survival rate of the cultivated animals needs to be detected regularly, the sperm survival rate is an important index for evaluating the semen quality, and the detection under an optical microscope is simple and convenient to operate but has poor detection precision, so that a new method for the sperm survival rate is proposed by the person skilled in the art to solve the technical problems.
Disclosure of Invention
Aiming at the defects of the prior art, the application provides a novel method for sperm survival rate, which solves the problem of poor detection precision when the existing optical microscope is adopted for detection.
In order to achieve the above purpose, the application is realized by the following technical scheme: a novel method for sperm viability, comprising the following detection steps:
s1: reagent preparation
Preparing eosin Y dye liquor and neutral red dye liquor according to the using dosage, and then preparing buffer liquor for later use after the eosin Y dye liquor and neutral red dye liquor are prepared;
s2: semen collection and treatment
Collecting semen from cultured animals, placing the collected semen in a constant-temperature water bath tank for heat preservation treatment, diluting the heat-preserved semen by high power, and estimating the heat-preserved semen to obtain an estimated fraction;
s3: survival rate detection
Dripping 10l semen on a glass slide by adopting a microsampler, detecting the survival rate of sperms on the glass slide by respectively using the eosin Y dye solution and the neutral red dye solution prepared in the step S1, and carrying out statistical analysis by using SPSS software after the detection is finished to obtain a detection result;
s4: conclusion analysis
Combining the estimated score in the step S2 and the detection result in the step S3, thereby obtaining the sperm viability condition of the detected animal.
Preferably, in the step S1, both the eosin Y dye solution and the neutral red dye solution are purchased in three factories of Shanghai reagent, and the eosin Y dye solution is prepared by distilled water.
Preferably, in the step S1, the concentration of the eosin Y dye solution is 0.15%, the PH value of the neutral red dye solution is 7.4, and the concentration of the buffer solution is 0.01%.
Preferably, in the step S2, the temperature of the constant-temperature water bath tank is 37 ℃, and the semen is subjected to high-power dilution and the sperm contour and the sperm interval are determined by means of a light microscope.
Preferably, in the step S2, when estimating the sperm under the visual field, the motility of sperm with 10% of straight advancing is 0.1,20% of straight advancing is 0.2, and so on, and 1 is divided into full.
Preferably, in the step S3, 200 sperm are counted per sample, and the sperm survival rate is calculated, and the calculation formula of the sperm survival rate is as follows: survival = (total number of sperm-number of non-linear motile sperm)/total number of sperm 100%.
Preferably, in the step S3, when the viability of the sperm on the slide glass is detected by using the eosin Y dye solution, 10ta0.15% eosin Y dye solution is added to the slide glass and mixed uniformly, 2011% nigrosine or aniline blue is then added as a background color, then another slide glass is used for making a smear, after natural drying, the living sperm is observed under a microscope without staining, the cytoplasm is transparent, and dead sperm is stained red.
Preferably, in the step S3, when the survival rate of the sperm on the glass slide is detected by adopting neutral red dye solution, the neutral red dye solution and the buffer solution are added dropwise on the glass slide, and after being uniformly mixed, the glass slide is dyed for 2 minutes, and then a cover plate is added, and the dead sperm is dyed into red, the living sperm is not dyed, and the cytoplasm is transparent under a bright field microscope of 400 times for half an hour.
The application provides a new method for sperm survival rate. The beneficial effects are as follows:
1. the application detects the sperm survival rate of the cultured animals by combining the estimated score of the semen during the collection and processing and the detection data of various sperm survival rates, has high detection precision and more convenient detection, is convenient for the livestock breeding institutions to detect the sperm survival rate of the cultured animals, and has higher popularization and use values.
2. According to the application, the sperm survival rate of the cultured animals in the culture mechanism is detected regularly, and the optimal bred animals in the cultured animals are screened out according to the data condition of the sperm survival rate, so that the breeding success rate of the culture mechanism is improved, and meanwhile, the economic benefit of the culture mechanism is also improved.
Detailed Description
The technical solutions of the embodiments of the present application will be clearly and completely described below in conjunction with the embodiments of the present application, and it is apparent that the described embodiments are only some embodiments of the present application, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
Embodiment one:
the embodiment of the application provides a novel method for sperm survival rate, which comprises the following detection steps:
s1: reagent preparation
Preparing eosin Y dye liquor and neutral red dye liquor according to the using dosage, and then preparing buffer liquor for later use after the eosin Y dye liquor and neutral red dye liquor are prepared;
s2: semen collection and treatment
Collecting semen from cultured animals, placing the collected semen in a constant-temperature water bath tank for heat preservation treatment, diluting the heat-preserved semen by high power, and estimating the heat-preserved semen to obtain an estimated fraction;
s3: survival rate detection
Dripping 10l semen on a glass slide by adopting a microsampler, detecting the survival rate of sperms on the glass slide by respectively using the eosin Y dye solution and the neutral red dye solution prepared in the step S1, and carrying out statistical analysis by using SPSS software after the detection is finished to obtain a detection result;
s4: conclusion analysis
Combining the estimated score in the step S2 and the detection result in the step S3, thereby obtaining the sperm viability condition of the detected animal.
In the S1 step, both the eosin Y dye liquor and the neutral red dye liquor are purchased in Shanghai reagent three factories, and the eosin Y dye liquor is prepared by distilled water.
In the S1 step, the concentration of the eosin Y dye solution is 0.15%, the PH value of the neutral red dye solution is 7.4, and the concentration of the buffer solution is 0.01%.
And S2, in the step of performing high-power dilution on the semen by means of a light microscope, determining the sperm outline and the sperm spacing, wherein the temperature of the constant-temperature water bath is 37 ℃.
In the step S2, when estimating the sperm under the visual field, the motility of the sperm which is 10 percent and advances in a straight line is 0.1,20 percent and advances in a straight line is 0.2, and the like, and 1 is divided into full divisions.
In the step S3, 200 sperms are counted in each sample, the sperm survival rate is calculated, and the calculation formula of the sperm survival rate is as follows: survival = (total number of sperm-number of non-linear motile sperm)/total number of sperm 100%.
In the step S3, when the viability of the sperms on the glass slide is detected by adopting the eosin Y dye solution, 10tA0.15% eosin Y dye solution is added on the glass slide and is uniformly mixed, 2011% aniline black or aniline blue is then added as a background color, then another glass slide is used for making a smear, after natural drying, the viable sperms are observed under a microscope to be uncolored, the cytoplasm is transparent, and dead sperms are colored red.
The detection of sperm viability is an important content of semen detection, the sperm quality and the sperm viability are directly reflected, and the sperm viability is obviously affected by the reduction of sperm viability.
Neutral red is a water-soluble dye, and can be stored in cells through the cell membranes of the live sperms to cause the cells to be colored; when the sperm cell membrane and lysosome membrane are destroyed, dye cannot be taken up, and therefore dead sperm is not stained.
And S3, when the neutral red dye solution is adopted to detect the survival rate of the sperms on the glass slide, after the neutral red dye solution and the buffer solution are added dropwise on the glass slide and mixed uniformly, a cover plate is added after dyeing for 2min, and the dead sperms are dyed into red, the viable sperms are not dyed and the cytoplasm is transparent after observation in half an hour under a microscope with 400 times of the bright field.
The step is obtained by comparing two methods for measuring the sperm viability, the difference between the two methods does not appear, but the method finds that the measurement of the sperm viability by the eosin dye solution is influenced by the concentration of the eosin dye solution in the test process, the loss of sperm and even the killing of sperm can be caused by the larger concentration, and the neutral red dyeing method can perform morphological description and identification on the viable sperm under an optical microscope, which is superior to the eosin Y dyeing and heating methods, and has popularization and application values in the detection of semen quality.
In the step S3, a dripping method can be adopted for detection, and the dripping method comprises the following operation steps: the same amount of semen, eosin water solution and aniline black solution are firstly dripped into a small culture dish, penicillin small bottle or a small centrifuge tube (generally below 1.5 ml), after 3min of staining, one drop is taken out on a glass slide, then a cover slip is covered, direct microscopic examination is carried out, dead sperms are stained red, living sperms are not stained, and cytoplasm is transparent, 200 sperms are counted for each specimen, and the sperm viability is calculated.
Embodiment two:
examples of the present application experimental comparative analysis examples are provided on the basis of the above examples.
Three cows are randomly selected from a cow breeding factory in Ningxia Longde county, semen is collected, the collected semen is placed in a constant-temperature water bath box for constant-temperature preservation according to the treatment mode of the embodiment, the semen is diluted by a high power by means of an optical microscope, and then the semen is smeared on a glass slide for detection, wherein the detection modes are three detection modes of evaluation scores, eosin Y dye liquor and neutral red dye liquor, and detection information data are shown in table 1:
TABLE 1
As can be seen from the information data in Table 1, when three different detection modes are adopted to detect semen of three cows respectively, the evaluation score, the eosin Y dye liquor and the neutral red dye liquor are all less than 0.1 for detecting data of the same cow, so that the detection mode has high detection precision and can be popularized and used in livestock raising institutions.
Although embodiments of the present application have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the application, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A novel method for sperm viability, comprising the steps of:
s1: reagent preparation
Preparing eosin Y dye liquor and neutral red dye liquor according to the using dosage, and then preparing buffer liquor for later use after the eosin Y dye liquor and neutral red dye liquor are prepared;
s2: semen collection and treatment
Collecting semen from cultured animals, placing the collected semen in a constant-temperature water bath tank for heat preservation treatment, diluting the heat-preserved semen by high power, and estimating the heat-preserved semen to obtain an estimated fraction;
s3: survival rate detection
Dripping 10l semen on a glass slide by adopting a microsampler, detecting the survival rate of sperms on the glass slide by respectively using the eosin Y dye solution and the neutral red dye solution prepared in the step S1, and carrying out statistical analysis by using SPSS software after the detection is finished to obtain a detection result;
s4: conclusion analysis
Combining the estimated score in the step S2 and the detection result in the step S3, thereby obtaining the sperm viability condition of the detected animal.
2. The new method for sperm viability as described in claim 1, wherein in step S1, both eosin Y dye liquor and neutral red dye liquor are purchased from Shanghai reagent three factories and eosin Y dye liquor is prepared using distilled water.
3. The method according to claim 1, wherein in the step S1, the concentration of eosin Y dye solution is 0.15%, the PH of neutral red dye solution is 7.4, and the concentration of buffer solution is 0.01%.
4. A new method for sperm viability as described in claim 1, wherein in step S2, the temperature of the thermostatic waterbath is 37 degrees celsius, and the sperm is subjected to high-power dilution to determine sperm outline and sperm spacing by means of a light microscope.
5. A new method of sperm viability as described in claim 1, wherein in step S2, when estimating sperm in the visual field, 10% of sperm motility proceeds straight at 0.1,20% and proceeds straight at 0.2, and so on, 1 is divided into full.
6. The method according to claim 1, wherein in step S3, 200 sperm cells are counted per sample, and the sperm cell viability is calculated, and the sperm cell viability calculation formula is: survival = (total number of sperm-number of non-linear motile sperm)/total number of sperm 100%.
7. The method according to claim 1, wherein in step S3, when the viability of sperm on the slide is detected by using eosin Y dye solution, 10ta0.15% eosin Y dye solution is added to the slide and mixed uniformly, 2011% nigrosine or aniline blue is added as a background color, then a smear is made from another slide, after natural drying, living sperm is observed under a microscope without staining, cytoplasm is transparent, and dead sperm is stained red.
8. The method according to claim 1, wherein in step S3, when detecting the sperm viability on the slide glass by using neutral red dye solution, the neutral red dye solution and buffer solution are added dropwise to the slide glass, and after mixing uniformly, the slide glass is stained for 2min, and then covered with a cover plate, and the dead sperm is stained red, the living sperm is not stained, and the cytoplasm is transparent under a bright field of 400 times of microscope for half an hour.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311119420.7A CN117147548A (en) | 2023-08-31 | 2023-08-31 | Novel method for sperm survival rate |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311119420.7A CN117147548A (en) | 2023-08-31 | 2023-08-31 | Novel method for sperm survival rate |
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| CN117147548A true CN117147548A (en) | 2023-12-01 |
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| CN202311119420.7A Withdrawn CN117147548A (en) | 2023-08-31 | 2023-08-31 | Novel method for sperm survival rate |
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- 2023-08-31 CN CN202311119420.7A patent/CN117147548A/en not_active Withdrawn
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