CN116735897A - Method for early detection of gender of ewe fetus - Google Patents
Method for early detection of gender of ewe fetus Download PDFInfo
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- CN116735897A CN116735897A CN202310832510.4A CN202310832510A CN116735897A CN 116735897 A CN116735897 A CN 116735897A CN 202310832510 A CN202310832510 A CN 202310832510A CN 116735897 A CN116735897 A CN 116735897A
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- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 42
- 239000000243 solution Substances 0.000 claims description 39
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- 229910052737 gold Inorganic materials 0.000 claims description 27
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 21
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- 239000003085 diluting agent Substances 0.000 claims description 12
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 claims description 12
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- 238000007789 sealing Methods 0.000 claims description 9
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 6
- 241000283707 Capra Species 0.000 claims description 6
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 6
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 6
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- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 3
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 3
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- 235000013855 polyvinylpyrrolidone Nutrition 0.000 claims description 3
- 229920000036 polyvinylpyrrolidone Polymers 0.000 claims description 3
- 239000001509 sodium citrate Substances 0.000 claims description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims description 3
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- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 claims description 3
- 230000001737 promoting effect Effects 0.000 abstract 1
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- 230000020509 sex determination Effects 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
- 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
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
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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
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
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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
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
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Abstract
The invention relates to a method for early detecting the gender of a ewe fetus, which comprises the following steps: collecting sheep blood and separating serum 28-35 days after the breeding of the ewes; inserting the sheep Muller tube inhibition factor AMH test paper into serum for 5-10 seconds, taking out, and observing whether a quality control line C and a detection line T in the test paper develop or not after 15-18 minutes; the color development results were as follows: the test paper quality control line C does not develop color, the test line T develops color, and the test result is invalid; the quality control line C develops color, the detection line T does not develop color, and the ewe fetus is a ewe; and the quality control line C and the detection line T are both developed, which indicates that the female sheep fetus has a male lamb. The sex of the fetuses of the ewes can be safely, conveniently, simply, quickly and accurately detected, the fetuses are not damaged, no pollution is caused, the problem that the supply quantity and the market demand of the ewes of the farm are disjointed is solved, the market demand of the pregnant ewes is balanced and consistent with the high-efficiency slaughtering is met, and reliable technical support is provided for promoting the quick development of the breeding ewes and fattening ewes.
Description
Technical Field
The invention relates to a method for early detecting the sex of a ewe fetus, belonging to the technical field of animal breeding.
Background
In the production process of male and female sheep, due to the obvious difference in growth speed, application and the like of male and female sheep, two different industrial forms and market demands of breeding female sheep and fattening male sheep are formed. In general, after natural hybridization, the proportion of male lambs and female lambs is half, and the conventional sex control semen freezing technology and frozen embryo transfer technology of sheep are not mature, so that the method cannot be applied to the mass production process. Ultrasonic technology can only judge whether a ewe is pregnant or not, but cannot identify the sex of a fetus in gestation period. The growth speed of the male lambs is obviously better than that of the female lambs, so that the feeding cost and income of the male and female lambs are different, classified feeding is required according to the requirements, the feeding cost is reduced as much as possible, and the market requirements are met. For example: when the ewes are required to be bred and expanded, only the fetuses of the ewes are expected to be all the ewes, and even if the breeding cost is slightly high, the final ewe income is not influenced; and the female sheep carrying the male lambs can lead the male lambs carried by the female sheep to enter the production of fattening male sheep under the condition of the same feeding cost, so that the feeding economic benefit is ensured to the greatest extent. Thus, early fetal sex identification in sheep is of great significance in animal husbandry.
At present, the main methods for sex identification of sheep are as follows: 1) The cytogenetic method has 100% accuracy, but the difficulty of obtaining high-quality metaphase chromosome disperse phase is great, the disperse phase obtaining rate is low, and the identification process is long. 2) Biochemistry not only reduces fetal viability due to potential cytotoxicity, but also makes it easy to misjudge a female fetus as male once the X-chromosome of some female fetuses has been inactivated in advance. 3) The cytotoxicity analysis method has high accuracy and is easy to cause death of male fetuses. 4) Although the indirect immunofluorescence method does not damage the fetus and has a sex identification accuracy, it has a disadvantage that the assessment of fluorescence intensity has a certain subjectivity, which makes the sex identification result of the fetus unstable. 5) The blastula formation inhibition method is practical and simple, but is easy to misjudge a part of female fetuses with retarded development as male, and the fetuses just in the mulberry stage are difficult to obtain at the same time without in vitro insemination. 6) The male specific DNA probe method has high accuracy in identifying the sex of the fetus, but the method is limited in popularization and application because the fetus is easy to be damaged due to the large amount of fetal cells (15 or more) used for detection and the identification time is long, and each livestock needs to use the same male specific DNA probe. 7) The PCR amplified DNA fragment method has high discrimination rate, simple operation and high applicability, but the method amplifies large repeated fragments, and the discrimination accuracy rate still has instability in a certain proportion due to the high crossing rate between mammals. 8) The PCR amplification SRY sequence method has high sensitivity and correctness for identifying the sex of the fetus, simple and quick operation, but serious pollution, and needs strict operation program to control the pollution of DNA.
In addition, early fetal sex determination in sheep is limited to the blastocyst stage of 7-8 days of breeding, and has various defects and shortcomings, and no detection means is available for the fetal sex 28-35 days after breeding, so that improvement of the prior art is needed.
Disclosure of Invention
The invention aims to provide a method for early detecting the gender of the ewe fetus, which further improves the accuracy of the identification of the ewe fetus in early stage, ensures that the operation is simpler, more convenient and quicker, does not hurt the existing fetus, avoids pollution, ensures that the technology for early sex identification enters the practical and productive stages, and provides reliable technical support for the high-quality and high-volume production of animal husbandry.
The invention is completed by the following technical scheme: a method for early detection of the gender of a ewe fetus, comprising the steps of:
1) Collecting 1-10 ml ewe blood 28-35 days after ewe breeding, standing in shade for 1-2 hours, and separating serum;
2) Inserting the sheep Muller tube inhibition factor AMH test paper into the serum in the step 1) for 5-10 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 15-18 minutes:
3) The color development results were as follows:
the test paper quality control line C does not develop color, and the detection line T develops color, so that the detection result is invalid and the detection needs to be repeated;
the test paper quality control line C develops color, and the detection line T does not develop color, so that the female sheep fetus is a female lamb;
the test paper quality control line C and the test line T are both developed, which shows that the foetus of the ewe is a male lamb, and the following comparison is carried out by using the developed color and an AMH colorimetric card:
31 When AMH is more than or equal to 2-5mIU/ml, indicating that the female sheep fetus is a male lamb;
32 When AMH is more than or equal to 5-10 mIU/ml, indicating that the fetuses of the ewes are two male lambs;
33 When AMH is more than or equal to 10-25mIU/ml, indicating that the fetuses of the ewes are three male lambs;
34 When AMH is more than or equal to 25-40mIU/ml, indicating that the fetuses of the ewes are four male lambs;
35 When AMH is more than or equal to 40-80mIU/ml, indicating that the fetuses of the ewes are five male lambs.
The step 2) of the Muller tube inhibition factor AMH test paper for sheep is prepared by the following method:
21 The components were prepared as follows:
gold solution: adding 20ml of chloroauric acid solution with the mass concentration of 2% and sodium citrate with the mass concentration of 1% into purified water to 100ml, heating until the solution shows purple red, and cooling to room temperature to obtain a gold solution;
sealing liquid: dissolving 0.005g bovine serum albumin in 300ul purified water, adding 30ul potassium carbonate solution, and uniformly mixing to obtain 330ul sealing solution;
sample pad treatment fluid: 0.605g of tricarboxymethylamino methane and 1.5g of sodium chloride are completely dissolved in 100ml of purified water to obtain sample pad treatment liquid;
colloidal gold conjugate dilution: uniformly mixing 0.605g of tricarboxymethylamino methane, 0.2g of polyvinylpyrrolidone, 5g of sucrose, 0.9g of sodium chloride, 0.5g of bovine serum albumin, 3ml of sheep serum, 0.15ml of Tween 20 and 100ml of purified water to obtain a colloidal gold conjugate diluent;
AMH coating liquid: dissolving 1ml of 100-200mg goat Miaole tube inhibition factor monoclonal alpha sub-level antibody AMHmAbCoating and 1% trehalose solution in mass concentration in 100ml of 0.01M PBS buffer solution, and uniformly mixing to obtain AMH coating solution;
IgG coating solution: 100mg of goat anti-mouse IgG antibody and 1ml of trehalose solution with the mass concentration of 1% are dissolved in 100ml of 0.01M PBS buffer solution, and the mixture is uniformly mixed to obtain IgG coating solution;
22 Film drawing on the bottom plate
22A) Pasting an NC film on the middle part of the upper surface of the PVC plate to obtain a bottom plate;
22B) Uniformly marking the upper end of the NC film on the bottom plate of the step 22A) with IgG coating liquid at the speed of 500mm/s by using an XYZ3010 metal spraying film marking instrument, taking the upper end of the NC film as a quality control line C, uniformly marking the lower end of the NC film with AMH coating liquid as a detection line T, wherein the distance between the quality control line C and the detection line T is 3 mm+/-1 mm, the distance between the quality control line C and the upper end of the NC film is 8 mm+/-1 mm, the distance between the detection line T and the lower end of the NC film is 7 mm+/-1 mm, and then placing the NC film in a drying box, and drying for 18 h+/-2 h at the temperature of 25+/-2 ℃ and the humidity of less than or equal to 30% to obtain a film marking plate;
23 Gold pad preparation
23A) Adding 1.2ml of potassium carbonate solution and 1.5-2mg of AMHmb4 into 100ml of gold solution in the step 21), stirring for 40min, dropwise adding 330ul of sealing solution, stirring for 10min, centrifuging for 40min at the temperature of 4 ℃ and under the condition of 12000r/min until the supernatant becomes water, discarding the supernatant, taking 5.3 ml of residual liquid, adding 40ml of colloidal gold conjugate diluent, and uniformly mixing to form an AMHmb2 composite colloidal gold conjugate diluent solution;
23B) Uniformly spreading 40ml of AMHmb2 composite colloidal gold conjugate diluent on 1080cm 2 Placing the glass cellulose film RB65 on a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a gold pad;
24 Sample pad preparation)
24A) Uniformly spreading the sample pad treatment liquid in the step 21) on a glass cellulose film SB06 according to the amount of 30 ml/sheet, then placing the sample pad treatment liquid in a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 ℃ plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a sample pad;
25 Plate for sticking
25A) Attaching the gold pad in the step 23) to the lower end of the NC film of the film-dividing plate in the step 22B), and enabling the rear end of the gold pad with the thickness of 1-2mm to be attached to the NC film;
25B) Attaching the sample pad of the step 24) to the front end of the gold pad of the step 25A), and enabling the rear end of the sample pad to be completely overlapped on the gold pad, wherein the front end of the sample pad is attached to the front end of the PVC plate;
25C) Attaching a water absorption pad to the rear end of the PVC plate of 25B), and enabling the front end of the water absorption pad with the thickness of 1-2mm to be attached to the rear end of the NC film;
26 Cutting the veneer in the step 25C) into test strips with the thickness of 3.0+/-0.5 mm to obtain the Mueller tube inhibition factor AMH test paper for sheep.
The invention has the following advantages and effects: by adopting the scheme, the sex of the ewe fetus can be safely, conveniently, simply, quickly and accurately detected, the fetus is not damaged, the pollution is avoided, low-cost and high-quality guarantee is provided for subsequent feeding, and the development of animal husbandry is promoted. The detection result of the invention can meet the market demands of male lambs and female lambs, and can detect the number of male lambs born by the female sheep, so that pregnant female sheep can be priced and sold or fed differently according to the gender of the fetus, thereby reducing the breeding cost and increasing the breeding income. Not only realizes the quick turnover and virtuous circle of invested funds, but also solves the problem of the dislocation of the actual supply and market demand of the ewes in the existing production process, fundamentally meets the market demand of pregnant ewes and quick fattening rams, and provides reliable technical support for improving the supply demand of breeding ewes and fattening rams in the mutton sheep industry in China.
Drawings
FIG. 1 is a schematic diagram of a structure of a sheep Muller tube inhibition factor AMH test paper;
FIG. 2 is an AMH colorimetric card.
Detailed Description
The invention is further described below with reference to examples.
The Muller tube inhibition factor AMH test paper for sheep of example 1 was prepared by the following method:
21 The components were prepared as follows:
gold solution: adding 20ml of chloroauric acid solution with the mass concentration of 2% and sodium citrate with the mass concentration of 1% into purified water to 100ml, heating until the solution shows purple red, and cooling to room temperature to obtain a gold solution;
sealing liquid: dissolving 0.005g bovine serum albumin in 300ul purified water, adding 30ul potassium carbonate solution, and uniformly mixing to obtain 330ul sealing solution;
sample pad treatment fluid: 0.605g of tricarboxymethylamino methane and 1.5g of sodium chloride are completely dissolved in 100ml of purified water to obtain sample pad treatment liquid;
colloidal gold conjugate dilution: uniformly mixing 0.605g of tricarboxymethylamino methane, 0.2g of polyvinylpyrrolidone, 5g of sucrose, 0.9g of sodium chloride, 0.5g of bovine serum albumin, 3ml of sheep serum, 0.15ml of Tween 20 and 100ml of purified water to obtain a colloidal gold conjugate diluent;
AMH coating liquid: dissolving 1ml of 100-200mg goat Miaole tube inhibition factor monoclonal alpha sub-level antibody AMHmAbCoating and 1% trehalose solution in mass concentration in 100ml of 0.01M PBS buffer solution, and uniformly mixing to obtain AMH coating solution;
IgG coating solution: 100mg of goat anti-mouse IgG antibody and 1ml of trehalose solution with the mass concentration of 1% are dissolved in 100ml of 0.01M PBS buffer solution, and the mixture is uniformly mixed to obtain IgG coating solution;
22 Film drawing on the bottom plate
22A) Pasting an NC film on the middle part of the upper surface of the PVC plate to obtain a bottom plate;
22B) Uniformly marking the upper end of the NC film on the bottom plate of the step 22A) with IgG coating liquid at the speed of 500mm/s by using an XYZ3010 metal spraying film marking instrument, taking the upper end of the NC film as a quality control line C, uniformly marking the lower end of the NC film with AMH coating liquid as a detection line T, wherein the distance between the quality control line C and the detection line T is 3 mm+/-1 mm, the distance between the quality control line C and the upper end of the NC film is 8 mm+/-1 mm, the distance between the detection line T and the lower end of the NC film is 7 mm+/-1 mm, and then placing the NC film in a drying box, and drying for 18 h+/-2 h at the temperature of 25+/-2 ℃ and the humidity of less than or equal to 30% to obtain a film marking plate;
23 Gold pad preparation
23A) Adding 1.2ml of potassium carbonate solution and 1.5-2mg of AMHmb4 into 100ml of gold solution in the step 21), stirring for 40min, dropwise adding 330ul of sealing solution, stirring for 10min, centrifuging for 40min at the temperature of 4 ℃ and under the condition of 12000r/min until the supernatant becomes water, discarding the supernatant, taking 5.3 ml of residual liquid, adding 40ml of colloidal gold conjugate diluent, and uniformly mixing to form an AMHmb2 composite colloidal gold conjugate diluent solution;
23B) Uniformly spreading 40ml of AMHmb2 composite colloidal gold conjugate diluent on 1080cm 2 Placing the glass cellulose film RB65 on a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a gold pad;
24 Sample pad preparation)
24A) Uniformly spreading the sample pad treatment liquid in the step 21) on a glass cellulose film SB06 according to the amount of 30 ml/sheet, then placing the sample pad treatment liquid in a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 ℃ plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a sample pad;
25 Plate for sticking
25A) Attaching the gold pad in the step 23) to the lower end of the NC film of the film-dividing plate in the step 22B), and enabling the rear end of the gold pad with the thickness of 1-2mm to be attached to the NC film;
25B) Attaching the sample pad of the step 24) to the front end of the gold pad of the step 25A), and enabling the rear end of the sample pad to be completely overlapped on the gold pad, wherein the front end of the sample pad is attached to the front end of the PVC plate;
25C) Attaching a water absorption pad to the rear end of the PVC plate of 25B), and enabling the front end of the water absorption pad with the thickness of 1-2mm to be attached to the rear end of the NC film;
26 Cutting the veneer in the step 25C) into test strips with the thickness of 3.0+/-0.5 mm to obtain the Mueller tube inhibition factor AMH test paper for sheep.
Example 2a method for early detection of fetal gender in ewes comprising the steps of:
1) Collecting 2ml of ewe blood 28 days after the ewe is bred, standing in a shady place for 12 hours, and separating serum;
2) Inserting the sheep Muller tube inhibition factor AMH test paper into the serum in the step 1) for 52 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 15 minutes:
3) The color development results were as follows:
the test paper quality control line C does not develop color, and the detection line T develops color, which indicates that the detection result is invalid and the detection needs to be repeated 2.
Example 3a method for early detection of fetal gender in ewes comprising the steps of:
1) Collecting 10 ml ewe blood 30 days after the ewe is bred, standing in a shady place for 2 hours, and separating serum;
2) Inserting the sheep Muller tube inhibition factor AMH test paper into the serum in the step 1) for 10 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 15 minutes:
3) The color development results were as follows:
and (3) developing color by a test paper quality control line C, and not developing color by a detection line T, so as to show that the ewe fetus is a ewe.
Example 4a method for early detection of fetal gender in ewes comprising the steps of:
1) Collecting 5ml ewe blood 33 days after the ewe is bred, standing in a shady place for 1.5 hours, and separating serum;
2) Inserting the sheep Muller tube inhibition factor AMH test paper into the serum in the step 1) for 8 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 16 minutes:
3) The color development results were as follows:
the test paper quality control line C and the test line T are developed, and after the developed color is compared with an AMH colorimetric card:
AMH was 4mIU/ml, indicating that the foetus of the ewe was a male lamb.
Example 5a method for early detection of fetal gender in a ewe comprising the steps of:
1) Collecting 8 ml ewe blood 35 days after the ewe is bred, standing in a shade place for 2 hours, and separating serum;
2) Inserting the sheep Muller tube inhibition factor AMH test paper into the serum in the step 1) for 8 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 17 minutes:
3) The color development results were as follows:
the test paper quality control line C and the test line T are developed, and after the developed color is compared with an AMH colorimetric card:
AMH was 10 mIU/ml, indicating that the foetus of the ewe was two male lambs.
Example 6 a method for early detection of fetal gender in a ewe comprising the steps of:
1) Collecting 6 ml ewe blood 30 days after the ewe is bred, standing in a shady place for 2 hours, and separating serum;
2) Inserting the sheep Muller tube inhibition factor AMH test paper into the serum in the step 1) for 8 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 16 minutes:
3) The color development results were as follows:
the test paper quality control line C and the test line T are developed, and after the developed color is compared with an AMH colorimetric card:
AMH was 20mIU/ml, indicating that the foetus of the ewe was three male lambs.
Example 7 a method for early detection of fetal gender in a ewe comprising the steps of:
1) Collecting 4ml of ewe blood 31 days after the ewe is bred, standing in a shady place for 1 hour, and separating serum;
2) Inserting the sheep Muller tube inhibition factor AMH test paper into the serum in the step 1) for 6 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 18 minutes:
3) The color development results were as follows:
the test paper quality control line C and the test line T are developed, and after the developed color is compared with an AMH colorimetric card:
AMH was 35mIU/ml, indicating that the fetuses of the ewes were four male lambs.
The effect of the invention is demonstrated by comparative experiments:
36 ewes are detected by the method of the embodiment of the invention, and the detection results are as follows:
the fetuses of 17 ewes are female lambs; the fetuses of 16 ewes were male lambs, in which: the fetuses of the 4 ewes are detected to be respectively four male lambs, the fetuses of the 3 ewes are respectively three male lambs, the fetuses of the 6 ewes are respectively two male lambs, and the fetuses of the 3 ewes are respectively one male lamb; only 3 fetuses have no detected sex, and the result of ultrasonic detection is that the fetuses of the ewes stop developing and the fetuses die.
Claims (2)
1. A method for early detection of the gender of a ewe fetus, comprising the steps of:
1) Collecting 1-10 ml ewe blood 28-35 days after ewe breeding, standing in shade for 1-2 hours, and separating serum;
2) Inserting the sheep Muller tube inhibition factor AMH test paper into the serum in the step 1) for 5-10 seconds, taking out, and observing whether the quality control line C and the detection line T in the test paper develop or not after 15-18 minutes:
3) The color development results were as follows:
the test paper quality control line C does not develop color, and the detection line T develops color, so that the detection result is invalid and the detection needs to be repeated;
the test paper quality control line C develops color, and the detection line T does not develop color, so that the female sheep fetus is a female lamb;
the test paper quality control line C and the test line T are both developed, which shows that the foetus of the ewe is a male lamb, and the following comparison is carried out by using the developed color and an AMH colorimetric card:
31 When AMH is more than or equal to 2-5mIU/ml, indicating that the female sheep fetus is a male lamb;
32 When AMH is more than or equal to 5-10 mIU/ml, indicating that the fetuses of the ewes are two male lambs;
33 When AMH is more than or equal to 10-25mIU/ml, indicating that the fetuses of the ewes are three male lambs;
34 When AMH is more than or equal to 25-40mIU/ml, indicating that the fetuses of the ewes are four male lambs;
35 When AMH is more than or equal to 40-80mIU/ml, indicating that the fetuses of the ewes are five male lambs.
2. The method for early detecting the gender of the fetuses of the ewes according to claim 1, wherein the step 2) of the test paper for detecting the AMH (Muller tube inhibition factor) of the sheep is prepared by the following method:
21 The components were prepared as follows:
gold solution: adding 20ml of chloroauric acid solution with the mass concentration of 2% and sodium citrate with the mass concentration of 1% into purified water to 100ml, heating until the solution shows purple red, and cooling to room temperature to obtain a gold solution;
sealing liquid: dissolving 0.005g bovine serum albumin in 300ul purified water, adding 30ul potassium carbonate solution, and uniformly mixing to obtain 330ul sealing solution;
sample pad treatment fluid: 0.605g of tricarboxymethylamino methane and 1.5g of sodium chloride are completely dissolved in 100ml of purified water to obtain sample pad treatment liquid;
colloidal gold conjugate dilution: uniformly mixing 0.605g of tricarboxymethylamino methane, 0.2g of polyvinylpyrrolidone, 5g of sucrose, 0.9g of sodium chloride, 0.5g of bovine serum albumin, 3ml of sheep serum, 0.15ml of Tween 20 and 100ml of purified water to obtain a colloidal gold conjugate diluent;
AMH coating liquid: dissolving 1ml of 100-200mg goat Miaole tube inhibition factor monoclonal alpha sub-level antibody AMHmAbCoating and 1% trehalose solution in mass concentration in 100ml of 0.01M PBS buffer solution, and uniformly mixing to obtain AMH coating solution;
IgG coating solution: 100mg of goat anti-mouse IgG antibody and 1ml of trehalose solution with the mass concentration of 1% are dissolved in 100ml of 0.01M PBS buffer solution, and the mixture is uniformly mixed to obtain IgG coating solution;
22 Film drawing on the bottom plate
22A) Pasting an NC film on the middle part of the upper surface of the PVC plate to obtain a bottom plate;
22B) Uniformly marking the upper end of the NC film on the bottom plate of the step 22A) with IgG coating liquid at the speed of 500mm/s by using an XYZ3010 metal spraying film marking instrument, taking the upper end of the NC film as a quality control line C, uniformly marking the lower end of the NC film with AMH coating liquid as a detection line T, wherein the distance between the quality control line C and the detection line T is 3 mm+/-1 mm, the distance between the quality control line C and the upper end of the NC film is 8 mm+/-1 mm, the distance between the detection line T and the lower end of the NC film is 7 mm+/-1 mm, and then placing the NC film in a drying box, and drying for 18 h+/-2 h at the temperature of 25+/-2 ℃ and the humidity of less than or equal to 30% to obtain a film marking plate;
23 Gold pad preparation
23A) Adding 1.2ml of potassium carbonate solution and 1.5-2mg of AMHmb4 into 100ml of gold solution in the step 21), stirring for 40min, dropwise adding 330ul of sealing solution, stirring for 10min, centrifuging for 40min at the temperature of 4 ℃ and under the condition of 12000r/min until the supernatant becomes water, discarding the supernatant, taking 5.3 ml of residual liquid, adding 40ml of colloidal gold conjugate diluent, and uniformly mixing to form an AMHmb2 composite colloidal gold conjugate diluent solution;
23B) Uniformly spreading 40ml of AMHmb2 composite colloidal gold conjugate diluent on 1080cm 2 Placing the glass cellulose film RB65 on a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a gold pad;
24 Sample pad preparation)
24A) Uniformly spreading the sample pad treatment liquid in the step 21) on a glass cellulose film SB06 according to the amount of 30 ml/sheet, then placing the sample pad treatment liquid in a drying oven, and drying for 18 hours plus or minus 2 hours at the temperature of 25 ℃ plus or minus 2 ℃ and the humidity of less than or equal to 30 percent to obtain a sample pad;
25 Plate for sticking
25A) Attaching the gold pad in the step 23) to the lower end of the NC film of the film-dividing plate in the step 22B), and enabling the rear end of the gold pad with the thickness of 1-2mm to be attached to the NC film;
25B) Attaching the sample pad of the step 24) to the front end of the gold pad of the step 25A), and enabling the rear end of the sample pad to be completely overlapped on the gold pad, wherein the front end of the sample pad is attached to the front end of the PVC plate;
25C) Attaching a water absorption pad to the rear end of the PVC plate of 25B), and enabling the front end of the water absorption pad with the thickness of 1-2mm to be attached to the rear end of the NC film;
26 Cutting the veneer in the step 25C) into test strips with the thickness of 3.0+/-0.5 mm to obtain the Mueller tube inhibition factor AMH test paper for sheep.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310832510.4A CN116735897A (en) | 2023-07-08 | 2023-07-08 | Method for early detection of gender of ewe fetus |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310832510.4A CN116735897A (en) | 2023-07-08 | 2023-07-08 | Method for early detection of gender of ewe fetus |
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