CN111562397A - Method for detecting mating opportunity of cattle - Google Patents
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- CN111562397A CN111562397A CN202010502250.0A CN202010502250A CN111562397A CN 111562397 A CN111562397 A CN 111562397A CN 202010502250 A CN202010502250 A CN 202010502250A CN 111562397 A CN111562397 A CN 111562397A
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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
- G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- 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
- G01N2333/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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Abstract
The invention provides a method for detecting the breeding opportunity of cattle, which comprises the following steps: collecting urine of cow which urinates again 1-2 hours after last urination; inserting bovine luteinizing hormone LH test paper into the urine obtained in the step 1) for 5-10 seconds, and observing whether a quality control line C and a detection line T in the test paper are colored or not after 15-18 minutes: the quality control line C is not colored, the detection line T is colored, and the detection result is invalid; the quality control line C is colored, the detection line T is not colored, and the luteinizing hormone LH level of the cow is low; the quality control line C and the detection line T are developed and compared with an LH colorimetric card; LH < 10mIU/ml, immature egg cells of the cow, and incapability of mating; detecting the cow with LH at 10-25 mIU/ml in ovulation period, and repeating the steps 1) -3) every 4 hours until LH reaches 45-100 mIU/m, and the ovulation time of the cow is 5-10 hours, and mating.
Description
Technical Field
The invention relates to a detection method, in particular to a method for detecting the mating opportunity of cattle, belonging to the technical field of animal detection methods.
Background
Currently, in the breeding process of cattle, the breeding or insemination time of cattle is usually selected in the natural estrus of cows. In the actual production, the ovulation time of the cow is judged mainly by trying the situation, observing the behavior, checking the vagina and the secretion thereof, checking the touch ovary of the rectum and the like, and natural mating or artificial insemination is guided. Generally, sperm is inseminated 1-2 times in one estrus, because sperm reaches the ampulla of the oviduct to obtain capacitation and finally has fertilization capability, generally several hours are needed, and the survival time in the reproductive tract is longer than the time for maintaining the fertilization capability of the ovum, the first inseminating is carried out by estimating the hour before ovulation, the second inseminating is carried out before estimating the loss of the fertilization capability of the ovum, but in the actual production process, the time is different from individual to individual and is not easy to be accurately mastered, so the time is fixed, the interval time between two inseminations is fixed at 8-10 hours, the ovulation of the cow occurs 4-16 hours after estrus stops, the individual difference is large, the cow can be pregnant by carrying out inseminations for multiple times, but the multiple inseminations can also cause antibodies for resisting the sperm to be generated in the cow, and the infertility is formed in the later period. The maturation of cow ova and the rupture of follicles are regulated by the peak of luteinizing hormone LH, ovulation 20-24 hours after the peak of luteinizing hormone LH, and the closer to the ovulation time, the mating or insemination is performed, the higher the mating success rate of cows is.
Disclosure of Invention
In view of the physiological characteristics of the cattle, the invention provides a method for detecting the mating time of the cattle, so as to accurately predict the ovulation time of the cattle within 5-10 hours, thereby improving the success rate of single mating of the cattle.
The invention is completed by the following technical scheme: a method for detecting mating opportunity of cattle is characterized by comprising the following steps:
1) collecting 1-10ml of urine of cow which urinates again 1-2 hours after last urination;
2) inserting bovine luteinizing hormone LH test paper into the urine obtained in the step 1) for 5-10 seconds, taking out, and observing whether a quality control line C and a detection line T in the test paper are colored or not after 15-18 minutes:
3) the color development results were as follows:
the test paper quality control line C is not colored, and the detection line T is colored, so that the detection result is invalid and needs to be detected again;
the test paper quality control line C is colored, and the detection line T is not colored, which indicates that the luteinizing hormone LH level of the cow is low and needs to be detected every other day;
the test paper quality control line C and the detection line T are developed, and the development and the LH colorimetric card are used for carrying out the following comparison;
31) when the luteinizing hormone LH is less than or equal to 10mIU/ml, the luteinizing hormone LH of the cow is at a basic level, the egg cells are not mature and can not be bred;
32) when the luteinizing hormone LH is 10-25 mIU/ml, indicating that the cow starts to enter the ovulation period, the steps 1) -3) are repeated every 4 hours for three times every day until the luteinizing hormone LH of the cow reaches 45-100 mIU/m, indicating that the peak of the luteinizing hormone LH of the cow comes 5-10 hours away from the ovulation time;
4) and (4) carrying out natural or artificial hybridization according to the detection result of the step 3).
During natural or artificial breeding in the step 4), the survival time of the sperms of the bulls entering the bodies of the cows is as follows: 24-36 hours.
The luteinizing hormone LH test paper for the cattle in the step 2) is prepared by the following method:
21) the components were prepared as follows:
gold solution: adding 2ml of chloroauric acid solution with the mass concentration of 1% and 2ml of sodium citrate with the mass concentration of 1% into purified water to 100ml, heating until the solution is purple red, and cooling to room temperature to obtain gold solution;
sealing liquid: dissolving 0.005g bovine serum albumin in 300ul of purified water, adding 30ul of potassium carbonate solution, and uniformly mixing to obtain 330ul of confining liquid;
sample pad treatment solution: completely dissolving 0.605g of tricarboxymethylaminomethane and 1.5g of sodium chloride in 100ml of purified water to obtain a sample pad treatment solution;
colloidal gold conjugate dilution: uniformly mixing 0.605g of tricarboxymethylaminomethane, 0.2g of polyvinylpyrrolidone, 5g of sucrose, 0.9g of sodium chloride, 0.5g of bovine serum albumin, 3ml of bovine serum and 0.15ml of Tween 20 with 100ml of purified water to obtain a colloidal gold conjugate diluent;
LH coating liquid: dissolving 1ml of trehalose solution with the LHmABCcuring antibody of 100-200mg and the mass concentration of 1% in 100ml of PBS buffer solution of 0.01M, and uniformly mixing to obtain LH coating solution;
IgG coating solution: dissolving 1ml of trehalose solution with the mass concentration of 1 percent in 100ml of 0.01M PBS buffer solution by using 100-200mg bovine luteinizing hormone monoclonal alpha sub-level antibody LHmAlbCoting, and uniformly mixing to obtain IgG coating solution;
22) scribing a film on a substrate
22A) Attaching an NC film to the middle part of the upper surface of the PVC plate to obtain a bottom plate;
22B) uniformly scratching the IgG coating solution in the step 21) on the upper end of the NC membrane on the bottom plate in the step 22A) at the speed of 500mm/s according to the amount of 0.8 mu l/cm to be used as a quality control line C, uniformly scratching the LH coating solution on the lower end of the NC membrane to be used as a detection line T, wherein the distance between the quality control line C and the detection line T is 6mm +/-1 mm, the distance between the quality control line C and the upper end of the NC membrane is 8mm +/-1 mm, the distance between the detection line T and the lower end of the NC membrane is 7mm +/-1 mm, then putting the test line T into a drying box, and drying the test line T for 18h +/-2 h at the temperature of 25 +/-2 ℃ and the humidity of less than or equal to;
23) preparation of gold pad
23A) Adding 1.2ml of potassium carbonate solution and 1.5-2mg of LHmb2 into 100ml of the gold solution obtained in the step 21), stirring for 40min, dropwise adding 330ul of confining liquid, stirring for 10min, centrifuging for 40min at 4 ℃ and 12000r/min until the supernatant becomes water, removing the supernatant, taking 5.3ml of residual liquid, adding 40ml of colloidal gold conjugate diluent, and uniformly mixing;
23B) uniformly spreading 40ml of colloidal gold conjugate diluted solution on 1080cm2Putting the glass cellulose membrane on a drying oven, and drying at 25 +/-2 ℃ for 18 +/-2 h at the humidity of less than or equal to 30% to obtain a gold pad;
24) sample pad preparation
24A) Uniformly spreading the sample pad treatment solution obtained in the step 21) on a glass cellulose membrane according to the amount of 30 ml/sheet, then putting the glass cellulose membrane into a drying oven, and drying the glass cellulose membrane for 18h +/-2 h at the temperature of 25 +/-2 ℃ and the humidity of less than or equal to 30% to obtain a sample pad;
25) pasting board
25A) Attaching the gold pad of the step 23) to the lower end of the NC film of the film scribing plate of the step 22B), and lapping the rear end of the gold pad of 1-2mm on the NC film;
25B) attaching the sample pad of the step 24) on the front end of the gold pad of the step 25A), and completely lapping the rear end of the sample pad on the gold pad, wherein the front end of the sample pad is attached to the front end of the PVC plate;
25C) attaching the water absorption pad to the rear end of the PVC plate of 25B), and lapping the front end of the water absorption pad with the diameter of 1-2mm on the rear end of the NC film;
26) cutting the pasting plate in the step 25C) into test strips with the thickness of 3.0 +/-0.5 mm to obtain the test strips for detecting bovine luteinizing hormone LH.
The invention has the following advantages and effects: by adopting the scheme, serum or urine of the cow can be conveniently extracted in the estrus period of the cow, the luteinizing hormone LH detection test paper provided by the invention is used for detecting the luteinizing hormone LH, the ovulation time of the cow can be accurately predicted within 5-10 hours by detecting the luteinizing hormone LH level of the cow and comparing the level with a colorimetric card, and the survival time of sperms of a bull entering the cow is 24-36 hours, so that the mating success rate of the sheep can be greatly improved, and the successful mating can be realized by one-time natural mating or artificial insemination. Fundamentally has solved prior art and can only be through the visual inspection cow oestrus, and carry out natural mating or artificial insemination and lead to needing to carry out many times mating or insemination and can succeed drawback, has practiced thrift a large amount of manpowers, material resources, financial resources, time, improves the reproductive rate of sheep.
Drawings
FIG. 1 is a schematic structural diagram of an LH test strip;
FIG. 2 is an LH color chart.
Detailed Description
The invention is further illustrated by the following examples.
Example 1
The test paper for detecting bovine luteinizing hormone LH is prepared by the following steps:
1) the components were prepared as follows:
11) gold solution: adding 2ml of chloroauric acid solution with the mass concentration of 1% and 2ml of sodium citrate with the mass concentration of 1% into purified water to 100ml, heating until the solution is purple red, and cooling to room temperature to obtain gold solution;
12) sealing liquid: dissolving 0.005g bovine serum albumin in 300ul of purified water, adding 30ul of potassium carbonate solution, and uniformly mixing to obtain 330ul of confining liquid;
13) sample pad treatment solution: completely dissolving 0.605g of tricarboxymethylaminomethane and 1.5g of sodium chloride in 100ml of purified water to obtain a sample pad treatment solution;
14) colloidal gold conjugate dilution: uniformly mixing 0.605g of tricarboxymethylaminomethane, 0.2g of polyvinylpyrrolidone, 5g of sucrose, 0.9g of sodium chloride, 0.5g of bovine serum albumin, 3ml of sheep serum and 0.15ml of Tween 20 with 100ml of purified water to obtain a colloidal gold conjugate diluent;
15) LH coating liquid: dissolving 1ml of 200mg bovine luteinizing hormone monoclonal alpha-subunit antibody LHmABCOting and 1% trehalose solution in 100ml of 0.01M PBS buffer solution, and mixing uniformly to obtain LH coating solution;
16) IgG coating solution: dissolving 100mg of goat anti-mouse IgG antibody and 1ml of trehalose solution with the mass concentration of 1% in 100ml of 0.01MPBS buffer solution, and uniformly mixing to obtain an IgG coating solution;
the amount of each component is used for 5 PVC plates of 6cm multiplied by 30cm, and each PVC plate is cut into 100 test strips of 3mm multiplied by 6 cm;
2) scribing a film on a substrate
2A) Respectively sticking NC films to the middle parts of the upper surfaces of the 5 PVC plates to obtain a bottom plate;
2B) uniformly scratching the IgG coating liquid in the step 16) on the upper end of an NC film on the bottom plate in the step 2A) as a quality control line C and the LH coating liquid in the step 15) on the lower ends of 5 NC films on the bottom plate in the step 2A) as a detection line T at the speed of 500mm/s by using an XYZ3010 gold spraying and film scratching instrument, wherein the distance between the quality control line C and the detection line T is 6mm +/-1 mm, the distance between the quality control line C and the upper end of the NC film is 8mm +/-1 mm, the distance between the detection line T and the lower end of the NC film is 7mm +/-1 mm, then putting the test line T into a drying box, and drying for 18h +/-2 h at the temperature of 25 +/-2 ℃ and the humidity of less than or equal to 30% to obtain a film scratching plate;
3) preparation of gold pad
3A) Adding 1.2ml of potassium carbonate solution and 1.5-2mg of LHmb2 into 100ml of the gold solution obtained in the step 11), stirring for 40min, dropwise adding 330ul of the confining liquid obtained in the step 12), stirring for 10min, centrifuging for 40min at 4 ℃ and 12000r/min until the supernatant becomes water, removing the supernatant, taking 5.3ml of residual liquid, adding 40ml of the colloidal gold conjugate diluent obtained in the step 14), and uniformly mixing to obtain 45.3ml of mixed liquid of the colloidal gold conjugate diluent;
3B) uniformly spreading 45.3ml of the mixed solution of the colloidal gold conjugate diluent in the step 3A) on 5 pieces of glass cellulose membranes RB65, wherein the size of each glass cellulose membrane RB65 is 6mm multiplied by 30cm, then putting the glass cellulose membranes RB65 into a drying oven, and drying the glass cellulose membranes RB65 for 18h +/-2 h at the temperature of 25 +/-2 ℃ and the humidity of less than or equal to 30 percent to obtain a gold pad;
4) sample pad preparation
4A) Uniformly spreading the sample pad treatment solution obtained in the step 13) on 5 pieces of glass cellulose membranes SB06 according to the amount of 30 ml/piece, wherein the size of each glass cellulose membrane RB65 is 20mm multiplied by 30cm, then putting the glass cellulose membranes RB65 into a drying oven, and drying the glass cellulose membranes RB65 for 18h +/-2 h at the temperature of 25 +/-2 ℃ and the humidity of less than or equal to 30 percent to obtain a sample pad;
5) preparation of each flitch
5A) Attaching the gold pad in the step 3) to the lower end of the NC film of the film scribing plate in the step 2B), and lapping the rear end of the gold pad with the diameter of 1-2mm on the NC film;
5B) attaching the sample pad in the step 4) to the front end of the gold pad in the step 5A), and completely lapping the rear end of the sample pad on the gold pad, wherein the front end of the sample pad is attached to the front end of the PVC plate;
5C) attaching the water absorption pad to the rear end of the PVC plate of 5B), and lapping the front end of the water absorption pad with the diameter of 1-2mm on the rear end of the NC film;
6) cutting each patch plate obtained in the step 5C) into 100 test strips with the size of 6cm multiplied by 3mm, and boxing to obtain 500 test strips for detecting luteinizing hormone LH, wherein the structure of each test strip for detecting luteinizing hormone LH is shown in figure 1.
Example 2
A method for detecting mating timing of cattle, comprising the steps of:
1) sampling, and collecting 10ml of urine of the cow which urinates again 1 hour after the last urination;
2) detecting, inserting a test paper for detecting luteinizing hormone LH in example 1 into the urine in the step 1), taking out the test paper for 10 seconds, and observing whether the quality control line C and the detection line T in the test paper are colored after 15 minutes:
3) the detection results are as follows:
the test paper quality control line C is not colored, and the detection line T is colored, which indicates that the detection result is invalid;
4) repeating the steps 1) and 2) again to obtain the following detection results:
the test paper quality control line C and the detection line T are developed, and the development on the test paper is compared with an LH colorimetric card (shown in figure 2) in the following way;
41) luteinizing hormone LH < 10mIU/ml, indicating that the cow has basal luteinizing hormone LH, immature egg cells and can not be bred.
Example 3
A method for detecting mating timing of cattle, comprising the steps of:
1) sampling, and collecting 8ml of urine obtained by re-urination of the cow for 1 hour after the last urination;
2) detecting, inserting a test paper for detecting luteinizing hormone LH in example 1 into the urine in the step 1), taking out the test paper for 8 seconds, and observing whether the quality control line C and the detection line T in the test paper are colored after 16 minutes:
3) the detection results are as follows:
the test paper quality control line C is colored, the detection line T is not colored, the luteinizing hormone LH level of the cow is low, and the steps 1) and 2) are repeated after every other day: the test paper quality control line C and the detection line T are developed, and the development on the test paper is compared with an LH color card (shown in figure 2) as follows;
luteinizing hormone LH of 20mIU/ml, which indicates that the cow starts to enter the ovulation period, and then the steps 1) and 2) are repeated every 4 hours, the detection is carried out three times every day, and the luteinizing hormone LH of the cow reaches 60mIU/m in the next day, which indicates that the peak of the luteinizing hormone LH of the cow is generated at the following ovulation time: 5-10 hours;
4) according to the detection result of the step 3), natural mating is carried out, estrus is not produced, the cow is pregnant, and then a calf is born.
Example 4
A method for detecting mating timing of cattle, comprising the steps of:
1) collecting 5ml of urine of the cow which urinates again 2 hours after last urination;
2) detecting, inserting a test paper for detecting luteinizing hormone LH in example 1 into the serum in the step 1), taking out for 8 seconds, and observing whether the quality control line C and the detection line T in the test paper are colored after 16 minutes:
3) the detection results are as follows:
the test paper quality control line C and the detection line T are developed, and the development on the test paper is compared with an LH colorimetric card (shown in figure 2) in the following way;
luteinizing hormone LH of 10mIU/ml, which indicates that the cow starts to enter the ovulation period, and then the steps 1) and 2) are repeated every 4 hours, the detection is carried out three times every day, and the luteinizing hormone LH of the cow reaches 80mIU/m in the next day, which indicates that the peak of the luteinizing hormone LH of the cow is generated at the following ovulation time: 5-10 hours;
4) according to the detection result of the step 3), natural mating is carried out, estrus is not produced, the cow is pregnant, and then a calf is born.
Example 5
The method for detecting the hybridization time of the sheep comprises the following steps:
1) sampling, and collecting 10ml of urine of the cow which urinates again 1 hour after the last urination;
2) detecting, inserting a test paper for detecting luteinizing hormone LH in example 1 into the urine in the step 1), taking out the test paper for 7 seconds, and observing whether the quality control line C and the detection line T in the test paper are colored after 16 minutes:
3) the detection results are as follows:
the test paper quality control line C and the detection line T are developed, and the development on the test paper is compared with an LH colorimetric card (shown in figure 2) in the following way;
luteinizing hormone LH of 20mIU/ml, which indicates that the cow starts to enter the ovulation period, and then the steps 1) and 2) are repeated every 4 hours, the detection is carried out three times every day, and the luteinizing hormone LH of the cow reaches 60mIU/m in the next day, which indicates that the peak of the luteinizing hormone LH of the cow occurs, and is far from the ovulation time: 5-10 hours;
4) according to the detection result of the step 3), artificial insemination is carried out conventionally, and then the cow does not have heat again, which indicates that the cow is pregnant and then produces a calf.
Example 6
The method for detecting the hybridization time of the sheep comprises the following steps:
1) sampling, and collecting 8ml of urine of the ewe which urinates 2 hours after the last urination;
2) detecting, inserting a test paper for detecting luteinizing hormone LH in example 1 into the urine in the step 1), taking out the test paper for 7 seconds, and observing whether the quality control line C and the detection line T in the test paper are colored after 17 minutes:
3) the detection results are as follows:
the test paper quality control line C and the detection line T are developed, and the development on the test paper is compared with an LH colorimetric card (shown in figure 2) in the following way;
luteinizing hormone LH of 20mIU/ml, which indicates that the cow starts to enter the ovulation period, and then the steps 1) and 2) are repeated every 4 hours, the detection is carried out three times every day, and the luteinizing hormone LH of the sheep ewe reaches 100mIU/m on the next day, which indicates that the peak of the luteinizing hormone LH of the cow occurs, and is far from the ovulation time: 5-10 hours;
4) according to the detection result of the step 3), artificial insemination is carried out conventionally, and then the cow does not have heat again, which indicates that the cow is pregnant and then produces a calf.
The LH colorimetric cards are all conventional products.
The effect of the invention is demonstrated by comparative experiments as follows:
80 cows were each tested by the method of examples 3-6, randomly divided into four groups, and tested by the same method as in examples 3-6, and the test results were as follows:
the first group had 16 pregnancies and 4 estrus returns;
the second group had 18 pregnancies and 2 estrus returns;
the third group had 15 pregnancies and 5 return events;
the fourth group has 17 pregnancies and 3 returning events, and the mating success rate is more than 80%.
Comparative example 1
Taking 30 cows, and naturally breeding according to the conventional method, wherein 15 cows are pregnant, and the rest 15 cows return to the estrus, and the success rate of breeding is 50%.
Comparative example 2
Taking 30 cows, and carrying out artificial hybridization according to the conventional method, wherein 16 cows are pregnant, and the rest 14 cows return to the estrus, and the success rate of the hybridization is 53%.
Claims (2)
1. A method for detecting mating opportunity of cattle is characterized by comprising the following steps:
1) collecting 1-10ml of urine of cow which urinates again 1-2 hours after last urination;
2) inserting bovine luteinizing hormone LH test paper into the urine obtained in the step 1) for 5-10 seconds, taking out, and observing whether a quality control line C and a detection line T in the test paper are colored or not after 15-18 minutes:
3) the color development results were as follows:
the test paper quality control line C is not colored, and the detection line T is colored, so that the detection result is invalid and needs to be detected again;
the test paper quality control line C is colored, and the detection line T is not colored, which indicates that the luteinizing hormone LH level of the cow is low and needs to be detected every other day;
the test paper quality control line C and the detection line T are developed, and the development and the LH colorimetric card are used for carrying out the following comparison;
31) when the luteinizing hormone LH is less than or equal to 10mIU/ml, the luteinizing hormone LH of the cow is at a basic level, the egg cells are not mature and can not be bred;
32) when the luteinizing hormone LH is 10-25 mIU/ml, indicating that the cow starts to enter the ovulation period, the steps 1) -3) are repeated every 4 hours for three times every day until the luteinizing hormone LH of the cow reaches 45-100 mIU/m, indicating that the peak of the luteinizing hormone LH of the cow comes 5-10 hours away from the ovulation time;
4) and (4) carrying out natural or artificial hybridization according to the detection result of the step 3).
During natural or artificial breeding in the step 4), the survival time of the sperms of the bulls entering the bodies of the cows is as follows: 24-36 hours.
2. The method for detecting bovine mating timing according to claim 1, wherein the bovine luteinizing hormone LH test strip of step 2) is prepared by the following method:
21) the components were prepared as follows:
gold solution: adding 2ml of chloroauric acid solution with the mass concentration of 1% and 2ml of sodium citrate with the mass concentration of 1% into purified water to 100ml, heating until the solution is purple red, and cooling to room temperature to obtain gold solution;
sealing liquid: dissolving 0.005g bovine serum albumin in 300ul of purified water, adding 30ul of potassium carbonate solution, and uniformly mixing to obtain 330ul of confining liquid;
sample pad treatment solution: completely dissolving 0.605g of tricarboxymethylaminomethane and 1.5g of sodium chloride in 100ml of purified water to obtain a sample pad treatment solution;
colloidal gold conjugate dilution: uniformly mixing 0.605g of tricarboxymethylaminomethane, 0.2g of polyvinylpyrrolidone, 5g of sucrose, 0.9g of sodium chloride, 0.5g of bovine serum albumin, 3ml of bovine serum and 0.15ml of Tween 20 with 100ml of purified water to obtain a colloidal gold conjugate diluent;
LH coating liquid: dissolving 1ml of trehalose solution with the LHmABCcuring antibody of 100-200mg and the mass concentration of 1% in 100ml of PBS buffer solution of 0.01M, and uniformly mixing to obtain LH coating solution;
IgG coating solution: dissolving 1ml of trehalose solution with the mass concentration of 1 percent in 100ml of 0.01M PBS buffer solution by using 100-200mg bovine luteinizing hormone monoclonal alpha sub-level antibody LHmAlbCoting, and uniformly mixing to obtain IgG coating solution;
22) scribing a film on a substrate
22A) Attaching an NC film to the middle part of the upper surface of the PVC plate to obtain a bottom plate;
22B) uniformly scratching the IgG coating solution in the step 21) on the upper end of the NC membrane on the bottom plate in the step 22A) at the speed of 500mm/s according to the amount of 0.8 mu l/cm to be used as a quality control line C, uniformly scratching the LH coating solution on the lower end of the NC membrane to be used as a detection line T, wherein the distance between the quality control line C and the detection line T is 6mm +/-1 mm, the distance between the quality control line C and the upper end of the NC membrane is 8mm +/-1 mm, the distance between the detection line T and the lower end of the NC membrane is 7mm +/-1 mm, then putting the test line T into a drying box, and drying the test line T for 18h +/-2 h at the temperature of 25 +/-2 ℃ and the humidity of less than or equal to;
23) preparation of gold pad
23A) Adding 1.2ml of potassium carbonate solution and 1.5-2mg of LHmb2 into 100ml of the gold solution obtained in the step 21), stirring for 40min, dropwise adding 330ul of confining liquid, stirring for 10min, centrifuging for 40min at 4 ℃ and 12000r/min until the supernatant becomes water, removing the supernatant, taking 5.3ml of residual liquid, adding 40ml of colloidal gold conjugate diluent, and uniformly mixing;
23B) uniformly spreading 40ml of colloidal gold conjugate diluted solution on 1080cm2Putting the glass cellulose membrane on a drying oven, and drying at 25 +/-2 ℃ for 18 +/-2 h at the humidity of less than or equal to 30% to obtain a gold pad;
24) sample pad preparation
24A) Uniformly spreading the sample pad treatment solution obtained in the step 21) on a glass cellulose membrane according to the amount of 30 ml/sheet, then putting the glass cellulose membrane into a drying oven, and drying the glass cellulose membrane for 18h +/-2 h at the temperature of 25 +/-2 ℃ and the humidity of less than or equal to 30% to obtain a sample pad;
25) pasting board
25A) Attaching the gold pad of the step 23) to the lower end of the NC film of the film scribing plate of the step 22B), and lapping the rear end of the gold pad of 1-2mm on the NC film;
25B) attaching the sample pad of the step 24) on the front end of the gold pad of the step 25A), and completely lapping the rear end of the sample pad on the gold pad, wherein the front end of the sample pad is attached to the front end of the PVC plate;
25C) attaching the water absorption pad to the rear end of the PVC plate of 25B), and lapping the front end of the water absorption pad with the diameter of 1-2mm on the rear end of the NC film;
26) cutting the pasting plate in the step 25C) into test strips with the thickness of 3.0 +/-0.5 mm to obtain the test strips for detecting bovine luteinizing hormone LH.
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| CN113281525A (en) * | 2021-04-14 | 2021-08-20 | 昆明天沃生物科技有限公司 | Method for detecting sow egg discharge amount |
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Application publication date: 20200821 |