CN113311176A - Method for detecting pregnancy and gestation number of sows - Google Patents
Method for detecting pregnancy and gestation number of sows Download PDFInfo
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- CN113311176A CN113311176A CN202110402314.4A CN202110402314A CN113311176A CN 113311176 A CN113311176 A CN 113311176A CN 202110402314 A CN202110402314 A CN 202110402314A CN 113311176 A CN113311176 A CN 113311176A
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61D—VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS
- A61D17/00—Devices for indicating trouble during labour of animals ; Methods or instruments for detecting pregnancy-related states of animals
- A61D17/006—Devices for indicating trouble during labour of animals ; Methods or instruments for detecting pregnancy-related states of animals for detecting pregnancy of animals
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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
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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
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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Abstract
The invention relates to a method for detecting pregnancy and gestation number of sows, which comprises the following steps: collecting pig blood or urine from 7-14 days after sow hybridization; the method comprises the steps of inserting a test paper for detecting HCG-LIKE of a pig into serum or urine for 5-10 seconds, after 15-18 minutes, enabling a quality control line C and a detection line T to be colored, comparing the colored test paper with an HCG-LIKE colorimetric card, detecting whether a sow is pregnant, and detecting the pregnant number according to the HCG-LIKE level, so that the problems that whether the pig is pregnant or not can be judged, but whether embryos survive or not and the pregnant number cannot be reflected in the prior art, so that false pregnancy or non-pregnant pregnancy is caused, economic loss caused by extremely low pregnant number is caused, and the LIKE are solved. So that the problem sows can be eliminated as early as possible, the normal pregnant sows are correspondingly raised and managed, and the breeding efficiency and the breeding benefit of the sows are improved.
Description
Technical Field
The invention relates to a detection method, in particular to a method for detecting the pregnancy and the pregnancy number of sows, belonging to the technical field of animal detection methods.
Background
At present, the pregnancy detection of the pigs is mainly completed by a boar estrus test method, namely, adult boars with vigorous sexual desire are used for estrus test 18 to 24 days after the mating of sows, if the sows refuse to approach the boars and oestrus does not occur 3 to 4 days after the estrus test of the boars, the pregnancy of the sows can be preliminarily determined, and the judgment accuracy of the method is 90.34 percent, but the judgment accuracy of non-pregnant sows is lower and is only 36.07 percent.
In the prior art: 1) the ultrasonic detection is detection by closely combining the physical characteristics of ultrasonic waves with the acoustic characteristics of animal tissue structures, the early pregnancy diagnosis is carried out 22 days after hybridization, the accuracy rate can reach 100%, but the ultrasonic detection is high in price and is not applied to pig pregnancy diagnosis. 2) The D-type ultrasonic diagnosis method, also called ultrasonic Doppler exploration method, is a detection method which utilizes the physical characteristics that the frequency of sound waves changes when an object moves, and the accuracy rate reaches 100 percent. 3) The rectal palpation method is to select a female with a short stature, a handle is inserted into the rectum of a large-sized multiparous sow, excrement is drawn out, the uterus is touched, amniotic fluid exists in the uterus of the pregnant sow, the uterine artery is strong, the uterine artery of the non-pregnant sow is free of amniotic fluid, the elasticity is poor, the uterine artery is weak, whether the sow is pregnant or not is easy to judge, the pregnancy can be diagnosed within 25 days after the sow is bred, the accuracy rate reaches 95-100%, but the rectal palpation method is only suitable for large-sized multiparous sows such as large white sows, long white sows and the like, the palm of an operator is small, the physical strength is high, and the method is not adopted in most pig farms at present. 4) The vaginal examination method is that the vagina is opened by a vagina prolapsus appliance after 3-4 weeks of sow hybridization, the color, texture, mucus and other changes of the vagina intima are observed, the vagina of the pregnant sow is pale, dry and astringent, the uterine orifice is tightly closed, and extremely strong jelly sealing is realized; the endometrium of the non-pregnant sow is moist in the period close to the follicular phase, has shallow red or flush and thin secretion, so whether the sow is pregnant or not can be distinguished, but the method is often low in accuracy in diagnosing vaginitis, metritis and pseudopregnant sow, so that the method is not applied in production. 5) Radioimmunoassay (RIA) is a microanalysis method combining the principle of an immune reaction with the radiation measurement of an isotope, wherein a sow starts to produce estrogen, mainly estrone, which is converted into conjugated estrone sulfate (E1S) in a fetus in 12 days of pregnancy and enters maternal blood circulation, so that early pregnancy diagnosis of the pig can be carried out by taking blood from 25-30 days after mating with RIA to determine the content of E1S, and has the limitation that the Radioimmunoassay (RIA) is labeled with an isotope 3H, must be measured by a liquid flash apparatus, is easily polluted by radioactive substances, has high requirements on instruments, has strict laboratory requirements, cannot be carried out in a production site, and cannot see results on the day. 6) The enzyme immunoassay method (EIA) is characterized in that progesterone is labeled by enzyme, qualitative test is carried out according to the depth of color reaction generated by enzyme acting on a substrate through visual observation, the optical density value of a substrate solution can be read by a spectrophotometer or an enzyme-labeled reader, then the concentration of P4 is calculated for quantitative diagnosis, and the content of E1S in serum can be measured for pregnancy diagnosis. 7) The reproductive hormone diagnostic method, the dominant hormone in the sow is progesterone, it can antagonize proper amount of exogenous reproductive hormone, make it not react, before the 1 st estrus cycle after mating comes, inject proper amount of exogenous reproductive hormone, according to pregnant sow have certain exogenous reproductive hormone to react to judge whether pregnant: pregnant sows do not show oestrus symptoms, and non-pregnant sows act on target organs together with exogenous hormones and ovarian hormones, so that oestrus is more obvious in external manifestation, limited by various factors (such as instruments, medicines and the like), and cannot be effectively popularized and applied in production practice.
Therefore, there is a need for developing a technique for detecting the pregnancy of a pig which is rapid, accurate and easy to handle at the production site. An ideal and efficient early pregnancy diagnosis method is produced, thereby improving the fertility of sows and the economic benefit of the pig industry and promoting the rapid development of the pig industry. Is a necessary trend of high-quality pig production development.
Therefore, there is a need for improvements in the prior art.
Disclosure of Invention
The invention aims to solve the defects in the prior art, and provides a method for detecting early pregnancy of pigs, so as to accurately master whether the pigs are pregnant and whether embryos survive, and the number of the embryos, reduce economic loss caused by false pregnancy and empty pregnancy of the pigs, shorten the non-breeding days of sows, reasonably feed and manage pregnant sows in time, improve the breeding efficiency, promote intensive management of the pigs and improve the production benefit of the pigs.
The invention is completed by the following technical scheme: a method for detecting early pregnancy of a pig, comprising the steps of:
1) collecting 1-10ml of sow blood on 7-14 days after sow hybridization, standing in shade for 1-2 hours, separating out serum, or collecting 1-10ml of urine of sow which urinates 1-2 hours after last urination;
2) inserting the porcine human chorionic gonadotropin HCG-LIKE test paper into the serum or 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 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 quality control line C of the test paper is colored, and the detection line T is not colored, which indicates that the chorionic gonadotropin HCG-LIKE level of the sow is low and the detection needs to be carried out every other day;
the test paper quality control line C and the detection line T are developed, and the development is compared with a chorionic gonadotropin HCG-LIKE colorimetric card in the following way;
31) when the HCG-LIKE is less than 10mIU/ml, repeating the steps 1) -2) again, and detecting the HCG-LIKE continuously less than 10mIU/ml for two times, wherein the result shows that the sow is not pregnant or the embryo is stopped;
32) when the HCG-LIKE is not less than 10mIU/ml, indicating that the sow is possibly pregnant, repeating the steps 1) -2) every other day for one detection, and continuously detecting twice; when the chorionic gonadotropin HCG-LIKE of the two detection results rises and the chorionic gonadotropin HCG-LIKE of the next time is 1-2 times of the chorionic gonadotropin HCG-LIKE of the previous time, indicating that the sow is pregnant and is a live fetus, and then judging the number of the fetus according to the following sequence;
when HCG- - -LIKE is 15-30 mIU/ml, it indicates that the sow has a single fetus;
when HCG- - -LIKE is 35-60 mIU/ml, it indicates that the sow has double births;
when HCG- - -LIKE is 65-125 mIU/ml, it indicates that the sow carries three births;
when HCG- - -LIKE is 130-250 mIU/ml, it indicates that the sow carries four fetuses;
when HCG- - - -LIKE is 255-500 mIU/ml, it indicates that the sow carries five fetuses,
when HCG- - - -LIKE is 505- -1000mIU/ml, it indicates that the sow has six births,
when HCG- - - -LIKE is 1005 to 2000mIU/ml, it indicates that the sow carries seven fetuses,
when HCG- - -LIKE is 2005-4000 mIU/ml, it indicates that the sow has eight fetuses,
when HCG- - -LIKE is more than 4000mIU/ml, the result shows that the sow has nine or more fetuses.
The test paper for detecting the human chorionic gonadotropin HCG- - -LIKE of the pig 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% into 100ml of sodium citrate solution with the mass concentration of 1%, 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 sheep serum and 0.15ml of Tween 20 with 100ml of purified water to obtain a colloidal gold conjugate diluent;
HCG coating solution: dissolving 1ml of trehalose solution with the mass concentration of 1% of HCG-LIKE mABCOting, which is a monoclonal alpha sub-antibody of 100-200mg pig, in 100ml of PBS buffer solution of 0.01M, and uniformly mixing to obtain HCG-LIKE coating solution;
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 0.01M PBS buffer solution, 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 IgG coating liquid on the upper end of the NC film on the bottom plate in the step 22A) as a quality control line C at the speed of 500mm/s by using an XYZ3010 gold spraying and film scratching instrument, uniformly scratching HCG- - -LIKE coating liquid on the lower end of the NC film as a detection line T at the speed of 0.8 mul/cm, 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 percent to obtain a film scratching plate;
23) preparation of gold pad
23A) Adding 1.2ml of potassium carbonate solution and 1.5-2mg of LH monoclonal antibody 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, removing 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 RB65 on 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 percent 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 SB06 according to the amount of 30 ml/sheet, then putting the sample pad treatment solution into a drying box, and drying the sample pad 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 porcine chorionic gonadotropin HCG-LIKE test strip.
The invention has the following advantages and effects: by adopting the scheme, a blood sample or a urine sample can be taken on the 7 th to 14 th days after the mating of the sow, whether the sow is pregnant or not and the number of fetuses are simply, conveniently, quickly and accurately detected, whether the sow is pregnant or not, whether the embryo is alive or not and the number of fetuses are known earlier, and the problems that whether the sow is pregnant or not can only be judged, but whether the embryo is alive or not and the number of fetuses cannot be reflected, so that false pregnancy or non-pregnant fetus is brought, economic loss caused by extremely low number of fetuses and the like are solved. So that the problem sows can be eliminated as early as possible, the normal pregnant sows are correspondingly raised and managed, and the breeding efficiency and the breeding benefit of the sows are improved.
Drawings
FIG. 1 is a schematic structural diagram of a HCG- -LIKE test strip;
FIG. 2 is a chart of HCG- - -LIKE color chart.
Detailed Description
The invention is further illustrated by the following examples.
Example 1
The test paper for detecting HCG-LIKE of porcine chorionic gonadotropin 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% into 100ml of sodium citrate solution with the mass concentration of 1%, 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 medical-grade 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) HCG- - -LIKE coating liquid: dissolving 1ml of 100-200mg of porcine chorionic gonadotropin monoclonal alpha-subunit antibody HCG- - -LIKE mABCOting and 1% of trehalose solution in mass concentration in 100ml of 0.01M PBS buffer solution, and uniformly mixing to obtain HCG- - -LIKE 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 0.01M PBS buffer solution, and uniformly mixing to obtain 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 6cm multiplied by 3 mm;
2) scribing a film on a substrate
2A) Respectively sticking NC film pastes to the middle parts of the upper surfaces of the 5 PVC plates to obtain a bottom plate;
2B) using an XYZ3010 gold spraying membrane scribing instrument, uniformly scribing the IgG coating liquid of the step 16) on the upper ends of 5 NC membranes on the bottom plate of the step 2A) at a speed of 500mm/s according to the quantity of 0.8 mu l/cm to serve as a quality control line C, uniformly scribing the HCG- - -LIKE coating liquid of the step 15) on the lower ends of the 5 NC membranes on the bottom plate of the step 2A) to serve 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 detection line T into a drying box, and drying the detection line T for 18h +/-2 h at the temperature of 25 +/-2 ℃ and the humidity of less than or equal to 30% to obtain a membrane scribing plate;
3) preparation of gold pad
3A) Adding 1.2ml of potassium carbonate solution and 2mg of LH monoclonal antibody 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, removing 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 a mixed solution of 45.3ml of the colloidal gold conjugate diluent;
3B) uniformly spreading 45.3ml of mixed solution of colloidal gold conjugate diluent on 5 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 box, 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;
25) preparation of each flitch
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 each pasting board in the step 5C) into 100 test strips with the size of 6cm multiplied by 3mm, and boxing to obtain 500 pieces of human chorionic gonadotrophin HCG-LIKE test paper, wherein the structure of each human chorionic gonadotrophin HCG-LIKE test paper is shown in figure 1.
Example 2
The method for detecting the pregnancy and the number of fetuses of the sow comprises the following steps:
1) collecting 1ml of sow blood from the neck vein of the sow by using a blood collecting needle and an anticoagulant vessel on the 7 th day after the sow is bred, standing for 1 hour in a shade place, and separating out serum;
2) inserting a human chorionic gonadotropin HCG- - -LIKE test strip of example 1 into the serum of step 1) for 5 seconds, taking out, and observing whether the quality control line C and the detection line T in the test strip develop color after 15 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, which indicates that the detection result is invalid;
when the steps 1) -2) are repeated, observing whether the quality control line C and the detection line T in the detection test paper are developed, wherein the development result is as follows:
the test paper quality control line C is colored, and the test line T is not colored, which indicates that the human chorionic gonadotrophin HCG- - -LIKE level of the sow is low, the sow is not pregnant, and the sow is waited for the next estrus to be re-bred.
Example 3
The method for detecting the number of pregnancies and fetuses of the sows comprises the following steps:
1) collecting 1ml of sow blood from the neck vein of the sow by using a blood collecting needle and an anticoagulation blood vessel on the 8 th day after the sow is bred, standing for 1 hour in a shade place, and separating out serum;
2) inserting a human chorionic gonadotropin HCG- - -LIKE test strip of example 1 into the serum of step 1) for 5 seconds, taking out, and observing whether the quality control line C and the detection line T in the test strip develop color after 15 minutes:
3) the color development results were as follows:
the test paper quality control line C is colored, the detection line T is not colored, only shows that the HCG- - -LIKE level of the porcine chorionic gonadotropin is normal, and the steps 1) -2) are repeated after two days for detection, and the results are as follows:
the test paper quality control line C and the detection line T are developed, and the development and HCG- - -LIKE colorimetric card are used as shown in figure 2 to carry out the following comparison;
31) detecting HCG- - -LIKE < 10mIU/ml twice continuously, indicating that the embryo of the sow is stopped and waiting for the re-hybridization in the next estrus.
Example 4
The method for detecting the pregnancy and the number of fetuses of the sow comprises the following steps:
1) collecting 5ml of urine which is re-urinated by the sow 2 hours after the last urination on the 12 th day after the sow is bred;
2) inserting a human chorionic gonadotropin HCG- - -LIKE test strip of example 1 into the urine of step 1) for 9 seconds, taking out, and observing whether the quality control line C and the detection line T in the test strip develop color after 16 minutes:
3) the color development results were as follows:
the test paper quality control line C and the detection line T are developed, and the development and HCG- - -LIKE colorimetric card are used as shown in figure 2 to carry out the following comparison;
31) the human chorionic gonadotropin HCG- - -LIKE is more than 10mIU/ml, which indicates that the sow is possibly pregnant, the steps 1) and 2) are repeated every other day, the detection is carried out twice continuously, and the HCG- - -LIKE of the next time is 40 mIU/ml which is 2 times of the HCG- - -LIKE of the previous time which is 20 mIU/ml, which indicates that the sow is pregnant and is live fetus, and the HCG- - -LIKE is 40 mIU/ml which indicates that the sow is pregnant and is double fetus and can only be eliminated.
Example 5
The method for detecting the pregnancy and the number of fetuses of the sow comprises the following steps:
1) collecting 5ml of urine which is re-urinated by the sow 1 hour after the last urination on the 10 th day after the sow is bred;
2) inserting a human chorionic gonadotropin HCG- - -LIKE test strip of example 1 into the urine of step 1) for 8 seconds, taking out, and observing whether the quality control line C and the detection line T in the test strip develop color after 16 minutes:
3) the color development results were as follows:
the test paper quality control line C and the detection line T are developed, and the development and the HCG colorimetric card are shown in figure 2 for comparison;
31) the human chorionic gonadotropin HCG- - -LIKE is more than 10mIU/ml, which indicates that the sow is possibly pregnant, the steps 1) and 2) are repeated every other day, the detection is carried out twice continuously, and the HCG- - -LIKE of the next time is 1000mIU/ml which is 2 times of the HCG- - -LIKE of the previous time which is 500mIU/ml, which indicates that the sow is pregnant and is live fetus, and the HCG- - -LIKE is 1000mIU/ml which indicates that the sow has six fetuses, so the method can be eliminated only in consideration of economic benefit.
Example 6
The method for detecting the pregnancy and the number of fetuses of the sow comprises the following steps:
1) on 14 days after the mating of the sows, 5ml of urine which is re-urinated by the sows 1 hour after the last defecation is collected;
2) inserting a human chorionic gonadotropin HCG- - -LIKE test strip of example 1 into the urine of step 1) for 8 seconds, taking out, and observing whether the quality control line C and the detection line T in the test strip develop color after 17 minutes:
3) the color development results were as follows:
the test paper quality control line C and the detection line T are developed, and the development and the HCG colorimetric card are shown in figure 2 for comparison;
31) the human chorionic gonadotropin HCG- - -LIKE is more than 10mIU/ml, which indicates that the sow is possibly pregnant, the steps 1) and 2) are repeated every other day, the detection is carried out twice continuously, the HCG- - -LIKE of the next time is 4000mIU/ml which is 2 times of the HCG- - -LIKE of the previous time which is 2000mIU/ml, which indicates that the sow is pregnant, and the sow is live, and the HCG- - -LIKE is 4000mIU/ml which indicates that the sow is pregnant with eight fetuses, the sow is fed by pregnancy according to the conventional method until 8 piglets are produced after 115 days.
Example 7
The method for detecting the pregnancy and the number of fetuses of the sow comprises the following steps:
1) collecting 5ml of urine which is re-urinated by the sow 1 hour after the last urination on the 10 th day after the sow is bred;
2) inserting a human chorionic gonadotropin HCG- - -LIKE test strip of example 1 into the urine of step 1) for 8 seconds, taking out, and observing whether the quality control line C and the detection line T in the test strip develop color after 16 minutes:
3) the color development results were as follows:
the test paper quality control line C and the detection line T are developed, and the development and the HCG colorimetric card are shown in figure 2 for comparison;
31) the human chorionic gonadotropin HCG- - -LIKE is more than 10mIU/ml, which indicates that the sow is possibly pregnant, the steps 1) and 2) are repeated every other day, the detection is continuously carried out twice, the HCG- - -LIKE of the next time is 5000 mIU/ml, which is 2 times of the HCG- - -LIKE of the previous time, which indicates that the sow is pregnant, and the sow is live, and the HCG- - -LIKE is 5000 mIU/ml, which indicates that the sow is pregnant with more than nine fetuses, the sow is fed by pregnancy according to the conventional method until the piglet produces 12 heads after 115 days.
Example 8
The method for detecting the pregnancy and the number of fetuses of the sow comprises the following steps:
1) collecting 5ml of urine which is re-urinated by the sow 1 hour after the last urination on the 10 th day after the sow is bred;
2) inserting a human chorionic gonadotropin HCG- - -LIKE test strip of example 1 into the urine of step 1) for 8 seconds, taking out, and observing whether the quality control line C and the detection line T in the test strip develop color after 16 minutes:
3) the color development results were as follows:
the test paper quality control line C and the detection line T are developed, and the development and the HCG colorimetric card are shown in figure 2 for comparison;
31) the human chorionic gonadotropin HCG- - -LIKE is more than 10mIU/ml, which indicates that the sow is possibly pregnant, the steps 1) and 2) are repeated every other day, the detection is carried out twice continuously, the HCG- - -LIKE of the next time is 8000 mIU/ml, which is 2 times of the HCG- - -LIKE of the previous time, which indicates that the sow is pregnant, and the sow is live, the HCG- - -LIKE is 1000mIU/ml, which indicates that the sow is pregnant with more than nine fetuses, and the HCG- - -LIKE is 8000 mIU/ml, which indicates that the sow is pregnant with more than nine fetuses, the sow is fed by the conventional method until 15 piglets are born after 115 days.
The HCG- - -LIKE colorimetric cards are all conventional products.
The effect of the invention is demonstrated by comparative experiments as follows:
210 sows were tested by the methods of examples 2-8, wherein 210 sows were divided into seven groups of 30 pigs each, and the tests were performed by the same methods as in examples 2-8, and the test results were as follows:
the first group has 28 pregnant sows, eliminates 2 sows which are not pregnant, and the 28 pregnant sows are live sows but 4 sows with the gestation number lower than 8 and can only be eliminated, the rest 24 sows give 264 piglets in total, and the average number of piglets is 11;
in the second group, 24 sows are pregnant, 6 sows which are not pregnant are eliminated, the pregnant sows are live, but 6 sows with the gestation number lower than 8 are eliminated, the rest 18 sows give 216 piglets and the average number of the piglets is 12;
in the third group, 25 pregnant sows, 20 live fetuses, 5 fetuses and 5 non-pregnant sows are eliminated, the number of the 20 sows is 200, and the average number of the sows is 10;
the fourth group had 27 pregnancies, 25 live births, 2 embryonic births, 3 non-pregnancies, eliminated, 255 piglets for 25 sows, and 10.2 average piglets.
In the fifth group, 29 sows are pregnant, 1 sow is not pregnant, the number of fetuses of 3 sows is less than 8, the sows are eliminated, 263 piglets are born in total by 26 sows, and 10.11 piglets are born on average.
In the sixth group, 29 pregnant pigs, 28 live fetuses, 1 non-pregnant pig, 1 fetuses stop to be pregnant, and 3 fetuses with less than 8 fetuses are eliminated, and the number of the born fetuses is 276 in total for 25 sows, and the average number of the born fetuses is 11.04.
In the seventh group, 26 sows are pregnant, 4 sows are not pregnant, 5 fetuses are less than 8 fetuses, and the sows are eliminated, and the average farrowing rate is 11.23 and 236.
The method can be used for detecting the number of the pregnant sows, and the sows with less than 8 fetuses can be selected to be eliminated, so that the economic benefit of a farm is ensured, and the problem that the breeding cost is increased due to too few fetuses is solved.
Claims (2)
1. A method for detecting the pregnancy and the gestation number of sows is characterized by comprising the following steps:
1) collecting 1-10ml of pig blood on 7-14 days after the sow is bred, standing for 1-2 hours in a shade place, separating serum, or collecting 1-10ml of urine of the sow which urinates 1-2 hours after the last defecation;
2) inserting the chorionic gonadotropin HCG-LIKE detection test paper of the sow into the serum or 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 detection test paper are colored 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 quality control line C of the test paper is colored, and the detection line T is not colored, which indicates that the chorionic gonadotropin HCG-LIKE level of the sow is low and the detection needs to be carried out every other day;
the test paper quality control line C and the detection line T are developed, and the development is compared with a chorionic gonadotropin HCG-LIKE colorimetric card in the following way;
31) when the HCG-LIKE is less than 10mIU/ml, repeating the steps 1) -2) again, and detecting the HCG-LIKE continuously less than 10mIU/ml for two times, wherein the result shows that the sow is not pregnant or the embryo is stopped;
32) when the HCG-LIKE is not less than 10mIU/ml, indicating that the sow is possibly pregnant, repeating the steps 1) -2) every other day for one detection, and continuously detecting twice; when the chorionic gonadotropin HCG-LIKE detected at the next time rises and is 1-2 times of the chorionic gonadotropin HCG-LIKE detected at the previous time, the sow is pregnant and is a live fetus, and then the number of the fetus is judged according to the following steps;
when HCG- - -LIKE is 15-30 mIU/ml, it indicates that the sow has a single fetus;
when HCG- - -LIKE is 35-60 mIU/ml, it indicates that the sow has double births;
when HCG- - -LIKE is 65-125 mIU/ml, it indicates that the sow carries three births;
when HCG- - -LIKE is 130-250 mIU/ml, it indicates that the sow carries four fetuses;
when HCG- - - -LIKE is 255-500 mIU/ml, it indicates that the sow carries five fetuses,
when HCG- - - -LIKE is 505- -1000mIU/ml, it indicates that the sow has six births,
when HCG- - - -LIKE is 1005 to 2000mIU/ml, it indicates that the sow carries seven fetuses,
when HCG- - -LIKE is 2005-4000 mIU/ml, it indicates that the sow has eight fetuses,
when HCG- - -LIKE is more than 4000mIU/ml, the result shows that the sow has nine or more fetuses.
2. The method of claim 1, wherein the test strip for detecting HCG-LIKE in the step 2) is prepared by the following steps:
21) the components were prepared as follows:
gold solution: adding 2ml of chloroauric acid solution with the mass concentration of 1% into 100ml of sodium citrate solution with the mass concentration of 1%, 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 tris (carboxymethyl amino) methane 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 sheep serum and 0.15ml of Tween 20 with 100ml of purified water to obtain a colloidal gold conjugate diluent;
HCG- -LIKE coating liquid: dissolving 1ml of trehalose solution with the mass concentration of 1% of 100-200mg of monoclonal alpha-sub-antibody HCG-LIKE mABCOting of porcine chorionic gonadotropin into 100ml of 0.01M PBS buffer solution, and uniformly mixing to obtain HCG-LIKE coating solution;
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 0.01M PBS buffer solution, 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 surface of the PVC plate to obtain a bottom plate;
22B) uniformly scratching IgG coating liquid on the upper end of the NC film on the bottom plate in the step 22A) as a quality control line C by using an XYZ3010 gold spraying film scratching instrument at the speed of 500mm/s according to the amount of 0.8 mu l/cm, uniformly scratching HCG-LIKE coating liquid on the lower end of the same NC film 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 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 detection 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 percent to obtain a film scratching plate;
23) preparation of gold pad
23A) Adding 1.2ml of potassium carbonate solution and 1.5-2mg of LH monoclonal antibody 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, removing supernatant, taking 5.3ml of residual liquid, adding 40ml of colloidal gold conjugate diluent, and uniformly mixing;
23B) uniformly spreading the uniformly mixed colloidal gold conjugate diluent in the step 23A) on 1080cm2Putting the glass cellulose membrane RB65 on 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 percent 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 SB06 according to the amount of 30 ml/sheet, then putting the sample pad treatment solution into a drying box, and drying the sample pad 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 strip for detecting the HCG-LIKE of the porcine chorionic gonadotropin.
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| CN111596073A (en) * | 2020-06-04 | 2020-08-28 | 昆明天沃生物科技有限公司 | Method for detecting early pregnancy of sheep |
| CN111596075A (en) * | 2020-06-04 | 2020-08-28 | 昆明天沃生物科技有限公司 | Method for detecting early pregnancy of cattle |
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2021
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| EP0250137A2 (en) * | 1986-06-09 | 1987-12-23 | Ortho Diagnostic Systems Inc. | Colloidal gold immunoassay |
| US20190183963A1 (en) * | 2003-10-03 | 2019-06-20 | Thorn Bioscience Llc | Process for the synchronization of ovulation for timed breeding without heat detection |
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| US9857373B1 (en) * | 2016-10-17 | 2018-01-02 | Reliant Immune Diagnostics, LLC | Pregnancy test to assess disease risk |
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