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Detection method for antisperm antibody in cervical mucus of dairy cows

A technology of antisperm antibody and cervical mucus, which is applied in the field of genetic engineering, can solve problems such as inability to quantify and exclude false positive results, and achieve high application value

Inactive Publication Date: 2011-11-23
TARIM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it cannot be quantified, nor can it rule out false positive results caused by fertility-related AsAbs

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034]1) According to the breeding records and rectal examination results, it is determined that the infertile cows have been bred repeatedly. After the direct examination, stimulate the cervix of the cows and wait for the cervical mucus of the cows to be in estrus to be frozen at -20°C for later use;

[0035] 2) Take 0.25ml with activity rate ≥ 0.40 and density ≥ 10 8 Sperm / ml bull fine tubes were frozen, thawed in a water bath at 38°C, centrifuged at 700g for 8 minutes, discarded the supernatant, added 1ml of EBSS to suspend, spread the suspension on the bottom of the test tube previously placed with 2ml of EBSS containing 10% fetal bovine serum, Tilt 45° at 37°C, 5% CO 2 Incubate in the incubator for 45min, and take the supernatant;

[0036] 3) Add 500 μl bromelain solution with a concentration of 80 μg / ml to 500 μl thawed cervical mucus at room temperature, incubate at 37° C. for 30 minutes, and centrifuge at 2600 g for 15 minutes;

[0037] 4) Dilute the supernatant of s...

Embodiment 2

[0039] 1) According to the breeding records and rectal examination results, it is determined that the infertile cows have been mated repeatedly, and the cervical mucus of the cows that are in estrus is stimulated after the direct examination and frozen at -20°C for later use;

[0040] 2) Take 0.25ml with activity rate ≥ 0.40 and density ≥ 10 8 Sperm / ml is frozen in a bull tube, thawed in a water bath at 38°C, centrifuged at 700g for 8 minutes, discarded the supernatant, added 1ml of EBSS to suspend, and spread the suspension on the bottom of the test tube previously placed with 2ml of EBSS containing 10% fetal bovine serum. Tilt 45° at 37°C, 5% CO 2 Incubate in the incubator for 45min, and take the supernatant;

[0041] 3) Add 500 μl of bromelain solution with a concentration of 120 μg / ml to 500 μl of thawed cervical mucus at room temperature, incubate at 37°C for 30 minutes, and centrifuge at 2600 g for 15 minutes;

[0042] 4) Dilute the supernatant of step 3) with EBSS sol...

Embodiment 3

[0044] 1) Take the cervical mucus of dairy cows in the peak estrus period and freeze them at -20°C for future use;

[0045] 2) Take 0.25ml with activity rate ≥ 0.40 and density ≥ 10 8 Sperm / ml is frozen in a bull tube, thawed in a water bath at 38°C, centrifuged at 700g for 8 minutes, discarded the supernatant, added 1ml of EBSS to suspend, and spread the suspension on the bottom of the test tube previously placed with 2ml of EBSS containing 10% fetal bovine serum. Tilt 45° at 37°C, 5% CO 2 Incubate in the incubator for 45min, and take the supernatant;

[0046] 3) Add 500 μl of bromelain solution with a concentration of 160 μg / ml to 500 μl of thawed cervical mucus at room temperature, incubate at 37°C for 30 minutes, and centrifuge at 2600 g for 15 minutes;

[0047] 4) Dilute the supernatant of step 3) with EBSS solution in 8 gradients of 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128, 1 / 256 to 15 μL, then add 15 μL of the supernatant prepared in step 2) and mix well, cover with 20...

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Abstract

The invention discloses a detection method for an antisperm antibody in the cervical mucus of dairy cows, comprising the steps of: 1) taking cervical mucus of the dairy cow in estrus; 2) taking bull fistula frozen sperm, centrifuging and removing supernatant, adding EBSS (Earle's Balanced Salt Solution) suspension, laying the suspension at the bottom of a test tube in which the EBSS containing 10% fetal calf serum is pre-arranged, incubating for 45 min in an incubator, taking the supernatant; 3) adding pineapple protease liquid in the cervical mucus defrosted at room temperature and centrifuging; and 4) diluting the supernatant in the step 3) by using EBSS, adding 15 microliters of supernatant prepared in the step 2) and mixing, covering 20 microliters of paraffin oil, incubating for 30 min, wherein the number of the agglutinated sperm is recorded as N, the total number of sperms is recorded as Nz, the antisperm antibody is judged to be positive when N / Nz is more than and equal to 10%, and the antisperm antibody is judged to be negative when N / Nz is less than 10%. According to the detection method, the antisperm immune infertility cows in the dairy cows can be easily and rapidly detected.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for detecting antisperm antibodies in cervical mucus of dairy cows. Background technique [0002] Antisperm antibody (antisperm antibody, AsAb) can combine with sperm to produce immune reaction and affect reproductive ability. Sperm are antigenic to males and sperm are alloimmune antigens to females, and each mating is equivalent to an immunization. Local and serum antisperm antibodies in the reproductive tract are the product of the body's own or allogeneic immune response to sperm antigens, and are the main cause of immune infertility. The core of dairy cow production is reproduction, and preventing infertility is the primary problem to be solved. The impact of antisperm antibodies on infertility is common in dairy cows, and a considerable part of antisperm immune infertile cows can be prevented by appropriate targeted technical measures. Imm...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/53
Inventor 高庆华马瑛韩春梅
Owner TARIM UNIV
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