Method for diagnosing pregnancy in ruminant

A ruminant and cell technology, applied in biochemical equipment and methods, microbial measurement/inspection, biological testing, etc., can solve the problems of unreliable judgment results, fluctuations in gene expression, etc., to achieve pregnancy rate, The effect of shortening the interval between births

Pending Publication Date: 2020-04-21
HOKKAIDO UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in blood diagnosis, the level of gene expression fluctuates for each

Method used

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  • Method for diagnosing pregnancy in ruminant
  • Method for diagnosing pregnancy in ruminant
  • Method for diagnosing pregnancy in ruminant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1. Interference in extrauterine tissues (cervical canal and ectopic os) of pregnant and non-pregnant cattle Expression analysis of hormone-inducible factors

[0067] 1) Collection of mucous membrane

[0068] Among the milking cows of the Holstein breed raised at the Dairy Product Production Research Facility of the Bioproduction Research Farm of the Northern Biosphere Field Science Center, Hokkaido University, 18 days after artificial insemination were selected, the pregnancy was confirmed through the subsequent pregnancy test. For domestic cattle and cattle that have not been artificially inseminated, wipe the vulva with an inverse soap solution. Open the vestibule of the vagina directly or using a colposcope, insert a commercially available measuring spoon, and scrape and collect the mucous membranes from the endocervical canal and the periphery of the ectopic os. It was confirmed by visual observation of the collected sample that blood was mixed, and b...

Embodiment 2

[0079] Example 2. Extrauterine tissues of pregnant and non-pregnant cattle (cervical canal, peripheral portion of the uterine ostium, and prevaginal Expression analysis of interferon-inducible factors in

[0080] Except for using a cotton swab instead of a measuring spoon as a collection tool, proceed in the same manner as in Example 1, and collect samples from the endocervical canal, the peripheral part of the uterine ostium, and the vaginal vestibule of pregnant cows, and from the endocervical canal of non-pregnant cows. mucous membranes. The collected cotton swabs were soaked in 500 μL of ISOGEN II (Nippon Gene) and ice-cooled 1.5mL tubes, and the cells in the mucosa were recovered, and the total RNA was extracted in the same manner as in Example 1. cDNA synthesis and real-time PCR by transcription reaction.

[0081] The results of the relative gene expression level of ISG15 are shown in image 3 . The expression of ISG15 was observed in the mucous membranes of the c...

Embodiment 3

[0082] Example 3. Extrauterine tissue (cervical canal) of pregnant and non-pregnant cows by quantitative RTLAMP method Expression analysis of interferon-inducible factors in

[0083] For the RNA samples from the cervical canal mucosa of pregnant cows and non-pregnant cows collected and prepared by the same method as 1) and 2) of Example 1, the total RNA amount was adjusted to 100ng respectively and then DNase treatment was carried out. DNA digestion. Use 100 ng of RNA samples from pregnant cattle and non-pregnant cattle cervical canal mucosa treated with DNase, 100 ng of RNA samples from pregnant cattle and non-pregnant cattle cervical canal mucosa without DNase treatment, and 100 ng of RNA samples from non-pregnant cattle without DNase treatment. The RNA sample 1000ng of pregnant bovine cervical canal mucosa was directly subjected to RTLAMP reaction.

[0084] The reaction conditions are as follows. Add 2.9 μl of sterilized distilled water, 12.5 μl of reaction solution, ...

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Abstract

The invention aims to diagnose pregnancy in a livestock animal by a method which can be carried out at a breeding site while minimizing invasion of the subject animal and fetuses thereof. The method according to the present invention for diagnosing pregnancy in a ruminant comprises: a measurement step of using a sample containing mucosal epithelial cells collected from the genitals ranging from the uterine cervical canal to the vulvar orifice of the subject ruminant, said subject ruminant being in the state corresponding to the early stage of pregnancy, and measuring the expression level of amolecule, which is induced in response to IFN-[tau], in the mucosal epithelial cells; and a diagnosis step for comparing the expression level with an expression level of the aforesaid molecule in corresponding mucosal epithelial cells of a non pregnant ruminant of the same species and thus diagnosing pregnancy in the subject ruminant. According to the present invention, pregnancy can be quickly diagnosed before a new ovulation cycle starts after breeding, artificial insemination or fertilized egg transplantation.

Description

technical field [0001] The present invention relates to methods for determining pregnancy in ruminants. Background technique [0002] In recent years, regardless of whether it is for meat, milk or other purposes, the breeding of livestock animals such as cattle, sheep, and goats is almost always carried out through artificial insemination or fertilized egg transplantation. However, the pregnancy rate brought about by artificial insemination and fertilized egg transplantation is not high, for example, the pregnancy rate of dairy cows is lower than 50%. The low pregnancy rate has brought serious economic burdens to livestock farmers, such as the increase in the number of artificial insemination or fertilized egg transplantation, the period of empty fetuses without pregnancy, and the prolongation of delivery intervals, which lead to increased production costs. [0003] The weakening of estrus signs in livestock animals, the negative impact of genetic improvement on reproductiv...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/6883G01N33/68
CPCC12N15/09C12Q1/68G01N33/50G01N33/68C12Q2600/158C12Q2600/124C12Q1/6876C12Q1/6851C12Q1/686
Inventor 高桥昌志国井宏树伊藤月乃川原学呗花子铃木惇文
Owner HOKKAIDO UNIVERSITY
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