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A method for sex identification of early bovine embryos

An embryonic and gender-based technology, applied in the direction of embryonic cells, biochemical equipment and methods, cell culture active agents, etc., can solve the problems of easy contamination, affecting accuracy, and high sensitivity

Active Publication Date: 2021-12-07
天津力牧生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] As we all know, the loop-mediated isothermal amplification method used to identify the sex of mammalian embryos has extremely high sensitivity, is easily contaminated, and has very strict requirements on the operation process. As long as a very small amount of genes other than the target gene or other test items are mixed Additions may cause misjudgment
[0015] However, as mentioned above, embryos obtained by in vitro fertilization usually need to be obtained from embryo culture fluid, or embryos undergo cryopreservation, and embryo culture fluid, cryopreservation used freezing liquid (for example, containing PBS+FBS+ethylene glycol+ sucrose) and embryo thawing solution used before embryo sex identification (for example, including PBS+FBS+sucrose), usually contains animal-derived substances such as FBS (fetal bovine serum), which may seriously affect the accuracy of the loop-mediated isothermal amplification method
It has been found that the most common problem with the LMA method, which may be caused by this type of embryo culture, freezing, and thawing fluid, is that the reaction to the shared primers for both sexes is negative and requires retesting

Method used

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  • A method for sex identification of early bovine embryos
  • A method for sex identification of early bovine embryos
  • A method for sex identification of early bovine embryos

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0089] Embodiment 1: the segmentation method of bovine in vitro fertilization embryo (ex vivo egg picking)

[0090] Cattle used in the experiment: Chinese Yellow Cattle (Nanyang Cattle, a working breed).

[0091] Step (1), collection and in vitro maturation of oocytes

[0092] In vitro collection: Take the ovary from the slaughterhouse and put it in an insulated bucket with double-antibiotic saline, and transport it back to the laboratory within 3 hours at 31-33°C; extract the follicles with a surface size of 2-8mm, collect the precipitate, and examine it under a stereomicroscope Next, pick out oocyte COCs (that is, cumulus-oocyte complexes) that contain at least three layers of cumulus cells, and wash them twice in egg washing liquid to remove excess impurities;

[0093] The COCs collected above were washed once in the oocyte maturation medium, and then transferred to a new maturation medium for 22–24 h. The culture conditions were 38.8°C, 5.5–6.5% CO2, and saturated humidit...

Embodiment 2

[0117] Embodiment 2: the segmentation method of bovine in vitro fertilization embryo (ex vivo egg collection)

[0118] Cattle used in the test: Holstein cattle (dairy cow breed).

[0119] Step (1) to step (6) are carried out with reference to step (1) to step (6) of Example 1, respectively.

[0120] Step (7), loop-mediated isothermal amplification reaction, detection and result determination

[0121] Put 6 reaction centrifuge tubes in the ring-mediated isothermal amplification real-time turbidity detection system and amplify at 63°C for 40min. After the reaction, record the results represented by "+" and "-" in each tube, according to Example 1 Male and female were judged according to the criteria shown in the table.

[0122] Use the method of this embodiment 2 to carry out gender identification, altogether 138 samples obtained by dividing 19 morulae were measured, the result: the results of the negative control group and the positive control group were all correct, from 9 e...

Embodiment 3

[0123] Embodiment 3: the segmentation method of bovine in vitro fertilization embryo (ex vivo egg collection)

[0124] Cattle used in the experiment: Simmental cattle (beef breed).

[0125] Step (1) to step (6) are carried out with reference to step (1) to step (6) of Example 1, respectively.

[0126] Step (7), loop-mediated isothermal amplification reaction, detection and result determination

[0127] Put 6 reaction centrifuge tubes in the ring-mediated isothermal amplification real-time turbidity detection system and amplify at 63°C for 40min. After the reaction, record the results represented by "+" and "-" in each tube, according to Example 1 Male and female were judged according to the criteria shown in the table.

[0128] Using the method of this embodiment 3 to carry out gender identification, 158 samples obtained by dividing 23 morulae were measured, the result: the results of the negative control group and the positive control group were all correct, and from 12 emb...

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Abstract

The invention relates to a method for gender identification of early bovine embryos, comprising the following steps: performing in vitro fertilization of oocytes and sperm to obtain morulae; cutting the morulae in the segmentation liquid droplet with a segmentation knife to obtain embryonic tissues containing 10 to 12 cells As a sample for gender identification; the ring-mediated isothermal amplification method gender identification kit makes samples and prepares Master Mix; after the samples are mixed, put the reaction tube in the ring-mediated isothermal amplification real-time turbidity detection system for reaction, and determine the sample according to the results One of the three cases needs to be redone for male, female, uncertain. The method of the invention presents excellent technical effects as described in the specification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a sex identification method, in particular to a method for sex identification of bovine early embryos. In particular, it relates to a method for sex identification of bovine early embryos using a loop-mediated isothermal amplification method. Background technique [0002] Animal husbandry uses the physiological functions of livestock and poultry and other animals that have been domesticated by humans, or wild animals such as deer, musk deer, fox, mink, otter, quail, etc., through artificial breeding and breeding, so that they can transform plants such as pasture and feed into Animals can obtain livestock products such as meat, eggs, milk, wool, cashmere, hides, silk and medicinal materials. Different from self-sufficient livestock breeding, animal husbandry has the characteristics of centralization and scale, and has become a pillar industry to ensure basic people's livelihood. [00...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6879C12Q1/6888C12Q1/6844C12N5/075C12N5/076C12N5/073
CPCC12Q1/6879C12Q1/6888C12Q1/6844C12N5/0609C12N5/061C12N5/0604C12Q2600/166C12N2501/31C12N2501/02C12N2501/11C12N2500/12C12N2500/14C12N2500/16C12N2500/30C12N2501/91C12N2501/998C12N2501/999C12Q2531/119C12Q2537/1376C12Q2545/113
Inventor 王小武王娜陈建美郝少强赵明礼郭春明李琳许晓椿
Owner 天津力牧生物科技有限公司
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