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Method for improving embryo development rate of in vitro fertilization of river buffalo

A technology of in vitro fertilization and development rate, applied in embryonic cells, biochemical equipment and methods, microorganisms, etc., can solve the problems of difficult access and difficult extraction of follicular fluid, and achieve the effect of improving embryonic development rate and production efficiency

Active Publication Date: 2019-11-12
GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INST
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Problems solved by technology

The effect of in vitro maturation in many existing in vitro culture technologies has not been very satisfactory, it is difficult to combine this technology with embryo transfer and living oocyte collection (OPU) technology, apply it to production practice, and promote its application; The research on the in vitro fertilization effect of oocyte mature buffalo oocytes, for example, the paper "Influence of different breeds of bovine follicular fluid on the in vitro fertilization effect of buffalo oocytes" published by the applicant (Chinese Animal Husbandry and Veterinary Medicine 2010 Volume 37 No. Phase 4); in this paper, the research on the in vitro fertilization effect of bovine follicles of different varieties on buffalo oocytes, the paper pointed out that the optimal amount of follicular fluid is 5%-10%, and pointed out that adding 5% and 10% follicles The fluid can significantly promote the development of embryos after in vitro fertilization of oocytes collected in slaughterhouses. Too high or too low a concentration will inhibit the development of buffalo IVF embryos. Obtained, for this reason, the applicant studies how to improve the formula and operation method on the basis of the original research, so that follicular fluid can achieve the purpose of promoting the development of buffalo IVF embryos at a lower concentration: 1%-4%

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  • Method for improving embryo development rate of in vitro fertilization of river buffalo

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Experimental program
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Effect test

Embodiment 1

[0025] In vitro culture method of eggs collected in slaughterhouses:

[0026] 1. In vitro maturation culture:

[0027] Collect buffalo ovaries from the slaughterhouse, keep them warm in an insulated jug containing 30°C saline, and send them to the laboratory within 2 hours. It is best to ensure that the temperature in the insulated jug is still at 30°C after arriving at the laboratory. Pour out the ovaries, remove redundant tissues such as mesentery and fallopian tubes, and then wash them twice with normal saline containing double antibodies. After cleaning, extract follicles with a diameter of 2mm with a 10mL syringe, slowly inject them into a plate, and quickly select them under a stereomicroscope For oocytes with uniform cytoplasm and more than 3 layers of granulosa cells outside the cells, wash twice, culture on a plate, and place in maturation solution A at 39°C and 5% CO 2 Cultivate in a saturated air humidity incubator. The preparation method of maturation solution A i...

Embodiment 2

[0050] 1. In vitro maturation culture:

[0051] Collect buffalo ovaries from the slaughterhouse, keep them warm in an insulated jug containing 39°C saline, and send them to the laboratory within 3 hours. It is best to ensure that the temperature in the insulated jug is still at 33°C after arriving at the laboratory. Pour out the ovaries, remove redundant tissues such as the mesentery and fallopian tubes, and then wash 3 times with normal saline containing double antibodies. After cleaning, use a 10mL syringe to extract follicles with a diameter of 6mm, slowly inject them into a plate, quickly select oocytes with uniform cytoplasm and more than 3 layers of extracellular granulosa cells under a stereomicroscope, wash 3 times, and culture them in a plate. In mature solution A at 39°C, 5% CO 2 Cultivate in a saturated air humidity incubator. The preparation method of maturation solution A is as follows: take 50mL of base solution and add 200μL of FSH-LH storage solution, 45μL of ...

Embodiment 3

[0074] 1. In vitro maturation culture:

[0075] Collect buffalo ovaries from the slaughterhouse, keep them warm in an insulated jug containing 33°C saline, and send them to the laboratory within 2.5 hours. It is best to ensure that the temperature in the insulated jug is still at 32°C after arriving at the laboratory. Pour out the ovaries, remove redundant tissues such as the mesentery and fallopian tubes, and then wash 3 times with normal saline containing double antibodies. After cleaning, use a 10mL syringe to extract follicles with a diameter of 5mm, slowly inject them into a plate, quickly select oocytes with uniform cytoplasm and more than 3 layers of extracellular granulosa cells under a stereomicroscope, wash 3 times, and culture them in a plate. In mature solution A at 39°C, 5% CO 2 Culture in a saturated air humidity incubator, wherein the preparation method of maturation solution A is as follows: take 50mL of base solution and add 200μL of FSH-LH storage solution, ...

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Abstract

The invention relates to the technical field of buffalo embryo development, in particular to a method for improving the embryo development rate of in vitro fertilization of a river buffalo. Accordingto the present invention, the improvement of buffalo oocyte mature culture solution is mainly researched, and the cleavage rate and blastocyst rate of buffalo fertilized eggs can still be increased inlow concentration of follicular solution (1%-4%), so as to improve the in vitro embryo development rate and produciton efficiency of buffaloes. In the formula, in order to activate the activity of oocytes, the applicant does a large number of studies, and finds that the effect of follicular solution on the promotion of embryo development rate can be significantly improved by dissolving cysteine,methionine, alanine, lysine, tryptophan and glycine in TCM199 solution and then chelating the above solution with the follicular soluiton.

Description

【Technical field】 [0001] The invention relates to the technical field of buffalo embryo development, in particular to a method for improving the development rate of in vitro fertilized embryos of river buffalo. 【Background technique】 [0002] Embryo culture technology is of great value for the excavation of reproductive potential, rapid multiplication and protection of germplasm resources of domestic animals. There are many factors affecting embryo in vitro culture and in vitro embryo preservation, among which oocyte maturation in vitro, in vitro fertilization, in vitro culture of early embryos and embryo freezing are the four main links in the process of in vitro embryo production. The effect of in vitro maturation in many existing in vitro culture technologies has not been very satisfactory, it is difficult to combine this technology with embryo transfer and living oocyte collection (OPU) technology, apply it to production practice, and promote its application; The resear...

Claims

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Application Information

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IPC IPC(8): C12N5/073
CPCC12N5/0604C12N2500/84C12N2500/32C12N2501/31C12N2501/11
Inventor 庞春英梁贤威陈明棠梁莎莎马小娅谭正准陆杏蓉段安琴邓廷贤
Owner GUANGXI ZHUANG AUTONOMOUS REGION BUFFALO INST
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