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A method for increasing the developmental rate of in vitro fertilized embryos in river buffalo

An in vitro fertilization and developmental rate technology, applied in embryonic cells, biochemical equipment and methods, microorganisms, etc., can solve the problems of difficulty in extracting follicular fluid, not easy to obtain, etc., to improve the division rate and blastocyst rate, improve embryonic Effects of developmental rates and productivity

Active Publication Date: 2022-01-07
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%

Method used

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  • A method for increasing the developmental rate of in vitro fertilized embryos in river buffalo

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Experimental program
Comparison scheme
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 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 o...

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Abstract

The invention relates to the technical field of buffalo embryo development, in particular to a method for improving the developmental rate of river-type buffalo in vitro fertilized embryos. %), it can still achieve the effect of increasing the division rate and blastocyst rate of buffalo fertilized eggs, thereby improving the buffalo in vitro embryo development rate and production efficiency; in the formula, in order to activate the activity of oocytes, the applicant has done a lot of research It was found that the use of cysteine, methionine, alanine, lysine, tryptophan and glycine dissolved in TCM199 solution and then mixed with follicular fluid for chelation can significantly improve the effect of follicular fluid on the promotion of embryonic development rate.

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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Patent Type & Authority Patents(China)
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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