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Method for producing in-vitro calf embryo

A technology for calves and embryos, applied in the field of in vitro embryo production, can solve problems such as individual differences in calves, and achieve the effect of high blastocyst development rate

Inactive Publication Date: 2011-02-02
北京奶牛中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] 4. Individual differences in calves

Method used

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  • Method for producing in-vitro calf embryo
  • Method for producing in-vitro calf embryo

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Calf Hormone Superovulation and Egg Collection

[0039] The day of superovulation treatment of calves was day 0, CIDR for sheep (for sheep, place of origin New Zealand) was implanted vaginally with a suppository, and FSH was injected at intervals of 12 hours on day 5 (40 mg, 30 mg) and day 6 (30 mg, 30 mg) respectively ( FSH, trade name Folltronpin-V, purchased from Canadian Bioniche Animal Health company), on the 7th day eggs were collected, and the thrombus was withdrawn at the same time.

[0040] The number of usable oocytes obtained in each calf super-rowing was 48 pieces.

Embodiment 2

[0041] Example 2 In vitro maturation of calf oocytes

[0042]2 hours before the in vitro maturation operation, put an appropriate amount of maturation culture medium in each well of a 6-well conical culture plate (average 10 μl of maturation culture medium per oocyte), cover with mineral oil, and put it in 38.5 ° C, 5% CO 2 , pre-equilibrated in a humidified incubator. Among them, the composition of the mature medium is: TCM199+10mM HEPES+5%FBS+0.5μg / ml FSH+5μg / mlLH+1μg / ml E 2 + 27.5 μg / ml sodium pyruvate + 100 IU / ml penicillin + 100 μM cysteamine.

[0043] Put the identified and usable oocytes into the preheated maturation medium, wash them gently 4 times, transfer them into the maturation medium of a 6-well conical culture plate that has been equilibrated in the incubator for 2 hours, and place them in a constant temperature incubator Cultivate for 22h-24h. Culture conditions are 38.5°C, 5% CO 2 , saturated humidity.

Embodiment 3

[0044] Example 3 In vitro fertilization of calf oocytes

[0045] Take a thin tube (0.25ml) of frozen semen (the semen comes from the frozen semen of bulls from the Beijing Dairy Cow Center), put it in a water bath at 37°C for about 10 seconds, take it out until there are bubbles floating up, dry it with a facial tissue, and disinfect it with an alcohol cotton ball Thin tube; pre-equilibrated for 2 hours with 6ml fertilization solution (114.0mM NaCl+4.02mM KCl+2.25mM CaCl 2 .2H 2 O+0.52mMMgCl 2 6H 2 O+0.83mM NaH 2 PO 4 h 2 O+37.0mM NaHCO 3 + 13.9mM glucose + 0.5mM sodium pyruvate + 3mg / ml BSA + 10μg / ml phenol red + 100IU / ml penicillin + 10μg / ml heparin) was taken out from the constant temperature incubator, and one end of the semen thin tube Cut off, insert into the centrifuge tube and cut off the other end until all the semen enters the centrifuge tube, gently turn the centrifuge tube to mix. 300-400g, centrifuge for 5min. Use a pipette to suck off the supernatant, le...

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Abstract

The invention establishes an efficient method for producing an in-vitro calf embryo, and the method comprises the technology of efficient oocyte in-vitro maturation, in-vitro fertilization, in-vitro embryo cultivation and the like. In the production of the in-vitro calf embryo, the oocyte in a calf ovary can be completely taken as abundant embryo sources, and superovulation and collection embryo are carried out on the calf. The method is combined with the cow female-controlled technology and uses a female-controlled X or Y sperm selectively to perform in-vitro fertilization according to a breeding object, so as to produce a fine cow embryo with definite blood relationship; and after transplantation, the produced calf can be directly supplemented to a high-yield cow core group or be used for breeding a reserved bull.

Description

technical field [0001] The invention relates to a method for in vitro production of embryos, in particular to a method for in vitro production of calf embryos. Background technique [0002] JIVET (Juvenile In Vitro Embryo Transfer / Technology) technology is the in vitro production technology of young animal embryos. Biological high-tech system. JIVET technology can not only greatly improve the reproductive potential of animals, but also shorten the generation interval and accelerate the process of genetic improvement. [0003] The research on calf in vitro embryo production technology originated in the 1950s. After injecting gonadotropin into immature calves, Black et al. induced the development of ovarian follicles and obtained normally developed eggs, thus proving that hormone induction can also make calves Follicular development, ovulation and fertilization. Afterwards, many scholars conducted a lot of research on the developmental ability of calf oocytes. Since the 19...

Claims

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Application Information

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IPC IPC(8): C12N5/075A61D19/02C12N5/073
Inventor 薛建华吕小青朱玉林宣柏华梁鸿斌李艳华曹福存吴胜权王彦平王海浪
Owner 北京奶牛中心
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