Application of octanoylated ghrelin in promoting bovine oocyte maturation in vitro

An oocyte and in vitro maturation technology, applied in the field of livestock gamete embryo engineering, can solve problems such as uncertain effects, achieve the effect of less toxic effect, promote cleavage rate and blastocyst rate, and promote the effect of synchronous maturation

Active Publication Date: 2020-02-21
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] However, the role of octanoylated Ghrelin in in vitro segmental maturation of bovine oocytes and its further use in in vitro fertilization and embryo production is still uncertain, and there is no research report on this aspect so far

Method used

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  • Application of octanoylated ghrelin in promoting bovine oocyte maturation in vitro
  • Application of octanoylated ghrelin in promoting bovine oocyte maturation in vitro

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1 oocyte maturation culture

[0026] (1) Oocyte collection

[0027] Use a 10ml syringe containing 2ml egg extraction solution (containing 25mM hepes M199+1%PVA+200uM IBMX+1% double antibody) to extract follicles with a diameter of 3-8mm from the surface of bovine ovaries obtained from the slaughterhouse. Put the egg-absorbing liquid after absorbing the oocytes into the 7cm 3 In domestic petri dishes, cumulus-oocyte omplexes (COCs) with uniform cytoplasm and three or more layers of dense cumulus granulosa cells were selected under a stereo microscope for pre-maturation in vitro to cultivate.

[0028] (2) Oocyte pre-maturation treatment

[0029] The selected COCs were washed 3 times in the egg washing solution (containing hepes M199+1%PVA+200uM IBMX+1% double antibody), and washed in the pre-maturation solution containing octanoylated Ghrelin (adding 1-1000ng / ml concentration of octane Acylated Ghrelin's M199 culture medium) was washed 3 times, and then put...

Embodiment 2

[0044] Example 2 In vitro fertilization and in vitro embryo production

[0045] Further experiments were carried out with the oocytes matured and cultured in Example 1.

[0046] (1) In vitro fertilization

[0047] Using the petri dish micro-drop method, first wash the mature oocytes in fertilization fluid (BO solution + 20mg / ml BSA + 20mg / mL heparin sodium + 100mg / mL streptomycin + 100IU / mL penicillin) for 2-3 times Put it into the balanced fertilization fluid (the amount of 60 μl of fertilization fluid, 20 oocytes), and the oocytes cultured in the pre-maturation fluid without octanoylated Ghrelin as the control; then use the flotation method to process the frozen semen , the sperm floated up in 2ml of semen washing solution (BO solution + 3.38mg / mL caffeine) for 30min, then took 600-800μl of the supernatant and put it into a 1.5ml centrifuge tube for centrifugation (1800 rpm, 5min) twice, after centrifugation, remove the supernatant and add The final volume of fertilized fl...

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Abstract

The invention provides a bovine oocyte in vitro pre-maturation liquid, the pre-maturation liquid uses the bovine oocyte in vitro culture liquid as a matrix, and the matrix contains octanoylated Ghrelin. The invention suppresses the recovery of bovine oocyte meiosis through in vitro culture of octanoylated Ghrelin, thereby making oocyte nuclei and cytoplasm mature synchronously, and further improving the quality and in vitro development ability of bovine oocytes. It can be popularized and applied in animal husbandry production as a new type of meiosis preparation drug, and has good market effect and social effect.

Description

technical field [0001] The invention relates to the technical field of livestock gamete embryo engineering, in particular to the application of octanoylated ghrelin in promoting bovine oocyte maturation in vitro. Background technique [0002] Bovine oocyte in vitro maturation (In Vitro Maturation, IVM), as one of the three major technical links of embryo in vitro production technology (InVitro Production, IVP), is an important reproductive biotechnology and a key technology that determines in vitro embryo production. It obtains a large number of usable eggs through in vitro maturation and culture of oocytes derived from antral follicles of abandoned ovaries in slaughterhouses, which can provide a rich source of mature eggs for breeding, cloning, sex control, nuclear transfer, transgenic animal production, etc., turning waste into treasure. [0003] In vitro maturation of oocytes refers to the ability of oocytes to spontaneously resume meiosis and obtain the ability of normal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2501/392C12N2501/30C12N2501/31C12N2501/91C12N2500/32
Inventor 许晓玲刘彦白佳桦冯涛宋玉清肖霖力肖银霞
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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