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Method for improving bovine oocyte maturation in vitro

A technology for in vitro maturation of oocytes, applied in the field of improved in vitro maturation of bovine oocytes, which can solve the problems of low production efficiency of in vitro embryos

Active Publication Date: 2020-06-05
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

In vitro embryo production in cattle is less efficient than in other mammals

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1: live bovine ovum collection

[0066] Refer to the method of the inventor's team (CN110251660A, Chinese patent application number 2019106516223, Y19032) to collect live bovine eggs, as follows:

[0067] 1. Superovulation treatment before egg retrieval

[0068] Select cattle with follicles ≥ 20 with a diameter of 2-8mm on both ovaries as oocyte donor cattle, and the age of the cattle is 2-5 years old; inject FSH preparations in 4 equal doses within two days for superovulation, each time The injection interval is 12 hours, and the dose of each injection is 0.5 μg / kg cattle body weight in terms of FSH;

[0069] 2. Live egg collection

[0070] 48 hours after the last injection of FSH preparation, live egg collection is carried out, and the live egg collection operation is as follows:

[0071] (21) Preoperative preparation: adjust the equipment to the working state (the pressure of the egg suction vacuum pump is 50-90mmHg), inject about 5ml of egg suction li...

Embodiment 2

[0078] Example 2: In Vitro Maturation of Bovine Oocytes

[0079] By adding EGCG (10 μmol / L) in the in vitro maturation solution and referring to Li Minling's literature method to investigate the in vitro maturation of bovine oocytes, to investigate the methodological performance of the present invention with indicators such as maturation rate and cumulus expansion, using in vitro fertilization and parthenogenesis Two methods of activation method were used to investigate the developmental ability of oocytes after maturation, in order to improve the efficiency of in vitro culture of bovine embryos and improve the quality of development. Unless otherwise specified, the cattle involved in the operation are Chinese (Nanyang) yellow cattle.

[0080] 1. Materials

[0081] (1) Frozen semen (Nanyang yellow cattle, thin tube) is stored by the company itself. TCM-199 was purchased from GIBCO Company, Dulbecco'sPBS was purchased from GIBCO Company, fetal bovine serum (FBS) was purchas...

Embodiment 3

[0105] Example 3: In Vitro Maturation of Bovine Oocytes

[0106] Carry out with reference to embodiment 1 and embodiment 2, difference is only that the composition of mature culture fluid (M liquid) used in embodiment 2 is: 20mg / L sodium octanoate, 5mg / L polyvinyl alcohol, 10%ECS (estrus bovine serum) , 26.2mmol / L NaHCO3, 10μmol / L EGCG, 5mmol / L Hepe, 1μg / mL E2 (estradiol), 3% BFF (bovine follicular fluid), 10μg / mL LH (luteinizing hormone) , 100 μmol / L cysteine, 10 μg / mL FSH (follicle stimulating hormone), and TCM-199 were added to 100%.

[0107] With reference to the test process of Example 2, the results are as follows:

[0108] (1) After the oocytes were matured in vitro for 22-24 hours, the total number of eggs counted was 221, and the cumulus expansion index CEI (%±S.E.)=2.81±0.09**.

[0109] (2) After the oocytes were matured in vitro and cultured for 22-24 hours, the total number of eggs counted was 235, and the maturation rate (%±S.E.)=69.33±2.13**.

[0110] (3) Af...

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Abstract

The invention relates to a method for improving bovine oocyte maturation in vitro. Particularly, the method for improving bovine oocyte maturation in vitro comprises the following steps: (1) providing oocytes; and (2) maturation of oocytes in vitro: putting the occytes in mature culture solution to perform maturation cultivation in vitro for 22-24 h under culture conditions of 100% humidity, 38.5DEG C and 5% CO2. The mature culture solution comprises: ECS, NaHCO3, EGCG, Hepe, estradiol, BFF, luteinizing hormone (LH), cysteine, follicle stimulating hormone (FSH) and TCM-199. The method for improving bovine oocyte maturation in vitro can effectively promote performance of bovine occyte maturation in vitro.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a method for in vitro maturation of oocytes, in particular to an improved method for in vitro maturation of bovine oocytes. Through the method of the invention, the performance of bovine oocyte maturation in vitro can be obviously improved, for example, cumulus expansion index, oocyte maturation rate in vitro, cleavage rate and blastocyst rate can be significantly improved. Background technique [0002] In vitro fertilization of oocytes refers to the process of fertilizing oocytes through special treatment of sperm. The completion of in vitro fertilization requires a culture system that is conducive to the survival and metabolism of sperm and eggs. In vitro fertilization is the basis for in vitro embryo production. At present, the in vitro production technology of embryos is developing rapidly and is mainly used in the reproduction of various domestic animals, exotic breeds, wild anim...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075C12N5/073A61D19/00
CPCC12N5/0609C12N5/0604A61D19/00C12N2517/10C12N2500/12C12N2501/392C12N2501/31C12N2500/32C12N2500/50C12N2500/30C12N2501/999C12N2500/60
Inventor 郭晶王小武王娜张月桥郝少强赵明礼郭春明许晓椿
Owner 天津力牧生物科技有限公司
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