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Culture solution for improving development potential of in-vitro cultured porcine oocytes and its cultivating method

A technology for in vitro maturation and cultivation of oocytes, which is applied in the field of culture medium for improving the developmental potential of porcine oocytes in vitro, can solve the problems of the gap between maturation quality and developmental potential, and achieve improved developmental potential, great application prospects, and improved oocyte growth potential. crack rate effect

Inactive Publication Date: 2017-08-11
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a commonly used model organism, the research on in vitro maturation of pig oocytes began in the late 1980s. After years of development, although great progress has been made, the porcine oocytes matured in vitro, the There is still a large gap between the mature quality and developmental potential compared with the body

Method used

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  • Culture solution for improving development potential of in-vitro cultured porcine oocytes and its cultivating method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 In vitro culture of porcine oocytes

[0030] 1. Preparation of solution and culture medium

[0031] (1) Forskolin (Forskolin): Dissolve 1 mg Forskolin in 2.44 mL DMSO to obtain a stock solution with a final concentration of 1 mM, store at -20°C; 2 O was diluted to 10 μM working solution and stored at 4°C for later use.

[0032] (2) Nuclear inhibitor Roscovitine: Dissolve 1 mg Roscovitin in 2.83 ml DMSO to obtain a stock solution with a final concentration of 1 mM, store at -20°C; then use ddH 2 O was diluted to 10 μM working solution and stored at 4°C for later use.

[0033] (3) 0-22 h culture solution: take 9.87 g TCM-199, 2.2 g NaHCO 3 , 0.5496 g D-glucose, 0.1 g sodium pyruvate, 0.075 g penicillin sodium salt, 0.05 g streptomycin sulfate, 1 g polyvinyl alcohol dissolved in 1 L ddHO 2 O, after mixing well, add 0.5 μg / mL LH, 0.5 μg / mL FSH, 10 ng / mL EGF, 0.57 mM L-cysteine, 10% PFF; placed in CO 2 The incubator was equilibrated overnight.

[0034] (4) 2...

Embodiment 2

[0049] Example 2 In vitro culture of porcine oocytes

[0050] 1. Preparation of solution and culture medium

[0051] Same as Example 1.

[0052] 2. In vitro culture of porcine oocytes

[0053] (1) Forskolin and nuclear inhibitor Roscovitine were added to the 0-22 h culture medium respectively, so that the final concentration of Forskolin was 3 μM, and the final concentration of Roscovitine was 7 μM; the porcine oocytes were added with Forskolin and Roscovitine were cultured in the 0-22 h culture solution for 24 hours, then transferred to the 22-44 h culture solution for 24 hours, and the subsequent parthenogenetic activation experiment was carried out.

[0054] (2) Set up a control group, which is the same as in Example 1.

[0055] 3. Results

[0056] The results are shown in Table 4. By examining the morphologically normal rate, cleavage rate, and blastocyst rate of porcine oocytes, it was found that porcine oocytes had normal cell morphology after being cultured in the m...

Embodiment 3

[0060] Example 3 In vitro culture of porcine oocytes

[0061] 1. Preparation of solution and culture medium

[0062] Same as Example 1.

[0063] 2. In vitro culture of porcine oocytes

[0064] (1) Forskolin and nuclear inhibitor Roscovitine were added to the 0-22 h culture medium respectively, so that the final concentration of Forskolin was 7 μM, and the final concentration of Roscovitine was 3 μM; the porcine oocytes were added with Forskolin and Roscovitine were cultured in the 0-22 h culture solution for 24 hours, then transferred to the 22-44 h culture solution for 24 hours, and the subsequent parthenogenetic activation experiment was carried out.

[0065] (2) Set up a control group, which is the same as in Example 1.

[0066] 3. Results

[0067] The results are shown in Table 5. By examining the morphologically normal rate, cleavage rate, and blastocyst rate of porcine oocytes, it was found that porcine oocytes had normal cell morphology after being cultured in the m...

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Abstract

The invention discloses a culture solution for improving maturity quality and development potential of in-vitro cultured porcine oocytes and its cultivating method. The culture solution is prepared from, by weight, 3-7 mu mol / L of forskolin, 3-7 mu mol / L of Roscovitine, 8-12g / L of TCM-199, 1-4g / L of NaHCO3, 0.4-0.7g / L of D-glucose, 0.05-0.15g / L of sodium pyruvate, 0.05-0.1g / L of penicillin sodium, 0.03-0.07g / L of streptomycin sulphate, 1-4g / L of polyving akohol, 0.3-0.7mu g / mL of luteotropin, 0.3-0.7mu g / mL of follicle-stimulating hormone, 8-12ng / mL of epidermal growth factor, 0.3-0.7mmol / L of L- cysteine, and 8-12% of follicular fluid. The culture solution has the advantages of improving cleavage rate, blastaea number and blastaea cell population of porcine oocytes, and effectively improving the development potential of the in-vitro cultured porcine oocytes; so the culture solution has great application prospect.

Description

technical field [0001] The invention belongs to the technical field of embryo engineering, and in particular relates to a culture solution and a culture method for improving the developmental potential of porcine oocytes cultured in vitro. Background technique [0002] Mammalian oocyte in vitro culture technology (In vitro maturation, IVM) is an important part of embryo engineering, which means that the immature oocytes taken out of follicles are matured and cultured in vitro, and developed to the second meiotic division metaphase. A technique in which in vitro fertilization is performed and split into embryos. As a commonly used model organism, the research on in vitro maturation of pig oocytes began in the late 1980s. After years of development, although great progress has been made, the porcine oocytes matured in vitro, the There is still a large gap in the mature quality and developmental potential compared with the body. Therefore, we still need to continue our effort...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2501/405C12N2501/31C12N2501/70C12N2501/11C12N2500/32C12N2500/34C12N2500/30C12N2500/12
Inventor 丛佩清牛惠然张冰月蔡青青何祖勇
Owner SUN YAT SEN UNIV
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