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Method for improving in-vitro fertilization effect of unfrozen pig sperms

A technology of in vitro fertilization and pig sperm, applied in the preservation of human or animal bodies, biochemical equipment and methods, embryonic cells, etc., can solve the problems of poor embryo quality, poor oocyte maturation quality, and high fertilization rate of polysperm. Achieve the effect of improving quality, improving the effect of in vitro fertilization, and improving activity

Active Publication Date: 2020-10-30
JILIN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Poor oocyte maturation quality, high polysperm fertilization rate and poor embryo quality are the main problems faced by pig IVP

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] A method for improving the in vitro fertilization effect of thawed pig sperm, comprising the following steps:

[0026] (1) The frozen semen is taken out and then thawed, wherein the frozen base liquid used for semen freezing is made of the following components by weight: 200 parts of glucose, 300 parts of lactose, 10 parts of sodium bicarbonate, 5 parts of penicillin sodium, 8 parts of streptomycin sulfate, 1800 parts of egg yolk, 7000 parts of triple distilled water, wherein the frozen base liquid also includes phenethyl caffeate and polyvinylpyrrolidone, wherein the molar concentration of caffeic acid phenethyl in the frozen base liquid is 30 μmol / L, the mass fraction of polyvinylpyrrolidone in the frozen base fluid is 0.4%;

[0027] (2) Wash the thawed semen with the fertilization solution and centrifuge twice, then wash and centrifuge the centrifuged pellet once with mTBM capacitation solution, add mTBM capacitation solution to the pellet after centrifugation and r...

Embodiment 2

[0033] A method for improving the in vitro fertilization effect of thawed pig sperm, comprising the following steps:

[0034] (1) The frozen semen is taken out and then thawed, wherein the frozen base liquid used for semen freezing is made of the following components by weight: 250 parts of glucose, 350 parts of lactose, 13 parts of sodium bicarbonate, 6 parts of penicillin sodium, 10 parts of streptomycin sulfate, 2000 parts of egg yolk, 8000 parts of triple distilled water, wherein the frozen base liquid also includes caffeic acid phenethyl and polyvinylpyrrolidone, wherein the molar concentration of caffeic acid phenethyl in the frozen base liquid is 40 μmol / L, the mass fraction of polyvinylpyrrolidone in the frozen base fluid is 0.5%;

[0035] (2) Wash the thawed semen with the fertilization solution and centrifuge twice, then wash and centrifuge the centrifuged pellet once with mTBM capacitation solution, add mTBM capacitation solution to the pellet after centrifugation ...

Embodiment 3

[0041] A method for improving the in vitro fertilization effect of thawed pig sperm, comprising the following steps:

[0042] (1) The frozen semen is taken out and then thawed, wherein the frozen base liquid used for semen freezing is made of the following components by weight: 300 parts of glucose, 400 parts of lactose, 15 parts of sodium bicarbonate, 7 parts of penicillin sodium, 12 parts of streptomycin sulfate, 2200 parts of egg yolk, 9000 parts of triple distilled water, wherein the frozen base liquid also includes phenethyl caffeate and polyvinylpyrrolidone, wherein the molar concentration of caffeic acid phenethyl in the frozen base liquid is 50 μmol / L, the mass fraction of polyvinylpyrrolidone in the frozen base fluid is 0.6%;

[0043] (2) Wash the thawed semen with the fertilization solution and centrifuge twice, then wash and centrifuge the centrifuged pellet once with mTBM capacitation solution, add mTBM capacitation solution to the pellet after centrifugation and ...

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PUM

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Abstract

The invention discloses a method for improving the in-vitro fertilization effect of unfrozen pig sperms. The method comprises the following operation steps: (1) taking out frozen sperms, and unfreezing the frozen sperms; (2) cleaning the unfrozen seminal fluid with fertilization operating fluid, centrifuging twice, cleaning centrifugal precipitate with mTBM energetic fluid, centrifuging once, resuspending, and culturing to obtain energetic seminal fluid; (3) picking out a cumulus-oocyte complex from the ovary from which the follicular fluid is extracted under a microscope, culturing, taking out oocytes, and removing the cumulus; and (4) dropwise adding the obtained seminal fluid into each small fertilization cell, putting the small fertilization cells into an incubator for culture, and then washing off sperms attached to the oocytes to finish in-vitro fertilization culture. According to the invention, caffeic acid phenethyl ester and polyvinylpyrrolidone with corresponding concentrations are jointly added into the freezing base solution I, and the caffeic acid phenethyl ester and polyvinylpyrrolidone generate a synergistic effect so that the vitality, plasma membrane integrity rateand DNA integrity rate of the unfrozen pig sperms can be effectively improved, and the in-vitro fertilization effect of the unfrozen pig sperms is further improved.

Description

technical field [0001] The invention relates to the technical field of cryopreservation of pig sperm, in particular to a method for improving the in vitro fertilization effect of thawed pig sperm. Background technique [0002] The cryopreservation of semen can effectively preserve the genetic breeding and germplasm resources of organisms [8]. With the rapid development of pig frozen semen production technology and artificial insemination technology, the conception rate and number of piglets of breeding pigs bred with frozen semen have been effectively improved, which is close to the breeding level of fresh semen. However, due to many reasons such as the difficulty in the production of pig frozen semen, the low survival rate after freezing and thawing, and the low conception rate, the research on pig semen freezing technology is still not fully mature. In general, there are still many problems to be solved urgently to perfect the preparation technology of pig frozen semen. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073C12N5/076A01N1/02
CPCC12N5/061C12N5/0604A01N1/0226A01N1/0221A01N1/0215A01N1/021C12N2500/14C12N2500/12C12N2500/34C12N2500/30C12N2500/32C12N2500/60C12N2501/998
Inventor 钱芙蓉李兆华张志彬于永生高一菅芯蕊王珺曹阳张立春昭日格图孙华王俊恒李彦春刘向光金庆国柳海星高见红马腾飞孟庆祝丁德李闯张鑫陆永辉祝永凯
Owner JILIN ACAD OF AGRI SCI
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