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Spermin-vitro capacitation solution, kit applied to in-vitro fertilization and in-vitro fertilization method of mammals

A mammalian, in vitro fertilization technology, applied in biochemical equipment and methods, animal cells, vertebrate cells, etc., can solve the problems of low fertilization efficiency and high cost, and achieve the effect of improving fertilization efficiency, fertilization efficiency and convenient use.

Inactive Publication Date: 2018-06-12
ZHEJIANG ACAD OF MEDICAL SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The present invention aims at the low efficiency of in vitro fertilization of gerbils in the prior art and the need for expensive instruments for microinjection, and provides a sperm in vitro capacitation solution, a kit for in vitro fertilization and a method for in vitro fertilization of mammals , improve the efficiency of in vitro fertilization

Method used

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  • Spermin-vitro capacitation solution, kit applied to in-vitro fertilization and in-vitro fertilization method of mammals
  • Spermin-vitro capacitation solution, kit applied to in-vitro fertilization and in-vitro fertilization method of mammals
  • Spermin-vitro capacitation solution, kit applied to in-vitro fertilization and in-vitro fertilization method of mammals

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Effect test

Embodiment 1

[0039] The computer-assisted semen analysis (CASA) system was used to analyze the movement parameters of gerbil sperm in different culture medium and different culture time, aiming to explore the time of hyperactivation of spermatogenesis in gerbils, and to screen suitable gerbils Capabilization medium. A total of 4 kinds of capacitation culture medium were screened, among which HTF was SAGE auxiliary germ cell medium (Quinns Fertilization Medium, U.S. SAGE company), when using, add 10% serum (SAGE company); IVF is commercial in vitro fertilization and split culture fluid (Vitrolife company, product number 503938); "In vitro capacitation fluid" means the present invention In vitro capacitation liquid of the formula; "in vitro fertilization liquid" means the in vitro fertilization liquid of the formula of the present invention.

[0040] Sperm collection in gerbils: ether anesthetized 6 gerbils, opened the abdominal cavity of gerbils, exposed the epididymis, removed the surroun...

Embodiment 2

[0059] 1.1 Droplet Equilibrium

[0060] Get in vitro capacitation fluid and in vitro fertilization fluid and make capacitation fluid drop (300 μ L / drop) and fertilization fluid drop (200 μ L / drop) respectively in 35mm petri dish, use mineral oil 4ml (the present invention uses paraffin oil, the same below.) Cover all droplets and equilibrate 30 min before use.

[0061] 1.2 Sperm acquisition, culture and determination of sperm capacitation time in gerbils

[0062] At 8:00 in the morning, take 4-month-old male mice with reproductive ability, kill them by cervical dislocation, disinfect the abdomen with 75% alcohol, open the abdominal cavity, take the cauda epididymis, use ophthalmic scissors to peel off the fat around the cauda epididymis, and remove it on sterile filter paper For blood and fat, gently squeeze the cauda epididymis with your thumb and index finger, and at the same time puncture the cauda epididymis with a 1mL syringe needle. After milky white sperm clusters emer...

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Abstract

The invention discloses a spermin-vitro capacitation solution, a kit applied to in-vitro fertilization and an in-vitro fertilization method of mammals. The spermin-vitro capacitation solution comprises 110-140mmol of NaCl, 2.40-2.95mmol of KCl, 22.5-27.5mmol of NaHCO3, 4.8-6.0mmol of glucose, 0.22-0.30mmol of sodium pyruvate, 0.22-0.30mmol of taurine, 0.04-0.06mmol of epinephrine, 14-16mg / ml of BSA, 0.4-0.6mmol of MgCl2, 1.6-2.0mmol of CaCl2 and 8-12mmol of a DL-sodium lactate solution, wherein the pH value of the spermin-vitro capacitation solution is 7.35+ / -0.10. The spermin-vitro capacitation solution is capable of obviously improving the in-vitro fertilization efficiency of mongolian gerbil; after the method is used, the in-vitro fertilization efficiency of mongolian gerbil is greatlyimproved; two-cell development rate reaches 65% or above; the maximum two-cell development rate can reach up to 85%; the in-vitro fertilization rate obtained by the method is equivalent to that of a microinjection method and is even higher than that of the microinjection method; the method is convenient to use and has a good application prospect.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a sperm in vitro capacitation solution, a kit for in vitro fertilization and a mammalian in vitro fertilization method. Background technique [0002] The long-clawed gerbil (Mongolian gerbil, Meriones unguiculatus) is a research tool for neurology, parasitology, virology, bacteriology, endocrinology, genetics, hematology, lipids, glucose metabolism, oncology, pharmacology, radiation biology It is a good model animal for , reproduction and toxicology, and has high scientific research value and application prospect. The traditional method of conservation consumes a lot of manpower and material resources, and natural disasters and accidental microbial contamination may destroy these precious strains, and special phenotypes may also be lost due to genetic drift during the breeding process. Embryo cryopreservation is the main way to preserve gerbil resources in the future, which requires...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076C12N5/073
CPCC12N5/0604C12N5/061C12N2500/12C12N2500/14C12N2500/16C12N2500/30C12N2500/34C12N2500/38C12N2501/31C12N2501/81
Inventor 郭红刚褚晓峰李莉卢领群杜江涛石巧娟戴方伟应华忠
Owner ZHEJIANG ACAD OF MEDICAL SCI
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