Buffalo oocyte enucleation method

Abstract

The present invention provides a buffalo oocyte enucleation method which comprises the following steps: 1) a cumulus oophorus oocyte cell complex is taken form ovary of a slaughter house for in vitro maturation culture for 22-24h under the conditions of 38.5 DEG C, 5% CO2 and saturated humidity; 2) the maturation-cultured COCs are gently and slowly blew and beaten by 100muL pipette tip for removal of cumulus oophorus cells to left oocytes; and 3) selecting MII oocytes containing first polarbody for enucleation; 4) transferring the buffalo oocytes into a culture solution containing 7.5 mug / mL cytochalasin B (CB) for culture for 30min under the conditions of 38.5 DEG C, 5% CO2 and saturated humidity; 5) transferring the treated buffalo oocytes into 30-40 muL of an enucleation operating liquid covered with paraffin oil; and 6) enucleating in an inverted microscope provided with a spindle body imaging system. By use of the cytochalasin B (CB) for culture of the oocytes and use of the spindle body imaging system for auxiliary enucleation, the efficiency of enucleation can be effectively improved.

Description

【Technical field】 [0001] The invention belongs to the field of biotechnology, in particular to a method for enucleation of buffalo oocytes. 【Background technique】 [0002] During somatic cell nuclear transfer in buffalo, enucleation of oocytes is a crucial step, and the main methods used for enucleation are blind aspiration and fluorescent staining. Blind aspiration is to remove the polar body and a small amount of oocyte cytoplasm around it. Its biggest disadvantage is that the enucleation efficiency is low and the absorbed cytoplasm is too much, which affects the development of the recombinant embryo. The fluorescent staining method is to first stain the oocytes with 5 μg / mL Hoechest 33342 for 15 minutes, and specify enucleation under a fluorescent microscope, which has a high enucleation efficiency. It is mainly used for the detection of enucleation efficiency. In recent years, a spindle imaging system has been developed, which utilizes the principle of polarization of ...

AI Technical Summary

Problems solved by technology

There is no damage to the enucleation and less removal of cytoplasm. However, for novices, it takes a long time to practice to see the flickering spindle, and because the bright light flickers weakly, there are many fat droplets in the buffalo oocyte cytoplasm and poor light transmission. Oocytes can't see the blinking spindle, and can't accurately enucleate