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Method for isolating and identifying sperm cells of Buffalo testis

A technology of sperm cells and identification methods, which is applied in the field of separation and identification of buffalo testicular sperm cells, can solve the problems of low cell purity, high requirements, and vulnerability to damage, and achieve good results in identification

Inactive Publication Date: 2019-01-11
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the commonly used methods for separating sperm cells include density gradient centrifugation and gravity sedimentation, but the steps are cumbersome and require high operator experience, and the purity of the separated cells is low, and the separation time is long, and the cells are kept in the environment for a long time. Under non-culture conditions, susceptible to damage

Method used

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  • Method for isolating and identifying sperm cells of Buffalo testis
  • Method for isolating and identifying sperm cells of Buffalo testis
  • Method for isolating and identifying sperm cells of Buffalo testis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment one: Buffalo sperm cell separation method

[0038] (1) Buffalo testes were collected from the slaughterhouse, placed in 37°C normal saline containing double antibodies, and transported back to the laboratory within 3 hours.

[0039] (2) Use tweezers and surgical scissors to peel off the testis albuginea and adipose tissue in PBS to expose the albuginea; select a testis without damage and internal bleeding, weigh it, disinfect it with 75% alcohol for 10 minutes, repeat twice, Transfer to ultra-clean bench;

[0040] (3) Put the testis with albuginea into the beaker of 500mL PBS and wash three times;

[0041] (4) Put the testis into a dish containing 100mL DMEM / F12, cut the tunica albuginea, tear off as much testicular tissue as possible with pointed tweezers and put it into a pre-prepared petri dish containing DMEM / F12, record testicular the net weight of the tissue;

[0042] (5) Use tweezers to tear up the tissue as much as possible into a paste;

[0043] ...

Embodiment 2

[0052] Example 2: Identification method of isolated buffalo sperm cells

[0053] (15) Use an appropriate amount of culture medium to resuspend the cells, count them, and adjust the cell concentration according to the counting results.

[0054] (16) Cell counting: take 10 μL of the filtrate, mix with 10 μL of 0.4% trypan blue, and count the number of unstained living cells under the microscope. 4 ;

[0055] (17) Take an appropriate amount of cells and use DM / F12(+) to resuspend, transfer the cell suspension to a 100mm dish covered with 0.1% gelatin, 37°C, CO2 Cultivate in an incubator with a concentration of 5.5% for 4 to 6 hours, collect the suspended cells in the upper layer, 2000rpm, 5min, carefully aspirate and discard the supernatant, resuspend and culture for 4 to 6 hours, repeat three times, and collect the suspension for later use.

[0056] (18) Add Hoechst33342 to the previous testis cell suspension to adjust to a final concentration of 5 μg / mL. Add 3mL staining medi...

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Abstract

The invention belongs to the technical field of sperm cell treatment, and specifically relates to a method for isolating and identifying sperm cells of Buffalo testis, including the following steps: obtaining Buffalo testis, digest with collagenase IV, hyaluronidase and DNase I combined enzyme, sieving, filtering, and taking suspension cells after differential adherence; 2) taking the suspend cells for live cell stain; and 3) separating by flow cytometry. Beneficial effect: the technical scheme is an effective method for separating buffalo sperm cells, By digesting bovine testis tissue with two enzymes, so that a single cell suspension is obtain, the interference of somatic cells is removed by the method of continuous differential adherence, according to the fluorescence intensity of non-adherent cells stained by flow cytometry, the DNA content of cells is judged, and the interference of other non-target cells such as diploid spermatogonia and tetraploid primary meningoblasts is effectively digested and removed to achieve the successful sorting of a large number of haploid spermatozoa.

Description

technical field [0001] The invention belongs to the technical field of sperm cell processing, and in particular relates to a method for separating and identifying buffalo testicular sperm cells. Background technique [0002] Sperm cells are haploid cells that belong to one of the testicular germ cell types and are produced when the second meiotic division is completed. It is the result of the second mature division in the process of spermatogenesis, maturation, and formation of functional sperm, which undergoes deformation to form sperm. In recent years, sperm cell microinjection insemination technology has become an important technology for assisted reproduction, and the acquisition of high-viability and high-purity sperm cells has a broader prospect in the future research of reproductive medicine. [0003] At present, the commonly used methods for separating sperm cells include density gradient centrifugation and gravity sedimentation, but the steps are cumbersome and req...

Claims

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Application Information

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IPC IPC(8): C12N5/076C12Q1/02
CPCC12N5/061C12N2509/00G01N33/5005
Inventor 张明张鹏飞黄愉淋侯振
Owner GUANGXI UNIV
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