A method of removing defective sperms of mammal through immunomagnetic beads

A mammalian and immunomagnetic bead technology, applied in the field of removing mammalian defective sperm, can solve the problems of high motility or deformity rate, waste of manpower, unreturned material resources, etc., and achieve the effect of less sperm damage and less impact.

Inactive Publication Date: 2016-05-04
INNER MONGOLIA SAIKEXING LIVESTOCK BREEDING & SEED IND BIOTECH RES INST CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] There are certain requirements on the quality of sperm when making mammalian frozen semen, especially the production of sexually controlled frozen semen has stricter requirements on the original semen. If the quality of semen is too low, it cannot be separated normally on the machine.
Some sire breeds have good genetic quality but high sperm quality such as motility or deformity rate, so they cannot produce sexually controlled frozen semen to meet the needs of customers, wasting a lot of manpower and material resources without getting returns

Method used

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  • A method of removing defective sperms of mammal through immunomagnetic beads
  • A method of removing defective sperms of mammal through immunomagnetic beads
  • A method of removing defective sperms of mammal through immunomagnetic beads

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Screening of Defective Sperm Surface Specific Membrane Protein Antibodies

[0026] Select fresh semen with deformed and high dead sperm rate, use a density meter to detect the actual concentration of the original semen, add 1ml to 2ml of 0.1% PBS according to about 10 million fresh semen, mix well, centrifuge at room temperature, remove the supernatant, and add 1ml of 0.1% PBS %PBS, 10 μg ~ 15 μg Anti-Ubiquitin (MBL, MK-12-3) were mixed evenly, and incubated at room temperature in the dark for 30 min ~ 45 min with rotation. After the culture, centrifuge at room temperature at 1500prm for 5-8min, remove the supernatant, add 0.5ml-1ml0.1%PBS, suspend the sperm pellet, add 1.5-2μl Goatanti-MouseIgGsecondaryAntibodyFITCconjugate, and rotate at room temperature for 30min-45min in the dark. After culturing, centrifuge at 1500 rpm for 5-8 minutes, remove the supernatant, and observe under a fluorescent microscope. Such as figure 1 , by immunofluorescence detection,...

Embodiment 2

[0027] Embodiment 2 A method for removing mammalian defective sperm by immunomagnetic beads is completed according to the following steps:

[0028] 1. Anti-Ubiquitin combined with defective sperm

[0029] ①Choose fresh semen with deformity and high dead sperm rate as the experimental sample and use a densitometer to detect the actual concentration of the original semen;

[0030] ② Add 1 million fresh essence to 1ml of 0.1% PBS, mix gently, then centrifuge at 1500rpm for 5min at room temperature;

[0031] ③Remove the supernatant, add 100 μl 0.1% PBS, 1 μg Anti-Ubiquitin, mix well, and rotate at room temperature for 30 minutes in the dark;

[0032] ④ After the culture, centrifuge at 1500 rpm for 5 minutes at room temperature, and remove the supernatant;

[0033] ⑤ Add 1ml 0.1% PBS to suspend the sperm pellet;

[0034] 2, Preparation of PanMouseIgG

[0035] ① Take 25μl Add PanMouseIgG to 1ml 0.1% PBS, shake and mix well, then place in DynaMag TM Stand on -5Magnet for 2mi...

Embodiment 3

[0041] Embodiment 3 A method for removing mammalian defective sperm by immunomagnetic beads is completed according to the following steps:

[0042] 1. Anti-Ubiquitin combined with defective sperm

[0043] ①Choose fresh semen with deformity and high dead sperm rate as the experimental sample and use a densitometer to detect the actual concentration of the original semen;

[0044] ② Add 10 million fresh essence to 2ml of 0.1% PBS, mix gently, then centrifuge at 1500rpm for 5min at room temperature;

[0045] ③Remove the supernatant, add 1ml 0.1% PBS, 10μg Anti-Ubiquitin, mix well, and rotate at room temperature for 30min in the dark;

[0046] ④ After the culture, centrifuge at 1500 rpm for 5 minutes at room temperature, and remove the supernatant;

[0047] ⑤ Add 1ml 0.1% PBS to suspend the sperm pellet;

[0048] 2, Preparation of PanMouseIgG

[0049] ① Take 50μl Add PanMouseIgG to 1ml 0.1% PBS, shake and mix well, then place in DynaMag TM Stand on -5Magnet for 2min;

[00...

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Abstract

A method of removing defective sperms of mammal through immunomagnetic beads is removed. The method includes 1) bonding the defective sperms with an antibody, 2) bonding the immunomagnetic beads with the defective sperm-antibody composite, and 3) separating the defective sperms from normal sperms. The defective-sperm specific antibody is utilized and coupled to the immunomagnetic beads to form an immunomagnetic bead-antibody-defective sperm composite, thus finally removing the defective sperms from the normal sperms. Compared with other manners, the immunomagnetic bead technique is low in influence on sperm activity, simple in operation and more suitable for frozen sperms manufactured by mechanical separation after the defective sperms are removed.

Description

technical field [0001] The invention relates to a method for removing defective sperm in mammals, in particular to a method for removing defective sperm in mammals with immunomagnetic beads. Background technique [0002] When making mammalian frozen semen, there are certain requirements on the quality of sperm, especially the production of sex-controlled frozen semen has stricter requirements on the original semen. If the quality of semen is too low, it cannot be separated normally on the machine. Some sire breeds have good genetic quality but high sperm quality such as motility or deformity rate, so they cannot produce sexually controlled frozen semen to meet the needs of customers, wasting a lot of manpower and material resources without getting returns. [0003] The current method of removing dead sperm is mainly the float method or the percoll method. The float method needs to centrifuge and put the sperm in a 37-degree water bath for a period of time, and then the live ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076
CPCC12N5/0081
Inventor 李喜和苏杰张健赵高平
Owner INNER MONGOLIA SAIKEXING LIVESTOCK BREEDING & SEED IND BIOTECH RES INST CO LTD
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