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Method for carrying out ICSI (intracytoplasmic sperm injection) on pig

A lipase and solution technology, applied in the field of pig ICSI operation, can solve the problems of hindering sperm, affecting the depolymerization of sperm nucleus, delaying the oscillation of calcium ions in the cytoplasm by the sperm plasma membrane, and achieving the effect of increasing the formation rate and reducing the adhesion problem.

Active Publication Date: 2015-05-20
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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Problems solved by technology

However, when PVP is injected into the cytoplasm together with the sperm, it will surround the sperm, which not only makes the sperm plasma membrane tough, affects the rupture and delays the calcium ion oscillation in the cytoplasm, affects the depolymerization of the sperm nucleus, but also prevents the sperm from releasing the oocyte to the cytoplasm Cell activating factor, which affects the activation of oocytes, which seriously affects the success rate of ICSI

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  • Method for carrying out ICSI (intracytoplasmic sperm injection) on pig
  • Method for carrying out ICSI (intracytoplasmic sperm injection) on pig
  • Method for carrying out ICSI (intracytoplasmic sperm injection) on pig

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preparation example Construction

[0042] The preparation method of in vitro maturation medium (IVM): in vitro maturation medium is made up of solvent and solute, and solvent is pure water; Solute and its concentration in culture medium: Medium 199 powder (Gibco, 31100-035) 9.5g / L , 10% (volume fraction) PFF, 10ng / ml EGF, 0.5mg / ml FSH, 0.5mg / ml LH, 70ng / ml Cys.

[0043] The preparation method of the activation solution: the activation solution is composed of a solvent and a solute, and the solvent is pure water; the solute and its concentration in the culture medium: 0.3M D-Mannitol, 0.5mM Hepes, 1.0mM CaCl 2 2H 2 O, 0.1mM MgCl 2 ·6H 2 O.

[0044]The preparation method of PZM-3 culture fluid: PZM-3 culture fluid is made up of solvent and solute, and solvent is pure water (Singma); Solute and its concentration in culture fluid: 6.3115g / L NaCl, 0.7455g / L KCl, 0.0476g / L KH 2 PO 4 , 0.0986g / L MgSO 4 ·7H 2 O, 2.1061g / L NaCO 3 , 0.022g / L sodium pyruvate, 0.6166g / L CaC 2 o 4 ·5H 2 O, 0.5% L-Glutamine, 0.05...

Embodiment 1

[0048] Embodiment 1, the method for improving pig ICSI operating efficiency and effect detection

[0049] 1. Methods for Improving Operational Efficiency of ICSI in Pigs

[0050] Experimental materials: Pig ovaries were collected from local slaughterhouses, placed in normal saline (38°C) with double antibodies (penicillin and streptomycin), and transported back to the laboratory within three hours.

[0051] 1. Wash the retrieved ovaries three times with normal saline with double antibodies, use a 10ml syringe and a 18-gauge needle to extract the follicle fluid with a diameter of 3-6mm follicles, and pick out 3-5 layers of eggs with a suction pipette. Cumulus-oocyte complexes (COCs) of cumulus cells.

[0052] 2. Wash the COCs selected in step 1 twice with the egg washing solution (PVA-TL-HEPES), and after cleaning the impurities, select the cumulus cells to wrap 3-5 layers, and the cumulus cells to wrap the complete oocyte densely and evenly Cells were transferred into in vit...

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Abstract

The invention discloses a method for carrying out ICSI (intracytoplasmic sperm injection) on a pig. The method for carrying out ICSI on the pig comprises the following steps: (1) hydrolyzing sperms in vitro animal semen with lipoidase or lipoidase solution, and collecting a hydrolysis product, so that the sperms used for preparing ICSI fertilized ova; (2) injecting the sperms used for preparing the ICSI fertilized ova into in vitro oocytes to obtain ICSI ova; and (3) carrying out electric activation on the ICSI ova and cultivating, so that the ICSI fertilized ova are obtained. The method for carrying out ICSI on the pig has the advantages that bad effects on depolymerization of pig sperms, formation of male pronucleus and later development due to addition of PVP can be avoided, and regions such as plasma membranes and perforatorium of the pig sperms can not be damaged, so that release of oocyte activation promoting factors from the pig subjected to ICSI operation can be facilitated, and the formation rate of the male pronucleus and the formation rate of blastocysts of embryos after fertilization are increased.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for pig ICSI operation. Background technique [0002] ICSI (Intracytoplasmic sperm injection) is intracytoplasmic sperm injection technology, which is the second generation of "test-tube baby". This technology uses a microscopic operating system to inject a single sperm into an egg to fertilize it. Compared with mice and humans, the membrane surface of pig sperm contains more lipids, glycolipids and glycoproteins and the acrosome region is rich in phospholipids and cholesterol. Therefore, during the ICSI operation, pig sperm tends to adhere to the injection needle, thus increasing the difficulty of micromanipulation. [0003] At present, a high molecular substance polyvinylpyrrolidone (PVP) is usually added to the operating solution to reduce the adhesion of the sperm to the injection needle and facilitate the operation. However, when PVP is injected into the c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/076C12N15/89A01K67/027
Inventor 李奎刘志国魏迎辉
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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