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Method for avoiding harm to porcine oocyte in vitro development through zearalenone

A zearalenone and oocyte technology, applied in the fields of reproductive biology and developmental biology, can solve the problems of high cost, incompleteness, poor effect, etc., and achieve the effects of easy operation, improved maturation rate, and simple operation

Active Publication Date: 2018-11-06
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But these methods are not perfect enough, or the cost is higher, or the effect is relatively poor.

Method used

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  • Method for avoiding harm to porcine oocyte in vitro development through zearalenone
  • Method for avoiding harm to porcine oocyte in vitro development through zearalenone
  • Method for avoiding harm to porcine oocyte in vitro development through zearalenone

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1: LPC rescue effect test on in vitro maturation of pig cumulus-oocytes poisoned by ZEA

[0023] Main reagents used in this experiment: Zearalenone (Zearalenone, ZEA, 32935) was purchased from Sigma Company, lysophosphatidylcholine (Lysophosphatidylcholine, LPC, L5254) was obtained from Sigma Company, dimethylsulfoxide (Dimethylsulfoxide) sulfoxide, DMSO, D8371) was purchased from Beijing Suo Laibao Technology Company, and M199 medium (Medium, 11150067) was purchased from Gibco Company.

[0024] Preparation of porcine follicle fluid: use a 20mL syringe to extract antral follicles with an ovarian diameter > 6mm, put the follicle fluid in a 50mL imported centrifuge tube, collect as much as possible at a time, centrifuge at 3000rpm for 15 minutes to retain the supernatant, and then use the supernatant in sequence Filter through 0.45 and 0.22 filters, dispense into 1.5mL centrifuge tubes, and store at -20°C.

[0025] Preparation of lotion: Use sterile ultrapure wat...

Embodiment 2

[0043] Example 2: Test of the rescue effect of LPC on parthenogenic activation of in vitro matured porcine oocytes poisoned by ZEA

[0044] CB preparation: cytochalasin B (Cytochalasin B, CB), prepare a 1000-fold concentrated stock solution of CB with DMSO, and a solution with a concentration of 5 mg / mL, aliquot in sterile centrifuge tubes, 1 μL for each tube, and protect from light at -20 °C save.

[0045] Hyaluronidase preparation: Hyaluronidase (Hyaluronidase) can be dissolved in PBS, operating solution, or M2, and the working solution concentration of hyaluronidase is 0.003g / mL. Store at -20°C.

[0046] Preparation of oocyte activation solution: The preparation of the activation solution is based on the PEM-5 solution, adding 0.6mML-cysteine, 4mg / mL BSA, 7.5μg / mL CB, filtering through a 0.22 filter, and preparing and using immediately.

[0047] Preparation of electrical activation solution (parthenogenetic activation): the electrical activation solution is prepared with ...

Embodiment 3

[0058] Embodiment 3: LPC is to the rescue effect test of the parthenogenetic early embryo of the pig poisoned by ZEA

[0059] Preparation of in vitro culture medium for porcine parthenogenetic early embryos (PEM-5): Prepare 500 mL of in vitro culture medium for porcine parthenogenetic early embryos with sterile ultrapure water. The ingredients include: sodium chloride (108mM), potassium chloride (10mM), phosphoric acid Potassium dihydrogen (0.35mM), magnesium sulfate heptahydrate (0.4mM), sodium bicarbonate (25.07mM), sodium pyruvate (0.2mM), calcium lactate pentahydrate (2.0mM), glutamine (2.0mM ), hypotaurine (5.0mM), appropriate amount of phenol red, pH: 7.2-7.4, dilute to 500mL with sterile ultrapure water, filter through a 0.22 filter and store at 4°C.

[0060] Specific steps:

[0061] 1. Balance porcine parthenogenetic early embryo in vitro culture medium: use 24-well plate, add 700 μL porcine parthenogenetic early embryo in vitro culture medium to each well, cover with...

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Abstract

The invention relates to a method for avoiding harm to porcine oocyte in vitro development through zearalenone, mainly LPC (lysophosphatidylcholine) is added to a porcine oocyte in vitro maturation culture solution, the concentration of the added LPC working solution is 10 mu g / mL, the solution is prepared and used currently, and 0.2 mu L of the solution is added to each 1 mL of the porcine oocytein vitro maturation culture solution. It is discovered that when the LPC is added to the in vitro maturation culture solution, the maturing rate of the porcine oocyte suffering from ZEA poison can beimproved remarkably, the porcine oocyte parthenogenetic activation rate can be improved remarkably, the cleavage of the early parthenogenetic embryo is promoted, and an operation step for completelyavoiding the ZEA poison by adding the LPC to the porcine oocyte in vitro culture is put up for the first time. According to the method for avoiding the harm to porcine oocyte in vitro development through the zearalenone, it is proved that the LPC really has a resistance action to the ZEA poison, and a research direction which is entirely different from the prior art is provided for studying ZEA detoxification methods.

Description

technical field [0001] The invention relates to the fields of reproductive biology and developmental biology, in particular to a method for rescuing the damage of zearalenone to the in vitro development of pig oocytes. Background technique [0002] Zearalenone (ZEA) is a non-steroidal mycotoxin produced by Fusarium graminearum, Fusarium equiseti, and Fusarium nivalum. It mainly exists in corn, wheat, sorghum, oats and other grains that are susceptible to fungal contamination, and is the most widely polluted mycotoxin in the world. Due to abundant rainfall and high relative humidity in most areas of my country, grain and animal feed are more susceptible to mycotoxin contamination. Studies have shown that ZEA has an estrogen-like effect, and the ketone group at the C8 position is easily reduced to generate α-zearalenol (α-ZOL) and β-zearalenol (β-ZOL). All have estrogen-like activity. The estrogen-like activity of α-ZOL is three times that of ZEA, while the estrogen-like act...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2500/30
Inventor 李兰刘雪莲李娜沈伟
Owner QINGDAO AGRI UNIV