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A method for improving the quality and efficiency of in vitro maturation of an oocyte

A technology of in vitro maturation and oocytes, applied in the field of bioengineering, can solve the problems of low quality and efficiency of oocyte maturation, achieve great practical application value, high repeatability, and high blastocyst rate

Pending Publication Date: 2019-01-11
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a method that can effectively improve the quality and efficiency of oocyte maturation in vitro to solve the problem of low oocyte maturation quality and efficiency caused by in vitro maturation methods in the prior art

Method used

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  • A method for improving the quality and efficiency of in vitro maturation of an oocyte
  • A method for improving the quality and efficiency of in vitro maturation of an oocyte
  • A method for improving the quality and efficiency of in vitro maturation of an oocyte

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The acquisition of embodiment 1 oocyte and in vitro maturation method

[0029] 1. Oocyte Acquisition and In Vitro Maturation

[0030] The ovaries obtained from the slaughterhouse were placed in saline at 37°C and transported to the laboratory within 2 hours. The cumulus-oocyte complexes (COCs) in the follicles with a diameter of 2-8mm were drawn out by a vacuum pump, and the IVM fluid was washed and detected under a microscope, and then the COCs were put into the IVM fluid at 38.5°C and 5% CO 2 cultured in an incubator for 24 h. Among them, the composition of IVM solution is as follows: TCM199 basal culture solution + 0.01IU·mL -1 FSH+10μg·mL -1 Heparin+40ng·mL -1 IGF+50ng·mL -1 EGF+0.01IU·mL -1 LH+1μg·mL -1 E. 2 +10% FBS.

[0031] 2. Oocyte nuclear maturation statistics

[0032] After 24 hours of IVM for oocytes in each group, hyaluronidase was used to remove cumulus cells, and MII stage oocytes were selected, and then the proportion of MII stage oocytes was ...

Embodiment 2

[0047] Hormone levels at different times in the IVM fluid of embodiment 2 blank group and COCs group

[0048] 1. Experimental Design

[0049] The blank group was the IVM solution without COCs at 38.5°C, 5% CO 2 cultured in an incubator for 24 h.

[0050] The COCs group consisted of IVM solution containing COCs at 38.5°C, 5% CO 2 cultured in an incubator for 24 h.

[0051] The components of the above IVM solution are as follows: TCM199 basal culture solution + 0.01IU·mL -1 FSH+10μg·mL -1 Heparin+40ng·mL -1 IGF+50ng·mL -1 EGF+0.01IU·mL -1 LH+1μg·mL -1 E. 2 +10% FBS.

[0052] At 0, 4, 8, 12, 16, 20, and 24 hours after the start of IVM, the IVM fluids of the blank group and the COCs group were taken and stored at -80°C for hormone detection.

[0053] 2. Test results of hormone levels at different times in the blank group and COCs group

[0054] The test results of hormone levels in the blank group are shown in Table 1. The 8h blank group FSH, LH, E 2 Levels (5.75±0.52...

Embodiment 3

[0062] Example 3 Influence of replacing in vitro maturation solution on in vitro maturation of oocytes and IVF efficiency

[0063] 1. Experimental Design

[0064] Normal maturation group: COCs were matured in IVM solution for 24 hours, and the medium was not changed during the maturation period.

[0065] 12h medium change group: COCs were put into IVM solution for 12h to mature, and then transferred to freshly prepared IVM solution for 12h to mature.

[0066] 8h medium change group: put COCs into IVM solution for maturation, and transfer them into freshly prepared IVM solution at 8h and 16h after the start of IVM to continue maturation.

[0067] The components of the above IVM solution are as follows: TCM199 basal culture solution + 0.01IU·mL -1 FSH+10μg·mL -1 Heparin+40ng·mL -1 IGF+50ng·mL -1 EGF+0.01IU·mL -1 LH+1μg·mL -1 E. 2 +10% FBS.

[0068] After maturation, the oocyte rates of MII stage oocytes in the three groups were counted, and then the MII stage oocytes in...

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Abstract

The invention provides a method for improving the quality and efficiency of in vitro maturation of an oocyte, which includes the step of changing an in vitro maturation fluid at least once during in vitro maturation, and further comprising adding an amount of FGF to the in vitro maturation fluid. The oocyte prepared by the method of the invention has remarkably improved nuclear maturation efficiency and cytoplasmic maturation efficiency, the MII oocyte is up to 95%, the proportion of oocytes with normal distribution of cortical granules and mitochondria, the levels of glutathione and ATP are significantly higher than those of the normal maturation group; Adding a certain amount of FGF to IVM can further improve the maturation quality and efficiency of oocytes. After in vitro fertilization,the in vitro fertilization efficiency of the oocytes prepared by the method of the invention is significantly higher than that of the normal maturation group, which is embodied in higher cleavage rate, blastocyst rate and more blastocyst cells, and has great application value.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for in vitro maturation efficiency of mammalian oocytes Background technique [0002] In vitro maturation (IVM) of oocytes is a key link in livestock embryo biotechnology and an important part of in vitro fertilization (IVF). However, compared with the in vivo environment, the existing oocyte IVM system is still imperfect, which leads to the lower quality of IVM oocytes. A large number of studies have shown that the quality of IVM oocytes in the methods of the prior art, such as fertilization ability, development ability, and ability to produce children after embryo transfer, are significantly lower than those of mature oocytes in vivo (Shi JM et al., JPineal Res, 2009 ; 47(4):318-323; Lonergan P et al., Annu Rev Anim Biosci, 2016; 4:255-268). The low quality and developmental potential of IVM oocytes has become a bottleneck limiting the efficiency of embryonic ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075
CPCC12N5/0609C12N2501/105C12N2501/11C12N2501/115C12N2501/31C12N2501/392C12N2501/91
Inventor 赵学明朱化彬郝海生杜卫华庞云渭张燕刘岩赵亚涵
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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