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Medium supplement to increase the efficiency of oocyte maturation and embryo culture in vitro

a technology of oocyte maturation and medium supplement, which is applied in the field of media and methods for oocyte maturation or embryo culture, can solve the problems of poor quality of initiating oocytes, low rate of in vitro development of porcine oocytes and embryos to blastocysts, and low efficiency

Inactive Publication Date: 2017-09-14
UNIVERSITY OF MISSOURI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides a method for improving the developmental competence of oocytes and embryos by contacting them with a medium containing fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF-1). This method can lead to better outcomes for assisted reproduction techniques.

Problems solved by technology

However, the rate of in vitro development of porcine oocytes and embryos to blastocysts is very low (10-15%) when compared to oocytes that are matured and fertilized in vivo.
This low efficiency can, therefore, be attributed to the poor quality of the initiating oocytes as a result of inadequate in vitro maturation.

Method used

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  • Medium supplement to increase the efficiency of oocyte maturation and embryo culture in vitro
  • Medium supplement to increase the efficiency of oocyte maturation and embryo culture in vitro
  • Medium supplement to increase the efficiency of oocyte maturation and embryo culture in vitro

Examples

Experimental program
Comparison scheme
Effect test

example 1

Evaluation of Cytokines Individually In IVM Medium

[0047]The individual effects of FGF2, LIF, and IGF-1 on maturation of oocytes derived from pre-pubertal pre-pubertal gilts after commercial slaughter were examined after each had been added to an otherwise standard, chemically-defined medium comprised of TCM 199 supplemented with 10 ng / ml EGF, 0.5 μg / ml LH, 0.5 μg / ml FSH, 3.05 mM glucose, 0.91 mM sodium pyruvate, 0.57 mM cysteine, 10 ng / mL gentamicin, and 0.1% polyvinyl alcohol (PVA). After 42 hr in the oocyte maturation medium with the single cytokine addition, oocytes were in vitro fertilized and the resulting zygotes were cultured under standard in vitro culture conditions for 6 days. While each of FGF2, LIF, and IGF-1, individually improved the efficiency of producing nuclear matured, i.e. metaphase II (MII), oocytes, none of the cytokines improved oocyte developmental competence, as determined by the ability of the fertilized oocytes (zygotes) to form blastocysts after IVF. (FIG...

example 2

Evaluation of Cytokines In Combination In IVM Medium

[0048]Optimal concentrations of FGF2 (40 ng / ml) and LIF (20 ng / ml) were added in combination to the standard IVM medium. The combination of FGF2 and LIF improved nuclear maturation beyond that achieved with the single factors in Example 1 (Table 1). In addition, these matured oocytes had improved developmental competence to form blastocysts, as demonstrated by the increased number of blastocyst-stage embryos (Table 1, column 5, 6).

[0049]The effects of adding the IGF-1 at the optimal concentration of 20 ng / ml in combination with the other two cytokines to the IVM medium was then examined. The combination of all three cytokines (FGF2, LIF, and IGF-1) increased oocyte nuclear maturation to 89%, as compared to 55% in controls (FIG. 5 and Table 2, column 4). After IVF, zygotes from oocytes cultured in medium comprising FGF2, LIF, and IGF-1 advanced to the blastocyst stage more efficiently than controls (49.7% versus 38%). This protocol,...

example 3

Evaluation of Cytokines In Embryo Culture Medium

[0051]When the same cytokine cocktail (40 ng / ml FGF2, 20 ng / ml LIF, and 20 ng / ml IGF-1) was added to a standard in the embryo culture medium at Day 4 after IVF, more embryos were able to develop to blastocyst stage (Table 4, column 6), indicating that the cocktail can also benefit embryo development when they were added to the embryo culture medium.

TABLE 4Effects of FGF2, LIF and IGF on blastocyst formationwhen added to the culture medium on day 4 after IVF.*Zy-Cleavage / Blastocysts / Blastocyst / Repli-gotesZygoteCleavageZygoteTreatment**cate(n)(%)(%)(%)Control417258.1 ± 8.054.3 7.8 a30.2 ± 4.0 aFGF2 + LIF +417160.7 ± 3.167.5 ± 4.4 b41.0 f 3.9 bIGF-1*Data are reported as means ± SEM.**Oocytes were matured in chemically defined TCM-199 maturation medium. After maturation, MII oocytes were used for IVF. Presumptive IVF zygotes were cultured in standard MU1-PZM till day 4, when the FGF2, LIF, and IGF-1 cocktail was added in the culture medium...

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Abstract

The present invention provides a novel oocyte maturation medium or / and embryo culture medium with a chemically defined supplement to produce matured oocytes at high efficiency. The inventive medium or supplement comprises three growth factors, namely, fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), and insulin-like growth factor 1 (IGF-1) in a synergistic combination. Methods for oocyte and embryo culture are also provided.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of priority of U.S. Provisional Application No. 62 / 301,309, filed on Feb. 29, 2016, the disclosure of which is hereby incorporated by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH[0002]This invention was made with Government support under Grant No. RO1 HD069979 awarded by the National Institutes of Health. The Government has certain rights in the invention.FIELD OF THE INVENTION[0003]The present invention relates to methods and compositions for reproductive technology, more particularly to media and methods for oocyte maturation or embryo culture.BACKGROUND OF THE INVENTION[0004]The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.[0005]Cloning pigs by somatic cell nuclear transfer (SCNT) or in vitro fertilization (IVF) has wide applications in basic research, human medicine and agricultural p...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61B17/435C12N5/075
CPCA61B17/435C12N5/0609C12N2501/105C12N2501/10C12N2501/115C12N2517/10C12N2501/11C12N5/0604C12N2517/04C12N2501/235C12N2501/727C12N2501/31
Inventor YUAN, YESPATE, LEE D.PRATHER, RANDALL S.ROBERTS, R. MICHAEL
Owner UNIVERSITY OF MISSOURI
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