# US 2020/0399587 A1 · Culture medium for improving development of bovine in vitro fertilized embryos and cloned embryos

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- Number: US 2020/0399587 A1
- Document ID: US20200399587A1
- Status: Published application
- Kind: A1
- Application number: 17/008,676
- Filed: 2020-09-01
- Published: 2020-12-24
- Earliest claimed: 2020-05-09
- Assignees: Northeast A&F University
- Applicants: Northeast A&F University
- Inventors: Jingcheng ZHANG; YUEMENG HUANG; MIN ZHANG; DEBAO WANG; YONG WANG; YONG ZHANG
- Web page: https://patentgrep.com/patent/US20200399587A1

## CPC

- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- C12N 5/0604: {Whole embryos; Culture medium therefor}
- C12N 2500/32: Amino acids
- C12N 2500/38: Vitamins
- C12N 2501/105: Insulin-like growth factors [IGF]
- C12N 2501/11: Epidermal growth factor [EGF]
- C12N 2501/115: Basic fibroblast growth factor (bFGF, FGF-2)
- C12N 2501/119: Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
- C12N 2501/235: Leukemia inhibitory factor [LIF]
- C12N 2501/39: Steroid hormones
- C12N 2501/392: Sexual steroids
- C12N 2501/999: Small molecules not provided for elsewhere
- C12N 2523/00: Culture process characterised by temperature
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A culture medium for improving the development of bovine in vitro fertilized embryos and cloned embryos includes a SOF basal medium, progesterone, vitamin E, L-carnitine, FGF, corticosterone, citrulline, EGF, IGF and LIF.

## Claims

### Claim 1

A culture medium for improving development of bovine in vitro fertilized embryos and cloned embryos, comprising: a synthetic oviduct fluid (SOF) basal medium, citrulline, progesterone, vitamin E, L-carnitine, fibroblast growth factor (FGF), corticosterone, epidermal growth factor (EGF), insulin growth factor (IGF) and leukemia inhibitory factor (LIF).

### Claim 2 (depends on 1)

The culture medium of claim 1, comprising: 1-100 ng/mL of progesterone, 10-1000 μM of vitamin E, 0.1-30 mM of L-carnitine, 1-100 ng/mL of FGF, 1-100 ng/mL of corticosterone, 0.01-1 mg/mL of citrulline, 1-100 ng/mL of EGF, 1-100 ng/mL of IGF and 1-100 ng/mL of LIF.

### Claim 3 (depends on 1)

The culture medium of claim 1, further comprising: an essential amino acid, a non-essential amino acid, fatty acid-free bovine serum albumin (BSA), insulin-transferrin-selenium (ITSG), β-mercaptoethanol, hypotaurine, 1-oleoyl-sn-glycero-3-phosphate (sodium salt) and salidroside.

### Claim 4 (depends on 3)

The culture medium of claim 3, comprising: 0.1-2% by volume of the essential amino acid, 0.1-2% by volume of the non-essential amino acid, 1-10 mg/mL of the fatty acid-free BSA, 0.1-2% by volume of the insulin-transferrin-selenium, 10-100 μM of β-mercaptoethanol, 1-10 mM of hypotaurine, 0.1-1 μg/mL of 1-oleoyl-sn-glycero-3-phosphate (sodium salt) and 0.01-0.2 mM of salidroside

### Claim 5 (depends on 4)

The culture medium of claim 4, wherein the SOF basal medium comprises 261.68 mg/L of CaCl2.2H2O, 99.99 mg/L of C6H5Na3O7.2H2O, 29.23 mg/L of glutamine, 372.18 mg/L of MgSO4.7H2O, 499.04 mg/L of inositol, 10.00 mg/L of phenolsulfonphthalein, 533.78 mg/L of KCl, 161.95 mg/L of KH2PO4, 44.02 mg/L of sodium pyruvate, 2.10025 g/L of NaHCO3, 6.29399 g/L of NaCl and 0.757 mg/L of sodium lactate.

### Claim 6 (depends on 5)

The culture medium of claim 5, further comprising: 12 μg/mL of gentamicin.

### Claim 7 (depends on 1)

A culture method for improving the development of bovine in vitro fertilized embryos and cloned embryos, comprising:
culturing the bovine in vitro fertilized embryos or the cloned embryos in the culture medium of claim 1.

### Claim 8 (depends on 7)

The culture method of claim 7, comprising:
preheating the culture medium for at least 2 h; and
culturing the bovine in vitro fertilized embryos or the cloned embryos in the preheated culture medium in a CO2 incubator for 7-8 days;
wherein the bovine in vitro fertilized embryos are obtained through 12-16 h of bovine sperm-egg binding; and the cloned embryos are somatic cell cloned embryos which are chemically activated with 6-DMAP.

### Claim 9 (depends on 9)

The culture method of claim 9, wherein the culture of the bovine in vitro fertilized embryos or the cloned embryos is performed at 38.5° C., 5% CO2 and 7% O2 under saturated humidity.