# US 11,297,831 B2 · Cryopreserving ungulate embryos

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 11,297,831 B2
- Document ID: US11297831B2
- Status: Granted
- Kind: B2
- Application number: 15/743,893
- Filed: 2016-07-13
- Published: 2018-12-13
- Granted: 2022-04-12
- Earliest claimed: 2015-07-14
- Assignees: ABS Global, Inc.
- Applicants: Genus plc
- Inventors: Bruno Valente Sanches
- Web page: https://patentgrep.com/patent/US11297831B2

## CPC

- A01N 1/162: Temperature processes, e.g. following predefined temperature changes over time
- A01N 1/147: Carriers for immersion in cryogenic fluid for slow freezing or vitrification
- A61D 19/04: for embryo transplantation
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A61D: VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS

## Abstract

Technologies for cryopreserving ungulate embryos for implantation into recipient females are described.

## Claims

### Claim 1

A method for cryopreserving an in vitro produced ungulate embryo comprising the steps of:
culturing an immature oocyte in vitro under a first culture condition to obtain a mature oocyte;
fertilizing said mature oocyte to generate a zygote;
denuding said zygote;
culturing said zygote in a second culture condition comprising an atmosphere of 5% oxygen (O2) and 5% carbon dioxide (CO2) to prepare said in vitro produced ungulate embryo;
freezing said in vitro produced ungulate embryo wherein the freezing comprises the steps of:
(1) incubating said embryo in a freezing solution comprising of 1.0 to 4 molar (M) ethylene glycol for 5 to 30 minutes at a first temperature between 10° C. and 38° C.;
(2) loading said embryo in a receptacle comprising the steps of adding:
i) a first thawing solution to said receptacle;
ii) a first air bubble and a second thawing solution, wherein said first air bubble separates said first and second thawing solutions;
iii) a second air bubble and said in vitro produced ungulate embryo in a freezing solution, wherein said second air bubble separates said second thawing solution and said in vitro produced ungulate embryo in a freezing solution;
iv) a third air bubble;
v) a third thawing solution, wherein said third air bubble separates said in vitro produced ungulate embryo in a freezing solution and said third thawing solution;
vi) a fourth air bubble; and
vii) a fourth thawing solution, wherein said fourth air bubble separates said third and fourth thawing solutions and said first, second, third, and fourth thawing solutions comprise ethylene glycol in an isotonic diluent medium;
(3) exposing the receptacle prepared in step (2) to a temperature of −2 to −10° C. for a time period of 1 min to 60 minutes;
(4) lowering said temperature at a rate of −0.2 to −0.8° C. per minute until reaching a second temperature of −30 to −36° C.; and
(5) immersing the receptacle in liquid nitrogen for storage.

### Claim 2 (depends on 1)

The method of claim 1, further comprising the step of inducing ovulation in a female and aspirating said immature oocyte from said female reproductive tract.

### Claim 3 (depends on 1)

The method of claim 1, wherein said culturing of said immature oocyte in said first culture condition in vitro comprises incubating said immature oocyte at 35 to 40° C. in the presence 3 to 9% CO2 and saturated humidity.

### Claim 4 (depends on 1)

The method of claim 1, wherein said step of fertilizing said mature oocyte is for 20 to 24 hours.

### Claim 5 (depends on 4)

The method of claim 4, wherein said step of fertilizing said mature oocyte is in the presence of 5% O2.

### Claim 6 (depends on 1)

The method of claim 1, wherein said zygote culturing is for 5 to 9 days and said second culture condition comprises medium supplemented with bovine serum albumin (BSA) under mineral oil.

### Claim 7 (depends on 6)

The method of claim 6, wherein said medium is selected from the group consisting of C4 medium, synthetic oviductal fluid (SOF) medium, synthetic oviductal fluid with amino acids (SOFaa) medium, and combinations thereof.

### Claim 8 (depends on 1)

The method of claim 1, wherein said in vitro produced ungulate embryo is in a developmental stage selected from the group consisting of morula, early blastocyst, blastocyst, and expanded blastocyst.

### Claim 9 (depends on 1)

The method of claim 1, wherein said in vitro produced ungulate embryo or immature oocyte is an ungulate embryo or oocyte selected from the group consisting of Bos taurus, Bos indicus and crossed breed Bos indicus-taurus.

### Claim 10 (depends on 1)

The method of claim 1, further comprising thawing said in vitro produced ungulate embryo comprising:
exposing said receptacle that contains said frozen embryo in freezing solution to a first thawing environment for a first period of time sufficient to thaw said thawing solutions, exposing said receptacle to a second thawing environment at a thawing temperature between 10° C. to 38° C. for a second period of time, sufficient to thaw the freezing solution; and
mixing the thawing solutions, freezing solutions and embryo within the receptacle.

### Claim 11 (depends on 10)

The method of claim 10, wherein said method includes at least one of the following conditions: wherein the first thawing temperature is from 10° C. to 38° C.; the second thawing temperature is from 20° C. to 38° C.; the thawing environment is or comprises a liquid bath; or the step of mixing is achieved through gentle agitation of the receptacle.

### Claim 12 (depends on 11)

The method of claim 11, further comprising transferring said ungulate embryo to a recipient ungulate.

### Claim 13 (depends on 1)

The method of claim 1, wherein the thawing solution is a 1:1 dilution of the freezing solution in isotonic diluent medium.

### Claim 14 (depends on 1)

The method of claim 1, wherein the thawing solution is a 1:1 dilution of the freezing solution in isotonic diluent medium comprising DPBS.

### Claim 15 (depends on 1)

The method of claim 1, wherein the ethylene glycol is present in the thawing solution at 0.75 M.