# US 4,380,997 A · Embryo transfer method and apparatus > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 4,380,997 A - Document ID: US4380997A - Status: Granted - Kind: A - Application number: 06/251,969 - Filed: 1981-04-07 - Granted: 1983-04-26 - Earliest claimed: 1981-04-07 - Assignees: Rio Vista International, Inc. - Inventors: Stanley P. Leibo - Web page: https://patentgrep.com/patent/US4380997A ## CPC - A61D 19/04: for embryo transplantation - A61D: VETERINARY INSTRUMENTS, IMPLEMENTS, TOOLS, OR METHODS ## Abstract A method for storing, thawing and transferring frozen embryos to recipient animals is disclosed which provides the advantage of a one step dilution, to elminate damage from the cryoprotective solution after thawing, and immediate transfer to the recipient animal. Also disclosed is an apparatus for freezing, storing, thawing and transferring frozen embryos to recipients which comprises a container having first and second chambers wherein a volume of cryoprotective solution containing the embryo can be stored in the first chamber and an effective volumetric amount of a diluent solution can be stored in the second chamber the chambers being divided by removable separation means so as to provide for a dilution process within a single field container. ## Claims ### Claim 1 A method for thawing and transferring frozen embryos to recipients comprising: (a) thawing a volume of frozen cryoprotective agent containing the embryo; (b) combining said thawed volume with an effective volumetric amount of a diluent solution for diluting the cryoprotective agent, said diluent solution having a concentration of a nontoxic, nonpermeating diluent sufficient to result in a dilution mixture which is substantially isosmolal; and (c) transferring said dilution mixture and embryo directly into a recipient where washing of said dilution mixture from said embryo occurs. ### Claim 2 (depends on 1) The method of claim 1 wherein said diluent is sucrose. ### Claim 3 (depends on 1) The method of claim 1 wherein said cryoprotective agent is selected from the group consisting of dimethyl sulfoxide, glycerol, and low molecular weight glycols. ### Claim 4 (depends on 2) The method of claim 2 wherein said cryoprotective agent is glycerol. ### Claim 5 (depends on 1) The method of claim 1 wherein said thawing of said volume is allowed to occur in an uncontrolled manner by allowing same to reach ambient temperature. ### Claim 6 (depends on 1) The method of claim 1 wherein said volumetric amount of diluent solution is at least about six times as great as the volume of said volume of cryoprotective agent. ### Claim 7 (depends on 1) The method of claim 1 wherein said transfer of said dilution mixture is performed surgically. ### Claim 8 (depends on 1) The method of claim 1 wherein said transfer of said dilution mixture is performed nonsurgically. ### Claim 9 (depends on 1) The method of claim 1 wherein said cryoprotective agent is glycerol in a PBS solution in a concentration of from about 1.0 M to about 2.0 M and wherein said diluent solution is sucrose in a PBS solution in a concentration of from about 0.5 M to about 1.0 M. ### Claim 10 In a method for thawing and transferring embryos frozen in a volume of a cryoprotective solution containing a cryoprotective agent to a suitable recipient at a synchronized time wherein the cryoprotective solution is diluted in a onestep process, the improvement comprising: (a) preparing a premeasured diluent solution for combining with said volume to form a dilution mixture, said diluent solution having a volume effective, when combined with the volume of cryoprotective solution, to reduce the concentration of the cryoprotective agent in solution to a nonharmful level, said diluent solution having a concentration of a nontoxic, nonpermeating compound sufficient to be substantially isosmolal with the cryoprotective solution; (b) thawing the volume of cryoprotective agent; (c) combining the thawed volume with the premeasured diluent solution to form said dilution mixture; (d) allowing sufficient time for dilution to occur; and (e) delivering said dilution mixture to the recipient where washing of said dilution mixture from said embryo occurs. ### Claim 11 (depends on 10) The improved method of claim 10 wherein said diluent is sucrose. ### Claim 12 (depends on 11) The improved method of claim 11 wherein said cryoprotective agent is glycerol. ### Claim 13 (depends on 12) The method of claim 12 wherein the volumetric ratio of cryoprotective solution to diluent solution is in the range of from approximately 1:6 to approximately 1:10. ### Claim 14 (depends on 10) The method of claim 10 and further comprising imparting gentle mixing action to said dilution mixture during step (d) thereof. ### Claim 15 A method for storing, thawing and transferring an embryo frozen in a volume of cryoprotective agent comprising: (a) freezing said volume containing said embryo in a container containing an effective amount of a diluent solution separated from said volume; (b) storing said container at freezing temperatures until such time as transfer is desired; (c) thawing said diluent solution and said volume and combining said diluent solution with said volume of cryoprotective agent to effect dilution of said agent; and (d) transferring the contents of said container to the recipient. ### Claim 16 (depends on 15) The method of claim 15 wherein said container comprises a tubular structure and means for separating said tubular structure into a first chamber for containing said volume and embryo and a second chamber for containing said effective amount of diluent solution. ### Claim 17 (depends on 16) The method of claim 16 wherein said separation means comprises an air bubble. ### Claim 18 (depends on 15) The method of claim 15 wherein combination of said diluent solution and volume of cryoprotective agent is achieved by imparting centrifugal force to said container. ### Claim 19 (depends on 15) The method of claim 15 wherein said diluent solution is a solution of sucrose in a phosphate-buffered saline solution. ### Claim 20 (depends on 15) The method of claim 15 wherein said volume of cryoprotective agent comprises a phosphate-buffered solution of glycerol. ### Claim 21 (depends on 15) The method of claim 15 wherein the container is thawed at the situs of the recipient animal. ### Claim 22 (depends on 21) The method of claim 21 wherein said transferring of the contents of said container to the recepient is performed nonsurgically. ### Claim 23 (depends on 22) The method of claim 22 wherein said nonsurgical transfer is effected with an artificial insemination gun.