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Agriculture and Natural Resources

FSA3119

Purebred Cattle Series

Embryo Transfer in Cattle

Tom R. Troxel

Professor and AssociateDepartment Head - AnimalScience

Arkansas IsOur Campus

Visit our web site at:http://www.uaex.edu

Introduction

Embryo transfer is one step in theprocess of removing one or moreembryos from the reproductive tract of a donor female and transferring themto one or more recipient females.Embryos also can be produced in thelaboratory via techniques such as

in vitro

fertilization or somatic cellcloning. But the actual transfer of anembryo is only one step in a series of processes that may include some or allof the following: superovulation andinsemination of donors, collection of embryos, isolation, evaluation andshort-term storage of embryos, micromanipulation and genetic testing of embryos, freezing of embryos andembryo transfer.Embryo transfer in cattle hasrecently gained considerable popularity with seedstock dairy and beef producers. Most of the applicableembryo transfer technology was developed in the 1970s and 1980s; however,the history of the concept goes backmuch farther. Embryo transfer wasfirst performed and recorded byWalter Heape in 1890. He transferredtwo Angora rabbit embryos into agestating Belgian doe. The Belgiandoe produced a mixed litter of Belgianand Angora bunnies. Embryo transferin food animals began in the 1930swith sheep and goats, but it was notuntil the 1950s that successfulembryo transfers were reported incattle and pigs by Jim Rowson atCambridge, England.The first commercial embryotransfers in this country were done inthe early 1970s. Initially, embryoswere recovered from valuable donorsand transferred to recipient animalsusing surgical procedures. It was notuntil nonsurgical methods weredeveloped in the late 1970s thatembryo transfer grew in popularity.

Potential Value of EmbryoTransfer in Cattle

The reproductive potential of eachnormal newborn calf is enormous.There are an estimated 150,000potential “eggs” or ova in the cow andbillions of sperm produced by eachbull. By natural breeding, only a fraction of the reproductive potential of anoutstanding individual is realized.The average herd bull will sire 15 to50 calves per year, and the averagecow will have one calf per year. Withartificial insemination, it is possible toexploit the vast number of sperm produced by a genetically superior bull;however, the reproductive potential of the female has been largely unutilized. Under normal managementprograms, a cow produces an averageof eight to ten calves in her lifetime.Like artificial insemination has donefor the bull, embryo transfer is a technique that can greatly increase thenumber of offspring that a geneticallyimportant cow can produce.

Steps for Embryo Transferin Cattle

Virtually all commercial embryotransfers use nonsurgical recovery of the embryos rather than surgical techniques. The process involves severalsteps and considerable time as well as variable expense.

1) Selection of the Donor Cow

The first step is selecting a donorcow. Beef producers will differ in theiropinions regarding the criteria forselecting a genetically outstanding

University of Arkansas, United States Department of Agriculture, and County Governments Cooperating

cow. Whether the criteria is performance records,show ring appeal or both, consideration must begiven to potential dollar value of her calves. There isconsiderable expense incurred in achieving a successfully transferred pregnancy. Therefore, the sale valueof the newborn calf should be high enough to warrantthe added expense of the embryo transfer procedure.Because dairy cattle are selected more routinely onone major trait (milk production), the decisions concerning donor cows are somewhat less complicatedthan in beef cattle. However, the economic considerations are equally important. Embryo transfer is not a“cure-all.” It does not make average cattle good orgood cattle better. It is suitable for a limited numberof seedstock producers with beef or dairy cattle thatcan be breed or species “improvers” for one or moreeconomically important traits.The potential donor cow should be reproductivelysound to produce the desired results. The cow shouldhave a normal reproductive tract and postpartumhistory, especially with regard to estrous lengths of 18 to 24 days. Both beef and dairy cows should be atleast 60 days postpartum before the transfer procedure begins. It has been suggested that prospectivedonor cows in embryo transfer programs be selectedon the following criteria:• Regular estrous cycles commencing at ayoung age• A history of no more than two breedingsper conception• Previous calves with approximately 365-dayintervals• No parturition difficulties or reproductiveirregularities• No conformational or detectable genetic defectsThe cow should be maintained at a nutritionlevel appropriate for her size and level of milk production. Both the very obese cow and the thin cowwill have reduced fertility, so it is important that thedonor be in an appropriate body condition score atthe time of embryo transfer.

2) Superovulation of the Donor Cow

Superovulation of the donor cow is the next stepin the embryo transfer process. Superovulation isthe release of multiple eggs at a single estrus. Cowsor heifers properly treated can release as many asten or more viable eggs at one estrus. Approximately85 percent of all normal fertile donors will respondto superovulation treatment with an average of fivetransferable embryos. Some cows that are repeatedlysuperovulated at 60-day intervals may produce fewernumber of eggs over time. The basic principle of superovulation is to stimulate extensive follicular development through the use of follicle-stimulating hormone(FSH). Superovulation protocols may differ amongembryo technicians, but generally, FSH preparationsare injected twice daily for four days at the middleor near the end of a normal estrous cycle, while afunctional corpus luteum (CL) is on the ovary. Aprostaglandin injection given on the third day of thetreatment schedule will cause CL regression andestrus to occur approximately 48 to 60 hours later.

3) Insemination of the Cow

Because of the release of many ova from multiplefollicles, there is a greater need for viable sperm cellsto reach the oviducts of the superovulated females.Therefore, many embryo transfer technicians willchoose to inseminate the cow several times duringand after estrus. One scheme is to inseminate thesuperovulated cow at 12, 24 and 36 hours after theonset of standing estrus. Using high-quality semenwith a high percentage of normal, motile cells is a very critical step in any embryo transfer program.The correct site for semen placement is in the body of the uterus. This is a small target (1/2 to 1 inch) justin front of the cervix.

4) Flushing the Embryos

To collect the embryos nonsurgically, a smallsynthetic rubber catheter is inserted through thecervix of the donor cow, and a special medium isflushed into and out of the uterus to collect theembryos seven or eight days after estrus (Figure 1).

Figure 1. Diagram of theembryo flushing andrecovery procedure.

This collection procedure is relatively simple and canbe completed in 30 minutes or less without harm tothe cow. A presterilized stylet is placed in the lumenof the catheter to offer rigidity for passage throughthe cervix into the body of the uterus. When the tipof the catheter is in the body of the uterus, the cuff is slowly filled with approximately 2 ml of normalsaline. The catheter is then gently pulled so that thecuff is seated into the internal os of the cervix. Additional saline is then added to the cuff to completelyseal the internal os of the cervix. A Y-connector withinflow and outflow tubes is attached to the catheter. A pair of forceps is attached to each tube to regulatethe flow of flushing fluid. The fluid is sequentiallyadded and removed by gravity. The fluid in theuterus is agitated rectally, especially in the upperone-third of the uterine horn. The uterus is finallyfilled with medium to about the size of a 40-daypregnancy. One liter of fluid is used per donor. Manyembryo transfer technicians use a smaller volume andflush one uterine horn at a time. Each uterine horn isfilled and emptied five to ten times with 30 to 200 mlof fluid each time, according to size of the uterus. Theembryos are flushed out with this fluid into a largegraduated cylinder. After about 30 minutes, embryos

settle and can be located under a stereomicroscope bysearching through an aliquot from the bottom of the cylinder.

5) Evaluation of the Embryos

As the individual embryos are located using amicroscope, they are evaluated for their quality andclassified numerically as to the potential likelihood of success if transferred to a recipient female. The majorcriteria for evaluation include:• Regularity of shape of the embryo• Compactness of the blastomeres (the dividingcells within the boundaries of the embryo)• Variation in cell size• Color and texture of the cytoplasm (the fluidwithin the cell wall)• Overall diameter of the embryo• Presence of extruded cells• Regularity of the zona pellucida (the protectivelayer of protein and polysaccharides around thesingle-celled embryo)• Presence of vesicles (small bubble-likestructures in the cytoplasm)Embryos are classified according to thesesubjective criteria as:Grade 1: Excellent or goodGrade 2: FairGrade 3: PoorGrade 4: Dead or degeneratingEmbryos also are evaluated for their stage of development without regard to quality. These stagesare also numbered:Stage 1: UnfertilizedStage 2: 2 to 12 cellStage 3: Early morulaStage 4: MorulaStage 5: Early blastocystStage 6: BlastocystStage 7: Expanded blastocystStage 8: Hatched blastocystStage 9: Expanding hatched blastocystThere is apparently no difference in pregnancyrates of fertilized cells in different stages of development, assuming they are transferred to the recipientfemale in the appropriate stage of the estrous cycle.Stage 4, 5 and 6 embryos endure the freezing andthawing procedures with the greatest viability.Embryo quality is also of utmost importance in thesurvival of the freezing and thawing stress. Grade 1embryos generally are considered the only ones tofreeze. Grade 2 embryos can be frozen and thawed,yet pregnancy rates typically are reduced. In aLouisiana study involving 1,116 beef and dairy cowsof 15 breeds, 58 percent of all embryos were transferable, 31 percent were unfertilized and 11 percentwere degenerated.

Figure 2. Morphologically normal bovine embryosrecovered at various stages of development.

6) Selection and Preparation ofRecipient Females

Proper recipient herd management is critical toembryo transfer success. Cows that are reproductivelysound, that exhibit calving ease and that have goodmilking and mothering ability are recipient prospects.They must be on a proper plane of nutrition (bodyconditionscore6forbeefcowsanddairybody conditionscore 3 to 4). These cows also must be on a soundherd health program.It is difficult to know how many recipient cowsare needed per each donor flushed. To establish anaverage figure for the number of embryo transfercalves from a single donor cow in a year is difficult. Variations in conditions are wide, but if a cow isflushed every 90 days over a 12-month period andfive pregnancies are obtained per collection, an average of 20 pregnancies per year could result. Somecows have produced more than 50 pregnancies peryear by embryo transfer and probably could haveproduced more if economically feasible. In theLouisiana study previously mentioned, the averagenumber of embryos found per cow was 7.4. With only58 percent of these being transferable, the averagewas 4.3 transferable embryos per flush.To maximize embryo survival in the recipientfemale following transfer, conditions in the recipientreproductive tract should closely resemble those inthe donor. This requires synchronization of theestrous cycles between the donor and the recipients,optimally within one day of each other. Synchronization of the recipients can be done in a similar mannerand at the same working time as the donor cows.There are a number of different estrous synchronization protocols with advantages and disadvantages foreach protocol. The critical point regarding recipientcow estrous synchronization is the timing must matchthe time of insemination of the donor cow so that thedonor and the recipients have a similar uterine environment seven days later when the transfer takes

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