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Q:HowCanIMaximizetheGeneticPotentialof
MyBestMare?
A:EquineEmbryoTransfer!!

Introduction
Embryotransfer(ET)inequineshastakenconsiderablylongertobecomecommonplacethaninother
species,butitisnowbecomingamorepopularprocedure.Thiscanbeattributedtothefactthatmanybreed
registries,mostnotablytheQuarterHorsehaveraisedtheirlimitsonthenumberoffoalsborntomareeach
year.TheexceptiontothisistheJockeyClub,whichstilldoesnotallowanimalsbornusingthisprocedure
toberegistered.Inadditiontobreedregistriesopeninguptofoalsbornusingthismethod,thesuccessrates
ofequineembryotransferhavebeensteadilyincreasing,triggeringmoreinterestanduseoftheprocedure
(Allen,WR,2005).
Donormares,fromwhichtheoocytepartoftheembryoistaken,areusedintheembryotransferprocedure
formanyreasons.Maresusedforshowingorracingcanbeusedasdonorsandcontinuetocompetewhile
anotherlessvaluablemarecancarrythepregnancy.Animalsthatareeithertooyoungortoooldtocarrya
pregnancycanbeusedasdonorsaswell.Embryotransferalsoincreasesthenumberofoffspringthatcan
bebornperyeartoasinglemare.TheETinmareshasbeenanimportanttoolabletoimprovethe
reproductiveefficiencyandgeneticofthisspecie,itimprovestheprogenyofmareswithhighgenetic
potential.(Carnevalle,1996Squiresetal.,1985aEastetal.,1998AguilarandWoods,1997Allen,WR,
2005).TheleadingcountriesinvolvedinequineembryotransferincludetheUnitedStates,Argentina,and
Brazil(Squiresetal.2003).

HistoryofEmbryoTransfer
1890s:Firstsuccessfulembryotransferdoneinrabbits
1971:Firstsuccessfulembryotransferbetweendonkeysandhorses
1974:Birthofhorsefoalfollowingnonsurgicalembryotransfertorecipientmare
1984:FoalsborninJapanproducedusingfrozenembryos
1984:Firstsuccessfulembryotransferbetweentwoequinespecies(zebrahorse)
1990s:EmbryotransferprocedurestartstotakeoffinArgentineanpoloponies
2002:UniversityofSydneyclaimsfirstsexsortedpregnancybyembryotransfer
CostsofEquineEmbryoTransferinUSAEquineembryotransferiscostly.Thechartbelowshowsthecost
of5differentequinereproductiveclinicsin2006(Table1).Theownersofthedonormaretypicallyleaseor
purchasetherecipientmarefromtheclinic.Alloftheclinicshaveanentryfeerangingfrom$600to$1500.
Thistakescareofsynchronizingtherecipientmareandaspecifiednumberofflushes.Whentherecipient
mareisconfirmedpregnanttheleasingfeeisthandue.Thisfeevariedbetween$2600and$4200forthese
clinics.Thetotalpricerangedfrom$3200to$4200.Thesecostsdonotincludeveterinariancostswhich
canbesubstantial.Whenthefoalisweanedtheleasedrecipientmareneedstobereturnedtotheclinic
(Martin,EF,2006).

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Embryocollectionandtransfer
EmbryoRecovery
Collectionismostsuccessfulwhen:1)performedseventoeightdaysafterovulationforfreshtransfer,and
2)performedatsixdayspostovulationforfreezing.Atypicalprocedureforembryocollectionisas
follows:
1.Thedonormareisplacedinastockhertailiswrappedandmovedoutoftheway
2.Theexternalgenitaliaandbuttocksarecleanedandsterilized
3.AFoleycatheterisinsertedpastthecervix,andacuffisinflatedwithabout60mLofsterilesaline
4.Thetwowaycatheteristhendrawnbackagainsttheinternalcervicalos,formingatightseal
5.1literofprewarmed(~35C)flushfluid,usuallyphosphatebufferedsaline(PBS),isinfusedintothe
mare
6.Thefluidisdrainedthroughafilterthatcollectstheembryo
7.Theflushisrepeatedatleasttwomoretimes
8.Thecollectedembryosareidentified,measured,andgraded.Mostembryoscollectedareearlyto
expandedblastocysts,ranginginsizefrom0.4mm1.0mmindiameter.

Figure1ThedonormareisplacedinastockhertailiswrappedandmovedoutofthewayTheexternal
genitaliaandbuttocksarecleanedandsterilized

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Figure2AFoleycatheterisinsertedpastthecervix,andacuffisinflatedwithabout60mLofsterile
saline

Figure31literofprewarmed(~35C)flushfluid,usuallyphosphatebufferedsaline(PBS),isinfused
intothemarethefluidisdrainedthroughafilterthatcollectstheembryotheflushisrepeatedatleasttwo
moretimes*notethatintheleftpictureitisshownaYtubingcircuitandintherightasimpletubing.

Figure4Thecollectedembryosareidentified,measured,andgraded.Mostembryoscollectedareearlyto
expandedblastocysts,ranginginsizefrom0.4mm1.0mmindiameter.Thisisaembryo(expanded
blastocyst)recovery9daysafterovulation,grade1.
Tabela2.EffectofthedayoftheflushingonembryorecoveryratebetweenDay6to9(Day0=ovulation).

Day6

Reference

n
(%)

Castleberryetal.(1980)

3/13
(23)

Bowenetal.(1985)

12/23

Day7
n
(%)
15/22
(68)

(52)
Iulianoetal.(1985)

21/32
(66)

Wade&Gallagher(1989)

n
(%)
4/8

Day9
n
(%)

(50)
25/31

(81)
68/90
(76)
26/45
(58)

Squiresetal.(1990)

Day8

50/61

(82)
31/47

(66)

86/137 73/96 218/293 43/53

(63)

(76)

(74)

(81)

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Meiraetal.(1993)

Fleury&Alvarenga

Mean

70/127 23/41

(55)

(56)

106/215 388/669 18/33

(49)
(58)
(55)

192/332 311/509 716/1109 61/86

(58)

(61)

(65)

(71)

(AdaptedfromAGUILAR&WOODS,1997Vanderwall,2002).
Table3.Diameterofequineembryosrecoveredfromtheuterinelumen.

Embryodiameter(mm)
DayPostovulation
Noofembryos
Mean
Range
6
121
0.208
0.1320.756
7
144
0.406
0.1361.460
8
142
1.132
0.1203.980
9
41
2.220
0.7304.520
AdaptedfromVanderwall,2002.

EmbryoTransfer
Surgical
Ingeneral,surgicalembryotransfer(vs.nonsurgicaltransfer)ismoretimeconsuming,expensive,labor
intensive,andriskiertotherecipientmare.However,surgicalproceduresaremoresuccessfulthannon
surgicalmethodsbecausetheyofferimprovedsterilityandmoreaccurateplacementoftheembryointhe
reproductivetract.Asurgicalincisionismadeintoeithertheventralmidlineortheflankoftherecipient
mare.

MidventralLaparotomy
1.Mareplacedundergeneralanesthetic
2.Positionedintodorsalrecumbence,hairremoved>
3.Siteofincision(10cmcranialtoudder)scrubbedanddraped
4.Thetipoftheuterinehornipsilateraltoovulationisexposed
5.Incisionmadeintolumenofuterinehorn
6.EmbryoisimplantedintheuterinehornusingasterilizedPasteurpipette

FlankIncisionProcedure
1.Mareisrestrainedandtranquilized
2.Localanestheticappliedatsiteofincision(flank)
3.Flankscrubbedanddraped
4.Proximalthirdofuterinehornipsilateraltoovulationisexposed
5.Uterinehornpenetratedatmostavascularsite
6.EmbryoimplantedintheuterinehornusingasterilizedPasteurpipette

Nonsurgical
1.Positionembryoinsterilepipetteinmediumbetweentwoairspaces(asshowninfigurebelow.Note
thatintherightsideastrawisfilledwiththreecolumns(pink),theembryomustcomeinthemiddle

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column).

2.Manuallyguidepipettethroughcervixintouterus

3.Depositembryoinuterinebody.

SurgicalversusNonSurgicalProcedure
Bothtablesbelowfromtheirrespectivestudyshowagreater50daypregnancyratewithSurgicalTransfer.
Thefirsttableseparatesday7andday8embryos(Iulianoetal.,1985).Surgicaltransferratesare69%and
75%fortheirperspectivedayswhilenonsurgicalis50%and40%.Thesecondtablefromamorerecent
studyin2000(Carnevaleetal.,2000)doesnotseparatetheageoftheembryosandconsistsofmoredata.It
showsasurgicaltransferrateof57.9%andnonsurgicalrateof39.7forthe50daypregnancyrate.
Furthermore,itdemonstratesa10%dropinpregnancyratesfromday12today50withtheembryodeath
ratesbeingnegligible.
Table4.Factorsaffectingembryotransferinhorses

(Iulianoetal.,1985)
Table5.TransferfactorsaffectingpregnancyratesatDays12and50andembryodeath(ED)rates

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(Carnevaleetal.,2000)

Estrussynchronization
ThedegreeofsynchronybetweendonorsandrecipientisveryimportantinequineETprograms.Usually,
therecipientsareselectedwithsynchronybetween1(ovulationonedaybeforedonor)to+3(ovulation3
daysafterdonor).Thepregnancyratebetweenthesedayshasnotbeendifferentintervalo(Douglas,1982
Imel,1981Imeletal.,1981Iuliano,1983Iulianoetal.,1985Squiresetal.,1982,1985).
Additionally,Jacobetal.(2002)concludedrecently,instudycarriedwithagreatnumberofanimals
(n=384),thatthesynchronydegreecanbeusedwithbiggerflexibilityinnonsurgicalembryotransfers,
beingabletobeuserecipientswithsynchrony1to+5withoutsignificantinterferencesinpregnancyrate.
TheadministrationofPGF2?isthemethodoftenusedtosynchronizeestrus.Itisdependentofpresence
andfunctionalityofcorpusluteum(CL).MareswithoutCLorovulatedinlessthanfivedaysshouldnotbe
includedinthistreatment.Itisindicatedtworecipientstoonedonormare.Therecipientshouldbe
synchronizedonetotwodaysafteradministrationofPGF2?inthedonor.(Squiresetal.,1985Bergfelt,
2000).
Anotherwaytosynchronizeestrusisusingprogestagens.Thesehormonesareadministeredforaperiodof
10to15days.Whenremoved,theestrusisexhibit.TheuseofPGF2?allowadecreaseofprogestagen
treatmentfrom15daystoaround8days.RemovingtheprogestinandadministeringPGF2?,themarewill
exhibitestrusin3days(Bristol,1993Bergfelt,2000).Whentworecipientsareselected/donor,itmightbe
necessaryinduceovulationwithHCGintherecipient.Theadministrationof2000a2500UIofhCGin
estrus,afterdetectionofafollicleof35mm,hastentheovulationwithin24to48hpostadministration
(Squiresetal.,1985aPalmer,1993).
Table6.Relationshipofsynchronybetweendonorsandrecipientandpregnancyrateat50days.

Synchrony NoTranfers NoPregnant

3
2
1
0
+1

17
26
62
79
20

11
22
42
56
13

%Pregnant
50days
64.7
84.6
67.7
70.9
65.0

AdaptedfromVanderwall,2002.

Recipientselection
ThesuccessofanequineETprogramisverydependentofthequalityoftherecipient.Thereproductive
historical,theroutineexaminationofthereproductivetractand,ifpossible,cytology,cultureanduterine
biopsyareimportantinordertochosethebestrecipient.Besides,thephysicalstatusandthephenotypic
characteristicsalsohavetobeconsidered(Squiresetal.,1985Squiresetal.,1999Rieira,2000).The
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evaluationofCLmorphology,uterineandcervicaltonus,presenceofedema,cists,fluidorairintheuterus
arealsoimportantfactorstobeconsideredinselectionoftherecipient(Carnevaleetal.,2000).

Superovulation
ItisimportanttorememberthattheefficiencyofETinhorsesislowercomparedwiththebovinespecie.
Manlybecauseitisstillhardtoimprovesatisfactorilytheembryorecoveryratebythecurrentmethodsof
superovulation(Palmeretal.,1993McCue,1996,Squiresetal.,2003).Whilethemeanofovulationsper
cycleinsuperestimulatedcowsisaround11to12,inmaresisaround3to7whenstimulatedwithequine
pituitaryextract(Aguilar&Woods,1997Alvarengaetal.,2000)or1to4using1225mgofeFSHtwicea
day(reviewedinSquiresEL,2006).Butitisstillusefultoobtainmultipleovulations,seekingmore
embryosinagivenbreedingseasonfromonemare.Additionally,stimulatingmultiplefolliclesallow
availabilityforoocytecollection,extraembryosforfreezing,enhancementoffertilityinsubfertilemares,
andadvancementofthefirstovulationoftheyear(SquiresEL,2006).

HorseAgrobusinessinBrazil(Additionaltopic)
AccordingwiththeBrazilianInstituteofGeographyandStatistic(IBGE),current,thereare8.4millionsof
equidsinBrazil.Ofthose,5.9millionsofequines,1.3millionsofmulesand1.2millionsofdonkeys.The
equineBrazilianherdisthelargerofSouthAmericaandthethirdoftheworld,loosingonlyforChinaand
Mexico(7.9and6.3millionsequinesrespectively.)In2003,theCAPB/CNA(ConfederacaodaAgricultura
ePecuariadoBrasil)installedtheNationalCommissionoftheHorse(CNA)withtheobjectivetooffer
technicalandpoliticalsupportforBrazilianhorseowners.TheCNAsupportedabigstudyfromtheEscola
deSuperiordeAgriculturaLuizQueirozEsalq/USP,EstudodoComplexodoAgronegocioCavalo.
Accordingtothisstudy,about30sectorsofthehorseindustrymakeannuallyR$7.5billions(~US$4.3
billions),seetable2(Limaetal.,2006).Thesaleseventsareinastrongexpansionmoment.Current270
horsesaleseventsareregistered.Thenumberofanimals,embryosandbreedingsoldin2004sum10,374
thefinancialbusinessincreased430%since1995(Guerra&Medeiros,2007Limaetal.,2006).The
Brazilianhorseindustryhasincreasedalotandithasabigpotentialtokeepgrowing(seefigure1,Horse
meatexportation).Atthispoint,thereproductivelybiotechnologieshaveanimportantfunctioninthe
equinegeneticimprovement,andtheembryotransferisoneofthemostappliedtechnique.
Table6.
ACTIVITIE
BUSINESSMOVMENT
HorseIndustry
R$7,5bilhes(~US$4.3bi)
AutomobilistIndustry R$39,6bi(~US$22.6bi)
AviationIndustry
R$8,9bilhes(~US$5.0bi)
EMBRAER
R$4,4bilhes(~US$2.5bi)
Adaptedfrom(Limaetal.,2006).
Figure1.Brazilianexportationofhorsemeat.

(Guerra&Medeiros,2007)

Websitecreatedby:JasonBenzine,AndrewDietsche,Reno
Dearaujo,ChelseaCrawford

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