http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.

0088861 s2

Production of Hybrids between Western Gray Wolves and Western Coyotes

!."#a$id"%ech"&ail' (ruce")."*hristensen' *her+l",."Asa' %argaret"*allahan' -ulie".."/oung 0u1lished:"Fe1ruar+"22'"2013 #45:"10.1371/journal.pone.0088861

Abstract 6sing " artificial " inse&ination " we " atte&pted " to " produce " h+1rids " 1etween " capti$e' " &ale' " western' " gra+ " wol$es"7Canis lupus8"and"fe&ale'"western"co+otes"7Canis latrans8"to"deter&ine"whether"their"ga&etes" would"1e"co&pati1le "and"the "co+otes"could"produce"and"nurture"offspring. "9he"results"contri1ute "new" infor&ation"to"an"ongoing "contro$ers+"o$er"whether"the "eastern"wolf"7 Canis lycaon8"is"a"$alid"uni:ue" species"that"could"1e"su1ject"to"the"6.",.";ndangered",pecies"Act."Atte&pts"with"transcer$icall+"deposited" wolf"se&en"into"nine"co+otes"o$er"two"1reeding"seasons"+ielded"three"co+ote"pregnancies."4ne"co+ote"ate" her"pups'"another"produced"a"resor1ed"fetus"and"a"dead"fetus"1+"*<section'"and"the"third"produced"se$en " h+1rids'"si="of"which"sur$i$ed."9hese"results"show"that'"although"it"&ight"1e"unli>el+"for"&ale"western " wol$es"to"successfull+"produce"offspring"with"fe&ale"western"co+otes"under"natural"conditions'"western< gra+<wolf"sper&"are"co&pati1le"with"western<co+ote"o$a"and"that"at"least"one"co+ote"could"produce"and" nurture"h+1rid"offspring."9his"finding"in"turn"de&onstrates"that"ga&ete"inco&pati1ilit+"would"not"ha$e" pre$ented " western' " gra+ " wol$es " fro& " inse&inating " western " co+otes " and " thus " producing " h+1rids " with" co+ote"&t#?A'"a"clai&"that"counters"the"$iew"that"the"eastern"wolf"is"a"separate"species."@owe$er'"so&e " of"the"difficulties"e=perienced"1+"the"other"inse&inated"co+otes"tend"to"te&per"that"finding"and"suggest" that"&ore"e=peri&entation"is"needed'"including"deter&ining"the"1eha$ioral"and"ph+sical"co&pati1ilit+"of" western"gra+"wol$es"copulating"with"western"co+otes."9hus"although"our"stud+"adds"new"infor&ation"to" the"contro$ers+'"it"does"not"settle"it."Further"stud+"is"needed"to"deter&ine"whether"the"putati$e Canis lycaon"is"indeed"a"uni:ue"species. Figures

Citation: %ech"!#'"*hristensen"()'"Asa"*,'"*allahan"%'"/oung"-."720138"0roduction"of"@+1rids"1etween")estern" Ara+")ol$es"and")estern"*o+otes."0!o,"4?;"B728:"e88861."doi:10.1371/journal.pone.0088861 Editor: 0awel"%ichala>'"Cirginia"9ech"Cirginia'"6nited",tates"of"A&erica Received:"?o$e&1er"7'"2013D"Accepted:"-anuar+"12'"2013D"Published:"Fe1ruar+"22'"2013 9his"is"an"open<access"article'"free"of"all"cop+right'"and"&a+"1e"freel+"reproduced'"distri1uted'"trans&itted'"&odified'" 1uilt " upon' " or" otherwise " used" 1+" an+one " for" an+" lawful " purpose. " 9he " wor>" is " &ade " a$aila1le " under " the " *reati$e " *o&&ons"**0"pu1lic"do&ain"dedication. Funding: 9his " stud+ " was " supported " 1+ " the " ?orthern " 0rairie " )ildlife " Eesearch " *enter' " 6. " ,. " Aeological " ,ur$e+" 7http://www.npwrc.usgs.go$/8D " the " 6. " ,. " #epart&ent " of " Agriculture " )ildlife " ,er$ices " F " ?ational " )ildlife " Eesearch" *enter " 7http://www.aphis.usda.go$/wildlifeGda&ag " "e/nwrc/ " "8D " the " )ildlife " ,cience " *enter" 7http://www.wildlifesciencecenter.org/8'"and"the",aint"!ouis"Hoo"7http://www.stlIoo.org/8."9he"funders"had"no"role"in" stud+"design'"data"collection"and"anal+sis'"decision"to"pu1lish'"or"preparation"of"the"&anuscript. Co peting interests:"9he"authors"ha$e"declared"that"no"co&peting"interests"e=ist.

!ntroduction
9wo"schools"of"thought"do&inate"the"&olecular<genetics"literature"on"Canis"sp."7wol$es8"in"the"Areat"!a>es"region"of" the"6.",."and"*anada:"the+"are"h+1rids"1etween"Canis lupus"!."7gra+"wolf8"and"Canis latrans",a+"7co+ote8"J1K'"or"the+" are " h+1rids " 1etween " the " gra+ " wolf " and"Canis lycaon",chre1er " 7the " eastern " wolf8' " a " putati$e " uni:ue " species"J2K." Eesearchers " ha$e " presented " &olecular<genetic " e$idence " for " a " new " interpretation " of " the " ta=ono&+ " of " ?orth " A&erican"Canisspecies."9he+"ha$e"concluded"that"the"gra+<wolf"su1species"Canis lupus lycaon'"is"a"separate"species'" si&ilar"to"Canis rufus"Audu1on"and"(ach&an"7red"wolf8'"that"should"1e"na&ed"Canis lycaon",chre1er'"the"eastern" wolf"J2K."9he"stud+"was"1ased"on"anal+ses"of"8"&icrosatellite "loci"and"&itochondrial "#?A"7&t#?A8"control<region " se:uences"fro&"wol$es"of"southeastern"4ntario"fro&"the"1B60s."?one"showed"gra+"wolf"&t#?A'"and"esti&ates"were" that"&t#?A"se:uences"fro&"1oth"the"eastern"wolf"and"red"wolf"di$erged"120'000F300'000"+ears"ago"fro&"co+otes"as" co&pared" to "a "di$ergence " fro& " the "gra+ " wolf " of" around" 2" &illion " +ears. "(ased "on"this " e$idence " the " researchers " suggested"that"1oth"the"red"wolf"and"eastern"wolf"e$ol$ed"in"?orth"A&erica"along"with"the"co+ote'"as"opposed"to"the " gra+"wolf"which"e$ol$ed"in"the"4ld")orld."9hese"wor>ers"J2K"also"co&pared"their"&icrosatellite"allele"fre:uencies"with" pu1lished"fre:uencies"of"wol$es"and"co+otes"fro&"other"areas"J3K'"J3K"and" found"1oth"&icrosatellite "e$idence"and" &t#?A"e$idence"of"eastern"wol$es"as"far"west"as"%anito1a. 0re$iousl+ " others"J1K'"J2K"had " considered " the " sa&e " &t#?A " haplot+pes " as " e$idence " of " gra+ " wolf " = " co+ote" h+1ridiIation' " and " that " school " continued " to " interpret " the& " that " wa+"J6K'"J7K. " 9he " challengers"J2K"1ased " their" interpretation"on"the"fact"that"those"haplot+pes"ha$e"not"1een"found"in"e=tant"non<h+1ridiIing"co+ote"populations' " and"those"whose"wor>"was"challenged"J8K"agreed"that"the"new"interpretation"J2K"should"1e"further"tested.",e$eral" other"authors"accepted"the"eastern"wolf"as"a"species"JBKFJ16K."@owe$er'"wor>ers"J17K"using"38'000"single<nucleotide" pol+&orphis&s " 7,?0s8 " rejected " that " interpretation " and " fa$ored " the " wolf " = " co+ote " interpretation' " although" others"J18K'"J1BK"disagreed. Eegarding " the " current " wolf " population " in " the "Areat "!a>es " region' " fro& "northeastern "4ntario " west "across " 4ntario' " %ichigan'")isconsin'"%innesota'"and"at"least"part"of"%anito1a'"the"disparate"schools"of"thought"are"that"those"wol$es" are"either"h+1rids"1etween"gra+"wolf"="eastern"wolfJ13K'"J20K"or"gra+"wolf"="co+ote"J1K'"J7K."9he"gra+"wolf"has"1een" on"the"6.,.";ndangered",pecies"!ist"since"1B67"and"protected"1+"the";ndangered",pecies"Act"of"1B73'"was"delisted"in " 2012'"1ut"that"delisting"is"1eing"legall+"contested."5n"addition'"clai&s"ha$e"1een"&ade"1ased"on"genetic"anal+ses"that "

the"nati$e"Areat"!a>esL"wol$es"were"not"restored"J6K"1ut"cf"J12K'"J21Kand"that"the"Areat"!a>esL"wol$es"are"a"Muni:ue" population " or " ecot+pe " of " gra+ " wol$esN"J7K"1ut"cfJ22K. " 9he " 6.,. " Fish " and " )ildlife " ,er$ice " has " accepted " the" interpretation"that"the"eastern"wolf"is"a"$alid"new"species"J18K"that"&ight"1e"considered"endangered"J23K."9herefore" it"is"i&portant"to"tr+"to"deter&ine"the"correct"interpretation"of"the"co+ote<li>e"&t#?A"haplot+pes"in"gra+"wol$es"1+ " e=a&ining"e$idence"other"than"genetic"data. 9he"cru="of"the"contro$ers+"rests"on"the":uestion"as"to"whether"&ale"gra+"wol$es"can"h+1ridiIe"with"fe&ale"co+otes. " )here"definiti$el+<identified"gra+"wol$es"and"co+otes"are"s+&patric"7western"?orth"A&erica8"there"is"no"docu&ented " h+1ridiIation " 1etween " the " two' " and " wol$es " >ill " co+otes"J23K. " Further&ore' " where " the " putati$e " eastern " wolf " and" eastern"co+otes"are"s+&patric"7eastern"?orth"A&erica8"the+"do"h+1ridiIe"J22K'"J26K. ;=peri&ental"h+1ridiIation "1etween">nown"gra+"wol$es"and"western"co+otes"has"not"1een"atte&pted'"1ut"a"&ale " eastern"co+ote"and"putati$e"eastern"wolf"did"h+1ridiIe"in"capti$it+"J22K."@owe$er'"for"co+ote"&t#?A"to"appear"in"a" wolf'"a"fe&ale"co+ote"and"&ale"wolf"would"ha$e"to"&ate.",uch"a"&ating"could"present"significant"1eha$ioral'"ph+sical' " and " possi1l+ " ph+siological " i&pedi&ents. " (ecause " there " is " no " docu&entation " that " gra+<wolf " se&en " can " fertiliIe" western"co+ote"o$a'"we"used"artificial"inse&ination"to"test"this"possi1ilit+.

"ethods ;ight"&ale'"western"gra+"wol$es"fro&"a"den"in"northern"(ritish"*olu&1ia"used"for"se&en"collection"were " housed"together"along"with"a"proestrous"fe&ale"in"a"0.3<"hectare"enclosure"at"the")ildlife",cience"*enter" in " *olu&1us' " %? " and " fed" road<>illed " white<tailed " deer " 7 Odocoileus virginianus8 " ad " li1itu&. " 9he+" were" $accinated"annuall+"with"#ura&ane"%a="2"and"Ea1$ac"7(oehringer"5ngelhei&8.")e"collected"se&en"either " &anuall+"or"1+"electroejaculation"7;;8"on"16"and"18"-anuar+"2012"and"on"23"and"22"-anuar+"2013"79a1le" 18.

#able $% Captive western& gray wolf se doi:10.1371/journal.pone.0088861.t001 %anual",e&en"*ollection

en analysis results at ti

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9he"handler"sti&ulated"the"penis"of"the"wolf"$ia"&anual"stro>ing"through"the"prepuce.")hen"engorge&ent" of"the"1ul1ous"glandis"was"detected'"the"prepuce"was"retracted"pro=i&al"to"the"1ul1ous"glandis"and"the" penis"grasped"pro=i&al"to"the"1ul1ous"glandis"1etween"the"thu&1"and"fingers"of"one"hand."9he"tip"of"the" glans"penis"was"directed"into"a"glass"funnel"with"a"plastic"12<cc"centrifuge"tu1e"attached"to"the"1ase"and" secured"inside"a"20<&l"tu1e"as"a"war&<water"1ath"at"appro=i&atel+"37O*"to"pre$ent"cold<shoc>"to"the " sper&. ;lectroejaculation )e"collected"se&en"fro&"the"wol$es"under"general"anesthesia"76.22"&g/>g"5%">eta&ine"h+drochloride:" .etaset'"(oehringer"5ngelhei&"Cet&edica"5nc.'",t."-oseph'"%4'"6,A'"and"=+laIine:"Eo&pun'"(a+er"*orp.'" ,hawnee " %ission' " .,' " 6,A8 " and " &aintained " 1+ " supple&ental " >eta&ine. " -ust " prior " to " sti&ulation " we" flushed"the"urinar+"1ladder"with"sterile"saline"to"&ini&iIe"urine"conta&ination"of"sa&ples.")e"passed"an " 8<French"pol+prop+lene"catheter"7,o$ereign'"9+co".endall"@ealthcare'"%ansfield'"%A'"6,A8"coated"with" sterile"lu1rication"through"the"urethra"into"the"1ladder."After"urine"was"aspirated'"we"repeatedl+"infused" saline"and"aspirated"until"the"effluent"was"clear. )e"collected"se&en"1+";;"7%odel"12"electroejaculator:"A"P","5nstru&ents'"%idlothian'"9Q'"6,A8"using"a" 3<c&<dia&eter"pro1e"with"three'"7.2<c&"long'"linear"electrodes"709";lectronics'"(oring'"4E'"6,A8"placed" $entrall+"in"the"rectu&"a1o$e"the"prostate"gland"and"pudendal"ner$e.")e"increased"the"sti&ulation"slowl+"

6 until"the"hind<li&1s"e=tended'"returned"to"Iero'"and"repeated"rh+th&icall+"at"graduall+"increasing"$oltage'" with"a"c+cle"of"appro=i&atel+"2"seconds'"until"ejaculation."%a=i&u&"current"did"not"e=ceed"300"&A&p' " the"&easure"of"current"reaching"the"tissues.")e"repeated"sti&ulation"as"long"as"the"sa&ple"appeared" cloud+'"an"indicator"of"sper&"presence. ,e&en"Anal+sis"and"@andling 5&&ediatel+ " following " collection' " we " separated " a " drop " of " se&en " for " anal+sis. " )e " esti&ated " percent" &otilit+ " and " status " using " a " phase<contrast " &icroscope " at " 200Q " and " calculated " concentration " using " a" %a>ler "*ounting "*ha&1er "7,efi<%edical "5nstru&ents' "@aifa' " 5srael8. ")e "spun "the "re&aining "sa&ple "at" 700Rg"for"10"&inutes'"discarded"the"supernatant"7se&inal"fluid8'"and"resuspended"the"sper&"pellet"in"a" roo&<te&perature' " *hill<2 " e=tender " 7%initu1e " of " A&erica' " Cerona' " )5 " 232B38 " de$eloped " to " preser$e" do&estic"dog"se&en"for"o$ernight"ship&ent."4n"the"da+"of"collection"we"pac>aged"the"sa&ple"plus"*hill"2" in"a"*hill<2",hipping".it"7%initu1e"of"A&erica8'"an"insulated"container"with"ice"pac>s"placed"so"that"the " sper& " cool " at " a " controlled " rate " during " transport' " and " shipped " the& " o$ernight " to " the " 6,#A " ?ational" )ildlife"Eesearch"*enterLs"0redator"Eesearch"Facilit+"in"%ill$ille'"69'"6,A."9here"the+"were"to"1e"used"to" inse&inate " western " co+otes " in " the " capti$e " colon+ " whose " ancestors " were " wild<caught " in " such " western" states"as"5daho'"6tah'")+o&ing'"and"%ontana. 2012"fe&ale"co+ote"estrus"s+nchroniIation"and"artificial"inse&ination ,i="fe&ale"co+otes"located"at"the"0redator"Eesearch"Facilit+'"used"in"2012'"ranged"fro&"3F2"+ears"old" 73.8S0.38.")e"placed"deslorelin"i&plants"72.1"&g'"4$uplantT'"0eptech"Ani&al"@ealth"0t+"!i&ited'"?,)'" Australia8"su1cutaneousl+"in"the"inguinal"region"of"the"si="fe&ales"on"3"-anuar+'"2012.",i="adult"fe&ale" co+otes"that"were"1<7<+ears<old"73.7U0.B8"and"housed"at"the"sa&e"facilit+"did"not"recei$e"i&plants"and " were " &onitored " as " controls " 79a1le " 28. " )e " &easured " seru& " progesterone " concentrations " 1+" che&ilu&inescent " &icroparticle " i&&unoassa+ " on " 16 " -anuar+ " 7treated " ani&als8 " and " 1B " -anuar+ " 71oth" treated"and"control"ani&als8."Four"treated"co+otes"with"rising"$alues"a1o$e"1aseline"7V2"ng/&l8"were" selected"for"inse&ination"7two"3<+ear"olds'"one"3<+ear"old'"and"one"2<+ear"old8.

#able '% (eru progesterone concentrations fro doi:10.1371/journal.pone.0088861.t002

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)e " perfor&ed " inse&inations " on " 17 " and " 1B " -anuar+ " 2012 " after " ph+sicall+ " restraining " co+otes'" anesthetiIing"the&"with"=+laIine"71.22"&g/>g"5C8"and">eta&ine"76.22"&g/>g"5C8'"and"placing"the&"in" sternal"recu&1enc+"with"the"hind"legs"in"fle=ion. )e"inserted"a"cotton"swa1"into"the"$agina'"turned"and"re&o$ed"the"swa1'"and"then"rolled"it"on"a"glass" slide"su1se:uentl+"stained"with"a"&odified")right"Aie&sa"stain"protocol"for"$aginal"c+tological"e$aluation." )e"re&o$ed"the"su1cutaneous'"deslorelin"i&plant"$ia"a"s&all"incision"in"the"s>in.")e"$isualiIed"the"cer$i= " 1+ " passing " a " rigid " uretero<renoscope " 733 " c&RB.2 " FrenchD " .arl " ,torI " Ceterinar+ " ;ndoscop+<A&erica'" Aoleta' " *A8 " through " the " $aginal " canal. " )e " then " ad$anced " a " 2<French' " fle=i1le " pipette " through " the" endoscope"and"through"the"e=ternal"os"of"the"cer$i="for"2"c&.")e"injected"3F2"&l"of"e=tended'"gra+<wolf" se&en"7concentration"un>nown8"through"the"pipette"slowl+"into"the"uterus"1efore"withdrawing"the"pipette " and"endoscope.")e">ept"the"hind"end"of"the"fe&ale"slightl+"ele$ated"for"20"&inutes"during"the"reco$er+" fro& " anesthesia " to " pre$ent " retrograde " flow " of " the " se&en. " )e " used " a " co&&ercial " rela=in " assa+ "J27K' J28K"to"diagnose"pregnanc+"at"appro=i&atel+"30<da+s"gestation"716"Fe1ruar+"20128.")e"too>"radiographs" at"appro=i&atel+"26<da+s"gestation"712"%arch"20128"to"count"fetuses. 2013"fe&ale"co+ote"estrus"s+nchroniIation"and"artificial"inse&ination )e"i&planted"10"fe&ale"co+otes"aged"3F6"+ears"7&ean"age'"3.28"+ears"S0.3",;8"with"a"su1cutaneous' " deslorelin" i&plant "72.1 " &g' "4$uplantT8 "in"the "inguinal " region"on" -anuar+ "10'"2013 "79a1le "38. ",eru&"

7 progesterone"concentrations"were"e$aluated"1+"che&ilu&inescent"&icroparticle"i&&unoassa+"on"21'"23'" and"22"-anuar+.")e"used"a"rising"trend"in"seru&"progesterone"a1o$e"1aseline"7V2"ng/&l8"as"a"guide"for" deciding"when"to"inse&inate"and"selected"fi$e"co+otes"7three"3<+ear"olds'"one"2<+ear"old'"and"one"6<+ear" old8"for"inse&ination.")e"inse&inated"one"fe&ale"on"22"-anuar+"2013'"three"on"22"-anuar+"2013'"and " two"on"26"-anuar+"2013'"once"each'"e=cept"one"fe&ale"inse&inated"on"22"-anuar+"and"22"-anuar+.")e " tested " for " pregnanc+ " 1+ " seru&<rela=in " assa+ " on " 6 " %arch' " 2013' " and " radiographicall+ " e$aluated " the" fe&ales"on"1B"%arch"2013"to"count"fetuses.")e"perfor&ed"i&plant"re&o$al"and"inse&ination"as"descri1ed" for"2012.

#able *% (eru progesterone concentrations fro doi:10.1371/journal.pone.0088861.t003 41ser$ing"Fe&ales"during"0arturition

captive& western& coyote fe

ales in ')$*%

)e"&aintained"all"pregnant"fe&ales"indi$iduall+"in"clo$er"pens"70.1"ha8"with"an"infrared"ca&era"in"den" 1o=es"to"&onitor"the&"for"signs"of"la1or"without"distur1ing"the&"starting"at"60"da+s"esti&ated"gestation. " #uring"the"da+'"the"pens"were"within"$iew"of"areas"nor&all+"used"1+"staff'"and"we"used"1inoculars"and " spotting"scopes"to"assess"their"status."At"night'"staff"chec>ed"on"the"fe&ales"at"hourl+"inter$als"in"2012 " and"at"3<hour "inter$als"in"2013"$ia"the "$ideo "feed"fro&"the "infrared"ca&era"or"1+" using"night "$ision " 1inoculars. ;thics",tate&ent 9his"stud+"was"carried"out"in"strict"accordance"with"the"reco&&endations"in"the"Auide"for"the"*are"and" 6se"of"!a1orator+"Ani&als"of"the"?ational"5nstitutes"of"@ealth."9he"protocol"7WA<1B23"and"A&end&ent"18" was"appro$ed"1+"the"5A*6*"of"the"6.",."#epart&ent"of"Agriculture'"Ani&al"and"0lant"@ealth"5nspection " ,er$ice' " )ildlife " ,er$ice " ?ational " )ildlife " Eesearch " *enter. " 5nsertion " of " deslorelin " i&plants' " artificial" inse&ination'"and"*<section"were"perfor&ed"under">eta&ine"and"=+laIine"anesthesia'"and"all"efforts"were" &ade"to"&ini&iIe"pain"or"disco&fort. ,tatistics @+pothesiIing " that " the " deslorin<i&planted " fe&ales " would " show " ele$ated " progesterone " le$els' " we " log< transfor&ed " seru&<progesterone " concentrations " and " co&pared " the " &eans " of " the " treated " sa&ple " with" those " of " the " controls " 1+ " two<sa&ple " t<test. " )e " also " co&pared " proportion " of " co+otes " with " ele$ated" progesterone"7V2.0"ng/&l8"in"the"treated"sa&ple"with"that"proportion"in"the"control"sa&ple"1+"one<tailed" FisherLs"e=act"test. Results )e"o1tained"&otile"sper&"fro&"three"wol$es"in"2012"and"eight"in"2013'"1ut"the":ualit+"$aried"a&ong " &ales"1ased"on"assess&ent"of"side<to<side"&otion"and"degree"and"speed"of"forward"progress"7 9a1le"18." 9wo"sa&ples"contained"so&e"1lood"conta&ination'"perhaps"due"to"irritation"of"the"1ladder"or"urethra"1+" the"catheterD"two"were"conta&inated"1+"urineD"and"two"contained"sper&"that"were"agglutinating. 5n"2012'"seru&"progesterone"concentrations"were"ele$ated"a1o$e"1aseline"7V2.0"ng/&lD"range"of"1.0"to" 30.2"ng/&l'"&edian"X"12.3"ng/&l8"in"2"of"6"treated"fe&ales"co&pared"with"an"ele$ation"in"1"of"6"fe&ales " in " the " control " group " 7range " of " 0.3 " to " 3.8 " ng/&l' " &edian " X " 0.B " ng/&l8 " 79a1le " 28. " *o+otes " recei$ing" i&plants "had"higher"seru&"progesterone"concentrations"than"control"co+otes"70"X"0.0168"and"treated " ani&als"had"ele$ated"seru&"progesterone"le$els"o$er"1aseline"co&pared"to"control"ani&als"70"X"0.038." Caginal"c+tological"e$aluation"in"the"treated"fe&ales"1oth"in"2012"and"2013"re$ealed"cells"consistent"with" estrogen"influence.

8 5n " 2012 " rela=in " assa+s " indicated " that " 2<+ear<old' " nulliparous " fe&ale " 7,hadow8 " was " pregnant. " At" appro=i&atel+"26"da+s"gestation"we"&o$ed"her"to"a"s&aller"pen"to"facilitate"&onitoring'"ultrasound'"and " radiograph+"and"found"she"had"a"single"fetus.",he"did"not"use"her"den"1o="after"the"&o$e'"so"we"relied" on"night<$ision"1inoculars"e=clusi$el+"to"o1ser$e"her.",he"went"into"la1or"after"63"da+s"gestation"722" %arch"20128."After"she"underwent"2F3"hours"of"la1or"without"deli$er+'"we"transported"her"to"a"$eterinar+" hospital"for"an"e&ergenc+"c<section."9he"fetus"did"not"sur$i$e"and"was"esti&ated"to"ha$e"1een"dead"for " se$eral"hours."9here"was"also"sign"of"a"resor1ed"fetus."9he"fe&ale"reco$ered"without"further"incident. 5n"2013"rela=in"assa+s"indicated"that"two"fe&ales"1eca&e"pregnant. ?ulliparous'"6<+ear<old"Fe&ale"("was"esti&ated"to"ha$e"at"least"6"fetuses.",he"used"her"den"1o="and" deli$ered"at"least"7"pups"on"da+"60"723"%arch"20138."Cia"$ideo"&onitoring"we"saw"that"the"first"pup " whelped"was"still1orn.")hen"Fe&ale"("left"the"den"for"food'"we"saw"that"si="pups"were"ali$e'"although" one"appeared"wea>'"and"the"still1orn"pup"was"&issing."9his"fe&ale"cared"for"and"nursed"si="pups"for"13 " da+s"1efore"we"re&o$ed"the&"for"hand"rearing. Four<+ear<old"Fe&ale"5"had"gi$en"1irth"to"7"co+ote"pups"in"2011"and"8"in"2012."5n"2013'"she"had"at"least " 8"h+1rid"fetuses.",he"did"not"use"her"den"1o=.")e"o1ser$ed"through"a"spotting"scope"Fe&ale"5"going"into " la1or"during"dus>"the"e$ening"of"da+"61'"and"she"deli$ered"four"pups"1efore"it"was"too"dar>"to"o1ser$e " 723"%arch"20138."9he"first"two"pups"1orn"were"&o$ing'"and"the"fe&ale"placed"the&"in"separate"ground" di$ots."9he"ne=t"pup"&a+"ha$e"1een"still1orn.")e"ne$er"saw"it"&o$e'"and"the"fe&ale"carried"it"around"to" &ultiple"di$ots."(efore"deli$er+"of"the"fourth"pup'"she"ate"at"least"two"of"the"three"pups"including"the " third<one"1orn.")e"confir&ed"that"the"fourth"pup"was"ali$e"1ased"on"pup"sounds."At"that"ti&e"it"was"too " dar>"to"&onitor'"e$en"with"night<$ision"1inoculars'"without"distur1ing"the"fe&ale."(+"&orning"there"were" no"pups. (ased"on"2<da+"litter"counts"of"all"pups"1orn"to"co+otes"at"the"69"facilit+"fro&"2002"to"2012"7n"X"8B8'"the" fre:uenc+"of"singleton"1irths"is"low"70.018"while"the"fre:uenc+"of"producing"6"pups"is"high"7Figure"18." ;ating"pups"had"1een"e=tre&el+"rare"to"non<e=istent"at"this"facilit+"and"did"not"occur"with"either"of"the " two"pre$ious"litters"of"Fe&ale"5"that"ate"her"pups"in"2013.

Figure $% +u ber of litters and litter si,es of western& coyote pups produced at the - (% .epart ent of Agriculture Wildlife (ervices +ational Wildlife Research Center Predator Research Facility between '))/0')$'% doi:10.1371/journal.pone.0088861.g001 )e " transported " the " 6 " sur$i$ing " pups " when " 13<da+s " old " to " the " )ildlife " ,cience " *enter' " *olu&1us'" %innesota " for " rearing " 1+ " hu&ans' " and " as " of " this " writing " 7-anuar+ " 20138 " all " were " sur$i$ing " and " were " health+."(+"4cto1er"the"h+1rid"pups"had"characteristics"of"1oth"gra+"wol$es"and"co+otes"7Figures"2'"3'"38." As"of"6"?o$e&1er"2013'"the"two"fe&ale"h+1rids"weighed"17.6"and"21.8">g'"and"the"four"&ales"$aried" 1etween"20.1"and"26.0">g."For"co&parison'"10"western"fe&ale"co+otes"1<7<+ears"old"fro&"our"colon+ " weighed"10.1F16.2">g"a1out"the"sa&e"ti&e"7&ean"X"12.3S0.26">g",;8'"and"20"&ales"2F7<+ears"old" weighed " 10.1F17.1 " >g " 7&ean " X " 13.3S0.31 " >g " ,;8. " 9hus " the " h+1rid " 6<7<&onth " old " &ales " weighed" significantl+"&ore"than"the"adult"&ale"co+otes"70V0.00018"and"o$erlapped"in"weight"with"the"range"of " weights"of"&ale"wolf"pups"fro&"south<central"Alas>a"7&ean"X"26.2S2.3">g",;8"J2BK."(ecause"we"onl+"had" two"fe&ale"h+1rids"we"did"not"test"the"significance"of"the"weight"difference"with"co+otes'"1ut"the"sa&e " trend"is"apparent.

Figure '% #hree 1023 onth3old& litter ate& F$ hybrids between a a fe ale& western coyote resulting fro artificial inse ination% doi:10.1371/journal.pone.0088861.g002

ale& western& gray wolf and

Figure *% Facial view of two 1023 onth3old F$ hybrids between a a fe ale& western coyote resulting fro artificial inse ination% doi:10.1371/journal.pone.0088861.g003

ale& western& gray wolf and

10

Figure 4% (ide view of a 1023 onth3old F$ hybrid between a fe ale& western coyote resulting fro artificial inse ination% doi:10.1371/journal.pone.0088861.g003 .iscussion

ale& western& gray wolf and a

A"logical"hierarch+"of":uestions"see>ing"to"deter&ine"whether"free<ranging"western"co+otes"and"western" gra+"wol$es"&ight"ha$e"h+1ridiIed"in"the"past"would"include"the"following: 1. #o"fe&ale"western"co+otes"&ate"with"&ale"western"wol$es"and"produce"fertile"offspringY 2. )ill"fe&ale"western"co+otes"&ate"with"&ale"western"wol$es"and"produce"fertile"offspringY 3. *an"fe&ale"western"co+otes"&ate"with"&ale"western"wol$esY 3. 5f"the+"&ate'"can"the+"produce"fertile"offspringY 2. 0h+siologicall+' " 1eha$iorall+' " and " ph+sicall+ " can " fe&ale " western " co+otes " &ating " with " western" wol$es"produce"fertile"offspringY 6. 0h+siologicall+'"1eha$iorall+'"and"ph+sicall+"can"fe&ale"western"co+otes"i&pregnated"with"western" wolf"se&en"produce"offspringY 9o"date"there"is"no"infor&ation"to"answer":uestions"1F2'"and"there"is"e$idence"against"1"and"2J23K."4ur" results"show"that"western"gra+<wolf"se&en"can"inse&inate"at"least"so&e"western"co+otes"and"that"those " co+otes " can " deli$er " $ia1le " pups. " @owe$er' " our " o$erall " results " were " e:ui$ocal " enough " to " &a>e " it" worthwhile"to"consider"the"e=tent"to"which"the"failures"li>el+"resulted"fro&"our"&ethods"$ersus"fro&"so&e" inherent"pro1le&s"in"co&pati1ilit+"1etween"western"gra+"wolf"se&en"and"western"co+ote"production"of" pups"resulting"fro&"that"inse&ination. )e " used " se&en " fro& " eight " indi$idual " gra+ " wol$es " and " atte&pted " to " inse&inate " nine " capti$e " western" co+otes"o$er"two"1reeding"seasons'"which"resulted"in"three"co+ote"pregnancies."4f"those'"one"had"trou1le" deli$ering"a"single"pup"and"had"to"1e"gi$en"a"*<section'"1ut"the"fetus"had"died."9here"was"also"e$idence" of"a"resor1ed"fetus."A"second"fe&ale"produced"at"least"se$en"pups'"the"first"of"which"was"still1orn'"1ut"si= " sur$i$ed."9he"third"fe&ale"produced"at"least"8"pups"1ut"consu&ed"the&"all"within"23"hours. A"su&&ar+"of"the"$arious"failures"at"least"so&e"co+otes"e=perienced"1etween"atte&pted"inse&ination" and"nor&al"pup"deli$er+"and"care"follow:"718"lac>"of"conception"or"earl+"e&1r+onic"death'"728"low"litter " siIe'"738"resor1ed"fetus'"738" ina1ilit+ "to "deli$er'" 728"still1orn"fetus' "and"768"consu&ption "of"pups. "9he" co+ote"that"ate"her"pups"showed"&ore"difficult+"than"nor&al"in"deli$er+'"1eha$ing"as"though"she"was"in" se$ere"pain.

11 9he"following"possi1le"&ethodological"pro1le&s"could"ha$e"caused"these"failures:"718"inade:uate":uantit+" or " :ualit+ " of " gra+<wolf " se&en' " 728 " fault+ " ti&ing " of " the " inse&ination' " and " 738 " artificial " conditions " of" capti$it+."4n"the"other"hand'"we"cannot"rule"out"reproducti$e "inco&pati1ilit+ "for"at"least"so&e"of"the " failures."(ased"on"our"e=perience"with"assessing"sper&":ualit+"and"artificiall+"inse&inating"do&estic"dogs'" we"can"onl+"judge"that"&ost"of"the"failures"to"concei$e"were"pro1a1l+"caused"1+"poor"se&en":ualit+"or" :uantit+"or"1+"&isti&ing"of"inse&inations.",o&e"68F78%"of"do&estic"dogs"induced"with"4$uplant"in"late" anestrus"1eca&e"pregnant"J30K'"J31K. 5n"1oth"+ears'"se&en"was"of"generall+"poorer":ualit+"7percent"&otilit+"and"nor&al"&orpholog+8"than"is" t+pical"later"in"the"wolf"1reeding"season"7*.",."Asa"et"al."unpu1lished8."Fe&ale"wol$es"at"the"%innesota " facilit+"are"recepti$e"to"&ating"during"the"first"wee>"in"Fe1ruar+"through"earl+"%arch"7earliest"&ating:"1" Fe1ruar+D " &ean: " 16 " Fe1ruar+8"J32K. " %ales " onl+" produce " sper& " around " the " 1reeding " season' " with " the" highest":ualit+"o1ser$ed"in"the"&iddle"of"that"period"J33K."5n"contrast'"co+otes"at"the"6tah"facilit+"&ate"as" earl+" as" &id<-anuar+' "two<si=" wee>s" earlier " than " the " wol$es" in" our "stud+ " 7-. " .. "/oung' " unpu1lished8." (ecause"we"needed"to"induce"estrus"and"o$ulation"in"the"fe&ale"co+otes"to"ti&e"the"inse&inations'"it"was " i&portant"to"sti&ulate"the&"$er+"earl+"in"their"1reeding"season"to"&ini&iIe"the"chances"that"the+"would" o$ulate"naturall+"1efore"we"could"inse&inate"the&."9hus"at"the"ti&e"of"those"earlier"o$ulation"dates'"our" wol$es"were"not"+et"producing"high<:ualit+"sper&. @owe$er'"so&e"of"our"other"findings"could"&ore"li>el+"ha$e"1een"a"result"of"interspeciesL"reproducti$e" inco&pati1ilit+: "resorption "of"a"fetus' "production "of"onl+" a"single " pup'" death"of"that "pup' "and" difficult " deli$er+"that"could"ha$e"led"to"pup"consu&ption."9he"difficult"deli$er+"in"2012"leading"to"the"need"for"a"*< section"pro1a1l+"resulted"fro&"the"fact"that"the"fetus"was"e=tra"large'"a"condition"co&&on"in"do&estic " dogs"1earing"a"single"pup."9he"h+1rid"fetus"weighed"271"g&'"2B%"larger"than"a"t+pical"co+ote"pup"at" 1irth"79a1le"38."9he"successful"deli$er+"in"2013"included"si="pups"whose"a$erage"weights"at"2"wee>s"were" si&ilar"to"the"a$erage"weight"of"si&ilarl+<aged"co+ote"pups"fro&"si&ilarl+<siIed"litters"79a1le"28."5t"is"of" interest'"howe$er'"that"one"of"the"2<wee><old"h+1rids"at"720"g&"was"8%"hea$ier"than"the"largest"pup"of" fi$e"litters"of"si="co+otes"each"at"the"sa&e"age."9he",;"of"the"&ean"weight"7630.7"g&8"of"the"h+1rid"litter " 731.18 " was " B% " greater " than " the " highest " ,; " of " each " of " the " fi$e " co+ote " litters " 79a1le " 28. " 9his " greater" $ariation"in"h+1rid"pup"siIe'"if"t+pical'"&ight"cause"deli$er+"pro1le&s"in"so&e"co+otes.

#able 4% (i,e of a single hybrid fetus at C3section co at birth 5*46% doi:10.1371/journal.pone.0088861.t003

pared with a typical western coyote pup

12

#able /% Weights of $*3day3old& western3gray wolf 7 western3coyote hybrid pups co with si7 litters of $43day3old& western3coyote pups% doi:10.1371/journal.pone.0088861.t002

pared

5n"an+"case'"one"of"the"nine"co+ote"fe&ales"did"produce"a"nor&al"litter"of"si="li$e"pups"and"nursed"the&' " de&onstrating "that " gra+<wolf "se&en "can " inse&inate " co+ote " o$a' "and " that "a " fe&ale " co+ote "can " carr+'" deli$er'"and"nurse"offspring"fro&"the"interspecies"conception.")hether"&ore"than"an"occasional"co+ote" can " do " so " is " an " open " :uestion' " so " there " &ight " 1e " so&e " e$idence " fro& " our " stud+ " that " successful" h+1ridiIation"in"the"wild"1etween"&ale"gra+"wol$es"and"fe&ale"co+otes'"e$en"if"the+"did"copulate'"&ight " 1e"unco&&on. 9o"definiti$el+"deter&ine"how"li>el+"it"is"for"&ale"gra+"wol$es"and"western"co+otes"to"h+1ridiIe"naturall+'" se$eral"&ore"in$estigations"&ust"1e"done."9his"stud+"onl+"addresses"ite&"6"a1o$e."@owe$er'"our"results" do"suggest"that"one"of"the"first"steps"is"to"deter&ine"whether"our"h+1rids"are"fertile"and"to"repeat"our " in$estigations " with " the " highest " :ualit+ " and " :uantit+ " of " gra+<wolf " se&en " and " opti&iIing " the " ti&ing " of" inse&inations"with"se$eral"&ore"fe&ale"co+otes."5f"such"studies"result"in"se$eral"&ore"failures'"that"would" tend"to"reduce"the"need"for"researching"ite&s"1F2. Ac8nowledg ents

)e"appreciate"the"assistance"of"the"following:".aren"(au&an"with"the",aint"!ouis"Hoo"and",tace+"(ru&&er'"-eff " ,chultI' " ;ri>a " ,te$enson' " (onnie " %ac#onald' " and " %i>e " #a$is " with " the " ?ational " )ildlife " Eesearch " *enter " and " the " ad$ice " and " suggestions " of " re$iewer " Eichard " 0. " 9hiel. " An+ " use " of " trade' " fir&' " or " product " na&es " does " not " i&pl+" endorse&ent"1+"the"6.",."Ao$ern&ent.

Author Contributions
*oncei$ed"and"designed"the"e=peri&ents:"!#%."0erfor&ed"the"e=peri&ents:"()*"*,A"%*"-./."Anal+Ied"the"data:" !#%"()*"*,A"-./."*ontri1uted"reagents/&aterials/anal+sis"tools:"()*"*,A"%*"-./.")rote"the"paper:"!#%"()*"*,A" -./.

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$%!eh&an"?'";isenhawer"A'"@ansen".'"%ech"!#'"0eterson"E4'"et"al."71BB18"5ntrogression"of"co+ote"&itochondrial " #?A"into"s+&patric"?orth"A&erican"gra+"wolf"populations.";$ol"32:"103F11B."doi:"10.2307/230B386 '%)ilson"0-'"Arewel",'"!awford"5#'"@eal"?-%'"Aranac>i"AA'"et"al."720008"#?A"profiles"of"the"eastern"*anadian"wolf" and"the"red"wolf"pro$ide"e$idence"for"a"co&&on"e$olutionar+"histor+"independent"of"the"gra+"wolf."*an"-"Hool"78:" 2126F66."doi:"10.113B/cjI<78<12<2126 *%Eo+"%,'"Aeffen";'",&ith"#'"4strander";A'")a+ne"E."71BB38"0attern"of"differentiation"and"h+1ridiIation"in"?orth " A&erican"wolfli>e"canids"re$ealed"1+"anal+sis"of"&icrosatellite"loci."%ol"(iol"and";$ol"11:"223F270. 4%Eo+"%,'"Aeffen";'",&ith"#'")a+ne"E."71BB68"%olecular"genetics"of"pre<1B30Ls"red"wol$es."*onser$"(iol"10:"1313F 1323."doi:"10.1036/j.1223<173B.1BB6.10021313.= /%)a+ne"E.'"!eh&an"?"71BB28"%itochondrial"#?A"anal+sis"of"the"eastern"co+ote:"origins"and"h+1ridiIation."5n:"(oer " A@' " editor. " ;colog+ " and " %anage&ent " of " the " ;astern " *o+ote.Fredericton' " ?(' " *anada: " )ildlife " Eesearch " 6nit'" 6ni$ersit+"of"?ew"(runswic>."pp."BF22. 1%!eonard " -A' " )a+ne " E. " 720088 " ?ati$e " Areat " !a>es " wol$es " were " not " restored. " (iol " !ett " 3: " B2FB8" doi:10.10B8/E,(5.2007.0323. 2%.o1l&uller",'"?ord"%'")a+ne"E.'"!eonard"-A"7200B8"4rigin"and"status"of"the"Areat"!a>es"wolf."%ol";col"18:"2313F 2326"doi:10.1111/j.1362<2B3Q.200B.03176.=. 9%)a+ne"E.'"Cila"*"720038"%olecular"genetic"studies"of"wol$es."5n"%ech"!#'"(oitani"!'"editors.")ol$es:"(eha$ior' " ;colog+'"and"*onser$ation."*hicago:"6ni$ersit+"of"*hicago"0ress."pp."218F238

:%.+le " *-' " -ohnson "AE' " 0atterson "(E' " )ilson " 0-' " ,ha&i" .' " et"al. " 720068 " Aenetic "nature "of "eastern" wol$es: " 0ast' " present"and"future."*onser$"Aenet"7:"273F287."doi:"10.1007/s102B2<006<B130<0 $)%Fain",E'",traughan"E-'"9a+lor"(F"720108"Aenetic"outco&es"of"eastern"ti&1er"wolf"reco$er+"in"the"western"Areat" !a>es",tates."*onser$"Aenet"11:"1737F1762."doi:"10.1007/s102B2<010<0068<= $$%%urra+"#!'")aits"!"720078"9a=ono&ic"status"and"conser$ation"of"the"endangered"red"wolf:"a"response"to".+le'"et " al."720068."*onser$"Aenet"8:"383F1382."doi:"10.1007/s102B2<007<B307<1 $'%Eutledge"!/'"(os".5'"0earce"E-'")hite"(?"720108"Aenetic"and"&orpho&etric"anal+sis"of"si=teenth"centur+"*anis " s>ull " frag&ents: " i&plications " for " historic " eastern " and " gra+ " wolf " distri1ution " in " ?orth " A&erica. " *onser$ " Aenet " 11:" 1273F1281"doi:10.1007/s102B2<00B<BB27<2. $*%)a+ " -A' " Eutledge " !' " )heeldon " 9' " )hite " (? " 720108 " Aenetic " characteriIation " of " eastern " co+otes " in " eastern" %assachusetts."?ortheastern"?at"17:"18BF203."doi:"10.1626/032.017.0202 $4%)heeldon"9"7200B8"Aenetic"characteriIation"of"*anis"populations"in"the"western"Areat"!a>es"region."0eter1orough'" 4?'"*anada:"%,"9hesis'"9rent"6ni$. $/%)heeldon"9'")hite"(?"7200B8"Aenetic"anal+sis"of"historic"western"Areat"!a>es"region"wolf"sa&ples"re$eals"earl+ " *anis"lupus/l+caon"h+1ridiIation."(iol"!ett"2:"101F103."doi:"10.10B8/rs1l.2008.0216 $1%)heeldon"9'"0atterson"(E'")hite"(?"720108"*oloniIation"histor+"and"ancestr+"of"northeastern"co+otes."(iol"!ett"6:" 236F237."doi:"10.10B8/rs1l.200B.0822 $2%$on@oldt " (%' " 0ollinger " -0' " ;arl " #A' " .nowles " -*' " (o+>o " AE' " et " al. " 720118 " A " geno&e<wide " perspecti$e " on " the" e$olutionar+"histor+"of"enig&atic"wolf<li>e"canids."Aeno&e"Ees"21:"12B3F1302."doi:"10.1101/gr.116301.110 $9%*ha&1ers","%'"Fain",E'"FaIio"('"A&aral"%"720128"An"account"of"the"ta=ono&+"of"?orth"A&erican"wol$es"fro& " &orphological"and"genetic"anal+ses."?orth"A&"Fauna"77:"1F67."doi:"10.3BB6/nafa.77.0001 $:%Eutledge"!/'")ilson"0-'".lutsch "*F*'"0atterson"(E'")hite "(?"720128 "*onser$ation"geno&ics"in"perspecti$e: "A" holistic " approach " to " understanding " *anis " e$olution " in " ?orth " A&erica. " (iol " *onser$ " 122: " 186F1B2. " doi:" 10.1016/j.1iocon.2012.02.017 ')%)ilson " 0-' " Arewal" ,' " %allor+ " FF' " )hite " (?" 7200B8 " Aenetic "characteriIation "of "h+1rid "wol$es " across " 4ntario. "-" @ered"100:"7,upple&ent"18",80F,8B."#oi:10.10B3/jered/esp033. '$%%ech " !# " 7200B8 " *r+ing " wolf: " concluding " that " wol$es " were " not " restored. " (iol " !ett " 2: " 62F66. " doi: " 10.10B8/rs1l.2008.0330 ''%*ronin"%A'"%ech"!#"7200B8"0ro1le&s"with"the"*lai&"of";cot+pe"and"9a=on",tatus"of"the")olf"in"the"Areat"!a>es " Eegion."%ol";col"18:"3BB1F3BB3."doi:"10.1111/j.1362<2B3=.200B.03331.= '*%6.,."Fish"and")ildlife",er$ice"720138"0roposed"Eule:";ndangered"and"9hreatened")ildlife"and"0lants:"Ee&o$ing" the"Ara+")olf"7Canis lupus8"fro&"the"!ist"of";ndangered"and"9hreatened")ildlife"and"%aintaining"0rotections"for"the " %e=ican")olf"7Canis lupus baileyi8"1+"!isting"5t"as";ndangered."-une"13'"2013. '4%%ech"!#"720118"?on<genetic"data"supporting"genetic"e$idence"for"the"eastern"wolf."?ortheastern"?at"18:"221F 226."doi:"10.1626/032.018.030B '/%.olenos>+"A("71B718"@+1ridiIation"1etween"wolf"and"co+ote."-"%a&&al"22:"336F33B."doi:"10.2307/137868B '1%.olenos>+"A('",tandfield"E4"71B728"%orphological"and"ecological"$ariation"a&ong"gra+"wol$es"7 Canis lupus8"of" 4ntario'"*anada."5n"Fo="%)'"editor."9he")ild"*anids:"9heir",+ste&atics'"(eha$ioral";colog+"and";$olution."?ew"/or>: " Con"?ostrand"Eeinhold."pp."62F72. '2%(au&an"-;'"*lifford"#!'"Asa"*,"720088"0regnanc+"diagnosis"in"wild"canids"using"a"co&&erciall+"a$aila1le"rela=in " assa+."Hoo"(iol"27:"306F313."doi:"10.1002/Ioo.201B6 '9%*arlson"#A'"Aese";%"720078"Eela=in"as"a"diagnostic"tool"for"pregnanc+"in"the"co+ote"7 Canis latrans8."Ani&"Eeprod" ,ci"101:"303F312."doi:"10.1016/j.anireprosci.2006.07.011 ':%(utler " %-' " (allard " )(' " )hitlaw " @A " 720068 " 0h+sical " characteristics' " he&atolog+' " and" seru& " che&istr+ " of " free< ranging"gra+"wol$es'"7Canis lupus8'"in"southcentral"Alas>a."*an"Field"?at"120:"202F212. *)%Fontaine";'"%ir"F'"Cannier"F'"Aerardin"A'"Al1ou+"%'"?a$arro"*'"et"al."720118"5nduction"of"fertile"oestrus"in"the"1itch " using " #eslorelin' " a " AnE@ " agonist. " 9heriogenolog+ " ?o$ " 76: " 1261F1266 " doi:10.1016/j.theriogenolog+.2011.06.031. " ;pu1"2011"Aug"26. *$%$on"@ei&endahl"A'"%iller"*"720128"*linical"e$aluation"of"deslorelin"to"induce"oestrus'"o$ulation"and"pregnanc+"in " the"1itch."Eeprod"#o&est"Ani&"#ec"37",uppl"63B8F3BB"doi:10.1111/rda.12080. *'%Asa"*,'"(au&an".!'"*allahan"0'"(au&an"-'"Col>&ann"#@'"et"al."720068"AnE@<agonist"induction"of"fertile"estrus " with " either " natural " &ating " or " artificial " inse&ination " followed " 1+ " 1irth " of " pups " in " gra+ " wol$es " 7 Canis lupus8." 9heriogenolog+"66:"1778F1782."doi:"10.1016/j.theriogenolog+.2006.01.032 **%%itsuIu>a"%"71B878"*ollection'"e$aluation"and"freeIing"of"wolf"se&en."5n"Fran>'"@."editor."Ad$ances'"5ssues"and " 0ro1le&s"in"*apti$e")olf"Eesearch."(oston:"#r.")."-un>"0u1lishers."pp."127F131. *4%Aier"@9"71B688"*o+otes"in".ansas."%anhattan'".ansas:"(ulletin"3B3'"Agric";=p",tation'".ansas",tate"*ollege"of " Agriculture"and"Applied",cience."118"p.

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