Proposed Protocol for Field Testing of Endurance Fitness of Young Labrador Retrievers

This is a sel -a chi ed e sion o an o iginal a icle. This e sion may di e om he o iginal in pagina ion and ypog aphic de ails. Au ho (s): Ti le: Yea : Ve sion: Copy igh : Righ s: Righ s u l: Please ci e he o iginal e sion: CC BY 4.0 h ps://c ea i ecommons.o g/licenses/by/4.0/ P oposed P o ocol o Field Tes ing o Endu ance Fi ness o Young Lab ado Re ie e s © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land Published e sion Söde lund, Ella-E ika; Ky öläinen, Heikki; Lai inen-Vapaa uo i, Ou i M.; Hyy iäinen, Heli K. Söde lund, E.-E., Ky öläinen, H., Lai inen-Vapaa uo i, O. M., & Hyy iäinen, H. K. (2023). P oposed P o ocol o Field Tes ing o Endu ance Fi ness o Young Lab ado Re ie e s. Me hods and P o ocols, 6(4), 61. h ps://doi.o g/10.3390/mps6040061 2023 Ci a ion: Söde lund, E.-E.; Ky öläinen, H.; Lai inen-Vapaa uo i, O.M.; Hyy iäinen, H.K. P oposed P o ocol o Field Tes ing o Endu ance Fi ness o Young Lab ado Re ie e s. Me hods P o oc. 2023,6, 61. h ps://doi.o g/10.3390/ mps6040061 Academic Edi o : Fe nando Albe icio Recei ed: 25 May 2023 Re ised: 26 June 2023 Accep ed: 27 June 2023 Published: 28 June 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). P o ocol P oposed P o ocol o Field Tes ing o Endu ance Fi ness o Young Lab ado Re ie e s Ella-E ika Söde lund 1, Heikki Ky öläinen 2, Ou i M. Lai inen-Vapaa uo i 1and Heli K. Hyy iäinen 1,* 1Depa men o Clinical Equine and Small Animal Medicine, Facul y o Ve e ina y Medicine, Uni e si y o Helsinki, 00014 Helsinki, Finland; [email p o ec ed] (E.-E.S.); [email p o ec ed] (O.M.L.-V.) 2Neu omuscula Resea ch Cen e , Facul y o Spo and Heal h Sciences, Uni e si y o Jy äskylä, 40014 Jy äskylä, Finland; [email p o ec ed] *Co espondence: [email p o ec ed] Abs ac : The numbe o dogs and, wi h i , dog spo s a e g owing in popula i y, and he aining o dogs begins a an ea ly age. Al hough i ness es ing is an impe a i e pa o pu pose ul aining and spo s, o ou knowledge, no objec i e ield es s a e a ailable o measu ing young dogs’ endu ance i ness. The aim o his s udy is o desc ibe a simple, easy- o- epea , and inexpensi e way o es aining in e en ion e ec s on endu ance i ness in young Lab ado Re ie e s. Heal hy clien - owned 16-week-old Lab ado Re ie e s will be ec ui ed and di ided in o es and con ol g oups. The es g oup will ha e an eigh -week aining p og am ollowed by a ou -week de aining pe iod, while he con ol g oup will li e a no mal puppy li e. All dogs will be es ed o endu ance i ness ou imes a ou -week in e als: a baseline, one mon h la e , wo mon hs la e a he end o he aining pe iod, and one mon h a e ending he aining p og am. Each o he ou es ing sessions will be iden ical and will consis o ou measu emen s o hea a e (HR) and blood lac a e (BL): a baseline, a e o ing 1000 m, a e sp in ing 200 m, and a eco e y 5–8 min a e he sp in . The aining-induced changes in endu ance i ness a e e alua ed by changes in HR and hea a e eco e y imes (HRR), BL, and unning imes. Keywo ds: physical i ness; i ness es ; ae obic i ness; endu ance es ; dog 1. In oduc ion In humans, physical i ness is de ined by he Ame ican College o Spo Medicine (ASCM) as “a se o a ibu es o cha ac e is ics ha people ha e o achie e ha ela es o he abili y o pe o m physical ac i i y” [ 1 ]. Physical i ness is u he di ided in o skill- and heal h- ela ed componen s. One o he heal h- ela ed componen s is ca dio espi a o y endu ance, also known as ae obic i ness o endu ance i ness [ 2 ]. Endu ance i ness e e s o he capaci y o he ci cula o y and espi a o y sys ems o adminis e oxygen o skele al muscles, hus enabling hem o p oduce ene gy du ing physical ac i i y [ 3 , 4 ]. When he e is insu icien oxygen a ailable, anae obic p ocesses (glycolysis) occu o allow o ene gy p oduc ion, and lac ic acid is o med [ 5 ]. Accumula ion o lac a e leads o acidosis, which in u n causes muscle a igue, a enua ing muscle ac ions, and o ce p oduc ion [6]. Ae obic aining has been shown o dec ease es ing hea a e (HR) in humans and ho ses [ 7 – 9 ]. HR moni o ing is used o assess he e ec i eness o aining p og ams and he le el o ca dio ascula i ness, as well as o es ima e indi ec maximal oxygen up ake (VO2max) du ing exe cise and o measu e he in ensi y o physical pe o mance in humans, ho ses, and dogs [ 7 , 10 – 17 ]. HR da a can be compiled wi h an elec oca diog am by auscul a ion a es o wi h wea able HR senso s [ 18 ]. In ield es s, di e en HR senso bel s a e used o measu e HR du ing physical pe o mance [ 16 , 19 , 20 ]. The e a e HR senso s and senso bel s designed speci ically o ho ses; o example, Pola Elec o Oy (Kempele, Finland) has de eloped HR senso s sui able o use unde he saddle while iding and a Me hods P o oc. 2023,6, 61. h ps://doi.o g/10.3390/mps6040061 h ps://www.mdpi.com/jou nal/mps Me hods P o oc. 2023,6, 61 2 o 10 handheld senso o quick HR moni o ing. To he au ho s’ knowledge, he e a e no HR senso s designed o dogs, bu Essne e al. [ 20 , 21 ] ha e alida ed one human HR senso model in dogs. In humans and ho ses, hea a e eco e y ime (HRR) has been hough o indica e changes in i ness le el and aining s a us [ 22 – 26 ]. In eli e human a hle es, HRR is only ela ed o ae obic i ness du ing he i s 10–30 s a e pe o mance [ 25 ]. In non-a hle e humans, HRR has been ound o dec ease signi ican ly a e 8 weeks o aining bu o e u n o almos baseline le els a e 2 weeks o de aining [ 24 ]. Racing ho ses wi h lowe es ing HR be o e exe cise has been shown o ha e as e HRR [ 27 ], which, when no ed a e in e al exe cise, is ela ed o be e ace pe o mance [ 26 ]. Ne e heless, he e is a lack o in o ma ion a ailable ega ding dogs’ HRR and i s ela ion o hei pe o mance capabili ies. One key ma ke o endu ance i ness in humans is he concen a ion o lac a e in he blood ela i e o exe cise in ensi y [ 2 , 28 , 29 ]. Du ing exe cise, he poin whe e he o ma ion o lac a e exceeds he elimina ion o lac a e om he blood is called he lac a e h eshold o anae obic h eshold [ 2 , 29 ]. Physical pe o mance below his poin is conside ed ae obic exe cise [ 2 , 29 ]. Analysis o blood lac a e (BL) can also e lec exe cise in ensi y [ 2 , 30 ]. The e o e, BL analysis is ou inely pe o med in human spo s medicine [ 31 ]. BL analyses a e also used in dogs and ho ses [ 19 , 32 – 35 ]. Fo example, endu ance- ained dogs ha e signi ican ly lowe BL concen a ions a e long-dis ance unning han un ained dogs [ 36 ]. Endu ance- ained dogs ha e been ound o each lac a e h eshold a speeds o 13.9 km/h ( ± 2.9 km/h) [ 35 ]. A po able, minimally in asi e BL measu emen de ice is eadily a ailable o dogs, and i has been shown o be eliable in measu ing hei BL le els [ 37 ]. In his p o ocol, his me hod will be used o young Lab ado Re ie e s. In addi ion o he abo e-men ioned me hods, endu ance i ness can be es ed in humans wi h se e al alida ed ield es ba e ies [ 38 – 43 ]. One o he mos widely used ield es s is he 20-min shu le un es (20 mSRT) [ 39 , 44 ]. In his es , he pa icipan s un a an inc easing pace o 20 m back and o h un il hey can no longe keep up wi h he demanded speed. This es has been shown o be alid and eliable o measu ing ca dio espi a o y i ness in bo h adul s and child en [ 39 , 43 , 45 – 47 ]. The e a e also ime- limi ed es s, such as he 6-min walking es (6 MWT) and he 12-min unning es [ 48 , 49 ], in which a dis ance a eled o e a ce ain pe iod o ime is measu ed. These wo es s a e no eliable and alid o child en, and hus, 20 mSRT should be used [ 50 ]. Ano he op ion is he dis ance-limi ed es , which measu es he ime spen a eling a ce ain dis ance [ 48 , 50 ]. A dog wi h good physical i ness has a be e capabili y o pe o m i s ask, which also educes he isk o inju y; hence, he abili y o measu e he dog’s i ness le el is impe a i e. Wi h he inc easing numbe o pe s and wo king dogs, owne s’ in e es in he well-being o hei dogs is also on he ise [ 51 – 55 ]. P o iding i ness- ela ed in o ma ion o dog owne s can a ec hei dog handling and exe cising habi s and he eby con ibu e o he well-being o dogs. Mo eo e , he popula i y o dog spo s is g owing, and dogs’ aining begins a an ea ly age. This may lead o an inc easing a e o musculoskele al inju ies i he dogs’ physics is no app op ia ely es ed and ained. Field es s o assessing endu ance i ness in dogs a e sca ce. The six MWT is used in non-heal hy dogs o cha ac e ize he se e i y o espi a o y and ca diac diseases [ 6 , 15 , 56 ] and o cha ac e ize golden e ie e muscula dys ophy [ 57 ]. The same es has been applied o sea ch-and- escue dogs oo [ 58 ]. Howe e , he es does no apply o endu ance i ness es ing. Ano he i ness es o assessing dogs’ physical i ness has been published [ 59 ], bu his es does no include an objec i e measu emen o endu ance i ness; ins ead, i concen a es on a ious aspec s o locomo ion. Despi e i ness es ing being an impo an pa o pu pose ul aining and spo s in o he species, he e a e cu en ly no ield es s a ailable o es ing young dogs’ endu ance i ness. In e e ina y medicine, cu en ield es s o measu ing endu ance i ness a e designed o ho ses [8,9,16,60]. Me hods P o oc. 2023,6, 61 3 o 10 The aim o his s udy is hus o design a simple, easy- o- epea , and inexpensi e ield es o measu e he e ec s o a aining in e en ion on endu ance i ness in young dogs. This p o ocol will no only be use ul o e e ina y medicine p o essionals bu is also sui able o be used by non-p o essionals (i.e., owne s and dog aine s). 2. P ocedu e P i a ely-owned Lab ado Re ie e sibling pai s aged 16 weeks wi h no known heal h issues and wi h a heal hy gene ic backg ound based on he Finnish Kennel Club heal h eco ds will be ec ui ed. Exclusion c i e ia will be any de ec ed abno mali ies in lameness assessmen o in he gene al examina ion. These assessmen s will be epea ed a each es session igh be o e physical pe o mance. In addi ion, he dogs will be unde he e e ina ian’s supe ision a all imes. All dog owne s will sign a w i en in o med consen o m be o e pa icipa ing in he s udy. Owne s will be ad ised ha hey may cancel hei pa icipa ion a any ime. Fu he mo e, he e e ina ian can wi hd aw he dog o any jus i ied eason (i.e., heal h- ela ed conce ns, owne - ela ed beha io al issues, e c.). In he e en o wi hd awal, any esea ch da a collec ed up o ha poin will be used. App o al o he s udy p o ocol will be sough om he Regional S a e Adminis a i e Agency o Sou he n Finland. The dogs will be di ided in o wo g oups: om each sibling pai , one o he siblings will be in he aining g oup, and he o he in he con ol g oup. The dogs will be andomly assigned o hese g oups. The aining g oup will ha e an eigh -week aining p og am, including exe cises on an unde wa e eadmill and a home exe cise p og am, and a de aining pe iod o ou weeks a e inishing he aining p og am. The con ol g oup will li e a no mal puppy li e, i.e., puppy managemen will be conduc ed acco ding o each indi idual owne ’s p e e ences, wi h no es ic ions o added exe cises ela ed o esea ch. Bo h g oups will unde go he same ou come measu emen s a he same ime poin s (desc ibed below), and o bo h g oups, he s udy pe iod will las a o al o 12 weeks. 2.1. S udy P o ocol The es will be pe o med in an indoo hall wi h a con olled en i onmen and empe a u e (15–20 ◦ C) and on a good, non-slip su ace (Agili y So ex, Lappse , Helsinki, Finland) o ensu e a s anda dized es ing en i onmen . P io o each es session, he dog will be aken ou o a calm walk as a wa m-up and o allow i o de eca e and u ina e. The dog will be allowed o acclima e o i s su oundings o 5–15 min be o e he es ing. The leng h o he acclima ion will be based on clinical expe ience. The ime o a es session will emain consis en h oughou he s udy. The measu emen s o he sibling pai will be conduc ed consecu i ely on he same day. The es p o ocol is pe o med in he same way each ime (Figu e 1). A clinical examina ion and lameness e alua ion will be conduc ed p io o e e y es session, igh be o e pe o mance. Lameness e alua ion will be conduc ed by palpa ion and manipula ion as well as gai obse a ion o any indica ions o unsoundness. Each o he i ness es sessions will consis o wo physical pe o mance se s and ou measu emen s o HR and BL (Figu e 2). The physical pe o mance will be e alua ed by o ing o 1000 m and a sp in es o 200 m. Du ing he “ o ” phase, a unning o will be u ilized, de ined wi h a unning gai wi h a suspension phase be ween each diagonal suppo phase. Du ing o , he dog will be mo ing o wa d wi h ela i e ease a a cons an “jogging” speed. Du ing he “sp in ” phase, he dog will be conside ed o sp in when i is galloping. Bo h o a o y and ans e se gallops will be accep ed. Du ing a sp in , he dog will be mo ing o wa d as as as i seems o be able o do so, wi h he aim o cons an ly inc easing o main aining he op speed ha i can be mo i a ed o achie e. HR and BL measu emen s will be aken be o e physical pe o mance es s (baseline, BASE), a e o ing o 1000 m (POST1), a e sp in ing o 200 m (POST2), and a eco e y (REC) 5–8 min a e he las un. These measu emen s will ake abou 10 min o comple e. All measu emen s will be eco ded on he es o m (Supplemen a y Figu e S1). Me hods P o oc. 2023,6, 61 4 o 10 Me hodsP o oc.2023,6,xFORPEERREVIEW4o 11   a e  o ing o 1000m(POST1),a e sp in ing o 200m(POST2),anda  eco e y(REC) 5–8mina e  helas  un.Thesemeasu emen swill akeabou 10min ocomple e.All measu emen swillbe eco dedon he es  o m(Supplemen a yFigu eS1). Figu e1.Thes uc u eo  heendu ance i ness es p ocedu e.Thep ocedu ewillbeca iedou in hesameway o each es session.HR=hea  a e,BL=bloodlac a e,App=Pola EquineApp.  Figu e2.Thes uc u eo  he i ness es session.Eacho  he i ness es sessionswillconsis o  ou  measu emen so hea  a e(HR)andbloodlac a e(BL):baselinea  es (BASE),a e  o ing1000 m(POST1),a e sp in ing200m(POST2),and eco e yo 5–8mina e  helas  un(REC). Runningdis anceswillbemeasu edwi ha ange inde andma kedclea lywi h cones.Du ing he es , hedogwillwea anon-slipcolla o ha ness,andi willha ewa e  a ailablea e  he es .Eachdogwilluse hei ownindi idually i ed es ain s(i.e.,ha - nesso colla andlead).App op ia e i willbecon i medby hepe son esponsible o  es ing.In hecaseo inapp op ia egea ,anindi iduallyadjus ablecolla willbep o- idedby he esea ch eam. The es willbein e up edi  hedogbecomeslameo showsanysignso excessi e a igue.Signso low-le el a igueinclude, o example,sudden eluc ance omo eo ex- cessi epan ing[35,37].Signso se e e a igueincludedogsgoingaw yo collapsing [35,37].As hedogsin hiss udywillbeyoung, he es canno becon inuedun ilse e e signsa eno iced,bu  he es mus bediscon inueda low-le elsigns.Thesignswillbe obse edby heowne aswellas he e e ina ianand heassis an (a e e ina ynu se), all amilia wi h heb eed. Thedogswill o alongside hei owne o anassis an  o 1000m.I acons an  o  ismain ained, hespeedo  he o willno becon olled.A e  o ing o 1000m, he dogwillbes oppedandPOST1measu emen swillbepe o med.Immedia ely,HRwill be eco ded om hepulsemoni o ’smobileapplica ion(app),andblood om heinne  Gene al examina ion O hopedic assessmen Connec ing he HR senso o App Placing he HR senso Ensu ing he igh place o HR senso wi h palpa ion Con i ming he HR in he App is cong uen wi h he auscul a ion Measu ing BL T o ing 1000 m Reco ding o HR Measu ing BL Sp in ing 200 m Reco ding HR Measu ing BL Res o 5–8 min, eco ding HR all he ime Measu ing BL a e 5–8 min es Figu e 1. The s uc u e o he endu ance i ness es p ocedu e. The p ocedu e will be ca ied ou in he same way o each es session. HR = hea a e, BL = blood lac a e, App = Pola Equine App. Me hodsP o oc.2023,6,xFORPEERREVIEW4o 11   a e  o ing o 1000m(POST1),a e sp in ing o 200m(POST2),anda  eco e y(REC) 5–8mina e  helas  un.Thesemeasu emen swill akeabou 10min ocomple e.All measu emen swillbe eco dedon he es  o m(Supplemen a yFigu eS1). Figu e1.Thes uc u eo  heendu ance i ness es p ocedu e.Thep ocedu ewillbeca iedou in hesameway o each es session.HR=hea  a e,BL=bloodlac a e,App=Pola EquineApp.  Figu e2.Thes uc u eo  he i ness es session.Eacho  he i ness es sessionswillconsis o  ou  measu emen so hea  a e(HR)andbloodlac a e(BL):baselinea  es (BASE),a e  o ing1000 m(POST1),a e sp in ing200m(POST2),and eco e yo 5–8mina e  helas  un(REC). Runningdis anceswillbemeasu edwi ha ange inde andma kedclea lywi h cones.Du ing he es , hedogwillwea anon-slipcolla o ha ness,andi willha ewa e  a ailablea e  he es .Eachdogwilluse hei ownindi idually i ed es ain s(i.e.,ha - nesso colla andlead).App op ia e i willbecon i medby hepe son esponsible o  es ing.In hecaseo inapp op ia egea ,anindi iduallyadjus ablecolla willbep o- idedby he esea ch eam. The es willbein e up edi  hedogbecomeslameo showsanysignso excessi e a igue.Signso low-le el a igueinclude, o example,sudden eluc ance omo eo ex- cessi epan ing[35,37].Signso se e e a igueincludedogsgoingaw yo collapsing [35,37].As hedogsin hiss udywillbeyoung, he es canno becon inuedun ilse e e signsa eno iced,bu  he es mus bediscon inueda low-le elsigns.Thesignswillbe obse edby heowne aswellas he e e ina ianand heassis an (a e e ina ynu se), all amilia wi h heb eed. Thedogswill o alongside hei owne o anassis an  o 1000m.I acons an  o  ismain ained, hespeedo  he o willno becon olled.A e  o ing o 1000m, he dogwillbes oppedandPOST1measu emen swillbepe o med.Immedia ely,HRwill be eco ded om hepulsemoni o ’smobileapplica ion(app),andblood om heinne  Gene al examina ion O hopedic assessmen Connec ing he HR senso o App Placing he HR senso Ensu ing he igh place o HR senso wi h palpa ion Con i ming he HR in he App is cong uen wi h he auscul a ion Measu ing BL T o ing 1000 m Reco ding o HR Measu ing BL Sp in ing 200 m Reco ding HR Measu ing BL Res o 5–8 min, eco ding HR all he ime Measu ing BL a e 5–8 min es Figu e 2. The s uc u e o he i ness es session. Each o he i ness es sessions will consis o ou measu emen s o hea a e (HR) and blood lac a e (BL): baseline a es (BASE), a e o ing 1000 m (POST1), a e sp in ing 200 m (POST2), and eco e y o 5–8 min a e he las un (REC). Running dis ances will be measu ed wi h a ange inde and ma ked clea ly wi h cones. Du ing he es , he dog will wea a non-slip colla o ha ness, and i will ha e wa e a ailable a e he es . Each dog will use hei own indi idually i ed es ain s (i.e., ha ness o colla and lead). App op ia e i will be con i med by he pe son esponsible o es ing. In he case o inapp op ia e gea , an indi idually adjus able colla will be p o ided by he esea ch eam. The es will be in e up ed i he dog becomes lame o shows any signs o excessi e a igue. Signs o low-le el a igue include, o example, sudden eluc ance o mo e o excessi e pan ing [ 35 , 37 ]. Signs o se e e a igue include dogs going aw y o collapsing [ 35 , 37 ]. As he dogs in his s udy will be young, he es canno be con inued un il se e e signs a e no iced, bu he es mus be discon inued a low-le el signs. The signs will be obse ed by he owne as well as he e e ina ian and he assis an (a e e ina y nu se), all amilia wi h he b eed. The dogs will o alongside hei owne o an assis an o 1000 m. I a cons an o is main ained, he speed o he o will no be con olled. A e o ing o 1000 m, he dog will be s opped and POST1 measu emen s will be pe o med. Immedia ely, HR will be eco ded om he pulse moni o ’s mobile applica ion (app), and blood om he inne side o he ea lap will be d awn o assess he BL le el. HR will also be measu ed wi h a s e hoscope i he e is suspicion o senso - o-app connec ion ailu e (no ed as an excep ionally low o high HR o no HR). The es will con inue immedia ely a e he measu emen s wi h a 200 m sp in , unning as as as possible. A e sp in ing he 200 m, he dog will again be s opped, and HR and BL measu emen s will be epea ed (POST 2). Me hods P o oc. 2023,6, 61 5 o 10 Once he POST2 measu emen s a e conduc ed, he dog will be encou aged o lie down and ha e a es , wi h no s imulus p o ided. This will be accomplished h ough e bal and non- e bal ins uc ions and he dog’s mo i a ion. The dog needs o lie down as i needs o calm down and be elaxed. Once he dog is calm, nei he he owne no he esea ch pe sonnel will speak o i . A e 5–8 min, HR and BL will be measu ed again (REC). The HR is moni o ed h ough he app so ha he dog will no be dis u bed. HR will be moni o ed un il i is back o he base alue. The ime aken o achie e he le el o es ing HR will be eco ded and conside ed HRR. The unning speeds o he 1000 m o and 200 m sp in will be ob ained om he app. 2.2. Desc ip ion o Measu emen Technique All he i ness es measu emen s will be conduc ed immedia ely, which means wi hin a ew seconds a e exe cise. Du ing he measu emen s, an assis an will hold he dog. In cases o beha io al easons (i.e., a sensi i e dog), he owne can handle he dog. In his case, owne s will be ins uc ed o handle he dogs in a s anda dized manne o ensu e p ope and consis en managemen o he dogs h oughou he es and hus o e i y he accep able epea abili y o he p o ocol. I will be allowed o s and o si bu no o lie down, excep o he REC measu emen s, when he dog will be encou aged o lie down. The HR o he dog will be eco ded by auscul a ion wi h a s e hoscope placed on he le side o he dog a he i h o six h in e cos al space. The hea will be auscul a ed o 15 s. The esul ob ained will be mul iplied by ou o ob ain bea s pe minu e (bpm). The esul will be e e ed o as he baseline HR. Auscul a ion will be conduc ed p io o placing he HR senso , which will be se o he same, abo e-desc ibed place. The HR senso (Pola H10 senso , Pola Elec o Oy, Kempele, Finland, Ca . No.: 92075957) will be a ached o i s ches s ap. To imp o e con ac be ween he ches s ap and he skin, he dog’s coa will be wa e ed on he le side o he dog a he i h o six h in e cos al space, and e e ina y lub ican gel (K uuse Bo i e Gel, K uuse, Langesko , Denma k) will be applied o he measu emen a ea o he ches s ap. I will hen be a ached o he dog’s ches . The co ec place o he HR senso will be con i med by palpa ing he hea h ough he senso . I needed, he dog is allowed o acclima e o he ches s ap o up o 15 min. P io o a aching he HR senso o he dog, he senso will be connec ed o an app (Pola Equine app, e sion 1.2.1, Pola Elec o Oy, Kempele, Finland), which can be downloaded om Google Play (Google I eland Limi ed, Dublin, I eland) o a sma phone. Fo he app o wo k, he de ice mus be an And oid phone wi h an ope a ing sys em o 6.0 o newe , and he phone mus be a leas Blue oo h 4.0 compa ible. The app will display HR in eal- ime, so HR can be moni o ed om a dis ance wi hou dis u bing he dog. The app will also collec da a on unning speed and dis ance. The e ec s o a ious ac ions du ing he es (such as aking blood samples o BL measu emen s) on he HR can be seen h ough he con inuous collec ion o HR da a. A e a aching he HR senso o he dog, he applica ion is con i med by auscul a ing he HR simul aneously. While he owne o assis an holds he dog, he inne su ace o he dog’s ea lap will be wiped wi h an an isep ic and allowed o d y o 5–10 s. I he ea lap is s ill we , i can be wiped wi h a clean, d y gauze. A s e ile 22-gauge needle will be used o make a small punc u e on he inne su ace o he ea lap. A blood d op will be d awn and applied immedia ely o a eac an s ip placed on a handheld lac a e analyze (Lac a e Scou 4, SensLab Gmbh, Leipzig, Ge many, Ca . no.: 7023-0441-0246) o de e mine BL. 3. Expec ed Resul s The sample size calcula ions will employ esul s o p e iously published simila s udies [ 6 , 15 , 56 – 58 ] and equi ed es ima es based on clinical expe ience. A s a is ician specialized in medical esea ch will be consul ed in he assessmen o he equi ed numbe o dogs. Powe analysis wi h a leas 80% powe will be used wi h a s a is ical signi icance le el o p= 0.05. Me hods P o oc. 2023,6, 61 6 o 10 P ima y ou come a iables o he es p o ocol will be changes in BL, HR, and HRR induced by an eigh -week aining in e en ion. Changes in hese pa ame e s will be e alua ed by compa ing measu emen s a baseline, a e he in e en ion, and a e he de aining pe iod. Main enance o he changes will be assessed by measu ing he pa am- e e s a he end o he in e en ion and a e a ou -week de aining pe iod. Desc ip i e s a is ics will be calcula ed o he measu ed HR, BL, and HRR alues. Fo BL, change om POST2 a REC will also be calcula ed. The s a is ical calcula ions will be pe o med using SAS so wa e e sion 9.4 (SAS Ins i u e Inc., Ca y, NC, USA). 4. Discussion Al hough no es s a e a ailable o young dogs, some i ness es s o adul dogs ha e been published [ 58 , 59 , 61 – 63 ]. Mos o he published i ness es s a e labo a o y ypes pe o med on a eadmill, o i hey a e ac ual ield es s, hey do no speci ically measu e endu ance i ness bu ocus mo e on o e all physical i ness [ 58 , 59 ]. One s udy has assessed he eliabili y o a eadmill exe cise es agains BL, HR, and body empe a u e esponses o inc emen al exe cise in dogs [ 61 ]. This es o i s modi ica ions ha e been used in o he s udies measu ing endu ance i ness in dogs [ 61 – 63 ]. Howe e , eadmills may no be eadily a ailable o e e yone needing o es dogs’ endu ance i ness. Thus, es s wi hou eadmills could be mo e easible, al hough hey migh ha e hei challenges. Fo example, in ou s udy, as he puppies g ow, hei unning speed du ing sp in s migh be oo as o a human o keep up wi h. The e o e, wi hou a eadmill, he p oposed p o ocol would no be sui able o adul dogs. In ou p oposed p o ocol, he dis ances o be o ed and sp in ed a e chosen acco ding o he bes clinical knowledge om bo h human and e e ina y medicine. In humans, he e a e no s udies o uni ied opinions abou a sui able unning dis ance o child en. Acco ding o Finley e al. [ 64 ], mos o he child en’s c oss-coun y coaches conside a sui able unning dis ance in compe i ions o 6- o 9-yea -olds o be 800–1600 m. Based on clinical expe ience and common mu ual unde s anding, a sui able unning dis ance o young dogs is highly dependen on he b eed, bu a puppy o a non-chond odys ophic b eed should be able o un o a leas 5 min pe mon h o age, wo imes a day. In ou p o ocol, o ing o 1000 m ep esen s ae obic pe o mance, and sp in ing o 200 m ep esen s anae obic pe o mance. A challenge o hese unning dis ances may be ha hey a e oo sho o ele a e a young dog’s HR and BL. In humans, nume ic ables a e used in ield es s o quan i y endu ance i - ness [ 48 , 49 , 65 , 66 ]. These ables ac o in he ime used o he pe o mance o dis ance co e ed in a ce ain ime [ 48 , 49 , 65 , 66 ]. In ho ses, ypically measu ed physical pa ame- e s in i ness ield es s a e HR, HRR, and BL [ 26 , 27 , 67 , 68 ]. These also apply o canine endu ance es s pe o med on a eadmill, wi h he addi ion o body empe a u e as a ypically measu ed a iable [ 36 , 59 , 69 , 70 ]. In ou p oposed p o ocol, BL, HR, and HRR will be used o e alua e he changes in endu ance i ness. We will no use body empe a u e because wi h young dogs i migh ake oo much ime o measu e be- ween he ae obic and anae obic pe o mances, gi ing he dogs oo much es be ween pe o mance se s. A speci ic HR senso , o iginally designed o humans, has also been alida ed o dogs [ 20 , 21 ]. Howe e , his pa icula de ice is no longe a ailable, and he e o e, he Pola H10 senso will be used in his es . Wi h his HR senso and he Pola Equine app, HR as well as unning imes and speeds can be measu ed. Possible challenges may be ha he HR senso bel a ound he dog’s ches can dis u b he dog while unning. Also, i he dog has a e y hick coa o is e y obese, he con ac o he HR senso bel migh be insu icien . I possible, hai could be sha ed om he senso a ea, bu his is no obliga o y. Al hough sha ing hai would imp o e senso con ac , as he dogs included in he s udy will be p i a ely owned, he decision on clipping he hai is he owne ’s decision. Mo eo e , manda o y hai clipping migh a ec he owne ’s willingness o use he HR senso bel and hus he whole es . Me hods P o oc. 2023,6, 61 7 o 10 The e a e only a ew HRR- ela ed s udies in animals, and mos o hem ha e been conduc ed in ho ses [ 26 , 27 , 60 ]. In his s udy, we will use HRR as a pe o mance indica o in endu ance es ing because i is a simple, inexpensi e, and non-in asi e me hod. The beha io o young dogs may a y ma kedly, and i he dog is e y sensi i e, i migh also ha e an in luence on he HRR. Fo BL measu emen s, a handheld Lac a e Scou 4 analyze will be used in ou s udy. Lac a e Scou analyze measu emen s ha e been shown o be compa able wi h labo a o y me hods [ 34 , 37 ]. The analyze is simple, po able, and apid, making i ideal o ield use. In addi ion o he esul s being eadable in 10 s, only a e y small amoun o blood (0.5 µ L) is needed o he sample. The analyze also has a wide ange o measu emen s (0.5–25.0 mmol/L) (14). In he p o ocol p oposed he e, he blood sample will be aken om he inne su ace o he ea lap, bu i his is no possible, he sample can be aken om he me aca pal pad. In human medicine, physiological esponses o exe cise a e known o change du ing g ow h and de elopmen , and hus, pedia ic es s ha e hei own ou come e e ence alues ins ead o sha ing he adul no ms [ 71 , 72 ]. The musculoskele al sys em o young dogs is somewha di e en om ha o an adul dog. The bigges di e ences a e seen in he unc ions o he hea and ci cula o y sys em. In young dogs, he hea size is la ge in ela ion o he body weigh han in adul dogs [ 73 ]. On a e age, g owing dogs ha e lowe ed blood cell, hemoglobin, and hema oc i alues han adul dogs, which weakens he anspo o oxygen in he bloods eam [ 74 , 75 ]. The e o e, he esul s o i ness es s on young dogs a e no compa able o hose o adul dogs. To he au ho s’ knowledge, he e a e no published a icles abou i ness es ing in young dogs. The p oposed p o ocol o ield es ing he endu ance i ness o young dogs is use ul because he equipmen and en i onmen needed o he es a e inexpensi e and eadily a ailable, and he p o ocol is non-in asi e. Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a : h ps: //www.mdpi.com/a icle/10.3390/mps6040061/s1, Supplemen a y Figu e S1, Fo m o use in eco ding measu emen esul s. Au ho Con ibu ions: Concep ualiza ion, E.-E.S., H.K., O.M.L.-V. and H.K.H.; w i ing—o iginal d a p epa a ion, E.-E.S.; w i ing— e iew and edi ing, E.-E.S., H.K., O.M.L.-V. and H.K.H.; isualiza- ion, E.-E.S. and H.K.H.; supe ision, H.K., O.M.L.-V. and H.K.H.; p ojec adminis a ion, E.-E.S. and H.K.H.; unding acquisi ion, H.K.H. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This esea ch was unded by he Academy o Finland, g an numbe 315673. Open access unding p o ided by Uni e si y o Helsinki. Ins i u ional Re iew Boa d S a emen : The animal s udy p o ocol was app o ed by he Ins i u ional Re iew Boa d o Regional S a e Adminis a i e Agency o Sou he n Finland (ESAVI/11887/2019, da e o app o al 14 May 2019). In o med Consen S a emen : No applicaple. Da a A ailabili y S a emen : No new da a we e c ea ed o analyzed in his s udy. Da a sha ing is no applicable o his a icle. Con lic s o In e es : The au ho s decla e no con lic o in e es . 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