Departament de Ciència Animal i dels Aliments, Facultat de Veterinària, Universitat Autònoma de Barcelona, Bellaterra, Spain;

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1 Articl Modlling for th inhritanc of ndangrd quid multipl births and frtility: dtrmining risk factors and gntic paramtrs in donkys (Equus asinus) Francisco Javir Navas Gonzálz 1,6, Jordi Jordana Vidal 2,6, Amy Kathrin McLan 3,6, Jos Manul Lón Jurado 4,6, Ccilio José Barba Capot 5,6, Andr Arando Arbulu 1,6, Juan Vicnt Dlgado Brmjo 1,6 1 Dpartmnt of Gntics, Faculty of Vtrinary Scincs, Univrsity of Córdoba, Spain; fjng87@hotmail.com; andrarando@hotmail.com; juanviagr218@gmail.com 2 Dpartamnt d Ciència Animal i dls Alimnts, Facultat d Vtrinària, Univrsitat Autònoma d Barclona, Bllatrra, Spain; jordi.jordana@uab.cat 3 Dpartmnt of Animal Scinc, Univrsity of California Davis, Davis, CA, USA; acmclan@ucdavis.du 4 Cntro Agropcuario Provincial d Córdoba, Diputación Provincial d Córdoba, Córdoba, Spain; jomaljur@yahoo.s 5 Dpartmnt of Animal Production, Faculty of Vtrinary Scincs, Univrsity of Córdoba, Spain; cjbarba@uco.s 6 Th Worldwid Donky Brds Projct, Faculty of Vtrinary Scincs, Univrsity of Córdoba, Córdoba, Spain; worldwiddonkybrdsprojct@hotmail.com * Corrspondnc: fjng87@hotmail.com; Tl.: Simpl Summary: Th consquncs of multipl births in quids compromis th lif of th dam and rsulting offspring. Howvr, in ndangrd spcis, thos individuals pron to dvlop multipl ovulations rsult in th collction of mor frtilisd mbryos from th oviduct, what incrass th productivity and profitability of mbryo prsrvation tchniqus. Embryo prsrvation is spcially important in highly ndangrd quin populations such as crtain donky (Equus asinus) brds; for which convntional rproductiv tchniqus hav prviously faild. For instanc, bcoming an ffctiv altrnativ to artificial insmination with frozn smn to prsrv th individuals gntic matrial. Th prsnt rsults nabl a bidirctional slction stratgy. On on hand, th possibility of dvloping and maintaining spcializd lins rlying on th ability of particular donkys to dvlop multipl births, hnc, incrasing th productivity of assistd rproduction tchniqus for th consrvation of ndangrd spcis. On th othr hand, whn mbryo collction is not th purpos aimd at, slction could focus on th obtntion of thos individuals that may b lss pron to dvlop multipl births, thus, avoiding th risks of multipl gstations, what in th nd translats in th improvmnt of th rproductiv wlfar of th individuals. Abstract: Multipl births or twinning in quids ar dangrous, undsirabl situations that compromis th lif of th dam and rsulting offspring. Howvr, mbryo vitrification and frzing tchniqus tak advantag of individuals whos multipl ovulations allow flushing mor frtilisd mbryos from th oviduct to b collctd, incrasing th productivity and profitability of such tchniqus. Embryo prsrvation is spcially important in highly ndangrd populations such as crtain donky (Equus asinus) brds; for which convntional rproductiv tchniqus hav prviously faild. For instanc, bcoming an ffctiv altrnativ to artificial insmination with frozn smn to prsrv th individuals gntic matrial. Th objctiv of this study was to xamin th historical foaling rcords of Andalusian donkys to stimat gntic paramtrs for multipl births, assssing th historical foal numbr born pr animal, maximum foal numbr pr birth and multipl birth numbr pr animal. W dsignd an Animal Modl with singl rcords considring th fixd ffcts of birthyar, birth sason, sx, farm, and husbandry systm, and ag as 2018 by th author(s). Distributd undr a Crativ Commons CC BY licns.

2 a linar and quadratic covariat. Rstrictd maximum liklihood rportd hritability stimats ranging from 0.18±0.01 to 0.24±0.01. Gntic and phnotypic corrlations rangd from 0.01±0.01 to 0.83±0.01 and 0.12±0.01 and 0.53±0.01, rspctivly. Ths stimats nabl th potntial for slction against/for ths traits, offring a nw prspctiv for donky brding and consrvation. Kywords: Donky; brding; twinning; hritability; REML; risk factors Introduction Th occurrnc of multipl births has bn addrssd on of th principal causs of ftal and nonatal loss in quids [1]. Th majority of twin prgnancis in horss (73%) trminats in abortion or stillbirth of both twins from ight months to trm. Th liklihood of on or both twin foals to b born aliv or to surviv aftr birth complications rducs to around 11%. Th foals ar usually born stuntd or maciatd, what dos not allow thm to surviv furthr from 2 wks of ag [1]. In th cas of th donky spcis, Quarsma t al. [2] addrssd th ovrall nonatal mortality for th first month of lif to b of nar 9%. Ths authors would also rport that th prcntag of twin foaling at full trm was only around 3%, with a nonatal foal mortality rat of 40%. Hnc, th slction of individuals that may b lss pron to prsnt multipl ovulation could b a prvntiv altrnativ to dcras th risks attachd. By contrast, th donky is a spcis for which th most of its brd populations hav bn classd as ndangrd [3] and that has bn rportd to b highly rproductivly compromisd as it happns to many othr ndangrd populations [4] what may b attributd to th dltrious ffcts of inbrding in such populations [5]. Th long gstation cycl (a norm of 12 months to giv birth in th 13 th month [6]), a frtility that stadily dcrass ovr gnrations [2] and th highly inbrd status of donky brd populations [5, 7] only contribut to worsning th ndangrmnt risk situation that donky brds fac worldwid. Furthrmor, highly standardisd rproduction tchniqus in horss and othr quids [8] such as artificial insmination with frozn smn and mbryo transfr still rprsnt a challng in donkys [9-11]. Undr this contxt, mbryo vitrification and frzing aris as nw possibilitis that may nabl th prsrvation of th gntic matrial of donkys blonging to populations for which th numbrs rarly xcd 1000 individuals. This is supportd as th prgnancy rats of 50% and 36% aftr th transfr of frsh and vitrifid mbryos, rspctivly [12], ovrcom th bst currntly rportd rsults for prgnancy rat (28%) obtaind for utrin horn insmination using frozn-thawd smn [13]. Th fficincy of such rproductiv tchniqus could b improvd rlying on th highr ability of crtain animals to dvlop multipl ovulations, vn mor, whn thos animals may b gntically pron to dvlop thm mor oftn. Studis of th gntic background of multipl prgnancis ar ancdotal as frtility, in gnral, has a vry low hritability as common to othr rproductiv traits. Ths studis ar vn mor limitd whn w focus on studying quids such as horss [14, 15] or donkys, for which no study has bn rportd. This was a rtrospctiv study ovr a priod of 38 yars (th birth yar of th oldst animal rgistrd in th studbook was 1980) aimd to invstigat th occurrnc of multipl prgnancis in th historical population of Andalusian donkys and th influnc that non-gntic factors such as farm, husbandry systm, yar of birth, birth sason, birth month or ag may hav had on th prvalnc of ths multipl gstations. Scond, w stimatd th gntic paramtrs of frtility and multipl births through th analysis of historical foal numbr born pr animal, maximum foal numbr pr birth and multipl birth numbr pr animal using REML mthods. Last, w stimatd prdictd brding valus for all th traits as a way to assss th potntial implmntation of a bidirctional brding schm. This schm may simultanously consist of animals slctd against multipl births bcaus of th gstation complications that thy involv, whil othr individuals may promot th

3 occurrnc of multipl births, sking highr consrvation profitability basd upon an incrasd numbr of mbryos to collct whil implmnting assistd rproduction plans. 2. Matrials and Mthods 2.1. Sampl siz and background W studid th foaling rcordings of 765 individuals rgistrd in th historical pdigr rcord of th Andalusian donky brd (181 jacks and 584 jnnis). As ag rang was not normally distributd (P 0.01 for both Kolmogorov-Smirnov and Shapiro-Wilk s tsts for normality), w usd minimum, Q1, mdian, Q3 and maximum to dscrib th ag rang in our sampl. Minimum ag in th rang was six months, Q1 ag was six yars, mdian ag was tn yars, Q3 ag was 14 yars, and th maximum ag was 29 yars. Such wid ag rang was considrd, givn th fact that w assss an ndangrd brd from which th information blonging to ach individual is indispnsabl, using Rstrictd Maximum Liklihood REML mthods and aftr including ag of birth in our modl to corrct for th cass in which th animals wr too young to hav givn birth to thir first foal/s. Th youngst ag at which both jacks and jnnis gav birth for th first tim was thr and four yars old, rspctivly [5]. It is oftn a dcision of ownrs in particular not to brd th animals until thy hav bn rcognisd as apt for rproduction and includd in th studbook of th brd what taks plac whn th animals turn 3 yars old. Th donkys in th sampl wr th progny of 93 jackstocks and 253 jnnis. All th donkys wr rgistrd in th brd s Spanish studbook. Th rlationships in th pdigr of th brd ar routinly gntically tstd through microsatllit-assistd gnotyping and parntag tsts for th rsulting offspring of ach mating. Parntag tsts for ach offspring had prviously bn prformd with microsatllit molcular markrs to nsur th rliability of th information in th pdigr as a way to countract th small siz of th sampl tstd. Th DNA usd for parntag tsts was obtaind from hair sampls that ar routinly takn whn th inscription of ach nw animals taks plac and from th historical bank of sampls of th brd kpt at th laboratory of applid molcular gntics of th Univrsity of Córdoba. All tsts wr carrid out using a pdigr fil providd by th Union of Andalusian Donky Brdrs (UGRA). Th pdigr fil includd 1017 animals (272 mals and 745 fmals) born btwn January 1980 and July 2015 from which only 914 donkys, 246 mals, and 668 fmals, wr aliv during th dvlopmnt of th study. Th pdigr of th donkys in our sampl was tracd back six gnrations providing indirct information from 930 connctd ancstors (91% of th historical population rgistrd) and rporting an avrag inbrding of 0.7% Birth rlatd traits First, w studid multipl birth occurrnc assssing thr diffrnt traits for ach jack or jnny. To obtain this information, w contrastd th rgistris of th pdigr fil with intrviws with th 145 ownrs whos animals participatd in th study. W dcidd to intrviw th ownrs as du to th arly abortion of multipl gstations it is vry likly for thm not to b includd in th pdigr. Scond, from this initial sampl of ownrs, w only considrd th ons who affirmativly rspondd to th qustion in block 2 for th stimation of gntic paramtrs (90 out of 145 ownrs intrviwd) as a vtrinarian or thriognologist had issud an official gstation diagnosis (simpl or multipl). This xcluding critrion was applid as a way to considr thos cass whn abortions had occurrd. Many twin (and triplt) prgnancis in quids ar alrady lost at vry arly stags and th abortd matrial stays mostly undtctd by th ownrs, what could hav distortd th tru numbr of prgnancis with multipl concptuss. First, w summarizd th historical foal numbr born pr animal. That is for th total of 765 individuals, th numbr of offspring foald (rsulting from natural mating or artificial insmination) by ach of 584 jnnis or born to ach of 181 jacks, ithr ovr thir rproductiv liftim or up to July

4 Scond, th maximum foal numbr pr birth, or th maximum numbr rsulting at any of all th dlivris through th lif of ach jnny, considring which jack was usd to brd. That is, for th sam 765 animals, th maximum numbr of offspring born in a singl foaling vnt in which th individual (mal or fmal) was part of ithr ovr its rproductiv liftim or up to July Third, multipl birth numbr pr animal, that is for th sam 765 animals, th sum of all mating vnts rsulting in multipl gstations ithr ovr th rproductiv liftim of th individual (mal or fmal) or up to July Th units of study considrd for dscriptiv statistics and populational data wr th births occurring in th Andalusian donky population and thir charactristics. Hnc, births wr only computd onc pr coupl of brding animals to avoid rdundancis, instad of computing thm twic (on pr jack and on pr jnny involvd). Howvr, for gntic analyss, th unit of analysis that w considrd was th liftim parntship rcord of ach animal sparatly. That is to say; w summd vry molcularly confirmd jack and jnny s birth rgistris sparatly as a singl valu for ach so that for th data considrd rliabl and irrdundant. Givn th BLUP mthodology was applid [16], data can ithr blong dirctly from fild obsrvations and rgistris or indirct, whr individuals ar dirctly gnalogically linkd to common individuals Intrviw dscription A tlphon survy was carrid out to 145 diffrnt ownrs whos farms wr locatd in Andalusia (southrn Spain). Th survy took plac in Jun W intrviwd ownrs rgarding th spcific foaling rgistry of all th animals historically prsnt at thir farms sinc th 1980s until 2017 and rgistrd in th stud-book of th brd at th momnt that th survy took plac. Th oldst donky from which thr was information availabl had bn born in All th intrviws comprisd a battry of 18 qustions that wr askd by th sam intrlocutor and ach intrviw lastd for a man tim of 10 minuts. Dspit th lack of prvalnc of multipl births or gstation in thir farms statd by th ownrs, all th qustions wr askd indistinctly. A dscription of th qustions and options askd to th ownrs is shown in Tabl S1. Tabl S2, dfins th catgoris (lvls) of th husbandry systm lvl. Thr wr opn qustions (rgarding th location of th farms, th ag of th animals or th numbr of animals prsnt in th farms at th momnt that th intrviws took plac) and closd qustions (rgarding th sx, th husbandry systm undr which th animals wr handld, and th prvalnc of this condition from th Past up to th dat whn th intrviw was prformd). All th information providd by th ownrs was contrastd with th information providd by UGRA and th information prsnt in th official stud-book of th brd Rcords dscription and scals W organizd th qustions into thr blocks (Tabl S1). Th first block aimd at dscribing th farms of th ownrs intrviwd in ordr to statistically assss th possibl ffcts that may condition th prvalnc of multipl gstations or births. W includd th qustions askd to th ownrs to classify th husbandry systm undr which thir farms wr managd in Tabl S2. Ths qustions basd on th xtnsion of trritory to which th donkys had accss, whthr th donkys wr rproductivly handld and whthr th ownr hld daily contact with thm or thy wr handld just for minimum punctual halth inspction and stud book inclusion. Th scond block comprisd a singl qustion rlatd to whthr th diagnosis by a vtrinarian or thriognologist had bn rqustd. Th scond block comprisd an xcluding qustion as only th ownrs affirmativly rsponding to it wr includd in th statistical and gntic analyss. Th third block consistd of qustions rgarding th assrtiv diagnosis of th multipl births, and th car and prvntiv masurs takn in ach cas. Whn th animals had nvr givn birth, had suffrd from an undtctd arly mbryonic loss nor had carrid any mbryo, w gav thm a scor of 0.

5 Prvious statistical analysis (scrning) Th avrag numbr of foals born pr yar was 28.19, raching th highst numbr (71) in Th man prvalnc of multipl births pr hundrd births in th Andalusian donky population was 9.73%. Th 11.18% of th population had not givn birth to any foal whn th rgistris wr studid. Th proportion of singl, twins and triplts prgnancis dtctd (all tripl prgnancis wr intrruptd) was 58.28%, 30.46%, and 0.08%, rspctivly. Th prcntag of fmals with progny slctd for brding was 10.76% and 25% for mals in th historical population. Historically brding jacks wr 2.98 yars oldr than brding jnnis on avrag. Th avrag ag of parnts whn thir offspring was born was 8.08 yars (8.03 for jnnis and 8.16 for jacks). Th avrag gnration intrval or th avrag ag of th parnts at th birth of thir offspring that in thir turn will produc th nxt gnration of brding animals for th historical population was 7.40 yars [5]. A Shapiro-Wilk tst was applid to th data to chck th fitnss dgr of th variabls in th modl to a normal distribution. Scond, th highly statistical significanc of all th lmnts in th modl (P<0.001), rvald that th data significantly dviatd from a normal distribution. Kurtosis valus supportd ths rsults (Tabl S3). Thus, w carrid out a cross-sctional study mploying Chi-squar analysis to dtrmin whthr th catgorical indpndnt ffcts of birth yar, birth sason, birth month, sx, farm/ownr, and husbandry systm and th covariat of th ag may randomly influnc th dpndnt variabls of historical foal numbr born pr animal, maximum foal numbr pr birth and multipl birth numbr pr animal. W prformd a Kruskal-Wallis H tst to study th potntially xisting diffrncs btwn lvls of th sam factor xcpt for ag, as it is masurd on a continuous scal (Tabl 1). W prsnt Kruskal Wallis H Ranks for all th lvls of th factors affcting historical foal numbr born pr animal, maximum foal numbr pr birth and multipl birth numbr pr animal in Tabl S4. Tabl 1. Summary of th rsults for th Kruskal-Wallis H tst for fixd ffcts and th covariat includd in th modl to tst for birth rlatd traits in Andalusian donkys. Facto r Itm Historical foal numbr born pr animal Maximum foal numbr pr birth Multipl birth numbr pr animal for a givn donky χ Yar of birth df p-valu Lvls Man rank Eta χ df p-valu Mont h of birth Lvls January, Fbruary, March, April, May, Jun, July, August, Sptmbr, Octobr, Novmbr, January, Fbruary, March, April, May, Jun, July, August, Sptmbr, Octobr, Novmbr, January, Fbruary, March, April, May, Jun, July, August, Sptmbr, Octobr, Novmbr, Dcmbr Dcmbr Dcmbr Man rank Eta

6 χ df p-valu Sas on of birth Lvls Win tr Spri ng Sum mr Aut umn Win tr Spri ng Sum mr Aut umn Win tr Spri ng Sum mr Aut um n Man rank , , , , , , , , , 01 Eta χ df Sx p-valu Lvls Jack Jnny Jack Jnny Jack Jnny Man rank ,74 411,3 374,23 Eta χ Farm /Ow nr df p-valu <0.001 <0.001 <0.001 Lvls Man rank Eta χ df p-valu < Husb Sm Sm Sm Sm Sm Sm andr Int i i Ext Int i i Ext Int i i Ext y Lvls int xt int xt int xt syst m Man rank , , , , , , , , , 02 Eta Ag (in Sparman s rho yars ) p-valu < <

7 Simultanously, w studid th pairwis comparisons for any dpndnt variabls for which th Kruskal-Wallis tst was significant, aiming at assssing whthr thr wr diffrncs btwn groups of th sam factor affcting thm. W usd th Mann-Whitny U Tst for sx, as it only has two lvls, jack and jnny, and Dunn s tst for th rst of th factors. If w tst multipl comparisons (hypothss), th liklihood of incorrctly rjcting a null hypothsis incrass, that is rjcting th xistnc statistically of significant diffrncs btwn two or mor groups (for instanc, making a Typ I rror). Th Bonfrroni corrction compnsats for that incras by tsting ach hypothsis (comparison) at a significanc lvl of α/m, whr α is th dsird ovrall alpha lvl, and m is th numbr of hypothss. Onc w tst for th diffrncs in th distribution of th lvls for ach catgory, an indpndnt-sampl mdian tst was carrid out to assss th diffrncs in th mdian btwn lvls within th sam factor. Aftr conducting a Kruskal-Wallis H with thr or mor groups (k), w computd th strngth ffct of th factors on th variabls tstd. Kruskal-Wallis H producs chi² valus with k-1 dgrs of frdom. Thn, w can transform chi² into an F valu with k-1 numrator dgrs of frdom (dfn) and N-k dnominator dgrs of frdom (dfd) using th xprssion F(dfn,dfd)=chi²/(k-1), modifid from Murphy t al. [17]. From F(dfn,dfd) w calculat partial ta squard [18] from th following quation ηp²=(f dfn)/(f dfn + dfd). Eta was computd to masur th strngth of association btwn ach catgorical indpndnt factor from th first st with th numrical dpndnt variabls of historical foal numbr born pr animal, maximum foal numbr pr birth and multipl birth numbr pr animal using th Crosstabs procdur from SPSS Statistics for Windows, Vrsion 24.0, IBM Corp. (2016) (Tabl 1) [19] [20] [20] [20] [20] [20] [20] [20] [20] [20] [20] [20]. Similarly, for th cas of ag, Sparman s rho was computd to masur th strngth of association btwn it and th numrical dpndnt variabls of historical foal numbr born pr animal, maximum foal numbr pr birth and multipl birth numbr pr animal using th Bivariat procdur from SPSS Statistics for Windows, Vrsion 24.0, IBM Corp. (2016) (Tabl 1). Th Sparman s corrlation, dnotd by th symbol ρ (rho), is a nonparamtric masur of th strngth and dirction of association that xists btwn two variabls masurd on at last an ordinal scal. Th tst is usd for ithr ordinal variabls or for continuous data that has faild th assumptions ncssary for conducting th Parson's product-momnt corrlation, as ag in our study (Tabl 1) [19] [20] [20] [20] [20] [20] [20] [20] [20] [20] [20] [20]. All non-paramtrical tsts wr carrid out using th indpndnt sampls packag from th non-paramtrical task of SPSS Statistics for Windows, Vrsion 24.0, IBM Corp. (2016). Catgorical rgrssion (CATREG) was usd to dscrib how th variabls in our study dpndd on th factors considrd (Tabl 2 and 3). Th rsulting rgrssion quation could b usd to prdict historical foal numbr born pr animal, maximum foal numbr pr birth and multipl birth numbr pr animal for any combination of th svn indpndnt factors. Catgorical Rgrssion was carrid out using th Optimal Scaling procdur from th Rgrssion task from SPSS Statistics for Windows, Vrsion 24.0, IBM Corp. (2016).

8 263 Tabl 2. Modl summary of stpwis linar rgrssion with transformd variabls. Variabl Multipl R R Squar Adjustd R Squar Apparnt Prdiction Error Estimat Std. Error 264 Historical foal numbr born pr animal Maximum foal numbr pr birth Multipl birth numbr pr animal Tabl 3. Standardizd Cofficints and significanc of CATREG modl. Variabl Historical foal numbr born Maximum foal numbr pr birth Multipl birth numbr pr animal pr animal Paramtr Standardizd Significanc Standardizd Significanc Standardizd Significanc Cofficints (β) Cofficints (β) Cofficints (β) Factor Birth month Birth yar Birth sason Sx Ownr/Farm Husbandry systm Ag (in yars) Gntic modl, phnotypic and gntic paramtrs As w only considrd on masur pr animal, th modl usd in th analysis of birth rlatd variabls was a simpl Animal Modl with singl rcords. Th fixd ffcts that wr submittd to th abov dscribd statistical procdurs and comprisd th mixd modl consistd of th birth yar (from 1984 to 2017); birth sason (summr, spring, autumn and wintr); birth month (from January to Dcmbr); sx (jack or jnny); th farm (90 farms/ownrs) and husbandry systm (int, smiint, smi-xt and xt). At a prvious stag of th study, w computd th doubl intraction btwn hrd and yar of birth (Hrd*Birthyar) and th tripl intraction btwn th hrd, th yar of birth and sason of birth (Hrd*Birthyar*Birth sason) as ths wr th most rgularly includd in litratur for th sam kind of studis in othr spcis such goats or shp. Adjustd R-squard was computd both including and without including th intraction, and w includ a summary of th rsults in Tabl S4. W computd xpctd prdiction rror of rgrssion with Bootstrap ( lav-on-out bootstrap ) from 200 bootstrap sampls [20, 21]. Adjustd R-squard tlls bttr than R squar

9 (prcntag of varianc xplaind by th modl) about whthr w should add a nw variabl or not as it tris to corrct for th fact that th improvmnt in R squar could b attributd to chanc alon, considring th ratio (N-1)/(N-k-1) whr N = numbr of obsrvations and k = numbr of variabls (prdictors). Th tripl intraction was statistically nonsignificant (P>0.05) so that it was not includd in th modl. Although, th Hrd*Birth yar doubl intraction was statistically significant P<0.01, its inclusion within th modl distortd th rsults in th following way so that w dcidd not to includ such intraction. Th modl for historical foal numbr born pr animal xplaind a highr prcntag of th varianc in th sampl whn w includd th intraction. Howvr, th stimation of th gntic paramtrs rportd almost thr tims th standard rror of th sam modl without including th intraction as statd blow, that may hav its basis on th high amount of possibl lvls of th intraction matchd to a proportionally small sampl. For maximum foal numbr pr birth, thr was a rduction in Adjustd R squard from to and th xpctd prdiction rror incrasd from to whn w includd th Hrd*Birth yar intraction. For multipl birth numbr pr animal, on or mor lvls for th catgoris wr supplmntary (only usd by cass that do not occur in th sampl). Ths rsults suggstd that th inclusion of this intraction in th modl may rsult in potntially distorting ffcts which wr highlightd at th statistical lvl as xpctd prdiction rror could not b computd. Th rsults of th gntic and phnotypic paramtrs stimatd by a prliminary modl including Hrd*Birth yar itration supportd such distorting ffcts, as thr was an incras in th standard rrors from th currnt modl (without th intraction) to to to (including th intraction). As th prvious statistical analysis had rportd, th basis for such distorting ffcts may b th fact that th numbr of catgoris considrd for hrd*yar intraction was 441, whil th whol sampl siz was 765. This data may gnrat an statistical imbalanc that may rsult in an ovrstimation of th ffct of th intraction as it has bn rportd by litratur [22], making it impossibl to tst for its ffcts proprly, du to th lack of nough animals in th pdigr btwn whom to compar. Th multi-trait animal modl usd for th analyss is as follows: Yijklmop=μ+aij+Yak+Monl+Sam+Sxn+Faro+Sysp+b1Aq+b2A 2 q+ijklmnop whr Yijklmnop is th sparat rcord of ith trait for jth donky (historical foal numbr born pr animal (1 in matrix blow), maximum foal numbr pr birth (2 in matrix blow) and multipl birth numbr pr animal for a givn donky (3 in matrix blow)); μ is th ovrall man for th trait; aij is th additiv gntic ffct of th jth donky for ith trait, Yak is th fixd ffct of th kh birth yar (k= ); Monl is th ffct of th lth birth month (January, Fbruary, March, April, May, Jun, July, August, Sptmbr, Octobr, Novmbr, Dcmbr); Sam is th fixd ffct of th mth birth sason (m=summr, spring, autumn, wintr); Sxn is th fixd ffct of th nth sx (n=jack, jnny); Faro is th fixd ffct of th oth farm/ownr (o=1-90); Sysp is th fixd ffct of th pth husbandry systm (p=int, smi-int, smi-xt, xt); b1 and b2 ar th linar and quadratic rgrssion cofficints on ag whn th tsts took plac (Aq and A 2 q) and ijklmnop is th random rsidual ffct associatd with ach rcord. No matrnal ffct was computd bcaus of th low compltnss lvl found in th pdigr, as 53.36% of th dams in th study wr unknown [5]. Such a lack of information could hav rprsntd a problm whn prforming th gntic analyss. Howvr, as our sampl provids dirct or indirct information from 91% of th animals includd in th pdigr, w could sav th possibl drawback mant by th missing information. Thn, th quality of th prdictd gntic valus stimatd was quantifid by rporting thir rliability. W includd th ag of th animals xprssd in yars as a linar and quadratic covariat to corrct th variabls masurd according to th liftim of ach animal and spcifically th cass in which th animals wr too young to hav givn birth to thir first foal/s. W includd th ffct of sx on our modl to sav th imbalanc btwn sxs, vn mor, whn w considr th vast diffrncs btwn th offspring of mals and fmals givn th long duration of th gstation of th spcis. In matrix notation, th multi-trait modl usd was: y X.... β Z.... α ε y =.. X.. β +.. Z.. α + ε y.... X β.... Z α ε

10 whr y1 to y3 rprsnt th phnotypical obsrvation for ach trait and animal. Th vctors of fixd ffct for th thr diffrnt traits considrd (β1 to β3,) includ all th ffct rlatd in th modl dscribd abov and th vctors α1 to α2 and ε1 to ε2, ar random additiv gntics and rsidual ffcts for ach trait, rspctivly. Th incidnc matrics X1 to X3 and Z1 to Z3 associat lmnts of β1 to β3 and α1 to α2 with th rcords in y1 to y2. If A is th matrix of additiv gntic rlationships among individuals, th mixd modl quation (MME) usd is as follows: X X X Z Z X Z Z + A k β = X y α Z y Proxis of prolificacy (i.. numbr of offspring producd in a singl parturition) ar calculatd as sums ovr random tim priods vntually cnsord by natur, and/or th will of th ownr, and/or th timfram of th study (ach donkys liftim, spcially in animals that ar too young to hav givn birth). Hnc th importanc of including, assssing and controlling factors such as ownr and ag of birth as rportd abov Gntic assssmnt softwar Th first stag of our analyss was to obtain stimats of gntic paramtrs and corrlations (phnotypic and gntic) for historical foal numbr born pr animal, maximum foal numbr pr birth and multipl birth numbr pr animal for a givn donky in Andalusian donkys. Estimations wr prformd using th MTDFREML Multipl Trait Drivativ Fr Rstrictd Maximum Liklihood softwar [23], a packag mploying th simplx procdur to locat th maximum of th log liklihood (logl). W considrd having rachd convrgnc whn th varianc of function valus (-2 logl) in th simplx was lss than W run th modl svral tims rstarting th program with initial valus from th prvious round (run) to nsur convrgnc of logl to a global maximum. Whn th -2 logl and stimats wr similar in succssiv analyss, w concludd that convrgnc rachd th global maximum. Aftr w rachd convrgnc, th scond stag aimd at prdicting brding valus (BLUP, bst linar unbiasd prdictors for animal random ffcts), thir accuracis and rliabilitis for ach animal sortd by sx. Th analyss wr carrid out including th rlationship matrix of animals with dirct rcords rlatd through at last on known ancstor, considring th 1017 donkys in th historical pdigr. Covariat and fixd ffcts postrior mans (BLUES, bst linar unbiasd stimators for fixd ffcts and covariabl) wr computd with MTDFREML, as wll. Th standard rrors of gntic corrlations among frtility and multipl birth traits wr obtaind dirctly from th MTDFREML analyss. 3. Rsults 3.1. Intrviw rsults Out of th 145 ownrs intrviwd, w considrd th information from 90 farms/ownrs. Ths ownrs had affirmativly rspondd to th qustion in th scond block as thy wr thy only who had rqustd information concrning diagnosis by thir vtrinarians or thriognologists and thrfor, wr th only ons providing rliabl information. Du to th particularitis of th spcis and th brding routins carrid by th ownrs, th artificial insmination with frsh smn of th animals rgistrd in th studbook was infrqunt, and almost all th matings wr prformd naturally. No productiv artificial insmination using frozn smn was rgistrd. Th matings of only 66 animals out of th 765 donkys from which thr was information (8.63% of th total sampl) had rsultd in multipl gstations. Out of this prcntag, 1.04% of th animals dvlopd multipl gstations in mor than on occasion through thir livs and only on of th animals was rsponsibl

11 for 0.13% of multipl gstations in th population (fiv multipl births out of 40 births through his lif). Shapiro-Wilk Tst (P<0.001) and highr or lowr kurtosis valus than thr on all th fixd ffcts, th covariat and intraction showd that thy highly significantly did not fit a normal distribution. Th variability obsrvd for th two traits analyzd was from modrat to high, with a cofficint of variation of 21.3% for th husbandry systm ffct and 82.2% for th ffct of th farm/ownr Statistical analyss Th rsults of Chi-Squar, Eta (for ach indpndnt catgorical-dpndnt numrical pair of variabls) and Sparman s rho corrlation cofficint (for th ffct of ag on th numrical dpndnt variabls studid), tsting for th xistnc of linar corrlation ar shown in Tabl 1. Eta ffctivly and statistically significantly masurd th strngth of collinarity that th sx and farm factors hav on continuous variabls of historical foal numbr born pr animal, maximum foal numbr pr birth and multipl birth numbr pr animal for a givn donky. Husbandry systm rportd highly statistically significant collinarity with th historical foal numbr born pr animal (Tabl 1). Kruskal-Wallis H tst and Chi squar rportd th ffcts birth yar, birth sason and birth month to b statistically nonsignificant (P>0.05) for th thr dpndnt variabls considrd. Th sam tst rportd th rst of indpndnt variabls (sx, ownr/farm and husbandry systm) to b statistically significant (P<0.05) for all dpndnt variabls xcpt for husbandry systm on maximum foal numbr pr birth and multipl birth numbr pr animal for a givn donky (P>0.05) (Tabl 1). From th rsults of th Mann-Whitny U Tst, w can conclud that historical foal numbr born pr animal and maximum foal numbr pr birth in jacks was statistically significantly highr than in jnnis (U= , P<0.001 and U= , P<005). Howvr, th opposit trnd was dscribd by multipl birth numbr pr animal for a givn donky, which was statistically significantly highr in jnnis than in jacks (U= , P<0.05) (Tabl S5). Th rsults of th Dunn tst in our study rportd th fact that thr wr highly statistically significant diffrncs for 14.89% of pairwis comparisons of farms/ownrs (Tabl S5). Th sam tst rportd statistically significant diffrncs btwn xt, smixt and smiint husbandry systms (P<0.05) for maximum foal numbr pr birth. CATREG was prformd to th 7 qualitativ indpndnt variabls (birth yar, birth month, birth sason, sx, farm, husbandry systm) and ag as a covariabl with th thr birth-rlatd continuous variabls (historical foal numbr born pr animal, maximum foal numbr pr birth and multipl birth numbr pr animal for a givn donky) as dpndnt variabls. Thn stpwis linar rgrssion to th data with th rsultd quantifications was applid, and w prsnt th summary rsults with th significant variabls in Tabls 2 and 3. Th standardizd cofficints (β) ar listd in Tabl 3. CATREG rportd all of th indpndnt variabls xcpt for birth yar and sx to b significant for historical foal numbr born pr animal. Sx was nonsignificant for maximum foal numbr pr birth. Birth sason and husbandry systm wr nonsignificant for Multipl birth numbr pr animal. Thr was a small to modrat monotonic (whthr linar or not) significant (P<0.05) corrlation btwn ag and th thr variabls tstd (Tabl 1). This corrlation was invrs (-0.137) in historical foal numbr born pr animal, that is if ag incrass th historical numbr of foals pr donky dcrass, whil it was dirct, for maximum foal numbr pr birth (0.085) and multipl birth numbr pr animal for a givn donky (0.339), which paralllly incrasd with ag. Th numbr of standard dviations a dpndnt variabl will chang, pr standard dviation incras in th ag or ag CATREG (β) standardizd cofficints is shown in Tabl 3. CATREG (β) standardizd cofficints rangd from to for maximum foal numbr pr birth and multipl birth numbr pr animal, rspctivly. For th month of birth, Chi squar valus wr non-significant (P>0.05). Thus, thr was not any statistical diffrnc btwn th valus of th dpndnt variabls for ach of th twlv lvls of

12 birth month. Eta valus rangd from to what rportd a modrat association btwn month of birth and th dpndnt variabls of multipl birth numbr pr animal for a givn donky and historical foal numbr born pr animal. Th low to modrat CATREG standardizd cofficints rportd a low to modrat incras of th standard dviation ndd to incras a unit of standard dviation of ach of th thr variabls on this factor (from to 0.170, for historical foal numbr born pr animal and multipl birth numbr pr animal, rspctivly). For birthyar, Chi squar valus wr non-significant (P>0.05). Thus, thr was not any statistical diffrnc btwn th valus of th dpndnt variabls for ach of th 31 lvls of birthyar. Eta valus rangd from to what rportd a modratly high association btwn birth yar and th dpndnt variabls of multipl birth numbr pr animal for a givn donky and historical foal numbr born pr animal. CATREG standardizd cofficint for birth yar and historical foal numbr born pr animal was non-significant. Howvr, CATREG standardizd cofficints for maximum foal numbr pr birth (0.201) and multipl birth numbr pr animal (-0.289) suggstd that a modrat incras in th standard dviation of th birth yar may b ndd to incras a unit of standard dviation in maximum foal numbr pr birth. By contrast, a modrat dcras in th standard dviation of th birth yar was ndd to incras a unit in th standard dviation of multipl birth numbr pr animal. For th birth sason, Chi squar valus wr non-significant (P>0.05). Thus thr was not any statistical diffrnc btwn th valus of th dpndnt variabls for ach of th four lvls of birth sason. Eta valus rangd from to what rportd a low association btwn birth sason and th dpndnt variabls of maximum foal numbr pr birth and multipl birth numbr pr animal for a givn donky. CATREG standardizd cofficint for th birth sason and multipl birth numbr pr animal was non-significant. Howvr, CATREG standardizd cofficints for maximum foal numbr pr birth (0.100) and historical foal numbr born pr animal (0.071) rportd a low incras of th standard dviation of th birth yar was ndd to incras a unit of standard dviation in both dpndnt variabls. For sx, Chi squar valus wr all significant (P<0.05), thus thr wr statistical diffrncs btwn th valus of th dpndnt variabls for ach of th two lvls of sx. Eta valus rangd from to what rportd a low to modratly high association btwn sx and th dpndnt variabls of maximum foal numbr pr birth and multipl birth numbr pr animal for a givn donky. CATREG standardizd cofficint for sx and historical foal numbr born pr animal and maximum foal numbr pr birth wr non-significant. Howvr, CATREG standardizd cofficints for multipl birth numbr pr animal (0.445) rportd a high incras of th standard dviation of sx was ndd to incras a unit of standard dviation in multipl birth numbr pr animal. Ownr/Farm, Chi squar valus, wr all significant (P<0.001), thus thr wr highly significant statistical diffrncs btwn th valus of th dpndnt variabls for ach of th 90 lvls of th farm/ownr factor. Eta valus rangd from to what rportd a high association btwn ownr/farm and th dpndnt variabls of historical foal numbr born pr animal and multipl birth numbr pr animal for a givn donky. CATREG standardizd cofficint for ownr/farm wr all highly statistically significant (P<0.001). CATREG standardizd cofficints ranging from to rportd a high incras of th standard dviation of ownr/farm was ndd to incras a unit of standard dviation in all thr dpndnt variabls masurd. Husbandry systm Chi squar valu was only significant (P<0.001) for historical foal numbr born pr animal, thus thr wr highly significant statistical diffrncs btwn th valus of that dpndnt variabl for ach of th four lvls of th husbandry systm factor. Eta valu for this dpndnt variabl was what rportd a modratly low association btwn husbandry systm and th indpndnt variabls of historical foal numbr born pr animal and multipl birth numbr pr animal for a givn donky. CATREG standardizd cofficint for ownr/farm was statistically significant (P<0.05) for historical foal numbr born pr animal and maximum foal numbr pr birth. CATREG standardizd cofficints ranging from to rportd a modratly high

13 incras of th standard dviation of th husbandry systm was ndd to incras a unit of standard dviation in all thr dpndnt variabls masurd. W show th factors affcting th thr birth rlatd variabls in ordr of importanc according to th CATREG standardizd cofficints (β) in Tabl 4. Sinc w usd th stpwis mthod, thr was no multicollinarity problm. Th standardizd solution for th rgrssion quations can b found in Tabl 4 as wll. Tabl 4. Rgrssion quations for maximum foal numbr pr birth, multipl birth numbr pr animal for a givn donky and historical foal numbr born pr donky. Gnral Modl Rgrssion quation Z`Ymaxmulhis= βfarm*zfarm + βbirthyar*zbirthyar +βbirthmonth*zbirthmonth+βbirthsason*zbirthsason + βsx*zsx + βhusbandrysystm*zhusbandrysystm + βag*zag Spcific rgrssion quations Lgnd Z ymaxmulhis= Z scor for ach variabl (maximum foal numbr pr birth, multipl birth numbr pr animal for a givn donky and historical foal numbr born pr donky). β=standardizd cofficint for ach of th factors apparing in th subindx. Z=Z scor for ach of th factors apparing in th subindx. Lgnd Z'ymax= Z scor for maximum foal numbr pr birth. Maximum foal numbr pr birth Z`ymax= 0.867(Zfarm) (Zbirthyar) (Zbirthmonth) (Zbirthsason) (Zsx) (Zhusbandrysyst m) (Zag) βfarmzfarm=0.867(zfarm) βbirthyarzbirthyar=0.201(zbirthyar) βbirthmonthzbirthmonth=0.094(zbirthmonth) βbirthsasonzbirthsason=0.100(zbirthsason) βsxzsx=0.145(zsx) βhusbandrysystmzhusbandrysystm=0.532(zhusbandrysyst m) βagzag=0.150(zag) Multipl birth numbr pr animal for a givn donky Z`ymul=0.598(Zfarm) + ( ) (Zbirthyar) (Zbirthmonth) (Zsx) (Zag) Z'ymul= Z scor for multipl birth numbr pr animal for a givn donky. βfarmzfarm=0.598(zfarm) βbirthmonthzbirthmonth=0.170(zbirthmonth) βbirthyarzbirthyar=-0.289(zbirthyar) βsxzsx=0.445 (ZSx)

14 βagzag=0.159(zag) Historical foal numbr born pr donky Z`yhis=0.873(Zfarm) (Zbirthsason) (Zbirthmonth) (Zhusbandrysyst m) (Zag) Z'yhis= Z scor for historical foal numbr born pr donky. βfarmzfarm=0.873(zfarm) βbirthmonthzbirthmonth=0.092(zbirthmonth) βbirthsasonzbirthsason=0.071(zbirthsason) βhusbandrysystmzhusbandrysystm=0.553 (ZHusbandrysystm) βagzag=0.155(zag) 490 Non-significant ffcts for ach variabl wr not includd (P>0.05) Modl prdictiv powr CATREG R squard cofficint obtaind rangd from to for multipl birth numbr pr animal and maximum foal numbr pr birth, rspctivly (Tabl 2) 3.4. Gntic modl, varianc componnts, gntic and phnotypic corrlations, prdictd Brding Valus and prdiction accuracy W show th stimats for hritability, gntic and phnotypic varianc stimatd with REML in Tabl 5. Tabl 6 shows th gntic and phnotypic corrlation chart. Th rsults for th stimats of prdictd brding valus (PBV) for both jacks and jnnis ar shown in Tabl 7. Tabl 5. Estimatd componnts of varianc, hritability (h 2 ) and standard rror (SE) for maximum foal numbr pr birth, multipl birth numbr pr animal for a givn donky and historical foal numbr born pr donky obtaind from multivariat analyss using REML mthods in Andalusian donkys. Trait σ σ σ h 2 ±SE Maximum foal numbr pr birth Multipl birth numbr pr animal for a givn donky Historical foal numbr born pr donky ± ± ±

15 Tabl 6. Estimatd phnotypic (rp) (abov diagonal) and gntic (rg) (blow diagonal) corrlations for maximum foal numbr pr birth, multipl birth numbr pr animal for a givn donky and historical foal numbr born pr donky obtaind in bivariat analyss using REML mthods in Andalusian donkys. 511 Traits Maximum foal numbr pr birth Multipl birth numbr pr animal for a givn donky Historical foal numbr born pr donky Multipl birth numbr Historical foal Maximum foal pr animal for a givn numbr born pr numbr pr birth donky donky ± ± ± ± ± ± Tabl 7. Dscriptiv statistics of prdictd brding valus (PBV), standard rror of prdiction (SEP), accuracy (rti) and rliability (RAP) for maximum foal numbr pr birth, multipl birth numbr pr animal for a givn donky and historical foal numbr born pr donky for all th donkys includd in th pdigr sortd by sx. Sx Jacks (N=272) Variabl Maximu m foal numbr pr birth Multipl birth numbr pr animal for a givn donky Historica l foal numbr born pr donky Paramt Minimu Maximu Skwn Man SEM SD r m m ss a Kurtosi PBV SEP rti RAP PBV SEP rti RAP PBV SEP rti RAP PBV s a

16 Maximu SEP m foal rti numbr pr birth RAP Multipl PBV birth SEP numbr pr rti Jnnis animal (N=745) for a RAP givn donky Historica PBV l foal SEP numbr rti born pr donky RAP a Standard rror for Skwnss statistic was and and standard rror for Kurtosis statistic was and for jacks and jnnis, rspctivly for all factors assssd Covariat and fixd ffcts postrior mans W show th rsults for th bst linar unbiasd stimators (BLUEs) obtaind from th REML quantitativ gntic analysis, including ag as a linar and quadratic covariat, th fixd ffcts of birth yar, birth month, birth sason, sx, farm/ownr and husbandry systm in Tabl S8. 4. Discussion According to litratur, donkys hav a 13% highr frtility than horss [24], raching an incidnc for multipl ovulations of 61% in Mammoth and standard jnnis. This highr incidnc of multipl ovulation in donkys translats in twinning occurring mor frquntly, spcially in mammoth and standard donkys. Although rsarchrs hav found th incidnc of twins to b as high as 40% (via ultrasound at day 21 in standard donkys), for ndangrd donky brds such as Asinina d Miranda, th prcntag of twin foaling at full trm was 2.85% [2]. According to our rsults, only 8.75% of th Andalusian donky brd population has producd multipl offspring, and only around 10% of total births considrd in th study (controlld) wr multipl. Th rat of multipl ovulations in th donky spcis varis with th rports from litratur, ranging from 5.3% to almost 70% [7] so that our rsults fall within th rang rportd for othr donky brds. Th rproductiv trnds of this polygynous spcis hav bn rportd to highly dpnd on th ownr tasts for crtain morphological or coat charactristics and local availability of th animals. Navas t al. [5] suggstd th typical xcssiv contribution of fw ancstors to th gn pool of small critically ndangrd donky populations may lad to narrow bottlncks shortly whos hiddn ffcts can only b controlld by tracking th populations. Among such hiddn ffcts, th compromiss xrtd on th rproductiv and immunity systm of th animals hav bn addrssd to b som of th dtrminants of th difficultis xprimntd to conciv by individuals [25]. Such rproductiv compromiss hav bn suggstd to b a dirct caus of inbrding dprssion in donkys. Howvr, th lack of compltnss of th pdigr of ndangrd donky populations and th irrgular distribution through grat xtnsions of trritory maks th stimation

17 of this paramtr littl rliabl [5]. Quarsma t al. [2] rportd th numbrs obtaind in 40 captiv mammalian populations indicatd an avrag valu of 3.14 of lthal quivalnts with 50% du to rcssiv lthal allls. Tabrnr t al. [26] statd that multipl ovulations tnd to rpat in svral strous cycls, what may support th xistnc of animals that prsnt a crtain cyclical prdisposition towards multipl births. Th rlativ frquncis for multipl prgnancis of crtain donkys wr highr than for othrs, what suggstd a gntic background bhind multipl births, as it had prviously bn rportd by Ginthr [27]. Similarly, Quarsma t al. [2] suggstd an indirct slction of crtain family lins may hav bn carrid out in th Mammoth donky, what may hav rsultd in th highr incidnc of multipl ovulations rportd by Blanchard t al. [28]. Spcific studis hav assssd th possibl rprcussion of crtain nvironmntal factors on th frtility of donkys. For xampl, in our study, th Chi squar valus for th birth sason wr nonsignificant (P>0.05). Thus thr was not any statistical diffrnc btwn th valus of th dpndnt variabls for ach of th four lvls of birth sason. Th findings by Contri t al. [29] support our rsults. Ths authors would rport strous cycl can b dtctd during th whol yar in jnnis, with no diffrncs in th strous cycl lngth among sasons. Parallly, th pattrn of th plasma concntration of crtain hormons such as E2 and P4 during th strous cycl did not rport any diffrnc among sasons, although a largr diamtr of th ovulating follicl was rportd for spring and summr. Brding sason and month significantly affctd gstation and strous cycl lngth in donkys [30]. Howvr, ths authors did not study whthr th ffct of th month may condition th occurrnc of multipl births and frtility. Quarsma and Payan-Carrira [31] rportd th incidnc of singl, doubl, and tripl ovulations to b 57.58%, 36.36%, and 6.06%, rspctivly. Th sam authors statd, multipl ovulations affctd nithr th lngth of th introvulatory intrval nor th individual cycl stags (P > 0.05) but lngthnd th intrval from th bginning of strus to th last ovulation (P = 0.01), what may support th rsults found by our study and thos found by Galisto and Prz-Marin [30] as wll. No papr has rportd th highr prvalnc of multipl births or a highr liklihood of prsnting a highr maximum numbr of foals dpnding on th husbandry tchniqus carrid in th farms. Th rsults found in our study for Dunn s and indpndnt sampls mdian tsts suggstd donkys locatd at smixt farms prsntd a highr liklihood of prsnting highr maximum foal numbrs pr birth, followd by smiint farms and xt farms, rspctivly. Th critria usd to classify th husbandry systms of th farms in th study (Tabl 2) may suggst that th accss to mor xt trritoris, whn ownrs provid rgular rproductiv car to th animals and th daily contact with th ownrs may hav an incrasing importanc in th occurrnc of a highr numbr of foals pr birth. Th highr strngth ffct of th farm factor on all th variabls tstd ranging from to 0.873, for multipl birth numbr pr animal and historical foal numbr born pr animal, rspctivly supportd th finding. A highr rlvanc was attributd to jnnis in having a historically highr numbr of foals, a highr numbr of multipl offspring and a highr maximum numbr pr birth. Ths valus balancd (providing an qual rlvanc to jacks and jnnis) as th numbr of foals and multipl births incrasd, as w can obsrv in th charts in Tabl S5. Howvr, still thr sm to b a vry slight ffct of spcific jacks on promoting th obtntion of a highr historical numbr of foals. This could b attributd to th rproductiv charactristics of th jnny and brding stratgis of donky ownrs, as it has alrady bn suggstd by Brsińska t al. [14] and is addrssd by th rsults of th Mann-Whitny U tst of our study (Tabl S5). According to our rsults, th fact that foal numbr born pr animal and maximum foal numbr pr birth in jacks was statistically significantly highr than in jnnis could b attributd to th fact that jacks can act as th sir for svral jnnis at th sam tim, whil jnnis ar going to b rproductivly blockd for a whol yar whn thy hav bcom prgnant. Th sam tst suggstd that although jacks wr likly to significantly rach a highr numbr of foals on a crtain gstation through thir livs whn compard to jnnis, jnnis wr statistically significantly mor pron to dvlop multipl gstations through thirs. This could b