Genome-wide association studies for sperm traits in Assaf sheep breed

Genome-wide associa ion s udies o spe m ai s in Assa sheep b eed M. Se ano a, ⁎,M. Ramón b , J.H. Cal o c, , M.Á. Jiménez a , F. F ei e d , J.M. Vázquez d ,J.J. A anz e a Depa amen o de Mejo a Gené ica Animal, INIA, 28040 Mad id, Spain b IRIAF-CERSYRA, Valdepeñas 13300, Ciudad Real, Spain c Unidad de Tecnología en P oducción Animal, CITA, 59059 Za agoza, Spain d OVIGEN, G anja Flo encia s/n, C a. Villalazán-Peleagonzalo, 49800 To o, Zamo a, Spain e Depa amen o de P oducción Animal, Uni e sidad de León, 24007 León, Spain ARAID, 50004 Za agoza, Spain abs ac a icle in o A icle his o y: Recei ed 6 May 2020 Recei ed in e ised o m 20 Augus 2020 Accep ed 21 Augus 2020 A ailable online xxxx Keywo ds: Associa ion s udy Pseudo-pheno ypes Rams Single-nucleo ide polymo phisms geno ypes Spe m pa ame e s Spe m quali y ai s ou inely collec ed by a ificial insemina ion (AI) cen e o ams p ogeny es a e ela ed wi h he capaci y o p oduce spe m doses o AI and, in mo e o less g ade, wi h males' e ili y. Low-quali y ejac- ula es a e unuse ul o pe o m AI spe m doses, which suppose high economic loses o he AI cen e . Mo eo e , spe m quali y ai s ha e low he i abili y alues which make adi ional gene ic selec ion li le e ficien o i s im- p o emen . In his wo k, a genome-wide associa ion s udy (GWAS) was conduc ed by using spe m quali y ai s da a and 50 K A yme ix cus omchipgeno ypeso 429 ams o Assa b eed om OVIGENAI cen e. Fu he mo e, 47 o hese ams we e also geno yped wi h he Illumina HD O ine BeadChip, and he e o e HD geno ypes we e impu ed o all ams wi h pheno ype da a. P e ious o he GWAS, a linea eg ession model was fi ed including spe m ai s as dependen a iables; he flock o o igin, da e o spe m collec ion, and jump numbe as fixed e - ec s; ams age a collec ion in mon hs as co a ia e; and am pe manen e ec as andom. Pseudo-pheno ypes ob ained om his model we e used as inpu o GWAS. Associa ions a he ch omosome-wise le el (FDR 10%) o 76 single-nucleo ide polymo phisms (SNPs) in 4 ch omosomes o ejacula e concen a ion (CON), 20 SNPs in 3 ch omosomes o ejacula e olume (VOL), 32 SNPs in 1 ch omosome o ejacula e numbe o spe ma ozoa (SPZ), and 23 SNPs o spe ma ozoa mass mo ili y (MOT) in 17 ch omosomes we e ound. Only SNPs associa ed wi h MOT o e came he genome-wide significance le el. Some candida e genes o spe m ai s a iabili y we e SLC9C1 (OAR1), TSN (OAR2), and FUT10 (OAR26) o MOT;. DOCK2,CPLANE1,SPEF2, and RAI14 (OAR16) o CON; SCAPER and PSMA4 (OAR18) o VOL; and PARM1 and LOC101110593 (OAR6) o SPZ. SNPs associa ed wi h spe m ai s we e no ound o be co ela ed wi h milk p oduc ion gene ic a ia ion; howe e , he high equencies o some SNPs wi h nega i ee ec o e spe m ai s ound in animals a he op milk yieldes ima edb eeding alues (EBVs) anking would allow o exe some selec i e p esu e o imp o e ams spe m pe o mances. E ec s and equencies o some o he SNPs de ec ed o e spe m quali y ai s make hese a ian s good candida es o be used in ma ke -assis ed selec ion o imp o e spe m cha ac e is ics o Assa ams and AI cen e e ficiency o p o- duce spe m doses. © 2020 The Au ho s. Published by Else ie Inc. on behal o The Animal Conso ium. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). Implica ions Finding ou he gene ic basis o spe m quali y ai s in he o ine spe- cie is a e y impo an issue since hei mode a e o low he i abili y makes adi ional selec ion me hods li le e ficien o i s imp o emen . Spe m ai s a e highly ela ed o a ificial insemina ion cen e s e fi- ciency o pe o m ams p ogeny es in sheep dai y gene ic p og ams. The genome mu a ions associa ed wi h he a iabili y o spe m ai s he e de ec ed could allow o pe o m a ma ke -assis ed selec ion o imp o e ams spe m cha ac e is ics, and he e o e, hei ep oduc i e abili y and capaci y o p oduce spe m doses. In oduc ion A ificial insemina ion (AI) is an essen ial ep oduc i e ool in ge- ne ic b eeding p og ams o dai y uminan s. The use o his echnique enables p ogeny es s o p edic es ima ed b eeding alues (EBVs)o males, con ibu es o connec flocks, and is he bes s a egy o dissem- ina e he gene ic imp o emen achie ed by he gene ic p og am using eli e ams. In sheep insemina ion cen e s, he e ilizing capaci y o he spe ma ozoa and he sui abili y o he semen o p oducing AI doses a e assessed using h ee pa ame e s: he olume o he ejacula e Animal xxx (2021) xxx ⁎Co esponding au ho . E-mail add ess: [email protected] (M. Se ano). ANIMAL-100065; No o Pages 9 h ps://doi.o g/10.1016/j.animal.2020.100065 1751-7311/© 2020 The Au ho s. Published by Else ie Inc. on behal o The Animal Conso ium. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). Con en s lis s a ailable a ScienceDi ec Animal The in e na ional jou nal o animal biosciences Please ci e his a icle as: M. Se ano, M. Ramón, J.H. Cal o, e al., Genome-wide associa ion s udies o spe m ai s in Assa sheep b eed, Animal, h ps://doi.o g/10.1016/j.animal.2020.100065 (VOL), he concen a ion o spe ma ozoa (CON), and he spe ma ozoa mass mo ili y (MOT) measu ed on a subjec i e scale. VOL and CON phe- no ypes a e ela ed o he e ec i e numbe o doses ha can be elabo- a ed om one ejacula e, while spe m mo ili y has been desc ibed as a good indica o o spe m e ilizing abili y (Gadea, 2005;B oekhuijse e al., 2012). Howe e , a di ec ela ionship be ween hese spe m pa- ame e s and am's e ili y is no e iden . The mode a e he i abili y es ima es ound o spe m cha ac e is ics in some sheep b eeds (Da id e al., 2007; Pelayo e al., 2019)join o he ac ha hese ai s a e only collec ed in a e y limi ed numbe o males (only ams om AI cen e s) makes con en ional selec ion ine - ec i e o imp o e hem. Fu he mo e, he gene ic co ela ion be ween dai y ai s, milk yield and a and p o ein con en s, he main selec ion objec i e in mos dai y sheep b eeds, and spe m quali y ai s is un- known. I his co ela ion was nega i e, selec ion o eli e ams o milk ai s may lead o an impai men o am's spe ma ic quali y. Cu en genomic ools, such as medium and high-densi y geno yp- ing single-nucleo ide polymo phism (SNP) chips, could help o iden i y genomic egionsassocia ed wi h such low he i abili y ai s, enabling o conduc ma ke assis ed selec ion o spe m quali y ai s in li es ock. In las yea s, some wo ks dealing wi h genome-wide associa ion s udies (GWAS) o spe m quali y ai s ha e been conduc ed in ca le (Suchocki and Szyda, 2014; He ing e al., 2014; Fonseca e al., 2018; Yin e al., 2019;Qin e al., 2017), pigs (Diniz e al., 2014; Ma ques e al., 2018), and goa s (Wang e al., 2020). All o hem we e pe o med by using medium-densi y SNP geno yping a ays and a a iable numbe o geno yped animals. Some pu a i e candida e genes we e associa ed wi h mo e o less deg ee wi h spe m mass mo ili y, olume, concen a- ion, and numbe o spe ma ozoa. In goa s (Wang e al., 2020), a s ong associa ion o he DSCAML1 gene wi h spe m mo ili y and densi y was ound. Howe e , o da e, no s udies dealing wi h genome-wide associa- ion o spe m ai s in sheep ha e been conduc ed. The main objec i e o his wo k is o iden i y SNPs and genomic e- gions associa ed wi h spe m quali y ai s by using geno ypes om he 50 K A fiyme ix cus om chip and he Illumina HD O ine BeadChip. Ma e ial and me hods Da a Spe m quali y ai s we e ob ained om 27 635 ejacula es o 429 ma u ed ams p esen in OVIGEN AI cen e , eco ded be ween 2006 and 2018. Ejacula es we e ob ained a e na u al ejacula ion in an a ifi- cial agina. Fo a gi en am, 1 o 2 successi e ejacula es we e collec ed o e a 2–5 min pe iod and e alua ed immedia ely a e collec ion. Th ee ai s we e assessed o each ejacula e: VOL (ml), which was mea- su ed using a g adua ed collec ion ube, CON (spe ma ozoa × 10 6 /ml), which was de e mined using a s anda d spec opho ome e , and MOT, which was assessed o undilu ed semen unde a mic oscope. MOT was sco ed subjec i ely, based on wa e mo ion on a con inuous scale om 0 (no mo ion) o 5 ( equen apid and igo ous wa es) acco ding o he o iginal me hod desc ibed by E ans and WMC (1987). A ou h de i ed ai , he numbe o spe ma ozoa (SPZ) was compu ed as he p oduc o VOL and CON and also included in he analyses. Da a ega d- ing hese measu emen s a e shown in Table 1. S a is ics Pheno ypes used in GWA s udies we e ob ained om he spe m quali y da a desc ibed abo e. Fo ha , in a fi s s ep, we pe o med a mixed linea eg ession analysis ha allowed us o ob ain a pseudo- pheno ype o he GWAS o he second s ep. The ou spe m ai s, VOL, CON, SPZ, and MOT desc ibed abo e, e alua ed in a o al 27 635 ejacula es belonging o 429 ma u ed ams, we e conside ed as depen- den a iables in he analyses. A mixed linea eg ession analysis, in- cluding he am flock o o igin (51 le els), he da e o spe m collec ion (1806 le els), and he ejacula e numbe (2 le els, fi s o second jump) as fixed e ec s; he age o he am a spe m collec ion in mon hs as a co a ia e; and he am pe manen en i onmen al e ec as andom (429 le els), was fi ed. Male's indi idual es ima es adjus ed by ac o s included in he model we e ob ained and used as pseudo-pheno ypes (inpu s) in he GWAS. BLUPF90 sui e (Misz al e al., 2002) was used o un he mixed linea eg ession analyses. Once pseudo-pheno ypes we e ob ained, a GWAS was ca ied ou in a second s ep. F om he 429 pheno yped ams, only 342 ha e 50 K SNP A yme ix mic oa ay geno ypes (47 702 SNPs) in he cu en geno- yped Assa popula ion. F om hese 342 males geno yped wi h he 50 K mic oa ay, 47, we e also geno yped wi h he HD Illumina O ineBeadChip (604 317 SNPs). Medium densi y 50 K and HD geno yp- ing pla o ms ha e in common 43 511 SNPs. Impu a ion o 50 K o HD geno ypes was conduc ed wi h BEAGLE4.0 so wa e (B owning and B owning, 2007). Only geno ypes wi h an impu a ion p obabili y highe o equal o 95% we e e ained. A e impu a ion and fil e ing by SNPs call a e (0.9), indi idual call a e (0.9), and MAF (0.001), a o al o 540 411 SNPs dis ibu ed on he 26 o ine au osomes and X ch omo- some om 342 ams we e included in subsequen analyses. GWAS was conduc ed wi h he mixed linea model based associa ion analysis (MLMA) o he genome-wide complex ai analysis so wa e (Yang e al., 2011) o he whole genome and including a gene ic ela ionship ma ix o con ol o he andom e ec s o gene ic simila i y and exclud- ing he ch omosome on which he candida e SNP is loca ed (lea ing- one-ch omosome-ou LOCO) applying he ollowing model: yj¼uþSNPiþgjþej whe e y j is he pseudo-pheno ype o he spe m ai analyzed o he geno yped animal j;uis he o e all mean; SNP is he e ec o he i SNP (assumed as a co a ia e coded as 0, 1, o 2, espec i ely, o Table 1 Basic s a is ics o spe m ai s da a collec ed in he fi s and second ejacula es om Assa ams. T ai N Mean SD Min Max CV Volume (ml) 27 635 1.23 0.57 0.1 8 0.46 Volume 1s ejacula e (ml) 21 643 1.31 0.57 0.1 5 0.43 Volume 2nd ejacula e (ml) 5 887 0.91 0.40 0.9 5 0.44 Concen a ion (×10 6 spz.ml −1 ) 26 414 3 978.64 1 328.49 120 9 701 0.33 Concen a ion 1s ejacula e (×10 6 spz.ml −1 ) 20 858 4 088.13 1 358.45 120 9 636 0.33 Concen a ion 2nd ejacula e (×10 6 spz.ml −1 ) 5 453 3 569.43 1 119.33 120 9 701 0.31 Numbe o spe ma ozoa (×10 6 ) 26 411 5 191.88 3 165.30 109 37 968 0.61 Numbe o spe ma ozoa 1s ejacula e (×10 6 ) 20 857 5 657.60 3 261.03 109 25 170 0.58 Numbe o spe ma ozoa 2nd ejacula e (×10 6 ) 5 451 3 433.42 1 950.33 109 37 968 0.57 Mo ili y 25 699 4.82 0.58 1 5 0.12 Mo ili y 1s ejacula e 20 339 4.80 0.62 1 5 0.13 Mo ili y 2nd ejacula e 5 360 4.89 0.42 1 5 0.09 N: numbe o eco ds; Min: minimum alue; Max: maximum alue; Spz = spe ma ozoa numbe . M. Se ano, M. Ramón, J.H. Cal o e al. Animal xxx (2021) xxx 2 geno ypes aa, Aa, and AA); g j is he andom addi i e gene ic e ec ; and e j is he esidual e o . Ch omosome-wise significance associa ion was assessed using a alse disco e y a e (FDR mul i- es co ec ion h esh- old o 10%). The choice o a h eshold alue o 10% is jus ified because his wo k is mainly explo a o y and a fi s a emp o find a ian s asso- cia ed wi h spe ma ic cha ac e s in sheep, so we decided o be mo e conse a i e. Visualiza ion o he associa ion esul s was pe o med in Manha an plo s and quan ile-quan ile plo s using R so wa e (R co e Team, 2019). Va ian E ec P edic o om Ensembl (www.emsembl.o g)was used o anno a e significan SNPs de ec ed a he genome and ch omo- some le els. The posi ional candida e genes we e iden ified in he 250Kb egion on bo h sides o he significan SNPs acco ding o he sheep genome assembly Oa _ 3.1 and based on he Ensembl elease 81. Fo unc ional p o ein in e ac ion ne wo ks, he STRING so wa e was used (Szkla czyk e al., 2019;h ps://s ing-db.o g/). To examine he equencies o he SNPs associa ed wi h spe m ai s de ec ed in he GWAS analysis in an inc eased numbe o Assa animals, we used he whole popula ion cu en ly geno yped in his b eed,which consis ed o 1503 (1077 males and 426 emales) geno yped o he 50 K cus om pla o m and impu ed o he HD o ine BeadChip ( he 1077 males included he 342 pheno yped ams used in he GWAS analysis). Milk yield EBVs o he 10% op and 10% bo om anking o hese 1503 animals o he significan SNPs associa ed wi h spe m ai s we e s ud- ied in o de o deciphe hepu a i e ela ionship be ween milk p oduc- ion e ficiency and he equencies o hese a ian s. Resul s The mixed-e ec s linea model o spe m ai s All he fixed e ec s included in he model, he am flock o o igin, he da e o spe m collec ion, he ejacula e numbe , and he age o am a he collec ion showed a significan e ec on spe m ai s. Fig. 1 shows he es ima es and IC95% om he mixed-e ec s linea model o he da e o spe m collec ion (exp essed as mon h o collec ion, in Fig. 1), ejacula e numbe and age o am in mon hs o he ou spe m ai s conside ed. Ejacula es showed la ge VOL and SPZ (al hough CON dec ease, in pa due o highe VOL) du ing he ep oduc i e season ( om July o nex Janua y). A dec ease in VOL, CON, and SPZ om he fi s o he second ejacula e was obse ed bu showing he second ejacu- la e highe mo ili y han he fi s one. Rega ding he age o am, al hough significan , he magni ude o he e ec was always e y close o 0. Genome-wide associa ion s udy o spe m ai s A e he QC o geno ype da a om 342 ams, a o al o 540 411 SNPs dis ibu ed on he 26 au osomes and X o ine ch omosome we e included in subsequen analyses. Supplemen a y Table S1 shows genome-wide complex ai analysis esul s o significan SNPs a he ch omosome and genome-wide le el o he spe m ai s s udied. Genome-wide 10% FDR h eshold o MOT was se on a P- alue o 2.4 × 10 −5 . Ch omosome-wide 10% FDR h esholds co esponded o P- alues anging om 5.8 × 10 −6 o 1.2 × 10 −4 o MOT; om 2.3 × 10 −5 o 4.5 × 10 −5 o VOL; om 5.7 × 10 −6 o 4.8 × 10 −4 o CON; and om 2.3 × 10 −4 o 1.3 × 10 −4 o SPZ. Twen y- h ee SNPs dis ib- u ed in 17 di e en ch omosomes o e came he genome-wide signifi- cance le el o MOT ai . Fo VOL, CON, and SPZ, significan associa ionswe e oundonlya hech omosomele elinch omosomes: OAR6 (11 SNPs), OAR18 (6 SNPs), and OAR23 (3 SNPs) o VOL; OAR10 (3 SNPs), OAR13 (1 SNP), OAR16 (71 SNPs), and OAR26 (1 SNP) o CON; and OAR6 (32 SNPs) o SPZ. MLMA and LOCO app oaches yielded simila esul s o all ai s an- alyzed, and only esul s o he LOCO app oach a e shown. Fig. 2 shows Manha an and quan ile-quan ile plo s o he GWAS analyses o he ou spe m ai s s udied. quan ile-quan ile was plo ed o explo e ha ac ual dis ibu ion o P_ alues ob ained ollowed he expec ed dis- ibu ion and no o he e ec s, such popula ion s uc u e, we e missed. Supplemen a y Table S2 shows allele equencies and e ec s o signifi- can SNPs in he 342 ams used o he GWAS and in he cu en Assa geno yped popula ion (1503 animals). Fo VOL, in mos cases, he less equen allele (A1) o he associa ed SNP showed a nega i e e ec o e he ai wi h allele subs i u ion alues (b) anging be ween −0.07 and −0.15 and equencies om 0.06 o 0.49. In some cases, he A1 allele had a low posi i e e ec o e VOL (0.07 o 0.09) wi h an in e media e allele equencies ( om 0.32 o 0.49) (Supplemen a y Table S2). In gene al, he A1 alleles showed a nega i e e ec o e CON ai wi h b alues om −407 o −179 and equencies anging be ween 0.05 and 0.5. Howe e , also posi i e e ec s o he A1 alleles on CON we e obse ed ( om 181 o 242) a A1 equencies be ween 0.25 and 0.50 (Supplemen a y Table S2). In mos cases, he A1 e ec o e SPZ ai was highly posi i e ( om 426 and 760) o nega i e ( om −451 o −700) wi h e y simila e- quencies (be ween 0.08 and 0.50) (Supplemen a y Table S2). In e es ingly, he A1 allele exe s a nega i e subs i u ion e ec wi h magni udes anging be ween −1.62 and −0.24 and equencies be- ween 0.03 and 0.34, o all significan SNPs de ec ed in he GWA s udy o e he MOT ai (Supplemen a y Table S2). Supplemen a y Table S3 shows a ian e ec p edic ion esul s o significan SNPs associa ed wi h spe m ai s. Table 2 shows pu a i e causalgenes loca ed in he 250Kb egion on bo h sides o hesignifican SNPs o each ai , and he biological p ocesses and molecula unc ions in which hey a e in ol ed. Pheno ypes, i exis , a e also shown. Fo VOL ai , associa ions a he ch omosome le el o 20 SNPs lo- ca ed in OAR6, OAR18, and OAR 23 we e ound. Mos a ian s we e Fig. 1. Es ima es (poin s) and IC95% (segmen s) om mixed-e ec s linea model o he da e o spe m collec ion (exp essed he e as mon h o collec ion, Feb o Dec), ejacula e numbe (2nd ejacula e) and age o am in mon hs o he ou spe m ai s conside ed: spe m olume (VOL; ml, spe m concen a ion (CON; spe ma ozoa ×10 6 /ml, spe m mo ili y (MOT; 0–5 scale), and o al numbe o spe ma ozoa (SPZ; spe ma ozoa × 10 6 ). Le el o significance is ep esen ed by as e isks (***: P≤0.001; **: P≤0.01; *: P≤0.05). M. Se ano, M. Ramón, J.H. Cal o e al. Animal xxx (2021) xxx 3 loca ed in in ons om genes such as ARHGEF38 (Rho Guanine Nucleo ide Exchange Fac o 38)andPCDH7 (P o ocadhe in 7)inOAR6andCABLES1 (Cdk5 Abl enzyme subs a e 1) in OAR23. Howe e , wo missense a ian s (oa 3_OAR18_30765484, and oa 3_OAR18_30789799) wi h mode a e e ec (SIFT alues 0.41 and 0.12, espec i ely) we e ound in he SCAPER (S-phase cyclin A-associa ed p o ein in he ER)geneinOAR18,wi hallele equencies a ound 6% in bo h cases. Fo CON, 76 SNPs significan associa ed wi h he ai we e ound in OAR10, OAR13, OAR16, and OAR26, being he mos sig- nifican hose ound in OAR16 (71 SNPs). Also in his case, mos a ian s we e loca ed in in ons, ups eam and downs eam egions o genes such as DLEU7 (Dele ed in lymphocy ic leukemia 7)inOAR10; OSMR (Oncos a in M ecep o ), RANBP3L (RAN binding p o ein 3 like), NADK2 (NAD kinase 2, mi ochond ial), SKP2 (S-phase kinase associ- a ed p o ein 2), CAPSL (Calcyphosine like), IL7R (In e leukin 7 ecep o ), SPEF2 (Spe m flagella p o ein 2), and RAI14 (Re inoic acid induced 14) in OAR16; and SORBS2 (So bin and SH3 domain con aining 2)in OAR26. Two synonymous a ian s wi h low e ec , oa 3_OAR16_ 1971417 and oa 3_OAR16_36979589, we e de ec ed in DOCK2 (Dedica o o cy okinesis 2)andCPLANE1 (Ciliogenesis and plana pola - i y e ec o 1) genes o OAR16 wi h allele equencies o 13 and 5.6%, espec i ely. Fig. 2. Manha an and Q-Q plo s om genome-wide associa ion s udy (GWAS) o spe m ai s in Assa sheep. T ai s conside ed a e ejacula e olume (ml; Figs. A and B); spe m concen a ion (spe m ×10 6 /ml; Figs. C and D); spe m mo ili y (0–5 scale, Figs. E and F); and o al numbe o spe ma ozoa (spe m × 10 6 ; Figs. G and H). Dashed line in Manha an plo s (A, C, E, and G) co esponds o a e age h eshold alue o a FDR o 10% e alua ed a he ch omosome le el. M. Se ano, M. Ramón, J.H. Cal o e al. Animal xxx (2021) xxx 4 Fo he ai SPZ, 32 associa ed SNPs we e loca ed in in ons, up- s eam and downs eam egions o genes a OAR6. Some o he a ge genes we e CCSER1 (coiled-coil se ine ich p o ein 1), KCNIP4 (po assium ol age-ga ed channel in e ac ing p o ein 4), GBA3 (cy osolic be a-glucosidase), STIM2 (s omal in e ac ion molecule 2), and PARM1 (P os a e and ogen- egula ed mucin-like p o ein 1). Finally, 23 SNPs associa ed a he genome-wise le el wi h MOT we e loca ed dispe sed ac oss he whole genomea ch omosomes 1, 2,3,4, 6, 7, 9, 10, 12, 13, 15, 17, 19, 20, 22, and 26. All SNPs we e loca ed in in- ons, 3′UTR, splice and downs eam egions o genes such as SLC9C1 (Solu e ca ie amily 9 membe C1) in OAR1; TSN (T anslin) in OAR2; GRM8 (Glu ama e me abo opic ecep o 8) in OAR4; BMP2K (BMP2 in- ducible kinase)andPAQR3 (P oges in and adipoQ ecep o amily membe 3)inOAR6;NCALD (Neu ocalcin del a) in OAR9; DIP2C (Disco in e ac ing p o ein 2 homolog C)inOAR13;CMIP (c-Ma inducing p o ein) in OAR14; CUL9 (Cullin 9), CFB (Complemen ac o B) and C2 (Complemen C2)in OAR20; and FUT10 (Fucosyl ans e ase 10) in OAR 26. Discussion Among he spe m ai s collec ed ou inely in AI cen e s, only mass spe m mo ili y seems o be di ec ly ela ed wi h o e all e ili y (Colenb ande e al., 2003; Foo e, 2003;Theau-Clémen e al., 2011; Da id e al., 2015). Howe e , spe m olume and concen a iona e ai s wi h some in e es ega ding AI cen e s e ficiency o p oduce semen doses. He i abili y o o ine spe m ai s anges om e y low o mode a e. Fo MOT he i abili y in Lacaune and Assa b eeds akes alues o 0.07 and 0.03, espec i ely (Da id e al., 2007; Pelayo e al., 2019). Mode a e es ima es o 0.19 and 0.12 ha e been ound o CON and VOL, espec i ely, in Assa b eed. Highe es ima es o 0.27 and 0.18 o CON and VOL, espec i ely, we e ound in Lacaune b eed (D a ide al., 2007; Pelayo e al., 2019). Es ima es o gene ic co ela ion among hese ai s a e a iable ac oss sheep b eeds. In Manchega and Assa dai y b eeds, es ima es o gene ic co ela ions be ween VOL and CON we e −0.49 and −0.25, espec i ely; be ween VOL and MOT, −0.29 and −0.33, espec i ely; and be ween CON and MOT, 0.32 and 0.20, e- spec i ely (Pelayo e al., 2019). In addi ion, i has been desc ibed ha in ensi e selec ion in milk p oduc ion in ca le has been associa ed wi h an impai men o he e - ilizing capaci y (Be y e al., 2009). The e o e, he de ec ion o genes as- socia ed wi h he pheno ypes measu ed in AI cen e s is key o y o imp o e he ep oduc i e p ope ies o he spe m o AI ams. These ea- u es a e mo e impo an in pheno ypes such as MOT due o hei low he i abili y and i s ela ionships wi h he e ili y o he seminal doses. In he s udy he e conduc ed, some genomic egions ha e been associ- a ed wi h spe m quali y ai s, finding significan SNPs wi hin o close o genes unc ionally ela ed o ep oduc i e ai s in o he species. Ejacula e concen a ion Fo CON, a s ong signal has been de ec ed a ch omosome 16. The SNP oa 3_OAR16_1971417 is a synonymous a ian G > A loca ed a Table 2 Pu a i e causal genes de ec ed in he genome-wide associa ion s udy o spe m ai s in Assa ams. T ai Ch Gene symbol Gene name Biological p ocess, molecula unc ion, pheno ype CON 16 DOCK2 Dedica o o cy okinesis 2 Memb ane a pola iza ion, immune esponse, chemo axis, ac in cy oskele on o ganiza ion 16 CPLANE1 Ciliogenesis and plana pola i y e ec o 1 T ansmemb ane p o ein, ubiqui ous exp ession in es is (RPKM 2.7) 16 RANBP3L RAN binding p o ein 3 like In acellula anspo , benign p os a ic hype plasia 16 NADK2 NAD kinase 2, mi ochond ial NAD me abolic p ocess, egula ion o sys emic a e ial blood p essu e, calcium-media ed signaling 16 LMBRD2 LMBR1 domain con aining 2 In eg al componen o memb ane 16 CAPSL Calcyphosine like Calcium ion binding, high exp ession in es is (RPKM 3.9) 16 IL7R In e leukin 7 ecep o Cell g ow h, homeos asis o numbe o cells, se e e combined immunodeficiency 16 SPEF2 Spe m flagella 2 Spe ma ogenesis, e iliza ion, spe m mo ili y, immo ile sho - ail spe m 16 RAI14 Re inoic acid induced 14 Nucleoplasm, mi ochond ion, ac in cy oskele on, spe ma ogenesis MOT 1 SLC9C1 Solu e ca ie amily 9 membe C1 Spe m mo ili y, spe ma ogenesis, high exp ession in es is (RPKM 3.5) 2TSN T anslin DNA-binding p o ein, e y high exp ession in es is (RPKM 26.5) 4GRM8 Glu ama e me abo opic ecep o 8 Neu oac i e ligand– ecep o in e ac ion, glu ama e gic synapse 6 PAQR3 P oges in and adipoQ ecep o amily membe 3 Nega i e egula ion o p o ein phospho yla ion, mos exp essed in es is (RPKM 20.7) 6BMP2K BMP2 Inducible kinase Skele al de elopmen and pa e ning, p o ein phospho yla ion 9NCALD Neu ocalcin del a Neu onal calcium senso , egula ion o sys emic a e ial blood p essu e 13 DIP2C Disco in e ac ing p o ein 2 homolog C Plasma u ic acid le els, AMP binding 14 CMIP c-Ma inducing p o ein T-cell signaling pa hway, in u e o emb yonic de elopmen 20 CUL9 Cullin 9 Mic o ubule cy oskele on o ganiza ion, egula ion o mi osis, mos exp essed in es is (RPKM 6.1) 20 CFB Complemen ac o B Complemen ac i a ion, egula ion o he immune eac ion 20 C2 Complemen C2 ac o B Complemen ac i a ion, au oimmune diseases, esponse o nu ien 26 FUT10 Fucosyl ans e ase 10 Fe iliza ion, p o ein olding, wound healing VOL 6 ARHGEF38 Rho guanine nucleo ide exchange ac o 38 Regula ion o Rho p o ein signal ansduc ion, mos exp essed in p os a e (RPKM 3.4) 6TIGD2 Tigge ansposable elemen de i ed 2 DNA binding 6PCDH7 P o ocadhe in 7 Cell–cell ecogni ion and adhesion, calcium ion binding 18 SCAPER S-phase cyclin A associa ed p o ein in he ER Nucleic acid binding, zinc ion binding, mos exp essed in es is (RPKM 4.5) 18 PSMA4 P o easome subuni alpha 4 Co e alpha subuni o he 20S p o eosome, ubiqui in-dependen p o ein ca abolic p ocess 23 CABLES1 Cdk5 and Abl enzyme subs a e 1 P o ein binding, egula ion o cell cycle, Body Mass Index SPZ 6 CCSER1 Coiled-coil se ine ich p o ein 1 DNA me hyla ion, gas oin es inal mic obiome, 6KCNIP4 Po assium ol age-ga ed channel in e ac ing p o ein 4 Regula ion o po assium ion ansmemb ane anspo 6GBA3 Cy osolic be a-glucosidase Be a-glucoside ca abolic p ocess, p o ein s abiliza ion 6STIM2 s omal in e ac ion molecule 2 Cellula calcium ion homeos asis, ac i a ion o s o e-ope a ed calcium channel ac i i y 6OCIAD1 OCIA domain con aining 1 Regula ion o s em cell di e en ia ion, p o ein binding, o a ian cance 6HOPX HOP homeobox Chape one-media ed p o ein assembly, nega i e egula ion o cell di e en ia ion, his one deace yla ion 6LOC101110593 RE1-silencing ansc ip ion ac o -like Nega i e egula ion o cell p oli e a ion, cellula esponse o elec ical s imulus 6PARM1 P os a e and ogen- egula ed mucin-like p o ein 1 Posi i e egula ion o elome ase ac i i y Ch = ch omosome; CON = spe m concen a ion; MOT = mass spe m mo ili y; VOL = ejacula e olume; SPZ = numbe o spe ma ozoa. M. Se ano, M. Ramón, J.H. Cal o e al. Animal xxx (2021) xxx 5 exon 32 o he DOCK2 gene. Thisgeneencodes a la ge p o ein o 180 kDa in ol ed in in acellula signaling ne wo ks and highly ela ed o he immune esponse. DOCK2 deficiency has been ela ed o placen al ab up ion and p os a e cance . Fu he mo e, Liu e al. (2019) e ealed ha he me hyla ion o DOCK2 in ca le was associa ed wi h a po en ial ole in he egula ion o male e ili y, sugges ing ha spe m me hyla- ion pa e ns influence spe m e ili y. Then, his SNP could be in linkage disequilib ium wi h o he mu a ions in he p omo e o egula ion e- gions o he gene ha could a ec CpG islands. The SNP oa 3_OAR16_36979589 ( s421824327) is a synonymous a ian G > A loca ed a exon 11 o he CPLANE1 (ciliogenesis and plana pola i y e ec o 1) gene which is in ol ed in se e albiological p ocesses such as cilium assembly and p o ein localiza ion o he cilia y ansi ion zone. Mu a ions o his gene (also known as Jb s17) p oduce he Joube synd ome, Meckel synd ome, and o al- acial-digi al synd ome and cause ciliogenesis and cilia y a ficking de ec s esul ing in a dec eased o he SHH (Sonic Hedgehog Signaling Molecule) a ge gene exp ession (To iyama e al., 2016). The sonic hedgehog signaling pa hway is impo - an in he mu ine epididymis o he de elopmen o spe m mo ili y (Tu ne e al., 2006). The SNPs oa 3_OAR16_38356302 C > A, oa 3_OAR16_38429385 A > G, oa 3_OAR16_38445552 C > A, and oa 3_OAR16_38480186 A > G a e in- on a ian s loca ed a he SPEF2 gene. SPEF2 plays an impo an ole in spe ma ogenesis and flagella assembly (Si onen e al., 2011) and is exp essed in all cilia ed cells and equi ed o cilia unc ion (Si onen e al., 2010). Fig. 3 (a) shows he unc ional p o ein associa ion ne wo k o he SPEF2 gene. This ne wo k in ol es se e al genes ela ed o spe m cells p ope ies. In humans, a ian s o SPEF2 gene a e associa ed wi h many spe m anomalies such as oligozoospe mia, sho spe m flagellum, and abno mal spe ma ogenesis and also wi h in e ili y (Liu e al., 2020). P obably, hese in on a ian s a e no causal mu a ions a ec ing CON bu should be in linkage disequilib ium wi h o he mu a ions in coding egions o he SPEF2 gene, no included in he geno yping pla o m. Fou SNPs, oa 3_OAR16_39293892 G > A, oa 3_OAR16_39321712 G > A, oa 3_OAR16_39334360 G > A, and AX-124358876 ( s410614862) T > C, loca ed a in ons 1 and 2 o he RAI14 (Re inoic acid induced 14, also named NORPEG) gene, we e associa ed wi h he a iabili y on CON. Rai14 is an ac in binding p o ein which is exp essed in he es is by bo h Se oli and ge m cells in he semini e ous epi helium. Rai14 egu- la esspe ma idpola i yandspe ma id anspo du ingspe miogenesis (Qian e al., 2013) and is also in ol ed in he eo ganiza ion o ac in fil- amen s in Se oli cells du ing he epi helial cycle, pa icipa ing in con- e ing spe ma id cell adhesion in he es is (Qian e al., 2013b). Ejacula e olume Two missense a ian s in OAR18, oa 3_OAR18_30765484 G > A and oa 3_OAR18_30789799 A > G loca ed in exons 24 and 23 o he SCAPER gene, espec i ely, p oduce and aminoacid change o Th eonine o Isoleucine and Phenylalanine o Leucine, espec i ely, ha a e p edic ed as ole a ed changes wi h SIFT alues o 0.41 and 0.12, espec i ely. The SCAPER (S phase cyclin A-associa ed p o ein in he endoplasmic e iculum) gene encodes a ansmemb ane anspo p o ein which is highly exp essed in es icle (P o eomicsDB, h ps://www.p o eomicsdb.o g/). In humans, SCAPER has been associa ed wi h e ini is pigmen osa (Ta ou e al., 2017) and also wi h hypogonadism (a dec eased unc- ionali yo hegonad)and abno mal es ismo phology (Human Pheno- ype On ology, h ps://www.geneca ds.o g/cgi-bin/ca ddisp.pl?gene= SCAPER). Fig. 3 (b) shows he unc ional p o ein associa ion ne wo k o he SCAPER gene. The SNPs oa 3_OAR18_29981728 A > C ( s415094151) and oa 3_OAR18_29985155 G > A a e downs eam gene a ian s o he PSMA4 (P o easome 20S subuni alpha 4) gene loca ed a OAR18. PSMA4 is a componen o he 20S co e p o easome complex in ol ed in he p o eoly ic deg ada ion o mos in acellula p o eins. The 20S p o easome media es ubiqui in-independen p o ein deg ada ion, which is equi ed in se e al pa hways including spe ma ogenesis (20S-PA200 complex). Many p o eins such asmeio ic p o eins, co e his- ones, and unnecessa y o ganelles a e deg aded du ing spe ma ogene- sis. I has been demons a ed ha he exp ession o PSMA8 (P o easome 20S Subuni Alpha 8), which also be pa o he 20S p o ea- some, in spe ma ocy es, is essen ial o mice male e ili y (Zhang e al., 2019). Howe e , emale e ili y does no equi e PSMA8 (Zhang e al., 2019). Al hough he e a e no da a abou PMSA4 in ol emen in males' e ili y, i should sha e some o he p ope ies desc ibed o PMSA8, because i 's high exp ession in es is and ole in he 20S p o easome. The TET2 (Te me hylcy osine dioxygenase 2) gene is loca ed 332 bp downs eam he oa 3_OAR6_19723487 a OAR6 (ARHGEF38). The p o- ein encoded by his gene is a me hylcy osine dioxygenase ha ca a- lyzes he con e sion o me hylcy osine o 5-hyd oxyme hylcy osine and plays a key ole in ac i e DNA deme hyla ion. Exp ession le els o his gene du ing human spe ma ogenesis a e pi o al o male e ili y (Ni e al., 2016). Au ho s showed ha le els o mRNA iso o ms o he TET genes in spe ma ozoa associa ed wi h spe m pa ame e s and we e significan ly educed in sub e ile men. Ejacula e numbe o spe ma ozoa Fo he ejacula e numbe o SPZ all significan associa ed SNPs we e loca ed a OAR6. The SNP oa 3_OAR6_89628167 G > A is an in on a - ian loca ed a PARM1 (P os a e and ogen- egula ed mucin-like p o ein 1) gene. Pa m1 is a highly glycosyla ed, mucin-like ype 1 ansmemb ane p o ein in ol ed in he posi i e egula ion o elome ase ac i i y, in he su i al o p os a e cells (Co ne e al., 2003), and con ibu es o o ula- ion and/o lu eal unc ion by ac ing as a egula o o p oges e one me- abolism (Pa k e al., 2013). In ca le, a SNP ( s111027720) loca ed in hePARM1 gene has been associa ed wi h he de elopmen o clea ed emb yos o he blas ocys s age in “in i o” e iliza ion p ocedu es Fig. 3. Func ional p o ein associa ion ne wo ks o SPEF2 (a) SCAPER (b) and SLC9C1 (c) genes. M. Se ano, M. Ramón, J.H. Cal o e al. Animal xxx (2021) xxx 6 (Coch an e al., 2013a). The same au ho s ound an SNP in he PARM1 gene associa ed wi h some e ili y ai s such as hei e s' concep ion a e, daugh e p egnancy a e, and bull p oduc i e li e in Hols ein ca le (Coch an e al., 2013b). Th ee SNPs, oa 3_OAR6_72193906 A > G, oa 3_OAR6_72207209 G > A, and oa 3_OAR6_72211624 A > G loca ed a in on, ups eam and downs eam he LOC101110593 (RE1-silencing ansc ip ion ac o -like) gene, we e posi i ely associa ed wi h SPZ. LOC101110593 is in ol ed in whole o ganism dec eased e ili y and emb yo de elopmen educed a e (Thako e-Shah e al., 2015). Spe ma ozoa mass mo ili y Fo MOT ai , SNPs associa ions we e ound dispe sed ac oss he whole genome. The SNP oa 3_OAR1_175397925 C > A a ch omosome 1 is an in on 6 a ian o he SLC9C1 (Solu e ca ie amily 9 membe C1, also named Spe m-Specific Na(+)/H(+) Exchange and Spe m-NHE) gene. SLC9C1 is a membe o he sodium-hyd ogen exchange (NHE) amily and is equi ed o male e ili y and spe m mo ili y (Wang e al., 2003). Spe m-specific sodium/hyd ogen exchange is in ol ed in in acellula pH egula ion o spe ma ozoa and equi ed o spe m mo ili y and e ili y. Also has a ole in spe m cell hype ac i a ion, a c i ical s ep o spe m mo ili y essen ial in he p epa a ion o spe m o e iliza ion. SLC9C1 is an in eg al componen o he plasma mem- b ane as a channel anspo o ions, glucose, o he suga s, sal s, o ganic acids, me al ions, and amine compounds. Fig. 3 (c) shows he unc ional p o ein associa ion ne wo k o he SLC9C1 gene. The ADCY10 (Adenyla e cyclase ype 10, Epididymis Sec e o y Spe m Binding P o ein Li 7a)gene,a p edic ed unc ional pa ne , in e ac s and is co-exp essed wi h SLC9C1. ADCY10 (also named Tes icula Soluble Adenylyl Cyclase SAC)playsac i - ical ole in mammalian spe ma ogenesis by p oducing he cAMP which egula es cAMP- esponsi e nuclea ac o s indispensable o spe m ma u a ion in he epididymis. Gene ic app oaches ha e demons a ed ha Adcy10 is necessa y o male e ili y and specifically o spe m mo- ili y and capaci a ion (Xie e al., 2006). The a ian oa 3_OAR2_185913749 G > A is a 3′-UTR loca ed a exon 6o heTSN (T anslin, also named Tes is B ain-RNA Binding P o ein) gene. The 3′-UTR egion plays a c ucial ole in gene exp ession by influencing he localiza ion, s abili y, expo , and ansla ion e ficiency o an mRNA. The 3′-UTR can influence polyadenyla ion, ansla ion e ficiency, localiza ion, and s abili y o he mRNA since i con ains bo h binding si es o egula o y p o eins as well as mic oRNAs. This gene is highly exp essed in b ain and es is and is in ol ed in DNA damage epai and in mRNA anspo (Cho e al., 2004; Wang e al., 2004). The p o ein T anslin is in ol ed in ansla ional egula ion du ing spe ma ogenesis, and binds o specific mRNAs in he es is, o ming an RNP complex (Mo ales e al., 2002). Mice lacking he TB RBP gene ( he TSN mouse o hologue) can si e o sp ing, bu ha e educed spe m p oduc ion (Chenna hukuzhi e al., 2003). The SNP oa 3_OAR26_27800594 C > A SNP is an in on 2 a ian o he FUT10 (Fucosyl ans e ase 10)gene.FUT10 encodes a ucosyl ans e ase p o ein wi h oles in p o ein olding, glycosyla ion, ne ous sys em de el- opmen , and e iliza ion. FUT10 is highly exp essed in es is (6.67 TPM) and in ca le has been associa ed wi h he ejacula e olume (Qin e al., 2016). Despi e he mode a e posi i eco ela ion exis ing be weenCON and MOT in Assa sheep b eed (0.20), any common gene o genomic egion was ound in he GWAS o bo h ai s. Rela ionship o candida e genes equencies and es ima ed b eeding alues o milk yield Rela ionship be ween spe m ai s s udied and milk yield was ex- plo ed. Fo ha , 1503 animals wi h geno ypes men ioned abo e we e anked based on hei EBVs o milk yield (pe sonal communica ion, Assa sheep b eeding p og am). Then, op and bo om 10% animals we e chosen, and allele equencies o he mos significan SNPs ound in his wo k we e compa ed be ween bo h, op and bo om ani- mals. Table 3 shows allele equencies o he mos significan SNPs ound o spe m quali y ai s in he 10% op and 10% bo om anking o EBVs o milk yield in 1503 Assa animals, and hedi e ence be ween hem in bo h g oups (A1 EBVnega i e-A1 EBVposi i e). In gene al o all ai s, equencies o he A1 alleles a e e y simila in bo h g oups o animals. Howe e , wo SNPs o CON in he RAI14 gene wi h nega i e e ec o e he ai and 2 SNPs o VOL in he PSMA4 gene wi h posi i e e ec o e he ai showed significan highe A1 equen- cies in he g oup o animals wi h nega i e EBVs o milk yield. Fo he ejacula e numbe o SPZ he 3 SNPs loca ed in he LOC101110593 gene, which exe a posi i e e ec o e he ai , ha e highe A1 Table 3 Allele equencies o mos significan single-nucleo ide polymo phisms o spe m quali y ai s in he op and bo om 10% anking o he milk yield es ima ed b eeding alues (EBV) o 1 503 Assa animals geno yped wi h he 50 K A yme ix cus om chip and impu ed o he HD Illumina BeadChip. EBV milk yield nega i e EBV milk yield posi i e Ma ke ID A1 > A2 Gene b T ai N eq A1 eq A2 N eq A1 eq A2 Di oa 3_OAR16_36979589 G > A CPLANE1 −407.02 CON 75 0.13 0.87 75 0.11 0.89 0.02 oa 3_OAR16_1971417 G > A DOCK2 −269.76 CON 75 0.21 0.79 75 0.13 0.87 0.08 oa 3_OAR16_39293892 G > A RAI14 −255.00 CON 75 0.34 0.66 75 0.29 0.71 0.05 oa 3_OAR16_39321712 G > A RAI14 −207.40 CON 75 0.37 0.63 75 0.29 0.71 0.07 oa 3_OAR16_39334360 G > A RAI14 −229.23 CON 75 0.41 0.59 75 0.26 0.74 0.15 AX-124358876 T > C RAI14 −224.85 CON 75 0.46 0.54 75 0.30 0.70 0.16 oa 3_OAR16_38429385 A > G SPEF2 −290.53 CON 75 0.23 0.77 75 0.21 0.79 0.02 oa 3_OAR16_38480186 A > G SPEF2 −350.15 CON 75 0.25 0.75 75 0.21 0.79 0.05 oa 3_OAR16_38356302 C > A SPEF2 −206.52 CON 75 0.37 0.63 75 0.31 0.69 0.06 oa 3_OAR16_38445552 C > A SPEF2 −188.11 CON 75 0.42 0.58 75 0.47 0.53 −0.05 AX-169032210 A > G CFB/C2 −0.63 MOT 75 0.06 0.94 75 0.06 0.94 0.00 oa 3_OAR26_27800594 C > A FUT10 −0.73 MOT 75 0.18 0.82 75 0.22 0.78 −0.04 oa 3_OAR1_175397925 C > A SLC9C1 −1.36 MOT 75 0.06 0.94 75 0.02 0.98 0.04 oa 3_OAR2_185913749 G > A TSN −0.94 MOT 75 0.08 0.92 75 0.06 0.94 0.02 oa 3_OAR6_72193906 A > G LOC101110593 659.10 SPZ 75 0.13 0.87 75 0.16 0.84 −0.03 oa 3_OAR6_72211624 A > G LOC101110593 676.75 SPZ 75 0.06 0.94 75 0.13 0.87 −0.07 oa 3_OAR6_72207209 G > A LOC101110593 647.38 SPZ 75 0.11 0.89 75 0.16 0.84 −0.05 oa 3_OAR6_89628167 G > A PARM1 −538.34 SPZ 75 0.33 0.67 75 0.39 0.61 −0.06 oa 3_OAR18_29981728 A > C PSMA4 0.07 VOL 75 0.51 0.49 75 0.39 0.61 0.13 oa 3_OAR18_29985155 G > A PSMA4 0.08 VOL 75 0.56 0.44 75 0.43 0.57 0.13 oa 3_OAR18_30789799 A > G SCAPER −0.15 VOL 75 0.10 0.90 75 0.04 0.96 0.06 oa 3_OAR18_30765484 G > A SCAPER −0.15 VOL 75 0.11 0.89 75 0.07 0.93 0.04 A1 = less equen allele; b = allele subs i u ion e ec ; N = numbe o eco ds; Di = equency A1 in he EBV nega i e g oup – equency A1 in he EBV-posi i e g oup. T ai = spe m ai (CON: spe m concen a ion; MOT: mass spe m mo ili y; SPZ: numbe o spe ma ozoa; VOL: ejacula e olume). M. Se ano, M. Ramón, J.H. Cal o e al. Animal xxx (2021) xxx 7 equencies in he op EBVs g oup o animals, bu also ha loca ed in he PARM1 gene wi h nega i e e ec o e SPZ. Despi e he small numbe o animals analyzed, i seems ha hese SNPs a e no linked wi h genes in- ol ed in milk p oduc ion. Howe e , he high equencies ound o some SNPs wi h nega i e e ec o e spe m ai s in he op milk yield EBVs g oup, would allow exe ing some selec ion p essu e o e hem o imp o e spe m cha ac e is ics in he high me i animals o milk p oduc ion. Mos SNPs ha we e significan ly associa ed wi h some spe m ai belongs exclusi ely o he HD o ine Illumina BeadCheap. This ac indi- ca es ha high-densi y SNP pla o ms a e mo e sui able o de ec genes o genomic egions ela ed o animal cha ac e is ics, a leas in his sheep b eed. In gene al, sheep b eeds show a low linkage disequilib- ium han o he li es ock species, such as ca le and pigs, among ma ke s om low densi y geno yping chips, as he 50K A yme ix pla - o m. Some o hegenes he e de ec ed in associa ion wi h spe m quali y ai s could be used in ma ke assis ed selec ion o ams o be used in AI cen e s o p ogeny es in dai y b eeding p og ams. In summa y, he GWA s udies conduc ed in his wo k ha e e ealed he exis ence o genomic egions and some pu a i e causal genes asso- cia ed wi h spe m quali y ai s. Among hem, SPEF2 o ejacula e con- cen a ion, SCAPER and PSMA4 o ejacula e olume, PARM1 o ejacula e numbe o spe ma ozoa, and SLC9C1 and FUT10 o spe m mass mo ili y seem o be good candida e genes o imp o e spe m qual- i y ai s in sheep. Mo e s udies inc easing he numbe o geno yped and pheno yped animals will be necessa y o alida e he magni ude o he associa ions he e de ec ed and o es ablish he possibili y o using hese genes o imp o e ams’spe m ai s. Supplemen a y ma e ials Supplemen a y da a o his a icle can be ound online a h ps://doi. o g/10.1016/j.animal.2020.100065. E hics app o al No applicable. Da a and model a ailabili y s a emen OVIGEN AI cen e is he owne o pheno ypic da a and geno ypes om Assa animals. None o he da a we e deposi ed in an o ficial epos- i o y bu a ailable upon eques s which mus be accompanied by a de- sc ip ion o wha he da a will be used o . Au ho ORCIDs Malena Se ano ORCID 0000–0002–1621-3102. Au ho con ibu ions Malena Se ano: Concep ualiza ion, o mal analysis, in es iga ion, esou ces, w i ing –o iginal d a , w i ing – e iew & Edi ing. Manuel Ramón: Fo mal analysis, W i ing –O iginal D a .Jo ge H Cal o: w i ing – e iew & edi ing, isualiza ion. Mª Ángeles Jiménez: Da a cu a ion. Fe nando F ei e: Funding acquisi ion, esou ces. José Manuel Vázquez: Resou ces. Juan José A anz: W i ing – e iew & edi ing. Decla a ion o in e es None. 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