Seasonal changes of nuclear DNA fragmentation in boar spermatozoa in Spain

animals A icle Seasonal Changes o Nuclea DNA F agmen a ion in Boa Spe ma ozoa in Spain Raquel Ausejo 1,2, Juan Manuel Ma ínez 3,*, Ped o Sole -Llo ens 2, Al onso Bola ín1, Te esa Tejedo 4 and Ma ía Vic o ia Falce o 1   Ci a ion: Ausejo, R.; Ma ínez, J.M.; Sole -Llo ens, P.; Bola ín, A.; Tejedo , T.; Falce o, M.V. Seasonal Changes o Nuclea DNA F agmen a ion in Boa Spe ma ozoa in Spain. Animals 2021, 11, 465. h ps://doi.o g/10.3390/ ani11020465 Academic Edi o s: Es e Bel án F u os and Luis Miguel Pas o Ga cía Recei ed: 31 Decembe 2020 Accep ed: 5 Feb ua y 2021 Published: 9 Feb ua y 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 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/). 1 Depa men o Animal Pa hology, Obs e ics and Rep oduc ion, Facul y o Ve e ina y Medicine, Uni e si y o Za agoza, 50013 Za agoza, Spain; s e e ina ios@magapo .com (R.A.); [email p o ec ed] (A.B.); alce o@uniza .es (M.V.F.) 2Depa men o Bio echnology R&D, Magapo S.L., 50600 Ejea de los Caballe os, Spain; bio ecnologia@magapo .com 3Depa men o Resea ch and De elopmen , Magapo A.I.E., 50013 Za agoza, Spain 4 Depa men o Ana omy, Emb yology and Gene ics, CIBERCV, Facul y o Ve e ina y Medicine, Uni e si y o Za agoza, 50013 Za agoza, Spain; ejedo @uniza .es *Co espondence: in es igacion@magapo .com Simple Summa y: A i icial insemina ion is widely used in pig p oduc ion and cu en ly a boa pe o ms se e al housand ma ings pe yea . T adi ionally spe m quali y is ocused on he numbe o spe ma ozoa, hei mo ili y and mo phology. Howe e , he quali y o spe m DNA, which con ains gene ic in o ma ion, is also ela ed o e ili y p oblems. The aim o his esea ch was o s udy he e ec o na u al ligh hou s and age o he boa on he s a us o he spe m DNA. A e a powe ul s a is ical analysis, i was ound ha he pe cen age o spe ma ozoa wi h agmen ed DNA dec eases wi hin he obse ed age ange as he boa ge s olde . On he o he hand, he amoun o spe ma ozoa wi h agmen ed DNA was he lowes in au umn while i was he highes in summe . This s udy demons a es he emaining seasonali y o boa s in Spain and highligh s he impo ance o con olling he en i onmen al condi ions in he a ms. Spe m DNA es ing p o ides a basis o imp o ing he selec ion o AI boa s by excluding males wi h highe DNA agmen a ion due o hei e y young ep oduc i e age ha may pose a po en ial sub e ili y. Abs ac : The e a e nume ous cases when con en ional spe miog am pa ame e s a e all wi hin an accep able ange bu boa sub e ili y pe sis s. The o al spe m nuclea DNA agmen a ion index ( DFI) is a ai ela ed o e ili y and p oli icacy p oblems ha is no ou inely e alua ed in comme cial AI boa s. The aim o his esea ch was o s udy he e ec o he pho ope iod, season and ep oduc i e age o he boa on DFI (measu ed by SCSA) o 1279 ejacula es om 372 di e en boa s belonging o 6 di e en b eeds loca ed in 6 AI s uds in Spain. DFI da a anged om 0.018% o 20.1%. Al hough he e was a signi ican single boa e ec in he DFI occu ence, a nega i e co ela ion be ween he DFI and he age o he boa was ound (p< 0.001). DFI would dec ease due o aging o he boa 0.66% each yea old wi hin he obse ed age ange. A e including age as a co a ia e in he ANCOVA, no di e ences we e ound in DFI be ween pho ope iods when he spe m collec ion da e was e alua ed. Howe e , when he da e o he p oduc ion o semen in he es is was e alua ed, he o al pe cen age o spe ma ozoa wi h agmen ed nuclea DNA was 1.46% highe in he inc easing pho ope iod in compa ison o he dec easing pho ope iod (p< 0.0001). On he o he hand, o bo h da es, he lowes DFI alues co esponded o minimum day leng h o dec easing pho ope iod phase (au umn), while he highes DFI alues we e ound in summe (maximum day leng h o dec easing pho ope iod phase). Keywo ds: pho ope iod; boa ; spe m; DNA agmen a ion; spe ma ogenesis Animals 2021,11, 465. h ps://doi.o g/10.3390/ani11020465 h ps://www.mdpi.com/jou nal/animals Animals 2021,11, 465 2 o 13 1. In oduc ion The compe i i eness o indus ial pig p oduc ion depends, among o he ac o s, on he ep oduc i e ou pu ha is go e ned by many elemen s a ec ing bo h emales and males. The e is a la ge a ia ion in he e ili y esul s, mainly caused by a m- and sow- ela ed pa ame e s ( e iewed by Knox, 2016), and a i icial insemina ion (AI), he mos impo an ool used o sp ead he gene ic imp o emen in he pig indus y [ 1 – 3 ]. Based on he cu en expe ience o he boa AI cen e s in Spain (AIM Ibé ica (Topigs No s in Spain), pe sonal communica ion), 2000 sows and/o gil s a e being insemina ed wi h ejacula es om jus one boa . Ne e heless, he aim o 6000 insemina ions pe yea wi h one boa ’s ejacula es is an expec a ion no ye accomplished ( e iewed in Roca e al., 2016 [ 4 ]). The con en ional app oach o e alua ing spe m quali y is mainly ocused on spe m mo ili y, he o al coun o spe m cells in he dose and spe m mo phology [ 5 ]. These pa ame e s ha e been adi ionally associa ed wi h male e ili y. When spe m mo ili y o mo phology a e poo , he boa is no longe conside ed as a alid b eeding animal, o he numbe o spe m cells in he AI dose is inc eased in he comme cial AI dose. Ne e heless, he e a e some semen ai s a ec ing e ili y ha a e no compensable by adding mo e spe m cells in he AI dose [ 6 ] o hey a e no iden i ied in he con en ional spe miog am [ 4 ] hus occu ing a a molecula le el [7]. Howe e , he e a e nume ous cases whe e semen pa ame e s a e all wi hin an ac- cep able ange, bu he male ac o pe sis s o educed e ili y [ 8 ]. When good quali y semen doses a e used, he e is s ill a ound a 6% e ili y a ia ion a he a m le el due o boa - ela ed ac o s [ 9 , 10 ]. The e o e, i is c ucial o iden i y hese o he boa - ela ed ac o s ha de ine sub e ile boa s, ideally be o e en e ing he b eeding he d. This would allow pig p oduce s an oppo uni y o enhance o e all ep oduc i e e iciency and imp o e he gene ic dissemina ion o high me i animals. Damaged spe m ch oma in may impai he capabili y o he spe ma ozoa o e ilize an oocy e, dec ease insemina ion success, cause abo ion o e al abno mali ies and e en educe o sp ing i ali y [ 11 ]. The spe m ch oma in s abili y assay (SCSA) is a simple es ha assesses spe m nuclea ch oma in s a us by low cy ome y using ac idine o ange (AO) luo och ome [ 12 ]. Boa spe m nuclea DNA is no mally highly condensed and s able; he SCSA de ec s he abno mal ch oma in s uc u e and po en ial DNA damage ha depends on he suscep ibili y o DNA o dena u a ion [ 13 , 14 ]. The esul s ob ained om he SCSA a e no co ela ed wi h o he con en ional semen pa ame e s and can be conside ed an independen pa ame e [15]. Spe m quali y can be in luenced by mul i ac o ial gene ic ac o s [ 16 ] bu also en i on- men al ac o s such as s ess and seasonali y [ 17 – 19 ]. Especially in empe a e clima es, empe - a u e and pho ope iod show he mos impo an e ec s on pig ep oduc ion [20–22] . P e ious s udies de o ed o e alua ing he ela ionship be ween semen quali y in boa s and he in lu- ence o seasonali y a e ambiguous [ 23 , 24 ]. In indus ial pig p oduc ion ( Sus sc o a domes ica ), a clea d op in male e ili y has been obse ed in he wa m summe mon hs, possibly due o hea s ess [25]. In indus ial pig p oduc ion, i is hough ha spe m olume and concen a- ion a e lowes in sp ing; hey g adually inc ease du ing he summe and each a peak in la e au umn [ 26 ]. Likewise, a daily a ia ion in empe a u e g ea e han 10 ◦ C has been iden i ied as a dep esso o spe m p oduc ion [ 21 ]. In any case, animals ea ed in indus ial AI s uds a e no comple ely exposed o he en i onmen al condi ions, bu ha e hea ing, a i icial ligh and ans so he season in luence could be diminished. Rega ding molecula spe m quali y, while boa -spe ma ozoa collec ed in sp ing– summe appea ed o ha e a ela i ely highe pe cen age o spe ma ozoa wi h damaged nuclea DNA, a signi ican inc ease was only e iden in ac iona ed ejacula es om wo ou o i e boa s [ 25 ]. Con a ily, Pe ocelli e al. (2015) [ 27 ] epo ed ha season, pho ope iod o gene ic line did no a ec spe m nuclea DNA agmen a ion. Howe e , Peña e al. (2019) [ 14 ] desc ibed ha a opical summe induces nuclea DNA damage and educes spe m concen a ion wi hou dep essing mo ili y sugges ing ha adi ional me hods o e alua ing spe m mo ili y may no de ec inhe en ly comp omised spe ma ozoa. Animals 2021,11, 465 3 o 13 To he bes o ou knowledge, all he s udies abou pho ope iod and spe m nuclea DNA agmen a ion in ol ed a limi ed numbe o boa s and we e pe o med wi h di e en analysis me hods o DNA agmen a ion index (DFI) de e mina ion o ha e been pe - o med in he sou he n hemisphe e wi h e y di e en clima es and seasonali y o Spanish la i ude. On he o he hand, he p esen esea ch is based on a e ospec i e s udy o a la ge numbe o ejacula es p oduced be ween 2014 and 2019 in di e en AI s uds loca ed in di e en pa s o Spain, all o hem analyzed wi h he same sys em. Besides, p e ious s udies do no ake in o accoun o he boa a iables ha could in luence DFI, as is he case o he age o he boa [28]. The aim o his esea ch was o s udy he e ec o pho ope iod, season and age o he boa on he spe m nuclea DNA agmen a ion (measu ed by SCSA), a pa ame e ha is no ou inely e alua ed in comme cial AI boa s. 2. Ma e ial and Me hods The s udy included app o ed me hods and s anda d ope a ing p ocedu es o boa semen p ocessing. The AI s uds in his s udy complied wi h he Council Di ec i e, 2008/120/EC ou lining minimum s anda ds o he p o ec ion o pigs and Di ec i e, 2010/63/EU o he Eu opean Pa liamen and he Council o 22 Sep embe 2010 on he p o ec ion o animals used o scien i ic pu poses. 2.1. Boa s and AI S uds In ol ed Ba ns and Animals To al DNA agmen a ion index ( DFI, %) o 1279 ejacula es om 372 di e en boa s we e s udied. Da a we e collec ed in 6 di e en comme cial AI s uds loca ed in 6 di e en p o inces o Spain. AI s uds we e selec ed based on known common p oduc ion p oce- du es, simila housing sys ems and simila boa managemen and popula ion. The size o he AI s uds anged om 80 o 300 boa s. Boa s we e chosen andomly a he beginning o he s udy. They we e always alloca ed in indi idual c a es o >6 m 2 . All acili ies had wa e a ailable ad libi um, and animals we e ed 1–2 imes pe day a o al o 2.5–3.5 kg wi h a comme cial boa eed (9.2 MJ/kg ME, 15.5% p o ein, 4.5% ibe , 12,500 IU/kg i A and 170 IU/kg i E ). Al hough wea he condi ions di e ed among he s a ions, ligh exposu e was con olled in all o hem, wi h mo e han 150 lux a eye le el du ing 14–16 h pe day and limi ed con ac wi h na u al ligh . Tempe a u es inside he acili ies we e also con olled in all cases, anging om 11 (coldes , in win e ) o 28 ◦ C (ho es , in summe ). The bi h da e o each boa was eco ded. The dis ibu ion (mean ± SD) o he age o he boa s a collec ion was 19.6 ±9.5 mon hs. We s udied bo h he da e o collec ion and he assumed da e o es icula p oduc ion o semen (de ined as 50 days be o e collec ion; [ 28 ] in he es icles o classi y in each one o he pho ope iods. Da es o semen collec ion we e chosen andomly om 10 Ma ch 2014 o 21 Janua y 2019. The es ima ed da es o semen p oduc ion in he es is we e om 15 Janua y 2014 o 26 No embe 2018. The s udied 372 boa s belonged o six pig b eeds: Du oc (63), Land ace (39), La ge Whi e (21), Pie ain (156), syn he ic hyb id lines (70) and Yo k (23). They co esponded o se e al AI s uds in Spain dis ibu ed as ollows A (82), B (147), C (23), D (14), E (14) and F (92). 2.2. Pho ope iod Classi ica ion The s udy in ol ed boa s wi h a leas one alid DFI da um in bo h pho ope iods (inc easing pho ope iod: om 21 Decembe 21 o 21 June; dec easing pho ope iod: om 22 June o 20 Decembe ). As ega ds semen collec ion, he e we e 574 da a om 372 boa s o inc easing pho ope iod (1–3 da a/boa ) and 705 da a om 372 boa s o dec easing pe- iod (1–4 da a/boa ). As ega ds semen p oduc ion, he e we e 688 da a om 372 boa s o inc easing pe iod (1–4 da a/boa ) and 591 da a om 372 boa s o dec easing pho ope iod (1–3 da a/boa ) (Tables 1and 2). Animals 2021,11, 465 4 o 13 Table 1. Means and a iabili y o age a semen collec ion and unadjus ed and adjus ed means and a iabili y o LN DFI wi h age o semen p oduc ion as co a ia e. LN DFI alues we e e alua ed in 654.25 days o semen p oduc ion (adjus ed means). n: numbe o da a; SD: s anda d de ia ion; SE: s anda d e o . Pho ope iod nAge o Semen Collec ion (Days) LN DFI (Unadjus ed) LN DFI (Adjus ed) Mean SD Mean SD Mean SE Inc easing 574 654.43 279.270 −0.2706 1.11866 −0.389 0.038 Dec easing 705 633.05 278.738 −0.3255 1.18808 −0.370 0.035 Table 2. Means and a iabili y o age a expec ed semen p oduc ion in es is, and unadjus ed and adjus ed means and a iabili y o LN DFI wi h age o semen p oduc ion as a co a ia e. LN DFI alues we e e alua ed in 598.40 days o semen p oduc ion (adjus ed means). n: numbe o da a; SD: s anda d de ia ion; SE: s anda d e o . Pho ope iod nAge o Semen P oduc ion (Days) LN DFI (Unadjus ed) LN DFI (Adjus ed) Mean SD Mean SD Mean SE Inc easing 688 527.12 251.505 0.0931 0.989900 −0.206 0.039 Dec easing 591 681.30 286.310 −0.7602 1.169810 −0.584 0.043 In an addi ional app oach, bo h pho ope iods we e di ided in wo subpe iods: Min- imum day leng h du ing inc easing pho ope iod phase (win e : om 21 Decembe o 20 Ma ch ), maximum day leng h du ing inc easing pho ope iod phase (sp ing: om 21 Ma ch o 20 June 20), maximum day leng h o dec easing pho ope iod phase (summe : om 21 June o 20 Sep embe ) and minimum day leng h o dec easing pho ope iod phase (au umn: om 21 Sep embe o 20 Decembe ). Table 3shows he numbe o da a (n), numbe o indi iduals (N) and da a pe indi idual o each subpe iod when conside ing he da e o semen collec ion. Table 4includes his in o ma ion when conside ing he da e o semen p oduc ion. Table 3. Means and a iabili y o age a semen collec ion and he unadjus ed and he adjus ed means and a iabili y o LN DFI wi h age o semen p oduc ion as co a ia e. LN DFI alues we e e alua ed in 654.25 days o semen p oduc ion (adjus ed means). n: numbe o da a; N: numbe o indi iduals; SD: s anda d de ia ion; SE: s anda d e o . Means wi h di e en supe sc ip a e di e en (p< 0.05). Season n (Da a) N (Indi iduals) Da a/Indi idual Age o Semen Collec ion (Days) LN DFI (Unadjus ed) LN DFI (Adjus ed) Mean SD Mean SD Mean SE Win e 352 324 (1–3) 735.70 14.668 −0.6366 1.12147 −0.403 a0.050 Sp ing 222 212 (1–2) 525.57 15.701 0.3082 0.83535 −0.352 b0.073 Summe 442 340 (1–4) 615.55 12.387 0.0159 1.09573 −0.187 b0.043 Au umn 263 262 (1–2) 718.75 18.283 −0.8991 1.11579 −0.690 c0.058 Table 4. Means and a iabili y o age a expec ed semen p oduc ion in es is, and unadjus ed and adjus ed means and a iabili y o LN DFI wi h age o semen p oduc ion as co a ia e. LN DFI alues we e e alua ed in 598.40 days o semen p oduc ion (adjus ed means). n: numbe o da a; N: numbe o indi iduals; SD: s anda d de ia ion; SE: s anda d e o . Means wi h di e en supe sc ip a e di e en (p< 0.05). Season n (Da a) N (Indi iduals) Da a/Indi idual Age o Semen P oduc ion (Days) LN DFI (Unadjus ed) LN DFI (Adjus ed) Mean S anda d De ia ion Mean S anda d De ia ion Mean S anda d E o Win e 315 280 (1–3) 523.71 14.506 0.1855 0.81631 −0.086 b0.056 Sp ing 362 297 (1–2) 528.54 12.980 0.0334 1.11823 −0.257 a0.046 Summe 151 148 (1–2) 550.97 21.229 0.2241 0.89280 0.072 b0.077 Au umn 451 340 (1–2) 722.42 13.233 −1.0864 1.05103 −0.813 c0.046 Animals 2021,11, 465 5 o 13 2.3. Collec ion and Semen Sampling In all cases, ejacula e collec ion was pe o med in a sepa a e collec ion oom. Double glo es and hygienic measu es we e aken wi h p e ious d y cleaning o he p epu ium and penis be o e collec ion. Ejacula e collec ion was pe o med wi h semiau oma ic de ices in all cases. All he ejacula e was collec ed, including ich and pos - ich spe m ac ions. Bac e iology was checked o each ejacula e in blood aga pla es (37 ◦ C du ing 48 h) wi h always less han 300 UFC/mL (da a no shown). Each AI s ud collec ed semen samples o DFI e alua ion egula ly om he beginning o he ial un il he inaliza ion, e e y 12–16 weeks. All boa s we e sampled each ime in each AI s ud ega dless o he spe m quali y. Ejacula es we e aken o a p oduc ion labo a o y and samples we e p edilu ed wi h TNE bu e (0.01 M T is-HCl, 0.15 M NaCl and 1 mM disodium EDTA; pH 7.4) wi hin 5 min a e collec ion, aiming a concen a ion o 20 million spe m cells pe mL. Dilu ed samples we e s o ed immedia ely a − 20 ◦ C and hen sen o he e e ence labo a o y in s y o oam boxes wi h con olled eezing empe a u e in he ollowing 24 h. 2.4. DNA F agmen a ion Spe m nuclea DNA damage was assessed by he SCSA as desc ibed by E enson, (2016) [ 8 ]. Semen samples p edilu ed wi h TNE s o ed a − 20 ◦ C we e sen o ou labo a o y a ha empe a u e wi hin 24 h. A e a i al, samples we e s o ed a − 80 ◦ C un il u he p ocessing. Fo analysis, immedia ely a e hawing an aliquo o 200 µ L o each sample was ea ed wi h 400 µ L o a solu ion con aining 0.1% T i on X-100, 0.15 mol/L NaCl and 0.08 mol/L HCl pH 1.2. Then, 1.2 mL o s aining bu e (6 µ g/mL ac idine o ange, 100 mmol/L ci ic acid, 200 mmol/L Na 2 HPO 4 , 1 mmol/L disodium EDTA and 0.15 mol/L NaCl, pH 6.0) was mixed wi h he semen sample a e 30 s. The esul ing sample was analyzed by FACS (Bec on Dickinson) using he so wa e CellQues 3 (Bec on Dickinson), coun ing mo e han 2000 spe m cells (aiming 5000). Equi alen ins umen se ings we e used o all samples ollowing he p ocedu e desc ibed by Ma inez-Pas o e al. (2010) [ 12 ]. His og am plo s ( o al spe m cells s. DFI) and DFI eadings we e calcula ed o each spe m cell. DFI is used o e e o he a io be ween he ed luo escence and he o al luo escence ( ed + g een) o indi idual spe ma ozoa. A se ies o h eshold alues a e se o DFI o di ide be ween mode a e DFI (mDFI; cu -o a 0.25 DFI) and high DFI (hDFI; cu -o a 0.75 DFI ) [ 12 ]. Spe ma ozoa ha indi idually su passed hese alues we e classi ied as mDFI o hDFI as well. Howe e , bo h mode a e and high DNA agmen a ion could lead o e ili y p oblems while he cu -o is mo e heo e ical han based on e ili y consequences. Thus, o al DFI ( DFI), he sum o mDFI plus hDFI was conside ed o his s udy. 2.5. S a is ical Analyses All a iables s udied ( DFI, age a semen collec ion/p oduc ion) we e es ed o no mali y dis ibu ion by he Shapi o–Wilks es . In case when signi ican depa u e om no mali y was de ec ed a loga i hmic ans o ma ion (e.g., LN DFI) was used o co ec his p oblem. A Pea son’s co ela ion ( ) was un o assess he ela ionship be ween LN DFI and age a semen collec ion/p oduc ion. An ANCOVA (analysis o co a iance) was un o de e mine he e ec o pho ope iod o subpe iod ( ixed e ec ) and boa ( andom e ec ) on LN DFI a e con olling o age a semen collec ion/p oduc ion (co a ia e). This co a ia e is linea ly ela ed o he dependen a iable (LN DFI) and i s inclusion in o he analysis can inc ease he abili y o de ec di e ences be ween subpe iods (independen a iable). This ANCOVA was used o de e mine whe he he e a e any s a is ically signi ican di e ences be ween he adjus ed means o ou subpe iods. These adjus ed means esul om adjus ing he c ude pho ope iod o subpe iod means o chance imbalance in he dis ibu ion o he age among pho ope iod o subpe iods. In his way, he e ec o age on LN DFI was emo ed. p- alues < 0.050 we e conside ed as s a is ically signi ican . When a signi ican e ec was de ec ed, pa ial η was calcula ed as a measu e o e ec size (pe cen age o he Animals 2021,11, 465 6 o 13 a iance in LN DFI explained by he signi ican ac o ). Reg ession models we e applied o he p edic ion o he e olu ion o DFI as a unc ion o he indi idual age. The coe icien o de e mina ion R 2 measu es he a iabili y o LN DFI explained by i s ela ionship o age a semen collec ion/p oduc ion; also, R2is a measu e o goodness o linea model i . 3. Resul s DFI da a anged om 0.018% o 20.1%. As hey signi ican ly depa ed om no mal dis ibu ion (Shapi o–Wilk es , p> 0.05), DFI alues we e subjec ed o a loga i hmic ans o ma ion ha imp o ed he esul s. The Q–Q (quan ile–quan ile) plo compa ing he dis ibu ion o LN DFI bo h o inc easing and dec easing pho ope iod agains he no mal dis ibu ion showed linea i y o he poin s on he line y = x sugges ing ha LN DFI was no mally dis ibu ed (da a no shown). The same, signi ican and nega i e alue was obse ed o bo h LN DFI and age a semen collec ion and LN DFI and age a semen p oduc ion ( = − 0.426, p< 0.001); when age inc eased, LN DFI gene ally dec eased. Linea eg ession o LN DFI on age a semen collec ion/p oduc ion explained 18.1% o obse ed LN DFI a ia ion (coe icien o de e mina ion R 2 = 0.181). Figu es 1and 2plo he da a o he 1279 ejacula es acco ding o hei LN DFI and he age a collec ion o expec ed spe m p oduc ion in he es is, espec i ely. As i is shown in he igu es, some da a change om one pho ope iod o ano he depending on he da e conside ed (collec ion o spe m p oduc ion in he es is). Animals 2021, 11, x 6 o 13 means esul om adjus ing he c ude pho ope iod o subpe iod means o chance imbal- ance in he dis ibu ion o he age among pho ope iod o subpe iods. In his way, he e - ec o age on LN DFI was emo ed. p- alues < 0.050 we e conside ed as s a is ically sig- ni ican . When a signi ican e ec was de ec ed, pa ial η was calcula ed as a measu e o e ec size (pe cen age o he a iance in LN DFI explained by he signi ican ac o ). Re- g ession models we e applied o he p edic ion o he e olu ion o DFI as a unc ion o he indi idual age. The coe icien o de e mina ion R2 measu es he a iabili y o LN DFI explained by i s ela ionship o age a semen collec ion/p oduc ion; also, R2 is a measu e o goodness o linea model i . 3. Resul s DFI da a anged om 0.018% o 20.1%. As hey signi ican ly depa ed om no mal dis ibu ion (Shapi o–Wilk es , p > 0.05), DFI alues we e subjec ed o a loga i hmic ans o ma ion ha imp o ed he esul s. The Q–Q (quan ile–quan ile) plo compa ing he dis ibu ion o LN DFI bo h o inc easing and dec easing pho ope iod agains he no mal dis ibu ion showed linea i y o he poin s on he line y = x sugges ing ha LN DFI was no mally dis ibu ed (da a no shown). The same, signi ican and nega i e alue was obse ed o bo h LN DFI and age a semen collec ion and LN DFI and age a semen p oduc ion ( = −0.426, p < 0.001); when age inc eased, LN DFI gene ally dec eased. Linea eg ession o LN DFI on age a semen collec ion/p oduc ion explained 18.1% o obse ed LN DFI a ia ion (coe icien o de e - mina ion R2 = 0.181). Figu es 1 and 2 plo he da a o he 1279 ejacula es acco ding o hei LN DFI and he age a collec ion o expec ed spe m p oduc ion in he es is, espec i ely. As i is shown in he igu es, some da a change om one pho ope iod o ano he depend- ing on he da e conside ed (collec ion o spe m p oduc ion in he es is). Figu e 1. Rela ionship be ween spe m nuclea DNA agmen a ion index and age o he boa a he collec ion o he ejac- ula e. F agmen a ion is exp essed as he LN DFI and age o he boa is exp essed in days. Da a a e ep esen ed acco ding o i s classi ica ion in inc easing (⬤) o dec easing (△) pho ope iod. -4.00 -3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 0 200 400 600 800 1000 1200 1400 1600 1800 2000 LN DFI Age a semen collec ion (days) R2Lineal = 0.181 y = 0.86 - 1.77E - 3*x Figu e 1. Rela ionship be ween spe m nuclea DNA agmen a ion index and age o he boa a he collec ion o he ejacula e. F agmen a ion is exp essed as he LN DFI and age o he boa is exp essed in days. Da a a e ep esen ed acco ding o i s classi ica ion in inc easing (•) o dec easing (4) pho ope iod. Animals 2021,11, 465 7 o 13 Animals 2021, 11, x 7 o 13 Figu e 2. Rela ionship be ween spe m nuclea DNA agmen a ion index and age o he boa a he expec ed ime o p oduc ion o he ejacula e in he es is. F agmen a ion is exp essed as he LN DFI and age o he boa is exp essed in days. Da a a e ep esen ed acco ding o i s classi ica ion in inc easing (⬤) o dec easing (△) pho ope iod. Two ANCOVA we e un o de e mine he e ec o pho ope iod ( ixed ac o ) on LN DFI using he boa as a andom e ec and conside ing he age a semen collec ion o he expec ed age a semen p oduc ion in he es icles as co a ia es. Table 1 shows he ob- ained esul s o LN DFI (unadjus ed and adjus ed means) and age a semen collec ion in bo h pho ope iods. LN DFI alues we e e alua ed o 654.25 days o boa age a semen collec ion (adjus ed means). A e con olling o age a semen collec ion, he e was a s a- is ically signi ican e ec o boa on LN DFI (F (371, 904) = 2.457; p < 0.0001, pa ial η2 = 0.502). Howe e , he e was no s a is ically signi ican di e ence in LN DFI be ween pho- ope iods (F (1, 904) = 0.140; p = 0.708). Table 2 shows he ob ained esul s o LN DFI (unadjus ed and adjus ed means) and age a expec ed semen p oduc ion in he es is in bo h pho ope iods. LN DFI alues we e e alua ed o 598.40 days o boa age a semen p oduc ion (adjus ed means). A e con- olling o age a semen p oduc ion, simila ly o age a collec ion, he e was a s a is ically signi ican e ec o male on LN DFI (F (371, 904) = 2.221; p < 0.0001, pa ial η2 = 0.477). In addi ion, a s a is ically signi ican di e ence in LN DFI be ween pho ope iods was ound (F (1, 904) = 34.349; p < 0.0001, pa ial η2 = 0.037). The dec easing pho ope iod had he lowes LN DFI (mean di e ence o 0.378; 95% CI: 0.251–0.505; p < 0.001). A e s udying he e ec o inc easing o dec easing pho ope iod, in an addi ional app oach, i was e alua ed whe he he numbe o sunligh hou s a ec s he seasonali y o boa spe m DNA agmen a ion. Da a we e classi ied in ou seasons (win e , sp ing, summe and au umn). Two ANCOVA we e un o de e mine he e ec o seasons ( ixed ac o ) on LN DFI using he boa as a andom e ec and conside ing he age o he boa as a co a ia e. Table 3 shows he ob ained esul s o LN DFI (unadjus ed and adjus ed means) and age a semen collec ion in he seasons. LN DFI alues we e e alua ed in 654.25 days o semen p oduc ion (adjus ed means). A e con olling o age a semen collec ion, he e was a s a is ically signi ican e ec on LN DFI o bo h boa (F (371, 902) = 2.186; p < 0.0001, pa ial η2 = 0.473) and subpe iod (F (3, 902) = 15.875; p < 0.0001, pa ial η2 = 0.050). The adjus ed mean o LN DFI was signi ican ly lowe in au umn han in he es -4.00 -3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 0 200 400 600 800 1000 1200 1400 1600 1800 2000 LN DFI Age a semen p oduc ion (days) R2Lineal = 0.181 Y = 0.76 - 1.77E - 3*x Figu e 2. Rela ionship be ween spe m nuclea DNA agmen a ion index and age o he boa a he expec ed ime o p oduc ion o he ejacula e in he es is. F agmen a ion is exp essed as he LN DFI and age o he boa is exp essed in days. Da a a e ep esen ed acco ding o i s classi ica ion in inc easing (•) o dec easing (4) pho ope iod. Two ANCOVA we e un o de e mine he e ec o pho ope iod ( ixed ac o ) on LN DFI using he boa as a andom e ec and conside ing he age a semen collec ion o he expec ed age a semen p oduc ion in he es icles as co a ia es. Table 1shows he ob ained esul s o LN DFI (unadjus ed and adjus ed means) and age a semen collec ion in bo h pho ope iods. LN DFI alues we e e alua ed o 654.25 days o boa age a semen collec ion (adjus ed means). A e con olling o age a semen collec ion, he e was a s a is ically signi ican e ec o boa on LN DFI (F (371, 904) = 2.457; p< 0.0001, pa ial η2 = 0.502). Howe e , he e was no s a is ically signi ican di e ence in LN DFI be ween pho ope iods (F (1, 904) = 0.140; p= 0.708). Table 2shows he ob ained esul s o LN DFI (unadjus ed and adjus ed means) and age a expec ed semen p oduc ion in he es is in bo h pho ope iods. LN DFI alues we e e alua ed o 598.40 days o boa age a semen p oduc ion (adjus ed means). A e con- olling o age a semen p oduc ion, simila ly o age a collec ion, he e was a s a is ically signi ican e ec o male on LN DFI (F (371, 904) = 2.221; p< 0.0001, pa ial η2 = 0.477). In addi ion, a s a is ically signi ican di e ence in LN DFI be ween pho ope iods was ound (F (1, 904) = 34.349; p< 0.0001, pa ial η2 = 0.037). The dec easing pho ope iod had he lowes LN DFI (mean di e ence o 0.378; 95% CI: 0.251–0.505; p< 0.001). A e s udying he e ec o inc easing o dec easing pho ope iod, in an addi ional app oach, i was e alua ed whe he he numbe o sunligh hou s a ec s he seasonali y o boa spe m DNA agmen a ion. Da a we e classi ied in ou seasons (win e , sp ing, summe and au umn). Two ANCOVA we e un o de e mine he e ec o seasons ( ixed ac o ) on LN DFI using he boa as a andom e ec and conside ing he age o he boa as a co a ia e. Table 3shows he ob ained esul s o LN DFI (unadjus ed and adjus ed means) and age a semen collec ion in he seasons. LN DFI alues we e e alua ed in 654.25 days o semen p oduc ion (adjus ed means). A e con olling o age a semen collec ion, he e was a s a is ically signi ican e ec on LN DFI o bo h boa (F (371, 902) = 2.186; p< 0.0001, pa ial η2 = 0.473) and subpe iod (F (3, 902) = 15.875; p< 0.0001, pa ial η2 = 0.050). The adjus ed mean o LN DFI was signi ican ly lowe in au umn han in he es o subpe iods. Animals 2021,11, 465 8 o 13 Addi ionally, he adjus ed mean in summe and sp ing was signi ican ly highe han in win e . No o he signi ican di e ences among subpe iods we e ound. Table 4shows he ob ained esul s o LN DFI (unadjus ed and adjus ed means) and age a expec ed semen p oduc ion in he es is in he di e en seasons. LN DFI alues we e e alua ed in 598.40 days o semen p oduc ion (adjus ed means). A e con olling o age a semen p oduc ion, simila ly o age a collec ion, he e was a s a is ically signi ican e ec on LN DFI o male (F (371, 902) = 2.217; p< 0.0001, pa ial η2 = 0.477) and subpe iod (F (3, 902) = 49.609; p< 0.0001, pa ial η2 = 0.142). The adjus ed mean o LN DFI was signi ican ly lowe in au umn han in he es o subpe iods. Addi ionally, he adjus ed mean in summe and win e was signi ican ly highe han in sp ing. No o he signi ican di e ences among subpe iods we e ound. 4. Discussion The seasonali y o ep oduc ion is undispu able in in ensi e pig p oduc ion in em- pe a e clima e coun ies [ 29 ]. E en when good quali y semen doses a e deli e ed om AI s a ions ega ding spe m coun s, spe m mo ili y, spe m abno mali ies and spe m longe i y, esul s in a ms can be impai ed in ce ain pe iods o he yea , ela ed o empe a u e and pho ope iod [ 30 ]. This ac has a s ong a m e ec [ 31 ] bu also has a boa - ela ed e ec [10]. High DFI alues ha e been ela ed o educed e ili y, longe imes o p egnancy and highe spon aneous misca iage a es in humans [ 11 , 32 ] and i has shown a ela ion wi h p egnancy a e and o al pigle s bo n in boa s [ 33 ]. Mo eo e , i is no ela ed o o he classic spe m quali y pa ame e s [ 8 ] and is a candida e o explain pa o he boa - ela ed e ec o sub e ili y [ 9 ]. The e o e, he e ec o pho ope iod, season and age o he boa on DFI was e alua ed. Ou i s inding was ha age o he boa was nega i ely co ela ed o DFI. Boa s in AI s uds a e subjec ed o a high eplacemen a e due o he sho pig gene a- ion in e al and he compe i i e and as gene ic imp o emen o each new gene a ion, su passing he p oduc i e cha ac e is ics o i s ances o s. This means ha he e is a ush in young animals o en e he AI s uds and be ained o be included in he p oduc ion o seminal doses a ea ly ages. On he o he hand, he e a e boa s conside ed old (namely mo e han 3 yea s old), bu s ill exploi ed due o he p e e ence o local b eede s o hei ou s anding gene ic me i . I is well known ha ejacula e olumes and p oduc i i ies a e lowe in young boa s [ 28 ]. Semen olume inc eases om pube y un il abou 24 mon hs age and emains ela i ely cons an he ea e . Spe m concen a ion inc eases sha ply om pube y o he i s yea o age, ollowed by a long- e m mode a e dec ease un il 3 yea s o age and i s abilizes he ea e [ 34 ]. Pa icula ly o spe m agmen a ion, [ 27 ] ound ha he age o he boa had a signi ican e ec on DNA s abili y and ch oma in s uc u e. These au ho s ound lowe pe cen age o spe ma ozoa wi h DNA agmen a ion in he g oup o boa s aged 20–26 mon hs old in compa ison o he young animals (7–8 mon hs old). In he boa popula ion o ou s udy, DFI anged om 0.018 o 20.1% (LN DFI: − 4 o 3). The big numbe o andomly chosen ejacula es analyzed in he p esen s udy explains he bigge ange o DFI in compa ison o o he au ho s [ 27 ]. Didion e al. (2009) [ 7 ] p oposed ha boa spe ma ozoa wi h g ea e han 6% DFI migh nega i ely a ec bo h he a owing a e and o al pigle s bo n. This h eshold would co espond in ou igu e wi h LN DFI alues abo e 1.8 in which mos o he boa s a e aged below 2 yea s. This esul ag ees wi h he imeline o he ma u a ion o boa es icles desc ibed by Knech e al., 2017 [ 31 ]. In addi ion, Boe-Hansen e al. (2008) [ 33 ] epo ed ha he o al numbe o pigle s bo n o Hampshi e, Land ace and Danish La ge Whi e boa s was, espec i ely, 0.5, 0.7 and 0.9 pigle s less pe li e when he % DFI alues we e abo e 2.1%. This cu -o would coincide app oxima ely in Figu e 1wi h he LN DFI o 1, abo e which mos o he boa s had in any case less han 2 yea s old. Animals 2021,11, 465 9 o 13 The nega i e co ela ion o he DFI and he age o collec ion and expec ed p oduc ion o semen ound in ou s udy illus a es one po en ial hidden e ili y oublesome ela ed o he high u no e o boa s a he AI s uds. Acco dingly, he a e age age o he boa s s udied a he momen o he ejacula e collec ion was 19.6 mon hs. I is wo h no ing ha he managemen o he inpu s and de ia e o boa s in he AI s ud could p oduce a bias in he in e p e a ion o he esul s. The ejacula es o young boa s ha sco ed poo ly in he con en ional semen analysis we e empo ally disca ded o seminal doses p oduc ion and i he low p oduc i i y las ed o a ce ain ime (namely 3–4 mon hs) he animal was e en ually culled. In pa allel, he animals whose seminal doses p oduced p o en low e ili y esul s in epea ed occasions we e also emo ed om he AI s ud. As DFI is co ela ed wi h low semen quali y o poo e ili y esul s [ 7 , 11 , 33 ], he AI s ud managemen c i e ia we e indi ec ly selec ing boa s ha sus ain spe m pa ame e s abo e he s anda d and show good e ili y esul s and would p e en boa s wi h high DFI alues o ge old in he AI s uds. The inclusion o indi idual boa as a andom e ec in he ANCOVA and he la ge num- be o boa s and ejacula es s udied ensu e he soundness o he esul s ob ained. Resul s show ha he pe cen age o spe ma ozoa wi h agmen ed nuclea DNA in young animals could dec ease along wi h he aging o he boa being especially no o ious un il wo yea s old. The e o e, hese esul s suppo ha he exploi a ion o young boa s wi hou es ing hei spe m nuclea DNA s abili y could bea o poo indi idual ep oduc i e esul s, as p e iously sugges ed by [ 27 ]. Ea ly diagnosis o spe m nuclea DNA agmen a ion would allow o s ic e selec ion and elimina ion o males wi h po en ial e ili y diso de s om b eeding. Spe m DNA agmen a ion could be a aluable p ognos ic ool o p edic inal e ili y ou comes in pigs [ 9 , 35 ]. As i is shown in Figu e 1, he equa ion ob ained ( y = 0.86 −1.77E −3x ) explains 18% o he a iabili y o DFI. Acco ding o his equa ion, DFI would dec ease due o aging o he boa 0.66% each yea old. Ou second inding was ha he pho ope iod did no in luence DFI when he semen collec ion da e was conside ed and age was included in he ANCOVA analysis as a co a i- a e o con ol i s e ec (Table 1). Al hough, he e was no s a is ically signi ican di e ence in LN DFI be ween pho ope iods o he collec ion da e (p= 0.708), he e was a s a is ically signi ican e ec o boa on LN DFI (p< 0.0001). These esul s show he la ge p opo ion o a iabili y ha is caused by he indi idual e ec o boa . This high a iabili y ound among indi iduals ag ees wi h o he au ho s. Fo example, Didion e al. (2009) [ 7 ] ound a ange o % DFI among 18 boa s o 1.2–28.3%; Pe ocelli e al. (2015) obse ed an indi idual a iabili y in DFI o 80% and Zasiadczyk e al. (2015) [ 25 ] ound ha boa was a signi ican sou ce o a iabili y in DNA agmen a ion (p< 0.039). Al hough he e we e no s a is ically signi ican di e ences o pho ope iod in DFI conside ing he collec ion da e, om a biological poin o iew i is easonable o di ide he pho ope iod conside ing he da e when he spe m o he ejacula es was p oduced in he es is. In boa s, each spe ma ogenic cycle and he en i e spe ma ogenic p ocess las s 8.6–9.0 and 40 days, espec i ely. The spe m ansi h ough he epididymis akes app oxima ely 10 days [36]. The e o e, in o de o conside he pe iod ha in luenced he samples collec ed, 50 days we e sub ac ed om he da e o collec ion o calcula e he da e o p oduc ion (beginning o spe ma ogenesis). Boa e ec also in luenced on LN DFI when he esul s we e analyzed conside ing he da e o semen p oduc ion in he es icles in pho ope iod di ision (p< 0.0001). Howe e , in his case, he e we e also s a is ically signi ican di e ences be ween he means o he pho ope iods (p< 0.0001) (Table 2). On a e age DFI was 1.46% highe in inc easing pho ope iod compa ed o dec easing pho ope iod. The e o e, o pu i b ie ly, he da e o he collec ion had no signi ican e ec on DFI, while on he con a y, he expec ed semen p oduc ion da e in he es is did ha e a signi ican e ec on DFI. These esul s ag eed wi h he highe ac i i y obse ed in la e au umn and win e (dec easing pho ope iod) in wild pigs, he ances o s o he Sus sc o a domes ica [ 37 ]. O he au ho s ound con adic o y esul s o pho ope iod in di e en cha ac e is ics o indus-