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-
