Animal Rep oduc ion Anim Rep od. 2019;16(4):887-894
h ps://doi.o g/10.21451/1984-3143-AR2018-0100
O iginal A icle
Copy igh © The Au ho (s). This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License,
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E ec o glyce ol, n, n-dime hyl o mamide and n-me hyl-2-py olidone on abbi
spe m s o ed a 4 °C and 16 °C
Paula Domingo1* , Mai e Olaci egui1 , Noelia González1 , Ignacio De Blas2 , Lydia Gil1 
1Uni e sidad de Za agoza, Facul ad de Ve e ina ia, Á ea de Obs e icia y Rep oducción Animal, Depa amen o de Pa ología
Animal, España
2Uni e sidad de Za agoza, Facul ad de Ve e ina ia, Á ea de Salud Animal, Depa amen o de Pa ología Animal, España
a
*Co espondence: [email p o ec ed]
Recei ed: Augus 9, 2018
Accep ed: Augus 2, 2019
Abs ac
A i icial insemina ion wi h cooled semen is
he mos common p ac ice in abbi a ms and any
imp o emen on i helps o inc ease he e iciency and
p oduc i i y o abbi mea a ms. The e o e, he aim o
his s udy was o assess whe he di e en c yop o ec an
agen s (CPA) as glyce ol, N, N-Dime hyl o mamide
(DMF) and N-Me hyl-2-Py olidone (NMP) can imp o e
cooled abbi spe m quali y s o ed a 4ºC and 16ºC. Spe m
samples we e dilu ed wi h INRA 96

(Ex ende A),
INRA 96 wi h 6% glyce ol (Ex ende B) o 6% DMF
(Ex ende C) o 6% NMP (Ex ende D) espec i ely and
s o ed a 4ºC and 16ºC. Samples we e hen analysed a
4, 24, 48 and 72 hou s a e e ige a ion by in eg a ed
spe m analysis sys em (ISAS

), eosin-nig osin s ain
( i ali y), hypo-osmo ic swelling es (HOS es ) and
ac osome in eg i y es . Ex ende C showed highe
pe cen age o mo ili y, i ali y and HOS es han
ex ende B and D (p<0.05). Whe eas spe m quali y
dec eased o e ime (p<0.05), da a showed ha he
addi ion o DMF kep he mo ili y and spe m plasma
memb ane in eg i y a e 24 hou s o s o age be e han
o he diluen s. These esul s sugges ha he addi ion
o DMF o INRA 96

exe s a p o ec i e e ec on he
memb ane o spe ma ozoa imp o ing seminal quali y.
Keywo ds: c yop o ec an , dime hyl o mamide, n-me hyl-
2-py olidone, glyce ol, abbi spe m p ese a ion.
In oduc ion
Spe ma ozoon su i al is a ec ed by s o age
empe a u e and ex ende (Ca luccio e al., 2004). Cu en ly,
he mos common empe a u e used o p ese e abbi spe m
in a ms is a ound 16 °C due o he e ili y is conside ably
lowe a e 48 hou s o s o age (López and Al a iño,
1998; Roca e al., 2000; Rosa o e al., 2006; Johinke e al.,
2014). Likewise, he ex ende a ec s spe m quali y. Many
ex ende s (López and Al a iño, 2000; Ca luccio e al., 2004)
and subs ances (T ejo e al., 2013; Johinke e al., 2014;
Sa ıözkan e al., 2014) ha e been e alua ed in o de o
p ese e semen he mos ime possible o ca y i o ano he
a ms. Ne e heless, Ca luccio e al. (2004) demons a ed
ha INRA 96 kep abbi spe m quali y be e han o he s
ex ende s e en hough INRA 96 was made speci ically
o he p ese a ion o s allion spe m.
Mo eo e , because o abbi spe m cha ac e is ics
(high ac i a ion ene gy and low wa e pe meabili y
coe icien ), c yop o ec an agen s (CPA) wi h lowe
molecula weigh and highe pe meabili y, such as amides
o me hyl g oups, a e sui able o be used (Cu y e al.,
1995; Mocé and Vicen e, 2009). N, N-Dime hyl o mamide
(DMF) is an amide sol en used in chemical eac ions.
P e ious s udies in boa (Malo e al., 2012), canine
(Fu ino e al., 2010; Lopes e al., 2009; Mo a e al., 2011),
goa (Beze a e al., 2011) and owl spe m (Chalah e al.,
1999) demons a ed ha DMF is no be e CPA han
glyce ol. Howe e , s allion spe m (Al a enga e al.,
2005; Olaci egui e al., 2014; Pukazhen hi e al., 2014)
dilu ed wi h DMF as an al e na i e o glyce ol showed
be e esul s. N-Me hyl-2-Py olidone (NMP) is also an
amide sol en commonly used in chemical eac ions as an
al e na i e o DMF. Bu unlike DMF, NMP has no been
s udied be o e o s o age spe m samples.
On he o he hand, glyce ol is he main CPA used
o p ese e domes ic o wild animal spe m (Cu y e al.,
1995). Ne e heless, glyce ol has no been he i s CPA o
choice o p ese e abbi semen due o i s oxici y, which
may esul in osmo ic s ess, p o ein dena u a ion, al e a ion
o ac in in e ac ions and induc ion o p o ein- ee memb ane
blis e ha leads o ge wo se e ili y (Al a iño, 1993;
Gilmo e e al., 1995; Okuda e al., 2007; Ia aldano e al.,
2012). The lack o p e ious s udies using DMF o NMP
as a CPA in abbi spe m p ese a ion and he supposed
oxici y o glyce ol, leads o s udy mo e hese CPA. The aim
o his s udy was o assess he quali y o cooled abbi
spe m s o ed a 4 °C and 16 °C and dilu ed wi h INRA 96
supplemen ed wi h glyce ol, DMF o NMP. Mo eo e ,
e alua e he e ec o s o age ime du ing 72 hou s and
decide whe he glyce ol, DMF o NMP can be used as
CPA on cooled abbi semen p ese a ion.
Me hods
Chemicals
Unless no ed o he wise, all chemicals we e om
Pan eac Quimica S.L.U (Ba celona, Spain).
Domingo e al. C yop o ec an e ec on abbi spe m s o age
Anim Rep od. 2019;16(4):887-894 888
Animals, semen collec ion and p ocessing
The s udy was pe o med ollowing app o al by he
Ve e ina y E hical Commi ee o Uni e si y o Za agoza.
The ca e and use o animals we e pe o med acco ding
o he Spanish Policy o Animal P o ec ion RD1201/05,
which mee s he Eu opean Union Di ec i e 86/609 on
he p o ec ion o animals used o expe imen al and o he
scien i ic pu poses.
Rabbi spe m samples we e collec ed om eigh
sexually ma u e bucks p e iously selec ed om a comme cial
AI cen e (Técnicas Cunícolas S.A., Za agoza, Spain) and
used as semen dono s. Males we e housed in indi idual
cages wi h 12 hou s o ligh and 12 hou s o da kness a
a oom empe a u e o 22-24 °C and a ela i e humidi y
o 60-70%. All abbi s we e ed a comme cial pelle die
acco ding o hei ep oduc i e condi ion and esh wa e
was p o ided ad libi um.
Rabbi spe m samples we e collec ed using a i icial
agina (IMV Technologies, L’Aigle, F ance). A e semen
collec ion, any gel plug was emo ed and a mac oscopic
analysis was pe o med assessing he colou and he olume
o he sample. The i s mic oscopic analysis o he mo ili y
in he a m was made as well. Only ejacula es wi h whi e
colou , mo e han 0,2ml and good wa e mo ion (a leas
85% o mo ili y) we e used o he esea ch.
All ejacula es we e pooled wi h he pu pose o
elimina e indi idual di e ences, he ea e we e di ided
in ou ac ions and each one was dilu ed wi h a di e en
ex ende : INRA 96

(IMV Technologies, L’Aigle, F ance)
(Ex ende A) as con ol, and INRA 96

supplemen ed wi h
6% glyce ol (Ex ende B), 6% DMF (Ex ende C) o 6%
NMP (Ex ende D).
Subsequen ly, each semen sample was placed
in o wo Eppendo ube in o de o s o e each sample a
4 °C and a 16 °C. Samples we e cooled p og essi ely
om 37 °C o 16 °C and 4 °C in a ime pe iod among
90 and 120 minu es (Mocé and Vicen e, 2009) and inally
hey we e s o ed.
E alua ion o spe ma ozoa
Spe m mo ili y, i ali y, memb ane in eg i y and
ac osome in eg i y we e assessed a 4, 24, 48 and 72 hou s
a e collec ion o bo h empe a u e s o age and all
ex ende s.
Spe m mo ili y and kinema ics
Spe m mo ili y and kinema ics pa ame e s we e
e alua ed by ISAS so wa e (PROISER R+D, Valencia,
Spain) ollowing he de aul se ing speci ically o abbi s.
Fi e mic oli e s we e placed on a slide and co e ed wi h
20 × 20mm co e slip. Fi e ñields we e andomly cap u ed
a ×10 magni ica ion by phase-con as mic oscope. Up o
200 ames pe second we e acqui ed selec ing pa icles wi h
an a ea o be ween 10 and 70µm2. The cu -o alues used o
de e mine spe m eloci y we e 10<slow<25<medium<50< apid
(µm/s). The linea ly mo ile spe m we e de ia ed <45%
om a s aigh line. The analyses p o ided in o ma ion
abou he pe cen age o mo ile spe ma ozoa (MOT, %),
cu ilinea eloci y (VCL, µm/s), s aigh -line eloci y
(VSL, µm/s), a e age pa h eloci y (VAP, µm/s), linea i y
(LIN=VSL/VCL, %), s aigh ness (STR=VSL/VAP, %),
wobble (WOB=VAP/VCL, %), ampli ude o la e al head
displacemen (ALH, µm) and bea c oss equency (BCF, Hz).
Vi ali y
Eosin-nig osin s ain was used o e alua e he i ali y
o he spe ma ozoa ollowing he p o ocol desc ibed by
Bjö ndahl e al. (2003). Acco ding o he s ain pene a ion
ough he damaged memb ane he dead spe ma ozoa had
ed o da k pink heads (s ained) and he li e spe ma ozoa
had whi e heads (uns ained) (WHO, 2010).
Spe m plasma memb ane in eg i y
Hypo-osmo ic swelling es (HOS es ) was pe o med
ollowing he p o ocol es ablished by Jeyend an e al.
(1984). Ten mic oli e s o semen we e mixed wi h 90µl
o HOS es solu ion (100 mM o Sodium Ci a e) and
kep a 37 °C a leas 30 minu es. Subsequen ly 100 µl
o glu a aldehyde 2% was added o he sample o ix i .
Each sample was analysed by phase-con as mic oscope
a x400 magni ica ion. Spe ma ozoa wi h in ac memb anes
allowed an in lux o wa e inside he cell gi ing place o
swollen spe ma ozoa wi h coiling ail (Amo im e al.,
2009; WHO, 2010).
Ac osome in eg i y
Ac osome in eg i y es is based on he ixa ion o
he spe ma ozoa. Ten mic oli e s o semen was immedia ely
ixed in 90µl o glu a aldehyde 2% solu ion, igh a e each
sample was analysed by phase-con as mic oscope a x1000
magni ica ion (Pu sel and Johnson, 1974). Ac osomes we e
di e en ially ca ego ized in o wo classes: in ac ac osome
(no mal apical idge) and damaged ac osome (damaged
apical idge and/o missing apical idge).
S a is ical analysis
The s udy was eplica ed h ee imes. Da a we e
analysed using IBM SPSS S a is ics 23 o Windows.
Resul s we e exp essed as mean ± SEM. The e ec o he
ex ende , s o age empe a u e and ime o s o age on he
s udied pa ame e s we e analysed by ANOVA. Duncan’s
pos -hoc es was used o e alua e he e ec o he ex ende
and ime o s o age. The in e ac ions among he ex ende
used, s o age empe a u e and ime elapsed we e assessed
using GLM p ocedu e. The le el o signi icance was se
a p<0.05.
Resul s
The e ec o he ex ende played an impo an
ole in seminal p ese a ion, in ac , all pa ame e s s udied
Domingo e al. C yop o ec an e ec on abbi spe m s o age
Anim Rep od. 2019;16(4):887-894 889
we e signi ican ly signi ican (p<0.05), excluding ac osome
in eg i y pa ame e (Table 1 and Figu e 1). When he
samples we e dilu ed wi h ex ende C, he da a ob ained
we e simila o con ol samples (ex ende A), mo eo e
ex ende C go highe pe cen age (p<0.05) o MOT, i ali y
and HOS es . A ew wo se esul s o MOT, i ali y and
HOS es we e ob ained by ex ende D, hough he wo s
esul s we e obse ed in samples dilu ed wi h ex ende B.
O he kinema ic pa ame e s we e s udied, showing ex ende
A g ea e esul s on eloci y and ajec o y.
When he e ec o s o age empe a u e on cooled
abbi spe m was analysed no signi ican di e ences we e
obse ed on MOT, kinema ic pa ame e s, i ali y, HOS
es and ac osome in eg i y. Wi h he excep ion o BCF
pa ame e (p<0.001), which was highe when spe m samples
we e s o ed a 4 °C (8.307 ± 0.189 Hz) ins ead o 16 °C
(7.475 ± 0.203 Hz). In spi e o no s a is ically di e ences
we e obse ed be ween s o age samples a 4 °C o 16 °C,
i should be no ed ha s udying ho oughly he in e ac ion
be ween he diluen s and he empe a u e s a is ically
di e ences (p=0.027) on MOT we e shown (Figu e 2).
Rega ding all spe m pa ame e s s udied, spe m
quali y dec eased o e s o age ime (p<0.05) (Table 2).
The pe cen age o o al spe m mo ili y d opped o om
64.7 ± 2.3% a 4 hou s a e collec ion o 44.5 ± 2.3%
passing 48 hou s. Ne e heless, he sha pes decline o
mo ili y was om 72 hou s o s o age (28.6 ± 2.6% o
mo ile spe ma ozoa). The i ali y o spe ma ozoa was kep
ill 72 hou s a e s o age, om 87.5 ± 1.8% o 75.3 ± 1.7%
o li e spe ma ozoa. Almos all kinema ic pa ame e s and
ac osome in eg i y shown a conside able decline a e
24 hou s o s o age. Las ly, spe m plasma memb ane was
s a ed o damage as o 48 hou s.
The in e ac ion be ween he ex ende and he
hou s ha elapsed since he collec ion o he samples a e
p esen ed in Figu e 3. All he pa ame e s s udied, excep
ac osome in eg i y, we e s a is ically signi ican (p<0.05)
showing ha as ime goes by, semen quali y dec eases.
Ex ende A ob ained he highes MOT up o
24 hou s, bu a e ha pe iod o ime he addi ion o DMF
o INRA 96 o e ed highe MOT. Ex ende C ob ained
he bes da a on MOT a 72 hou s (40.8 ± 4.6%). MOT
pa ame e was simila o ex ende C and D un il 24 hou s
Table 1. E ec o he ex ende on kinema ic pa ame e s de e mined by ISAS. Di e en le e s wi hin each column
indica e s a is ically signi ican di e ences (p<0.05). Da a a e mean ± SEM.
MOT VCL VSL VAP LIN STR WOB ALH BCF
(%) (µm/s) (µm/s) (µm/s) (%) (%) (%) (µm) (Hz)
Ex ende A 59.9 ± 2.4a73.7 ± 2.4ab 26.7 ± 1.1a43.9 ± 1.4a35.1 ± 1.1a59.1 ± 1.0bc 58.1 ± 1.0a2.9 ± 0.1b7.3 ± 0.3b
Ex ende B 35.4 ± 2.4c66.9 ± 2.3b22.1 ± 1.1b36.1 ± 1.4b34.9 ± 1.9a61.1 ± 1.0b55.9 ± 1.0a3.1 ± 0.1b7.6 ± 0.3ab
Ex ende C 59.3 ± 2.2a75.5 ± 2.3a21.5 ± 1.1bc 37.2 ± 1.3b29.5 ± 1.0b58.0 ± 1.0c50.3 ± 1.0b3.4 ± 0.1a8.4 ± 0.3a
Ex ende D 47.5 ± 2.2b56.7 ± 2.3c18.9 ± 1.1c28.3 ± 1.3c33.9 ± 1.0a65.9 ± 1.0a51.5 ± 1.0b3.0 ± 0.1b8.3 ± 0.3a
p 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
Figu e 1. Pe cen age o i ali y, HOS es and ac osome in eg i y o abbi spe ma ozoa dilu ed wi h ex ende A,
ex ende B, ex ende C and ex ende D om he 16 °C and 4 °C g oups. Da a a e mean ± SEM. Di e en le e s wi hin
each diagnos ic es deno e s a is ical di e ences (p<0.05).
Domingo e al. C yop o ec an e ec on abbi spe m s o age
Anim Rep od. 2019;16(4):887-894 890
Table 2. E ec o ime o s o age on MOT, kinema ic pa ame e s, i ali y, HOS es and ac osome in eg i y. Di e en le e s wi hin each column indica e s a is ically signi ican
di e ences (p<0.05). Da a a e mean ± SEM.
Time o
s o age MOT VCL VSL VAP LIN STR WOB ALH BCF Vi ali y HOS es Ac osome
in eg in
(hou s) (%) (µm/s) (µm/s) (µm/s) (%) (%) (%) (µm) (Hz) (%) (%) (%)
464.7 ± 2.3a76.9 ± 2.3a31.6 ± 1.0a46.4 ± 1.3a41.3 ± 1.0a68.0 ± 1.0a60.2 ± 1.0a3.1 ± 0.1b9.3 ± 0.3a87.5 ± 1.8a79.2 ± 1.4a79.7 ± 2.2a
24 60.8 ± 2.3a79.7 ± 2.3a22.5 ± 1.0b39.7 ± 1.3b28.7 ± 1.0c57.1 ± 1.0b50.0 ± 1.0c3.5 ± 0.1a8.4 ± 0.3b84.8 ± 1.9a75.3 ± 1.4a70.8 ± 2.2b
48 44.5 ± 2.3b64.5 ± 2.3b18.3 ± 1.0c31.2 ± 1.3c29.4 ± 1.0c59.2 ± 1.0b49.2 ± 1.0c3.1 ± 0.1b7.9 ± 0.3b85.5 ± 1.7a68.8 ± 1.4b66.8 ± 2.2b
72 28.1 ± 2.6c48.5 ± 2.6c15.5 ± 1.2c26.0 ± 1.5d33.9 ± 1.1b60.1 ± 1.1b56.1 ± 1.1b2.8 ± 0.1b5.7 ± 0.3c75.3 ± 1.7b56.7 ± 1.5c57.0 ± 2.2c
p 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.023 0.001 0.017 0.001 0.001
Domingo e al. C yop o ec an e ec on abbi spe m s o age
Anim Rep od. 2019;16(4):887-894 891
Figu e 2. E ec o he empe a u e on he mo ili y pa ame e o abbi spe ma ozoa dilu ed wi h ex ende A, ex ende B,
ex ende C and ex ende D. Da a a e mean ± SEM. Di e en le e s wi hin each diagnos ic es deno e s a is ical
di e ences (p<0.05).
Figu e 3. Pe cen age o MOT, i ali y, HOS es and ac osome in eg i y o abbi spe ma ozoa a 4, 24, 48, and 72 hou s
om he ou ex ende (A-D) g oups. Da a a e mean ± SEM. Means ma ked wi h an as e isk wi hin each ime poin
deno e s a is ical di e ences (p<0.05).
o s o age, he ea e MOT d opped eaching less han 20%
a 72 hou s a e collec ion. Ex ende B ob ained he wo s
MOT in ela ion o o he ex ende s (p<0.05) (Figu e 3a).
The pe cen age o ali e spe ma ozoa ob ained wi h
ex ende A was simila han ex ende C du ing 72 hou s
(Figu e 3b). Ex ende B o D showed simila alues ill
48 hou s o s o age, a e 48 hou s o s o age he i ali y
o spe ma ozoa dilu ed wi h ex ende B came down om
82.6 ± 3.5% a 48 hou s o 56.9 ± 3.5% a 72 hou s o
s o age (p<0.05).
The e alua ion o he damage o he spe m plasma
memb ane is syn hesise in Figu e 3c. As i is shown,
ex ende C o e ed be e p o ec ion on he spe m plasma

Domingo e al. C yop o ec an e ec on abbi spe m s o age
Anim Rep od. 2019;16(4):887-894 892
memb ane, obse ing s a is ically di e ences (p<0.05)
a e 72 hou s o s o age.
Discussion
I is known ha he addi ion o CPA o an ex ende
is pe o med wi h he goal o p o ec he spe m om
c yodamage, which causes de as a ing consequences in
spe m su i al du ing spe m cooling p ocess. Though
also, CPA, such as glyce ol, could ha e a oxic e ec on
spe m (memb ane des abiliza ion, p o ein and enzyme
dena u a ion, osmo ic s ess, al e a ion o ac in in e ac ions
and induc ion o p o ein- ee memb ane blis e ) di ec ly
ela ed o he concen a ion used and he ime o cell exposu e
(Al a iño, 1993; Gilmo e e al., 1995; Okuda e al., 2007;
Ia aldano e al., 2012). In e es ingly, his s udy on abbi
spe m showed same esul s as p e ious s udies in s allion
spe m p ese a ion (Olaci egui e al., 2014; Pukazhen hi e al.,
2014): he addi ion o DMF main ained cooled abbi spe m
quali y be e han glyce ol. In addi ion, samples dilu ed
wi h INRA 96 plus DMF go , along wi h samples dilu ed
wi h INRA 96, he bes spe m quali y du ing cooled spe m
p ese a ion. Su p isingly, spe m quali y dec eased when
samples we e dilu ed wi h INRA 96

plus NMP. Reminding
o abbi spe m cha ac e is ics, he mos app op ia e
sol en s o use in abbi spe m p ese a ion should ha e
low molecula weigh and high pe meabili y, such as amides
(Cu y e al., 1995; Mocé and Vicen e, 2009). The e o e,
he di e ences ound be ween DMF and NMP could be
owing o hei molecula weigh s and densi ies. Despi e
DMF and NMP a e bo h an amide sol en s, NMP has highe
molecula weigh (99.13g/mol) and densi y (1.028g/cm3)
han DMF (73.09g/mol o molecula weigh ; 0.944g/cm3
o densi y). These small di e ences in hei composi ion
migh sugges ha a e esponsible o NMP no being a
good CPA o s o age cooled abbi spe m. Anyhow, u he
s udies should be ca ied ou o de e mine he op imal
concen a ion o NMP.
On he o he hand, as expec ed glyce ol go he
wo s da a due o i s oxici y p e iously demons a ed
by o he au ho s (Al a iño, 1993; Gilmo e e al., 1995;
Okuda e al., 2007), no ge ing no 30% o mo ile abbi
spe m s o ing i a 16 °C.
P e ious s udies ha e shown he abili y o abbi
spe m o esis en i onmen al s ess. Because o he
d ople s and esicles p esen in abbi spe m; hey a e ich
in choles e ol and phospholipids, assis ing o modula e he
memb ane luidi y and in his way o wi hs and g ea e cold
shock (Spa e al., 2002; Cas ellini e al., 2006; Mocé and
Vicen e, 2009). Mo eo e , he high choles e ol:phospholipid
a io o abbi spe m plasma memb ane allows he abili y
o e ilize oocy es om cooling spe m a 4 °C is simila
o esh spe m (Mocé and Vicen e, 2009). In he p esen
s udy on cooled abbi spe m, we demons a ed ha he e
a e no di e ences be ween s o age abbi spe m a 4 °C
o 16 °C when INRA 96 is he ex ende used, con a ily
o he s udy published by Johinke e al. (2014) which using
T is-ci ic-acid ex ende wi h wo di e en concen a ions o
glucose, ob ained be e esul s when s o ed abbi spe m a
15 °C ins ead o 5 °C. Conside ing his, we can app ecia e
ha he ex ende and CPA play an impo an ole, mo eo e
INRA 96 ex ende can be used sa is ac o ily among 4 °C
and 16 °C.
As expec ed, in his s udy he spe m quali y o all
he samples dec eased h oughou s o age ime as o he
au ho s p e iously epo ed (López and Al a iño, 1998;
Roca e al., 2000), showing a e 48 hou s a conside able
d op on MOT pa ame e . Ne e heless p e ious s udies
demons a ed ha MOT is no he only good pa ame e o
e alua e spe m quali y, o he pa ame e s ela ed wi h MOT
such as VCL, VSL, VAP, LIN, STR, WOB, ALH and BCF
a e also use ul o p edic male in i o e ili y (Liu e al.,
1991; Hagen e al., 2010). To da e, all kinema ic pa ame e s
in abbi spe ma ozoa ha e ne e been s udied p e iously by
o he au ho s. Su p isingly, in his s udy we can app ecia e
ha almos all kinema ic pa ame e s only we e main ained
ill 24 hou s, unlike i ali y pa ame e which ha dly showed
a decline du ing 72 hou s o s o age. Mo eo e , e en
hough he quali y o all he samples dec eased h oughou
s o age ime, da a showed ha he addi ion o DMF kep
he mo ili y and spe m plasma memb ane in eg i y a e
24 hou s o s o age be e han o he ex ende s.
Summa ising, hese esul s demons a ed ha he
addi ion o DMF o INRA 96 diluen exe s a p o ec i e
e ec on he memb ane o spe ma ozoa and he e o e i
imp o e seminal quali y. Mo eo e NMP could no be
used o eplace DMF conside ing ha wo se esul s we e
ob ained. Also we a e in ag eemen wi h p e ious esul s
epo ed o he oxici y o glyce ol, which made i no a
good c yop o ec o (Al a iño, 1993; Gilmo e e al., 1995;
Okuda e al., 2007). Finally, he empe a u e o s o age
among 4 °C and 16 °C did no a ec he quali y o abbi
spe m dilu ed wi h INRA 96.
Acknowledgemen s
The au ho s would like o hank Miguel Domingo,
chie o Técnicas Cunicolas S.A. a m, o his incalculable
help in he ieldwo k. This s udy was suppo ed by
Go e nmen o A agon Resea ch G oups (Fondo Social
Eu opeo, DGA) and IA2.
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Financial suppo : None.
Au ho con ibu ions: PD: W i ing - o iginal d a , Da a cu a ion, Fo mal analysis, Me hodology, and Concep ualiza ion,
MO: Re iew and edi ing; NG: Re iew and edi ing; IB: Da a cu a ion and Fo mal analysis; LG: Concep ualiza ion,
Supe ision, Re iew and edi ing.
Con lic s o in e es : The au ho s ha e no con lic o in e es o decla e.
Abb e ia ions
CPA: C yop o ec an agen s
DMF: N, N-Dime hyl o mamide
NMP: N-Me hyl-2-Py olidone
AI: A i icial insemina ion
MOT: Pe cen age o mo ile spe ma ozoa
VCL: Cu ilinea eloci y
VSL: S aigh -line eloci y
VAP: A e age pa h eloci y
LIN: Linea i y
STR: S aigh ness
WOB: Wobble
ALH: Ampli ude o la e al head displacemen
BCF: Bea c oss equency
HOS es : Hypo-osmo ic swelling es