Effect of glycerol, n, n-dimethylformamide and n-methyl-2-pyrrolidone on rabbit sperm stored at 4 °C and 16 °C

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, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed. 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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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