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Author's personl opy Animl Reproution Siene 133 (212) 123 128 Contents lists ville t SiVerse SieneDiret Animl Reproution Siene jou rn l h om ep ge: www.elsevier.om/lote/nireprosi The effet of temperture n on the motility n viility of ostrih sperm Mu Bonto,, Chrlie K. Cornwllis, Irek A. Mleki,, Pulin K. Rynik-Trzskowsk e, Shlk W.P. Cloete,f Deprtment of Animl Sienes, University of Stellenosh, Mtieln 76, South Afri Deprtment of Biology, Lun University, SE-223 62 Lun, Sween Shool of Animl Biology M85, Fulty of Nturl n Agriulturl Sienes, University of Western Austrli, 35 Stirling Highwy, Crwley, WA 69, Austrli UWA Institute of Agriulture, University of Western Austrli, 35 Stirling Highwy, Crwley, WA 69, Austrli e Deprtment of Genetis n Animl Breeing, Wrsw University of Life Sienes, Ciszewskiego 8, 2-786 Wrsw, Poln f Institute for Animl Proution: Elsenurg, Privte Bg X1, Elsenurg 767, South Afri r t i l e i n f o Artile history: Reeive 26 Jnury 212 Reeive in revise form 27 My 212 Aepte 15 June 212 Aville online 21 June 212 Keywors: Semen iluent Sperm storge Sperm viility Ostrih Motility CASA system s t r t As the hemil environment of semen iluents n hve profoun effet on sperm qulity, we exmine the effet of temperture n on the motility n viility of sperm in the ostrih. Semen ws ollete from four mles, eh mle eing replite three times. Ejultes were ilute n inute for 1 min t 2 C n 4 C in four ifferent uffers, temperture juste t 6, 7, 8 n 9 respetively. Averge pth veloity (VAP), urviliner veloity (VCL), stright-line veloity (VSL), linerity (LIN), et ross frequeny (BCF) n mplitue of lterl isplement (ALH) were then reore for eh smple using CASA. The viility of sperm ws ssesse using nigrosin eosin stining. Sperm inute t 4 C h higher motility prmeters, exept for ALH. At 4 C, VAP, VSL n LIN inrese with while VCL, BCF n ALH were higher for lower s. The viility of sperm ws not ffete y temperture ut erese t vlues > 7. A in the neutrl rnge ppere to yiel higher qulity sperm fter in vitro storge t 2 C. However, the effet of ifferent levels n tempertures on sperm longevity nees to e investigte further to evelop vile ostrih speifi iluents. 212 Elsevier B.V. All rights reserve. 1. Introution Assiste reproutive tehnologies like rtifiil insemintion (AI), semen holing n ryopreservtion re neee to preserve n isseminte livestok germplsm. In omesti irs, these tehniques were first estlishe in hikens, n then extene to other poultry speies (Blesois n Brillr, 27; Donoghue n Wishrt, 2; Lke, 1986). Commeril ostrih frming is onstrine y Corresponing uthor t: Deprtment of Animl Sienes, University of Stellenosh, Privte Bg X1, South Afri. Tel.: +27 44 272 677; fx: +27 44 279 191. E-mil ress: monto@sun..z (M. Bonto). low fertility, high levels of emryo n hik mortlity n inequte geneti improvement (Cloete et l., 1998; Cloete n Mleki, 211; Mleki et l., 28). The vent of niml n humn frienly methos to ollet semen from mle ostrihes n rtifiilly inseminte femles (Mleki et l., 28; Rynik et l., 27) resulte in new interest in eveloping ssiste reproutive tehnology to ugment inustry evelopment. Reently, Bonto et l. (211) estlishe tht semen n e ollete frequently without epleting mle sperm reserves or impiring their liio. This mple supply of semen llows AI tehnology to move to the next phse, whih is to esign protool for semen holing n preservtion. Sperm motility is ritil ftor in etermining semen qulity n fertilizing ility. In femle irs, spermtozo 378-432/$ see front mtter 212 Elsevier B.V. All rights reserve. http://x.oi.org/1.116/j.nireprosi.212.6.8
Author's personl opy 124 M. Bonto et l. / Animl Reproution Siene 133 (212) 123 128 re store for extene perios of time in speilize strutures, the sperm storge tuules (SSTs) lolize minly in the utero-vginl juntion of the oviut (Bkst, 211). These strutures hve evolve to relese sperm over time to insure suffiient sperm numers t the site of fertiliztion, even in the sene of mles. For instne, femle ostrihes were foun to ly fertilize eggs for up to 4 weeks fter the lst opultion (Mleki et l., 24). A single insemintion le to mximum fertile perios of 12 ys in Jpnese quils (Reish et l., 1996) n 7 ys in turkeys (Lorenz, 195). Chnges in the externl meium re elieve to regulte sperm motility y omplex intertions mong ftors like temperture, n ion omposition. For instne, n inrese of temperture from 3 C to 4 C resulte in the intivtion of sperm motility in omesti hikens (Gllus gllus). An lkliniztion of the externl, following n ition of lium, then restore sperm motility t 4 C (Ashizw n Wishrt, 1987; Ashizw et l., 1994). Similr results were otine in the turkey n quil (Holm n Wishrt, 1998), wherey the perentge of motile sperm n sperm veloity ws stimulte y n lkline prtiulrly t 4 C (the verge vin oy temperture) ompre to 3 C. Chnges in of the vginl muos fter oviposition hve lso een oserve in oth roilers n turkeys (Bkst, 198), initing tht suh vrition in my hnge the motility of sperm fter insemintion. Therefore, the effet of storge meium, storge temperture, n their intertion nees onsiertion uring the evelopment of speies speifi semen iluent to mintin sperm viility n tivity in vitro. To te, only few stuies hve een onute on sperm motility n survivl in the ostrih fter short term storge. Unilute ostrih semen ogultes rpily t room temperture, leing to eteriortion of sperm qulity (Ciereszko et l., 21; Mleki et l., 28). Furthermore, hnges in motility n viility of ostrih sperm were investigte y storing ilute ostrih semen t 5 C in Miniml Essentil Meium iluent (Y-jie et l., 21), in EK, Lke s n NCl iluents (Mleki et l., 28) or Ovoyl n EK (Ciereszko et l., 21). Sperm motility n viility oul only e mintine for 4 12 h in these stuies, suggesting the nee for the evelopment of suitle iluent speifi for ostrih sperm. The next ruil step in eveloping workle AI progrm for ostrihes is esigning storge meium tht prolongs sperm viility. A key to eveloping suh meium involves etermining the optiml for mximl preservtion of sperm viility n motility in vitro. Chnges in sperm motility n viility over iologilly relevnt rnge were thus ssesse t tempertures of 2 C n 4 C, using omputer-ssiste semen nlysis (CASA) for sperm veloity n nigrosin eosin stining for sperm viility. 2. Mteril n methos 2.1. Stuy popultion, semen olletion n evlution The stuy ws rrie out t the Outshoorn Reserh Frm, South Afri, in Deemer 28 on four South Afrin lk ostrih mles (2 4 yers of ge). Mles were trine prior to the experiment to mount ummy femle n ejulte into n rtifiil lo (Rynik et l., 27). Three ejultes were ollete on onseutive ys (24 h intervl) from eh mle. Ejulte volume ws mesure with n utomti pipette, n sperm onentrtion ws etermine with hemoytometer in 2 L of semen ilute 1:4 (v/v) with phosphte uffere sline solution ontining 1% formlin. The totl numer of sperm per ejulte ws then lulte y multiplying ejulte volume n onentrtion. Finlly, smples of net semen were mounte onto glss slie n the proportion of live norml (memrne intt with norml morphology, eosin non-permele), live norml (memrne intt, eosin impermele ut with ltertion to norml morphology) n e sperm (memrne mge eosin permele) ws estimte fter ounting 5 sperm stine with nigrosin eosin (Lke n Stewrt, 1978) on uplite slies. Sperm forming omplete unit of slightly urve he, mi-piee n til were onsiere s live norml (s esrie y Soley n Roerts, 1994), while ny sperm showing evine from this struture ws onsiere s live norml (i.e. gint sperm, oule hee sperm, he ent on the til, swollen he, roken he, stright sperm). 2.2. Smple preprtion Net semen ostrih ejulte hs een previously estimte to hve of 7. (Y-jie et l., 21), hene for this experiment, eh ejulte ws teste t four levels t the two tempertures s follows: 2 L of sperm ws ilute in 8 L (1:4) of eh of four uffers, temperture juste to 2 C n 4 C. All uffers ontine 15 mm of NCl n one of the following ompouns: 2 mm of 2-(N-morpholino) ethne sulphoni i (MES) for 6; 2 mm of N-tris-[hyroxymethyl]- methyl-2-minoethnesulfoni i (TES) for 7; 2 mm of Tris[hyroxymethyl] minomethne hyrohlorie (TRIZMA) for 8 n; 2 mm of 3[ylohexylmino]-2- hyroxyl-1-propne-sulfoni i for 9. Smples were then inute for 1 min t either 2 C or 4 C. The orer of the tretments ws rnomise ut our experimentl proeure i not llow for rnomising temperture tretments. 2.3. Assessment of sperm motility After inution, 1 L of the 1:4 sperm solutions were further ilute to 1 million sperm/ml with their originl uffer hel t the sme temperture. Sperm veloity ws mesure with 1 ojetive uner negtive phse ontrst using BX 41 Olympus mirosope (Olympus Corportion, Tokyo, Jpn). For eh smple, sperm veloity ws reore y loing 5 L of solution onto mirosope slie ple on hete stge t either 2 C or 4 C n tking three reings from three seprte fiels of view. This proeure ws repete twie for eh smple. The igitl vieo files were nlyze using Sperm Clss Anlyzer (SCA v.3, Miropti S.L., Spin). Averge pth veloity (VAP, veloity over lulte smoothe
Author's personl opy M. Bonto et l. / Animl Reproution Siene 133 (212) 123 128 125 pth), urviliner veloity (VCL, veloity over the tul sperm trk, inluing ll evitions of sperm he movement), stright line veloity (VSL, veloity over the stright line istne etween the eginning n the en of the sperm trk), linerity (LIN, eprture of sperm trk from stright line [VSL/VCL] 1), et-ross frequeny (BCF, frequeny with whih the sperm rosses the smoothe pth), n mplitue of lterl isplement (ALH, time verge of solute vlues of the instntneous turning ngle of the sperm he long its urviliner trjetory) were lulte for eh smple from the two reorings of t lest 1 sperm trks eh. The meins for ll prmeters were strongly orrelte with their mens (Person s orreltion oeffiient R >.95), ut we use the mein to ensure tht entrl teneny of ll mesures on ejultes were not influene y non-norml istriution of motility. Finlly, smples of eh of the sperm suspensions were stine with nigrosin eosin n mounte onto glss slie to etermine the proportion of live norml, live norml n e sperm in the sme wy s for net sperm. 2.4. Sttistil nlysis To investigte the effets of temperture n on sperm veloity, Generl Liner Moels (GLM) were performe on ll CASA prmeters (meins of VAP, VCL, VSL, LIN, BCF n ALH lulte from ll trke sperm over the 3 fiels mesure) entere s response vriles, while temperture, n their intertion were entere s fixe ftors. Mle numer ws entere s fixe ftor to ontrol for iniviul vrition. Similr GLMs were performe on men perentge of live norml, live norml n e sperm to evlute the effet of temperture n on viility of sperm. All sttistil nlyses were performe using Genstt version 13 (VSN Interntionl Lt., UK). (1) Men VAP (µm/s) (2) (3) Men VSL (µm/s) Men VCL (µm/s) 4 35 3 25 2 15 1 5 6 5 4 3 2 1 3 25 2 15 1 2ºC 4ºC 3. Results 3.1. Desriptive sttistis of ejulte prmeters The men ejulte volume (±SEM) ross the whole experiment ws 1.11 ±.26 ml, with n verge onentrtion of 3.2 ±.29 1 9 sperm/ml, n ontine 4.13 ± 1.36 1 9 sperm. The men proportion of live norml, live norml n e sperm ws 72.63 ± 4.93%, 18.15 ± 3.57% n 9.22 ± 2.29% respetively. While no ge effet ws etete for ny of the vrile mesure (P >.5), signifint mle effet ws foun for ll ejulte prmeters (P <.1). 3.2. Effet of temperture n on the motility of sperm The intertion etween temperture n ws signifint for ll CASA trits (P <.5; Tle 1). Veloity prmeters (VAP, VSL n VCL) s well s LIN n ALH were inepenent of t 2 C (P >.5; Fig. 1), ut the BCF of sperm showe n inrese s inrese from 6 to 7 (F 3,41 = 3.81, P =.2; Fig. 2). Furthermore, we etete mle effet on LIN (F 3,41 = 5.98, P =.2) n ALH (F 3,41 = 4.81, P =.6), with one mle showing higher 5 Fig. 1. Effet of n temperture on the veloity of sperm of four mle ostrihes: (1) men verge pth veloity (VAP); (2) men urviliner veloity (VCL) n; (3) men stright line veloity (VSL). Vertil lines out the mens enote stnr errors. For ny temperture n, those results whih re signifintly ifferent (P <.5) re ssigne ifferent letter. sperm LIN n lower sperm ALH ompre to the rest of the mles. Sperm in ll tretments inute t 4 C h higher (P <.5) veloity thn t 2 C. In ontrst to the teneny t 2 C, ll veloity prmeters t 4 C were ffete y, ut to ifferent extents (Tle 1). VAP n VSL inrese with with mximum t 8 (men ± SE = 32.61 ± 1.71 m/s, F 3,41 = 1.98, P =.35 n men ± SE = 25.38 ± 1.79 m/s, F 3,41 = 2.84, P =.4, respetively; Fig. 1), while VCL ws highest t lower vlues, (F 3,41 = 8.2, P =.1). In ition, LIN rehe mximum t 8 (men ± SE = 67.34 ± 3.3%, F 3,41 = 18.16, P =.1; Fig. 2), while BCF n ALH of sperm were higher t 6 (men ± SE = 5.5 ±.2 Hz, F 3,41 = 1.67, P =.1 n
Author's personl opy 126 M. Bonto et l. / Animl Reproution Siene 133 (212) 123 128 Tle 1 Degrees of freeom, F-vlues n signifine levels enoting the effet of temperture n on the motility of the ostrih sperm. Vrile Temperture Temperture f F P f F P f F P VAP 1,85 134.14.1 3,85 1.68.17 3,85 2.34.8 VCL 1,85 11.15.1 3,85 4.1.1 3,85 7.31.1 VSL 1,85 122.28.1 3,85 2.78.46 3,85 2.93.38 LIN 1,85 295.24.1 3,85 15.52.1 3,85 19.2.1 BCF 1,85 11.8.1 3,85 1.11.348 3,85 14.47.1 ALH 1,85 144.22.1 3,85 4.59.1 3,85 28.15.1 Bol vlues re signifint in the nlysis. Sperm motility ws ssesse using omputer-ssiste sperm nlysis system (CASA system). VAP: verge pth veloity; VCL: urviliner veloity; VSL: stright line veloity; LIN: linerity; BCF: et-ross frequeny; n ALH: mplitue of lterl isplement. men ± SE = 2.17 ±.6 m, F 3,41 = 61.82, P =.1, respetively) n lower t 9 (men ± SE = 4.1 ±.2 Hz, men ± SE = 1.4 ±.6 m respetively). No mle effet ws etete for ny of the CASA prmeters t 4 C (P >.5). (1) Men LIN (%) (2) Men BCF (Hz) 8 7 6 5 4 3 2 1 6 5 4 3 2 1 6 6 7 7 8 8 9 e 9 2ºC 4ºC 3.3. Effet of temperture n on the viility of sperm Sperm viility ws not ffete y temperture (F 1,85 =.1, P =.9). However, with n inrese in, the perentge of live norml sperm erese (F 3,85 = 8.19, P =.1; Fig. 3), while the perentge of e sperm inrese (F 3,85 = 6.85, P =.1) n the perentge of norml sperm remine unhnge throughout tretments (F 3,85 =.52, P =.672). Furthermore, we foun vrition etween mles, oth in the perentge of live norml n live norml sperm (F 3,41 = 4.18, P =.1 n F 3,41 = 15.61, P =.1 respetively), ut not in the perentge of e sperm (F 3,41 =.98, P =.41). % of live norml spermtozo 6 5 4 3 2 1 e e e (3) 2.5 Men ALH (µm) 2 1.5 1.5 Fig. 2. Effet of n temperture on the motility of sperm of four mle ostrihes: (1) men linerity (LIN); (2) men et ross frequeny (BCF) n; (3) men mplitue of lterl isplement (ALH). Vertil lines out the mens enote stnr errors. For ny temperture n, those results whih re signifintly ifferent (P <.5) re ssigne ifferent letter. % of e spermtozo 7 6 5 4 3 2 1 Fig. 3. Effet of n temperture on the perentges of live norml n e sperm of four mle ostrihes. Vertil lines out the mens enote stnr errors. For ny temperture n, those results whih re signifintly ifferent (P <.5) re ssigne ifferent letter.
Author's personl opy M. Bonto et l. / Animl Reproution Siene 133 (212) 123 128 127 4. Disussion Our finings revel tht omintion of high temperture (4 C) n 8 pper to mximize ll veloity prmeters, with the exeption of urviliner veloity. LIN showe similr pttern ut BCF n ALH h n opposite tren with lower vlues oserve with higher. Temperture i not ffet the viility of sperm, ut higher proportions of live norml sperm were oserve t lower vlues. Motility prmeters hve een extensively mesure y CASA to investigte sperm motility in ors (Holt et l., 1997), ulls (Amnn, 1989) n turkeys (Bkst n Ceil, 1992) n hve een foun to orrelte well with fertility in vin speies (hikens: Blesois n Brillr, 27; turkeys: King et l., 2). For instne, fertiliztion suess ws foun to e orrelte to VAP in the hiken (Whishrt n Plmer, 1986), n in the turkey, VSL, LIN n BCF esrie the overll sperm moility phenotype, n VSL, VAP, VCL, LIN n BCF were higher in mles esrie s hving high moility sperm, ompre to mles with low moility sperm (King et l., 2). Ostrih sperm pper to e stimulte y ner oy temperture (in the ostrih: verge: 38.3 C with rnge etween 37.9 n 4.7 C: Whittow, 1976) n slightly lkline onitions. Previous reserh on hikens, turkeys n quils show similr response wherey sperm motility eme more vigorous for vlues higher thn 7 (Holm n Wishrt, 1998). However, no sperm movement ws reporte for ny of these speies in ii onitions ( 5 6) n sperm motility ws only initite t 4 C from of 7.8 for turkey sperm n 7.2 for quil sperm. Ostrih sperm seems to iffer from this pttern, s sperm movement n veloity were etete for -vlues s low s 6 t oth tempertures. Originlly, sperm were thought to remin motionless uring their sty in the femle s SSTs (hiken n turkey: Zvlet n Ogswr, 1987), until their motility ws reinitite upon relese from the SSTs n their migrtion to the fertiliztion site. However, it hs een rgue (Fromn, 23) tht sperm shoul sty motile uring their sty in the SSTs s they hve to mintin their position ginst onstnt urrent generte y the epithelil ells of the SSTs. Our results re onsistent with this eing possiility in ostrihes, s sperm motility ws oserve, even for low vlues. In the ostrih, roni nhyrse, n enzyme tht my regulte through ironte n hyrogen seretion, ws etete in the surfe of the epithelium of the UVG n the SSTs (Holm et l., 2) suggesting tht femles might e le to regulte sperm tivity n storge through hnging the. At 6 n 4 C, high VCL n low VSL n LIN vlues tht we reore inites sperm movement my e eome ner irulr, suggesting epenent regultory mehnism tht my enle sperm to turn roun insie SST n move out. In vitro stuies hve lso emonstrte tht the motility n metoli rte of sperm, n onsequently the lifespn of sperm, n e ltere y the of iluents. For instne, in the hiken (Bogonoff n Shffner, 1954) low ereses motility, lti i proution n oxygen uptke of sperm wheres high my inrese metoli rte n susequently ffet sperm motility n viility. Further eviene rises from experiments showing tht sperm from oth hikens n turkeys n e kept immotile t n ii n hve their motility restore y iluting the meium with n lkline uffer (Holm n Wishrt, 1998). Our results pper to e in orne with this, s the omintion of higher temperture n lkline signifintly inrese the motility of sperm, while n ii seems to impir sperm veloity. Interestingly, while temperture i not ffet sperm viility, we oserve greter proportion of live sperm for low to neutrl vlues (6 7). is known to ffet metoli rte n it is generlly epte tht reution in of iluents is require to store semen in vitro to mintin viility n fertilizing ility. For instne, in turkeys, sperm mixe with iluents t 6.5 in omprison to 7.5 h higher rtes of fertility fter 6 h in vitro storge t 15 C (Giesen n Sexton, 1982). Although the ext mehnisms wherey low uring in vitro storge promotes viility re unknown, it is thought tht the reution in sperm metolism onserves energeti resoures promoting lifespn. This is onsistent with the results from our stuy, s higher vlues seeme to stimulte sperm motility, ut sperm viility ws ompromise. This highlights the nee to further investigte if the ition of ompouns to iluents tht my e metolize y sperm n llevite the negtive effets of higher on sperm viility. In onlusion, oy temperture n slightly lkline onitions seem to stimulte ostrih sperm motility, ut it ppers tht n ii to neutrl rnge for the ostrih-speifi iluents is require to ensure etter sperm survivl for rtifiil insemintion uring in vitro storge. These results re therefore the first milestone in our unerstning of the sperm funtion in the in vitro environment for this speies. However, the proesses of in vivo sperm storge in the oviut re hllenge to the evelopment of pproprite methos to preserve sperm in vitro. Semen iluents must provie reltively onstnt physio-hemil stility to sperm uring in vitro storge, inluing iso-osmoti n nerly neutrl onitions, s well s pproprite energy sustrtes (Blesois n Brillr, 27; Donoghue n Wishrt, 2). However, s the fertilizing ility of sperm kept in vitro t vin oy temperture nnot e mintine for long, sperm metolism must e reue y progressively eresing temperture n potentilly levels. Our results suggest tht to mximize fertility in rtifiil insemintion progrms of ostrihes, ifferent temperture storge might e require: lower temperture for long term storge tht mximizes viility n longevity, n oy-like storge temperture for insemintion purposes tht stimultes sperm motility to mximize sperm storge. Aknowlegments We grtefully thnk the Outshoorn Reserh Frm n espeilly Anel Engelreht, Stefn Engelreht, Znell Brn n Bsie Pfister for ssistne in tking re of the irs, s well s Ceeli Mutlow t the Klein Kroo Lortory for ssistne with lortory equipment. Funing ws provie y the Western Cpe Agriulturl Reserh Trust
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