Standardization of sample preparation, staining and sampling methods for automated sperm head morphometry analysis of boar spermatozoa

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1 interntionl journl of ndrology ISSN ORIGINAL ARTICLE Stndrdiztion of smple preprtion, stining nd smpling methods for utomted sperm hed morphometry nlysis of or spermtozo M. Grí-Herreros,* I. M. Apriio, F. J. Brón,à L. J. Grí-Mrín nd M. C. Gil* *Deprtments of Mediine & Animl Helth, nd Physiology, Fulty of Veterinry Mediine, University of Extremdur, Cáeres, nd àdeprtment of Biosttisti, Fulty of Mediine, University of Málg, Málg, Spin Summry Keywords: utomted sperm morphometry nlysis, or, morphometry, sperm morphology, stining proedures Correspondene: M Cruz Gil Any, Unity of Reprodution nd Ostetris, Fulty of Veterinry, Avd. de l Universidd s/n, Cáeres, Spin. E-mil: rgil@unex.es Reeived 2 Deemer 2005; revised 2 Ferury 2006; epted 14 Ferury 2006 doi: /j x The ury of omputer-ssisted sperm morphometry nlysis (CASMA) depends on the reful preprtion, fixtion nd stining of spermtozo. The effiieny of CASMA my e enhned y developing optimized protools. The im of the present study ws to evlute the influene of sperm wshing nd the use of three stining tehniques [rpid Pnopti, Hemolor nd Hrris s Hemtoxylin (HH)] on imge-proessing ury nd or sperm hed morphometry. Sperm wshing hd signifint effet on smples stined with rpid Pnopti, inresing the perentge of orretly inrized sperm heds nd the ontrst etween ells nd kground. However, rpid Pnopti yielded the lowest perentge of properly digitized sperm heds. HH provided the highest ell/kground ontrst, nd lso greter sperm hed stining intensity, ut disrimintion of sperm midpiees ws onsidered insuffiient. Hemolor oupied n intermedite position, providing eptle olour intensity nd stisftory ell/kground ontrst. Use of different stining proedures prompted dimensionl differenes in sperm hed morphometry. Signifint differenes etween nimls were oserved for ll morphometri prmeters. Low within-niml vrition oeffiients refleted homogeneous sperm hed popultion. Between-niml vrition oeffiients were reltively high for Hemolor nd HH, nd signifintly high for the rpid Pnopti stin. Using Pnopti nd HH, stle morphometri mesurements required t lest 100 properly digitized sperm heds rther thn 200, while Hemolor required only 50 spermtozo. These results indite tht oth wshing of semen nd stining proedures signifintly ffet the ury of imge proessing nd sperm hed dimensions. Hemolor nd HH proved to e the est stining tehniques for evluting sperm hed dimensions in or. Introdution Morphologil lssifition of spermtozo hs eome n integrl prt of routine semen nlysis. The reson is tht poor sperm morphology is n importnt inditor of deresed fertility oth in men nd nimls (Bostofte et l., 1982; Kruger et l., 1988; Crrell et l., 1994; Leroy-Mrtin et l., 1994). Sperm morphology hs, moreover, een used s n inditor of iologil (Ks & Thwits, 1992) nd toxiologil stress (Wyroek et l. 1983; Dvis et l. 1995). To redue the sujetivity of sperm hed morphology ssessment omputer-ssisted sperm morphometry nlysis (CASMA) hs een previously desried (Jgoe et l., 1986; Moruzzi et l., 1988; Boyle et l., 1992; Dvis et l., 1992), systems ompletely utomti. These systems imge spermtozo through omputer nd perform numer of morphometri mesurements of sperm hed geometry. These progrmmes hve een shown to e urte, preise nd repetle (Clmer et l., 1994), nd they revel sutle differenes mong individuls interntionl journl of ndrology 29 (2006) ª Journl ompiltion 2006 Blkwell Pulishing Ltd 553

2 Morphometril evlution of or sperm heds M. Grí-Herreros et l. whih nnot e deteted with sujetive methods (Jgoe et l., 1986; Dvis et l., 1992; Dvis & Grvne, 1994; Bll & Mohmmed, 1995). However, on the morphometri nlysis of spermtozo numer of nlytil vriles n influene the results of the nlysis nd n e ttriuted to the CASMA system itself, to the method of smple preprtion or it n e the result of iologil vrition. Preprtion of the semen smple inludes the wshing or not of the semen nd the use of different stining proedures. Aording to some uthors, semen wshing, with the purpose of eliminting seminl plsm, improves the performne of the tehnique (Dvis et l., 1992; Dvis & Grvne, 1993; Grvne & Dvis, 1995). As regrds stining, we hve to tke into onsidertion tht spermtozo of individul speies inorporte stins unevenly. Therefore, it is essentil to find the most suitle stining tehnique for eh speies. Proper stining is fundmentl to otin good sperm ontrst nd therey minimize mistkes in hed digitiztion (Kruger et l., 1988; Dvis & Grvne, 1993). Biologil vrition is nother vrile tht n ffet the results of the morphometri sperm nlysis. If n insuffiient numer of spermtozo re nlysed, this vrition in smple popultion my onfuse the interprettion of quired dt. For exmple, humn sperm nlysis requires t lest 200 ells to ensure representtive smple for evluting the perentge of norml sperm (Dvis & Grvne, 1993). The existene of ll these vriles n influene the results of CASMA. Therefore, for omprison of results otined y different lortories it is neessry to stndrdize the morphometri evlution protool of the spermtozo. In men (Dvis & Grvne, 1993, 1994; Kruger et l., 1993; Lquet et l., 1996) nd in some domesti speies (Grvne et l., 1996,; Csey et l., 1997; Snho et l., 1998; Buendí et l., 2002; Soler et l., 2005) reserh hs stndrdized sperm nlysis n venue hitherto unexplored in the pig. Although severl studies ddressed the use of CASMA longside other sperm ssessment tehniques in the or (Shmidt, 1997; Hiri et l., 2001), this study ppers to e the first pproh to CASMA stndrdiztion in this speies. Hitherto, sperm morphometry nlysis hs een performed using modifition of protools developed for other niml speies. The present study therefore sought to nlyse the effets of smple preprtion, stining tehniques nd numer of sperm ells nlysed on the results otined, s first step towrds the stndrdiztion of morphometri nlysis protools essentil for ensuring the ury of CASMA tehnology. Mterils nd methods Colletion nd preprtion of seminl smples We used ommeril seminl doses for rtifiil insemintion of 4 Ierin ors (2.5 3 yers of ge; kg of ody weight) from the Stmoek Ieri, S.A. rtifiil insemintion entre (Mejord, Toledo, Spin). They re proven fertility ors, going through proess of semen olletion one week in the lst 9 months. Whole ejultes were mnully olleted on the sme dy nd then immeditely pled in wter th t 37 C. All ejultes fulfilled the minimum requirements for use in rtifiil insemintion (minimum 70% of motile spermtozo nd 80% of morphologilly norml spermtozo nd totl numer of spermtozo higher thn ). The semen ws immeditely diluted to onentrtion of spermtozo per ml in n extender (modified BTS) for refrigerted semen nd distriuted in ommeril ontiners of 100 ml for rtifiil insemintion. One their temperture ws redued to 17 C the diluted ejulted ones were trnsported to the lortory within 12 h. The experiments were rried out immeditely fter the reeption of the smples in the lortory. Semen from eh individul seminl dose ws treted s follow: (i) semen smples of 1 ml were diluted 10x in phosphte-uffered sline (PBS) (finl onentrtion sperm ells per ml) nd entrifuged t 600 g for 10 min (wshed semen) or (ii) semen smples of 1 ml (onentrtion sperm ells per ml) were entrifuged t 600 g for 10 min (unwshed semen). After entrifugtion, smples were resuspended in the pproprite volume of PBS to give finl ell onentrtion of sperm ells per ml. Slides were prepred with 7-lL drop nd the drop drgged ross the slide using the edge of nother slide s desried y Zneveld & Polkoski (1977); slides were llowed to ir dry for 30 min. Stining proedures Rpid Pnopti (Pnre Quimi S.A., Brelon, Spin), Hemolor (Merk KGA, Drmstdt, Germny) nd Hrris s Hemtoxylin (HH) (Pnre Quimi S.A.) were used to stin four slides from eh individul ejulte (two from wshed semen nd two from unwshed semen). Rpid Pnopti stining ws performed y 10-min fixed smple in methnol follow y 13-min stin in eosin nd 11 min in methylene lue. For rinsing the slides distilled wter ws used. Hemolor stining ws pplied following kit reommendtions: fixtion (methnol) for 5 se in solution 1 follow y sinking in 554 interntionl journl of ndrology 29 (2006) Journl ompiltion ª 2006 Blkwell Pulishing Ltd

3 M. Grí-Herreros et l. Morphometril evlution of or sperm heds solution 2 (red olour retive) for 3 se, in solution 3 (lue olour retive) for 6 se nd finlly in the uffer solution for 45 se. Hemtoxylin stining ws performed y 10-min fixed smple in methnol follow y sinking in Hemtoxylin solution during 30 min. All slides were exposed to dry ir nd kept permnently mounted with Eukitt (O. Kindler GmH & Co., Freiurg, Germny). The totl numer of stined slides ws 24 for the wshed semen seletions (per stin: two slides per or, four ors, eight slides; three stins ¼ 24 slides) nd 24 for the unwshed semen ones (per stin: two slides per or, four ors, eight slides; three stins ¼ 24 slides). Morphometri nlysis Morphometri evlution of the semen smples ws performed using the morphometri module of ISASÒ (Proiser R+D S. L., Buñol, Spin), omputer-ided sperm nlysis system. A Nikon Lophot mirosope (Nikon, Tokyo, Jpn), with rightfield 100x oil immersion ojetive nd 3.3x photo-oulr ws used. Video signl ws quired y Sony CCD AVC-D7CE video mer (Sony Corportion, Tokyo, Jpn) mounted over the mirosope. The rry size of the video frmegrer ws its providing digitized imges of pixels nd 256 grey levels. Resolution of imges ws lm per pixel oth in horizontl nd vertil xes. Before oservtion, the illumintion soure ws entred, nd green filter ws pplied on the light soure to inrese the ontrst etween sperm imges nd the kground light. The intensity of the ul, the gin nd the offset of the mer were stndrdized ross ll smples. Spermtozo lose to the distnt edge of the origin were exluded to void the possiility of ising sperm morphometry nlysis (Soler et l., 2003). Sperm ells were displyed on the monitor nd ll the spermtozo whih did not present ny overlp with deris or other ells were onsidered for nlysis. The differentition of sperm heds ws sed on imge segmenttion within predetermined rnge of grey level vlues. If ny inorret definition of the sperm hed oundry ws deteted, it ws orreted y using n editing fility of the system. Misdigitiztion errors ourred when the sperm hed omputer generted outline did not urtely orrespond to the rel edge of the sperm hed or even when n outlined portion of the midpiee ws onsidered s ontinution of the sperm hed. Both the systemti serh nd pture of the spermtozo in the slide nd the morphometri nlysis were rried out y the sme person. With the oundry informtion of eh sperm hed, mesurement of the size nd shpe ws mde utomtilly y using the ISAS progrm. The following morphometri prmeters were seleted: length (L) nd width (W) (in lm, the lrgest nd smllest vlues, respetively, of the Feret dimeters mesured t ngles of 0, 30, 60, 90, 120 nd 150 ; L nd W re not neessrily orthogonl), re (A) (in lm 2, s the sum of ll pixel res ontined within the oundry), perimeter (P) (in lm, s the sum of externl oundries) nd four derived prmeters of hed shpe: elliptiity (E), ttending the expression L/W, rugosity (R) s 4 p A/P 2, elongtion (El) s (L ) W)/(L + W) nd regulrity (Rl) s p L W/4 A. Three hundred sperm hed imges were otined from eh slide in the mnul quisition mode of the progrm. After nlysis, the numer of misdigitized sperm hed ws reorded for eh nlysis. Dt were ompiled nd stored for further nlysis. Influene of stining tehnique nd wshing on imge proessing ury A totl of 4800 spermtozo per stining proedure (2400 spermtozo from wshed semen nd 2400 from unwshed semen) were digitized nd nlysed. The numer of sperm heds orretly onverted to mesurle inry imges nd the numer of indequtely digitized sperm heds were reorded to estimte the effiieny of the system nd method used. Influene of stining tehnique on sperm morphometry After evluting the effet of sperm wshing on imge proessing ury, the following slides were seleted: wshed + rpid Pnopti stin, unwshed + Hemolor stin nd unwshed + HH. At lest 250 properly-digitized sperm heds were otined from eh slide, two per niml nd stining method, nd finlly nlysed using the ISAS morphometri module. Sperm hed vlues for the three stining tehniques were then ompred: men vlues for morphometri prmeters were reorded for eh or; the effet of stining proedure, of or, nd of stining proedure within eh or ws then nlysed. Influene of the numer of spermtozo Two hundred properly digitized sperm heds from slides stined with rpid Pnopti (wshed semen), Hemolor (unwshed semen) nd HH (unwshed semen) were nlysed from eh niml. Susets of 50, 100, 150 nd 200 rndomly seleted spermtozo were ompred to determine the minimum smple size needed to hrterize the whole popultion. interntionl journl of ndrology 29 (2006) ª Journl ompiltion 2006 Blkwell Pulishing Ltd 555

4 Morphometril evlution of or sperm heds M. Grí-Herreros et l. Sttistil nlysis The men nd stndrd error of the men were lulted for desriptive sttistis. Kolmogorov Smirnov nd Levene tests were used to hek the normlity of dt distriution nd vrine homogeneity respetively. Nonprmetri tests were used for ll the lultion s the dt did not djust to norml distriution. For eh morphometril prmeter, differenes etween stining proedure, individul ors nd stining proedure within ors were evluted y the Kruskll Wllis test, followed y the posteriori Mnn Whitney U-test to ompre pirs of vlues (s men vlues). Moreover, the within-niml oeffiient of vrition (CV) ws lulted s the men of eh individul CV, nd the etween-niml CV ws estimted s the CV of within-niml CVs; this lst prmeter, in prtiulr, ws intended s mens of mesuring the disrimintion power of eh stin etween nimls. To determine the minimum smple size needed to hrterize the whole popultion, 200 spermtozo from eh niml were nlysed. Susets of 50, 100, 150 nd 200 rndomly seleted spermtozo were ompred using the Mnn Whitney U-test. All sttistil nlyses were performed using the sttistil pkge SPSS version 11.0 for Windows softwre (SPSS In., Chigo, IL, USA). Results Influene of stining tehnique nd wshing on imge proessing ury The HH stin yielded the lowest perentge, nd rpid Pnopti the highest perentge of misdigitized sperm heds (Tle 1). For Hemolor nd HH, the perentge of orretly inrized sperm heds ws very similr, oth for wshed nd unwshed sperm, while for the rpid Pnopti stin the perentge ws muh lower for unwshed smples. The effet of wshing on eh individul niml (Tle 2) ws prtiulrly strong when using the rpid Pnopti stin, with mrked inrese in CV vlues for unwshed smples. Therefore, utomted sperm hed morphometry nlysis ws performed using the rpid Pnopti stin with wshed sperm nd the Hemolor nd HH stining tehniques with unwshed sperm. Effet of stining tehnique on sperm morphometry Comprison of sperm morphometri prmeters using rpid Pnopti (wshed smples), Hemolor (unwshed smples) nd HH (unwshed smples) is shown in Fig. 1. The stining method signifintly influened ll prmeters. With regrd to diret morphometri prmeters (length, width, re nd perimeter), the lowest vlues were found using the HH stin; Hemolor nd rpid Pnopti yielded firly similr vlues for ll prmeters exept width, lthough differenes were sttistilly signifint (p < 0.005). Within-niml omprison of the effet of stining on diret morphometri prmeters displyed similr results (Tle 3): signifint differenes were oserved etween stining proedures for ll prmeters (p < 0.01). With regrd to rugosity nd regulrity, rpid Pnopti nd HH yielded similr results, while lower vlues were otined with Hemolor. For elliptiity nd elongtion, the three stining tehniques provided different results (p < 0.005), vlues rising in the order rpid Pnopti < Hemolor < HH. Within eh stining proedure, signifint etweenniml differenes were oserved for ll prmeters (p < 0.01) (Tle 3). Additionlly, within-niml CVs rnged from 3.02% (rugosity) to 12.53% (elongtion) for rpid Pnopti, 2.62% (rugosity) to 10.33% (elongtion) for Hemolor, nd 2.45% (regulrity) to 9.38% (elongtion) for HH. There ws no signifint differene etween CVs for the three stins (p > 0.40). The etween-niml CV (estimted s the CV of within-niml CVs) ws lulted to determine, for eh stin, the degree of differene in the CV prmeter etween the four ors. The highest vlues were found for the rpid Pnopti stin, rnging etween 24.30% (elliptiity) nd 90.1% (regulrity), ompred with rnge from 1.81% (elliptiity) to 28.26% (rugosity) for Hemolor nd from 7.0% (length) to 26.26% (rugosity) for HH. Tle 1 Effet of stin nd wshing on perentge of orretly inrized sperm heds Stin N Unwshed Wshed Rpid Pnopti ± ± 5.3 Hemolor ± ± 2.5 Hrris s Hemtoxylin ± ± 0.6 Vlues, in perentge, re men ± SD. Influene of the numer of ells nlysed No sttistilly signifint differenes (p > 0.10) in morphometri vlues were reorded within eh niml for eh stining proedure, when using 100, 150 or 200 ells, with the exeption of or 1 [50 vs. 200 ells, HH, length (p ¼ 0.029) nd elliptiity (p ¼ 0.025)]; in or 4 (rpid Pnopti) there were smll ut signifint differenes in perimeter (p ¼ 0.042). 556 interntionl journl of ndrology 29 (2006) Journl ompiltion ª 2006 Blkwell Pulishing Ltd

5 M. Grí-Herreros et l. Morphometril evlution of or sperm heds Tle 2 Effet of niml, stin nd wshing on the perentge of orretly inrized sperm heds Rpid Pnopti Hemolor Hrris s Hemtoxylin Bor Unwshed Wshed Unwshed Wshed Unwshed Wshed Men ± SD 81.4 ± ± ± ± ± ± 0.6 CV (%) SD, stndrd devition; CV, oeffiient of vrition. Disussion Morphologil lssifition of spermtozo hs eome n integrl prt of modern semen nlysis in humn (World Helth Orgniztion, 1999) nd severl domesti speies (Dott, 1975; Ske, 1982; Evns & Mxwell, 1987). A reltionship hs een reported etween the perentge of morphologilly norml spermtozo nd fertility, oth in men nd in other nimls (Bne, 1961; Sklet et l., 1988; Jsko et l., 1990; Krinus et l., 1990; Leroy-Mrtin et l., 1994). Studies in or hve found sesonl vrition in sperm morphology nd negtive effet of high tempertures nd humidity on sperm morphology (Suriysomoon et l., 2005). In the sujetive evlution of semen hrteristis, onsiderle vritions re reported oth etween tehniins nd etween lortories (Ske, 1982; Bker & Clrke, 1987). CASMA ws developed to redue this sujetivity in sperm hed morphology ssessment (Ktz et l., 1988; Dvis et l., 1993; Grvne et l., 1995, 1996,; Buendí et l., 2002; Soler et l., 2003). This tehnology, sed on the use of omputer-ssisted imge-nlysis tehniques thus reduing soures of vrition hs reveled sutle differenes etween individuls whih nnot e deteted through the use of sujetive methods (Anos & Isoun, 1980; Dvis & Grvne 1994). There hs een some reserh into the stndrdiztion of semen nlysis in humns (Dvis & Grvne, 1993; Lquet et l., 1996; Soler et l., 2003) nd in numer of domesti speies (Dvis et l., 1993; Grvne et l., 1995, 1998; Ggo et l., 1998; Snho et l., 1998; Buendí et l., 2002; Rijsselere et l., 2004; Soler et l., 2005). Nevertheless, even in those studies ddressing the use of CASMA for the morphometri evlution of porine spermtozo, there hs een no referene to the optimum onditions for morphometri hrteriztion in this speies; insted, the protools pplied hve een derived from other speies. The present study ompred rpid Pnopti, Hemolor nd HH stins, s ll three re widely used for sperm morphometri nlysis in other speies nd the stining proedures re simple nd provide relile results (Grvne et l., 1995, 1997; Csey et l., 1997; Ggo et l., 1998; Snho et l., 1998; Buendí et l., 2002; Soler et l., 2003, 2005). The ury of CASMA tehnology depends on reful preprtion, fixtion nd stining of spermtozo (Dvis & Grvne, 1993; Grvne et l., 1995, 1998; Soler et l., 2003). Smple preprtion methods inlude sperm wshing, to remove seminl plsm nd ensure n pproprite ontrst etween ells nd kground, followed y resuspension in simple medium nd stining using one of rnge of tehniques. While some uthors report tht wshing semen smples enhnes CASMA performne (Dvis et l., 1993; Dvis & Grvne, 1993; Grvne & Dvis, 1995; Grvne et l., 1995), others note the opposite effet: greter ontrst etween the ells nd kground in fresh, unwshed smples, nd fewer properly digitized nd nlysed ells in wshed smples using vriety of stining proedures (Soler et l., 2003). In the present study, wshing hd prtiulrly signifint effet on smples stined with rpid Pnopti, inresing y up to 11% the perentge of orretly inrized sperm heds nd the ontrst etween ells nd kground. In nimls with the more turid seminl plsm (nos 2 nd 4), wshing onsiderly inresed the perentge of orretly inrized ells when using the rpid Pnopti stin (CV 6 vs. 10 for wshed nd unwshed smples respetively). Using Hemolor nd HH stins, the numer of orretly inrized sperm heds ws similr in wshed nd unwshed smples, so it ws deemed unneessry to wsh smples when using these stins; this simplifies the method nd lso voids mnipultion of the semen. Clerly, ll protools for the morphometri evlution of porine spermtozo must stte whether semen is wshed or not, s ertin morphometri prmeters were modified y wshing, irrespetive of the stining tehnique (dt not shown). Differenes in vlues depending on whether sperm were wshed or not hve lso een reported y reserhers in humn smples (Soler et l., 2003). interntionl journl of ndrology 29 (2006) ª Journl ompiltion 2006 Blkwell Pulishing Ltd 557

6 Morphometril evlution of or sperm heds M. Grí-Herreros et l. Length 10 Width Are 26 Perimeter Elliptiity 3 0,9 Rugosity 2 0,7 2 0,6 0,4 Elongtion Regulrity 1 0,4 0,3 N = PP HC HH 0,9 0,8 N = PP HC HH Figure 1 Box-nd-whisker plots showing vritions in sperm hed morphometri ording to stin used (PP, rpid Pnopti; HC, Hemolor; HH, Hrris s Hemtoxylin); N represents totl numer of sperm ells nlysed. Eh ox enloses the 25th perentile, the line in the middle is the medin nd the vertil rs extend to 5th nd 95th perentiles of the men vlues. Boxes lelled with different supersripts re sttistilly different to eh other (p < 0.005). 558 interntionl journl of ndrology 29 (2006) Journl ompiltion ª 2006 Blkwell Pulishing Ltd

7 M. Grí-Herreros et l. Morphometril evlution of or sperm heds Tle 3 Vlues (men nd oeffiient of vrition) of morphometri prmeters from four ors otined fter stining with rpid Pnopti (PP), Hemolor (HC) nd Hrris s Hemtoxylin (HH) Stin Bor N Length (lm) Width (lm) Are (lm 2 ) Perimeter (lm) Elliptiity Rugosity Elongtion Regulrity PP ,x (4.21) 4.33,x (3.81) 30.71,x (3.70) 22.85,x (2.08) 1.91,x (6.55) 0.74,x (2.00) 0.31,x (10.19) 0.92,x (2.34) ,x (4.63) 4.30,x (3.73) 30.45,x (4.20) 22.78,x (3.57) 1.94,x (6.66) 0.74,x (3.25) 0.32,x (10.13) 0.92,x (2.39) ,x (3.53) 4.12,x (4.02) 28.63,x (4.06) 22.10,x (2.56) 1.96,x (5.81) 0.74,x (2.66) 0.32,x (8.43) 0.91,x (2.16) d,x (6.62) 4.26 d,x (6.22) d,x (6.91) d,x (5.31) 1.91,x (9.73) 0.74,x (4.16) 0.31,x (21.38) 0.92,x (9.78) Men CV (WA) CV (BA) HC ,y (4.77) 4.34,x (3.90) 31.22,x (3.97) 23.16,y (2.32) 1.92,x (7.17) 0.73,y (2.06) 0.31,x (11.06) 0.91,y (2.39) ,y (4.61) 4.21,y (3.67) 29.67,y (3.59) 22.54,y (2.2) 1.97,y (6.94) 0.73,y (2.03) 0.32,x (10.30) 0.92,x (2.65) ,y (4.49) 4.15,y (4.38) 29.14,y (4.48) 22.34,y (2.91) 1.97.x (6.86) 0.73,y (2.90) 0.32,x (10.11) 0.91,x (2.45) ,x (5.07) 4.16 d,y (3.54) 29.16,y (4.34) d,y (3.32) 1.97,y (6.93) 0.73,y (3.50) 0.32,y (9.83) 0.92,x (3.48) Men CV (WA) CV (BA) HH ,z (4.19) 3.93,z (3.43) 25.92,z (3.59) 21.00,z (2.49) 1.96,z (6.24) 0.74,x (2.61) 0.32,z (8.96) 0.92,x (2.20) ,z (4.64) 3.90,z (4.80) 25.60,z (5.11) 20.97,z (3.26) 1.98,y (9.73) 0.73,y (3.94) 0.33,z (10.42) 0.92,x (2.54) ,z (4.42) 3.81,z (4.42) 24.53,z (4.21) 20.42,z (2.70) 1.98,x (6.75) 0.74,x (3.01) 0.33,x (9.62) 0.92,z (2.95) ,z (3.94) 3.78 d,z (3.59) d,z (3.90) d,z (2.29) 1.98,z (5.98) 0.74,z (2.14) 0.33,z (8.51) 0.92,x (2.09) Men CV (WA) CV (BA) WA nd BA, within-niml nd etween-niml oeffiient of vrition (%) respetively.,,, d: signifint, within-stining proedures, differenes etween ors. Vlues with different supersripts re signifintly different (p < 0.01). x, y, z: signifint, within-or, differenes etween stining proedures. Vlues with different supersripts re signifintly different (p < 0.01). interntionl journl of ndrology 29 (2006) ª Journl ompiltion 2006 Blkwell Pulishing Ltd 559

8 Morphometril evlution of or sperm heds M. Grí-Herreros et l. Tle 4 Vlues of morphometri prmeters (men ± stndrd devition) of four ors fter ounting 50 or 200 spermtozo. Within or, stin nd olumns, different supersripts re sttistilly different to eh other (p < 0.05) Bor Stin N Length (lm) Width (lm) Are (lm 2 ) Perimeter (lm) Elliptiity Rugosity Elongtion Regulrity 1 PP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 HC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 HH ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± PP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 HC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 HH ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± PP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 HC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 HH ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± PP ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 HC ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.03 HH ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.02 PP, rpid Pnopti; HC, Hemolor; HH, Hrris s Hemtoxylin. The ury of CASMA tehnology lso depends on the reful fixtion nd stining of these ells (Dvis & Grvne, 1993; Grvne et l., 1995; Ggo et l., 1998; Snho et l., 1998). Spermtozo of individul speies inorporte vrious fixtives nd stins unevenly (Hrsymowyz et l., 1976), nd proper stining is thus ruil oth for urte morphometri nlysis nd for minimizing errors in hed digitiztion (Dvis & Grvne, 1993). As in other studies, the numer of orretly inrized sperm heds ws the prmeter used here for evluting the suitility of the stining proedure for CASMA nlysis of porine spermtozo (Ggo et l., 1998; Soler et l., 2003). The stining tehnique providing the highest ell/kground ontrst nd the gretest stining intensity ws HH; however, mjor drwk of this stin is tht it does not dequtely disriminte sperm midpiees, finding lso reported in epididyml red deer sperm y Soler et l. (2005); this stin is therefore not reommended when oth hed nd midpiee re to e evluted. The Hemolor stin oupied n intermedite position etween rpid Pnopti nd HH, providing dequte stining intensity nd suffiient ell/kground ontrst. This is therefore onsidered the most suitle tehnique for evluting or sperm hed nd midpiee. Evlution of these results lso indites dimensionl differenes in sperm hed morphometry etween stining proedures, thus hindering relile omprison of studies performed using different stining tehniques. The mesurements otined here re signifintly lower thn those reported for porine spermtozo using fixtion in 96% methnol followed y 5 se in 0.5% methyl violet (Shmidt, 1997, ited y Hiri et l., 2001) nd those otined using modified Frelly stin (niline lue, 5 min; rystl violet, 1 min) y Hiri et l. (2001). With regrd to the effet of eh niml, for eh stin proedure, on morphometri prmeters, signifint differenes were oserved etween nimls for ll prmeters. Within-niml CVs for morphometri prmeters (exept elongtion) were similr to those reorded for rit (Grvne & Dvis 1994), stllion (Dvis et l., 1993), got (Grvne et l., 1995) nd rm spermtozo (Snho et l., 1998) using CASMA. These smll CVs reflet homogeneous sperm hed popultion, with little evidene of the onsiderle polymorphism found in humn smples (Omelet et l., 1995) nd lp spermtozo (Buendí et l., 2002). In the present study, perimeter, rugosity nd regulrity for Hemolor nd HH, nd perimeter nd rugosity for rpid Pnopti, were the most suitle prmeters for use in the identifition of 560 interntionl journl of ndrology 29 (2006) Journl ompiltion ª 2006 Blkwell Pulishing Ltd

9 M. Grí-Herreros et l. Morphometril evlution of or sperm heds individul ors, inditing the mrked onstny of sperm morphometri prmeters for individul nimls, s lso reported for lp (Buendí et l., 2002) nd ynomolgus monkey (Ggo et l., 1998), where perimeter nd shpe my e the most importnt prmeters. The reson for the ury of the CASMA system in enling the detetion of smll ut signifint differenes etween pprently norml spermtozo in given individul is prtiulrly interesting, s the existene of supopultions of norml spermtozo presenting different fertility profiles in sme smple hs een reported (Pérez-Sánhez et l., 1994; Hiri et l., 2001). Thurston et l. (2001), using Fourier shpe desriptors to lssify sperm heds, deteted morphologilly distint supopultions of spermtozo, whose rtio within fresh ejulte orrelted with semen qulity ssessments mde following ryopreservtion. Semen nlysis should therefore e performed to estlish the presene of eh of these supopultions, nd not only to provide verge vlues for the semen popultion s whole (Snho et l., 1998). Between-niml CVs present reltively low vlues for Hemolor, medium vlues for HH nd high vlues for rpid Pnopti stin. The high vlue using rpid Pnopti might e due to the effet of individuls with very turid seminl plsm (e.g. or 4) on etween-niml CVs. This result supports the suggestion of wshing sperm from mles with very turid seminl plsm if smples re to e stined with rpid Pnopti. Stle morphometri sperm hed mesurements were hieved with Pnopti nd Hemtoxylin stining using t lest 100 rther thn 200 properly digitized sperm heds. With Hemolor, only 50 spermtozo were required to otin stle morphometri mesurements, thus onsiderly reduing nlysis time without ffeting ury. In summry, this reserh reveled signifint differenes depending on sperm wshing nd on the stining proedure used. Results otined under different onditions nnot therefore e fully extrpolted. Hemolor (nlysis of 50 properly digitized sperm ells) nd HH (nlysis of 100 properly digitized sperm ells) were found to e the most suitle stining proedures for morphometri nlysis of porine sperm heds; HH did not effiiently disriminte sperm midpiees, nd is therefore not suitle for midpiee mesurements in porine spermtozo. Aknowledgements M. Grí-Herreros ws supported y Dotorl grnt from the Deprtment of Edution, Universities nd Investigtion of the Bsque Government-Eusko Jurlritze, Spin. Referenes Anos, V. O. & Isoun, T. T. (1980). Further oservtions on the testiulr pthology in Tripnosom vivx infetion of sheep nd gots. Reserh in Veterinry Siene 28, Bker, H. W. G. & Clrke, G. N. (1987) Sperm morphology: onsisteny of ssessment of the sme sperm y different oservers. Clinil Reprodution nd Fertility 5, Bll, B. A. & Mohmmed, H. O. (1995) Morphometry of stllion spermtozo y omputer-ssisted imge nlysis. Theriogenology 49, Bne, A. (1961). Arosoml normlity ssoited with sterility in or. Proeedings 4th Interntionl Congress of Animl Reprodution & Artifiil Insemintion, The Hgue, pp Bostofte, E., Serup, J. & Ree, H. (1982) Reltion etween morphologilly norml spermtozo nd pregnnies otined during twenty yer follow-up period. Interntionl Journl of Andrology 5, Boyle, C. A., Khoury, M. J., Ktz, D. F., Annest, J. L., Kresnow, M., DeStefno, F. & Srder, S. M. (1992). The reltionship of omputer-sed mesures of sperm morphology nd motility to mle infertility. Epidemiology 3, Buendí, P., Soler, C., Polihi, F., Ggo, G., Urquiet, B., Pérez-Sánhez, F. & Bustos-Oregón, E. (2002) Morphometri hrteriztion nd lssifition of lp sperm heds using the Sperm-Clss Anlyzer Ò omputer-ssisted system. Theriogenology 57, Clmer, J. C., Quiros, M. C., Olmedo, S. B., Sánhez, I. & Botti, C. (1994) Development of n ojetive nd mnul tehnique to study humn sperm morphology. Andrologi 26, Crrell, D. T., Zoell, R. L., Middleton, R. G. & Urry, R. L. (1994) A funtionl nlysis nd the potentil linil signifine of 7 tegories of sperm morphology. Journl of Urology 151, Csey, P. J., Grvne, C. G., Dvis, R. O., Chot, D. D. & Liu, I. K. M. (1997) Morphometri differenes in sperm hed dimensions of fertile nd sufertile stllions. Theriogenology 47, Dvis, R. O. & Grvne, C. G. (1993) Stndrdiztion of speimen preprtion, stining nd smpling methods improves utomted sperm-hed morphometry nlysis. Fertility nd Sterility 59, Dvis, R. O. & Grvne, C. G. (1994) Consisteny of sperm morphology lssifition methods. Journl of Andrology 15, Dvis, R. O., Bin, D. E., Siemers, R. J., Thl, D. M., Andrew, J. B. & Grvne, C. G. (1992) Aury nd preision of the CellForm-Humn utomted sperm morphometry system. Fertility nd Sterility 58, Dvis, R. O., Grvne, C. G. & Csey, P. J. (1993) Automted morphometri nlysis of stllion spermtozo. Amerin Journl of Veterinry Reserh 54, interntionl journl of ndrology 29 (2006) ª Journl ompiltion 2006 Blkwell Pulishing Ltd 561

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11 M. Grí-Herreros et l. Morphometril evlution of or sperm heds on the type nd ourrene of sperm normlities in Nuin uks. Amerin Journl of Veterinry Reserh 49, Soler, C., de Montserrt, J. J., Gutiérrez, R., Núñez, J., Núñez, M., Snho, M., Pérez-Sánhez, F. & Cooper, T. G. (2003) Use of the Sperm-Clss Anlyzer Ò for ojetive ssessment of humn sperm morphology. Interntionl Journl of Andrology 26, Soler, C., Gde, B., Soler, A. J., Fernández-Sntos, M. R., Esteso, M. C., Núñez, J. et l. (2005) Comprison of three different stining methods for the ssessment of epididyml red deer sperm morphometry y omputerized nlysis with ISAS Ò. Theriogenology 64, Suriysomoon, A., Lundeheim, N., Kunvongkrit, A. & Einrsson, S. (2005) Effet of temperture nd humidity on sperm morphology in Duro ors under different housing systems in Thilnd. Journl of Veterinry Medil Siene 67, Thurston, L. M., Wtson, P. F., Milehm, A. J. & Holt, W. (2001) Morphologilly distint sperm supopultions defined y Fourier shpe desriptors in fresh ejultes orreltes with vrition in or semen qulity following ryopreservtion. Journl of Andrology 22, World Helth Orgniztion (1999) WHO Lortory Mnul for the Exmintion of Humn Sperm nd Sperm Cervil Muus Intertion, 4th edn. Cmridge University Press, Cmridge. Wyroek, A. J., Gordon, L. A., Burkhrt, J. G., Frnis, M. W., Kpp, R. W., Letz, G., Mlling, H. V., Tophm, J. C. & Whorton, M. D. (1983) An evlution of humn sperm s inditor of hemilly indue ltertions of spermtogeni funtion. Muttion Reserh 115, Zneveld, L. J. D. & Polkoski, K. L. (1977) Colletion nd physil exmintion of the ejulte. In: Tehniques in Humn Andrology (ed. E. S. E. Hfez), pp , Elsevier, Amsterdm. interntionl journl of ndrology 29 (2006) ª Journl ompiltion 2006 Blkwell Pulishing Ltd 563