Effects of Zearalenone, α-zearalenol, and Genistein on Boar Sperm Motility In Vitro

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1 Originl Pper Effects of Zerlenone, α-zerlenol, n Genistein on Bor Sperm Motility In Vitro Aél Krejcárková 1 *, Petr Folková 1, Onřej Šimoník 1, Mrtin Ššková 1, Romn Krejčířová 1, Onřej Dráek 2, Rko Rjmon 1 1 Deprtment of Veterinry Sciences, Fculty of Agriculture, Foo n Nturl Resources, Czech University of Life Sciences Prgue, Prgue, Czech Repulic 2 Deprtment of Soil Science n Soil Protection, Fculty of Agriculture, Foo n Nturl Resources, Czech University of Life Sciences Prgue, Prgue, Czech Repulic *Corresponing uthor: el.krejcrkov@gmil.com ABSTRACT Krejcárková A., Folková P., Šimoník O., Ššková M., Krejčířová R., Dráek O., Rjmon R. (17): Effects of zerlenone, α-zerlenol, n genistein on or sperm motility in vitro. Czech J. Anim. Sci., 62, Genistein (GEN) n zerlenone (ZEA), environmentl oestrogens commonly present in feestuff for pigs, re known for their effects on reprouctive functions. The im ws to verify the in vitro effects of.5 µm concentrtions of GEN, ZEA n its metolite α-zerlenol (α-zol) on pig sperm motility. A ose-epenent increse of the immotile sperm mount ginst fst n meium-fst sperm clusters ws oserve with ll three oestrogens from the lowest concentrtions teste. Iniviul CASA (computer-ssiste sperm nlysis) prmeters of motile sperms seeme to e less sensitive inictors. This shoul e consiere especilly in toxicologicl reserch on sperm moel. Bckgroun of inconsistencies in to te-pulishe ppers is iscusse. The results shift the effective concentrtions of ZEA, α-zol, n GEN to vlues chievle in vivo n rises the questions of risk ssessment of these compouns in pig reprouction. Keywors: pig; spermtozo; environmentl oestrogens; motility; CASA; cluster nlysis The environment commonly comprises sustnces tht cn ffect hormonl homeostsis in n orgnism thnks to their ility to in on enogenous receptors, so-clle enocrine isruptors (Ceerroth et l. 1). Mycotoxin zerlenone (ZEA) n its metolites or soy phytoestrogens, minly glycones genistein (GEN) n izein (DAI), elong mong these sustnces; espite their non-steroil structure they cn in to the oestrogen receptors in mmmlin cells (Mjic 1). Given the populrity of soyen mel in the current pig nutrition n the high sensitivity of pigs (Benzoni et l. 8), the question regring the effects of these sustnces on pig reprouction is still relevnt. Bor exposition to ZEA resulte repetely in generlly negtive effects on their reprouctive system (Benzoni et l. 8; Knor n Mes 9; Sutkeviciene et l. 9; Bht et l. 1), wheres GEN h rther miguous effects (Yun et l. 12). Surprisingly, the semen qulity ws rther peripherl interest in these in vivo experiments. Lower sperm motility ws recore fter long-term feeing of ZEA t low concentrtions (Young n King 1986). On the contrry, 2 ppm of GEN in fee stimulte the fructose level in Supporte y the Ministry of Euction, Youth n Sports of the Czech Repulic (S grnt) n y the Internl Grnt Agency of the University of Life Sciences Prgue (CIGA) (Project No. 1627). 435

2 Originl Pper Czech J. Anim. Sci., 62, 17 (1): semen compre to the control group, resulting in its etter qulity (Yun et l. 12). Uner in vitro conitions, the inhiitory effects on the or sperm motility were recore for ZEA s well s for its mjor metolite lph-zerlenol (α-zol) t quite high concentrtions of µm (Tskmkiis et l. 6, 7). Lower concentrtions seeme to e without ny sustntil effect (Benzoni et l. 8; Tskmkiis et l. 8; Smuu et l. 13). On the other hn, fter incution with 31.2 µm of α-zol, Gry et l. (16) oserve reuction in the percentge of totl sperm motility, percentge of rpi motility, percentge of progressive motility, n CASA (computer-ssiste sperm nlysis) prmeters, such s VCL (curviliner velocity), VSL (stright-line velocity), VAP (velocity on verge pth), ALH (mplitue of lterl he isplcement), n LIN (linerity). The effects of GEN on or sperm motility were lso miguous. Wheres Oh et l. (11) recore reuction in totl motility in the.1 µm concentrtion rnge, Kim et l. (14) i not oserve ny ifferences in the presence of 1,, n µm GEN compre to the control group, n furthermore, µm GEN even h stimultory effects. It is ovious tht the reporte effects of ZEA, α-zol, n GEN iffer mong stuies. Furthermore, ue to the ifferences in the use methoologies they re not esy to compre. There re ifferences in tretment conitions s well s in the evlution of sperm motility. Some stuies use sujective oservtion only (Tskmkiis et l. 8; Kim et l. 14). Moreover, the pproch to the ojective ssessment of sperm motility y CASA hs chnge over recent yers (Simonik et l. 15). The im of our stuy ws to ojectively ssess the effects of these commonly ville environmentl oestrogens on the motility of or ejculte spermtozo in the rnge of concentrtion where the minimum effect of these sustnces cn e hypothesize, n using up-to-te sperm motility ssessment methos, to elucite the possile resons for inconsistent results from pulishe ppers. MATERIAL AND METHODS Chemicls. All chemicls were purchse from Sigm Alrich (USA) unless specifie otherwise. Stock solutions (1 mm) of ZEA (Z2125), α-zol (Z166) or GEN (G6776) were prepre y issolving lyophilize power in imethyl sulfoxie (DMSO; D5879) n store t C. Susequently, working solutions were prepre every y using the sperm extener (SUS; Meichimic, Itly) s iluent. Animls n smple collection. Semen of five ors with verifie fertility (insemintion sttion ProAgro Nymurk, Czech Repulic) ws use for experiments. Proven sperm-rich frctions with t lest 75% of motile spermtozo were extene immeitely fter collection y sperm extener (SUS; Meichimic) to spermtozo per ml, trnsporte n store susequently in stnr insemintion ose tues t 17 C for further processing n nlyses. Experiments were execute in the lortory of the Deprtment of Veterinry Science, Czech University of Life Sciences Prgue no lter thn 48 h fter collection. Before ech experiment, sperm motility ws checke gin n only semen with > 75% of motile spermtozo (sujective evlution) t tht time were use. Experimentl esign. Three single experiments, ech with oestrogen (ZEA, α-zol, n GEN) were crrie out seprtely y the sme operting proceure: liquots (9 μl) of semen were rewrme in plstic Eppenorf microtues to 38 C in wter th (SUB 6; Grnt Instruments, UK). Then μl of ZEA, α-zol or GEN solution in DMSO with extener were e to ech microtue to rech finl concentrtions of the prticulr oestrogen s follows: μm (control),.5 μm, 1 μm, 2.5 μm, 5 μm, 1 μm, n μm. The content of DMSO per smple ws 2 μl. All semen liquots were then incute uner the sme conitions (38 C in wter th) n the sperm motility ws exmine fter 2 n 4 hours of incution. All tools n lortory equipment use uring the experiment were hete t 38 C. The experiment for ech oestrogen ws repete t lest six times. Sperm motility nlysis. The sperm motility ws evlute y the CASA moule (Nis Elements, Version 3.; Lortory Imging, Czech Repulic). More specificlly, 2 μl of the smple were plce into µm eep chmer (Lej glss, the Netherlns) n the sperm motility ws cpture t six fiels per chmer y microscope with heting tle (Eclipse E (Nikon Corp., Jpn), mgnifiction, negtive phse contrst; ojective Ph 1 BM (Nikon Corp.); cmer ProgRes CT1 (Jenoptik, Germny)). Sperm movement ws recore in 2-s time lp with frequency of

3 Originl Pper frmes/s, i.e. 69 igitlize imges were processe. The trck of ech sperm ws nlyze for the following kinemtic prmeters: curviliner velocity (VCL (µm/s)), verge-pth velocity (VAP (µm/s)), stright-line velocity (VSL (µm/s)), mplitue of the lterl he isplcement (ALH (µm)), et cross frequency (BCF (Hz)), linerity (LIN; VSL/ VCL (%)), strightness (STR; VSL/VAP (%)), n wole (WOB; VAP/VCL (%)). Spermtozo with VAP > 1 µm/s were clssifie s motile. Mein of the CASA kinemtic prmeters mentione ove ws expresse from the popultion of motile spermtozo, n the supopultion structure ws lso nlyze. Finlly, the percentge of IMMOTILE sperm (VAP 1 µm/s) ws expresse. Sttisticl nlysis. A sttisticl nlysis ws performe using STATISTICA softwre, Version 12 (SttSoft CR, Czech Repulic). The following sttisticl tests were use: cluster nlysis, chi-squre test, ANOVA, Scheffé s test. With regr to the wie rnge of the nlyze files (n > 6 in ech experimentl group) n the vriility oserve mong semen liquots efore the tretment, the level of significnce in ll sttisticl tests ws set t P <.1. To ientify sperm supopultions, cluster nlysis ws performe. Motile spermtozo CASA t sets from iniviul experiments were ssesse y k-mens clustering se on Euclien istnces of the chosen sperm motility escriptors (VCL, VSL, VAP, ALH, n BCF). Thus, motile spermtozo were ssigne to three specific isjointe clusters corresponing to three sperm supopultions: SLOW, MEDIUM FAST, n FAST. For the purpose of etecting the sperm istriution in the iniviul supopultion, fourth ctegory of IMMOTILE sperm (VAP 1 µm/s) ws e. Differences in the sperm istriution into supopultions were checke using the chi-squre test. The effects of environmentl oestrogens on the iniviul sperm kinemtic prmeters were evlute y mens of n nlysis of vrince (ANOVA) with interctions. Then the vlue of men ± SEM ws expresse. Any sttisticlly significnt ifferences mong the otine vlues were consequently specifie y the Scheffé s test. RESULTS AND DISCUSSION The im of this stuy ws to ojectively ssess the effects of three nturlly occurring oestrogenic compouns (ZEA, -ZOL, GEN) on the motion ctivity of or ejculte spermtozo. Sperm motility is wiely use s sic mrker of semen or spermtozo qulity (Dzyu et l. 15; Zhng et l. 15; Petelk n Krylov 16; Simonik et l. 16). Previous stuies focusing on the effects of the ove-mentione sustnces iffer in their conclusions s well s in their methoologicl pproches. But the metho of motility ssessment my significntly influence otine results (Broekhuijse et l. 11). We pplie ojective motility evlution y mens of computer-ssiste sperm nlysis (CASA), using the cluster nlysis metho, which is suitle for resolving heterogeneity of the sperm motility t in iscrete supopultions (Mrtinez- Pstor et l. 11). This ws comine with the evlution of the immotile sperm proportion n the iniviul CASA prmeters. The kinemtic chrcteristics of motile sperm supopultions (SLOW, MEDIUM FAST, FAST) in experiments on ZEA, α-zol, or GEN re shown in Tles 1, 2, n 3, respectively. A significnt ecrese in the men vlues ws oserve etween 2 n 4 hours of incution for most of the prmeters. Only the VCL n ALH prmeters in α-zol n GEN experiments showe opposite trens in some supopultions (Tle 2 n 3). The IMMOTILE supopultion ws efine s sperm with VAP 1 µm/s n these sperm were not inclue in the clustering proceure. Evluting the istriution of spermtozo mong supopultions (IMMOTILE, SLOW, MEDIUM FAST, n FAST), significnt ifferences were recore s erly s fter 2 hours of incution for ll three compouns t ll concentrtions teste (Figures 1A, 2A, 3A). With the higher concentrtion of ech compoun, the percentge of sperms in supopultions IMMOTILE (especilly) or SLOW increse to the etriment of the FAST or MEDIUM FAST supopultions generlly. After 4-hour incution (Figures 1B, 2B, 3B) the effects were more pronounce. The effects cn e orere ZEA < α-zol < GEN. Although the effects of these three oestrogenic compouns were teste seprtely, giving priority to testing the concentrtion series, which ment the comprison coul only e me very pproximtely, this is to our knowlege the only pper evluting the effects of these compouns on sperm uner the sme protocol. Evluting iniviul kinemtic prmeters, the efficcy of iniviul compouns hs mnifeste 437

4 Originl Pper Czech J. Anim. Sci., 62, 17 (1): Tle 1. Kinemtic prmeters chrcterizing motile or sperm supopultions in experiment on zerlenone effects (men ± SEM) Two-hour incution Four-hour incution SLOW MEDIUM FAST FAST SLOW MEDIUM FAST FAST VCL (µm/s) 49.6 ± ± ± ± ± ±.3 VAP (µm/s) 26.1 ± ± ± ± ± ±.19 VSL (µm/s) ± ± ± ± ± ±.23 ALH (µm) 4.44 ± ±.2 8. ± ± ± ±.3 LIN (%) ± ± ± ± ± ±.2 WOB (%) 57.3 ± ± ± ± ± ±.17 STR (%) ± ± ± ± ± ±.18 BCF (Hz) 6.62 ± ± ± ± ± ±.4 VCL = curviliner velocity, VAP = velocity on verge pth, VSL = stright-line velocity, ALH = mplitue of lterl he isplcement, LIN = linerity, WOB = wole, STR = strightness, BCF = et cross frequency supopultions were efine y cluster nlysis of computer ssiste semen nlysis t sets (k-mens clustering se on Euclien istnces); motile spermtozo of ll tretment (,.5, 1, 2.5, 5, 1, n µm of zerlenone) n incution vrints (2 or 4 hours) were involve, vlues with ifferent superscripts in row within the sme supopultion significntly iffer etween 2 n 4 hours of incution (P <.1, Scheffé s test) rther ifferently (Figures 4 6). Similr courses of chnges were seen for the velocity prmeters (VCL, VAP, n VSL) n for the clculte prmeters (LIN, STR, n WOB) n BCF s well. Therefore, the results for VCL, ALH, n LIN re presente only. For GEN t on BCF re lso shown. The trens iffere mong compouns. Vlues similr to the control with only few exceptions chrcterize the ZEA experiment (Figure 4). Dt on α-zol show much wier vrition with significnt evitions from the control, especilly t concentrtions ove 2.5 μm (Figure 5). The Tle 2. Kinemtic prmeters chrcterizing motile or sperm supopultions in experiment on α-zerlenol effects (men ± SEM) 438 Two-hour incution Four-hour incution SLOW MEDIUM FAST FAST SLOW MEDIUM FAST FAST VCL (µm/s) ± ± ± ± ± ±.34 VAP (µm/s) ± ± ± ±.6.9 ± ±.24 VSL (µm/s) ±.6.55 ± ± ± ± ±.3 ALH (µm) 4. ± ±.2 8. ± ± ± ±.4 LIN (%) ± ± ± ±.19.6 ± ±.24 WOB (%) ± ±.9.51 ± ± ± ±. STR (%).4 ± ± ± ± ± ±.31 BCF (Hz) 6.65 ± ± ± ± ± ±.4 VCL = curviliner velocity, VAP = velocity on verge pth, VSL = stright-line velocity, ALH = mplitue of lterl he isplcement, LIN = linerity, WOB = wole, STR = strightness, BCF = et cross frequency supopultions were efine y cluster nlysis of computer ssiste semen nlysis t sets (k-mens clustering se on Euclien istnces); motile spermtozo of ll tretment (,.5, 1, 2.5, 5, 1, n µm of α-zerlenol) n incution vrints (2 or 4 hours) were involve, vlues with ifferent superscript letters in row within supopultion significntly iffer etween 2 n 4 hours of incution (P <.1; Scheffé s test)

5 Originl Pper Tle 3. Kinemtic prmeters chrcterizing motile or sperm supopultions in experiment on genistein effects (men ± SEM) Two-hour incution Four-hour incution SLOW MEDIUM FAST FAST SLOW MEDIUM FAST FAST VCL (µm/s) ± ± ± ± ± ±.31 VAP (µm/s) ± ± ± ± ± ±.24 VSL (µm/s) ±.7. ± ± ± ± ±.28 ALH (µm) 3.85 ±.2 5. ± ± ± ± ±.4 LIN (%) ± ± ± ± ± ±.23 WOB (%) ± ± ± ± ± ±.19 STR (%) ± ± ± ± ± ±.31 BCF (Hz) 6.66 ± ± ± ± ±.3 6. ±.4 VCL = curviliner velocity, VAP = velocity on verge pth, VSL = stright-line velocity, ALH = mplitue of lterl he isplcement, LIN = linerity, WOB = wole, STR = strightness, BCF = et cross frequency supopultions were efine y cluster nlysis of computer ssiste semen nlysis t sets (k-mens clustering se on Euclien istnces); motile spermtozo of ll tretment (,.5, 1, 2.5, 5, 1, n µm of genistein) n incution vrints (2 or 4 hours) were involve, vlues with ifferent superscripts in row within the sme supopultion significntly iffer etween 2 n 4 hours of incution (P <.1, Scheffé s test) sme concentrtion seems to e just s criticl for the GEN effects (Figure 6). Zerlenone is lrey known for its negtive effects on the reprouctive orgns of ors from the ose of ppm in fee (Knor n Mes 9). This correspons to pp in tissues, i.e. roun 1 μm (Prelusky 1994; own unpulishe t), which is much less thn effective concentrtions (125 μm ZEA) reporte from in vitro experiments on sperm motility y Tskmkiis et l. (6). However, our cluster nlysis inictes significnt reistriution of spermtozo mong supopultions even from the concentrtion of.5 μm (Figure 1). In ition, the men vlues of some kinemtic prmeters fter 2-hour incution inicte possile effects of this concentrtion, lthough the ifference from the control is consiere to e significnt (P <.1) t ALH only (Figure 4). Inhiitory effects of ZEA concentrtions ove 5 μm re clerly evient with oth methos of sperm motility evlution. All of these finings emonstrte tht the effective ZEA Distriution of sperm into supopultions (%) c /c e f (B) / c e f Zerlenone ose (μm) Zerlenone ose (μm) Figure 1. Percentge istriution of sperm into IMMOTILE (lck r), SLOW (spotte r), MEDIUM FAST (grey r), n FAST (stripe r) supopultion fter 2-hour n 4-hour (B) incution with ifferent zerlenone oses f ifferent superscripts inicte sttisticlly significnt ifferences etween the tretment groups t P <.1 (chisqure test) 439

6 Originl Pper Czech J. Anim. Sci., 62, 17 (1): Distriution of sperm into supopultions (%) c e f g (B) c e f g α-zerlenol ose (μm) α-zerlenol ose (μm) Figure 2. Percentge istriution of sperm into IMMOTILE (lck r), SLOW (spotte r), MEDIUM FAST (grey r), n FAST (stripe r) supopultion fter 2-hour n 4-hour (B) incution with ifferent α-zerlenol oses g ifferent superscripts inicte sttisticlly significnt ifferences etween the tretment groups t P <.1 (chisqure test concentrtion on sperm motility is much lower thn ny yet pulishe. The lph-zerlenol, resulting from the liver iotrnsformtion of ZEA (Benzoni et l. 8), is consiere to e 1 times more potent thn ZEA lone (Agg 4). Tskmkiis et l. (8) i not oserve ny ifferences compre to the control group up to concentrtion of 94 μm fter 4 h of incution. Nevertheless, Gry et l. (16) recore negtive influence of 31.2 μm of α-zol on sperm motility even fter 3 min of incution, n Benzoni et l. (8) oserve ecrese in VSL in or spermtozo uner in vitro conitions t concentrtion of μm of α-zol. Our cluster nlysis gin inictes effects even from the lowest α-zol concentrtion teste, i.e. from.5 μm (Figure 2). Compre to ZEA, the chnges re much more pronounce t the level of sperm supopultions s well s with the evlution of kinemtic prmeters (Figure 5) where significnt evitions from the control were foun fter 2-hour incution with most of the concentrtions, incluing the lowest one. This is in ccornce with generlly higher oestrogenic ctivity of α-zol (Fink-Gremmels n Mlekinej 7). Wheres ZEA n its metolites re ssocite with rther negtive effects on reprouction (Zineine et l. 7), phytoestrogens (such s GEN) cn potentilly ct eneficilly (Whitten n Ptisul 1). There were oserve no negtive ut even positive effects of GEN on or semen uner in vivo conitions (Yun et l. 12). Uner Distriution of sperm into supopultions (%) c /c e f (B) / c e f Figure 3. Percentge istriution of sperm into IMMOTILE (lck r), SLOW (spotte r), MEDIUM FAST (grey r), n FAST (stripe r) supopultion fter 2-hour n 4-hour (B) incution with ifferent genistein oses g ifferent superscripts inicte sttisticlly significnt ifferences etween the tretment groups t P <.1 (chisqure test) 4

7 Originl Pper in vitro conitions the reporte GEN effects on or semen re heterogeneous. Wheres Oh et l. (11) recore negtive effects on sperm motility, Kim et l. (14) oserve no negtive effects in most cses, or even some stimultion. Our t inicte similr chnges to those seen with ZEA or α-zol significnt reistriution of sperms from the lowest teste concentrtion ue to ecrese in the percentge of the two fstest supopultions minly in fvour of immotile sperms. The effects seem to e even more pronounce y cluster nlysis (Figure 3) s well s iniviul kinemtic prmeter evlution (Figure 6). Compring results of oth nlytic methos use, the cluster nlysis seems more sensitive n lso the most consistent. However, the comintion of oth methos cn elucite some of the non-complince of our results with previously pulishe work. Although the rtio of sperm supopultions exhiite cler epenency on the ZEA ose s erly s fter 2-hour incution from the lowest ZEA concentrtion of.5 μm, the velocity prmeters of motile spermtozo i not significntly iffer from the control group in ny of the teste concentrtions. Moreover, the vlues for the lowest concentrtion of.5 μm were even higher (for VCL significntly) compre to the other ZEA concentrtions (Figure 4A). The ALH curve looks very similr (Figure 4B). Oviously, the lowest ZEA concentrtion ecrese the proportion of motile spermtozo with slight increse in the sperm movement ctivity. Oh et l. (11) i not oserve ny effect on the iniviul CASA prmeters in their genistein stuy either, ut in most cses they etecte n increse in the percentge of immotile spermtozo, s we i in our cse. It is, therefore, possile tht lthough the rtio of immotile sperm increses, the chrcteristics of the so fr motile spermtozo o not chnge for efine perio. This cn e cuse y the fct tht stress cn evoke trnsient stimultion of the sperm efore its susequent exhustion. Given the heterogeneity of the sperm popultions n the spee of their rection to stress, this effect my e mnifeste s the sence of ny impct on the verge vlues of kinemtic prmeters or even s the increse in the verge sperm velocity. This mechnism is inicte y increses in VCL men vlues t the lowest ZEA concentrtion fter 2-hour incution or in the FAST sperm cluster fter prolonge 4-hour cultivtion (Tle 1) oth situtions were ccompnie y the simultneous losses of motile spermtozo, or more precisely the losses of spermtozo with the FAST n MEDIUM FAST motion. This reltion- LIN (%) VCL (μm/s) (C) / / / / Zerlenone ose (μm) / / / / / c Zerlenone ose (μm) (μm) / / (B) ALH (μm/s) / Figure 4. Effects of zerlenone 2- n 4-hour tretments on or sperm kinemtic prmeters: curviliner velocity (VCL), mplitue of lterl he isplcement (ALH) (B), n linerity (LIN) (C) ifferent superscripts inicte sttisticlly significnt ifferences etween the tretment groups t P <.1 (Scheffé s test) / / / / Zerlenone ose (μm) 441

8 Originl Pper Czech J. Anim. Sci., 62, 17 (1): ship is more visile in the VCL t s mong the velocity prmeters VCL cn e more influence ue to its coinciing with function of the lterl he isplcement (ALH). Such ehviour of CASA t sets might explin the negtive results of some previous ppers. For exmple, Benzoni et l. (8) lso evlute VCL n VSL in or spermtozo. After sperm exposition to ZEA (2 pm μm) they oserve no effects of ZEA; however, uthors i not provie the percentge of totl motility. Dt on α-zol ress nother pitfll of kinemtic prmeters men vlues. Contrry to the experiment with ZEA, ose-epenent ecrese in VCL vlues (Figure 5A) ws foun. The course of the curve for ALH (Figure 5B) men vlues fter 2-hour incution shows the sme tren s for VCL. However, the finings fter 4 hours of incution look strnge, ecuse contrry to the ecrese in VCL with incresing α-zol concentrtion, the ALH vlues were stle or (t the concentrtions of 5 n μm) even increse. These normlities lso ppere (in mirrorlike mnner) with the BCF prmeter n the prmeters reflecting the strightness of motion LIN (Figure 5C), STR, WOB (t not shown). Thus, spermtozo exhiite higher evition of lterl he isplcement upon slower frequency n lrger curvture of their pthwy. Mechnicl ssessment of iniviul prmeters might le to conclusions out the non-liner ction of α-zol, stimultory effects on ALH or progressivity of movement. However, compring ll ville t it cn e ssume tht the men vlues were ctully ffecte y the presence of higher mount of spermtozo with more or less locl movement in the slow sperm ctegory, which with the overll smll mount of motile sperm mrkely shifte the men kinemtic vlues. An increse in the ALH men vlues chrcterizing the slow sperm cluster fter 4 hours of incution lso correspons with this suggestion. Similrly, surprising increse of LIN prmeter in GEN experiment is etter unerstnle compring t on sperm supopultions mong low numer of motile spermtozo slow ones re preominnt. As LIN = VSL/VCL n or spermtozo show physiologiclly higher percentge of circultory movement (Johnson et l. ), slow spermtozo hve higher LIN compre to fst ones even on the sme trjectory. 11 (B) 7.5 VCL (μm/s) (C) / / /c c e / /c /c c/ α-zerlenol ose (μm) / ALH (μm/s) / /c c/ / α-zerlenol α-zerlenol ose (μm) LIN ( %) c / //c / / c /c α-zerlenol ose (μm) Figure 5. Effects of α-zerlenol 2- n 4-hour tretments on or sperm kinemtic prmeters: curviliner velocity (VCL), mplitue of lterl he isplcement (ALH) (B), n linerity (LIN) (C) ifferent superscripts inicte sttisticlly significnt ifferences etween the tretment groups t P <.1 (Scheffé s test) 442

9 Originl Pper It is ovious tht the inhiition effects of GEN were most ominnt mong ll three teste contminnts e.g. μm of GEN cuse n increse in the immotile sperm rtio compre to the control group y out 65.3% (vs 28.34% for α-zol n 22.81% for ZEA). Also the ynmics of the men vlue of kinemtic prmeters ws clerly steeper. This istinct imge cn e relte to slightly ifferent impcts of GEN on metolic pthwys compre to ZEA n α-zol. Nevertheless, the oestrogenic ctivity of GEN is regre s much lower thn tht of ZEA n its metolites (e.g. Gromzk et l. 8). It is interesting tht Oh et l. (11) i not recor ny chnges in the selecte CASA prmeters in the rnge of concentrtion from.1 to μm of GEN, lthough they oserve significnt increse in the numer of immotile spermtozo. As our experiments on iniviul oestrogenic compouns were performe one fter the other in time, the ejculte qulity s fctor cn remin in the ckgroun. It is evient tht sperm resistnce to the incution conitions (see ech sperm ctegory in the control rtio, Figures 1 3) iffere with every experiment. However, Oh et l. (11) oserve ecrese in the totl motility fter GEN ition with concentrtion of s little s.1 μm even fter 15 or 3 min of incution. Following on from the informtion mentione ove, no similr fining hs yet een pulishe for ZEA or α-zol. On the other hn, Kim et l. (14) i not oserve ny effects on sperm motility t the sme concentrtions (1 μm of GEN) fter 3 hours of incution. This iscrepncy might e ue to the use of ifferent evlution methoologies Kim et l. (14) use sujective evlution, Oh et l. (11) the more ojective CASA. But there my lso e nother explntion. GEN, s well s ZEA n its metolites, re sustnces solule in ethnol or DMSO, ut very poorly solule in wter. Nevertheless, ll proceures testing their effects on sperm re conucte in wtery solutions, wherey these sustnces re issolve in polr solvent n then e to the wtery sperm smple. Our experience (not pulishe) shows tht even smll chnges in the composition of the cultivtion meium cn le to the precipittion of these sustnces or to their ining to other components which ffect the vilility of these sustnces n their effects. Nevertheless, none of the cite stuies exmine the 9 /c c (B) 7 65 VCL (μm/s) 7 e c f LIN (%) /e e c/ e / c c c c (C) LIN (%) /e e c/ e / c c c c (D) BCF (Hz) / / /c c/ / /c c/ Figure 6. Effect of genistein 2- n 4-hour tretments on or sperm kinemtic prmeters: curviliner velocity (VCL), mplitue of lterl he isplcement (ALH) (B), linerity (LIN) (C), n et cross frequency (BCF) (D) f ifferent superscripts inicte sttisticlly significnt ifferences etween the tretment groups t P <.1 (Scheffé s test) 443

10 Originl Pper Czech J. Anim. Sci., 62, 17 (1): rel concentrtion of contminnts in the meium. Therefore, it is necessry to tke cutious pproch to the stuies with negtive finings. The issue of vilility of the effective sustnces my lso unerlie the not fully liner reltionship of the oserve effects n oses (e.g. the effects of our lowest concentrtion of.5 μm of ZEA s well s of α-zol), lthough, the quntittive interction receptor effective sustnce (Schmieer et l. 3) shoul not e unerestimte either. The vilility of sustnces itself coul not explin the trnsitions etween the inhiitory n stimultory effect e.g. in the cite stuy of Kim et l. (14) where they oserve higher motile sperm rtio compre to the control group fter 6 hours of incution. Such effects cn e relte to the so-clle non-monotonic ose-response curves efine s nonliner reltionship etween ose n effect where the slope of the curve chnges sign somewhere within the rnge of oses exmine (Vnenerg et l. 12). CONCLUSION Our t inicte tht the effects of ZEA, α-zol, or GEN re lrey emonstrle with the lowest teste concentrtions, i.e..5 μm of the compoun in ose-epenent mnner, which shifts the effective concentrtions of ZEA, α-zol, n GEN to vlues rechle in the fiel conitions n rises the question of risk ssessment of these compouns in pig reprouction. Repete finings of shifte proportions of sperm supopultions without ny significnt chnges in the men vlues of kinemtic prmeters suggest tht uner miler stress these men CASA vlues nee not necessrily e sensitive enough s n inictor of chnges in the sperm motility. This shoul e consiere especilly in toxicologicl reserch on sperm moel where even mil chnges cn e inictive. The iscusse ckgroun to the inconsistency of so fr pulishe ppers shoul e tken into ccount in future similrly focuse stuies. REFERENCES Agg B.I. (4): Mycotoxins in foo n fees 3 zerlenone. Assiut University Bulletin for Environmentl Reserches, 7, Benzoni E., Minervini F., Ginnoccro A., Fornelli F., Vigo D., Visconti A. (8): Influence of in vitro exposure to mycotoxin zerlenone n its erivtives on swine sperm qulity. Reprouctive Toxicology, 25, Bht R., Rvishnkr R.B., Ri V., Krim A.A. (1): Mycotoxins in foo n fee: present sttus n future concerns. Comprehensive Reviews in Foo Science n Foo Sfety, 9, Broekhuijse M.L.V.J., Sostric E., Feitsm H., Gell B.M. (11): Aitionl vlue of computer ssiste semen nlysis (CASA) compre to conventionl motility ssessment in pig rtificil insemintion. Theriogenology, 76, Ceerroth C.R., Auger J., Zimmermnn C., Eustche F., Nef S. (1): Soy, phyto-oestrogens n mle reprouctive function: review. Interntionl Journl of Anrology, 33, Dzyu V., Cosson J., Dzyu B., Roin M. (15): Oxitive stress n motility in tench Tinc tinc spermtozo. Czech Journl of Animl Science,, Fink-Gremmels J., Mlekinej H. (7): Clinicl effects n iochemicl mechnism ssocite with exposure to the mycoestrogen zerlenone. Animl Fee Science n Technology, 137, Gry S.L., Lckey B.R., Boone W.R. (16): Effects of Pnx ginseng, zerlenol, n estriol on sperm function. Journl of Ginseng Reserch,, Gromzk K., Wskiewicz A., Chelkowski J., Golinski P. (8): Zerlenone n its metolites: occurrence, etection, toxicity n guielines. Worl Mycotoxin Journl, 1, 9 2. Johnson L.A., Weitye K.F., Fiser P., Mxwell W.M.C. (): Storge of or semen. Animl Reprouction Science, 62, Knor A., Mes D. (9): The role of mycotoxins in pig reprouction: review. Veterinrni Meicin, 12, Kim T.H., Yuh I.S., Prk I.C., Cheong H.T., Kim J.T., Prk C.K., Yng B.K. (14): Effects of quercetin n genistein on or sperm chrcteristics n porcine IVF emryo evelopments. Journl of Emryo Trnsfer, 29, Mjic G. (1): Enocrine isrupting chemicls n omestic nimls. Slovenin Veterinry Reserch, 47, Mrtinez-Pstor F., Tizo E.J., Gre J.J., Anel L., e Pz P. (11): Sttisticl series: opportunities n chllenges of sperm motility supopultion nlysis. Theriogenology, 75, Oh S.A., Prk Y.J., Song W.H., Mohme E.S.A., Png M.G. (11): Effects of estrogenic xoniiotics on or sperm motility n motion kinemtics. Reprouctive n Developmentl Biology, 35, Petelk A., Krylov V. (16): Cryopreservtion of fluorescence ctivte cell sorte or spermtozo se on 444

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