Bull Vet Inst Pulwy 5, 581-589, 8 FFCT OF DXAMTHASON ON TH CHANGS OF SMN QUALITY INDUCD BY NDOTOXIN IN STALLION JANUSZ DANK Deprtment of Animl Reproduction nd Animl Helth Protection, University of Technology nd Life Sciences, 85-8 Bydgoszcz, Polnd jdnek@wp.pl Received for publiction December 1, 7 Abstrct The effect of steroidl nti-inflmmtory drug - dexmethsone on the chnges of semen qulity in stllions fter endotoxin dministrtion ws studied. Three cliniclly helthy stllions were injected intrvenously with endotoxin (LPS) from scherichi coli 55:B5 t the dose of.3 µg/kg b.w.; while four stllions were treted with dexmethsone (.9 mg/kg b.w., i.m.) 1 min before the dministrtion of LPS. The dministrtion of the endotoxin elicited significnt (P<.5) decrese in the gel-free semen volume (GFSV), totl spermtozoon motility (TSM), nd spermtozoon concentrtion (SC). As regrds to ech of the morphologicl defects of the spermtozo, the percentge of those with cytoplsmic droplets droplet in distl (form 1), proximl nd typicl position (defects 1-3); til loops single loop (form ), double loop, loop of the end prt of the til, spirlling of the til, nd til looped round the hed (defects -8); two (or more) heds (form 13), nd smll ("dwrf") heds (form 15) were clerly higher in endotoxin treted stllions. The dministrtion of dexmethsone (DX) hd different effect on endotoxin-induced chnges of the semen s qulity. A positive influence of DX ws noticed in certin mcro- nd microscopic chrcteristics of the semen. There were noted smller chnges in TSM, SC, defects 1-3, form 1, nd form 13. Dexmethson hd no influence on GFSV. A negtive influence of the dministrtion of LPS+dexmethsone, in reltion to the stllions treted only with LPS, ws especilly noticed in forms nd 15. This dt suggest tht the dexmethsone does not prevent nd to certin degree enhnces n dverse influence of endotoxin on the semen qulity in stllions. Key words: stllion, endotoxin, dexmethsone, semen qulity. The effect of endotoxin on the reproductive functions of mles of domestic nimls hs been prtly elucidted. In rms (, 8) nd bors (7) exposed to S. typhimurium endotoxin, there ws number of clinicl, endocrinologicl nd seminologicl chnges, including the morphology of spermtozo. Studies in bors showed tht endotoxin induced infiltrtion of polymorphonucler neutrophils (PMN) into the testiculr interstitium nd morphologicl chnges of Leydig cells (9, 3). In stllions, the dministrtion of scherichi coli endotoxin hd negtive influence on the levels of blood serum nd seminl plsm testosterone, qulity of semen, especilly on the sperm motility nd morphology of spermtozo, nd on the concentrtion of seminl plsm lbumin nd ctivity of sprtte minotrnsferse (5, 7, 9). Steroidl nti-inflmmtory drugs re widely used in equine prctice. Dexmethsone is long-cting, synthetic nlogue of hydrocortisone (cortisol) widely used in vrious equine disorders, including llergic, cutneous, circultory, intestinl, nd musculoskeletl diseses (15, 1). Aprt from mny other effects chrcteristic for steroidl nti-inflmmtory drugs, dexmethsone hs n bility to prevent dverse effects of endotoxin on number of body systems in horses nd other nimls (11, 1, 1,, 5). In lipopolyscchride-chllenged swine (19), dexmethsone decresed blood plsm tumour necrosis fctor (by pproximtely %), nd interleukin-, but did not lter interleukin-1 levels. Co-incubtion with 1 µm dexmethsone (this concentrtion gretly exceeds the therpeutic dose of the drug for horses) suppresses the production of TNFα by lipopolyscchride-stimulted equine peritonel mcrophges (18). Dexmethsone lso significntly inhibited TNF production by equine mmmry exudte mcrophges when the gent ws dded one hour before lipopolyscchride dministrtion (17). Dexmethsone tretment hd differentil effects on LPS-induced testiculr inflmmtion nd steroidogenesis inhibition in dult rts (13). There re no such studies concerning stllions. In the previous pper (7), it ws shown tht non-steroidl nti-inflmmtory drug - flunixin meglumine hd positive effect on most semen chrcteristics, which hd been chnged under the influence of endotoxin. The im of this study ws to determine the effects of dexmethsone on the chnges of semen qulity in stllions induced by endotoxin dministrtion.
58 Mteril nd Methods Animls. Seven cliniclly helthy stllions (Polish Primitive Horses) were investigted during the mting seson (April-July). The stllions were divided into two groups: (three stllions ged 8-1 yers, nd weighing 37- kg nd +DX (four stllions ged -1 yers nd weighing 8- kg). Tretment with endotoxin nd dexmethson. Lipopolyscchride (LPS) from scherichi coli (serotype 55:B5; Sigm Chemicl Co.) dissolved in 5 ml of pyrogenic physiologicl sline solution ws infused (i.v.) t the dose of.3 µg/kg b.w. The experimentl stllions (group +DX) received intrmusculrly single dose (.9 mg/kg b.w.) of dexmethsone in the form of preprtion Dexfort (Intervet, Hollnd). The presence of bi-esters of dexmethsone cuses Dexfort to ct immeditely nd t lest for 8 d fter its dministrtion. Dexfort contins mg of dexmethsone phenylpropionte nd 1 mg of dexmethsone sodium phosphte. Dexfort ws injected 1 min before the infusion of endotoxin. Collection nd exmintion of semen. Semen ws smpled by mens of n rtificil vgin (Missouri model) twice week during four weeks nd 7 nd h before tretment (men mrked s time ), nd nd 7 h nd twice week during 9 weeks fter the LPS injection. The gel-free semen volume nd motility of spermtozo were recorded nd the concentrtion of spermtozo ws counted in hemocytometer. Semen morphology (eosin-nigrosin stining) ws studied ccording to Bielński et l. (). The percentge of bnorml forms of spermtozo, especilly the spermtozo with cytoplsmic droplet in distl (form 1), proximl nd typicl position (defects 1-3), with loops of the til: single loop (form ), double loop, loop of the end prt of the til, spirlling of the til, nd til looped round the hed (defects -8), two (or more) heds (form 13), nd smll ( dwrf ) heds (form 15), ws determined. Sttisticl nlysis. The dt were nlysed sttisticlly using the Sttistic SttSoft progrmme, with n nlysis of vrince (ANOVA). Differences between men vlues were nlysed with the Tuky test. Results re expressed s mens ± SD nd significnce ws defined s P<.5. Results The present work clims tht the dministrtion of endotoxin hd negtive influence on the qulity of stllion semen. The chnges in the gel-free semen volume, motility, nd concentrtion of spermtozo were observed (Figs 1-3). In group, there ws sttisticlly significnt decrese in the gel-free semen volume up to 1.5 ml ( GFSV - 1.%) t week fter the dministrtion of LPS. In the group +DX, there ws significntly incresed GFSV, up to 19.7 ml, GFSV - 11.%. Greter chnges were relted to the motility of spermtozo. In reltion to the initil time (time ) nd the experimentl group, the stllions receiving endotoxin demonstrted sttisticlly significnt decrese in the motility of spermtozo: in group t weeks 1 nd 3 nd between 5-8 (with the mximl decrese t week, up to 53.1%, TSM - 7.%), nd in group +DX t week 3, up to.1% ( TSM -.8%). In reltion to the initil vlue, the concentrtion of spermtozo ws significntly lower in the stllions from both groups. After endotoxin dministrtion, the concentrtion of spermtozo decresed between weeks -8 (mximum t week, up to 95. x 1 /ml, SC - 18.%) in the group, nd t week nd between weeks 5-8 (mximum t week 5, up to 13 x 1 /ml, SC -53.1%) in the +DX group. Figs -5 show the percentge of spermtozo with cytoplsmic droplet (defects 1-3), nd with til loop (defects -8). The percentge of spermtozo with cytoplsmic droplet (defects 1-3) ws higher in the experimentl stllions semen. In the groups nd +DX, there ws significnt increse between weeks -9 (mximum t week 3, up to 9.%, Defects 1-3 - 311.%), nd between weeks 3-9 (mximum t week, up to.8, Defects 1-3 - 13.%), respectively. When nlysing the chnges relted to ech defect, the experimentl stllions demonstrted prticulrly high rise of spermtozo with cytoplsmic droplet in distl position (Fig. ); in the group, it incresed between weeks -9 (mximum t week 5, up to.5% ( Form 1-377.9%), nd in the group +DX between weeks 3-8 (mximum t week, up to 5.8%, Form 1-19.3%. The percentge of spermtozo with til loops (defects -8) ws lso higher in both groups of experimentl stllions. In the groups nd +DX, the number of these defects ws significntly incresed between hour 7 nd week 9 (mximum t week 3, up to 1.%, Defects -8-1.%), nd t weeks 1-, nd between weeks -9 (mximum t week 8, up to 7.%, Defects -8-5.%), respectively. Among these defects, the experimentl stllions demonstrted the lrgest increse in the percentge of spermtozo with single til loop (Fig. 5). The increse of spermtozo with this defect ws noted in group, between hour nd week 9 (mximum t week 3, up to 5. % ( Form - 157.1%). nd in group +DX between hour 7 nd week 9 (mximum t week 8, up to 5.3% ( Form - 11.7%). After the dministrtion of LPS nd LPS+DX, there ws n increse in the percentge of spermtozo with two (or more) heds (form 13), nd smll ("dwrf") heds (form 15). In the group, there ws n increse in form 13 of spermtozo t week 8, up to 1.%, Form 13-3.%, nd form 15, t weeks -7, with mximum increse t week, up to.7%, Form 15-35.%. In the group +DX, the percentge of form 13 spermtozo incresed t week 5, up to 1.%, Form 13-1.%, nd of form 15 spermtozo t week 5, up to.1%, Form 15 -.%. (Figs -7).
583 8 7 +DX 5 GFSV (ml) 3 1 Time of exmintion Fig. 1. Gel-free semen volume of stllions fter DX nd LPS dministrtion (men ±SD). - group of stllions with LPS, +DX group of stllions with LPS+DX, *- significnt difference to the time, : no significnt difference between groups, t P<.5 9 8 7 b +DX TSM (%) 5 3 Time of exmintion Fig.. Totl motility of spermtozo in the semen of stllions fter DX nd LPS dministrtion (men ±SD). - group of stllions with LPS, +DX group of stllions with LPS+DX, *- significnt difference to the time, :b - significnt difference between groups, t P<.5
58 5 SC (x1 /ml) 35 3 5 15 1 *b +DX 5 h 8h 1wk wk 3wk wk 5wk wk 7wk 8wk 9wk Time of exmintion Fig. 3. Concentrtion of spermtozo in the semen of stllions fter DX nd LPS dministrtion (men ±SD). Symbols re explined in the footnotes to Fig.. 1 Defects 1-3 (%) 1 1 8 +DX - Time of exmintion Fig.. Spermtozo with cytoplsmic droplet (distl, proximl, nd typicl position) in the semen of stllions fter DX nd LPS dministrtion (men ±SD). Symbols re explined in the footnotes to Fig. 1.
585 Form 1 (%) 9 8 7 5 3 +DX 1-1 Time of exmintion Fig.. Spermtozo with cytoplsmic droplet in distl position in the semen of stllions fter DX nd LPS dministrtion (men ±SD). Symbols re explined in the footnotes to Fig. 1. 18 Defects -8 (%) 1 1 1 1 8 *b +DX - Time of exmintion Fig. 5. Spermtozo with til loops (single loop, double loop, loop of the end prt of the til, spirlling of the til, nd til looped round the hed) in the semen of stllions fter DX nd LPS dministrtion (men ±SD). Symbols re explined in the footnotes to Fig..
58 9 Form (%) 8 7 5 +DX 3 1 Time of exmintion Fig. 5. Spermtozo with single til loops in the semen of stllions fter DX nd LPS dministrtion (men ±SD). Symbols re explined in the footnotes to Fig. 1. Frorm 13 (%),,, 1,8 1, 1, 1, 1,,8,,,, -, -, -, +DX Time of exmintion *b Fig.. Spermtozo with two (or more) heds in the semen of stllions fter DX nd LPS dministrtion (men ±SD). Symbols re explined in the footnotes to Fig..
587 5 +DX Form 15 (%) 3 1-1 Time of exmintion Fig. 7. Spermtozo with smll ("dwrf") heds in the semen of stllions fter DX nd LPS dministrtion (men ±SD). Symbols re explined in the footnotes to Fig. 1. Discussion In the present work, it ws demonstrted tht the dministrtion of endotoxin hd negtive influence on the qulity of stllion semen. A sttisticlly significnt decrese in the gel-free semen volume, motility, nd concentrtion of spermtozo fter LPS dministrtion ws noted. As regrds ech of the morphologicl defects of the spermtozo, the percentge of those with cytoplsmic droplets, til loops, loose heds, two (or more) heded, nd smll "dwrf" heds ws clerly higher. The stllions (5, 7) receiving endotoxin demonstrted decrese in sperm concentrtion nd increse in the percentge of spermtozo with secondry chnges, i.e. with cytoplsmic droplet nd with til loops nd loose heds. Among the primry defects, spermtozo with dwrf nd gigntic heds were previling. The obtined results re close to the observtions of other uthors in mles of different niml species. In rms (8), treted with single doses (3. µg/kg b.w.), of S. typhimurium LPS s well s in the cse of repeted endotoxin dministrtion (.3 µg/kg b.w., 1 times, t 1 h intervls) nd in dose of. µg/kg b.w. (five times, t h intervls), the semen volume decresed in week 5, nd the motility of spermtozo decresed in weeks nd -, without ny negtive influence on the overll number of spermtozo in the semen. In weeks 1 nd 3-, ccording to the LPS dose pplied, there ws sttisticlly significnt increse in the percentge of spermtozo with bnorml heds. In these nimls, prt from primry chnges, there ws n increse in the percentge of spermtozo with cytoplsmic droplets in proximl position (week -). The dministrtion of endotoxin S. typhimurium to bors (7) hd no ny significnt influence on the semen volume, motility, nd totl number of spermtozo, but the percentge of spermtozo with morphologicl chnges ws higher. Prticulrly mrked increse in spermtozo with cytoplsmic droplets took plce in weeks -3 fter injection of. µg LPS/kg b.w., nd with coiled til in week fter injection of.5 µg LPS/kg b.w. Other defects such s nucler pouch formtion nd bnormlly shped sperm heds occurred in weeks 3- fter the dministrtion of the endotoxin. rlier reports lso showed tht steroidogenesis nd spermtogenesis re ffected during bcteril LPSinduced cute inflmmtion. Studies in bors showed tht endotoxin induced infiltrtion of polymorphonucler neutrophils into the testiculr interstitium nd morphologicl chnges of Leydig cells (9). In dult rts treted with LPS, it ws found tht the dmge to the seminiferous epithelium during inflmmtion is more likely to be due to direct effects of the inflmmtion, rther thn disturbnces in ndrogen production (). Oxidtive stress is mjor cusl fctor in ltered steroidogenesis nd spermtogenesis, nd perhps the infertility during endotoxin-induced cute inflmmtion (3). Dexmethsone belongs to group of drugs with nti-inflmmtory nd ntipyretic properties. The effects of corticosteroids were ssessed in endotoxemi, especilly during endotoxin shock, in horses (1, 1, 5). The role of DX in testiculr steroidogenesis or its influence on sperm production nd seminl chrcteristics is prtilly known (1, 8, 1). However, in vivo evidence suggests tht glucocorticords (DX) my decrese testosterone production indirectly vi the hypothlmic-pituitry-gondl xis (). Dexmethsone, so s IL-1β, inhibits LH-stimulted testosterone relese from mouse testiculr cells (3). In the present study, DX tretment prevented n endotoxin-induced decrese in the concentrtion of
588 spermtozo nd hd no cler influence on the gel-free semen volume nd on percentge of form spermtozo. A negtive influence of DX dministrtion ws noticed especilly, in percentge of forms 9 nd 15 of spermtozo nd seminl plsm lbumin concentrtion in the stllions treted with endotoxin. A previous pper () showed tht injection of DX only hd negtive influence on the morphologicl properties of spermtozo in the stllions. As regrds ech of the morphologicl defects of the spermtozo, the percentge of those with loose heds nd smll ("dwrf") heds ws clerly higher. Flunixin meglumine (non-steroidl ntiinflmmtory drug) t the dose of 1.1 mg/kg given fter the injection of scherichi coli LPS (.3 µg/kg b.w.) hd positive influence on the seminologicl chnges occurring during endotoxemi in the stllions (7). This pper showed tht the effects of flunixin were especilly visible for the motility nd concentrtion of spermtozo nd for morphologicl defects of spermtozo in stllions, which received the compound injection 5 min fter the infusion of endotoxin. In dult rts (13), it ws found tht co-dministrtion of dexmethsone inhibited the systemtic inflmmtory response, but did not prevent the locl inflmmtion in the testes. Dexmethsone did not prevent the lipopolyscchrideinduced erly inhibition of testosterone, but reversed lter inhibition of this hormone. In conclusion, the dministrtion of glucocorticoid - dexmethsone hd different effect on endotoxin-induced chnges of semen qulity in the stllions. A positive influence of DX ws noticed in certin mcro- nd microscopic chrcteristics of semen prmeters. There were noted smller chnges in totl motility nd concentrtion of spermtozo nd in the percentge of spermtozo with cytoplsmic droplet nd two (or more) heds, without influence on the gelfree semen volume. Greter, negtive chnges in the qulity of stllions semen fter dministrtion of endotoxin+dx, in reltion to the group of stllions with lone LPS, were noticed in the percentge of spermtozo with til loops, loose hed, nd smll ("dwrf") hed. Dexmethsone in ccepted therpeutic doses did not prevent nd to certin degree enhnced the dverse influence of endotoxin on the semen qulity in the stllions. Acknowledgments: This study ws grnted by the Polish Stte Committee for Scientific Reserch (grnt No. 5PK 18). The study ws executed in the Ntionl Veterinry Reserch Institute in Pulwy, Polnd. References 1. Brth A.D., Bowmn P.A.: The sequentil ppernce of sperm bnormlities fter scrotl insultion or dexmethsone tretment in bulls. Cn Vet J 199, 35, 93-1.. Bielński W., Dudek., Bittmr A., Kosinik K.: Some chrcteristics of common bnorml forms of spermtozo in highly fertile stllions. J Reprod Fert Suppl 198, 3, 1-. 3. Cover P.O., Bnh-Jones F., John C.D., Buckinghm J.C.: Annexin 1 (lipocortin 1) mimics inhibitory effects of glucocorticoids on testosterone secretion nd enhnces effects of interleukin-1β. ndocrine 18, 33-39.. Dnek J.: Biochemicl chnges in seminl plsm fter endotoxin injection in stllions. Bull Vet Inst Pulwy,, 193-199. 5. Dnek J.: ffect of scherichi coli endotoxin on the levels of testosterone nd estrdiol-17β in blood serum nd seminl plsm nd on the semen chrcteristics in the stllion. Bull Vet Inst Pulwy 3, 7, 191-9.. Dnek J.: ffect of dexmethsone nd flunixin on the blood serum testosterone nd estrdiol-17β concentrtions nd on the morphology of spermtozo in the stllion. Medycyn Wet, 139-133. 7. Dnek J.: ffects of dministrtion of scherichi coli lipopolyscchrides nd flunixin meglumine on the semen qulity in the stllion. Bull Vet Inst Pulwy, 8, 1-. 8. Dnek J.: ffect of dexmethsone on the concentrtions of testosterone, estrone, 17β-estrdiol, nd biochemicl prmeters in stllion semen. Medycyn Wet 5, 1, 335-337. 9. Dnek J.: ffects of flunixin meglumine on selected clinicopthologic vribles, nd serum testosterone concentrtion in stllions fter endotoxin dministrtion. J Vet Med A,, 53, 357-33. 1. Dnek J.: ffect of dexmethsone on model endotoxemi in the stllion. Bull Vet Inst Pulwy, 5, 89-9. 11. wert K.M., Fessler J.F., Templeton C.B., Bottoms G.D., Ltshw H.S., Johnson B.S.: ndotoxin-induced hemtologic nd blood chemicl chnges in ponies: ffects of flunixin meglumine, dexmethsone, nd prednisolone. Am J Vet Res 1985,, -3. 1. Fruenfelder H.C., Fessler J.F., Moore A.B., Bottoms G.D., Boon G.D.: ffects of dexmethsone on endotoxin shock in the nesthetized pony: hemtologic, blood gs, nd cogultion chnges. Am J Vet Res 198, 3, 5-11. 13. Gow R.M., O'Bryn M.K., Cnny B.J., Ooi G.T., Hedger M.P.: Differentil effects of dexmethsone tretment on lipopolyscchride-induced testiculr inflmmtion nd reproductive hormone inhibition in dult rts. J ndocrinol 1, 18, 193-1. 1. Juhsz J., Ngy P., Kulesr M., Huszenic Gy.: A review: Fctors influencing semen qulity nd endocrinologicl sex function in stllions-with emphsis on glucocorticoids. Foli Vet 1, 5, 3-8. 15. Klus A.M., Hpke H.J.: Ntűrliche und synthetische Glukokortikoide beim Sportpferd: eine Literturűbersicht. Dt Tierrztl Woch 199, 13, 89-58. 1. Lne P.J., Lees P.: Action of dexmethsone in n equine model of cute non-immune inflmmtion. Res Vet Sci 199, 8, 87-95. 17. Milm S.B., Mcky R.J., Skelley L.A.: Secretion of tumor necrosis fctor by endotoxin-treted equine mmmry exudte mcrophges: effect of dexmethsone nd pentoxifylline. Cornell Vet 199, 8, 35-. 18. Morris D.D., Moore J.N., Cowe N., Fischer J.K.: Dexmethsone reduces endotoxin-induced tumor
589 necrosis fctor ctivity production in vitro by equine peritonel mcrophges. Cornell Vet 1991, 81, 7-75. 19. Myers M.J., Frrell D.., Plmer D.C., Post L.O.: Inflmmtory meditor production in swine following endotoxin chllenge with or without co-dministrtion of dexmethsone. Int Immunophrmcol 3, 3, 571-579.. O Bryn M.K., Schltt S., Phillips D.J., Krester D.M., Hedger M.P.: Bcteril lipopolyscchride-induced inflmmtion compromises testiculr function t multiple levels in vivo. ndocrinology, 11, 38-. 1. Olson N.C., Brown T.T. Jr., Anderson D.L.: Dexmethsone nd indomethcin modify endotoxininduced respirtory filure in pigs. J Appl Physiol 1985, 58, 7-8.. Olson N.C., Brown T.T. Jr.: Dexmethsone-induced ttenution of crdiopulmonry dysfunction in endotoxemic clves. Am J Vet Res 198, 7, 187-19. 3. Reddy M.M., Mhipl S.V., Subhshini J., Reddy M.C., Roy K.R., Reddy G.V., Reddy P.R., Reddnn P.: Bcteril lipopolyscchride-induced oxidtive stress in the impirment of steroidogenesis nd spermtogenesis in rts. Reprod Toxicol,, 93-5.. Sokkr S.M., Drwiesh G., Mdbooly A.: Study of the pthologicl effect of scherichi coli endotoxin in rms. J Vet Med B 3, 5, -3. 5. Templeton C.B., Bottoms G.D., Fessler J.F., wert K.M., Roesel O.F., Johnson M.A., Ltshw H.S.: ndotoxininduced hemodynmic nd prostglndin chnges in ponies: effects of flunixin meglumine, dexmethsone, nd prednisolone. Circ Shock 1987, 3, 31-.. Thibier M., Rollnd O.: The effect of dexmethsone (DXM) on circulting testosterone (T) nd luteinizing hormone (LH) in young postpubertl bulls. Theriogenology 197, 5, 53-. 7. Wllgren M.: Clinicl, endocrinologicl, nd spermtologicl studies fter endotoxin injection in the bor. J Vet Med A 1989, 3, -75. 8. Wllgren M., Kinndhl H., Lrsson K.: Clinicl, endocrinologicl, nd spermtologicl studies fter endotoxin in the rm. J Vet Med A 1989, 3, 9-13. 9. Wllgren M., Kindhl H., Rodriguez-Mrtinez H.: Altertions in testiculr function fter endotoxin injection in the bor. Int J Androl 1993, 1, 35-3. 3. Wllgren M., Kindhl H., Rodriguez-Mrtinez H.: Modultion of endotoxin-induced ndrologicl ltertions by flunixin meglumine in the bor. J Vet Med A 1995,, 357-39.