Influenza outbreak causes reduction in semen quality of boars

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1 Peer reviewed Cse report Influenz outrek cuses reduction in semen qulit of ors Drew W. Lugr, MS; Drrl Rglnd, DVM, PhD; Kr R. Stewrt, MS, PhD Summr An influenz outrek occurred t Purdue Universit s swine rn, resulting in infection of 28 ors with influenz A virus (H3N2) nd cusing the deth of two ors. The 28 ors, pproximtel 35 weeks of ge, were enrolled in stud t the time of the outrek nd the cse report herein descries the effects of the unintended influenz outrek on sperm production. Semen ws collected from the ors once week nd evluted for totl sperm production nd concentrtion, semen volume, nd reltive motilit. Compred to previous collections, totl sperm production ws sustntill decresed (26% reduction) pproximtel 4 weeks fter the first oserved clinicl signs nd remined low for 6 susequent weeks. Semen production then returned to pre-outrek levels nd ws mintined for the durtion of the oservtion period. Sperm motilit nd percent norml sperm production were lso slightl reduced 2 weeks fter infection. Kewords: swine, influenz A virus, or, semen qulit Received: Decemer 20, 2016 Accepted: April 25, 2017 Resumen - Brote de influenz cus reducción en l clidd de semen en mchos Un rote de influenz sucedió en un sl de l grnj porcin de l Universidd de Purdue, resultndo en l infección de 28 mchos con el virus de l influenz tipo A (H3N2 por sus sigls en inglés), cusndo l muerte de dos mchos. Los 28 mchos de proximdmente 35 semns de edd, se inclueron en un estudio l momento del rote, el reporte descrito en este cso, sí como los efectos de un rote de influenz no intenciondo sore l producción de semen. Un vez l semn, se recolectó semen de los mchos, se evluó l concentrción l producción totl, el volumen, l motilidd reltiv del semen. Comprándolo con ls colecciones previs, l producción totl de semen este disminuó sustncilmente (26% de reducción) proximdmente 4 semns después del los primeros signos clínicos oservdos, este permneció jo por ls siguientes 6 semns. L producción de semen regresó los niveles previos l rote, se mntuvo durnte del periodo de oservción. L motilidd del semen el porcentje de producción norml tmién disminueron ligermente 2 semns después de l infección. Résumé - Une épidémie d influenz cuse une réduction de l qulité de l semence de verrts Une épidémie d influenz est survenue à l porcherie de l université Purdue, entrinnt l infection de 28 verrts vec le virus de l influenz A (H3N2) et cusnt l mort de deux verrts. Les 28 verrts, âgés d environ 35 semines, fisient prtie d une étude u moment de l épidémie et le présent rpport de cs décrit les effets de cette épidémie inttendue d influenz sur l production de sperme. De l semence étit prélevée des verrts une fois pr semine et évluée pour l production totle de sperme et l concentrtion, le volume de semence, et l moilité reltive. Comprtivement ux collectes ntérieures, l production totle de sperme étit diminuée de mnière sustntielle (26% de réduction) environ 4 semines près l oservtion des premiers signes cliniques et est demeurée file pour les 6 semines suséquentes. L production de semence retourn ensuite ux niveux pré-épidémie et fut mintenue pour l durée de l période d oservtion. L moilité du sperme et le pourcentge de production de sperme norml étient églement réduits 2 semines près l infection. Intensive frming strtegies crete opportunities for pthogens to enter nd rpidl spred throughout herds, cusing reduced growth rtes, overll performnce, nd helth compred to unchllenged herds. These reductions in growth nd performnce lso hve consequentil economic impct DWL, KRS: Deprtment of Animl Sciences, Purdue Universit, West Lfette, Indin. DR: Deprtment of Veterinr Clinicl Sciences, Purdue Universit College of Veterinr Medicine, West Lfette, Indin. Corresponding uthor: Dr Kr R. Stewrt, 915 W Stte Street, Room 3-235, West Lfette, IN ; Tel: ; Fx: ; E-mil: krstewrt@purdue.edu. This rticle is ville online t Lugr DW, Rglnd D, Stewrt KR. Influenz outrek cuses reduction in semen qulit of ors. J Swine Helth Prod. 2017;25(6): Journl of Swine Helth nd Production Volume 25, Numer 6 on producers. In ddition to nutrition-relted mesures of performnce, reproductive performnce nd efficienc re lso kes to producer profitilit nd re susceptile to reductions cused phsiologicl stressors like het stress nd diseses. However, there is lck of literture on the effects of disese on semen qulit in ors. Porcine reproductive nd respirtor sndrome virus is thought to impir motilit nd morpholog of sperm without mjor chnge in sperm production, though most reserch is focused on its trnsmission through semen. 1,2 The Torque teno virus 3 nd porcine circovirus tpe 2 4 re lso trnsmitted through the semen, ut do not pper to lter semen qulit in ors. Influenz A virus is pthogen tht induces clinicl signs, resulting in fever, which leds to elevted core od temperture, ut there is lck of pulished informtion on the effects of this disese on semen qulit in ors. Choi et l 5 reported tht 22.8% of pigs in the United Sttes test positive for 303

2 swine influenz virus vi hemgglutintion inhiition nd rel time-polmerse chin rection. 5 Gloll, influenz is one of the most widespred respirtor viruses ffecting the swine industr nd is considered endemic throughout most of the world. 6 One of the min issues with influenz infections in swine is tht pigs re susceptile to swine, vin, nd humn strins of influenz virus. 6 Influenz-infected nimls generll displ clinicl signs shortl fter infection, which include, ut re not limited to fever, dirrhe, nd sneezing nd (or) coughing. 7 Influenz is primril spred vi nose-to-nose contct etween pigs nd vi erosolized, infectious respirtor excretions. 7 To the uthors knowledge, this is the first report on the effects of influenz A virus infection on the susequent semen qulit of ors. The purpose of this cse report is to elucidte the effects of influenz A virus infections on sperm production nd semen qulit in ors. Influenz outrek nd tretment timeline Twent-eight 35-week-old ors were eing evluted for semen qulit prmeters t the Purdue Universit Animl Sciences Reserch nd Eduction Center when n influenz outrek occurred. The first oservtion of mild clinicl signs of illness (intermedite coughing nd lethrg) in three ors occurred April 22, 2016, three weeks into the evlution of semen qulit. On the morning of April 24, 2016 (strt of week 4), one or ws found ded nd mild clinicl signs were oserved in eight to 10 ors. B April 25, 2016, moderte clinicl signs (persistent coughing nd lethrg) were oserved in over 75% of the ors present. At this point, ll of the ors were treted with single injection of tulthromcin t dosge of 2.5 mg per kg to minimize the potentil for secondr infections, nd with single intrmusculr injection of flunixin meglumine t dosge of 2.2 mg per kg to mnge prexi. On April 26, 2016, second or ws found ded in the erl fternoon nd moderte clinicl signs were oserved in ll of the remining ors. These moderte clinicl signs persisted for 2 to 3 ds post tretment in mjorit of the ors, nd mild clinicl signs lsted up to 10 ds in some ors. B d 11 post tretment, 2 weeks fter initil clinicl signs were oserved (end of week 5), no clinicl signs of illness were oserved. 304 The ors tht died on April 24 nd April 26 were necropsied licensed veterinrin nd lung smples were collected nd sumitted for dignostic testing. The necropsies reveled consolidtion of the crnil nd ventrl lung loes nd ccessor loes, with some firinous exudte in the pleurl cvit. The lungs hd vring degrees of pulmonr edem nd congestion nd there ppered to e incresed fluid in the pericrdil sc of the ffected ors. Lung tissue ws sumitted to the Indin Animl Disese Dignostic Lortor (ADDL; West Lfette, Indin) nd the cuse of the influenz outrek ws confirmed. Testing of the disesed lung tissue vi polmerse chin rection reveled the presence of influenz tpe A nucleic cids elonging to the H3N2 strin of influenz virus. Blood ws collected nd serum ws hrvested from ll of the ors during week 5 nd smples were sumitted to the Indin ADDL. The serum ws tested for influenz tpe A virus ntiodies vi enzme-linked immunosorent ss (ELISA), nd the results showed tht ll ors tested positive for influenz A virus ntiodies. Effects on reproductive performnce The influenz outrek hd mjor impct on the semen qulit prmeters mesured on freshl collected semen in this incident. Prior to the influenz outrek, the ors were housed in individul stlls nd semen ws routinel collected one time per week vi the doule-gloved hnd technique nd n rtificil sow. Semen ws evluted on site immeditel fter ech collection for sperm concentrtion, semen volume, reltive motilit, nd totl sperm production (concentrtion volume). Semen ws evluted on site strting t week 1 (April 4, 2016) through week 17 ( Jul 29, 2016). At weeks 5 through 11, semen ws diluted with commercil semen extender on site nd lso evluted computer-ssisted sperm ssessment (CASA; CEROS II, IMV Technologies USA; Mple Grove, Minnesot) for sperm totl motilit nd progressive motilit, s well s microscopic exmintion for percent norml sperm morpholog, once weekl for ech or. Totl motilit refers to n movement of the sperm, wheres progressive motilit, suset of totl motilit, refers to sperm movement in mostl stright mnner. To determine percent norml morpholog, phse contrst microscope ws used to count minimum of 200 sperm. Sttisticl nlsis ws performed using the PROC MIXED function of SAS (version 9.4; SAS Institute Inc, Cr, North Crolin). Sttisticl nlsis of semen prmeters utilized repeted mesures criteri of or week. Different covrince structures (compound smmetr, heterogeneous utoregressive, nd unstructured) were tested in order to minimize Akike informtion criterion. Where pproprite, collection nd lortor technicin were utilized s potentil rndom effects. A P vlue <.05 ws considered sttisticll significnt nd P vlue <.10 ws considered tendenc. The influenz outrek occurred t the end of week 3 of the stud nd lsted through the end of week 5. Norml semen production ws seen in weeks 1 through 6 (70.74, 65.14, 70.89, 65.31, 63.98, nd ± sperm per ejculte, respectivel). Sperm production ws significntl reduced during weeks 7 through 12 (54.22, 49.45, 49.45, 48.43, 53.41, nd ± sperm per ejculte, respectivel), returning to norml during weeks 13 through 17 (69.23, 67.45, 72.82, 66.79, nd ± sperm per ejculte). For this reson, totl sperm production, totl motile sperm production, semen volume, nd semen concentrtion were nlzed in three phses: Phse 1 (weeks 1 to 6), Phse 2 (weeks 7 to 12), nd Phse 3 (weeks 13 to 17). Totl sperm production dt re summrized in Tle 1. Totl sperm production did not differ in phses 1 nd 3 (P >.05). Totl sperm production ws greter in phses 1 nd 3 compred to Phse 2 (67.92 nd versus ± sperm; P <.001). Averge totl sperm production in phses 1 nd 3 ws sperm compred with sperm in Phse 2, difference of sperm representing 26% reduction in sperm production. Totl motile sperm production dt re summrized in Tle 1. Totl motile sperm production ws lower in Phse 2 thn in phses 1 nd 3 (P <.001), nd did not differ in phses 1 nd 3 (P >.05). Semen volume ws greter in Phse 3 thn in phses 1 nd 2 (P <.001 nd P <.01, respectivel). Semen volume ws greter in Phse 2 thn in Phse 1 (P <.05). Sperm concentrtion ws lower in Phse 3 thn in Phse 1 (P <.001), nd lower in Phse 2 thn in phses 1 nd 3 (P <.001). Sperm motilit nd progressive motilit dt re summrized in Figure 1. Sperm motilit ws lower in week 7 thn in weeks 9 through 11 (P <.05), weeks 5, 6, nd 8 through 11 did not differ (P >.05), nd Journl of Swine Helth nd Production Novemer nd Decemer 2017

3 Tle 1: Sperm production dt phse from 28 ors unintentionll infected with influenz A virus t universit reserch frm, with pre-influenz phse (Phse 1), n influenz-ffected phse (Phse 2), nd post-influenz phse (Phse 3)* Sperm production Phse Totl sperm production <.01 Reltive motilit (%) <.01 Totl motile sperm <.01 * Clinicl signs oserved during the influenz outrek included coughing nd lethrg. The outrek resulted in the deths of two ors during the oservtion period. Lung smples from the ded ors were sumitted to dignostic lortor, which confirmed polmerse chin rection the presence of tpe A influenz virus (H3N2). Following the lortor dignosis, lood ws collected nd serum hrvested nd sumitted for dignosis, confirming tpe A influenz n enzme-linked immunosorent ss. Phse 1 (weeks 1-6) where first clinicl signs were oserved t week 3 nd were no longer oserved week 5; Phse 2 (weeks 7-12); Phse 3 (weeks 13-17). Phses were sed on the weekl nlsis of the dt tht showed sustntil reduction in totl sperm production in weeks Totl motile sperm = totl sperm production (totl sperm production = semen volume sperm concentrtion) reltive motilit (s ssessed phse contrst microscop). Within row, vlues with different superscripts re different (P <.05; ANOVA). SE = stndrd error. SE P weeks 5 through 8 did not differ (P >.05). Progressive motilit did not differ for week 5 compred to other ll weeks (P >.05). Progressive motilit tended to e lower for week 6 thn for week 10 (P <.10). Week 7 progressive motilit ws lower thn in weeks 8 through 11 (P <.05, P <.01, P <.01, P <.01; respectivel), nd within weeks 8 to 11 progressive motilit did not differ (P >.05). Percent norml morpholog dt re summrized in Figure 2. Percent norml morpholog ws lower for weeks 5 nd 6 thn for weeks 10 nd 11 (P <.05). Week 5 percent norml morpholog did not differ from tht in weeks 6 to 9 (P >.05), nd percent norml morpholog in week 6 did not differ from tht in weeks 5, 7, nd 8 (P >.05). Week 6 percent norml morpholog tended to e lower thn week 9 percent norml morpholog (P <.10). Discussion The results of this report indicte tht influenz A infection hd significnt impct on the semen qulit of ors, including totl sperm production, motilit, nd morpholog. To the uthors knowledge, this is the first reported cse of influenz A infection ffecting semen qulit in ors, lthough there re reports of influenz s impct on semen qulit in humns nd mice. 8,9 Reserch suggests tht other diseses cn impct semen qulit of ors; however, most reserch on disese in ors is primril focused on the trnsmission of pthogens Journl of Swine Helth nd Production Volume 25, Numer 6 through semen. This report suggests tht 4 weeks fter the onset of clinicl signs of infection, totl sperm production is reduced in the or. This reduction resulted from reduced sperm production within the testes nd ws confirmed reduction in sperm concentrtion. Semen volume incresed during the oservtion period, s is normll seen in growing, mturing ors. These dt suggest tht infection with influenz virus cuses phsiologicl response to the extent tht there is reduction in the production of sperm, likel cused fever nd elevted core od temperture. This reduced sperm production hs een seen in men infected with ferile (fever cusing) diseses. 10,11 It is unlikel tht influenz A virus directl impcted semen production in the testes due to its locliztion in lung tissue; insted, influenz A virus likel hd n indirect effect inititing fever response which could cuse reduction in sperm production. Further reserch should investigte the effects of ferile infection on semen qulit prmeters in ors to determine the direct cuse of the reduced sperm production. During the 17-week oservtion period, sperm motilit nd morpholog were nlzed for weeks 5 through 11. Dt suggest sperm motilit nd progressive motilit m decline t minimum of 2 to 4 weeks fter the onset of clinicl signs of fever or infection with influenz virus nd return to norml fter 5 to 6 weeks. The percentge of norml spermtozo ws reduced t minimum of 2 to 3 weeks fter the initil onset of clinicl signs nd stedil returned to norml 7 to 8 weeks. This cse report did not nlze dt on CASA prmeters nd percent norml sperm morpholog during the first 4 weeks of the oservtion period nd thus the entire impct on motilit nd morpholog prmeters cnnot e estimted. However, the results of nlsis suggest tht these prmeters re temporril decresed few weeks fter influenz A infection, which grees with results in men infected with ferile diseses. 12 The semen nlsis results presented in this report re common in ors tht hve undergone stressful event le to increse core od temperture, such s het stress. Bors tht re het stressed, for exmple, tpicll hve reduced sperm motilit nd incresed sperm normlities within 2 weeks of the het stress event. 13 The length of time the stressor impcts the ors lso pls role, s motilit nd morpholog prmeters do not return to norml until 4 to 6 weeks fter the stressor is removed. 13 Similr results were seen in this cse, where there ws cler del in ffected sperm reching the cudl epididmis nd hving n effect on spermtogenesis. Influenz nd (or) the consequentil phsiologicl responses to influenz A virus infection pper to ffect spermtogenesis to the extent tht n entire spermtogenic ccle (pproximtel 41 ds) is required for sperm production to return to norml. 14 To the uthors knowledge, this is the first reported cse of impired semen qulit due to n influenz outrek in ors. While it is unlikel tht influenz virus directl impirs sperm production, the residul effects of 305

4 Figure 1: An unintentionl outrek of influenz A (H3N2) occurred t universit reserch frm, ffecting totl sperm motilit nd progressive motilit of 28 ors. This outrek cused clinicl signs consisting of coughing nd lethrg, s well s the deths of two ors. Lortor dignosis confirmed the presence of this virus in lung tissue smples, s well s in serum smples from the ors. Clinicl signs were oserved in weeks 3-5 nd cused ltent effects on semen production nd qulit. The figure shows the effect of influenz on sperm totl motilit nd progressive motilit s ssessed computer ssisted sperm ssessment weeks Blue rs represent totl motilit where rs with differing letters (, ) re sttisticll different (P <.05). Red rs represent progressive motilit where rs with differing letters (x, ) re sttisticll different (P <.05). Error rs for oth totl motilit nd progressive motilit represent the stndrd error. This grph shows tht oth totl motilit nd progressive motilit of sperm were decresed t week 7 nd then returned to norml eginning t week 8. Sperm motilit prmeters were not mesured prior to week 5, so the totl extent of the effects of influenz on sperm motilit prmeters cnnot e estimted. Sttisticl nlses on totl nd progressive motilit were performed using repeted mesures in the MIXED procedure of SAS (version 9.4) with collection nd lortor technicin s rndom effects. Significnt differences were determined t P vlue <.05 nd tendenc t P vlue < Totl motilit Progressive motilit x Motilit (%) 65 x x Time (week) virl infections m e seen in the reproductive function of ors. Implictions Influenz nd its effects on the od cn negtivel impct norml sperm production in ors. The consequences of influenz on totl sperm production re deled due to the nture of spermtogenesis. Conflict of interest None reported. Disclimer Scientific mnuscripts pulished in the Journl of Swine Helth nd Production re peer reviewed. However, informtion on medictions, feed, nd mngement techniques m e specific to the reserch or commercil sitution presented in the mnuscript. It is the responsiilit of the reder to use informtion responsil nd in ccordnce with the rules nd regultions governing reserch or the prctice of veterinr medicine in their countr or region. References 1. Prieto C, Surez P, Butist JM, Snchez R, Rillo SM, Simrro I, Soln A,Cstro JM. Semen chnges in ors fter experimentl infection with porcine reproductive nd respirtor sndrome (PRRS) virus. Theriogenolog. 1996;45: Prieto C, Cstro JM. Porcine reproductive nd respirtor sndrome virus infection in the or: review. Theriogenolog. 2005;63: Journl of Swine Helth nd Production Novemer nd Decemer 2017

5 Figure 2: An unintentionl outrek of influenz occurred t universit reserch frm, ffecting the percentge of morphologicll norml sperm of 28 ors. This outrek cused clinicl signs which consisted of coughing nd lethrg, s well s the deths of two ors. Lortor dignosis confirmed the presence of tpe A influenz virus, specificll H3N2, in lung tissue smples from the ded ors, s well s in serum smples from the remining ors. Clinicl signs of the outrek were oserved weeks 3-5 nd cused ltent effects on semen production nd qulit. The figure illustrtes the effect of influenz virus on the percentge of morphologicll norml sperm. Sperm morpholog ws ssessed weeks 5-11 using phse contrst microscop, where minimum of 200 sperm were counted nd ctegorized s norml or norml. Weeks with differing letters (, ) re different (P <.05) nd error rs re represented the stndrd error. This figure revels tht the percentge of morphologicll norml sperm ws low t weeks 5 nd 6, nd then incresed over time. It is likel tht percent norml morpholog during weeks 1-3 would hve een similr to weeks 9-11, where plteu ws oserved. Since morphologicl ssessment ws not conducted weeks 1-4, the full effect of influenz A virus on sperm morpholog cnnot e completel estimted. Sttisticl nlsis on sperm morpholog ws performed using repeted mesures in the MIXED procedure of SAS (version 9.4) with collection nd lortor technicin s rndom effects. Significnt differences were determined t P vlue <.05 nd tendenc t P vlue < Norml morpholog (%) Time (week) 3. Kekrinen T, Lopez-Sori S, Segles J. Detection of swine Torque teno virus genogroups 1 nd 2 in or ser nd semen. Theriogenolog. 2007;68: Rose N, Opriessnig T, Grslnd B, Jestin A. Epidemiolog nd trnsmission of porcine circovirus tpe 2 (PCV2). Virus Res. 2012;164: Choi YK, Gol SM, Joo HS. Prevlence of swine influenz virus sutpes on swine frms in the United Sttes. Arch Virol. 2002;147: Brown IH. The epidemiolog nd evolution of influenz viruses in pigs. Vet Microiol. 2000;74: Lnge E, Klthoff D, Blohm U, Teifke J, Breithupt A, Mresch C, Strick E, Fereidouni S, Hoffmnn B, Mettenleiter T, Beer M, Vhlenkmp T. Pthogenesis nd trnsmission of the novel swineorigin influenz virus A/H1N1 fter experimentl infection of pigs. J Gen Virol. 2009;90: Evenson DP, Jost LK, Corzett M, Blhorn R. Chrcteristics of humn sperm chromtin structure following n episode of influenz nd high fever: cse stud. J Androl. 2000;21: Shrm G, Pols H. Ctogenetic effects of influenz virus infection on mle germ cells of mice. Hum Genet. 1978;45: McLeod, J. Effect of chickenpox nd of pneumoni on semen qulit. Fertil Steril. 1951;6: Soehdi K. Azoospermi cused tphoid fever A: cse report. Andrologi. 1982;14: Crlsen E, Andersson AM, Petersen JH, Skkkeek NE. Histor of ferile illness nd vrition in semen qulit. Hum Reprod. 2003;10: Wettemnn RP, Wells ME, Johnson RK. Reproductive chrcteristics of ors during nd fter exposure to incresed mient temperture. J Anim Sci. 1979;49: Almeid FFL, Lel MC, Frnc LR. Testis morphometr, durtion of spermtogenesis, nd spermtogenic efficienc in the wild or (Sus scrof scrof). Biol Reprod. 2006;75: Journl of Swine Helth nd Production Volume 25, Numer 6 307