Surgical stress influences cytokine content in autologous conditioned serum

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1 s_s_nner Surgicl stress influences cytokine content in utologous conditioned serum C. T. FJORDBAKK*, G. M. JOHANSEN, A. C. LØVÅS, K. L. OPPEGÅRD nd A. K. STORSET Deprtment of Compnion Animl Clinicl Sciences, Norwegin School of Veterinry Science, Oslo, Norwy Deprtment of Food Sfety & Infection Biology, Norwegin School of Veterinry Science, Oslo, Norwy. Equine Veterinry Journl ISSN 5-6 DOI:./evj.77 *Correspondence emil: Received: 9.9.; Accepted: 5.. Summry Resons for performing study: No recommendtions hve een mde regrding the reltive timing of lood collection for utologous conditioned serum (ACS) preprtion nd surgicl procedures. Ojectives: ) To identify effects of surgicl stress on cytokine levels in ACS, ) identify hemtologicl mrkers for prediction of cytokine production in ACS nd ) investigte the necessity for specilised ACS continers when prepring cytokine-rich serum. Study design: Experimentl in vitro study. Methods: Blood ws drwn from 5 stllions dmitted for elective cstrtion preopertively nd h post opertively nd incuted in ACS continers nd plstic vcutiner tues contining Z Serum Clot Activtor. Concentrtions of interleukin (IL)- receptor gonist (IL-R), IL-, IL-β, tumour necrosis fctor (TNF)-α, insulin-like growth fctor (IGF)- nd trnsforming growth fctor (TGF)-β were determined in ll serum smples nd compred etween preprtion methods nd smpling time y ANOVA. Chnges in cytokine levels induced y incution, defined s delt cytokine, were clculted y sutrcting the seline levels from the levels in incuted smples. Bsed on post opertive serum myloid A (SAA), horses were grouped into mild, moderte nd mrked surgicl stress; delt cytokine levels in post opertive smples were compred etween these groups y ANOVA. Results: Delt IGF- ws significntly lower in post opertive smples compred with preopertive. Horses in the mrked surgicl stress group hd significntly lower delt IL-R nd delt TGF-β thn the moderte group nd significntly lower delt IGF- thn the mild group. No ssocition etween cytokine levels nd hemtology vriles were identified. Cytokine levels were comprle etween serum prepred in lood tues nd in specilised ACS continers. Conclusions: Surgicl stress influences the cytokine content in ACS. Useful predictors of cytokine production in ACS were not identified. Specilised ACS continers my not e necessry for preprtion of cytokine-rich serum. Keywords: horse; regenertive medicine; utologous conditioned serum Introduction In recent yers, iologicl therpy for joint disese hs gined considerle interest in oth humn nd veterinry medicine []. This term usully implies ny form of tretment tht exploits the ody s nturl ilities to fight disese; for exmple y utilising inflmmtory cytokine ntgonists. As interleukin (IL-) is considered one of the pivotl inflmmtory cytokines in osteorthritis [], the endogenous interleukin receptor ntgonist (IL-R) hs een focl point of reserch in iologicl therpy for joint disese in horses []. The potentil eneficil effects of this protein in disesed joints were demonstrted in model of experimentlly induced osteorthritis in horses, where intr-rticulr tretment with IL-R gene trnsfer reduced clinicl signs nd improved histologicl ppernce of the rticulr crtilge []. Autologous conditioned serum (ACS) represents prcticl lterntive for iologicl tretment of joint disese. Autologous conditioned serum refers to the cytokine-rich serum hrvested fter exposing circulting leucocytes to ctivting surfces, therey stimulting de novo synthesis of severl nti-inflmmtory cytokines nd growth fctors [,5]. Compred with unmnipulted horse lood, ACS contins incresed levels of nti-inflmmtory cytokines such s IL-R nd IL-, s well s the growth fctors trnsforming growth fctor (TGF)-β nd insulin-like growth fctor (IGF)- [6]. However, the exct composition of cytokines nd growth fctors in ACS is undetermined [] nd considerle inter-individul differences in cytokine levels hve een documented [6]. Also, s incution of equine whole lood in plstic vcutiner tues contining Z Serum Clot Activtor results in comprle levels of IL-R nd IL-, the need for expensive specilised ACS hrvesting systems my e questioned [6]. Intr-rticulr tretment with ACS resulted in improved clinicl signs such s reduced lmeness nd joint effusion in n experimentl model of crpl osteorthritis in horses [7]. However, histologicl ppernce of the rticulr crtilge ws not different from the untreted plceo group. A recent study investigting the effects of ACS on equine chondrocytes in vitro, found only miniml eneficil effect of ACS vs. unconditioned serum on chondrocyte metolism [8]. Despite ovious pucities in clinicl evidence, ACS is populr mong equine prctitioners nd is commonly used in joints unresponsive to other intr-rticulr remedies or s first-line tretment when cost is of no concern [9]. Prophylctic nd post rthroscopic use hve lso een reported []. No recommendtions hve een mde regrding the timing of lood collection for ACS preprtion in reltion to surgicl procedures such s rthroscopy. Elective surgicl procedures induce n cute-phse response in the horse quntifile s n increse in the cute-phse protein serum myloid A (SAA) peking pproximtely h post opertively []. As the cute-phse response interferes with leucocyte function [], ACS prepred from lood drwn post opertively my e inferior in content of cytokines nd growth fctors compred with ACS prepred from lood drwn preopertively. Therefore, the ojectives of the study were ) to identify potentil effects of surgicl stress on the content of cytokines nd growth fctors in ACS, ) identify potentil hemtologicl or inflmmtory mrkers for prediction of cytokine production in ACS nd ) investigte the necessity of specilised ACS continers when prepring cytokine-rich serum. Our hypotheses were tht ) cytokine nd growth fctor content of ACS prepred h post opertively would e lower compred with ACS prepred preopertively, ) cytokine levels in ACS re ssocited with leucocyte levels nd ) lood incution in plstic vcutiner tues contining Z Serum Clot Activtor results in comprle cytokine nd growth fctor levels s incution in specilised ACS systems contining medicl-grde glss eds. Mterils nd methods Horses Fifteen stllions (ge 8 yers, men. yers) dmitted to the hospitl for elective cstrtion or cryptorchidectomy were recruited to the study. These horses comprised 7 Stndrdreds, Norwegin/Swedish Coldlooded Trotters, Icelndic horses, one Pur Rz Espñol nd one Americn Qurter Horse. All stllions were considered to e helthy sed Equine Veterinry Journl 7 (5) 7 EVJ Ltd

2 C. T. Fjordkk et l. Surgicl stress influence cytokine content in utologous conditioned serum 5 IL-R IL-β IGF-.6.. Bse h ACS I ACS II Bse h ACS I ACS II Bse h ACS I ACS II IL-. TNF-α TGF-β Bse h ACS I ACS II Bse h ACS I ACS II Bse h ACS I ACS II Fig : Levels of cytokines nd growth fctors (men nd 95% confidence intervl) determined y commercilly ville ELISA tests in preopertive serum smples of 5 helthy stllions dmitted for elective cstrtion/cryptorchidectomy. Different letters indicte significnt differences (P<.5) etween the preprtion methods. ACS = utologous conditioned serum; IGF = insulin-like growth fctor; IL = interleukin; IL-R = IL- receptor gonist; TGF = trnsforming growth fctor; TNF = tumour necrosis fctor. on physicl exmintion nd preopertive hemtology nd iochemistry nlyses including SAA (reference rnge mg/l). Surgicl procedures Surgicl procedures were performed septiclly under generl nesthesi; normlly descended testes were removed through scrotl incisions wheres inguinlly or dominlly retined testes were removed using n inguinl pproch s previously descried []. Spermtic cords were ligted prior to emscultion nd primry closure of ll surgicl incisions ws performed. Penicillin (, iu/kg wt i.v.) nd flunixin meglumine (Findyne c,. mg/kg wt i.v.) were dministered periopertively. Smple collection nd preprtion Blood ws collected septiclly preopertively nd h post opertively from n indwelling jugulr vein ctheter (BD Seclon guge ctheter) d plced using stndrd hospitl routines. Preopertively, one Equine Veterinry Journl 7 (5) 7 EVJ Ltd plstic EDTA vcutiner tue ws sumitted for routine hemtology nlysis within h of smple collection. At oth smpling times, the reminder of the lood ws distriuted into different continers. One plstic vcutiner tue contining Z Serum Clot Activtor ws llowed to cogulte t room temperture for h prior to centrifugtion (5 g, min) nd serum collection (seline serum). One identicl plstic vcutiner tue contining Z Serum Clot Activtor nd ACS continers (ACS I: 6 ml polypropylene syringe; ACS II: ml polypropylene tue) e,f oth contining medicl-grde glss spheres were incuted t 7 C for h, prior to centrifugtion (5 g, min for the vcutiner tue nd 7 g, min for the ACS continers, respectively) nd serum collection ( h serum, ACS I nd ACS II, respectively). Serum from the ACS continers ws filtered through. μm filter (Sterifix. μm Luer Lock) g ; ll serum smples were stored in 5 ml cryovils t - C until nlysis. Smple nlyses Serum myloid A nlysis ws performed in pre- nd post opertive seline serum smples using n nti-saa coted ltex gglutintion

3 Surgicl stress influence cytokine content in utologous conditioned serum C. T. Fjordkk et l. 6 IL-R IL-β IGF Bse h ACS I ACS II Bse h ACS I ACS II Bse h ACS I ACS II IL-. TNF-α TGF-β Bse h ACS I ACS II Bse h ACS I ACS II Bse h ACS I ACS II Fig : Levels of cytokines nd growth fctors (men nd 95% confidence intervl) determined y commercilly ville ELISA tests in post opertive serum smples of 5 helthy stllions dmitted for elective cstrtion/cryptorchidectomy. Different letters indicte significnt differences (P<.5) etween the preprtion methods. ACS = utologous conditioned serum; IGF = insulin-like growth fctor; IL = interleukin; IL-R = IL- receptor gonist; TGF = trnsforming growth fctor; TNF = tumour necrosis fctor. photometric immunossy. Commercilly ville ELISA kits were used to determine serum concentrtion of cytokines nd growth fctors in seline serum, h serum, ACS I nd ACS II. Equine-specific kits for IL-R h, IL- h, IL-β i nd tumour necrosis fctor (TNF)-α h nd humn-specific kits for IGF- h nd TGF-β i were used; the ltter kits hve previously een vlidted for cross-rectivity in equine serum [,]. All ssys were run ccording to the mnufcturer s instructions. Dt nlysis The rithmetic men nd 95% confidence intervl (CI) ws clculted for ll vriles. Due to positive skewness, vriles were trnsformed using either log trnsformtion (preopertive SAA) or squre root trnsformtion (post opertive SAA, cytokines nd growth fctors) until norml distriution ws confirmed visully nd y the Shpiro Wilk test prior to further nlyses. All vriles including the IL-R:IL-β rtio were compred etween the time points (pre- nd post opertively) nd serum preprtions (seline serum, h serum, ACS I nd ACS II) y repeted mesures ANOVA with post hoc Tukey s HSD tests. The chnge in cytokine nd growth fctor levels induced y incution ws defined s delt cytokine nd clculted s follows: delt cytokine = incuted level - seline level; these vriles were compred pre- nd post opertively using ANOVA nlyses. Bsed on explortory dt nlysis, the vrile post opertive SAA ws grouped into ctegories nd defined s mild surgicl stress (< mg/l); moderte surgicl stress ( mg/l) nd mrked surgicl stress (> mg/l); delt cytokine in post opertive smples were susequently compred etween these groups using ANOVA nlyses with post hoc Tukey s HSD tests. Associtions etween the hemtology vriles plus SAA nd ech of the cytokines nd growth fctors in ll incuted preopertive smples were determined y Person correltion coefficients with 95% CIs nd y liner regression modelling. Vriles ssocited with ech of the cytokines/growth fctors in univrite regression nlysis with P. were included in multivrite nlysis; oth purposeful forwrd/ckwrd selection ws performed. For ll nlyses, P<.5 ws considered significnt; nlyses were performed using commercil sttisticl softwre j. Equine Veterinry Journl 7 (5) 7 EVJ Ltd

4 C. T. Fjordkk et l. Surgicl stress influence cytokine content in utologous conditioned serum ΔIL-R ΔIL-β ΔIGF- - Pre op Post op Pre op Post op Pre op Post op ΔIL- ΔTNF-α ΔTGF-β. Fig : Chnge in cytokine nd growth fctor content induced y incution (delt cytokine = incuted level - seline level) in pre- nd post opertive serum smples (men nd 95% confidence intervl). Different letters indicte significnt difference (P<.5) etween the timepoints. IGF = insulin-like growth fctor; IL = interleukin; IL-R = IL- receptor gonist; TGF = trnsforming growth fctor; TNF = tumour necrosis fctor Pre op Post op Pre op Post op Pre op Post op Results Routine cstrtion ws performed in stllions. Unilterl inguinl cryptorchidectomy ws performed in stllions nd unilterl dominl cryptorchidectomy performed in stllions. In the cryptorchid horses, the descended testis ws routinely removed through scrotl incision. All surgicl procedures went without complictions nd ll horses were dischrged from the hospitl on the first post opertive dy. Serum myloid A A significnt increse in SAA ws seen post opertively in ll horses (preopertive. mg/l, 95% CI.. mg/l; vs. post opertive 6.6 mg/l, 95% CI mg/l, respectively, P<.). Five horses (norml stllions only) hd post opertive SAA levels < mg/l (men 5 mg/l, 95% CI -5.8 to 5.9 mg/l) nd were susequently ctegorised in the mild surgicl stress group. Four horses (norml stllions only) hd post opertive SAA levels mg/l nd were ctegorised in the moderte surgicl stress group (men mg/l, 95% CI mg/l), wheres 6 horses ( cryptorchid nd norml stllions) hd post opertive SAA > mg/l nd were ctegorised in the mrked surgicl stress group (men. mg/l, 95% CI mg/l). Preopertive serum nlyses Due to kit vilility, ACS I ws used in 8 horses only. Levels of cytokines nd growth fctors determined in preopertive serum smples re displyed in Figure. Compred with seline serum incution resulted in significntly incresed levels of IL-R in ll serum preprtions (P<.) nd significntly incresed levels of IL-β in ACS I nd ACS II (P =. nd P =., respectively). Twenty-four h serum contined significntly more Equine Veterinry Journl 7 (5) 7 EVJ Ltd IGF- thn seline serum (P =.). There were no significnt differences etween the preprtion methods ( h serum, ACS I nd ACS II) in cytokine or growth fctor content or in IL-R:IL- β rtio. Post opertive serum nlyses Due to kit vilility, ACS I ws used in 8 horses only. Levels of cytokines nd growth fctors determined in post opertive serum smples re displyed in Figure. Bseline cytokine levels were comprle with preopertive levels except for IGF- which ws significntly lower (6.5 pg/ml, 95% CI -. to 5.5 pg/ml post opertively vs.. pg/ml, 95% CI 65.. pg/ml preopertively, P =.7). Compred with seline serum, incution of post opertive smples resulted in significntly incresed levels of IL-R nd IL-β in ll serum preprtions (P<. nd P<.5, respectively). Autologous conditioned serum I contined significntly more IGF- nd TGF-β thn seline serum (P =. for oth growth fctors, respectively). There were no significnt differences etween the serum preprtion methods ( h serum, ACS I nd ACS II) in cytokine or growth fctor content or in IL-R:IL- β rtio. Effect of surgicl stress Post opertively, incution resulted in significntly lower delt IGF- compred with preopertive smples (P<.; Fig ). Horses ctegorised in the mrked surgicl stress group hd significntly lower delt IL-R nd delt TGF-β vs. horses ctegorised in the moderte surgicl stress group (P =. nd P =.9, respectively, Fig ) nd significntly lower delt IGF- vs. horses ctegorised in the mild surgicl stress group (P =.9). Considerle interindividul differences in cytokine levels were found in ll serum preprtions. No ssocition etween cytokine levels nd hemtology vriles or post opertive SAA were identified. A strong 5

5 Surgicl stress influence cytokine content in utologous conditioned serum C. T. Fjordkk et l ΔIL-R < ΔIL-β 8 6 > - < ΔIGF > -. < > 5 ΔIL- ΔTNF-α ΔTGF-β < -. > < - > < > Fig : Dimond plot (men with 95% confidence intervl) of delt cytokine determined in post opertive serum smples fter grouping the horses into ctegories sed on post opertive serum myloid A (SAA) levels; mild surgicl stress = post opertive SAA < mg/l; moderte surgicl stress = post opertive SAA mg/l; mrked surgicl stress = post opertive SAA > mg/l. Different letters indicte significnt differences (P<.5) etween groups. IGF = insulin-like growth fctor; IL = interleukin; IL-R = IL- receptor gonist; TGF = trnsforming growth fctor; TNF = tumour necrosis fctor. positive correltion ws found etween thromocyte count nd the level of TGF-β in ll preopertive serum smples (r =.79; 95% CI.66.88). This reltionship ws confirmed using liner regression modeling, where 6% of the vrince in TGF-β level ws ccounted for y thromocyte count. The level of TGF-β ws found to increse with 7 pg/ml for every 9 /l increse in thromocyte count nd the model showed significnt fit (P<.). Discussion Elective surgery resulted in significnt lower seline serum content of IGF- nd significntly reduced de novo synthesis of IGF- in incuted serum smples. The finding of reduced circulting level of IGF- post opertively ws not surprising, s similr findings hve een reported during cute-phse responses in mn [5]. However, the reduced de novo synthesis of IGF- oserved in incuted serum smples post opertively hs, to the uthors knowledge, not previously een descried nd demonstrtes tht surgicl stress my influence constituents of ACS. This ws lso evidenced y the significnt decrese in delt IL-R, delt IGF- nd delt TGF-β seen in horses with post opertive SAA > mg/ml vs. horses with lower post opertive SAA. A proposed mechnism of SAA is ltertions in leucocyte recruitment nd function [] tht might explin these findings. However, the exct physiologicl effects of SAA re poorly understood s contrdicting effects regrding leucocyte function hve een descried []. Acute-phse protein levels increse with incresing tissue dmge [6], which formed the sis for ctegorising the horses in the current study into groups of mild, moderte nd mrked surgicl stress. Surgicl stress level corresponded with the cstrtion method performed; not surprisingly, cryptorchidectomy elicited higher SAA levels thn regulr cstrtions. However, high SAA levels were seen in stllions routinely cstrted lso; this might reflect differences in tissue hndling nd surgicl technique. Surgeon effect ws not evluted s ll routine cstrtions were performed y finl yer veterinry students under supervision. Although considered minimlly invsive, elective rthroscopy lso induces significntly elevted post opertive SAA levels evident from the first to the fifth post opertive dy [6]. The results from the current study re therefore relevnt to clinicl prctice where ACS tretment is recommended fter joint surgery. In instnces of high post opertive SAA, postponing lood collection for ACS preprtion until SAA is within norml rnge my optimise ACS qulity. The considerle interindividul differences in cytokine levels in ACS identified in the current study nd s reported y others [6] proly contriuted to our filure in predicting cytokine levels in incuted smples. Assuming tht the clinicl effects of ACS re due to the nti-inflmmtory cytokines nd/or growth fctors investigted in the current study, these results should rise some concern when using ACS in clinicl prctice s to dte there is no method of identifying nimls with poor de novo synthesis of these proteins in ACS nd which susequently will respond poorly to tretment. Determintion of cytokine content in ACS y ELISA-nlyses prior to tretment could provide this informtion; however, such n rrngement would e imprcticl nd cost prohiitive in most instnces. Serum from lood incuted in plstic vcutiner tues contining Z Serum Clot Activtor contined similr levels of the cytokines nd growth fctors investigted s serum incuted in specilised ACS continers. These results corroorte those of previous study [6] nd indicte tht cytokine production my e consequence of whole lood incution rther thn n effect of specilised ACS continers. One of the ACS continers used in the current study is mrketed for humn use wheres the other ACS system is prototype continer not yet commercilly ville. Both continers re, however, designed using the sme principles s ACS systems mrketed for use in equine prctice. Significnt differences in cytokine production etween commercilly ville ACS systems hve previously een demonstrted [6]. There were only minor differences etween ACS I nd ACS II in the current study nd the clinicl relevnce of these my e questioned. However, the purpose of our study ws not to compre the efficcy of these products nd we do not recommend using products not mrketed for veterinry use in clinicl prctice. 6 Equine Veterinry Journl 7 (5) 7 EVJ Ltd

6 C. T. Fjordkk et l. Surgicl stress influence cytokine content in utologous conditioned serum In conclusion, lood collection for ACS preprtion during n cute-phse response my influence the resultnt ACS qulity nd should e done with cution. Specilised ACS continers my not e necessry for preprtion of cytokine-rich serum. Authors declrtion of interests No competing interests hve een declred. Ethicl niml reserch Owners gve informed consent for their horses inclusion in the study. All procedures were pproved y the Norwegin School of Veterinry Science nd in ccordnce with ntionl legisltion concerning ethicl niml reserch (FOR ). Sources of funding The Swedish/Norwegin Foundtion for Equine Reserch, the Norwegin School of Veterinry Science, nd VetLine Bio AB. Authorship C.T. Fjordkk contriuted to study design, study execution, dt interprettion nd preprtion of the mnuscript. A.K. Storset contriuted to study design, dt interprettion, nd preprtion of the mnuscript. A.C. Løvås contriuted to study execution, dt nlysis, dt interprettion nd preprtion of the mnuscript. K.L. Oppegård contriuted to study execution, dt nlysis nd dt interprettion nd preprtion of the mnuscript. G.M. Johnsen contriuted to dt nlysis nd dt interprettion. All uthors hve given finl pprovl of the mnuscript. Mnufcturers ddresses Greiner Bio-One Ltd, Stonehouse, UK. Actvis; Oslo, Norwy. c MSD Animl Helth; Bergen, Norwy. d CNC Medicl Devices, Mumi, Indi. e Onoccomed; Plsmconcept AG, Köln, Germny. f Em-Cyte Corportion, Fort Myers, Florid, USA. g Brun, Meslungen, Germny. h R&D Systems Europe Ltd; Aingdon, Oxfordshire, UK. i Kingfisher Biotech; St. Pul, Minnesot, USA. j Stt Corp, College Sttion, Texs, USA. References. Textor, J. () Autologous iologic tretment for equine musculoskeletl injuries: pltelet-rich plsm nd IL- receptor ntgonist protein. Vet. Clin. Equine 7, Todhunter, P.G., Kincid, S.A., Todhunter, R.J., Kmmermnn, J.R., Johnstone, B., Bird, A.N., Hnson, R.R., Wright, J.M., Lin, H.C. nd Purohit, R.C. (996) Immunohistochemicl nlysis of n equine model of synovitis-induced rthritis. Am. J. Vet. Res. 57, Frisie, D.D., Ghivizzni, S.C., Roins, P.D., Evns, C.H. nd McIlwrith, C.W. () Tretment of experimentl equine osteorthritis y in vivo delivery of the equine interleukin- receptor ntgonist gene. Gene Ther. 9, -.. Hnnum, C.H., Wilcox, C.J., Arend, W.P., Joslin, F.G., Dripps, D.J., Heimdl, P.L., Armes, L.G., Sommer, A., Eisenerg, S.P. nd Thompson, R.C. (99) Interleukin- receptor ntgonist ctivity of humn interleukin- inhiitor. Nture, Meijer, H., Reinecke, J., Becker, C., Tholen, G. nd Wehling, P. () The production of nti-inflmmtory cytokines in whole lood y physico-chemicl induction. Inflmm. Res. 5, Hrh, T.H., Doremus, K.M., McIlwrith, C.W. nd Frisie, D.D. () Autologous conditioned serum: the comprtive cytokine profiles of two commercil methods (IRAP nd IRAP II) using equine lood. Equine Vet. J., Frisie, D.D., Kwck, C.E., Werpy, N.M., Prk, R.D. nd McIlwrith, C.W. (7) Clinicl, iochemicl, nd histologic effects of intr-rticulr dministrtion of utologous conditioned serum in horses with experimentlly induced osteorthritis. Am. Vet. J. Res. 68, Crlson, E.R., Stewrt, A.S., Crlson, K.L., Durgm, S.S. nd Pondenis, H.C. () Effects of serum nd utologous conditioned serum on equine rticulr chondrocytes treted with interleukin- β. Am. J. Vet. Res. 7, Ferris, D.J., Frisie, D.D., McIlwrith, C.W. nd Kwck, C.E. () Current joint therpy usge in equine prctice: survey of veterinrins 9. Equine Vet. J., Pollock, P.J., Prendergst, M., Schumcher, J. nd Bellenger, C.R. (5) Effects of surgery on the cute phse response in cliniclly norml nd disesed horses. Vet. Rec. 56, Crismn, M.V., Scrrtt, W.K. nd Zimmermn, K.L. (8) Blood proteins nd inflmmtion in the horse. Vet. Clin. Equine, Schumcher, J. () Cryptorchid cstrtion. In: Equine Surgery, th edn., Eds: J.A. Auer nd J.A. Stick, Elsevier Sunders, St. Louis, Missouri. pp De Kock, S.S., Rodgers, J.P., Swnepoel, B.C. nd Guthrie, A.J. () Administrtion of ovine, porcine nd equine growth hormone to the horse: effect of insulin-like growth fctor-i nd selected IGF inding proteins. J. Endocrinol. 7, Dhlgren, L.A., Mohmmed, H.O. nd Nixon, A.J. (5) Temporl expression of growth fctors nd mtrix molecules in heling tendon lesions. J. Orthop. Res., Nedelec, B., de Oliveir, A. nd Grrel, D.R. () Acute phse modultion of systemic insulin-like growth fctor- nd its inding proteins fter mjor urn injuries. Crit. Cre Med., Jcosen, S., Nielsen, J.V., Kjelgrd-Hnsen, M., Toeloell, T., Fjeldorg, J., Hlling-Thomsen, M., Mrtinussen, T. nd Thoefner, M.B. (9) Acute phse response to surgery of vrying intensity in horses: preliminry study. Vet. Surg. 8, Equine Veterinry Journl 7 (5) 7 EVJ Ltd 7