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CSIRO PUBLISHING www.pulish.siro.u/journls/rfd Reprodution, Fertility nd Development, 2011, 23, 285 296 Effets of ntioxidnt vitmins on neworn nd plentl trits in gesttions t high ltitude: omprtive study in high nd low ltitude ntive sheep Vítor H. Prrguez A,C,E, Miljenko Atlgih A, Osr Arned B, Crlos Grí B, Andrés Muñoz D, Móni De los Reyes A nd Bessie Urquiet A A Fulty of Veterinry Sienes, University of Chile, Csill 2, Correo 15, L Grnj, Sntigo, Chile. B Fulty of Mediine, University of Chile, Csill 2, Correo 15, L Grnj, Sntigo, Chile. C Interntionl Center for Anden Studies (INCAS), Universty of Chile, Csill 2, Correo 15, L Grnj, Sntigo, Chile. D Deprtment of Genetis, Gregor Mendel Blg., Cmpus Rnles, University of Córdo, 14071 Córdo, Spin. E Corresponding uthor. Emil: vprrgu@uhile.l Astrt. The present study evluted the hypothesis tht the effets of hypoxi on sheep pregnnies t high ltitude (HA) re medited y oxidtive stress nd tht ntioxidnt vitmins my prevent these effets. Both HA ntive nd newomer ewes were mintined t n ltitude of 3589 m during mting nd pregnny. Control low ltitude (LA) ntive ewes were mintined t se level. Hlf of eh group reeived dily orl supplements of vitmins C (500 mg) nd E (350 IU) during mting nd gesttion. Ner term, mternl plsm vitmin levels nd oxidtive stress iomrkers were mesured. At delivery, lms were weighed nd mesured, nd plents were reovered for mrosopi nd mirosopi evlution. Vitmin onentrtions in supplemented ewes were two- or threefold greter thn in non-supplemented ewes. Plsm ronyls nd mlondildehyde in non-supplemented ewes were onsistent with stte of oxidtive stress, whih ws prevented y vitmin supplementtion. Vitmin supplementtion inresed lm irthweight nd otyledon numer in oth HA ntive nd newomer ewes, lthough plentl weight nd otyledon surfe were diminished. Plents from vitmin-supplemented HA ewes were similr to those from ewes t se level, mking these plentl trits (weight, numer nd dimeter of otyledons) similr to those from ewes t se level. Vitmin supplementtion hd no effet on LA pregnnies. In onlusion, supplementtion with vitmins C nd E during pregnny t HA prevents oxidtive stress, improving pregnny outomes. Additionl keywords: hypoxi, ovine, oxidtive stress, pregnny. Introdution At high ltitude (HA), hypori hypoxi hs signifint effets on severl hrteristis of the ovine reprodutive proess. Reently, we demonstrted sustntil differenes in the mgnitude of the effets of HA on pregnny nd neworn trits etween ewes tht hve lived t HA for severl genertions, those ntive to low ltitude (LA) tht hd een exposed to HA only from the eginning of the pregnny nd LA ewes tht gestted t se level. These effets inluded lower irthweight t HA (y,23% in HA ntive sheep nd 29% in LA ntive sheep) (Prrguez et l. 2005), inresed otyledon dimeter in oth HA nd LA ntive ewes (y 70 nd 26%, respetively), inresed plentl weight (y 41%) nd redution in the numer of otyledons (y 25%) in HA ntive ewes, nd tendeny for inresed plentl weight (,8%) nd redution in the numer of otyledons (,4%) in LA ntive ewes. In ddition, the otyledon runle ontt surfe re ws inresed y 128 nd 36% in HA nd LA ntive ewes, respetively, wheres the otyledon surfe re oupied y the vsulture ws inresed y 135 nd 75% in HA nd LA ntive ewes, respetively (Prrguez et l. 2006). Humn pregnny is lso ffeted y HA, with low irthweight one of the most ommonly oserved effets (Yip 1987; Jensen nd Moore 1997; Mortol et l. 2000; Giussni et l. 2001; Keyes et l. 2003; Moore et l. 2004; Hrtinger et l. 2006). In lssi study performed in Colordo (USA), it ws estlished tht irthweight dereses y 102 g per 1000 m ltitude nd tht the perentge of neworns with low irthweight inreses y 54% t ltitudes etween 2744 nd 3350 m ove se level (.s.l.; Jensen nd Moore 1997). Pregnnies t 4300 m.s.l. show the effets of ltitude on fetl growth from 25 weeks gesttion onwrds; 21% of fetuses hd n estimted Ó CSIRO 2011 10.1071/RD10016 1031-3613/11/020285

286 Reprodution, Fertility nd Development V. H. Prrguez et l. odyweight elow the fifth perentile of the popultion t se level, nd irthweight ws 12% less thn irthweight t se level (Krmpl et l. 2000). The inidene of low irthweight t 3600 m.s.l. ws lmost threefold greter thn tht oserved t LA (Keyes et l. 2003). Humn pregnny t HA is lso hrterised y inresed presenttion of symmetril fetl growth, fetl distress, preterm lour, premture rupture of the memrnes, hypertension, pre-elmpsi nd neworn respirtory distress (Krmpl et l. 2000; Giussni et l. 2001; Keyes et l. 2003; Moore et l. 2004). Consistent with our oservtions in sheep, ies from mothers who were long-term residents t HA exhiited only 30 70% of the irthweight redution oserved in those ies from mothers who hd reently rrived in n HA environment (Moore et l. 2001, 2004; Hrtinger et l. 2006). Severl studies in pregnnt women t HA hve shown hnges in plentl morphology nd, lthough humn nd ovine plents differ in struture nd development, n inrese in villous vsulristion nd plentl weight ppers to e ommon feture, t lest in HA ntive popultions (Zmudio 2003). However, reent study in HA newomer pregnnt women t different ltitudes reported onserved vsulrity nd redution in plentl weight (Tissot vn Ptot et l. 2009). Exposure to nturl or rtifiil hypori hypoxi environment onstitutes physiologil insult tht leds to inresed ellulr prodution of retive oxygen speies (ROS), whih my result in stte of oxidtive stress (Jonny et l. 2001; Møller et l. 2001; Askew 2002; Mglhães et l. 2004, 2005). Emerging evidene suggests lose ssoition etween ute mountin sikness nd oxidtive stress (Biley et l. 2001; Arned et l. 2005). Furthermore, lthough pregnny ould e defined s stte of oxidtive stress (Mytt nd Cui 2004), pthologil pregnnies, suh s those omplited y preelmpsi, present severl-fold inrese in oxidtive stress iomrkers (Khlil nd Grnger 2002; Mytt nd Cui 2004); the inidene of this pthology is pproximtely fivefold higher t HA thn t LA (Plmer et l. 1999; Keyes et l. 2003). Antioxidnt vitmin therpy hs een shown to redue hypoxiindued oxidtive dmge (Ilvzhgn et l. 2001; Mglhães et l. 2005) nd help prevent ute mountin sikness (Biley nd Dvies 2001) nd, lthough there is no onsensus on this regrd, there is onsiderle evidene showing it to e enefiil in preventing pre-elmpsi (Chppell et l. 1999, 2002; Rodrigo et l. 2005; Rumiris et l. 2006). In the present study, we explored the hypothesis tht the effets of HA hypori hypoxi on sheep pregnnies re medited y oxidtive stress y exmining the ility of ntioxidnt vitmin therpy to improve plentl hrteristis nd neworn odyweight in HA nd LA ntive sheep. Mterils nd methods The present study ws performed in ordne with the Interntionl Guiding Priniples for Biomedil Reserh Involving Animls (Counil for Interntionl Orgnistion of Medil Sienes, World Helth Orgniztion) nd ws pproved y the Bioethis Review Committee of the Fulty of Veterinry Sienes, University of Chile, s well s y the Bioethis Committee of Chile s Ntionl Ageny for Sientifi Reserh (Comisión Nionl de Investigión Científi y Tenológi). Animl mngement Forty time-mted (known gesttionl ge) singleton pregnnt Creole ewes (Chilen mix reed developed from the Churr nd Mnheg Spnish reeds), whih hd history of two previous pregnnies nd norml prturitions, were used in the HA experiment. Twenty ewes were HA ntive (3500 4000 m.s.l.). These nimls were desendnts of the sheep herds introdued to the HA re y the Spnird settlers nd hve thus een dpted to hypori hypoxi for over 500 genertions. The other 20 ewes were LA ntive (o500 m.s.l.). Thirty dys efore mting, the LA ewes were tken to our experimentl sttion t HA (Interntionl Center for Anden Studies (INCAS), University of Chile; 3589 m.s.l., rometri pressure (P) ¼ 667 kp), where they joined the HA group. Animls were provided with lflf hy dily (,2kgdy 1 ; dry mtter (DM) ¼ 90.2%, metolisle enegery (ME) ¼ 9.9 MJ kg 1, rude protein (CP) ¼ 13.5%) nd fresh wter d liitum. The food supply ws lulted to stisfy dily ovine requirements in lte gesttion (Ntionl Reserh Counil 1985). Ten ewes eh from the HA nd LA groups were hosen t rndom to reeive ntioxidnt vitmin supplementtion, dministered dily from 30 dys efore mting until the end of the experiment. To hieve supplementtion, 0.5 kg lflf supplemented with 500 mg vitmin C nd 350 IU vitmin E per niml ws dministered in individul feeding troughs erly eh morning. After the onsumption of this rtion, the remining lflf ws given to the ewes. As ontrol, n dditionl LA group of 10 Creole ewes kept t LA were evluted. Hlf the LA ewes lso reeived ntioxidnt vitmin supplementtion. These nimls were mintined using sme mngement nd feeding protools s t HA. Thus, totl of six groups of pregnnt ewes were studied: highlnd origin, HA pregnny with (HHv) or without (HH) vitmin supplementtion; lowlnd origin HA pregnny with (LHv) or without (LH) vitmin supplementtion; nd lowlnd origin LA pregnny with (LLv) or without (LL) vitmin supplementtion. Mting t HA nd LA ws hieved y introduing two proven mles into the femles pens. Mles were lternted dily etween vitmin-treted nd non-treted femles. The hests of the rms were pinted dily with mixture of vegetle oil nd oloured powder, so tht ny ewe in oestrous tht ws mounted y mle ould e deteted y oservtion of oloured rump. Ewe odyweight t the eginning of the mting period did not differ mong the groups. The length of the mting period ws 35 dys. Twenty-five dys fter detetion of mting, the ewes were sreened for pregnny y trnsretl ultrsound. Mesurements in mternl lood At 110 120 dys gesttion, one lood smple (5 ml) ws otined from eh pregnnt ewe from the left jugulr vein to enle determintion of plsm onentrtions of vitmins C nd E. In ddition, these lood smples were used to determine plsm onentrtions of ronyl groups (CO) nd mlondildehyde (MDA) to estimte protein nd lipid oxidtive dmge, respetively. Totl plsm ntioxidnt pity (TAC)

Vitmins C nd E in sheep pregnny t high ltitude Reprodution, Fertility nd Development 287 ws lso ssessed to omplete the evlution of oxidtive stress (Griffiths et l. 2002). Vitmin onentrtions were mesured y high-performne liquid hromtogrphy (HPLC; Wters Alline 2695; Wters, Milford, MA, USA). Vitmin C ws mesured y HPLC with mperometri detetion s desried y Phl nd Kissinger (1979). Briefly, plsm smples were diluted 10-fold in ultrpure wter, homogenised nd filtered through 0.45-mm pore memrne. The hromtogrphi onditions were s follows: nlytil olumn 250 4.6 mm (Symetry Shield C18, 5 mm; Wters); moile phse (metphosphori id 4 g L 1 ultrpure wter) flow rte 1 ml min 1 ; injetion volume 10 ml; nd working temperture 308C. The detetor ws equipped with glssy ron eletrode operted t 800 mv nd n Ag/AgCl referene eletrode. Vitmin E ws mesured y HPLC with fluoresene detetion ording to the methods of Zho et l. (2004). For vitmin E extrtion, plsm smples (100 ml) were diluted 1 : 1 : 6 in ethnol nd dihloromethne, respetively, then entrifuged t 10 000g for 10 min t room temperture nd the pellet ws dried under nitrogen tmosphere. The dried residue ws reonstituted in methnol (200 ml). The hromtogrphi onditions were s follows: nlytil olumn 150 4.6 mm (Symetry C18, 5 mm; Wters); moile phse (methnol HPLC 100%) flow rte 1 ml min 1 ; injetion volume 20 ml; nd working temperture 308C. The spetrofluorimeter ws set t exittion nd emission wvelengths of 290 nd 330 nm, respetively. Plsm CO ws determined y spetrophotometri method, following the protool desried y Reznik nd Pker (1994). Plsm MDA onentrtions were mesured y HPLC with fluoresene detetion using the thiorituri id ssy desried y Lästrd et l. (2002). TAC ws ssessed in plsm using the totl rdil-trpping ntioxidnt prmeter tehnique desried y Wyner et l. (1985) nd modified y Lissi et l. (1992). Immeditely fter venous lood smples hd een olleted, one rteril lood smple ws otined per niml for mesurement of lood gses. The rteril lood smple ws drwn y punture of the left rotid rtery using sterile, heprinised 1-mL syringe. Mesurements of P O 2, P CO 2, hemtorit (Ht), hemogloin (H), H oxygen sturtion (S H ), nd ph were performed immeditely in n IL Synthesis 25 gs nlyser (Instrumenttion Lortory, Lexington, MA, USA), lirted to lol tmospheri pressure nd ovine temperture. Neworn mesurements Immeditely fter irth, neworn lms were weighed on n eletroni sle (Simplex 150, Shnghi Yohu Weighing System, Shnghi, Chin). In ddition, iprietl dimeter (BPD), dominl dimeter (AD) nd thorx height (TH) were determined mnully using lipers. BPD ws mesured s the gretest distne etween the prietl ones; AD ws mesured trnsverse to the longitudinl plne, immeditely rostrl to the lst ri; nd TH ws mesured s the vertil distne etween the sternum nd the spinl olumn t the level of the elows. Plentl reovery nd proessing From Dy 140 of pregnny onwrds, nimls were oserved frequently to detet externl nd ehviourl signs of prturition. Immeditely fter delivery, nturl expulsion of the plents, inluding memrnes, ws wited. Reovered plents were rinsed with physiologil uffered sline, pled in plsti olnder for 1 min to drin nd weighed on n eletroni sle (Exell KS-500, Mnufturs Triunfo, Sntigo, Chile). Plents were then fixed for 48 h in 10% uffered formlin efore the numer of otyledons ws determined. In ddition, otyledon dimeter ws lulted nd n estimte of the surfe re of otyledon runle ontts ws mde. Cotyledon dimeter ws determined y verging the distne of the longest xis nd its trnsverse xis t the entre of the struture. Cotyledon surfe re ws estimted y ssuming irumferentil surfe, s desried y Prrguez et l. (2006). Plentl surfe re for eh niml ws lulted y dding up individul otyledon runle ontt surfe res. An estimte of ross-setionl plentl vsulr re ws otined s follows. After n dditionl overnight fixtion period in 10% uffered formlin, three otyledons per niml were proessed for light mirosopi histology. Seril 6-mm setions from the middle zone through the trnsverse plne were otined nd stined with hemtoxylin eosin. Mesurement of the otyledon re oupied y the vsulture ws determined in four setions nd four mirosopi fields of 151 386 mm 2 per setion using n Elipse E-600 mirosope (Nikon, Tokyo, Jpn). Imges were ptured with 100 ugmenttion using digitl video mer (Cool Snp-Pro; Medi Cyernetis, Bethesd, MD, USA) oupled to the mirosope. In eh imge, ross-setionl otyledon re oupied y the vsulture s frtion of the otyledon tissue re ws lulted using Imge Pro-Plus morphometri nlysis softwre (Medi Cyernetis). This vlue ws then multiplied y the plentl surfe re for eh niml, otining vlue for ross-setionl plentl vsulr re per niml. These vlues were verged to otin ross-setionl plentl vsulr re per group. In this nlysis, medium to lrge vessels were minly onsidered euse most of the mirovessels hd ollpsed or were destroyed t the time of delivery. Sttistil nlyses Dt were ompred y nlysis of vrine using the generl liner model (GLM) proedure in SAS (SAS, Cry, NC, USA). Comprisons were mde using two sttistil models. The first model ws used for gesttions t HA nd onsisted of ftoril model with two ftors: origin (ple where the ewes tht gve irth were orn) nd the vitmin supplementtion (presene or sene). The seond model took into onsidertion the previous two ftors in ddition to the ple in whih gesttions took ple. Intertions mong ftors were lso nlysed. When signifint differenes were found, Dunn s test ws used to determine signifint differenes mong groups. P 0.05 ws onsidered signifint. Results re expressed s the men s.e.m. Results No effet of sire ws found on mternl or neworn lm vriles.

288 Reprodution, Fertility nd Development V. H. Prrguez et l. Mternl vriles All pregnnies resulted in norml prturition nd helthy neworns. Mternl odyweight on Dy 140.2 0.7 of gesttion in the HH, HHv, LH, LHv, LL nd LLv groups ws 46.7 1.1, 47.5 1.7, 47.2 1.3, 46.4 1.5, 48.3 1.1 nd 47.9 1.5 kg, respetively. There were no signifint differenes mong the six groups. Plsm onentrtions of vitmins C nd E re shown in Fig. 1,. Antioxidnt vitmin supplementtion signifintly inresed plsm onentrtions of oth vitmins C nd E. Plsm vitmin C levels in treted ewes were pproximtely twofold greter thn levels in untreted ewes (Po0.05). No effet of origin or gesttion ltitude on plsm vitmin C levels ws found, lthough signifint intertion etween origin nd vitmin tretment ws oserved (i.e. levels in the HH group were higher thn those in the LH group; Po0.03). No intertion ws oserved when the ltitude t whih the pregnny took ple ws inluded in the sttistil model. This is due to the ft plsm vitmin C levels in the LL group were the sme s in the HH nd LH groups. Compred with levels in untreted ewes t the sme ltitude, plsm vitmin E levels were twofold greter in the vitmin-supplemented HA gesttion groups nd threefold greter in the vitmin-supplemented LA gesttion group (Po0.05). These differenes ppered to e due to intertions () Cronyl groups (nmol mg 1 prot.) 4.5 3.0 1.5 0.0 () Plsm vitmin C (µg ml 1 ) 5 4 3 2 1 () MDA (µm) 0.8 0.6 0.4 0.2 0 0.0 () Plsm vitmin E (µg ml 1 ) 3.5 3.0 2.5 2.0 1.5 1.0 0.5 () TAC (uri eq. mg dl 1 ) 1.5 1.0 0.5 0.0 0.0 Fig. 1. Plsm onentrtions of vitmins C () nd E () in pregnnt ewes t high ltitude either supplemented dily with vitmins C nd E (HHv, high ltitude ntives; LHv, low ltitude ntives) or not (HH, high ltitude ntives; LH, low ltitude ntives). LLv nd LL, ontrol pregnnies t low ltitude with nd without vitmin supplementtion, respetively. Columns with different letters differ signifintly (Po0.05, Dunn s test). Dt re presented s men s.e.m. Fig. 2. Plsm onentrtions of ronyl groups () nd mlondildehyde (), s well s totl ntioxidnt pity () in pregnnt ewes t high ltitude either supplemented dily with vitmins C nd E (HHv, high ltitude ntives; LHv, low ltitude ntives) or not (HH, high ltitude ntives; LH, low ltitude ntives). LLv nd LL, ontrol pregnnies t low ltitude with nd without vitmin supplementtion, respetively. Columns with different letters differ signifintly (Po0.05, Dunn s test). Dt re presented s men s.e.m.

Vitmins C nd E in sheep pregnny t high ltitude Reprodution, Fertility nd Development 289 etween origin, ltitude of gesttion nd vitmin E dministrtion (Po0.01). In pregnnies developed t LA, no signifint effet of vitmin dministrtion ws oserved on vriles of oxidtive sttus (Fig. 2) or on lood gses (Tle 1). Mternl plsm onentrtions of iomrkers of oxidtive dmge re shown in Fig. 2,. Plsm CO nd MDA levels were higher in HA pregnnies (Po0.05). However, vitmin supplementtion signifintly ttenuted the inreses in iomrkers of oxidtive dmge, with vlues in vitmin-treted HA ewes similr to those seen in oth vitmin-treted nd untreted LA ewes. TAC levels (mesured s mg ml 1 uri id equivlents) were the lowest in the LH nd LL groups (Po0.05). Vitmin dministrtion inresed TAC levels in LHv ewes (Po0.05; Fig. 2). No differenes were deteted when ompring origin nd ltitude of pregnny. Plsm CO nd TAC levels showed signifint intertions etween origin nd vitmin dministrtion (for plsm CO, levels in the LH group were higher thn those in the HH group, while for TAC, levels in the HH were higher in those in the LH group; Po0.01), s well s etween these ftors nd the ltitude where gesttion took ple (Po0.004). Arteril lood gs mesurements otined ner term re given in Tle 1. A hypoxemi stte ws oserved in ll HA groups. However, P O 2 levels were higher in oth HA vitmintreted groups (Po0.05). P CO 2 levels were lower in ewes gestting t HA (Po0.05) nd there were no signifint differenes mong the HA three groups; tht is, vitmin supplementtion hd no effet on plsm P CO 2 levels. Both Ht nd H onentrtions were higher in pregnnies t HA (Po0.05). Vitmin supplementtion tended to inrese Ht in the ewes, lthough this differene did not reh sttistil signifine. H onentrtions were ffeted y n intertion etween origin nd vitmin dministrtion (Po0.05), wheres Ht onentrtions were ffeted y n intertion etween these ftors nd the ltitude t whih the pregnny took ple (Po0.05). Vlues of S H were lower in the HA groups, with vlues in the LH ewes eing the lowest (Po0.05). There were no signifint differenes in ph etween the six groups. Pregnny outomes Pregnny hrteristis nd outomes re listed in Tle 2. Vitmin supplementtion hd no signifint effet on pregnnies t LA. The length of gesttion ws longer in ntive HA Tle 1. Blood gses in high ltitude ntive nd newomer ewes gestting t high ltitude (110]120 dys gesttion) nd either supplemented or not with vitmins C nd E Dt re the men s.e.m. Different supersript letters within olumns indite signifint differenes etween groups (Po0.05, Dunn s test). Ht, hemtorit; H, hemogloin onentrtion; S H, hemogloin oxygen sturtion; HH, high ltitude (HA) ntive ewes tht gestted t HA without vitmin supplementtion; HHv, HA ntive ewes tht gestted t HA with vitmin supplementtion; LH, low ltitude (LA) ntive ewes tht gestted t HA without vitmin supplementtion; LHv, LA ntive ewes tht gestted t HA with vitmin supplementtion; LL, LA ntive ewes tht gestted t LA without vitmin supplementtion; LLv, LA ntive ewes tht gestted t LA with vitmin supplementtion Group P O 2 (mmhg) P CO 2 (mmhg) Ht (%) H (mg dl 1 ) S H (%) ph HH 53.0 1.2 23.8 0.9 32.2 0.7 12.5 0.5 82.6 2.8 7.510 HHv 60.4 1.3 25.8 0.9 33.3 0.7 12.9 1.1 85.9 3.9 7.458 LH 49.5 1.4 25.0 1.0 35.0 0.7 14.3 0.4 77.5 3.4 7.497 LHv 58.8 1.4 25.4 1.0 33.2 0.8 13.4 1.4 85.5 3.2 7.474 LL 95.9 1.6 39.2 1.1 28.3 0.8 d 10.5 0.6 96.8 2.0 7.482 LLv 98.3 1.6 40.3 1.2 30.5 0.9 d 10.9 0.7 98.2 2.2 7.495 Tle 2. Effets of vitmins C nd E on pregnny outomes in high nd low ltitude ntive ewes gestting t high ltitude Dt re the men s.e.m. Different supersript letters within olumns indite signifint differenes etween groups (Po0.05, Dunn s test). BPD, iprietl dimeter; TH, thorx height; AD, dominl dimeter; HH, high ltitude (HA) ntive ewes tht gestted t HA without vitmin supplementtion; HHv, HA ntive ewes tht gestted t HA with vitmin supplementtion; LH, low ltitude (LA) ntive ewes tht gestted t HA without vitmin supplementtion; LHv, LA ntive ewes tht gestted t HA with vitmin supplementtion; LL, LA ntive ewes tht gestted t LA without vitmin supplementtion; LLv, LA ntive ewes tht gestted t LA with vitmin supplementtion Group Length of gesttion (dys) Neworn weight (kg) Neworn BPD (m) Neworn TH (m) Neworn AD (m) HH 152.1 4.1 3.26 0.15 6.38 0.14 10.87 0.49 9.08 1.68 HHv 151.9 4.2 3.83 0.13 7.02 0.13 11.29 0.15 9.26 2.32 LH 145.0 7.6 2.98 0.14 6.29 0.15 11.23 0.53 8.74 1.60 LHv 150.6 4.5 3.60 0.15 6.66 0.15 11.00 0.14 8.69 1.11 LL 145.4 2.5 4.38 0.20 6.86 0.20 10.50 0.24 10.82 0.71 LLv 146.2 3.8 4.26 0.20 7.04 0.17 10.66 0.28 10.50 1.03

290 Reprodution, Fertility nd Development V. H. Prrguez et l. Tle 3. Effets of vitmins C nd E on plentl hrteristis in high nd low ltitude ntive ewes gestting t high ltitude Dt re the men s.e.m. Different supersript letters within olumns indite signifint differenes etween groups (Po0.05, Dunn s test). HH, high ltitude (HA) ntive ewes tht gestted t HA without vitmin supplementtion; HHv, HA ntive ewes tht gestted t HA with vitmin supplementtion; LH, low ltitude (LA) ntive ewes tht gestted t HA without vitmin supplementtion; LHv, LA ntive ewes tht gestted t HA with vitmin supplementtion; LL, LA ntive ewes tht gestted t LA without vitmin supplementtion; LLv, LA ntive ewes tht gestted t LA with vitmin supplementtion Group Plentl weight (g) No. otyledons Cotyledon dimeter (m) HH 380.9 16.7 55.2 4.5 2.4 0.5 HHv 305.2 15.9 83.4 4.3 1.4 0.3 LH 285.5 18.7 56.6 5.1 1.9 0.3 LHv 313.0 17.6 78.6 4.5 1.3 0.2 LL 308.5 23.6 82.0 6.1 1.5 0.3 LLv 323.4 24.6 79.4 9.8 1.4 0.3 ewes thn in ntive LA ewes (Po0.05), wheres gesttionl length ws intermedite in the LHv group. Neither vitmin supplementtion nor the ltitude t whih gesttion took ple ffeted gesttion length. The weight of neworn lms ws signifintly lower in HA pregnnies (Po0.05), with the lowest vlues reorded for lms orn to LH ewes. The weight of neworn lms ws inresed signifintly following vitmin supplementtion of HA groups ompred with the untreted HA groups (Po0.05). The gretest differene in the weight of neworn lms ws etween the LH nd LHv groups. The BPD of neworn lms ws shorter for the HA groups (Po0.05). Vitmin supplementtion inresed BPD to vlues oserved t in lms orn t LA. There were no signifint differenes in TH etween lms in the six groups. The AD of neworn lms ws only ffeted y the ltitude t whih gesttion took ple, with AD eing lowest in the LH nd LHv groups (Po0.05). () 300 200 100 0 () 40 Plentl surfe re (m 2 ) d Plentl hrteristis Vitmin supplementtion hd no effet on ny plentl hrteristis in pregnnies developed t LA (Tle 3; Fig. 3). Plentl weight, the numer of otyledons nd otyledon dimeter re listed in Tle 3. Plentl weight ws greter in HA ntive ewes (Po0.05). Vitmin supplementtion deresed plentl weight in this group, resulting in plentl weight tht ws similr to tht seen in LA ntive ewes. Intertions for this trit were oserved etween origin nd vitmin dministrtion (Po0.004), s well s etween these ftors nd gesttionl ltitude (Po0.008). The numer of otyledons ws deresed signifintly in pregnnies t HA (Po0.05). Vitmin supplementtion inresed the numer of otyledons in these groups to mth vlues otined in ewes t LA. An intertion (Po0.02) for this trit ws found when origin, gesttionl ltitude nd vitmin effets were inluded in the sttistil model. Cotyledon dimeter ws highest in HH ewes (Po0.05), followed y LH ewes. Vitmin supplementtion signifintly deresed otyledon dimeter in HA ewes (Po0.05) to vlues similr to those in LA pregnnies. Plentl surfe re nd ross-setionl plentl vsulr re re shown in Fig. 3,. The plentl surfe re ws inresed y oth HA origin nd HA gesttion (Po0.05). Thus, the HH group hd the gretest plentl surfe re, followed y the LH group. Vitmin supplementtion deresed the Cross-setionl plentl vsulr re (m 2 ) 30 20 10 0 Fig. 3. Plentl surfe re () nd ross-setionl plentl vsulr re () in pregnnt ewes t high ltitude either supplemented dily with vitmins C nd E (HHv, high ltitude ntives; LHv, low ltitude ntives) or not (HH, high ltitude ntives; LH, low ltitude ntives). LLv nd LL, ontrol pregnnies t low ltitude with nd without vitmin supplementtion, respetively. Columns with different letters differ signifintly (Po0.05, Dunn s test). Dt re presented s men s.e.m. plentl surfe re in oth groups of ewes t HA (Po0.05). Signifint intertions were found etween origin nd vitmin dministrtion (Po0.001), s well s etween these ftors nd the ltitude t whih gesttion took ple (Po0.001). Crosssetionl plentl vsulr re ws inresed in HA ntive

Vitmins C nd E in sheep pregnny t high ltitude Reprodution, Fertility nd Development 291 ewes (Po0.05), wheres vitmin tretment deresed this trit in ewes from the sme origin (Po0.05). Neither the ltitude t whih gesttion took ple nor vitmin supplementtion hd ny effet in LA ewes. Signifint intertions etween origin nd vitmin tretment were found for plentl vsulrity (Po0.01), s well s etween these ftors nd the gesttionl ltitude (Po0.009). Disussion To our knowledge, this is the first study to hrterise redox homeostsis nd the effet of ntioxidnt vitmin supplementtion on sheep pregnny t HA. The present study demonstrtes signifint effet of ntioxidnts on preventing oxidtive stress nd its undesirle onsequenes on fetl irthweight nd plentl hrteristis in hypoxi pregnnies, therey olishing most of the differenes etween pregnnies in nimls tht hve resided t HA for long or short time. Mternl lood vriles It is ommonly epted tht ruminnts synthesise their own vitmin C to stisfy dietry needs. However, under ertin stressful environmentl onditions, suh s t old tempertures, exogenous supplementtion is neessry to void the effets of vitmin C defiieny (Blk nd Hidiroglou 1996). The suess of orl dministrtion of vitmin C in inresing serum onentrtions in ruminnts is ontentious. It hs een reported tht lood levels of vitmin C did not inrese in ruminnts following orl dministrtion (Knight et l. 1941). However, more reent work hs demonstrted signifint inreses in vitmin C plsm onentrtions fter dministrtion in ows (Hidiroglou 1999). The results of the present study re in greement with this lter work, inditing tht dily orl dministrtion of vitmin C to pregnnt ewes over longer period (,150 dys) signifintly inreses the plsm levels of this vitmin. In the se of vitmin E, the inrese oserved in plsm levels in supplemented ewes in the present study ws generlly onsistent with tht reported previously (Oho et l. 1992; Njeru et l. 1994; Cpper et l. 2005). The inreses in plsm vitmin onentrtions in supplemented pregnnt ewes re of onsiderle importne in terms of vitmin trnsfer to the fetus. In ft, the results of fetl lm growth nd neworn outomes, s reported in the present study, re strongly supported y previous demonstrtion of diret reltionship etween mternl nd neworn plsm levels of vitmin E (Cpper et l. 2005), refleting trns-plentl trnsfer of this vitmin. Furthermore, evidene of erly trns-plentl trnsfer of vitmins C nd E hs een reported in humn pregnnies (Juniux et l. 2004). Tking into ount the ntomil nd developmentl differenes etween humn nd sheep plents, this informtion supports our results in neworn lms. A stte of oxidtive stress ws oserved in pregnnies t HA, s demonstrted y the signifint inrese in plsm onentrtions of CO nd MDA. This stte ws entuted in ewes new to the HA environment reltive to ntive HA ewes. The differene in the mgnitude of the hypoxi-indued oxidtive stress etween the two groups my e due to n dptive mehnism present in ntive HA ewes. A similr finding ws reported reently in ntive highlnder nd sojourner humns fter long exposure to HA (Sinh et l. 2009). Despite the CO nd MDA results in the present study, no onordnt hnges in TAC levels were oserved. This pprent inonsisteny hs een desried in other experiments t HA. For exmple, it hs een demonstrted in humns tht while working in hypori hypoxi environment, hnges in oxidtive stress iomrkers re not neessrily ssoited with hnges in TAC levels (Cho et l. 1999), proly due to vritions in the timing of the expression of the different iomrkers in plsm (Vij et l. 2005). Furthermore, it is of note tht supplementtion with vitmins C nd E in the present study signifintly lowered levels of iomrkers of oxidtive stress in HA pregnnies to vlues oserved in pregnnies t LA, nd signifintly inresed TAC levels in the plsm of ewes tht were newomers to the HA environment. These results demonstrte tht ntioxidnt therpy in gesttionl ewes t HA is effetive in preventing oxidtive stress. Despite the effets desried ove, n evident hypoxemi stte ws present in the ewes t HA, nd vlues for ll lood prmeters were similr to those reported previously (Prrguez et l. 2006). The dministrtion of ntioxidnt vitmins hd n interesting effet on the lood fetures involved in oxygen trnsport in HA nimls. Signifint inreses in P O 2 (13.9 18.8%) nd in S H (4.4 10.3%) were oserved, suggesting protetive effet of the vitmins on pulmonry funtion under HA hypoxi. It hs een demonstrted tht ute or hroni exposure to hypoxi, s well s oxidtive stress, indue lung injury nd diminish gs exhnge (Tuder et l. 2007; Prk et l. 2009). It hs lso een demonstrted tht the dministrtion of ntioxidnts in rts exposed to hypori hypoxi prevents hypoxi-indued pulmonry dmge nd results in signifintly deresed MDA levels in the lood s well s n inrese in totl ntioxidnt sttus (Uzun et l. 2006). Although we did not exmine lung struture nd funtion s prt of the present study, further work my explin the improvement in lood oxygen trnsport fetures oserved in our study; there my e preventtive effet of the vitmins on hypoxi/oxidtive stress-indued pulmonry dmge. The oserved differenes in lood oxygen pressure nd H sturtion in response to ntioxidnt vitmin therpy my provide etter oxygention to the fetl plentl unit nd thus e one of the ftors responsile for irthweight reovery. This effet would e inresed y the rightwrd shift in the H ffinity urve, s hs een demonstrted under hypori hypoxi onditions (West 2002). Pregnny outome In previous study of pregnnt sheep, we reported the effets of nturl HA hypori hypoxi on neworn outomes (Prrguez et l. 2005), whih were the result of slow fetl growth resulting in low irthweight. The present study onfirms our previous findings nd demonstrtes tht vitmins C nd E, supplied dily during pregnnies t HA, diminished the effet of hypori hypoxi on neworn irthweight nd BPD, resulting in vlues tht were omprle to those oserved t LA (Prrguez et l. 2005). From our point of view, one of the most

292 Reprodution, Fertility nd Development V. H. Prrguez et l. impressive effets of the ntioxidnt vitmin supplementtion in pregnnt ewes t HA, independent of the time of exposure to this environment, ws the signifint inrese (,20%) in lm odyweight t irth. This inrese ws speifi effet of the vitmin tretment t HA, euse vitmin supplementtion t LA hd no effet. Considering the improvement in the mortlity, moridity (Nsh et l. 1996) nd growth rtes of the lms s funtion of irthweight (Villete nd Theriez 1981), this vitmin supplementtion my hve signifint enefiil effet on the helth nd prodution of HA sheep livestok, with susequent eonomi enefits for HA sheep reeders. There is no informtion on the effet of ntioxidnt therpy on fetl growth in pregnnies under hypori hypoxi. However, different types of experiments hve demonstrted the enefiil effets of ntioxidnt therpy on severl physiologil responses to HA or hypoxi (Inn et l. 1995; Kelly nd Rihrdson 1996; Tn et l. 1996; Mohnrj et l. 1998; Purkysth et l. 1999; Biley nd Dvies 2001; Ilvzhgn et l. 2001; Miur et l. 2006). In ddition, severl studies hve shown tht, in pthologil gesttions in whih oxidtive stress is evident, s in preelmpsi (Chmy et l. 2006) or dieti pregnnies (Cedererg et l. 2001), vitmins C nd E re suessful in preventing oxidtive fetl dmge nd improving fetl outome (Cedererg et l. 2001; Chppell et l. 2002; Rodrigo et l. 2005). However, reent lrge multientre linil studies hve not een le to onfirm the enefits of vitmins C nd E in pregnnies t risk of pre-elmpsi (Poston et l. 2006; Villr et l. 2009; Xu et l. 2010). Moreover, n inresed rte of low irthweight ies (Poston et l. 2006) nd inresed risk of fetl loss, perintl deth or preterm lour hs een reported (Xu et l. 2010). However, there re some methodologil drwks in these ltter studies (i.e. popultions with different risks of pre-elmpsi, oexisting illness, unknown sttus of plsm vitmin onentrtions, lood onentrtions hieved with vitmin supplementtion not determined, no mesurements of iomrkers of oxidtive stress in the plsm, et.), limiting the interprettion of their results. Conversely, there is omplementry ody of evidene tht lends support to the role of ntioxidnt sttus on fetl outome. Thus, one study in women with low irthweight ies deteted high onentrtions of oxidtive stress iomrkers in the lood showed tht diminished fter vitmin C tretment (Krowiz-Bilińsk et l. 2002). It is importnt to note tht in humn pregnnies t term there is positive orreltion etween mternl nd fetl iomrkers of oxidtive stress (Argüelles et l. 2006). Furthermore, in other studies performed in helthy pregnnt women, diret reltionship etween plsm onentrtions of vitmins C nd E t 24 28 weeks gesttion nd irthweight ws seen (Lee et l. 2004; Min et l. 2006), in ddition to n inverse reltionship etween plsm vitmin C onentrtions t 37 weeks gesttion nd tropholst poptoti tivity (Ahn et l. 2007). These reltionships suggest key role for ntioxidnt sttus on mternl oxidtive stress, plentl integrity nd fetl growth. Plentl hnges Ovine plentl tissue is very suseptile to environmentl stressors, inluding HA hypoxi (Prrguez et l. 2006), poor nutrition (Vonnhme et l. 2003) nd high temperture (Gln et l. 1999). In previous study, we demonstrted severl plentl hnges (inresed plentl weight, otyledon dimeter, plentl surfe re nd otyledon re oupied y the vsulr lumin, s well s deresed numer of otyledons per plent) s ompenstory response to mternl fetl hypoxi stress. Moreover we demonstrted tht, in our experimentl model, the effets of poor nutrition nd high environmentl tempertures on these prmeters were negligile (Prrguez et l. 2006). Reently, we reported tht these plentl hnges were ssoited with the plentl overexpression of the prongiogeni ftors vsulr endothelil growth ftor (VEGF) nd endothelil nitri oxide synthse (enos; Prrguez et l. 2010). In the present study, we oserved tht, in generl, supplementtion with vitmins C nd E hd no effet on LA pregnnies, ut it prevented the effets of HA hypoxi on most of the plentl hrteristis desried ove in HA-dpted ewes. A less signifint effet of vitmin supplementtion ws seen in HA newomer ewes, in whih only derese in the numer of plentomes nd plentl re, s well s tendeny for deresed otyledon dimeter, were evident. Indeed, in oth groups supplemented with vitmins t HA, there ws trend for the plentl prmeters to move towrds those seen in ewes t LA (Prrguez et l. 2006). During pregnny, the development nd funtion of the plent depend primrily on the growth of its vsulr ed, whih is ontrolled y severl ngiogeni ftors (Reynolds et l. 2005). Thus, the min effets of vitmins on hypoxi plents my e explined y redution in the lol expression of ngiogeni ftors, resulting in redution in plentl vessels nd mss. As shown in the present study, ntioxidnt vitmin supplementtion inreses oxygen trnsport nd redues oxidtive stress, ompensting for the effet of the hypoxi environment. Under these onditions, hypoxi induile ftor (HIF)-1, the protein responsile for the tivtion of severl ngiogeni ftors, inluding VEGF (Crmeliet et l. 1988), my e diminished s result of redued expression of the HIF-1 suunit, thus deresing expression of the ngiogeni ftors. As result of deresed VEGF (Bouloumié et l. 1999) or s diret effet of meliorted oxygen tension (Sldek et l. 1997), enos my e lso redued. There is no informtion ville on the effet of ntioxidnt vitmins on plentl ngiogenesis nd its regulting ftors. In hiken model of ngiogenesis, inhiition of ngiogenesis y ntioxidnts hs een reported (Polytrhou nd Ppdimitriou 2004), whih my support our hypothesis. However, this should e onfirmed with future experiments. Compring the results of vitmin supplementtion on the weight of neworns nd plentl hnges, n pprent ontrdition emerges. If we onsider tht plentl surfe re nd its ross-setionl vsulr re should e diretly ssoited with the mternl fetl trnsfer of oxygen nd other nutrients, we my expet tht the improvement of neworn weight, s oserved in ewes reeiving ntioxidnt vitmin supplementtion in the present study, would e the result of n inrese in these plentl trits. However, in the present study, ntioxidnt vitmin supplementtion resulted in the opposite plentl response. Even though the method used to mesure vsulrity in the present study did not enle us to determine plentome

Vitmins C nd E in sheep pregnny t high ltitude Reprodution, Fertility nd Development 293 mirovsulrity, nd so the funtionl exhnge re, hnges deteted in the medium nd lrge vessels ould reflet the existene of stimuli for vritions in totl vsulr growth. Our interprettion of these findings is tht, despite the redution in plentl surfe re nd ross-setionl vsulr re, the ntioxidnt vitmin tretment indued some mehnism tht inresed overll plentl vsulr lood flow. There is no pulished informtion on the effet of ntioxidnt vitmins on ovine plents in norml or pthologil pregnnies. However, the enefiil effet of ntioxidnt vitmin therpy in humn pregnnies omplited y hypertension (e.g. s result of pre-elmpsi or dietes) my e ssoited with reovery of vsulr responses to vsodiltor gents, whih were previously diminished y oxidtive stress (Kossenjns et l. 2000). Severl plentl hnges hve een reported in humn pregnnies t HA (Zhng et l. 2002; Tissot vn Ptot et l. 2003, 2004; Zmudio 2003) tht re onsistent with those oserved in ewes t HA (Prrguez et l. 2006). We hve not found ny study in whih ntioxidnts were dministered to pregnnt women t HA to prevent oxidtive stress. However, in pre-elmpsi, pthologil ondition hrterised y plentl hypoperfusion, inresed vsulr resistne, proteinuri nd, frequently, low irthweight ies (Huel 1999), it hs een postulted tht oxidtive stress is the fundmentl normlity leding to endothelil dmge nd the presenttion of linil symptoms (Noris et l. 2005). Furthermore, gretly elevted risk of pre-elmpsi in pregnnt women t HA hs een reported (Keyes et l. 2003). Most of the evidene in these studies indites lose ssoition etween low plsm ntioxidnt sttus, espeilly plsm vitmin C onentrtions, nd development of the syndrome (Huel 1999; Chppell et l. 2002; Rodrigo et l. 2005; Chmy et l. 2006; Mehendle et l. 2008; Kry et l. 2010). In ddition, deresed onentrtions of vitmin C in umilil venous plsm nd in plentl tissue homogentes in pre-elmpti women hs een reported (Kim et l. 2006) nd enefiil role of vitmins C nd E in preventing pre-elmpsi hs een demonstrted (Chppell et l. 1999, 2002; Rodrigo et l. 2005; Rumiris et l. 2006). The effets of ntioxidnt therpy in pre-elmpsi re ssoited with deresed endothelil dmge, meliortion of plentl lood flow nd n improvement of other symptoms (Noris et l. 2005). This informtion supports our interprettion of the dt nd onstitutes strong sis on whih to onsider the effetiveness of supplementtion with vitmins C nd E in preventing the effets of HA oxidtive stress on the plentl fetl environment. Conlusion The present results llow us to onlude tht supplementtion with vitmins C nd E during ovine gesttion t HA prevents mternl hypoxi-indued oxidtive stress nd its effets on plentl hrteristis. Moreover, oth HA nd LA ntive pregnnt ewes respond stisftorily to the effet of these ntioxidnt vitmins, with inresed neworn odyweight nd n olition of the differenes oserved etween these groups of nimls in the sene of dily ntioxidnt dministrtion. Even though we knowledge tht the sene of highlnd origin lowlnd pregnny group is prole limittion of the present study, the results llow us to highlight the possiility of using supplementtion with vitmins C nd E s n eonomi tool to improve ovine prodution t HA. Aknowledgements The uthors thnk Mr Gino Lluso for his ssistne with niml mngement nd re, nd Mr Luis Pizrro nd Mr Miguel Sepúlved for their tehnil ssistne. This work ws supported y grnts FONDECYT 1020706 nd 1070405 from CONICYT, Chile. Referenes Ahn, Y. M., Kim, Y. J., Prk, H., Prk, B., nd Lee, H. (2007). Prentl vitmin C sttus is ssoited with plentl poptosis in norml-term humn pregnnies. Plent 28, 31 38. doi:10.1016/j.placenta. 2006.01.018 Arned, O. F., Grí, C., Lgos, N., Quirog, G., Cjigl, J., Slzr, M. P., nd Behn, C. (2005). Lung oxidtive stress s relted to exerise nd ltitude. Lipid peroxidtion evidene in exhled reth ondenste: possile preditor of ute mountin sikness. Eur. J. Appl. Physiol. 95, 383 390. Argüelles, S., Mhdo, M. J., Ayl, A., Mhdo, A., nd Herís, B. (2006). Correltion etween irulting iomrkers of oxidtive stress of mternl nd umilil lood t irth. Free Rdi. Res. 40, 565 570. doi:10.1080/10715760500519834 Askew, E. W. (2002). Work t high ltitude nd oxidtive stress: ntioxidnt nutrients. Toxiology 180, 107 119. doi:10.1016/s0300-483x(02) 00385-2 Biley, D. M., nd Dvies, B. (2001). Aute mountin sikness; prophylti enefits of ntioxidnt vitmin supplementtion t high ltitude. High Alt. Med. Biol. 2, 21 29. doi:10.1089/152702901750067882 Biley, D. M., Dvies, B., Young, I. S., Hullin, D. A., nd Seddon, P. S. (2001). A potentil role for free rdil-medited skeletl musle soreness in the pthophysiology of ute mountin sikness. Avit. Spe Environ. Med. 72, 513 521. Blk, W. D., nd Hidiroglou, M. (1996). Phrmokineti study of sori id in sheep. Cn. J. Vet. Res. 60, 216 221. Bouloumié, A., Shini-Kerth, V. B., nd Busse, R. (1999). Vsulr endothelil growth ftor up-regultes nitri oxide synthse expression in endothelil ells. Crdiovs. Res. 41, 773 780. doi:10.1016/s0008-6363(98)00228-4 Cpper, J. L., Wilkinson, R. G., Kspidou, E., Pttinson, S. E., Mkenzie, A. M., nd Sinlir, L. A. (2005). The effet of dietry vitmin E nd ftty id supplementtion of pregnnt nd ltting ewes on plentl nd mmmry trnsfer of vitmin E to the lm. Br. J. Nutr. 93, 549 557. doi:10.1079/bjn20051376 Crmeliet, P., Dor, Y., Herert, J. M., Fukumur, D., Brusselmns, K., et l. (1988). Role of HIF-1 lph in hypoxi-medited poptosis, ell prolifertion nd tumour ngiogenesis. Nture 394, 485 490. doi:10.1038/ 28867 Cedererg, J., Bsu, S., nd Erikson, U. J. (2001). Inresed rte of lipid peroxidtion nd protein ronyltion in experimentl dieti pregnny. Dietologi 44, 766 774. doi:10.1007/s001250051686 Cedererg, J., Simán, C. M., nd Erikson, U. J. (2001). Comined tretment with vitmin E nd vitmin C dereses oxidtive stress nd improves fetl outome in experimentl dieti pregnny. Peditr. Res. 49, 755 762. doi:10.1203/00006450-200106000-00007 Chmy, V. M., Lepe, J., Ctln, A., Retml, D., Esor, J. A., nd Mdrid, E. M. (2006). Oxidtive stress is losely relted to linil severity of pre-elmpsi. Biol. Res. 39, 229 236. doi:10.4067/s0716-97602006000200005

294 Reprodution, Fertility nd Development V. H. Prrguez et l. Cho, W., Askew, E. W., Roerts, D. E., Wood, S. M., nd Perkins, J. B. (1999). Oxidtive stress in humns during work t moderte ltitude. J. Nutr. 129, 2009 2012. Chppell, L. C., Seed, P. T., Briley, A. L., Kelly, F. J., Lee, R., et l. (1999). Effet of ntioxidnts on the ourrene of pre-empsi in women t inresed risk: rndomized tril. Lnet 354, 810 816. Chppell, L. C., Seed, P. T., Kelly, F. J., Briley, A., Hunt, B. J., Chrnok- Jones, D. S., Mllet, A., nd Poston, L. (2002). Vitmin C nd E supplementtion in women t risk of preelmpsi is ssoited with hnges in indies of oxidtive stress nd plentl funtion. Am. J. Ostet. Gyneol. 187, 777 784. doi:10.1067/mob.2002.125735 Gln, H. L., Hussey, M. J., Brer, A., Ferrzzi, E., Chung, M., Hoins, J. D., nd Bttgli, F. C. (1999). Reltionship of fetl growth to durtion of het stress in n ovine model of plentl insuffiieny. Am. J. Ostet. Gyneol. 180, 1278 1282. doi:10.1016/s0002-9378(99)70629-0 Giussni, D. A., Phillips, P. S., Anstee, S., nd Brker, J. P. (2001). Effets of ltitude versus eonomi sttus on irth weight nd ody shpe t irth. Peditr. Res. 49, 490 494. doi:10.1203/00006450-200104000-00009 Griffiths, H. R., Møller, L., Brtosz, G., Bst, A., Bertoni-Freddri, C., et l. (2002). Biomrkers. Mol. Aspets Med. 23, 101 208. doi:10.1016/ S0098-2997(02)00017-1 Hrtinger, S., Tpi, V., Crrillo, C., Bejrno, L., nd Gonzles, G. F. (2006). Birth weight t high ltitudes in Peru. Int. J. Gyneol. Ostet. 93, 275 281. doi:10.1016/j.ijgo.2006.02.023 Hidiroglou, M. (1999). Tehnil note: forms nd route of vitmin C supplementtion for ows. J. Diry Si. 82, 1831 1833. doi:10.3168/ JDS.S0022-0302(99)75414-7 Huel, C. A. (1999). Oxidtive stress in the pthogenesis of preelmpsi. Pro. So. Exp. Biol. Med. 222, 222 235. doi:10.1046/j.1525-1373. 1999.D01-139.X Ilvzhgn, G., Bnsl, A., Prsd, D., Thoms, P., Shrm, S. K., Kin, A. K., Kumr, D., nd Selvmurthy, W. (2001). Effet of vitmin E supplementtion on hypoxi-indued oxidtive dmge in mle lino rts. Avit. Spe Environ. Med. 72, 899 903. Inn, C., Kili, I., Kilin, K., Klyi, O., nd Kotiloglu, E. (1995). The effet of high dose ntentl vitmin E on hypoxi-indued hnges in neworn rts. Peditr. Res. 38, 685 695. doi:10.1203/00006450-199511000-00009 Juniux, E., Cindrov-Dvies, T., Johns, J., Dunster, C., Hempstok, J., Kelly, F. J., nd Burton, G. J. (2004). Distriution nd trnsfer pthwys of ntioxidnt moleules inside the first trimester humn gesttionl s. J. Clin. Endorinol. Met. 89, 1452 1458. doi:10.1210/jc.2003-031332 Jensen, G. M., nd Moore, L. G. (1997). The effet of high ltitude nd other risk ftors on irthweight: independent or intertive effets? Am. J. Puli Helth 87, 1003 1007. doi:10.2105/ajph.87.6.1003 Jonny, P., Steinerg, J., Roh, P., Rihlet, J. P., Gortn, C., Grdette, B., nd Jmmes, Y. (2001). Opertion Everest III (Comex 97): the effet of simulted severe hypori hypoxi on lipid peroxidtion nd ntioxidnt defense systems in humn lood t rest nd fter mximl exerise. Resusittion 49, 307 314. doi:10.1016/s0300-9572(00)00373-7 Kry, Ö., Sepii-Dinel, A., Krltin, D., Shin, D., Ylvç, S., Akyol, M., Kndemir, Ö., nd Altn, N. (2010). A quntittive evlution of totl ntioxidnt sttus nd oxidtive stress mrkers in preelmpsi nd gesttionl dieti ptients in 24 36 weeks of gesttion. Dietes Res. Clin. Prt. 89, 231 238. doi:10.1016/j.diabres.2010. 04.015 Krowiz-Bilińsk, A., Suzin, J., nd Sieroszewski, P. (2002). Evlution of oxidtive stress indies during tretment in pregnnt women with intruterine growth retrdtion. Med. Si. Monit. 8, CR211 CR216. Kelly, M. J., nd Rihrdson, W. (1996). Chrteristis of the protetive tion of lph-toopherol in vsulr hypoxi. Phrmol. Toxiol. 78, 209 213. doi:10.1111/j.1600-0773.1996.tb00207.x Keyes, L., Armz, F., Niermeyer, S., Vrgs, E., Young, D., nd Moore, L. (2003). Intruterine growth restrition, preelmpsi, nd intruterine mortlity t high ltitude in Bolivi. Peditr. Res. 54, 20 25. doi:10.1203/01.pdr.0000069846.64389.dc Khlil, R. A., nd Grnger, J. P. (2002). Vsulr mehnisms of inresed rteril pressure in preelmpsi: lessons from nimls models. Am. J. Physiol. Regul. Integr. Comp. Physiol. 283, R29 R45. Kim, Y. H., Kim, C. H., Cho, M. K., Kim, K. M., Lee, S. Y., Ahn, B. W., Yng, S. Y., Kim, S. M., nd Song, T. B. (2006). Totl peroxyl rdiltrpping ility nd ntioxidnt vitmins of the umilil venous plsm nd the plent in pre-elmpsi. J. Ostet. Gyneol. Res. 32, 32 41. doi:10.1111/j.1447-0756.2006.00348.x Kossenjns, W., Eis, A., Shy, R., Brokmn, D., nd Mytt, L. (2000). Role of peroxynitrite in ltered fetl-plentl vsulr retivity in dietes or preelmpsi. Am. J. Physiol. Hert Cir. Physiol. 278, H1311 H1319. Knight, C. A., Duther, R. A., Guerrnt, N. B., nd Behtel, S. (1941). Destrution of sori id in the rumen of diry ows. J. Diry Si. 24, 567 577. doi:10.3168/jds.s0022-0302(41)95436-x Krmpl, E., Lees, C., Blnd, J. M., Espinos Dordo, J., Mososos, G., nd Cmpell, S. (2000). Fetl iometry t 4300 m ompred to se level in Peru. Ultrsound Ostet. Gyneol. 16, 9 18. doi:10.1046/j.1469-0705. 2000.00156.X Lästrd, M., Ljungkvist, G., Olin, A. C., nd Torén, K. (2002). Determintion of mlondildehyde in reth ondenste y high-performne liquid hromtogrphy with fluoresene detetion. J. Chromtogr. B Anlyt. Tehnol. Biomed. Life Si. 766, 107 114. doi:10.1016/s0378-4347(01)00437-6 Lee, B., Hong, Y. C., Lee, K. H., Kim, W. K., Chng, N. S., Prk, E. A., nd Hnn, H. J. (2004). Influene of mternl serum levels of vitmins C nd E during the seond trimester on irth weight nd length. Eur. J. Clin. Nutr. 58, 1365 1371. doi:10.1038/sj.ejcn.1601976 Lissi, E., Psul, C., nd Del Cstillo, M. D. (1992). Luminol luminesene indued y 2,2 0 -zo-is(2-midinopropne) thermolisys. Free Rdi. Res. Commun. 17, 299 311. doi:10.3109/10715769209079523 Mglhães, J., Asensão, A., Visor, G., Sores, J., Oliveir, J., Mrques, F., nd Durte, J. (2004). Oxidtive stress in humns during nd fter 4 hours of hypoxi t simulted ltitude of 5500 m. Avit. Spe Environ. Med. 75, 16 22. Mglhães, J., Asensão, A., Mrques, F., Visor, G., Sores, J. M. C., Ferreir, R., Neuprth, M. J., nd Durte, J. A. (2005). Effet of highltitude expedition to Himlyn pek (Pumori, 7,161 m) on plsm nd erythroyte ntioxidnt profile. Eur. J. Appl. Physiol. 93, 726 732. doi:10.1007/s00421-004-1222-2 Mehendle, S., Kilri, A., Dnngt, K., Trlekr, V., Mhdik, S., nd Joshi, S. (2008). Ftty ids, ntioxidnts, nd oxidtive stress in preelmpsi. Int. J. Gyneol. Ostet. 100, 234 238. doi:10.1016/j.ijgo. 2007.08.011 Min, J., Prk, H., Prk, B., Kim, Y. J., Prk, J., Lee, H., H, E., Prk, E., nd Hong, Y. C. (2006). Proxne gene polymorphism nd vitmin levels during pregnny: reltionship with mternl oxidtive stress nd neontl irthweights. Reprod. Toxiol. 22, 418 424. doi:10.1016/ J.REPROTOX.2006.01.003 Miur, S., Ishid, A., Nkjim, W., Ohmur, A., Kwmur, M., nd Tkd, G. (2006). Intrventriulr sori id dministrtion dereses hypoxi ishemi rin injury in neworn rts. Brin Res. 1095, 159 166. doi:10.1016/j.brainres.2006.04.045 Mohnrj, P., Merol, A. J., Wright, V. P., nd Clnton, T. L. (1998). Antioxidnts pretet rt diphrgmti musle funtion under hypoxi onditions. J. Appl. Physiol. 84, 1960 1966. Møller, P., Loft, S., Lundy, C., nd Olsen, N. V. (2001). Aute hypoxi nd exerise indue DNA strnd reks nd oxidtive DNA dmge in humns. FASEB J. 15, 1181 1186. doi:10.1096/fj.00-0703com