130 riial artileriial artile Nitrate detoxification using antioxidants and probiotics in the water for rabbits ¤ Detoxificación de nitrato usando antioxidantes y probióticos en

Rev Colomb Cienc Pecu 2018; 31(2):130-138

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Revista Colombiana de Ciencias Pecuarias

Original articles

Nitrate detoxification using antioxidants and probiotics in the water for rabbits¤

Detoxificación de nitrato usando antioxidantes y probióticos en el agua para conejos

Detoxificação de nitrato usando antioxidantes e probióticos na água para coelhos

Youssef A Attia1, AS, PhD; Abd El Hamid E1, AS, PhD; Ahmad M Ismaiel2, AS, PhD; Maria C de Oliveira3, MV, PhD; Mohammed A Al-Harthi1, AS, PhD; Asmaa S El-Naggar2, AS, PhD; Gustavo A Simon4, Eng Agr, PhD.

1Arid Land Agriculture Department, Faculty of Meteorology, Environment and Arid Land Agriculture, King Abdulaziz University, Jeddah, Saudi Arabia.

2Department of Animal and Poultry Production, Faculty of Agriculture, Damanhour University, Damanhour, Egypt.

3Faculty of Veterinary Medicine, University of Rio Verde, Rio Verde, Brazil.

4Faculty of Agronomy, University of Rio Verde, Rio Verde, Brazil.

(Received: June 19, 2016; accepted: September 11, 2017)

doi: 10.17533/udea.rccp.v31n2a06

¤ Tocitethisarticle:AttiaYA,ElHamidEA,IsmaielAM,deOliveiraMC,Al-HarthiMA,El-NaggarAS,SimonGA.Nitratedetoxificationusingantioxidantsandprobioticsinthewaterforrabbits.RevColombCiencPecu2018;31(2):130-138.

* Correspondingauthor:MariaCdeOliveira.FaculdadedeMedicinaVeterinária,UniversidadedeRioVerde,CampusUniversitários/n,CEP:75901-910,RioVerde,GO,Brazil.E-mail:[email protected]

Abstract

Background: Agriculturalpracticesincreasegroundwaterpollutionfromnitrate.Highnitrateintakecouldnegativelyaffectanimalgrowth.Objective: Todeterminetheeffectsofdifferentlevelsofnitrateindrinkingwaterondigestive,liver,andkidneyfunctions,andonwaterandfeedintake,andtodeterminetheabilityofvitaminC,vitaminE+selenium(Se)orprobioticstoovercometheeffectsofnitrateinNewZealandWhiterabbitbucks.Methods: Forty-twomalerabbitswererandomlydistributedintosixtreatmentgroups:1)controlgroup(withnonitrate);2)350ppmnitrate;3)700ppmnitrate;4)700ppm+200ppmvitaminC;5)700ppm+200ppmvitaminE+Se;and6)700ppm+1000ppmprobiotic.Productiveperformance,digestive,liver,andkidneyfunctions,andhepaticandrenalhistologywereevaluated.Results: Waterintakewasreduced(p<0.05)bythe 350ppm nitratetreatment.Rabbitsgiven700ppmnitrateshowedlower(p<0.05)drymatterintake,nutrientdigestibility,andincreased(p<0.05)waterandnitrateintake,aswellasureaconcentration,andaspartateaminotransaminase (AST)andalanineaminotransferase (ALT)activity.VitaminC,vitaminE+Seandprobioticsimproved(p<0.05)feedintakeandnutrientdigestibility,andreduced(p<0.05)water

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Attia YA et al. Nitrate detoxification for rabbits

andnitrateintake,ureaconcentration,andASTandALTactivities.Conclusion: Rabbitsmaytolerateupto350ppmofnitrate,but700ppmofnitratenegativelyaffectdigestive,liver,andkidneyfunctions,whichareimprovedbyvitaminC,vitaminE+Seandprobioticsupplementation.

Keywords: additives, animal nutrition, water composition and quality, water pollution, water purification.

Resumen

Antecedentes: Lasprácticasagrícolasaumentanlapolucióndeaguassubterráneasconnitratos.Unaaltaingestióndenitratosposiblementeafectanegativamenteelcrecimientoanimal.Objetivo: Determinarlosefectosdediferentesnivelesdenitratoenelaguadebebidasobrelasfuncionesdigestivas,hepáticasyrenales,ingestióndeaguayalimento,ydeterminarlahabilidaddelavitaminaC,vitaminaE+Selenio(Se)oprobióticosensuperarlosefectosdelnitratoenconejosNuevaZelandaBlanco.Métodos: Cuarentaydosconejosmachossedistribuyeronalazarentreseistratamientos:1)grupocontrol(sinnitrato),2)350ppmnitrato,3)700ppmnitrato,4)700ppm+200ppmvitaminaC,5)700ppm+200ppmvitaminaE+Se,y6)700ppm+1000ppmprobiótico.Seevaluóeldesempeñoproductivo,lasfuncionesdigestivas,hepáticasyrenales,ylahistologíarenalyhepática.Resultados: La ingestiónde350ppmdenitratodisminuyó(p<0,05)laingestióndeagua.Losconejosqueconsumieron700ppmdenitratopresentaronmenor(p<0,05)ingestióndemateriasecaydigestibilidaddenutrientes,ymayor(p<0,05)ingestióndeaguaynitrato,concentracióndeurea,ymayoresactividadesdeaspartatoaminotransferasa(AST)yalaninaaminotransferasa(ALT).LasvitaminasC,E+Seyelprobióticomejoraron(p<0,05)elconsumodealimentoyladigestibilidaddenutrientes,yredujeron(p<0,05)laingestióndeaguaynitrato,laconcentracióndeurea,ylasactividadesdeASTyALT.Conclusión: Los conejospueden tolerarhasta350ppmdenitrato,pero700ppmdenitratoafectannegativamente lasfuncionesdigestivas,hepáticasyrenales,lascualesmejoranconlasuplementacióndevitaminaC,vitaminaE+Seydelprobiótico.

Palabras clave: aditivos, composición y calidad del agua, nutrición animal, polución del agua, purificación del agua.

Resumo

Antecedentes: Aspráticasagrícolasaumentamacontaminaçãodeáguassubterrâneasapartirdenitratoeaaltaingestãodestasubstânciapodeafetarnegativamenteocrescimentoanimal.Objetivo: Determinar os efeitosdediferentesníveisdenitratonaáguadebebidasobreasfunçõesdigestivas,hepáticaserenais,ingestãodeáguaeraçãoedeterminarahabilidadedavitaminaC,vitaminaE+Selênio(Se)ouprobióticoemsobreporosefeitosdonitratoemcoelhosNovaZelândiaBranco.Métodos: Quarentaedoiscoelhosmachosforamdistribuídosaoacasoemseistratamentos:1)grupocontrole(semnitrato),2)350ppmdenitrato,3)700ppmdenitrato,4)700ppm+200ppmdevitaminaC,5)700ppm+200ppmvitaminaE+See6)700ppm+1000ppmdeprobiótico.Foramavaliadosodesempenhoprodutivo,asfunçõesdigestivas,hepáticaserenaisalémdahistologiahepáticaerenal.Resultados: Aingestãode350ppmdenitratodiminuiu(p<0,05)aingestãodeágua.Coelhosquereceberam700ppmdenitratoapresentarammenor(p<0,05)ingestãodematériaseca,menordigestibilidadedenutrientes,emaior(p<0,05)ingestãodeáguaedenitrato,maiorconcentraçãoplasmáticadeureiaemaioresatividadesdeaspartatoaminotransferase(AST)ealaninaaminotransferase(ALT).AadiçãodevitaminasCouvitaminaE+Seeprobióticomelhorou(p<0,05)oconsumoderação,adigestibilidadedenutrientesereduziram(p<0,05)aingestãodeáguaedenitrato,aconcentraçãodeureiaeasatividadesdeASTeALT.Conclusão: Coelhospodemtoleraraté350ppmdenitrato,mas700ppmdenitratoafetanegativamenteasfunçõesdigestivas,hepáticaserenais,asquaissãomelhoradasapartirdasuplementaçãodevitaminaC,vitaminaE+Seouaindaprobiótico.

Palavras-chave: aditivos, composição e qualidade da agua, nutrição animal, poluição da agua, purificação da água.

Introduction

Agriculturalpracticesmayincreasegroundwaterpollutionfromnitrate(Burrowet al.,2010),andhigh

nitrate intake adversely affects rabbit productiveperformance (Shehata, 2005). Ingestion of highconcentrationsofnitratesbuildsupnitrite, and themajorbiologicaleffectofnitrite is its involvement

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in the transformation of normal hemoglobin to methemoglobin, which is unable to transportoxygentothetissues(NdounlaandPulgarin,2014).In ruminants, the rumen iswell suited for nitratereductase activity, and only 10-20% of nitrite isabsorbedinthebloodstream,asthebulkisrapidlymetabolized by rumenmicroflora into ammonia(Cockburnet al.,2013).However,non-ruminantsareunabletocarryoutthisconversion(Robson,2007).

The common symptoms of chronic nitratepoisoningareareductioninweightgain,decreasedmilk production, depressed appetite, and greatersusceptibility to infections incattle(Gulmezogluet al.,2010).Symptomsincludeabluishcolorinthemucousmembranes, rapid and difficult breathing,rapidpulse,tremors,staggering,collapse,anddeath(Hancock, 2010). In addition, nitrites reactwithsecondary amines to form nitrosamines,many ofwhicharecarcinogenicinexperimentalanimals(Hordet al.,2009).Sharmaet al.(2011)evaluatedtheeffectofnitratelevels(45,100,200,400,and500mg/L)indrinkingwateronthelungsofrabbitsandfoundlungswith no congestion, inflammatory cells, andbreakdownof alveoli, andnormal bronchiolewithconcentrationsofonly45mg/L.

Sharma et al. (2013; 2014) reported that 200,400,or500ppmofnitrateindrinkingwatercausedcyanosis and lethargy in rabbits.Tachycardia andtachypneaprecededcyanosisandwereproportionalto thenitrateconcentration inwater.Nitrate intakeat200and400ppmresulted in focalcollectionoflymphocytes in interstitial tissues of the kidney.Congestion of blood vessels and damage to tubular epitheliumwasobservedwith500ppmnitrateintake.

Shehata(2005)showedvitaminC(VitC)efficacyin nitrate andmercury detoxification. Selimet al. (2008)reportedantioxidanteffectsofvitaminsE(VitE)andVitC.Probioticscanalsoconvertnitriteintoammoniaand,ultimately,toaminoacidsandproteins(Robson,2007).

VitaminEisafat-solublevitaminwithantioxidantproperties.Ascorbicacid,orVitC,isapotentwater-soluble antioxidant (Attiaet al., 2011a). Selenium(Se)istheactivecenterofglutathioneperoxidase,andthiscatalyzesthebreakdownofhydrogenperoxide

(Attia et al., 2010).Moreover, dietary probioticsdemonstrateantioxidantactivityandprotectiveeffectagainstspermdamageinducedbyhigh-fatdiets(Chenet al.,2013).

Considering that high nitrate concentration indrinkingwaterhasnegativeeffectsonanimalhealthand performance, and the importance of waterdetoxificationtomaintainthehealthandproductivityofrabbits,thisstudywascarriedouttodeterminetheeffectsofdifferentlevelsofnitrateindrinkingwaterondigestive,liverandkidneyfunctions(digestibilitycoefficients,nutritivevalues),waterandfeedintake,andalsotheabilityofVitC,VitE+Seorprobioticsto overcome the effects of nitrate inNewZealandWhite(NZW)rabbitbucks.

Materials and methods

Animals

Forty-two,16-week-oldNewZealandWhite(NZW)malerabbits,withanaveragebodyweightof2,180±51g,wererandomlydistributedamongsixtreatmentgroupsofsevenrabbitseach,at16to61weeksofage.TheexperimentwasconductedattheRabbitResearchUnit,Al-Bostan,FacultyofAgriculture,DamanhourUniversity, during the period of June to July.Therabbitswere kept under the samehygienic (healthandvaccinationprogram),andenvironmental(27˚Ctemperature,58%relativehumidity,anddaylength)conditionsduringtheexperimentalperiod.Thelight-darkcyclewas14:10hdaily.

The treatments included threemain groups, inwhichnitrateconcentrationswere0(tapwater),350or700ppm.Duetoexpectednegativeeffects(Attiaet al., 2013),onlythehighernitratedose(700ppm)wassupplementedwithvitaminsandprobioticstotesttheeffectsonnitratedetoxification.Withinthe700ppmgroup, therewere four subgroups, fromwhichonegroupwasusedasthecontrolandthewaterfortheotherthreewassupplementedwitheither200ppmVitC(UnitedCompanyforChemicalsandMedicalPreparation,Cairo,Egypt),200ppmVitE+0.2ppmof Se as sodium selenite (United Company forChemicalsandMedicalPreparation,Cairo,Egypt),and1000ppmofaprobioticcontainingLactobacillus

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acidophilus, Saccharomyces cerevisiae, sodium chloride,monobasic potassiumphosphate, sodiumbicarbonate, and dextrose (VetapharmCompany,Cairo,Egypt).Nitratewassupplementedassodiumnitrate (MisrCompany forChemical andMedicalPreparation,Cairo,Egypt).Thecommercialpelleteddietusedwascomposedofbarleyhay,wheatbran,maize,soybeanmeal,dicalciumphosphate,sodiumchloride,andvitaminandmineralpremix.Chemicalanalysisofthedietsonas-fedbasiswasconductedaccording to theAOAC (2004) specifications,exceptbythecontentsofNDF,ADF(VanSoestandWine,1967),andhemicellulose(bysubtractionofADFfromNDF;Table1).

Table 1. Chemical composition of the commercial pelleted diet.

everyday.Theamountconsumedwascalculatedbysubtractingtheresidualwaterfromtheamountoffered.

Both rabbits and rationswereweighed at thebeginningandat theendof theexperimentalperiodtodeterminethebodyweightandrationconsumption,respectively.At32and60weeksofage,adigestibilitytrialwas performed to determine the digestibilitycoefficients and nutritive values using six animalspertreatment.Theanimalswerehousedindividuallyinmetabolic cages that allowedcollectionof fecesthroughout thedigestibility trial (5d).Quantitativecollectionof feceswas startedat24hafterofferingthedailyfeed.Thefecesofeachrabbitwerecollectedonceaday,at9:00am.Feedintakewasrecordedatthesametimeeverymorningfora5-dcollectionperiod.All collected feces of each rabbitweremixed forhomogeneity, then subsampledand storedat -18°C untilanalysis.Feedsampleswerealsocollectedandpreparedforproximateanalysis.Fecalsamplesweredriedinaforced-ventilationovenat60°Cfor72handthengroundthrougha1mmscreenonaWileygrinder.Chemical analyses (drymatter, crudeprotein, etherextract,andcrudefiber)of thefeedsandfeceswereconductedaccordingtoAOAC(2004)specifications.Theobtainedmeanwasusedtocalculatethedigestibilitycoefficient,expressedondrymatter(DM).

At32and56weeksofage,sixbloodsamplespertreatmentwererandomlycollectedfromthemarginalearveinundervacuumincleanheparinizedtubesfordeterminationofbiochemicalconstituents inplasma.Bloodsampleswerecentrifugedfor20minat2000× g andtheplasmawasfrozenat-18°C for analysis. Plasma ureaandcreatinine concentrationsweredeterminedusingcommercialkits (DiamondDiagnostics,Cairo,Egypt;N.S.BIOTEC,Alexandria,Egypt),respectively.Theactivitiesofaspartateaminotransferase(AST)andalanineaminotransferase(ALT)as(U/L)inplasmaweredeterminedwithcommercialkitsproducedbyPasteurLab (Tehran, Iran).Totalantioxidantcapacity (TAC)wasdeterminedaccordingtoKoracevicet al.(2001).

Statistical analysis

Statisticalanalysiswascarriedoutusingone-wayanalysisofvariance(Ferreira,2009),withorthogonalcontrasts:0vs350ppm;0vs700ppm;350vs700ppm;

Parameter Level (%)Dry matter 89.74

Organic matter 90.59

Crude protein 16.17

Crude fiber 13.71

Ether extract 2.51

Nitrogen-free extract 58.20

Ash 9.41

Neutral detergent fiber 32.31

Acid detergent fiber 15.90

Hemicellulose 16.41

Theselectednitratedoseswerebasedontheresultsobtainedbyotherresearchers(Gilmanet al.,1998;Shehata,2005),whoshowedthatnitrateconcentrationat 600-700 ppmhad deleterious effects on rabbitperformance.Nitrate content in thedrinkingwaterandfeedwasdeterminedaccordingtoPappenhagen(1958),andvalueswere14ppmintapwater,7ppminwellwater,12ppminagriculturaldrainage,and50 ppm in the feed.Nitrate intakewas calculatedwiththedeterminedvaluesoffeedandwater,andthesupplemented nitrate dose.

Therabbitswereraisedinanopen-systemrabbitryandhousedinagalvanizedwirecagebattery(50cmwidth×40cmdepth×45cmheight).Allcageswereprovidedwithamanualfeederandcleanfreshwaterthatwascontinuouslyavailablethroughanautomaticsystemofnippledrinkers.Each rabbitwasofferedafixedamountofwater (500mL)at the same time

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750ppmvs.VitC+VitE+Se+Prob;VitCvsVitE+Se; Vit C vs Prob; and Vit E+Se vs Prob.

Results

Neither nitrate level nor additives affected(p>0.05) bodyweight.However, compared to thecontrolgroup,350ppm(p<0.05)reducedwaterintakeandincreasednitrateintake,and700ppm(p<0.05)

increasedwaterandnitrateintake.Comparingbothnitratelevels,700ppmincreased(p<0.05)waterandnitrateintakes.Fornitrateadditionat700ppm+,alladditivesresultedinhigher(p<0.05)feedintakeandlowerwater and nitrate intakes.Water and nitrateintakewerereduced(p<0.05)whenVitCwasaddedcomparedtotheVitE+Seaddition,butcomparingtheadditionofVitE+Se toprobiotic,water andnitrate intakeswere increased (p<0.05) due to theprobiotic(Table2).

Table 2. Effect of different nitrate levels in drinking water and the supplementation with Vit C or Vit E + Se or probiotic on productive performance, water, and nitrate intake of New Zealand White rabbit bucks during 16-61 weeks of age.

Nitrate (ppm) Supplementation Bodyweight at 61 weeks (g)

Feed intake (g/d) Waterintake (mL/d)

Nitrateintake (mg/d)

0 - 2634 103 326 8.11

350 - 2733 104 312 114

700 - 2727 95 365 256

700 Vit C 2799 105 324 229

700 Vit E + Se 2934 107 284 206

700 Probiotic 3033 108 325 232

SEM 186.7 3.76 2.45 1.52

Orthogonal contrast – p value

Control vs 350 ppm 0.211 0.818 0.001 0.001

Control vs 700 ppm 0.647 0.117 0.001 0.001

350 vs 700 ppm 0.358 0.074 0.001 0.001

700 vs 700 + all additives 0.764 0.005 0.001 0.001

700 Vit C vs Vit E + Se 0.156 0.736 0.001 0.001

700 + Vit C vs Probiotic 0.155 0.612 0.757 0.720

700 + Vit E + Se vs Probiotic 0.920 0.865 0.001 0.001n = 7 rabbits per treatment. SEM: Standard error of the mean.

Forbothageclasses,notreatmenteffect(p>0.05)wasobservedonetherextractdigestibility.Comparedtothecontrolgroup,350ppmnitrateintakereduced(p<0.05) crude protein (CP) digestibility, and at700ppmitreduced(p<0.05)drymatterintake,anddigestibilityofCPandcrudefiber(CF).Increasingnitratelevelsfrom350to700ppmresultedinlower(p<0.05)digestibilityofCPandCF.However,whenalladditiveswereused,DMIanddigestibilityofCP,CF,andDM(thelatteronlyat32weeks)wereincreased(p>0.05).SupplementationwithVitE+Sereduced(p<0.05) CP and CF digestibility compared tosupplementationwithVitCorprobiotic.Compared

toVitCsupplementation,probioticadditionincreasedCPdigestibility(Table3).

Therewasnoeffectofthetreatmentsoncreatininelevelat32weeksofage.Nitrateadditionandincreasingnitrate inwater from350ppm to700ppmresulted(p<0.05) inhigherureaconcentration,ASTandALTactivities,andlowerTAC.The700ppm+alladditivestreatment reduced (p<0.05) urea and creatinineconcentrations, andAST andALT activities, butincreased(p<0.05)TAC.At56weeksofage,probioticsupplementationresultedinhigher(p<0.05)creatininelevelsandTACcomparedtoVitCinclusion(Table4).

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Table 3. Effect of different nitrate levels in drinking water and the supplementation of Vit C or Vit E + Se or probiotic on dry matter intake and digestibility coefficients of NZW rabbit bucks at 32 and 60 weeks of age.

Nitrate (ppm) Supplementation DMI Digestibility coefficient (%) DMI Digestibility coefficient (%)

(g/d) DM CP EE CF (g/d) DM CP EE CF

32 weeks of age 60 weeks of age

0 - 110 64.4 75.4 48.6 43.8 108 60.7 71.4 44.5 39,6

350 - 101 63.3 72.7 48.6 42.0 98 69.3 68.7 44.9 38,0

700 92 60.5 63.2 45.6 33.4 88 56.4 59.3 41.7 29,6

700 Vit C 103 68.5 76.6 48.6 45.4 109 64.1 72.7 44.7 41,0

700 Vit E + Se 111 66.7 74.4 46.0 39.3 109 62.6 70.3 42.0 35,4

700 Probiotic 113 66.7 78.7 47.5 49.1 101 62.7 74.6 43.3 45,0

SEM 3.24 1.63 0.56 1.57 2.28 6.02 4.81 0.58 1.63 2,29

Orthogonal contrasts – p value

Control vs 350 ppm 0.083 0.622 0.005 1.000 0.581 0.278 0.230 0.008 0.876 0.617

Control vs 700 ppm 0.002 0.116 0.001 0.204 0.007 0.042 0.536 0.001 0.237 0.009

350 vs 700 ppm 0.073 0.259 0.001 0.204 0.021 0.278 0.082 0.001 0.186 0.023

700 vs 700 + all additives 0.001 0.003 0.001 0.366 0.001 0.026 0.247 0.001 0.393 0.001

700 Vit C vs Vit E + Se 0.106 0.467 0.016 0.273 0.082 1.000 0.829 0.015 0.270 0.114

700 + Vit C vs Probiotic 0.043 0.467 0.022 0.631 0.274 0.347 0.840 0.034 0.546 0.234

700 + Vit E + Se vs Probiotic 0.619 1.000 0.001 0.524 0.010 0.347 0.988 0.001 0.602 0.012

n = 3 animals per treatment. DMI: Dry matter intake; DM: Dry matter; CP: Crude protein; EE: Ether extract; CF: Crude fiber. SEM: Standard error of the mean.

Discussion

Thelackofasignificanteffectofnitrateonbodyweight of rabbit bucks is because animals becomeadapted to thehighnitrate concentrationsandmaybe able to use a portion of nitrate as a non-protein nitrogensource.Thesupplementsdecreasednitrateintake as a result of the lowerwater intake andincreasedfeedintake.ThesupplementationwithVitC,VitE+Se,andprobioticreducedwaterintakein11,22,and11%, respectively,compared to thecontroltreatmentwith700ppmofnitrate,showingthatVitE+SehadastrongereffectthanVitCandprobioticonwaterandnitrateintakes.ThiscouldbeduetothesynergeticeffectsofVitEandSeasantioxidants(Attiaet al.,2010).Kritaset al.(2008)notedthatprobioticsupplementationimprovedgrowthperformanceanddecreasedmorbidity and/ormortality of rabbits,whichcouldbeattributedtoimprovedgutfunction.Differencesamongtheabove-mentionedresultscouldbe attributed to the age of the rabbits, the type of supplementationandhygienicconditions.

TheresultsofthisstudyarealsoinagreementwiththosereportedbyAbdEl-Khaleket al.(2008),who foundthatVitCwithorwithoutVitEsignificantlyincreasedfeedintake.Thiscouldpartiallyexplaintheroleofantioxidantagentsinovercomingthenegativeeffects of nitrate and eliminating free radicals, inagreement with the increased total antioxidantcapacityinbloodplasmafoundinthepresentstudywithVitC,VitE+Seorprobioticsupplementation.SimilarresultswereobservedbyAttiaet al.(2011b)withprobioticsupplementation,andDokoupilovaet al.(2007)withVitC(200or400mg/L)indrinkingwatersupplementedwith729mgnitrate/Linrabbits.

Highnitrateintake,suchas350ppmor700ppm,mayinhibitthehypothalamic-pituitary-thyroidaxisandcontributetoreducedbloodplasmaconcentrationsofT3andT4(Zakiet al.,2004).Restingmetabolicrateandenergyexpendituremightbereducedbyhighnitrateintake,asdemonstratedbyLarsenet al.(2014)inhumans,whichmayexplainthelowerDMIandconsequent lower nutrient digestibility in animals

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Table 4. Effect of different nitrate levels in drinking water and the supplementation of Vit C or Vit E + Se or probiotic on blood plasma constituents of NZW rabbit bucks collected at 32 and 56 weeks of age.

Nitrate (ppm)

Supplementation Urea-N (mg/dL)

Creatinine (mg/dL)

AST (U/L)

ALT (U/L)

TAC (μMol/L)

Urea-N (mg/dL)

Creatinine (mg/dL)

AST (U/L)

ALT (U/L)

TAC (μmol/mL)

32 weeks of age 56 weeks of age

0 - 43.16 0.90 24.98 23.42 1.30 42.98 1.14 26.40 27.51 1.13

350 - 49.74 1.02 30.42 30.64 1.00 45.43 1.00 34.51 31.08 0.83

700 57.33 1.03 47.07 42.05 0.34 53.80 0.72 41.50 38.41 0.58

700 Vit C 38.70 1.30 30.45 29.12 1.57 39.80 1.32 27.31 24.38 1.32

700 Vit E + Se 40.78 1.18 29.12 25.24 1.60 39.23 1.64 26.38 21.88 1.64

700 Probiotic 39.56 1.28 26.26 25.00 1.50 39.21 1.74 27.03 23.51 1.74

SEM 0.89 0.19 1.52 1.79 0.09 1.92 0.11 2.19 2.41 0.13

Orthogonal contrasts – p value

Control vs 350 ppm 0.0001 0.6700 0.0187 0.0090 0.0273 0.3757 0.4085 0.0137 0.3048 0.1198

Control vs 700 ppm 0.0001 0.6360 0.0001 0.0001 0.0001 0.0004 0.0137 0.0001 0.0033 0.0062

350 vs 700 ppm 0.0001 0.9622 0.0001 0.0002 0.0001 0.0045 0.0850 0.0318 0.0400 0.1893

700 vs 700 + all additives 0.0001 0.3423 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001 0.0001

700 Vit C vs Vit E + Se 0.1116 0.6700 0.5431 0.1395 0.8463 0.8367 0.0557 0.7655 0.4700 0.1014

700 + Vit C vs Probiotic 0.5012 0.9433 0.0638 0.1177 0.5622 0.8320 0.0137 0.9278 0.8015 0.0338

700 + Vit E + Se vs Probiotic 0.3423 0.7223 0.1971 0.9256 0.4410 0.9952 0.5343 0.8354 0.6361 0.5975

n = 6 samples per treatment. AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; TAC: Total antioxidant capacity. SEM: Atandard error of the mean.

receivingnitrateinwater,mostlyat700ppm.Theseresults are similar to those reported by Shehata (2005),whofoundthatadditionofnitratesignificantlydecreasedfeedintakeandnutrientdigestibility.

Digestibility coefficients of CP andCFwerereduced in animals receiving nitrate in drinkingwater,andthiscoincidedwithincreasedwaterintakeat the highest nitrate dose by 21%.The increaseinwater intake at the highest nitrate level couldbe due to frequentmicturition (Cockburn et al., 2013)indicatingthatrabbitstrytoexcretetheextranitrogenouscompoundsthroughtheurine.AccordingtoCockburnet al.(2013),highnitrateintakeresultsinmore peristalticmovements and, consequently,less time for nutrient absorption. In addition, nitrate andnitritemaydecreasethenumberofclonogeniccells, which initiatemucosal cell regenerationandcancauseapoptosis in the intestinalcryptandatrophy of intestinal villi (Grudzinski and Law,1998ab).However,nochangesintherelativeweight

of thesmall intestinewereseen.VitC,VitE+Seorprobioticsupplementationovercamethenegativeeffectsofnitrateonfeedanddigestibilitycoefficients,improvinggutfunction.

Althoughnitratehadnonegativeeffectonorganweights,changesinliverenzymes(ALTandAST),kidneyfunction(ureaconcentration)andantioxidantstatus(TAC)wereobserved.TheincreaseofASTandALTinthe700-ppmnitrategroupindicateshepatocytenecrosis,whichwasconfirmedbyhistopathology.Thenegativeeffectonkidneyfunctionwasconfirmedbytheincreaseinbloodplasmaureaandthechangeinthe urea/creatinine ratio, and further confirmedbyhistopathological examination. Similarly, Shehata (2005) found thatNZW female rabbits that drankwatersupplementedwith729mgnitrate/Lshowedhigher blood urea concentrations.These findingsagreewiththosebySharmaet al.(2013;2014)whoindicated thatnitrate impairs livermetabolismandkidneyfunction.

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VitaminC,VitE+Se,andprobioticsovercomethe negative effect of 700 ppm nitrate on liverenzymes(ALTandAST)andkidney(urea,creatinineconcentration)function,andtheseresultsagreewiththose reported by Selim et al.(2008).TheimprovedliverandrenalfunctionduetoVitC,VitE+Se,andprobioticscoincidedwithincreasedtotalantioxidantcapacity, showing their role as antioxidants.Theseboostingeffectsofprobioticsagreewiththosereportedby Selim et al.(2008),andcouldbeattributedtotheimprovement ingutmicrobiota,whichmight haveutilizedordegradednitrateforgrowthandperformednitratedetoxification.Moreover,theeffectsofVitC,VitE+Se,orprobioticsongut, liver, andkidneyfunctionscoincidedwiththedecreasingseverityofpathologicchangesintheliverandkidney,supportingthehypothesisthatVitC,VitE+Seandprobioticsimprovethestructureandfunctionofliverandkidney.

Inconclusion,rabbitsmaytolerateupto350ppmnitrate,but700ppmnitratenegativelyaffectdigestive,liver,andkidneyfunctions,whichareimprovedwithVitC,VitE+Se,andprobioticsupplementation.

Conflicts of interest

Theauthorsdeclaretheyhavenoconflictsofinterestwithregardtotheworkpresentedinthisreport.

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130 riial artileriial artile Nitrate detoxification using antioxidants and probiotics in the water for rabbits ¤ Detoxificación de nitrato usando antioxidantes y probióticos en