Study of seasonal weight loss tolerance in small ruminants-an NMR-metabolomics approach

! ii! The wo k p esen ed in his disse a ion was ca ied ou unde he ITQB PhD P og am a Ins i u o de Tecnologia Química e Biológica An ónio Xa ie o Uni e sidade No a de Lisboa, unde supe ision o Doc o Manolis Ma zape akis and co-supe ision o P o esso And é Ma inho de Almeida. Financial suppo was awa ded h ough a doc o al ellowship a ibu ed o Ma iana Miguel Rebelo da Palma wi h he e e ence SFRH/BD/85391/2012, and he P ojec “Lac a ion and milk p oduc ion in goa (Cap a hi cus): iden i ying molecula ma ke s unde lying adap a ion o seasonal weigh loss” wi h he e e ence PTDC/CVT/116499/2010, bo h om FCT - Fundação pa a a Ciência e Tecnologia (Lisbon, Po ugal). The animal ial componen o he goa expe imen was suppo ed by ICIA – Ins i u o Cana io de In es igaciones Ag a ias (Valle Gue a, Tene i e, Spain), as well as he Ve e ina y Facul y o he Uni e si y o Las Palmas de G an Cana ia (A ucas, G an Cana ia, Spain). The animal ial componen o he sheep expe imen was suppo ed wi h esea ch unds om DAFWA – Depa men o Ag icul u e and Food Wes e n Aus alia (Pe h, WA, Aus alia). Pa o his wo k was p esen ed in COST Ac ions FA 1002 – P o eomics in Fa m Animals and FA 1308 – Dai yCa e mee ings, as well as o he na ional and in e na ional scien i ic con e ences in Eu ope and he USA. The goa expe imen componen was awa ded he Bes PhD S uden Pos e P ize a he VI Cong esso da Sociedade Po uguesa de Ciências Ve e iná ias ha ook place in Oei as in Ap il 2014. ! iii! ACKNOWLEDGMENTS Aos meus pais, i mão e a ó, a quem dedico es a ese. É g aças a eles que es ou aqui hoje! Aos meus amigos, dos elhos empos: Ana e Susana, po oda a amizade e apoio ao longo de an os anos; Ma alda e Ana Luísa, po me p o a em que udo se esol e po mui o que pa eça impossí el; Filipa pela mo i ação pa a conco e ao ITQB; e Luís pela gene osa amizade e companhia nos meus p imei os anos em Lisboa. E aos amigos mais ecen es, que conheci ao longo des es 4 anos: Cláudia, And eia e Ri a, e aos hon osos memb os da unidade de queimados. Com ocês udo oi mais ácil e bem mais di e ido. To And é M. Almeida o us ing me om he beginning, augh me so aluable hings, and o all oppo uni ies o p esen ou wo k and mee new people. We p o ed dis ance is eally ela i e. To Manolis Ma zape akis o accep ing me a his g oup and allow me o de elop his wo k while lea ning on he ascina ing (and complex) NMR wo ld. To he membe s o he Goa Team: Lo enzo He nández-Cas ellano, Noemí Cas o, Anas asio A güello and Juan Capo e; and he Sheep Team: Tim Scanlon, Tanya Kilmins e , John Mil on, Ch is Oldham and Johan G ee , o hei key con ibu ions o wo k imp o emen . And a specially hank o Joana Lé ias, José Sal ado and Ana Fe ei a o all iendship, suppo and unny momen s along hese yea s. To Susana Al es and Rui Bessa o hei essen ial con ibu ion in he a y acids ask and o helping me lea n a bi abou his huge subjec . Gonçalo G aça and Luís Gonçal es o all p ecious sugges ions and suppo on he me abolomics and s a is ical analyses. To all echnicians om CERMAX, Communica ion O ice, Main enance and Glass O ice, specially o João Pi es and Luís Mo gado o helping me sol e all he daily small d amas. To all membe s and o me membe s o BioMolecula NMR, Ino ganic Biochemis y and NMR, Bac e ial Ene gy and Me abolism and Biological Ene gy and T ansduc ion Labo a o ies. ! i ! ! ! ABSTRACT Small uminan s a e e y impo an in he opics and he Medi e anean. In hese egions, pas u e sca ci y du ing he d y season leads o Seasonal Weigh Loss (SWL), one o he majo p oblems in uminan p oduc ion. Howe e , some b eeds p esen highe ole ance o SWL. Unde s anding he physiological mechanisms by which b eeds a e able o cope wi h SWL is o u mos impo ance in animal selec ion. We s udied wo dai y goa b eeds (Majo e a and Palme a) and h ee b eeds o mea - p oducing sheep (Me ino, Do pe and Dama a), wi h di e en le els o ole ance o SWL. The aim o his s udy was o cha ac e ize he me abolome o he mamma y gland and milk in hese wo goa b eeds, as well as he me abolome o muscle and li e in sheep in o de o ob ain a b oad iew o SWL ole ance physiology in animals wi h di e en p oduc ion ap i udes. We used Nuclea Magne ic Resonance (NMR) o assess me abolome p o iles and Gas Ch oma og aphy o de e mine he a y acid composi ion. Goa s and sheep we e s udied in wo di e en wo k asks. In bo h species, animals we e di ided in con ol and es ic ed- ed g oups o he di e en b eeds in ol ed. The NMR-me abolomics app oach was shown o be adequa e o he ep oducible assessmen o he NMR- isible aqueous me abolome o milk, mamma y gland, muscle and li e o small uminan s leading o he iden i ica ion o espec i ely 50, 46, 51 and 46 me aboli es. In eg a ion o he NMR esul s wi h da a om o he me hodological app oaches was essen ial o he c ea ion o a mo e comp ehensi e analysis. Conce ning he dai y goa esul s, signi ican di e ences we e obse ed in he me aboli es o mamma y gland and milk be ween con ol and ! i! es ic ed g oups, albei wi h no di e ences be ween b eeds. Obse ed di e ences in me abolic pa hways a e ela ed o adap a ion o he low- ene gy die . Fa y acid analysis e ealed in e ac ion be ween b eed and eed- es ic ion in mamma y gland. Majo e a b eed showed mo e a ia ions in me aboli es, howe e no a ia ions we e obse ed in a y acid p o ile. This b eed seems o ha e highe ole ance o he low nu i ional condi ions while main aining he body a y acid depo s. In he con ex o SWL ole ance, he Majo e a b eed was con i med o be mo e ole an o eed- es ic ion and should hus be p e e able o b eed selec ion in SWL p one egions. Rega ding he sheep esul s, Do pe showed ewe changes on bo h issues, whe eas Me ino p esen ed mo e di e ences be ween con ol and es ic ed- ed g oups, mainly ela ed wi h a and p o ein mobiliza ion. Dama a p esen ed an in e media e esponse, wi h di e ences in amino acids composi ion in muscle and in ene gy- ela ed pa hways in li e . Me abolic a ia ions in Dama a b eed a e a e lec ion o i s, unique body cha ac e is ic, he a - ail. Do pe and Dama a b eeds a e mo e e icien ly adap ed o eed- es ic ion, changing he nu i ional ene gy sou ce wi hou comp omising he o e all muscle s uc u e. These b eeds a e mo e ole an o SWL condi ions, and hus mo e sui able o selec ion in ha sh en i onmen al condi ions. Resul s con i m he in luence o eed- es ic ion, as a majo p omo e o SWL, in small uminan ’s milk and mea p oduc ion. In gene al, ole an b eeds pe o med di e en ly unde eed- es ic ion and p o ed o be mo e sui able o b eed selec ion, especially in ha sh en i onmen al condi ions. Unde eed- es ic ion condi ions, ole an dai y goa b eed (Majo e a b eed) kep he mamma y gland s uc u e and unc ion, ! ii! whe eas mea p oduce sheep b eeds (Do pe and Dama a b eeds) main ained muscle s uc u e. ! iii! RESUMO Os pequenos uminan es são um impo an e ecu so alimen a e económico nas egiões opicais e medi e ânicas. Du an e a es ação seca as pas agens o nam-se escassas e pouco nu i i as, le ando os animais à Pe da de Peso Sazonal (PPS). Nes as egiões a PPS é a maio limi ação na p odução de pequenos uminan es. No en an o algumas aças ap esen am maio ole ância à PPS, o nando-as pa icula men e in e essan es pa a a comp eensão dos mecanismos isiológicos de adap ação a es a condição. Os esul ados con ibui ão pa a a de inição de es a égias de seleção de aças mais adequadas às egiões á idas. Nes e abalho o am es udadas duas aças de cab as lei ei as ( aça Majo e a e aça Palme a) e ês aças de o inos p odu o es de ca ne (Me ino, Do pe e Dama a), com di e en es ní eis de adap ação à PPS. Com o obje i o de es uda os mecanismos isiológicos de ole ância à PPS, o am ca ac e izados os me abolomas da glândula mamá ia e do lei e das aças cap inas, e do ígado e músculo-esquelé ico das aças o inas p odu o as de ca ne. Fo am u ilizadas écnicas de Ressonância Magné ica Nuclea (RMN) pa a a de e minação dos pe is me abólicos, e de C oma og a ia Gasosa pa a ca ac e ização dos pe is de ácidos go dos. As aças cap inas e o inas o am es udadas sepa adamen e, e os animais de cada aça di ididos em g upos con olo e de es ição alimen a . A abo dagem me abolómica po RMN con i mou se adequada pa a a ca ac e ização do me aboloma aquoso do lei e, glândula mamá ia, ! ix! músculo e ígado em pequenos uminan es, pe mi indo a iden i icação de 50, 46, 51 e 46 me aboli os espe i amen e. Rela i amen e às aças cap inas, o am iden i icadas di e enças nos pe is me abólicos en e g upos con olo e em es ição alimen a , em ambas as amos as. As p incipais di e enças obse adas es ão elacionadas com adap ações me abolómicas à es ição nu icional. No en an o, não se obse a am di e enças en e aças. Foi obse ada in e ação en e a aça e a es ição alimen a nos pe is de ácidos go dos da glândula mamá ia, sendo a aça Palme a mais susce í el ao a amen o. A aça Majo e a não ap esen ou a iações nos pe is de ácidos go dos, no en an o oi a aça com mais a iações nos pe is me abólicos. Es es esul ados pode ão indica uma maio ole ância à es ição alimen a , uma ez que não o am solici adas as p incipais ese as ene gé icas co po ais. Em ge al, a aça Majo e a é mais ole an e à es ição alimen a , sendo po isso mais adequada à o imização de aças. Rela i amen e às aças o inas, a aça Do pe ap esen ou menos a iações em ambos os ecidos. Po ou o lado, a aça Me ina oi a que ap esen ou mais a iações en e o g upo con ol e o g upo de es ição alimen a , es ando na sua maio ia elacionadas com a mobilização de p o eínas e de ecido adiposo. A aça Dama a ap esen ou uma espos a in e média en e as an e io men e mencionadas, com di e enças na composição muscula e nas ias me abólicas elacionadas com a p odução de ene gia. As di e enças obse adas nes a aça e le em a sua p incipal ca ac e ís ica dis in i a, a ese a adiposa na cauda (cauda-go da). As aças Do pe e Dama a demons a am maio capacidade adap a i a à es ição alimen a a a és da p odução de ! 4! Milk composi ion can also be a ec ed by umen diges ion p oduc s h ough blood s eam and mamma y gland. Mamma y gland has a e y complex ana omy and physiology (Figu e 1.2). B ie ly, milk is sec e ed by al eola epi helial cells in o he lumen and hen eleased hough he duc s. Milk cons i uen s could a ise om i e pa hways: I) he exocy o ic pa hway, esponsible o he sec e ion o wa e , lac ose, oligosacha ides, phospha e, calcium and ci a e; II) he lipidic sec e ion pa hway esponsible o he syn hesis o iacylglyce ides and phospholipids; III) he anscy o ic pa hway, whe eby p o eins and mac omolecules a e anspo ed; IV) he memb ane anspo pa hway whe eby ions and small molecules a e ans e ed om blood o milk; and V) he pa acellula anspo pa hway, esponsible o di ec mo emen o molecules be ween milk and in e s i ial space, bu could be inac i e du ing lac a ion in mos o he species12. Fo mo e de ails on mamma y gland mo phology and physiology, pa icula ly in small uminan s, kindly e e o he e iew by Lé ias e al. (2013)13. ! ! ! ! ! ! ! ! ! 5! Figu e 1.2. Diag am o he al eola epi helial cell and he pa hways o milk sec e ion. Pa hways: I) exocy olic sec e ion, II) a sec e ion, III) esicula anscy osis, IV) memb ane anspo , V) pa acellula anspo . Key: (SV) sec e o y esicle; (RER) ough endoplasmic e iculum; (BM) basemen memb ane; (N) nucleus; (PC) plasma cell; (FDA) a deple ed adipocy e; (JC) junc ional complex including he igh and adhe ens junc ions; (GJ) gap jun ion; (ME) myoepi helial cell; (MFG) milk a globule. Adap ed om McManaman and Ne ille, 2003. 1.3 SEASONAL WEIGHT LOSS AND ANIMAL PRODUCTION In he opics and he Medi e anean a eas, animal p oduc ion a ies conside ably h oughou he yea . In hose egions, he exis ence o wo di e en seasons ( ainy and d y), egula e pas u e a ailabili y, a ec ing animal p oduc ion. In ac , du ing he d y season, due o he sca ci y o pas u es, animals may lose up o 40% o hei body weigh , a condi ion usually known as Seasonal Weigh Loss (SWL)14,15. SWL is one o he majo d awbacks in animal p oduc ion16–21, as had been demons a ed in Sou h A ica16,17, Wes e n A ica18,19, Wes e n Aus alia20 and he Cana y Islands22–24. Mea and milk p oduc ion is s ongly a ec ed by SWL. In gene al, quan i y and quali y dec ease and educe income. To coun e ! 6! he e ec s o SWL, a me s use supplemen a ion o balance he nu i ional need o he animals. Howe e , supplemen a ion is expensi e and di icul o implemen in ex ensi e p oduc ion sys ems in de eloping coun ies o emo e loca ions. An al e na i e me hod o add essing he e ec s o SWL is he use o b eeds na u ally adap ed o his cons ain o selec ed by domes ica ion, ha a e able o h i e and mo e e ec i ely p oduce in such di icul en i onmen s. SWL opic was ex ensi ely e iewed ecen ly and he e ec s o SWL in uminan s, pig and abbi s was summa ized. As majo sou ces o p o ein p oduc s, hese species we e mainly s udied by p o eomics app oaches. Feed- es ic ion was used as majo p omo e o he SWL condi ions. Iden i ied bioma ke s candida es o SWL ole ance in hese animals a e ela ed wi h p o ein (muscle b eakdown) and a y acid me abolisms. Du ing lac a ion and mea p oduc ion animals can expe ience nega i e ene gy balances ha a e exace ba ed by he pas u e sca ci y, inc easing SWL a ec s25. Howe e , depending on he species and b eed, animals had de eloped di e en adap a ions o cope wi h SWL. 1.3.1 Goa s and Seasonal Weigh Loss The goa e sa ili y o adap and p oduce in ha sh en i onmen al condi ions a e due o speci ic a ibu es o he species ha can dis inguish i om he o he uminan s. Goa s ha e a ela i ely small body size, low me abolic equi emen s, capaci y o educe me abolism, abili y o change eeding s a egies, an e icien use o high- ib e o ages, he capaci y o educe ni ogen needs, and hey can make an e icien use o wa e 26. Besides, goa eeding beha iou seems also o a y be ween ! 7! b eeds and geno ypes, enhancing hei adap a ion capaci y27. The e o e, goa s seems o be he domes ic uminan specie mo e ole an o ha sh en i onmen s27,28 and can inc ease he esilience o popula ions mo e exposed o he e ec o se e e changes in clima e, economy and heal h, and p o ide a way o main ain asse s29, pa icula ly in Wes e n and Sou he n A ica30. The in e es o goa dai y p oduc s has inc eased signi ican ly wo ldwide31, especially in de eloped coun ies as a heal hie subs i u e o bo ine milk, wi h low alle genic isks and impo an nu i ional p ope ies32,33, and as gou me ing edien s8. 1.3.2 Sheep and Seasonal Weigh Loss The capaci y o sheep o ole a e ha sh en i onmen s is mainly ensu ed by e icien a deposi ion and adap abili y o di e en eed sou ces. Animals can s o e ene gy supplies in adipose issues, in pe iods du ing which ood is abundan and o good quali y20,34,35. Du ing sca ci y pe iods, hese supplies can be mobilized o mee ene gy demands, allowing animals o cope and su i e d y seasons, p egnancy and lac a ion34. Fa - ailed and a - ump sheep b eeds a e he mos ep esen a i es o his adap a ion. Since ail and ump a depo s a e he mos a ec ed by nu i ional a ia ions, hey can be used as indica o s o he ole ance le el o he animal34. Some wo ks ollowed his app oached o s udy he e ec o nu i ional es ic ion in sheep20,21,35–37. Sheep p oduc ion is one o he majo comme cial ac i i ies in some coun ies o he Sou he n hemisphe e, such as Aus alia, New Zealand, A gen ina o Sou h A ica10,38. ! 8! Unde s anding he biochemical and physiological mechanisms by which, such b eeds a e able o cope wi h SWL, could esul in an e icien p oduc ion p ocess ha could include aspec s such as inc easing o p oduc ion yields, ea lie de ec ion o pa hologies and a mo e sensi i e quali y con ol. A good managemen o hese b eeds could also con ibu e o be e policies o wa e and land use and clima e change con ol. Fa ming animals mo e adap ed o he en i onmen al condi ions will also con ibu e o enhanced animal wel a e. In his con ex , me abolomics-based s udies allow he analysis o he me abolome o a gi en sample and he co ela ion wi h he o he Omics a eas, con ibu ing o a mo e comp ehensi e o e iew o he animal physiology and p oduc ion po en ial39. 1.4 THE USE OF METABOLOMICS AS AN ANALYTICAL TOOL Me abolomics s udies a e gene ally conce ned wi h he iden i ica ion and quan i ica ion o small me aboli es (compounds o low-molecula weigh , MW<1.5 kDa) ha esul om se e al me abolic pa hways. The me abolome is di ec ly ela ed o gene exp ession, p o ein ac i i y and/o he me abolic ac i i ies40–42, and is highly complemen a y o o he Omics- based s udies such as P o eomics and T ansc ip omics, con ibu ing o a mo e comp ehensi e o e iew o he animal physiology and p oduc ion po en ial39. Mos o he me abolomics s udies a e de eloped using Mass Spec ome y (MS) o Nuclea Magne ic Resonance (NMR) echniques. Each me hodology has speci ic ea u es ha could ep esen ad an ages o disad an ages. The selec ion o he echnique ha be e i s each ! 9! wo k is hen depended on he objec i es o be achie ed43. Usually, due o hei complemen a y cha ac e , bo h echniques a e used. In his s udy we selec ed he NMR echnique o assess aqueous NMR- isible me abolome o ou samples, and a compa ison wi h MS will no be pu sued. NMR echnique is based on he physical p ope ies o a omic nuclei, speci ically spin and nuclea magne ism. The spin o a nucleus is a quan um p ope y ha can exis in wo s a es ha can be hough o as a ba magne poin ing up o down. Spin can be nai ely isualized, like a plane o a ing along i s axis. Nuclea magne ism is he capaci y o he nuclea pa icles o ac as magne s and in e ac wi h o he magne ic ields. These ex e nal magne ic ields can come om he molecula en i onmen (o he nuclei o elec ons o he same molecule), o ex e nal sou ces like he magne ic ield p oduced by an NMR spec ome e . The magne ic s a es gene a ed by he wo spin o ien a ions a e a ec ed by he magne ic ield in opposi e ways and his esul s in an ene gy di e ence be ween hem. These di e en spin s a es can be in e - con e ed by abso bing and e-emi ing elec omagne ic adia ion o a speci ic equency. These physical p ope ied can be ansla ed o chemical in o ma ion in he o ms o esonance equency (chemical shi ) and spin coupling (J-coupling). Chemical shi ep esen s he sligh di e ences in esonance equency due o he magne ic mic oen i onmen gene a ed by he neighbou ing nuclei and elec ons while J-couplings a e due o he di ec magne ic in e ac ions o chemically bonded nuclei. The e o e, chemical shi ca ies in o ma ion abou he chemical su oundings o each nucleus, and he J-coupling con ains in o ma ion o he di ec ly bonded nuclei. In addi ion, he signal in ensi y is di ec ly p opo ional o he numbe o a oms ha gi e ise o i ! 10! and he signal can he e o e be used o quan i a ion44. Finally i should be no ed ha he biologically ele an iso opes ha a e ideal o NMR a e 1H, 13C, 15N and 31P o which we will only be dealing o he i s wo, wi h he as majo i y o he da a being o 1H. 1.5 NMR-BASED METABOLOMICS Nume ous me abolomics s udies ha e been published since he debu o he concep . Since hen, he numbe o Nuclea Magne ic Resonance (NMR)-based s udies inc easing s eadily43. NMR spec oscopy has impo an ad an ages ha highligh i s use ulness in me abolic s udies. They include o ins ance being quan i a i e, quali a i e, non-des uc i e, non-equilib ium dis u bing45, highly ep oducible and ha ing he abili y o de ec compounds wi hin a wide ange o physiochemical p ope ies in he mic omola ange42,45. NMR-based me abolomics s udies ha e been used in a m animal sciences o : in quali y analysis46, milk me abolomics47,48, sample p o enance disc imina ion49, and diagnosis50,51. The use o he NMR-me abolomics app oach o small uminan s eme ged ecen ly and has been specially applied o diagnosis52–55, bioma ke s iden i ica ion24,56–58 and animal wel a e59. These p incipal ad an ages o NMR make i e y in e es ing o me abolomics s udies, ei he applied by i sel o combined wi h o he app oaches. I s simul aneous quan i a i e and quali a i e capaci y, oge he wi h he non-in asi e and non-des uc i e na u e u ns NMR in o a p omising echnique o be used in a m animal science. ! 11! 1.6 OBJECTIVES OF THE STUDY The ele ance o di e en small uminan b eeds wi h ole ance o SWL e ec s is ecognized in animal selec ion p og ams. These b eeds could be o c ucial impo ance in de eloped coun ies, o op imize p oduc ion and con ol he nega i e aspec s o seasonal weigh loss, and in mo e ulne able egions, o inc ease p oduc ion and income. Addi ional in o ma ion abou he physiology o hese b eeds will help unde s and he mechanisms o adap a ion in a mo e in eg a ed iew and de ine he selec ion s a egies. The e o e, wi h his wo k we aim o: • Op imize me hods o : sample p epa a ion o di e en issues and luids o small uminan o igin (goa milk and mamma y gland; sheep muscle and li e ), and o es ablish me hods able o adequa ely p o iling hei me abolomes using NMR. • P o ile he me abolome and he a y acid con en o mamma y gland and milk o wo dai y goa b eeds, wi h di e en le els o ole ance o SWL as a way o help unde s and which a e he biochemical pa hways ha a e de e minan o such ai in dai y goa s. • P o ile he me abolome o muscle and li e o h ee sheep b eeds, wi h di e en le els o ole ance o SWL, unde s anding which a e he biochemical pa hways ha a e de e minan o seasonal weigh loss ole ance in mea p oducing sheep. • Rende a ailable he in o ma ion gene a ed abo e o he scien i ic communi y, speci ically in he ields o animal science and me abolomics. • Rende he in o ma ion gene a ed abo e o he gene al public. ! 12! 1.7 THESIS OUTLINE To accomplish he objec i es, h ee majo asks we e designed: “NMR Me abolomics: Me hods Op imiza ion o small uminan samples”, “S udy o he eed- es ic ion e ec s in dai y goa s” and “NMR Me abolomics and eed- es ic ion e ec s in sheep”. Figu e 1.3 ep esen s a schema ic summa y o he main objec i e, me hods and ou comes om his wo k. The i s ask, desc ibed in chap e 2, includes he op imiza ion o me hods and p ocedu es needed o ul il he ollowing asks. P incipal s eps o he p ocedu es we e es ed and selec ed o be simple, quick and e ec i e. In chap e 3, he e ec s o eed- es ic ion in dai y goa s, wi h special ocus in mamma y gland and milk aqueous me abolome and a y acid p o ile a e s udied. In his pa , majo in luence o eed- es ic ion is discussed. Chap e 4 desc ibes he e ec s o eed- es ic ion in mea -p oduce sheep. Muscle and li e me abolome was discussed in he con ex o SWL. In chap e 5 majo conclusions o each chap e will be highligh ed. Resul s om chap e 3 and 4 will be in eg a ed in he con ex o SWL. Fu u e pe spec i es o his wo k a e also discussed. ! 13! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! Figu e 1.3. G aphical abs ac . Summa y o he key objec i es, me hods and ou comes om he hesis. ! 20! 2.1 SUMMARY NMR-based me abolomics s udies in ol e he analysis o biological issues and luids h ough a speci ic se o p oceedings. He e, we desc ibe he me hods we ha e es ablished o sample p epa a ion, NMR da a acquisi ion, da a collec ion and da a analysis ha allowed us o success ully p o ile he me abolomes o a ious samples o majo in e es o esea ch in animal sciences, speci ically in uminan s. We ha e es ablished alid me hodologies o s udy he me abolomes o wo king samples: aqueous ex ac s o mamma y gland and he milk se um o dai y goa s; and aqueous ex ac s o muscle and li e o sheep. We also desc ibe impo an in o ma ion on he alida ion p ocedu es we ha e used and how hese p ocedu es may be o use by o he esea che s in his no el ield. ! 21! 2.2 INTRODUCTION Li es ock is an essen ial esou ce o o e come he wo ldwide inc easing need o ood p oduc ion. Unde s anding he physiology o di e en species is de e minan o p oduc ion op imiza ion. Me abolomics-based s udies p o ide in o ma ion abou he me abolome, ha combined wi h o he Omics-based s udies allow an enhanced o e iew o he animal physiology ha can help imp o e i s p oduc ion po en ial1. In insic a ibu es o Nuclea Magne ic Resonance (NMR) spec oscopy such as i ’s quan i a i e, quali a i e, non-des uc i e and non-equilib ium dis u bing na u e2 make i ideal o me abolic s udies. Mo eo e , i is highly ep oducible and has he abili y o de ec compounds wi hin a wide ange o physicochemical p ope ies in he mic omola o millimola ange2,3. The wide ange o applica ions o NMR ha e been demons a ed by he inc easing numbe o NMR-based s udies4. In animal and human sciences5–16, NMR-me abolomics s udies ha e been applied o se e al ypes o biological samples such as u ine17, ea s18, sali a19, se um20, amnio ic luid21, exhaled b ea h condensa e22, and se e al issues6,7,23. In a m animals, NMR echniques ha e been applied o assess ood quali y and sa e y pa ame e s23–27, and de e mine i s o igin28–30, p o ing i s b oad ange o applica ions in his a ea. Howe e , i s applica ion in small uminan s is qui e ecen and has been specially applied o diagnosis31-34, bioma ke s iden i ica ion35–38 and animal wel a e39. Mos o he NMR-me abolomics s udies a e based on 1D spec a (1H NOESY) o liquid samples ha allow he iden i ica ion and quan i ica ion o me aboli es, equi e low op imiza ion o acquisi ion pa ame e s and a e ! 22! usually as o acqui e. Addi ional echniques can be used o assis me abolome p o iling, depending on he ype o sample and he kind o analysis equi ed. Al e na i ely, he high- esolu ion magic angle spinning (HR-MAS) app oach can also be applied o we solid samples, and equi es only a small in ac piece o issue, wi hou any p e ious p epa a ion such as ex ac ions4,40. Fo liquid s a e NMR, issue ex ac ion and sample p epa a ion is usually s aigh o wa d, done in a ew s eps and in a way ha allows keeping he in eg i y o he sample as much as possible. The Me hanol/chlo o o m (M/C) me hod2,7,23,41p o ed o be mo e sui able han pe chlo ic acid ex ac ion, especially due o he low a iabili y o he echnique, he use o small sol en olumes and he highe me aboli e yields ob ained42. M/C ex ac ion me hod also a oids oxida ion o sample me aboli es and p o eins43. The me hod has been used in se e al NMR-based me abolomics s udies6,7,15,44. A e acquisi ion, da a in e p e a ion can be achie ed ia a comple e spec al assignmen and quan i ica ion - a ge ed app oach (p o iling) o ia an un a ge ed app oach (chemome ic me hod). P o iling equi es he iden i ica ion and quan i ica ion o all de ec able me aboli es in a mix u e45. Al hough mo e ime consuming, his app oach is conside ed o be mo e eliable han he chemome ic. Signal assignmen is achie ed using spec al decomposi ion, assis ed by da abases o compound spec al da a o allow peak iden i ica ion. Quan i ica ion is achie ed by compa ison o peak olumes wi h hose o s anda ds46. Ta ge ed analysis yields in o ma ion o he composi ion o each sample, howe e i is limi ed by he numbe o compounds ha can be eliably iden i ied. NMR me abolomics employing p o iling has been used in small uminan s, ! 23! especially o diagnosis and bioma ke iden i ica ion37,39,47. This app oach has he added ad an age o p o iding in o ma ion abou he physicological me abolome p o ile o he s udied animals, which can be use ul o u u e wo ks. In con as , chemome ic analysis is based on he quan i ica ion o signals in disc e e egions o he spec a wi hou any signal in e p e a ion p io o he analysis. This is only possible when esul s a e analysed wi h s a is ical ools. While chemome ics is as and con enien i has he possible d awbacks o missing low concen a ed spec al signals ha a e loca ed nea by high concen a ed ones48 and is also a ec ed by small a ia ions in chemical shi s43. The chemome ic app oach does no allow sample p o iling, howe e i is a use ul and as e op ion when he p o ile has al eady been es ablished, o when only he a ia ion o ew compounds is needed. I s use was al eady desc ibed in p oduc ion animals o diagnosis and quali y con ol35,49. Sample p epa a ion o NMR-me abolomics is usually an easy and swi p ocess3 al hough, o each new sample ype, se e al op imiza ion s eps ha e o be conduc ed o ensu e sample p epa a ion ep oducibili y and maximize esul s. Hence, de elopmen and adap a ion o me hodologies ha can acili a e he implemen a ion o hese s udies a e equi ed. Al hough, NMR-based me abolomics s udies a e an al eady es ablished a ea, i s use in a m animals is s ill limi ed. As such, p o ocol op imiza ions could help u he de elop his opic. In his wo k we desc ibe he sample p epa a ion me hods ha be e sui ed o ou s udies on milk, mamma y gland, muscle and li e samples om small uminan s. We u he highligh majo op imiza ions needed o comple e and ho oughly p o ile he me abolome. Ou comes a e o impo ance no only o me abolomics esea ch in he con ex o bioma ke s iden i ica ion and physiological/pa hological condi ions, bu also o ood composi ion ! 24! de e mina ion and quali y con ol. Resul s could also be use ul o o he p oduc ion species such as pig, poul y o ca le and ul ima ely o a mo e gene al use o NMR-based me abolomics in animal and ag icul u al sciences. 2.3 MATERIAL AND METHODS 2.3.1 Biological ma e ial Goa mamma y gland samples we e ob ained om wo dai y b eeds (Majo e a and Palme a), om Cana y Islands, and we e sepa a ed in a con ol (N=9) and an expe imen al g oup (N=10). Animals and nu i ional ials we e desc ibed by Lé ias e al.50. Sheep muscle and li e samples we e ob ained om am lambs o Me ino b eed and we e di ided in o a con ol (N=11) and an expe imen al g oup (N=10). Animals and sample collec ion we e desc ibed by Scanlon e al.51 and Al es e al.52. A e collec ion, samples we e immedia ely ozen in liquid ni ogen and kep a -80ºC un il p epa a ion. Milk samples we e ob ained om he dai y goa s desc ibed abo e50. A e collec ion, samples we e immedia ely ozen in liquid ni ogen and kep a -80ºC un il p epa a ion. 2.3.2 Tissue sample p epa a ion Mamma y gland, muscle and li e we e indi idually g ound o a ine powde , keeping he samples ozen du ing he whole p ocess wi h liquid ni ogen, wi h a po celain mo a and pes le. Tissue ac ion ex ac ion ! 25! p ocedu es we e pe o med in glasswa e, and samples and sol en s (me hanol, chlo o o m and wa e ) we e kep on ice h oughou he p ocess. Tissue ac ion ex ac ions we e pe o med ollowing he me hanol/chlo o o m (M/C) me hod41, wi h some adjus men s. Mamma y gland and muscle samples Mamma y gland samples weigh ed 200 mg and muscle 500 mg. A o al o 1.2 ml o a chlo o o m/me hanol mix u e (1:2, / ) was added o each sample and o exed o 1 minu e. Then 0.4 ml o chlo o o m we e added and o exed o 1 minu e, and subsequen ly 0.4 ml o wa e was added and o exed again o 1 minu e. The homogena e was cen i uged a 1935 g o 20 minu es a 4°C in a Beckman Coul e A an i J25I (Pasadena, CA, USA) cen i uge. The me hanol/wa e ac ion was sepa a ed by pipe ing and d ied in a acuum concen a o Labconco Cen iVap Concen a o (Kansas Ci y, MO, USA). E apo a ion las ed be ween 2 and 3 hou s. D ied esidue was ozen a -80°C un il NMR expe imen s. Li e samples Li e samples weigh ed app oxima ely 175 mg. A o al o 3 ml o a wa e /me hanol mix u e (1:2, / ) we e added o each sample and o exed du ing 1 minu e. Then 2 ml o chlo o o m we e added and o exed o 1 minu e, and ano he 1 ml o wa e was added and o exed again o 1 minu e. Homogena e was cen i uged a 1935 g o 20 minu es a 4°C in a Beckman Coul e A an i J25I (Pasadena, CA, USA) ! 26! cen i uge. The me hanol/wa e ac ion was sepa a ed by pipe ing and d ied in a acuum concen a o Labconco Cen iVap Concen a o (Kansas Ci y, MO, USA). E apo a ion las ed be ween 5 and 7 hou s. D ied esidue was ozen a -80°C un il NMR expe imen s. 2.3.3 Milk sample p epa a ion Milk samples (~1 ml) we e hawed on ice and weighed (925.4 o 1243.0 mg). Samples we e ul acen i uged a 110.000 g o 75 minu es a 4°C. P o ein pelle (bo om laye ) and he lipidic ac ion ( op laye ) we e emo ed and he milk se um (middle laye ) was u he cen i uged a 21.600 g o 20 minu es a 4°C. Bo h cen i uga ions we e conduc ed in a Beckman TL-100 (Pasadena, CA, USA) ul acen i uge. Milk se um was il e ed using a Vi aspin2 3 kDa cu -o spin il e du ing 2 hou s, o 3.220 g, a 4°C in an Eppendo 5810R (Hambu g, Ge many) cen i uge. Se um sample was ozen a -80°C un il p epa a ion o NMR expe imen s. 2.3.4 NMR expe imen s Sample p epa a ion All samples we e hawed on ice jus be o e p epa a ion o NMR expe imen s. Sample p epa a ion was made as ollows: o he d ied ex ac o mamma y gland and muscle samples we e added 600 µl o phospha e bu e (150 mM; pH 7.0 (pD 7.4); wi h 1 mM DSS); o he d ied ex ac o li e we e added 800 µl o phospha e bu e (100 mM; pH 7.4 (pD 7.8); wi h 0.5 mM DSS); and o 300 µl o milk se um we e added 300 ! 27! µl phospha e bu e (150 mM; pH 7.0 (pD 7.4); wi h 26.7 µM TSP). A e esuspension, all issue samples we e cen i uge a 141 000 g o 10 minu es, and he supe na an was ans e ed o an NMR ube. Milk samples we e cen i uged inside he NMR ube, o 1 minu e in a homemade cen i uge o NMR ubes. NMR da a acquisi ion P o on (1H) NMR spec oscopy was pe o med on an 800 MHz B uke A anceII+ (E lingen, Ge many) spec ome e equipped wi h a oom empe a u e iple esonance HCN Z-g adien p obe, a 298 K. 1H 1D- NOESY spec a we e collec ed as ollows: pulse sequence: “noesyp 1d”; spec al wid h: 12 ppm; mixing ime: 0.1 s; elaxa ion delay: 1 s; acquisi ion ime: 4 s. Fo all sample ypes, J-Resol ed and 2D 1H-1H COSY spec a we e also collec ed on selec ed samples using he s anda d B uke pa ame e s o p o iling, in o de o assis in esonance assignmen . All NOESY spec a we e p ocessed wi h TopSpin3.2 using exponen ial apodiza ion wi h 1Hz line b oadening and ze o illing up o 128K poin s (B uke Biospin, Bille ica, MA, USA). 2.3.5 Da a analysis Ta ge ed analysis - Me aboli e P o iling Me aboli e p o iling was pe o med using Chenomx NMR Sui e 8.0 so wa e, wi h he in e nal e e ence lib a y e sion 9, ollowing he gene al ecommenda ions o he so wa e manual. ! 28! The concen a ion o he in e nal s anda d, DSS, was used o calib a e peak olumes. No pH ma ke s we e used since he solu ions we e bu e ed. Fo he issue samples, mass co ec ed concen a ions we e ob ained by no maliza ion wi h he mass o each issue sample. Assignmen ambigui ies we e examined using he combina ion o J- Resol ed and COSY spec a oge he wi h da a om ex e nal sou ces such as published li e a u e and he me aboli e da abase o he Biological Magne ic Resonance Bank (www.bm b.wisc.edu). Un a ge ed analysis - Da a Binning Spec al binning was pe o med wi h Chenomx NMR Sui e 8.0 using uni o m binning wi h size o 0.04 ppm, om 0 o 9.5 ppm. The egions o u ea (5.65-5.88 ppm), wa e (4.68-5.15 ppm) and DSS (0.01-0.08 ppm) we e excluded. 2.3.6 S a is ical analysis S a is ical analysis was pe o med using SIMCA-P 13.0.3.0 so wa e (Ume ics AB, Umeå, Sweden). Mul i a ia e analysis es s we e pe o med o e alua e any clus e ing beha iou o he samples’ g oups. The i s app oach was made using he unsupe ised P incipal Componen Analysis (PCA), ha allows e alua ion o sample clus e ing (sco es) and iden i y me aboli es esponsible o g oup clus e ing (loadings). When PCA could no e eal any clus e ing be ween g oups, da a was analysed h ough a supe ised Pa ial Leas Squa es Disc iminan Analysis (PLS-DA) and an O hogonal Pa ial Leas Squa es ! 29! Disc iminan Analysis (OLPS-DA). These me hods e alua e epea ed a iables be ween g oups and so hey a e be e gea ed a highligh ing di e ences be ween g oups. To compa e pai ed g oups such as con ol e sus es g oups o example, we es ed he s a is ical signi icance o he esul s using a - es in Mic oso Excel. We used es ype-3 wi h 2 ails, conside ing p<0.05 o ejec null-hypo hesis. A schema ic summa y o he me hodologies used in hese samples is p esen ed in Figu e 2.1. Figu e 2.1. Summa y o he me hodology o NMR-based me abolomics o small uminan s, o issue samples (muscle, mamma y gland and li e ) and milk se um. ! 36! Figu e 2.3. Mul i a ia e analysis o he goa mamma y gland (aqueous ac ion). A) PCA sco es plo o chemome ics app oach da a (NC = 2; PC1 = 63.8%; PC2 = 12.5%), B) PCA sco es plo o p o iling app oach da a (NC = 3, PC1 = 39.5%; PC2 = 13.9%), C) PLS sco es plo o chemome ics app oach da a (NC = 3; PC1 = 57.5 %; PC2 = 15.9%; R2 = 0.811; Q2 = 0.701; pCV-ANOVA = 0.016; pe mu a ion es : 100 pe mu a ions, R2 = (0.0, 0.454), Q2 = (0.0, -0.296)), D) PLS sco es plo o p o iling app oach da a (NC = 3; PC1 = 16.8%; PC2 = 35.1%; R2 = 0.606; Q2 = 0.540; pCV-ANOVA = 0.211; pe mu a ion es : 100 pe mu a ions, R2 = (0.0, 0.736), Q2 = (0.0, -0.173)). The same compa ison was made wi h li e samples, whe e uni a ia e analysis o he p o iled da a led o he iden i ica ion o 10 me aboli es wi h di e ences be ween he wo expe imen al g oups. PCA sco es plo o he binning da a e ealed no sepa a ion be ween he wo g oups (Figu e 2.4A). PCA sco es plo o he p o iling da a shown some sepa a ion o he wo g oups in he second componen , albei wi h wo ou lie s in each ! 37! s udy g oup (Figu e 2.4B). PLS sco es plo o he binning da a (Figu e 2.4C) showed some sepa a ion o he s udy g oups by he second p incipal componen , albei wi h some dispe se poin s in bo h b eeds. Quali y pa ame e s a e no accep able and he pe mu a ion es does no alida e he model. The PLS sco es plo o he p o iling da a (Figu e 2.4D) p esen ed sepa a ion o he wo s udy g oups by he i s p incipal componen , al hough wi h ew dispe se poin s. Quali y pa ame e s a e no accep able and he pe mu a ion es does no alida e his model. ! 38! Figu e 2.4. Mul i a ia e analysis o he sheep li e (aqueous ac ion). A) PCA sco es plo o chemome ics app oach da a (NC = 2; PC1 = 63.0%; PC2 = 6.94%), B) PCA sco es plo o p o iling app oach da a (NC = 2, PC1 = 19.6%; PC2 = 16.6%), C) PLS sco es plo o chemome ics app oach da a (NC = 2; PC1 = 60.8%; PC2 = 7.93%; R2 = 0.688; Q2 = 0.305; pCV-ANOVA = 0.1285; pe mu a ion es : 100 pe mu a ions, R2 = (0.0, 0.502), Q2 = (0.0, 0.085)), D) PLS sco es plo o p o iling app oach da a (NC = 2; PC1 = 15.4%; PC2 = 9.51%; R2 = 0.249; Q2 = 0.502; pCV-ANOVA = 0.011; pe mu a ion es : 100 pe mu a ions, R2 = (0.0, 0.707), Q2 = (0.0, -0.239)). These esul s sugges ha bo h app oaches (p o iling and chemome ics) a e able o pick up di e ences be ween g oups. Howe e , in his case, he p o iling app oach seems o be mo e e ined and obus in ha analysis. This disc epancy could be due o se e al easons. Fi s ly, he numbe o a iables used in he chemome ic app oach, is much la ge ! 39! and i includes many poin s wi h jus noise. Also, chemome ics analysis can be sensi i e o sligh a ia ions in he baseline, di e ences in peak posi ions and o he ac ha in ense peaks will o e shadow he in luence o weake ones ha a e o e lapped wi h hem. All he abo e a e easily deal wi h by he p agma ic analysis pe o med du ing p o iling and hus a e no as much o an issue. On he o he hand, he analysis using chemome ics is e y as as i does no equi e p io in e p e a ion o he spec a. Fac o s esponsible o he sepa a ion o he da a g oups, by PCA o PLS, can be e alua ed by he loading plo o each p incipal componen . In chemome ics app oach, o bo h samples, i was possible o iden i y some egions o he spec a wi h impo an di e ences. Wi hin hese, we iden i ied he egions co esponding o he me aboli es ha had in u n being p e iously iden i ied o ha e signi ican di e ences in he uni a ia e analysis o p o iled da a. O he a ia ions we e o igina ed om compounds wi h isoelec ic poin nea he bu e pH and some uniden i ied peaks. Wi hin he la e some single peaks ha we e no possible o iden i y h ough he p o iling app oach we e included. Ano he limi a ion o using he quan i a i e analysis is he numbe o compounds in he da abases ha could limi he iden i ica ion o some less common me aboli es, especially in uncommon samples. In gene al, and in o de o obse e di e ences be ween sample g oups, i is necessa y o pe o m ei he uni a ia e o mul i a ia e analysis o he da a. The p o iled da a a e amenable o uni a ia e analysis which will e eal changes on speci ic me aboli es. Mul i a ia e me hods ep esen a mo e powe ul way o disce n di e ences be ween da ase s. They can be applied o bo h a ge ed (p o iling analysis) and un a ge ed da a (chemome ic analysis) and can e eal co ela ed a ia ions wi hin g oups o da a. Fo a mo e de ailed explana ion abou he mul i a ia e ! 40! me hods and he di e ences among hem kindly e e o he wo ks o Wo ley and Powe s 48 and B o and Smilde 60. Di e ences in me aboli e composi ion be ween issues could be obse ed in he 1D NMR ep esen a i e spec a in Figu e 2.5 (A – sheep li e ; B – sheep muscle; C – goa mamma y gland). Figu e 2.5. Rep esen a i e spec a a 800 MHZ, wi h a HCN Z-g adien p obe a 298 K. A) aqueous ac ion o sheep li e , B) aqueous ac ion o sheep muscle, C) aqueous ac ion o goa mamma y, D) goa milk se um. Milk Using he p o iling app oach he signals o 50 me aboli es we e iden i ied in milk se um. These me aboli es accoun o 93% o he o al peak a ea in spec a o he se a. A ep esen a i e 1D NMR spec um om goa milk ! 41! is p esen ed in Figu e 2.5D. Following his app oach, and conside ing wo s udy g oups 36 we iden i ied signi ican di e ences in 17 me aboli es. Using he binning in o ma ion, in he chemome ics app oach, he PCA analysis was able o sepa a e oughly he con ol and he expe imen al samples (Figu e 2.6A). PCA sco es o he p o iling da a (Figu e 2.6B) showed a sha p sepa a ion o he g oups, indica ing ha bo h app oaches came o simila conclusions. This obse a ion e ealed ha , in his sample, bo h me hods a e capable o ex ac ing essen ial in o ma ion o g oup samples, al hough hey a e based on di e en le els o in o ma ion. Loading plo s showed he bins esponsible o hese di e ences. As in mamma y gland, some a e om compounds sensi i e o minimal a ia ions in pH; o he s co espond o some uniden i ied peaks, especially in he a oma ic egion o he spec a; and o he s o he peaks o he compounds iden i ied wi h signi ican di e ences be ween g oups in he quan i a i e app oach. Figu e 2.6. Mul i a ia e analysis o he goa milk se um. A) PCA sco es plo o chemome ics app oach da a (NC = 3; PC1 = 25.0%; PC2 = 24.3%), B) PCA sco es plo o p o iling app oach da a (NC = 3, PC1 = 32.8%; PC2 = 13.5%). ! 42! 2.5 CONCLUSIONS Wi h his wo k, we o e guidelines and ecommenda ions ega ding he s udy o me abolomics o mamma y gland, muscle, li e and milk samples om small uminan s. Conce ning he numbe o compounds iden i ied we ollowed he mean (20 o 60) o he NMR de ec able me aboli es in a ypical issue 4,43, so we can conclude ha he p esen ed me hods a e adequa e o he na u e o hese s udies. Rega ding da a analysis, we can conclude ha bo h quan i a i e and chemome ic analysis e ie e eliable esul s, e en wi h small g oups o samples. The choice o one o hem should be aken conside ing he speci ic objec i e o each wo k. Gene ally, o a comple ely new app oach o a speci ic sample, we ecommend he p o iling o he me abolome, conside ing ha he me abolome i sel is a b and new and use ul piece o in o ma ion. A e a ce ain deg ee o knowledge is ob ained o a gi en sample, o when some ype o speci ic in o ma ion is pu sued, such as he p esence/absence o a gi en me aboli e o example, i is simple o pe o m a chemome ic analysis and check in o ma ion abou i s speci ic egion in he spec a. Also, when only he su eillance o gene al di e ences be ween g oups is equi ed, he chemome ics app oach seems o be he be e choice, gi en he educed ime needed o da a analysis and he lack o need o p e- exis en da abases. This app oach could be especially help ul in s udies conce ning diagnosis h ough a speci ic bioma ke , quali y con ol and au hen ici y es s, pa icula ly in mea and dai y p oduc s. ! 43! 2.6 ACKNOWLEDGEMENTS This wo k was suppo ed by Fundação pa a a Ciência e Tecnologia (FCT, Lisbon, Po ugal) h ough esea ch p ojec PTDC/CVT/116499/2010 (Lac a ion and milk p oduc ion in Goa (Cap a hi cus): iden i ying molecula ma ke s unde lying adap a ion o seasonal weigh loss); Doc o al g an SFRH/BD/85391/2012 (M. Palma), he Ciência 2007 (AM Almeida and M Ma zape akis); he In es igado FCT (M Ma zape akis) esea ch con ac p og ams; he Na ional NMR Facili y (RECI/BBB-BQB/0230/2012) co e unding; and P ojec LISBOA-01-0145- FEDER-007660 (Mic obiologia Molecula , Es u u al e Celula ) unded by FEDER unds h ough COMPETE2020 - P og ama Ope acional Compe i i idade e In e nacionalização (POCI) and na ional unds h ough FCT - Fundação pa a a Ciência e a Tecnologia. ! 44! 2.7 REFERENCES 1. Kada mideen, H. Genomics o sys ems biology in animal and e e ina y sciences: P og ess, lessons and oppo uni ies. Li es Sci. 166, 232–248 (2014). 2. Beckone , O. e al. Me abolic p o iling, me abolomic and me abonomic p ocedu es o NMR spec oscopy o u ine, plasma, se um and issue ex ac s. Na P o oc. 2, 2692–2703 (2007). 3. Be nini, P. e al. S anda d ope a ing p ocedu es o p e-analy ical handling o blood and u ine o me abolomic s udies and biobanks. J. Biomol. NMR. 49, 231–243 (2011). 4. Gowda, G. A. & Ra e y, D. Can NMR sol e some signi ican challenges in me abolomics? J Magn Reson. 260, 144–160 (2015). 5. Se ko a, N. e al. Me abolic p o iling o li e s and blood om obese Zucke a s. J Hepa ol. 44, 956–62 (2005). 6. Se ko a, N. J., Rose, J. C., Eppe son, L. E., Ca ey, H. V. & Ma in, S. L. Quan i a i e analysis o li e me aboli es in h ee s ages o he ci cannual hibe na ion cycle in 13-lined g ound squi els by NMR. Physiol. Genomics. 31, 15–24 (2007). 7. Mannina, L. e al. NMR me abolic p o iling o o ganic and aqueous sea bass ex ac s: implica ions in he disc imina ion o wild and cul u ed sea bass. Talan a. 77, 433–44 (2008). 8. Ma ínez-G anados, B. e al. Me abolic p o ile o ch onic li e disease by NMR spec oscopy o human biopsies. In J Mol Med. 27, 111–7 (2010). 9. Mazzei, P. & Piccolo, A. 1H HRMAS-NMR me abolomic o assess quali y and aceabili y o mozza ella cheese om Campania bu alo milk. Food Chem. 132, 1620–1627 (2012). 10. Wang, L. e al. Me abonomic P o iling o Se um and U ine by 1H NMR- Based Spec oscopy Disc imina es Pa ien s wi h Ch onic Obs uc i e Pulmona y Disease and Heal hy Indi iduals. PLosOne. 8, e65675 (2013). 11. Sundekilde, U. K., Poulsen, N. A., La sen, L. B. & Be am, H. C. Nuclea magne ic esonance me abonomics e eals s ong associa ion be ween milk me aboli es and soma ic cell coun in bo ine milk. J. Dai y Sci. 96, 290–299 (2013). 12. Diaz, S. e al. Following Heal hy P egnancy by Nuclea Magne ic Resonance (NMR) Me abolic P o iling o Human U ine. J P o eome Res. 12, 969–979 (2013). 13. P a icò, G. e al. Explo ing human b eas milk composi ion by NMR- based me abolomics. Na . P od. Res. 28, 95–101 (2014). 14. Yang, Y. e al. Sys em esponses o ch onic cold s ess p obed ia 1 H NMR spec oscopy in plasma and u ine ma ices. Mol Biosys . 11, 1425–1433 (2015). 15. Lu, J. e al. NMR-based me abolomic analysis o Halio is di e sicolo exposed o he mal and hypoxic s esses. Sci To al En i on 545, 280–288 (2016). 16. Xu, C. e al. (1)H-Nuclea Magne ic Resonance-Based Plasma ! 45! Me abolic P o iling o Dai y Cows wi h Fa y Li e . Asian-aus alas. J. Anim. Sci. 29, 219–229 (2016). 17. Emwas, A.-H. e al. S anda dizing he expe imen al condi ions o using u ine in NMR-based me abolomic s udies wi h a pa icula ocus on diagnos ic s udies: a e iew. Me abolomics. 11, 872–894 (2015). 18. Galbis-Es ada, C. e al. Di e en ial E ec s o D y Eye Diso de s on Me abolomic P o ile by 1H Nuclea Magne ic Resonance Spec oscopy. BioMed Resea ch In e na ional. 2014: 542549 (2014). 19. San one e al. Sali a me abolomics by NMR o he e alua ion o spo pe o mance. J Pha m Biomel Anal. 88, 441–446 (2014). 20. Aus dal, M. e al. Me abolomic Bioma ke s in Se um and U ine in Women wi h P eeclampsia. PLosOne, 9, e91923 (2014). 21. G aça, G. e al. (1)H NMR based me abonomics o human amnio ic luid o he me abolic cha ac e iza ion o e us mal o ma ions. J P o eome Res. 8, 4144–4150 (2009). 22. San ini, G. e al. Elec onic Nose and Exhaled B ea h NMR-based Me abolomics Applica ions in Ai ways Disease. Cu Top Med Chem. 16, 1610– 1630 (2016). 23. Bonne on , C. M. M. e al. Me abolomic s udy o a y li e s in ducks: Iden i ica ion by 1H-NMR o me abolic ma ke s associa ed wi h echnological quali y. Poul . Sci. 93, 1542–52 (2014). 24. Be am, H., S ødkilde-Jø gensen, H., Ka lsson, A. & Ande sen, H. Pos mo em ene gy me abolism and mea quali y o po cine M. longissimus do si as in luenced by s unning me hod — A 31P NMR spec oscopic s udy. Mea Sci. 62, 113–119 (2002). 25. Hu, F., Fu iha a, K., Ka o, Y. & Tanoku a, M. Nondes uc i e Quan i ica ion o O ganic Compounds in Whole Milk wi hou P e ea men by Two-Dimensional NMR Spec oscopy. J Ag ic Food Chem. 55, 4307–4311 (2007). 26. Coco, L. e al. Compa ison among Di e en Gil head Sea B eam (Spa us au a a) Fa ming Sys ems: Ac i i y o In es inal and Hepa ic Enzymes and 13C-NMR Analysis o Lipids. Nu ien s. 1, 291–301 (2009). 27. Beaucle cq, S. e al. Se um and Muscle Me abolomics o he P edic ion o Ul ima e pH, a Key Fac o o Chicken-Mea Quali y. J P o eome Res. 15, 1168–1178 (2016). 28. Rezzi, S. e al. Classi ica ion o gil head sea b eam (Spa us au a a) om 1H NMR lipid p o iling combined wi h p incipal componen and linea disc iminan analysis. J Ag ic Food Chem. 55, 9963–9968 (2007). 29. S andal, I., P aël, A., McE oy, L., Axelson, D. & Au sand, M. Disc imina ion o Cod Li e Oil Acco ding o Wild/Fa med and Geog aphical O igins by GC and 13C NMR. J Am Oil Chem Soc. 85, 105–112 (2007). 30. Sacco e al. Disc imina ion be ween Sou he n I aly and o eign milk samples using spec oscopic and analy ical da a. Food Chem. 114, 1559–1563 (2009). 31. Mahe , A. D. e al. 1H NMR spec oscopy o se um e eals unique me abolic inge p in s associa ed wi h sub ypes o su gically induced os eoa h i is in sheep. J. P o eome Res. 11, 4261–4268 (2012). ! 52! Figu e 3.1. Rep esen a i e emale specimens o Palme a (A) and Majo e a (B) goa b eeds. As in he Cana y Islands, Palme a and Majo e a goa b eeds a e mainly aised o milk p oduc ion, we ha e a ge ed he milk and he mamma y gland as s udy samples. The aim o his wo k was o cha ac e ize he me abolome and he a y acid p o ile o he mamma y gland and milk, and how hey a e a ec ed by eed- es ic ion. This wo k complemen s ecen ly s udies on he e ec o eed- es ic ion in li e weigh and milk p oduc ion in he same animals7 and on he blood me aboli es and ho monal p o iles8. Resul s will be o impo ance in animal selec ion in d ough -p one egions. Mo eo e , since a is he milk componen wi h highe in luence in o ganolep ic quali ies and he mo e a ec ed by en i onmen al and physiological condi ions9, ou comes will also help de ine s a egies in dai y p oduc s op imiza ion and selec ion, wi h applica ion o o he uminan species in d ough -p one egions. ! 53! 3.3 MATERIAL AND METHODS Animals In his expe imen , we used 19 o he animals al eady desc ibed in a companion pape (see de ails in Lé ias e al.7). He ein we used nine Majo e a b eed and en Palme a b eed adul dai y goa s om he expe imen al lock o Pico esea ch s a ion (ICIA, Valle Gue a, Tene i e, Spain). Animals we e di ided in wo se s pe b eed: a es ic ed- ed g oup (Majo e a n=5, Palme a n=4) and a con ol g oup (Majo e a n=4, Palme a n=6). Animals we e clinically heal hy du ing he s udy. S udy The s udy was conduc ed a he Facul y o Ve e ina y Medicine o he Uni e sidad de Las Palmas de G an Cana ia (A ucas, G an Cana ia, Spain) du ing 23 days, in May-June 2012. As desc ibed by Ma ínez-De La Puen e e al.10, animals om con ol g oups we e ed ollowing he guidelines ecommended by he Ins i u Na ional de la Reche che Ag onomique (INRA). This die was composed by maize, soy 44 (44% c ude p o ein), dehyd a ed luce ne, dehyd a ed bee oo , luce ne hay, and a i amin-mine al supplemen . Animals we e also ed ad-libi um wi h s anda d whea s aw. Goa s om es ic ed- ed g oups we e ed ad- libi um wi h s anda d whea s aw (low-le el o c ude p o ein, high-le el o ibe and low ene gy)11 and i amin-mine al supplemen . This die es ic ion was op imized o educe 15 – 20% o he ini ial body weigh (day 0) a he end o he expe imen al pe iod (day 23). Animals o each ! 54! nu i ional g oup we e housed oge he . Fo mo e de ails, kindly e e o Lé ias e al. 7,8. Sample collec ion Mamma y gland biopsies (27-268 mg esh issue) we e collec ed om he le hal udde . Be o e biopsy collec ion, he whole udde was cleaned and disin ec ed using po idone-iodine. Local analgesia and anaes hesia was induced by in amuscula injec ion o Xylazine (Xilagesic 20%, Calie , Ba celona, Spain). A 2 mm incision was pe o med, allowing he exposi ion o he sec e o y issue. Then biopsies we e collec ed using a scalpel blade. A e sample collec ion, mamma y gland was su u ed using Sa il 2/0 (B aun, Ba celona, Spain) and hen sp ayed wi h Te amicin (Te amicina sp ay, P ize , Mad id, Spain, con aining oxy e acycline HCl and pa en blue). Finally, p ophylac ic ea men was adminis e ed by in amuscula injec ion o Te amicin (Te amicina L.A., P ize , Mad id, Spain). Goa s we e milked daily ollowing he milking pa ame e s desc ibed by To es e al.12 and He nández-Cas ellano e al.13. Milk samples we e collec ed om he a ailable milk o he las day o expe imen be o e biopsy collec ion. All samples we e immedia ely ozen in liquid ni ogen and s o ed a -80°C un il use. 3.3.1 NMR-Me abolomics Sample p ocessing F ozen mamma y gland samples we e powde ed wi h po celain pes le and mo a con aining liquid ni ogen. Me aboli es we e hen ex ac ed ! 55! ollowing Bligh and Dye me hod wi h some modi ica ions14–17. B ie ly, o e e y mamma y gland sample, 1.2 ml cold chlo o o m/me hanol mix u e (1:2, / ) we e added and mixed. An addi ional olume o 0.4 ml o cold chlo o o m was added and mixed again. Then 0.4 ml o cold wa e was added and mixed by o exing. The homogena e was cen i uged a 1935 g o 20 minu es a 4°C in a Beckman Coul e A an i J25I (Pasadena, CA, USA) cen i uge. The chlo o o m and me hanol/wa e ac ion was sepa a ed by pipe ing and d ied in a acuum concen a o Labconco Cen iVap Concen a o (Kansas Ci y, MO, USA). The wa e -soluble esidue was dissol ed in 600 µl phospha e bu e (150 mM, pH 7.0, wi h 1 mM sodium-2,2-dime hyl-2-silapen ane-5-sul ona e (DSS), in 90% H2O and 10% D2O) and ans e ed in o a 5 mm NMR ube. Milk samples we e hawed on ice, cen i uged and il e ed ollowing he p ocedu e desc ibed by Lu e al.18 and Sundekilde e al.19 wi h some modi ica ions. Samples we e ul acen i uged a 110.000 g o 75 minu es a 4°C. P o ein pelle and he lipidic ac ion we e emo ed and he milk se um was u he cen i uged a 21.600 g o 20 minu es a 4°C. Bo h cen i uga ions we e conduc ed in a Beckman TL-100 ul acen i uge (Pasadena, USA). Milk se um was il e ed using a Vi aspin2 3-kDa cu - o spin il e (Sa o ius S edium Bio ech GmbH, Goe ingen, Ge many) cen i uged a 3.220 g o 2 hou s a 4°C in an Eppendo 5810R cen i uge (Hambu g, Ge many). 300 µl o he milk se um we e mixed wi h 295 µl phospha e bu e (300 mM, pH 7.0, in deu e ium oxide) and 5 µl 3-( ime hylsilyl)-2,2',3,3'- e adeu e op opionic acid (TSP) (0.05% w in D2O) and ans e ed o a 5 mm NMR ube. ! 56! NMR Spec oscopy P o on (1H) NMR spec oscopy was pe o med on an 800 MHz B uke A anceII+ (E lingen, Ge many) spec ome e equipped wi h a oom empe a u e iple esonance HCN Z-g adien p obe, a 298 K. 1H 1D-NOESY spec a (spec al wid h: 20 ppm; mixing ime: 0.01 s; elaxa ion delay: 4 s; acquisi ion ime: 4 s) we e collec ed o each mamma y gland sample using he “noesygpp 1d” pulse sequence. Fo he mos concen a ed sample o each g oup, addi ional spec a we e collec ed o assis wi h assignmen , namely J-Resol ed and 1H-1H COSY. The spec a acquisi ion condi ions used we e di e en om hose sugges ed by Chenomx, howe e , compa ison o he collec ed da a wi h es s acqui ed unde e e ence condi ions e ealed no signi ican di e ences and hus we e used wi hou co ec ions. Spec a we e p ocessed and analysed using TopSpin2.1 so wa e (B uke , E lingen, Ge many). The same se o expe imen s we e used o collec he spec a om he milk se um samples as ollows: 1H 1D-NOESY (spec al wid h: 12 ppm; mixing ime: 0.1 s; elaxa ion delay: 1 s; acquisi ion ime: 4 s), J- Resol ed and 1H-COSY. Spec a we e p ocessed and analysed using TopSpin3.2 (B uke , E lingen, Ge many). Me aboli e P o iling Me aboli e iden i ica ion and quan i ica ion was pe o med using Chenomx NMR Sui e 8.0 so wa e (Chenomx Inc., Edmon on, Canada), ! 57! using he in e nal e e ence lib a y (Ve sion 9), and by compa ison wi h published li e a u e o o he animals20,21. Da a Analysis The se s o concen a ions o each me aboli e o each animal we e analyzed using SIMCA 13.0.3.0 so wa e (Ume ics AB, Umeå, Sweden). S a is ical signi icances we e es ed hough uni a ia e analysis, using - es wi h 2 ails and es ype 3, conside ing p<0.05 o ejec null- hypo hesis, using Mic oso Excel. To e alua e any clus e ing beha iou hough mul i a ia e analysis, an unsupe ised P incipal Componen s Analysis (PCA) was applied o he all he se s o da a o each sample ype (mamma y gland and milk se um) o: 1) clus e he samples (sco es), and 2) iden i y me aboli es esponsible o he g oup clus e ing (loadings). Fu he mo e, when PCA did no e eal any clus e ing be ween g oups, a supe ised Pa ial Leas Squa es Disc iminan Analysis (PLS-DA) and O hogonal Pa ial Leas Squa es Disc iminan Analysis (OPLS-DA) we e pe o med. These es s e alua e epea ed a iables be ween g oups and highligh di e ences be ween g oups as p e iously used in o he me abolomics app oaches19,22–24. The small sample size ha is ypical in such s udies and he inhe en la ge numbe o a iables ob ained may a ec he consis ency o he mul i a ia e analysis used. To e alua e he consis ency o he esul s, SIMCA-P pe o ms a numbe o es s and epo s he pa ame e s Q2, pCV-ANOVA and R2 as quali y pa ame e s o he models. Q2 indica es he p edic i e abili y o he model, while R2 is he indica o o he goodness o he i . Bo h should be as closed o 1 as possible. pCV-ANOVA indica es he signi icance o PLS/OPLS and can help in he choice o he op imal numbe o ! 58! componen s. Howe e , his alue can a y du ing he alida ion p ocess24, so we will p esen i bu conside only Q2 and R2 as quali y pa ame e s. The ellipses in he sco es plo s a e d awn a he 95% con idence le el. 3.3.2 Fa y Acids P o ile Sample p ocessing Lipids om mamma y gland we e ex ac ed as desc ibed25 bu using dichlo ome hane and me hanol (2:1, ol/ ol). To al lipids we e measu ed g a ime ically, weighing he esidue a e e apo a ion o sol en s a 37ºC. Ex ac ed lipids we e con e ed o a y acid me hyl es e s (FAME) using sodium me hoxide in anhyd ous me hanol (0.5N) ollowed by hyd ochlo ic acid in me hanol (1:1, ol/ ol) and using 1 mg o nonadecanoic acid as in e nal s anda d. FAME om lyophilized milk a samples we e p epa ed by di ec ans-es e i ica ion using KOH in me hanol (2N) and ex ac ed wi h hexane26. FAME om mamma y gland and milk samples we e hen analyzed by gas ch oma og aphy using a Shimadzu 2010Plus (Shimadzu, Kyo o, Japan), equipped wi h a lame-ioniza ion de ec o a used silica capilla y column (SP-2560, 100 m, 0.2 mm in e nal diame e , and 0.20 µm ilm hickness; Supelco Inc., Belle on e, PA, USA). Ini ial o en empe a u e o 50°C was held o 1 min, inc eased a 50°C/min o 150°C and held o 20 min, inc eased a 1°C/min o 190°C and hen inc eased a 2°C/min o 220°C and held o 18 min. The injec o and de ec o empe a u es we e main ained a 250ºC. Helium was used as ca ie gas a a low a e o 1 mL/min and 1 µL o sample was injec ed. Iden i ica ion o FAME was ! 59! achie ed by compa ison o he FAME e en ion imes wi h hose o comme cial s anda d mix u es (FAME mix 37 componen s om Supelco Inc., Belle on e, PA, USA) and by elec on impac mass spec ome y using a Shimadzu GC-MS QP2010 Plus (Shimadzu, Kyo o, Japan). Da a Analysis Fo uni a ia e analysis, da a om bo h samples we e e alua ed using P oc MIXED o SAS (SAS Ins ., Ca y, NC, USA) wi h a model ha included he ea men (con ol s. es ic ed) and he b eed and hei in e ac ion as ixed e ec s. Signi icance was conside ed o p < 0.05. Mul i a ia e analysis was pe o med o mamma y gland and milk samples, using SIMCA 13.0.3.0 so wa e (Ume ics AB, Umeå, Sweden). To e alua e sample clus e ing was applied he unsupe ised P incipal Componen Analysis (PCA). Animal Wel a e Disclaime Spanish and Eu opean Union guidelines and legisla ion on ca e, use and handling o expe imen al a m animals we e ollowed. This expe imen was au ho ized by he scien i ic commi ee o he acul y o e e ina y medicine o he Uni e sidad de Las Palmas de G an Cana ia (G an Cana ia, Spain). Au ho AM Almeida holds a FELASA g ade C ce i ica e, enabling he design and conduc ion o animal expe imen a ion in he Eu opean Union. ! 60! 3.4 RESULTS 3.4.1 NMR-Me abolomics Mamma y Gland A ep esen a i e 1H 1D NOESY spec um o he mamma y gland issue, wi h examples o iden i ied me aboli es is shown in Figu e 3.2. In his speci ic example a spec um o he Majo e a b eed is p esen ed (con ol g oup). Me aboli e concen a ions in he mamma y gland om he ou expe imen al g oups a e shown in Table 3.1. A o al o 46 me aboli es we e iden i ied in he aqueous ac ion o he mamma y gland ex ac . The majo cons i uen s o he mamma y gland in all g oups a e lac ose, glu ama e, glycine, lac a e and glucose. C ea inine is he only me aboli e whose concen a ion is signi ican ly di e en be ween b eeds (p < 0.01), being wo imes highe in Majo e a b eed con ol g oup han in Palme a b eed con ol g oup. Fo he o he me aboli es, no s a is ically signi ican di e ences we e ound be ween b eeds nei he o con ol no o es ic ed- ed g oups. Addi ionally, some di e ences in me aboli e concen a ions we e obse ed be ween con ol and unde ed g oups wi hin each b eed: ace a e, AMP/ADP/ATP, IMP and phosphocholine. Changes in di e en me aboli e concen a ions a e highligh ed in Table 3.1. Among hese, he mos e iden a ia ion occu ed in adenosine- phospha es g oup (AMP/ADP/ATP), which dec eased abou 19 imes om he con ol o he es ic ed- ed g oup in Majo e a, wi hou changes in Palme a. ! ! 61! PCA o he me aboli e concen a ions in all g oups was pe o med. The sco es plo did no show any clea g ouping o he samples (Fig. 3.3A), bu a sligh disc imina ion be ween con ol and es ic ed- ed g oups (in bo h b eeds) was obse ed on he second p incipal componen . PLS-DA was pe o med in pai ed g oups: Majo e a con ol e sus Palme a con ol, Majo e a con ol e sus Majo e a es ic ed- ed, and Palme a con ol e sus Palme a es ic ed- ed. This es was able o sepa a e Majo e a con ol and Palme a con ol (Fig. 3.3B), Majo e a con ol and Majo e a es ic ed- ed g oups (Fig. 3.3C), wi h good quali y pa ame e s, wi h only wo componen s, in each es . On he o he hand, i was only possible o sepa a e Palme a con ol and Palme a g oups applying he OPLS-DA model (Fig. 3.3D). These da a indica e ha animals o di e en g oups ha e di e en esponses o he weigh loss induc ion. Loadings plo s o he PLS-DA o Majo e a b eed (con ol e sus es ic ed- ed) and he OPLS-DA o Palme a b eed (con ol e sus es ic ed- ed) a e p esen ed as supplemen a y ma e ial (Supplemen a y Figu e S3.1). ! 68! Simila ly o mamma y gland samples, iden i ied me aboli es om he ou expe imen al g oups a e shown in Table 3.2. A o al o 50 me aboli es we e iden i ied in milk se um. No di e ences we e obse ed be ween b eeds, ei he when compa ing con ol o when compa ing es ic ed- ed g oups. In con ol g oups he i s i e mos concen a ed me aboli es a e he same: lac ose, ci a e, phosphocholine, au ine and glycine. In he es ic ed- ed g oups, only he i s h ee o he ela i e mos concen a ed me aboli es a e common: lac ose, ci a e and c ea ine. Al hough wi h only signi ican di e ence in ci a e and c ea ine, hese esul s sugges highe a ia ion in ela i e concen a ion o me aboli es in es ic ed- ed g oups han in he con ol ones. Di e ences in me aboli es concen a ion be ween con ol and es ic ed- ed g oups o each b eed a e shown in Table 3.2. Se e al me aboli es showed a ia ion be ween expe imen al g oups such as ace yl-L-ca ni ine, adenosine, alanine, ca ni ine, ci a e, c ea ine, dime hylglycine, ucose, uma a e, hippu a e, lac a e, me hylmalona e, N-aceylglucosamine, succina e, ime hylamine oxide (TMAO), UDP-galac ose and UDP-glucose. Among hese, he highe a ia ion occu ed in uma a e ha dec eased 15 old in Majo e a and app oxima ely 7 imes in case o Palme a b eed. ! 69! Table 3.2. Iden i ied me aboli es in milk se um o Majo e a and Palme a b eeds ( es ic ed and con ol g oups). A e age concen a ion (mM) and s anda d de ia ion a e shown o each expe imen al g oup. In hese samples he limi o compound iden i ica ion in Chenomx so wa e was 0.0004 mM. Key: (a) me aboli es wi h signi ican di e ences be ween con ol and es ic ed g oups in one/bo h b eeds; (moie y) only he a oma ic pa o he molecule was con i m; (TMAO) ime hylamine oxide; (*) p < 0.05 when compa ed wi h con ol o he same b eed; (**) p < 0.01 when compa ed wi h con ol o he same b eed. ! 70! PCA pe o med wi h me aboli e concen a ions o samples om all g oups, e ealed an exis ing sepa a ion be ween con ol and es ic ed- ed g oups. PLS-DA models we e conduc ed o cla i y his sepa a ion be ween g oups in he ollowing se s o da a: Majo e a con ol and Palme a con ol, Majo e a con ol and Majo e a es ic ed- ed, and Palme a con ol and Palme a es ic ed- ed. This es was able o sepa a e he h ee da a se s, as displayed in Figu e 3.5, wi h good quali y pa ame e s. Figu e 3.5. Mul i a ia e analysis o milk se um me aboli es concen a ions. The ellipses in he sco es plo s a e d awn a he 95% con idence le el.. (A) PCA sco es plo o he 4 expe imen al g oups (NC= 3, PC1=32.8%, PC2=13.5%); (B) PLS sco es plo o Majo e a con ol and Palme a con ol g oups (NC=3, Q2=0.68, R2=0.52, ρCV-ANOVA=0.48, PC1=25.3%, PC2=14.6%); (C) PLS sco es plo o Majo e a con ol and Majo e a es ic ed g oups (NC=2, Q2=0.91, R2=0.59, ρCV-ANOVA=0.03, PC1=44.4%, PC2=14.2%); (C) PLS sco es plo o Palme a con ol and Palme a es ic ed g oups (NC=2, Q2=0.82, R2=0.48, ρCV-ANOVA=0.62, PC1=35.7%, PC2=12.5%). ! 71! I is no ewo hy o men ion ha only wo componen s we e needed o sepa a e con ol and es ic ed- ed g oups, in bo h b eeds, wi h good quali y pa ame e s o he model. Majo e a con ol and Palme a con ol g oups we e sepa a ed by h ee componen s. Loadings plo s o he PLS-DA o Majo e a b eed (con ol e sus es ic ed- ed) and Palme a b eed (con ol e sus es ic ed- ed) a e p esen ed in supplemen a y ma e ial (Supplemen a y Figu e S3.2). 3.4.2 Fa y Acids P o ile The lipid and a y acid con en (mg/g d y issue) and a y acid composi ion o mamma y gland issue, exp essed in pe cen age o he o al a y acids, is p esen ed in Supplemen a y Table S3.1. The mos ep esen a i e a y acids o mamma y gland issue in all expe imen al g oups we e oleic acid (18:1cis-9), palmi ic acid (16:0) and s ea ic acid (18:0), ep esen ing 69% o he o al a y acids in Palme a con ol g oup and 77% in Palme a es ic ed- ed g oup. I is no ewo hy ha 16:0 and 18:1cis-9 we e he only wo a y acid ha p esen ed signi ican (p ≤ 0.05) in e ac ions be ween es ic ed- ed and b eed. Al hough he p opo ions o 16:0 and 18:1cis-9 nume ically dec eased and inc eased espec i ely wi h eed es ic ion in bo h b eeds, only in Palme a he changes we e signi ican . In he mamma y gland, mos o he di e ences obse ed we e due o he es ic ed- ed-e ec and no b eed-e ec di e ences (p > 0.05) we e obse ed besides 16:0 and 18:1cis-9. Conce ning he e ec o eed es ic ion, i was obse ed ha he p opo ions o 12:0, 14:0, 14:1cis-9, 15:0, iso-16:0, 16:0, 18:3n-3, 18:2 cis-9, ans-11 and a ew 18:1- ans we e lowe , and only he ! 72! 17:1cis-9, 18:1cis-9 and 20:4n-6 we e highe in es ic ed- ed g oup compa ed o con ol. Thus, he e ec o eed es ic ion was also obse ed in SFA and MUFA sums, wi h he cis-MUFA p esen ing also g ea e p opo ions in es ic ed- ed g oup han in con ol. Howe e , looking o he indexes used o es ima e he SCD ac i i y only SCD-17 (17:1cis-9/(17:1cis-9+17:0) showed a endency (p=0.078) o p esen highe alues o he es ic ed- ed g oup compa ed o con ol. The PCA o he ou expe imen al g oups did no e eal any clea g oup sepa a ion (Figu e 3.6). Al hough a sligh endency o sample clus e ing by b eed along he PC1, esul s a e no obus o discuss his sepa a ion. Loadings lis o his model is p esen ed in Supplemen a y Table S3.2. Figu e 3.6. Mul i a ia e analysis o mamma y gland a y acids pe cen ages: P incipal Componen Analysis sco es o he ou expe imen al g oups (NC = 4; PC1 = 31.4%; PC2 = 18.4%; ellipse: Hos elling’s T2 (95%)). The a y acid composi ion o milk, exp essed in pe cen age o he o al a y acids, is p esen ed in Supplemen a y Table S3.3. F om he en mo e abundan a y acids in his sample, eigh a e sa u a ed a y acids ep esen ing a ound 75% o he o al a y acids. Oleic acid (18:1cis-9) ! 73! and linoleic acid (18:2n-6) a e he o he wo compounds in his bulk, no ing ha 18:1cis-9 is he second mos abundan a y acid in he sample. Milk p esen ed only wo mino a y acids wi h signi ican in e ac ion (p < 0.05) be ween b eed and ea men , namely ma ga ic acid (17:0) and hep adecenoic acid (17:1cis-9). In bo h b eeds he p opo ions o 17:0 and 17:1cis-9 we e highe in es ic ed- ed g oups compa ed o con ol g oups, howe e Majo e a showed he g ea es inc ease in bo h a y acids. The majo i y o he 46 iden i ied a y acids p esen ed di e ences due o eed es ic ion, while 10 a y acids did no show any (p > 0.05) e ec wi h ea men . Some mino a y acids also showed signi ican di e ences (p < 0.05) be ween b eeds, wi h he sho chain- a y acids, iso-14:0, 18:0, 20:0 and 22:0 p esen ing he highes p opo ions in he Palme a b eed and he 14:0, 14:1cis-9, 16:1cis-9 and 18:2 isome s he highes p opo ions in he Majo e a b eed. A dec ease in he o al sa u a ed a y acids (SFA) om con ol o es ic ed- ed g oups was also obse ed. Conce ning he cis-monounsa u a ed a y acids (cis-MUFA), es ic ed- ed g oups p esen ed highe alues han con ol g oups. The opposi e esponse was obse ed in he o al ans- MUFA es ic ed- ed g oups p esen ed a lowe pe cen age o o al ans- MUFA han con ol g oups. No signi ican di e ences we e obse ed be ween ea men s in he o al polyunsa u a ed a y acids (PUFA). Rega ding he indexes ha es ima e SCD ac i i y, excep SCDi-14, all indexes we e highe (p < 0.001) in es ic ed- ed animals compa ed o con ol. In addi ion, Majo e a b eeds showed highe (p < 0.05) indexes compa ed o Palme a b eed. No in e ac ion (p > 0.05) be ween b eed and eed es ic ion was de ec ed in any o he es ima ed indexes. A PCA o he ou expe imen al g oups e ealed sepa a ion by ea men , along o he i s p incipal componen (PC1) (Figu e 3.7). Analysing he ! 74! loadings alues o his model (Supplemen a y Table S3.4), i was possible o iden i y ha majo di e ences we e due o a ia ions in 18:1cis-9, 17:1cis-9, 15:0 and 10:0 be ween con ol and es ic ed g oups. I is no ewo hy o men ion ha he a iables ela ed as ele an in loadings we e all a y acids wi h signi ican a ia ion due o eed es ic ion (18:1cis-9 and 10:0) and due o in e ac ion be ween b eed and eed es ic ion (17:1cis-9 and 17:0) in he uni a ia e analysis. Figu e 3.7. Mul i a ia e analysis o milk a y acids pe cen ages: P incipal Componen Analysis sco es o he ou expe imen al g oups (NC = 3; PC1 = 51.3%; PC2 = 16.1%; ellipse: Hos elling’s T2 (95%)). ! 75! 3.5 DISCUSSION 3.5.1 NMR-Me abolomics NMR-based me abolomics p o ed o be a s aigh o wa d app oach o in es iga e he esponses o eed- es ic ion in mamma y gland and milk in dai y goa s. A simple 1D spec um (Fig. 3.2) enabled he iden i ica ion o me aboli es, and a p elimina y pa e n ecogni ion analysis. In his wo k, NMR also me o he impo an ad an ages o me abolomics s udies: simple sample p epa a ion, quick spec a acquisi ion and he non-des uc i e na u e o he me hod, allowing samples o be used wi h o he echniques, i necessa y. Since i is he i s ime ha mamma y gland and milk is s udied in hese goa b eeds, we ollowed bo h he uni a ia e and he mul i a ia e me hods o analyse he da a, as sugges ed in a e iew abou da a analysis in me abolomics27 and ollowed by some au ho s in speci ic me abolomics s udies19,23. Gi en he explo a o y cha ac e o he wo k i is no possible o p e iew i he s udied condi ion (SWL) will exp ess hough di e ences in a ia ions o single me aboli es o in he ela ion o mul iple me aboli es. S a is ical analysis e ealed di e ences be ween con ol and es ic ed- ed g oups and also he b eed esponse- endency o he ea men . Conce ning uni a ia e analysis, we obse ed di e ences in me aboli es be ween con ol and es ic ed- ed g oups, in bo h mamma y gland and milk. In mos o he cases, he a ia ion in uni a ia ed analysis was con i med by he loadings plo o he mul i a ia e analysis (PCA). In he mamma y gland, PCA was no able o sepa a e he ou s udy g oups. In ac , con ol and es ic ed- ed g oups, we e only sepa a ed ! 76! using PLS in he Majo e a b eed and OPLS in he Palme a b eed. Gi en he na u e o hese es s, ha o e - i models o da a, analysis equi es special a en ion. Fo his eason hese models need alida ion28. In ou s udy, we accep ed he esul s o PLS and OPLS when he quali y pa ame e s o hese es s (Q2) was abo e 0.6, al hough some au ho s ha e conside ed 0.541 and 0.430 as accep able alues. Ne e heless, his beha iou in PLS and OPLS es s sugges s ha he me abolome o he indi iduals om he Palme a b eed (con ol and es ic ed- ed g oups) a e simila , e lec ing a slowe o less exp essi e esponse o eed es ic ion on he me aboli e composi ion o his issue. In he Majo e a b eed, es ic ed- ed and con ol g oups we e easily disc imina ed sugges ing ha , in he same expe imen al ime, hese animals had a sha pe , and p omp esponse o he ea men . This ac could be a possible indica ion o an adap i e eac ion ia a s onge esponse o SWL om his b eed. This indica ion howe e should be con i med wi h mo e me abolomics expe imen s o wi h complemen a y ansc ip omic and p o eomic analysis. PCA analysis o se um milk was able o disc imina e con ol and es ic ed- ed g oups, bu no he b eeds, which by i sel , could mean ha milk is a sample wi h mo e ma ked a ia ions han he mamma y gland. Mo eo e PLS was capable o sepa a e b eeds be ween con ol animals (bo h b eeds), and con ol and es ic ed- ed g oups in each b eed. PLS analysis guided by he p e ious PCA disc imina ion ( i s g ouping) is expec ed o p oduce mo e ele an esul s28. The e o e, i seems ha he me aboli e con en o he milk is mo e sensi i e o eed es ic ion han hose om he mamma y issue, o ha he a ia ion in milk is sha pe and p omp , conside ing he expe imen al pe iod. ! 77! Mamma y gland Mos o he iden i ied compounds in he aqueous ac ion o mamma y gland ex ac ha e been p e iously desc ibed as cons i uen s o he mamma y gland29,30, as well as o o he animal issues, such as goa plasma, ed mea , squi els li e and ish muscle14,31–33. C ea inine is he only me aboli e ha signi ican ly a ies be ween con ol g oups o he wo b eeds, being wo imes highe in Majo e a han in he Palme a b eed. C ea inine is cons an ly p oduced in he body and, al hough i s concen a ion in blood depend o some ac o s (die , gende ), in gene al a ies along wi h muscle mass34. Lé ias e al.7 measu ed he li e body weigh o he s udy animals, and obse ed a signi ican highe body weigh in Majo e a con ol g oup han in Palme a con ol g oup du ing he whole expe imen al pe iod (23 days). This ac could explain he highe concen a ion o c ea inine in his g oup. Ano he publica ion wi h he same animals8, e ealed signi ican di e ences be ween c ea inine concen a ion in blood plasma, al hough in he ci ed s udy, Palme a b eed had highe c ea inine concen a ion han Majo e a b eed (con ol g oups). These a ia ions wi h appa en ly opposi e ends could be due o he milking p ocess and i s subsequen nu ien s mobiliza ion and syn hesis, o some b eed- ela ed ac o s, especially when conside ing hei di e en milk p oduc ion yields7. Signi ican di e ences we e obse ed be ween con ol and es ic ed- ed g oups in ace a e, adenosine-phospha es (AMP/ADP/ATP), IMP and phosphocholine. Inc eased concen a ion o ace a e was obse ed in he Majo e a es ic ed- ed g oup. In mammals, ace a e can be p oduced by he body, bu can also be in oduced by exogenous mechanisms such as bac e ial e men a ion in he diges i e ac 35. Fo uminan s, gu ! 84! Indeed i was epo ed ha changes in umen mic obio a could a ec he gas oin es inal communi y, since a la ge popula ion in umen easily deple e suga s and ni ogen-compounds om eeds u s71. Howe e , a ia ion in ucose in he Majo e a b eed (7 imes highe han con ol) is mo e ma ked han in Palme a b eed (4 imes highe han con ol), wha could be indica i e o di e en adap i e s a egies be ween he wo b eeds. TMAO and hippu a e, among o he compounds, we e e e ed ecen ly as a p oduc o gu mic obio a ac i i y72,73 in humans. Al hough hey we e al eady desc ibed in milk20, hei a ia ions be ween con ol and es ic ed- ed g oups could be a consequence o adap a ion o he mic obio a o he new die . As men ioned be o e, mic obio a communi ies could be a ec ed by se e al ac o s such as weigh and die and he combina ion o gu lo a in each indi idual c ea e a unique me abolome72. Indeed, he con ibu ion o mic obio a o he whole me abolome dynamics is gaining impo ance, and i is now conside ed as an essen ial ac o in physiologic in e p e a ion72,74. In his s udy, hese me aboli es only a ied in he Majo e a b eed. This ac could be pe cei ed as a di e en and quicke adap a ion o he es ic ed- ed condi ion by he Majo e a b eed compa ed o he Palme a b eed. Majo e a b eed shown a highe numbe o me aboli es wi h signi ican a ia ions (be ween con ol and es ic ed- ed g oups) han Palme a. As explained abo e, mos o hese a ia ions may be ela ed o di e en mic obio a bulks, o o mic obio a communi ies wi h di e en adap abili y o die changes. As seen in mamma y gland, all a ia ions we e consis en in bo h b eeds, sugges ing ha indeed, he Palme a b eed could ha e a di e en me abolic eac ion when exposed o die es ic ion. ! 85! Di e ences in milk composi ion migh no always be ela ed o di e ences in mamma y gland composi ion, sugges ing ha he animal ends o main ain i s egula unc ion du ing lac a ion, adjus ing he milk composi ion o he new die condi ions. Milk se um showed mo e me aboli es’ a ia ion han mamma y gland, suppo ing he idea ha milk is no only mo e suscep ible o change, bu also can e lec hese changes quickly han sec e o y issue. Also, conside ing ha we used he sec e o y issue o mamma y gland, changes in milk composi ion could be ela ed o di e ences in sec e ion egula ion no a ec ing he composi ion o he gland i sel . Mo eo e , some compounds could pass di ec ly om blood o milk wi hou any egula ion a he mamma y gland le el. This e ec o die composi ion in small uminan milk composi ion, as p e iously men ioned57,75,76, could be u he explo ed o die op imiza ion o milk p oduc ion, especially in d ough -p one egions. Conce ning he bioma ke iden i ica ion, no me aboli e seems o be a clea bioma ke candida e o SWL ole ance, as signi ican di e ences we e obse ed when compa ing es ic ed- ed g oups o bo h b eeds. Al hough wi hou signi ican di e ences be ween b eeds, some me aboli es such as AMP/ADP/ATP in mamma y gland and uma a e in milk se um, could be co ela ed wi h an adap i e esponse o SWL ac o s. 3.5.2 Fa y Acids P o ile Mamma y gland Mamma y gland p esen ed signi ican in e ac ions be ween b eed and eed es ic ion o 18:1cis-9 and 16:0, he wo majo a y acids o his ! 86! issue. Appa en ly, Majo e a goa s we e able o main ain he a y acid composi ion o mamma y issue when subjec ed o eed es ic ion, whe eas he Palme a b eed goa s clea ly inc eased he concen a ion o 18:1cis-9 and dec eased he concen a ion o 16:0 in he same si ua ion. Due o he impo ance o he mamma y gland o new-bo n su i al9, i is likely ha his issue has signi ican ole ance o ex e nal in luence, keeping i s in eg i y in o de o p ese e unc ions, namely lac opoiesis. Thus, he la ge abili y o Majo e a goa s o main ain he a y acid composi ion o mamma y issues when compa ed o Palme a goa s migh be a e lex o i s highe ole ance o SWL. On he con a y, because Palme a goa s a e mo e suscep i e o SWL, he dec ease in 16:0 and inc ease o 18:1cis-9 ound in eed es ic ed Palme a b eed could esul om ex ensi e a mobiliza ion in o de o compensa e he eed es ic ion and main ain milk p oduc ion as p e iously sugges ed77. Milk The milk a y acid composi ion seems o be la gely a ec ed by eed- es ic ion i espec i e o he b eed. In ou expe imen , an in e ac ion be ween b eed and eed es ic ion was only obse ed o wo mino a y acids (17:0 and 17:1 cis-9). These wo a y acids ep esen only abou 2% o he o al a y acids iden i ied, indica ing he small in luence o he b eed in milk a ia ions. In con as , he main a y acids p esen in milk such as 10:0, 12:0, 14:0, 16:0, 18:0 and 18:1 cis-9 we e only a ec ed by eed es ic ion. These esul s indica e ha milk a y acid composi ion seems o be easily a ec ed by eed- es ic ion bu ha bo h b eeds espond simila ly as also obse ed in he mul i a ia e analysis o milk a pe cen ages (Figu e 3.7). Milk a can a ise om wo sou ces, de no o ! 87! syn hesis in mamma y gland (mainly he sho and medium chain a y acids including abou 50% o he 16:0) and up ake o exogenous a y acids om ci cula ion (mainly 16:0 and C18 a y acids). Supply o exogenous a y acids o he mamma y gland comp ise mos ly he ci cula ing lipop o eins, de i ed ei he om diges i e ac o om hepa ic eassembly o non-es e i ied a y acids mobilized om he body a ese es. U iliza ion o a depo s a e especially impo an in ea ly lac a ion gi en ha up o 40% o he milk a is de i ed om mobilized a y acids78. Du ing ea ly lac a ion and unde nu i ion pe iods, adipose issues no only con ibu e o milk a sec e ion, bu also o supply ene gy o o he issues and spa e glucose and amino acids o use in he mamma y gland77. Thus, milk a y acid composi ion is qui e suscep ible o he ene gy balance s a us9. In pa icula , when he ene gy balance is nega i e, animals mobilize lipids s o ed in adipose issues o be used o ene gy p oduc ion, mainly he 16:0, 18:0 and 18:1cis-9 79. In ou s udy, he la ge dec eased in he p opo ions o de no o syn he ized a y acids including he 16:0 and he inc eased p opo ions o 18:1cis-9 and 18:0 in milk o es ic ed- ed g oup compa ed o con ols a e consis en wi h an ex ensi e a issue mobiliza ion and lack o die a y de i ed p ecu so s o he de no o a y acid syn hesis. P e ious s udies on he same animals, analysed he in luence o eed es ic ion in blood me aboli es and p o ein exp ession in he mamma y gland sec e o y issue. Bo h s udies e ealed esul s ha suppo hese conclusions. Namely, i was obse ed an inc ease in non-es e i ied FA in blood, om con ol o es ic ed- ed g oups due o highe me aboliza ion o a y acids depo s8. Conce ning p o ein exp ession, bo h b eeds exhibi ed a dec ease in exp ession o p o eins ela ed wi h a biosyn hesis80. ! 88! We also obse ed an inc ease in cis-MUFA alues in milk, om con ol o es ic ed- ed g oups, in pa icula in he 16:1cis-9, 17:1cis-9 and 18:1cis- 9. This sugges s a high s ea oyl-Coa desa u ase (SCD) ac i i y, which is esponsible o syn hesizing mos ly he cis-9 MUFA om hei espec i e a y acid. In ac , he SCD p oduc /subs a e a io compu ed wi h he a y acid p esen in milk used o es ima e he o e all SCD ac i i y suppo s hese indings. Mo eo e , he SCD seems o be mo e ac i e in Majo e a han in he Palme a b eed. Obse ed alues o linoleic acid (18:2n-6) and 18:1cis-9 we e signi ican ly highe in es ic ed- ed g oups o bo h b eeds. This esul is o pa icula ly in e es o milk p oduc ion managemen and p oduc op imiza ion, due o i s impo ance in human nu i ion and heal h9,81. In summa y, he di e en ial esponse pa e n obse ed be ween mamma y gland issue and milk could be due o he high le el o o ganiza ion o he mamma y epi helium, whose cells a e specialized in con e ing ci cula ing nu ien s in milk componen s, and he se e al sec e o y and egula o y pa hways p esen in his issue9,58. Indeed, we obse ed an in luence o b eed in eed- es ic ion condi ions in mamma y gland a y acid composi ion, wi h Majo e a goa s, an obse a ion which migh indica e a highe ole ance o SWL. In milk, he in luence o b eed in es ic ed- ed condi ions was no signi ican , a ec ing less han 2% o he o al FA in he sample. Milk a composi ion p esen ed se e al signi ican esponses o eed es ic ion in bo h b eeds. Conside ing ha a can in luence milk cha ac e is ics and e en cheese quali y and p ope ies, i opens some possibili ies o p oduc op imiza ion. The Majo e a b eed shows highe ole ance o eed es ic ion appea ing o be hen he be e b eed o wo k in a p oduc managemen con ex . ! 89! 3.6 CONCLUSIONS In gene al, he wo b eeds had mo e simila esul s in milk han o he mamma y gland. Milk composi ion is mo e a ec ed by he eed- es ic ion han by he b eed. On he o he hand, esul s om mamma y gland e ealed b eed speci ic esponses. Majo e a b eed p esen ed mo e a ia ions due o eed- es ic ion in he me aboli es p o ile han in he a y acids p o ile. Indeed, i was obse ed in luence o he b eed wi h he eed- es ic ion in he a y acids. Resul s sugges ha , du ing he ime o expe imen , Majo e a, esponse o eed- es ic ion wi h some changes in he me abolic pa hways ela ed o ene gy p oduc ion, wi h no signi ican use o body a depo s. The Palme a b eed p esen ed ewe a ia ions in he small me aboli es bu highe in e ac ion o b eed wi h eed- es ic ion in he a y acids p o ile. This b eed seems o s a using he body a esou ces as ene gy sou ce, ea lie han Majo e a b eed. These esul s could con i m ha he Majo e a b eed can cope wi h eed- es ic ion condi ions du ing longe pe iods han Palme a b eed, being hen mo e ole an o his condi ion. Milk a composi ion p esen ed se e al signi ican esponses o eed es ic ion in bo h b eeds. Conside ing ha a can in luence milk cha ac e is ics and e en cheese quali y and p ope ies, i opens some possibili ies o p oduc op imiza ion. Majo e a b eed shows highe ole ance o eed es ic ion being hen he be e b eed o wo k in he p oduc managemen con ex . ! 90! 3.7 ABBREVIATIONS LIST ADP adenosine diphospha e AMP adenosine monophospha e ATP adenosine iphospha e DSS sodium-2,2-dime hyl-2-silapen ane-5-sul ona e FA a y acid(s) IMP inosine monophospha e MUFA monounsa u a ed a y acids MW molecula weigh NMR nuclea magne ic esonance OPLS-DA o hogonal pa ial leas squa es disc iminan analysis PCA p incipal componen analysis PLS pa ial leas squa es PUFA polyunsa u a ed a y acids SFA sa u a ed a y acids SWL seasonal weigh loss TMAO ime hylamine oxide TSP 3-( ime hylsilyl)-2,2',3,3'- e adeu e op opionic acid UDP-(…) u idine diphospha e (…) 1D-COSY one dimension co ela ion spec oscopy 1D-NOESY one dimension nuclea O e hause e ec spec oscopy ! 91! 3.8 ACKNOWLEDGEMENTS Au ho s acknowledge Gonçalo G aça and Luís Gonçal es (Ins i u o de Tecnologia Química e Biológica An ónio Xa ie - UNL, Po ugal) o he suppo wi h s a is ic analysis and Chenomx So wa e. Au ho s acknowledge Susana P. Al es and Rui Bessa o he a y acids analysis. This wo k was unded by esea ch p ojec PTDC/CVT/116499/2010 om Fundação pa a a Ciência e Tecnologia (FCT, Lisbon, Po ugal). The NMR spec ome e is pa o The Na ional NMR Facili y (RECI/BBB- BQB/0230/2012). Au ho s acknowledge he collabo a ion o A. Mo ales- deLaNuez, A Sua ez-T ujillo, P. Delgado-delOlmo, D. Ma ell-Jaizme and G Cugno o hei collabo a ion in sample collec ion. Au ho s a e membe s o COST ac ion FA1002 – P o eomics in Fa m Animals and FA1308 – Dai yCa e o which ne wo king suppo is acknowledged. Au ho s M Palma and SP Al es a e unded by FCT g an s SFRH/BD/85391/2012 and SFRH/BPD/76836/2011, espec i ely. ! 92! 3.9 REFERENCES 1. He e a, R., Puyol, D., Ma Ín, E., P esa, L. & Rod íguez, P. In luence o he No h A lan ic Oscilla ion on he Cana y Islands P ecipi a ion. J Clima e. 14, 3889–3903 (2001). 2. F esno, M. R. e al. Es udio p elimina del endimien o p oduc i o de la cab a Majo e a. 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NMR-Based Milk Me abolomics. Me aboli es 3, 204–222 (2013). 21. P a icò, G. e al. Explo ing human b eas milk composi ion by NMR- based me abolomics. Na . P od. Res. 28, 95–101 (2014). 22. Yang, Y. e al. Sys em esponses o ch onic cold s ess p obed ia 1 H NMR spec oscopy in plasma and u ine ma ices. Mol Biosys 11, 1425–1433 (2015). 23. Sun, Y. e al. Cha ac e iza ion o he se um me abolic p o ile o dai y cows wi h milk e e using 1H-NMR spec oscopy. Ve Q. 34, 159–163 (2014). 24. T iba, M. N. e al. PLS/OPLS models in me abolomics: he impac o pe mu a ion o da ase ows on he K- old c oss- alida ion quali y pa ame e s. Mol Biosys 11, 13–19 (2015). 25. FOLCH, LEES & STANLEY, S. A simple me hod o he isola ion and pu i ica ion o o al lipides om animal issues. The Jou nal o biological chemis y 497–509 (1957). 26. Molken in, J. & P ech , D. Valida ion o a gas‐ch oma og aphic me hod o he de e mina ion o milk a con en s in mixed a s by bu y ic acid analysis. Eu J Lipid Sci Tech. 102, 194–201 (2000). 27. Saccen i, E., Hoe sloo , H., Smilde, A., Wes e huis, J. & Hend iks, M. Re lec ions on uni a ia e and mul i a ia e analysis o me abolomics da a. Me abolomics. 10, 361-374 (2014). 28. Wo ley, B. & Powe s, R. Mul i a ia e Analysis in Me abolomics. Cu Me abolomics 1, 92–107 (2013). 29. Rohl s, E. M., Ga ne , S. C., Ma , M. H. & Zeisel, S. H. Glyce ophosphocholine and phosphocholine a e he majo choline me aboli es in a milk. J. Nu . 123, 1762–8 (1993). 30. Rudolph, M. C. e al. Me abolic egula ion in he lac a ing mamma y gland: a lipid syn hesizing machine. Physiol. Genomics 28, 323–36 (2007). 31. Se ko a, N. J., Rose, J. C., Eppe son, L. E., Ca ey, H. V. & Ma in, S. L. Quan i a i e analysis o li e me aboli es in h ee s ages o he ci cannual hibe na ion cycle in 13-lined g ound squi els by NMR. Physiol. Genomics 31, 15–24 (2007). 32. Williams, P. G. Nu i ional composi ion o ed mea . Nu i ion & Die e ics 64, S113–S119 (2007). 33. Nagasawa, T., Hashiguchi, N. & Onode a, R. E ec o S a a ion on he Plasma Nτ-Me hylhis idine Concen a ion in Goa s. Biosci Bio ech Bioch. 57, 517 (2014). 34. Ban i, G., Fabb o, M. & Lippi, G. Rela ion be ween se um c ea inine and body mass index in eli e a hle es o di e en spo disciplines. B i J Spo Med ! 100! Figu e 4.1. Rep esen a i e male specimens o Me ino (A), Do pe (B) and Dama a (C) sheep b eeds. The aim o his wo k was o cha ac e ize he me abolome o he muscle and li e o Me ino, Dama a and Do pe sheep b eeds, and s udy he e ec o eed es ic ion in hese issues, which a e impo an om he p oduc i e and me abolic pe spec i es. We used an NMR-me abolomics based app oach, which, o he bes o ou knowledge was o he i s ime applied o hese b eeds. The esul s will be o impo ance o unde s and which biochemical pa hways a e associa ed wi h SWL ole ance in sheep and ha may bene i b eeding p og ams. 4.3 MATERIAL AND METHODS Animal expe imen The ial was ca ied ou a he Me edin Resea ch S a ion in Wes e n Aus alia, ollowing he expe imen al design and nu i ional ea men s p e iously desc ibed3. B ie ly, a o al o 72 six-mon h-old am lambs om each o he Me ino, Do pe and Dama a b eeds we e di ided in o he expe imen al die g oups (12 animals pe g oup: Me ino g ow h, Me ino es ic ed, Do pe g ow h, Do pe es ic ed, Dama a g ow h and Dama a es ic ed). All animals we e ed on comme cial pelle s and had ee ! 101! access o d inking wa e as desc ibed3. Indi idual nu i ional ea men s we e calcula ed so ha animals in he g ow h g oups gained weigh (100g/day) and animals in he es ic ed g oups los weigh (100g/day). The ial las ed 42 days, a e which animals we e slaugh e ed in a comme cial aba oi , ollowing comme cial p ac ices. Fo u he in o ma ion, kindly e e o Scanlon e al. (2013)3 and Almeida e al. (2013) 9. By he end o he nu i ion ial, gas ocnemius muscle and li e issues we e sampled and p ese ed a -80°C o u he analysis. NMR-Me abolomics Sample p ocessing Fo muscle issue we analysed 11 samples in all expe imen al g oups, wi h he excep ion o he Me ino Res ic ed g oup whe e 10 samples we e used. Fo he li e issue we used 12 samples o all g oups, wi h he excep ion o he Me ino Res ic ed g oup whe e 11 samples we e used. F ozen issues we e powde ed indi idually wi h po celain mo a and pes le wi h liquid ni ogen. Me aboli es we e ex ac ed ollowing he Bligh and Dye me hod10 wi h adap a ions. Muscle samples we e p ocessed as p e iously desc ibed o goa mamma y gland samples11. Fo li e samples he sol en olumes used we e modi ied as ollows: an ini ial 3 ml cold chlo o o m/me hanol mix u e (1:2, / ) was added o he issue and mixed, ollowed by he addi ion and mixing o 2 ml o cold chlo o o m. Then 1 ml o cold wa e was added and mixed by o exing. Fo bo h issues, he mix u e was inally cen i uged and he ! 102! me hanol/wa e ac ion was sepa a ed and d ied as p e iously desc ibed11. NMR Spec oscopy The aqueous ac ion o he muscle samples was e-suspended in 600 µl phospha e bu e (150 mM, pH 7.0/ pD 7.4, wi h 1 mM sodium-2,2- dime hyl-2-silapen ane-5-sul one (DSS), in D2O), while he wa e -soluble ac ion o he li e samples was dissol ed in 800 µl phospha e bu e (100 mM, pH 7.4/pD 7.8, wi h 0.5 mM DSS, in D2O). Samples we e ans e ed in o 5 mm NMR ubes. P o on (1H) NMR spec oscopy was conduc ed on an 800 MHz B uke A anceII+ (E lingen, Ge many) spec ome e , wi h a iple esonance HCN Z-g adien p obe, a 298 K. 1H 1D-NOESY spec a we e collec ed o each sample using he “noesyp 1d” pulse sequence (spec al wid h: 12 ppm; mixing ime: 0.1 s; elaxa ion delay: 1 s; acquisi ion ime: 4 s), ollowing he pa ame e s o p o iling ecommended om Chenomx NMR Sui e so wa e (Chenomx Inc., Edmon on, Canada). All spec a we e p ocessed wi h a line b oadening (lb) o 0.5 Hz and a inal numbe o 128K poin s. Addi ional J- esol ed spec a we e collec ed o assis wi h assignmen . All spec a we e acqui ed, p ocessed and analysed using TopSpin 3.2 (B uke , E lingen, Ge many). ! 103! Me aboli e P o iling Me aboli e iden i ica ion and quan i ica ion was ca ied ou using Chenomx NMR Sui e 8.12 so wa e (Chenomx Inc., Edmon on, Canada), using he in e nal e e ence lib a y (Ve sion 10), and wi h suppo o published da a o o he animals12–14. Da a Analysis Bo h uni a ia e and mul i a ia e analysis we e pe o med o he ob ained me aboli e concen a ions, ollowing he app oach p e iously desc ibed11. B ie ly, o uni a ia e analysis we pe o med a - es wi h 2 ails and es ype 3, conside ing p < 0.05 o ejec null-hypo hesis (equal means be ween g oups), using Mic oso Excel. Mul i a ia e analysis was pe o med using he SIMCA 13.0.3.0 so wa e (Ume ics AB, Umeå, Sweden) o unsupe ised P incipal Componen s Analysis (PCA) and supe ised Pa ial Leas Squa es Disc iminan Analysis (PLS-DA). In PLS analysis, Q2 (p edic i e abili y o he model) and R2 (goodness o he i ) we e conside ed as quali y pa ame e s o he model. Resul s we e accep ed o Q2 abo e 0.5 15. Fo PLS models, a pe mu a ion es was addi ionally pe o med, using 100 pe mu a ions and accep ing he model as “ alid” when R2Y-in e cep < 0.4 and Q2Y-in e cep < 0.05. All ellipses in he sco es plo s we e d awn a he 95% con idence le el. Animal Wel a e Disclaime All wo k in ol ing animals was conduc ed acco ding o ele an in e na ional guidelines (Eu opean Union p ocedu es on animal ! 104! expe imen a ion—Di ec i e 2010/63/EU) ha egula e he use o p oduc ion animals in animal expe imen a ion. These de ine ha in he case o expe imen s ca ied ou unde s anda d p oduc ion condi ions, no app o al om an e hics commi ee is equi ed. Ne e heless, his expe imen was conduc ed wi h he app o al o he E hics Commi ee o he Depa men o Ag icul u e and Food Wes e n Aus alia (DAFWA, Pe h, WA, Aus alia) egis e ed as p ocess 07ME06. The en i e ial was conduc ed unde he supe ision o he e e ina y au ho i y in he S a e o Wes e n Aus alia. Au ho AM Almeida holds a FELASA (Fede a ion o Eu opean Labo a o y Animal Socie y Associa ions) g ade C ce i ica e ha enables designing and ca ying ou animal expe imen a ion unde Eu opean Union egula ions. Animal managemen , handling, anspo and slaugh e we e all conduc ed eplica ing app o ed s anda d comme cial p ac ices in he Commonweal h o Aus alia and in he S a e o Wes e n Aus alia. ! 105! 4.4 RESULTS NMR-Me abolomics Muscle issue A ep esen a i e 1H 1D NOESY spec um o he sheep gas ocnemius muscle (Me ino b eed, g ow h g oup) is shown in Figu e 4.2. ! 106! Figu e 4.2. Rep esen a i e 800 MHz 1H 1D NOESY spec um o gas ocnemius muscle aqueous ac ion in Me ino b eed, g ow h g oup. Key: (1) ace yl-L-ca ni ine; (2) ADP/AMP/ATP; (3) alanine; (4) anse ine; (5) be aine; (6) ca ni ine; (7) ca nosine; (8) c ea ine/c ea ine phospha e; (9) uma a e; (10) glucose; (11) glucose-1-phospha e; (12) glucose-6-phospha e; (13) glu amine; (14) glycine; (15) IMP; (16) inosine; (17) lac a e; (18) malona e; (19) me hylmalona e; (20) myo-inosi ol; (21) NAD+/NADP+; (22) nico inu a e; (23) phenylalanine; (24) succina e; (25) au ine; (26) y osine. ! 107! A o al o 51 me aboli es we e iden i ied in he aqueous ac ions o he muscle, and he me aboli e concen a ions om he six expe imen al g oups a e p esen ed in Supplemen a y Table S4.1. The mos abundan me aboli es a e lac a e and c ea ine/c ea ine-phospha e in all b eeds, ollowed by au ine, anse ine, ca ni ine and glu amine in he Do pe and Dama a b eeds; and ca ni ine, malona e, au ine and anse ine in he Me ino b eed. Signi ican di e ences (p < 0.05) be ween g ow h and es ic ed g oups o each b eed a e p esen ed in Table 4.1. Among hese, he mo e ma ked di e ences we e obse ed in glyce ophosphocholine in he Me ino b eed, which dec eased 4.1 imes be ween g ow h and es ic ed g oups; and adenine in he Do pe b eed, which inc eased 2.5 imes om g ow h o es ic ed g oups. In he Me ino b eed, we iden i ied di e ences be ween g ow h and es ic ed g oups in ci a e, glucose-6-phospha e, glu a hione, glyce ophosphocholine, glycine, ace yl-L-ca ni ine, au ine and y osine. In he Dama a b eed, di e ences be ween g ow h and es ic ed g oups we e obse ed in glucose-1-phospha e, inosine monophospha e (IMP), isoleucine, leucine, y osine, aline, phenylalanine and au ine. In he Do pe b eed only ou me aboli es show signi ican di e ences be ween g oups: adenine, o ma e, glycine and au ine. ! 108! Table 4.1. Me aboli es in gas ocnemius muscle o Me ino, Do pe and Dama a sheep b eeds, wi h signi ican di e ences be ween g ow h and es ic ed g oups, in a leas one b eed. A e age concen a ion (mmol/ g issue) and s anda d de ia ion a e shown o each expe imen al g oup. Mul i a ia e analysis was applied o me aboli e concen a ions. PCA sco es plo o all g oups does no show any speci ic sepa a ion by g oup (Supplemen a y Figu e S4.1A), howe e i is possible o see some dis inc ion by b eed. In he PCA sco es plo o he g ow h g oups (Fig. ! 109! 4.3A) i is possible o see some sepa a ion be ween b eeds, especially Me ino and Do pe . On he o he hand, he PCA sco es plo o he es ic ed g oups e ealed no speci ic sepa a ion be ween b eeds (Supplemen a y Figu e S4.1B). Howe e , i is no ewo hy ha he Dama a es ic ed g oups show a less b oad dis ibu ion when compa ed o he dis ibu ions o he o he wo b eeds. Mul i a ia e analysis was also applied pe b eed. PCA sco es o Me ino g ow h and es ic ed g oups e ealed he wo g oups could be sepa a ed by he second p incipal componen (PC2) (Fig. 4.3B). Analysis o he loadings (Supplemen a y Table S4.2) indica es ha he me aboli es ha con ibu e mo e o his sepa a ion we e glyce ophosphocholine, ci a e, ace yl-L-ca ni ine, myo- inosi ol, glu a hione and glucose-6-phospha e. Fo Dama a and Do pe b eeds i was only possible o sepa a e hei g ow h and es ic ed g oups applying PLS analysis. Dama a g ow h and es ic ed g oups we e sepa a ed by he i s p incipal componen (PC1) wi h accep able quali y pa ame e s (Supplemen a y Figu e 4.1C). Howe e , he pe mu a ion es (Supplemen a y Figu e S4.1D) ailed he model alida ion. Conce ning he Do pe g ow h and es ic ed g oups, PLS was no able o sepa a e he g oups wi h accep able quali y pa ame e s (Supplemen a y Figu e 4.1E). Fu he mo e, pe mu a ion es (Supplemen a y Figu e S4.1F) do no alida e he model. ! 116! The dis inc a ia ions among b eeds, iden i ied by bo h s a is ical app oaches, could be indica i e o b eed-speci ic esponse o he eed- es ic ion ea men . In e es ingly, we obse ed ha some muscle me aboli es could explain di e ences be ween ea men g oups in bo h s a is ical analyses in Me ino and Dama a. In he li e , he same was obse ed in Me ino b eed. Howe e , a mo e de ailed analysis o hese a ia ions is needed o unde s and he physiological signi icance o hese obse a ions. Figu e 4.6 summa ises he majo esul s o each b eed and includes some esul s om p e ious s udies on hese animals, o help in eg a e and e alua e all he in o ma ion. ! 117! Figu e 4.6. Schema ic ep esen a ion o majo esul s in muscle and li e me abolome o es ic ed g oups, o Me ino, Dama a and Do pe b eeds, when compa ed wi h he espec i e g ow h g oup. Resul s om p e ious wo k in he same animals a e also shown, and ma ked wi h uppe case le e s (a / b). Me aboli es wi h signi ican a ia ion a e ma ke wi h s a s (* / **). Key: (a) Almeida el al., 2016. PLoS One. 11(2):e0146367; (b) an Ha en e al., 2013. Animal. 7:439-445; (*) p < 0.05; (**) p < 0.01; (FAS) a y acid syn hase; (G6Pase) glucose 6-phospha ase; (PEPCK) phosphoenolpy u a e ca boxykinase; (PFK) phospho uc okinase; (PK) py u a e kinase; (PGM) phosphoglucomu ase; (GS) glycogen syn hase; (=) no a ia ion in he pa hway; (↑) pa hway inc eased; (↓) pa hway dec eased. ! 118! 4.5.1 Me ino b eed me abolomes In muscle samples, dec eased le els o amino acids like y osine, glycine and au ine du ing es ic ion could be an indica ion o a educ ion o muscle g ow h. Tau ine le els can also be a ec ed by a educ ion in die a y cys eine le els17, making i di icul o sepa a e he e ec o die es ic ion and he inne esponse o he animal. Glycine also has an addi ional ole as a p ecu so o glucagon in glycogenolysis18, and can a ec glu a hione p oduc ion19,20 in he an ioxidan de ence mechanism. Howe e , glu a hione syn hesis can also be limi ed by die ela ed cys eine in ake21, mixing he e ec s o he es ic ed eed o hose o he me abolomics esponse. Since glu a hione is p oduced in he li e and eleased o he muscle22, i s dec eased le els in he li e could be ela ed wi h i s inc ease in muscle o he same animals. The lowe le els o asco ba e (Vi amin C) in he li e could also be ela ed o glu a hione le els23. Howe e , uminan s a e able o syn hesize i om glucose, and i s educ ion could also be ela ed o he die es ic ion. Low le els o glu a hione and asco ba e could be indica i e o oxida i e s ess in he issue24 likely as a consequence o weigh loss. Sa cosine is a p ecu so o c ea ine, so a ia ions in hei concen a ions in li e could be ela ed wi h his pa hway25,26. Le els o c ea ine/c ea ine- phospha e, wo me aboli es ela ed o ene gy p oduc ion in muscle, a e in ac inc eased in he es ic ed me ino g oup as well as in he o he b eeds. As p e iously men ioned, glycine is a p ecu so o glucagon, which, du ing low glucose le els and unde s ess condi ions, p omo es gluconeogenesis and glycogenolysis. Indeed, signi ican di e ences in some me aboli es ela ed wi h such hese me abolic pa hways we e ! 119! obse ed. Glucose-6-phospha e and UDP-glucose/UDP-gluco ona e a e in e media e p oduc s in he glycogenolysis pa hway. Thei lowe le els in muscle o he es ic ed g oup could be due o he deple ion o glucose and glycogen s ock in hese animals, due o he die limi a ion. P e ious p o eome analysis o he same muscle samples e ealed an inc eased exp ession o phosphoglucomu ase, an enzyme in ol ed in glycogenolysis and glycogenesis6. The o e exp ession occu s only in he Me ino b eed and no in he SWL ole an b eeds, con i ming ha his b eed is mo e suscep ible o eed es ic ion and has a speci ic esponse o his ea men . an Ha en e al. (2013)5 de e mined he gene exp ession o egula o y enzymes in he li e o he same animals (Do pe and Me ino), and no changes we e obse ed in he enzymes o he gluconeogenesis (phosphoenolpy u a e ca boxylase) and glycolysis (phospho uc okinase and py u a e kinase) pa hways. The exp ession le el o glucose-6-phospha ase, essen ial in glucose supply du ing eed- es ic ion, was de e mined and i s alue dec eased in es ic ed g oups. Du ing die es ic ion animals end o educe he glycoly ic pa hway and p omo e gluconeogenesis5. These esul s could hen be indica i e o a mino adap a ion o he eed- es ic ion. In he li e s o he es ic ed g oup we also obse ed lowe le els o alanine and lac a e. These wo me aboli es a e ela ed in bo h muscle and li e o he Co i and he Alanine-Glucose Cycles. A dec ease on hei concen a ions could be an indi ec consequence o he lowe le els o glucose in bo h issues. Alanine is also a s uc u al amino acid and i s concen a ion is usually low du ing die es ic ion when muscle b eakdown occu s. Glucose le els in he li e o he same animals we e de e mined in a p e ious s udy and was obse ed a signi ican dec ease be ween g ow h and es ic ed animals5, suppo ing his hypo hesis. ! 120! Ene gy om nu ien s in he die could be ob ained h ough p ocesses o he han glycolysis, especially du ing eed- es ic ion pe iods. Va ia ions in some me aboli es a e indica i e o such p ocess. The inc eased le els o ace yl-L-ca ni ine in he muscle o he es ic ed me ino g oup a e an indica ion o a mobiliza ion. This me aboli e is esponsible o he anspo o a y acids in o mi ochond ia o be oxidized and used as ene gy sou ces du ing high ene gy demanding o glucose s a a ion pe iods. P e ious esul s showed lowe exp ession le els o a y acid syn hase, an enzyme esponsible o a y acids syn hesis, in he li e o es ic ed animals5. These esul s sugges ha es ic ed- ed animals a e using a y acids as an ene gy sou ce. Du ing as ing pe iods and low ca bohyd a e die s, ke one bodies (ace oace a e and 3-hyd oxybu y a e) a e p oduced in li e . We ha e indica ions o ke one body p oduc ion in he li e o he Me ino ed- es ic ed animals, as 3-hyd oxybu y a e le el is highe in he es ic ed g oup han in he con ol g oup. Howe e , in uminan bo h ke one compounds could be a esul o umen ac i i y11,27 due ei he o changes in mic obio a p o ile o in he die composi ion. Succina e and ci a e show a ia ions be ween g oups in li e and muscle, espec i ely. Bo h me aboli es a e in e media es in he K ebs cycle wi h associa ions o o he me abolic pa hways. These a ia ions could be indica i e o a ia ions in he K ebs cycle o egula ion o o he seconda y pa hway, as he inhibi ion o glycolysis and p omo ion o gluconeogenesis by ele a ed le els o ci a e. The inc eased le els o glyce ophosphocholine in he muscle could also be ela ed wi h some egula o y p ocess, since his me aboli e is a s o age o m o choline in cy osol, wi h unc ions as muscle con ol and sou ce o me hyl g oup28. ! 121! I is no ewo hy ha , as wool p oduce s hese animals will use impo an nu i ional esou ces o wool p oduc ion, making hem una ailable o be used in o he pa hways in ha sh condi ions. Also, he wool p oduc ion con inued du ing he ial, channelling nu i ional esou ces o i and in luencing he gene al pa hways. 4.5.2 Dama a b eed me abolomes Le els o isoleucine, leucine, y osine, aline, phenylalanine and au ine dec eased in he muscle o he es ic ed g oup. All hese amino acids a e ela ed o muscle de elopmen and hei lowe concen a ions could be di ec ly linked o muscle p oduc ion dec ease. Howe e , a p e ious s udy on muscle o hese animals e ealed a unique indi idual esponse o his b eed, when compa ed wi h he Me ino and Do pe b eeds6. In he Dama a b eed he le els o desmin, a muscle-speci ic p o ein esponsible o cell a chi ec u e, inc eased, ensu ing he s uc u e and unc ion o he muscle e en i some issue mobiliza ion occu s6. Isoleucine and leucine a e also ela ed wi h o he me abolic pa hways, ela ed o ke onic bodies p oduc ion and cell g ow h egula o espec i ely, ha can in luence hei concen a ions. The educ ion o UDP-glucose/UDP-guco ona e and glucose-1-phospha e in li e and muscle espec i ely, as obse ed in he Me ino b eed, is indica i e o changes in he glycogenolysis/glycogenesis pa hway. IMP and adenine le els a e bo h lowe in es ic ed g oup, in muscle and li e espec i ely. IMP is a nucleoside and an in e media e in pu ine me abolism, om which adenine is one o he examples. This esul could be a consequence o a slowe muscle de elopmen o an imbalance in ! 122! hese issues. Choline could also be connec ed wi h cell de elopmen and balance28, and i is also educed in he li e o he es ic ed- ed animals. Va ia ions in K ebs cycle in e media es (ci a e), in umen- ela ed me aboli e ( o ma e), and in asco ba e we e obse ed in his b eed, simila ly o he Me ino b eed. Conside ing he special adap a ion o his b eed o s o e a in he ail, i is in e es ing ha changes obse ed in he me abolism a e in gene al no di ec ly ela ed o a me abolism. P e ious s udies wi h he same animal g oups sugges ha he Dama a b eed has a unique lipid me abolism, mos ly due o he pu a i e con ibu ion o he a ail as supplie o odd and b anched-chain a y acids (BCFA) o he muscle29. Howe e , i is also sugges ed ha his ole ance o eed es ic ion could also be due o some kind o peculia i ies in umen ac i i y29. Speci ically, i he Dama a b eed has some diges i e adap a ion ha can inc ease he e iciency o ib e diges ion, he ace a e-p opiona e a io will be a ec ed29,30. Indeed, in he p esen s udy, le els o ace a e in he li e o he es ic ed g oup we e lowe han in he g ow h g oup. 4.5.3 Do pe b eed me abolomes Va ia ions in glycine and sa cosine in his b eed show he same pa e n as obse ed in Me ino b eed. Highe le els o sa cosine in li e o he es ic ed g oups could be indica i e o an inc ease glu a hione p oduc ion. A he same ime, lowe le els o glycine in muscle o es ic ed g oups sugges a dec ease o glycogenolysis. P e ious esul s5 on he enzyme exp ession le els show ha enzymes ela ed wi h glycolysis (phospho uc okinase and py u a e kinase) ha e lowe ! 123! exp ession in he es ic ed g oup. Simul aneously, enzyme ela ed o glycogenesis (glycogen syn hase) did no a y be ween ea men s. Mo eo e , he same s udy showed ha le els o glucose in li e o es ic ed g oup did no di e om he alues o g ow h g oup5, sugges ing an e icien esponse o his b eed o eed es ic ion condi ion. Since ca ni ine is essen ial o a mobiliza ion and ene gy p oduc ion du ing as ing pe iods and eed es ic ion, highe le els o his me aboli e in he li e could help explain he glucose homeos asis o his b eed. P e ious esul s on he enzymes ela ed wi h a y acid syn hesis ( a y acid syn hase)5 e ealed a dec ease on i s exp ession in he es ic ed g oup, ein o cing he hypo hesis o a y acids eques o ene gy p oduc ion. As obse ed in he o he wo b eeds, au ine le els a e lowe in he muscle o he es ic ed g oup, being indica i e o lowe muscle p oduc ion and de elopmen . Howe e , p e ious esul s on enzymes in ol ed in p o ein ca abolism (glu ama e dehyd ogenase) show a dec ease in es ic ed g oups. These esul s sugges ha in he Dama a, muscle p oduc ion could be educed due o eed es ic ion, while he Do pe b eed can main ain issue unc ion and s uc u e. Adenine concen a ion was highe in muscle o he es ic ed g oup, opposi e o wha was obse ed in he li e o he Dama a b eed. This me aboli e is a nucleoside wi h unc ions in p o ein syn hesis and ene gy p oduc ion. The Do pe b eed also showed di e ences in umen- ela ed me aboli es ( o ma e) be ween he es ic ed and g ow h g oups ha could be indica i e o adap a ions in umen mic obio a as esponse o he es ic ion- ed egime. ! 124! 4.5.4 Me abolomics esul s, g ow h, ca cass ai s, lep in and insulin concen a ions In gene al, we obse ed a dec ease in muscle de elopmen , an inc ease in an ioxidan ac i i y and di e ences in ene gy p oduc ion pa hways be ween he di e en b eeds as each b eed cope wi h eed es ic ion. Do pe and Dama a b eeds seem o be mo e ole an o eed es ic ion. P e ious s udies on hese animals2,6 (Supplemen a y Table S4.8) al eady sugges ed his endency. Va ia ions in he ca cass weigh and yields and in he dimensions o eye muscle we e simila in Do pe and Dama a b eed, and bo h di e en o wha was obse ed in Me ino. Conce ning plasma pa ame e s, Dama a b eed p esen ed di e ences in lep in and insulin concen a ions, when compa ed wi h he o he b eeds. Highe le els o lep in and insulin in Dama a could be jus i ied by he exis ence o he ail a depo . Lep in is p oduced by adipose cells and is highly co ela ed wi h body a , whe eas insulin s imula es lipogenesis and a y acids es e i ica ion31. In uminan s, insulin s imula es he lep in exp ession32, which oge he wi h he body a con en , could explain he highe le el o lep in concen a ions in his b eed. In he con ex o he b eed selec ion owa ds SWL ole ance, ou esul s inally con i m ha he Do pe and Dama a b eeds ha e pe o med be e unde SWL condi ions. ! 125! 4.6 ABBREVIATIONS LIST DSS sodium-2,2-dime hyl-2-silapen ane-5-sul ona e IMP inosine monophospha e MW molecula weigh NC numbe o componen s NMR nuclea magne ic esonance PCA p incipal componen analysis PC1/PC2 p incipal componen 1/2 PLS pa ial leas squa es SWL seasonal weigh loss UDP-(…) u idine diphospha e (…) 1D-NOESY one dimension nuclea O e hause e ec spec oscopy ! 132! physiology o i s majo body cha ac e is ic, he a - ail. In he con ex o he b eed selec ion owa ds SWL ole ance, ou esul s con i m ha he Do pe and Dama a b eeds pe o med be e unde SWL condi ions. Thei adap a ion seems o be linked o a mo e e icien me abolic adap a ion o eed- es ic ion, which changes he nu i ional ene gy sou ce wi hou comp omising he o e all muscle s uc u e. A possible adap a ion a he umen le el should also be conside ed in hese b eeds, since hey p esen ed some a ia ions ela ed wi h umen mic obio a composi ion and ac i i y. 5.4 SMALL RUMINANTS AND SEASONAL WEIGHT LOSS Seasonal weigh loss has an impo an in luence in small uminan b eeding, ei he o milk o mea p oduc ion. Di e en species and di e en b eeds seem o espond in di e en ways o his condi ion. Dai y goa s end o keep he mamma y gland s uc u e and unc ion o ensu e milk p oduc ion despi e he condi ions. Milk e lec s eadily he adap a ion mechanisms and he a ia ions in die composi ion, being he e o e an excellen sample o be used in s udies o his na u e. Mea -p oducing sheep, namely he b eeds ole an o SWL, end o keep he muscle s uc u e, appealing o al e na i e ene gy sou ces bu hei s uc u al p o eins. In e es ingly, he umen mic obio a seems o be in ol ed and has an impo an ole in adap a ion p ocess in bo h species and almos all b eeds. Changes in some me aboli es a e indica i e o di e en mic obio a p o ile o kine ics, e ealing c i ical a ia ions in umen. I was al eady desc ibed22 ha uminan s could ake ad an age o he quicke ! 133! adap a ion capaci y o he umen mic obio a o adap o se e al condi ions. This capaci y is also e e ed as essen ial o small uminan es ablishmen in di e en clima e egions, and o adap a ion o he changing en i onmen du ing mig a ions (clima e, habi a and pas u e bo anical composi ion). Indi idual a iabili y wi hin g oups (he ds and b eeds) seems o ha e been essen ial o hese p ocesses22. Ou esul s, mainly on he goa s, e lec his indi idual a iabili y wi hin g oups, e en in con ol g oups. Se e al uminan species show some capaci y o selec and so ood acco ding o a ailabili y and nu i ional needs ha con e hem a wide ange o oppo uni ies22. ! 5.5 FUTURE PERSPECTIVES Acco ding o he main objec i e o his wo k, we de e mined he majo e ec s o eed- es ic ion in small uminan s. The adap i e p ocess o an en i onmen depends no only on he animal ole ance capaci y bu also on he a ailable esou ces. Along he wo k de elopmen some ques ions and hypo hesis a ise, opening he oppo uni y o con inue and expand he s udy: • In u u e s udies, an inc ease in he ime o expe imen , especially in he goa s udy could be conside ed. This could allow o iden i y addi ional a ia ions be ween b eeds. I is howe e a pe spec i e wi h ce ain limi a ions, pa icula ly om he animal wel a e poin o iew. Fu he mo e, and pa icula ly o he goa expe imen , inc easing he expe imen al pe iod could lead o an an icipa ed lac a ion end. • Me abolomic analysis o issues ela ed wi h body a depo s as adipose issue, li e and muscle should be conside ed, especially in goa s. This ! 134! in o ma ion could be help ul o comple e a sys ema ic app oach in his specie. As shown in he Dama a b eed, speci ic adap a ions a e speci ically ela ed o a ail ha in u n is essen ial o cope wi h pe iods o eed es ic ion. • Some indica ions ha he s udied di e ences could be ela ed o umen kine ics and p o ile we e ound in bo h species. The s udy o he umen mic obiome p o ile could u he elucida e mechanisms o ole ance o seasonal weigh loss. Fis ula ed animals could be used o accomplish ha . Applica ion o Omics app oaches, such as genomics and me abolomics could also allow he s udy he whole mic obiome and i s dynamics du ing he diges i e p ocess. • Cha ac e iza ion o he en i onmen s whe e he b eeds e ol ed could gi e some clues abou he adap i e p ocess. The cha ac e is ics o he endemic plan s, as chemical composi ion and he wa e e en ion capaci y, could also be de e minan o he e olu iona y success o small uminan s. • Conside ing he mos ecen guidelines in animal science, non in asi e samples should be p e e ed, no only o physiological s udies bu also o bioma ke s iden i ica ion. In his con ex , u he me abolomics s udies on milk, and sali a could also be conside ed. • Gi en he complemen a y na u e o NMR and MS, a ollow up analysis by MS could be conside ed. A use ul app oach could be he use o LC- MS/MS o iden i y pep ides ha could be esidually p esen in his ac ion a e ex ac ion. ! 135! • Implemen a ion o NMR asks as complemen a y o o he Omics app oaches could be essen ial o complemen analysis, and allow an in eg a ed and b oade sys ema ic e iew. • The p esen me hods and esul s could be a use ul s a ing poin o explo e SWL in o he li es ock species. 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E olu iona y s eps o ecophysiological adap a ion and di e si ica ion o uminan s: a compa a i e iew o hei diges i e sys em. Oecologia 78, 443–457 (1989). ! 138! ! 139! APPENDIX ! 140! SUPPLEMENTARY MATERIAL CHAPTER 3 Supplemen a y Figu e S3.1 Loadings om mamma y gland aqueous ac ion: (A) PLS loadings plo o Majo e a con ol and Majo e a es ic ed g oup; (B) OPLS loadings plo o Palme a con ol and Palme a es ic ed g oup. Key: (1) 3-me hylhis idine; (2) ace a e; (3) ace yl-L-ca ni ine; (4) adenine; (5) adenosylhomocys eine; (6) alanine; (7) AMP/ADP/ATP; (8) anse ine; (9) aspa a e; (10) benzoa e; (11) be aine; (12) ca ni ine; (13) ca nosine; (14) choline; (15) ci a e; (16) c ea ine; (17) c ea ine phospha e; (18) c ea inine; (19) o ma e; (20) uma a e; (21) glucose; (22) glu ama e; (23) glu a hione; (24) glyce ophosphocholine; (25) glycine; (26) IMP; (27) inosine; (28) isoleucine; (29) lac a e; (30) lac ose; (31) leucine; (32) me hylmalona e; (33) myo-inosi ol; (34) NAD+; (35) NADP+; (36) nico inu a e; (37) phenylalanine; (38) phosphocholine; (39) py u a e; (40) succina e; (41) au ine; (42) y osine; (43) UDP-glucose; (44) u idine; (45) aline; (46) xan hine. ! 141!