Maternal diet determines milk microbiome composition and offspring gut colonization in Wistar rats

Ci a ion: Ma ínez-Oca, P.; Alba, C.; Sánchez-Ronce o, A.; Fe nández-Ma celo, T.; Ma ín, M.Á.; Esc i á, F.; Rod íguez, J.M.; Ál a ez, C.; Fe nández-Millán, E. Ma e nal Die De e mines Milk Mic obiome Composi ion and O sp ing Gu Coloniza ion in Wis a Ra s. Nu ien s 2023,15, 4322. h ps://doi.o g/ 10.3390/nu15204322 Academic Edi o : Ma loes Dekke Ni e Recei ed: 14 Sep embe 2023 Re ised: 7 Oc obe 2023 Accep ed: 9 Oc obe 2023 Published: 10 Oc obe 2023 Copy igh : © 2023 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). nu ien s A icle Ma e nal Die De e mines Milk Mic obiome Composi ion and O sp ing Gu Coloniza ion in Wis a Ra s Paula Ma ínez-Oca 1,† , Claudio Alba 2,† , Alicia Sánchez-Ronce o 3, Tama a Fe nández-Ma celo 4, Ma íaÁngeles Ma ín4,5 , Fe nando Esc i á3,4, Juan Miguel Rod íguez 2, Ca men Ál a ez 3,4 and Elisa Fe nández-Millán3,4,* 1Ins i u o de In es igación en Ciencias de la Alimen ación (CIAL), Campus de Excelencia Cien í ica, Consejo Supe io de In es igaciones Cien í icas-Uni e sidad Au ónoma de Mad id (CSIC-UAM), 28049 Mad id, Spain; [email p o ec ed] 2Depa men o Nu i ion and Food Science, Facul y o Ve e ina y Sciences, Uni e si y Complu ense o Mad id, 28040 Mad id, Spain; [email p o ec ed] (C.A.); jm [email p o ec ed] (J.M.R.) 3 Depa men o Biochemis y and Molecula Biology, Facul y o Pha macy, Complu ense Uni e si y o Mad id, 28040 Mad id, Spain; [email p o ec ed] (A.S.-R.); [email p o ec ed] (F.E.); [email p o ec ed] (C.Á.) 4Cen o de In es igación Biomédica en Red (CIBERDEM), ISCIII, 28029 Mad id, Spain; [email p o ec ed] (T.F.-M.); [email p o ec ed] (M.Á.M.) 5Depa men o Me abolism and Nu i ion, Ins i u e o Food Science and Technology and Nu i ion (ICTAN), Consejo Supe io de In es igaciones Cien í icas (CSIC), 28040 Mad id, Spain *Co espondence: [email p o ec ed] †These au ho s con ibu ed equally o his wo k. Abs ac : Mo he ’s milk con ains a unique mic obiome ha plays a ele an ole in o sp ing heal h. We hypo hesize ha ma e nal malnu i ion du ing lac a ion migh impac he mic obial composi ion o milk and a ec adequa e o sp ing gu coloniza ion, inc easing he isk o la e onse diseases. Then, Wis a a s we e ed ad libi um (Con ol, C) ood es ic ion (Unde nou ished, U) du ing ges a ion and lac a ion. A e bi h, o sp ing eces and milk s omach con en we e collec ed a lac a ing day (L)4, L14 and L18. The V3–V4 egion o he bac e ial 16S RNA gene was sequenced o cha ac e ize bac e ial communi ies. An analysis o be a di e si y e ealed signi ican dispa i ies in mic obial composi ion be ween g oups o die a L4 and L18 in bo h milk, and ecal samples. In o al, 24 phyla we e iden i ied in milk and 18 we e iden i ied in eces, wi h Fi micu es, P o eobac e ia, Ac inobac e oido a and Bac e oido a collec i ely ep esen ing 96.1% and 97.4% o hose iden i ied, espec i ely. A highe abundance o Pas eu ellaceae and Po phy omonas a L4, and o Gemella and En e- ococcus a L18 we e egis e ed in milk samples om he U g oup. Lac obacillus was also signi ican ly mo e abundan in ecal samples o he U g oup a L4. These mic obial changes comp omised he numbe and a ie y o milk– eces o eces– eces bac e ial co ela ions. Mo eo e , inc eased o sp ing gu pe meabili y and an al e ed exp ession o goble cell ma ke s TFF3 and KLF3 we e obse ed in U pups. Ou esul s sugges ha al e ed mic obial communica ion be ween mo he and o sp ing h ough b eas eeding may explain, in pa , he de imen al consequences o ma e nal malnu i ion on o sp ing p og amming. Keywo ds: milk mic obiome; me abolic p og amming; gu coloniza ion; ood es ic ion; lac a ion 1. In oduc ion Ea ly li e ad e se e en s play an impo an ole in he e iology o many diseases. In pa icula , lac a ion is conside ed a c i ical p og amming window, being a sensi i e pe iod in which o es ablish neu al connec ions, beha io esponses and me abolic ci cui s [1,2]. Acco ding o his, al e ed ma e nal nu i ion has been he mos ex ensi ely s udied p o- g amming challenge o o sp ing me abolic diseases, including obesi y and ype 2 diabe es (T2D) [ 3 – 6 ]. Unde op imal condi ions, ma e nal milk is ega ded he bes eeding sou ce o neona es, p o iding all nu ien equi emen s o ensu e adequa e g ow h and ma u a ion. Nu ien s 2023,15, 4322. h ps://doi.o g/10.3390/nu15204322 h ps://www.mdpi.com/jou nal/nu ien s Nu ien s 2023,15, 4322 2 o 26 Howe e , a hypocalo ic o low-p o ein die du ing lac a ion can ha e a nega i e impac on b eas milk p oduc ion [ 7 ], as well as on i s con en o nu ien s [ 8 , 9 ], and he milk in ake by young pups [ 8 – 10 ], exe ing nega i e e ec s on hei de elopmen . O he s udies in oden s, unde mode a e o mild calo ic es ic ion du ing lac a ion (a 20% es ic ion o ad libi um eeding), ia me abolomic analysis, iden i ied changes in 29 me aboli es ela ed o a ious me abolic pa hways [ 11 ]. The mechanisms by which b eas milk p og ams in an s o a low o high isk o becoming obese o glucose in ole an a adul hood a e no ully de ined bu his me abolic dys unc ion has been associa ed wi h al e ed insulin sensi i i y [ 12 ], hepa ic glycogen me abolism [ 13 ], a dis up ed lep in p oduc ion p o ile [ 14 ] and damaged hypo halamic ci cui s [ 14 , 15 ]. Mo eo e , we and o he s ha e also desc ibed ha ma e nal ood es ic ion leads o al e ed in es inal ba ie ma u a ion and he al e ed unc ion o he o sp ing [ 4 , 16 , 17 ], p omo ing he de elopmen o local o ex a-in es inal in lamma o y e en s [16]. I is wo h no ing ha ma e nal milk composi ion e e s no only o essen ial nu ien s (lipids, ca bohyd a es, p o eins, i amins o mine als) bu also o o he bioac i e ac o s, such as ho mones (insulin o lep in), cy okines, immunoglobulins, and nume ous ypes o oligosaccha ides, among o he s [ 2 ]. Fo a long ime hough o be a s e ile body luid, nowadays i is widely accep ed ha b eas milk has also i s own mic obio a, able o in lu- ence newbo ns’ immune sys ems [ 18 ]. The o igin o his mic obio a is s ill unce ain. One hypo hesis sugges s ha , h ough he p ocess o b eas eeding, skin bac e ia may be ans- e ed o b eas milk o di ec ly o he pe son lac a ing. Ano he hypo hesis sugges s ha a signi ican milk e lux om he in an ’s o al ca i y h ough he nipple in o he mamma y gland may also occu . Finally, an en e o-mamma y a icking model has been p oposed as well [ 19 ]. Howe e , ega dless o he ou e ia which bac e ia en e he mamma y gland, b eas milk cons i u es he key sou ce o mic obes o neona es’ gu coloniza ion. Human s udies ha e e ealed ha he milk o heal hy women con ains a g ea di e si y o bac e ia, bu wi h a co e o gene a including S ep ococcus and S aphylococcus, ollowed by Lac obacillus,Bi idobac e ium and En e ococcus [ 20 , 21 ]. In e es ingly, he gene a S ep ococcus and S aphylococcus, along wi h Bi idobac e ium, ha e been desc ibed as pionee anae obic bac e ia colonizing he gu niche in he i s days o li e [ 20 , 22 ], demons a ing e ical mo he - o-child bac e ial ans e h ough b eas eeding [ 20 ]. The b eas milk mic obiome can a y depending on he phase o lac a ion we a e in, he ma e nal me abolic s a us o he body mass index as well as he weigh gain she expe ienced du ing p egnancy o he ype o deli e y [ 23 , 24 ]. In his ega d, i has been seen ha obese mo he s ha e a di e en and less di e se milk mic obial composi ion han ha o no mal-weigh mo he s [ 24 ], which is cha ac e ized by low le els o Bi idobac e ium bu high le els o S aphylococcus [ 25 ]. In ag eemen , lowe le els o Bi idobac e ium ha e been epo ed in eces om obese child en compa ed o hose o no mal weigh o he same age [ 26 ]. Mo eo e , animal s udies, includ- ing ou s, ha e also demons a ed ha he die es ic ion o mo he s impac s milk mic obial di e si y [ 27 ] and causes a shi in he composi ion o o sp ing gu mic obio a [ 16 , 27 ], which p ecedes he onse o me abolic synd ome [ 4 , 16 ], sugges ing a causal ela ionship. Thus, gi en he signi ican associa ion be ween mic obiome composi ion and me abolic disease ou comes, he ma e nal die a y in luence on o sp ing disease de elopmen may be pa ially explained by he die a y modula ion o milk mic obial componen s impac ing he o sp ing gu ’s p ima y coloniza ion. Howe e , o ou knowledge, no s udies ha e pe o med a de ailed cha ac e iza ion o he milk mic obiome du ing lac a ion in pa allel o he s udy o he empo al p og ess o gu mic obial coloniza ion. The e o e, he p ima y pu pose o his s udy is o de e mine he in luence o ma e nal die (a 65% ood es ic ion o Wis a a s) on he milk mic obiome and whe he o no a ia ions in he milk mic obiome a e ela ed o a ia ions in he neona e ecal mic obiome. To his end, he e we analyzed longi udinal ma e nal milk and o sp ing ecal samples ia me a axonomic sequencing, om a ew days a e bi h o he ime close weaning. Nu ien s 2023,15, 4322 3 o 26 2. Ma e ials and Me hods 2.1. Animals and Die s The Ins i u ional Animal Ca e and Use Commi ee o he Communi y o Mad id ap- p o ed all me hods o his s udy (PROEX 349/15 and PROEX 215.8/21). The in es iga o s we e acc edi ed, and he animal acili ies we e app o ed by he Gene al Adminis a ion o Ag icul u e and Li es ock o he Au onomous Go e nmen o Mad id (Spain). Ten- week-old Wis a a s, ob ained om Jan ie Labs (F ance), we e housed unde speci ic pa hogen- ee and s anda d labo a o y condi ions (12:12 h ligh cycle; 21 ◦ C) wi h ad lib access o wa e and a no mal chow die (2.9 kcal/g; 12% a , 60% ca bohyd a es and 28% p o ein; A04, SAFE). A e 10 days o habi ua ion, emales we e housed wi h males o e nigh , and wo weeks a e ma ing, p egnan a s we e andomly assigned o one o he wo ea men g oups. The i s g oup was ed ad lib wi h he men ioned s anda d chow die (con ol (C), n= 6), and he second g oup was 65% ood es ic ed du ing he las hi d o ges a ion and pos na ally (unde nou ished (U), n= 6), as p e iously desc ibed [ 16 ]. To educe he e ec o li e size on pos na al g ow h and milk in ake, li e s we e made uni o m a bi h, o eigh pups pe nu sing dam. Food es ic ion was main ained in he mo he s du ing lac a ion, and pups we e sac i iced ia decapi a ion a 4, 14 and 18 days o li e (L4, L14 and L18) o collec he di e en ypes o samples. The weigh gain o pups was moni o ed h oughou lac a ion and he body and gas oin es inal sys em leng h ( om he pylo us o he anus) we e measu ed o de e mine he nu i ional es ic ion e ec s on he o gan sizes. This animal model is designed o moni o lac a ion un il L18, which is 5 days be o e weaning, o p e en he pups om beginning o inges he mo he ’s chow die by hemsel es and hus ensu e he e was no mixed die . 2.2. Sample Collec ion Milk collec ion o i s mic obiological analysis was always conduc ed a he same ime (9:00 a.m.) and unde s e ile condi ions. Al hough a dams p oduce copious amoun s o milk ela i e ha p oduced by smalle oden s, he impac o long- e m ood es ic ion, om mid-ges a ion h ough o lac a ion, signi ican ly limi ed he a ailabili y o milk om mo he s’ b eas s. P io esea ch on oden s has sugges ed ha he composi ion o s omachal milk is simila o ha o b eas milk [ 28 ], ep esen ing e ical ansmission be ween mo he and child. Thus, a e sac i ice, he pups’ s omachs we e dissec ed, and he con en s we e kep a 4 ◦ C and immedia ely p ocessed. Likewise, ecal colonic con en was collec ed asep ically om pups in o a s e ile ube, 0.5 mL o TE50 bu e (10 mM T is-HCl and 50 mM EDTA, pH 8) was added, he mix u e was o exed un il i was homogeneous, and hen i was ozen a − 80 ◦ C un il DNA ex ac ion and mic obial s udies we e pe o med. Fo gene exp ession analysis, he dis al colon was opened longi udinally and cleansed wi h saline solu ion. Fo his ological s udies, he colon was dissec ed and, main aining i s cylind ical s uc u e and ecal con en , imme sed in Ca noy ixa i e solu ion. 2.3. Analy ical De e mina ions Be o e sac i ice ia decapi a ion, he neona es we e weighed and hen blood samples we e collec ed; hei se um was sepa a ed and s o ed ozen a − 20 ◦ C un il analysis. Se um insulin and glucagon le els we e de e mined wi h a speci ic a insulin and glucagon adioimmunoassay ki (LINCO Resea ch, Inc., Me ck Millipo e, S . Louis, MI, USA), in acco dance wi h he manu ac u e ’s p o ocol. A sensi i i y o 0.1 ng/mL and o 20 pg/mL was achie ed wi h o e nigh equilib ium using 100 µ L o he se um samples, espec i ely. The coe icien s o a ia ion wi hin and be ween assays we e 10% o insulin and 4.0 and 7.3% o glucagon, espec i ely. Se um glucose and lipid pa ame e s ( o al choles e ol and iglyce ides) we e de e mined ia he enzyma ic colo ime ic glucose oxidase/pe oxidase me hod (Biosys ems, Ba celona, Spain). Nu ien s 2023,15, 4322 4 o 26 2.4. DNA Ex ac ion 2.4.1. Milk The samples unde wen cen i uga ion a 4 ◦ C o 15 min a a ela i e cen i ugal o ce o 11,000 × g. DNA was hen ex ac ed om he esul an pelle s using QIAamp DNA S ool Mini Ki (Qiagen, Hilden, Ge many), in acco dance wi h he manu ac u e ’s guidelines, albei wi h p e iously documen ed modi ica ions [ 29 ]. The ex ac ed DNA was elu ed in 20 µ L o nuclease- ee wa e and subsequen ly s o ed a − 20 ◦ C pending addi ional analyses. The DNA concen a ion was es ima ed u ilizing NanoD op ND-1000 UV-Vis Spec opho ome e (NanoD op Technologies, Wilming on, DE, USA). The DNA ex ac ion and pu i ica ion p o ocol delinea ed abo e was also applied o wo nega i e con ol blanks, which did no con ain any sample ma e ial. 2.4.2. Feces The ozen ecal samples we e apidly hawed using a d y hea block se a 37 ◦ C, ollowed by o exing o achie e e-homogeniza ion. DNA was ex ac ed u ilizing QI- Aamp Fas DNA S ool Mini Ki (Qiagen, Ge man own, MD, USA) in conjunc ion wi h 0.1 mm diame e zi conia/silica beads (BioSpec P oduc s, Inc., Ba les ille, OK, USA) and a Fas P ep FP120A-115 homogenize (Qbiogene, Ca lsbad, CA, USA). Du ing each ound o DNA ex ac ion, 500 µ L o TE50 bu e om he same aliquo was used in he sample and 500 µ L o nuclease- ee wa e (Ambion, Wal ham, MA, USA) was used o nega i e con ols. The ex ac ed DNA samples we e elu ed in 20 µ L o ATE bu e , as p o ided in he ex ac ion ki , and s o ed a −80 ◦C un il u he analysis. 2.5. DNA Quali y, Quan i ica ion, and Sequencing DNA quali y and quan i ica ion we e assessed as p e iously desc ibed [ 20 ]. B ie ly, he concen a ion o he DNA samples was de e mined using he Quan -iT PicoG een eagen (The mo Fishe Scien i ic, Inc., Wal ham, MA, USA). Ampli ica ion a ge ed he V3–V4 egion o he 16S RNA gene. Bo h nega i e and posi i e con ol PCR p oduc s we e subjec ed o aga ose gel elec opho esis a 80 V o 30 min using 1% aga ose gel and TAE (T is-Ace a e-EDTA) bu e . The gel was s ained wi h GelRed ™ (10X, Bio ium, F emon , CA, USA) and isualized using Ul aCam Digi al Imaging Sys em (Bio-Rad, He cules, CA, USA). Amplicon quali y was assessed wi h a QIAxcel DNA Sc eening ca idge (Qiagen, Hilden, Ge many) in acco dance wi h he manu ac u e ’s guidelines. High- esolu ion capilla y elec opho esis on a QIAxcel Ad anced Sys em (Qiagen) was employed o isualiza ion. Samples demons a ing a peak a he desi ed amplicon size and minimal p ime dime o ma ion we e deemed o be o su icien quali y. Subsequen o p ima y PCR ampli ica ion ( he esul ing amplicons being app ox- ima ely 450 bp in leng h), he indi idual amplicon lib a ies we e cha ac e ized using Bioanalyze 2100 (Agilen Technologies, Palo Al o, CA, USA). The lib a ies we e hen pooled in equimola concen a ions, ollowed by pu i ica ion and quan i ica ion. The p e- cise concen a ion o he pooled sample was con i med ia eal- ime PCR (Kapa Biosys ems, Wilming on, MA, USA). Finally, he pooled DNA samples unde wen sequencing on an Illumina MiSeq ins umen , employing 2 × 300 bp pai ed-end ead sequencing a he Genomics Uni o Pa que Cien í ico de Mad id, Spain. In acco dance wi h manu ac u e speci ica ions, sequences unde wen demul iplexing acili a ed by Illumina’s so wa e pla o m ( . 2.6.2.3). Subsequen bioin o ma ic anal- ysis was conduc ed u ilizing Quan i a i e Insigh s in o Mic obial Ecology 2 (QIIME 2, e sion 2022.2) in conjunc ion wi h R S a is ical Compu ing En i onmen ( e sion 3.5.1; URL: h ps://www. -p ojec .o g/ accessed on 13 Janua y 2023). Fo he op imiza ion o sequence in eg i y, he DADA2 algo i hm was employed unde speci ic pa ame e s: o wa d eads we e unca ed pos -posi ion 295 while excising he ini ial 15 nucleo ides; con e sely, e e se eads we e unca ed a posi ion 258 wi h he p elimina y 7 nucleo ides elimina ed. This p ocedu e was implemen ed o ob ia e posi ions demons a ing a median nucleo ide quali y sco e in e io o Q20. Taxonomic a ilia ion o indi idual amplicon Nu ien s 2023,15, 4322 5 o 26 sequence a ian s (ASVs) was asce ained h ough he q2 ea u e classi ie , u ilizing a nai e Bayes axonomic classi ica ion schema agains he SILVA 138.1 e e ence da abase. Las ly, po en ial con aminan s we e iden i ied, isualized and pu ged om he da ase ia he decon am package ( e sion 1.2.1), inco po a ing da a om wo nega i e con ol samples o igo ous e i ica ion. 2.6. Me a axonomic Analysis o Bac e ial Mic obio a A ma ix de ailing ASVs speci ic o each espec i e sample was sys ema ically gene - a ed. The subsequen no maliza ion o bac e ial axa abundances was pe o med u ilizing he o al sum scaling (TSS) me hod, whe e in each disc e e ASV coun was di ided by he cumula i e lib a y size, he eby ende ing he ela i e p opo ion o indi idual ASV coun s pe sample. Fo he examina ion o alpha di e si y, bo h Shannon and Richness di e si y indices we e employed, acili a ed h ough he egan package wi hin R S a is ical Compu ing En i onmen (Ve sion 2.5.6). To p o ide a comp ehensi e assessmen o communi y s uc u e, be a di e si y was analyzed using bo h B ay–Cu is and bina y Jacca d dissimila i y me ics. These me ics se e o quan i y he composi ional dissimila i y be ween mic obial communi ies ac oss samples. All compu a ions and s a is ical manipula ions we e execu ed wi hin he R amewo k, ensu ing me hodological consis ency and analy ical igo . 2.7. Immuno luo escence S aining The immuno luo escence s aining o igh junc ion p o ein zonula occludens-1 (ZO-1) was pe o med on colonic sec ions as p e iously desc ibed [ 16 ]. B ie ly, a e being depa a - inized and ehyd a ed, he an igens we e e ie ed wi h sodium ci a e bu e (10 mM Na 3 C 6 H 5 O 7 and Tween-20 0.05%, pH 6) a 97 ◦ C o 30 min. Non-speci ic backg ound was blocked ia incuba ion wi h 2% BSA, 1% NGS and 0.2% T i on X-100 in PBS o 1 h a oom empe a u e. Sec ions we e incuba ed o e nigh a 4 ◦ C wi h abbi an i-ZO-1 (1:50, The moFishe Scien i ic, Inc., Wal ham, MA, USA) and hen p obed in he da k wi h a 1:100 dilu ion o Alexa Fluo 488-conjuga ed goa an i- abbi IgG an ibody and a 1:1000 dilu ion o 10 mg/mL o DAPI solu ion (The mo Fishe Scien i ic, Wal ham, MA, USA) o 1 h a oom empe a u e. Finally, he sec ions we e co e ed wi h P oLong Gold An- i ade Moun an (In i ogen, The mo Fishe Scien i ic, Inc., Wal ham, MA, USA). Nega i e con ol slides we e ob ained by incuba ing hem wi hou he p ima y an ibody bu wi h a seconda y Alexa Fluo an ibody. Fo quan i ica ion, images we e cap u ed using a Leica SP-2 AOBS con ocal mic oscope a 630 × magni ica ion. A leas wo non-adjacen sec ions o colon pe a we e s ained and ou images pe sec ion we e used. Two independen expe imen s we e conduc ed, wi h 2–3 a s pe g oup in each expe imen . To measu e he in ensi y o he s ained a ea, Fiji (ImageJ so wa e . 2.14.0) was employed [30]. 2.8. Inne Mucus Laye Thickness Analysis Samples om he dis al colon we e ha es ed and immedia ely p ese ed in Ca noy’s ixa i e (d y me hanol: chlo o o m: glacial ace ic acid in he a io 60:30:10) as desc ibed ea lie [ 16 ]. A e 4 h in Ca noy’s solu ion, he colons we e washed in d y me hanol and e hanol 100% o 1 h, cleaned wi h xylene and embedded in pa a in wax. Then, he Alcian blue pH 2.5 (AB) (AB-8GX, PanReac Applichem; Schi ’s eagen , VWR Chemicals) s aining o 5 µ m sec ions was pe o med. Only egions in which he mucus laye was sandwiched be ween he epi helium on one side and luminal con en on he o he we e used o measu emen s o hickness. Ca e was aken o conside only hose egions ha ep esen ed egula hickness; 6 o 8 measu emen s we e aken pe image, a e aged o e he o al usable a ea o he colon. His ological de e mina ions we e pe o med using a ligh mic oscope (Eclipse 80i, Nikon, Tokyo, Japan) connec ed o a digi al came a (XCD- U100CR, Sony, Tokyo, Japan). Mo phome ic cha ac e is ics we e analyzed using His olab so wa e . 7.0 (Mic o ision Ins umen s, EVRY Cedex, F ance. Measu emen s we e always single-blinded. Nu ien s 2023,15, 4322 6 o 26 2.9. RNA Ex ac ion and Quan i a i e RT-PCR To al RNA was ex ac ed om ozen colonic issues using TRIzol Reagen (In i ogen, The mo Fishe Scien i ic, Inc., Wal ham, MA, USA) and e e se ansc ibed using a high- capaci y cDNA e e se ansc ip ion ki (Applied Biosys ems, The moFishe Scien i ic, Inc., Wal ham, MA, USA). Real- ime quan i a i e PCR analyses we e pe o med using o wa d and e e se p ime s (Table 1) o de e mine he ela i e abundance o Muc2,T 1, T 3 and Kl 3 genes. The compa a i e h eshold cycle me hod was used o calcula e ela i e exp ession. The a ge gene alues we e no malized o he exp ession o he endogenous e e ence (18S) (Table 1). Table 1. Lis o p ime s (Sigma-Ald ich). Gene Symbol Sequence (50–30) MUC2 Muc2 AAGCCAGATCCCGAAACCAT ATGGCCCCATTCACAACTGCC TFF1 T 1 CAAGGTGACCTGTGTCCTC CTTGCTGGTTCTCAATGACC TFF3 T 3 GACTCCAGCATCCCAAATGT GCAGATCAGGGGTGAGTGTT KLF3 Kl 3 TCATGTACACCAGCCACCTG TAGTCAGTCCTCTGTGGTTC 18S ARN Rps18 GTAACCCGTTGAACCCCATT CCATCCAATCGGTAGTAGCG 2.10. S a ical and Bioin o ma ic Analyses The dis ibu ion o he da a was ini ially assessed using he Shapi o–Wilk no mali y es . Da a con o ming o a no mal dis ibu ion we e exp essed as he mean ± s anda d e o o he mean (SEM) o he mean alongside he 95% con idence in e al (95% CI). When da a we e no no mally dis ibu ed, hey we e p esen ed as he median coupled wi h he in e qua ile ange (IQR). Fo he compa ison o wo g oups, signi icance was assessed using a 2- ailed S uden ’s - es o no mally dis ibu ed da a and Wilcoxon ank sum es s o non-pa ame ic da a, wi h a signi icance le el se a p< 0.05 o all es s. Fo compa isons in ol ing mo e han wo g oups, analysis o a iance (ANOVA) was used o no mally dis ibu ed da a, whe eas he K uskal–Wallis es was employed o non-pa ame ic da a. Pos hoc analyses included pai wise - es s o ANOVA and Wilcoxon ank sum es s wi h con inui y co ec ion o he K uskal–Wallis es . Fu he specialized analyses included he e alua ion o median ela i e abundances o dominan axa using K uskal–Wallis es s o Wilcoxon ank es s, ollowed by app o- p ia e mul iple compa ison co ec ions such as he Bon e oni me hod. Alpha di e si y was measu ed using he Shannon di e si y index, which conside s bo h he numbe and e enness o mic obial species. Be a di e si y was isualized h ough p incipal coo di- na es analysis (PCoA) based on dis ance ma ices, wi h B ay–Cu is and bina y Jacca d indices used o quan i a i e and quali a i e analyses, espec i ely. PERMANOVA wi h 999 pe mu a ions was pe o med o e eal s a is ically signi ican di e ences in be a di- e si y (p< 0.05). Pea son ank co ela ion analyses we e conduc ed o e alua e po en ial co ela ions be ween bac e ial gene a in milk and ecal samples, as well as wi hin ecal samples alone. All s a is ical and bioin o ma ic analyses we e conduc ed using R so wa e, combining e sion 3.3.2 and e sion 4.0.3 (R-p ojec , h p://www. -p ojec .o g, accessed on 13 Janua y 2023), along wi h QIIME pipelines ( . 1.8.0). 3. Resul s 3.1. E ec o Nu i ional Res ic ion on he Biochemical P o ile o O sp ing Ra s du ing Lac a ion Bo h nu i ional es ic ion and he pa icula me abolic adap a ions ha expe ience he mo he du ing ges a ion and lac a ion may con ibu e o he ea ly p og amming o an o sp ing. In his con ex , ma e nal ood es ic ion du ing he las hi d o p egnancy Nu ien s 2023,15, 4322 7 o 26 esul ed in pups wi h a signi ican lowe body weigh a bi h compa ed o ha o C newbo ns, bu wi hou s a is ical di e ences in he li e size and only a sligh endency o inc ease in he male- o- emale a io (p= 0.09) (Table 2). Consis en wi h p e ious da a [ 12 ], nu i ional es ic ed lac a ing a s (U) showed a signi ican educ ion in body weigh gain compa ed o ha o C animals, eaching a di e ence o almos a 50% a he end o he pe iod (L18) (Table 3). Simila ly, se um glucose, insulin and glucagon le els emained signi ican ly lowe h oughou lac a ion in U animals. In he case o he se um lipid p o ile, he e we e no di e ences be ween he wo g oups (C and U) a any age. Howe e , while choles e ol and iglyce ides le els we e high in bo h C and U pups a he beginning o lac a ion (L4), hese le els dec eased and became s able wi h inc easing age (Table 3). Table 2. Cha ac e is ics o he li e s a bi h. Con ol Unde nou ished Newbo ns weigh (g) 6.16 ±0.14 5.69 ±0.09 ** Li e size 11.5 ±1.30 9.86 ±0.72 Numbe o male newbo ns 6.25 ±11.11 5.71 ±0.65 Numbe o emale newbo ns 5.25 ±0.63 4.00 ±0.67 Ra io males/ emales 1.23 ±0.24 1.88 ±0.25 Da a a e means ± SEM (6–8 animals). S uden ’s analysis was used o e alua e di e ences be ween he wo g oups. ** p< 0.01 compa ed o C g oup. Table 3. Se um biochemical p o ile o he o sp ing du ing lac a ion. Con ol Unde nou ished L4 L14 L18 L4 L14 L18 Body weigh (g) 11.36 ±0.23 27.94 ±0.4 34.65 ±1.02 8.35 ± 0.15 *** 16.5 ± 0.41 *** 16.62 ±0.36 *** Glycemia (mg/dL) 119.1 ±1.43 107.8 ±1.03 146.19 ±1.19 85.9 ± 4.02 *** 85.3 ±5.29 ** 93.15 ±4.94 *** Insulinemia (ng/mL) 0.67 ±0.06 0.70 ±0.08 0.78 ±0.17 0.29 ± 0.04 *** 0.25 ±0.06 ** 0.36 ±0.09 * Glucagonemia (pg/mL) 252.3 ±10.6 298 ±11.2 129.1 ±6.9 103.9 ± 4.4 *** 97 ±1.3 *** 83.2 ±4.3 *** Choles e ol (mg/dL) 154.66 ±4.11 127.48 ±5.81 119.95 ±4.5 144.38 ±3.28 119.62 ±4.51 116.86 ±5.2 T iglyce ides (mg/dL) 157.08 ±5.26 79.94 ±4.75 63.41 ±5.42 160.17 ±8.57 74.02 ±3.07 68.42 ±3.51 Da a a e mean ± SEM (n= 6–8 animals). Biochemical and ho monal pa ame e s o con ol (C) and unde nou ished (U) a s a 4, 14 and 18 days o lac a ion (L). S uden ’s analysis was used o e alua e di e ences be ween g oups in e ms o nu i ional ype. * p< 0.05; ** p< 0.01; *** p< 0.001 compa ed o C g oup a he same age. 3.2. E ec o Ma e nal Die on he Milk and Gu Mic obiome o O sp ing h oughou Lac a ion 3.2.1. Me a axonomic Analysis o he Milk Samples The compa a i e me a axonomic analysis o mic obial p o iles in milk samples, s a i- ied acco ding o nu i ional s a us (C s. U) and days o lac a ion (L4, L14 and L18), was conduc ed u ilizing 35 o he samples as delinea ed in Sec ion 2.5. F om hese 35 milk samples, a cumula i e o al o 811,904 high-quali y il e ed sequences was ob ained. The sequence coun pe sample oscilla ed be ween 12,569 and 33,787 (median [IQR]: 22,540 [18,872.5–28,013]), which could be clus e ed in o 2586 dis inc ASVs. The alpha di e si y wi hin hese milk samples, i espec i e o whe he hey we e om C o U g oups, exhibi ed compa able Richness and Shannon di e si y indices ac oss all ime poin s o lac a ion (L4, L14 and L18), as depic ed in Figu e 1a,b. Nu ien s 2023,15, 4322 8 o 26 Nu ien s 2023, 15, x FOR PEER REVIEW 8 o 26 wi hin hese milk samples, i espec i e o whe he hey we e om C o U g oups, exhib- i ed compa able Richness and Shannon di e si y indices ac oss all ime poin s o lac a ion (L4, L14 and L18), as depic ed in Figu e 1a,b. Figu e 1. Mic obial alpha di e si y in milk samples exp essed by (a) he numbe o obse ed gene a (gene a ichness) and (b) he Shannon index. Box plo s ep esen median and in e qua ile anges oge he wi h maximum and minimum alues. K uskal–Wallis analysis was used o e alua e diffe - ences be ween g oups (in e ms o lac a ional age and ype o die ). The Be a di e si y analysis o milk samples was measu ed as (c) he p esence/absence (Binna y Jacca d me hod) o (d) he ela i e abundance (B ay–Cu is simila i y analysis) o he diffe en species quan i ied in con ol g oups (yellow: C4; o ange: C14; ed: C18) and unde nou ished g oups (ligh blue: U4; cyan blue: U14; da k blue: U18). The alue gi en on each axis label ep esen s he pe cen age o he o al a iance ex- plained by ha axis. Pe mano a analysis was used o e alua e diffe ences be ween g oups (in e ms o lac a ional age and ype o die ). N = 5–6 pe condi ion. C: con ol; U: unde nou ished. L: lac a ion. An analysis o be a di e si y was conduc ed o compa e he C and U g oups a each designa ed lac a ional ime poin . Two-dimensional p incipal coo dina es analysis (2D- PCoA) u ilizing Jacca d dis ance me ics p o ided a isual pla o m o assessing he ex- en o sample clus e ing based on bo h nu i ional s a us and lac a ional s age. No ably, dis inc clus e ing pa e ns eme ged a day 4 and day 18 (Figu e 1c), sugges ing a signi i- can di e gence in mic obial p o iles be ween C and U coho s a hese sampling poin s. Figu e 1. Mic obial alpha di e si y in milk samples exp essed by ( a ) he numbe o obse ed gene a (gene a ichness) and ( b ) he Shannon index. Box plo s ep esen median and in e qua ile anges oge he wi h maximum and minimum alues. K uskal–Wallis analysis was used o e alua e di e ences be ween g oups (in e ms o lac a ional age and ype o die ). The Be a di e si y analysis o milk samples was measu ed as ( c ) he p esence/absence (Binna y Jacca d me hod) o ( d ) he ela i e abundance (B ay–Cu is simila i y analysis) o he di e en species quan i ied in con ol g oups (yellow: C4; o ange: C14; ed: C18) and unde nou ished g oups (ligh blue: U4; cyan blue: U14; da k blue: U18). The alue gi en on each axis label ep esen s he pe cen age o he o al a iance explained by ha axis. Pe mano a analysis was used o e alua e di e ences be ween g oups (in e ms o lac a ional age and ype o die ). N = 5–6 pe condi ion. C: con ol; U: unde nou ished. L: lac a ion. An analysis o be a di e si y was conduc ed o compa e he C and U g oups a each designa ed lac a ional ime poin . Two-dimensional p incipal coo dina es analysis (2D- PCoA) u ilizing Jacca d dis ance me ics p o ided a isual pla o m o assessing he ex en o sample clus e ing based on bo h nu i ional s a us and lac a ional s age. No ably, dis inc clus e ing pa e ns eme ged a day 4 and day 18 (Figu e 1c), sugges ing a signi ican di e gence in mic obial p o iles be ween C and U coho s a hese sampling poin s. This obse a ion was subs an ia ed s a is ically ia a subsequen PERMANOVA analysis based on Jacca d simila i y me ics, which e ealed signi ican di e ences in bac e ial composi ion be ween he wo nu i ional g oups (p= 0.009 and p= 0.014). Addi ionally, a sepa a e PCoA plo gene a ed using B ay–Cu is simila i y indices, which we e no malized ela i e o he Nu ien s 2023,15, 4322 9 o 26 abundance o dis inc ASVs, demons a ed signi ican clus e ing p edica ed on nu i ional s a us a L4 (p= 0.029), bu no a L14 o L18 (p> 0.05) (Figu e 1d). A he axonomic le el, disce nible di e ences in bac e ial ela i e abundances we e obse ed ela ed o die ype a speci ic ime poin s. Taxa belonging o he Fi micu es and P o eobac e ia phyla domina ed in milk samples om bo h C and U animals. O he phyla such as Bac e oido a and Ac inobac e oido a we e also p esen bu a a lowe ela- i e abundance. Al hough Fi micu es showed a endency o dec ease and P o eobac e ia showed a endency o inc ease hei ela i e abundance in he U g oup a L18, no s a is ical signi icance was de ec ed when compa ed o ha o hei C coun e pa s (Figu e 2a–d). On he o he hand, he ela i e abundance o sequences belonging o he amily Pas eu ellaceae was signi ican ly highe in he samples om he U g oup (median [IQR] = 5.57 [3.33–8.2]) han in hose om C animals (median [IQR] = < 0.01 [<0.01–<0.01]) a L4 (p= 0.0055). Simila ly, he gene a Gemella and En e ococcus mani es ed ele a ed ela i e abundances in he U samples a L18, wi h median [IQR] alues o 0.94 [0.52–2.19] s. 0.64 [0.41–0.85] (p= 0.007) and 0.08 [<0.01–2.61] s. < 0.01 [<0.01–0.03] (p= 0.030), espec i ely. In con as , he genus Rombou sia was mo e abundan in he C g oup a L18 (p= 0.015). Addi ion- ally, Po phy omonas was mo e p e alen in U samples a L4, wi h a median [IQR] o 0.95 [0.67–1.66] s. 0.1 [0.01–0.25] (p= 0.024). No signi ican di e ences in he ela i e abundances o se e al o he axa, such as Lac obacillus,Roden ibac e , Unclassi ied_gene a, S ep ococcus,Esche ichia-Shigella,Tu icibac e ,Ro hia and Veillonella, we e de ec ed be ween C and U a any o he ages conside ed (Figu e 2e and Table S1). Nu ien s 2023, 15, x FOR PEER REVIEW 9 o 26 This obse a ion was subs an ia ed s a is ically ia a subsequen PERMANOVA analysis based on Jacca d simila i y me ics, which e ealed signi ican diffe ences in bac e ial composi ion be ween he wo nu i ional g oups (p = 0.009 and p = 0.014). Addi ionally, a sepa a e PCoA plo gene a ed using B ay–Cu is simila i y indices, which we e no mal- ized ela i e o he abundance o dis inc ASVs, demons a ed signi ican clus e ing p ed- ica ed on nu i ional s a us a L4 (p = 0.029), bu no a L14 o L18 (p > 0.05) (Figu e 1d). A he axonomic le el, disce nible diffe ences in bac e ial ela i e abundances we e obse ed ela ed o die ype a speci ic ime poin s. Taxa belonging o he Fi micu es and P o eobac e ia phyla domina ed in milk samples om bo h C and U animals. O he phyla such as Bac e oido a and Ac inobac e oido a we e also p esen bu a a lowe ela i e abundance. Al hough Fi micu es showed a endency o dec ease and P o eobac e ia showed a endency o inc ease hei ela i e abundance in he U g oup a L18, no s a is ical signi icance was de ec ed when compa ed o ha o hei C coun e pa s (Figu e 2a–d). On he o he hand, he ela i e abundance o sequences belonging o he amily Pas eu el- laceae was signi ican ly highe in he samples om he U g oup (median [IQR] = 5.57 [3.33– 8.2]) han in hose om C animals (median [IQR] = < 0.01 [<0.01–<0.01]) a L4 (p = 0.0055). Simila ly, he gene a Gemella and En e ococcus mani es ed ele a ed ela i e abundances in he U samples a L18, wi h median [IQR] alues o 0.94 [0.52–2.19] s. 0.64 [0.41–0.85] (p = 0.007) and 0.08 [<0.01–2.61] s. < 0.01 [<0.01–0.03] (p = 0.030), espec i ely. In con as , he genus Rombou sia was mo e abundan in he C g oup a L18 (p = 0.015). Addi ionally, Po phy omonas was mo e p e alen in U samples a L4, wi h a median [IQR] o 0.95 [0.67– 1.66] s. 0.1 [0.01–0.25] (p = 0.024). No signi ican diffe ences in he ela i e abundances o se e al o he axa, such as Lac obacillus, Roden ibac e , Unclassi ied_gene a, S ep ococcus, Esche ichia-Shigella, Tu icibac e , Ro hia and Veillonella, we e de ec ed be ween C and U a any o he ages conside ed (Figu e 2e and Table S1). Figu e 2. Compa ison o he ela i e abundance o sequences belonging o main bac e ial phyla (a) Fi micu es, (b) P o eobac e ia, (c) Bac e oido a and (d) Ac inobac e io a, and (e) he mos abundan gene a ob ained ia me a axonomic analysis in milk samples a lac a ing days L4, L14 and L18 om Figu e 2. Compa ison o he ela i e abundance o sequences belonging o main bac e ial phyla ( a ) Fi micu es, ( b ) P o eobac e ia, ( c ) Bac e oido a and ( d ) Ac inobac e io a, and ( e ) he mos abundan gene a ob ained ia me a axonomic analysis in milk samples a lac a ing days L4, L14 and L18 om con ol (C) and unde nou ished (U) animals. Box plo s ep esen median and in e qua ile anges oge he wi h maximum and minimum alues. Wilcoxon analysis was used o e alua e di e ences be ween g oups in e ms o ype o die . N = 5–6. The F p eceding Pas eu elleceae in milk samples e e s o (e) amily axon. Nu ien s 2023,15, 4322 16 o 26 animals; (iii) ha he co ela ions we e pa icula ly nume ous a he end o lac a ion in bo h g oups o animals al hough in he C g oup associa ions we e mo e equen among bac e ia exclusi ely om eces, while die a y es ic ion led o a g ea e numbe o as- socia ions among milk bac e ia. Despi e his dynamism, a ecu ing end eme ged: he p esence o Esche ichia-Shigella in milk samples o en mi o ed i s p esence in ecal samples. Fu he mo e, he analysis b ough o ligh a gene al end whe e Esche ichia-Shigella and Lac obacillus exhibi ed a nega i e co ela ion ac oss a ious ime poin s, a L4 o C samples ( = − 0.91) and a L14 and L18 o U samples ( = − 0.98 a bo h ages). This sugges s a pa e n whe e a high p e alence o one genus migh be associa ed wi h a dec eased p e alence o he o he . While his ecu ing end was obse ed, i wa an s u he in es iga ion o explo e he po en ial implica ions and unde lying mechanisms d i ing hese obse a ions. Rega ding he posi i e associa ions be ween ecal anae obic bac e ia, an indica o o in es inal ma u i y, hese we e mo e equen in C animals a he di e en imes s udied han hey we e in U pups (Pa abac e oides,Bac e oides,Tu icibac e ,Gemella, Veillonella,Esche ichia-Shigella,Fusobac e ium,Lac obacillus,Rombus ia and Roden ibac e ), which again e idenced he in luence o die in gu coloniza ion. 3.4. Nu i ional Res ic ion du ing Lac a ion Dis up s he Colonic Ba ie In eg i y o he O sp ing Gi en he shi in he bac e ial p o ile o malnou ished pups, hei in es ine s uc u al and mo phome ic cha ac e is ics we e analyzed. As shown in Table 4, he body ( om nose o annus) and o al in es inal leng h ( om pylo us o cecum) inc eased p og essi ely om 4 o 18 days o lac a ion in bo h expe imen al g oups C and U; howe e , he g ow h expe i- enced by U o sp ing a s was less p onounced and he measu emen s we e signi ican ly lowe han hose in C animals. The leng h o he small in es ine in pups om es ic ed mo he s was always lowe han he C alues bu i only had s a is ical signi icance a L4 and L18. Rega ding he leng hs o he la ge in es ine, including colon and cecum, he highes di e ences wi h C alues we e obse ed a L4 whe eas no a ia ions we e de ec ed a L14 and L18 o he colon. Su p isingly, he U cecum was ound o be longe han he cecum o C a s a L14; howe e , i s leng h dec eased again owa ds he end o lac a ion (L18), emo ing he s a is ical signi icance. Table 4. In es inal mo phome ic cha ac e is ics o he o sp ing du ing lac a ion. Con ol Unde nou ished L4 L14 L18 L4 L14 L18 Body leng h (cm) 6.23 ±0.07 8.89 ±0.09 9.36 ±0.114 6.28 ±0.17 7.4 ±0.09 *** 8.01 ±0.13 *** To al in es ine leng h (cm) 33.47 ±0.37 48.0 ±0.90 48.43 ±1.53 29.54 ±0.67 41.0 ±2.30 39.41 ±0.72 *** Small in es ine leng h (cm) Colon leng h (cm) Cecum leng h (cm) 29.15 ±0.38 40.6 ±0.81 41.47 ±1.34 25.27 ±0.64 ** 35.0 ±2.20 33.14 ±0.70 *** 3.64 ±0.13 5.83 ±0.14 5.90 ±0.24 3.16 ±0.12 * 5.17 ±0.19 5.52 ±0.14 0.68 ±0.04 1.35 ±0.06 1.38 ±0.05 0.65 ±0.03 ** 1.43 ±0.08 1.26 ±0.05 Small In es. Leng h/Body Leng h 4.68 ±0.14 4.57 ±0.12 0.48 ±0.19 4.64 ±0.10 5.23 ±0.32 4.49 ±0.20 Colon Leng h/Body Leng h 0.56 ±0.03 0.67 ±0.01 0.63 ±0.03 0.57 ±0.02 0.76 ±0.03 * 0.75 ±0.03 ** Quan i ica ion o in es inal pa ame e s o he o sp ing a 4, 14 and 18 days o lac a ion (L4, L14 and L18). S uden ’s -analysis was used o e alua e di e ences be ween g oups in e ms o nu i ion ype. Da a ep esen mean ±SEM (n= 6–8). * p< 0.05; ** p< 0.01; *** p< 0.001 compa ed o C g oup. Mo eo e , in o de o e alua e i body p opo ions we e main ained despi e nu i ional es ic ion, we adjus ed he leng h o he small in es ine and colon based on he o al body leng h. The measu emen s o he small in es ine in he U g oup did no di e om hose o he C a s. Howe e , nu i ional es ic ion esul ed in signi ican changes in he ac ional leng h o he colon, which was highe a L14 and L18 compa ed o ha in he age-ma ched C g oup. This sugges s ha he e is some p ese a ion o colon g ow h du ing his s age o de elopmen (Table 4). As he in es inal ba ie is imma u e a bi h and unde goes impo an s uc u al and unc ional changes induced by he ab up ansi ion in nu ien supply om ma e nal um- Nu ien s 2023,15, 4322 17 o 26 bilical co d blood o en e al milk in ake, in es inal pe meabili y and mucus laye hickness we e analyzed. These s udies ocused on he colonic issue because his is he main mic obi- ological ese oi o he o ganism. To assess he i s pa ame e , we measu ed ZO-1 le els, which is a igh junc ion p o ein loca ed in he apical zone o colonic epi helial cells ha egula es pa acellula pe meabili y and con ibu es o he p ese a ion o in es inal ba ie in eg i y [ 31 ]. Immuno luo escence analysis pe o med a ea ly (L4) and la e lac a ion (L18) showed an inc ease in signal in ensi y o ZO-1 in C animals, o ming a ne wo k o inc easingly consolida ed in eg i y in epi helial cells (Figu e 8a). Howe e , ZO-1 le els we e signi ican ly educed in U a s a L18 compa ed o hose in he C g oup (Figu e 8b), sugges ing a loss o in es inal ba ie in eg i y a his age. Nu ien s 2023, 15, x FOR PEER REVIEW 17 o 26 ac ional leng h o he colon, which was highe a L14 and L18 compa ed o ha in he age-ma ched C g oup. This sugges s ha he e is some p ese a ion o colon g ow h du - ing his s age o de elopmen (Table 4). As he in es inal ba ie is imma u e a bi h and unde goes impo an s uc u al and unc ional changes induced by he ab up ansi ion in nu ien supply om ma e nal um- bilical co d blood o en e al milk in ake, in es inal pe meabili y and mucus laye hickness we e analyzed. These s udies ocused on he colonic issue because his is he main mic o- biological ese oi o he o ganism. To assess he i s pa ame e , we measu ed ZO-1 le - els, which is a igh junc ion p o ein loca ed in he apical zone o colonic epi helial cells ha egula es pa acellula pe meabili y and con ibu es o he p ese a ion o in es inal ba ie in eg i y [31]. Immuno luo escence analysis pe o med a ea ly (L4) and la e lac- a ion (L18) showed an inc ease in signal in ensi y o ZO-1 in C animals, o ming a ne - wo k o inc easingly consolida ed in eg i y in epi helial cells (Figu e 8a). Howe e , ZO-1 le els we e signi ican ly educed in U a s a L18 compa ed o hose in he C g oup (Figu e 8b), sugges ing a loss o in es inal ba ie in eg i y a his age. Figu e 8. Cha ac e is ics o he colonic ba ie in eg i y and i m mucus laye in he offsp ing a s a 4 and 18 days o lac a ion. (a) Immuno luo escence o ZO-1 (g een) in colon sec ions. Nucleic acids we e s ained wi h DAPI (blue) and whi e a ows indica e a eas whe e he ZO-1 s ain was dis up ed a he epi helial cell memb ane (magni ica ion: 63×). (b) Quan i ica ion o he ZO-1 luo escence in- ensi y exp essed in a bi a y uni s. (c) Alcian Blue (AB)-s ained colonic sec ions. The i m mucus laye was ma ked wi h whi e b acke s (magni ica ion: 20×). (d) Quan i ica ion o he i m mucus hickness. Da a ep esen mean ± SEM (n = 3–4; 4 sec ions pe animal). S uden ’s analysis was used o e alua e diffe ences be ween g oups in e ms o he ype o die . * p < 0.05 compa ed o C g oup wi hin each age. C: con ol; U: unde nou ished; L: lac a ion. Figu e 8. Cha ac e is ics o he colonic ba ie in eg i y and i m mucus laye in he o sp ing a s a 4 and 18 days o lac a ion. ( a ) Immuno luo escence o ZO-1 (g een) in colon sec ions. Nucleic acids we e s ained wi h DAPI (blue) and whi e a ows indica e a eas whe e he ZO-1 s ain was dis up ed a he epi helial cell memb ane (magni ica ion: 63 × ). ( b ) Quan i ica ion o he ZO-1 luo escence in ensi y exp essed in a bi a y uni s. ( c ) Alcian Blue (AB)-s ained colonic sec ions. The i m mucus laye was ma ked wi h whi e b acke s (magni ica ion: 20 × ). ( d ) Quan i ica ion o he i m mucus hickness. Da a ep esen mean ± SEM (n= 3–4; 4 sec ions pe animal). S uden ’s analysis was used o e alua e di e ences be ween g oups in e ms o he ype o die . * p< 0.05 compa ed o C g oup wi hin each age. C: con ol; U: unde nou ished; L: lac a ion. The colonic lumen con ains a gel-like s uc u e called mucus ha ac s as a i s line o de ense agains mic oo ganisms in ading he lamina p op ia. I is p oduced by specialized sec e ing cells called goble cells, which o m pa o he in es inal epi helium [ 32 ]. Because mic obial changes also play an impo an ole in mucus p oduc ion, we hen quan i ied he inne mucus hickness wi h AB s aining (Figu e 8c). Mo phome ic measu emen s o he mucus laye showed a end o enhanced hickness unde ood- es ic ed condi ions a bo h L4 (1.9- old inc ease s. C) and L18 (1.6- old inc ease s. C); howe e , his did no each s a is ical signi icance (p= 0.068, o bo h ages) (Figu e 8d). To u he cha ac e ize he e ec Nu ien s 2023,15, 4322 18 o 26 o ma e nal nu i ion on gu ba ie p ope ies, we nex analyzed he gene exp ession o key ma ke s o specialized mucus-sec e ing goble cells: Muc2,T 3,T 1 and Kl 3 (Figu e 9). Nu i ional es ic ion did no induce any signi ican change in he exp ession o Muc2, he main goble cell ma ke , ela i e o C g oup le els a any age conside ed (Figu e 9a). On he con a y, he exp ession o T 3, a ac o co-sec e ed wi h MUC2 and in ol ed in cell mig a ion, p oli e a ion and he epai o he mucosal epi helium, was signi ican ly highe a 4 (1.56- old inc ease) and 18 days o li e (1.48- old inc ease) compa ed o he C alues (Figu e 9b). Al hough he mucosal p o ec i e unc ion o TFF1 is simila o ha o TFF3, i s exp ession was no al e ed in U o sp ing a s ela i e o ha o C animals du ing lac a ion (Figu e 9c). Finally, Kl 3 exp ession, ano he ac o in ol ed in he egene a ion o epi helial cells, was signi ican ly down egula ed a la e lac a ion (L18) unde nu ien - es ic ed condi ions (Figu e 9d). Nu ien s 2023, 15, x FOR PEER REVIEW 18 o 26 The colonic lumen con ains a gel-like s uc u e called mucus ha ac s as a i s line o de ense agains mic oo ganisms in ading he lamina p op ia. I is p oduced by special- ized sec e ing cells called goble cells, which o m pa o he in es inal epi helium [32]. Because mic obial changes also play an impo an ole in mucus p oduc ion, we hen quan i ied he inne mucus hickness wi h AB s aining (Figu e 8c). Mo phome ic meas- u emen s o he mucus laye showed a end o enhanced hickness unde ood- es ic ed condi ions a bo h L4 (1.9- old inc ease s. C) and L18 (1.6- old inc ease s. C); howe e , his did no each s a is ical signi icance (p = 0.068, o bo h ages) (Figu e 8d). To u he cha ac e ize he effec o ma e nal nu i ion on gu ba ie p ope ies, we nex analyzed he gene exp ession o key ma ke s o specialized mucus-sec e ing goble cells: Muc2, Tff3, Tff1 and Kl 3 (Figu e 9). Nu i ional es ic ion did no induce any signi ican change in he exp ession o Muc2, he main goble cell ma ke , ela i e o C g oup le els a any age conside ed (Figu e 9a). On he con a y, he exp ession o Tff3, a ac o co-sec e ed wi h MUC2 and in ol ed in cell mig a ion, p oli e a ion and he epai o he mucosal epi he- lium, was signi ican ly highe a 4 (1.56- old inc ease) and 18 days o li e (1.48- old in- c ease) compa ed o he C alues (Figu e 9b). Al hough he mucosal p o ec i e unc ion o TFF1 is simila o ha o TFF3, i s exp ession was no al e ed in U offsp ing a s ela i e o ha o C animals du ing lac a ion (Figu e 9c). Finally, Kl 3 exp ession, ano he ac o in ol ed in he egene a ion o epi helial cells, was signi ican ly down egula ed a la e lac a ion (L18) unde nu ien - es ic ed condi ions (Figu e 9d). Figu e 9. Changes induced by ma e nal nu i ion in he exp ession p o ile o goble cell ma ke s om he offsp ing colon a 4 and 18 days o lac a ion. Quan i a i e RT-qPCR analysis o Muc2 (a), Tff3 (b), Tff1 (c) and Kl 3 (d) no malized wi h he 18S ibosomal gene. Da a ep esen mean ± SEM (n = 5–6). S uden ’s -analysis was used o e alua e diffe ences be ween he wo g oups in e m so he ype o die . * p < 0.05; ** p < 0.001 compa ed o g oup C wi hin each age. C: con ol; U; Unde - nou ished; L: lac a ion. Figu e 9. Changes induced by ma e nal nu i ion in he exp ession p o ile o goble cell ma ke s om he o sp ing colon a 4 and 18 days o lac a ion. Quan i a i e RT-qPCR analysis o Muc2 ( a ), T 3 ( b ), T 1 ( c ) and Kl 3 ( d ) no malized wi h he 18S ibosomal gene. Da a ep esen mean ± SEM (n= 5–6). S uden ’s -analysis was used o e alua e di e ences be ween he wo g oups in e m so he ype o die . * p< 0.05; ** p< 0.001 compa ed o g oup C wi hin each age. C: con ol; U; Unde nou ished; L: lac a ion. 4. Discussion Lac a ion is conside ed a pe iod o majo ele ance in me abolic p og amming because milk bac e ia a e among he i s mic obes o en e he neona al gas oin es inal ac and, consequen ly, hey ac as d i e s in he acquisi ion and de elopmen o a heal hy mic obio a [ 18 , 33 ]. Mo he - o-in an bac e ial ans e h ough ma e nal milk has been epea edly desc ibed a he species and/o he s ain le el, ia bo h cul u e-dependen and cul u e-independen echniques [ 18 , 34 ]. Wo h no ing is ha app oxima ely a qua e o he bac e ia de ec ed in in an eces du ing ea ly li e seems o de i e om milk [ 35 ] and he bac e ial gene a accoun ing o mos o he abundance (>70%) in in an eces a e sha ed wi h human milk [ 36 ]. In he same di ec ion, i has been ecen ly desc ibed ha Nu ien s 2023,15, 4322 19 o 26 ma e nal milk is closely associa ed wi h o sp ing mic obiome di e si y [ 37 ]. The e o e, as die has been shown o be a majo de e minan o he ype and abundance o mic obio a in he gas oin es inal ac , in he p esen s udy we ha e in es iga ed how ma e nal die du ing p egnancy and lac a ion a ec his mic obial communica ion be ween mo he and o sp ing. Ou da a show ha he diso de o milk mic obio a induced ia ma e nal ood es ic ion seems o change he p o ile o o sp ing in es inal mic obiome and hen p omo e an imma u e in es inal mic obio a and gu ba ie unc ion. E en hough no die a y e ec was obse ed o alpha di e si y, nei he in milk no in ecal samples, be a di e si y was signi ican o age and die , in bo h ypes o samples, wi h he bac e ia ound in U samples a L4 displaying he highes dis ance o all o he samples. I is wo h no ing ha bac e ia iden i ied in ecal samples om UL14 we e mo e simila o CL4 han o hose collec ed om animals o he same age bu consuming a di e en die . These esul s ein o ce he idea ha ma e nal die in luence milk mic obial composi ion and his e ec is mi o ed in o sp ing eces, p omo ing an imma u e gu mic obiome. Likewise, se e ely unde nou ished child en om Bangladesh [ 38 ] showed ma ked gu mic obio a imma u i y ha could no be e e sed wi h die a y in e en ions, which is in ag eemen wi h ou p e ious s udies pe o med wi h a ca ch-up g ow h a model [ 16 ] o in humanized mouse models o malnu i ion [39]. The bac e ia ound in milk and o sp ing eces du ing lac a ion we e mainly ep e- sen ed by he phyla Fi micu es, P o eobac e ia, Bac e oido a and Ac inobac e ia, he la e axa being less abundan han he o me wo. The mos abundan ASVs ound in he milk we e iden i ied as Lac obacillus, which accoun ed o mos o he eads wi hin he phylum Fi micu es. The con ibu ion o Lac obacillus spp. o milk is highly conse ed ac oss species and known o p oduce bac e iocins, which a e able o inhibi he g ow h o pa hogens [ 40 ]. Mo eo e , as a acul a i e anae obe, Lac obacillus is conside ed a pionee bac e ial specie ha du ing he i s days o lac a ion deple es oxygen om gu and he eby acili a es he subsequen coloniza ion o he gu en i onmen ia obliga e anae obes [ 41 ]. Wa en e al. [ 27 ] epo ed ha a ia ions in he p o ein con en o ma e nal die in luenced he ela i e abundance o he genus Lac obacillus no only om ma e nal milk bu also om o sp ing eces. In ag eemen , we obse ed Lac obacillus spp. o show g ea e ela i e abundance a he beginning o lac a ion in he eces om pups o mo he s subjec ed o ood es ic ion, despi e no pa allel e ec o die being obse ed in milk. In e es ingly, i has been sugges ed ha highe coloniza ion o he gu wi h Lac obacillus spp. in ea ly li e p edic s an inc eased isk o being o e weigh and me abolic al e a ions in he u u e [ 42 ]. Gi en ha Lac obacillus spp. a e no mally ound in he aginal niche, i is possible ha he high le els o Lac obacillus axa in he o sp ing ecal samples om U mo he s o igina ed in pa , du ing deli e y, om changes in he aginal mic obio a composi ion o mo he s. The composi ion o he aginal mic obio a is unde he con ol o ho monal pa e ns [ 43 ] wi h highe abundance in Lac obacillus species wi h inc easing ges a ional age. This e en seems o be pa o a s a egy o ensu e lac ic acid p oduc ion, educe pH in he aginal ca i y and p o ec he e us om in ec ions. Howe e , he in luence o en i onmen al ac o s, such as s ess o die , on aginal communi y s abili y is poo ly known. Al hough ch onic s ess induced by se e e ood es ic ion, like in pa ien s wi h ano exia ne osa, has been associa ed wi h bac e ial aginosis, mens ual cycle i egula i y and ameno hea [ 44 ], he in e ac ion be ween die , mens ual dys unc ion and aginal communi y s abili y emain unexplo ed. Thus, i is possible ha ma e nal die du ing ges a ion may al e he aginal mic obio a ansmi ed o o sp ing and subsequen ly in luence hei gu coloniza ion and ma u a ion. In addi ion o Lac obacillus,Esche ichia-Shigella, included in he phylum P o eobac e ia, is also conside ed a pionee bac e ium wi h high abundance h oughou lac a ion in he eces o well-nou ished pups. Howe e , i s p oli e a ion appea s o be condi ioned by nu i ional s a us gi en i s almos absolu e absence a he beginning o lac a ion in he milk o U mo he s as well as in hei o sp ing eces, bu he subsequen sha p inc ease in eces. This e en migh a ec he es ablishmen o a sui able in es inal mic oen i onmen Nu ien s 2023,15, 4322 20 o 26 o ensu e he coloniza ion o s ic anae obic bac e ia and consequen ly, delay gu mic o- bio a ma u a ion. Al hough Esche ichia-Shigella is a non-pa hogenic genus, unde speci ic ci cums ances, ce ain se o ypes can igge symp oms such as dia hea, wi h inc eased in es inal pe meabili y and dis up ed epi helial ba ie o e en ex a-in es inal in lamma- o y p ocesses [ 45 – 48 ]. The low numbe o samples analyzed in his s udy implies ha he mic obial shi s de ec ed o his genus did no each a s a is ically signi ican h eshold bu migh , in ac , be ele an o homeos asis and heal h, and should be ca e ully e alua ed in u u e s udies in ol ing a la ge numbe o animals. Wi hin he phylum P o eobac e ia, ou me a axonomic analysis also success ully de ec ed sequences belonging o he amily Pas eu ellaceae. Mos o hem we e assigned o he genus Roden ibac e , while a mino ac ion was assigned o o he gene a, such as Haemophilus and Mu ibac e . Howe e , i is impo an o no e ha ou sequencing app oach aced some limi a ions in dis inguishing be ween sequences belonging o di e en gene a wi hin his amily. Consequen ly, al hough he ele a ed abundance o Pas eu ellaceae in milk samples om U animals a L4 migh p ima ily be a ibu able o Roden ibac e , he po en ial con ibu ion o o he gene a wi hin his amily should no be disca ded. I is no ewo hy ha in he p esen s udy, mo he s’ milk was no collec ed om he mamma y gland as is ca ied ou in o he simila app oaches [ 27 ], bu om he pup’s s omach, which is indeed he same ou e as ha o suckling o sp ing. Undoub edly, he o sp ing mic obio a, pa icula ly ha om he o al ca i y, will modi y he bac e ia exp essed in milk [ 19 , 49 ], bu will also con ibu e o he es ablishmen o he i s gu colonize s. In line wi h his concep , he p esence o he genus Po phy omonas in he milk o nu i ionally es ic ed mo he s d ew a en ion in ou s udies. Po phy omonas is an anae obic bac e ium ound in he o al ca i y and is associa ed wi h he de elopmen o pe iodon al disease. I s p esence in he o al ca i y and consequen ly, i s inges ion du - ing ood in ake causes in es inal dysbiosis by c ea ing an en i onmen a o able o he coloniza ion o p o-in lamma o y mic oo ganisms [ 50 ] and e en ually leading o ulce - a i e coli is [ 51 ] o colo ec al cance [ 52 ]. Likewise, me abolic s udies pe o med wi h s ep ozo ocin- ea ed mice ( o gene a e a diabe ic si ua ion) and subsequen Po phy omonas adminis a ion showed a wo sening o he glycemic con ol oge he wi h local and sys- emic in lamma o y e en s [ 53 ]. Thus, he p esence o Po phy omonas in milk migh be conside ed a bioma ke o u u e gu dysbiosis, in lamma o y p ocesses and long- e m me abolic al e a ions. Acco dingly, we ha e p e iously desc ibed he a o emen ioned e iopa hological iangle in he same animal model o ood es ic ion used he ein [ 16 ]. Howe e , he e y ea ly igge ing e en s o his pheno ype and i s ela ionship wi h b eas eeding and ma e nal die needed u he in es iga ions. On he o he hand, i is known ha in es inal pe meabili y a bi h is high in newbo ns and hen g adually dec eases o ensu e ull unc ionali y and p o ec ion om pa hogens [ 54 ]. Mo eo e , e idence sugges ha ce ain bac e ia and bioac i e compounds in b eas milk play a key ole in egula ing mucosal in eg i y and in es inal epi helial pe meabili y. Hu- man s udies ha e shown ha ull- e m b eas ed in an s ha e lowe in es inal pe meabili y han do o mula- ed in an s [ 55 , 56 ]. These obse a ions a e consis en wi h animal s udies showing ha ge m- ee mice ha e many mo phological in es inal de ec s compa ed o hei no mally colonized coun e pa s, sugges ing ha he de elopmen o ba ie unc ion is dependen on he p esence o mic oo ganisms [ 57 ]. Howe e , he speci ic mechanisms by which a ia ions in he gu mic obio a shape he ea ly plas ici y o in es inal pe meabili y and how hese ac o s con ibu e o heal h s a us in la e li e emain poo ly unde s ood. The apical epi helial ne wo k o igh junc ion p o eins is he p incipal de e minan o pa acel- lula pe meabili y and immune sys em homeos asis [ 31 ]. In he p esen s udy, we obse ed ha ma e nal ood es ic ion inc eased colonic pe meabili y in he lac a ing o sp ing, as e idenced by a educ ion in ZO-1 p o ein le els, which was no ully amelio a ed when unde nu i ion was co ec ed pos weaning [ 16 ]. In ag eemen , se e e p o ein de iciency in animal models led o s uc u al and unc ional changes o he in es ine, including malab- so p ion and he de elopmen o a leaky gu wi h inc eased pe meabili y [ 17 , 58 ]. In es inal Nu ien s 2023,15, 4322 21 o 26 biopsies om se e ely unde nou ished child en wi h en e opa hy also showed a educed exp ession o he igh junc ions claudin-4 and E-cadhe in [ 59 ], al hough he molecula mechanism ela ing unde nu i ion o changes in pa acellula p o eins emains unclea . In line wi h his, he dis up ion o epi helial igh junc ion p o eins is consis en wi h he ansloca ion o bac e ial componen s o he ci cula ion and de elopmen o endo oxemia, as we [ 16 ] and o he s [ 60 ] ha e p e iously desc ibed. These changes in igh junc ion p o ein le els pa alleled changes in he exp ession o epi helial cell componen s such as he e oil ac o amily o pep ides (TFF), which a e no mally co-sec e ed wi h mucins. In pa icula , TFF1 and TFF3 ac i ely pa icipa e wi h immune sys em molecules in p o ec ing he in es inal mucosa om mic oo ganisms [ 61 ]. In ou animal model, we did no obse e di e ences in T 1 exp ession associa ed wi h die ype. Howe e , a he beginning and end o lac a ion, he exp ession o T 3 was inc eased in o sp ing subjec ed o die a y es ic ion. In his ega d, ecen s udies in animals and pedia ic pa ien s wi h in lamma o y bowel disease ha e shown ele a ed se um le els o TFF3, an e en associa ed wi h inc eased in es inal pe meabili y and in lamma ion ha is no seen in heal hy subjec s o pa ien s in emission [ 62 ]. The e o e, conside ing he lack o di e ences in mucin (Muc2) exp ession be ween C and U lac a ing pups, bu he inc ease in T 3 in he la e , we hypo hesize ha he in lamma o y esponse is ac i a ed in he colon o his popula ion. In line wi h his, Fança-Be hon e al. [ 17 ] p e iously desc ibed ha he o sp ing o mo he s ed a low-p o ein die showed dis u bed exp ession o T 3 in he colon du ing lac a ion. Like- wise, he KLF (K üppel-like ac o ) amily o ansc ip ion ac o s is also in ol ed in he con ol o cell p oli e a ion and di e en ia ion in bo h heal hy and pa hological si ua ions, as well as in in lamma o y esponses [ 63 ]. Mo eo e , s udies in humans wi h colo ec al cance ha e desc ibed an un a o able p ognosis a low exp ession le els o KLF3 [ 64 ]. The e o e, he educed exp ession o his ac o in he colon o UL18 a s compa ed o ha in CL18 animals could be conside ed a ma ke o incipien in lamma ion. Taken oge he , hese esul s sugges ha inc eased pe meabili y may p o ide ansi ional bene i s o U pups, such as imp o ed nu ien up ake and he de elopmen o sys emic ole ance, which migh a o hei su i al. Howe e , i may also imply some disad an ages, including he inc eased ansloca ion o mic obes and o eign pa icles leading o he de elopmen o in ec ion, in lamma ion and me abolic disease. Finally, we used Pea son’s co ela ions o suppo e ical mo he - o-child ansmis- sion and he po en ial in luence o nu i ional s a us in his p ocess. I should be no ed ha e ical ansmission can be di ec , when he p esence o a pa icula bac e ium in he milk de e mines he p esence o he same genus in he eces, o indi ec , when he p esence o a pa icula genus in he milk de e mines he p esence o absence o o he di e en gene a in he eces. Thus, a ea ly lac a ion, he p esence o Esche ichia-Shigella in he milk om well- ed animals posi i ely co ela ed wi h he abundance o hese axa in he o sp ing’s eces, bu wi h a lowe in es inal g ow h o Lac obacillus spp. This nega i e co ela ion be ween milk-de i ed Esche ichia-Shigella and he ecal con en o Lac obacillus was no obse ed in U animals, p obably due o he disc iminan abundance o Esche ichia-Shigella in he milk o ood- es ic ed mo he s a L4 which migh ha e allowed he ini ial coloniza ion o he gu o be domina ed by Lac obacillus spp. Howe e , he la e educ ion o his axon in he milk om ood- es ic ed animals, s ongly co ela ed wi h lowe p esence o Lac obacillus in eces, bu he highe ela i e abundance o Esche ichia-Shigella suppo ed he nega i e co ela ion be ween he ela i e abundance o hese wo gene a in eces as p e iously de- sc ibed du ing he p og ession om ulce a i e coli is o colo ec al cance [ 65 ]. In e es ingly, Pea son’s co ela ions also e idenced ha he numbe o posi i e associa ions de ec ed du ing lac a ion be ween ecal anae obic bac e ia, including Fusobac e ium,Roden ibac e , Bac e oides,Rombus ia o Veillonella was signi ican ly mo e abundan in con ol animals han in hose exposed o ma e nal ood es ic ion, sugges ing ha he o ma ion o an anae obic en i onmen , cha ac e is ic o an adul gu mic obial p o ile, was delayed in he o sp ing as a esul o ma e nal die . Thus, ou indings indica e ha gu mic obial composi ion is Nu ien s 2023,15, 4322 22 o 26 no only de e mined by he pa icula cha ac e is ics o each mic obial communi y, bu ha en i onmen al changes also shape he inal mic obio a. Se e al ad an ages and limi a ions o he p esen s udy should be no ed. The main s eng h is ha by collec ing sequen ial samples om each li e , we could examine longi u- dinal changes in milk mic obio a composi ion o e ime and co ela e hem wi h a ia ions in he ecal mic obiome. In addi ion, he use o a a model o e s he ad an age o be- ing able o ca e ully con ol he ma e nal die in he absence o o he ex e nal in luences o s udy he in es inal ou comes o he o sp ing. Howe e , his in i sel is a limi a ion when we conside ha a s a e poly ocous and al icial mammals, meaning ha a s gi e bi h o la ge li e s bo n a an imma u e s age a e ela i ely sho p egnancies, whe eas women gene ally bea a single e us a an ad anced s age o me abolic de elopmen . How- e e , compa a i e physiology be ween species p o ides an oppo uni y o unde s and he molecula , cellula and biochemical mechanisms in ol ed in he ea ly o igin o me abolic p og amming. On he o he hand, die and age we e no able o explain all he changes obse ed in milk and ecal bac e ial composi ion, sugges ing ha o he ac o s may ha e con ibu ed o he la ge in e -indi idual a ia ions obse ed in he mic obio a p o iles. In his ega d, sex di e ences in he gu mic obio a ha e been epo ed in adul indi iduals and a e usually a ibu ed o ho monal di e ences be ween males and emales. Howe e , he e is no consensus on his ela ionship in ea ly li e coloniza ion o in ela ion o b eas - eeding [ 42 , 49 ]. Thus, he absence o gende -s a i ied analysis in he p esen s udy could possibly explain why ou da a showed some inconclusi e esul s on he associa ion o he die -induced shi in he milk mic obio a wi h a ia ions in he ecal composi ion o he o sp ing. Finally, as 16S RNA gene sequencing has limi ed abili y o esol e axa beyond he genus le el, u he me agenomic and cul u ing analysis will be equi ed o con i m and alida e he esul s o his s udy as well as o quan i y bac e ial load and he iabili y o he bac e ia iden i ied in ou samples. 5. Conclusions In conclusion (Figu e 10), ma e nal malnu i ion is able o modi y he mic obial com- posi ion o b eas milk, a phenomenon ha is e lec ed in he in es inal mic obio a o he o sp ing. This ac suppo s he idea o a e ical ansmission o bac e ia om mo he o child. One o he e ec s o his nu i ional condi ion is he delay in he ma u a ion o he in- es inal mic obio a, as e idenced by he gap ha exis s bo h, in he e olu ion o he bac e ial di e si y and he appea ance o he main colonizing gene a du ing lac a ion. These mic o- bial changes we e accompanied by al e a ions in o sp ing gu pe meabili y, pa icula ly a he end o lac a ion, as well as in he goble cell ma ke s T 3 and Kl 3, associa ed wi h cell enewal and ch onic in lamma o y p ocesses. I is wo h men ioning ha ou p e ious s udies [ 16 ] ha e al eady demons a ed ha his mic obio a imma u i y is main ained beyond weaning and e en un il adul hood, which poin s ou he po en ial con ibu ion o al e ed gu coloniza ion o he associa ed mo bidi ies and he sequelae o malnu i ion in ea ly li e, including an inc eased isk o obesi y and glucose in ole ance. The e o e, ou da a highligh he impo ance o b eas eeding as a c i ical window o he apeu ic in e en ion ia he modula ion o he in an mic obio a as pa o p e en i e medicine. Nu ien s 2023,15, 4322 23 o 26 Nu ien s 2023, 15, x FOR PEER REVIEW 23 o 26 he apeu ic in e en ion ia he modula ion o he in an mic obio a as pa o p e en i e medicine. Figu e 10. Ma e nal nu i ion de e mining milk mic obio a and in luencing offsp ing gu coloniza- ion. Nu i ional es ic ion du ing he ea ly s ages o de elopmen al e s he mic obial composi ion o ma e nal milk, a phenomenon e lec ed in he in es inal mic obio a o he offsp ing. As a esul , i al e s he abundance o majo axa in milk and eces, delaying he ma u ing o offsp ing gu mi- c obio a. Addi ionally, hese changes in mic obial composi ion a e associa ed wi h changes in gu pe meabili y and in he goble cells ma ke s, which a e associa ed wi h ch onic egene a ion o cells and in lamma o y p ocesses. Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a www.mdpi.com/xxx/s1. Table S1: Rela i e abundance o he mos abundan gene a in he milk o con ol (C) and unde nou ished (U) a s a 4, 14 and 18 days o lac a ion.; Table S2: Rela i e abun- dance o he mos abundan gene a in he eces o con ol (C) and unde nou ished (U) offsp ing a s a 4, 14 and 18 days o lac a ion. Au ho Con ibu ions: Concep ualiza ion: E.F.-M. and C.Á.; me hodology: P.M.-O., C.A., T.F.-M. and A.S.-R.; in es iga ion: E.F.-M., P.M.-O. and C.Á.; da a analysis: P.M.-O., C.A. and T.F.-M.; und- ing Acquisi ion: E.F.-M.; C.Á., M.Á.M. and J.M.R.; isualiza ion: E.F.-M., C.A., F.E. and M.Á.M. w i - ing—o iginal d a p epa a ion: P.M.-O., C.A. and E.F.-M.; w i ing— e iew and edi ing: E.F.-M., C.Á., F.E., M.Á.M. and J.M.R. All au ho s ha e ead and ag eed o he published e sion o he man- usc ip . Funding: This wo k was suppo ed by he g an s PID2020-116134RB-I00, PID2019-105606RB-I00 and CIBER-Conso cio Cen o de In es igación Biomédica en Red-(CB07/08/0013), Ins i u o de Salud Ca los III, om Minis e io de Ciencia e Inno ación (MINECO). Ins i u ional Re iew Boa d S a emen : All he expe imen s we e conduc ed in acco dance wi h Eu opean Union (2010/63/EU) and Spanish (RD 53/2013) legisla ion and wi h he app o al o he Animal Ca e and Use Commi ee o he Communi y o Mad id (PROEX 349/15 and PROEX 215.8/21). In o med Consen S a emen : No applicable. Da a A ailabili y S a emen : Da a a e a ailable upon eques o he au ho s. Con lic s o In e es : The au ho s decla e no con lic o in e es . Figu e 10. Ma e nal nu i ion de e mining milk mic obio a and in luencing o sp ing gu coloniza ion. Nu i ional es ic ion du ing he ea ly s ages o de elopmen al e s he mic obial composi ion o ma e nal milk, a phenomenon e lec ed in he in es inal mic obio a o he o sp ing. As a esul , i al e s he abundance o majo axa in milk and eces, delaying he ma u ing o o sp ing gu mic obio a. Addi ionally, hese changes in mic obial composi ion a e associa ed wi h changes in gu pe meabili y and in he goble cells ma ke s, which a e associa ed wi h ch onic egene a ion o cells and in lamma o y p ocesses. Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a h ps:// www.mdpi.com/a icle/10.3390/nu15204322/s1. Table S1: Rela i e abundance o he mos abundan gene a in he milk o con ol (C) and unde nou ished (U) a s a 4, 14 and 18 days o lac a ion.; Table S2: Rela i e abundance o he mos abundan gene a in he eces o con ol (C) and unde nou ished (U) o sp ing a s a 4, 14 and 18 days o lac a ion. Au ho Con ibu ions: Concep ualiza ion: E.F.-M. and C.Á.; me hodology: P.M.-O., C.A., T.F.-M. and A.S.-R.; in es iga ion: E.F.-M., P.M.-O. and C.Á.; da a analysis: P.M.-O., C.A. and T.F.-M.; unding Acquisi ion: E.F.-M.; C.Á., M.Á.M. and J.M.R.; isualiza ion: E.F.-M., C.A., F.E. and M.Á.M. w i ing— o iginal d a p epa a ion: P.M.-O., C.A. and E.F.-M.; w i ing— e iew and edi ing: E.F.-M., C.Á., F.E., M.Á.M. and J.M.R. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This wo k was suppo ed by he g an s PID2020-116134RB-I00, PID2019-105606RB-I00 and CIBER-Conso cio Cen o de In es igación Biomédica en Red-(CB07/08/0013), Ins i u o de Salud Ca los III, om Minis e io de Ciencia e Inno ación (MINECO). 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