gy, Part A 145 (2006) 502–508www.elsevier.com/locate/cbpa

Comparative Biochemistry and Physiolo

Maternal diet influences gene expression in intestineof offspring in chicken (Gallus gallus)

Johanna M.J. Rebel ⁎, Saskia Van Hemert, Arjan J.W. Hoekman, Francis R.M. Balk,Norbert Stockhofe-Zurwieden, Dirk Bakker, Mari A. Smits

Animal Sciences Group, PO Box 65, 8200 AB Lelystad ,The Netherlands

Received 15 December 2005; received in revised form 10 August 2006; accepted 18 August 2006Available online 1 September 2006

Abstract

The diet of the mother during pregnancy influences the onset of different diseases and health-related traits in the offspring. We investigated theinfluence of the mother hen diet on the intestinal gene expression pattern in the offspring. Hens received for 11 weeks either a commercial feed ora commercial feed supplemented with vitamins and minerals. The offspring of the two groups showed no changes in growth rate or feedconversion. Of this offspring, gene expression patterns in the intestine were measured at 3 and 14 days of age with an intestinal cDNA-microarray.Between the two groups, 11 genes were found to be differentially expressed both at 3 and 14 days of age. Thus, these genes were differentlyregulated when the intestine is developing as well as when the intestine is more mature. Genes that are differentially expressed at day 3 and/or day14 affect intestinal turnover, proliferation and development, metabolism and feed absorption. To confirm that differences in gene expression arerelated to intestinal development, we investigated intestinal proliferation. This indeed also showed differences in proliferation between the twogroups at day 3 and day 14 of age. The gene expression and proliferation results indicate that feed of the hens influences the functionality ofintestine of the offspring at day 3 and 14 of age.© 2006 Elsevier Inc. All rights reserved.

Keywords: Development; Feed; Gene expression; Intestine; Mother diet; Offspring

1. Introduction

Genetic mutations and selection within populations maypromote individual survival. However, genetic mutations re-quire long, evolutionary time periods to change the populations.These mutations are not likely to be reversible or rapidlyadaptable to changing environmental conditions. Prenatal pro-gramming, however, may provide populations or individualsto adapt to the environmental changing conditions rapidly.Programming is a process whereby a stimulus or stress at acritical or sensitive period in development has lasting or lifelongsignificance. The in utero environment may therefore have animpact on the homeostatic regulatory mechanisms in adult life. Ithas been proposed that poor foetal nutrition leads to program-ming of metabolism in a manner beneficial to survive under

⁎ Corresponding author. Tel.: +31 320 238108; fax: +31 320 238264.E-mail address: Annemarie.Rebel@WUR.NL (J.M.J. Rebel).

1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved.doi:10.1016/j.cbpa.2006.08.035

conditions of poor nutrition, representing an adaptive response(Gluckman and Hanson, 2004).

Early life nutrition in humans has been suggested toinfluence the progression of chronic diseases later in life. Inhumans, a low birth weight has been associated with increasedrisks of diseases later in life including obesity, diabetes andcardiovascular diseases (Leeson et al., 1997; Jaquet et al., 2000;Godfrey and Barker, 2001; Hales and Barker, 2001; Godfrey,2002; Verkauskiene et al., 2005). There is, however, conflictingevidence on the impact of early life nutrition and overweight inhumans (Scaglioni et al., 2000; Huxley et al., 2002; Huxley,2004). The offspring of animals in which maternal nutrition wasrestricted during early life exhibited changes later in life. Forexample, undernutrition in utero leads to elevated bloodpressure in adult guinea pigs and rats (Persson and Jansson,1992; Woodall et al., 1996; Lillycrop et al., 2005). In a differentstudy, maternal dehydration or maternal undernutrition in sheepand rats resulted in long-term effects in adulthood as measuredby plasma osmolality, sodium concentration in blood, body fat

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and blood pressure (Ross and Desai, 2005). This suggests thatthe nongenetic effects for onset of different diseases and health-related traits in the offspring could be “imprinted” in early life.

Although the effects of undernutrition have been documen-ted in epidemiologic studies and animal models, the effects ofmaternal diet without energy restriction have not been studied inextent. However, foetal development can be affected by normalrange of nutritional variation in diets of the mothers.

In chicks, it is known that in ovo (in the egg) supplemen-tation of nutrition can influence intestinal development asmeasured by villus length in the intestines of the offspring (Takoet al., 2004; Uni et al., 2005). The mother diet can influence theegg yolk compositions, and hatchability and embryonicmortality in chickens and quail (Wilson, 1997; Karadas et al.,2005). The selenium intake of the mother hen could influencethe level of selenium in different organs of the chick (Pappaset al., 2005). The effects of the mother diet on the intestinaldevelopment of the offspring, however, are not often reported.Rebel et al. reported effects of the breeder diet on the sus-ceptibility in the offspring for Malabsorption syndrome and theeffects on the percentage of circulating lymphocytes in 1-day-old broiler offspring (Rebel et al., 2004).

Here we investigated intestinal development as shown bygene expression profiles and proliferation in relation to the dietof the mother hens.

2. Material and methods

2.1. Chickens

2.1.1. Breeder hensFour hundred hybro breeder pullets (Gallus gallus) were

placed in two pens, within each pen 14 Hybro cockerels fromweek 18 of age onward (breeder and cockerels were obtainedfrom Nutreco®, Boxmeer, The Netherlands). From week 18 ofage until week 30 of age, one group of breeders was offered arelative low level of vitamins and trace minerals via the premix(in the text named “low mix”) (Table 1). The other group ofbreeders was offered a relative high level of vitamin and traceminerals (in text named “high mix”). The following amounts ofadded vitamins and trace minerals were chosen, and included in

Table 1Composition of breeder feed

Breeder low mix Breeder high mix

Vitamin A 7500 IU 15,000 IUVitamin D 2500 IU 3000 IUVitamin E 10 IU 200 IUVitamin C Not added 100 mg/kgVitamin B1 3 mg/kg 6 mg/kgVitamin B2 8 mg/kg 16 mg/kgZinc 40 mg/kg 70 mg/kgCopper 7.5 mg/kg 15 mg/kgSelenium 0.125 mg/kg 0.25 mg/kg

Other vitamin and trace mineral supplemental levels were not differentiated.Breeder feed: vit. K3 6.0 mg/kg, vit B6 7.0 mg/kg, vit. B12 40 μg/kg, niacin60 mg/kg, folic acid 3.0 mg/kg, biotin 0.25 mg/kg, choline 300 mg/kg, Fe65 mg/kg, I 1.0 mg/kg, antioxidants 100 mg/kg.

a commercial corn–wheat–soybean meal basal diet. No growthpromoters and coccidiostatics were added. Body weight, layingpercentage, feed intake, egg weight, hatchability did not differbetween the group of breeder hens.

Fertilised eggs were obtained from 29-week-old breeders,collected during 3 days and both group of eggs were hatched atthe same time under standard conditions. Hatchability rate forthe low mix group was 85% and for the high mix group 82%.

2.1.2. Broiler chicksThe chickens were reared on a litter floor and feed and water

was provided ad libitum. All broilers received the same kind offeed which consisted of Vit A 3.44 mg/kg; Vit D 50 μg/kg; Vit E10 mg/kg; Vit K3, 2 mg/kg; Niacine, 40 mg/kg; Vit B1 2 mg/kg;Vit B2 6 mg/kg; Vit B6, 3.0 mg/kg; Vit B12, 20 mg/kg; Folicacid, 1 mg/kg; Biotin, 0.10 mg/kg, Choline chloride 300 mg/kg;Fe, 50 mg/kg; Mn, 80 mg/kg; I, 1 mg/kg; Co, 0.20 mg/kg; Zn40 mg/kg; Cu 7.5 mg/kg; Se 0.15 mg/kg added in a commercialcorn–wheat–soybean meal basal diet, in grower feed 80% ofthese levels. The light schedule was 3 h dark 1 h light. Allchickens were immunised at day 1 with infectious bronchitis(IB) and New Castle disease virus (NDV) spray vaccine(Intervet®, Boxmeer, The Netherlands). The experiments weredone in the Netherlands at ID-Lelystad. The study wasapproved by the Institutional Animal Experiment Commissionin accordance with the Dutch regulations of animal experimen-tation. At day 3 and 14, 5 chicks of both breeder groups werekilled by cervical dislocation and necropsied. Pieces of thejejunum were removed and immediately frozen in liquidnitrogen. Adjacent parts were taken and were collected inbuffered 4% formaldehyde. The intestinal sections were cutopen for better fixation and were fixed for a maximum of 24 hbefore processing for paraffin embedding. Serial sections werecut for proliferating cell nuclear antigen (PCNA) staining.

2.2. PCNA staining

PCNAwas detected by immunohistochemistry using a mono-clonal antibody against recombinant rat PCNA (PC-10)(DAKO®, Glostrup, Denmark) according to the manufacturer'sinstructions. The number of PCNA positive cells in the crypt area,transition area and the villus area of the small intestine werescored in a blindly fashioned matter using a semi-quantitativescale. Score: 0=no staining, 1= few cells with staining,2=approximately 10% of cells with staining, 3=approximately25%with staining, 4=50%with staining, 5=N50%with staining.

2.3. RNA isolation

Pieces of the jejunum were crushed under liquid nitrogen.50–100 mg tissues of the different chicks were used to isolatetotal RNA using TRIzol reagent (Invitrogen, Breda, the Nether-lands), according to instructions of the manufacturer with anadditional step as described (van Hemert et al., 2003). For thearray hybridisation, pools of RNA were made in which equalamounts of RNA from five different chickens of the same groupand timepoint were present.

Fig. 1. Mean mass of five chickens of each group.

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2.4. Hybridising of the microarray

The microarrays were constructed as described earlier (vanHemert et al., 2003). The microarrays contained 3072 cDNAsspotted in duplicate from a subtracted intestinal library(described in van Hemert et al., 2003) . Before hybridisation,the microarray was pre-hybridised in 5% SSC, 0.1% SDSand 1% BSA at 42 °C for 30 min. To label the RNA, theMICROMAX TSA labelling and detection kit (Perkin-Elmer,Wellesley, MA) was used. The TSA probe labelling and arrayhybridisation were performed as described in the instructionmanual with minor modifications. Biotin- and fluorescein-labelled cDNAs were generated from 5 μg of total RNA fromthe chicken jejunum pools per reaction. The cDNA synthesistime was increased to 3 h at 42 °C, as suggested (Karsten et al.,2002). Post-hybridisation washes were performed according tothe manufacturer's recommendations. Hybridisations wereperformed in duplicate with the fluorophores reversed. Aftersignal amplification, the microarrays were dried and scannedfor Cy5 and Cy3 fluorescence in a Packard Bioscience BioChipTechnologies apparatus. The image was processed withGenepix pro 5.0 (Genomic Solutions, Ann Arbor, MI) andspots were located and integrated with the spotting file of therobot used for spotting. Reports were created of total spotinformation and spot intensity ratio for subsequent dataanalyses.

2.5. Analysis of the microarray data

For each of the two timepoints, the pools of RNA from fivedifferent chickens of offspring of breeder low mix washybridised against RNA from offspring of breeder high mix.The hybridisation was repeated with the fluorphores reserved.From each RNA four values were obtained, two for one slideand two for the dye-swap. Genes with two or more missingvalues were removed from further analysis. Missing valueswere possibly due to a bad signal-to-noise ratio. A gene wasconsidered to be differentially expressed when the mean valueof the ratio log2 (Cy5/Cy3) was N1.58 or b−1.58 and thecDNA was identified with significance analysis of microarrays(based on one class analysis in SAM; Tusher et al., 2001) with aFalse discovery rate b2%. Because the ratio was expressed in alog2 scale, a ratio of N1.58 or b−1.58 corresponded to a morethan threefold up- or downregulation, respectively, which is theexpression difference limit indicated by the manufacturer of theMICROMAX TSA labelling and detection kit (Perkin-Elmer,Wellesley, MA).

2.6. Quantitative lightcycler real-time PCR

A quantitative PCR on the RNA samples of the jejunumobtained during postmortem was done as described previously(van Hemert et al., 2003). Briefly, 200 ng of RNA was reversetranscribed with random hexamers. Generated cDNA wasstored at −20 °C until use. PCR amplification and analysisfor the Retinol binding protein II was done with the primers: 5′-ATTGCATTCACATAGCTGTTTC-3′ and 5′-ACCAGGTG

TGCCATCAG-3′. The reaction mixture consisted of 1 μlcDNA (1:10 diluted), 1 μl of each primer (10 μM solution), 2 μlLightCycler FastStart DNA Master SYBR Green mix, 2 mMMgCl2 in a total volume of 20 μl. All templates were amplifiedwith a preincubation for 10 min at 95 °C followed by ampli-fication for 40 cycles: (5 s 95 °C, 10 s 59 °C, 15 s 72 °C). Forthe gene Calbindin 1 3 mM MgCl2 was used and amplificationwas performed with annealing at 56 °C and two specific primers5′-CATGGATGGGAAGGAGC-3′ and 5′-GCTGCTGGCACCTAAAG-3′ ForG. gallus similar to chromosome 10 3mMMgCl2was used and amplification was performed with annealing at64 °C and two specific primers 5′-TCTTCCCAGGCTGTGAG-3′ and 5′-GTCACCAGCTTGTTCTTC-3′. For the gene liverfatty acid binding protein 3 mM MgCl2 was used and ampli-fication was performed with annealing at 59 °C and two specificprimers 5′-CCAATGAGTTCACCATTGG-3′ and 5′-AAATGATAAGTGCATGCAGG-3′. In each run, four standards of thegene of interest were included with appropriate dilutions of theDNA to determine the cDNA concentration in the samples. AllRT-PCRs amplified a single product as determined by meltingcurve analysis.

3. Results

The mass of the chicks did not differ between both groupsfrom day 1 until day 32 (Fig. 1) No differences were observed inthe morphology of the jejunum when the intestines were inves-tigated for cystic crypt formation, villus atrophy and infiltrationof polymorphonuclear cells.

A comparison of gene expression in the intestine was madebetween chicks of which the mother hens received a relative lowlevel of vitamins and trace minerals via the premix (low mix)with offspring of mother hens that received a relative high levelof vitamin and trace minerals (high mix) (Table 1). Geneexpression was investigated in the chicks at 3 and 14 days ofage. The analysis of the micro-array showed different numbersof up- and down-regulated genes between the two groups andbetween different ages (Table 2).

Of the 31 genes found to be differentially expressed at one ofthe two time points investigated, 11 genes were differentiallyexpressed at both time points. Of these 11 genes 5 were higher

Table 2Genes and expressed sequenced tags differentially expressed in one of theoffspring groups at days 3 and 14 of age

Accession number Description Ratioday 3

Ratioday 14

XM_421801 Gallus gallus similar toCRP-ductin-alpha

2.35 3.19

NM_001006290 Gallus gallus adenosinedeaminase (ADA)

2.34 2.08

XM_422715.1 Gallus gallus similar to Fcfragment of IgG binding protein

1.98 2.63

L11147.2 Gallus gallus cognin/prolyl-4-hydroxylase/proteindisulfide isomerase

1.84 2.13

NM_001001751.1 Gallus gallus cytochrome P450 A 37 1.7 1.77XM_420282.1 Gallus gallus similar to DNA

segment, Chr 10−1.61 −2.81

AF421549 Gallus gallus CDH1-D −1.71 −2.56XM_419273.1 Gallus gallus similar

to ENSANGP00000007226−2.03 −2.17

XM_418698.1 Gallus gallus similar to GOB-4 −2.24 −3.01XM_414486.1 Gallus gallus similar to fatty acid

binding protein 6−2.56 −3.04

XM_419952.1 Gallus gallus similar to proteindisulfide isomerase A6 precursor

−2.57 −3.02

XM_417389.1 Gallus gallus similar to cellularapoptosis susceptibility protein (CASP)

2.63 ns

NM_204192.1 Gallus gallus fatty acid bindingprotein 1, liver

2.48 ns

CR353495.1 Gallus gallus finished cDNA,clone ChEST250m1

2.36 ns

XM_416504. Gallus gallus similar to putativeG protein-coupled receptor 92

1.99 ns

XM_416748. Gallus gallus similarto phosphodiesterase 9A isoform

1.94 ns

M18421 Chicken apolipoprotein B 1.93 nsXM_422636.1 Gallus gallus similar to Retinol-binding

protein II1.88 ns

BX932092. Gallus gallus finished cDNA,clone ChEST157n7

1.78 ns

NM_001030730 Gallus gallus similar toN-acyl-phosphatidylethanolamine-hydrolyzing phospholipase D

−1.79 ns

NM_205513.1 Gallus gallus calbindin 1 −2.53 nsXM_426327. Gallus gallus similar to

aminopeptidase Ans 2.45

XM_422360.1 Gallus gallus similar tocalcium-activated chloride channel

ns 2.32

XM_417652.1 Gallus gallus similar toguanylin precursor

ns 1.9

AF110982.1 Gallus gallus T-cell receptor betachain constant region

ns 1.7

BX950422.2 Gallus gallus finished cDNA,clone ChEST730b4

ns 1.7

NM_001006438.1 Gallus gallus similar to sterolC-4 methyl oxidase-like

ns 1.7

XM_422811.1 Gallus gallus similar toMaltase-glucoamylase, intestinal

ns 1.69

AJ851452.1 Gallus gallus mRNA forhypothetical protein

ns 1.62

XM_420563.1 Gallus gallus similar to onzin ns −1.66XM_424890.1 Gallus gallus similar to

fructose-bisphosphate aldolasens −2.08

Log2 ratios, positive ratios mean that the genes are higher expressed in theintestines of the offspring of which the mothers received a high mix.na: not available. ns: not significantly differentially expressed.

Table 3Amplified PCR products

Gene name Ratio PCR high/low

liverFAPB 3.14⁎

Retinol bp 1.95Segment chr 10 −2.56⁎Calbindin −2.11⁎

The mean is given of five individual chickens of each group.liverFAPB; liver fatty acid binding protein.Retinol bp: Retinol binding protein II.Segment chr 10; Gallus gallus similar to DNA segment chromosome 10.Calbindin; Calbindin 1.⁎ Pb0.07 based on results of the lightcycler PCR on jejunum obtained fromindividual chickens of each group when the offspring of the high mix groupwere compared to the offspring of the low mix group.

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expressed in the offspring from the mothers that received a highmix compared to the offspring group of the mothers with a lowmix and the other 6 were lower expressed in the offspring of thehigh mix group.

At day 3 or 14, the chicks of which the mother received ahigh mix had a higher expression of 20 genes compared to thechicks of the mother low mix group while only 9 genes werelower expressed.

Due to the fact that array results could be influenced by eachstep of the assay validation of expression differences is neces-sary. For this validation the lightcycler quantitative PCR waschosen. Four differentially expressed genes at day 3 werechosen for validation. For these four genes, the results of geneinduction or suppression between the groups of the individualchickens were similar to that obtained by the microarray(Table 3). Conformation of our microarray data with the RT-PCR was also shown in earlier studies (van Hemert et al., 2004,2003).

After analysis of the differentially expressed genes, differ-ences in gene expression in the intestine could be attributed orlead to differences in proliferation of intestinal cells. Therefore,we performed a PCNA staining on the adjacent part of theintestine that we had used for RNA isolation. Differences infrequency of PCNA positive cells were found between thegroups and between day 3 and day 14 of age (Fig. 2). Especiallythe frequency of PCNA positive cells is lower in the villus areaof the chicks of which the mothers received a high diet at day 3of age compared to the high mix group. In the transition area,

Fig. 2. Mean PCNA score in the intestine of broiler offspring.

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the difference in PCNA staining between day 3 and day 14 issmaller in the mother high mix group when compared to theoffspring of the breeders low mix. Also in the crypt area nodifference in PCNA positive cell frequency was observedbetween day 3 and day 14 for the chicks of the mother high mixgroup.

4. Discussion

The two groups of chicks showed differences in intestinalgene expression at day 3 and day 14 of age, while the chicksreceived the same diet. The two groups were kept in the samestable under identical conditions, same feed and the geneticbackground of the two broiler groups was identical. The onlydifference between the two groups was the diet of the motherhens during 11 weeks. The high mix mother group did receivean additional amount of vitamins and minerals. The energy andprotein levels between the groups were equal. It is known thatVitamin A or vitamin D in pregnant women could affect thedevelopment and growth of the foetus and children (Gazalaet al., 2003; Javaid et al., 2006; Mannion et al., 2006). However,no difference in body weight between the group offspring wasobserved. It is not known what the effects of these vitamins areat the development of the intestine of the offspring. Here, themother diet influence gene expression of the intestine in theoffspring at day 3 and day 14 of at least 11 genes. These genesare probably influenced for a longer period of time due to thediet of the mothers.

Genes that are higher expressed at day 3 and day 14 of age inthe chicks of which the mothers received the higher mix are:cognin/prolyl-4-hydroxylase/protein disulfide isomerase, CRP-ductin-alpha, cytochrome p450 A37, ADA and Fc fragment ofIgG binding protein. These genes are involved in epithelialturnover and maturation (Cheng et al., 1996; Al-Awqati et al.,2000; Ourlin et al., 2000; Myllyharju, 2003; Wildhaber et al.,2003). The offspring of the breeders with a high mix have alower persistent expression at day 3 and 14 of CDH-1, fatty acidbinding protein 6; protein disulfide isomerase A6 precursor andGOB-4 compared to the low mix group. These genes areinvolved in metabolic regulation (Komiya et al., 1999; Wan andKirschner, 2001; Agellon et al., 2002; Besnard et al., 2002).

Thus, the offspring of the breeders with a high diet expresspersistently higher, as measured at day 3 and 14, intestinal genesthat affect the turnover/proliferation of intestinal cells comparedto the chicks of which the mothers received a low diet. Toconfirm the possible effect of mother diet on intestinal cellturnover or proliferation in the offspring, we investigated thenumber of intestinal cells that proliferate. The offspring of thebreeders with a high diet had at day 14 a higher proliferation inthe transition area compared to the offspring of the low mix. Inall three areas investigated, the decrease in number ofproliferating cells from day 3 to day 14 is less in the offspringof the high mix group. Thus, the proliferation data confirm thegene expression data, there are differences between groups atintestinal turnover due to mother diet. The high mix of themothers resulted in the offspring to a persistent lowerexpression of genes involved in intestinal metabolic function

when compared to the offspring of mothers with a low mix; thiscould be due to differences in maturation of the intestine. Whenin time the intestine is proliferating more, it could be arguedthat this led to less differentiated villus area and difference inmetabolism (Potten and Loeffler, 1990). The offspring of bothbreeder groups received equal feed as described.

Most of the intestinal genes of the offspring that aredifferentially expressed between the two mother mix groups atday 3 are related with intestinal development, utilisation oflipids and nutritional absorption (Lazier et al., 1994; Fareseet al., 1996; Basque et al., 1998; Behrens et al., 2003; Renteroet al., 2003; Wang et al., 2003; Ogura et al., 2005). At day 14,these 10 genes are not differentially expressed between thegroups. At day 3 of age, the intestine of the broiler is rapidlydeveloping in length and function. As shown here, thefrequency of proliferating cells at day 3 is higher when com-pared to day 14 in the villus and transition area. The first daysafter hatch, the function of the intestine is mainly to absorb theyolk and this has to change towards absorption of feed. At day14 of age, the organisation of the intestinal epithelium and theimmune system are matured and only the length of the intestineis increasing (Uni et al., 2003). The regulation of the genes atday 3 could affect the yolk utilisation and maturation of theintestine. At day 3, the intestine is proliferating mostly in thecrypt area, although between the groups most differences inthe number of proliferating cells were found in the villus area.The gene expression and the proliferation data could indicatethat the differentiation or maturation of intestinal cells differbetween the two groups at day 3.

The maternal diet also affects genes at day 14 of age andthese genes are involved in feed absorption and metabolism ofthe intestine. At day 14 also, the T-cell receptor beta chainregion mRNA is increased. This could lead to the conclusionthat the immune system of the two groups is differentdeveloped. At day 3, these genes did not differ between thegroups which mean that these genes are only affected in themature intestine.

Thus, mother diet influences gene expression that couldaffect the metabolism, feed absorption and development of theintestine and intestinal immune system of the offspring at day 3and day 14 of age. The gene expression data are partlyconfirmed with the PCNA staining. It was shown that themother diet influence the number of proliferating cells in thevillus and transitional area which indeed confirms that motherdiet influence intestinal development. This is analogous to whatis thought in humans, namely, that prenatal nutrition affectsmetabolic pathways in the offspring. It was found that motherdiet influences the development of the intestine of the offspringover time. This can have implications on chronic disease issuesthat are related to foetal development. At this time, it is notknown which diet leads to chicks with a better health-relatedtrait. The offspring in this experimental setup, however, reactedalso different at a malabsorption (MAS) challenge, which is asyndrome that affects the small intestine (Rebel et al., 2004).Offspring of breeders with a high mix reacted more severely3 days after MAS challenge, while the intestine of the samegroup recovered faster. Thus, the mother diet influences

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persistent in time the functionality of the intestine of theoffspring and could therefore also influence intestinal health.

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