Wheat and bread samples
The four samples of double haploid lines included in this study were from the Berkut × Krichauff doubled haploid (DH) population, previously used to map quantitative trait loci (QTL) for high AX in this population . The population was grown in 2009 at Roseworthy in South Australia, at an adequate-rainfall site. The lines were chosen based on their fructan and arabinoxylan content (the lines with the highest and the lowest fructan and arabinoxylan amounts were included). These samples were compared with commercially bought white and wholemeal flour mixes and their baked products (Laucke Flour Mills; http://www.laucke.com.au).
Bread and chapatti sample preparation
Bread loaves (1 kg, standard size) were baked using an automatic bread maker Breville BBM100 (Sydney, Australia). An identical baking program was established for the preparation of all breads.
The endpoint of baking was set as “Medium Crust.” The total baking time was 3 h and 5 min, including 25 min kneading, 1 h and 40 min fermentation and 1 h baking at 155°C.
The average amount of the flour mix used for preparation of a loaf of bread was 600 g.
Weight of other ingredients (yeast and water) was 420 g for white and 550 g for wholemeal bread. Once the baking was completed bread loaves were removed from the oven, left to cool on a rack and subsequently frozen to −25°C.
Chapatti breads were prepared using 120 g of the flour mix and 80 ml of water. Dough was kneaded by hand for 20 min than left to rest for a further 30 min (room temperature) before cooking. The dough was shaped into a flat 6-inch disc and cooked on a hot skillet (Sunbeam Classic Skillet SK 4200P 25 cm; 210°C) for 1–2 min until brown spots formed on both sides of the dough. Cooked chapatti was left to cool to room temperature and put at −25°C freezer.
The samples (both yeast leavened and chapatti breads) were freeze-dried for 48 h (Dynavac freeze drying unit) to remove all moisture before grinding (Retch 2M1000b; Retch, Ochten, The Netherlands). All milled samples were stored in plastic containers at room temperature until analysis.
Sugar, phytate and micronutrient analyses
Sugar and phytate analyses were performed using High Performance Liquid Chromatography (HPLC) on a Dionex ICS-3000 unit as described by Huynh et al.  for fructans, Nguyen et al.  for AXs and by Lehrfeld et al.  for phytate. The micronutrient concentration of samples was determined by inductively coupled plasma optical emission spectrometry (ICP-OES)  using an ARL 35B ICP analyser.
Extraction of the wheat samples
Procedure was conducted as previously described by Vidanarachchi et al. . Briefly, the wheat exudate was dissolved in distilled water (50 g/L) and the solution was filtered through a 600 μm screen to remove particulate matter and then centrifuged at 12,000 g for 10 min at 4°C. The supernatant was collected and dialysed (MWCO 12–14 kDa, Medicell International Ltd., London, UK) exhaustively against distilled water (48 h). The dialysate was collected and then lyophilized to yield a fine off-white powder (wheat extract). In addition, in order to avoid embryonic dehydration, the samples were diluted prior to in-ovo injections (solution concentration was not higher than 320 OSM).
In vitro iron bioavailability assessment
An in vitro digestion/Caco-2 cell culture model  was used to assess in vitro Fe bioavailability. With this method, bread/flour samples (0.5 g) were subjected to simulated gastric and intestinal digestion.
Briefly, the intestinal digestion is carried out in cylindrical inserts closed on the bottom by a semipermeable membrane and placed in wells containing Caco-2 cell monolayers bathed in culture medium. The upper chamber was formed by fitting the bottom of Transwell insert ring (Corning) with a 15000 Da molecular weight cut off (MWCO) membrane (Spectra/Por 2.1, Spectrum Medical, Gardena, CA). The dialysis membrane was held in place using a silicone ring (Web Seal, Rochester, NY).
Iron uptake by the cell monolayers was assessed by measuring ferritin concentrations in the cells. Three replicates of each Fe bioavailability measurement were performed, each utilizing a separate sample of the wheat extract.
In terms of materials for the study, Caco-2 cells were obtained from the American Type Culture Collection (Rockville, MD) at passage 17 and used in experiments at passage 29. Cells were seeded at densities of 50,000 cells/cm2 in collagen-treated 6 well plates (Costar Corp., Cambridge, MA). The integrity of the monolayer was verified by optical microscopy. The cells were cultured at 37°C in an incubator with 5% CO2 and 95% air atmosphere at constant humidity, and the medium was changed every 48 h.
The cells were maintained in Dulbecco’s modified Eagle medium plus, 25 mmol/L HEPES, and 10% fetal bovine serum. 48 h prior the experiment, the growth medium was removed from culture wells, the cell layer was washed, and the growth medium was replaced with minimum essential media (MEM) at pH 7.0.
The MEM was supplemented with 10 mmol/L PIPES, 1% antibiotic/antimycotic solution, 4 mg/L hydrocortisone, 5 mg/L insulin, 5 μg/L selenium, 34 μg/L triiodothyronine, and 20 μg/L epidermal growth factor. This enriched MEM contained less than 80 μg Fe/L.
All ingredients and supplements for cell culture media were obtained from Gibco (Rockville, MD). The cells were used in the Fe uptake experiment at 13 days post seeding. In these conditions, the amount of cell protein measured in each well was highly consistent between wells. In experiment day, 1.5 mL of the digested sample was added to the insert’s upper chamber and incubated for 2 h. Then, inserts were removed and 1 mL of MEM was added. Cell cultures were incubated for 22 h at 37°C.
Harvesting of Caco-2 cells for ferritin analysis
The protocols used in the ferritin and total protein contents analyses of Caco-2 cells were similar to those previously described [8, 29, 9, 28, 30].
Briefly, growth medium was first removed from the culture well by aspiration and the cells were washed twice with a solution containing 140 mmol/L NaCl, 5 mmol/L KCl, and 10 mmol/L PIPES at pH 7.0. The cells were harvested by adding an aliquot of deionized water and placing them in a sonicator (Lab-Line instruments, Melrose Park, IL). The ferritin and total protein concentrations were determined on an aliquot of the harvested cell suspension with a one-stage sandwich immunoradiometric assay (FER-IRON II Ferritin assay, Ramco laboratories, Houston, TX) and a colorimetric assay (Bio-Rad DC Protein assay, Bio-Rad, Hercules, CA), respectively.
Caco-2 cells synthesize ferritin in response to increases in intracellular iron concentration. Therefore, we used the ratio of ferritin/total protein (expressed as ng ferritin/mg protein) as an index of the cellular Fe uptake. All glassware used in the sample preparation and analyses was acid washed.
In-vivo (intra amniotic administration procedure) bioavailability analysis
Birds, diets, and study design
One hundred and sixty Cornish-cross fertile broiler eggs were obtained from a commercial hatchery (Moyer’s chicks, Quakertown, PA), from a maternal flock 35 weeks in lay. The eggs were incubated under optimal conditions at the Cornell University Animal Science poultry farm incubator.
The intra amniotic administration procedure was previously described [9, 31]. Briefly, at 17 days of embryonic incubation, eggs containing viable embryos (n = 150) were weighed and divided into 13 groups (n = 12), each with an average egg weight of 49 ± 1.15 g.
All treatment groups were assigned eggs of similar weight-frequency distribution.
A group of 144 eggs was then injected with the specified solution (1 ml per egg) with a 21-gauge needle into the amniotic fluid, which was identified by candling . The intra-amniotic treatment solution included the following: ‘Control 1’: non injected eggs; ‘Control 2’: inulin solution (4%-inulin/ddH2O); ‘Control 3’: arabinose solution (4%-arabinose/ddH2O) and 10 samples (extracted in ddH2O).
‘Control 1’ (n = 12) was non injected group that paralleled routine procedures in commercial hatcheries. After all the eggs were injected, the injection holes were sealed with cellophane tape and the eggs placed in hatching baskets such that each treatment was equally represented at each incubator location.
Hatchability was similar in all treatment groups and was approximately 90%. Immediately after hatch (21 d) and from each treatment group, 10 chicks were euthanized by carbon dioxide exposure. The digestive tracts and livers were quickly removed from the carcass and separated into various sections for tissue (small intestine and liver ~ 1-2 cm; ~2-3gr, respectively) and cecum.
Samples were collected for relative mRNA abundance of intestinal Fe related transporters, relative liver ferritin amounts and bacterial analysis of cecal content, respectively. The samples were immediately frozen in liquid nitrogen, and then stored in a −80°C freezer until analysis. All animal protocols were approved by the Cornell University Institutional Animal Care and Use Committee.
Blood analysis and hemoglobin (Hb) measurements
Blood samples were collected weekly from the wing vein (n = 10, ~50 μL) using micro-hematocrit heparinized capillary tubes (Fisher, Pittsburgh, PA). Samples were collected immediately after hatch. Blood Hb concentrations were determined spectrophotometrically using the cyanmethemoglobin method (H7506-STD, Pointe Scientific Inc. Canton, MI) following the kit manufacturer’s instructions.
Isolation of total RNA
Total RNA was extracted from 30 mg of the duodenal tissue (tissue was harvested from the proximal duodenum, n = 10) using QiagenRNeasy Mini Kit (RNeasy Mini Kit, QiagenInc.,Valencia, CA) according to the manufacturer’s protocol. Briefly, tissues were disrupted and homogenized with a rotor-stator homogenizer in buffer RLT®, containing β-mercaptoethanol. The tissue lysate was centrifuged for 3 minutes at 8,000 g in a micro centrifuge. An aliquot of the supernatant was transferred to another tube, combined with 1 volume of 70% ethanol and mixed immediately.
Each sample (700 μL) was applied to an RNeasy mini column, centrifuged for 15 s at 8000 g, and the flow through material was discarded. Next, the RN easy columns were transferred to new 2-mL collection tubes, and 500 μL of buffer RPE® was pipetted onto the RNeasy column followed by centrifugation for 15 s at 8000 g. An additional 500 μL of buffer RPE were pipetted onto the RNeasy column and centrifuged for 2 min at 8000 g.
Total RNA was eluted in 50 μL of RNase free water. All steps were carried out under RNase free conditions. RNA was quantified by absorbance at A 260/280. Integrity of the 28S and 18S ribosomal RNAs was verified by 1.5% agarose gel electrophoresis followed by ethidium bromide staining.
DNA contamination was removed using TURBO DNase treatment and removal kit from AMBION (Austin, TX, USA).
DMT1, DcytB and Ferroprtin gene expression analysis
As previously described [8, 29, 32, 9], briefly, PCR was carried out with primers chosen from the fragment of the chicken (Gallus gallus) duodenal DMT1 gene (GeneBank database; GI 206597489) (forward: 5’-AGC CGT TCA CCA CTT ATT TCG-3’; reverse: 5’-GGT CCA AAT AGG CGA TGC TC-3’), DcytB gene (GI 20380692) (forward: 5’-GGC CGT GTT TGA GAA CCA CAA TGT T-3’; reverse: 5’-CGT TTG CAA TCA CGT TTC CAA AGA T-3’) and Ferroportin gene (GI 61098365) (forward: 5’-GAT GCA TTC TGA ACA ACC AAG GA’; reverse: 5’-GGA GAC TGG GTG GAC AAG AAC TC-3’). Ribosomal 18S was used to normalize the results (GI 7262899) (forward: 5’- CGA TGC TCT TAA CTG AGT-3’; reverse: 5’-CAG CTT TGC AAC CAT ACT C-3’).
Determination of the linear phase of the PCR amplification was performed (Access RT-PCR system, Promega, Madison, WI) with pooled aliquots removed at 15, 20, 25, 30, 35, 40, 45, 50, and 55 cycles.
Amplification of the chicken duodenal DMT1, DcytB and Ferroportin genes were performed for 32, 40 and 30 cycles respectively, which consisted of denaturation (95°C, 30 s), annealing (48°C, 1 min),
and extension (72°C, 1 min); ribosomal 18S was amplified at 32 cycles under identical conditions in a different tube. All PCR products were separated by electrophoresis on 2% agarose gel, stained with ethidium bromide, and quantified using the Quantity One 1-D analysis software (Bio-Rad, Hercules, CA).
Ferritin and Fe in the liver
Liver samples were treated as described by Mete et al. . Briefly, 1 g of sample was diluted into 1 mL of 50 mM Hepes buffer, pH 7.4, and homogenized on ice at 5000 g and for 2 min. One mL of each homogenate was subjected to heat treatment for 10 min at 75°C to aid isolation of ferritin since other proteins are not stable at that temperature [33, 34, 32, 9]. After heat treatment the samples were immediately cooled down on ice for 30 min.
Thereafter, samples were centrifuged at 13000 g for 30 min at 4°C until a clear supernatant was obtained and the pellet containing most of the insoluble denaturated proteins was discarded. All tests were conducted in duplicates for each animal (n = 6).
Electrophoresis, staining and measurement of gels
Native polyacrylamide gel electrophoresis was conducted using a 6% separating gel and a 5% stacking gel. Samples were run at a constant voltage of 100 V. After electrophoresis, the gels were treated with either of the two stains: Coomasie blue G-250 stain, specific for proteins, or potassium ferricyanide (K3Fe(CN)6) stain, specific for iron.
The corresponding band found in the protein and iron stained gel was considered to be ferritin [32, 35, 33, 34]. The gels were scanned with Bio-Rad densitometer. Measurements of the bands were conducted using the Quantity-One 1-D analysis program (Bio-Rad, Hercules, CA). The local background was subtracted from each sample.
Horse spleen ferritin (Sigma Aldrich Co., St. Louis, MO) was used as a standard for calibrating ferritin protein and iron concentrations of the samples. Dilutions of the horse spleen ferritin were made and treated similarly to the liver supernatant samples in order to create a reference line for both protein and iron-stained gels. Iron levels were determined using the same calibration since approximately 20% of the weight of horse spleen ferritin is iron.
Saturation levels of ferritin with iron were calculated as the percentage of the iron present in the protein to the maximum amount of iron atoms (4500 iron atoms/ferritin molecule) ferritin can incorporate [32, 35, 33, 34].
Collection of microbial samples and DNA isolation
The cecum was removed and treated as described previously (Zhu et al., 2002; Tako et al., 2008; Tako and Glahn, 2012). The contents of the cecum were squeezed out into a sterile 50-mL tube containing 9 mL of sterile PBS and homogenized by vortexing with glass beads (3-mm diameter) for 3 min.
Debris was removed by centrifugation at 700 × g for 1 min, and the supernatant was collected and centrifuged at 12,000 × g for 5 min. The pellet was washed twice with PBS and stored at −20°C until DNA extraction. For DNA purification, the pellet was resuspended in 50 mM EDTA and treated with lysozyme (Sigma Aldrich Co., St. Louis, MO; final concentration of 10 mg/mL) for 45 min at 37°C. The bacterial genomic DNA was isolated using a Wizard Genomic DNA purification kit (Promega Corp., Madison, WI). The DNA concentration was determined spectrophotometrically.
Primer design and PCR amplification of bacterial 16S rDNA
Primers for Lactobacillus (forward: 5’- CAT CCA GTG CAA ACC TAA GAG-3’; reverse: 5’- GAT CCG GTG CAA ACC TAA GAG-3’) and Bifidobacterium (forward: 5’- GGG TGG TAA TGC CGG ATG-3’; reverse: 5’- CCA CCG TTA CAC CGG GAA-3’) were designed according to previously published data [36–38, 9, 35]. Universal primers identifying all known bacteria were designed using the invariant region in the 16 s rDNA of the bacteria (forward: 5’- CGT GCC AGC CGC GGT AAT ACG -3’; reverse: 5’- CGT GCC AGC CGC GGT AAT ACG-3’). The universal primer set was used for determining the total microflora population. For PCR amplification of the bacterial targets from cecal contents, 5 μL of DNA extract was added to 45 μL of PCR mixture containing 27.5 μL of nuclease-free water, 5 μL of each primer (10 μg/mL), 1.5 μL of nucleotide (dNTP) mix, 5 μL of PCR buffer, and 1 μL of Taq polymerase (Go-Taq, Promega). The PCR thermal conditions were as follows: 1 cycle of 94°C for 3 min, 35 cycles of 94°C for 30 s, 60°C for 1 min, 68°C for 2 min, and finally 1 cycle of 68°C for 7 min. The PCR reaction was run with different numbers of cycles (25, 30, 35, 40, 45, or 50) for each primer set, and 35 cycles was in the center of the exponential increase in PCR products.
The PCR products were separated by electrophoresis on 2% agarose gel, stained with ethidium bromide, and quantified using a Gel-Pro analyzer version 3.0 (Media Cybernetics, Bethesda, MD). To evaluate the relative proportion of each examined bacteria, all products were expressed relative to the content of the universal primer product and proportions of each bacterial group are presented, where the total of the examined bacteria was set at 100%.
Results were analyzed by one way ANOVA using the SPSS statistical software (SPSS 17.0; SPSS Inc, Chicago, IL). Differences between treatments were compared by Tukey’s test and significance level of p < 0.05 was used for all comparisons. Values in the text are means ± SD.