Meckel Diverticulum

The experimental diets were provided to the hens after placing them in the metabolism units at week 27. Samples of each diet were obtained before and after the excreta collection phase to determine the DM content of the diets. The animals were monitored at least twice daily. Feed was weighed at the beginning and feed and birds were weighed at the end of the excreta collection phase. In week 30, total excreta were collected from the trays at 24-h intervals for four consecutive days. Feathers, skin scales, and spilled feed were carefully removed before each excreta collection. Feed residues from the trays were weighed, and feed intake was corrected for these residues. Excreta were immediately frozen after each collection at −20 °C. During the excreta collection phase, the eggs of all hens were collected and maintained at room temperature until further processing.
In week 31, the blood and intestinal contents were sampled on four consecutive days beginning at 9 AM each day, whereby treatments were equally distributed among the days. Feed was deprived for 2 h before slaughtering followed by 1 h ad libitum access to the feed to standardize intestinal fill. The hens were individually stunned with a gas mixture of 35% CO₂, 35% N₂, and 30% O₂ and killed by decapitation. The trunk blood was collected in tubes containing sodium fluoride for MI analysis or lithium heparin for P and Ca analysis. Blood samples were then centrifuged for 10 min at 2500 × g to obtain the plasma. Digesta from the crop, gizzard, jejunum, the terminal part of the ileum, defined as the last two thirds of the section between Meckel’s diverticulum and 2 cm prior to the ileo-ceco-colonic junction, and both ceca were collected. The crop and gizzard were clamped with an arterial clamp to prevent emptying and were then opened and upended. The crop and gizzard digesta was gently removed with a spatula without scraping the mucosa. The jejunum, terminal ileum, and ceca were cut lengthwise, and digesta was gently removed with a spatula without scraping the mucosa. Digesta samples were immediately frozen at −20 °C, freeze-dried, and pulverized (PULVERISETTE 9; Fritsch GmbH, Idar-Oberstein, Germany). Pulverized samples were stored in airtight containers until further analysis.
From each bird a 2 cm long segment of the ileum was sampled 10 cm distal to the Meckel’s diverticulum. This section was cut open and rinsed with NaCl (0.9%). The ileal mucosa scrapings were retrieved, frozen on dry ice, and stored at −80 °C until RNA extraction. Other samples from different tissues were obtained for other measurements which will be published separately.
Yolk and albumen from one egg per hen were separated, weighed, freeze-dried, weighed again, pulverized with pestle and mortar, and stored until further analysis.

A total of 12 (group HI-0) and 14 (groups HI-2.5 and HI-5.0) broilers per group (two broilers from each cage), whose body weights represented the mean body weight of the whole group, were selected for sample collection in order to avoid that effects were biased by random selection of broilers with very low or very high body weights. All analyses described below were carried out in these animals. The animals were killed by bleeding (opening of Vena jugularis and Arteria carotis) under electrical anesthesia using a BTG-40A stunning device (Westerhoff Geflügeltechnik, Hoogstede, Germany) in accordance with the European legislation for euthanasia of animals [36 ]. Whole blood was collected into ethylenediaminetetraacetic acid-coated polyethylene tubes (9 mL S-Monovette, Sarstedt, Nümbrecht, Germany). Plasma was prepared by centrifugation (1100 × g, 10 min) at 4 °C and stored at −20 °C. The liver was excised, washed in ice-cold NaCl solution (0.9%), weighted and small aliquots were snap-frozen in liquid nitrogen and stored at −80 °C. The gastrointestinal tract was removed and digesta from the ileum (segment between Meckel´s diverticulum and the ileo-cecal junction) and the cecum was collected. Tissue and digesta samples were snap-frozen in liquid nitrogen and stored at −80 °C pending analysis.