Mast cells in the jejunum of birds fed the coarsely ground mash diet that are clustered in the upper end of the villi. L = lumen; V = villus. Original magnification × 100. 

Context in source publication

Context 1

... study was performed with 288, day-old male Arbor Acre broiler chicks. All birds were raised in an environmentally controlled room with continuous light (10 to 20 lux). According to normal management practices, the room temperature was maintained at 33 ° C from d 0 to 5 and then gradually reduced to 22 ° C by the end of the experiment. Feed and water were supplied ad libitum throughout the experiment. The broilers were randomly allocated to 1 of 4 dietary treatments in a 2 × 2 factorial design. The factors included particle size (coarse vs. fine) and diet form (mash vs. pellet). The birds were housed in 90 × 60 cm pens containing 12 birds, and each treatment was fed to 6 pens of birds from d 1 to 22 of age. All experimental corn-soy diets were formulated to meet or exceed the nutrient requirements of the NRC (1994). The coarse mash diet was prepared by mixing corn and soybean meal ground through a 5-mm screen with all other ingredients to yield a final particle size of 953 ␮ m. The fine mash diet was prepared by mixing corn and soybean meal ground using a 3-mm screen and then mixed with other dietary ingredients to yield a final particle size of 594 ␮ m. The actual particle size of each diet was determined by a standard method (Ensor et al., 1970). The pelleted diets were processed at a temperature of 85 ° C in a pellet mill (Muyang Corp., Yangzhou, China) using a 38-mm thick die with 2-mm diameter holes. On d 22, six birds per treatment (1 bird/pen) were selected randomly and slaughtered by cervical disloca- tion. Segments of the intestine were removed from the duodenum (5 cm from the pylorus), jejunum (5 cm poste- rior to the yolk stalk), and ileum (2 cm anterior to the ileocecal valve). The intestinal contents were removed by flushing with saline. One sample of each of the intestinal tissues, about 2 cm in length, was fixed immediately by immersion in Carnoy’s fluid (60 mL of 100% ethanol, 30 mL of chloro- form, and 10 mL of glacial acetic acid) and kept at room temperature for 12 h until needed for histochemistry staining. Another sample of each intestinal tissue, about 10 cm in length, was immediately immersed in liquid N and preserved at − 80 ° C for later histamine and stem cell factor analysis. The intestine samples previously fixed in Carnoy’s fluid were dehydrated in a graded series of alcohol and xylene and then embedded in paraffin wax. Paraffin sections (6 ␮ m thick) were made and mounted on poly- L -Lys-coated slides, dewaxed in xylene and then rehydrated. Two or 3 copies of each paraffin-embedded segment were rinsed with 0.5 M HCl (pH 0.5) for 5 min and stained with toluidine blue (Gurr, Poole, UK). The sections were then dehydrated and mounted (Xu et al., 2001). Mast cells were observed using a Microcheck Grid (Wuhan Optical Ltd, Wuhan, China), following the proce- dures described by Xu et al. (2001). The average number of mast cells in a single intestinal villus was acquired by randomly counting more than 10 intestinal villi per section. For the measurement of mast cell numbers in the intestinal serosa, stained mast cells of the whole serosa were counted using 3 sections per sample (Xu et al., 2001) in high-power fields ( × 40) with a Microcheck Grid containing 100 microchecks (0.25 mm 2 ). Mast cell numbers in the intestinal serosa were expressed as a cell number per squared millimeter of the intestine serosa. The HPLC method used for the determination of histamine was that described by Veciana-Nogues et al. (1995). Following tissue homogenization and extraction, the samples transited a cation exchange column (Symmetry, C 18 , 5 ␮ m, 4.6 × 150 mm, Waters Ireland, Dublin) and were eluted by a gradient elution program at a flow rate of 1 mL/min. The eluate was then reacted with o-phthalalde- hyde (Sigma, Chicago, IL) at 40 ° C and examined using a fluorescence detector (Waters 2475 Multi λ Fluorescence Detector, Waters Ireland) at excitation 340 nm and emis- sion 450 nm. The data were analyzed using a data inte- grator (Waters TM 600, Waters Ireland). All reagents were filtered through a 0.45- ␮ m dialyzer and degassed before use. Postcolumn derivatizating reagents were prepared fresh daily and protected from light. Samples of the different intestinal segments weighing approximately 0.5 g were homogenized at 4 ° C in 1 mL of a buffer containing 1 M NaCl-Tris, 0.25% Triton X-100, and protease inhibitors, as described by Ga ̧a et al. (1999). Samples were then centrifuged for 10 min at 10,000 × g at 10 ° C, and the supernatant was obtained. Stem cell factor protein concentrations were measured in duplicate using a commercial ELISA kit (enzyme immunoassay for the quantitative determination of goat anti-chicken stem cell factor, Dalianfanbang Biochemical Technology Ltd, Dal- ian, China) according to the manufacturer’s instruction. Sensitivity was 45 pg/mL. Intra- and interassay CV were 6.8 and 11.0%, respectively. The recovery was 98 to 110%. Data were analyzed by ANOVA using the GLM proce- dures of SAS (SAS Institute Inc., Cary, NC) appropriate for a 2 × 2 factorial design. The statistical model included the effects of diet particle size (coarse vs. fine), diet form (mash vs. pellet), and their interactions. Differences between treatments were analyzed using a t -test following a significant F -test. Results were expressed as least squares means and SEM. Probability values of < 0.05 were considered to be significant. Particle size affected the location and quantity of mast cells in the intestinal villi. Mast cells in the intestinal villi of broilers fed the finely ground mash diet were distributed relatively evenly throughout the intestine, with mast cells visualized in the mucosa, submucosa, and serosa (Figure 1). In contrast, mast cells in the intestinal villi of birds fed the coarsely ground mash diet were clustered in the upper end of the villi (Figure 2). Diet form did not affect the distribution of mast cells in the small intestine of birds. Mast cells were observed throughout the intestine for both finely ground and coarsely ground pelleted diets with the pattern similar to that observed for the finely ground mash diet (Figures 3 and 4). There was a significant ( P = 0.02) particle size × diet form interaction for the number of mast cells in the duodenum, with the number of mast cells dramatically greater in finely ground mash diets compared with coarsely ground mash diets (4.65 vs. 1.95), whereas the number of mast cells was similar for coarsely and finely ground pelleted diets (3.15 vs. 3.01). In addition, there was a remarkable effect of particle size on mast cell numbers in the small intestinal villus, whereby the number of mast cells in the duodenum ( P = 0.04), jejunum ( P < 0.01), ...