In addition, main phyla found in the jejunum and ileum post-weaning were comparable to the results from another study [52]. to be elucidated. = 8 per treatment group, at each time point). Open in a separate window Physique 1 Study timeline (A). From day 2 until day Fluorometholone 44, all piglets orally received either -glucans or a control treatment (tap water) every other day (1). A sub-selection of piglets was weaned on day 28 (3). Pigs were vaccinated with Salmoporc? on day 21 (primary vaccination; (2) and on day 45 (booster vaccination; (4). Faecal samples were collected on days 4, 8, 14, 26, 35, 43, 59 and 69 (squares; ). Blood samples were taken on days 14, 26, 35, 43, 59, and 69 (circles; ). A subset of animals were sacrificed on days 27, 44, and 70 (cross; ?). Schematic representation of the experimental design showing the remaining number of animals per treatment over time as determined by deselection or dissection of animals (B). The control and the -glucan groups are presented as yellow and green, respectively. The dashed line separates the pre-weaning and post-weaning time points. 2.3. Experimental Procedures 2.3.1. Dietary Intervention and Oral Vaccination From day 2 until day 44 of the study, piglets received either -glucans (MacroGard?) or a control treatment (tap water) every other day. Piglets received the dietary interventions orally using disposable syringes (Discardit II, BD). MacroGard? (Orffa Additives B.V., Werkendam, The Netherlands), which consisted of 100% yeast (Typhimurium. The vaccine suspension, made up of 5 108C5 109 CFU/mL of the live attenuated enterica serovar Typhimurium, was Fluorometholone freshly prepared according to manufacturers instructions prior to oral administration. 2.3.2. Blood and Faecal Sampling Blood and faecal samples were collected at different time points during the study for the evaluation of immune function and microbiota composition, respectively. Faecal samples were collected on days 4 (= 24 per treatment group), 8 (= 24), 14 (= 24), 26 (= 24), 35 (= 16), 43 (= 16), 59 (= 8), and 69 (= 8). Rectal stimulation was performed by inserting the tip of a wetted (with sterilized H2O) cotton swab (PurFlock Ultra, Puritan) into the rectum, by making small, gentle movements (circular and back- and forward). These fresh faecal samples were collected in cryotubes, immediately placed on dry ice and stored at ?80 C until further processing. DNA isolation from faeces was done on all faecal samples from animals that were dissected. Blood was collected on days 14, 26, 43, and 69 from the jugular vein of the pig using Natrium Heparin tubes (S-monovette?, Sarstedt, Germany). Blood samples were either kept at room heat (RT) until further processing for cell analysis or centrifuged at Fluorometholone 2000 for 10 min to collect serum. Serum was stored at ?20 C until further use. Piglets were sampled in a random order and weighed directly before sample collection. 2.3.3. Dissection After euthanasia and exsanguination, the ileocecal mesenteric lymph node (MLN) was removed and stored on ice-cold (4 C) RPMI 1640 Medium (with GlutaMAX? supplement, Gibco?), made up of 10% fetal calf serum (FCS, Gibco?) and 1% L-Glutamine (Gibco?). Subsequently, the GI tract was removed from the abdominal cavity of the piglet, and the jejunum, ileum, and caecum were identified and segmented accordingly. Digesta samples from these GI segments were taken on day 27 (pre-weaning), and day 44 and 70 (post-weaning) by gently squeezing the segment content into a plastic container. Next, the collected digesta was completely homogenized using a clean spatula. Approximately 1 PROCR g of homogenized digesta was stored in a sterile cryogenic vial, snap-frozen on dry ice and Fluorometholone stored at ?80 C until further processing. The remainder of the digesta from each GI segment was mixed with H2O for pH measurement (Physique S1) using a pH meter (ProLine B210). 2.4. Measurements 2.4.1. Microbiota Analysis DNA was extracted from faecal and digesta samples using a customized Maxwell 16 Total RNA protocol (Promega Corp., Madison,.
Categories