In ovo feeding

A total of 144 fertilized eggs from Cobb 500 hens were obtained from Asagi Hatchery Inc. (Honolulu, HI, USA) on the 15^th day of embryonic development. Then, the eggs were carefully transferred to incubators set at 37.5 °C and a relative humidity of 58%. The eggs were then divided into three treatment groups based on the type of in ovo feeding they would receive: XOS2, XOS3, or no in ovo feeding (CON). The XOS2 (purity > 95%) and XOS3 (purity > 90%) were sourced from Megazyme International Ireland Ltd., Bray, Ireland. The solution for the in ovo feeding was prepared at a concentration of 6 mg of XOS2 or XOS3 in each mL of 0.85% normal saline, following the same methodology of a previous study that investigated the effects of in ovo xylotriose and xylotetraose [10]. Our study did not include a vehicle control group, such as a saline-injected group. Previous studies demonstrated no significant changes between the in ovo saline feeding and non-injection control groups [10, 16, 17]. Considering the result of these studies, we excluded the saline group from the current study. As the in ovo feeding at the later stage of embryonic development could affect the gut microbiota development [5], in ovo XOS feeding of embryos was carried out on the 17^th embryonic day in a biosafety cabinet as previously described [10]. The broad end of eggs was chosen for the injection of XOS solution and was disinfected with 10% povidone iodine prior to stabbing with a disinfected awl. Then, 0.5 mL of XOS solution that contained 3 mg of XOS was injected using blunt tip 21 gauze sterile needles to the amniotic sac of the embryos, and the process ended by attaching a piece of parafilm over the site of injection.