2.3. Sampling

In the morning of d 19 and 24 post-weaning, after the meal, blood samples of all pigs were collected from jugular vein to obtain serum. Blood was collected in vacutainers without anticoagulant (BD, Franklin Lakes, NJ). Serum samples were collected after centrifuging (3,000 × g for 15 min at 4 °C) and stored at −80 °C until they were analyzed for concentration of malondialdehyde (MDA) and tumor necrosis factor α (TNFα) as biological indicators of systemic oxidative stress and inflammatory responses, respectively (Duarte et al., 2019). On d 25 post-weaning, all pigs were stunned by an electric device and euthanized by exsanguination. Then the gastric intestinal tract was quickly removed and the small intestine was dissected. The middle sections of jejunum and ileum were isolated and flushed with distilled water. Half of the sections were fixed in 10% formaldehyde-phosphate buffer and kept for microscopic assessment of mucosal morphology. The other half of the sections were then opened for scraping the mucosal layer of the intestine. The mucosa of the jejunum and ileum was scraped into a 2-mL tube and frozen in liquid nitrogen. Mucosa samples were then stored in −80 °C until analyzing for MDA and TNFα concentrations. One tube of digesta samples (50 mL) from jejunum, ileum and colon was also collected, and digesta pH was measured using a pH meter immediately. Digesta were directly put on ice, and then stored in −20 °C until analyzing. Spleen weight was also measured as an indicator of the expression of pro-inflammatory cytokines such as TNFα and interleukin-β (Touchette et al., 2002).