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Vitamin D signaling is important for intestinal homeostasis. An increase in vitamin D receptors in immune cells can modulate cell phenotype and cytokine secretion. Cytokines regulate both pro- (interleukin 17; IL-17) and anti-inflammatory (IL-10) responses triggered by external stimuli. Inflammation in intestinal tissues can disrupt the structure and the remodeling of epithelial tight junction complexes, thus, compromising the protective barrier. The objective of the study was to determine the impact of dietary supplementation with 25-hydroxycholecalciferol ( 25 OHD 3 ), a hydroxylated metabolite of vitamin D, on intestinal cytokine abundance and epithelial barrier integrity over time in broilers. A randomized complete block design experiment was conducted to evaluate the effect of dietary 25 OHD 3 inclusion on relative protein expression of the cytokines, IL-17 and IL-10, and tight junction proteins, Zona Occludens 1 (ZO-1), and Claudin-1 (CLD-1), in broiler chicken duodenum and ileum from 3 to 21 days post-hatch. On day 0, male chicks ( n = 168) were randomly assigned to raised floor pens. Experimental corn-soybean meal-based treatments were as follows: (1) a common starter diet containing 5,000 IU of D 3 per kg of feed (VITD 3 ) and (2) a common starter diet containing 2,240 IU of D 3 + 2,760 IU of 25 OHD 3 per kg of feed ( 25 OHD 3 ) fed from days 0 to 21. On days 3, 6, 9, 12, 15, 18, and 21, 12 birds per treatment were euthanized to collect tissue samples for quantitative, multiplex, and fluorescent Western blot analysis. Target proteins were quantified using Image Quant TL 8.1 and expressed relative to total protein. Feeding 25 OHD 3 post-hatch decreased ileal IL-10 (anti-inflammatory) protein expression in 21-day-old broilers compared with VITD 3 only ( P = 0.0190). Broilers fed only VITD 3 post-hatch had greater IL-17 (pro-inflammatory) protein expression in the ileum at 18 and 21 days-of-age ( P = 0.0412) than those that fed 25 OHD 3 . Dietary inclusion of 25 OHD 3 lowered the abundance of key inflammatory cytokines in the ileum of young broilers., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Abascal-Ponciano, Leiva, Flees, Avila, Starkey and Starkey.)

As antibiotic-free (ABF) broiler production continues to increase, understanding the development and local immune response in the intestines of ABF broilers is essential. Mitotically active cells, the majority of which will become enterocytes, help maintain the intestinal epithelial barrier. Macrophages prevent pathogen invasion by their phagocytic activity, functioning as immune response amplifying cells to aid in the recruitment of additional immune cells, and stimulating cytokine production in other adjacent cells. The objective of this experiment was to evaluate commonly used practical production practices on intestinal cell mitotic activity and local intestinal immunological responses. A randomized complete block design experiment with a 3 × 2 factorial treatment structure was conducted. The 3 dietary protein sources were: soybean meal (SBM), a mix of 50% poultry by-product meal and 50% feather meal (PFM), and porcine meat and bone meal (MBM) and broilers were reared on either new litter (NL) or used litter (UL). On d 3, 8, 11, 15, and 21, 6 birds per treatment from 6 blocks (total n = 36 per d) were randomly selected for sampling. Broilers were injected intraperitoneally with 5'-bromo-2'-deoxyuridine (BrdU) 1 h prior to sampling to label mitotically active cells. Samples were analyzed using cryohistology and immunofluorescence to determine the density of mitotically active cells and macrophages. Mitotically active cell and macrophage densities changed in both the duodenum and ileum over time. Neither dietary protein source nor litter condition affected mitotically active cell or macrophage densities in the duodenum on d 11 and 21 or in the ileum on d 3, 8, 11, and 15. However, on d 3 and 15 in the duodenum ( P ≤ 0.0126) and d 21 in the ileum ( P ≤ 0.0009), broilers reared on UL had greater mitotically active cell densities than those reared on NL. On d 8 in the duodenum, broilers fed MBM had increased macrophage density compared with those fed PFM and SBM ( P ≤ 0.0401). These results indicate dietary protein source and litter condition may impact the physiology of the broiler small intestine, though additional work with this model is necessary to understand the underlying mechanisms., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Keel, Calderon, Tejeda, Starkey and Starkey.)

The previous work has demonstrated that maternal supplementation of the circulating metabolite of vitamin D3 (D3), 25-hydroxycholecalciferol (25OHD3), enhances the immunocompetence of broiler chick offspring. In post-hatch broiler diets, 25OHD3 has been shown to affect intestinal morphology and improve the immune status of broilers. An experiment with a 2 × 2 factorial treatment arrangement was conducted to assess the effects of combining maternal (MDIET) and post-hatch (PDIET) dietary 25OHD3 inclusion on duodenal crypt and macrophage cell populations and mitotic activity in young broiler chickens. All diets were formulated to provide 5,000 IU of vitamin D. Broiler breeder hens were offered 1 of 2 MDIET: 5,000 IU D3 per kg of feed (MCTL) or 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (M25OHD3) from week 25 to 41. Male broiler offspring ( n = 480) hatched from eggs collected during week 41 of breeding age were allotted in raised floor pens (4 birds per pen from day 0 to 7 and individually allotted from day 8 to 21). Chicks were fed 1 of 2 PDIET (starter day 0 to 21): 5,000 IU D3 per kg of feed (PCTL) or 2,240 IU D3 + 2,760 IU 25OHD3 (P25OHD3). DUO samples ( n = 12 birds per treatment per day) were collected on days 3, 6, 9, 12, 15, 18, and 21 for cryohistological and immunofluorescence analysis to facilitate the enumeration of the total macrophages, CD80+ macrophages (pro-inflammatory macrophages), and mitotically active cells (BrdU+) to calculate the proportion of proliferating cells (PPC) per duodenal crypt. Bird age impacted crypt PPC with the greatest PPC per duodenal crypt observed on days 3 and 9, and the lowest PPC per crypt was observed on day 21 ( P < 0.0001). Broilers from the M25OHD3:PCTL treatment had a greater PPC ( P =.002) than birds from the MCTL:PCTL treatment at day 3. An interaction among MDIET and PDIET was observed for proliferating macrophages at day 21 ( P = 0.029) where M25OHD3:P25OHD3 birds had more proliferating macrophages than M25OHD3:PCTL-fed birds. These results indicate that combined MDIET and PDIET 25OHD3 supplementation may alter early post-hatch duodenal development and innate immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Leiva, Avila, Abascal-Ponciano, Flees, Sweeney, Wilson, Starkey and Starkey.)