Impact of Reduced Dietary Crude Protein in the Starter Phase on Immune Development and Response of Broilers Throughout the Growth Period

Abstract

Crude protein (CP) levels in commercial broiler (Gallus gallus) diets, optimized for maximum yield production vs. feed cost, have only begun to be assessed for impact on immune function. In order to study immune effects of dietary CP levels, different starter phase (day 1-14) diets were fed to 230 Ross 708 male broiler chicks randomly assigned at 1 day of age into two treatment groups. Group 1: Standard diet (STD) contained 3,000 kcal AMEn/kg energy and 23.78% CP; and Group 2: Reduced crude protein diet (RCP) contained 3,000 kcal AMEn/kg energy and 21.23% CP. From day 15-35 a common standard grower/finisher diet (3,150 kcal AMEn/kg energy and 22.18% CP) was allocated to both groups. Zymosan, a glycan derived from yeast cell walls that binds to TLR 2 and Dectin-1, was used for intra-abdominal challenge. Results demonstrated that a reduced crude protein starter diet (21.23 vs. 23.78% CP) between age 1-14, while maintaining the same levels of metabolizable energy and essential amino acids, did not affect broilers growth performance or lymphoid organ weights (P > 0.05). Interestingly, basal leukocyte levels in the RCP group significantly (P < 0.01) increased in the blood compartment at d35 in the unchallenged birds. Significant enhancements to leukocyte infiltration into the abdominal cavity were also detected post-immune challenge with zymosan (day 14 and day 35; P < 0.01). Post-challenge levels of TNF-α, IL-1β, and CXCL8 gene expression cells collected from the abdominal cavity were not affected by the diets (P > 0.05). Moreover, dietary treatments did not influence percentage of ROS producing cells in the abdominal cavity (P > 0.05). To our best knowledge, this is the first study that reports the impacts of reduced crude protein diet on the innate immune response of poultry to an acute inflammation model in the abdominal cavity. Overall, our results highlight that reduced crude protein diets can be used without negatively impacting broiler performance and may enhance the capacity of broilers to recruit leukocytes upon infection.

Keywords: broiler; cytokines; dietary crude protein; inflammation; innate immunity; leukocyte.

Figure 1

Impact of reduced crude protein diet on chicken lymphoid organs relative weights (g/100 g BW). Bursa (A), thymus (C); spleen (E), femur (G), and lymphoid organs absolute weight (g), Bursa (B), thymus (D); spleen (F), femur (H), on day 7, 14, 28, and 35 of trial (n = 5 per group). RCP, Reduced Crude Protein; STD, Standard diet.

Figure 2

Impact of reduced crude protein diet on leukocyte migration following an in vivo zymosan challenge. Recruitment into the chicken abdominal cavity when in vivo challenge was performed on d 14 (A) or d 35 (B). Cells were harvested by abdominal lavage at 0 h (without injection) and at 4 and 12 h post intra-abdominal administration of zymosan (2.5 mg), (n = 5 for time 0 and 12, and n = 6 for time 4 h). Corresponding changes to the number of leukocytes in the bone marrow on day 14 (C) and 35 (D), and the peripheral blood on day 14 (E) and 35 (F). **P < 0.01 and ***P < 0.001 between dietary treatments and ##P < 0.01 and ###P < 0.001 among different time points. RCP, Reduced Crude Protein; STD, Standard diet.

Figure 3

Impact of reduced crude protein diet on heterophil, lymphocyte, and monocyte/macrophage intra-abdominal leukocyte composition following an in vivo zymosan challenge. Changes to intra-abdominal heterophil percentages [d 14 (A); d 35 (B)] and absolute numbers [d 14 (C); d 35 (D)]. Parallel changes to the percentage of lymphocytes d 14 (E) and d 35 (F), and monocyte/macrophages d 14 (G) and d 35 (H). Heterophils are the dominant cells recruited after in vivo challenge and most affected by RCP diet (N = 5 for time 0 and 12, and N = 6 for time 4 h). *P < 0.05, **P < 0.01, and ***P < 0.001 between dietary treatments and ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 among different time points. RCP, Reduced Crude Protein; STD, Standard diet.

Figure 4

Impact of reduced crude protein diet on ROS production in abdominal leukocytes following an in vivo zymosan challenge. Total percentage of ROS producing cells are shown following in vivo challenges on day 14 (A) or 35 (B). Higher resolution evaluation shows ROS production among distinct phagocyte subsets [heterophil d14 (C) and d35 (D); monocyte/ macrophages d14 (E) and d35 (F)] (n = 5 for time 0 and 12, and n = 6 for time 4 H). ##P < 0.01 and ###P < 0.001 among different time points. RCP, Reduced Crude Protein; STD, Standard diet.

Figure 5

Impact of reduced crude protein diet on heterophil, lymphocyte, and monocyte/macrophage on peripheral blood leukocyte composition following an in vivo zymosan challenge. Changes to blood heterophil percentages [d 14 (A); d 35 (B)] and absolute numbers [d 14(C); d 35 (D)]. Parallel changes to the percentage of lymphocytes d 14 (E) and d 35 (F), and monocyte/macrophages d14 (G) and d 35 (H). **P < 0.01 between different treatments. RCP, Reduced Crude Protein; STD, Standard diet; n = 5.

Figure 6

Impact of reduced crude protein diet on heterophil, lymphocyte, and monocyte/macrophage on bone marrow leukocyte composition following an in vivo zymosan challenge. Bone marrow leukocyte subpopulation after 0, 4, and 12 h post intra-abdominal administration of zymosan (2.5 mg). Changes to bone marrow heterophil percentages [d 14 (A); d 35 (B)] and absolute numbers [d 14 (C); d 35 (D)]. Parallel changes to the percentage of lymphocytes d 14 (E) and d 35 (F), and monocyte/macrophages d14 (G) and d 35 (H). *P < 0.05 between different treatments. RCP, Reduced Crude Protein; STD, Standard diet; n = 5.

Figure 7

Impact of reduced crude protein diet on gene expression of pro-inflammatory markers following an in vivo zymosan challenge. Fold change gene expression in TNF-α on d 14 (A) and d 35 (B), IL-1β on d14 (C), and d 35 (D), CXCL8 on d 14 (E), and d 35 (F) in abdominal leukocytes after 0 and 4 h intra-abdominal challenge with zymosan. *P < 0.05 between different treatments and #P < 0.05 and ##P < 0.01 among different time points. RCP, Reduced Crude Protein; STD, Standard diet; n = 6.