Effects of stocking density and dietary vitamin C on performance, meat quality, intestinal permeability, and stress indicators in broiler chickens

The experimental protocol of the current study was approved by the Institutional Animal Care and Use Committee at Chung-Ang University (IACUC approval no. 2019-00086). The study was performed using a completely randomized design with a 2 × 2 factorial arrangement consisting of 2 different SD and 2 supplemental levels of dietary vitamin C. A total of 1,368 mixed-sex Ross 308 broiler chickens (1:1 ratio of males and females) of 21 days of age with similar body weights (BW) were randomly allotted to 1 of 4 treatments with 6 replicates each. The study lasted for 14 days from 21 to 35 days of age. The experimental period was determined based on the fact that growing chickens are more sensitive to high SD than young chickens [14,15]. Different numbers of birds per floor pen were used to create 2 different SD levels, namely low SD (9 birds/m²) and high SD (18 birds/m²) as reported in our previous study [6]. The final floor space (4.216 m²) was calculated by subtracting the space of immobile objects (0.584 m² for 2 feeders and 1 bell-shaped drinker per pen) from the floor space (2.0 m × 2.4 m). Each floor pen was covered with a 10-cm thick layer of rice hulls. The commercial-type basal diet was prepared without dietary supplementation of vitamin C as the control group (Table 1). The nutrient and energy concentrations in the basal diet were formulated to meet or exceed nutrient recommendations of the Ross 308 manual [16]. Vitamin C (> 970 g/kg coated vitamin C, Zhejiang Mingzhu Animal Health Products, Hangzhou, China) was then supplemented to the basal diet at the inclusion level of 200 mg/kg. The supplemental level of dietary vitamin C was determined based on previous studies reporting that the beneficial effect of supplemental vitamin C in broiler diets was observed at 200 mg/kg [12,18].

Before the initiation of the study, all chickens were raised under the same environmental conditions and were fed the same commercial starter and grower diets for 20 days. Experimental diets and water were provided ad libitum for 14 days. All experimental pens were placed in an environmentally controlled room. The average room temperature was maintained at 22.3 ± 2.5℃ during overall experimental period. The study was performed under 24-hour lighting conditions. Body weight gain (BWG) and feed intake (FI) were recorded at the end of the study. Feed efficiency (FE) was calculated as BWG divided by FI after adjusting for mortality [19].

At the end of the study, 3 male birds with a BW close to average BW in each replicated pen were selected. One bird was used to analyze stress indicators including blood heterophil (H):lymphocyte (L) and the concentrations of corticosterone (CORT) in the feather. The remaining 2 birds were euthanized by CO₂ asphyxiation. One bird was used for analyzing breast meat quality and liver antioxidant status, whereas the other bird was used for analyzing intestinal permeability in the jejunum by a Ussing chamber.

Blood samples were collected from wing veins. Blood H:L was analyzed using the method of Lentfer et al. [20] with a minor modification. Briefly, a standard microscope slide with approximately 7 μL of blood was prepared and dried at room temperature. The slides were then prefixed for 5 minutes after immersion in methanol. Afterwards, the slides were stained with 0.2 mL wright stain solution (Wright stain solution, Muto pure chemicals, Tokyo, Japan) for 2 minutes at room temperature and then rinsed with water until the edges of the slides turned pinkish red. The slides were again stained with 0.2 mL giemsa stain (Giemsa’s staining solution, Duksan pure chemicals, Ansan, Korea) for 5 minutes at room temperature and then rinsed with water until the edges of the slides turned pinkish red. After air drying, the hemocyte smears were inspected under a microscope. Blood H and L were counted by the same 2 people to 100 cells per individual smear and blood H:L was calculated based on the method of Cengiz et al. [21].

Feather samples were collected from the same birds used for blood H:L analysis. The primary flight feathers were collected and stored at −80°C before analysis. Feather CORT concentrations were analyzed using the method of Bortolotti et al. [22] with a minor modification [23]. In short, feathers without calamus were minced into pieces of < 5 mm² by scissors. Feather pieces were placed in 50-mL tubes with 10 mL methanol (HPLC grade, Honeywell, NC, USA). The tubes were sonicated in a water bath at room temperature for 30 minutes, and then incubated in a shaking water bath at 50°C overnight. The methanol was then separated from the remaining feather by syringe filters (HyunDai Micro, Anseong, Korea) in a filtration funnel. The feather remnants were washed twice again with 2 mL methanol and the washes were added back to the original extract. The methanol extract was then concentrated by evaporation in a water bath at 50°C for 7 hours. The extract residues were mixed with 1 mL phosphate-buffered saline (pH 7.4), and centrifuged at 17,900×g for 15 minutes. The resulting extracts were used for analyzing feather CORT concentrations with a CORT competitive ELISA kit according to the manufacturer’s protocol (Thermo Fisher scientific, Waltham, MA, USA). The feather CORT concentrations were expressed as a function of feather length (pg/mm).

Both sides of the breast muscle were excised to measure meat quality and stored at 4°C until further analysis. The right portion was used for pH and meat color assays, whereas the left portion was used to determine water holding capacity (WHC) and thiobarbituric acid-reactive substance (TBARS) values. The detailed procedure of the meat quality analysis was reported previously [6].

Antioxidant status in the liver such as malondialdehyde (MDA) and total antioxidant capacity (TAC) was determined using a commercially available OxiSelectTM TBARS assay Kit (MDA Quantitation, STA-330, Cell Biolabs, San Diego, CA, USA) and OxiSelectTM Total Antioxidant Capacity (TAC) Assay Kit (STA-360, Cell Biolabs), respectively, according to the manufacturer’s protocol. Protein concentrations were also analyzed using a commercial kit (Thermo Fisher Scientific) [24]. The relative concentrations of MDA and TAC to protein concentrations were calculated and expressed as µmol/mg and U/mg protein, respectively [25].

Intestinal permeability in the jejunal mucosa as a measure of intestinal barrier function was determined based on the transepithelial electrical resistance (TER) values measured in a dual-channel self-contained Ussing chamber system (U2500, Warner Instruments, Hamden, CT, USA). The detailed procedure was reported previously [6].

All data were analyzed by 2-way ANOVA as a completely randomized design using the PROC MIXED procedure (SAS Institute, Cary, NC, USA). Each replicate was considered an experimental unit. The statistical model included SD, dietary supplementation of vitamin C, and their interaction. The LSMEANS procedure was used to calculate treatment means and the PDIFF option in SAS was used to separate the means if the difference was significant. Statistical significance was considered at p < 0.05.

During 14 days of the study (from 21 to 35 days of age), no interaction between different SD and dietary supplementation of 200 mg/kg vitamin C was observed for growth performance of broiler chickens (Table 2). For the main effects, birds raised at high SD had less (p < 0.01) BW, BWG, and FI than those raised at low SD, whereas dietary supplementation of vitamin C had no effects on growth performance of broiler chickens, regardless of SD.

There was no interaction between SD and dietary supplementation of 200 mg/kg vitamin C for blood H:L and feather CORT concentrations (Table 3). However, birds raised at high SD had greater (p < 0.01) blood H:L and feather CORT concentrations than those raised at low SD. Dietary supplementation of 200 mg/kg vitamin C had no effects on blood H:L and feather CORT concentrations, regardless of SD.

No interaction between SD and dietary supplementation of 200 mg/kg vitamin C was observed for breast meat quality (Table 4) and antioxidant status such as MDA and TAC concentrations in the liver (Table 5). In addition, there were no main effects of SD and dietary supplementation of vitamin C on meat quality and antioxidant status in the liver.

No interaction between SD and dietary supplementation of 200 mg/kg vitamin C was observed for TER values in the jejunal mucosa as a measure of intestinal permeability (Table 6). However, birds raised at high SD had less (p < 0.05) TER values than those raised in low SD. However, TER values were not affected by dietary supplementation of vitamin C.