The Institutional Animal Care and Use Committee approved all animal handling procedures (case number 08/2020), and the experiment was conducted according to the experimental protocol for the use of live birds from the Brazilian College of Animal Experimentation.

The experiment was conducted in Viçosa, MG, Brazil (20°45'57.19" S, 42°51'35.42" W, and 682 m altitude). Male broiler chickens (Cobb 500) used in the experiment were obtained from a commercial hatchery (Rivelli Alimentos SA, Matheus Leme, MG, Brazil), and they were vaccinated against bursal disease and Marek’s disease (Serotype 3, Live Marek’s Disease Vector, Merial Inc., Athens, GA). From one day of age until the beginning of the experiment, the birds were reared on floor pens (200 × 100 cm) equipped with two nipple drinkers and a feed dispenser. They had free access to water and were fed ad libitum with a corn/soybean meal-based diet in mashed form that was formulated to meet the nutritional recommendations of Rostagno et al. (2017).

At 18 days of age, a total of 120 male broilers were assigned based on their body weight to a completely randomized experimental design with three treatments, eight repetitions, and five birds per experimental unit. The birds were housed in 24 experimental units consisting of wire cages (600 cm²/bird) in a four-level battery equipped with a trough feeder and a nipple drinker.

The treatments were a basal diet or a basal diet with the addition of 1 or 2 mg of capsaicinoids/kg of diet. The corn/soybean meal basal diet was formulated to meet the nutritional recommendations of Rostagno et al. (2017; Table 1). The source of capsaicinoids used was Capcin^® (ID4Feed, France), which had a concentration of 5 g capsaicinoids/kg; this product was used at a concentration of 200 and 400 mg/kg of basal diet. The diets were prepared in mashed form. The birds had free access to water and feed throughout the experimental period (18 to 26 days of age).

The ambient temperature was maintained at 22 °C and the birds were exposed to 18 h of continuous light daily during the experimental period.

Broiler performance was evaluated at 26 days of age by determining body weight gain (BWG), feed intake (FI), and feed conversion rate (FCR).

At 26 days of age, one male bird with a weight closest to the average weight of the experimental unit was selected for sample collection. Blood was collected from this bird through the wing vein. The blood was centrifuged at 3,600 × g at 4 °C for 10 min for separation; serum samples were stored at −20 °C until analysis. After blood collection, the bird was euthanized by cervical displacement. Samples of the jejunum of 2 cm in length were collected, stored individually in cryogenic tubes, and then placed in liquid nitrogen. Subsequently, they were transferred to freezer storage at −80 °C until the RNA extraction process.

The collected blood was used to analyze serum levels of glucose, triglycerides, cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL; Cobas c 311; Roche Diagnostics GmbH, Basel, Switzerland) following the manufacturer’s instructions. To measure the serum level of malondialdehyde (MDA), 2.5 mL of 20% trichloroacetic acid and 1.0 mL of 0.67% thiobarbituric acid were added to 0.5 mL of serum, and the mixture was heated for 30 min in boiling water. The resulting chromogen was extracted with 4.0 mL of n-butyl alcohol. The absorbance of the organic phase was determined at a wavelength of 530 nm.

Total RNA was extracted from 50 mg of jejunum samples using TRIzol^® (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The resulting precipitate was rehydrated with 25 μL of UltraPure DNase/RNase-Free water. RNA concentration was estimated using a NanoDrop^TM Lite Spectrophotometer (Thermo Fisher Scientific, Beverly, MA, USA), and RNA integrity was determined in a 1.0% agarose gel. The first cDNA strand was synthesized using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Thermo Fisher Scientific, Beverly, MA, USA). The primer sets used are shown in Table 2. β-actin was used as the reference gene for data normalization because of its higher expression and stability. The following target genes were assessed: nuclear factor-κB (NF-κB), glutathione peroxidase (GPX), and Cu, Zn-superoxide dismutase (SOD1). The RT-qPCR analyses were performed in duplicate using Applied Biosystems™ QuantStudio Real-Time PCR Systems (Applied Biosystems, Thermo Fisher Scientific, Beverly, MA, USA) with the relative quantification method, and applying the SYBR^® Green system (Applied Biosystems - Foster City, CA, USA) and the GoTaq^® qPCR Master Mix kit (Promega Corporation, Madison, USA). Polymerase chain reactions were conducted using the following the program: 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s, and 60 °C for 1 min. The threshold cycle (Ct) values obtained were later normalized (ΔCt) based on the Ct values of the endogenous control gene β-ACT. The calculation of the relative gene expression levels was performed according to the 2^−ΔCt method described by Livak and Schmittgen (2001).

For each variable, the analysis of variance was performed according to the following general model:

in which Y_ij is the measured dependent variable, μ is the overall mean, α_i is the effect of treatments, and ε_ij is the random error.

The average FI and BWG of the five birds in each cage were considered for the statistical analysis of the growth performance variables. For serum and mRNA expression analyses, one bird per replicate was considered the experimental unit. Analysis of variance for each variable was performed under a completely randomized design using the GLM procedure of SAS (Statistical Analysis System, 9.4). The analysis of variance assumptions given by normality of residuals and homogeneity of variances were evaluated using the Shapiro-Wilk and Hartley tests, respectively. For the mRNA expression of GPX and SOD1, since the analysis of variance assumptions were not verified, the original data were transformed by Box-Cox transformation. The comparisons between the means of treatments were performed through Tukey’s test, considering a 5% probability of type I error.

There was no effect of capsaicinoid supplementation on the performance of broilers from 18 to 26 days of age (P>0.05; Table 3).

There was no effect of treatment on serum levels of glucose, triglycerides, cholesterol, HDL, LDL, VLDL, or MDA (P>0.05; Table 4).

Broilers supplemented with 1 and 2 mg/kg capsaicinoids showed higher mRNA expression of SOD1 (P<0.001) in the jejunum compared with birds fed the basal diet (Figure 1). However, the mRNA expression of GPX (P>0.05) was not influenced by the treatments. The capsaicinoid supplementation of 2 mg/kg reduced the mRNA expression of NF-κB (P = 0.031) in the jejunum of broilers compared with the basal diet.