Effects of dietary supplementation with encapsulated capsaicinoids on growth performance and cecal microbiota in broiler chickens

Abstract

Capsaicinoids (CAPs) are widely investigated as natural alternatives to antibiotic growth promoters; however, most studies have examined capsaicin (CAP) alone, and the combined effects of CAP and dihydrocapsaicin (DHC) in encapsulated form remain largely unexplored in broilers. This study evaluated dietary encapsulated capsaicinoids (E-CAPs; 0.47% CAP and 0.22% DHC w/w) on growth performance, meat quality, serum biochemistry, antioxidant status, organ indices, intestinal morphology, and gut microbiota in Arbor Acres broilers. A total of 384 broilers were randomly assigned to a basal diet (CON) or diets supplemented with 150 mg/kg (LDC) or 450 mg/kg (HDC) E-CAPs. Compared with CON, HDC increased average daily gain and reduced the feed-to-gain ratio during the grower and overall periods (P < 0.05), whereas LDC showed limited effects. HDC also decreased serum alanine aminotransferase and total bile acid levels relative to CON (P < 0.05), without differences in other biochemical or antioxidant parameters or in organ indices and intestinal morphology, indicating good metabolic safety. Both LDC and HDC reduced crude fat content and thiobarbituric acid reactive substances levels in breast meat during refrigerated storage, without affecting meat color, compared with the CON group (P < 0.05). Relative to CON, HDC reduced the relative abundance of Actinomycetota and increased the relative abundances of Clostridium_innocuum, Alistipes, and Turicimonas (q < 0.05), accompanied by predicted enrichment of pathways related to bacterial secretion systems and antimicrobial resistance. Overall, supplementation with 450 mg E-CAPs/kg diet (≈3.1 mg total CAPs/kg) enhanced growth performance, improved meat quality, and beneficially modulated gut microbiota while maintaining metabolic and intestinal health, supporting the combined encapsulated CAP - DHC formulation as a safe and effective feed additive for broilers.

Introduction

In recent decades, antibiotics have been widely applied in livestock and poultry production to promote growth, improve feed efficiency, and control diseases (Low et al., 2021). However, their excessive use has raised serious public health and environmental concerns (Ghimpețeanu et al., 2022). Consequently, many countries have restricted or banned the use of antibiotics as growth promoters in animal feed (Phillips, 2007; Brüssow, 2017; Low et al., 2021), driving a growing interest in safe and effective natural alternatives (Millet and Maertens, 2011; Tang et al., 2025). Among these, plant-derived bioactive compounds have attracted considerable attention due to their antimicrobial, antioxidant, anti-inflammatory, and immunomodulatory activities, and have been increasingly used as functional feed additives in livestock production (Rossi et al., 2020; Hu et al., 2025).

Capsaicinoids (CAPs), the major pungent alkaloids in chili peppers, represent a promising botanical feed additive for antibiotic-free or antibiotic-replacement applications (Govindarajan, 1986; Naves et al., 2019). Accumulating evidence indicates that CAPs exert a broad range of physiological effects, including antioxidant, antimicrobial, anti-inflammatory, and lipid metabolism–regulating activities (Luo et al., 2011; Uarrota et al., 2021). Among naturally occurring CAPs, capsaicin (CAP) and dihydrocapsaicin (DHC) together account for approximately 80–90% of the total CAPs content and are considered the principal contributors to their biological activity (Barbero et al., 2015; Alghamdi et al., 2025). Therefore, the combined presence of CAP and DHC, rather than CAP or DHC alone, more accurately reflects the natural composition and functional potential of CAPs.

Although numerous studies have demonstrated that dietary supplementation with CAP alone can improve growth performance, intestinal morphology, and gut health in livestock and poultry (Liu et al., 2021; Li et al., 2022), evidence regarding the biological effects of DHC remains scarce. More importantly, the potential synergistic or complementary effects of CAP and DHC when supplemented in combination, particularly in broiler diets, have received little attention. Given that CAP and DHC share overlapping yet distinct biological properties, their combined supplementation may elicit enhanced physiological responses compared with either compound alone (Almaghrabi et al., 2018; Wu et al., 2024). Despite these promising biological properties and potential synergistic effects, the practical application of CAPs in animal diets is often constrained by their pungency and susceptibility to degradation during feed processing. To overcome these limitations, encapsulated or coated formulations are commonly employed in commercial feed products to improve stability, handling, and delivery efficiency (Rollyson et al., 2014; Rezazadeh et al., 2023; Mehta et al., 2025).

Based on these considerations, the present study employed an encapsulated capsaicinoid formulation (E-CAPs) containing 0.47% CAP and 0.22% DHC (w/w) and systematically evaluated the effects of different dietary inclusion levels of E-CAPs on growth performance, meat quality, organ indices, serum biochemical parameters, antioxidant status, intestinal morphology, and cecal microbiota in Arbor Acres (AA) broilers. The findings aim to provide scientific evidence supporting the safe and effective use of combined CAP–DHC formulations as functional feed additives in poultry production.

Materials and methods

Experimental design, birds and management

All experimental procedures in this study strictly followed the Regulations for the Use of Experimental Animals in Zhejiang Province, China, and were approved by the Animal Care and Use Committee of Zhejiang University (permit code: ZJU20250213).

A total of 384 one-day-old white-feathered AA chicks (with an average body weight of approximately 41 g) were randomly assigned to three groups, with equal numbers of males and females. Each group contained 8 replicates, with 12 chicks per replicate. The control group (CON) received a basal diet, while the experimental groups were provided with the basal diet supplemented with an E-CAPs at either 150 mg/kg (low-dose E-CAPs, LDC) or 450 mg/kg (high-dose E-CAPs, HDC). The dietary supplementation levels were selected based on previous poultry studies using pure CAP at levels of 1.6 mg/kg and 2–6 mg/kg (Liu et al., 2021; Li et al., 2022), which correspond to approximately 1.0 and 3.1 mg/kg of total CAPs in the encapsulated product and represent low and high biologically relevant doses, respectively. The E-CAPs obtained from Lucta (Guangzhou) Flavours Co., Ltd. (Guangzhou, Guangdong, China), contained 0.47% CAP and 0.22% DHC (w/w). The chicks were fed their respective diets for 6 weeks, divided into two phases: the starter phase (days 1-21) and the grower phase (days 22-42). The experimental diets were formulated according to NRC standards, with the basic composition detailed in Table 1.

Table 1.

Basic composition of basal diet and nutritional level (%).

Days 1-21		Days 22-42
Ingredients, %		Ingredients, %
Corn	57.0	Corn	59.3
Soybean meal	22.50	Soybean meal	22.0
Rice bran	3.5	Rice bran	5.0
Corn gluten meal	6.0	Corn gluten meal	3.80
Extruded soybean	5.0	Extruded soybean	2.5
CaHPO4	1.60	Soybean oil	2.8
CaCO3	1.40	CaHPO4	1.30
Wheat flour	0.7	CaCO3	1.30
NaCl	0.3	Wheat flour	0.7
Soybean oil	1.00	NaCl	0.3
Premix1)	1.0	Premix1)	1
Total	100	Total	100
Nutrient levels2)		Nutrient levels2)
Metabolic energy (MJ/kg)	12.13	Metabolic energy (MJ/kg)	12.55
Crude protein (%)	20.9	Crude protein (%)	18.7
Crude fat (%)	4.62	Crude fat (%)	6.5
Calcium (%)	1.02	Calcium (%)	0.9
Total phosphorus (%)	0.68	Total phosphorus (%)	0.64
Crude fiber (%)	2.75	Crude fiber (%)	2.66
Lysine (%)	1.28	Lysine (%)	1.2
Methionine (%)	0.54	Methionine (%)	0.49
Methionine + Cystine (%)	0.91	Methionine + Cystine (%)	0.86

¹⁾Provided per kg of diet:lysine, 3.5 g; methionine, 5.6 g; vitamin A, 12000 IU; vitamin D3, 3000 IU; vitamin E, 36 IU; vitamin K3, 2 mg; vitamin B1, 3 mg; vitamin B2, 9 mg; vitamin B6, 5.4 mg; vitamin B12, 30 μg; niacin, 50 mg; pantothenate acid, 15 mg; folic acid, 0.6 mg; thiamin, 2 mg; riboflavin, 8.5 mg; biotin, 0.2 mg; Mn, 85.00 mg; Cu, 10.00 mg; Fe, 80 mg; Se, 0.50 mg; Zn, 80 mg; I, 0.7 mg.

²⁾The metabolic energy is calculated, while the levels of other nutrients are measured.

During the trial, environmental and feeding conditions were standardized for all groups, with ad libitum access to water and feed and continuous 24-h light exposure. For the first week, the temperature was maintained at 34 ± 1°C, and subsequently decreased by 2–3°C per week to ∼22°C for the remainder of the experiment. At the beginning and end of each phase, the chicks were individually weighed in the morning after an overnight fast.

Growth performance calculation

Feed intake was recorded every three days, and the initial body weight (IBW), final body weight (FBW), average daily feed intake (ADFI), average daily gain (ADG), and feed-to-gain ratio (F/G) were calculated for each growth phase.

Sample collection and preparation

At the end of the experiment, one male broiler chicken with a body weight close to the average was selected from each replicate, resulting in eight birds per group. Blood samples were first collected from the jugular vein into coagulation tubes containing separation gel, after which the birds were euthanized. The blood samples were allowed to clot at room temperature for 1 h, followed by centrifugation at 4°C and 3,000 rpm for 15 min. The resulting serum was aliquoted into centrifuge tubes and stored at −80°C until further analysis.

After blood collection, the chickens were euthanized with pentobarbital. Immediately afterward, cecal contents were aseptically collected into sterile centrifuge tubes, rapidly frozen in liquid nitrogen, and stored at −80°C for subsequent analysis. Concurrently, segments of intestinal tract tissue were excised, rinsed with phosphate-buffered saline (PBS), and fixed in 4% paraformaldehyde. Immediately after slaughter, breast muscle samples were collected for meat quality analysis. Portions of the same muscles were stored at 4°C and subjected to further meat quality analyses on days 3 and 7 postmortem.

Serum biochemical and antioxidant analysis

The determination of biochemical and antioxidant markers in the serum was performed according to the instructions provided by the supplier. The specific markers measured included glucose (Glu), alanine aminotransferase (ALT), total bile acid (TBA), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), total cholesterol (T-Chol), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), total protein (TP), urea (Urea), total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) level.

Organ indices

Immediately after slaughter, the liver, spleen, and kidneys were excised and weighed. Abdominal fat was also carefully collected and weighed. The relative weights of organs and abdominal fat (organ index and abdominal fat index) were calculated as grams per kilogram of live body weight (g/kg) by dividing the weight of each organ or fat sample by the live body weight of the bird.

Meat quality analysis

Crude fat (CF) content was determined by Soxhlet extraction (Luque de Castro and Priego-Capote, 2010). Drip loss (%) was calculated as (W1 − W2) / W1 × 100, where W1 and W2 represent the sample weights before and after storage at 4°C, respectively. Muscle pH was measured in a homogenate prepared by mixing 10 g minced meat with 90 mL distilled water. The homogenate was allowed to stand for 30 min, and pH was measured in triplicate using a FE20 pH meter (Mettler-Toledo, Zurich, Switzerland). Muscle color was recorded using a colorimeter (Minolta CR-10, Konica Minolta Sensing, Japan) based on the CIELAB system (L* = lightness, a* = redness, b* = yellowness). Total volatile basic nitrogen (TVB-N) and thiobarbituric acid reactive substances (TBARS) were determined according to Chinese National Standards GB 5009.228-2016 and GB 5009.181-2016, respectively.

Intestinal morphological analysis

The paraformaldehyde-fixed samples were embedded in paraffin, sectioned at 5 μm thickness, and stained with hematoxylin and eosin (H&E) following standard protocols (Ding et al., 2023). Intestinal morphology was examined under a light microscope (Olympus Corporation, Tokyo, Japan), and representative images were captured for subsequent morphometric analysis. All well-oriented villi with intact structure, without folding or breakage, and clearly identifiable crypts were selected for analysis. Villus height was measured from the tip to the villus–crypt junction, and crypt depth from the base to the junction. Morphometric measurements were performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA), and the mean values of all qualified villus–crypt units (at least 10 per sample, based on tissue integrity) were calculated for each sample.

Cecal microbiota 16S rRNA gene sequencing

Cecal content samples were submitted to Meiji Genomics (Shanghai, China) for bacterial 16S rRNA gene sequencing. The V3–V4 hypervariable regions of the 16S rRNA gene were amplified, and the PCR products were quantified using a Qubit fluorometer (Invitrogen, USA). Sequencing was performed on the Illumina NovaSeq PE250 platform (Illumina, San Diego, CA, USA). Primer sequences used for amplification were as follows: forward primer 338F (5ʹ-ACTCCTACGGAGGCACAG-3ʹ) and reverse primer 806R (5′–GGACTACVVGGGTATCTAATC–3′). Chimeric sequences were identified and removed during the DADA2 denoising process (Callahan et al., 2016). Amplicon sequence variants (ASVs) were inferred from high-quality sequences with a 100% sequence identity threshold, providing single-nucleotide resolution for downstream analyses. Downstream analysis was conducted using QIIME2 (version 2023.9), including assessments of α-diversity and β-diversity, relative abundance at the phylum and genus levels, and identification of differentially abundant taxa. Principal coordinates analysis (PCoA) based on weighted UniFrac distances was employed to visualize the compositional differences in cecal microbial communities among the experimental groups. LEfSe analysis was conducted to identify taxa with differential abundance among groups using the microeco R package (v1.16.0). Taxa with a linear discriminant analysis (LDA) score ≥ 2.0 and p < 0.05 were considered significantly different. Functional prediction of microbial communities was performed using PICRUSt2 (v2.6.2) based on the KEGG database.

Statistical analysis

All statistical analyses were performed using SAS (version 9.4, SAS Institute Inc., Cary, NC, USA). Differences among treatment groups for growth performance, organ indices, serum biochemistry, antioxidant status, intestinal morphology, and meat quality were evaluated by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test, while comparisons between two groups were conducted using independent-sample t-tests. Microbial relative abundance data were analyzed using the Wilcoxon rank-sum test. Data are presented as means ± standard error of the mean (mean ± SEM), and differences were considered statistically significant at P < 0.05. Figures were generated using GraphPad Prism (version 8.0, GraphPad Software, San Diego, CA, USA) and Adobe Illustrator CC 2018 (Adobe Systems Inc., San Jose, CA, USA).

Results

Growth performance

During the starter phase, no significant differences were observed in FBW, ADG, and ADFI among the groups (P > 0.05, Table 2), whereas the F/G of broilers in the HDC group was significantly lower than that of the CON group (P < 0.05). During the grower phase, broilers in the HDC group exhibited higher FBW and ADG than those in the CON and LDC groups (P < 0.05), and F/G was also significantly lower than those of the CON and LDC groups, while ADFI did not differ significantly among groups (P > 0.05). Throughout the trial, ADG was significantly increased in the HDC group compared with both the CON and LDC groups (P < 0.05), whereas ADFI tended to be higher in the HDC group (P = 0.097).

Table 2.

Effects of dietary supplementation with E-CAPs on growth performance in broilers.

Items	CON	LDC	HDC	P-value
Days 1-21
IBW (g)	41.07±0.10	41.09±0.16	41.10±0.09	0.985
FBW (g)	536.5 ± 8.4	537.9 ± 10.6	562.6 ± 12.4	0.170
ADG (g/d)	23.59±0.40	23.66±0.50	24.83±0.59	0.170
ADFI (g/d)	39.17±0.50	38.95±0.67	39.89±0.63	0.524
F/G	1.661±0.010a	1.648±0.014ab	1.609±0.017b	0.039
Days 22-42
FBW (g)	1882.6 ± 42.2b	1864.03±30.7b	2065.3 ± 74.5a	0.024
ADG (g/d)	64.10±1.86b	63.16±1.13b	71.55±3.25a	0.030
ADFI (g/d)	122.7 ± 2.6	121.6 ± 1.6	131.5 ± 5.0	0.103
F/G	1.918±0.025a	1.928±0.020a	1.843±0.023b	0.032
Days 1-42
ADG (g/d)	43.84±1.00b	43.41±0.73b	48.19±1.77a	0.024
ADFI (g/d)	80.83±1.36	80.29±1.04	85.68±2.68	0.097
F/G	1.846±0.019a	1.851±0.016a	1.781±0.016b	0.013

^a,bMeans within a row with different letters differed significantly (P < 0.05).

Abbreviations: E-CAPs, encapsulated capsaicinoids; CON, basal diet provided as the control; LDC, basal diet supplemented with 150 mg/kg E-CAPs; HDC, basal diet supplemented with 450 mg/kg E-CAPs; IBW, initial body weight; FBW, final body weight; ADG, average daily gain; ADFI, average daily feed intake; F/G, feed-to-gain ratio.

Serum biochemical and antioxidant parameters

Broilers in the HDC group showed marked reductions in serum ALT and TBA levels compared with those in the CON and LDC groups (P < 0.05, Table 3). Other measured biochemical parameters (including AST, glucose, HDL, LDL, LDH, total cholesterol, total protein, and BUN) remained unaffected (P > 0.05). Similarly, no significant differences were observed among treatments for the antioxidant indices GSH-Px, T-AOC, SOD, and MDA (P > 0.05).

Table 3.

Effects of dietary supplementation with E-CAPs on serum biochemical and antioxidant parameters in broilers.

Items	CON	LDC	HDC	P - value
Serum biochemical indices
ALT (U/L)	4.00±0.31a	4.25±0.41a	2.63±0.38b	0.011
AST (U/L)	432.0 ± 60.3	345.4 ± 19.6	439.4 ± 56.4	0.340
Glu (mmol/L)	13.97±0.60	15.03±0.54	13.73±0.36	0.188
HDL (mmol/L)	2.38±0.07	2.32±0.18	2.23±0.14	0.743
LDL (mmol/L)	1.70±0.07	1.68±0.15	1.49±0.12	0.402
LDH (U/L)	1969.6 ± 247.3	1743.4 ± 85.8	1507.2 ± 98.5	0.210
TBA (μmol/L)	14.87±2.42a	15.46±1.56a	8.74±1.52b	0.046
T-Chol (mmol/L)	4.08±0.12	4.03±0.35	3.69±0.25	0.508
TP (g/L)	19.90±0.45	21.35±1.48	20.77±0.71	0.587
BUN (mmol/L)	4.35±0.67	3.76±0.45	3.90±0.49	0.738
Antioxidant indices
GSH-Px (U/mL)	3542.5 ± 82.9	3477.6 ± 92.8	3750.8 ± 17.74	0.267
T-AOC (μmol/L)	0.77±0.04	0.70±0.04	0.82±0.02	0.078
SOD (U/mL)	88.6 ± 10.2	120.2 ± 9.2	116.80±15.8	0.177
MDA (nmol/mL)	1.81±0.33	2.38±0.40	1.66±0.15	0.247

^a,bMeans within a row with different letters differed significantly (P < 0.05).

Abbreviations: E-CAPs, encapsulated capsaicinoids; CON, basal diet provided as the control; LDC, basal diet supplemented with 150 mg/kg E-CAPs; HDC, basal diet supplemented with 450 mg/kg E-CAPs; ALT, alanine aminotransferase; AST, aspartate aminotransferase; Glu, glucose; HDL, high-density lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; LDH, lactate dehydrogenase; TBA, total bile acid; T-Chol, total cholesterol; TP, total protein; GSH-Px, glutathione peroxidase, T-AOC, total antioxidant capacity; SOD, superoxide dismutase; MDA, malondialdehyde.

Organ indices and abdominal fat percentage

CAPs supplementation did not significantly affect organ development or fat deposition (Table 4). Liver, spleen, and bursa of fabricius indices, as well as abdominal fat percentage, were similar among CON, LDC, and HDC groups (P > 0.05).

Table 4.

Effects of dietary supplementation with E-CAPs on organ indices and abdominal fat percentage in broilers.

Items	CON	LDC	HDC	P - value
Liver index (g/kg)	21.43±0.49	21.93±0.73	20.47±1.94	0.286
Spleen index (g/kg)	1.18±0.08	1.07±0.04	1.08±0.10	0.575
Bursa index (g/kg)	2.00±0.25	1.64±0.27	1.80±0.13	0.522
Abdominal index (g/kg)	10.92±1.10	11.38±1.32	10.41±0.59	0.807

Abbreviations: E-CAPs, encapsulated capsaicinoids; CON, basal diet provided as the control; LDC, basal diet supplemented with 150 mg/kg E-CAPs; HDC, basal diet supplemented with 450 mg/kg E-CAPs.

Breast meat quality during cold storage

As shown in Table 5, the CF content in breast meat was significantly lower in both the LDC and HDC groups than in the CON group (P < 0.01).

Table 5.

Effects of dietary supplementation with E-CAPs on breast meat quality and color parameters during cold storage.

Items	Days	CON	LDC	HDC	P - value
Crude Fat (%)	0	2.68±0.00a	1.87±0.09b	1.75±0.06b	< 0.001
TBARS (μg MDA/kg)	0	0.46±0.01	0.46±0.01	0.44±0.00	0.411
	3	0.49±0.01a	0.46±0.01ab	0.45±0.00b	0.042
	7	0.51±0.01a	0.48±0.01b	0.47±0.01b	0.003
TVB-N (mg/100 g)	0	13.43±0.72	14.04±0.27	12.46±0.47	0.122
	3	13.51±0.60	14.58±0.26	14.47±0.22	0.139
	7	15.02±0.60	14.71±0.24	14.55±0.31	0.719
Water loss rate (%)	0	3.16±0.90	2.81±0.25	2.82±0.17	0.512
	3	2.25±0.20	2.25±0.20	1.83±0.11	0.169
	7	1.87±0.15	1.86±0.13	1.75±0.18	0.838
pH	0	6.18±0.06	6.11±0.06	6.16±0.04	0.625
	3	6.05±0.05	6.07±0.08	6.13±0.03	0.631
	7	6.22±0.03	6.20±0.07	6.11±0.04	0.320
Brightness value (L*)	0	37.14±1.58	36.12±0.80	38.87±0.52	0.228
	3	38.26±1.13	37.33±0.53	39.55±0.89	0.229
	7	38.20±1.05	36.50±0.55	38.67±0.81	0.176
Redness value (a*)	0	−0.75±0.21	−0.98±0.52	−0.53±0.38	0.720
	3	0.75±0.33	0.82±0.15	0.16±0.18	0.114
	7	0.32±0.27	0.36±0.17	−0.06±0.19	0.326
Yellowness value (b*)	0	4.42±0.54	3.53±1.09	4.42±0.52	0.348
	3	5.85±0.45	5.58±0.22	6.19±0.34	0.488
	7	6.68±0.29	6.16±0.24	6.68±0.27	0.307

^a,bMeans within a row with different letters differed significantly (P < 0.05).

Abbreviations: E-CAPs, encapsulated capsaicinoids; CON, basal diet provided as the control; LDC, basal diet supplemented with 150 mg/kg of E-CAPs; HDC, basal diet supplemented with 450 mg/kg E-CAPs; TBARS, thiobarbituric acid reactive substances TVB-N, total volatile basic nitrogen.

The TBARS levels of breast meat were significantly lower in the HDC group than in the CON group on day 3 (P < 0.05). By day 7, this value was significantly lower in both the LDC and HDC groups compared to the CON group (P < 0.05). In contrast, no significant differences were detected among treatments for the TVB-N content, water loss rate, pH, or color parameters (L*, a*, b*) of breast meat throughout storage.

Intestinal morphology

Dietary CAPs did not significantly affect villus height, crypt depth, or villus-to-crypt ratio in either the ileum or jejunum (P > 0.05, Table 6).

Table 6.

Effects of dietary supplementation with E-CAPs on intestinal morphology in broilers.

Items	CON	HDC	P - value
Ileum
Villus height (μm)	543.6 ± 25.7	510.9 ± 34.9	0.463
Crypt depth (μm)	89.45±3.63	92.46±5.01	0.634
Villus/Crypt	6.34±0.27	5.78±0.30	0.185
Jejunum
Villus height (μm)	563.2 ± 19.7	532.0 ± 21.8	0.307
Crypt depth (μm)	101.60±3.26	97.75±5.36	0.549
Villus/Crypt	5.75±0.18	5.82±0.33	0.864

^a,bMeanswithin a row with different letters differed significantly (P < 0.05).

Abbreviations: E-CAPs, encapsulated capsaicinoids; CON, basal diet provided as the control; HDC, basal diet supplemented with 450 mg/kg of E-CAPs.

Cecal microbiota and predicted functions

Fig. 1 illustrated the effects of dietary E-CAPs supplementation on the cecal microbiota of broiler chickens. No significant differences in α-diversity were observed between the HDC and CON groups, as indicated by the Chao1, Simpson, Pielou’s evenness, and Faith’s phylogenetic diversity indices (Fig. 1A). In contrast, β-diversity analyses revealed distinct microbial community structures between the two groups, as demonstrated by PCA (P < 0.05; Fig. 1B) and PCoA (P < 0.05; Fig. 1C). At both the phylum and genus levels (top 20 taxa), noticeable shifts in microbial composition were detected (Fig. 1D–E)

Fig. 1.

Effects of dietary E-CAPs supplementation on intestinal microbiota composition and diversity in broilers. (A) Comparison of α-diversity indices between the CON and HDC groups. (B) Principal component analysis (PCA) based on microbial community composition. (C) Principal coordinates analysis (PCoA) based on Bray-Curtis distance. (D-E) Relative abundance of microbial taxa at the phylum and genus levels. (F) Linear discriminant analysis (LDA) scores of differential taxa identified by LEfSe. (G) LEfSe cladogram showing the phylogenetic distribution of significantly different taxa. (H) Differentially abundant genera between the two groups. E-CAPs, encapsulated capsaicinoids; CON, basal diet provided as the control; HDC, basal diet supplemented with 450 mg/kg E-CAPs. The data were evaluated using the Wilcoxon rank-sum test (n = 8). Asterisk (*) indicate significant differences between groups (P < 0.05).

LEfSe analysis identified several microbial biomarkers that distinguished the CON and HDC groups with an LDA score ≥ 2.0 (Fig. 1F–G). At the phylum level, Actinomycetota was identified as a biomarker for the CON group, whereas no phylum-level biomarkers were detected for the HDC group. At the genus level, Oscillospira was identified as a biomarker for the CON group, while the HDC group was characterized by genus-level biomarkers including the Clostridium_innocuum, Alistipes, Turicimonas, and Ligilactobacillus.

Subsequent Wilcoxon rank-sum tests with FDR correction further validated the LEfSe results (Fig. 1H). Compared with the CON group, the HDC group showed significantly reduced abundances of Actinomycetota at the phylum level and Oscillospira at the genus level, whereas the Clostridium_innocuum, Alistipes, and Turicimonas were significantly increased (P < 0.05 at the phylum level; q < 0.05 at the genus level).

Functional prediction using PICRUSt2 revealed no significant differences in overall pathway composition at Level 3 (Fig. 2D–E). Notably, HDC supplementation increased the relative abundance of specific pathways, including “Drug resistance: antimicrobial” at KEGG Level 2 and “Bacterial secretion system” at Level 3 (P < 0.05, Fig. 2F–G).

Fig. 2.

Predicted functional profiles of the intestinal microbiota using PICRUSt . (A-C) Relative abundances of KEGG pathways at Levels 1–3. (D) Principal Component Analysis (PCA) based on KEGG Level 3 pathway abundances. (E) Principal Coordinates Analysis (PCoA) based on KEGG Level 3 pathway abundances. (F-G) Differential KEGG pathways at Levels 2 and 3. E-CAPs, encapsulated capsaicinoids; CON, basal diet provided as the control; HDC, basal diet supplemented with 450 mg/kg E-CAPs. The data were evaluated using the Wilcoxon rank-sum test (n = 8). Asterisks (*) indicate significant differences between groups ( P < 0.05, P < 0.01).

Discussion

Previous studies have demonstrated that dietary supplementation with chili powder or chili extracts in various livestock species can enhance growth performance, primarily through mechanisms involving the stimulation of digestive secretions, modulation of intestinal function, and enhancement of antioxidant and immune capacities (Reda et al., 2020; Long et al., 2021; Su et al., 2023).

In the present study, E-CAPs was employed to mimic the natural composition of CAPs while reducing mucosal irritation and enabling controlled release in the distal intestine (Lu et al., 2020; Mehta et al., 2025). The results indicate that high-level E-CAPs supplementation enhanced growth performance in broilers, whereas low-level supplementation had no significant effect. These results suggest that a sufficient supplementation level is required for E-CAPs to exert growth-promoting effects, whereas the lower inclusion level may be inadequate to induce measurable improvements in growth performance. Notably, feed intake remained unchanged across broilers receiving different levels of E-CAPs supplementation. This finding is consistent with previous reports showing that dietary inclusion of single CAP formulations at varying levels increased ADG and/or reduced FCR without significantly affecting feed intake (Liu et al., 2021; Li et al., 2022). Such a response may be attributed to the inherent sensitivity of poultry to CAPs (Tewksbury and Nabhan, 2001). Importantly, the encapsulated formulation employed in this study likely mitigated mucosal irritation and ensured controlled release of CAP and DHC along the gastrointestinal tract, thereby allowing their biological effects to manifest primarily through post-absorptive metabolic regulation. Collectively, these findings suggest that E-CAPs function as metabolic efficiency enhancers rather than appetite stimulants, distinguishing them from conventional growth-promoting additives that rely on increased feed intake.

CAPs have been reported to modulate hepatic oxidative stress and inflammatory responses, thereby alleviating hepatocyte damage (Kang et al., 2010; Pang et al., 2024). In the present study, concomitant with the improvements in growth performance, HDC supplementation significantly decreased serum ALT levels, indicating enhanced hepatocyte membrane integrity and improved liver function (McGill, 2016; Ghorbanpour et al., 2023). Improved liver function may enhance the synthesis and conversion efficiency of nutrients (Alamri, 2018), thereby increasing feed efficiency and growth performance, which is consistent with the observed increases in ADG and reductions in FCR in the HDC group. In addition, E-CAPs supplementation significantly reduced serum TBA levels. As bile acids serve not only as detergents for lipid digestion but also as signaling molecules regulating glucose and lipid metabolism via FXR- and TGR5-dependent pathways, alterations in bile acid homeostasis can exert profound effects on whole-body energy utilization (Gong et al., 2022a; Du et al., 2025). The reduction in circulating TBA may indicate improved hepatic bile acid recycling or reduced metabolic burden on the liver, thereby contributing to enhanced metabolic efficiency. Moreover, bile acids are increasingly recognized as key mediators linking host metabolism and gut microbiota composition, suggesting that changes in bile acid dynamics may represent an important mechanistic bridge between hepatic metabolism and microbial modulation in response to E-CAPs supplementation (Hu et al., 2024). Other serum biochemical parameters, including AST, Glu, HDL, LDL, LDH, TP, T-Chol, and Urea, were not affected, indicating that HDC supplementation is metabolically safe and well tolerated.

With respect to meat quality, the present study demonstrated that high-dose E-CAPs significantly reduced CF content in muscle tissue. This effect may be associated with TRPV1-dependent activation of fatty acid β-oxidation and the regulation of lipid metabolism–related genes, as suggested by previous studies, thereby favoring lipid utilization for energy rather than muscle deposition and contributing to the observed reduction in muscle CF content (Luo et al., 2012; Liu et al., 2019; Chen et al., 2024). In parallel, HDC significantly decreased muscle TBARS levels during cold storage, indicating reduced local lipid peroxidation. This local antioxidative effect may be related to the enhanced fatty acid oxidation and the reduced substrate availability for lipid peroxidation, and possible direct ROS-scavenging effects of CAPs in muscle tissue. Moreover, HDC exerted no adverse effects on pH, water-holding capacity, TVB-N, or meat color, indicating that the physicochemical quality of breast meat during cold storage was not adversely affected by HDC supplementation.

Given that LDC supplementation did not significantly affect growth performance, serum biochemical parameters, or organ development compared with the CON group, and to optimize resource allocation and analytical focus, the subsequent intestinal morphology and microbiota analyses were conducted only for the CON and HDC groups. This approach allowed a clearer evaluation of the physiological effects associated with the efficacious dose. In the study by Li et al. (2022), supplementation with 2 mg/kg of a single CAP formulation increased villus height in the jejunum of broilers. In contrast, E-CAPs supplementation in the present study did not significantly affect the morphology of the jejunum or ileum. This discrepancy may be attributed to several factors. The encapsulation technology enabled E-CAPs to be gradually released in the small intestine, thereby reducing direct stimulation of the upper small intestinal epithelium (Sasako et al., 2022). Additionally, under the optimal rearing conditions of this study, intestinal morphology may have already been near its physiological maximum, limiting further improvement. Moreover, the observed growth-promoting effects may have been primarily mediated through metabolic and microbial regulation rather than structural changes in intestinal morphology. Consequently, we further examined cecal microbiota composition.

The gut microbiota plays a key role in maintaining intestinal and immune homeostasis by contributing to nutrient metabolism and generating bioactive metabolites with barrier-protective and anti-inflammatory properties (Fu et al., 2023; Ma et al., 2025). CAPs have been shown to modulate microbial diversity and metabolic activity; for instance, Mahalak et al. (2022) reported that pure CAP increased microbial diversity and butyrate production in an in vitro human gut model, while Gong et al. (2022b) demonstrated that CAP improved dyslipidemia in germ-free mice via alterations in bile acid composition. Together, these findings suggest that E-CAPs may influence host metabolic and immune homeostasis through regulation of gut microbial composition and metabolic potential. Based on these considerations, differences in gut microbiota composition between the CON and HDC groups were further examined. Although E-CAPs supplementation did not significantly affect α-diversity indices, β-diversity analysis revealed a clear separation of microbial community structures between the CON and HDC groups, indicating that high-dose E-CAPs selectively reshaped the cecal microbiota rather than broadly altering microbial richness or evenness. Such selective modulation is increasingly recognized as a hallmark of functional feed additives that fine-tune microbial ecological niches without disrupting overall community stability (Liu et al., 2020). Consistent with this observation, E-CAPs exerted a selective modulatory effect on intestinal microbiota at the genus level, characterized by increased relative abundances of Alistipes, Clostridium_innocuum, and Turicimonas, along with a reduced abundance of Oscillospira.

Specifically, Alistipes has been reported to produce short‑chain fatty acids (SCFAs) and indole-derived metabolites, thereby contributing to intestinal barrier integrity, lipid metabolic regulation, and anti-inflammatory responses (Haskey et al., 2023; Zhang et al., 2024; Lin et al., 2025). Clostridium_innocuum is capable of fermenting carbohydrates and generating SCFAs, which may enhance energy harvest and utilization efficiency (Bian et al., 2022; Petrov et al., 2022). Turicimonas has been associated with lipid metabolism and immune modulation in host–microbe interactions (Cao et al., 2025; Chen et al., 2025). In contrast, an increased abundance of Oscillospira has frequently been linked to reduced digestive efficiency and a pro-inflammatory state (Chen et al., 2020; Zhan et al., 2023). Taken together, these microbial alterations suggest that high-dose E-CAPs supplementation may indirectly improve nutrient utilization by promoting beneficial fermentative taxa while suppressing potentially unfavorable microbial populations.

Furthermore, PICRUSt2-based functional prediction analysis (Douglas et al., 2020) revealed that pathways related to antimicrobial mechanisms and bacterial secretion systems were enriched in the HDC group. These functional shifts may indicate enhanced microbial competitiveness and improved mucosal defense capacity, potentially influencing host lipid metabolism and oxidative stress pathways through microbial metabolites such as SCFAs, antimicrobial peptides, and signaling molecules (Gubatan et al., 2021; Nogal et al., 2021). Overall, the observed changes in cecal microbiota provide supportive evidence that microbiota-mediated modulation may contribute to the physiological effects of E-CAPs, complementing metabolic and growth responses observed in broilers.

Although the present study provides novel insights into the effects of dietary E‑CAPs supplementation in broilers, several limitations should be acknowledged. First, only two supplementation levels (150 and 450 mg/kg, corresponding to approximately 1.0 and 3.1 mg/kg total CAPs, respectively) were evaluated. The limited responses observed in the LDC may be related to the minimal effective concentration, controlled-release characteristics of the encapsulated product, or individual variation, highlighting the need for future gradient dose–response studies. Second, the E‑CAPs reflects the natural mixture of CAP and DHC, and no separate CAP or DHC groups were included, precluding direct evaluation of the independent or interactive effects of these components. Although the observed improvements in growth performance are consistent with previous reports using CAP alone (Long et al., 2021; Li et al., 2022), the specific contributions of CAP and DHC remain unclear. Third, functional predictions of the gut microbiota were based solely on 16S rRNA gene sequencing without direct measurements of key microbial metabolites, such as SCFAs or bile acids, which may mediate capsaicin effects potentially through TRPV1 and microbial modulation pathways (Wang et al., 2020). Although 16S rRNA sequencing provides valuable genus-level insights into gut microbial community structure, the absence of species- or strain-level functional validation, together with the lack of molecular or protein-level assessment of host responses (e.g., intestinal barrier function and immune regulation), limits deeper mechanistic interpretation of the predicted functional changes. Fourth, microbiota analysis was conducted only at the experimental endpoint, restricting understanding of temporal and spatial dynamics as well as potential post-withdrawal effects. Finally, the in vivo release kinetics and tissue distribution of CAPs were not directly measured, limiting insight into local versus systemic effects.

Future studies are warranted to address these limitations. Gradient dose–response trials, together with the inclusion of a non-encapsulated CAPs group and simulated digestion models to characterize release kinetics, would help clarify the dose–response relationship and the biological effects of E-CAPs. Targeted metabolomic profiling of microbial metabolites, including SCFAs and bile acids, combined with molecular and protein-level verification of host targets such as TRPV1 signaling, lipid metabolism–related genes (e.g., PPARα and PGC-1α), and intestinal barrier–associated proteins (e.g., ZO-1), would provide more direct and integrative mechanistic evidence for microbiota-mediated effects of E-CAPs (Chen et al., 2024). Time-series sampling across intestinal segments, together with metagenomic sequencing or culture-based functional characterization of key microbial strains, will further elucidate the spatiotemporal dynamics and functional roles of the gut microbiota. Such integrated approaches will enhance understanding of the mechanistic basis, dose–response relationships, and functional consequences of E-CAPs supplementation in broilers.

Conclusions

This study demonstrates that dietary supplementation with 450 mg/kg E-CAPs (≈3.1 mg total CAPs/kg) enhances growth performance in broilers by increasing average daily gain and feed efficiency, while reducing intramuscular fat. These benefits are likely mediated through synergistic interactions between gut microbiota and host metabolism, as reflected by the promotion of Alistipes, Clostridium_innocuum, and Turicimonas and the suppression of Oscillospira, which support liver function and nutrient utilization. Our findings provide novel evidence for the combined use of encapsulated CAP and DHC as a safe and effective functional feed additive and highlight the importance of gut microbial modulation in mediating growth-promoting effects, paving the way for further mechanistic studies.

CRediT authorship contribution statement

Kangwei Hou: Writing – review & editing, Writing – original draft, Validation, Software, Methodology, Investigation, Formal analysis, Data curation. Jun Li: Investigation. Yongxia Wang: Writing – review & editing, Resources, Project administration, Investigation, Formal analysis, Data curation. Maolong He: Formal analysis. Haifeng Wang: Writing – review & editing, Resources, Project administration, Methodology, Investigation, Funding acquisition, Conceptualization.

Disclosures

The authors declare that there are no conflicts of interest, financial or personal relationships with other people or organizations that could inappropriately influence or bias the content of this paper. All authors have reviewed and approved the final version of the manuscript.