Effects of purple corn anthocyanin on slaughter performance, immune function, the caecal microbiota and the transcriptome in chickens

Abstract

Poultry are susceptible to oxidative stress, which decreases immune function and negatively affects production performance under highly intensive feeding conditions. Moreover, anthocyanins can alleviate oxidative stress and improve immune functions in chickens. This study aimed to elucidate the effects of purple corn anthocyanin extract (PCE) on slaughter performance, immune function, the caecal microbiota and the transcriptome in chickens. A total of 180 female chickens were randomly divided into two groups, with one receiving a basal diet (CON) and one receiving a treatment (PCE) supplemented with 360 mg/kg PCE according to a completely randomized design. The results indicated that the levels of plasma immunoglobulin A, immunoglobulin G, immunoglobulin M, complement 3, and complement 4 in the PCE treatment group were greater (P < 0.05) than those in the CON group. The slaughter performance and caecal short-chain fatty acid parameters did not differ (P > 0.05) between the PCE and CON groups. The inclusion of PCE significantly increased (P < 0.05) the bursa of Fabricius/live weight value compared with those of the CON. Chickens receiving PCE had significantly (P < 0.05) increased relative abundances of norank_f_Muribaculaceae, Anaerofilum, Shuttleworthia, Brachyspira, and Tuzzerella but significantly decreased (P < 0.05) relative abundances of unclassified_f__Rikenellaceae, Oscillospira, norank_f__Barnesiellaceae, norank_f__Christensenellaceae, and Candidatus_Soleaferrea. A total of 2,846 differentially expressed genes (DEGs; P < 0.05), which consisted of 1,140 upregulated genes and 1,706 downregulated genes, were identified. Among them, 201 genes were annotated to the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes database for immune-related genes. Protein-protein interaction network analysis revealed that DEGs associated with the joining chain of multimeric IgA and IgM were significantly upregulated immune-related genes, and those associated with forkhead box P1, cathelicidin 1, cathelicidin 2, and cathelicidin 3 were significantly downregulated immune-related genes in chickens. The findings demonstrated that dietary supplementation with PCE has the potential to improve plasma immunoglobulin, immune organ, caecal potentially beneficial bacteria levels and immune-related gene expressions, which can increase the immune function of chickens.

Introduction

The chronic inflammation due to redox imbalance caused by an imbalance in the intestinal microbiota is an important factor affecting chicken production efficiency (Chen et al., 2021). Antibiotics are often used to improve gastrointestinal function in animals and increase production efficiency, but excessive use of antibiotics can lead to resistance and residue, posing a serious threat to consumer health (Tian et al., 2021). Many countries and regions, including China and Europe, have passed legislation to ban the addition of antibiotics to animal feed (Tian et al., 2021). Therefore, the search for viable alternatives to antibiotics has become a prominent research focus in the post-antibiotic era in chickens (Wickramasuriya et al., 2024).

The functions of the caecum in the body primarily include digesting feed, promoting bowel movements, maintaining balanced gut microbiota, promoting intestinal health, absorbing nutrients, and improving immunity in poultry (Bedford and Apajalahti, 2022). In addition, the caecum contains abundant lymphoid tissue, which can produce lymphocytes and antibodies, thereby enhancing the body's immunity (Taha-Abdelaziz et al., 2018). Inflammation in the caecum may lead to a decrease in lymphocytes and antibodies, increasing the susceptibility of the animals to bacterial or viral infections and resulting in symptoms such as fever and fatigue (Wickramasuriya et al., 2022). As a result, improving the microflora and immune-related genes in the caecum is becoming important for increasing the immune function of chickens (Bortoluzzi et al., 2017).

The effects of natural antioxidants on the immune function of chickens have been evaluated in terms of the immune organ index, individual immune indices, changes in cytokines, and apoptosis of immune cells (Zhou et al., 2019; Luo et al., 2022). Anthocyanins, a group of plant secondary metabolites widely found in the plant kingdom, serve as natural and safe oxidant sources and play important roles in modulating gastrointestinal health (Verediano et al., 2021; Tian et al., 2021). A previous study demonstrated that anthocyanins prevent intestinal inflammation by activating the antioxidant adaptive response of intestinal epithelial cells (Ershad et al., 2021) and improving the microbial population by increasing the number of beneficial bacteria (e.g., Bifidobacterium and Lactobacillus) in animals (Wang et al., 2020). Moreover, anthocyanins can also protect the intestine by regulating I-kappa-B-alpha phosphorylation by inhibiting nuclear kappa B (NF-κB), thereby reducing the expression of proinflammatory factors, such as tumour necrosis factor alpha, interferon gamma, and interleukins (Vendrame and Klimis-Zacas, 2015; Verediano et al., 2021). These cytokines can lead to intestinal barrier damage by adversely affecting the expression of tight junction proteins (Xiao et al., 2016). For example, Fan et al. (2018) revealed that adding dietary antioxidant-rich daidzein supplementation to broiler chickens can improve their immune function by transcriptome analysis. Similarly, Wang et al. (2021) reported that dietary supplementation with anthocyanin-rich plant extract could reduce free radicals (FRs) and decrease the mRNA levels of proinflammatory markers, thereby reducing inflammatory reactions and increasing the immune function of chickens. Thus, dietary supplementation with anthocyanins has the potential to improve immune function by modulating the caecal microbiota and immune-related genes in chickens.

Anthocyanins have various bioactivities, but few reports have focused on their immune function mechanism in chickens in vivo (Xie et al., 2018; Kalt, 2019). In addition, our previous study indicated that administering feed supplemented with 360 mg/kg anthocyanin-rich PCE improved the plasma antioxidant activity and production performance of Chishui black-bone chickens (Li et al., 2023). However, to the best of our knowledge, the effects of anthocyanins on the caecal microbiota, transcriptome sequencing, and immune function of chickens have rarely been reported until now. Therefore, the purpose of this study was to estimate the effects of purple corn anthocyanin extract (PCE) on slaughter performance, immune function, caecal short-chain fatty acid (SCFA) content, and bacterial communities. Moreover, we also employed RNA sequencing (RNA-seq) to detect the differentially expressed genes (DEGs) and underlying molecular mechanisms in caecal tissues influenced by anthocyanins.

Materials and methods

Ethics statement

This study was reviewed, and all experimental animal care procedures were approved by the Rules of Animal Welfare and Experimental Animal Ethics of Guizhou University (EAE-GZU-2021-P017), Guizhou, China.

Birds, diets, and management

The Chishui black-bone chicken is a famous dual-purpose meat and egg poultry breed in Guizhou Province, China (Zi et al., 2023; Li et al., 2023). A total of 180 Chishui black-bone female chickens with similar body weights (1717.56 ± 161.83 g, mean ± standard deviation) that were maintained at Guizhou Zhuxiang Breeding Co. Ltd., Chishui, China (28.590337 N, 105.697472 E) were chosen for this study. The chickens were randomly divided into two groups, with one receiving a basal diet (CON) and one receiving a diet supplemented with 360 mg/kg PCE according to a completely randomized design. The vaccination history for the birds were as following: all chickens were vaccinated against Marek's disease at the hatchery. Chickens were vaccinated against infectious bronchitis and Newcastle disease at 3 weeks. At 8 weeks, chickens were vaccinated against avian encephalomyelitis, avian infectious laryngotracheitis, and fowl pox. Chickens were vaccinated infectious bronchitis and Newcastle disease viruses booster at 10 weeks and 18 weeks. The PCE was obtained from Nanjing Herd Source Biotechnology Co., Ltd. (Nanjing, China) and contained 45.3 µg/g pelargonidin, 1975 µg/g cyanidin, 0.1 µg/g malvidin, 7.9 µg/g petunidin, and 591 µg/g delphinidin (Tian et al., 2022). Each group consisted of six replicates, and each replicate included 15 chickens. The chickens were housed in a 0.076 m³ cage with 3 chickens per cage. Previous studies showed that light length was not a major factor affecting chickens behavior and health, and 16L:8D had a better welfare status than control birds without affecting broiler performance (Bayram et al., 2010; Blatchford et al., 2012). Hence, all the chickens were subjected to identical environmental conditions, including 16 h of light per day, a temperature of 21°C, and humidity ranging from 45% to 65%. The feeding trial period consisted of a 74-d experimental period, including a 14-d adaptation period and a 60-d formal feeding period. Feed and water were provided ad libitum throughout the experimental period. The nutrient requirements of the chickens were determined according to the Chinese Standard (2004). The chemical composition and nutrient levels of the basal diet are shown in Table 1.

Table 1.

The chemical composition and nutrient levels of the basal diet.

Ingredients, g/kg of fed basis	Content	Nutrient levels, g/kg of DM	Content
Corn	615	Dry matter, g/kg of the as-fed diet	926
Soybean meal	261.9	Crude protein	163
Soybean oil	10.5	Metabolizable energy, MJ/kg2	122
Limestone	78.6	Calcium	32
Fishmeal	1	Total phosphorus	4.5
NaCl	3	Available phosphorus	2
Premix1	30	Lysine	8.7
		Methionine	4.2
		Methionin+cysteine	7.1

¹Premix provided per kg: 330,000 IU of vitamin A, 133,500 IU of vitamin D₃, 850 IU of vitamin E, 70 mg of vitamin B₁, 200 mg of vitamin B₂, 135 mg of vitamin B₆, 0.8 mg of vitamin B₁₂, 85 mg of vitamin K₃, 1,200 mg of nicotinamide, 350 mg of pantothenic acid, 9 mg of biotin, 12,000 mg of choline chloride, 340 mg of Cu, 2000 mg of Fe, 700 mg of Mn, 2,700 mg of Zn, and 12 mg of Se.

²Data was calculated value according to the feed database in China (2018).

Chemical composition

The chemical compositions of the basal diet for dry matter (AOAC 930.15), crude protein (AOAC 988.05), calcium (AOAC 927.02), and phosphorus (AOAC 964.06) were detected according to the Association of Official Analytical Chemists (2005). Moreover, the amino acid content of the basal diet was detected by using an amino acid analyser (Biochrom 30, Biochrom Ltd., Cambridge, United Kingdom) according to the methods by Li et al. (2023).

Blood immune parameters

The blood was collected as per the methods of Luo et al. (2022) and Maina et al. (2024). Briefly, at 1, 21, 41, and 61 d during the formal feeding period, two chickens were randomly selected from each replicate (n =12) before feeding, and approximately 5 mL of blood was harvested from their inferior wing veins. Next, the blood sample was centrifuged at 4,000 × g for 10 min at 4°C, and then the plasma was harvested, transferred to a 1.5-mL tube and stored at −80°C until analysis. The immune parameters used were immunoglobulin A (IgA, E027-1-1), immunoglobulin G (IgG, E026-1-1), immunoglobulin M (IgM, E025-1-1), complement 3 (C3, E032-1-1), and complement 4 (C4, E033-1-1), which were analysed according to the instructions from matched test kits provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Slaughter performance

Six replicates could lead to a 0.05 significance level and a power greater than 0.80 according to our previous study (Li et al., 2023), and the chicken was set at the experimental unit for slaughter performance, immune organs, SCFAs, bacteria and transcriptome sequencing. Therefore, the slaughter trial was performed at the end of the experiment, one chicken from each replicate was randomly selected, and six chickens from each treatment were slaughtered (n = 6). The slaughter performance parameters of the chickens were determined according to the methods of Liu et al. (2024). The live weight before slaughter was determined, after which the animals were euthanized by intravenous injection of sodium pentobarbitone and then scalded. Lastly, the feathers, heads, viscera, and feet were removed. After that, the chickens were euthanized by jugular vein bleeding. After evisceration, the carcass, semieviscerated and fully eviscerated carcasses, abdominal fat, breast muscle, and leg muscle were dissected and weighed separately. Next, the carcass rate, semieviscerated rate, eviscerated rate, breast muscle rate, leg muscle rate, and abdominal fat rate were calculated.

Relative weight of immune organ

For the above broilers, the liver, spleen, thymus and bursa of Fabricius were immediately dissected, blotted and weighed. Relative weight of immune organs = weight of organ/live weight × 100% (Wang et al., 2021; Wang et al., 2023).

Short chain fatty acids

The slaughter trial was performed at the end of the experiment, and the caecal contents were collected and stored in sterile tubes and then stored at −20°C for SCFA analysis. The SCFA was analysed according to the methods of Liao et al. (2020), with a minor modification. Briefly, approximately 25 mg of caecal content sample was weighed and transferred into a 2 mL grinding tube, and 500 μL of water (containing 0.5% phosphoric acid) was added. The sample was subsequently frozen and ground for 3 min, followed by ultrasonic extraction at 50 Hz for 10 min and then centrifugation at 13,000 × g for 15 min. The above supernatant was collected and transferred into a 1.5 mL centrifuge tube, and 0.2 mL of N-butanol solvent (containing internal standard 2-ethylbutyric acid 10 μg/mL) was added for extraction. The extract mixture was vortexed for 10 s, ultrasonicated for 10 min, and then centrifuged at 13,000 × g for 15 min at 4°C. The supernatant was used for SCFA determination. The individual SCFAs, including acetic acid (AA), propionic acid (PA), butyric acid (BA), isobutyric acid (isoBA), valeric acid (VA), isovaleric acid (isoVA), hexanoic acid (HA), and isohexanoic acid (isoHA), were analysed using a gas chromatography-mass spectrometry (GC/MS) system (8890B-5977B, Agilent Technologies Inc. California, USA) with an HP-FFAP column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, California, USA). The GC/MS conditions were as follows: helium was used as the carrier gas, the flow rate was 1.0 mL/min, the injection port temperature was 140°C, the injection volume was 1 μL, and the shaking ratio was 10:1. The programming temperature was as follows: the initial temperature of the column temperature was 80°C, the temperature was heated up to 120°C at a rate of 40°C/min, the temperature was increased to 200°C at a rate of 10°C/min, and then the temperature was increased to 230°C and held for 3 min. The electron impact ion power temperature was 230°C, the quadrupole rod temperature was 150°C, the transmission line temperature was 80°C, the ionization energy was 70 eV, and the scanning method was single ion monitoring. The individual SCFA concentration of each sample was calculated using the calibration curve method.

16S rRNA sequencing

Caecal contents were collected (1 bird per replicate cage) in a 5-mL sterile tube and stored in liquid nitrogen for DNA sequence analysis. The DNeasy® PowerSoil® Pro Kit (Qiagen, Hilden, Germany) was used to extract the total microbial DNA from 12 samples according to the manufacturer's instructions. The quality of the purified DNA was determined with a NanoDrop 2000 spectrophotometer and 2% agarose gel electrophoresis. We conducted PCR amplification using 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primers to amplify the V3 and V4 regions of the 16S rRNA gene. Each sample was run in triplicate. The PCR products were purified using a QIAquick gel extraction kit (Qiagen, Germany) after amplification. The purified PCR products were subsequently pooled at equimolar concentrations to construct DNA libraries, and the obtained products were subsequently sequenced on an Illumina MiSeq PE300 (Illumina, California, USA). Fastp (version 0.20.0) software was used to control the quality of the raw sequences, and Flash (version 1.2.7) software was used for paired-end sequence splicing, and the procedures were as followed: the 300 bp read was truncated at any site receiving an average quality score of <20 over a 50 bp sliding window, and the truncated read less than 50 bp was discarded; and then the overlapping sequence longer than 10 bp was assembled, and the maximum mismatch ratio of overlap region was 0.2; next, according to the barcode and primers, sample was distinguished, and the sequence direction was adjusted, and the maximum number of primer mismatches was 2. Uparse (version 7.1) software was used to cluster operational taxonomic units (OTUs) at 97% sequence identity. To minimize the effects of sequencing depth on alpha and beta diversity measure, and each of sample was rarefied to a sequencing depth of 45,058 16S rRNA reads for all downstream analysis. The taxonomy of each OTU representative sequence was analyzed by RDP Classifier version 2.2 against the 16S rRNA gene database (Silva v138), and the confidence threshold was 70%.

Transcriptome sequencing

The caecal tissue samples were collected in RNAse-free sterile tubes after being washed with sterile water, frozen in liquid nitrogen, and then stored at −80°C for RNA sequencing. TRIzol® reagent was used to extract the total RNA from the caecal tissue, and the RNA quality was determined with a 5300 Bioanalyzer (Agilent Technologies, California, USA) and quantified using a NanoDrop-2000 (Thermo Fisher Scientific, Delaware, USA). The caecal tissue RNA-seq transcriptome library was prepared following Illumina® Stranded mRNA Prep, Ligation from Illumina (San Diego, California, USA), using 1 μg of total RNA. In brief, messenger RNA was first isolated according to the poly(A) selection method with oligo (dT) beads and then fragmented with fragmentation buffer. Second, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, California, USA) with random hexamer primers (Illumina, San Diego, California, USA). The synthesized cDNA was subsequently subjected to end repair. Phosphorylation and ‘A’ base addition was done according to Illumina's library construction protocol. Libraries were size-selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantification with a Qubit 4.0, the paired-end RNA-seq sequencing library was sequenced with a NovaSeq 6000 sequencer (2 × 150 bp read length).

The raw paired-end reads were trimmed and quality controlled by Fastp with default parameters, after which the clean reads were separately aligned to the reference genome in orientation mode using the HISAT2 tool. The mapped reads of each sample were assembled by Stringtie via a reference-based approach. RSEM was used to analyse gene expression; subsequently, the gene length was homogenized, and the sequencing depth was subsequently normalized to obtain the transcripts per million reads (TPM). Gene Ontology (GO) annotation and enrichment analysis of the DEGs were performed using the Blast2GO and GO algorithms. KOBAS was used to analyse the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of the DEGs. The protein-protein interaction (PPI) networks of the DEGs with the highest confidence values were analysed using the String database.

Validation via real-time PCR

Total RNA was extracted from caecal tissue with TRIzol (Thermo Fisher Scientific, Waltham, MA). The RNA integrity was detected using a Universal Hood II imager and a Gel Doc™ XR system (Bio-Rad) using the electrophoresis (DYY-6C, Beijing Liuyi Biotechnology Co., Ltd., China) on a 2% agarose gel. cDNA synthesis was performed with a RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific, WI, USA). The joining chains of multimeric IgA and IgM (JCHAIN), forkhead box P1 (FOXP1), cathelicidin 1 (CATH1), cathelicidin 2 (CATH2), and cathelicidin 3 (CATH3) were detected, and the housekeeping gene used in this study was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Each gene sequence was collected from the NCBI database, and primers were designed using the TaqMan Primer Design Tool. The cDNA was analysed by real-time PCR in a 20 µL reaction mixture: 10 µL of SYBR Green qPCR master mix, 2 µL of cDNA, 1 µL of forward primer, 1 µL of reverse primer, and 6 µL of ddH₂O. The cycling conditions were as follows: preincubation at 95°C for 10 min, 40 cycles of amplification at 95°C for 30 s, and annealing for 1 min at 60°C (Table S1).

Statistical analysis

The effects of PCE on the slaughter performance, blood immune parameters, immune organs, and caecal SCFA parameters were analysed with SPSS (version 27, Chicago, IL, USA) using Student's t test. In addition, the relative muscle mRNA gene expression was calculated by 2^-△△Ct method, and the CON data were used as a calibrator. The free online Majorbio Cloud Platform (www.majorbio.com) was used to analyse the 16S rRNA bacterial parameters. The alpha diversity (Sobs, Shannon, Simpson, Ace, Chao, and Coverage) was measured with Mothur software (version 1.30.2), and multiple comparisons were performed using the Benjamini-Hochberg method. The QIIME2 package version 2020.2.0 was used to calculate the beta diversity distance matrix, principal coordinate analysis (PCoA) was performed on the basis of Bray-Curtis, and an analysis of similarity (ANOSIM) was used to test the differences between groups. The differences were analysed by a nonparametric test (Wilcoxon rank-sum test), and BH was used to correct for multiple tests. Confidence intervals were calculated by bootstrapping at 95%. For transcriptome data, DESeq2 (version 1.24.0) was used to analyse the differential expression between the two groups. The Benjamini Hochberg (BH) method was used to correct for multiple testing. DEGs with an absolute fold change (FC) of ≥2 and adjusted values of P < 0.05 were considered DEGs. The PPI network of the differentially expressed immune-related genes was explored (with a confidence score threshold set at 0.8) by using the Search Tool for the Retrieval of Interacting Genes/Proteins database, and the interaction network diagram was drawn with Cytoscape 3.9.1. A P value < 0.05 was considered significant.

Results

Blood immune parameter

The contents of plasma IgA, IgG, IgM, C3, and C4 in the PCE treatment group were greater (P < 0.05) than those in the CON group (Table 2).

Table 2.

Effect of purple corn anthocyanin extract on plasma immune content in chicken.

Item1	Treatments		SEM	P-value2
	CON	PCE
IgA, mg/L	548.00b	715.67a	24.520	0.008
IgG, mg/L	215.23b	267.33a	11.261	0.031
IgM, mg/L	339.67b	498.67a	13.703	0.001
C3, mg/L	83.57b	137.13a	3.680	0.001
C4, mg/L	26.63b	35.13a	1.056	0.005

¹IgA, immunoglobulin A; IgG, immunoglobulin G; IgM, immunoglobulin M; C3, complement 3; C4, complement 4.

²Values within each row with different small superscripts are significantly different (P < 0.05).

Slaughter performance and immune organs

There were no significant differences between the CON and PCE treatments for any slaughter performance parameter (Table 3; live weight, carcass rate, semieviscerated rate, eviscerated rate, breast muscle rate, leg muscle rate, and abdominal fat rate). Similarly, no significant differences (P > 0.05) were detected in the liver/live weight, spleen/live weight, or thymus/live weight between the two treatment groups (Table 4). In contrast, compared with the CON, the inclusion of PCE significantly increased (P < 0.05) the bursa of Fabricius/live weight value.

Table 3.

Effect of purple corn anthocyanin extract on slaughter performance in chicken.

Item	Treatments1		SEM	P-value2
	CON	PCE
Live weight, g	1963.00	1969.67	12.724	0.730
Carcass rate, %	90.48	90.08	0.445	0.565
Semi-eviscerated rate, %	81.06	81.27	0.460	0.760
Eviscerated rate, %	66.25	66.82	0.728	0.607
Breast muscle rate, %	15.29	15.56	0.417	0.667
Leg muscle rate, %	21.38	21.86	0.370	0.411
Abdominal fat rate, %	2.68	2.51	0.146	0.449

Table 4.

Effects of purple corn anthocyanin extract on relative weights of immune organs in chicken.

Organs	Treatments		SEM	P-value1
	CON	PCE
Liver/Body weight	2.82	2.78	0.041	0.562
Spleen/Body weight	0.18	0.19	0.003	0.158
Thymus/Body weight	0.39	0.40	0.004	0.109
Bursa of Fabricius/ Body weight	0.27b	0.31a	0.004	0.002

¹Values within each row with different small superscripts are significantly different (P < 0.05).

Short chain fatty acids

Feeding chickens with PCE had no effect (P > 0.05) on the total SCFA, AA, PA, BA, isoBA, VA, isoVA, HA, or isoHA content of the caecum (Table 5).

Table 5.

Effect of purple corn anthocyanin extract on caecal short chain fatty acids in chicken.

Item (μg/mL)1	Treatments		SEM	P-value2
	CON	PCE
Total SCFA	3.97	4.16	0.421	0.843
AA	1.37	1.59	0.142	0.490
PA	1.14	1.02	0.103	0.642
BA	0.25	0.19	0.124	0.513
isoBA	0.66	0.72	0.043	0.524
VA	0.26	0.29	0.062	0.833
isoVA	0.24	0.18	0.042	0.551
HA	0.01	0.01	0.000	0.672
isoHA	0.04	0.17	0.042	0.085

¹AA, acetic acid; PA, propionic acid; BA, butyric acid; isoBA, isobutyric acid; VA, valeric acid; isoVA, isovaleric acid; HA, hexanoic acid; isoHA, isohexanoic acid.

²Values within each row with different small superscripts are significantly different (P < 0.05).

Alpha and beta diversity analyses

To identify the specific caecum microbiota improved by anthocyanin-rich PCE, we used 16S rRNA to analyse the caecal microbes. After low-quality or chimeric sequences were removed, totals of 342,631 and 321,197 high-quality 16S rRNA bacterial gene sequences were obtained from the CON and PCE groups, respectively. We applied a sequencing depth (45,058) for rarefaction, and the rarefaction curves tended to plateau as the sequence number increased, indicating the full bacterial diversity is represented in this sequencing depth (Fig. 1A). A total of 1,050 OTUs were acquired and categorized into 16 phyla, 26 classes, 58 orders, 93 families, and 207 genera. The CON and PCE groups had 1,015 and 991 OTUs, respectively. Among them, 956 OTUs were common to both groups, while 59 OTUs were unique to the CON group, and 35 OTUs were unique to the PCE group (Fig. 1B).

Fig. 1.

Effect of purple corn anthocyanin extract on bacterial diversity in chickens. (A) Multi samples rarefaction curves of caecal bacterial 16S rRNA. The rarefaction curve demonstrates that the sequencing depth (45,058) was sufficient for almost all samples. (B) The Venn diagram. The Venn diagram showed that 59 OTUs were unique to the CON group, and 35 OTUs were unique to the PCE group, and 956 OTUs were common to both groups.

The results of the alpha diversity analyses revealed that PCE had no significant (P > 0.05) effect on the Coverage, Shannon, Simpson, Ace, Chao, or Sobs indices (Fig. S1A-F). The PCoA with the Bray-Curtis distance showed no significant (P = 0.302) segregation between the two groups (Fig. S1G), and the contribution values of principal component (PC) 1 and PC2 to the sample differences were 18.64% and 16.49%, respectively. Nonmetric multidimensional scaling (NMDS) with the Bray‒Curtis distance revealed that the stress value was 0.112 (Fig. S1H).

Caecal Bacteria

The results revealed that 7 phyla had relative abundances greater than 1% (Fig. 2A). The predominant phyla were Bacteroidota (56.67%, 57.90%) and Firmicutes (32.42%, 30.39%), followed by Desulfobacterota (2.56%, 2.63%), Spirochaetota (1.30%, 2.63%), Synergistota (2.13%, 1.70%), Proteobacteria (1.02%, 1.34%), and Actinobacteriota (1.17%, 1.09%). A total of 22 genera with relative abundances greater than 1% were observed (Fig. 2B). The top 5 genera were Bacteroides (23.54%, 22.64%), Rikenellaceae_RC9_gut_group (14.93%, 13.06%), unclassified_o_Bacteroidales (7.64%, 10.21%), Phascolarctobacterium (3.79%, 5.02%), and Ruminococcus_torques_group (2.88%, 2.37%).

Fig. 2.

Effect of purple corn anthocyanin extract on caecal dominant bacteria. (A) Top 15 caecal bacteria of chickens at phylum level. The predominant phyla were Bacteroidota, Firmicutes, Desulfobacterota, Spirochaetota, Synergistota, Proteobacteria, and Actinobacteriota. (B) Top 30 caecal bacteria of chickens at genus level. The top 5 genera were Bacteroides, Rikenellaceae_RC9_gut_group, unclassified_o_Bacteroidales, Phascolarctobacterium, and Ruminococcus_torques_group.

The results obtained from the Wilcoxon rank-sum test revealed no differences (P > 0.05) bacterial composition between the two treatments at the phylum level (Fig. 3A). Similarly, the PCE had no effect on any bacterial community with a relative abundance greater than 1% at the genus level, except for an increased (P = 0.013) relative abundance of norank_f_Muribaculaceae. However, PCE increased (P < 0.05) the relative abundances of Anaerofilum, Shuttleworthia, Brachyspira, and Tuzzerella at the genus level but decreased (P < 0.05) the relative abundances of unclassified_f_Rikenellaceae, Oscillospira, norank_f_Barnesiellaceae, norank_f_Christensenellaceae, and Candidatus_Soleaferrea at the genus level (Fig. 3B).

Fig. 3.

Analysis of the significant differences of bacterial composition base on Wilcoxon rank-sum test (relative abundance > 0.001). (A) Caecal bacteria at phylum level. There was no differences between the two treatments at the phylum level. (B) Caecal bacteria with significant change at genus level. The PCE treatment was significantly increased the relative abundances of Anaerofilum, Shuttleworthia, Brachyspira, and Tuzzerella, but decreased the relative abundances of unclassified_f_Rikenellaceae, Oscillospira, norank_f_Barnesiellaceae, norank_f_Christensenellaceae, and Candidatus_Soleaferrea at the genus level.

Quality of RNA-seq Reads

Transcriptome sequencing technology can reveal DEGs in specific biological states of tissues and lay a data foundation for subsequent differential analysis. To gain deeper insight into the effects of PCE on different immune-related genes in the caecum of chickens, we performed a transcriptome analysis of the caecum. The number of raw reads ranged from 42,999,448 to 58,539,938, with an average of 51,847,896 (Table S2). All samples had a Q20 (percentage of bases with a Phred value ≥ 20) greater than 98.03% and a Q30 (percentage of bases with a Phred value ≥ 30) greater than 94.54%, the error rate was 0.0243% on average, and the average GC content was 53.7%.

As shown in Fig. 4A, a total of 13,032 and 12,813 genes were identified in the CON and PCE groups, respectively. Among these genes, 706 genes were unique to CON, and 487 genes were unique to PCE. A total of 2,846 DEGs, including 1,140 upregulated genes and 1,706 downregulated genes, were identified (Fig. 4B). Cluster analysis of the DEGs revealed that the expression levels of genes in the samples within the group tended to be consistent, indicating good biological repeatability. However, there were differences in gene expression between the samples from the two treatment groups, indicating that dietary supplementation with PCE could affect gene expression (Fig. 4C). Moreover, the top 10 upregulated and downregulated DEGs are shown in Table S3.

Fig. 4.

Different genes in the caecal tissue of chickens. (A) Venn diagram of different genes. 708 genes were unique to CON group, and 706 genes were unique to PCE treatment. (B) Volcano plot of differentially expressed genes (DEGs). 1,140 upregulated genes and 1,706 downregulated genes were identified. (C) Cluster analysis. The cluster analysis showed that the expression levels of genes in the samples within the group tended to be consistent, and there were differences in gene expression between the samples.

Differentially Expressed Genes

A total of 2,846 DEGs were annotated using the GO analysis (Fig. 5A, Table S4) and KEGG analysis (Fig. 5B). There were 51 GO terms associated with the biological process (22 subclasses), cellular component (16 subclasses), and molecular function (13 subclasses) categories. The major categories in the biological process category were cellular process (GO:0009987), biological regulation (GO:0065007), and metabolic process (GO:0008152). For the CC category, cell part (GO:0044464), membrane part (GO:0044425), and organelle (GO:0043226) were the major categories. For molecular function, binding (GO:0005488), catalytic activity (GO:0003824), and molecular function regulator (GO:0098772) were the major categories.

Fig. 5.

Functional annotation analysis of differentially expressed genes (DEGs). (A) GO functional annotation analysis of DEGs. The major categories in the biological process category were cellular process, biological regulation, and metabolic process. (B) KEGG functional annotation analysis of DEGs. The major categories associated with metabolism were lipid metabolism, glycan biosynthesis and metabolism, and carbohydrate metabolism. (C) Venn diagram of immune function-related DEGs. 50 genes were annotated only as immune-related in GO, and 115 genes were annotated only as immune-related in KEGG.

The KEGG pathway analysis for annotation including the following six categories were metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases (Kanehisa et al., 2023). The major categories associated with metabolism in the KEGG pathway were lipid metabolism, glycan biosynthesis and metabolism, and carbohydrate metabolism (Fig. 5B). For the genetic information processing of the KEGG pathway, the major categories were folding, sorting and degradation, and translation. For the environmental information processing KEGG pathway, the major categories were signal transduction and signalling molecules and interactions. For the cellular processes of the KEGG pathway, the major categories were “cellular community-eukaryotes” and “cell growth and death”. For organismal systems in the KEGG pathway, the major categories were the endocrine system and the immune system. For human diseases in the KEGG pathway, the major categories were cancer-overview and infectious disease-viral. In addition, a total of 201 immune-related genes were annotated in the GO and KEGG databases. Among them, 86 genes were annotated as GO immune system processes, 151 genes were annotated as the KEGG immune system, 50 genes were annotated only as immune-related in GO, and 115 genes were annotated only as immune-related in KEGG (Fig. 5C).

GO enrichment analysis of DEGs

GO enrichment analyses were performed on the DEGs, and a total of 875 pathways were enriched (Table S5). The top 20 GO pathways are shown in Fig. 6. The multicellular organismal process, regulation of multicellular organismal process, anatomical structure development, regulation of cell population proliferation, extracellular region, ion transport, positive regulation of cell population proliferation, regulation of system process, and cell-cell junction organization were the main enriched pathways (adjusted P = 0.00431). Moreover, “cellular anatomical entity”, “biological regulation”, “intrinsic component of membrane”, and “integral component of membrane” were the main functional pathways for gene enrichment with greater than 700 genes. Moreover, a total of 85 genes were enriched in immune system processes, and the top 20 upregulated and downregulated DEGs are shown in Table S6.

Fig. 6.

Top 20 of GO enrichment analysis of differentially expressed genes. The colour indicates adjusted P-value and size of circles indicates number of differently expressed genes in the GO category.

KEGG Enrichment Analysis of DEGs

KEGG enrichment analyses were performed on the DEGs, and a total of 336 pathways were enriched (Table S7). The top 20 KEGG pathways are shown in Fig. 7. The amoebiasis, allograft rejection, autoimmune thyroid disease, asthma, calcium signalling pathway, intestinal immune network for IgA production, Staphylococcus aureus infection, African trypanosomiasis, viral myocarditis, haematopoietic cell lineage, and PI3K-Akt signalling pathways were the main enriched pathways with a Log10 (P adjust) greater than 4. Moreover, the PI3K-Akt signalling pathway, the calcium signalling pathway, and transcriptional misregulation in cancer were the main functional pathways for genes enriched with more than 50 genes. Among all the pathways, 21 were associated with the immune system (Table S8). Five pathways were significantly enriched, namely, the intestinal immune network for IgA production, the haematopoietic cell lineage, the B-cell receptor signalling pathway, the Fc epsilon RI signalling pathway, and Fc gamma R-mediated phagocytosis (Table S9).

Fig. 7.

Top 20 of KEGG enrichment analysis of differentially expressed genes. The colour indicates adjusted P-value and size of circles indicates number of differently expressed genes in the KEGG category.

Main Protein-Protein Interaction Network

The main PPI network of the DEGs was constructed to determine the interaction relationships among the differentially expressed immune-related genes (Fig. 8). The network contained 5 immune-related genes, including JCHAIN, whose expression was significantly upregulated, and FOXP1, CATH1, CATH2, and CATH3, whose expression was significantly downregulated (Table S6).

Fig. 8.

Main protein-protein interaction network of differentially expressed genes in the caecal tissues of chickens receiving purple corn anthocyanin extract. Network nodes and edges represented proteins and protein-protein associations, respectively. The size of the circle represents the values of log2 fold change. CASR, calcium sensing receptor; GPRC6A, G protein-coupled receptor class C group 6 member A; IL1RN, interleukin 1 receptor antagonist; JUN, jun proto-oncogene, AP-1 transcription factor subunit; FOS, fos proto-oncogene, AP-1 transcription factor subunit; IL15, interleukin 15; GSK3A, glycogen synthase kinase 3 alpha, transcript variant X1; JCHAIN, joining chain of multimeric IgA and IgM; FOXP1, forkhead box P1; F8, coagulation factor VIII; ADCY3, adenylate cyclase 3, transcript variant X1; IL7, interleukin 7; CCR2, C-C motif chemokine receptor 2; F2, coagulation factor II, thrombin; GNAS, GNAS complex locus, transcript variant X1; PIK3R3, phosphoinositide-3-kinase regulatory subunit 3, transcript variant X1; CD80, CD80 molecule; FOXO6, forkhead box O6; PIGR, polymeric immunoglobulin receptor; CX3CL1, C-X3-C motif chemokine ligand 1; CATH3, cathelicidin 3; CD28, CD28 molecule; AKT3, AKT serine/threonine kinase 3, transcript variant X1; AvBD1, avian beta-defensin 1; DEFB4A, defensin beta 4A, transcript variant 1; CATH1, cathelicidin 1; CATH2, cathelicidin 2.

Validation via real-time PCR

Among the identified DEGs, five immune-related genes, including one upregulated transcript (JCHAIN) and four downregulated transcripts (FOXP1, CATH1, CATH2, and CATH3), were validated in the caecum using real-time PCR. The results indicated that the trends in the expression of all the genes were similar between the RNA-seq and real-time PCR results (Fig. 9). Thus, these results showed that the RNA-seq results were accurate and reliable in this study.

Fig. 9.

Quantitative polymerase chain reaction (qPCR) validation of 5 differentially expressed immune-related genes identified using RNA sequencing. x-axis represents the genes, and y-axis represents their mRNA expression levels expressed in fold-change (FC) values. Expression levels determined via RNA-seq and qPCR are represented by blue and red fill columns, respectively. JCHAIN, joining chain of multimeric IgA and IgM; FOXP1, forkhead box P1; CATH1, cathelicidin 1; CATH2, cathelicidin 2; CATH3, cathelicidin 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Discussion

Immunoglobulins can mediate multiple protective functions through interactions with specific receptors and immune mediators, serving as the first line of defence against bacterial invasion (Woof and Kerr, 2006). Indeed, the immunoglobulins can hinder the invasion of pathogenic microorganisms into the body, and an increase in the immunoglobulin content indicates an increase in the body's immune function (Wlaźlak et al., 2023). A previous study revealed that dietary supplementation with antioxidants increased the blood levels of immunoglobulins, leading to increased humoral immunity in animals (Noh et al., 2019). For example, Feng et al. (2023) reported that the consumption of plant anthocyanins increased the plasma IgG and IgM contents of Hainan black goats. In the present study, dietary addition of PCE increased plasma IgA, IgM, and IgG levels, indicating that anthocyanins can promote the body's immune response and increase its ability to resist pathogen invasion in Chishui black-bone chickens. This observation might be because dietary anthocyanins increase immunoexpression in muscle (Table S8; Amer et al., 2022). Consistent with our results, Sharifian et al. (2019) reported that feeding with pomegranate peel extract could alleviate oxidative stress (OS) and enhance immune function by increasing the blood IgG content in broiler chickens.

The complement system plays an important role in the immune system, with C3 and C4 being important complement molecules that promote complement activation and mediate immune and inflammatory responses (Liu et al., 2025). In addition, a complement can label viruses for rapid degradation by proteasomes, thereby preventing their replication and subsequently enhancing immunity (Copenhaver et al., 2019). The results of the present study revealed that PCE increased plasma C3 and C4 contents, possibly because anthocyanins have broad-spectrum antibacterial and bactericidal effects, directly acting on and killing pathogens by cleaving cell membranes and destroying key intracellular targets, improving the composition and intestinal structure of the caecal microbiota (Dong et al., 2024). Our results are consistent with those of Amer et al. (2022), who reported that dietary supplementation with 200 mg/kg anthocyanin-rich roselle (Hibiscus sabdariffa L.) extract could increase the blood C3 content of broiler chickens.

Slaughtering performance is an important indicator for evaluating the meat performance of poultry and can intuitively reflect the composition of the animal body and its meat production performance (Wei et al., 2024). In this study, we found that anthocyanins did not affect the live weight, carcass rate, semieviscerated rate, eviscerated rate, breast muscle rate, leg muscle rate, or abdominal fat rate, suggesting that anthocyanins do not have side effects on slaughter performance in Chishui black-bone chickens. This result may be related to the fact that anthocyanins do not affect growth performance in chickens.

The degree of development of immune organs represents, to some extent, the developmental status of the body's immune system, and the developmental status of immune organs affects the body's immunity (Zhu et al., 2023). The thymus, spleen, and bursa of Fabricius are immune organs, and their relative weights are important indicators of immune function in poultry (Xu et al., 2023). Specifically, the bursa of Fabricius is the primary lymphoid organ, and an increase in its weight indicates an improvement in the body's immune function in poultry (Peng et al., 2009). A previous study indicated that the dietary addition of plant extracts to a broiler chicken diet has a positive effect on activating the immune system (Das et al., 2020). Hence, the addition of PCE increased the relative weight of the bursa of Fabricius, suggesting that anthocyanins promote the development of immune organs and affect the immune function and disease resistance ability of chickens. This result might have occurred because plant extracts can enhance the humoral and cellular immunity of broiler chickens, thereby enhancing the antiviral and lytic effects of natural killer cells, improving their antioxidant and immune capabilities and increasing the development and weight of immune organs (de Oliveira et al., 2024). Our results are consistent with those of Wang et al. (2021), who reported that dietary supplementation with anthocyanin-rich bilberry extract could increase the weight of the bursa of Fabricius in yellow-feathered chickens.

Bacterial metabolites such as SCFAs provide energy to the epithelial cells lining the intestine and inhibit the expression of virulence factors of pathogenic bacteria (Kogut, 2019). Additionally, SCFAs exert anti-inflammatory effects by inhibiting the NF-κB pathway, reducing vascular endothelial cytokines (Park et al., 2007), and enhancing intestinal barrier function by regulating the proliferation and differentiation of intestinal epithelial cells (Martin-Gallausiaux et al., 2021). Walton et al. (2006) reported that anthocyanins have low absorption in pigs because the total amount of anthocyanins absorbed was not significantly different among the three diets. Hence, in this study, dietary consumption of 360 mg/kg PCE did not affect caecal SCFAs, perhaps because anthocyanins are unstable in the gastrointestinal tract, resulting in the low bioavailability of anthocyanins (Du et al., 2023; Guo and Shahidi, 2024), and most anthocyanins arrive intact in the colon and are excreted in the faeces (Gonçalves et al., 2021). One study indicated that dietary supplementation with anthocyanins could improve ruminal fluid SCFA parameters in ruminants (Taethaisong et al., 2022). Another possible mechanism was that the ability of the microbiota in the caecum to digest carbohydrates in feed is lower than that of the microbiota in the rumen. However, this result must be confirmed further.

The gut microbiota plays an important role in maintaining intestinal health (Fan et al., 2023). The caecum, the longest feed retention area in the chicken gut, is characterized by high water absorption and bacterial diversity (Xiao et al., 2017); it plays a crucial role in detoxifying substances and inhibiting pathogens in animals (Vinardell and Lopera, 1987; Clench and Mathias, 1995). Various studies have demonstrated that the dietary addition of anthocyanins can improve the caecal microbiota, including by increasing beneficial bacteria and reducing pathogens in chickens (Verediano et al., 2022; Zhang et al., 2023). Interestingly, beneficial bacteria, such as Tuzzerella and Shuttleworthia, are beneficial for reducing tissue inflammation and enhancing immune function in animals (Lee et al., 2017; Bu et al., 2023). Thus, we found that PCE increased the abundances of taxa with potentially beneficial bacteria, including norank_f_Muribaculaceae, Tuzzerella, and Shuttleworthia, indicating that dietary supplementation with anthocyanins resulted in reduced caecal inflammation and improved immunity in Chishui black-bone chickens. Pathogens can cause serious nutritional and metabolic disorders and are associated with ageing and inflammation (Biagi et al., 2016; Tong et al., 2018; Carrasco et al., 2018). The results of this study revealed that dietary addition of PCE decreased the abundances of possible pathogens, such as norank_f_Barnesiellaceae, norank_f_Christensenellaceae, and Oscillospira, suggesting that anthocyanins can inhibit harmful bacteria and improve the caecal health state. Thus, chickens receiving PCE might increase potentially beneficial bacteria and decrease potentially harmful bacteria in their caecal contents, resulting in the alleviation of caecal inflammatory reactions and maintenance of the microbial balance. In brief, the results indicated that adding anthocyanin-rich PCE has the potential to be beneficial for regulating the caecal microflora to improve immune function in chickens.

In modern intensive livestock cultivation, poultry are susceptible to external environmental influences (Nardone et al., 2010), leading to OS status and decreased immune functions, which affect their health (Lee et al., 2019). The anthocyanins in poultry diets can not only eliminate FRs and reduce oxidative reactions but also stimulate the activity of immune cells, increasing their ability to recognize and clear pathogens and thereby improving the immune performance of the body (Changxing et al., 2018). Amer et al. (2022) reported that feeding with anthocyanin-rich roselle (Hibiscus sabdariffa L.) extract could enhance immune performance in broiler chickens. Moreover, Wang et al. (2021) reported that bilberry anthocyanin regulated a variety of biological processes, especially the defence response to bacteria and the humoral immune response, and suppressed cytokine–cytokine receptor interactions and the intestinal immune network for IgA production pathways by downregulating the expression of 6 immune-related proteins in yellow-feathered chickens. In the present study, GO enrichment analysis revealed that the differentially expressed immune-related genes were enriched primarily in multicellular organismal processes, regulation of multicellular organismal processes, etc., and KEGG enrichment analysis revealed that the differentially expressed immune-related genes were enriched primarily in the intestinal immune network for IgA production, the haematopoietic cell lineage, etc., signalling pathways. This enrichment might have occurred because anthocyanins have strong antioxidative and radical scavenging activities (López-Pedrouso et al., 2020). These results indicated that these signalling pathways may be the main signalling pathways involved in the differential regulation of the inflammatory response in anthocyanin-rich PCE.

PPI network analysis in the present study revealed that JCHAIN, FOXP1, CATH1, CATH2, and CATH3 may be the main factors involved in regulating immune function in chickens, which is worthy of study and is discussed below. JCHAIN is a small polypeptide expressed by mucosal and glandular plasma cells that regulates the polymer formation of IgA and IgM, and it is a key protein in secretory immunity (Johansen et al., 2000). Cheng et al. (2020) reported that feeding anthocyanin-rich fermented blueberry pomace could increase small intestine JCHAIN gene expression in high-fat diet-fed mice. In the present study, we found that dietary supplementation with anthocyanins upregulated JCHAIN gene expression in the caecum of chickens. This upregulation might have occurred because anthocyanin supplementation increased the abundances of potentially beneficial bacteria (such as Akkermansia and Lactobacillus), promoting the subsequent signal identification process and contributing to the generation of IgA-producing cells in the caecum (Cheng et al., 2020).

During the process of immune regulation, FOXP1 can regulate the differentiation and function of T cells and B cells, thereby affecting the normal function of the immune system (Wang et al., 2014). Zhuang et al. (2019) reported that FOXP1 can negatively regulate the inflammatory body complex Nlrp3-Caspase1-IL1β, thus delaying the formation and development of inflammatory reactions, and that antioxidants can decrease FOXP1 expression, reduce vascular inflammation and help improve immunity. Furthermore, Ahmed et al. (2017) reported that dietary supplementation with Ginkgo biloba leaf extract could increase immune function by downregulating FOXP1 gene expression in the livers of rats. Thus, the results of the present study revealed that PCE resulted in downregulated FOXP1 gene expression in the caecum, suggesting that anthocyanins could inhibit inflammatory reactions and enhance the immune function of chickens.

CATH is an important endogenous antimicrobial peptide that is a crucial component of the innate immune system and is widely expressed in various tissues of the body (Jiang et al., 2021). On the one hand, CATH directly activates different cell populations to produce and release different proinflammatory and immunoregulatory cytokines; on the other hand, CATH can stimulate the production and release of FRs, which take part in tissue damage (Agier et al., 2015). Various studies have shown that the gene expression of CATH increases when animals are under OS conditions (Zhang et al., 2014; Darwish et al., 2024). As a result, CATH has various immunomodulatory activities that strongly affect the course of inflammation (Agier et al., 2015). The results of the present study revealed that dietary supplementation with PCE decreased the gene expression of CATH1, CATH2, and CATH3, possibly because anthocyanins increase the immune function of chickens and inhibit their inflammatory processes. Interestingly, CATH can affect host defence against microorganisms by modulating epithelial responses to infecting pathogens and by influencing inflammatory processes at sites of pathogen entry (Agier et al., 2015). These results suggest that PCE could improve the caecal microbiota. In brief, these findings suggested that dietary supplementation with PCE could improve immune function by regulating caecal microbial and immune-related gene expression in broiler chickens.

Conclusions

The results of the present study indicate that supplementing the chicken diet with PCE would be a recommended nutritional strategy to increase immune function in the current chicken farming industry because (1) the inclusion of PCE enhances the plasma immunoglobulin content and bursa of Fabricius/live weight value; (2) PCE has the ability to increase caecal potentially beneficial bacteria and reduce possible pathogens in chickens; and (3) dietary supplementation with PCE modulates the expression of five candidate immune-related genes, including JCHAIN, FOXP1, CATH1, CATH2, and CATH3, which may play important roles in regulating chicken immune function. However, this study did not detect the caecal contents of immune-related metabolites or the histomorphology of the caecum of chickens. Therefore, further studies are necessary to determine the specific effects of anthocyanins on immune functions by modulating these metabolites and genes in chickens.

SUPPLEMENTARY MATERIALS

Transcriptomic sequencing files and microbial sequencing files containing raw data associated with each sample were submitted to the National Center for Biotechnology Information under study accession numbers PRJNA1081135 and PRJNA1081787, respectively.

Declaration of competing interest

The authors declare that we have no conflict of interest.