
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00744-2
10.1016/j.psj.2024.104165
104165
MICROBIOLOGY AND FOOD SAFETY
Investigation into the supplementation of a ferric sillen core-linked polymer on the health and physiological performance of broiler chickens
Christanseen Seán schristanseen@alltech.com
⁎1
Walls Dermot †¶
White Blánaid ‡§¶
Paul M.A. #
Ao Tuoying ⁎⁎
Ford M.J. ‖
Murphy Richard ⁎
Horgan Karina A. ⁎
Power Ronan ††
⁎ Alltech Ireland, Sarney Dunboyne, Co. Meath, Ireland
† School of Biotechnology, Dublin City University, Co. Dublin, Ireland
‡ School of Chemical Sciences, Dublin City University, Co. Dublin, Ireland
§ DCU Water Institute, Dublin City University, Co. Dublin, Ireland
¶ National Centre for Sensor Research, Dublin City University, Co. Dublin, Ireland
‖ Department of Animal and Food Sciences, University of Kentucky, KY
# Hill's Pet Nutrition, Kansas, MO
⁎⁎ Alltech-University of Kentucky Nutrition Research Alliance, Lexington, KY
†† Alltech, 3031 Catnip Hill Rd, Nicholasville, KY
1 Corresponding author: schristanseen@alltech.com
02 8 2024
11 2024
02 8 2024
103 11 1041659 4 2024
30 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Poultry is a ubiquitous and highly sought-after protein source valued for its accessibility, notable protein content, and lack of religious constraints. However, the demand for poultry has resulted in a surge in intensive production practices. The transition from subsistence agricultural practices to intensive food production resulted in the widespread adoption of antibiotics for both therapeutic and economic purposes. These interventions were intended to enhance meat yield, promote bird health, and enhance cost-effectiveness of production. However, this inadvertently contributed to the rise of antimicrobial resistance (AMR). Therefore, the need to explore alternative approaches to mitigate the problems associated with AMR has become increasingly pressing. In response, metal-based compounds have emerged as a promising substitute to conventional antibiotics. In this study, the effects of a water soluble metallo-antimicrobial supplement, ferric sillen core-linked polymer (FSCLP), on body weight gain, feed conversion, water intake, volatile fatty acid (VFA) production, cecal microbiome and intestinal morphology in broilers was examined. The findings of this study suggested that the addition of the FSCLP resulted in better bird performance, even during a period of heat stress. Volatile fatty acids analysis of cecal contents indicated that there were significantly higher levels (p < 0.05) of butyric and valeric acids. Cecal microbiome analysis confirmed significantly lower abundance (p < 0.05) of Proteobacteria (e.g., E. coli) and a significantly greater abundance of VFA-producing bacteria such as Intestinimonas butyriciproducens, Blautia and Lachnospiraceae. The intestinal morphology data showed supplementation with the FSCLP at 80 ppm resulted in a significantly higher (p < 0.05) villus height of the jejunum.

This study emphasises the potential of FSCLP as a feasible solution to the issues faced by AMR in chicken production, providing insights into its beneficial impacts on performance, microbial composition, and intestinal health.

Key words

metallo-antimicrobial
broiler performance
volatile fatty acid
cecal microbiome
==== Body
pmcKEY POINTS

• FSCLP supplementation improved body weight gain and VFA synthesis.

• A reduction in the prevalence of pathogenic bacteria, notably Escherichia coli, alongside an augmented abundance of VFA-producing bacteria within the cecal microbiome.

• Noteworthy improvements in villus height within the jejunum were observed following FSCLP supplementation, indicative of enhanced intestinal health

INTRODUCTION

Poultry meat is expected to experience a significant increase in demand in the coming years, with projections indicating a 17.8% increase by 2030 (Mak et al., 2022). Despite the ban on use of antimicrobial compounds for growth promotion in several countries, 73% of all antimicrobials sold globally are still utilised in animal agriculture for food production (Van Boeckel et al., 2019). The excessive use of antibiotics has resulted in selective pressure by clearing susceptible bacteria and accelerating the spread of antibiotic-resistant bacteria. As a result, antimicrobial resistance (AMR) represents a significant challenge to global public health and has the potential to become the leading cause of mortality worldwide, with estimates suggesting 1.27 million deaths were directly caused by bacterial AMR, and more than twice that number were associated with it (Murray et al., 2022). This trend highlights the vital need for a complete review of the effects of such intense production techniques on animal welfare, environmental sustainability, and public health, as well as the urgent need to develop alternative strategies of minimising antimicrobial resistance (Hafez and Attia, 2020).

Metal-based antimicrobials are receiving considerable attention as a promising strategy to address the growing issue of AMR (Frei et al., 2023). The appeal of these antimicrobials lies in the distinctive properties of certain metal ions, which have innate antibacterial properties. An advantageous mode of action of metal-based antimicrobials is their capability to circumvent bacterial defence and resistance mechanisms by exploiting bacterial transport systems to achieve intracellular delivery. This quality sets them apart from traditional antibiotics, which have become ineffective due to well-described resistance mechanisms such as enzymatic degradation, efflux pumps, and alterations in target sites (Frei et al., 2023).

Iron (Fe) is an essential trace mineral for all living organisms, necessary for DNA synthesis, energy metabolism, respiration, electron transport, and redox reactions (Golonka, Yeoh and Vijay-Kumar, 2019). However, the bioavailability of this essential nutrient is considerably low particularly, within the lower intestinal tract. This limitation is due to iron's poor solubility under aerobic conditions at physiological pH and due to the presence of iron-binding glycoproteins such as lactoferrin and transferrin in the host. Given the bacterial requirement for iron, antimicrobials that target iron uptake pathways are a promising approach.

One example of organic antimicrobials that have been widely used for decades within the agriculture industry, specifically in poultry farming, are organic acids. These compounds act by crossing the bacterial cell membrane due to the lipophilic nature of their undissociated form, altering the proton and associated anion concentrations in the bacterium's cytoplasm (Gómez-García et al., 2019). However, in poultry, the ceca are considered the main reservoir for pathogenic bacteria and due to most organic acids dissociating prior to reaching the lower GIT, organic acids alone have rarely been shown not to be effective in modulating the cecal microbiota. An approach to address this difficulty is to enhance the stability of organic acids by capitalising on their chelation potential with transition metals. Notably, recent investigations by Soliman et al., (2020) and Chandio et al., (2019), on the antimicrobial impacts of several ligands and their ferric complexes, observed that the ferric complex demonstrated heightened antimicrobial efficacy against the targeted bacteria and fungi, compared to the functional ligand. The development of novel metallo-antimicrobials based on the capacity of the active organic molecules to readily coordinate iron ions may offer a significant untapped resource that can serve as effective alternatives for managing infections in birds.

MATERIALS AND METHODS

Broiler Study Experimental Design, Sample Collection and Preservation

All procedures were approved by the University of Kentucky Institutional Animal Care and Use Committee. A Broiler trial was conducted in the Coldstream research facility, Lexington, Kentucky (between late July and August 2022). A total of 360 one-day-old Cobb male broiler chicks were randomly split into 3 groups of 8 pens, with 24 pens in total (15 birds/pen; 120 birds/group) using a randomized complete block design on built-up dirty litter with a coating of fresh pine shavings. Stocking density was 0.1486m2 (1.6 ft2) per chick. Prior to the start of the experiment, Fe content in each treatment group, basal starter, grower, and finisher diets, as well as feed components and water, was tested using ICP-OES. Basal diets were prepared by a commercial feed mill and consisted primarily of corn and soybean meal, as outlined in Table 1, Table 2. Starter diets were feed from d zero to d 9, grower diets from d 10 to d 21, and finisher diets from d 22 to d 42. Feed and water were provided ad-libitum throughout the study. All birds were weighed, and the values averaged by pen on d 0, 7, 14 21, 28, 35, and 42. Concurrently, feed and water intake were also assessed to evaluate feed conversion ratios, weight gain and to establish that the treatments had no effect on water consumption.Table 1 Composition of starter, grower, and finisher rations.

Table 1Ingredient	Starter (kg)	Grower (kg)	Finisher (kg)	
Corn	84.36	227.95	763.64	
Soy-bean meal (48% Cp)	45.81	96.51	299.37	
Vegetable oil	0.88	3.26	26.45	
Limestone	1.44	3.34	10.59	
Dicalcium phosphate	2.27	4.87	15.28	
Salt	0.56	1.33	4.41	
L-lysine	0.44	1.27	2.83	
DL-methionine	0.39	0.88	2.38	
Mineral premix (no Fe)	0.34	0.85	2.83	
Vitamin premix	0.34	0.85	2.83	
Cp = Crude protein

Table 2 Mineral premix.

Table 2Mineral	Form	Element content, %	Inclusion rate of premix, %	Minerals for 1 kg premix (g)	
Se	Na2SeO3	45.4	0.25	0.26	
Copper	CuSO4.5H2O	25	0.25	24	
Iodine	KIO3	59.31	0.25	0.67	
Manganese	MnSO4-H2O	29.5	0.25	135.59	
Zinc	ZnO	72	0.25	55.56	
Total Minerals				216.09	
Limestone				783.91	

The experimental groups for this study included a negative control and 2 FSCLP treatment groups which had a final concentration in the bird's water supply of 40 or 80 ppm Fe. The 80 ppm inclusion rate was in accordance with the National Research Council recommendations of maximal permissible levels of supplemental Fe. In contrast, the minimal level of iron supplementation is established at 40 ppm (National Research Council, 1994).

At 42 d post-hatch, the intact cecal pouches of 4 randomly caught birds per pen (32 birds per treatment) were removed immediately euthanized with argon gas followed by cervical dislocation. The cecal contents were aseptically transferred into sterile tubes, with one-half of the tubes containing 5 mL of DNA/RNA shield (Zymo Research, Germany), while the other half of the tubes were immediately flash frozen.

VFA Analysis

Sample Preparation

A modified version of the method described by Yang et al., (2021) was used in this investigation. Cecal samples were homogenized by grinding under liquid nitrogen to prevent any further freeze/thaw. The thawed digesta (0.5 g) was diluted with distilled water (1:4 w/v). Samples were then vortexed for 1 min until homogeneous, then centrifuged at 4,000 g for 10 min at 4°C. The clear supernatant (1 mL) was mixed with 0.2 mL of metaphosphoric acid solution (25% (v/v)) for deproteinization and Crotonic acid solution (40 μL of 434 per mmol; internal standard) was added as an internal standard. After incubation at 4°C for 30 min, samples were centrifuged at 18,500 × g (Centrifuge 5810 R, Eppendorf, Germany) for 10 min. The supernatant (0.1 mL) was blended with 0.9 mL of methanol and resulting solutions filtered through 0.45 μm microporous membranes (Merck) prior to gas chromatograph (GC) analysis (Agilent 7890A, Agilent 7693A Automatic Liquid Sampler, flame ionization detector, 10 μL micro-injector).

Preparation of Standards

Acetic, valeric, butyric, isobutyric, propanoic and isovaleric acid standards were accurately measured and diluted with ultrapure water to final concentrations of 25 mM, 1.5 mM, 10 mM, 1 mM, 10 mM and 1 mM respectively. The standard solution (1 mL) was mixed with 0.2 mL metaphosphoric acid solution (0.2 mL of 25% (v/v)) and crotonic acid solution (40 μL of 434 mM; internal standard). After incubation for 30 min at 4°C, samples were centrifuged at 12,000 rpm for 10 min. The supernatant was then blended with methanol at 1:9 (v/v) and resulting solutions were filtered through a 0.45 μm microporous membranes prior to GC analysis.

Chromatographic Conditions

Carryover prevention, fused-silica, free-fatty-acid phase (CP-FFAP) chromatographic column with a length of 25 m, internal diameter of 0.32 mm and film thickness of 0.3 μm was used for the analysis. The temperature program was set as follows: 100°C for 1 min, 100°C - 190°C increasing by 20°C/min, 190°C for 3 min. The total analysis time was 7.5 min with an injection port temperature of 220°C. The injection volume was 1 μL with a split ratio of 10:1.

Microbial Diversity and Community Composition Analysis of Cecal Microbiota

DNA Extraction, Library Preparation and Sequencing

DNA from cecal samples was isolated using the QIAGEN DNeasy PowerSoil Pro Kit, according to the manufacturer's protocol. Extracted DNA samples were quantified using a Qubit 4 fluorometer and Qubit dsDNA HS Assay Kit (Thermofisher Scientific, Waltham, MA).

DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) and IDT Unique Dual Indices with total DNA input of 1 ng. Genomic DNA was fragmented using a proportional amount of the Illumina Nextera XT fragmentation enzyme. Unique dual indices were added to each sample and this was followed by 12 cycles of PCR in order to construct libraries. DNA libraries were purified using AMpure magnetic Beads (Beckman Coulter) and eluted in QIAGEN EB buffer. DNA libraries were quantified using a Qubit 4 fluorometer and Qubit dsDNA HS assay kit. Libraries were then sequenced on an Illumina HiSeq X platform (2×150 bp).

Bioinformatic Analysis

Raw sequencing was performed by CosmosID Inc. (Rockville, MD) and data was analysed using the CosmosID-Hub Microbiome Analysis tool (“CosmosID,” n.d.). The system utilizes a high-performance data-mining k-mer algorithm that rapidly disambiguates millions of short sequence reads into the discrete genomes engendering the particular sequences. The pipeline has 2 separable comparators: the first consists of a pre-computation phase for reference databases and the second is a per-sample computation. The input to the pre-computation phase consists of databases of reference genomes, virulence markers and antimicrobial resistance markers that are continuously curated by CosmosID scientists. The output of the pre-computational phase is a phylogeny tree of microbes, together with sets of variable length k-mer fingerprints (biomarkers) uniquely associated with distinct branches and leaves of the tree. The second per-sample computational phase searches the hundreds of millions of short sequence reads, or alternatively contigs from draft de novo assemblies, against the fingerprint sets.

This query enables the sensitive yet highly precise detection and taxonomic classification of microbial NGS reads. The resulting statistics are analyzed to return taxonomic and relative abundance estimates for the microbial NGS datasets. To exclude false positive identifications the results are filtered using a threshold derived based on internal statistical scores that are determined by analyzing a large number of diverse metagenomes.

Jejunum and Ileum Morphology

A transverse jejunum and ileum section (2 cm) was collected from one bird per pen on d 42 and placed immediately into 10% neutral buffered formalin (Sigma-Aldrich, Saint Louis, MO) and later dehydrated by consecutive washes with ethyl alcohol (70–100%) using a Microm STP 120 Spin Tissue Processor (Thermo Fisher Scientific). Samples were then mounted and embedded in paraffin blocks using a Microm EC 350 Modular tissue embedding centre (Thermo Fisher Scientific). Embedded samples were cut into 3 longitudinal sections (5 µm) using a Rotary Microtome Microm HM 340 E (Thermo Fisher Scientific). Slides were deparaffinised with histolene and rehydrated with water through a series of decreasing concentrations of ethanol washes. Slides were then stained with 1% alcian blue (pH 2.5, 3% acetic acid) for 5 min and subsequently rinsed with water. Slides were immersed in 0.5% periodic acid solution for 10 min followed by 3 min washing with water, then stained with Schiff's reagent for 15 min and rinsed again with water. Finally, slides were cleaned with histolene and SubMount mounting medium (Statlab, McKinney, TX), a coverslip was carefully lowered in an effort to avoid any air bubbles and left to dry overnight. Each tissue sample was visualized with a Nikon Microscope Eclipse E400 (Microscope central, Feasterville, PA), and photographed using a SPOT Flex camera (Meyer Instruments, Houston, TX, US) with SPOT 5.0 software. Fifteen well-characterized, orientated and arbitrarily selected villi and corresponding crypts were identified and measured in each intestinal sample/slide and the mean measurement from the fifteen selected villi and crypt was used to determine villus height (VH), crypt depth (CD) and the VH:CD ratio. These measurements were carried out using ImageJ (Wan et al., 2022). Villus height was considered as the distance between the crypt mouth and villus tip, while crypt depth was considered as the infolding between 2 villi; 8 samples were analyzed per treatment. The VH:CD ratio was calculated as VH/CD. Jejunum parameters were measured at 40X magnification, while the ileum parameters were measured at 100X magnification.

Statistical Analysis

In accordance with standard statistical practice, the alpha significance level for this study was established at 0.05. Samples were compared across different treatment groups, namely control, FSCLP 40 ppm Fe, and FSCLP 80 ppm Fe. To determine the statistical significance of performance data and VFA content, a one-way ANOVA was employed followed by Tukey's test. To analyze the microbial community composition, box plots were utilized for quantitative visualization, while ANOVA was utilized to compare the relative abundances of taxa between treatment groups. Bacterial alpha diversity was estimated through the Shannon index, richness through Chao1, and evenness through Simpson. To test for statistical significance between treatment groups, the Wilcoxon Rank Sum Test was employed. The present study also evaluated beta diversity by calculating the dissimilarity matrix using the Bray-Curtis dissimilarity index. To assess differences in beta diversity among the gut microbial communities of distinct groups, principal coordinate analysis (PCoA) was employed as a tool for visualizing the relationships between multivariate datasets in a low-dimensional space. To determine statistical significance between groups, permutational multivariate analysis of variance (PERMANOVA) was conducted on the Bray-Curtis dissimilarity matrix.

RESULTS

Performance Results

The effects of FSCLP supplementation on the live performance of broiler chickens, are presented in Table 3. The results showed that at all sample points, the broilers provided with FSCLP at a concentration of 40 ppm and 80 ppm of Fe had significantly higher (p < 0.05) body weight (BW) and body weight gain (BWG) compared to the control group. Statistical analysis of feed intake (FI) data indicated that there was a significantly higher (p < 0.05) FI at all time points in birds supplemented with FSCLP (80 ppm). In contrast, the birds supplemented with FSCLP (40 ppm) only exhibited significantly higher (p < 0.05) FI at 28 and 35 d. FCR was similar among the 3 test groups, while the analysis of water intake (WI) showed that the birds treated with FSCLP (80 ppm) had significantly greater (p < 0.05) WI on d 28 which coincided with a period of high heat index.Table 3 Performance response of broilers supplemented with FSCLP at a concentration of 40 ppm and 80 ppm of Fe.

Table 3Experimental treatment	BW, g/b	BWG, g/b	FI, g/b	FCR	WI, g/b	
7 d:						
 0 ppm FSCLP	121A	76.9A	127A	1.66	305	
 40 ppm FSCLP	136B	92.0B	135 AB	1.5	303	
 80 ppm FSCLP	142B	97.7B	158B	1.63	315	
 SEM	3.11	3.26	7.87	0.08	9.94	
 P	<0.01	<0.01	0.04	0.39	0.67	
14 d:						
 0 ppm FSCLP	333A	287A	449A	1.57	969	
 40 ppm FSCLP	372B	326B	480A	1.48	999	
 80 ppm FSCLP	395B	350B	525B	1.59	1046	
 SEM	8.67	8.53	12.3	0.04	24.7	
 P	<0.01	<0.01	<0.01	0.31	0.12	
23 d:						
 0 ppm FSCLP	906A	856A	1249A	1.46	2616	
 40 ppm FSCLP	971B	914B	1307A	1.43	2693	
 80 ppm FSCLP	1008B	963B	1388B	1.44	2795	
 SEM	15.73	16.7	19.4	0.02	53	
 P	<0.01	<0.01	<0.01	0.45	0.09	
28 d:						
 0 ppm FSCLP	1326A	1273A	1905A	1.5	3733 A	
 40 ppm FSCLP	1422B	1362B	1998B	1.47	3861 AB	
 80 ppm FSCLP	1451B	1406B	2086 C	1.48	3955 B	
 SEM	20.4	21.1	27.2	0.01	66	
 P	<0.01	<0.01	<0.01	0.21	0.01	
35 d:						
 0 ppm FSCLP	1950A	1891A	2967A	1.57	5894	
 40 ppm FSCLP	2056B	1992B	3093 B	1.55	6129	
 80 ppm FSCLP	2081B	2035B	3184 B	1.57	6223	
 SEM	23.7	24.3	36.7	0.01	91	
 P	<0.01	<0.01	<0.01	0.21	0.06	
42 d:						
 0 ppm FSCLP	2763A	2698A	4306A	1.6	8588	
 40 ppm FSCLP	2873B	2805B	4457 AB	1.59	8899	
 80 ppm FSCLP	2880B	2826B	4528B	1.6	9001	
 SEM	31.9	33.9	55.2	0	140	
 P	0.04	0.04	0.04	0.2	0.13	
A-B Data is expressed as the means within a column and grouping with a common superscript do not differ significantly. Statistically significant differences were determined by one-way ANOVA followed by Tukey's test (p < 0.05). The trial was conducted using 8 replicate pens of 15 male broilers per treatment.

Volatile Fatty Acids Analysis of Cecal Contents

Analysis of the VFA levels in the cecal content (Table 4) of birds 42 d post-hatch showed significantly higher (p < 0.05) levels of butyric and valeric acid for bids provided 80ppm FSCLP. The remaining compounds examined were not significantly different among treatments.Table 4 Volatile fatty acid (VFA) levels in the cecal content of broilers supplemented with FSCLP.

Table 4	Treatments			
Fatty acids (mM/g)	Control	FSCLP (40 ppm)	FSCLP (80 ppm)	SEM	p-value	
Acetic acid	128.31A	139.39A	144.28A	4.727	0.181	
Butyric acid	16.83A	19.95A	21.25B	1.447	0.008	
Isobutyric acid	1.91A	1.80A	2.02A	0.034	0.652	
Isovaleric acid	1.34A	1.28A	1.41A	0.022	0.762	
Propionic acid	9.07A	8.90A	9.60A	0.215	0.526	
Valeric acid	1.12A	1.27A	1.37B	0.072	0.012	
A-B Data is expressed as the means within a column and grouping with a common superscript do not differ significantly. Statistically significant differences were determined by one-way ANOVA followed by Tukey's test (p < 0.05).

SEM = standard error of the mean.

Cecal Microbiome Analysis

The influence of FSCLP on microbial community development in the broiler caecum at 42 d post hatch was analyzed by shallow shotgun DNA sequencing. Sequencing of DNA in these samples yielded 354,529,000 raw reads, with 54,410,000 of these reads corresponding to bacterial hits. The median sequencing depth for these samples was determined to be 7,473,500 reads, which is slightly above our target depth of 6 million reads.

In this study no significant differences were observed between the control and either FSCLP supplemented group across all 3 alpha diversity measurements (Chao1, Shannon and Simpson; Figure S1-S3).

The PCoA plot based on Bray-Curtis dissimilarity index, revealed that the bacterial community composition at the species level differed significantly among groups, as determined through the use of permutational multivariate analysis of variance (PERMANOVA) (Figure 1). Analysis indicated that there were indeed significant differences in the composition of the bacterial community among the groups (p = 0.027), with a p-value of 0.007 being observed between the control group and the FSCLP (80 ppm) group however no difference was seen between FSCLP treatments (p = 0.263) or between the control group and the FSCLP (40 ppm) group (p = 0.137).Figure 1 Species level Bray–Curtis distance matrices (beta diversity) expressed as principal coordinate analysis (PCoA) comparing the cecum microbiota of an untreated control versus samples that have been supplemented with FSCLP at concentrations of 40ppm and 80ppm of Fe. Statistically significant differences were determined by PERMANOVA, with p < 0.05.

Figure 1

Microbial Community Composition

The 4 main bacterial phyla that were identified within the cecal samples were Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria. An overall summary of the phylum-level composition can be seen in Figure 2A. Bacteroidetes was the most abundant phylum within the control birds, with members comprising on average 48.17% of the total microbial population. However, following FSCLP (80 ppm) supplementation, Bacteroidetes were significantly less abundant (p < 0.05), with an average of 42.95% (Figure 2B). Firmicutes was identified as the second most abundant phylum in the control group, with an average of 45.22%. The abundance of Firmicutes was significantly higher number (p < 0.05), at 49.68% (Figure 2C) with FSCLP (80 ppm) supplementation. The third most abundant phylum was Actinobacteria, with no significant differences in abundance among the treatments. Actinobacteria averaged 4.91% in the control birds, 6.06% in the FSCLP (40 ppm) supplemented birds and 5.61% in the FSCLP (80 ppm) supplemented birds. Finally, Proteobacteria were significantly greater (p < 0.05) in the control (1.55%) and FSCLP (80 ppm) supplemented birds (1.49%) compared to FSCLP (40 ppm) supplemented birds (0.46%) (Figure 2D).Figure 2 Effects of 3 dietary treatments on the phylum-level composition. The phylum-level composition of average relative abundance cecum microbiota (A). The significant changes in the abundance of bacterial phyla found in cecum digesta (B: Bacteroidetes, C: Firmicutes, D: Proteobacteria). Data is expressed as boxplots. Statistically significant differences were determined by one-way ANOVA followed by Tukey's Test (denoted by *p < 0.05).

Figure 2

As significant differences (p < 0.05) were noted in Firmicutes, Bacteroidetes and Proteobacteria at a phyla level, these taxa were further analysed at a class level. Figure 3A effectively encapsulates the class-level taxonomic composition. Notably, within the Firmicutes phylum at this class level; Clostridia, Erysipelotrichia, Bacilli, and Negativicutes emerged as the most prevalent across both the control and supplemented groups. However, Negativicutes was the only group within this class whose abundance was significantly greater (p < 0.05) with FSCLP supplementation at both levels when compared to the control diet (Figure 3B). The results observed at the phylum level for Bacteroidetes were reflected at the class level by Bacteroidia, with a significantly lower abundance (p < 0.05) observed in response to supplementation with FSCLP (80 ppm) when compared to broilers on the control diet (Figure 3C). In both the control and supplemented groups, the most dominant classes within the Proteobacteria phylum were Deltaproteobacteria, Epsilonproteobacteria, Gammaproteobacteria, and Betaproteobacteria. The relative abundance of Gammaproteobacteria was the only group within this class whose abundance was significantly lower (p < 0.05) with FSCLP (40 ppm) supplementation when compared to the control diet (Figure 3D).Figure 3 Effects of 3 dietary treatments on cecal microbiota composition at the class-level . The class-level composition of average relative abundance cecum microbiota (A). The significant changes in the abundance of bacterial class found in cecum digesta (B: Negativicutes, C: Bacteroidia, D: Gammaproteobacteria). Data is expressed as boxplots. Statistically significant differences were determined by one-way ANOVA followed by Tukey's Test (denoted by *p < 0.05).

Figure 3

The relative abundance of several bacterial species was found to be significantly altered following supplementation with FSCLP. Specifically, the bacterial species Intestinimonas butyriciproducens and 2 unidentified species Lachnospiraceae and Blautia which belong to Clostridia class and Phascolarctobacterium faecium which belongs to the Negativicutes class (Table 5). Another important finding was that the bacterium Escherichia coli was less prevalent (lessened by 81.9%) in the birds that had received FSCLP (40 ppm) supplementation (Table 5).Table 5 Relative abundance of the dominant bacteria in the species classification in the cecum of control and supplemented broilers.

Table 5	Treatments			
Bacteria (Relative abundance %)	Control	FSCLP (40 ppm)	FSCLP (80 ppm)	SEM	p-value	
Escherichia coli	0.989A	0.168B	1.002A	0.267	0.004	
Blautia	3.346A	4.272B	4.715B	0.404	0.001	
Intestinimonas butyriciproducens	0.044A	0.068B	0.068B	0.008	0.02	
Phascolarctobacterium faecium	0.382A	1.124AB	1.243AB	0.217	0.043	
Lachnospiraceae	0.158B	0.201A	0.210A	0.016	0.024	
Phocaeicola vulgatus	5.163A	3.011B	2.390B	0.841	0.003	
Bacteroides thetaiotaomicron	0.273A	0.091B	0.055B	0.067	0.017	
Phocaeicola dorei	0.681A	0.137B	0.290AB	0.162	0.05	
A-B Data is expressed as the means within a column and grouping with a common superscript do not differ significantly. Statistically significant differences were determined by one-way ANOVA followed by Tukey's test (p < 0.05).

SEM = standard error of the mean.

Jejunum and Ileum Morphology

The statistical analysis of jejunum and ileum samples indicated that FSCLP supplementation at a concentration of 40 ppm resulted in a significantly greater (p < 0.01) villus height (VH) in the jejunum (Figure 4). However, no significant differences were found in crypt depth (CD) between the control and experimental groups in either tissue type. The villus height: crypt depth ratio in jejunum samples was approximately 8:1, while the ratio for the ileum samples was 4:1 with no significant differences (p ≤ 0.05) among the control and experimental groups in either tissue. No notable damage to the intestinal segments analyzed was observed (Figures S4-S5), with the intestinal villi from both the ileum and jejunum appearing tightly packed and long, while the crypts appeared shallow in both the supplemented and control birds.Figure 4 Impact of FSCLP supplementation on jejunum and ileum villus height of broilers 42 d old. Statistically significant differences were determined by one-way ANOVA followed by Tukey's Test (denoted by ** p < 0.01).

Figure 4

DISCUSSION

This study investigated the effect of an FSCLP on poultry performance including body weight, body weight gain, feed intake, feed conversion, water intake, VFA production, ceca microbiome, and intestinal morphology.

The production performance findings of this study concur with prior research indicating that supplementing poultry diets with organic acids leads to improved body weight and body weight gain (Baghban-Kanani et al., 2019). Despite temperatures reaching up to 30°C during weeks 3 and 4 of the trial, which is typically associated with detrimental consequences to bird productivity, (Vandana et al., 2020), a significantly higher body weight and body weight gain in both supplemented groups compared to the control was noted. Enhanced performance can result in earlier completion of production cycles potentially leading to economic benefits for producers. Being able to maintain performance under heat stress conditions is beneficial, as heat stress exerts adverse effects on feed efficiency, body mass, and feed consumption (Wasti et al., 2020). The significantly greater water intake was observed during the fourth week of the study may be attributed to the high heat index during this period. This result emphasized the benefit of developing a water-soluble product as it can be utilized during periods of environmental stress in poultry production when feed intake is also usually lowered (Nawaz et al., 2021). An additional advantage of water-soluble products is the convenience of administration in terms of simplicity and efficiency. Unlike feed additives, which require meticulous formulation and precise distribution, potentially requiring significant time and labor investments, water additives readily dissolve and uniformly disperse throughout the water supply, ensuring consistent and homogeneous intake by the birds.

The small intestine of chickens plays a crucial role in digestion and nutrient absorption. It is lined with a single layer of cells that contain the necessary enzymes, transporters, and hormones to break down and absorb nutrients. Additionally, this layer acts as a barrier against pathogens by producing glycoproteins and defensins (Zhang et al., 2019). The morphometric examination of broiler chicken jejunum and ileum on d 42 of this study gave insights into the influence of FSCLP supplementation, especially at a concentration of 40 ppm. The noticeable rise in villus height within the jejunum following such supplementation is of relevance. (Wang and Peng, 2008) study provided light on the crucial importance of villus height and crypt depth in the small intestine, stressing their pivotal function as indicators of gut development and animal health. This morphological adaptation indicates a beneficial relationship between FSCLP supplementation and jejunum structural development, underlining the potential for better nutrient use in broiler chickens. In contrast, our crypt depth analysis revealed no significant alterations due to FSCLP supplementation (Figure S6). This implies that FSCLP has a unique impact, largely altering architectural elements linked to villus shape rather than crypt depth. Also, it is critical to emphasize the lack of detectable negative effects related with FSCLP supplementation. This finding is critical from a safety standpoint, since it confirms that the inclusion of FSCLP at the examined concentration has no negative effects on the intestinal morphology of broiler chickens. This element adds to FSCLP's potential as a safe and effective dietary supplement for improving digestive physiology in chicken production systems.

High higher butyric and valeric acid detected in supplemented birds may also be related to the observed performance benefits. Significant increases in endogenously synthesized valeric acids have been shown in challenge models to stimulate intestinal villi development, digestive activity, and the reduction of intestinal lesions (Onrust et al., 2018). Butyric acid has also been proven to have an important function in maintaining gut health through a variety of ways such as, improving gut microbiota balance, improving intestinal barrier integrity, modulating intestinal epithelial cell proliferation, controlling epithelial inflammation via anti-inflammatory cytokine production, boosting the enteric immune system, and reducing oxidative stress (Salvi and Cowles, 2021). These findings connect well with the results obtained from the microbiome analysis, which demonstrated greater prevalence of specific bacterial species with FSCLP, including Intestinimonas butyriciproducens, Lachnospiraceae, Blautia and Phascolarctobacterium which are known for producing short chain fatty acids (Bui et al., 2020; Frolova et al., 2022). The results of this study highlight the potential benefits of supplementing with FSCLP to with regard to greater production of endogenous VFAs.

To evaluate differences in the microbial community composition of the treatment groups, alpha diversity was firstly analyzed using 3 different diversity measures Chao1, Shannon and Simpson. These diversity indices reflect the richness diversity, or evenness in a microbial sample or aim to reflect a combination of these properties. This study found that supplementation with FSCLP did not significantly impact alpha diversity of the microbial communities. However, the application of the metallo-antimicrobial at a concentration of 80 ppm of Fe had a significant effect on beta diversity indicating a difference in species composition compared to the control group. The compounds tested may shift the proportions of different species within the community. The most striking difference was the clear shift in the presence of short chain fatty acid bacteria in the supplemented birds. The use of metallo-antimicrobials altered the composition of the bacterial community in the caeca but did not significantly affect the overall diversity of the community.

The dominant bacterial phyla in both control and supplemented birds were Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria, with Firmicutes being the most prevalent in the supplemented birds. The addition of FSCLP at a concentration of 80 ppm led to significantly greater abundance of Firmicutes compared to the control group. This shift in Firmicutes abundance was also accompanied by a corresponding body weight gain of the chicks when compared to the control. These findings suggest a possible link between the abundance of Firmicutes in the cecal microbiota and efficient utilization of dietary energy, as Firmicutes are known to be involved in the degradation of polysaccharides and the production of butyrate (Herrmann et al., 2018). At phylum level, Proteobacteria were significantly lower in the FSCLP (40 ppm) supplemented birds compared to both the control and FSCLP (80 ppm) supplemented birds. Proteobacteria are a group of Gram-negative bacteria that are non-spore-forming and include several potential foodborne pathogens, such as Escherichia coli, Helicobacter pylori, and Campylobacter jejuni (Rychlik, 2020). Reducing the presence of potential foodborne pathogens is crucial for maintaining the integrity of the food chain and promoting One Health (Founou et al., 2021).

CONCLUSIONS

In conclusion, the findings of this study have important significance for the poultry industry, highlighting the beneficial effects of FSCLP supplementation. The observed improvements in various performance metrics, such as body weight, weight gain, and water intake, underscore the potential of FSCLP as a valuable additive in poultry nutrition. The shift in the ceca microbiota towards a higher prevalence of Firmicutes in the supplemented groups compared to the control group is notable. Firmicutes have been connected to higher levels of endogenous SCFA synthesis, which has been linked to better gut health and nutrient absorption. The adjustment of bacterial abundance within the ceca, particularly those involved in SCFA production, contributes to the poultry's overall well-being and may play a role in the observed improved performance. The reduction in the level of the zoonotic pathogen E. coli is a crucial finding, emphasizing the potential role of FSCLP in enhancing food chain integrity. This aspect is of particular interest to both poultry producers and consumers, as it addresses concerns related to food safety.

Author Contributions

SC: Analyzed data, wrote the manuscript, conducted experiments. BW, DW, MP, TA, RM, KH: Conceived and designed the research. MP and MF measured bird performance and conducted sample retrieval. All authors approved the manuscript.

Data Availability Statement: The data supporting the findings of this study are proprietary and cannot be publicly shared. For inquiries regarding access to the data, please contact the corresponding author, Seán Christanseen (schristanseen@alttech.com).

Ethical Statement: The experiment was conducted following protocols sanctioned by the Institutional Animal Care and Use Committee at the University of Kentucky, guaranteeing the welfare and ethical handling of the animals under investigation.

DISCLOSURES

At the time of research, SC, MP, TA RM, and KH were employed by Alltech. Alltech manufactures and markets animal feed supplements. The authors declare no other conflicts of interest.

Appendix Supplementary materials

Image, application 1

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104165.
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