Effects of dietary sodium diformate supplementation on growth performance, nutrient digestibility, and intestinal function of broiler chickens

Abstract

The present study evaluated the effects of dietary sodium diformate (NaDF) supplementation on growth performance, nutrient utilization, intestinal microbiota, immune function, and antioxidant status in broiler chickens. A total of 576 one-day-old male Arbor Acres broilers were randomly allocated to six dietary treatments (0, 1.0, 2.0, 3.0, 4.0, or 5.0 g/kg NaDF) with six replicates each for a 42-day trial. NaDF supplementation quadratically increased the average body weight at 42 days of age and the overall average daily gain of broilers (P < 0.05). The administration of NaDF induced both linear and quadratic decreases in the pH of the ileal contents (P < 0.05). The retention coefficients of crude protein were linearly improved by NaDF treatment (P < 0.05). Supplementation with NaDF modulated gut microbiota by linearly increasing ileal Lactobacillus counts while reducing jejunal and ileal Escherichia coli and Salmonella populations (P < 0.05). Additionally, NaDF supplementation quadratically decreased the concentration of jejunal tumor necrosis factor alpha. Collectively, these findings demonstrate that NaDF enhances broiler growth performance probably by lowering gut pH, promoting nutrient digestion, modulating microbiota, and reducing intestinal inflammation, supporting its potential as a promising alternative to antibiotic growth promoters in poultry production.

Introduction

The long-term use of in-feed antibiotics for growth promotion has fueled public health and environmental concerns, including antimicrobial resistance and gut microbiota disruption (Shao et al., 2021). In response, global regulations increasingly restrict or prohibit in-feed antibiotics, driving the search for sustainable alternatives capable of supporting animal productivity without relying on antimicrobials (Suresh et al., 2018). Among these, acidifiers have emerged as a particularly effective option, as they modulate the gastrointestinal pH to simultaneously suppress pathogenic bacteria while promoting beneficial microbial populations (Dittoe et al., 2018). Beyond their antimicrobial effects, these compounds have the potential to improve intestinal mucosal integrity and nutrient absorption efficiency, thereby establishing them as viable candidates for modern antibiotic-free animal production systems (Tugnoli et al., 2020; Cai et al., 2024).

One acidifier that has received considerable research interest is formic acid (FA). As a well-characterized organic acid, FA has been demonstrated to promote animal growth performance (Luise et al., 2020). FA exerts its antimicrobial effects by reducing gut pH and modulating gut microbiota (Ricke et al., 2020; Abd El-Hack et al., 2024). Extensive investigations have also supported beneficial roles for animal feed supplementation with FA to enhance feed digestion, improve nutrient digestibility, and protect the intestinal barrier function (Jedi Mostafaloo et al., 2021). Nevertheless, despite these benefits, concerns persist regarding certain disadvantages associated with FA, including its strong odor, corrosiveness, and rapid metabolism in the avian foregut (from crop to gizzard), which substantially reduce its growth-promoting efficacy (Lückstädt and Mellor, 2011; Luise et al., 2020; Ricke et al., 2020; Sun et al., 2024). The practical limitations of free FA application in animal nutrition have been overcome by adopting mineral salt derivatives (Chowdhury et al., 2008; Pearlin et al., 2020), such as potassium formate and potassium diformate (KDF). As a hydrogen-bonded complex of FA and potassium formate, KDF exhibits more favorable physicochemical properties than FA, including high water solubility, low volatility, non-corrosiveness, and odor neutrality (Huyghebaert et al., 2011; Chen et al., 2024). Supplementation with KDF triggers antimicrobial activity through pH reduction and gut microbiota modulation, leading to enhanced growth performance, increased nutrient availability, and improved gut function in multiple animal species (Paulicks et al., 2000; Øverland et al., 2009; Mikkelsen et al., 2009; Zhou et al., 2009). These documented benefits have prompted the European Community to approve KDF as the first non-antibiotic growth promoter in animal nutrition under Regulation EC 1334/2001.

Emerging evidence now indicates that another FA salt, sodium diformate (NaDF), may be a more promising gut acidifier than previously tested formate forms (Yusefi et al., 2022; Sun et al., 2024). NaDF is a white crystalline compound consisting of FA and sodium formate linked through hydrogen and covalent bonds. This chemical structure improves its physicochemical characteristics compared to KDF, while also providing additional advantages. For example, NaDF is a better acidifier, as evidenced by its greater H⁺ release per unit mass compared to KDF. NaDF also has a favorable buffering capacity that prevents H⁺ release at pH values below pH 3.5 (Sun et al., 2024). This pH-responsive property means that NaDF remains stable in the acidic gastric environment but dissociates to Na and formate under the neutral to slightly alkaline conditions occurring in the intestine. This pH responsiveness ultimately preserves gastric acid secretion while ensuring sustained antimicrobial efficacy throughout the entire gastrointestinal tract. Importantly, NaDF offers cost advantages in production, since it can replace a portion of the supplemental sodium chloride in the feed. For these reasons, NaDF is viewed as highly promising for improving animal growth and intestinal health (Reyshari et al., 2019; Sun et al., 2024).

Some studies have demonstrated improvements in the growth performance of different fish species following NADF supplementation (Reyshari et al., 2019; Jedi Mostafaloo et al., 2021; Wassef et al., 2021). Similarly, dietary NaDF supplementation linearly enhanced feed efficiency in nursery pigs at inclusion levels of 4.0–12.0 g/kg, with similar improvements observed in the average daily gain (ADG) and average daily feed intake (ADFI) of finishing pigs when supplemented with 2.5–7.5 g/kg NaDF (Gaffield et al., 2024). In broiler chickens, Sun et al. (2024) demonstrated that NaDF supplementation at 1 g/kg increased the final body weight and decreased the feed conversion ratio (FCR). However, critical gaps remain in understanding the optimal dietary inclusion levels of NaDF for broilers and how NaDF exerts its dose-dependent effects on digestive function, intestinal morphology, and immune responses. Based on the existing evidence, we hypothesized that the inclusion of NaDF in broiler diets may have beneficial effects on growth performance, nutrient digestibility, and intestinal health. Therefore, the aim of the present study was to apply NaDF supplementation at graded rates to broiler chickens to determine the effects on growth performance, nutrient digestibility, and intestinal antioxidant and immune function.

Materials and methods

Animals and regents

Broilers were purchased from Jiangsu Jinghai Poultry Industry Group Co., Ltd. (Nantong, Jiangsu, China). NaDF (≥98.0 %) was obtained from Jiangsu Zhongdan Chemical Technology Co., Ltd. (Taizhou, Jiangsu, China). Titanium dioxide (TiO₂) was purchased from Sigma-Aldrich (St. Louis, MO, USA).

Experimental Design, Diets, and Management

The protocols used in the animal experiments were approved by the Nanjing Agricultural University Institutional Animal Care and Use Committee (SYXK [Su] 2021-0086). A total of 576 one-day-old health male Arbor Acres broilers with a similar initial body weight (42.07 ± 0.51 g) were randomly assigned to six dietary treatments consisting of a basal diet supplemented with graded levels of NaDF at 0 (control), 1.0, 2.0, 3.0, 4.0, or 5.0 g/kg. Each treatment group consisted of six replicate cages, with 16 birds housed per cage. The feeding trial, which lasted for 42 d, was divided into a starter phase (age 1 to 21 d) and a grower phase (age 22 to 42 d). The nutrient levels of the basal diets used in both the starter and grower phases were formulated according to NRC (1994) recommendations, and the six experimental diets were prepared by adjusting the inclusion levels of corn, soybean meal, and soybean oil to ensure nutritional equivalence to the basal diet (Supplemental Tables 1 and 2). TiO₂ was added at 2.5 g/kg to the grower diet as an indigestible marker. All birds were raised in stainless-steel cages (280 cm × 90 cm × 45 cm) placed in a temperature-controlled room. Each pen was equipped with individual feeding troughs and nipple drinkers, allowing ad libitum access to feed and water throughout the 42-d trial. The room temperature was maintained at 32–34°C during the first week post-hatch and then gradually reduced by 2–3°C weekly until reaching approximately 22°C. A 23 h light:1 h dark photoperiod was maintained for the entire experimental period. Growth performance parameters, including average body weight (ABW) and cage-level feed consumption, were recorded at 21 and 42 days of age. These measurements were used to calculate each bird’s ADG, ADFI, and FCR.

Sample collection

Nutrient utilization was assessed by collecting excreta daily using collection trays in birds at 39 to 41 d of age (Nguyen et al., 2022). Excreta samples (100 g) were immediately mixed with 10 mL of 10 % sulfuric acid for nitrogen preservation and stored at −20°C (Zheng et al., 2025). The feed and excreta collected during the digestibility trial were dried at 60°C for 48 h and then ground for chemical analyses.

At 42 days of age, two birds with similar physiological status were selected from each replicate cage based on body weight proximity to the cage ABW. After euthanasia via cervical dislocation and decapitation, the birds were immediately eviscerated for tissue collection. Immediately after euthanasia, the contents of the proventriculus, gizzard, duodenum, jejunum, ileum, and cecum were collected for pH measurement according to previous research (Morgan et al., 2019). A digital pH meter (HI 8424; Hanna Instruments, Padova, Italy) equipped with a spear-tip piercing electrode (Sensorex S175CD) was used for the analysis.

Intestinal morphology was evaluated by excising approximately 2 cm long segments of the midjejunum and midileum, rinsing the segments with ice-cold phosphate-buffered saline (PBS; 0.1 M, pH 7.4), and immediately fixing them in 4 % paraformaldehyde. The remaining intestinal portions were longitudinally opened and rinsed with chilled PBS for mucosal layer collection using sterile glass slides (Sato et al., 1997). Pancreatic tissues were also harvested. Approximately 3 g of mid-jejunal and mid-ileal digesta were aseptically collected for the analysis of digestive enzyme activities and microbial populations. All samples were stored at −80°C until subsequent analysis.

Nutrient digestibility

The feed and excreta were analyzed according to the guidelines of the AOAC (2007) for dry matter (DM; method 930.15), crude protein (CP; method 984.13), and ether extracts (EEs; method 991.36; Soxhlet extraction with diethyl ether). Nitrogen was determined using a Kjeltec 8400 automatic nitrogen analyzer (Foss, Hilleroed, Denmark). The titanium dioxide marker was detected using the procedure of Short et al. (1996). The nutrient digestibility was calculated using the following equation:

$$\text{Digestibility}(\%)=1-\left(\left[\text{Ti}{\mathrm{O}}_{2\text{diet}}/\text{Ti}{\mathrm{O}}_{2\text{excreta}}\right]\times \left[\text{componen}{\mathrm{t}}_{\text{excreta}}/\text{componen}{\mathrm{t}}_{\text{diet}}\right]\right)\times 100\%$$

where the contents of TiO₂ and dietary components are given as g/kg.

Determination of digestive enzyme activities

The activities of digestive enzymes (trypsin, amylase, and lipase) were quantified using commercial colorimetric kits (Nanjing Jiancheng Institute of Bioengineering) following the manufacturer’s protocols. Briefly, the homogenate was incubated with enterokinase to catalyze the conversion of trypsinogen to trypsin. The activity of trypsin was then assayed by monitoring the hydrolysis of benzoyl-l-tyrosine ethyl ester, as previously described (Lainé et al., 1993), where one unit represented a 0.003 absorbance increase per min. Amylase activity was assessed using an iodometric assay, with one unit defined as starch hydrolysis of 10 mg per 30 min (Wen et al., 2012). Lipase activity was determined as fatty acid release during olive oil emulsion hydrolysis (Jin, 1995), with one unit corresponding to 1 μmol triglyceride hydrolysis per min at 37°C. Protein concentrations were measured using a commercially available bicinchoninic acid assay kit (Nanjing Jiancheng Institute of Bioengineering) for subsequent activity normalization.

Intestinal Morphology

The assessment of intestinal morphology was carried out in accordance with Tan et al. (2024), using standard histological procedures. Briefly, fixed intestinal samples underwent 24 h dehydration in graded ethanol (70−100 %), xylene clearing, and paraffin embedding. Serial 5 μm cross-sections were prepared and stained with hematoxylin and eosin for morphological evaluation. The villus architecture was quantified by measuring the villus height (VH) and crypt depth (CD) from fifteen intact villi per segment using a Nikon ECLIPSE 80i microscope (Nikon Corporation, Tokyo, Japan) equipped with digital morphometry software.

Intestinal Microbiota

The intestinal microbiota was quantified following the methodology established in our previous study (Li et al., 2018). Bacterial DNA was extracted from jejunal and ileal digesta samples using a Magnetic Stool DNA Extraction Kit (Vazyme, Nanjing, Jiangsu, China), with subsequent DNA concentration and quality assessed using ultraviolet spectrophotometry (NanoDrop ND-2000c, Thermo, USA). DNA integrity was verified using 1.5% agarose gel electrophoresis. The primer sequences used to target Lactobacillus, Escherichia coli, Salmonella, Clostridium perfringens, and total bacteria are presented in Supplemental Table 3. PCR amplification employed Taq DNA polymerase with subsequent product purification using a TIANgel Maxi Purification Kit, followed by cloning into Escherichia coli DH5α via the pMD18-T vector system. The target-containing plasmids were commercially sequenced by Tsingke Biotechnology (Nanjing, Jiangsu, China) to obtain positive clones.

Real-time PCR was conducted using QuantStudio 5 (Thermo Fisher Scientific, Waltham, MA, USA) with ChamQ Universal SYBR qPCR Master Mix (Vazyme) under the following standard cycling conditions: 95°C for 30 s, then 40 cycles of 95°C (5 s)/annealing (30 s), concluding with melting curve analysis. Standard curves generated from 10-fold serial dilutions of positive plasmids yielded the following linear relationships between Ct values and logarithmic gene copy numbers (Log₁₀ 16S rRNA gene copies/g contents):

$$Totalbacteria\text{Ct}=--0.3124\mathrm{X}+13.633,{\mathrm{R}}^2=0.9998$$

$$Lactobacillus\text{Ct}=--0.3071\mathrm{X}+12.892,{\mathrm{R}}^2=0.9995$$

$$Escherichiacoli\text{Ct}=--0.3214\mathrm{X}+14.158,{\mathrm{R}}^2=0.9991$$

$$Salmonella\text{Ct}=--0.3073\mathrm{X}+13.037,{\mathrm{R}}^2=0.9991$$

$$Clostridiumperfringens\text{Ct}=--0.2918\mathrm{X}+12.732,{\mathrm{R}}^2=0.9990$$

Intestinal antioxidant capacity

The antioxidant status was evaluated using commercial kits (Nanjing Jiancheng Institute of Bioengineering) for measuring superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities, along with reduced glutathione (GSH) and malondialdehyde (MDA) concentrations. SOD activity was determined as xanthine oxidase-mediated nitrite inhibition at 550 nm, with one unit defined as a 50 % inhibition rate per mL reaction solution. GSH-Px activity was assessed using the 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) reaction and monitoring at 412 nm, with enzymatic units representing 1 μmol GSH depletion per min. CAT activity was quantified based on hydrogen peroxide decomposition, with one unit equal to 1 mmol H₂O₂ processed per min. GSH content was spectrophotometrically analyzed at 412 nm using DTNB, while MDA levels were determined based on the formation of MDA-thiobarbituric acid adducts. All enzyme activities and metabolite concentrations were normalized to total protein content and expressed as units per mg protein.

Intestinal immune response

The levels of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interleukin 10 (IL-10) in intestinal mucosal homogenates were quantified using chicken-specific enzyme linked immunosorbent assay kits (CUSABIO, Wuhan, Hubei, China) with detection ranges of 0.27–200 pg/mL, 15.6–1000 pg/mL, and 1–200 pg/mL respectively. The sensitivity were 0.27 pg/mL for TNF-α, 3.9 pg/mL for IL-6, and 0.5 pg/mL for IL-10, with both inter- and intra-assay coefficients of variance maintained under 10 % for all analytes. All measurements were conducted in strict accordance with manufacturer protocols and normalized to total protein concentration for cross-sample comparison.

Statistical analysis

Statistical analyses were conducted using SPSS 26.0 software (SPSS Inc., Chicago, IL, USA) with one-way ANOVA and Duncan's post-hoc testing, where statistical significance was defined as P < 0.05. Growth performance and nutrient digestibility were statistically analyzed on a per-replicate basis, whereas the other parameters were calculated as the mean values obtained from two selected broilers per replicate. When the P-value of one-way ANOVA was less than 0.05, orthogonal polynomial contrasts were used to evaluate linear and quadratic responses to NaDF supplementation levels, with results presented as means with standard errors of the means.

Results

Growth performance

Supplementation of broiler feed with NaDF resulted in quadratic increases in ABW at 42 days of age (P = 0.005; Table 1) and overall ADG (P = 0.005). Supplementation levels ranging from 1.0 to 2.0 g/kg resulted in increases in ABW at 42 days of age (P = 0.016) and ADG throughout the entire experimental period (P = 0.015). NaDF supplementation had no effect on ADG, ADFI, or FCR during the starter or the grower phase (P > 0.05).

Table 1.

Effects of dietary NaDF supplementation on the growth performance of broiler chickens from 1 to 42 d of age.

Items	NaDF levels (g/kg)						SEM	P-values
	0	1.0	2.0	3.0	4.0	5.0		ANOVA	Linear	Quadratic
ABW
1 d	42.10	42.01	42.05	42.13	42.05	42.06	0.09	1.000	0.986	0.994
21 d	802.05	829.28	843.72	819.17	816.85	818.13	6.01	0.506	0.883	0.158
42 d	2565.65c	2728.55ab	2758.97a	2663.55abc	2608.38bc	2614.79bc	19.28	0.016	0.543	0.005
1 to 21 d
ADG (g/d)	36.19	37.49	38.17	37.00	36.90	36.96	0.29	0.501	0.882	0.157
ADFI (g/d)	52.15	53.74	54.23	53.22	54.22	53.42	0.47	0.839	0.509	0.381
FCR (g/g)	1.45	1.43	1.42	1.44	1.47	1.44	0.01	0.927	0.641	0.786
22 to 42 d
ADG (g/d)	83.98	90.44	91.20	87.83	85.31	85.55	0.87	0.074	0.511	0.020
ADFI (g/d)	151.36	155.66	157.66	152.55	147.94	146.55	1.45	0.197	0.078	0.090
FCR (g/g)	1.80	1.72	1.73	1.74	1.74	1.72	0.01	0.318	0.123	0.331
1 to 42 d
ADG (g/d)	60.09c	63.97ab	64.69a	62.42abc	61.10bc	61.26bc	0.46	0.015	0.542	0.005
ADFI (g/d)	101.75	104.70	105.94	102.89	101.08	99.99	0.79	0.241	0.159	0.070
FCR (g/g)	1.69	1.64	1.64	1.65	1.66	1.63	0.01	0.426	0.195	0.449

^a-cMeans within a row with different superscripts are different at P < 0.05.

Abbreviations: NaDF, sodium diformate; ABW, average body weight; ADFI, average daily feed intake; ADG, average daily gain; FCR, feed conversion ratio; SEM, standard error of the mean; ANOVA, one-way analysis of variance.

Gastrointestinal content pH

Supplementation with NaDF caused linear (P = 0.039; Table 2) and quadratic (P = 0.030) reductions in the pH of ileal digesta in 42-day-old broilers. The pH of ileal digesta was significantly lower in the NaDF-treated groups than in the untreated controls at NaDF inclusion levels ranging from 1.0 to 5.0 g/kg (P = 0.035). However, NaDF supplementation had no effect on the pH of digesta in the proventriculus, gizzard, duodenum, jejunum, or cecum (P > 0.05).

Table 2.

Effects of dietary NaDF supplementation on the pH of digesta in proventriculus, gizzard, duodenum, jejunum, ileum, and cecum of broilers.

Items	NaDF levels (g/kg)						SEM	P-values
	0	1.0	2.0	3.0	4.0	5.0		ANOVA	Linear	Quadratic
Proventriculus	5.60	5.37	5.31	5.32	5.21	5.38	0.06	0.671	0.262	0.233
Gizzard	3.32	3.23	3.34	3.30	3.26	3.08	0.11	0.988	0.632	0.680
Duodenum	6.14	5.79	5.78	5.72	5.80	5.75	0.05	0.196	0.064	0.111
Jejunum	6.62	6.32	6.16	6.21	6.10	6.18	0.06	0.092	0.014	0.094
Ileum	7.27a	6.67b	6.72b	6.70b	6.76b	6.73b	0.06	0.035	0.039	0.030
Cecum	6.56	6.19	6.16	6.20	6.33	6.27	0.07	0.677	0.546	0.210

^a-bMeans within a row with different superscripts are different at P < 0.05.

Abbreviations: NaDF, sodium diformate; SEM, standard error of the mean; ANOVA, one-way analysis of variance.

Nutrient digestibility

NaDF supplementation linearly (P = 0.002; Table 3) and quadratically (P = 0.037) increased the apparent total tract digestibility of CP in broilers aged from 39 to 41 d. Relative to the control group, birds fed the NaDF-supplemented diets (1.0–5.0 g/kg) showed a higher CP retention coefficient (P = 0.006).

Table 3.

Effects of dietary NaDF supplementation on nutrient digestibility in broilers from 39 to 41 d of age.

Items	NaDF levels (g/kg)						SEM	P-values
	0	1.0	2.0	3.0	4.0	5.0		ANOVA	Linear	Quadratic
DM (%)	64.86	69.88	70.37	69.33	69.67	70.17	0.62	0.078	0.042	0.080
CP (%)	57.38b	63.98a	64.50a	64.05a	64.75a	65.36a	0.72	0.006	0.002	0.037
EE (%)	77.04	79.25	80.97	80.79	80.46	81.94	0.91	0.725	0.155	0.576

^a-bMeans within a row with different superscripts are different at P < 0.05.

Abbreviations: NaDF, sodium diformate; CP, crude protein; DM, dry matter; EE, ether extract; SEM, standard error of the mean; ANOVA, one-way analysis of variance.

Digestive enzyme activities

All groups showed similar activities of trypsin, amylase, and lipase in both the pancreas and the jejunal contents (p > 0.05; Table 4).

Table 4.

Effects of dietary NaDF supplementation on the activities of digestive enzymes in the pancreas and jejunual contents of broilers.

Items	NaDF levels (g/kg)						SEM	P-values
	0	1.0	2.0	3.0	4.0	5.0		ANOVA	Linear	Quadratic
Pancreas
Trypsin (U/mg protein)	600.95	651.59	701.89	592.33	728.76	611.10	23.70	0.464	0.726	0.359
Amylase (U/mg protein)	325.42	425.80	484.54	447.43	333.97	377.50	22.00	0.215	0.905	0.050
Lipase (U/g protein)	564.30	560.41	604.37	610.92	708.42	645.93	30.68	0.770	0.198	0.913
Jejunal contents
Trypsin (U/mg protein)	4466.35	6983.43	7224.18	6774.43	7123.61	6271.33	309.81	0.081	0.136	0.016
Amylase (U/mg protein)	19.35	26.01	29.09	29.95	28.10	29.25	1.22	0.103	0.021	0.074
Lipase (U/g protein)	191.22	311.42	307.74	291.39	301.51	289.86	15.18	0.184	0.144	0.074

^a-bMeans within a row with different superscripts are different at P < 0.05.

Abbreviations: NaDF, sodium diformate; SEM, standard error of the mean; ANOVA, one-way analysis of variance.

Intestinal morphology

Supplementation with NaDF had no effect on VH, CD, or VH/CD in the small intestines (P > 0.05; Table 5).

Table 5.

Effects of dietary NaDF supplementation on the intestinal morphology of broilers.

Items	NaDF levels (g/kg)						SEM	P-values
	0	1.0	2.0	3.0	4.0	5.0		ANOVA	Linear	Quadratic
Jejunum
VH (μm)	770.62	928.38	939.27	934.28	910.54	891.84	21.62	0.200	0.209	0.037
CD (μm)	138.67	135.65	147.23	147.82	148.08	146.49	2.58	0.628	0.164	0.525
VH/CD (μm/μm)	5.84	7.10	6.42	6.54	6.34	6.39	0.22	0.744	0.897	0.418
Ileum
VH (μm)	706.21	810.79	820.00	849.34	793.42	800.55	19.16	0.393	0.259	0.091
CD (μm)	178.96	164.34	169.42	172.68	176.69	169.47	3.21	0.829	0.918	0.667
VH/CD (μm/μm)	4.06	5.07	5.03	5.02	4.73	4.87	0.16	0.433	0.357	0.136

Abbreviations: NaDF, sodium diformate; VH, villus height; CD, crypt depth; SEM, standard error of the mean; ANOVA, one-way analysis of variance.

Intestinal microbiota

Supplementation with NaDF caused a linear increase in the numbers of Lactobacillus bacteria in the ileal (P = 0.002) contents (Table 6). Conversely, NaDF supplementation resulted in a linear inhibition of the Escherichia coli (P = 0.003) and Salmonella (P = 0.004) populations in the jejunum and of the total bacteria (P < 0.001), Escherichia coli (P = 0.002), and Salmonella (P = 0.003) populations in the ileum. Specifically, the ileal populations of Lactobacillus were increased by NaDF supplementation at dosages from 2.0 to 5.0 g/kg (P = 0.022), while the ileal populations of total bacteria (P = 0.014) and Escherichia coli (P = 0.035) were reduced compared to the untreated controls. The ileal Salmonella population (P = 0.040) was reduced by supplementation with NaDF at 3.0 to 5.0 g/kg. NaDF supplementation had no significant effect on the populations of Clostridium perfringens in any groups (P > 0.05).

Table 6.

Effects of dietary NaDF supplementation on the bacterial populations in the jejunal and ileal contents of broilers.

Items	NaDF levels (g/kg)						SEM	P-values
	0	1.0	2.0	3.0	4.0	5.0		ANOVA	Linear	Quadratic
Jejunal content
Total bacteria (log10 copies/g)	8.15	7.86	7.84	7.84	7.86	7.86	0.04	0.236	0.095	0.099
Lactobacillus (log10 copies/g)	6.42	6.79	6.79	6.82	6.81	6.83	0.05	0.082	0.026	0.073
Escherichia coli (log10 copies/g)	6.15a	5.84ab	5.75b	5.67b	5.65b	5.67b	0.05	0.030	0.003	0.067
Salmonella (log10 copies/g)	3.61a	2.96b	3.12b	3.04b	2.97b	2.78b	0.07	0.023	0.004	0.347
Clostridium perfringens (log10 copies/g)	2.31	2.40	2.38	2.39	2.29	2.68	0.09	0.845	0.415	0.566
Ileal content
Total bacteria (log10 copies/g)	8.61a	8.39ab	8.20b	8.09b	8.09b	8.01b	0.06	0.014	<0.001	0.202
Lactobacillus (log10 copies/g)	6.50b	6.75ab	6.96a	7.10a	7.11a	7.04a	0.06	0.022	0.002	0.062
Escherichia coli (log10 copies/g)	6.32a	6.15ab	5.99b	5.97b	5.88b	5.93b	0.04	0.035	0.002	0.149
Salmonella (log10 copies/g)	3.79a	3.73ab	2.80abc	2.70bc	2.58c	2.69bc	0.15	0.040	0.003	0.199
Clostridium perfringens (log10 copies/g)	2.80	2.72	2.45	2.19	2.28	2.18	0.09	0.171	0.012	0.486

^a-cMeans within a row with different superscripts are different at P < 0.05.

Abbreviations: NaDF, sodium diformate; SEM, standard error of the mean; ANOVA, one-way analysis of variance.

Intestinal antioxidant capacity

Measurements of intestinal antioxidant indicators, including the activities of SOD, GSH-Px, and CAT and the contents of GSH and MDA, were not affected by NaDF treatment among the groups (P > 0.05; Table 7).

Table 7.

Effects of dietary NaDF supplementation on the intestinal antioxidant capacity of broilers.

Items	NaDF levels (g/kg)						SEM	P-values
	0	1.0	2.0	3.0	4.0	5.0		ANOVA	Linear	Quadratic
Jejunum
SOD (U/mg protein)	77.02	98.19	88.57	95.44	89.84	94.44	4.82	0.864	0.511	0.561
GSH-Px (U/mg protein)	188.01	236.92	207.05	237.84	228.85	222.46	8.80	0.569	0.337	0.344
CAT (U/mg protein)	23.91	25.92	26.62	27.36	27.85	25.09	0.55	0.316	0.269	0.050
GSH (mg/g protein)	3.77	5.69	5.99	5.60	5.67	5.12	0.24	0.075	0.169	0.011
MDA (nmol/mg protein)	2.76	1.79	1.65	2.03	1.98	2.05	0.12	0.117	0.275	0.034
Ileum
SOD (U/mg protein)	58.93	75.82	74.76	66.48	65.79	67.70	1.94	0.111	0.884	0.082
GSH-Px (U/mg protein)	85.60	117.16	121.47	124.18	106.92	110.00	5.33	0.339	0.390	0.062
CAT (U/mg protein)	19.04	19.85	20.72	22.20	21.19	20.90	0.58	0.728	0.235	0.338
GSH (mg/g protein)	5.39	5.71	5.34	4.48	4.64	5.78	0.25	0.594	0.694	0.289
MDA (nmol/mg protein)	2.21	1.43	1.35	1.33	1.54	1.61	0.10	0.095	0.168	0.015

^a-bMeans within a row with different superscripts are different at P < 0.05.

Abbreviations: NaDF, sodium diformate; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; CAT, catalase; GSH, reduced glutathione; MDA, malondialdehyde; SEM, standard error of the mean; ANOVA, one-way analysis of variance.

Intestinal immune response

Supplementation with NaDF resulted in a quadratic decrease in the jejunal TNF-α content (P = 0.011; Table 8). NaDF supplementation at levels ranging from 1.0 to 5.0 g/kg decreased the jejunal TNF-α concentration compared to the untreated controls (P = 0.016). NaDF supplementation did not alter the IL-6 or IL-10 contents in the small intestine (P > 0.05).

Table 8.

Effects of dietary NaDF supplementation on the intestinal immune response of broilers.

Items	NaDF levels (g/kg)						SEM	P-values
	0	1.0	2.0	3.0	4.0	5.0		ANOVA	Linear	Quadratic
Jejunum
TNF-α (pg/mg protein)	8.02a	3.71b	2.00b	4.27b	3.89b	3.91b	0.51	0.016	0.061	0.011
IL-6 (pg/mg protein)	6.03	5.58	5.11	5.38	6.08	5.19	0.43	0.983	0.797	0.809
IL-10 (pg/mg protein)	7.38	8.22	8.34	9.01	7.81	8.31	0.23	0.471	0.391	0.189
Ileum
TNF-α (pg/mg protein)	5.76	2.27	2.01	3.17	3.81	4.67	0.45	0.113	0.969	0.011
IL-6 (pg/mg protein)	2.82	2.89	2.84	2.54	2.68	2.78	0.11	0.953	0.632	0.731
IL-10 (pg/mg protein)	5.52	6.63	7.14	5.55	5.68	5.10	0.26	0.174	0.204	0.080

^a-bMeans within a row with different superscripts are different at P < 0.05.

Abbreviations: NaDF, sodium diformate; TNF-α, tumor necrosis factor alpha; IL-6, interleukin 6; IL-10, interleukin 10; SEM, standard error of the mean; ANOVA, one-way analysis of variance.

Discussion

The global phase-out of antibiotic growth promoters in livestock production has created a pressing need for sustainable alternatives that can enhance growth performance and disease resistance in animals. This study evaluated the potential of NaDF, a novel organic acid feed additive, to promote growth performance and improve the intestinal health of broiler chickens. Our results demonstrated that NaDF supplementation efficiently improved final ABW and overall ADG in broilers. Consistent with our findings, Sun et al. (2024) observed that NaDF supplementation at 1.0 g/kg significantly improved the ABW of broilers at 38 days. Several studies on KDF have supported the growth-promoting effects of formate-based acidifiers in broilers, with optimal dosages ranging from 4.0 to 6.0 g/kg providing significant improvements in ADG across different growth stages (Selle et al., 2004; Ragaa et al., 2016; Chen et al., 2024). The rapid growth of broilers demands high nutrient utilization, which may not be fully supported by the inherent levels of gastric acid secretion and digestive enzyme activity (Gao et al., 2021). NaDF supplementation appears to compensate for the physiological limitations that arise during this growth phase by enhancing gut acidity, thereby improving nutrient digestion and absorption.

The available research on poultry indicates that organic acids are predominantly absorbed and undergo metabolic processing in the proximal gastrointestinal regions, with only weak effects observed in the distal intestinal segments (Oakley et al., 2014; Sun et al., 2022). However, in the present study, NaDF supplementation effectively reduced the pH values in the ileal contents. The explanation for this effect most likely lies in differences in NaDF dissociation compared to conventional FA, as NaDF dissociates into FA and sodium ions in the gastric environment, thereby overcoming the inherent limitation that free FA hardly reaches the lower gastrointestinal tract. Importantly, NaDF possesses self-buffering properties that suppress acid release below pH 3.5, thereby preserving gastric acid secretion capacity. These properties may account for the negligible impact of NaDF observed in the highly acidic foregut pH and the significant pH-lowering effects in the ileal digesta. In agreement with our findings, Sun et al. (2024) reported that dietary supplementation of broilers with 1.0 g/kg NaDF effectively reduced the digesta pH in the duodenum and ileum, but had no obvious effects on proventricular or gizzard pH. This targeted pH modulation by NaDF in specific intestinal segments may contribute to improved gut microbiota composition and enhanced nutrient digestibility.

In the current study, NaDF supplementation of the basic broiler diet significantly improved the apparent total tract digestibility of CP. Beyond the documented reduction in pH, this improvement in CP retention coefficient after NaDF administration may also be associated with the numerically elevated jejunal trypsin activity, even though the increase was not statistically significant (P = 0.081). These observations align with studies on young beluga sturgeon, which responded to NaDF supplementation from 0.5 to 1.5 g/kg with significantly increased intestinal trypsin activity (Jedi Mostafaloo et al., 2021), as well as parallel findings in broilers by Chen et al. (2024), who demonstrated that supplementation with 4.0 g/kg KDF improved the apparent metabolizability of DM, CP, and gross energy. Overall, organic acid supplementation has emerged as an effective nutritional strategy to enhance nutrient utilization in monogastric animals, with particular benefits observed in young animals with immature digestive systems (Li et al., 2023; Ficagna et al., 2025). This enhanced efficacy stems from the ability of organic acids to compensate for physiological limitations in young animals, including insufficient gastric acid secretion and reduced digestive enzyme activity, while counteracting the alkalizing effects of conventional diets (Lingbeek et al., 2021).

Formate-based acidifiers also demonstrate significant efficacy in maintaining intestinal homeostasis through selective modulation of gut microbial ecology (Canibe et al., 2001; Mikkelsen et al., 2009; Sun et al., 2024). This is effected via a bifunctional mechanism that simultaneously inhibits pathogenic microorganisms (particularly Escherichia coli, Salmonella, and Clostridium spp.) while enhancing the proliferation of beneficial commensals, including Lactobacillus and Bifidobacterium species (Ragaa et al., 2016; Sun et al., 2022; Li et al., 2024). Our experimental results also revealed significant decreases in the pH-sensitive enteropathogenic populations (i.e., Escherichia coli and Salmonella) in the broiler jejunum and ileum in response to NaDF supplementation. This antimicrobial efficacy holds particular significance for poultry production systems, which are highly susceptible to incurring substantial economic losses due to enteropathogenic infections.

The mode of action of acidifiers involves both extracellular and intracellular mechanisms, as the reduced gastrointestinal pH creates an inhospitable environment for acid-sensitive microorganisms, while the lipophilic properties of the undissociated acid molecules enable them to permeate bacterial cell membranes (Pearlin et al., 2020). Their subsequent dissociation in the neutral cytoplasmic environment causes an intracellular proton accumulation that disrupts essential metabolic processes and ultimately inhibits the growth of pH-sensitive pathogens, such as Escherichia coli and Salmonella (Hassan et al., 2010). In the present study, Lactobacillus sp. demonstrated a linear increase in numbers in the ileum of the NaDF-treated broilers. Lactobacillus sp. are acidogenic microbes, and because they can inherently tolerate low-pH environments, they readily proliferate under conditions inhibitory to pH-sensitive pathogens. The NaDF-mediated reduction in pathogenic bacterial loads may also increase the ecological niche availability for acid-resistant symbionts like Lactobacilli, thereby preferentially enriching Lactobacillus populations.

Dietary acidification has now emerged as an effective nutritional strategy for immunomodulation, with formate-based acidifiers demonstrating favorable efficacy in regulating immune responses (Ragaa et al., 2016; Sun et al., 2024). In the present study, the observed reduction in jejunal TNF-α concentration in the NaDF-supplemented broilers further supports the anti-inflammatory potential of formate-based acidifiers. The avian immune system is known to serve as a fundamental regulator of poultry health and disease resistance (Gadde et al., 2017; Yang et al., 2018), and recent investigations have revealed that NaDF supplementation in broilers modulates intestinal immunity through the selective enrichment of immunostimulatory microbiota, including Jeotgalicoccus and Tetragenococcus species (Sun et al., 2024). These microbial shifts correlate with improved immune competence, as Jeotgalicoccus abundance demonstrates positive associations with a broad-spectrum immune response in broilers, encompassing anti-inflammatory signaling (interleukin 10), humoral immunity (immunoglobulin A and immunoglobulin G), and innate defense (lysozyme) (Song et al., 2022), while Tetragenococcus contributes to immune health through multiple mechanisms, including bacteriocin production and immunoregulatory effects via dendritic cell modulation (Islam et al., 2022; Kotake et al., 2022).

Conclusion

This study demonstrates that NaDF supplementation facilitates broiler productivity, probably through a combination of improved nutrient digestion, reduced intestinal pH, and attenuated intestinal inflammation. Notably, NaDF exhibits selective antimicrobial activity against enteropathogens while fostering the proliferation of beneficial microbiota. Collectively, our findings raise the possibility that NaDF may represent a promising alternative to antibiotic growth promoters in poultry production.

CRediT authorship contribution statement

Zhaoxing Li: Writing – original draft, Investigation. Huan Xu: Writing – original draft, Investigation, Data curation. Zichao Tan: Investigation. Hongyan Wang: Investigation, Data curation. Zongfu Li: Resources, Methodology, Investigation. Yang Luo: Resources, Methodology, Investigation. Yue Li: Data curation. Yanan Chen: Writing – review & editing, Methodology, Data curation. Tian Wang: Writing – review & editing, Methodology. Hao Zhang: Writing – review & editing, Supervision, Investigation, Funding acquisition, Data curation, Conceptualization.

Disclosures

The authors declare no conflict of interest.