Effects of Sodium Diformate on Immune Organs, Inflammatory Cytokines, Intestinal Morphology, and Cecal Microbiota of Hy-Line Brown Laying Hens

2.2 Feeding Management

The present study was conducted at the Experimental Farm of Shenyang Agricultural University. The laying hens were housed in three-tier stepped battery cages. The lighting schedule provided 16h of light and 8h of dark throughout the experimental period. All birds had ad libitum access to feed and water. The facility was heated by a water-circulating heating system and ventilated by axial-flow fans. The room temperature was maintained between 24 ℃ and 26 ℃. The feeding management, immunization protocols, and hygiene management of the experimental birds were carried out in accordance with the Hy-Line Brown laying hen management guidelines.

3.1 Immune Organ Indices

On the final day of the experimental period, two hens from each replicate were randomly selected and euthanized. The spleens were collected immediately after euthanasia, rinsed with physiological saline, blotted dry with absorbent paper, and weighed. The immune organ index was calculated as the percentage of spleen weight relative to live body weight (%).

3.2 Intestinal Mucosal Histomorphology

On the final day of the experimental period, two hens from each replicate were randomly selected and euthanized. Tissue samples were collected from the duodenum, jejunum, and ileum, and immediately fixed in 4% paraformaldehyde. The fixed intestinal samples underwent trimming, dehydration, embedding, sectioning, staining, and sealing using standard histological procedures. Target areas of the tissues were selected for photomicrography and imaging using CaseViewer image scanning software (CaseViewer 2.2, 3DHISTECH Ltd., Budapest, Hungary). Villus height (VH) and crypt depth (CD) were measured using Image Pro Plus 6.0 image analysis software (Image-Pro Plus 6.0, Media Cybernetics, Inc., Rockville, Maryland, USA), and the ratio of villus height to crypt depth (VH / CD) was subsequently calculated.

3.3 Intestinal Inflammatory Cytokines

On the final day of the experimental period, two hens from each replicate were randomly selected and euthanized. Total RNA was extracted from intestinal tissue samples using the TRIzol method according to the manufacturer's protocol. The RNA concentration and purity were assessed by measuring the OD260/280 ratio using a NanoDrop-2000 spectrophotometer (ThermoFisher Scientific Co., Waltham, MA, USA), and RNA integrity was verified using agarose gel electrophoresis. The extracted RNA was then reverse-transcribed into complementary DNA (cDNA) using a First-Strand cDNA Synthesis Kit (Tiangen Biotech Co., Ltd., Beijing, China) following the manufacturer's recommended protocol. Primers were designed using Primer-Blast (NCBI), and their specificity was verified. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). The cDNA samples were amplified by real-time quantitative polymerase chain reaction (qPCR) using the SuperReal PreMix (Probe) kit (Tiangen Biotech Co., Ltd., Beijing, China). The qPCR cycling conditions were as follows: 95 ℃ for 5 min, followed by 40 cycles of 95 ℃ for 10s, 60 ℃ for 30s, and a final extension step at 72 ℃ for 5 min. The specificity of the qPCR products was evaluated by melting curve analysis. β-Actin was used as the internal reference gene, and the relative expression levels of target genes were calculated using the 2^-ΔΔCt method. All kits were obtained from (Tiangen Biotech Co., Ltd., Beijing, China).

3.4 Cecal Microbiota

On the final day of the experimental period, two hens from each replicate were randomly selected and euthanized. Cecal contents were collected immediately after euthanasia, snap-frozen in liquid nitrogen, and stored for subsequent metagenomic analysis. All experimental procedures were performed according to the standard protocols provided by Illumina, including sample quality assessment, library preparation, library quality control, and sequencing. After confirming the quality of the genomic DNA extracted from the samples, the DNA was fragmented. The fragmented DNA then underwent end repair, A-tailing, adapter ligation, purification and size selection of ligation products, library amplification, and product purification to construct the sequencing library. Following library quality validation, sequencing was performed on an Illumina sequencing platform. The raw reads obtained from sequencing were subjected to quality control and filtered to obtain clean reads for subsequent bioinformatics analysis. The clean reads were assembled, coding genes were predicted, and a non-redundant gene set was constructed. Functional annotation and taxonomic analysis of the non-redundant gene set were performed using universal and specialized databases. The species composition and abundance information of the samples were statistically analyzed. All bioinformatics analyses were conducted using the BMKCloud platform (www.biocloud.net).

3.5 Statistical Analysis

Experimental data were collated and preliminarily calculated using Excel 2019, followed by one-way analysis of variance (ANOVA) using SPSS 26.0 statistical software (SPSS Inc., Chicago, IL, USA). Duncan's multiple range test was used for post-hoc comparisons among treatment groups. The results are presented as means with standard error of the mean (SEM). Statistical significance was declared at P < 0.05, and P < 0.01 was considered extremely significant.

4.Results and Analysis

4.1 Immune Organ Indices

As presented in Table 2, dietary supplementation with different levels of NaDF had no significant effect on the spleen index of laying hens (P = 0.350). A linear decreasing trend (P = 0.048) and a quadratic change (P = 0.092) were also observed. The highest spleen index was observed in the 2 g/kg NaDF supplementation group.

NOTE: NaDF: sodium diformate; ANOVA: analysis of variance; SEM: standard error of the mean.

4.2 Intestinal Mucosal Histomorphology

As presented in Table 3, compared with the control group, dietary supplementation with 1 g/kg to 4 g/kg NaDF significantly increased duodenal villus height (P < 0.05). The ratio of villus height to crypt depth was not significantly affected (P > 0.05), with a linear increasing trend (P = 0.040), and the highest value was observed at 4 g/kg. No significant differences were detected in jejunal parameters among all treatment groups (P > 0.05). The ileal VH / CD ratio in the 2 g/kg to 5 g/kg NaDF groups was significantly higher than that in the control group (P = 0.042), with linear and quadratic increases (P = 0.028, P = 0.004), and the highest ratio was observed in the 4 g/kg group. Ileal villus height also exhibited linear and quadratic increases (P = 0.004).

NOTE: a,b,c Within a row, means with different superscripts differ significantly (P < 0.05). VH: villus height; CD: crypt depth; VH / CD: ratio of villus height to crypt depth; SEM: standard error of the mean. Values are presented as means. Means in the same row without a common superscript letter differ significantly (P < 0.05). The same abbreviations and notations apply to subsequent tables unless otherwise stated.

4.3 Intestinal Inflammatory Cytokines

As presented in Table 4, compared with the control group, dietary supplementation with different levels of NaDF had no significant effect on the relative expression levels of inflammatory cytokines in the duodenum, jejunum, or ileum of laying hens (P > 0.05). No significant linear or quadratic regression relationships were observed between the expression levels of inflammatory cytokines in the duodenum, jejunum, or ileum and the increasing NaDF supplementation levels (P > 0.05).

NOTE: IL: Interleukin; TNF: Tumor Necrosis Factor.

4.4 Cecal Microbiota

4.4.1 Alpha Diversity Analysis

As shown in Figure. 1, compared with the control group, dietary supplementation with different levels of NaDF had no significant effect on the Abundance-based Coverage Estimator (ACE) index index, Chao1 index, Shannon index, or observed species (Sobs) (P > 0.05).

4.4.2 Beta Diversity Analysis

As shown in the Principal Coordinates Analysis (PCoA)and Non-metric Multidimensional Scaling (NMDS).analyses Figure. 2, the cumulative interpretation rate of PCoA was 43.62%, and the NMDS stress value (stress = 0.081) indicated that the ordination plot was reliable. No significant clustering was observed among the groups (P > 0.05), indicating that NaDF did not induce a major shift in overall cecal microbial community structure.

Figure. 2

4.4.3 Effects of Dietary NaDF Supplementation on Cecal Microbial Community Composition of Laying Hens

As shown in Figure. 3, at the genus level, the numbers of unique Operational Taxonomic Units (OTUs) in the cecal digesta microbiota of the control group and the 1 g/kg, 2 g/kg, 3 g/kg, 4 g/kg, and 5 g/kg NaDF groups were 19, 29, 23, 21, 20, and 34, respectively, with a total of 2,453 shared OTUs among all groups. At the species level, the numbers of unique OTUs in the cecal digesta microbiota were 121, 180, 133, 126, 112, and 241, respectively, with a total of 8,018 shared OTUs among all groups.

Figure. 3

4.4.4 Community Difference Analysis

As shown in Figure. 4, Figure. 5, and Figure. 6, at the phylum level, dietary NaDF supplementation had no significant effect on the cecal microbiota community composition of laying hens.

Figure. 4

Figure. 5

Figure. 6

At the genus level, compared with the control group, no significant differences were observed in the abundance of dominant genera such as Bacteroides and Phocaeicola among all groups (P > 0.05). Supplementation with 4 g/kg NaDF significantly reduced the abundance of Parabacteroides (P < 0.05). Supplementation with 3 g/kg NaDF increased the abundance of Alistipes and Prevotella, although the differences did not reach statistical significance (P = 0.083 and P = 0.055, respectively).

At the species level, compared with the control group, no significant differences were observed in the abundance of dominant species such as Bacteroides sp., Phocaeicola barnesiae, and Phocaeicola salanitronis among all groups (P > 0.05). Supplementation with 4 g/kg NaDF significantly reduced the abundance of Mordavella massiliensis (P = 0.027).

5.Discussion

5.1 Effects of NaDF on Immune Organ Indices of Laying Hens

Immune organ indices in livestock and poultry reflect feeding efficiency and immune status, with higher indices generally indicating more developed immune organs and stronger immune function. Previous studies have demonstrated that formate-based acidifiers exert species-specific and dose-dependent effects on the immune organs of poultry^[9]. Chen et al^[10]., reported that dietary supplementation with 0.60% potassium diformate significantly increased the thymus index of Ross 308 broilers. Similarly, Zou et al^[11]., found that supplementation with 2 g/kg potassium diformate enhanced the spleen index of Cobb broilers. In contrast, Chen et al^[12]., observed that dietary supplementation with 40 g/kg or 40 g/kg potassium diformate had no significant effect on immune organ indices in broilers.

In the present study, dietary supplementation with 0 g/kg to 5 g/kg NaDF had no significant effect on the spleen index of Hy-Line Brown laying hens, but a dose-dependent bidirectional trend (initially increasing and then decreasing) was observed. This finding is partially consistent with the results of Chen et al., but differs from the significant promoting effects reported by Chen et al., and Zou et al. The discrepancies among these studies may be attributed to differences in supplemental dosage, animal breed, and physiological stage. The nutrient allocation priority of laying hens favors egg production, with relatively lower priority assigned to immune organ development. Furthermore, after sexual maturity, organs such as the bursa of Fabricius undergo physiological regression, resulting in reduced responsiveness to exogenous stimuli^[13]. In the present study, although the spleen index did not reach statistical significance, the observed dose-dependent bidirectional trend suggests that the immunomodulatory effect of NaDF on laying hens may be relatively moderate. The optimal supplemental level and the underlying mechanisms of action require further investigation.

5.2 Effects of NaDF on Intestinal Mucosal Histomorphology

Intestinal morphology is a critical indicator for evaluating intestinal health in livestock and poultry, with VH, CD, and the ratio of VH / CD reflecting the digestive and absorptive capacity of the intestine^[14] (Liu et al., 2024). Numerous studies have demonstrated that formate-based acidifiers can improve intestinal morphology, although the effects vary by intestinal segment and acidifier type^[10]. For example, Chen et al^[10]., reported that 4 g/kg −8 g/kg potassium diformate (KDF) dose-dependently improved duodenal, jejunal, and ileal morphology in broilers. Sun et al^[8]., found that 1.0 g/kg NaDF significantly increased the duodenal VH / CD ratio in broilers. Similarly, Christian et al^[15]., observed that dietary supplementation with 0.30% NaDF significantly increased villus height in the jejunum and ileum of broilers. However, Li et al^[7]., reported that 1 g/kg to 5 g/kg NaDF had no significant effect on intestinal morphology in broilers, suggesting that the effects of formate-based acidifiers may be influenced by factors such as dosage and animal breed.

In the present study, dietary supplementation with 1 g/kg to 4 g/kg NaDF significantly increased duodenal villus height, and the VH / CD ratio showed a linear increasing trend with increasing NaDF levels. Supplementation with 2 g/kg to 5 g/kg NaDF significantly increased both ileal VH / CD ratio and villus height, exhibiting linear and quadratic increases, with the optimal effect observed at 4 g/kg NaDF. However, no significant differences were observed in any jejunal parameters among the treatment groups. These results are partially consistent with previous reports. Compared with KDF, NaDF did not significantly improve jejunal morphology, suggesting that different types of acidifiers exert segment-specific effects. Furthermore, compared with the studies of Sun et al., and Christian et al., in broilers, the effective dose of NaDF required to improve ileal morphology in the present study was higher. This discrepancy may be attributed to the longer intestinal development cycle and relatively delayed intestinal maturation in laying hens. It has been reported that the intestine of laying hens does not become essentially stable until 24 weeks of age^[16], whereas the intestinal development rate of broilers post-hatch is significantly faster than that of laying hens, allowing broilers to acquire high digestive and absorptive capacity at an early age. Therefore, the sensitivity of the laying hen intestine to formate-based acidifiers may be lower than that of broilers, requiring higher doses to produce significant morphological improvements. In the present study, dietary supplementation with 4 g/kg NaDF significantly improved villus height and VH / CD ratio in the duodenum and ileum of laying hens, indicating that an appropriate dose of NaDF exerts positive effects on intestinal morphology in laying hens.

5.3 Effects of NaDF on Intestinal Inflammatory Cytokines

Inflammatory cytokines, including Interleukin-6 (IL-6), Interleukin-1β (IL-1β), and Tumor Necrosis Factor-alpha (TNF-α), are known to participate in programmed cell death pathways such as apoptosis and pyroptosis, thereby exacerbating tissue damage^[17]. Specifically, IL-6 promotes leukocyte proliferation and antibody production, whereas TNF-α induces the cascade release of inflammatory mediators, amplifying the inflammatory response. Li et al^[7]., demonstrated that dietary supplementation with 1.0 g/kg to 5.0 g/kg NaDF significantly reduced jejunal TNF-α levels in broilers in a quadratic manner, while having no significant effect on IL-6 or IL-10 levels (P > 0.05). These findings suggest that NaDF exerts its immunomodulatory effects primarily through the suppression of pro-inflammatory cytokines rather than through the activation of anti-inflammatory cytokines.

These results are partially consistent with the findings of the present study. In this study, dietary supplementation with different levels of NaDF had no significant effect on the relative expression levels of inflammatory cytokines in the duodenum, jejunum, or ileum of laying hens. The discrepancy between our findings and those of Li et al^[7]., may be attributed to differences in animal breed. Broilers are characterized by rapid growth and high metabolic rates, which may render their intestinal immune system more responsive to dietary additives. In contrast, laying hens prioritize nutrient allocation for egg production and possess a stronger capacity to maintain intestinal immune homeostasis, resulting in a higher threshold for responsiveness to exogenous stimuli. Therefore, under healthy, non-challenged conditions, significant changes in inflammatory cytokine expression are less likely to be observed in laying hens.

Accumulating evidence indicates that the anti-inflammatory effects of formate-based acidifiers are condition-dependent. Liu et al^[18]., demonstrated in a mouse model that dietary supplementation with 10 g/kg potassium diformate significantly reduced serum levels of pro-inflammatory cytokines (IL-6, IL-12, and TNF-α) following Salmonella Typhimurium infection, indicating that KDF exerts broad-spectrum anti-inflammatory effects through the inhibition of the nuclear factor-κB (NF-κB) signaling pathway. Similarly, Sun et al^[8]., employed a Salmonella Pullorum infection model and confirmed that dietary NaDF pretreatment for 2 days effectively reduced bacterial loads in the cecum, liver, and spleen, and alleviated cecal histopathological damage, suggesting that NaDF protects against inflammatory injury along the gut-liver axis.

In the present study, the experimental animals were maintained under conventional, non-challenged conditions, and the intestinal inflammatory signaling pathways were not activated. This likely explains the lack of significant effects on inflammatory cytokine expression observed in our study. Taken together, our findings suggest that under conventional rearing conditions, NaDF exerts a neutral effect on basal intestinal immune status. The anti-inflammatory potential of NaDF warrants further validation under pathogen challenge or stress conditions.

5.4 Effects of NaDF on Cecal Microbiota

The normal physiological and metabolic processes of the body are regulated by the gut microbiota. The gastrointestinal microbiota of animals is composed of probiotics, pathogens, and commensal bacteria, and at the phylum level is primarily dominated by Firmicutes, Bacteroidetes, and Proteobacteria. Sun et al^[8]., reported that dietary NaDF supplementation had no significant effect on the cecal microbiota composition of broilers at the phylum level, nor did it alter alpha diversity indices. These findings are consistent with the results of the present study. In this study, dietary NaDF supplementation had no significant effect on cecal microbiota alpha diversity indices (ACE, Chao, Shannon, and Sobs) or β diversity in Hy-Line Brown laying hens. However, NaDF supplementation altered the overall microbial community structure in a dose-dependent manner, indicating that NaDF can regulate microbial community composition by changing the relative abundance of specific taxa while preserving species richness and diversity. Consistent with our findings, Li et al^[7]., demonstrated that NaDF supplementation linearly increased ileal Lactobacillus counts while reducing the colonization of Escherichia coli and Salmonella in broilers, without exerting destructive effects on the overall microbial community structure. Collectively, these studies indicate that NaDF exerts a "selective" regulatory effect on the gut microbiota — targeting the abundance of functional genera while maintaining microecological homeostasis. This characteristic is favorable for preserving gut stability when NaDF is used as a feed additive.

At the genus level, 4 g/kg NaDF significantly reduced the abundance of Parabacteroides. Additionally, 3 g/kg NaDF tended to increase the abundance of Alistipes and Prevotella, though these differences did not reach statistical significance. Alistipes and Prevotella are recognized as potential short-chain fatty acid (SCFA)-producing bacteria in the avian gut^[19], and SCFAs serve as key energy substrates for intestinal epithelial cells^[20]. While direct SCFA quantification was not performed in this study, the observed bacterial shifts are consistent with the improved intestinal morphology documented herein. At the species level, 4 g/kg NaDF significantly reduced the abundance of Parabacteroides and Mordavella massiliensis. Previous studies have reported that Parabacteroides is positively correlated with lipid metabolism in chickens^[21]. Organic acid salts such as sodium formate have been shown to improve feed conversion efficiency and nutrient digestibility in laying hens^[22], suggesting that similar modulatory effects on gut microbiota and host metabolism may contribute to the beneficial effects of NaDF observed in this study. Nevertheless, the causal relationships and underlying mechanisms remain to be elucidated through integrated metabolomic analyses.

Therefore, the modulatory effect of NaDF on gut microbiota, particularly the suppression of potentially harmful bacteria such as Parabacteroides, may represent one of the potential mechanisms by which NaDF improves intestinal health in laying hens. Collectively, these results indicate that NaDF improves intestinal health in laying hens by selectively regulating gut microbiota structure, optimizing intestinal morphology, and thereby promoting nutrient digestion and absorption.

6.Conclusion

In conclusion, dietary NaDF supplementation improved duodenal and ileal morphology, selectively modulated bacterial genera, and enhanced intestinal health in laying hens. However, it did not significantly affect spleen index, inflammatory cytokine expression, α-diversity, or phylum-level microbial composition. The 4 g/kg supplementation level consistently yielded the best intestinal morphology improvements. Future studies should elucidate the specific pathways underlying NaDF's effects on intestinal morphology and validate its efficacy across different physiological stages of laying hens.