Perennial ryegrass improves bone quality in geese: insights from the gut-bone axis

Abstract

Bone disorders are marked by leg deformities, and disruption in the gut microbiota have been associated with degradation of bone structure, which increases the susceptibility of fractures. Perennial ryegrass has antimicrobial, antioxidant and anti-inflammatory properties to improve bone health by regulating the gut microbiota. In this study, we investigated the anti-inflammatory potential of perennial ryegrass to modulate the inflammatory bone loss in meat geese through regulating gut microbiota. Perennial ryegrass (GD) significantly reduced (p < 0.05) the inflammatory response (IL-1β and TNF-α) by improving the gut barrier function. Perennial ryegrass (GD) improved tibia bone mineral density (BMD), ash% and tibia bone mineral contents (p < 0.05). Results of gut microbiota and micro-computed tomography analysis showed that the beneficial effect of GD on bone mass might be associated with higher relative abundance of short-chain fatty acids (SCFAs) producing gut microbiota in the cecum by improving microbial diversity as compared to commercial feeding (CD). Regarding bone turnover, GD increased bone formation markers such alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteoprotegerin (OPG), osteocalcin (OCN), mothers against decapentaplegic homolog (SMAD) expression by reducing the nuclear factor-kappa B (NF-κB) expression and bone resorption markers i.e. nuclear factor of activated T-cells, cytoplasmic 1 (NFATC1) and tumor necrosis factor receptor associated factor 6 (TRAF-6). Perennial ryegrass exhibited an increase in antioxidant potential by reducing the malondialdehyde (MDA) and reactive oxygen species (ROS) production leading to increased catalase (CAT) and glutathione per oxidase (GSH-PX) levels (P < 0.05). Western blot and Immunofluorescence analysis of tibia showed reduced expression of NF-κB in GD group. These results indicate the importance of perennial ryegrass in alleviating bone loss through gut microbiota via NF-κB signaling mechanism. These findings emphasize the significance of the gut-bone axis and offer new insights into the role of perennial ryegrass in promoting bone health.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13568-025-01996-1.

Introduction

Animal welfare concerns regarding such rapid growth have been rising, and a transition to the raising of slower-growing chickens has been suggested as a potential solution. Rapid growth increases prevalence of leg disorders, poorer ability to walk and perform various behaviors, increased prevalence of leg, skin, and cardiovascular disorders, increased susceptibility to heat stress, and higher mortality rates than chickens with slower growth rates compromising bird health and overall productivity (Korver 2023). Consequently, the poultry industry is increasingly confronted with various stressors, including environmental factors, nutritional deficiencies, and pathogen exposure. These challenges can lead to immune dysfunction (Li et al. 2015). Specifically, immune dysfunction manifests as chronic inflammation, adversely affecting poultry growth, reproduction, and overall health (Oke et al. 2024). Immune stress compromises the intestinal barrier and leads to digestive disorders, triggering inflammatory diseases, reduced production performance, and even mortality (Zhang et al. 2020). The inflammatory response can also disrupt normal physiological processes, leading to conditions such as skeletal disorders, a growing concern in poultry management, particularly in geese (Song et al. 2021). As a result, skeletal issues like valgus/varus deformities, tibial dyschondroplasia (TD), bone torsion, and fractures have become increasingly common in domestic birds with a prevalence rate of 20–30% (Hartmann and Flock 1979).

Moreover, bone disorders, including leg abnormalities, pose both welfare and production concerns. For instance, 14–21% prevalence rates in commercial meat ducks have been associated with impaired mobility, pain, and difficulty accessing food and water, ultimately compromising growth performance (Jones and Dawkins 2010). These disorders result from a complex interplay of pathological, nutritional, and environmental factors (Rath et al. 2000; Wang et al. 2022). Research indicates that these disorders may be exacerbated by inflammatory processes that alter bone metabolism, highlighting the need for a deeper understanding of the underlying mechanisms (Wang et al. 2022). The relationship between inflammation and bone health is complex, and addressing these challenges in poultry farming requires a comprehensive approach.

In this context, the gut microbiota plays a crucial role in maintaining bone health by influencing digestive processes, systemic inflammation and bone metabolism. Modulating the gut microbiota, whether through germ-free environments, antibiotics, or probiotics significantly affects bone quality and remodeling by influencing the interactions between the immune, endocrine, and calcium absorption (D’Amelio and Sassi 2018). A critical factor in this process is the production of SCFAs through gut microbiota fermentation, which benefits bone health by reducing inflammation and enhancing mineral absorption (Smith et al. 2013). Consequently, this connection between gut health and skeletal integrity highlights the need to explore further gut-bone interactions, particularly in poultry farming, to mitigate bone disorders through dietary interventions.

Naturally occurring inflammatory conditions in poultry often arise from bacterial infections, making lipopolysaccharide (LPS) a useful model to study the impact of chronic inflammation on bone formation. When LPS activates toll-like receptor 4 (TLR4), it triggers an immune response that releases pro-inflammatory cytokines, leading to inflammatory osteolysis (Lorenzo et al. 2008). This activation also involves NF-κB signaling pathway, which plays a significant role in regulating cytokine expression. Additionally, NF-κB promotes osteoclastogenesis by upregulating key differentiation genes and prolonging osteoclast survival (Yamashita et al. 2007). This interaction enhances osteoblastogenesis, inhibiting mesenchymal stem cells (MSCs) differentiation into adipocytes, ultimately promoting bone mass and strength (Cawthorn et al. 2012).

Short-chain fatty acids (SCFAs), metabolic byproducts of gut microbiota, have also been implicated in bone health. SCFAs such as butyrate have been shown to inhibit histone deacetylase (HDAC) and modulate osteoclast gene expression, thereby promoting bone homeostasis (Karakan et al. 2021; Whisner et al. 2016). Additionally, SCFAs facilitate calcium absorption by lowering intestinal pH, enhancing mineral availability for bone formation (Whisner et al. 2016). Butyrate promotes the expression of osteoblast-related genes, such as RUNX2 and bone morphogenic protein-2 (BMP-2). Then, it influences bone formation via the BMP/SMADs pathways (Zhong et al. 2021). This highlights the potential for dietary interventions that can modulate gut microbiota and SCFA production to support skeletal health in poultry.

Geese are herbivorous and possess a unique ability to utilize high-fiber feeds (Huang et al. 2017). In China, ryegrass is the most commonly available pasture. It is rich in bioactive compounds (Kagan 2021). Recent studies indicate that ryegrass can regulate intestinal microflora in geese (Zheng et al. 2021) and improve various ethno medical properties, such as antioxidant, antimicrobial, and anti-inflammatory effects in animals (Choi et al. 2017).

One of the critical interventions for enhancing poultry health is incorporating ryegrass into their diets. Its prebiotic properties can improve gut microbiota composition by increasing microbial diversity and increase SCFAs production (Tyagi et al. 2018; Zhang et al. 2021). By promoting a healthy gut environment, ryegrass is essential in modulating inflammatory responses, a key factor in maintaining poultry health (Ali et al. 2022). In this study, we aimed to investigate the role of perennial ryegrass (a fiber-rich, naturally available forage-as a novel dietary intervention) in alleviating bone loss in meat-type geese through modulating gut microbiota via NF-κB signaling mechanism. We evaluated changes in gut microbiota composition, SCFA levels, tibia bone structure, systemic inflammatory markers, and key signaling pathways such as NF-κB. This comprehensive approach explored how perennial ryegrass may mitigate inflammatory bone loss and promote skeletal health in geese.

Materials and methods

Animals and experimental design

All animal experimental procedures were done following guidelines of the Animal Welfare and Ethics Research Committee, College of Animal Science, Henan Agricultural University Zhengzhou, China. A total of 300 one-day-old mixed-sexed geese with an average of 90 g (± 5.00), were split into two equal categories: (1) the commercial feeding group (CD, n = 150) and (2) the perennial ryegrass feeding group (GD, n = 150) with six replicates of 25 geese each. For the commercial diet group (CD), geese were kept indoors throughout the experimental period and fed a commercial diet formulated according to company recommendations (Supplementary Table 1). For the grazing diet group (GD), geese had daily access to a fenced perennial ryegrass pasture (approximately 50 m² per replicate) from 06:00 to 18:00 h, allowing increased physical activity allowing natural foraging, walking, and social interactions. After grazing, the geese were returned to the indoor pens and provided with the same commercial diet once daily. The experimental design incorporated three dietary phases: the starter diet (days 1–20), the grower diet (days 21–40), and the finisher diet (days 41–60). Ryegrass nutritional profile was given in Supplementary Table 2. The trial was conducted for a period of sixty days.

Sample collections and preparations

Three geese per replicate on day 15, 30 and 60 were selected randomly and after following a 12-h fasting period were slaughtered, and blood samples were collected from the jugular vein for serum analysis. Tissues and cecal contents were separated and preserved in liquid nitrogen at -80 °C on day 15, 30 and 60 for subsequent experimental analysis including histology, ELISA and microbiome analysis.

Measurement of antioxidant defense indices

Levels of LPS, ROS, and MDA, as well as the enzymatic activities of GSH-Px and CAT, were determined using commercial colorimetric assay kits (Beyotime, China). Serum, bone marrow and cecal tissue samples were homogenized in nine volumes of the corresponding homogenization buffer at 4 °C. The homogenates were centrifuged at 10,000 × g for 10 min at 4 °C, and the resulting supernatants were collected for analysis according to the manufacturer’s instructions. Measured values of LPS, ROS, MDA, GSH-Px, and CAT were normalized to the protein concentration of the supernatant fraction.

Cecal Immunofluorescence and TJP measurement

Ceca underwent immunofluorescence analysis following the manufacturer guidelines (Elabscience, Wuhan, China) and TJPs were quantified (Claudin-1 and ZO-1) on day 15, 30 and 60. Images were taken, and TJPs were quantified using Image-J 6.0 Software (MD, USA). Quantification of Cytokines.

Inflammatory cytokines such as IL-1β, tumor necrosis factor (TNF-α) and anti- inflammatory (IL-10) cytokines were measured from tibia and ceca via ELISA kits using enzyme free centrifuge tubes provided by Jiangsu Meimian Industrial Co., Ltd.

Ileum and cecal histological analysis

Ileal and cecal samples were fixed in 4% formaldehyde for 48 h, subsequently embedded in paraffin, and sectioned at 4 μm thickness. The sections were subjected to hematoxylin-eosin (H&E) and Alcian blue-periodic acid-Schiff (AB-PAS) staining. Histopathological alterations were examined using a light microscope (Leica DM1000, Germany).

Measurement of morphological indices of tibia

Digital electronic balance and vernier caliper were used to measure the relative tibia weight (g), length (mm), and width (mm) of the geese bone. Tibia dyschondroplasia (TD) lesion score was evaluated according to the ratio of the total amount of cartilage plugs and the cut area upon cutting longitudinally at the proximal end, specifically, score 0 (normal bones with no cartilage lesions), score 1 (cartilage developed in up to 1/3 the cut area), score 2 (cartilage developed in 1/3 to 1/2 the cut area), and score 3 (cartilage developed in more than 1/2 the cut area). Mineral profile (calcium and phosphorus) from tibia were measured according to the method applied by David et al., 2022. Quantification of the bone makers, OCN, TRAF-6, and 1-NFATC-1 was done using the ELISA kit provided by Shangai MlBIO Biotechnology Co., Ltd China.

Micro-CT analysis and tibia staining

Micro-computed tomography (µ-CT) was utilized to evaluate the microarchitecture of the tibia. Before scanning, tibial samples were dried at 65 °C to remove any residual moisture, following procedures confirmed by preliminary tests. Scans were conducted using a µ-CT system (Bruker 1176, version 1.15.2.2+, Karlsruhe, Germany) operating at 70 kV with a 0.5 mm aluminum filter to minimize beam hardening artifacts. Two-dimensional images were reconstructed and compiled into three-dimensional datasets using the manufacturer’s software. The region of interest (ROI) was delineated as the metaphyseal area, spanning from 4 mm distal to 9 mm below the tibial condyle. Quantitative analyses included measurements of the bone volume-to-tissue volume ratio (BV/TV) and trabecular number (Tb.N), trabecular thickness (Tb.Th) and bone mineral density (BMD).

NF-κB Immunofluorescence (IF) imaging

Tibia sections were fixed in 4% paraformaldehyde for 10 min. Paraffin-embedded sections were dewaxed in xylene for 15 min, followed by dehydration through a graded ethanol series and rinsing with distilled water. Antigen retrieval was performed on rehydrated sections by boiling in citrate-EDTA buffer for 2 min. To block non-specific binding, sections were incubated with 10% fetal bovine serum for 30 min. The slides were then incubated overnight at 4 °C with primary antibodies: rabbit anti-NF-κB (CY2339, 1:500). The following day, sections were treated with HRP-conjugated goat anti-rabbit IgG (1:400, 50 min, room temperature, in the dark), followed by application of tyramine sodium fluorescein (PBST + 0.0003% H2O2) for 20 min at room temperature. Nuclear staining was performed with DAPI for 10 min, and slides were mounted with an antifade medium. The subcellular localization of NF-κB was observed using confocal fluorescence microscopy (TCS SP8 STED, Leica). Nuclei were stained blue with DAPI, while NF-κB was visualized as red signals using fluorescein-labeled antibodies.

Quantification of cecal SCFAs

Approximately 1 g of cecal digesta was homogenized with 3 mL of deionized water and centrifuged at 10,000 × g for 10 min. Subsequently, 1 mL of the supernatant was combined with 0.2 mL of 25% (w/v) metaphosphoric acid and stored at − 20 °C overnight. After thawing, the samples were centrifuged again, and 0.75 mL of the resulting supernatant was mixed with 0.15 mL of an internal standard solution to obtain a 0.9 mL mixture. To this, 1.8 mL of ethyl acetate was added, followed by vortexing for 15 s. The mixture was allowed to stand for at least 5 min to facilitate phase separation, after which approximately 1.2 mL of the upper organic layer was transferred into a glass vial. Short-chain fatty acids (SCFAs) were quantified using gas chromatography (GC-2010 Plus, Shimadzu Corporation, Japan) against known calibration standards, and concentrations were expressed in millimoles (mM).

Gene expression

Total RNA was isolated from tibia bone and cecal tissues (50–100 mg) using 1 mL of MagZol reagent (Magen Biotechnology, Guangzhou, China) following the manufacturer’s protocol. RNA concentration and purity were assessed using a NanoDrop 2000 spectrophotometer (Thermo Scientific). Gene-specific primers were designed using Primer3web, version 4.1.0 (Supplementary Table 3). Gene expression levels of target genes were calculated using the 2^−ΔΔCt method with GAPDH as the reference gene.

Western blotting

Western blotting of RUNX2 (Abclonal, A2817), OCN (Abcam, ab1080), SMAD (Abcam, ab1032), ALP (Pro-intech, 17250-1-PA), receptor activator of nuclear factor kappa beta-RANKL (Abcam, ab 10541-2-Ig), NFATC1 (Proteinintech, FR2011-37), plasma membrane calcium ATPase 1b-PMCA1b (HUABIO, ET 12009), calbindin-CaBP-28 K (Abcam, ab 511369), vascular endothelial growth factor A-VEGFA (Pro-Bio, 216634), hypoxia inducible factor-HIF (Huaxingbio, 3154), NF-κB p65 (Abclonal, ab1603-09), sodium-dependent phosphate cotransporter 2a-NPt2a (Pro-bio, LM1705-27), phosphate regulating endopeptidase X-linked -PHEX (ProBio, HX1827), FGF-23 (ProBio, BC021), HDAC (Huaxingbio, 45098), OPG (Proteinintech, NZ09), and β-actin (Abclonal, LA033) in tibial and duodenal tissues were done. Detection of immunoreactive bands was performed using an enhanced chemiluminescence (ECL) substrate, and band intensities were quantified by densitometric analysis. Target protein expression was normalized to β-actin as an internal loading control.

Analysis of 16 S rRNA gene sequencing

Extraction of DNA and PCR amplification

Genomic DNA, Illumina PE300/PE250 sequencing and Amplicon sequencing were carried out following the method used by Zulfiqar et al. 2025a, b. Bioinformatic analysis of gut microbiota was conducted using the Majorbio Cloud platform (https://cloud.majorbio.com). Alpha diversity indices, including observed ASVs, Simpson, Sob, and Ace indices, were computed using Mothur (v1.30.1). Beta diversity was assessed via principal coordinate analysis (PCoA) based on Bray-Curtis dissimilarity, implemented in the Vegan (v3.3.1) package in R. Circos diagram was generated to visualize microbial abundance at the genus level. Differentially abundant taxa (from phylum to genus level) were identified using linear discriminant analysis effect size (LEfSe) (http://huttenhower.sph.harvard.edu/LEfSe), with a significance threshold of LDA score > 2 and P < 0.05. Correlations between taxa were considered statistically significant if Spearman’s correlation coefficient exceeded 0.6 or was below − 0.6, with a P-value < 0.01 (NCBI accession number PRJNA1158539).

Statistical analyses

All statistical analyses were conducted using SPSS software (version 20.0, IBM Corp., Armonk, NY, USA), and data are presented as mean ± standard deviation (SD). A statistical power exceeding 0.80 was maintained, assuming a minimum effect size of 1.0. The assumptions of normality and homogeneity of variance were assessed using the Shapiro-Wilk and Levene’s tests, respectively. Subsequently, one-way analysis of variance (ANOVA) followed by unpaired T-test was employed to evaluate differences between the groups, based on the following statistical model:

In addition, Pearson correlation was used to identify the relationship between parameters. When p < 0.05 and p < 0.01, the variation was considered statistically significant respectively.

Results

Effect of pasture grazing system on growth performance of geese

The growth performance of meat geese was presented in Supplementary Table 3. Growth performance of meat geese during the starter phase (15 days), grower phase (30 days) and finisher phase (60 days) was evaluated using an unpaired Student’s t-test. The results revealed significant differences in average daily gain (ADG) and feed conversion ratio (FCR) between groups on day 30 and 60. Final body weight was higher in commercial feeding group as compared to the pasture grazing group.

Perennial ryegrass feeding improved tibia profile and bone markers

Perennial ryegrass had no effect on tibia index (relative tibia weight) as shown in Fig. 1a). Tibia Ca content exhibited a significant elevation on day 60 (Fig. 1b). Ryegrass supplementation led to a significant increase in tibia phosphorus (P) levels (Figs. 1c). CD group had higher growth plate width leadings towards increased TD lesion as compared to GD group (Figure d, i) (Fig. 2)Additionally, ryegrass feeding significantly enhanced (p < 0.05) the ALP and OCN levels by significantly reducing the bone resorption marker NFATC1 and TRAF6 compared to the CD group on days 15, 30, and 60 (Fig. 3-1e-h).

Fig. 1.

Perennial ryegrass improved the tibia mineral profile, TD lesions and bone markers in geese. a Tibia index. b Tibia Ca. c Tibia P. (d) TD score and arrows showing width of tibia growth plates. e Tibia ALP. f Tibia OCN. g Tibia TRAF6. h Tibia NFATC1. Values represent the mean ± standard deviation (SD) of n = 6 replicates per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. The asterisks symbol indicates significant differences *p < 0.05, **p < 0.01. Ca = calcium; P = phosphorus; TD = tibial dyschondroplasia; ALP = alkaline phosphatase; OCN = osteocalcin; TRAF6 = tumor necrosis factor receptor-associated factor 6; NFATC-1 = nuclear factor of activated t cells, cytoplasmic 1

Fig. 2.

Perennial ryegrass improved the tibia microstructure in geese. a Micro-CT analysis of trabecular bone. b BMD. (c) BV/TV%. d TS. e BS/BV. f BS/TV. (g) Tb. sep. h Tb. Th. i Tb. N. Values represent the mean ± standard deviation (SD) of n = 6 replicates per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. The asterisks symbol indicates significant differences *p < 0.05, **p < 0.01. BMD = bone mineral density; BV = bone volume; TS = tissue surface; BS = bone surface; TV = total volume; Tb. Th. Trabecular thickness; Tb. sep. = Trabecular separation; Tb. N = trabecular number

Fig. 3.

Perennial ryegrass improved the proteoglycan content and articular cartilage structure of tibia in geese. a Safranin staining (b) Proteoglycan content (c) Articular cartilage thickness on day 15. (d) Articular cartilage thickness on day 30. e Articular cartilage thickness on day 60. Values represent the mean ± standard deviation (SD) of n = 6 replicates per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. The asterisks symbol indicates significant differences *p < 0.05, **p < 0.01

Perennial ryegrass feeding improved the tibia microstructure

Figure 2a represented the micro-CT analysis of trabecular portion of tibia of meat geese. BMD was significantly increased (p < 0.05) on day 30 as a result of pasture grazing with perennial ryegrass feeding (Fig. 2b). Bone volume (BV/TV) exhibited an increase in GD group on day 15 (Fig. 2c). Tissue surface was also increased on day 15 and 60 as result of pasture grazing (Fig. 2d). Bone surface to bone volume (BS/BV) and bone surface to total volume (BS/TV) were significantly increased in GD group as compared to commercial feeding group (CD) (Fig. 2e-f). Perennial ryegrass feeding significantly reduced the trabecular separation in GD group (Fig. 2g). Additional, trabecular thickness (Tb. Th) and trabecular number (Tb. N) were also improved in GD group as compared to CD group (Fig. 2h-i).

Tibia histological analysis

Safranin O staining was to check the articular cartilage pattern and proteoglycan content (Fig. 3a). Perennial ryegrass significantly increased the proteoglycan content in GD group in comparison with CD group (Fig. 3b). There was no irregularity in the pattern of cartilage on both groups. On day 15, the GD group exhibited a significant increase (p < 0.05) in zone II and total articular cartilage thickness (Fig. 3c). By day 30, the thickness of zones I, II, III, and the total cartilage thickness were greater due to ryegrass grazing (Fig. 3d). Furthermore, on day 60, zone I and total articular cartilage thickness were significantly higher (p < 0.05) in the GD group (Fig. 3e).

Perennial ryegrass enhanced the antioxidant potential and immune response of meat geese

There was a significant increase (p < 0.01) in GSH-PX in the tibia bone marrow and ceca on day 15, 30, and 60 (Fig. 4a-b). CAT levels exhibited a significant increase (p < 0.05) in the tibia and ceca on days 15 and 60 (Fig. 4c-d). Oxidative stress marker (MDA) indicated a pronounced elevation in tibia, and ceca in CD group compared to GD at all three time points: 15, 30, and 60 days (Fig. 4e-f). GD group had significantly lower levels of IL-1β and TNF-α in tibia (BM) and ceca (Fig. 4g-j). On day 15, GD group had significantly higher (p < 0.05) levels of IL-10 levels in tibia. On days 30 and 60, IL-10 exhibited a significant increase in tibia and ceca in GD group as compared to CD group (Fig. 4k-l). LPS production was significantly more in commercial feeding groups. In terms of LPS production was highly significant (p < 0.01) on day 30 and 60 in CD group. In tibia, LPS was significantly higher on days 15, 30 and 60 in commercial feeding (Fig. 5). Same pattern was observed in ceca on day 30 and 60 (Fig. 8a-c). ROS production was lower (p < 0.01) in GD group in tibia on day 15 and 30. In serum, ROS production was significantly reduced in GD groups. ROS content in ceca was significantly higher (p < 0.01) in commercial feeding groups (CD) as compared to ryegrass grazing group (Fig. 8d-f).

Fig. 4.

Effect of perennial ryegrass on antioxidant potential and inflammatory indicators in geese. a GSH-PX BM (b) GSH-PX-C. c CAT BM. d CAT-C. e MDA BM (f) MDA-C. g IL-1β-BM. (h) IL-1β-C. i TNF-α-BM. j TNF-α-C. k IL-10-BM. l Il-10-C. Values represent the mean ± standard deviation (SD) of n = 6 replicates per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. The asterisks symbol indicates significant differences *p < 0.05, **p < 0.01. GSH-PX = glutathione peroxidase; CAT = catalase; C = ceca; BM = bone marrow; MDA = malondialdehyde; TNF-α = tumor necrosis factor alpha

Fig. 5.

a-b Immunofluorescence (IF) images of tight junction proteins (TJPs). c Protein expression of ZO-1. d protein expression of claudin-1. Blue: nucleus (DAPI); Red: ZO-1; Cerulean blue: merge of blue and red indicating nuclear localization of ZO-1, scale bar = 200 μm. Values represent the mean ± standard deviation (SD) of n = 6 replicates per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. The asterisks symbol indicates significant differences *p < 0.05, **p < 0.01

Fig. 8.

Oxidative stress mediators. a-c LPS in serum, tibia and ceca, d-f ROS in serum, tibia and ceca. (g) western blot of WNT10b, Runx2, OCN, SMAD, ALP and OPG with GAPDH as reference. (h) western blot of RANKL, NF-κB, NFATC-1 and HDAC. i western blot of CaBP-28 K, PMCA1b, NPt2a. j western blot of FGF-23, PHEX, VEGFA and HIF-1 with GAPDH as reference. Values represent the mean ± standard deviation (SD) of n = 6 replicates per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. The asterisks symbol indicates significant differences *p < 0.05, **p < 0.01. Runx2 = runt-related transcription factor2; SMAD = suppressor of mother against decapentaplegic; ALP = alkaline phosphatase; OPG = osteoprotegerin; RANKL = receptor activator of nuclear factor kappa beta ligand; NF-κB = nuclear factor kappa-light-chain-enhancer of activated B cells; HDAC = histone deacetylase; FGF-23 = fibroblast growth factor-23; PHEX = phosphate regulating endopeptidases X-linked; VEGFA = vascular endothelial growth factor; HIF-1 = hypoxia inducible factor-1; NFATC-1 = Nuclear factor of activated T cells. PMCA1b = Plasma membrane Ca + 2 ATPase; CaBP-28 = Ca binding protein 28

Perennial ryegrass improved gut morphology and intestinal barrier integrity

Perennial ryegrass (GD) improved the intestinal morphology by VH (villus height), VSA (villus surface area), VW (Villus width) and VH: CD (Villus height to crypt depth ratio) were significantly increased (p < 0.01) because of ryegrass feeding on day 15, 30 and 60 of the ileum section. GD exhibited a significant decline in CD (crypt depth) as shown (Supplementary Fig. 1a-e). Goblet cells were significantly higher in GD group (Supplementary Fig. 1f). Mucosal and muscularis thickness of the ceca were also improved (p < 0.01) in GD group (Supplementary Fig. 1g-h). GD significantly reduced diamine oxidase (DAO) levels (Supplementary Fig. 1i). Additionally, the expression of tight junction proteins was evaluated through immunohistochemical analysis (Fig. 5a-b). GD significantly upregulated ZO-1 expression, while Claudin-1 expression was markedly increased on last two phases of the experiment. Immunofluorescence analysis and expressions of ZO-1 and Claudin-1 were shown in Fig. 5 (c-d).

Gut microbiome composition at phylum level

High-throughput 16 S rRNA sequencing (targeting the V3-V4 variable region) was conducted to evaluate the impact of ryegrass intake on the gut microbiome of meat geese. GD had a strong effect on gut microbiome yielding a distinct cluster as compared to CD group. GD exhibited a significant change in gut microbiota as compared to CD group. GD increased the relative abundance of Bacteroides by notably decreasing the proportion of Firmicutes. At the phylum level on 15th day, the CD group was predominantly composed of Bacteroidota, Firmicutes, with Actinobacteriota surpassing Verrucomicrobiota. By 30th day, Firmicutes became the dominant phyla in the CD group, a trend that persisted on day 60th. Overall, Firmicutes and Bacteroidota remained the major phyla across both diet groups at all time points. Microbial diversity at the phylum level is depicted in Fig. 6(a-c).

Fig. 6.

a-i Microbiome analysis on day 15, 30 and 60. a-c phylum level, (d-f) alpha diversity (Simpson, Sobs and ace index), (g-i) represented the β-diversity

Perennial ryegrass modulated microbial diversity

Alpha diversity remained unaffected (Simpson, Shannon and Ace index) on all three phases as shown in Fig. 6(d-f). A significant difference in beta diversity (p < 0.05) was observed in the GD group on 15th day (Fig. 7g). However, no significant difference (p > 0.05) was observed between the two groups on 30th and 60th days (Fig. 6h-i).

Fig. 7.

SCFAs production. a acetic acid, (b) propionic acid, (c) butyric acid, (d) iso-butyric acid, (e) iso-valeric acid (f) valeric acid. g Integrated heatmap of SCFAs, tibia bone parameters, and immune indicators across dietary treatments. Values represent the mean ± standard deviation (SD) of n = 6 replicates per group. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test. The asterisks symbol indicates significant differences *p < 0.05, **p < 0.01

Gut Microbiome changes at genus level

Ryegrass intake modulated the gut microbiome community at the genus level. We sorted 20 bacteria at the genus level related to bone health. Streptococcus, Ruminococcus, Oscillospiracae, Faecalbacterium, Lactobacillus, Butyricoccus, Bifidobacterium, Lachnospiracae, Gastranaerophilales, Allistipes, Bacteroides, Barnesiella, Akkermansia, Blautia and Rombutsia were higher in terms of relative abundance in GD group. Compared with GD group, relative abundance of the microbes namely CUCG14, Erysipelatoclostridium, Subdoligranum, Rikenellaceae_RC_gut_group and E.coli were higher in CD group as shown in the Supplementary Fig. 2. The Circos diagram illustrated the diversity and relative abundance of bacterial genera in the two groups, facilitating the visualization of microbiome composition and the identification of bacterial distribution patterns in response to treatments. This graphical representation provides insights into microbial community dynamics, enabling the assessment of treatment or time dependent shifts in bacterial structure (Supplementary Fig. 3a-c).

Linear discriminant analysis effect size (LefSe) score analysis

LefSe analysis was conducted to identify differentially abundant bacterial taxa between the two groups. Relative abundance of Escherichia coli and CUCG-014. On Day 15 was higher in commercial feeding group, whereas Ruminococcus, Faecalibacterium, and Bifidobacterium were more prevalent in the GD group (Supplementary Fig. 3d). By Day 30, Enterococcus and E. coli were enriched in the CD group, while Alistipes, Blautia, and other taxa were more abundant in the GD group (Supplementary Fig.3e). On Day 60, the CD group showed increased levels of Enterococcus, Shigella, Subdoligranulum, and Shuttleworthia, whereas the GD group exhibited a greater abundance of Actinobacteria, Bifidobacteriales, and Oscillospiraceae (Supplementary Fig. 3f).

Perennial ryegrass increased the production of microbial SCFAs

SCFAs are the major metabolites produced by gut microbiota. In the present study, Ryegrass intake had a significant effect on SCFA’s levels. GD through natural grazing system significantly increased the acetate production at Day 15, 30 and 60 (Fig. 7a). The levels of propionate and butyrate were significantly higher in GD group (Fig. 7b-c). GD also increased (p < 0.01) the levels of other SCFAs namely Iso-butyrate, valeric acid and isovaleric acid (Fig. 7d-f).

Integrated heatmap of SCFAs, tibia bone parameters, and immune indicators across dietary treatments

Figure 7g illustrated the integrated effects of different dietary treatments on short-chain fatty acids (SCFAs), tibia bone characteristics, and immune or antioxidant indicators across the CD15, GD15, CD30, GD30, CD60, and GD60 groups. In the SCFA panel, the GD60 group exhibited the highest concentrations of acetate, propionate, and butyrate, suggesting that this treatment promoted a more mature and metabolically active gut microbiota. Intermediate increases were also observed in the GD30 group, whereas earlier time points showed comparatively lower SCFA production.

These increases in SCFAs corresponded with favorable changes in bone-related parameters. Groups with elevated SCFA levels, particularly GD30 and GD60, showed higher levels of alkaline phosphatase (ALP) and osteocalcin (OCN), both markers of bone formation. Structural tibia traits, including bone mineral density (BMD), trabecular number, and trabecular thickness were also enhanced in these groups. At the same time, trabecular separation was reduced, indicating improved trabecular integrity. Expression levels of NFATC1 and TRAF6, which are associated with osteoclast differentiation and bone resorption, tended to be lower in groups with higher SCFA production, further suggesting reduced osteoclast activity.

Immune and antioxidant indicators showed similar improvements in the later GD groups. Anti-inflammatory cytokine IL-10 and barrier associated proteins Claudin-1 and ZO-1 appeared elevated in these groups, indicating enhanced mucosal immune regulation and intestinal barrier integrity. Antioxidant enzymes such as catalase (CAT) and glutathione peroxidase (GSH-PX) were also higher, while pro-inflammatory markers including TNF-α, IL-1β and the oxidative stress marker MDA were reduced. Taken together, these results demonstrated that dietary treatments promoting SCFA production were associated with improved bone metabolism, reduced inflammation, strengthened epithelial barrier function, and enhanced antioxidant capacity. Overall, the heatmap revealed a coordinated physiological response, supporting the concept of a gut-bone-immune axis influenced by dietary modulation.

Effects of perennial ryegrass on mRNA expression of osteoblastic and osteoclastic genes

The production of pro-inflammatory cytokines play an important role in ongoing systemic inflammation. IL-1β, IL-18 and TNF-α expression levels were significantly higher (P < 0.01) in both tibia and ceca. GD increased the mRNA expression levels of IL-10 both in tibia and ceca (Table 1). The mRNA expression level of nuclear factor erythroid 2-related factor 2 (Nrf2) was significantly higher (p < 0.01) in GD group by reducing the expression level of kelch-like ECH-associated protein 1 (Keap1).

Table 1.

Systemic inflammatory genes (IL-1β, TNF-α, IL-10), angiogenesis genes (HIF-1, VEGFA), Ca and phosphorus transporters (PMCAIb, CaBP-D28K, NPt2a), Wnt-10b and its regulating genes, NF-κB and its regulating genes, local bone derived regulators (PHEX, MEPE, DMPI), SCFAs receptors (GPR43, GPR41, GPR109a) and MUC-2

		15d			30d			60d
Tissues	Gene	CD	GD	P-value	CD	GD	P-value	CD	GD	P-value
	IL-1β	1.00	0.05	P<0.01	1.62	0.08	P<0.01	0.25	0.11	P<0.01
	TNF-α	1.00	0.13	P<0.01	0.95	0.23	P<0.01	0.79	0.42	P<0.01
	IL-10	1.00	1.26	P<0.01	0.74	1.57	P<0.01	0.86	2.00	P<0.01
	GPR43	1.00	2.72	P<0.01	0.40	0.46	P>0.05	0.29	0.83	P<0.05
	GPR41	1.00	1.43	P<0.01	1.22	2.19	P<0.01	1.34	2.52	P<0.01
	GPR109a	1.00	1.40	P<0.01	1.16	1.72	P<0.01	1.32	2.08	P<0.01
	MUC-2	1.00	1.32	P<0.01	0.17	0.25	P>0.05	0.18	0.31	P<0.05
Duodenum	PMCAIb	1.00	2.70	P<0.01	0.17	1.49	P<0.01	0.59	0.75	P<0.05
	CaBP-D28K	1.00	1.15	P>0.05	2.52	3.07	P<0.01	0.92	1.61	P<0.01
	NPt2a	1.00	1.22	P<0.01	0.15	0.74	P<0.01	1.28	1.16	P>0.05
Tibia	IL-1β	1.00	0.32	P<0.01	1.40	0.53	P<0.01	1.79	0.65	P<0.01
	TNF-α	1.00	0.13	P<0.01	0.95	0.23	P<0.01	0.79	0.42	P<0.01
	IL-10	1.00	1.19	P>0.05	0.46	1.59	P<0.01	0.26	2.54	P<0.01
	HIF-1	1.00	1.27	P<0.01	0.54	0.85	P<0.01	0.84	1.10	P<0.05
	VEGFA	1.00	1.24	P<0.05	0.44	0.95	P<0.01	1.09	1.23	P<0.05
	ALP	1.00	3.03	P<0.01	1.54	4.97	P<0.01	2.60	3.82	P<0.05
	Wnt10b	1.00	3.18	P<0.01	1.88	3.79	P<0.01	1.50	4.30	P<0.01
	SOST	1.00	0.81	P<0.01	1.89	1.30	P<0.01	0.94	0.73	P<0.01
	OCN	1.00	1.95	P<0.01	0.34	1.93	P<0.01	1.21	1.89	P<0.01
	SMAD	1.00	1.53	P<0.01	1.14	1.64	P<0.01	0.97	1.45	P<0.01
	OPG	1.00	4.26	P<0.01	0.50	1.05	P<0.01	1.24	1.42	P>0.05
	RUNX2	1.00	1.44	P>0.05	1.21	2.59	P<0.01	1.74	2.07	P<0.05
	FGF23	1.00	1.41	P<0.01	0.71	0.62	P>0.05	1.34	2.07	P<0.01
	PHEX	1.00	1.47	P<0.01	0.75	2.07	P<0.01	0.89	3.16	P<0.01
	DMPI	1.00	1.48	P<0.01	0.40	5.15	P<0.01	1.24	3.15	P<0.01
	MEPE	1.00	4.74	P<0.01	1.33	5.22	P<0.01	1.51	3.89	P<0.01
	NF-κB	1.00	0.97	P>0.05	0.70	0.57	P<0.05	0.79	0.45	P<0.01
	RANKL	1.00	0.35	P<0.01	1.25	0.76	P<0.05	3.34	1.42	P<0.05
	TRAF6	1.00	0.32	P<0.01	1.35	1.03	P<0.05	0.71	0.53	P<0.05
	NFATC1	1.00	0.39	P<0.01	1.06	0.48	P<0.01	2.28	1.04	P<0.01
	HDAC	1.00	0.81	P<0.01	2.10	1.47	P<0.01	1.78	1.22	P<0.01

Mean values (n = 6 replicates per group) showed the significant difference between the groups (p < 0.05; p < 0.01)

We observed the major changes in the growth plates of the geese tibia bone due to inflammatory responses resulting in increased mRNA expression levels of VEGFA and HIF-1 in GD group (Table 1). The mRNA expression levels of calcium and phosphorus transporters namely PMCA1b, NPt2a and CaBP-D28 were significantly increased (p < 0.01) in GD group as compared to CD group (Table 1). ALP expression level was also higher (p < 0.01) in the tibia on all three phases. The mRNA expression of osteogenic pathways (SMAD) was higher in GD group (Table 1). GD increased the mRNA expression levels Runx2, OCN and OPG in GD group. Local bone derived regulators (PHEX, DMPI and MEPE) showed significantly higher mRNA expression levels in GD group. G-coupled protein receptors (GPRs) such as GPR43, GPR41 and GPR109a have been identified as receptors for SCFA’s, the molecular mechanisms underlying the role of SCFA’s in bone health have elucidated. These 3 receptors (GPR41, GPR 43 and GPR109a) showed higher mRNA expression in ceca on day 15 and 30 in GD group. NF-κB plays a very crucial role in bone resorption by increasing the inflammatory response. The genes responsible for bone resorption due to LPS induced inflammatory response are RANKL, NFATC-1, TRAF-6 and HDAC playing an important role in NF-κB regulation (Abu-Amer 2013). GD significantly reduced the mRNA expression of NF-κB on day 30 and 60. RANKL and TRAF6 were also downregulated on day 15, 30 and 60. GD group had significantly lower (P < 0.01) mRNA levels of NFATC-1 and HDAC on all three phases as compared to CD group (Table 1).

Perennial ryegrass affected protein expression of osteoblastic and osteoclastic genes

Western blotting was performed on the 60th day of the experiment. The protein expression of RUNX2, OCN, SMAD, ALP and OPG were higher in GD group (Fig. 8g). NF-κB and its regulating genes namely RANKL, HDAC and NFATC-1 exhibited higher protein expression in commercial feeding diet (CD) as compared to GD group (Fig. 8h). Western blot analysis of Ca and P transporters (CaBP-28 K, PMCA1b and NPt2a) showed significantly higher (p < 0.01) protein expression in GD group (Fig. 8i). Ryegrass intake had also significantly increased (p < 0.01) the protein expression of local bone derived regulators namely FGF-23 and PHEX. Protein expression of angiogenic genes (HIF-1 and VEGFA) were also performed to observe the blood vessel formation rate. These angiogenic genes showed high protein expression levels in GD group (Fig. 8j).

Immunofluorescence imaging of NF-κB

To further validate the activation of NF-κB signaling in the tibia of meat geese, immunofluorescence analysis was conducted to assess their nuclear translocation. Confocal fluorescence microscopy revealed a predominant localization of NF-κB in the cell nuclei of tibial tissue in the CD group compared to the GD group (Fig. 9). The results revealed reduced NF-κB in GD group as compared to CD group suggesting that ryegrass intake may involve actively in bone formation.

Fig. 9.

Immunofluorescence (IF) images of NF-κB. Rabbit Anti-NF-κB (CY3312) (1:500/1:200) showing nuclear translocation of NF-κB. Blue: nucleus (DAPI); Red: NF-κB staining; Merge; combination of blue and red indicating nuclear translocation of NF-κB, scale bar = 200 μm

Discussion

Intense selection pressure over the years has contributed to an increased prevalence of developmental disorders, especially those linked to growth rate and the development of the musculoskeletal system. TD is a condition which is associated with rapid growth and is characterized by an abnormal mass of cartilage in the proximal end of the tibia, resulting from inadequate vascularization and mineralization of the growth plate and bones (Leach and Monsonego-Ornan 2007). This abnormal mass can cause angular and rotational abnormalities in the leg which can contribute to impaired walking ability. We observed slower tibia growth in GD group that was associated with less TD lesions (Shim et al. 2012).

Calcium (Ca) concentration showed a significant difference between the groups. However, Ca content in the tibia were significantly higher in GD group. Perennial ryegrass resulted in increased bone calcium retention in geese, leading to enhanced bone calcium balance. These observations were in line with the study (Elkomy et al. 2023). Calcium plays a critical role in skeletal development and maintenance, and adequate dietary intake is essential for optimal bone mineralization (Noirrit-Esclassan et al. 2021). High dietary calcium intake has been positively correlated with increased bone mineral density, bone strength and multiple factors linked to bone mass (Tai et al. 2015; Viguet-Carrin et al. 2014; Yao et al. 2021). These mechanisms suggest that the elevated Ca in the GD group could have directly influenced the observed improvements in bone mineral content and structural metrics, highlighting the importance of perennial ryegrass in supporting optimal skeletal health.

There was significant increase in tibia P and tibia ash content in GD group which probably increased the intestinal phosphorus transport by upregulating the gene expression of NaPiIIb (potent intestinal P-transporter). Similar findings broiler chicken studies suggest that significant drop in gut pH resulted in optimized ileal nutrient digestibility and improved P utilization, contributing to better mineral absorption and bone health (Chen and Chen 2004).

There was a significant improvement in tibia microstructure in GD group. Tibia microstructure indicators e.g. BMD, BV, BV/TV, TS and Tb. N were significantly increased in GD as compared to CD group. GD had significantly lower trabecular separation (Tb.sp.) compared with commercial feeding (Johnson et al. 2011). A possible reason for increased bone mineralization and improved tibia microstructure is through increased physical activity because of grazing. The impact of physical activity in terms of exercise on bone biomechanical characteristics has been investigated in birds maintained under different housing conditions. High physical activity leads to increased bone mechanical strength and bone mass (Wang et al. 2021; Yu et al. 2024). Birds kept in conventional individual cages with limited movement exhibited reduced physical activity, whereas those housed in aviary systems experienced relatively unrestricted mobility (Fleming et al. 2006). Enhanced bone strength and increased bone mineral density (BMD) observed in aviary-housed birds were attributed to their higher levels of physical activity. Prolonged immobilization, as seen in caged hens, leads to increased bone resorption. In contrast, the elevated physical activity in aviary systems promotes bone formation and remodeling processes (Rodriguez-Navarro et al. 2018). Improved tibia microstructure observed in the GD group is likely attributed to increased physical activity associated with grazing. Enhanced mobility stimulates bone formation and remodeling, leading to greater bone strength and mineral density. These results reinforce the critical role of physical activity in promoting skeletal health in poultry.

Bone markers, including those of formation (e.g., Osteocalcin and ALP) and resorption (e.g. NFATC-1 and TRAF-6), play a crucial role in monitoring bone remodeling and maintaining skeletal homeostasis. These biochemical markers reflect the dynamic balance between osteoblast-mediated bone formation and osteoclast-driven bone resorption. Disruption in this balance can lead to bone disorders such as osteoporosis (Delmas 1993). ALP supports bone calcification by hydrolyzing phosphate esters (Lee et al. 2006). In the current experimental study, the perennial ryegrass lowered the serum TRAP, tibia TRAF-6 and NFATC-1 (bone resorption markers) and increased the level of serum and tibia ALP and OCN (markers of bone formation) suggesting that ryegrass supplementation downregulates bone resorption (Tang et al. 2020). These results support the idea that GD may offer therapeutic benefits for bone health, as indicated by studies.

Osteocytes represent the predominant cell type found within the mineralized matrix of bone tissue (Schaffler and Kennedy 2012). They play a crucial role in mechanosensing and are instrumental in regulating the activities of both osteoclasts and osteoblasts, in addition to facilitating the process of bone mineralization. Their exact contributions to these processes are complex and may vary depending on the physiological context. Dentin matrix protein 1 (DMP-1), PHEX and MEPE are key genes predominantly expressed in osteocytes, playing essential roles in bone mineralization and the regulation of mineral homeostasis (Choi et al. 2022). These results suggest that the impact of ryegrass consumption on bone health is probably facilitated by osteocytes. Perennial ryegrass upregulated mRNA expression of these local bone derived regulators. Our findings showed increased expression of MEPE and DMP-1 with ryegrass feeding. While MEPE functions as an inhibitor of bone crystal formation, its interaction with PHEX forms a complex that mitigates its inhibitory effects (Staines et al. 2012). Furthermore, elevated DMP-1 suggested a coordinated effort by osteocytes to modulate bone mineralization, ensuring balance in the mineralization process. These molecular changes highlight an adaptive dietary response to regulate bone health (Narayanan et al. 2003).

Oxidative stress, arising from excessive generation of free radicals and reactive oxygen species (ROS) and/or impaired antioxidant defense mechanisms, can damage key biological macromolecules such as DNA, lipids, and proteins. This disruption adversely affects cellular metabolism and physiological functions (Pizzino et al. 2017). Malondialdehyde (MDA) is widely recognized and extensively studied as a biomarker of lipid peroxidation and oxidative stress. Lipid peroxidation is a chain reaction initiated by free radicals that leads to oxidative degradation of polyunsaturated fatty acids in cellular membranes (Cordiano et al. 2023). Antioxidant enzymes represent the primary defense system against oxidative injury, functioning by inhibiting ROS formation or neutralizing their harmful effects (Birben et al. 2012). We found increased levels of antioxidant enzymes (CAT, GSH-PX) and reduced levels of oxidative mediator (ROS and MDA) in GD. It is well established that improved Nrf2 signaling contributes significantly to the activation and maintenance of cellular antioxidant defense systems. GD enhanced the protein expression of CAT and GSH-Px by decreasing MDA levels, thereby mitigating ROS-induced oxidative stress (Ali et al. 2023).

The present study demonstrates that bone health in animals is significantly influenced by dietary regimen, as evidenced by the superior bone parameters observed in the GD (perennial ryegrass) group compared to the CD (commercially feeding) group. One of the key contributing factors to this outcome appears to be the differences in systemic inflammation mediated by growth rate and energy intake. Animals in the GD group, which were allowed to graze on perennial ryegrass, exhibited lower systemic inflammation due to both the physical activity associated with grazing and the lower energy density of pasture-based diets. In contrast, the CD group, receiving a commercial high-energy diet, likely experienced faster growth rates and higher metabolic load, both of which are known to induce systemic inflammation (Kealy et al. 2002). Chronic low-grade inflammation has been well documented to impair bone remodeling by stimulating osteoclast activity while inhibiting osteoblast function, ultimately leading to increased bone resorption and decreased bone formation (Torres et al. 2023). This process is largely mediated by inflammatory cytokines such as TNF-α, IL-6, and IL-1β, which are upregulated in response to excessive energy intake and rapid weight gain (Gregor and Hotamisligil 2011). In the GD group, the lower energy availability from ryegrass and the slower growth rate likely mitigated the expression of these pro-inflammatory cytokines, thereby preserving bone homeostasis. Furthermore, physical activity associated with grazing may have contributed to mechanical loading on the skeleton, which is a critical factor for stimulating osteogenesis and improving bone strength (Saxon et al. 2005). Therefore, it can be proposed that the improved bone health observed in the GD group was not only a consequence of nutrient composition but also of systemic physiological responses to energy balance and physical activity. These findings support the hypothesis that dietary modulation through grazing systems may offer a natural and effective means to enhance skeletal health via reduction in metabolic inflammation warranting further investigation into its role in animal nutrition and disease resistance strategies.

Recent findings have proved the significant role of perennial ryegrass on gut microbiota in geese (Ali et al. 2022). Gut microbial diversity of geese was significantly altered by ryegrass intake. In the present study, geese were dominantly occupied by Firmicutes and Bacteroidota. Bacteroidota are associated with cellulose degradation (Berry 2016). We found that Bacteroidota increased with ryegrass intake. Firmicutes, an important phylum, is involved in protein and fat metabolism (Okazaki et al. 2016). Our results showed that Firmicutes were decreased because of ryegrass intake because commercial diet contained more calories. Concurrently, the relative abundance of Bacteroidota increased with dietary fiber intake (Kaoutari et al. 2013). There was significant reduction in Firmicutes: Bacteroidota ratio in GD group. In the present study, the GD group (perennial ryegrass) exhibited a gradual increase in the relative abundance of Proteobacteria compared with the CD group (commercial diet). While Proteobacteria includes many opportunistic microorganisms, it is a diverse phylum encompassing both commensal and functionally important taxa (Tewari 2024). Our data suggest that the observed increase was primarily attributable to fiber-fermenting and carbohydrate-metabolizing genera rather than pathogenic species (Hou et al. 2020). This likely represents an adaptive microbial response to the higher fiber content of perennial ryegrass, as these taxa can metabolize complex polysaccharides reaching the hindgut and contribute to short-chain fatty acid production (Liu et al. 2018a, b). Notably, no clinical signs of gut dysbiosis or adverse health effects were observed in the GD group, and overall microbial diversity and the abundance of beneficial taxa, such as Bacteroides and Firmicutes, were maintained or enhanced. These findings indicate that the relative increase in Proteobacteria reflects a functional adjustment of the gut microbiota to dietary fiber, rather than a pathological shift, highlighting the importance of interpreting changes in Proteobacteria within the context of community composition and functional capacity (Hou et al. 2020). These findings were in line with (Guo et al. 2019), providing evidence that that reduced Firmicutes/ Bacteroidota ratio was associated with reduction in bone loss (Huang et al. 2025). Furthermore, in collagen-induced arthritis models, mice receiving a resistant starch (RS) diet exhibited a significant shift in gut microbiota characterized by a lower abundance of Firmicutes and a higher abundance of Bacteroidota which correlated with reduced disease severity (Bai et al. 2021). It is possible that Firmicutes/ Bacteroidota ratio might serve as a critical mediator influencing tibia properties in meat geese warranting further studies.

An additional mechanism through which ryegrass may positively influence bone health is microbial metabolites production, specifically short-chain fatty acids and their receptors upregulations (GPR41 and GPR43). FOS (a prebiotic fiber) is linked with an increase in BMD, leading to improved Ca absorption (Takahara et al. 2000). Similarly, in our study, the positive effects of ryegrass improved the tibia microstructure in conjunction with SCFAs (acetate, propionate, and n-butyrate) through its prebiotic effects. Butyrate is a key energy source for colonocytes and has well-established anti-inflammatory properties, suggesting that the intervention may enhance gut epithelial health and reduce inflammation (Liu et al. 2018a, b). Butyrate and propionate can modulate the metabolic pathways of pre-osteoclasts differentiation, driving a shift toward glycolytic metabolism. This metabolic reprogramming induces cellular stress, which subsequently impairs osteoclast differentiation (Lucas et al. 2018). Furthermore, SCFAs may mitigate both intestinal and systemic inflammation, significantly contributing to the bone metabolism maintenance (Zaiss et al. 2019), which was further advocated by our findings. Ryegrass exerts beneficial effects on bone health through its prebiotic influence, promoting the production of SCFAs. These changes align with our hypothesis that the that SCFAs enhance tibia microstructure, regulate osteoclast differentiation via metabolic reprogramming, and mitigate inflammation, thereby supporting bone metabolism and maintaining bone mineral density.

In the present study, ryegrass intake significantly upregulated the expression of Wnt10b along with key regulatory genes, including Runx2, OCN, and BMP2. These findings align with the study by (Islam et al. 2024), reporting enhanced bone formation following ryegrass consumption. Additionally, the mRNA expression of NF-κB and its associated regulatory genes was significantly downregulated in the GD group. Similar findings were observed in the previous reported study (Zhang et al. 2022). Furthermore, pasture grazing reduced osteoclastogenesis indirectly by increasing osteoprotegerin (OPG) expression, which inhibits RANKL/NFκB-mediated osteoclast differentiation (Albers et al. 2013).

A schematic figure illustrating the molecular mechanism of perennial ryegrass in alleviating commercial diet-induced bone loss was shown in Supplementary Fig. 4. This figure depicted how perennial ryegrass influences gut microbiota composition, enhances SCFA production, strengthens gut barrier integrity, and modulates the NF-κB pathway to promote bone health. The proposed mechanism involves an increase in beneficial bacteria (Lactobacillus, Bifidobacterium, Akkermansia), reduced the relative abundance harmful bacteria (E.coli, Shigella, subdoligranum, Rikenellacae and Clostrium-UCG 14), SCFAs and their receptors expression, which enhance gut integrity via upregulation of tight junction proteins (ZO-1, Occludin) and improved the intestinal morphology thereby reducing the LPS production. This reduced systemic inflammation, as evidenced by decreased pro-inflammatory cytokines (IL-1β, TNF-α, IL-18) and increased IL-10. Antioxidant defenses (SOD, CAT, GPx) were also enhanced, further mitigating oxidative stress-induced bone resorption. At the molecular level, perennial ryegrass upregulates the expression bone formation genes (Runx2, SOST, OCN, SMAD, ALP and OPG), stimulating osteogenesis, while downregulating NF-κB and its regulating genes (RANKL, NFATC-1, TRAF6, HDAC), thereby reducing osteoclast activity.

In conclusion, supplementing perennial ryegrass advocates the significant potential for improving the bone health by positively influencing the growth rate, gut health through reduced systemic inflammation. Dietary perennial ryegrass contributes to the maintenance of balanced gut microbiota facilitating the production of SCFAs. SCFAs enhance osteocyte-driven mineralization by increasing mineral absorption further reinforced by upregulation of osteocytic genes. This balance ultimately supports bone integrity by modulating the immune response. Furthermore, perennial ryegrass appears to inhibit the NF-κB responsible for inflammatory bone loss. Collectively, these findings indicate that perennial ryegrass feeding represents a promising dietary strategy for maintaining bone homeostasis in meat geese through modulation of gut microbiota and key signaling pathway.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Zeshan Zulfiqar performed the experiment and wrote the original draft. Xiaoyan Zhu, Zhichang Wang and Hao Sun did formal analysis. Boshuai Liu supervised the experiment. Yinghua Shi and Yalei Cui conceptualized and structured the manuscript. Jiamin Sun and Li Zhaoyang supported lab analysis. Muhammad Arslan Asif revised the manuscript. All the authors have read and approved the final version of the manuscript.

Funding

Declaration.

This study was financially supported by the China Agriculture Research System of MOF and MARA (No. CARS-34) and the Science and Technology Innovation Leading Talent in Central Plains (No. 244200510010).

Data availability

Data will be available on request to authors.

Declarations

Consent for publication

All authors have reviewed and consented to the publication of this manuscript.

Competing interests

The authors declare no competing interests.