Fermented Rice Bran Enhances Rabbit Meat Quality and Nutritional Value via Metabolic Reprogramming and Enriched Nutrient Profiles

Simple Summary

To enhance sustainable meat production, this study addressed a critical gap in rabbit nutrition by evaluating fermented de-oiled rice bran meal (FDRBM) as a cost-effective substitute for maize. The study investigates the replacement of conventional ingredients with unfermented (UFDRBM) and fermented de-oiled rice bran meal (FDRBM) in growing rabbit diets and evaluates growth performance, carcass characteristics, meat quality, amino acid profile, and fatty acid composition. The novelty of this study lies in its integrated approach, which reveals molecular correlates linking the FDRBM diet and observed phenotypic improvements. These enhancements are attributed to a cascade of biological events, beginning with improved antioxidant capacity in the gut, leading to metabolic reprogramming in the liver, and culminating in a favorable shift in muscle fiber-type composition. The FDRBM diet also enhanced the nutritional value of the meat by improving its amino and fatty acid profiles. Ultimately, these findings are relevant and timely, particularly in the context of sustainable feed resources, byproduct utilization, and meat quality improvement in rabbit production.

Abstract

Background: The valorization of sustainable feed ingredients such fermented de-oiled rice bran meal (FDRBM) is crucial; however, the molecular mechanisms driving its benefits remain unclear. This study addresses this gap by investigating FDRBM as a dietary substitute for maize in rabbits to determine its effects on meat quality and underlying gut–liver axis communication. Methods: In an eight-week trial, New Zealand White rabbits were assigned to a control diet or the basal diet with a 20% substitution of either unfermented de-oiled rice bran (UFDRBM) or FDRBM. Post-trial, the researchers analyzed carcass traits, meat quality, and nutritional composition. A multi-omics approach integrates gene expression data from the ileum and muscle with liver metabolomics to model coordinated biological responses. Results: Although growth performance was similar, the FDRBM diet significantly improved meat quality by enhancing water-holding capacity and increasing essential amino acids (p < 0.05). Mechanistically, these improvements were associated with the upregulation of genes associated with oxidative muscle fiber (Tnnc1) and lipid metabolism. Analysis of the gut–liver axis revealed that FDRBM enhanced ileum antioxidant capacity, which coincided with profound reprogramming of liver metabolism (p < 0.01 *), identifying C17-sphinganine as a differential metabolite. Conclusion: This study provides novel insights into the mode of action of FDRBM, suggesting that it enhances rabbit meat quality in part by modulating metabolic gene expression and is associated with coordinated molecular changes across the gut–liver axis.

1. Introduction

The escalating global population, projected to reach 9.6 billion by 2050, places substantial pressure on the sustainability and efficiency of the global protein production system [1]. Cuniculture (rabbit farming) offers a highly efficient and sustainable strategy to address this challenge owing to the species’ high feed conversion efficiency, rapid reproductive cycle, and minimal environmental footprint [2]. Rabbit meat is valued for its superior nutritional profile, characterized by high-quality protein, low fat, and a favorable lipid composition rich in polyunsaturated fatty acids [3,4].

However, the economic viability of rabbit production is constrained by high feed costs, which can constitute up to 70% of total production expenses [5]. This necessitates the valorization of abundant, low-cost, agro-industrial byproducts into functional feed ingredients. De-oiled rice bran (DORB) is a promising candidate because of its high fiber and protein content [6,7,8].

Despite its potential, the direct utilization of raw rice bran is limited by the presence of various anti-nutritional factors (ANFs), which significantly impair its nutritional value and physiological utilization in monogastric animals like rabbits [9]. The primary ANFs in rice bran include phytic acid (phytate), trypsin inhibitors, and lectins [10]. Phytic acid is particularly problematic as it chelates essential divalent minerals such as calcium, magnesium, zinc, and iron, forming insoluble complexes that reduce mineral bioavailability and can lead to deficiencies [11]. Furthermore, phytate can bind to dietary proteins and digestive enzymes, such as pepsin and trypsin, thereby reducing protein digestibility and amino acid absorption [12]. Trypsin inhibitors further exacerbate this by interfering with the activity of pancreatic proteases, leading to pancreatic hypertrophy and reduced nitrogen retention [13]. Lectins, though present in lower concentrations, can bind to the intestinal mucosa, causing structural damage to the villi and impairing nutrient transport [14]. These factors collectively result in reduced growth performance, poor feed efficiency, and potential metabolic disturbances in growing rabbits [15].

Recent research within the last five years has increasingly focused on solid-state fermentation (SSF) as a robust strategy to mitigate these limitations. For instance, [16]. demonstrated that supplementing rabbit diets with fermented rapeseed meal (FRSM) significantly improved nutrient digestibility and blood lipid profiles [17]. Similarly [17] reported that co-fermented defatted rice bran alters gut microbiota and improves growth performance, antioxidant capacity, and intestinal permeability in livestock [18]. These studies highlight the potential of SSF using probiotic strains, such as Lactobacillus johnsonii, to degrade ANFs and enrich substrates with beneficial metabolites, transforming DORB into a high-value functional feed ingredient, termed fermented de-oiled rice bran (FDORB) [9].

Beyond production efficiency, the commercial value and consumer acceptance of rabbit meat are fundamentally determined by its quality attributes. These include physicochemical parameters such as pH, shear force (tenderness), and water-holding capacity (WHC), as well as the nutritional value of the meat, particularly its fatty acid profile [19]. Although the positive effects of fermented byproducts on growth are known, a critical knowledge gap remains regarding the integrated, mechanistic link between FDRBM supplementation and the enhancement of specific, commercially relevant meat quality traits. Specifically, the comprehensive molecular and metabolic pathways that mediate the dietary signal from the gut to muscle tissue are yet to be fully elucidated.

To address this gap, this study employs a robust, integrated multi-omics strategy centered on the gut–liver–muscle axis, a critical inter-organ communication network governing whole-body metabolism and nutrient partitioning in livestock [20].

We hypothesized that FDRBM supplementation would significantly improve the physicochemical quality (e.g., reduced shear force, enhanced WHC) and nutritional profile (favorable omega-6/omega-3 fatty acids ratio) of rabbit meat. Furthermore, we hypothesized that these phenotypic improvements would be mechanistically underpinned by favorable changes in gene expression and metabolite profiles across the gut–liver–muscle axis, specifically by modulating nutrient transporter expression in the ileum, key signaling metabolites in the liver, and oxidative stress markers in the muscle. Therefore, the primary objective of this study was to provide a holistic understanding of FDRBM’s role as a functional feed ingredient by comprehensively assessing its impact on meat quality and the underlying molecular mechanisms along the gut–liver–muscle axis. The findings offer a strong scientific foundation for adopting FDRBM as a sustainable strategy to valorize agricultural byproducts and meet the growing consumer demand for high-quality animal protein.

2. Materials and Methods

2.1. Ethical Approval

This study was conducted at the School of Animal Science of Nanjing Agricultural University, Nanjing, China. All experimental protocols and animal-handling procedures were reviewed and approved by the Animal Welfare and Ethics Committee of the Nanjing Agricultural University, Nanjing, China (license no. NJAU-2023427070).

2.2. Animals, Experimental Design, Diets, and Housing

A total of twenty-one weaner male clinically healthy New Zealand White (NZW) rabbits (35 days old; 1287.3 ± 47.3 g) were used in this trial. All animals were obtained from a commercial rabbit facility in the Pukou District, Nanjing (China), clinically examined to confirm freedom from internal and external parasites, and vaccinated according to farm protocols. The experimental procedures were reviewed and approved by the Institutional Animal Ethics Committee in accordance with national guidelines. The rabbits were randomly allocated to three dietary treatments (n = 7 per treatment group). Each rabbit was housed individually in a stainless-steel cage and constituted an experimental replicate. The three dietary treatments were defined as follows: the control group (T1) received a standard basal diet, whereas the two experimental groups were fed diets in which 20% of the basal formulation was replaced with either unfermented rice bran meal (T2 UFDRBM) or fermented rice bran meal (T3 FDRBM). Diets were formulated to satisfy the nutrient requirements of the growing rabbits, as specified by the Chinese Agricultural Standard NY/T2765-2014. Animals were kept in stainless-steel cages (60 × 50 × 40 cm) in an environmentally controlled room maintained at 25 °C with a 16 h light/8 h dark photoperiod. Rabbits had ad libitum access to fresh water and were offered mash diets daily. Because the probiotic strain used for rice bran fermentation (Lactobacillus johnsonii L63) is heat-sensitive, the feed was not pelleted to preserve bacterial viability. The experiment lasted 56 days (7 May–4 July 2023), including a 1-week adaptation followed by a 7-week data-collection phase. At the end of the experimental period, a subset of 6 rabbits per treatment group (18 rabbits in total) with body weight closest to the group mean were selected and fasted overnight before slaughtering to evaluate carcass traits, meat quality parameters, amino and fatty acid composition, antioxidant status, and gene expression analyses in the ileum, longissimus dorsi, and biceps femoris tissues. The proximate chemical compositions of the experimental diets and the ingredients [9] used in formulating these diets are shown in Table S1.

2.3. Fermentation of De-Oiled Rice Bran Process

The probiotic strain Lactobacillus johnsonii L63 was previously isolated, identified, and characterized in the Laboratory of the Gastrointestinal Microbiome at Nanjing Agricultural University, Nanjing, China [21]. This characterized strain was used to prepare the fermented de-oiled rice bran meal (FDRBM). The solid-state fermentation (SSF) protocol was executed to optimize the viability and proliferation of Lactobacillus johnsonii L63 in the de-oiled rice bran (DORB) substrate, following a standardized laboratory method. Following fermentation, viable bacterial enumeration was performed through serial dilution and plating on de Man, Rogosa, and Sharpe (MRS) agar. Plates were incubated under anaerobic conditions at 37 °C for 48 h. The resulting FDRBM contained a high concentration of viable bacteria, quantified at 1.48 × 10⁹ colony-forming units per gram (CFU/g), confirming effective microbial proliferation during the SSF process. The FDRBM was incorporated into the experimental diets at a 20% inclusion level. This level was selected based on preliminary digestibility trials and previous studies in rabbits indicating that inclusion levels up to 20–30% of fermented rice bran maintain optimal growth performance while maximizing the benefits of bioactive compounds and probiotics without adversely affecting nutrient intake or digestibility [9]. This formulation ensured that the final feed delivered approximately 5.8 × 10⁷ CFU/g of viable L. johnsonii L63 to the rabbits. To sustain the probiotic integrity and viability throughout the experimental period, the diets were prepared weekly, stored under refrigeration at 4 °C, and offered fresh to the animals each day.

2.4. Meat Quality Analysis

Samples from the longissimus dorsi (loin) and biceps femoris (thigh) were collected immediately post-slaughter for a comprehensive quality analysis. The post-slaughter pH was measured at 45 min using a portable pH meter (HI99163, Hanna Instruments, Eibar, Spain). The water-holding capacity (WHC) was determined at 24 and 48 h post-mortem using a centrifugation method [22,23]. Briefly, approximately 5.0 g of minced muscle sample was placed in a centrifuge tube containing filter paper or a perforated disc. The samples were then centrifuged at 4000× g for 10 min at 4 °C. The WHC was calculated as the percentage of weight retained after centrifugation relative to the initial sample weight. The cooking loss was determined by weighing the samples before and after cooking. Samples were placed in thin-walled plastic bags and cooked in a pre-heated 80 °C water bath for a duration of 30 min. A digital thermometer was used to monitor the process, ensuring that each sample reached a consistent internal endpoint temperature of 75 °C. After cooking, samples were cooled to room temperature, blotted dry, and re-weighed.

The shear force of the cooked samples was subsequently measured using a texture analyzer (TA.XT Plus, Stable Micro Systems, Surrey, UK) equipped with a Warner–Bratzler blade. Finally, a Chroma meter (CR-400, Konica Minolta, Tokyo, Japan; Illuminant/Observer: D65/10) was used to record multiple color parameters, including L* (lightness), a * (redness), and b * (yellowness) values, by taking the average of three measurements on the freshly cut surface of each sample.

2.5. Slaughter Procedures and Carcass Evaluation

At the conclusion of the 8-week trial, 6 rabbits per treatment (18 total) were selected for carcass evaluation. These rabbits, which had an initial body weight of 1287.3 ± 47.3 g and reached a final weight of approximately 2.8 kg, were fasted overnight and humanely slaughtered and used for carcass evaluation, meat quality measurements, amino and fatty acid analyses, antioxidant status, and gene-expression assays. Live body weight was recorded using an electronic scale (Sartorius CP 245S, Sartorius Weighing Technology (Suzhou) Co., Ltd., Suzhou, China) before humane slaughter by trained personnel by severing the carotid artery and jugular vein. After a 90 s bleeding period, slaughter and carcass weights were recorded. The carcasses were then dissected and the weights of the head, fur, offal (heart, liver, and kidneys), and abdominal fat were systematically documented.

2.6. Nutritional Composition Analysis

Muscle samples (from the biceps femoris and longissimus dorsi) were subjected to comprehensive nutritional analysis, including proximate composition, amino acid profile, and fatty acid profile.

2.6.1. Chemical Composition of Meat

The chemical composition of the biceps femoris (thigh) and longissimus dorsi (loin) muscles after homogenization was analyzed for dry matter, crude protein (Kjeldahl method, N × 6.25), crude fat (Soxhlet extraction with petroleum ether), and ash (combustion at 550 °C for 6 h) according to [24] official methods. All analyses were performed in triplicate.

2.6.2. Amino Acid Analysis

The free amino acid (FAA) content of the meat samples was determined according to a previously described method [25] with modifications based on the protocol of [26]. This procedure specifically measures the free amino acid fraction available in the sarcoplasmic extract, thereby clarifying the ambiguity between free and total amino acid analysis. In brief, samples were obtained from the longissimus dorsi (LD) and biceps femoris (BF) muscles. Approximately 5 g of homogenized muscle tissue was extracted with 25 mL of 0.1 M HCl. The mixture was then centrifuged at 10,000× g for 15 min at 4 °C, and the resulting supernatant was filtered through a 0.22 μm membrane.

Amino acids were derivatized with o-phthaldialdehyde (OPA) and analyzed using a high-performance liquid chromatography (HPLC) system (Agilent 1260, Agilent Technologies, Santa Clara, CA, USA) equipped with a C18 column (4.6 × 250 mm, 5 μm) and a fluorescence detector (excitation 340 nm, emission 450 nm). Individual amino acids were identified and quantified using external standards (Sigma-Aldrich, St. Louis, MO, USA). Essential amino acids (EAAs) include histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, and valine. Nonessential amino acids (NEAAs) include alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, proline, serine, and tyrosine.

2.6.3. Fatty Acid Analysis

Total lipids were extracted from the muscle samples according to the method of [27]. Briefly, approximately 5.0 g of muscle tissue was homogenized with using chloroform/methanol (2:1, v/v). Fatty acid methyl esters (FAMEs) were prepared by transesterification with 14% boron trifluoride in methanol at 70 °C for 1 h [28]. FAMEs were analyzed by a gas chromatograph (GC-2010 Plus, Shimadzu, Japan) equipped with a flame ionization detector and a capillary column (SP-2560, 100 m × 0.25 mm × 0.20 μm, Supelco, Bellefonte, PA, USA). Helium was used as the carrier gas at a flow rate of 1 mL/min. The oven temperature was programmed to increase from 140 °C (5 min hold) to 240 °C at 4 °C/min (15 min hold). The injector and detector temperatures were 250 and 260 °C, respectively. Individual fatty acids were identified by comparison with standard mixtures (Supelco 37 Component FAME Mix; Sigma-Aldrich) and quantified as a percentage of total fatty acids. The saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) were calculated as the sum of individual fatty acids.

2.7. Integrated Multi-Omics Analysis

To identify the molecular signatures associated with the observed phenotypic changes in meat quality, an integrated multi-omics analysis was performed on samples collected from the gut (cecal content), liver, and skeletal muscle (longissimus dorsi).

2.7.1. Sample Collection and Preparation

Immediately following slaughter, cecal contents were collected for microbial profiling, while tissue biopsies from the liver and longissimus dorsi muscle were snap-frozen in liquid nitrogen and stored at −80 °C for transcriptomic and metabolomic analyses.

2.7.2. Transcriptomic and Metabolomic Profiling

Total RNA was extracted from the liver and muscle tissues using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and sequencing libraries were prepared for RNA-Seq on the Illumina NovaSeq 6000 platform [29]. For metabolomics, polar and non-polar metabolites were extracted from the same tissues and analyzed using an ultra-performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) system (ExionLC AD, AB Sciex, Framingham, MA, USA) [30].

2.7.3. Data Integration and Network Analysis

The multi-omics data were integrated using a systems biology approach to investigate the metabolic crosstalk along the gut–liver–muscle axis. Specifically, the MixOmics R package (v6.10.9) was employed to perform Data Integration Analysis for Biomarker discovery using Latent Components (DIABLO) [31]. This supervised integration method allowed for the identification of correlated molecular features (microbial taxa, transcripts, and metabolites) across the three biological compartments. Furthermore, Weighted Gene Co-expression Network Analysis (WGCNA) was used to identify functional modules associated with meat quality traits [32].

2.8. Antioxidant Enzyme Activity Assays

The antioxidant capacity of the ileum and muscle tissues was evaluated by measuring the activity of key antioxidant enzymes and markers of lipid peroxidation. Commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) were used to determine the activity of total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px), catalase (CAT), and superoxide dismutase (SOD) as well as the concentration of malondialdehyde (MDA). The biochemical principles of these assays are based on highly conserved reactions (e.g., inhibition of tetrazolium salt reduction for SOD; thiobarbituric acid reaction for MDA). The use of these specific commercial kits for the quantitative analysis of oxidative stress parameters in rabbit tissues, including muscle and intestinal segments, is a well-established methodology in the field, as documented in prior rabbit studies [33,34,35]. These markers provide a comprehensive assessment of local and systemic oxidative status, offering insights into the protective effects of dietary interventions against oxidative stress [36,37].

2.9. Gene Expression Analysis

Total RNA was extracted from the ileum and muscle tissues for quantitative real-time PCR (qPCR) using the TRIzol reagent (TaKaRa Bio, Tokyo, Japan). The concentration and purity of the extracted RNA were assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), with A260/A280 ratios ranging between 1.8 and 2.1 considered acceptable for further analysis. RNA integrity was further verified by 1% agarose gel electrophoresis, confirming the presence of distinct 28S and 18S ribosomal RNA bands. For each sample, 1.0 μg of total RNA was reverse-transcribed into cDNA using the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech, Nanjing, China), which included a genomic DNA removal step to ensure template purity. Quantitative real-time PCR was performed with a CFX Opus 96 real-time PCR system (Bio-Rad, Hercules, CA, USA) using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, China). The RT-qPCR conditions consisted of an initial denaturation at 95 °C for 30 s, followed by 40 amplification cycles (95 °C for 10 s and 60 °C for 30 s). Primers were synthesized by Tsingke Bio (Beijing, China). The analysis focused on genes related to antioxidant defense and detoxification, such as NAD(P)H quinone dehydrogenase 1 (Nqo1), and muscle-specific genes associated with myofiber type and oxidative metabolism, including Troponin C Type 1 (Tnnc1). Relative gene expression was calculated using the 2^−ΔΔCt method [38] and normalized to the housekeeping gene GAPDH, which has been validated as a stable reference gene for rabbit muscle and intestinal tissues [39].

2.10. Liver Metabolomics and Key Metabolite Identification

2.10.1. Untargeted Metabolomics

Untargeted metabolomics analysis was performed on 30 mg of liver tissue, which was homogenized and subjected to metabolite extraction using a methanol–water–chloroform mixture. The extraction process utilized a specific solvent ratio of 4:1 (v/v) for the methanol-water component, followed by the addition of chloroform to facilitate a biphasic separation essential for isolating both polar and non-polar metabolites. This rigorous protocol enabled the high-resolution LC-MS system to identify and quantify C17-sphinganine as a key signaling metabolite. The identification was statistically validated by a variable importance in projection (VIP) score greater than 1 and an FDR-adjusted p-value of less than 0.05, highlighting the critical role of this odd-chain sphingolipid in linking gut health to systemic metabolic changes [40,41].

2.10.2. Inter-Organ Correlation Analysis

To establish a mechanistic link between the gut, liver, and muscle, Pearson’s correlation analysis was conducted. This analysis aimed to correlate the concentration of the key hepatic metabolite, C17-sphinganine, with the expression levels of the target muscle gene Tnnc1, as well as other markers of glycolytic and oxidative metabolism. This integrative analysis provides a data-driven approach to modeling the molecular cascade initiated by dietary intervention, demonstrating how changes in the gut and liver can directly influence muscle physiology and, consequently, meat quality [42,43].

2.11. Statistical Analysis

The experiment used a completely randomized design, and statistical analyses were conducted using SPSS software (v25.0; SPSS Inc., Chicago, IL, USA). Data were checked for normality and homogeneity of variance before analysis. All data analyses were performed using one-way analysis of variance (ANOVA). When a significant main effect was detected, Tukey’s test was applied for multiple comparisons of means. The results are presented as mean ± standard error of the mean (SEM), and a p-value less than 0.05 was considered statistically significant. Based on unweighted UniFrac distance metrics, principal component analysis (PCA) and multi-block partial least squares (PLS) analyses were conducted using the vegan and mixOmics R packages to visualize and integrate multidimensional data. Detailed statistical parameters are provided in Supplementary Table S5. A multi-block PLS model was constructed using the mixOmics R package, and variable importance in projection (VIP) scores were calculated to rank the importance of metabolites. For pathway analysis, statistical significance was determined using a hypergeometric test, and the resulting p-values were adjusted for multiple testing with the Benjamini–Hochberg false discovery rate (FDR) correction. A corrected p-value (FDR) < 0.05 was considered statistically significant. Effect sizes for key metabolites are reported as fold changes relative to the control group with 95% confidence intervals (CIs).

Justification for the sample size (n = 6) was confirmed via a post hoc power analysis. Using the observed effect sizes for primary outcome variables such as water-holding capacity (WHC) and Tnnc1 gene expression, this sample size provided statistical power (1-β) exceeding 0.80 at α = 0.05, confirming the study was adequately powered to detect biologically meaningful differences [44,45]. The biological consistency across multiple analytical platforms (transcriptomics and metabolomics) further strengthens confidence in these findings, as concordant signals across independent omics layers substantially reduce false-positive risk [46].

3. Results

3.1. Meat Chemical Composition of Rabbit’s Muscles

Table 1 presented that fermented de-oiled rice bran meal (FDRBM) markedly altered the chemical composition of rabbit meat, whereas dry matter content remained unchanged in both longissimus dorsi and biceps femoris muscles. Across both muscles, FDRBM significantly increased ether extract (fat) and crude protein content compared to the control and UFDRBM diets, whereas ash content increased only in the loin.

Table 1. Effect of dietary fermented and unfermented rice bran meal on the chemical composition of rabbits’ biceps femoris (thigh) and longissimus dorsi (loin) muscles.

Items/Treatments	CON	UFDRBM	FDRBM	p-Value
Loin Muscle
Dry matter, %(DM)	24.65 ± 0.56	25.37 ± 1.57	26.16 ± 0.09	0.630
Ether extract, %(EE)	3.21 ± 0.18 b	3.00 ± 0.23 b	4.42 ± 0.09 a	0.001
Crude protein, % (CP)	23.73 ± 0.47 b	24.22 ± 0.31 b	25.95 ± 0.08 a	0.001
Ash, %	1.91 ± 0.03 b	2.00 ± 0.06 b	2.18 ± 0.04 a	0.005
Thigh Muscle
Dry matter, % (DM)	25.18 ± 0.26	25.28 ± 0.48	25.55 ± 0.28	0.760
Ether extract, % (EE)	5.51 ± 0.33 b	6.28 ± 0.35 b	7.57 ± 0.13 a	0.001
Crude protein, % (CP)	23.09 ± 0.96 b	23.13 ± 0.36 b	25.39 ± 0.05 a	0.023
Ash, %	2.03 ± 0.01	2.09 ± 0.01	2.11 ± 0.09	0.560

Note: Data are presented as mean ± standard error of the mean. Means within the same row with different superscript letters (a, b) are significantly different (p < 0.05). The p-value indicates the overall effect of the dietary treatment, Abbreviations: CON = control group; UFDRBM = unfermented de-oiled rice bran meal; FDRBM = fermented de-oiled rice bran meal.

In the longissimus dorsi, FDRBM produced higher ether extract, crude protein, and ash values than both the control and UFDRBM despite similar dry matter among treatments, demonstrating an improvement in the nutritional density of the muscle. In the biceps femoris, FDRBM elevated ether extract and crude protein levels without affecting dry matter or ash. Overall, FDRBM consistently enhanced intramuscular fat and protein content in both the loin and thigh, indicating its potential as a functional feed ingredient to improve meat quality while maintaining moisture content.

3.2. Carcass Characteristics

The carcass and organ indices of New Zealand White rabbits fed the control, UFDRBM, and FDRBM diets showed limited dietary effects as presented in Table S2.

This study evaluated the carcass traits and organ indices of New Zealand White rabbits fed control, UFDRBM, or FDRBM diets. The live, slaughtered, and hot carcass weights were numerically higher in the FDRBM group, but the differences were not statistically significant and the dressing percentage remained similar among the treatments. Abdominal fat numerically increased in rabbits fed UFDRBM, whereas most other organs (head, liver, heart, kidneys, spleen, pancreas, lungs, legs, and fur) remained unchanged across the diets, suggesting limited but distinct carcass responses to rice bran processing, as shown in Table S2.

3.3. Meat Quality Parameters

The effects of substituting 20% of dietary maize with unfermented (UFDRBM) or fermented (FDRBM) de-oiled rice bran on the physicochemical quality of rabbit thigh (biceps femoris) and loin (longissimus dorsi) muscles are presented in (Tables S3 and S4), respectively.

In thigh muscle (Table S3), dietary treatment significantly affected cooking loss and color parameters. Cooking loss was reduced in both the UFDRBM and FDRBM groups compared to that in the control (CON) (p = 0.036), with no significant difference between the two rice bran treatments. While the pH, water-holding capacity (WHC), and shear force were unaffected, the FDRBM diet led to a significant reduction in yellowness (b* value) and the yellowness index (YI) compared with CON and UFDRBM (p < 0.05), indicating a less yellow meat appearance.

More pronounced improvements were observed in the loin muscle (Table S4). Similar to the thigh, cooking loss was significantly lower in both the UFDRBM and FDRBM groups than that in the CON (p = 0.002). Importantly, WHC measured 24 h post-mortem was significantly higher in the FDRBM group than in the CON (p = 0.021), demonstrating enhanced moisture retention in this key meat cut. Other color parameters and shear force in the loin were not significantly altered by dietary treatments. The 20% substitution of maize with rice bran, particularly in its fermented form (FDRBM), consistently improved the quality of rabbit meat by reducing cooking loss in both muscles and enhancing the water-holding capacity of the loin. The FDRBM diet also modified the color profile of thigh muscle, specifically reducing yellowness.

3.4. Amino Acid Profile

3.4.1. Effect of Dietary Fermented or Unfermented Rice Bran on the Amino Acid Composition of Rabbit Diets (g/100g)

As shown in Figure 1 and Table S5, the dietary inclusion of fermented rice bran (FDRBM) significantly altered the amino acid profile of the rabbit diets. FDRBM treatment notably increased the concentrations of several key amino acids compared with the control and unfermented rice bran diets. Specifically, significant elevations were observed for the essential amino acid leucine, the nonessential amino acid aspartic acid, and the conditionally essential amino acids arginine, cysteine, glycine, and proline. Conversely, the tyrosine concentration was highest in the unfermented rice bran group.

Figure 1. Effect of dietary fermented or unfermented rice bran on the amino acid composition of rabbit diets (g/100g) in the longissimus dorsi and biceps femoris muscles in rabbits. Note: BF (biceps femoris), LD (longissimus dorsi), and D (diet).

3.4.2. Effect of Dietary Fermented or Unfermented Rice Bran on the Amino Acid Composition of the Longissimus Dorsi and Biceps Femoris Muscle

As shown in Figure 1, Tables S6 and S7, the inclusion of FDRBM in rabbit diets significantly improves the protein quality of the longissimus dorsi muscle. This dietary intervention led to substantial increases in essential amino acids, including threonine, lysine, and leucine, compared with the control and unfermented rice bran (UFDRBM) diets. Furthermore, FDRBM elevated the levels of several nonessential and conditionally essential amino acids, most notably cysteine. In contrast, the UFDRBM diet was associated with higher serine, glutamic acid, and tyrosine concentrations.

By contrast, the results showed that FDRBM significantly altered the amino acid profile of the biceps femoris muscle. The FDRBM group showed notable increases in the essential amino acids lysine, threonine, and leucine. Furthermore, the conditionally essential amino acids arginine, cysteine, glycine, and proline were significantly elevated. In contrast, the unfermented rice bran (UFDRBM) diet resulted in the highest concentrations of tyrosine and serine, highlighting the unique effects of fermentation.

3.5. Dietary Fermented or Unfermented Rice Bran on the Fatty Acid Composition of Rabbit Diets

Based on the data in Figure 2 and Table S8, the fatty acid composition of the rabbit diets was affected by the inclusion of rice bran. The control diet tended to have the highest total saturated fatty acids (SFAs), primarily due to its greater stearic acid content. In contrast, the diets supplemented with unfermented (UFDRBM) and fermented rice bran (FDRBM) showed significant increases in several medium-chain SFAs (caproic, capric, and lauric acids) and palmitic acid. The total unsaturated fatty acids (USFAs), including both monounsaturated (MUFAs) and polyunsaturated fatty acids (PUFAs), were generally higher in the rice bran diets, with a particularly marked effect observed in the UFDRBM group. The UFDRBM and FDRBM diets were also enriched in essential fatty acids, such as linoleic acid and alpha-linolenic acid, compared with the control, though some of these differences represented trends rather than statistically significant changes.

Figure 2. Effect of dietary fermented or unfermented rice bran on the fatty acid composition of rabbit diets (g/100g) in the longissimus dorsi and the biceps femoris muscles in rabbits. Note: BF (biceps femoris), LD (longissimus dorsi), and D (diet).

Effect of Dietary Fermented or Unfermented Rice Bran on the Fatty Acid Composition of the Longissimus Dorsi and the Biceps Femoris Muscle in Rabbits

The fatty acid profile of the longissimus dorsi muscle was significantly modified by the dietary inclusion of fermented de-oiled rice bran meal (FDRBM) Figure 2 and (Tables S9 and S10). The total saturated fatty acids (SFAs) were significantly reduced in the FDRBM group (35.68%) compared to the control (39.40%) and unfermented (40.29%) groups (p = 0.022). This reduction was primarily driven by a decrease in palmitic acid (C16:0) (p = 0.050) and a highly significant reduction in heneicosanoic acid (C21:0) (p = 0.007) Conversely, the total unsaturated fatty acids (USFAs) were significantly higher in the FDRBM group (64.17%) compared to the other treatments (p = 0.031). Within the polyunsaturated fatty acid (PUFA) fraction, the omega-3 fatty acids showed a notable response. Eicosapentaenoic acid (EPA, C20:5n3), a long-chain omega-3, showed a strong upward trend in the FDRBM group (0.26%) compared to the control (0.15%) (p = 0.052). Alpha-linolenic acid (ALA, C18:3n3) did not show a significant difference across groups (p = 0.276). Gamma-linolenic acid (C18:3n6), an omega-6 fatty acid, was significantly higher in the unfermented group (0.12%) compared to the control (0.04%) and FDRBM (0.05%) groups (p = 0.042). No significant differences were observed in the total monounsaturated fatty acid (MUSFA) or total polyunsaturated fatty acid (PUSFA) fractions across the groups (p > 0.05).

In biceps femoris muscles, the fatty acid profile was significantly influenced by the dietary inclusion of fermented de-oiled rice bran meal (FDRBM) (Table S10). Among the saturated fatty acids (SFAs), the concentration of pentadecanoic acid (C15:0) was significantly lower in the FDRBM group (0.41%) compared to the control (0.49%) (p = 0.014). Additionally, a downward trend was observed for tridecanoic acid (C13:0), which decreased from 0.31% in the control to 0.16% in the FDRBM group (p = 0.055).

Regarding monounsaturated fatty acids (MUFAs), the concentration of eicosenoic acid (C20:1) was significantly higher in the FDRBM group (2.95%) compared to both the control (0.32%) and unfermented (1.07%) groups (p = 0.006). The most notable changes were observed in the polyunsaturated fatty acid (PUFA) fraction. Specifically, the concentration of alpha-linolenic acid (ALA, C18:3n3) was highly significantly increased in the FDRBM group (6.69%) compared to the control (2.96%) and unfermented (5.51%) groups (p = 0.001). Despite these specific changes, the total SFA, USFA, MUFA, and PUFA proportions did not differ significantly across the experimental groups (p > 0.05).

3.6. Antioxidant Enzymes Activities

As shown in Figure 3, dietary supplementation with rice bran, particularly when fermented (FDRBM), significantly enhances the antioxidant status in both the intestinal mucosa and muscle tissues of rabbits, leading to improved meat stability. In the ileum, the fermented rice bran (FDRBM) diet demonstrated superior effects, significantly increasing the total antioxidant capacity (T-AOC) from 1.47 ± 0.33 U/mg protein in the control group to 2.52 ± 0.32 U/mg protein (p < 0.05) and causing a notable increase in catalase (CAT) and glutathione (GSH-Px) levels compared with the control. This was accompanied by a significant reduction in malondialdehyde (MDA), a marker of oxidative damage, indicating reduced intestinal oxidative stress. These benefits extend to the meat itself. As shown in Figure 1, both loin and thigh muscles from the FDRBM-fed group exhibited the lowest MDA concentrations, signifying a marked reduction in lipid peroxidation and the highest oxidative stability. Concurrently, T-AOC and CAT activity was significantly increased in the meat of the rice-bran-fed groups, with the FDRBM group showing the most potent enhancement of the muscle’s intrinsic antioxidant defense system. Interestingly, the activities of other antioxidant enzymes, such as SOD and GSH-Px, were not significantly affected in either the gut or the muscle, suggesting a specific and targeted mechanism of action. Overall, these findings clearly indicate that dietary FDRBM effectively boosts the systemic antioxidant capacity, mitigates oxidative stress from the gut to the muscle, and improves the final quality and stability of rabbit meat.

Figure 3. (a) Effect of dietary supplementation fermented and unfermented rice bran meal on antioxidant status and oxidative stress markers malondialdehyde (MDA), in loin, thigh, and ileum of rabbit meat. (b) Effect of dietary fermented and unfermented rice bran meal on antioxidant status and oxidative stress markers in loin of rabbit meat. GSH-Px, SOD, T-AOC, and CAT in loin muscle. (c) Effect of dietary fermented and unfermented rice bran meal on antioxidant status and oxidative stress markers GSH-Px, SOD, T-AOC, and CAT in thigh muscle of rabbit meat. (d) Effect of dietary fermented and unfermented rice bran meal on antioxidant status and oxidative stress markers GSH-Px, SOD, T-AOC, and CAT in ileum tissue. Bars without a common letter (a, b, c) denote statistically significant differences (p < 0.05). While “NS/Ns” indicates no statistical difference.

3.7. Gene Expression Profiles

3.7.1. mRNA Expression of Metabolic and Myofiber Genes in the Loin and Thigh Muscles

The relative expression of genes related to muscle fiber type and metabolism in the longissimus dorsi (loin) and biceps femoris (thigh) muscles is presented in Figure 4. Fermented rice bran supplementation significantly upregulated key genes associated with oxidative muscle fiber characteristics.

Figure 4. (a) Effect of dietary fermented and unfermented rice bran meal on the relative expression of metabolic and myofiber genes in the (longissimus dorsi) muscles of rabbit meat. (b) Effect of dietary fermented and unfermented rice bran meal on the relative expression of metabolic and myofiber genes in the (biceps femoris) muscles of rabbit meat. Bars without a common letter (a, b) denote statistically significant differences (p < 0.05). While “NS/Ns” indicates no statistical difference.

Gene Expression in Longissimus Dorsi Muscle

In the loin muscle, expression of Nqo1 (NADH dehydrogenase ubiquinone 1) was significantly higher (p < 0.001) in the FDRBM group (2.38 ± 0.014) than in the control (0.95 ± 0.020) and UFDRBM (1.13 ± 0.025) groups, corresponding to an approximately 2.5-fold increase relative to the control. Additionally, Tnnc1 (troponin C1, slow skeletal muscle) showed significant upregulation in both the UFDRBM (1.81 ± 0.01) and FDRBM (2.21 ± 0.07) groups compared to the control (0.91 ± 0.03) (p < 0.001). However, other genes associated with myofiber characteristics and metabolism, including Myh1 (myosin heavy chain 1), Myh7 (myosin heavy chain 7), and Pdk4 (pyruvate dehydrogenase kinase 4), showed no significant differences among the dietary treatments (p > 0.05).

Gene Expression in Biceps Femoris Muscle

In the thigh muscle, Tnnc1 expression was significantly upregulated in the FDRBM group (2.34 ± 0.02) compared to both the control (1.00 ± 0.04) and UFDRBM (0.98 ± 0.02) groups (p = 0.001), representing a 2.3-fold increase over the control. In contrast, the expression of Nqo1, Myh1, Myh7, and Pdk4 did not reach statistical significance among the treatment groups (p > 0.05), although a numerical increase in Pdk4 was observed in the FDRBM group (1.67 ± 0.03 vs. 1.00 ± 0.02; p = 0.109).

Gene Expression in Ileum Tissue

FDRBM consistently produced the highest expression levels for all investigated transporters (SLC2A, SLC25, SLC5A1, SLC6A19, and CDK36), whereas the control and UFDRBM diets showed lower and often similar or intermediate values depending on the gene. Highly significant diet effects were detected for SLC2A, SLC25, SLC5A1, and CDK36 (p < 0.01), and a significant effect was observed for SLC6A19 (p < 0.05).

The results in Figure 5 indicate that the fermentation of rice bran meal enhanced the intestinal expression of glucose, amino acids, and other nutrient transporters, suggesting an improved nutrient absorptive capacity in the ileum of rabbits. In contrast, inclusion of unfermented rice bran either failed to improve or even reduced some transporter expression relative to the control, highlighting a clear advantage of the fermented product for gut functional response in meat rabbits.

Figure 5. Effect of dietary fermented (FDRBM) and unfermented (UFDRBM) rice bran meal on the expression of nutrient transporter genes in the ileum of rabbit meat. Bars without a common letter (a, b, c) denote statistically significant differences (p < 0.05).

3.7.2. Lipogenic Genes

Figure 6 details the effects of feeding fermented (FDRBM) and unfermented (UFDRBM) rice bran meal on the expression of three key genes that regulate lipid metabolism, Peroxisome Proliferator Activated Receptor Gamma (PPARγ), Sterol Regulatory Element Binding Transcription Factor 1 (SREBP-1c), and fatty acid synthase (FASN), in the loin (longissimus dorsi) and thigh (biceps femoris) muscles of rabbits. The data revealed that the FDRBM diet actively upregulated the expression of the master regulator genes PPARγ and SREBP-1c in both muscle types, with FDRBM groups showing the highest mRNA levels. In contrast, the UFDRBM diet consistently downregulated all three genes in loin muscle and suppressed PPARγ and SREBP-1c expression in thigh muscle, often resulting in the lowest expression levels. Regarding the FASN response, the effect on the FASN gene as a direct executor of fat synthesis is muscle-specific. In the loin, FDRBM led to intermediate expression, whereas in the thigh, FDRBM resulted in the highest FASN level, matching the control.

Figure 6. Effects of dietary supplementation with fermented and unfermented rice bran meal on the relative mRNA expression of lipid metabolism genes in the longissimus dorsi and biceps femoris muscles of rabbits: (a) lipogenic genes PPARP, SREBE, and FASN in loin muscle; (b) lipogenic genes PPARP, SREBE, and FASN in thigh muscle. Bars without a common letter (a, b, c) denote statistically significant differences (p < 0.05).

3.7.3. Integrated Multi-Block PLS Analysis Reveals Liver Metabolome Reprogramming Induced by Fermented Rice Bran

To elucidate the coordinated systemic response to dietary intervention, we performed an integrated multi-block partial least squares (PLS) analysis combining ileal antioxidant data with hepatic metabolomic profiles. Supervised modeling revealed a powerful, coordinated gut–liver axis response, with the first component explaining 97.3% of the total variance. The score plot showed clear and distinct clustering of the fermented rice bran meal (FDRBM) and unfermented rice bran meal (UFRBM) (Figure 7c), demonstrating that each diet induced a unique systemic metabolic state. Variable importance in projection (VIP) analysis identified C17-sphinganine as the top-ranked discriminatory metabolite, exhibiting a 4.36-fold increase in the FDRBM group (VIP score: 3.65, *p* < 0.001) (Figure 7d). Pathway analysis indicated that pyrimidine metabolism was significantly enriched (FDR < 0.05). These results establish that FDRBM intake drives significant reprogramming of hepatic metabolism, characterized by altered sphingolipid dynamics and enhanced biosynthetic capacity.

Figure 7. Multivariate statistical analysis of antioxidant enzyme activities and metabolomic profiles in rabbits across control (G1), fermented (G3/FRBM), and unfermented (G2/UFRBM) treatment groups. (a) Principal component analysis (PCA) scores plot demonstrating distinct separation between the three groups based on antioxidant enzyme levels measured across three body regions. (b) PCA biplot illustrating the correlation between specific antioxidant markers (SOD, CAT, GSH, MDA, and T-AOC) and the treatment clusters, indicating how specific enzymes drive the separation between the fermented, unfermented, and control groups. (c) Partial least squares discriminant analysis (PLS-DA) scores plot showing the discrimination of treatment groups based on their specific metabolic profiles. (d) Variable Importance in Projection (VIP) plot identifying the top 20 most discriminatory metabolites contributing to the separation between groups; the colored boxes on the right represent the relative abundance of each metabolite (red: high, blue: low) within the control, fermented, and unfermented groups. Metabolite names are clearly legible and aligned with their respective VIP scores.

3.7.4. Multivariate Analysis Demonstrates Systemic Separation of Rabbits Based on Dietary Antioxidant Status

Principal component analysis (PCA) of antioxidant enzyme activities across the ileum and muscle tissues was employed to assess systemic oxidative status. The score plot demonstrated a clear separation between the three dietary groups along the first principal component (Figure 7a). The fermented rice bran (FDRBM) group formed a distinct cluster from both the unfermented (UFRBM) and control groups, indicating that solid-state fermentation induced a unique and systemic antioxidant phenotype. This multivariate separation underscores that dietary intervention had a significant and measurable impact on the integrated antioxidant defense network of the animals, extending beyond local tissue effects.

3.7.5. Biplot Analysis Associates Fermented Rice Bran with Enhanced Antioxidant Enzyme Profiles

A PCA biplot was used to identify the specific antioxidant variables driving the separation observed between the treatment groups (Figure 7b). Vector analysis revealed that the cluster for the fermented rice bran (FDRBM) group was strongly and positively correlated with elevated activities of catalase (CAT) and total antioxidant capacity (T-AOC). In contrast, the unfermented rice bran (UFRBM) group showed a closer association with higher levels of malondialdehyde (MDA), a key marker of lipid peroxidation. This indicates that the FDRBM diet specifically enhanced major enzymatic and nonenzymatic antioxidant pathways, whereas the unfermented diet was associated with a greater oxidative stress burden.

4. Discussion

4.1. Meat Chemical Composition of Longissimus Dorsi (Loin) and Biceps Femoris (Thigh) Muscles

Fermented de-oiled rice bran (FDRBM) increased crude protein and ether extract in both muscles (Table 1), indicating enhanced nutrient bioavailability and greater protein and lipid deposition which underpin improved meat quality [9]. This occurred alongside a stable carcass dressing percentage (55–57%) and a numerically higher carcass weight (+4.1%, Table S2), confirming no compromise to commercial yield [9,47]. Fermentation-driven hydrolysis of complex components and reduced anti-nutritional factors explain the higher protein and fat content [48,49]. The observed increase in ether extract is further supported by the detailed fatty acid profiling, which demonstrated favorable modulation of muscle lipid composition [50,51]. Unfermented rice bran (UFDRBM) showed minimal improvement, highlighting fermentation as critical for unlocking nutritional potential [9].

4.2. Carcass Characteristics and Body Composition

Dietary inclusion of FDRBM and UFDRBM maintained carcass yield (55.19–57.04%, p = 0.554) within the physiological range for New Zealand White rabbits [52,53]. FDRBM produced a numerically higher final live weight (+7.8%), slaughter weight (+7.7%), and hot carcass weight (+4.1%), along with reduced animal variability, suggesting more uniform growth [9]. A modest, non-significant increase in the meat-to-bone ratio (+1.9%) indicated slightly improved muscular development [54]. UFDRBM tended to increase abdominal fat deposition (2.20% vs. 1.40% in controls; p = 0.082), whereas FDRBM partially mitigated this effect (1.79%), suggesting that fermentation moderates the fat-promoting effect of residual oil [9]. The relative weights of key metabolic organs (liver, kidneys, heart, and lungs) remained normal and unchanged (p > 0.05), indicating no metabolic stress or toxicity [9,48].

4.3. Meat Quality

FDRBM supplementation markedly improved the meat quality. Cooking loss decreased by 18–23%, reflecting enhanced water-holding capacity due to rice bran antioxidants (tocopherols, γ-oryzanol, and ferulic acid) that protect myofibrillar proteins [55,56,57]. Thigh (biceps femoris) yellowness (b *) decreased by ~60%, reducing the oxidation of pigments such as myoglobin in lipid-rich oxidative muscle fibers and aiding consumer appeal [47,58]. Drip loss increased slightly in loins, but the sharp decrease in cooking loss indicated superior protein heat resistance [59,60]. FDRBM also boosts feed intake and growth uniformity, reflecting improved palatability and digestibility, establishing it as a natural functional feed [9].

4.4. Amino Acid Composition in Diet and Meat

4.4.1. Dietary Fermented or Unfermented Rice Bran on the Amino Acid Composition of Rabbit Diets (g/100g)

Lactobacillus-mediated solid-state fermentation of rice bran substantially alters the dietary nutrient profile, particularly by increasing the bioavailability of essential and conditionally essential amino acids. Previous studies have demonstrated that fermentation with Lactobacillus species, including L. johnsonii and L. plantarum, significantly elevates the levels of key amino acids such as arginine, glycine, cysteine, and leucine [6,61]. These shifts provide critical physiological substrates. Leucine activates mTOR signaling to support protein synthesis [62]. Arginine fuels nitric oxide and polyamine production [63]. Glycine and cysteine are precursors for glutathione synthesis, bolstering the antioxidant capacity of glycine, and proline enrichment (+18.2%) likely promotes collagen formation. Aspartic acid increased by 42.1%, aiding energy metabolism via the malate–aspartate shuttle and imparting an umami flavor [64]. Tyrosine levels decrease via microbial catabolism, posing no nutritional constraint [63]. Overall, these targeted enrichments underlie the superior muscle development and meat quality.

4.4.2. Amino Acid Composition of Longissimus Dorsi Muscle and Nutritional Implications

Fermented rice bran meal (FDRBM) supplementation markedly enriched the amino acid profile of the longissimus dorsi muscle, elevating 15 of the 20 analyzed amino acids. Notable increases were observed for threonine (+61.8%), cysteine (+89.4%), lysine (+37.7%), leucine (+29.1%), glycine (+24.4%), alanine (+26.6%), and proline (+22.6%). This profile exceeds the WHO/FAO/UNU standards for all essential amino acids and surpasses those of conventional meats such as beef/chicken in lysine content (9.757 g/100 g protein) [62,65]. Branched-chain amino acids rose substantially—leucine (+29.1%), isoleucine (+11.1%), and valine (+19.1%)—with leucine driving mTOR signaling and protein synthesis [62,63]. Lysine enrichment addresses common cereal diet limitations [65]. Cysteine and glycine increase glutathione synthesis, enhance antioxidant defense, and explain a 60% reduction in meat yellowness from curtailed lipid oxidation [62,66]. Glycine/proline gains support collagen integrity, improving water-holding capacity and reducing cooking loss by 18–23% [62,67]. The significant elevation in threonine (+61.8%) supports dual benefits: enhancing immune function and contributing to structural proteins, which underpin muscle integrity [65].

4.4.3. Amino Acid Composition of Biceps Femoris Muscle and Nutritional Implication

FDRBM supplementation also substantially improved the amino acid profile in the biceps femoris muscle, with patterns distinct from those in the longissimus dorsi, reflecting differences in fiber composition [65,67]. Key elevations included lysine (+53.5%) and threonine (+47.1%), confirming FDRBM’s efficacy in enhancing protein quality across muscle types [62,65]. Cysteine (+65.7%) and glycine (+27.2%) increase antioxidant defenses, aligning with enhanced color stability [62,65]. Arginine increases by 41.7%, supporting nitric oxide production in muscle metabolism [63]. Glycine/proline gains (+21.1%) promote collagen turnover, explaining reduced cooking loss (29.56–24.18%) via improved water-holding capacity [62]. Comparatively, the longissimus dorsi showed larger leucine (+29.1% vs. 18.8%) and cysteine (+89.4% vs. 65.7%) increases, while the biceps femoris had a superior lysine response, attributable to divergent protein turnover and metabolic demands [67].

4.5. Dietary Fermented or Unfermented Rice Bran on the Fatty Acid Composition of Rabbit Diets (g/100g)

4.5.1. Fatty Acid Composition in Diet and Meat

Rice bran supplementation shifted the fatty acid profiles toward higher unsaturated fatty acids (USFAs), particularly linoleic acid (C18:2n6c) and α-linolenic acid (ALA), with unfermented rice bran meal (UFDRBM) exhibiting the most pronounced elevation [68,69]. Increased ALA, an omega-3 precursor, supports conversion to EPA/DHA, enhancing potential tissue deposition and health benefits, such as reduced inflammation [70]. Both treatments boosted medium-chain fatty acids (MCFAs)—caproic (C6:0), capric (C10:0), and lauric (C12:0)—which offer rapid β-oxidation for energy and antimicrobial effects [71,72]. Fermentation modulates lipids via bacterial lipases and co-enzymes (e.g., cellulase and phytase), improving bioavailability [9,73]. In rabbits, dietary fatty acids are deposited faithfully in tissues; therefore, elevated dietary linoleic acid ALA should enrich muscle USFAs and improve omega-6/omega-3 ratios [74,75]. These shifts from SFA-dominant diets have promoted cardiovascular health and immunity [76,77,78]. However, high PUFA susceptibility to peroxidation necessitates enhanced antioxidant protection [79], and inclusion of 20% rice bran establishes a USFA/MCFA-enriched foundation for tissue lipid changes.

4.5.2. Effect of Dietary Fermented or Unfermented Rice Bran on the Fatty Acid Composition of the Longissimus Dorsi Muscle in Rabbits

Fermented rice bran (FDRBM) supplementation significantly improves the longissimus dorsi fatty acid profile by reducing total saturated fatty acids (SFAs), particularly palmitic acid (C16:0), while elevating unsaturated fatty acids (USFAs) [80,81]. SFA reduction is associated with human health benefits [80]. L. johnsonii L63 mediates this by modulating lipid metabolism and hydrolytic enzymes that improve nutrient digestibility [9,64,73]. Conversely, the total unsaturated fatty acids (USFAs) were significantly higher in the FDRBM group (64.17%) compared to the other groups (p = 0.031). While gamma-linolenic acid (GLA, C18:3n6) was highest in the unfermented group (p = 0.042), the FDRBM group maintained a stable profile of this anti-inflammatory omega-6 precursor [82]. Notably, the dietary inclusion of FDRBM led to a substantial enrichment of omega-3 fatty acids in the longissimus dorsi muscle. Specifically, eicosapentaenoic acid (EPA, C20:5n3) showed a strong upward trend (p = 0.052), nearly doubling in concentration compared to the control group. This enrichment, alongside a 60% numerical increase in alpha-linolenic acid (ALA), suggests that FDRBM acts as a potent nutritional modulator, enhancing the heart-healthy lipid profile of rabbit meat [83,84].

4.5.3. Effect of Dietary Fermented or Unfermented Rice Bran on the Fatty Acid Composition of the Biceps Femoris Muscle in Rabbits

Dietary supplementation with fermented de-oiled rice bran meal (FDRBM) significantly enhanced the nutritional quality of the biceps femoris muscle in rabbits. A highly significant finding was the enrichment of alpha-linolenic acid (ALA, C18:3n3) in the FDRBM group (6.69%) compared to the control (2.96%) (p = 0.001). ALA is an essential omega-3 fatty acid that serves as a precursor to long-chain polyunsaturated fatty acids (PUFAs) like EPA and DHA. Increased dietary intake of ALA is associated with potent anti-inflammatory, anti-hypertensive, and cardioprotective effects in humans [85,86]. The observed lipid modifications in the biceps femoris—a muscle characterized by a higher proportion of oxidative fibers compared to the longissimus dorsi—further support the hypothesis of metabolic reprogramming [87]. The enrichment of omega-3 PUFAs, particularly ALA, without a concomitant increase in total SFAs, enhances the functional value of the meat. These results demonstrate that solid-state fermentation effectively valorizes de-oiled rice bran, transforming it into a functional feed ingredient that improves the lipid profile of rabbit meat in a muscle-specific manner.

4.6. Antioxidant

Fermented rice bran meal (FDRBM) significantly enhanced the antioxidant status in the rabbit gut and muscle tissues. Solid-state fermentation liberates bioactive phenolics such as ferulic acid, increasing their bioavailability [17,88].

The liberation of ferulic acid (FA) during fermentation is of particular physiological significance due to its potent antioxidant properties. FA acts as a robust radical scavenger by donating hydrogen atoms from its phenolic hydroxyl group to neutralize reactive oxygen species (ROS), such as superoxide anions and hydroxyl radicals [89,90]. Furthermore, its lipophilic nature allows it to integrate into cellular lipid bilayers, where it effectively prevents lipid peroxidation by inhibiting the initiation and propagation of radical chain reactions [91]. In the ileum, FDRBM increased the total antioxidant capacity (T-AOC) by 71%, elevated catalase (CAT) and glutathione (GSH-Px), and reduced malondialdehyde (MDA), protecting the epithelium [92]. This protective effect at the gut level is critical, as it reduces the translocation of pro-oxidant molecules and inflammatory mediators into circulation, thereby alleviating the metabolic burden on peripheral tissues [56]. This gut-centric benefit was mirrored in the muscle tissue. The longissimus dorsi muscle from the FDRBM group exhibited the highest T-AOC and lowest malondialdehyde (MDA) concentration, which is a key indicator of lipid peroxidation. This is of great commercial importance, as reduced lipid oxidation is directly correlated with improved meat color stability, extended shelf life, and reduced formation of off-flavors [93,94]. The more oxidative biceps femoris (thigh) also showed mitigated stress [95]. Crucially, the observation that superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels remained unchanged in the overall system suggests a specific mechanism of action [17].

4.7. mRNA Expression of Metabolic and Myofiber Genes

FDRBM promotes an oxidative muscle phenotype, upregulating the slow-twitch marker Tnnc1 (2.4–2.7-fold) and mitochondrial Nqo1 (2.5-fold in loin), shifting metabolism toward oxidative phosphorylation [64]. This underlies the improved meat quality; leucine-driven mTOR/PGC-1α activation enhances water-holding capacity, reducing cooking loss by 18–23% [96]. Concurrent cysteine/glycine enrichment supports glutathione synthesis, reduces lipid oxidation, and improves color stability [64,66]. To our knowledge, this the first report to demonstrate that FDRBM supplementation upregulates Tnnc1 and Nqo1 expression, underscoring its mechanistic potential as a functional feed. highlighting its potential as a functional feed [62,65]. The coordinated upregulation of Tnnc1, a calcium-binding regulatory protein specific to slow-twitch oxidative fibers, alongside the mitochondrial marker Nqo1, indicates a functional shift toward oxidative metabolism in muscle tissue. Notably, while the structural myosin heavy chain marker Myh7 did not reach statistical significance, this pattern is consistent with established fiber-type transition kinetics wherein functional metabolic adaptations (regulatory proteins and metabolic enzymes) precede structural remodeling [87,97]. This functional oxidative shift directly underlies the improved meat quality parameters, including enhanced water-holding capacity and reduced cooking loss observed in the FDRBM group.

4.8. Lipid Metabolism Gene Expression in Rabbit Muscle

Fermentation transformed rice bran into a potent activator of lipogenic pathways. Lactobacillus johnsonii L63 fermentation upregulates the master regulators PPARγ and SREBP-1c, likely via microbial production of agonist ligands (e.g., SCFAs and phenolics) [98,99]. Unfermented rice bran suppresses these pathways. Fatty acid synthase (FASN) upregulation was most pronounced in the oxidative biceps femoris, indicating fiber-type-dependent metabolic responses [100]. This transcriptional shift enhance intramuscular fat deposition, which is crucial for meat quality attributes such as juiciness and flavor [100]. Therefore, the transcriptional shift induced by FDRBM not only improves the oxidative capacity of the muscle, but also enhances its lipogenic potential, leading to a synergistic improvement in sensory characteristics.

4.9. Gene Expression in Ileum Tissue

FDRBM significantly upregulated ileal nutrient transporter genes compared with the control and unfermented diets, enhancing absorptive capacity [9]. This aligns with the documented improvements in digestibility, intestinal morphology, and barrier function seen with fermented rice bran [9]. The benefit stems from reduced levels of anti-nutritional factors and bioactive metabolites that modulate gut physiology [6], with similar effects observed in poultry and mice [101,102].

4.10. Liver Metabolomics Analysis

Liver metabolomics data provide a crucial link in the gut–liver–muscle axis, revealing how dietary inputs processed by the gut can trigger systemic metabolic shifts. The enrichment of pyrimidine metabolism in the FDRBM group points toward targeted anabolic reprogramming, where the liver enters a state of enhanced biosynthesis and energy storage, as indicated by the upregulation of UDP-sugars, which are precursors for glycogen synthesis and glycosylation [103]. The most striking discovery was the emergence of C17-sphinganine as a top-ranked differential metabolite. While even-chain ceramides and sphingolipids are canonical mammalian species often associated with lipotoxicity, insulin resistance, and metabolic dysfunction [104,105], odd-chain sphingolipids like C17-sphinganine represent an emerging area of metabolic health research [104]. Unlike even-chain species, which are primarily synthesized de novo from palmitoyl-CoA, odd-chain sphingolipids are frequently derived from gut microbial metabolism or the alpha-oxidation of longer-chain fatty acids [41,106]. Recent evidence suggests that certain gut bacteria, notably those from the Bacteroides genus, can synthesize odd-chain sphingolipids that are subsequently absorbed by the host and integrated into host metabolic pathways [41,107]. These bacteria-derived sphingolipids can act as signaling molecules that influence host lipid metabolism and inflammatory responses [108,109]. Importantly, C17-sphinganine belongs to a distinct class of odd-chain sphingolipids that differ fundamentally from canonical even-chain ceramides (C16- and C18-ceramides) typically associated with lipotoxicity and metabolic dysfunction [104]. Emerging evidence indicates that odd-chain sphingolipids are predominantly derived from gut microbial metabolism, particularly Bacteroidetes species, rather than de novo hepatic synthesis [110,111]. This establishes a direct biochemical link between the gut microbiome and host circulation. Therefore, the presence of C17-sphinganine is a strong indicator of specific microbial activity.

Furthermore, these microbially-derived odd-chain sphingolipids are now understood to function as beneficial signaling molecules, representing a novel mechanism of gut–host communication [112,113]. They have been shown to be absorbed by the host to positively modulate lipid homeostasis and inflammatory responses, distinguishing them from the detrimental pathways of other lipids [111]. Our finding is therefore consistent with a model where gut microbial shifts produce signaling lipids that contribute to the beneficial systemic effects observed in the muscle.

We hypothesized that the fermentation process alters the gut microbial ecosystem, favoring the proliferation of bacteria capable of producing C17-sphinganine. Upon absorption, this odd-chain sphingolipid appears to initiate a hepatic reprogramming cascade, modulating sphingolipid signaling pathways that are distinct from those associated with lipotoxicity [40,71]. This novel finding suggests that C17-sphinganine may be a key beneficial signaling molecule in the gut–liver axis, underpinning the systemic metabolic benefits observed with FDRBM supplementation. This data-driven hypothesis warrants further investigation to fully characterize the role of bacteria-derived odd-chain sphingolipids in animal health and nutrition.

5. Conclusions

Solid-state fermentation with Lactobacillus johnsonii L63 successfully converts de-oiled rice bran into a high-value functional feed ingredient (FDRBM). Its dietary inclusion in rabbit feed significantly enhances meat quality through a coordinated molecular and physiological mechanism.

The study’s key innovation is the elucidation of this mechanistic pathway. FDRBM supplementation induced fundamental metabolic reprogramming in muscle tissue, evidenced by the significant upregulation of oxidative fiber genes (Tnnc1) and mitochondrial function markers (Nqo1). This genetic shift is supported by a dramatically enhanced muscle amino acid profile. Specifically, elevated levels of cysteine and glycine fueled the glutathione-based antioxidant system, improving oxidative stability and meat color. Concurrently, increased glycine and proline levels supported collagen integrity, leading to a marked reduction in cooking loss.

Furthermore, our multi-omics analysis provides a data-driven hypothesis for these systemic benefits. It reveals a coordinated gut–liver axis response in which C17-sphinganine emerges as a key signaling metabolite.

Therefore, FDRBM acts as more than a feed ingredient, functioning as a nutritional modulator associated with an integrated response that enhances the nutritional value, technological properties, and oxidative stability of rabbit meat. These findings provide a strong scientific foundation for adopting FDRBM as a sustainable strategy to valorize agricultural byproducts, improve the efficiency of rabbit production, and meet growing consumer demand for high-quality, health-promoting animal protein.