Effects of Dietary Grape Branch and Leaf Silage on Growth Performance, Serum Biochemical Parameters, Gut Microbiota, and Metabolism in Kazakh Rams

TL;DR

Feeding grape branch and leaf silage to Kazakh rams improved their growth, reduced fat, and boosted health by altering gut microbes and metabolism.

Contribution

This study is the first to systematically evaluate grape branch and leaf silage as a feed alternative in rams, linking its effects to microbiota and metabolic changes.

Findings

Replacing corn silage with grape branch and leaf silage increased dressing percentage and reduced tail fat in rams.

The silage improved immune and antioxidant markers while lowering inflammation and oxidative stress.

It altered gut microbiota and enriched metabolic pathways like bile acid biosynthesis and glycerophospholipid metabolism.

Abstract

This study explored the use of grape branch and leaf silage as a feed ingredient in the diet of Kazakh rams. Sixty rams were divided into three groups: one fed Whole-crop Corn Silage, and the others with 50% or 100% replacement by grape branch and leaf silage over a 120-day feeding period. The results showed that replacing Whole-crop Corn Silage with grape branch and leaf silage improved slaughter performance by increasing dressing percentage and reducing tail fat deposition. It also enhanced immune function and antioxidant capacity while lowering inflammation and oxidative stress markers. Additionally, the grape branch and leaf silage positively influenced gut microbiota composition and metabolic pathways. These findings suggest that grape branch and leaf silage can be a beneficial feed alternative, supporting both production performance and health in sheep.

With the continuous development of the livestock industry, the availability of high-quality roughage is becoming increasingly constrained. Therefore, the exploitation of unconventional feed resources is crucial for the sustainable development of the sector. Grape branches and leaves are a major by-product of viticulture, abundant in supply but currently underutilised. Their ensilage presents potential feed value and ecological benefits. This study aimed to systematically evaluate the effects of dietary supplementation with grape branch and leaf silage on the growth performance, serum biochemical parameters, gut microbiota, and metabolomic profiles of Kazakh rams. Sixty Kazakh rams aged 6.0 ± 0.5 months with similar initial body weight (34.21 ± 2.13 kg) were randomly allocated to three dietary treatment groups: the control group (CG) fed whole-crop corn silage, the EG50 group where grape branch and leaf silage replaced 50% of the whole-crop corn silage, and the EG100 group where grape branch and leaf silage entirely replaced the whole-crop corn silage. Each treatment comprised four replicates with five rams per replicate. Following a 7-day adaptation period, a formal feeding trial was conducted for 120 days, after which relevant parameters were measured. Results: (1) Compared with the CG, the dressing percentage was significantly increased in the EG100 group (p < 0.05), while tail fat weight and tail fat percentage were both markedly decreased (p < 0.01). (2) Serum immunoglobulin (IgA, IgG) levels and antioxidant enzyme (SOD, CAT) activities were significantly elevated in the EG100 group (p < 0.05), accompanied by enhanced total antioxidant capacity. Concurrently, levels of inflammatory cytokines (IL-1β, TNF-α) and the oxidative damage marker malondialdehyde (MDA) were significantly reduced (p < 0.05). (3) Based on slaughter performance, jejunal microbiota analysis was performed for the CG and EG100 groups. The relative abundance of the phylum Firmicutes increased in the EG100 group, with beneficial genera such as Ruminococcus and Lactobacillus becoming predominant. (4) Metabolomic analysis revealed significant enrichment of pathways including primary bile acid biosynthesis and glycerophospholipid metabolism in the EG100 group, with 20 key differential metabolites identified. Dietary supplementation with grape branch and leaf silage may improve slaughter performance and reduce fat deposition in Kazakh rams, potentially by modulating the gut microbiota structure and its metabolic functions, thereby synergistically enhancing nutrient utilisation, anti-inflammatory capacity, and antioxidant status. This study provides a theoretical basis for the feed-oriented and value-added utilisation of grape processing by-products. Future research should further elucidate the molecular mechanisms underlying the interaction between its active components and host metabolism.

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Full text

1. Introduction

With the continuous development of the sheep farming industry in China, the shortage of high-quality roughage has become a key bottleneck restricting cost reduction and efficiency improvement within the sector. The limited sources and high cost of roughage directly constrain farming profitability and farmers’ income. Therefore, the exploitation and utilisation of unconventional feed resources to diversify feed sources is of urgent practical significance for ensuring stable regional livestock production and reducing costs. Vitis vinifera L. (grapevine) is a major economic crop cultivated worldwide. Global vineyard coverage is approximately 7.5 million hectares, generating substantial quantities of branches and leaves as primary agricultural waste during pruning [1]. It is estimated that tens of millions of tonnes of grape pruning residues are produced globally each year [2]. These by-products are rich in various nutrients; however, aside from limited disposal, field return, or on-site burning, most remain underutilised. This not only constitutes resource wastage, but their haphazard accumulation may also lead to environmental pollution and disease transmission. In fact, grape branches and leaves represent a highly promising unconventional feed resource. Studies indicate they contain abundant crude protein, crude fibre, minerals, and vitamins, and are rich in bioactive compounds such as polyphenols and flavonoids, which exert positive effects including antioxidant, anti-inflammatory, and immunomodulatory activities [3]. Research confirms that grape by-products (stems, leaves, pomace) are rich in nutrients and bioactive components, showing potential for application in ruminant feeding [4,5]. Recent in vivo trials have verified that dietary supplementation with grape stalks can safely modulate the milk microbiome in dairy cows, supporting their feasible utilisation in ruminants [6]. Zhao et al. [7] found that adding 10% grape branch and leaf meal to the diet increased lamb body weight and average daily gain while reducing the feed conversion ratio. Mu et al. [8] also reported a quadratic relationship between the supplementation level of grape seed proanthocyanidins and the final body weight, dry matter intake, and average daily gain in sheep. Notably, plant bioactive components can influence systemic immunity and inflammatory responses in the host by modulating the composition and function of the gut microbiota [9], providing an important ‘gut–immune axis’ perspective for explaining the impact of dietary interventions on animal health. Furthermore, growing evidence suggests that plant-derived bioactive compounds can synergistically regulate livestock growth performance, antioxidant status, immune response, and gut microecology [10], fresh grape branch and leaves contain high levels of anti-nutritional factors such as condensed tannins, which reduce feed palatability and impair the digestion and absorption of proteins and minerals, thereby directly limiting animal intake and nutrient utilisation. Additionally, their high moisture content presents storage challenges, increasing the risk of mould. Silage technology, as an effective method for preserving the nutritional value of fresh forage, offers an ideal solution to these challenges. Through anaerobic fermentation, the low pH environment and microbial activity during ensiling promote partial degradation or polymerisation of tannin molecules, reducing their protein-binding capacity, effectively improving palatability, and enabling long-term, safe storage. This overcomes the spatiotemporal limitations associated with utilising grape branches and leaves. The Kazakh ram is a premium indigenous breed in China, renowned for its hardiness, strong adaptability, and excellent meat quality. serving as a crucial pillar breed for the livestock industry in the Xinjiang region. Although existing research has focused on the effects of grape by-products on ruminant production performance, studies on the multi-dimensional effects of their silage products on Kazakh rams, particularly those systematically elucidating the mechanisms of action from the perspective of gut microbiota structure and metabolic function, remain insufficient. This study focuses on the resource recovery and feed utilisation of grape branches and leaves. It innovatively integrates traditional feeding trials with multi-omics technologies (16S rRNA sequencing and untargeted metabolomics), aiming to systematically analyse the effects and underlying mechanisms of grape branch and leaf silage across multiple levels: “phenotype–blood parameters–microbial structure–metabolic function”. This integrated strategy has been proven an effective framework for elucidating host–microbiota–metabolite interactions [11]. This research explores a feasible pathway for converting locally abundant grape pruning by-products into high-quality roughage suitable for Kazakh rams. We hypothesise that grape branch and leaf silage can improve the growth performance, immune and antioxidant status, and optimise carcass composition of Kazakh rams by modulating the gut microbiota and its metabolic functions. To this end, this study aims to systematically evaluate the effects of replacing whole-crop corn silage with different proportions of grape branch and leaf silage on the growth and slaughter performance, serum biochemical, immune, and antioxidant indices, jejunal microbial community structure, and jejunal metabolic profiles of Kazakh rams. This investigation aligns with the breed’s biological characteristic of hardiness and utilises regional resource advantages to achieve the goal of turning waste into treasure. This study aims to provide a systematic theoretical basis and technical reference for the high-value utilisation of grape by-products, holding significant practical importance for promoting sustainable regional agricultural development and implementing the rural revitalisation strategy.

2. Materials and Methods

2.1. Animals

The present trial was conducted using Kazakh rams from Xinjiang as experimental subjects. The study protocol was approved by the Animal Health and Utilization Committee of the College of Veterinary Medicine, Xinjiang Agricultural University (Approval No.: xjau-aw-2024-0948). The experiment was carried out at the Wugongtai Meat Sheep Cooperative in Hutubi County, Xinjiang Uygur Autonomous Region, China, from November 2024 to March 2025. The grape branch and leaf silage was sourced from Shanshan County, Turpan, and was prepared from locally harvested fresh grape branches and leaves at an approximate fresh weight ratio of 3:1 (branches to leaves). The silage material had a moisture content of 70% and was compacted at a density of 900 kg/m^3^. A mixed microbial feed additive (produced by Chris Hansen Czech s.r.o., containing Lactococcus lactis and Lactobacillus buchneri, with a viable count of 1.3 × 10^14^ CFU per kilogram) was incorporated into grape branch and leaf silage, along with 3% corn starch on a dry matter basis. The mixture subsequently underwent a 90-day fermentation period. The nutritional components of whole-crop corn silage and Grape branch-leaf silage are detailed in Table 1.

2.2. Experimental Design

Sixty healthy Kazakh rams (n = 60) aged 6.0 ± 0.5 months with an initial body weight (BW) of 34.21 ± 2.13 kg were randomly divided into three groups using a completely randomized design. Each treatment consisted of four replicates with five sheep per replicate. The control group (CG) was fed a roughage diet containing whole-crop corn silage. The EG50 group received a diet in which 50% of the whole-crop corn silage was replaced with grape branch and leaf silage, while the EG100 group was fed a diet with 100% replacement of whole-crop corn silage by grape branch and leaf silage. The experimental animals were fed according to the standard “Nutrient Requirements of Meat Sheep (NY/T 816-2021)” [12]. The trial consisted of a 7-day adaptation period followed by a 120-day formal feeding period.

2.3. Animal Husbandry Management

All Kazakh rams were housed under identical conditions and fed at 07:00 and 18:00 daily with free access to water. The experimental sheep were provided with a total mixed ration (TMR), and the detailed diet formulation is presented in Table 2.

2.4. Indicator Measurement

2.4.1. Analysis of Feed Chemical Composition

The chemical composition of the experimental diets was determined strictly in accordance with the standard methods of AOAC (2000) [13]. Raw material samples were collected at the same time point after the silage reached maturity following a 90-day fermentation period and were promptly stored at −20 °C. Prior to analysis, the samples were freeze-dried, uniformly ground to pass through a 40-mesh standard sieve, equilibrated in a desiccator, and then weighed. Crude protein (CP) was determined using the Kjeldahl method (AOAC 988.05) with a fully automated Kjeldahl nitrogen analyzer. Crude fat, expressed as ether extract (EE), was analyzed by Soxhlet extraction with petroleum ether (AOAC 920.39) for a standardized duration of 6 h. The contents of neutral detergent fiber (NDF) and acid detergent fiber (ADF) were sequentially determined according to the method of Van Soest et al. [14] using the same Ankom fiber analyzer. Calcium and phosphorus contents were measured by atomic absorption spectrometry (AOAC 968.08) and the ammonium vanadate-molybdate colorimetric method (AOAC 965.17), respectively, following a uniform dry ashing procedure and quantification against the same calibration curve. Crude ash content was obtained by incineration in a muffle furnace at 550 °C until a constant weight was achieved (AOAC 942.05). All analyses were performed in three analytical replicates. The metabolizable energy (ME) concentration of the experimental diets was calculated using a weighted summation method. This calculation was based on the dry matter proportion of each ingredient in the formulation, referencing the corresponding ingredient ME values from the “Chinese Feed Composition and Nutritional Value Tables [15]” and NRC [16]. The calculation formula was as follows:

$$[eqn]$$

ME_i_ represents the metabolizable energy value of the i-th ingredient (MJ/kg DM), and P_i_ denotes the proportion of that ingredient in the dietary dry matter.

2.4.2. Measurement of Growth Performance

The experimental sheep were weighed after fasting at the beginning and end of the trial to calculate the average daily gain (ADG). The amounts of feed offered and orts were recorded daily to determine the average daily feed intake (ADFI) and the feed conversion ratio (FCR). Upon completion of the 120-day trial, eight rams per group (two from each replicate) were randomly selected and transported to a commercial abattoir located 5 km from the experimental farm for slaughter. Animals were humanely slaughtered following the standardized commercial procedures stipulated by the Ministry of Agriculture and Rural Affairs of the People’s Republic of China [17], which involved exsanguination after electrical stunning. Following a 24-h fast and a subsequent 2-h water withdrawal period, the hot carcass weight and tail fat weight of all animals were immediately recorded post-slaughter using a calibrated electronic scale (accuracy 0.05 kg). The dressing percentage was expressed as the hot carcass weight (after removal of the viscera, head, hooves, and tail fat) as a percentage of the pre-slaughter live weight. It was measured after a 30-min resting period for equilibration, following the method described by Wu et al. [18]. The tail fat percentage was subsequently calculated. Average daily gain (ADG), average daily feed intake (ADFI), and the feed conversion ratio (FCR) were calculated according to standard formulas based on the total weight gain, total feed intake, and the number of experimental days during the trial period.

2.4.3. Determination of Serum Biochemical Parameters

Blood samples (5 mL) were collected from the jugular vein under fasting conditions at 0, 40, 80, and 120 days of the trial. The samples were centrifuged at 3000 r/min for 15 min. The resulting supernatant was aliquoted into 1.5 mL centrifuge tubes and sent to Beijing Huaying Biotechnology Co., Ltd. (Beijing, China)., for analysis. Serum concentrations of total protein (TP, 546 nm, g/L), total cholesterol (TC, 500 nm, mmol/L), triglycerides (TGs, 500 nm, mmol/L), high-density lipoprotein (HDL, 546 nm, mmol/L), low-density lipoprotein (LDL, 546 nm, mmol/L), glucose (GLU, 505 nm, mmol/L), blood urea nitrogen (BUN, 340 nm, mg/dL), total antioxidant capacity (T-AOC, 632 nm, U/mL), immunoglobulin A (IgA, 340 nm, g/L), immunoglobulin G (IgG, 610 nm, g/L), and immunoglobulin M (IgM, 340 nm, g/L) were determined using a Mindray BS-420 automatic biochemical analyzer [19]. The concentrations of interleukin-1β (IL-1β, 450 nm, pg/mL), tumour necrosis factor-α (TNF-α, 450 nm, pg/mL), superoxide dismutase (SOD, 405 nm, U/mL), catalase (CAT, 405 nm, U/mL), and malondialdehyde (MDA, 532 nm, nmol/mL) were measured using specific enzyme-linked immunosorbent assay (ELISA) kits (Beijing Sino-UK Institute of Biological Technology, Beijing, China) [20], with readings taken at the corresponding wavelengths on a Huawei Drum DR-200BS (Wuxi, China)microplate reader.

2.4.4. Gut Microbiota Analysis

To focus on the comparison yielding the greatest phenotypic differences (particularly in slaughter performance) and based on resource optimisation principles, this study selected the EG100 group, which demonstrated the most pronounced phenotypic effects, and the CG group for in-depth gut microbiota analysis [21]. Following slaughter, jejunal content samples were collected from each group and sent to Kaitai Biotechnology Co., Ltd. (Shanghai, China), for gut microbiota analysis. In this study, the hypervariable V3-V4 region of the bacterial 16S rRNA gene was amplified to analyze microbial diversity. PCR amplification was performed using the barcode-specific primers 338F (5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) with NEB Q5 High-Fidelity DNA Polymerase. The amplification protocol was as follows: initial denaturation at 98 °C for 30 s, followed by 25–27 cycles of denaturation at 98 °C for 15 s, annealing at 50 °C for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 5 min. PCR products were quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Omega Bio-Tek, Norcross, GA, USA) on a microplate reader (BioTek, FLx800, GA, USA) and pooled in equimolar ratios based on the sequencing depth required for each sample. Sequencing libraries were constructed using the Illumina TruSeq Nano DNA LT Library Prep Kit. The procedure included steps such as DNA end repair, 3′ adenylation, adapter ligation with index sequences, and library amplification. Purification at different stages was performed using BECKMAN AMPure XP beads. The final library quality was assessed on a LabChip system. Libraries passing quality control were sequenced on an Illumina NovaSeq 6000 platform using the NovaSeq 6000 SP Reagent Kit (500 cycles), generating 2 × 250 bp paired-end reads.

2.4.5. Gut Metabolomics

To focus on the comparison yielding the greatest phenotypic differences (particularly in slaughter performance) and based on resource optimisation principles, the EG100 group, which demonstrated the most pronounced phenotypic effects, and the CG group were selected for in-depth metabolomic analysis [22]. Following slaughter, jejunal content samples were collected from sheep in these groups and sent to Kaitai Biotechnology Co., Ltd. (Shanghai, China), for LC-MS analysis. Approximately 20 ± 5 mg of the jejunal tissue sample was precisely weighed and placed into a pre-cooled 2 mL centrifuge tube. Subsequently, 400 µL of pre-cooled 80% methanol (Cat. No. CAEQ-4-003302-4000) aqueous solution (containing 1 µg/mL L-2-chlorophenylalanine (CAS: 29909-00-0, Cat. No. BZP22-0492) as an internal standard), one large stainless-steel bead (5 mm diameter), and three small beads (2 mm diameter) were added. Cell disruption was performed using a cryogenic grinder at −50 °C, with the program set to 10 cycles (30 s of grinding at 30 Hz followed by a 30-s pause). After homogenisation, the mixture was centrifuged at 14,000× g and 4 °C for 10 min. The entire supernatant was transferred and dried using vacuum centrifugal concentration. The residue was reconstituted in 80 µL of 50% acetonitrile aqueous solution containing the internal standard, vortex-mixed for 30 s, and centrifuged again at 14,000× g and 4 °C for 10 min. Finally, 70 µL of the supernatant was collected for injection analysis. A pooled quality control (QC) sample was injected after every eight experimental samples in the sequence. Separation was achieved using a Thermo Vanquish UHPLC system coupled with a Waters ACQUITY UPLC BEH C18 column (1.7 µm, 2.1 × 100 mm) maintained at 40 °C. The injection volume was 2 µL and the flow rate was 0.3 mL/min. For positive ion mode, the mobile phases consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. For negative ion mode, the mobile phases were (A) 10 mM ammonium acetate with 0.1% ammonia and (B) pure acetonitrile (Cat. No. 1.00030.4008). The gradient elution program was as follows: 0–1.0 min, 98% A; 1.0–8.0 min, 98% to 1% A; 8.0–10.0 min, 1% A; 10.0–10.1 min, 1% to 98% A; 10.1–12.0 min, 98% A. Mass spectrometric detection was performed using a Thermo Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). The spray voltages were set to +3.8 kV and −3.2 kV for positive and negative ion modes, respectively. The ion transfer tube temperature was 320 °C and the vaporiser temperature was 300 °C. The sheath gas, auxiliary gas, and purge gas flow rates were set to 35, 10, and 0 arbitrary units (Arb), respectively. The S-lens RF level was 55%. Full scans were acquired over the range m/z 70–1050 with a resolution of 60,000 FWHM (at m/z 200). A data-dependent acquisition (DDA) mode was employed, where the top 10 most intense precursor ions were selected for fragmentation (normalised collision energy: 28 eV), with MS/MS spectra acquired at a resolution of 15,000 FWHM. Raw data files were processed using Compound Discoverer 3.3 software, performing peak detection (signal-to-noise ratio > 3), retention time alignment (0.2 min tolerance), and peak area integration. Compound identification was achieved by querying the mzCloud, MassBank and in-house databases, with mass error < 5 ppm and an MS/MS spectral match score > 70. Differential compounds were screened with the criteria of p-value < 0.05 and a false-discovery-rate-adjusted Q-value < 0.1.

Following peak extraction, alignment, and normalisation preprocessing, the normalised metabolite data were imported into SIMCA-P 16.0 software. PCA was first conducted, after which an OPLS-DA model was constructed. The validity of the model was evaluated via 200 permutation tests. Differential metabolites were screened using a VIP value > 1.0 from the OPLS-DA model, combined with a p-value < 0.05 derived from a two-tailed Student’s t-test. KEGG pathway enrichment analysis of the significant differential metabolites was performed using the MetaboAnalyst 5.0 platform, with a hypergeometric test and a significance threshold of p < 0.05.

2.5. Statistical Analysis

Data were initially organised using Microsoft Excel 2016 (Microsoft Corporation, Washington, DC, USA). All statistical analyses were performed using SPSS 27 (IBM, New York, NY, USA) software. Analysis of growth performance and serum biochemical parameters was conducted using a linear mixed model, with the treatment group included as a fixed effect and the replicate (batch) as a random effect. If a significant overall treatment effect was detected (p < 0.05), Tukey’s HSD test was applied for post hoc multiple comparisons. The false discovery rate (FDR) method was used to adjust all post hoc comparisons and series of hypothesis tests, with an adjusted q-value of <0.05 considered statistically significant. In addition to reporting significance, the analysis included estimates of effect sizes (e.g., Cohen’s d) and their 95% confidence intervals for key comparisons to provide a comprehensive assessment of the practical magnitude and estimation precision of the treatment effects. Final data are presented as mean ± standard error.

3. Results

3.1. Effects of Dietary Grape Branch and Leaf Silage on the Growth Performance of Kazakh Rams

As shown in the results of Table 3, the dressing percentage in the EG100 group was significantly higher than that in the CG group (p < 0.01). The tail fat weight in both the EG50 and EG100 groups was significantly lower than in the CG group (p < 0.01), with the EG100 group being significantly lower than the EG50 group (p < 0.01). The tail fat percentage in the EG50 and EG100 groups was significantly lower than in the CG group (p < 0.01), with the EG100 group showing the lowest value. The final body weight in the EG50 group was higher than in the CG and EG100 groups, though the difference was not significant (p > 0.05); the EG100 group had the lowest final weight. Both net weight gain and ADG were higher in the EG50 group compared to the CG and EG100 groups, but the differences were not significant (p > 0.05). The EG100 group exhibited the lowest net weight gain. The ADFI was higher in both the EG50 and EG100 groups compared with the CG group, although the difference was not statistically significant (p > 0.05), with the EG50 group showing the numerically highest value. The FCR in the CG group was lower than in the EG50 and EG100 groups, but this difference was not statistically significant (p > 0.05).

3.2. Effects of Dietary Grape Branch and Leaf Silage on the Serum Biochemical Parameters of Kazakh Rams

3.2.1. Effects of Dietary Grape Branch and Leaf Silage on the Serum Nutrient Profile in Kazakh Rams

According to the results presented in Table 4, at 120 d, the serum TC concentration in the EG100 group was lower than that in the CG group (p > 0.05). The LDL concentration in the EG50 group was significantly lower than in the CG group at 40 days (p < 0.05). By 120 days, both the EG50 and EG100 groups exhibited significantly lower LDL concentrations compared to the CG group (p < 0.05), with the EG100 group showing the lowest value. The serum GLU concentration in both the EG50 and EG100 groups was significantly lower than in the CG group at 40 days (p < 0.05), again with the EG100 group being the lowest. Furthermore, the BUN concentration in the EG100 group was significantly lower than that in the CG group (p < 0.05).

3.2.2. Effects of Dietary Grape Branch and Leaf Silage on the Serum Immune Parameters of Kazakh Rams

According to the results presented in Table 5, the serum IgA concentration in both the EG50 and EG100 groups was significantly higher than that in the CG group at 40 days (p < 0.01), with the EG100 group showing the highest value. The IgG level in the EG100 group was significantly higher than in the CG group at 40 days (p < 0.05). Similarly, the IgM concentration in the EG50 and EG100 groups was significantly higher than in the CG group at 40 days (p < 0.05), with the EG100 group again being the highest. The IL-1β level in the EG100 group was significantly lower than in the CG group at both 40 and 80 days (p < 0.05). By 120 days, IL-1β concentrations in both the EG50 and EG100 groups were significantly lower than in the CG group (p < 0.01). The TNF-α concentration in the EG50 and EG100 groups was significantly lower than in the control group at 40 and 120 days (p < 0.01), with the EG100 group having the lowest value. At 80 days, TNF-α levels in both the EG50 and EG100 groups were also significantly lower than in the CG group (p < 0.05).

3.2.3. Effects of Dietary Grape Branch and Leaf Silage on the Antioxidant Capacity of Kazakh Rams

As shown in the results of Table 6, at 40 and 120 d, the SOD activity in both the EG50 and EG100 groups was higher than that in the CG group. Notably, the activity in the EG100 group was significantly higher than that in the CG group (p < 0.05). The CAT activity in the EG50 and EG100 groups was significantly higher than that in the CG group (p < 0.01), with the EG100 group exhibiting the highest value. The MDA concentration in the EG100 group was significantly lower than that in the CG group at 40, 80, and 120 days (p < 0.05).

3.3. Effects of Dietary Grape Branch and Leaf Silage on the Jejunal 16S rRNA Microbial Profile of Kazakh Rams

3.3.1. Effects of Dietary Grape Branch and Leaf Silage on the Alpha Diversity of the Jejunal 16S rRNA-Based Microbial Community in Kazakh Rams

The results presented in Figure 1 show that the EG100 group exhibited a higher Chao1 index compared to the CG group (p = 0.658); a higher Simpson index compared to the CG group (p = 0.123); and a higher Shannon index compared to the CG group (p = 0.068).

3.3.2. Effects of Dietary Grape Branch and Leaf Silage on the Jejunal Microbiota Diversity of Kazakh Rams

Figure 2a shows that the EG100 group comprised 2861 genera, while the CG group comprised 2677 genera, with 589 core genera shared between the two groups, indicating the effect of dietary grape branch and leaf silage on the beta diversity of gut microbiota in Kazakh rams. As shown in Figure 2b, PCoA of the gut microbial communities revealed that PC1 and PC2 explained 53.18% and 18.01% of the total variance, respectively. Samples from both groups were clustered and exhibited a discernible separation trend, suggesting distinct differences between the two groups based on the structure of their gut microbial communities.

3.3.3. Effects of Dietary Grape Branch and Leaf Silage on the Jejunal Microbiota Diversity at the Phylum and Genus Levels in Kazakh Rams

Figure 3 presents the results. At the phylum level (Figure 3a), Firmicutes, Proteobacteria, and Actinobacteriota were the dominant phyla in the gut microbiota. Their relative abundances in the EG100 group were 38.01%, 41.11%, and 0.66%, respectively. Compared with the CG group, the EG100 group showed an increased relative abundance of Firmicutes, whereas decreased abundances were observed for Patescibacteria, Bacteroidota, Actinobacteriota, and Proteobacteria. At the genus level (Figure 3b), Ruminococcus and Limosilactobacillus were identified as the dominant genera. Their relative abundances in the EG100 group were 6.73% and 1.85%, respectively. Compared with the CG group, the abundances of these two genera were increased in the EG100 group.

3.4. Effects of Feeding Grape Branch and Leaf Silage on Jejunal Microbial Metabolism in Kazakh Ram

3.4.1. Multidimensional Analysis and Visualization of Metabolomics Data from the Longissimus Dorsi Muscle in Kazakh Rams Fed Grape Branch and Leaf Silage

Figure 4a shows that Dim 1 accounted for 27.6% of the metabolite variance and Dim 2 accounted for 22.5%. Within a 95% confidence interval, the CG and EG100 groups exhibited distinct separation, demonstrating strong dispersion and low inter-group similarity. These results indicate that feeding grape branch and leaf silage significantly altered the jejunal metabolic profile of Kazakh rams (p < 0.05). As shown in Figure 4b, a total of 1407 differentially expressed metabolites were identified, comprising 27 up-regulated compounds and 45 down-regulated compounds. Figure 4c reveals that the differential metabolites displayed a clear clustering pattern, which was further confirmed by the heatmap analysis.

3.4.2. KEGG Enrichment Analysis of Differential Metabolite Expression in Kazakh Rams Fed Grape Branch and Leaf Silage

The KEGG enrichment analysis of differentially expressed metabolites identified four significantly enriched pathways, as illustrated in Figure 5: Primary bile acid biosynthesis, Choline metabolism in cancer, Glycerophospholipid metabolism, and Sulfur metabolism.

3.4.3. Screening of Differential Metabolites in Kazakh Rams Fed with Grape Branch and Leaf Silage

Based on the results presented in Figure 6, the top 20 differential metabolites were selected for analysis according to the criteria of p < 0.05 and VIP > 1. Among these, seven metabolites, including Xestoaminool C, Lotaustralin, and Prolylleucine, were up-regulated, while thirteen metabolites, such as 3-amino-octanoic acid and 2-methyl-2E-heptenoic acid, were down-regulated.

4. Discussion

4.1. Effects of Dietary Grape Branch and Leaf Silage on the Growth Performance of Kazakh Rams

The EG50 group showed increases of 6.02% and 6.89% in net weight gain and ADG, respectively, compared to the CG group. Research by Ma J et al. [23] has demonstrated that dietary supplementation with grape seed extract improved the ADG of Kazakh sheep, which aligns with the findings of the present study. This suggests that feeding grape branch and leaf silage can effectively enhance growth performance. ADFI reflects both appetite and feeding capacity in animals. In the present study, the EG50 group exhibited a 14.15% increase in average daily feed intake compared to the CG group. Studies by Lu Zhenzhen et al. [24]. showed that dietary grape pomace improved the ADG and ADFI of meat sheep; similarly, research by Xia Cheng demonstrated that feeding grape pomace enhanced the ADG and ADFI of Kazakh rams [25]. It is noteworthy that the findings of this study suggest a potential trade-off in nutritional partitioning regarding the effects of grape branch and leaf silage on growth performance and carcass composition. Specifically, the EG100 group exhibited the highest dressing percentage and the lowest tail fat deposition, which may indicate that 100% substitution more effectively directed nutrients toward protein deposition (increasing dressing percentage) while suppressing lipid synthesis. In summary, the observed improvements in growth performance may be attributed to the enhanced palatability of the diet due to the addition of grape branch and leaf silage, thereby increasing feed intake.

The present study found that dietary grape branch and leaf silage significantly increased the dressing percentage in the experimental groups by 1.62% and 7.56%, respectively. It was also found that feeding grape branch and leaf silage could induce a highly significant decrease in both tail fat weight and tail fat percentage in the experimental groups. Studies have shown that grape seed proanthocyanidins can improve the slaughter performance of New Zealand rabbits [26]. Yang et al. demonstrated that dietary supplementation with grape seed proanthocyanidins, at doses of 10 or 20 mg per kg body weight, significantly increased carcass weight, longissimus dorsi muscle area, and dressing percentage in lambs [27]. Yao et al. [28] demonstrated that dietary supplementation with varying proportions of grape marc residue enhanced the meat yield in male crossbred lambs (Dorset × Small-tailed Han F1). These findings are consistent with the results of the present study, indicating that feeding grape branch and leaf silage can increase the carcass weight and dressing percentage while reducing tail fat weight in Kazakh rams. This effect may be associated with the bioactive compounds present in grape branches and leaves, such as grape seed proanthocyanidins. Furthermore, research has found that diets supplemented with grape seed proanthocyanidins (GSP) significantly reduced the abdominal fat percentage in broilers, which aligns with the reduction in tail fat weight observed in this study [29].

4.2. Effects of Dietary Grape Branch and Leaf Silage on the Serum Biochemical Parameters of Kazakh Rams

The results of this study revealed that, at day 120, serum TP levels in the experimental groups were 3.32% and 3.68% higher, respectively, than those in the CG group. In contrast, serum TC levels in the experimental groups were reduced by 8.7% and 20.6% compared with the CG group at day 120. Throughout the trial period, TG content in both the EG100 and EG50 groups remained lower than that in the CG group. Furthermore, LDL levels in the experimental groups were lower than those in the CG group at days 40, 80, and 120. The study by Zern et al. [30] found that, compared with the control group, guinea pigs fed a grape-supplemented diet showed no significant differences in plasma LDL-C concentrations, but plasma TG and VLDL-C were reduced by 39% and 50%, respectively. Therefore, from the perspective of serum biochemistry, the findings of the present study confirm that grape branch and leaf silage significantly improved the lipid metabolism status of Kazakh rams. The simultaneous decrease in serum TC, TG, and LDL directly reflects an overall improvement in lipid mobilisation, synthesis, and clearance at the systemic circulation level, which aligns with and corroborates the observed significant reductions in tail fat weight and percentage measured during slaughter. These parallel changes strongly suggest that the dietary intervention directly led to reduced terminal adipose tissue deposition by modulating systemic lipid metabolism. The underlying mechanism may be related to the bioactive components in grape branch and leaf inhibiting hepatic lipid synthesis, promoting peripheral lipolysis, or regulating lipoprotein metabolism, thereby contributing to the improved dressing percentage. This critical change may be the underlying mechanism responsible for the improved dressing percentage and reduced tail fat weight observed in this study.

The results of this study indicate that dietary supplementation with grape branch and leaf silage significantly increased the IgA, IgG, and IgM in the serum of Kazakh rams, suggesting an enhancement of humoral immune function [31]. Notably, this systemic immune enhancement coincided with alterations in the jejunal gut microbial community structure, particularly corresponding to an increase in the relative abundance of beneficial bacteria such as Lactobacillus. This finding aligns with the observations reported by Li et al. [32] and Li et al. [33], who documented improved immune function in ruminants fed distillers’ grains and grapevine forage, respectively. Existing theory posits a close interaction between the gut microbiota and the host immune system, known as the “gut–immune axis”. Specific beneficial microorganisms, such as Lactobacillus, have been shown to exert immunomodulatory effects by enhancing intestinal barrier function and regulating local and systemic immune responses. Research has confirmed that polyphenols, particularly flavonoids, can significantly modulate humoral immunity [34]. Therefore, the immune-enhancing effects observed in this trial may not only result from the direct action of polyphenols and flavonoids present in grape branches and leaves but may also be achieved through the following indirect pathway: these bioactive components first optimise the gut microbiota structure, promoting the proliferation of beneficial bacteria represented by Lactobacillus; subsequently, this remodelled microbial community amplifies regulatory signals to the host immune system via the gut–immune axis, ultimately leading synergistically to a systemic increase in serum immunoglobulin levels.

TNF-α is a key pro-inflammatory cytokine that promotes the secretion of various inflammatory mediators by T cells, thereby mediating inflammatory responses. MDA is a terminal product of lipid peroxidation, and its concentration reflects the extent of oxidative damage to cell membranes. The results of this study demonstrated that the levels of both IL-1β and TNF-α in the EG100 group were significantly lower than those in the control group. The study by Ma et al. [23] indicated that dietary supplementation with grape seed extract reduced IL-1β and TNF-α levels in weaned lambs. Zhu et al. [35], investigating the effects of grape seed proanthocyanidins on periodontal inflammation in diabetic periodontitis rats, found that grape seed proanthocyanidins could decrease serum TNF-α and IL-1β levels. These findings are consistent with the results of the present study. More importantly, the anti-inflammatory effects observed herein can be theoretically supported from the emerging perspective of nutritional immunology. This strategy aims to proactively modulate host immune homeostasis through specific dietary components [36]. The polyphenols and flavonoids abundantly present in grape branches and leaves are precisely such potential plant-derived immunomodulators. Therefore, the underlying reasons may not be attributed solely to the presence of bioactive components, but also to their role as dietary immunomodulators. They may systemically reduce the body’s inflammatory response levels, either by directly inhibiting inflammatory signalling pathways such as NF-κB, or indirectly by improving gut microecology and barrier function. This provides a novel perspective for developing grape branch and leaf silage as a functional feed to enhance the health and production resilience of ruminants.

Compared with the control group, the EG50 and EG100 groups exhibited higher activities of SOD and CAT, as well as enhanced T-AOC. Concurrently, both experimental groups showed lower MDA content than the control group. These coordinated yet distinct changes in indices comprehensively reflect an overall improvement in the body’s redox status. The significant increase in SOD and CAT activities indicates an enhanced capacity of the key enzymatic antioxidant defense system responsible for scavenging superoxide anion radicals and hydrogen peroxide. The decrease in MDA content, a terminal product of lipid peroxidation, provides direct evidence of reduced actual oxidative damage to biomacromolecules such as cell membranes. The elevated T-AOC serves as a composite indicator encompassing the overall level of both enzymatic and non-enzymatic antioxidant systems. The synergistic changes in the aforementioned indices demonstrate that grape branch and leaf silage not only increased the body’s antioxidant potential but also effectively mitigated the actual level of oxidative stress. Several previous studies corroborate these findings. Ma et al. [23] and Zhao et al. [7] reported that grape seed extract and white grape peel powder increased T-AOC and SOD activity in ruminants, respectively; Antunović et al. [37] also found that grape pomace enhanced SOD activity in lambs. The underlying mechanism may involve grape seed proanthocyanidins inhibiting oxidase activity (Kim et al. [38]) or components in grapevine extract such as quercetin upregulating antioxidant enzyme expression (Babri et al. [39]). Research on Cherry Valley ducks likewise confirmed the effect of grape seed proanthocyanidins in elevating CAT and SOD activities while reducing MDA content (Li et al. [40]). Therefore, the results of this study indicate that feeding grape branch and leaf silage can enhance the enzymatic antioxidant defense capacity and alleviate lipid peroxidation damage in Kazakh rams, thereby comprehensively improving their systemic redox balance.

This study found that dietary supplementation with grape branch and leaf silage significantly elevated serum antioxidant and immune indices in sheep but did not concurrently improve their growth performance (ADG, ADFI, FCR). This outcome may be explained by the nutritional partitioning theory, which posits a clear physiological priority for metabolic resources (maintenance > growth) within the organism [20]. Bioactive components such as plant polyphenols may be preferentially allocated to constructing defense systems—for instance, by activating the Nrf2-ARE pathway to enhance antioxidant capacity [41] or modulating the NF-κB pathway to optimise immune homeostasis. These investments are manifested as increased anti-stress potential, and their translation into growth performance may exhibit a lag. Secondly, under conditions where basal nutritional requirements are already met, the core effect of the supplement may lie in enhancing the body’s physiological buffering capacity. This contributes to maintaining long-term health and potentially improving product quality and feeding efficiency. Future research should extend the trial duration to verify the long-term production benefits of health improvements and integrate multi-omics analyses to systematically elucidate the molecular networks governing the regulation of nutritional partitioning and physiological homeostasis.

4.3. Effects of Dietary Grape Branch and Leaf Silage on the Jejunal Microbiota of Kazakh Rams

In this trial, based on slaughter performance, the EG100 and the CG group were selected for microbial diversity analysis of the jejunal contents in Kazakh rams. This study design is grounded in the core theory that the gut microbiome exhibits high regional specificity. Research indicates that significant differences in physiological conditions across various gastrointestinal tract segments lead to the formation of microbial communities with distinct compositions and functions [42]. Therefore, selecting the jejunum—a core site for nutrient absorption and mucosal immunity—for investigation allows for a more precise revelation of the impact of dietary interventions on host-microbe interactions at this critical interface, avoiding the informational interference from mixed samples such as rumen or faeces. The 16S rRNA sequencing results indicated that α-diversity indices, represented by the Shannon index, Simpson index, and Chao1 index, were higher in the EG100 group than in the CG group. The study by Klimenko et al. [43] demonstrated that gut microbial α-diversity was significantly lower in obese adolescents compared to their normal-weight peers.

In this study, the relative abundances of the phyla Firmicutes and Bacteroidota were significantly increased in the EG100 group, while the relative abundances of the phyla Proteobacteria, Actinobacteriota, and Acidobacteriota were significantly decreased. In the gastrointestinal tract of ruminants, Firmicutes and Bacteroidota exhibit the highest relative abundances and play crucial roles in feed digestion and utilisation. Firmicutes possess fibre-degrading functions [44]. The differing digestive capacities and characteristics of the hindgut and the rumen lead to variations in microbial community distribution across digestive tract segments. In cattle, the duodenal microbiota is primarily composed of Firmicutes and Proteobacteria, whereas the microbial communities of the jejunum and ileum are similar, dominated by Firmicutes followed by Bacteroidota [45]. Bacteroidaceae is a key bacterial group for fibre degradation, and its increased abundance may have promoted the fibre breakdown process [46]. The decrease in Proteobacteria within the gut microbiota may be associated with an improved intestinal microecological environment or a more stable community structure. The study by Zhao et al. [47] showed that the relative abundance of Proteobacteria decreased significantly with the age of sheep. Actinobacteriota can produce various bioactive secondary metabolites and play an important role in decomposing organic matter. The study by Zhan et al. [48] on the effects of different concentrate-to-forage ratio diets on the rumen microbiota of Hu sheep found that increasing the dietary concentrate ratio raised the proportion of Actinobacteriota in the rumen. This finding is consistent with the results of the present study. In summary, feeding grape branch and leaf silage reduced the abundances of Proteobacteria, Actinobacteriota, and Acidobacteriota in the jejunum of meat sheep, thereby potentially inhibiting fat synthesis.

In the present study, the relative abundances of Ruminococcus, Lactobacillus, and Limosilactobacillus were significantly increased in the EG100 group, whereas those of Butyricicoccus and Mogibacterium were significantly decreased. Wang Weiyun et al. [49] isolated two strains of Ruminococcus flavefaciens, H1 and H2, from the rumen of Inner Mongolian sheep. Strain H2 exhibited a filter paper enzyme activity of 0.21 µmol/(mL·min), demonstrating a strong cellulose-degrading capacity. Ruminococcus flavefaciens can degrade recalcitrant fibres such as cotton. The genus Lactobacillus is widely present in the jejunum and ileum of newborn ruminants and contributes to improving nutrient digestibility and immune function [50]. These findings collectively suggest that feeding grape branch and leaf silage enhanced the degradation rate of dietary crude fibre in meat sheep. Furthermore, the observed increase in serum immunoglobulin (IgA, IgG, IgM) levels may be associated with the elevated abundance of intestinal lactobacilli. Jin et al. [51] demonstrated that the abundance of Lactobacillus exhibited a negative correlation with IMF content. This finding is consistent with the observed reduction in fat content in the lamb meat in the present study.

4.4. Effects of Dietary Grape Branch and Leaf Silage on the Jejunal Metabolism of Kazakh Rams

The present study employed an untargeted metabolomics approach to systematically analyze the effects of dietary grape branches and leaves silage on the jejunal metabolic profile of Kazakh rams. The partial least squares-discriminant analysis (PLS-DA) model revealed a clear separation in metabolite distribution between the experimental and control groups. KEGG enrichment analysis of the differential metabolites identified four significantly enriched pathways: Primary bile acid biosynthesis, Choline metabolism in cancer, Glycerophospholipid metabolism, and Sulfur metabolism. The enrichment of Primary bile acid biosynthesis and Glycerophospholipid metabolism is associated with lipid digestion and membrane dynamics [52,53], while Choline metabolism in cancer may be related to cell proliferation [54,55]. Sulfur metabolism involves the metabolism of sulfur-containing amino acids (such as methionine and cysteine), leading to the production of glutathione (GSH), taurine, and sulfate. GSH serves as a major antioxidant and is involved in the oxidative stress response [56]. The top 20 key differential metabolites were selected based on variable importance in projection (VIP) scores for further analysis. The results indicated that the jejunal level of L-valine was significantly up-regulated in the EG100 group. As an essential branched-chain amino acid, L-valine serves not only as a fundamental substrate for protein synthesis but also plays a pivotal role in regulating protein turnover and energy metabolism [57]. This up-regulation may stem from the protective effect of polyphenolic compounds, such as tannins, present in grape branches and leaves against excessive ruminal degradation of dietary protein. Consequently, this increases the proportion of rumen-undegraded protein (RUP), allowing a greater amount of amino acids to be delivered to and absorbed in the hindgut [58]. The concurrent increase in serum total protein and decrease in urea nitrogen in the experimental groups collectively indicate a positive improvement in protein metabolism. This outcome can be explained by the observed upregulation of L-valine levels, which likely promoted protein synthesis by enhancing the body’s nitrogen utilization efficiency.

Alterations in lipid metabolites constituted another significant finding in this study. Several acylcarnitines, including hexanoylcarnitine, 2-methylbutyrylcarnitine, and 3-hydroxybutyrylcarnitine, were downregulated. Acylcarnitines are intermediate transport carriers for fatty acids entering the mitochondria for β-oxidation, and their decreased levels typically indicate a more efficient fatty acid oxidation process [59]. This finding can be interpreted from two interrelated perspectives. Firstly, from the viewpoint of the “microbiota–host” interaction, the gut microbiota has been established as a key upstream driver in remodelling host lipid metabolism [60]. In conjunction with the altered jejunal microbiota structure observed in this study, we speculate that grape branch and leaf silage may have influenced host fatty acid metabolism by modulating the composition and function of the intestinal microbiota. Secondly, from the broader pattern of “dietary intervention–metabolic response”, recent research indicates that dietary interventions can regulate lipid metabolism by systematically reshaping the host’s metabolomic profile, even across different animal models [61]. Therefore, the widespread downregulation of medium- and short-chain acylcarnitines observed herein is a specific manifestation of this cross-species conserved “diet–metabolic remodelling” effect, suggesting that grape branch and leaf silage may have enhanced mitochondrial function and promoted efficient fatty acid oxidation for energy production via the aforementioned pathways. This process likely reduced the accumulation of intermediate metabolites and optimised overall energy metabolic efficiency. This inference aligns with and corroborates the observed decrease in serum total cholesterol and triglyceride levels in the present study. Furthermore, the downregulation of N-stearoyl glutamate and upregulation of 2-methyl-2E-Heptenoic acid further revealed complex alterations in lipid metabolism. N-stearoyl glutamate belongs to the class of N-acyl amino acids, which are often involved in inflammatory signalling. Recent research indicates that the intestinal levels of many immunomodulatory lipid molecules, including N-acyl amino acids, are profoundly influenced by the microbiota and serve as crucial messengers in the “gut microbiota–host immunity” communication axis [62]. Consequently, the reduction in its levels may not only be directly attributed to the anti-inflammatory properties of grape branch and leaf polyphenols but may also stem from the remodelling of the gut microbiota by the silage, thereby reducing the synthesis of downstream pro-inflammatory lipid mediators. This change is consistent with the declining trend of serum pro-inflammatory cytokines IL-1β and TNF-α. Together, these findings indicate, across multiple levels of “microbiota–metabolite–immunity”, that grape branch and leaf silage can exert systemic anti-inflammatory effects by modulating the gut microecology and its metabolic output. In summary, feeding grape branches and leaves enhanced amino acid metabolic efficiency, down-regulated metabolites such as acylcarnitines, promoted protein synthesis while reducing fat synthesis, and alleviated intestinal inflammation in Kazakh rams, thereby achieving multi-pathway regulation at the metabolic level.

4.5. Study Limitations

Despite the promising results, this study has several limitations. Firstly, we did not quantitatively analyze the specific bioactive compounds, such as polyphenols and flavonoids, in the grape branch and leaf silage used. While the observed effects align with the known functions of these compounds cited in the literature, the lack of direct quantification prevents the establishment of a precise dose–response relationship and limits the mechanistic interpretation at the molecular level. Secondly, the trial was conducted over a fixed period (120 days) under controlled conditions; thus, the long-term effects and economic viability under practical farming settings require further investigation. Future research should focus on: (1) profiling the dynamic changes in key bioactive components during the ensiling process and their in vivo bioavailability; (2) elucidating the causal mechanisms using targeted metabolomics and multi-omics integration approaches; and (3) validating the optimal inclusion ratio and long-term benefits through large-scale field trials.

5. Conclusions

This study demonstrates that dietary supplementation with grape branch and leaf silage improves the production performance of Kazakh rams. Specifically, it enhances dressing percentage and reduces tail fat deposition, while simultaneously strengthening the body’s antioxidant and immune functions and lowering inflammation levels. By modulating the intestinal microbiota composition and metabolic function, grape branch and leaf silage represents a potential functional roughage resource for improving mutton yield and quality. These findings were obtained under specific experimental conditions and duration; therefore, the optimal inclusion level and long-term effects require further validation through expanded sample sizes and extended trial periods. Future research should focus on identifying the key active components (e.g., polyphenols) and employing multi-omics technologies to systematically elucidate the causal mechanisms along the “microbiota–metabolite–host phenotype” axis.