In Vitro and In Situ Evaluation of White Mulberry (Morus alba) Pomace and Leaf: Fermentation Kinetics, Digestibility, and Potential as Alternative Ruminant Feed Sources

Abstract

Mulberry (Morus alba) by-products represent underutilized feed resources with potential for ruminant nutrition. This study evaluated the rumen fermentation kinetics and rumen digestibility of dried mulberry pomace (MP) and leaf (ML) to determine optimal inclusion strategies in dairy cattle diets. Mulberry pomace (MP) and mulberry leaf (ML) were sun-dried and incorporated at 50% substitution levels into total mixed rations (TMR) with varying concentrations (30%, 35%, 40%, 45%, and 50%) of neutral detergent fiber (NDF). This created ten treatment groups: 30NP through 50NP (pomace-supplemented, where the number represents basal TMR NDF%) and 30NL through 50NL (leaf-supplemented), plus control groups containing only MP or ML and five basal TMR controls (30N through 50N). Rumen fluid was collected from two non-lactating Holstein cows fitted with ruminal cannulas. Chemical analysis revealed that ML contained 19% crude protein and 27.4% NDF, while MP contained 14.9% crude protein and 35.8% NDF. The highest gas production was observed in the 45NP (43.20 mL) and 50NL (43.50 mL) groups. Results demonstrated that MP achieved optimal fermentation when combined with 40–45% NDF TMR (maximum total volatile fatty acid (VFA): 88.86 mmol/L in 40NP at 48 h), whereas ML performed best with 45% NDF TMR (45NL: 88.03 mmol/L total VFA), indicating these as the most promising treatment combinations for ruminant feeding systems pending in vivo validation. Acetate proportions were higher in ML groups (84–96%), while propionate ratios were elevated in MP groups. Both materials maintained optimal ruminal pH (6.2–6.8). In vitro NDF digestibility was significantly higher for ML, with differences increasing from 2.97% at 2 h to 16.44% at 240 h. In situ degradation of MP was nearly complete at 48 h, while ML reached maximum degradation at 24 h. These findings indicate the potential of MP and ML as valuable alternative feed sources for ruminants, particularly in TMRs containing 40–45% NDF.

1. Introduction

Mulberry, an oval-shaped, nutritionally dense fruit of the Morus genus within the Moraceae family, exhibits considerable diversity with 24 recognized species, one subspecies, and over 100 identified varieties [1]. Paleobotanical, morphological, anatomical, and molecular evidence suggests that mulberry originated in the Himalayan foothills before dispersing across Asia, Europe, and the Americas. Historical cultivation records indicate Chinese domestication as early as 2200 BC, with contemporary global production concentrated in China (626,000 hectares) and India (280,000 hectares), predominantly featuring black mulberry (Morus nigra L.), white mulberry (Morus alba L.), and red mulberry (Morus rubra L.) [2].

The nutritional composition of mulberry components is influenced by cultivar, environmental conditions, and processing methods. Fresh mulberry fruits contain dry matter (9.45–28.50%), crude protein (0.51–2.98%), crude fat (0.34–7.21%), and ash (0.46–4.79%). Fresh mulberry leaves exhibit broader compositional ranges: dry matter (19.8–30.4%), crude protein (4.7–22.3%), crude fat (0.6–4.4%), ash (4.1–14.5%), carbohydrates (8.1–13.4%), and neutral detergent fiber (8.1–43.4%). Dried mulberry leaves demonstrate enhanced nutrient density, with crude protein (11.75–37.36%), neutral detergent fiber (19.38–36.66%), and acid detergent fiber (10.2–29.7%). Additionally, mulberry leaves provide essential micronutrients, including calcium (380–786 mg/100 g), ascorbic acid (200–280 mg/100 g), β-carotene (10,000–14,688 μg), iron (4.7–10.36 mg/100 g), and zinc (0.22–1.12 mg/100 g) [3,4].

Industrial processing generates mulberry pomace, comprising approximately 8% of fresh fruit weight and consisting primarily of peels and stems, which currently presents environmental disposal challenges despite its potential as ruminant feed [5]. Recent studies have established mulberry derivatives as effective protein sources across livestock species. Dietary inclusion of 3.5% mulberry leaf powder enhanced the growth performance of broiler chickens [6], while 4% supplementation modulated intestinal microbiota by promoting beneficial bacterial populations, including Bacteroides, Prevotella, and Megamonas [7]. Fermented mulberry leaf powder at 3% inclusion optimized daily weight gain and feed conversion efficiency in broiler chickens [8].

Ruminant studies have shown that mulberry leaf flavonoids ameliorate pathogenic Escherichia coli K99 infections through growth promotion, diarrhea reduction, and intestinal microbiota regulation [9]. Mulberry supplementation facilitates rumen papilla regeneration and enhances basal layer integrity in sheep [10], while 600 g/d mulberry leaf pellets improve dry matter intake, ruminal ammonia concentrations, and cellulolytic bacterial populations in cattle [11]. Furthermore, inclusion of 5–10% mulberry leaf in silage promotes rumen microflora development, enhances fermentation efficiency, and supports fiber digestion while maintaining milk fat-associated microbial communities [12].

Although extensive studies have documented the nutritional value and antioxidant properties of mulberry, a comprehensive understanding of mulberry pomace and leaf in ruminant nutrition remains limited. Therefore, this study was designed to evaluate the in vitro fermentation characteristics (total gas production, ammonia concentration, volatile fatty acid profiles, methane emissions, and CO₂ production) and in situ degradation kinetics of mulberry pomace and mulberry leaves to establish their potential as sustainable non-conventional ruminant feed resources.

2. Materials and Methods

2.1. Rumen Fluid Collection and Donor Cows

The study utilized two rumen-cannulated, non-lactating Holstein cows as sources of ruminal fluid. The animals, aged approximately three years with an average body weight of 530 kg, were maintained on a total mixed ration (TMR) formulated to meet maintenance-level nutritional requirements. The dietary composition on a dry matter basis included 13% crude protein (CP), 93% organic matter (OM), 45% neutral detergent fiber (NDF), 28% acid detergent fiber (ADF), and 6% acid detergent lignin (ADL). Freshwater was provided ad libitum to ensure proper hydration and physiological function.

Rumen fluid samples were collected three hours post-morning feeding to capture peak microbial activity and representative fermentation conditions. Approximately 1.5 L of ruminal fluid was obtained per session, with subsamples drawn from multiple sites within the rumen to ensure representativeness. Collection was performed using a flexible catheter inserted through the ruminal cannula. The fluid was immediately filtered through a double-layered cheesecloth to remove large feed particles while preserving the microbial population.

The filtered rumen fluid was transferred into preheated thermos flasks maintained at 39 °C, the optimal temperature for microbial viability and activity. To prevent alterations in microbial composition or fermentation parameters, samples were transported to the laboratory under strict anaerobic and temperature-controlled conditions within 15 min of collection. This protocol ensured the integrity of the rumen fluid for subsequent experimental procedures.

2.2. Basal Ration, Mulberry Pomace, and Mulberry Leaf

The mulberry pomace utilized in this study was sourced from a company specializing in the production of mulberry molasses located in Malatya, Turkey (latitude 35°54′39″ N, longitude 38°45′39″ E). The collected pomace was evenly distributed on a clean, flat surface in an open environment and sun-dried until a constant weight was achieved, which required approximately 3 to 4 days. Similarly, fresh white mulberry (Morus alba) leaves were procured from various sites in Erzincan, Turkey (latitude 39°44′47″ N, longitude 39°29′29″ E). These leaves were spread on a clean, flat surface in an open area and sun-dried under similar conditions until attaining a stable weight. Subsequently, the dried leaves were ground and stored at −20 °C for future use. For the experimental groups, total mixed rations (TMRs) with varying neutral detergent fiber (NDF) levels (30%, 35%, 40%, 45%, and 50%) were obtained from different farms. The TMR formulations were designed for a Holstein dairy cow weighing 600 kg, aged 55 months, with a body condition score of 3, producing 28 L of milk daily, and in the 10th week of lactation. Mulberry pomace and mulberry leaves were incorporated into the TMRs at a ratio of 50% TMR to 50% mulberry pomace or mulberry leaf, as detailed in

Table 1. Experimental treatment groups: total mixed rations with varying NDF levels supplemented with 50% mulberry pomace or mulberry leaf.

Treatment Code	Description
Basal TMR Controls
30N	Total mixed ration containing 30% NDF
35N	Total mixed ration containing 35% NDF
40N	Total mixed ration containing 40% NDF
45N	Total mixed ration containing 45% NDF
50N	Total mixed ration containing 50% NDF
Mulberry Pomace Treatments
MP	100% mulberry pomace (no TMR)
30NP	50% mulberry pomace + 50% TMR (30% NDF)
35NP	50% mulberry pomace + 50% TMR (35% NDF)
40NP	50% mulberry pomace + 50% TMR (40% NDF)
45NP	50% mulberry pomace + 50% TMR (45% NDF)
50NP	50% mulberry pomace + 50% TMR (50% NDF)
Mulberry Leaf Treatments
ML	100% mulberry leaf (no TMR)
30NL	50% mulberry leaf + 50% TMR (30% NDF)
35NL	50% mulberry leaf + 50% TMR (35% NDF)
40NL	50% mulberry leaf + 50% TMR (40% NDF)
45NL	50% mulberry leaf + 50% TMR (45% NDF)
50NL	50% mulberry leaf + 50% TMR (50% NDF)

TMR = Total mixed ration; NDF = neutral detergent fiber; MP = mulberry pomace; ML = mulberry leaf. The number in treatment codes indicates the NDF percentage of the basal TMR. All mulberry-supplemented treatments contain 50:50 (DM basis) mixtures of mulberry material and respective TMR.

.

2.3. Determination of Chemical Compositions

The basal and experimental total mixed rations (TMRs) underwent thermal desiccation at 55 °C for a 48 h period using a forced-air drying oven (VENTI-Line^®, VWR, Radnor, PA, USA). Following the drying process, the samples were subjected to mechanical grinding through a 1 mm screen mesh utilizing a laboratory mill (Retsch SM100, Haan, Germany) to achieve a uniform particle size distribution suitable for subsequent chemical analyses. The chemical constituents of both basal TMRs (designated as 30N, 35N, 40N, 45N, and 50N) and experimental TMRs (identified as MP and ML) are comprehensively presented in

Table 2. Feed chemical composition of experimental total mix rations.

	Diets
Chemical Composition (DM Basis)	MP	ML	30N	35N	40N	45N	50N
TDN, % *	55.7	49	66.6	59.7	63.1	61.2	60
NEL, Mcal/kg *	1.24	0.92	1.42	1.26	1.34	1.29	1.22
NEM, Mcal/kg *	1.16	0.76	1.47	1.25	1.36	1.28	1.2
NEG, Mcal/kg *	0.58	0.21	0.86	0.66	0.77	0.7	0.62
Dry matter, %	32.14	42.54	52.68	54.68	53.10	53.12	52.78
Crude protein, %	14.9	1.87	2.16	2.15	2.19	2.71	2.23
Ether extraction, %	14.9	1.87	2.16	2.15	2.19	2.71	2.23
NDF, %	35.8	27.4	30	35	40	45	50
ADF, %	35.9	31.3	19.9	35.3	27.3	31.4	32.3
NFC, %	13.9	43.8	48.9	29.4	37.5	32.5	31.2
Ash, %	21.7	19	5.78	7.66	7.6	7.58	7.24
A fraction, % CP	15.5	100	23.9	35.6	29.1	27.4	33.9

* Calculated by NRC (2001) equations [16]; TDN—Total digestible nutrient, ME—metabolizable energy, NEL—net energy lactation, NEM—net energy for maintenance, NEG—net energy growth, NDF—neutral detergent fiber, ADF—acid detergent fiber, and NFC—non-fibrous carbohydrate.

. Analysis was performed for ash, CP, and ether extract (EE) contents using The association of official analytical chemists (AOAC) methods [13]. Neutral detergent fiber (NDF) and acid detergent fiber (ADF) were analyzed by sequential detergent extraction according to Van Soest et al. (1991) [14]. The soluble N fraction in the borate-phosphate buffer (non-protein N, NPN, and fraction of the crude protein) was analyzed, following the procedure of Krishna-moorthy et al. [15]. Non-fiber carbohydrate (NFC) was calculated as per the National Research Council (NRC) equation [16].

NFC = 100 − (NDF% + CP% + EE% + Ash%)

(1)

2.4. In Vitro Experimental Design and Incubation Procedure

The study employed a robust, completely randomized design comprising 17 distinct treatments, each replicated 15 times. The replication number was determined through a prior power analysis (α = 0.05, power = 0.80, effect size = 0.35) based on preliminary fermentation trials, ensuring sufficient statistical power to detect biologically significant differences while accounting for inter-day variability in rumen inoculum characteristics. To address the potential variability associated with rumen fluid collection and donor animal physiological status, replicates were distributed across 12 experimental runs, with approximately 1–2 replicates per treatment per run. Blank controls (n = 5 per run) were integrated to quantify endogenous gas production, calibrate analytical determinations for volatile fatty acids and ammonia-nitrogen, and to assess the inoculum quality and microbial viability, with acceptance criteria defined as pH within 6.5–6.9 and gas production exceeding 10 mL within six hours. Runs failing to meet these criteria were excluded and repeated. Fermentation chambers consisted of 120 mL serum bottles containing 460 mg substrate (dry matter basis), 20 mL rumen fluid, and 40 mL anaerobic buffer medium (pH 6.8). Anaerobic conditions critical for microbial activity were maintained via continuous CO₂ flushing during inoculation. This methodological rigor ensures reliable and reproducible results in evaluating fermentation characteristics [17]. The buffer solution was prepared according to standard methods and contained the following ingredients to simulate the physiologic conditions of the rumen: (1) the macro-mineral solution consisted of magnesium sulfate, disodium phosphate, and potassium dihydrogen phosphate dissolved in bi-distilled water; (2) two bicarbonate buffer solutions containing ammonium bicarbonate and sodium bicarbonate as pH stabilizers; (3) a trace mineral solution containing manganese chloride, calcium chloride, ferric chloride, and cobalt chloride dissolved in distilled water; (4) redox indicator solution containing resazurin; and (5) a reducing agent solution prepared by dissolving sodium sulfide in distilled water containing sodium hydroxide [18,19]. All reagent-grade chemicals (sodium phosphate dibasic, sodium phosphate monobasic, sodium chloride, potassium chloride, magnesium sulfate, calcium chloride, all ≥ 99.0% purity; Sigma-Aldrich, St. Louis, MO, USA) were dissolved in ultrapure water (18.2 MΩ·cm resistivity). Buffer pH was verified before use (target 6.8 ± 0.1) and adjusted, if necessary, using dilute HCl or NaOH. Post inoculation, the bottles were sealed with rubber stoppers, crimped with aluminum caps, and incubated at 39 °C for 48 h. Fermentation bottles (n = 15 per treatment per run) were incubated at 39 ± 0.5 °C in a temperature-controlled environment on an orbital shaker (VWR Incubating Shaker Model IS-2073F, VWR International LLC, Radnor, PA, USA) set at 200 rpm with 2-inch orbital displacement. This standardized shaking regime was selected following preliminary optimization trials (n = 3 independent runs with identical substrate), which confirmed reproducible gas production dynamics (coefficient of variation = 3.8%) and was maintained continuously throughout the 48 h fermentation period. This mechanical agitation methodology ensures consistent substrate mixing and microbial inoculum contact, critical for reproducibility across experimental runs and for future replication by independent laboratories.

2.5. Total Gas Production and Fermentation Parameters

A high-resolution automated modular in vitro gas production system was used to measure total gas production and fermentation characteristics. The system was based on pressure-sensitive sensors, embedded in bottle caps, that recorded gas pressure and temperature at 5-min intervals during the entire incubation. All fermentation incubations were conducted in a temperature-controlled incubator room set to 39 ± 0.5 °C. Temperature was continuously monitored using calibrated thermometers (Durac Digital Thermometer ModernChef Inc., Xiamen, China, ±0.1 °C accuracy) placed within three representative fermentation bottles per run. Temperature recordings were documented every 4 h and maintained within the specified range throughout all 48 h incubations. Each 120 mL glass fermentation bottle contained a substrate–inoculum mixture to a consistent fill line, creating a uniform headspace of approximately 15 mL (12.5% of total bottle volume). Bottles were inspected every 12 h to verify valve function and prevent pressure-related complications. Prior to each experimental run, the incubator was pre-equilibrated for a minimum of 30 min to ensure thermal stability. Data acquisition in real time for 50 separate fermentation modules was controlled by the computer, processing and analyzing all the results with a dedicated software (In vitro semi-automatic modular gas system, V1) that generated data visualization, computational analysis, and results. Fermentation kinetics of total mixed ration (TMR) substrates were evaluated using mathematical modeling methods. In particular, the double-pool logistic equation model was applied together with the curve subtraction method as previously described, to estimate kinetic parameters [20,21]. Cumulative gas volumes were recorded at 24 and 48 h after inoculation. The duration of the lag phase was estimated according to the mathematical model given by Tunkala et al. (2023) [22].

y = A + Cexp{−exp [−B(X − M)]}

(2)

wherein A indicates the y-intercept, B = rate of gas production (mL/h), C = maximum gas produced (mL/g DM), X = total time (h) of incubation, and M = the time (h) at which the maximum rate of gas production was reached.

2.6. Determination of In Vitro Digestibility of Dry Matter and Neutral Detergent Fiber

In vitro digestibility coefficients were determined with the ANKOM DaisyII incubation system (ANKOM Technology, Macedon, NY, USA). Heat-sealed bags (F57 filter bag, 25 μm porosity) containing ground (1 mm screen) TMR samples (0.50 g) were incubated. These bags were then placed in an orderly manner into the glass fermentation vessel (containing 1800 mL of bicarbonate-phosphate buffer solution prepared with the two-part buffer system as outlined in the ANKOM procedure). To provide for an adequate buffer-to-inoculum ratio, 400 mL of fresh rumen fluid was added to each vessel. Digestibility was tracked at several time points (from 2 to 240 h) using duplicate fermentation vessels to describe temporal degradation patterns of feed components [23,24]. At each specified time interval, the bags were extracted, rinsed with tap water until the wash water was clear, and dried in an oven at 105 °C to a constant mass for DM residue analysis. Subsequently, the neutral detergent fiber (NDF) of the dried residues was analyzed using an ANKOM Fiber Analyzer (ANKOM 2000 Fiber Analyzer, ANKOM Technology, Macedon, NY, USA) with sequential detergent extraction, and NDF disappearance was calculated. To maintain microbial activity over long incubation periods, the fermentation medium (rumen fluid-buffer solution) was replaced at 120 h under anaerobic conditions to avoid exposure to oxygen [25]. The procedure was performed under aseptic conditions, and incubation continued at 39 °C with continuous rotation to simulate the ruminal environment. Strict anaerobic conditions were maintained throughout the experiment to ensure the survival of the microbial population. The in vitro dry matter digestibility (IVDMD) and in vitro NDF digestibility (IVNDFD) at each time interval were calculated using the following equations:

IVDMD (%, DM) = 100 − [(W3 − (W1 × C1)) × 100] (W2 × % DM_Feed)

(3)

IVNDFD (%, DM) = 100 × [(W2 × % NDF_Feed) − (W3 − (W1 × C1))]/(W2 × % DM_Feed)

(4)

uNDF calculation was performed using the NDF residue remaining in the bag at the end of incubation periods by incorporating the bag correction factor using the following equation:

uNDF (%, DM) = [W3 − (W1 × C)/D2] × W1 × 100

(5)

where W1 indicates the weight of the filter bag, W2 is the weight of the sample, W3 is the final weight (filter bag + sample), NDF_Feed is the % of NDF of feed (on DM basis), DM_Feed is the % of dry matter in the feed, and C1 is the correction factor (blank filter bag NDF value).

2.7. Determination of Volatile Fatty Acids (VFAs) and Ammonia-Nitrogen (NH₃-N)

To measure the volatile fatty acids (VFAs) and ammonia nitrogen (NH₃-N), fermentation fluid samples were taken at two time points, i.e., 24 and 48 h after incubation. A spectrophotometer for ammonia-nitrogen determination (Genesys 150 UV–Vis Spectrophotometer, Thermo Fisher Scientific Inc., Waltham, MA, USA). At each time point, three fermentation bottles were opened, and sterile techniques were used to collect supernatant aliquots for analysis. For VFA analysis, 1 mL of fermentation fluid from each bottle was mixed immediately with 0.2 mL of 25% (w/v) meta-phosphoric acid in microcentrifuge tubes to halt the microbial activity and to maintain the sample stability [26]. Simultaneously, another 1 mL aliquot was treated with 20 μL of concentrated sulfuric acid in separate tubes for NH₃-N quantification. These prepared samples were stored at −20 °C until further analysis. The concentrations of VFAs were determined using a gas chromatograph (Agilent 6890A GC System, Agilent Technologies, Santa Clara, CA, USA, equipped with a flame ionization detector and Agilent J&W GC column capillary column, 30 m × 0.25 mm × 0.25 μm). Before each daily run, the gas chromatograph was calibrated using certified gas standards (CO₂:CH₄:N₂ at 25:5:70% v/v; Linde Industrial Gases, Istanbul, Turkey) spanning the expected concentration ranges (CO₂: 10–90%, CH₄: 0.1–10%, N₂: remainder). Daily calibration curves (R² > 0.999) were generated and stored electronically. Standard gas injections were repeated every 8 samples to validate the calibration stability. An external standard mixture containing acetic, propionic, and butyric acids (≥99.5% pure, Chem Service Inc., West Chester, PA, USA) was used for calibration. The chromatographic method involved an initial temperature of 80 °C held for one minute, followed by a ramp up to 120 °C at 20 °C/min, then an increase to 230 °C maintained for three minutes. The injector port was set at 250 °C with a split ratio of 10:1 and an injection volume of 1 μL. The detector was also maintained at 250 °C. Hydrogen gas (≥99.998% pure) was used as the carrier gas at a flow rate of 30 mL/min, while air flow was set at 300 mL/min. Prior to analysis, frozen samples were thawed at room temperature and centrifuged at 14,500× g for 10 min at 4 °C. The supernatant was transferred to clean autosampler vials for injection. Ammonia nitrogen levels were measured using the phenol-hypochlorite method as outlined by Weatherburn [27].

2.8. Determination of In Situ Degradability

Ruminal degradation kinetics of feed samples were evaluated using the in situ nylon bag technique as originally described by Ørskov [28]. Two lactating Holstein dairy cows (body weight: 650 ± 25 kg; days in milk: 120 ± 15) fitted with permanent ruminal cannulae served as experimental donors. The animals were housed in individual tie-stalls and fed a total mixed ration formulated to meet the nutritional requirements for lactating dairy cows according to NRC (2001) guidelines [16]. Polyester bags (7 × 14 cm dimensions, 45 ± 10 μm pore size) were employed for ruminal incubation. Prior to use, all bags were machine-washed with non-ionic detergent, thoroughly rinsed with distilled water, and oven-dried at 55 °C for 48 h to achieve constant weight. After cooling in a desiccator, bags were individually weighed. About 3.0 to 3.5 g of air-dried ground sample (2 mm screen) was weighed into each bag. Separate bags were made for each time point (2, 4, 6, 8, 12, 24, 48, 72, 120, and 240 h) and for each animal, with duplicate samples per time point for each feed sample [29]. The bags were all tied to a nylon rope threaded through a hollow, rigid plastic tube (about 25 cm long and 2 cm in diameter) for simultaneous incubation and separation. A stainless-steel weight (500 g) was fixed to the far end of the tube to hold it in the ventral sac of the rumen and prevent floating. The whole apparatus was gently pushed into the rumen via the cannula with the proximal end of the rope firmly tied to the cannula plug so as not to lose the bags when subjected to ruminal contractions. At each designated time point, bags were retrieved from the rumen and immediately immersed in ice-cold water to arrest the microbial fermentation [30]. Bags were then rinsed under cold running tap water until the effluent became transparent, ensuring removal of loosely adhered microbial biomass and feed particles. Following manual rinsing, bags underwent three additional 5-min wash cycles in a portable washing machine (without detergent) to standardize the washing procedure across all samples. Subsequently, bags were oven-dried at 55 °C for 48 h to constant weight, cooled to ambient temperature in a desiccator, and weighed to determine residual dry matter content, followed by the calculation of degradability.

2.9. Determination of pH, Methane Emission, and Carbon Dioxide

The fermentation bottle caps were removed, and the pH was immediately determined at 0, 2, 6, 12, 24, and 48 h. Ruminal fermentation pH was measured using a calibrated pH meter (LAQUA F-72, HORIBA Scientific, Kyoto, Japan) at timepoints: 24 and 48 h. Measurements were performed on 1 mL samples from each replicate bottle in triplicate determinations; values were recorded to 0.01 pH unit precision. An acceptable pH range for continued fermentation was 6.0–7.0. Fermentation was considered compromised in case pH fell below 6.0. Stoichiometric models were used for estimating methane [31] and carbon dioxide [32] from VFA composition as follows:

Methane (CH₄), mmol/L = 0.45 × acetate − 0.275 × propionate + 0.40 × butyrate

(6)

Carbon dioxide (CO₂), mmol/L = acetate/2 + propionate/4 + 1.5 × butyrate

(7)

2.10. Statistical Analysis

All the statistical procedures were carried out using a computer-based statistical software package (SPSS version 25.0). To examine the differences between experimental groups, a Repeated Measures General Linear Model (Repeated Measures GLM) was applied for hourly gas production, IVDMD, NDF fractions, pH, NH₃-N, organic matter digestibility (OMD), methane production, and in situ degradation. For repeated measures analysis, the compound symmetry assumption was tested in terms of covariance structure and evaluated using Mauchly’s test of sphericity. In cases where the sphericity assumption was violated, the Greenhouse-Geisser correction was applied. The normality of data distribution was checked using Shapiro-Wilk’s test, and homogeneity of variances was evaluated by applying Levene’s test. Logarithmic transformation was applied to the non-normalized data. In the established model, experimental groups, time, and group–time interactions were determined as fixed factors. In case of significant interaction between fixed factors, an independent samples t-test was used to differentiate the significantly different means from 2 independent samples, a paired samples t-test when 2 samples are involved in case of a time-dependent case, and one-way analysis of variance in case of more than two factors. The Bonferroni multiple comparison test was employed to differentiate the significantly different means in multiple comparisons. The differences among the means were considered significant at a 95% confidence interval (p < 0.05).

3. Results

The chemical composition of both basal TMRs (designated as 30N, 35N, 40N, 45N, and 50N) and experimental TMRs (identified as MP and ML and their combinations with basal TMRs) was determined through comprehensive proximate and fiber analyses, with complete compositional data presented in Table 2.

Significant differences (p < 0.001) were seen between the groups in terms of gas production at different incubation hours (

Table 3. Gas production (mL/h) in mulberry pomace, mulberry leaf, and different TMR combinations.

Group	Hour				Mean
	6	12	24	48
MP	8.80	18.90	42.92	55.00	31.10 b
30NP	7.62	15.86	46.45	62.59	33.13 ab
35NP	7.04	22.23	49.68	62.76	35.43 ab
40NP	11.31	24.91	49.75	71.90	39.47 ab
45NP	14.42	29.65	61.74	66.97	43.20 a
50NP	8.88	17.59	37.84	63.27	31.90 ab
Mean	9.68 a	21.52 b	48.07 c	63.58 d	SEM: 1.078
ML	8.49	15.36	35.32	57.56	29.18 c
30NL	6.54	12.07	32.53	66.10	29.30 bc
35NL	11.67	22.78	49.63	75.84	39.98 ab
40NL	7.23	14.50	33.06	62.18	29.24 bc
45NL	10.36	20.90	45.14	66.07	35.62 abc
50NL	13.95 a	30.03 b	59.59 c	70.42 d	43.50 a
Mean	9.71 d	19.27 c	42.55 b	66.36 a	SEM: 1.085
30N	9.71	23.20	52.99	83.17	42.27
35N	10.85	27.47	59.83	68.97	41.78
40N	8.19	15.30	42.54	69.48	33.88
45N	8.95	26.23	57.55	70.65	40.85
50N	11.72	23.38	52.27	58.79	36.54
Mean	9.89 d	23.12 c	53.04 b	70.21 a	SEM: 1.388

MP—feed group with only mulberry pomace added; 30NP—group with feed containing 30% NDF added to 50% mulberry pomace; 35NP—group with feed containing 35% NDF added to 50% mulberry pomace; 40NP—group with feed containing 40% NDF added to 50% mulberry pomace; 45NP—group with feed containing 45% NDF added to 50% mulberry pomace; and 50NP—group with feed containing 50% NDF added to 50% mulberry pomace. ML—feed group with only mulberry leaf added; 30NL—group with feed containing 30% NDF added to 50% mulberry leaf; 35NL—group with feed containing 35% NDF added to 50% mulberry leaf; 40NL—group with feed containing 40% NDF added to 50% mulberry leaf; 45NL—group with feed containing 45% NDF added to 50% mulberry leaf; and 50NL—group with feed containing 50% NDF added to 50% mulberry leaf. 30N—feed containing 30% NDF; 35N—feed containing 35% NDF; 40N—feed containing 40% NDF; 45N—feed containing 45% NDF; and 50N—feed containing 50% NDF. SEM—Standard error of means. All values showed no significant difference (p > 0.05) except for the ones with different superscripts (^a;b;c;d) within the same line that are significantly different at p < 0.01.

). In the mulberry pomace supplemented groups, the highest gas production was observed in the 45NP group, while the lowest gas production was noted in the MP group. In the mulberry leaf-supplemented groups, the highest gas production was observed in the 50NL group, while the lowest gas production was observed in the ML group.

No difference was observed between the ratios of acetic acid to propionic acid (acetate/propionate, A/P). The acetic acid ratio was greater in groups with mulberry leaf (

Table 4. Comparative analysis of volatile fatty acid production and methane emissions across experimental treatments during in vitro fermentation.

	MP		30NP		35NP		40NP		45NP		50NP		SEM
Parameters (mmol/L)	24	48	24	48	24	48	24	48	24	48	24	48
AA	47.33	47.59	47.70	55.56	44.00	53.18	45.83	58.62	54.82	43.51	43.50	47.82	1.17
AA % *	66.69	74.56	64.45	76.92	64.67	79.40	64.34	83.11	68.31	65.72	65.66	73.44	1.93
PA	14.80	14.45	16.79	19.64	14.89	16.73	15.62	18.80	15.50	16.30	13.86	15.28	0.45
PA %	20.81	22.73	22.78	27.29	21.93	25.03	22.06	26.61	19.22	20.32	21.03	23.30	0.76
A/P	3.21	3.32	2.84	2.86	2.96	3.23	2.92	3.14	3.69	3.28	3.12	3.16	0.05
BA	6.09	5.73	6.78	7.92	6.50	7.31	6.78	7.80	7.11	6.92	6.28	7.78	0.15
BA %	8.70	9.01	9.20	10.89	9.57	10.98	9.58	11.06	8.81	8.63	9.51	12.34	0.32
Total VFA (mM)	70.97	70.48	73.90	86.59	68.00	80.43	71.09	88.86	80.39	78.98	66.16	74.37	1.79
Methane (mL) *	23.01	23.05	23.04	26.83	21.53	26.06	22.40	28.51	27.09	25.77	21.43	23.98	0.55
	ML		30NL		35NL		40NL		45NL		50NL		SEM
Parameters (mmol/L)	24	48	24	48	24	48	24	48	24	48	24	48
AA	41.53	51.85	33.95	48.54	42.94	58.98	43.19	55.32	44.58	56.95	42.17	53.21	0.92
AA %	67.57	91.82	67.14	96.44	66.58	91.44	64.30	86.90	66.03	84.47	64.93	86.88	2.13
PA	12.02	15.18	9.72	13.67	12.82	19.80	14.21	18.23	14.20	18.64	13.74	16.30	0.34
PA %	19.46	27.00	19.22	26.83	19.82	30.68	21.17	28.65	20.94	27.53	21.15	26.56	0.68
A/P	3.49	3.45	3.51	3.63	3.37	2.98	3.05	3.05	3.17	3.06	3.11	3.43	0.03
BA	5.71	7.29	4.71	6.90	6.25	9.35	6.71	8.05	6.45	8.77	6.56	8.86	0.10
BA%	9.38	12.90	9.37	13.79	9.65	14.51	10.04	12.64	9.54	13.01	10.23	14.41	0.31
Total VFA (mM)	61.47	77.33	50.56	72.17	64.56	92.03	67.15	85.30	67.59	88.03	64.83	82.05	1.36
Methane (mL)	20.61	25.78	16.90	24.30	21.39	29.22	21.40	27.13	21.96	28.20	20.93	26.96	0.42
	30N		35N		40N		45N		50N		SEM
VFA (mmol/L)	24	48	24	48	24	48	24	48	24	48
AA	42.66	50.39	48.83	60.58	46.78	56.52	47.99	62.05	51.82	55.90	0.98
AA %	64.34	76.56	63.50	79.52	64.86	80.06	64.10	82.85	64.28	69.85	1.31
PA *	14.86	17.93	17.06	20.19	15.50	19.62	16.36	21.31	17.15	19.07	0.41
PA %	22.11	27.09	22.23	26.56	21.31	27.70	21.85	28.52	21.29	23.74	0.45
A/P	2.97	2.82	2.87	3.00	3.07	2.87	2.94	2.93	3.02	2.95	0.03
BA	6.50	7.81	8.03	10.21	7.13	9.60	7.63	9.89	8.52	8.50	0.12
BA %	9.86	12.36	10.47	13.48	9.90	13.48	10.25	13.29	10.61	10.60	0.31
Total VFA (mM)	66.49	79.81	76.86	95.46	72.22	90.09	74.81	97.52	80.55	87.13	1.43
Methane (mL)	20.86	24.62	24.17	30.35	23.08	28.16	23.72	30.64	25.88	27.43	0.42
BA	7.16 b		9.12 a		8.37 a		8.76 a		8.51 a		<0.001
Methane	22.74 b		27.26 ab		25.62 ab		27.18 a		26.66 ab

AA—Acetic Acid; PA—propionic acid; A/P—acetic acid to propionic acid ratio; and BA—butyric acid. MP—feed group with only mulberry pomace added; 30NP—group with 30% NDF feed added to 50% mulberry pomace; 35NP—group with 35% NDF feed added to 50% mulberry pomace; 40NP—group with 40% NDF feed added to 50% mulberry pomace; 45NP—group with 45% NDF feed added to 50% mulberry pomace; and 50NP—group with 50% NDF feed added to 50% mulberry pomace. ML—feed group with only mulberry leaf added; 30NL—group with 30% NDF feed added to 50% mulberry leaf; 35NL—group with 35% NDF feed added to 50% mulberry leaf; 40NL—group with 40% NDF feed added to 50% mulberry leaf; 45NL—group with 45% NDF feed added to 50% mulberry leaf; and 50NL—group with 50% NDF feed added to 50% mulberry leaf. 30N—30% NDF feed; 35N—35% NDF feed; 40N—40% NDF feed; 45N—45% NDF feed; and 50N—50% NDF feed. ^ab: Statistically significant differences were observed between different letters in the same column in terms of experimental groups. * All values except the relevant parameters were statistically significant over time (24 h vs. 48 h; p < 0.001). All parameters showed no significant differences among treatment groups (p > 0.05) except for the ones with different superscripts (^a,b) within the same row that are significantly different at p < 0.01.

), whereas the propionic acid ratio was greater in groups with mulberry pomace compared to other groups. The butyric acid ratio was greater in groups where mulberry leaf was added. As the fermentation time extended (from 24 h to 48 h), significant increases were noted in VFA production (p < 0.05).

It is shown in

Table 5. Changes in pH and NH₃ levels over time in in vitro fermentation samples of groups.

		pH		NH3 (mM)
Group		24	48	24	48
MP		6.56	6.53	11.79	11.91
30NP		6.60	6.29	10.94	11.74
35NP		6.65	6.59	12.15	11.63
40NP		6.58	6.48	11.76	12.17
45NP		6.70	6.58	11.74	12.61
50NP		6.60	6.39	12.10	12.48
p	Group	–		**
	Time	***		**
	Group × Time	*		**
SEM		0.02		0.07
ML		6.68	6.40	11.41	11.65
30NL		6.68	6.49	11.88	12.03
35NL		6.78	6.45	9.43	11.72
40NL		6.76	6.34	11.65	11.19
45NL		6.58	6.42	12.39	11.98
50NL		6.63	6.43	10.48	11.70
p	Group	–		***
	Time	***		***
	Group × Time	**		***
SEM		0.02		0.07
p	Group	–		***
	Time	***		***
	Group × Time	–		*
SEM		0.01		0.11
30N		6.58	6.33	11.08	12.11
35N		6.51	6.44	11.65	12.35
40N		6.55	6.33	11.67	12.33
45N		6.57	6.32	12.01	12.42
50N		6.53	6.35	11.08	11.90
p	Group	–		–
	Time	***		***
	Group × Time	–		–
SEM		0.01		0.08

MP—feed group with only mulberry pomace added, 30NP—group with feed containing 30% NDF added to 50% mulberry pomace, 35NP—group with feed containing 35% NDF added to 50% mulberry pomace, 40NP—group with feed containing 40% NDF added to 50% mulberry pomace, 45NP—group with feed containing 45% NDF added to 50% mulberry pomace, and 50NP—group with feed containing 50% NDF added to 50% mulberry pomace. ML—feed group with only mulberry leaf added, 30NL—group with feed containing 30% NDF added to 50% mulberry leaf, 35NL—group with feed containing 35% NDF added to 50% mulberry leaf, 40NL—group with feed containing 40% NDF added to 50% mulberry leaf, 45NL—group with feed containing 45% NDF added to 50% mulberry leaf, and 50NL—group with feed containing 50% NDF added to 50% mulberry leaf. 30N—feed containing 30% NDF, 35N—feed containing 35% NDF, 40N—feed containing 40% NDF, 45N—feed containing 45% NDF, and 50N—feed containing 50% NDF. SEM—Standard error of means. –: p > 0.05; *: p < 0.05; **: p < 0.01; and ***: p < 0.001.

that the time factor has a significant effect on pH (p < 0.001). In groups with added mulberry pomace, the lowest mean ammonia was seen in the 30NP group (11.34), while the highest was noted in the 50NP group (12.29).

The mulberry pomace (MP) group exhibited a progressive increase in IVNDFDM from the 2nd hour to the 240th hour (

Table 6. In vitro dry matter digestibility (IVDMD) and NDF parameters of mulberry leaf and mulberry pomace.

Parameter	Group *	Hour										SEM
		2	4	6	8	12	24	48	72	120	240
IVNDFD, % NDF	MP	5.07 g	5.83 fg	11.21 ef	15.53 def	19.11 de	32.22 cd	35.74 c	52.72 b	53.53 ab	62.78 a	0.48
	ML	7.20 h	14.75 g	21.46 f	27.79 ef	39.09 de	44.32 cd	52.27 bc	54.59 abc	59.82 ab	67.28 a	0.61
IVDMD, %	MP	8.67 f	12.06 ef	18.76 e	22.00 e	25.15 e	41.03 d	48.29 cd	55.07 bc	63.27 ab	72.64 a	0.63
	ML	8.58 g	17.49 f	24.84 e	32.19 d	44.48 c	51.04 bc	57.29 b	61.46 ab	65.16 a	72.26 a	0.42
uNDF, % DM	MP	29.43 a	29.19 ab	27.53 bc	26.19 bcd	25.07 cd	21.01 df	19.92 f	14.66 g	14.41 g	11.62 h	0.15
	ML	34.34 a	31.55 b	29.06 c	26.72 cd	22.54 de	20.60 ef	17.66 fg	16.81 fgh	14.87 fgh	12.11 h	0.23
pdNDF, %	MP	1.98 g	2.22 fg	3.88 ef	5.22 def	6.33 de	10.40 cd	11.49 c	16.75 b	17.01 b	19.79 a	0.15
	ML	3.08 h	5.87 g	8.35 f	10.69 ef	14.87 de	16.81 cd	19.75 bc	20.61 abc	22.54 ab	25.30 a	0.23
uNDF, % NDF	MP	47.92 a	47.33 ab	44.79 abc	42.52 bc	41.13 c	34.14 d	32.39 d	23.78 e	23.50 e	18.86 f	0.25
	ML	46.83 a	43.08 b	39.57 c	36.42 cd	30.65 de	28.14 ef	24.04 fg	22.88 g	20.22 gh	16.53 h	0.29
uNDF, % DM	MP	94.93 a	94.17 ab	88.79 bc	84.47 bcd	80.89 cd	67.78 df	64.26 f	47.29 g	46.47 g	37.47 h	0.48
	ML	92.80 a	85.25 b	78.54 c	72.21 cd	60.91 de	55.68 ef	47.73 f	45.41 f	40.18 fg	32.72 g	0.61

MP—mulberry pomace; ML—mulberry leaf; IVNDF, % NDF—in vitro NDF digestibility; % IVDMD—in vitro dry matter uNDF; % uNDF, % DM—Undigested NDF; pdNDF—physically effective NDF. ^abcdefgh; Significant differences are observed between different letters in the same row (p < 0.001). * Statistically significant differences were observed between experimental groups in terms of the relevant variable (p < 0.05). SEM—Standard error of means.

). The mulberry leaf (ML) group showed significantly higher IVNDFDM values compared to the MP group at all time points. Within each group, all time points were significantly different from one another (p < 0.05) and showed a distinct temporal progression in fiber digestion. In the early period of incubation (2–8 h), mulberry leaf showed markedly higher digestibility than mulberry pomace. Although the amount of indigestible NDF was detected at similar levels in both groups, physically effective NDF (pdNDF) was found to be 62.2% higher in mulberry leaf compared to mulberry pomace.

The degrees of degradation of mulberry leaf and mulberry pomace in the in situ environment over time are indicated in

Table 7. Comparative in situ ruminal degradation parameters of mulberry by-products at different incubation intervals.

Time (Hour)	MP	ML	p
2	18.79 d	22.55 d	*
4	20.77 d	23.44 d	–
6	21.36 d	28.14 cd	**
8	21.59 d	31.63 c	***
12	49.41 c	52.57 b	–
24	66.52 b	73.51 a	***
48	71.29 ab	75.44 a	**
72	72.11 a	74.11 a	–
120	72.91 a	74.23 a	–
240	76.84 a	75.88 a	–

MP—mulberry pomace; ML—mulberry leaf; SEM—standard error of means; ^abcd—statistically significant differences were observed between different letters in the same column in terms of trial groups; –: p > 0.05; *: p < 0.05; **: p < 0.01; and ***: p < 0.001.

. Degradation rates of mulberry pomace and leaf increased after the 8th hour. Degradation of mulberry pomace was almost complete at the 48th hour, and that of mulberry leaf at the 24th hour (p < 0.05).

4. Discussion

Chemical composition of mulberry leaves and pomace demonstrates a nutritional profile consistent with leguminous forages, exhibiting greater crude protein and reduced fiber concentrations relative to gramineous species. Variations in the nutritional composition of mulberry-derived by-products reflect the influence of cultivar selection, environmental conditions, and harvest timing, with the analyzed samples conforming to established nutritional ranges documented in previous studies.

Fiber fraction analysis revealed NDF values within the globally reported range of 26.6–46.0% and ADF concentrations spanning 18.7–35% [33,34]. While crude fiber content aligned with the established range of 9.1–15.3% reported in previous studies [33,35,36]. The current study exhibited lower NDF concentrations compared to certain studies [37,38] yet was consistent with Vu et al. [39]. Ash content exceeded the ranges documented by some studies [40,41,42], while similar to others (2009) [43,44,45]. According to research findings, the higher ash content of MP (21.7% vs. 19%) affects fermentation dynamics in various ways. When evaluated in terms of mineral–microbial interactions, although certain minerals are necessary for cellulolytic bacteria, it is considered that excessive mineral presence may exhibit inhibitory effects and negatively affect microbial protein synthesis.

A previous study has established mulberry leaves as viable protein supplements for ruminants, with ensiled preparations demonstrating equivalent growth performance to conventional protein sources without adverse effects on dry matter intake [46]. Our findings support these notions since enhanced fermentation characteristics were noted in mulberry-supplemented treatments.

Gas production kinetics revealed distinct patterns between mulberry pomace and leaf. Mulberry pomace exhibited optimal fermentation when combined with 45% NDF substrates, while pomace alone had limited fermentation potential. In contrast, mulberry leaves achieved maximum gas production in high-NDF combinations (50NL group), with superior kinetics compared to those reported by Yao et al. for M. alba at 24 h [47]. The synergistic effect of mulberry by-products and the basal TMR is particularly noteworthy. This suggests that the combination of different fiber sources and carbohydrate types enhances overall fermentability, likely by supporting a more diverse and active microbial community. These variations reflect the influence of chemical composition and harvest-related factors on fermentation dynamics. According to the research results, methane production differences between the MP and ML groups are significant both statistically and biologically. The higher methane values measured in ML groups (20.61–25.78 mL) indicate greater energy loss in ruminal fermentation, which could potentially reduce feed efficiency in animals. From an environmental perspective, the lower methane production observed in MP groups provides advantages in terms of sustainability. The methane production differences identified in the study are directly related to VFA profiles; the high acetic acid/propionic acid ratio in ML groups increases hydrogen utilization and consequently methane synthesis.

The VFA profiles demonstrated time-dependent increases across all treatments, with acetic acid as predominant among the VFA (65–83% of total VFA in pomace groups, 64–96% in leaf groups). The 40NP treatment yielded maximum total VFA and acetic acid concentrations, while the 45NL combination produced optimal fermentation in leaf-supplemented groups. The elevated A:P ratios observed indicate predominant cellulolytic bacterial activity, consistent with Bach et al.’s findings linking fiber intake to increased acetate production [48]. The high acetate proportions in the mulberry-supplemented treatments (65–96%) indicate that both MP and ML support cellulolytic bacterial activity. This is further supported by the relatively high A:P ratios, which are typical of high-fiber diets. These patterns suggest that mulberry pomace incorporation at 50% in 40–45% NDF rations may optimize rumen fermentation efficiency. The VFA profile differences observed between MP and ML groups in the study originate from substrate structure and ruminal microbial ecosystem changes. The high acetic acid ratios detected in ML groups (84–96% versus 65–74%) can be associated with the structural characteristics of ML’s cellulose content. The data suggest that ML promotes the development of cellulolytic bacteria, while MP supports the proliferation of more amylolytic and succinate-producing bacteria. Additionally, flavonoids and other secondary metabolites present in ML likely cause changes in the VFA profile by affecting the metabolic activity of specific microbial populations. The rapid fermentation characteristic of ML may also contribute to increased acetic acid production, which is characteristic in early stages.

Maintaining a stable ruminal pH is critical for optimal microbial function and animal health. The pH values observed in this study (6.2–6.8) are within the optimal range for fiber digestion and microbial protein synthesis [49]. The NH₃-N concentrations observed in this study (9–13 mmol/L) are within the range considered optimal for microbial growth and protein synthesis [11]. Ammonia is a key nitrogen source for ruminal bacteria, and maintaining adequate levels is essential for efficient microbial fermentation. The similarity in NH₃-N concentrations between MP and ML treatments suggests that both by-products provide similar levels of rumen-degradable protein. This is consistent with their similar crude protein contents, further indicating that both by-products can contribute to meeting the nitrogen requirements of the rumen microbiota.

Digestibility data revealed temporal patterns characteristic of fibrous materials, with mulberry leaves exhibiting rapid initial degradation (first 12 h) followed by slower progression, while pomace degradation accelerated after 12 h. The observed IVDMD of 72.26% for mulberry leaves exceeded values reported by Doran et al. [37] but remained consistent with ranges documented by Sanchez [50] and Shayo [33]. NDF digestibility values (44.32% for leaves, 32.22% for pomace) were favorably comparable with alfalfa standards, supporting the potential of mulberry materials as quality roughage alternatives.

The in vitro dry matter digestibility of mulberry leaf observed in this study was greater than the value (61.3%) reported by Doran et al. [37]; however, it should be noted that the mulberry leaf used included both leaves and stems and was obtained from a digestion trial conducted on sheep [37]. In the same study, the CP content of dried mulberry leaf was found to be 16.2%, which may have caused a decrease in DM digestibility. Additionally, it has been reported that there are differences in the digestibility of roughages in different animal species [51]. In the current study, the IVDMD of mulberry leaf was determined as 72.26% (240 h). The in vitro true dry matter digestibility of mulberry leaf in this study is consistent with the data reported by Sanchez [50], who found the in vivo digestibility of mulberry leaf to be 78.4–80.8% and in vitro digestibility to be 80.2–95.0% [50]. Additionally, Shayo [33] reported the in vitro dry matter digestibility of mulberry leaves as 82.1% [33].

In the alfalfa hay, the 24 h IVDNDF (%NDF) and uNDF240 (%DM) were 31.2–40.2% and 15.5–18.5%, respectively [52]. The 24 h in vitro NDF digestibility (IVDNFD, %NDF) of mulberry pomace and mulberry leaf were 32.22% and 44.32%, respectively; the uNDF240 (%DM) values were 11.62% and 12.11%, respectively. An increase in the gap illustrates that ML was not only fermented faster in the beginning, but there was a larger pool of forage fiber that could potentially be fermented. These findings indicate that mulberry leaf has particularly higher early-stage digestibility, but both materials reach similar results in long-term incubation. Low digestibility can impair the dry matter intake because the cellulose particles remain in the rumen for a long time. The increment of acetic acid proportion was ascribed to the enhanced digestion of structural carbohydrate; this was also supported by increased NDF digestibility [53]. According to the presented data, it is observed that mulberry leaf exhibits superior performance compared to mulberry pomace in the early stages of fermentation. The basis of this superiority lies in ML’s lower NDF content (27.4% vs. 35.8%), higher crude protein ratio (19% vs. 14.9%), and higher physically effective NDF value. These characteristics facilitate easier access of ruminal microorganisms to the substrate, accelerating the colonization process. The temporal increase in IVNDFD values observed in the study (rising from 2.97% at 2 h to 16.44% at 240 h) reveals that ML maintains its advantage not only in the early period but also in long-term fermentation.

Saddul et al. (2004) found the DM degradation of whole mulberry leaf, leaf only, and stem only after a 72 h incubation period to be 66.7%, 77.9% and 55%, respectively [54]. In the study conducted by Liu et al. (2001), DM degradation of mulberry leaves was determined as 53.9% after 48 h incubation, whereas Singh and Makkar (2000) found this rate as 76% in the same hourly incubation [34,46]. It is noteworthy that the differences observed between the MP and ML groups at some incubation hours until the 72nd hour disappeared from the 72nd hour onwards. This situation indicates that the in situ degradation data of mulberry pomace and leaf groups can reach a similar level over time. In another study, they determined that DM digestibilities of white mulberry leaves added to sorghum at 2.5% and 5% rates after 12 h were 61.35% and 54.64%, respectively, and showed similar results to the present study [55].

In a previously conducted study, dry matter degradation of alfalfa hay after 48 and 72 h of incubation was reported as 65.72% and 70.89% [56], while in another study it was reported as 68.3% and 71.1% at 48 and 72 h, respectively [57]. In a study conducted by Yüksel and Kaya (2022), dry matter degradation of straw was determined as 49.64% and 62.41% at 48 and 72 h, respectively [58]. Dry matter degradation of corn silage after 48 h of incubation was found to be 63.6% [59]. The similarity of degradation rates of mulberry pomace and mulberry leaves at 48 and 72 h to alfalfa and corn silage indicates that they could be alternatives to quality roughages in rations.

However, it is important to note that the present study was conducted in vitro, and the results may not fully reflect in vivo performance. In vitro systems lack the complexity of the intact rumen, including the presence of rumen walls, the stratification of digesta, and the continuous absorption of VFAs. Therefore, in vivo feeding trials are necessary to validate these findings and to assess the effects of mulberry by-products on milk production, feed efficiency, and animal health under commercial conditions. Short-term laboratory studies cannot predict the potential effects of long-term use of mulberry by-products (adaptation, tolerance, or toxic effect accumulation). The translation of laboratory-scale results to field applications involves challenges such as storage stability, processing technologies, and cost factors. Furthermore, the possible effects of mulberry by-products on animal health and welfare cannot be comprehensively evaluated through in vitro methodologies. Considering all these limitations, despite the observed promising results, comprehensive field studies are needed to determine the optimal use of mulberry pomace and leaves in ruminant feeding.

The use of mulberry by-products as ruminant feeds offers significant environmental and economic benefits. Mulberry pomace is currently an underutilized waste product from the juice and molasses industry, and its disposal can pose environmental challenges. By converting this waste into a valuable feed resource, the livestock industry can contribute to a circular economy and reduce its environmental footprint. Similarly, mulberry leaves, which are abundantly available in many regions, can provide a low-cost alternative to conventional protein supplements. From an economic perspective, the use of locally available by-products can reduce feed costs, which account for the largest proportion of production expenses in dairy farming. This is particularly important in developing countries, where access to high-quality forages and protein supplements may be limited. By demonstrating the nutritional value and optimal inclusion strategies for mulberry by-products, this study provides a foundation for their adoption in practical feeding systems.

5. Conclusions

This comprehensive in vitro and in situ evaluation demonstrated that sun-dried white mulberry pomace and leaf represent nutritionally valuable by-products with distinct fermentation characteristics. The findings indicate that mulberry by-products exhibit favorable fermentation parameters, especially in combination with TMRs containing 40–45% NDF. Notably, mulberry leaf exhibited consistently higher NDF digestibility than pomace, with differences increasing from 2 h to 240 h. Both by-products maintained the optimal rumen pH range (6.2–6.8) and supported adequate NH₃-N concentrations for microbial protein synthesis. While these findings are promising, the limitations of in vitro methodologies necessitate long-term feeding trials to validate performance under commercial conditions. Nevertheless, this study provides compelling evidence that mulberry by-products can serve as valuable alternative feed resources for ruminants when strategically incorporated into diets, potentially reducing feed costs while contributing to agricultural sustainability through by-product valorization.