Comparative proximate composition, Van Soest fiber fractions, and phytochemical profiles of Moringa oleifera, Tamarindus indica, and Azadirachta indica foliage for potential use as ruminant feed resources

Introduction

Ruminant production systems throughout tropical and subtropical drylands operate under chronic nutritional stress caused by marked seasonal fluctuations in green biomass and escalating economic constraints surrounding conventional protein feeds (such as soybean meal and groundnut cake) and cereal grains (Makkar, 2018) [1] . Pastoralists and mixed crop-livestock smallholders frequently depend on mature cereal straws and stover as basal diets during extended dry spells. These crop residues are universally characterized by low crude protein (CP < 70g/kg dry matter [DM]), high cell-wall lignification, and poor trace mineral bioavailability, which suppress ruminal cellulolytic fermentation kinetics and depress voluntary dry matter intake (Van Soest, 1994; Leng, 1990) [3,2] . Perennial deep-rooted fodder trees and shrubs represent strategic feed assets capable of addressing this nutritional deficit. Due to extensive root architectures, these species maintain metabolic vigor, leafy canopy cover, and nutritional synthesis during severe drought conditions, retaining substantial concentrations of nitrogen, minerals, and soluble carbohydrates when herbaceous pastures senesce (Patra, 2010; Devendra et al., 1995) [5,4] . Among tropical arboreal foliages, Moringa oleifera Lam. (Moringaceae), Tamarindus indica L. (Fabaceae), and Azadirachta indica A. Juss. (Meliaceae) are widely naturalized, agronomic candidates with substantial biomass yields. Despite their common classification as multi-purpose fodder trees, their biochemical profiles and physiological integration into rumen metabolic pathways diverge significantly: Moringa oleifera (Drumstick tree): Displays rapid vegetative regeneration and exceptional crude protein content (220 -290g/kg DM), with an amino acid profile closely aligning with high-grade oilcakes (Makkar et al., 1996; Moyo et al., 2011) [6,7] . Its structural cell-wall carbohydrates are minimally lignified, promoting high ruminal organic matter degradability and post-ruminal nitrogen availability (Sultana et al., 2015) [8] .

Tamarindus indica (Tamarind tree):

A drought-hardy leguminous species yielding moderate nitrogen foliage (120 -160g/kg DM) coupled with substantial structural fiber. While useful as a roughage supplement, its vegetative matter contains varying amounts of polyphenols, tartaric acid, and condensed tannins that alter microbial protein degradation and gut passage rates (El-Siddig et al., 2006; Dey et al., 2008) [9] .

Azadirachta indica (Neem tree): Yields foliage with moderate crude protein (130 -180g/kg DM) and significant quantities of bioactive secondary triterpenoids, notably azadirachtin, salannin, and nimbidin (Ur-Rehman et al., 2023) [10] . Although these metabolites exert antimicrobial, anthelmintic, and defaunating activities that mitigate enteric methanogenesis, elevated concentrations impart marked bitterness, depressing voluntary intake and cellulolytic enzyme activity (Patra and Saxena, 2010; Garg, 2001) [11] . A significant limitation in applied ruminant nutrition is the lack of standardized, concurrent comparative evaluations of these three foliages harvested from identical agro-climatic boundaries. Previous reports present analytical disparities stemming from variable stages of maturity, disparate post-harvest processing regimes (sun versus shade drying), and uncoordinated analytical frameworks where proximate composition is reported without the necessary context of Van Soest detergent partitioning or quantified anti-nutritional profiles. To resolve these analytical gaps, this investigation tests the following research hypotheses: Hypothesis 1 (𝐻 1 ): Moringa oleifera foliage contains significantly higher CP and significantly lower acid detergent fiber (ADF) and acid detergent lignin (ADL) compared to T. indica and A. indica. Hypothesis 2 (𝐻 2 ): Anti-nutritional constituents (condensed tannins and total phenolics) are concentrated significantly higher in T. indica and A. indica, establishing biochemical constraints on dietary inclusion levels.

The specific objectives are: (1) to systematically quantify proximate composition across the three species using AOAC methods; (2) to delineate structural fiber fractions into neutral detergent fiber (NDF), ADF, and ADL using the Van Soest method; and (3) to measure major secondary compounds (total phenolics, condensed tannins, and phytates) to establish evidence-based feeding recommendations for ruminant diets.

Materials and Methods

Study site and sample collection

Foliage was harvested from mature, healthy trees established within the semi-arid tropical zone of Western India (20 ∘ 30 ′ N, 74 ∘ 32 ′ E, elevation 435m above sea level). Five representative, non-adjacent trees per species (𝑛 = 5 biological replicates) of uniform maturity and crown development were selected. Fully expanded, disease-free compound leaves were excised manually during the pre-flowering vegetative stage during early morning hours to minimize physiological transpirational stress.

Sample processing

Harvested leaves were sorted to exclude non-edible petioles and woody twigs ( > 3mm diameter), gently rinsed with distilled water to remove superficial dust and debris, and blotted dry. To preserve heatsensitive bioactive constituents and avoid non-enzymatic Maillard browning, samples were shade-dried on perforated stainless steel benches at ambient temperatures (26 -32 ∘ C) with active cross-ventilation until a constant weight was attained. The dried matrices were ground in a laboratory Wiley mill (Model 4, Thomas Scientific, Swedesboro, NJ) fitted with a 1.0mm stainless steel delivery screen, homogenized, and stored in airtight, dark high-density polyethylene (HDPE) containers at 4 ∘ C pending chemical analysis.

Proximate chemical analysis

Analyses were executed in analytical triplicates according to standard procedures (AOAC, 2016) [12]: Dry Matter (DM): Analytical method 934.01; desiccated in a forceddraft convective oven at 105 ± 2 ∘ C for 16h to constant mass. Total Ash: Analytical method 942.05; complete combustion in a programmable muffle furnace at 550 ± 10 ∘ C for 4h.

Crude Protein (CP):

Nitrogen-Free Extract (NFE):

Quantified mathematically by difference on a dry-matter basis:

Organic Matter (OM): Derived as:

OM ( %of DM ) = 100 -Ash ( %of DM )

Detergent fiber fractionation

Cell wall matrix constituents were determined sequentially using the filter bag technique in an ANKOM 200 Fiber Analyzer (ANKOM Technology, Macedon, NY) following Van Soest et al. (1991) [13]: Neutral Detergent Fiber (NDF): Digested with neutral detergent solution containing sodium dodecyl sulfate, disodium EDTA, and sodium borate, supplemented with heat-stable 𝛼-amylase (Sigma-Aldrich, A3306) to digest starch and sodium sulfite (Na 2 SO 3 ) to remove proteinaceous contaminants. Results are expressed inclusive of residual ash (NDF).

Acid Detergent Fiber (ADF): Digested in acid detergent solution containing cetyltrimethylammonium bromide (CTAB) dissolved in 0.5M H 2 SO 4 .

Acid Detergent Lignin (ADL):

Determined by solubilizing cellulose from the ADF residue via ambient immersion in 72% ( w/w ) H 2 SO 4 for 3h, followed by extensive washing, drying, and subsequent incineration in a muffle furnace at 525 ∘ C for 3h.

Cellulose and Hemicellulose: Calculated stoichiometrically:

Quantification of anti-nutritional compounds

Total Phenolics (TP): Extracted using aqueous methanol (70% v/v) via ultrasonic assistance at 40 ∘ C for 30min. Soluble phenolics were quantified spectrophotometrically using the Folin-Ciocalteu reagent (Makkar, 2003) at 𝜆 = 725nm. Concentrations were interpolated against a gallic acid standard calibration curve (𝑅 2 > 0.998) and reported as grams gallic acid equivalents per kilogram DM ( g GAE/kg DM).

Condensed Tannins (CT): Quantified using the butanol-HCl-Fe .

Statistical analysis

The experiment was structured as a single-factor Completely Randomized Design (CRD). The experimental unit was defined as an individual tree sample (𝑛 = 5 independent biological replicates per species). Data were assessed for normal distribution via the Shapiro-Wilk test and homogeneity of variance using Levene's test. Analysis of variance (ANOVA) was executed using the General Linear Model (GLM) procedure in SAS (Version 9.4, SAS Institute Inc., Cary, NC):

Where 𝑌 𝑖𝑗 is the dependent variable, 𝜇 is the overall population mean, 𝑇 𝑖 is the fixed effect of the foliage species (𝑖 = 1, 2, 3), and 𝜀 𝑖𝑗 is the random residual error term ∼𝑁 ( 0, 𝜎 2 ) . Significant differences among treatment means were separated using Tukey's Honestly Significant Difference (HSD) post-hoc test. Threshold significance was established at 𝛼 = 0.05.

Results

Proximate chemical composition

The proximate composition of M. oleifera, T. indica, and A. indica dried leaf foliage is summarized in Table 1.

Cell-wall fiber partitioning (Van Soest system)

The distribution of structural carbohydrates across the species is delineated in Table 2. ) differ significantly (𝑃 < 0.05). Hemicellulose calculated as ( NDF -ADF ) ; Cellulose calculated as ( ADF -ADL ) .

Moringa oleifera possessed the lowest structural fiber profile ( 𝑃 < 0.001), with NDF at 28.31%, ADF at 17.45%, and ADL at 5.12%.

Conversely, T. indica had the highest cell-wall concentrations, with NDF (46.22%) and ADF (31.40%) exceeding those of A. indica and M. oleifera. The lignification index, reflected by ADL, followed the descending order: T. indica (9.85%) > A. indica (7.60%) > M. oleifera (5.12%). Hemicellulose content was comparable between T. indica ( 14.82%) and A. indica (14.15%), but was significantly lower in M. oleifera (10.86%).

Anti-nutritional and phytochemical profiles

Phytochemical quantification of anti-nutritional constituents indicated pronounced species-dependent divergence (Table 3).

Discussion

Comparative protein matrix and nitrogen value

Crude protein is the primary limiting nutrient for ruminants fed lowquality tropical basal diets. Rumen cellulolytic microorganisms require a minimum dietary threshold of 70g CP/kg DM (7.0%) to maintain basic hydrolytic activity on structural polysaccharides; levels below this suppress voluntary intake through ruminal impaction (Van Soest, 1994) [3] . All three tested foliages exceeded this threshold, confirming their viability as supplemental protein sources. Moringa oleifera foliage exhibited marked protein accumulation ( 26.84%), confirming its role as a concentrated nitrogen source capable of substituting for conventional oilseed meals such as soybean meal or mustard cake (Sánchez et al., 2006; Moyo et al., 2011) [15,7] . The elevated ether extract fraction in M. oleifera (6.82%)-primarily composed of unsaturated fatty acids, including 𝛼-linolenic acid (Makkar et al., 1996) [6] -supplies readily available gross energy without exceeding the 6 -7% dietary lipid limit that impairs ruminal fiber digestion. While T. indica (14.22%) and A. indica (16.45%) yielded lower crude protein compared to Moringa, both provide sufficient nitrogen to function as maintenance or moderate production supplements for small ruminants consuming poor-quality roughages.

Cell-wall lignification and digestive mechanics

The voluntary intake and ruminal degradability of forages are largely governed by their NDF and ADF concentrations, which dictate rumen fill, chewing time, and gut clearance rates (Van Soest et al., 1991) [13] . The NDF fraction of M. oleifera (28.31%) was significantly lower than the 50 -60% threshold typically associated with voluntary intake depression in tropical ruminants. Coupled with its modest ADF (17.45%) and ADL (5.12%), Moringa provides low physical resistance to microbial enzymatic breakdown, correlating with elevated in vitro dry matter digestibility reported in previous studies (Soliva et al., 2005) [16] . In contrast, T. indica exhibited significant cell-wall development ( 46.22% NDF, 31.40% ADF), accompanied by higher core lignification (9.85% ADL). Because lignin is physically linked to hemicellulose via covalent ferulate cross-links, elevated ADL concentrations present a physical barrier that restricts bacterial cellulolytic access to structural polysaccharides. Consequently, while T. indica supplies functional structural fiber for rumination, its total organic matter digestibility is predictably lower than that of Moringa, suggesting its utility is best suited as a partial roughage replacer. Azadirachta indica occupied an intermediate position, presenting balanced fiber partitioning that supports ruminal function without inducing excessive physical fill.

Bioactive secondary metabolites and physiological thresholds

Anti-nutritional factors represent evolutionary chemical defense mechanisms against herbivory, yet their physiological impact on ruminants varies based on chemical structure and ingestion rate. The condensed tannin (proanthocyanidin) concentration observed in T. indica (24.36g/kg DM) falls within the biologically active range of 20 -40g/kg DM. In this range, condensed tannins form stable, pHdependent hydrogen-bonded complexes with dietary proteins within the near-neutral ruminal environment (pH6.0 -6.8), shielding them from microbial deamination and proteolysis (Patra et al., 2010) [11] .

These complexes dissociate in the acidic abomasum (pH < 3.0), releasing free amino acids for absorption in the duodenum, which enhances nitrogen utilization efficiency and mitigates urinary nitrogen excretion (Min et al., 2003) [17] . However, if Tamarindus foliage is fed in excess, condensed tannins can bind directly to microbial cell walls and extracellular cellulases, impairing structural carbohydrate digestion.

Azadirachta indica accumulated high concentrations of total phenolics (41.15g GAE/kg DM), coupled with its characteristic bitter limonoids (triterpenoids such as azadirachtin and nimbidin) (Ur-Rehman et al., 2023) [10] . While these secondary compounds possess broad-spectrum anthelmintic and protozoal-defaunating capabilitiesreducing enteric methanogenesis by suppressing associated methanogenic archaea (Dey et al., 2008)-their bitterness presents a palatability barrier. Unadapted livestock often reject unblended neem foliage, suggesting dietary inclusion should be limited to 10 -15% in complete feed formulations or balanced via gradual adaptation (Garg, 2001).

The elevated phytate concentration in M. oleifera (14.12g/kg DM) is of minor consequence in mature ruminants. Unlike monogastric species, the resident ruminal microflora synthesizes endogenous phytase enzymes capable of hydrolyzing the myo-inositol hexakisphosphate ring, releasing bioavailable inorganic orthophosphate and chelated divalent cations (Ca 2 + , Mg 2 + , Zn 2 +

) for systemic absorption (Yanke et al., 1998) [18] .

Synthesis and dietary strategic allocation

Synthesizing these nutritional parameters suggests distinct feed allocation strategies across the evaluated foliages: Moringa oleifera is an exceptional protein and energy resource that can be utilized as a direct substitute for high-cost commercial concentrates in high-producing lactating or rapidly growing livestock. Tamarindus indica supplies fermentable fiber and condensed tannins, making it suitable as an intermediate roughage supplement that reduces ruminal protein degradation when fed alongside highly soluble nitrogen sources. Azadirachta indica functions as a secondary phytogenic feed additive that provides baseline crude protein while contributing bioactive metabolites useful for internal parasite control and rumen fermentation modulation, provided dietary inclusion is managed to protect feed intake.

Conclusion

This comparative analysis demonstrates significant differences in proximate composition, structural fiber architecture, and secondary anti-nutritional profiles among Moringa oleifera, Tamarindus indica, and Azadirachta indica foliage: Moringa oleifera exhibits superior nutritional properties, characterized by high crude protein (26.84%), elevated ether extract (6.82%), and minimal core lignification (5.12%), making it a viable substitute for traditional vegetable protein meals. Tamarindus indica foliage is rich in structural cell-wall carbohydrates (46.22% NDF) and condensed tannins (24.36g/kg), serving as a moderate-energy forage that promotes protein bypass mechanics when fed in balanced proportions.

Azadirachta indica provides balanced crude protein (16.45%) alongside high phenolic concentrations (41.15g/kg), indicating significant functional value as a dietary supplement for microbial modulation, provided palatability limits are observed. Integrating these alternative tree foliages into ruminant feeding regimens offers smallholders an evidence-based approach to mitigate dryseason feed deficits, lower reliance on commercial concentrates, and enhance the environmental sustainability of tropical livestock operations.

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