DISCUSSION

Chemical composition of experimental feeds

The high CP content observed in LGP was primarily attributed to the inclusion of L. leucocephala, a widely available leguminous species in tropical regions that is commonly used as a high-quality protein feed for ruminants. This finding is consistent with Melesse et al. [7], who reported that L. leucocephala leaves contain high levels of CP (24.5%), total phenols (6.5%), and CT (2.3%), and provide an excellent supply of essential amino acids compared with other multipurpose tree foliage. Beyond its protein content, LGP was a rich source of phytonutrients, containing 6.8% CT and 2.4% flavonoids. These polyphenolic compounds, particularly tannins, play an important role in ruminal protein utilization. Zhang et al. [24] demonstrated that, under normal ruminal pH conditions, these active components bind with dietary proteins to form stable complexes, thereby reducing excessive microbial protein degradation in the rumen.

Feed intake and nutrient digestibility

Dietary chemical composition, physical characteristics, and other related factors typically influence DM intake, which subsequently affects livestock productivity. In this study, no significant differences in feed intake were observed among treatment groups, indicating that LGP supplementation maintained normal feed consumption. This sustained intake may be attributed to the synergistic formulation of LGP, which likely counteracted the potential negative effects commonly associated with high-tannin diets. While CT from L. leucocephala contributes to dietary protein protection, ginger provides bioactive compounds, including gingerols and shogaols, that stimulate digestive enzyme activity. This combination may enhance diet palatability and reduce the intake-depressing effects commonly associated with single-source high-tannin supplementation.

Throughout the in vivo trial, no clinical signs of mimosine toxicity, such as alopecia, excessive salivation, depressed appetite, or visible goiter, were observed in any experimental animal. This absence of clinical toxicity supports the safety of the formulated pellet and the thermo-mechanical processing approach used in the study. Furthermore, the significant improvements in DM and neutral detergent fiber digestibility may be mechanistically attributed to the combined effects of CT and ginger bioactives. The targeted suppression of rumen protozoa by CT and flavonoids in LGP likely reduced bacterial predation, allowing greater proliferation of cellulolytic and fibrolytic bacteria. Consequently, enhanced bacterial activity, together with stimulation of salivary secretion and microbial enzymatic activity by ginger bioactives, may have improved structural carbohydrate degradation and overall nutrient digestibility.

These results are consistent with those of Phesatcha et al. [25], who reported an increase in total DM intake after supplementation with Flemingia_ macrophylla_ pellets. Similarly, Totakul et al. [26] showed that crossbred bulls did not alter feed consumption when supplemented with Cnidoscolusa conitifolius leaf pellets. Increasing LGP supplementation levels improved palatability and influenced rumen fermentation. Improvements in feed conversion ratio, growth rate, nutrient digestibility, and palatability have also been reported when plants, particularly ginger, were used to stimulate feed intake [27]. In the present study, LGP enhanced DM and neutral detergent fiber digestibility, likely because reduced protozoal populations decreased predation on fibrolytic bacteria. The bioactive compounds naturally present in ginger may also enhance animal health and growth by stimulating gastric secretions and digestive enzyme activity, thereby supporting feed utilization and production.

The addition of CT and SP may negatively affect nutrient digestibility, particularly protein digestion, when included at excessive levels. However, a moderate reduction in ruminal protein degradation can be beneficial if tannin-CP complexes dissociate in the lower digestive tract, thereby allowing subsequent protein digestion. Matra et al. [28] reported improved CP digestibility in dairy cows fed dragon fruit peel pellets. Although significant increases in DM and neutral detergent fiber digestibility were observed in this study (p < 0.05), the relatively high standard error, particularly for DM digestibility, should be noted. Such variability is common in in vivo studies using low-quality basal roughages, such as rice straw, and reflects inherent individual variation among animals within a small Latin square design. Nevertheless, the significant treatment effect indicates that the synergistic action of phytogenic compounds in LGP was sufficient to improve rumen microbial activity and feed degradation.

Rumen fermentation characteristics, blood metabolites, and microbial populations

Rumen fermentation, microbial growth, and microbial activity were maintained within a pH range of 6.7-6.8. Improved rumen fermentation may be supported by maintaining ruminal pH through the strategic inclusion of feedstuffs containing phenolic acids [29]. Increasing LGP supplementation did not significantly alter NH3-N concentrations, which remained within the normal range of 13.7-18.5 mg/dL. BUN concentrations were closely associated with ruminal NH3-N concentrations, although no significant differences in BUN were observed among treatments. Phesatcha et al. [30] reported that CT improved nutrient utilization by forming protein-tannin complexes, decreasing ruminal feed protein degradation, and reducing ammonia production. In contrast, Matra et al. [28] found that ruminal NH3-N increased when Holstein crossbred bulls received dragon fruit peel pellets at 400 g/animal/day. In the present study, BUN concentrations ranged from 10.3 to 12.0 mg/dL and did not differ significantly among treatments, suggesting efficient use and absorption of available ruminal NH3-N for microbial synthesis. Similarly, Phesatcha et al. [25] reported normal BUN concentrations when tree leaf pellets were added to the diet as a CT source.

Patra and Saxena [31] reported that bioactive compounds can alter propionate formation when hydrogen is available in excess. Hydrogen is a major substrate for methanogenesis, whereas its use in propionate formation provides an alternative hydrogen sink. Therefore, inhibition of acetogenic bacteria and redirection of hydrogen toward propionate synthesis represent potential biological roles of tannins. A lower acetate-to-propionate ratio is generally associated with increased propionate concentration. Several studies have demonstrated that leguminous fodder shrubs can alter VFA profiles. Totakul et al. [26] reported that supplementation with Cnidoscolus leaf pellets increased propionate concentration and decreased the acetate-to-propionate ratio. Similar findings were reported by Phesatcha et al. [30], who observed increased propionate and reduced acetate proportions after leaf pellet supplementation.

Diets supplemented with CT and SP enhanced rumen fermentation, reduced CH4 production, decreased protozoal populations, and increased propionate production. Propionate concentration often increases when rumen methanogenesis is suppressed, as observed in this study. The shift in the VFA profile from acetate-to-propionate, along with reduced CH4 formation and redirected hydrogen utilization, may improve the energy availability to the host animal. These results are consistent with recent in vitro studies showing that Z. officinale mitigates CH4 emissions by altering rumen microbial activity [10]. Moreover, the synergistic use of phytogenic compounds has been shown to modify in vitro fermentation parameters and translate into reduced in vivo CH4 emissions while maintaining performance in dairy and beef cattle [32]. Similarly, phytogenic-based additives can suppress methanogenesis by shifting fermentation pathways toward propionate production [2].

The observed shift in rumen fermentation, particularly increased propionate and decreased CH4 production, is closely associated with the chemical properties of the phytogenic compounds. Rumen protozoa have a symbiotic relationship with methanogenic archaea, providing them with a habitat and hydrogen. The antiprotozoal actions of CT and flavonoids may disrupt this symbiosis and impair methanogenesis. Concurrently, to maintain thermodynamic balance in the rumen ecosystem, metabolic hydrogen may be redirected toward propionate synthesis. This competitive hydrogen sink can reduce CH4 emissions while providing more glucogenic energy to the host animal. Montoya-Flores et al. [8] reported a 14% reduction in CH4 production when Leucaena was included at a comparable level, which was attributed to the bioactive compounds present in Leucaena. Although formal correlation analysis between protozoal populations and CH4 production was not performed because CH4 was estimated indirectly using stoichiometric equations, the concurrent reduction in both parameters is consistent with established biological mechanisms. This parallel decline supports disruption of the symbiotic relationship between rumen ciliate protozoa and methanogenic archaea and further demonstrates the efficacy of LGP in favorably modifying the rumen microbiome.

Although LGP contained a relatively high CT concentration of 6.8%, the low daily supplementation rate, with a maximum of 150 g/head/day, ensured that total dietary CT remained below the threshold generally associated with impaired protein digestibility and well below commonly recommended safe inclusion limits. In addition, cattle in tropical regions, including Thai native beef cattle, may possess natural rumen adaptation. Their rumen microbiome often harbors mimosine-degrading bacteria, such as Synergistes jonesii, which can detoxify mimosine and its goitrogenic metabolites, including 3,4-dihydroxypyridine and 2,3-dihydroxypyridine. Thus, the formulation likely leveraged the beneficial effects of phytogenic compounds without approaching the threshold for mimosine-induced toxicity.

Essential oils in ginger may influence feed digestion and rumen fermentation. Saponins, which are among the bioactive constituents of ginger, may increase beneficial bacteria while decreasing protozoal populations. Higher LGP supplementation increased bacterial populations and reduced protozoal populations. Phesatcha et al. [30] similarly reported decreased protozoal and ruminal methanogen populations when beef cattle were supplemented with Mitragyna leaf pellets. The reduction of protozoa is a recognized mechanism by which bioactive compounds in both Leucaena and ginger suppress methanogenic archaea associated with protozoa, thereby improving nitrogen utilization and post-ruminal protein flow [5].

The reduction in protozoal populations, suppression of methanogenesis, and concurrent enhancement of microbial protein synthesis observed with LGP supplementation suggest a biological synergy among its constituent bioactive compounds. Leucaena is rich in CT, which can bind to the cell coat of ciliate protozoa and dietary proteins, impair cell membrane permeability, and inhibit enzyme activity. In parallel, the phytochemical profile of ginger, including flavonoids, gingerols, shogaols, and saponins, may complement this effect by disrupting lipid bilayers of methanogenic and protozoal cell membranes. This combination may create a dual-action inhibitory effect on cellular integrity, resulting in a more pronounced defaunation effect and greater bacterial proliferation and microbial protein synthesis than either ingredient alone. The rich phytochemical profile of these combined feedstuffs may therefore optimize fermentation kinetics and support higher microbial protein synthesis [4].

Ebeid et al. [33] reported that ginger supplementation improved digestion by increasing cellulolytic bacterial populations and enhancing salivary secretion, thereby increasing the secretion and activity of digestive enzymes. Ginger may benefit gastrointestinal ecology, improve feed stability, and limit the growth of pathogenic microorganisms, although excessive antimicrobial activity can reduce diet fermentability. Plant secondary metabolites and antiprotozoal actions of ginger components may therefore contribute to reduced total protozoal populations. Norrapoke and Pongjongmit [34] also reported that Mahad leaf pellet supplementation containing 15.6% CT significantly decreased protozoal populations in beef cattle.

The overall efficacy of LGP appears to be closely linked to dose-dependent synergy. At the optimal supplementation level of 150 g/head/day, which provided approximately 112.5 g of Leucaena leaves and 22.5 g of ginger powder, this study observed favorable ruminal outcomes. This dose increased propionate concentration and microbial protein synthesis while reducing protozoal populations and enteric CH4 emissions. Importantly, these effects occurred without adverse outcomes commonly associated with high-tannin interventions, such as reduced feed intake or compromised ruminal pH. Therefore, 150 g/head/day may represent a practical biological threshold at which complementary bioactives maximize fermentation efficiency while maintaining ruminal homeostasis.

Microbial protein synthesis

Nitrogen intake increased with higher LGP supplementation, likely due to protein complex formation. Hung et al. [35] demonstrated that microbial nitrogen synthesis efficiency was enhanced by supplementation with Leucaena leaf pellets containing 24% CP. Rumen bypass protein also improved when dietary protein and CT levels increased with LGP supplementation. Rumen microorganisms play a central role in protein production, and changes in microbial growth directly influence amino acid availability. In the present study, LGP significantly increased microbial protein synthesis and the efficiency of microbial nitrogen synthesis.

The enhancement of microbial protein synthesis and microbial nitrogen synthesis efficiency can be explained by two major mechanisms. First, reversible binding of CT to dietary proteins at normal ruminal pH forms stable CT-protein complexes. This process protects protein from rapid ruminal degradation and synchronizes nitrogen release with carbohydrate fermentation for optimal microbial growth. Second, reduced protozoal predation on bacteria increases the net flow of intact microbial cells from the rumen to the lower gut. Together, these mechanisms improve the overall efficiency of microbial nitrogen synthesis. Viennasay and Wanapat [36] reported that Flemingia supplementation enhanced microbial nitrogen synthesis and its efficiency in lactating dairy cows, whereas Phesatcha et al. [37] found that supplementing beef cattle with pellets containing insect protein and phytonutrient plants improved microbial nitrogen synthesis efficiency.

Beyond the direct suppression of methanogenesis, the synergy between ginger bioactives and Leucaena CT may involve specific physical and enzymatic interactions related to CT-protein complex formation. While appropriate CT levels protect dietary protein from ruminal degradation, excessive CT can irreversibly bind proteins, reducing lower-gut digestibility. Ginger bioactives may counteract this risk through two mechanisms. First, gingerols can stimulate salivary secretion, and saliva contains proline-rich proteins that have a high affinity for tannins. Increased salivary flow may therefore buffer excess CT and prevent excessive inhibition of dietary protein digestion in the rumen. Second, after reversible CT-protein complexes dissociate in the acidic environment of the abomasum, gingerols may stimulate lower-gut protease secretion, improving breakdown and absorption of liberated amino acids. This dual-action mechanism may explain the increased microbial nitrogen flow and sustained nutrient digestibility observed in LGP-supplemented cattle.

Practical relevance, limitations, and future perspectives

The synergistic efficacy of LGP should be interpreted in relation to previous studies that evaluated these phytogenic sources independently. Several previous in vivo studies using Leucaena alone reported a risk of reduced DM and structural carbohydrate digestibility at high inclusion levels due to excessive CT-protein binding [8]. In contrast, the present study demonstrated significantly improved digestibility of DM and neutral detergent fiber, suggesting that ginger bioactives in LGP may have counteracted potential CT-induced microbial inhibition. Similarly, although in vitro studies have documented the antimethanogenic and antimicrobial effects of ginger extracts, their practical in vivo application may be limited by the rapid ruminal degradation of volatile compounds. In contrast, the pelleted LGP matrix may help stabilize these bioactives and achieve a marked reduction in protozoal populations, along with increased microbial protein synthesis, at a conservative supplementation level of 150 g/head/day. Therefore, the combined formulation appears to provide greater rumen-modulatory benefits than either Leucaena or ginger alone.

From a practical perspective, LGP functions as a ready-to-use technology for resource-limited tropical farmers. Its reliance on standard pelleting equipment and locally available ingredients suggests strong potential for cooperative-level scale-up and broader commercialization. By using locally abundant materials from Roi Et Province and evaluating their efficacy in Thai native beef cattle fed a low-quality rice straw-based diet, this formulation directly addresses practical nutritional constraints in tropical smallholder systems. The innovation of this study extends beyond combining ingredients; it includes the development of a specific pelleted matrix. Unlike most previous studies that evaluated Leucaena as loose leaves or unprocessed meal, this study used cassava chips and molasses as binders, together with mechanical processing and sun drying. This thermo-mechanical approach may improve pellet integrity, storage stability, and ease of feeding, while potentially enhancing the bioavailability of volatile gingerols and further reducing residual mimosine within the pellet matrix.

Mimosine toxicity remains a recognized limitation in Leucaena utilization, but LGP may provide a dual mitigation strategy. In addition to physical mimosine reduction through drying and pelleting, enhanced fermentation and bacterial proliferation stimulated by ginger bioactives may further support ruminal detoxification of residual mimosine and its secondary metabolites. Consequently, this phytogenic formulation may promote sustainable and adoptable feeding practices in tropical regions.

The CT concentration must also be interpreted in relation to total dietary intake. Although LGP contained 6.8% CT, the maximum supplementation rate of 150 g/head/day diluted the net CT contribution to only a small fraction of total dietary DM. This level is likely within a beneficial range, allowing CT to act as a phytogenic rumen modifier without reaching concentrations that negatively affect feed intake or nutrient digestibility.

Despite these promising findings, several limitations should be acknowledged. First, the 4 × 4 Latin square design, although appropriate for evaluating ruminal fermentation, included only four animals, short experimental periods of 21 days, and exclusively female cattle. These factors limit direct extrapolation to long-term production outcomes, including average daily gain, final BW, carcass traits, and responses in male cattle. Second, although the physical integrity of LGP was adequate for handling and daily feeding, formal evaluation of pellet characteristics, including hardness, durability, and water stability, was not performed. Third, residual mimosine and specific ginger bioactives in the final pellet were not quantified because of analytical constraints. Finally, classical methods such as hemocytometer counting and VFA-based stoichiometric estimation do not identify specific microbial taxa or measure absolute CH4 emissions.

Future studies should therefore include long-term growth trials to validate practical application and economic benefits. Direct quantification of CH4 using respiration chambers or related technologies, comprehensive mimosine adaptation studies, quantification of residual mimosine and ginger bioactives, and advanced molecular microbiome analysis are also recommended to clarify the long-term physiological, microbial, environmental, and economic impacts of this synergistic pellet.