Search Results (7,793)

Insect farming has rapidly expanded in Europe following regulatory approval of insect-derived proteins in aquaculture feed and increasing interest in the valorization of insect by-products. Insect frass, consisting of excreta and exuviae, is a nutrient-rich material with beneficial microorganisms and potential as a sustainable alternative to conventional fertilizers, although its composition varies with insect species and feedstock. EU legislation requires thermal sanitization prior to market release, yet the effects of the thermal treatment on frass nutrient composition and biofertilizer performance remain poorly understood. Insect frass from black soldier flies (BSFFs) fed on a diet based on dairy industry byproducts was sanitized and mixed with sandy soil and used in two lettuce pot trials under greenhouse conditions. The aim of our study was to determine the effects of thermal sanitization on (1) macro- and micronutrient contents and dynamics (plant N and P uptake); and (2) biofertilizer potential, including plant physiology (chlorophyll, anthocyanins, flavonols, Fv/Fm), plant growth (biomass), and soil microbial activity (dehydrogenase and β-glucosaminidase). BSFF showed a clear potential to induce growth of lettuce plants by increasing chlorophyll content, biomass and microbial activity. Furthermore, the sanitization process did not significantly alter the measured agronomic performance of frass under the tested conditions or reduce its benefits on biomass growth, chlorophyll content, microbial enzyme activity and on nutrient uptake by the lettuce plants. These findings suggest that the mandatory sanitization does not compromise its agronomic functionality, supporting its strong potential within circular agricultural systems under the tested conditions. However, the results are valid under greenhouse conditions and for the specific frass, soil and crop combinations; field validation is needed to confirm these results under large-scale high-value crop production conditions. Full article

Coffee cherry pulp (CCP; cascara), a major by-product of coffee processing, has gained increasing attention as a sustainable source of phenolic compounds, dietary fiber, and other bioactive constituents with applications in food, nutraceutical, feed, and biomaterial industries. However, its utilization remains limited by compositional variability, anti-nutritional compounds, and inefficiencies in conventional processing. Controlled fermentation has emerged as a promising strategy to enhance the release, transformation, and bioavailability of CCP-derived bioactive through targeted microbial biotransformation and controlled bioprocessing. This review summarizes recent advances in enzymatic pretreatment, microbial fermentation, and metabolite-directed processing, with emphasis on their effects on phenolic transformation, antioxidant activity, and functional properties. The roles of selected lactic acid bacteria, yeasts, and microbial consortia in improving the nutritional, sensory, and biological characteristics of CCP-derived products are critically discussed. Potential applications of fermented CCP in functional foods and beverages, bioactive ingredients, biopolymers, and animal feed are also highlighted within the framework of an integrated circular bioeconomy. Finally, current challenges and future perspectives related to process scalability, metabolite control, regulatory approval, and AI-assisted bioprocess optimization are addressed. Full article

High-fat diet (HFD)-induced hyperlipidemia is an experimental metabolic condition characterized primarily by dysregulated serum lipid levels and hepatic lipid accumulation, with associated oxidative, inflammatory, mitochondrial, and cardiovascular alterations. This study investigated the therapeutic efficacy of epigallocatechin gallate (EGCG)-functionalized selenium nanoparticles (EGCG-SeNPs) against HFD-induced metabolic and hepatic injury, in comparison with free EGCG, sodium selenite (Na₂SeO₃), and Lipanthyl. EGCG-SeNPs were characterized by dynamic light scattering, zeta potential analysis, transmission electron microscopy, X-ray diffraction, and UV–visible spectrophotometry. Forty-two adult male rats were allocated into six groups: control, HFD, HFD/Lipanthyl, HFD/EGCG, HFD/Na₂SeO₃, and HFD/EGCG-SeNPs. High-fat diet (HFD) feeding induced pronounced dyslipidemia, elevated hepatic enzymes, increased cardiac injury biomarkers, enhanced lipid peroxidation and nitrosative stress, depletion of antioxidant defenses, and disruption of the Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2 (Keap1/Nrf2) regulatory axis. HFD also increased nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α), and interleukin-6 (IL-6), while altering mitochondrial apoptotic markers, including B-cell lymphoma 2 (Bcl-2), cytochrome c, and caspase-3. At the transcriptional level, HFD increased lipogenic gene expression and reduced the expression of genes related to fatty-acid oxidation, metabolic regulation, and mitochondrial homeostasis. EGCG-SeNPs showed the greatest overall improvement among the tested interventions, as indicated by an improved lipid profile, hepato-cardiac injury biomarkers, antioxidant status, inflammatory markers, apoptotic markers, hepatic architecture, and Nrf2 immunoreactivity. Collectively, EGCG-SeNPs may mitigate HFD-induced hepatic lipotoxicity and associated cardiac stress through coordinated modulation of lipid metabolism, redox balance, inflammation, and mitochondrial homeostasis. Full article

Plant-parasitic nematodes are a significant agricultural challenge, causing extensive damage to most essential crops globally. Predatory soil mites play a significant role as biocontrol agents against plant-parasitic nematodes and other pests in the soil ecosystem. A laboratory trial was conducted to evaluate the potential of the soil-dwelling predatory mite, Androlaelaps casalis (Berlese), to suppress the citrus nematode, Tylenchulus semipenetrans Cobb, and the root-knot nematode, Meloidogyne incognita (Kofoid and White). Our findings show that the predatory mite, A. casalis, completed development, oviposition, and survival when fed on second-stage juveniles of both T. semipenetrans (Ts-J2) and M. incognita (Mi-J2) and egg mass of M. incognita (Mi-EM) as prey in the lab in closed arenas at 30 °C, 55% RH. Survivorship was lower on (Mi-EM) than on (Ts-J2) and (Mi-J2). Individuals of A. casalis reared on (Ts-J2) and (Mi-J2) prey demonstrated enhanced performance as compared to (Mi-EM) prey. In addition, females laid a total of 48.72, 46.50, and 12.45 eggs on the three types of prey, respectively. Life table parameters showed that feeding of A. casalis on (Ts-J2) and (Mi-J2) led to the greatest intrinsic rate of increase per day (r_m = 0.286 and 0.279 females/female/day), while preying on (Mi-EM) offered the lowest reproduction rate (r_m = 0.092). In conclusion, this study provides novel insights into the biology and predatory performance of A. casalis under controlled laboratory conditions, providing foundational evidence that may inform the development of future sustainable nematode management strategies. Full article

The global shortage of high-quality protein feed resources continues to widen, and the development of high-value-added woody plants is a key strategy for alleviating feed shortage. The mulberry (Morus alba L.) is a recognized high-protein woody forage resource. However, the inconsistent quality of its natural silage and the unclear risk of mycotoxins represent the core bottlenecks limiting its widespread adoption as feed. Four treatments were set up in this study: (1) control; (2) lactic acid bacteria inoculant (LAB, Lactiplantibacillus plantarum); (3) cellulase enzyme (AC, Acremonium cellulolyticum); (4) a mixture of LAB + AC. After 60 days of ensiling, a systematic analysis was conducted to examine the effects of exogenous microbial inoculant and enzyme preparation on the fermentation quality, bacterial community, and mycotoxin in mulberry silages. Fresh mulberry exhibited a high crude protein content of 23% on a dry matter (DM) basis, making it a high-quality feed resource. Compared to the control, the addition of LAB and AC either alone or in combination, significantly improved (p < 0.001) the fermentation quality and safety of silages: lactic acid content increased from 0.85% DM to 1.41–2.03% DM; pH, ammonia nitrogen, and deoxynivalenol decreased from 4.85, 0.88% DM, and 3.92 μg/kg to 3.53–3.95, 0.40–0.55% DM, and 1.21–3.04 μg/kg, respectively. The combined LAB and AC treatment resulted in the most favorable fermentation performance of mulberry silage. Bacterial community analysis revealed that fresh mulberry exhibited high bacterial alpha diversity, with Gram-negative bacteria as the dominant bacterial community, and Sphingomonas roseiflava as a representative dominant species. After ensiling, bacterial alpha diversity decreased in all the silages. Furthermore, the Lactiplantibacillus plantarum eventually prevailed as the dominant bacteria and exhibiting the highest relative abundance in the LAB + AC-treated silage (57.23%). Bugbase functional prediction indicated that the proportion of potential pathogenic bacteria was significantly higher (p < 0.05) in fresh mulberry than silage. Thus, the synergistic action of LAB + AC treatment effectively optimized the ensiling fermentation process. Full article

Pig farming generates high-strength piggery wastewater (PWW) with extreme organic and nutrient concentrations. This research evaluated an integrated treatment system combining Vertical Flow Constructed Wetlands (VFCW), Microbial Fuel Cells (MFC), and Microalgae Photobioreactors (PBR) to enhance resource recovery, evaluate bio-electrochemical activity, and produce microalgal biomass. Findings showed that hydraulic saturation in the VFCW–MFC stage enhanced the open-circuit voltage response, reaching a maximum of 539 mV, indicative of bio-electrochemical activity. The optimized VFCW–MFC configuration, featuring pulsed feeding, achieved removals of total suspended solids (TSS, 83%) and chemical oxygen demand (COD, 69%). This integrated pretreatment mitigated ammonia toxicity and turbidity, enabling the subsequent cultivation of Tetradesmus obliquus microalga, reaching biomass yields of 1.1–1.3 g L⁻¹ while providing crucial tertiary polishing. Overall, the combined VFCW–MFC–PBR system achieved removal efficiencies exceeding 90% for total Kjeldahl nitrogen (TKN) and approximately 80% for COD. This synergistic approach successfully transforms PWW liabilities into valuable assets, including nutrient-rich biomass and bio-electrochemical activity, underscoring the potential of VFCW–MFC–PBR for sustainable wastewater management. Full article

Background: Appetite regulation is governed by a complex neuroendocrine network that integrates peripheral peptide signals with hypothalamic and brainstem circuits to coordinate energy intake and maintain energy homeostasis. Disruption of these pathways contributes to obesity and other disorders characterised by dysregulated feeding behaviour. Objective: To map and synthesise the current evidence on the role of appetite-regulating peptide hormones and central neural pathways in appetite control, obesity pathophysiology, and emerging therapeutic approaches. Methods: A scoping review of the literature was conducted to identify and synthesise evidence relating to the physiological and pathological mechanisms of appetite regulation. The review examined the actions of key peptide hormones, including ghrelin, glucagon-like peptide-1 (GLP-1), peptide YY (PYY), leptin, and insulin, their interactions within the gut–brain axis, and their effects on central appetite-regulating circuits. Results The evidence highlights the central role of the arcuate nucleus in integrating peripheral hormonal signals with neural pathways controlling feeding behaviour. Appetite regulation is mediated by the balance between orexigenic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons and anorexigenic pro-opiomelanocortin/cocaine- and amphetamine-regulated transcript (POMC/CART) neurons, with further modulation by the paraventricular, lateral, and ventromedial hypothalamic nuclei. The literature identifies hormone resistance, impaired satiety signalling, and altered neuroendocrine feedback as major contributors to obesity. Evidence on therapeutic interventions demonstrates the potential of GLP-1 receptor agonists, including liraglutide and semaglutide, and the dual incretin agonist tirzepatide, while also highlighting challenges related to treatment durability, adverse effects, and weight regain following discontinuation. Conclusions: Current evidence demonstrates that appetite regulation involves highly interconnected peripheral and central signalling pathways. The reviewed literature supports the development of multi-target and precision-based therapeutic strategies for obesity and identifies important areas for future research, including mechanisms of treatment resistance, long-term efficacy, and inter-individual variability in neuroendocrine responses. Full article

Background: Kidney disorders are strongly related to metabolic disturbances, including obesity and type 2 diabetes. Excessive intake of sugar and saturated fats promotes lipid accumulation, cellular energy issues and inflammatory responses. Cannabigerol (CBG), a non-psychotropic phytocannabinoid, has recently gained attention for its metabolic, anti-inflammatory and potential protective properties. Methods: The present study investigated the effect of two weeks of CBG administration (last 14 days of the experiment) on fatty acid (FA) composition, FA metabolic pathways and FA transporters in rats subjected to a high-fat high-sucrose diet (HFHS) for 6 weeks. Male Wistar rats were divided into four groups: Control, CBG, HFHS, and HFHS+CBG. Kidney tissue and urine samples were analyzed by gas–liquid chromatography (GLC) for lipid fractions and FA profiles, while protein expression of FA transporters and metabolic enzymes was assessed by immunoblotting. Polysaccharides and collagen fibers were visualized using Periodic Acid-Schiff (PAS) and AZAN staining, respectively. ELISA and colorimetric kits were used to measure urinary albumin and creatinine contents. Results: HFHS feeding altered renal lipid homeostasis, increasing saturated and monounsaturated fatty acids (SFA and MUFA, respectively) levels and affecting desaturation and elongation ratios. CBG supplementation affected renal lipid metabolism by lowering triacylglycerol (TAG) accumulation, restoring polyunsaturated fatty acids (PUFA) in phospholipid (PL) and altering FA ratios, suggesting an improvement in lipid balance. CBG also increased the expression of carnitine palmitoyltransferase 1 (CPT1) and lipoprotein lipase (LPL) and decreased the expression of stearoyl-CoA desaturase 1 (SCD1) and fatty acid synthase (FAS), suggesting a shift toward enhanced FA oxidation and reduced lipogenesis. Conclusions: Overall, CBG exerted good effects on renal lipid metabolism and may mitigate early lipid-mediated injury associated with metabolic kidney disorders. Full article

The replacement of fishmeal (FM) in aquafeeds for carnivorous fish remains challenging due to reduced palatability and adverse effects on liver health and intestinal microbiota. Marine by-products-based additives containing fish protein hydrolysates and seaweed polysaccharides have shown potential to overcome these limitations. This study evaluated the effects of graded supplementation of Haiweisu (HWS), a multi-marine by-product formulated with squid viscera hydrolysate, small-molecule components from fish protein hydrolysate, seaweed polysaccharides, and seaweed residue as a carrier, in a FM-free diet for juvenile largemouth bass. Four isonitrogenous and isolipidic diets were prepared: a FM-free control diet (CON) and three diets supplemented with 10, 20, or 30 g/kg HWS (designated S10, S20, and S30, respectively). Each diet was fed to triplicate groups of fish (29.26 ± 2.61 g) for 56 days. Results showed that HWS supplementation linearly increased final body weight, weight gain rate, and feed intake, while significantly reducing the feed conversion ratio (p < 0.05). All HWS-supplemented groups exhibited markedly lower hepatic lipid accumulation and plasma total cholesterol levels compared with the CON group, accompanied by alleviated hepatocellular steatosis and inflammatory infiltration as revealed by Oil Red O and H&E staining. Moreover, HWS significantly enhanced intestinal microbiota alpha diversity (Ace, Chao, Sobs, and Shannon indices), decreased the relative abundance of the dominant genus Mesomycoplasma, and enriched potentially beneficial genera including Methylobacterium, Delftia, and Sphingomonas (p < 0.05). In conclusion, dietary HWS supplementation effectively improved growth performance, alleviated hepatic steatosis and inflammation, and beneficially reshaped the intestinal microbiota in juvenile largemouth bass fed a FM-free diet. These findings support HWS as a promising functional additive for sustainable FM-free aquafeeds in carnivorous fish species. Full article

Enteric methane (CH₄) emissions from ruminants are a significant contributor to agricultural greenhouse gas emissions and represent an increasing concern in African livestock systems. This systematic review evaluates the effectiveness of tannin-rich plants as a dietary strategy for mitigating enteric CH₄ emissions in African ruminant production systems. The review followed PRISMA guidelines and included peer-reviewed original studies published between 2015 and 2025 that investigated tannin-rich plant interventions in cattle, sheep, or goats within African production systems. Eligible studies comprised both in vivo feeding trials and in vitro rumen fermentation experiments. Studies were included if they reported enteric CH₄ or greenhouse gas-related outcomes, while reviews, modeling studies, non-ruminant studies, and studies without CH₄-related outcomes were excluded. A total of eight eligible studies were identified, all conducted in South Africa despite the Africa-wide scope of the review. Overall, tannin-rich plant interventions showed potential to reduce CH₄ emissions, although the magnitude and consistency of responses varied depending on tannin type, source, inclusion level, form of administration, and dietary context. Purified and encapsulated tannin extracts generally produced more consistent CH₄ reductions than crude or whole-plant sources. Responses also differed between controlled total mixed ration systems and forage-based feeding systems. However, the small number of studies and their strong geographic concentration limit broader generalization across the continent. In conclusion, tannin-rich plants show promise as a natural CH₄ mitigation strategy in ruminants, but more regionally distributed and methodologically robust studies are needed across Africa to strengthen the evidence base. Full article

[Objective] To determine the control potential of Arma chinensis against major soybean pests Helicoverpa armigera and Spodoptera exigua, thereby providing theoretical and practical support for biological pest control in soybean fields. [Methods] Laboratory and field experiments were conducted to assess the predation capacity, feeding preference, and field control effect of third~fifth-instar nymphs and male/female adults of A. chinensis on first, third and fifth-instar larvae of these two pests. Predation functional responses were fitted to analyze predation characteristics and the relationship between searching efficiency and prey density. [Results] Both nymphs and adults of A. chinensis preyed on the larvae of H. armigera and S. exigua, with the predation functional responses conforming to the Holling Type II disk equation, which presented the highest predatory efficiency. The female adult of A. chinensis showed strong predation capacity against H. armigera (55.368) and S. exigua (50.699) larvae, with the highest daily prey consumption of 13.158 and 13.699 individuals, respectively. Searching efficiency of A. chinensis was negatively correlated with prey density, and significantly higher for first-instar than third-instar larvae. Under cooccurrence conditions, A. chinensis displayed an obvious feeding preference for H. armigera larvae. Field trials demonstrated that female adults of A. chinensis generated a 70% population decline rate of H. armigera. Meanwhile, the population decline rate of S. exigua reached over 80%. Female adults of A. chinensis achieved field control rates of 80% against H. armigera larvae and 70% against S. exigua larvae. [Conclusions] A. chinensis has strong predation and control potential against the larvae of H. armigera and S. exigua. Among these, females of A. chinensis demonstrated the highest efficacy in controlling the two types of Lepidoptera larvae, both indoors and in field conditions. It is a promising biological control agent for soybean fields and provides a scientific basis for large-scale application. Full article

Clonal hematopoiesis of indeterminate potential (CHIP) and the gut microbiota-derived metabolite trimethylamine N-oxide (TMAO) are both linked to NLRP3-mediated cardiovascular inflammation, but their interaction has not previously been explored. This work proposes the CHIDT axis (clonal hematopoiesis–dysbiosis–TMAO), a feed-forward mechanism in which TET2 loss-of-function CHIP- and TMAO-generating Gram-negative gut dysbiosis mutually enhance cardiovascular risk. The model proceeds in three nodes. CHIP-associated intestinal immune dysregulation promotes luminal expansion of Gammaproteobacteria, which produce both trimethylamine via CntA/CntB-mediated L-carnitine oxidation and ADP-heptose as an obligate LPS biosynthetic intermediate. TMAO amplifies NLRP3 inflammasome activation through the SIRT3 → SOD2 → mtROS pathway. The evidence base of the CHIDT model is strongest for TET2-CHIP; the proposed extension to DNMT3A-CHIP rests on indirect, associative data and requires dedicated experimental confirmation before it can be considered established. TXNIP cascade, with predicted disproportionate potency in macrophages epigenetically primed by TET2 haploinsufficiency. High concentrations of TMAO have also been shown to suppress TET2 expression in endothelial cells through CYTB promoter hypermethylation, inducing NLRP3–GSDMD-dependent pyroptosis, although it remains unclear whether physiological TMAO levels can trigger this effect. Concurrently, ADP-heptose activates the ALPK1–TIFA–NF-κB pathway in bone marrow progenitors, favoring the expansion of mutant hematopoietic stem and progenitor cells. The model identifies three potential therapeutic strategies: NLRP3 inhibition, microbial TMA lyase inhibition, and microbiome-targeted reduction in Gram-negative bacteria. None has been tested in CHIP carriers stratified by plasma TMAO. Further studies in preclinical models and human cohorts integrating CHIP genotyping and TMAO quantification are needed to validate the CHIDT axis as a target for precision cardiovascular prevention. Full article

Automatic milking systems (AMSs) are increasingly adopted in dairy farms, and barn design, particularly regarding cow traffic design (CowTD), plays a key role in system performance. This study evaluated the association between different CowTDs and operational and production indicators of AMS Brazilian dairy farms. The data were obtained from 149 commercial dairy farms equipped with AMS (average of 1.6 AMS per farm) encompassing approximately 14,642 lactating cows recorded between June and December 2025. Cow traffic designs were classified as free or smart-gated systems, including milk-first (MF) and feed-first (FF) configurations. Mixed models were used to evaluate the effects of regions, housing system, and CowTD on the number of lactating cows per AMS (NCowsAMS), milking frequency (MFreq), milk yield per milking (MYMilking), daily milk yield per cow (MYcow), daily milkings per AMS (MilkingsAMS), and daily milk yield per AMS (MYAMS). On average, farms milked 58 cows per AMS with a mean MFreq of 2.69 milkings/cow per day and produced 2227 kg of milk per AMS per day. Smart-gated CowTD supported a greater number of cows per AMS than free CowTD systems (FF = 57 and MF = 58 vs. Free = 53 cows/AMS). Although free CowTD increased MFreq to approximately three milkings/cow/day, this advantage did not translate into greater or equivalent MYAMS, despite MF and free CowTD exhibiting similar MYcow (37.0 and 37.2 kg/day, respectively). Even though free CowTD achieved the highest MilkingsAMS (Free = 156 vs. MF = 151 and FF = 143 milkings/day), it milked fewer cows per robot, resulting in lower MYAMS. Consequently, FF and MF systems produced 86 and 180 kg/day more MYAMS, respectively, than free CowTD. These results suggest that AMS performance is primarily driven by the NCowsAMS rather than MFreq alone. Under Brazilian commercial conditions, smart-gated CowTD systems appear to be more efficient, as evidenced by higher MYAMS, while allowing higher stocking densities, potentially without increasing labor requirements. Full article

Otters spend a considerable amount of time vocalizing within zoos and other captive settings. Although associations between these vocalizations and social activity have been investigated prior, it is not always clear how this is reflected in interactions with people under typical zoo conditions, including interactions with zoo visitors and staff. In addition, the implications for zoo welfare and their relation to vocalizing are also not clear, warranting greater investigation to identify potential future welfare metrics for otter species. We examined zoo Asian small-clawed otter vocalizations and general activity in the presence of people, including both visitors and staff. Our focus was on measuring vocalizations as a binomial response (yes/no) in relation to two variables: (a) people, and (b) time. In addition, we also measured differences in vocalizations during sessions, when visitors were present with or without staff. Finally, we measured general otter activity during sessions when 50% or more of the observation intervals had people present (More People), as opposed to sessions where less than 50% of all intervals had a person present (Less People). Overall, otters were statistically more active and less out of sight when more people were present; a weak to moderate correlation was observed between vocalizations and people, a weak correlation was observed between vocalizations and time, and there was no statistically significant difference in vocalizations of the otters in the presence of visitors regardless of staff presence. These results suggest that the measured variables showed no evidence of an overtly negative visitor effect for the otters and may have produced a potentially enriching effect. In addition, measuring multiple variables, including time of day, diurnal patterns, feeding patterns, weather, and providing experimental control could aid in identifying the potential visitor effect for otters and other zoo animals. Full article

This study evaluated the effects of dietary inclusion of free and protected acid protease on productive performance, milk composition, metabolic profile, nutrient digestibility, and ruminal environment in lactating Jersey cows. Fifteen multiparous cows (67 ± 7.5 days in milk; 27.5 ± 3.5 kg/day) were assigned to a 3 × 3 Latin square (5 squares) design with 21-day periods. Treatments consisted of: control (no enzyme), free protease (4.4 g/day), and protected protease (4.4 g/day). The protected form was developed using alginate-based encapsulation to enhance enzyme stability under ruminal conditions. Protease inclusion did not affect dry matter intake, milk yield, or feed efficiency (p > 0.05). However, free protease increased lactation persistency (p = 0.05) and improved fat-corrected and energy-corrected milk yields (p ≤ 0.02), with intermediate responses observed for protected protease. Milk fat and protein contents were higher in enzyme-fed cows (p ≤ 0.05), while other compositional parameters remained unchanged. Apparent crude protein digestibility was greater in cows receiving free protease (p = 0.037), with no effects on dry matter or fiber digestibility. Protease intake increased total volatile fatty acid concentrations and major fermentation products (acetate, propionate, and butyrate; p ≤ 0.01), indicating enhanced ruminal fermentation. Blood metabolites showed increased total protein and globulin levels in cows fed free protease (p ≤ 0.05), suggesting improved protein metabolism. Microbiota analysis revealed no differences in alpha or beta diversity; however, specific microbial taxa and predicted metabolic pathways were modulated by treatments, particularly in post-ruminal compartments. In conclusion, exogenous protease, especially in free form, improved protein utilization and corrected milk production without disrupting microbial stability. These findings highlight the potential of protease as a nutritional strategy to enhance efficiency in dairy systems through targeted modulation of ruminal function and nutrient metabolism. Full article