For their ability to enhance performance, immune response, and robustness to environmental stressors in both fish and crustaceans, phytogenic compounds are receiving increasing attention from the aquaculture industry as alternatives to traditional feed additives. Numerous studies have investigated the use of a specific combination of organic acids and botanicals (OA + B) in terrestrial animals, but their potential role in aquaculture remains unexplored. The objective of this study is to assess the feasibility of a blend of OA + B (microencapsulated in a lipid matrix; AviPlusAqua - Vetagro S.p.A.) to enhance the health of gilthead seabream. To better assess the potential of the selected blend, both in vitro and in vivo experiments were conducted. Head-kidney leukocytes (HKLs) were incubated with varying doses of OA + B, then viability and cellular immune parameters were evaluated after 30 min, 2 h, and 4 h. For the in vivo assay, 120 gilthead seabreams (body weight [BW]: 48.00 ± 5.00 g) received a diet supplemented with 0 (control [CTR]), 250, or 500 ppm of OA + B; then growth performance, humoral and cellular immunity, and gene expression of immune-related genes were evaluated after 15, 30, and 60 days. In vitro, data from gene expression, phagocytosis, and respiratory burst assays demonstrated that OA + B positively stimulate HKLs activity. In vivo results showed increased growth performance (+19% in overall BW; +0.31 specific growth rate [SGR]) from 30 days of supplementation onward, along with improved humoral and cellular immunity. Gene expression analysis of intestinal samples revealed a positive modulation of genes related to intestinal oxidative stress response and a balanced pro-/anti-inflammatory cytokine profile at both tested dosages. The results highlight that dietary OA + B supplementation modulates the immune response under homeostatic conditions, as evidenced by modulated expression of immune-related genes and enhanced phagocytic and respiratory burst activities.

Objectives This study investigated the relationship between the chemical composition of Yucca schidigera saponins and their antimicrobial and anticoccidial activities against Clostridium perfringens and Eimeria tenella.Methods Extracts of Y. schidigera were obtained using 100% methanol (MeOH), 50% MeOH, and water, and characterized by LC-HRMS to identify saponins. Multivariate analyses (PLS-DA and OPLS-DA) were performed to assess chemical differences among extracts. In vitro assays were conducted to evaluate antimicrobial activity against C. perfringens and anticoccidial activity against E. tenella. The most active extract was fractionated into three saponin-enriched subfractions, and ridge regression was applied to link chemical composition to bioactivity.Results LC-HRMS identified multiple saponins in all extracts, with chemical differences among solvents. The 100% and 50% MeOH extracts inhibited C. perfringens growth at 750 ppm, while the water extract was inactive. The 100% MeOH extract reduced E. tenella intracellular development by 77%, compared with 48% and 25% inhibition by the 50% MeOH and water extracts. Fractionation of this extract produced three subfractions, of which the first (fraction 1) showed the highest bioactivity (MIC = 500 ppm; 84% inhibition of E. tenella). Ridge regression highlighted schidigerasaponins D3/D4, smilagenin, sarsasapogenin, and hecogenin as key contributors to bioactivity.Conclusion Y. schidigera saponins exhibit significant antimicrobial and anticoccidial activity. The study establishes a data-driven framework for predicting structure-activity relationships, providing a rational basis for botanical screening and standardization.

Thyme oil (TO) is emerging as a promising candidate to counteract antimicrobial resistance due to its renowned antimicrobial and anti-inflammatory properties. However, rapid gastric absorption of its bioactive compounds limits its intestinal delivery, where its action is required, so the protection of these components is necessary. This pilot study optimized TO-loaded emulsions for targeted intestinal release. High-shear homogenization and membrane emulsification were compared to formulate single oil in water (O/W) and double water in oil in water (W/O/W) emulsions, screening emulsifiers (lecithin, Tween 20, Tween 80) and functional biopolymers (pectin, sodium alginate). High-shear homogenization with lecithin (0.5%), pectin (1.80%), and sodium alginate (0.2%) yielded stable submicron O/W emulsion (Span = 0.5; d(v,0.5) = 0.21 µm), achieving electrostatic stabilization (ζ-potential = −51.5 ± 1.5 mV) at a target poultry dosage. A pH-responsive behavior was observed: protective hydrogel formed in gastric conditions (d(v,0.5) = 2.64 µm) and maintained stability at intestinal pH (d(v,0.5) = 3.03 µm). Membrane emulsification enabled precise droplet control under mild conditions, producing monodisperse O/W emulsions (d(v,0.5) = 38–59 µm; Span ≤ 1.0) and W/O/W double emulsions (d(v,0.5) = 26.5 µm; Span = 0.6) with ultra-low interfacial tension (0.52 mN·m−1). Repeated membrane passes reduced droplet size to ~6.6 µm. These systems represent a foundational step toward bioactive intestinal delivery, providing a viable antibiotic-free strategy for sustainable livestock production.

Background/Objectives: Metabolic dysfunction-associated steatotic liver disease (MASLD) involves the interplay of hepatic lipid accumulation and immune-mediated inflammatory signaling, yet human-relevant in vitro systems that capture both processes simultaneously in a scalable format remain limited. The objective of this study was to develop and characterize a matrix-free 3D hepatocyte–macrophage co-culture model enabling simultaneous assessment of lipid accumulation and NF-κB-mediated inflammatory activation under glucolipotoxic stress. Methods: A 3D liver co-culture model was established by combining HepG2 hepatocyte-like cells with phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 macrophage-like cells stably expressing a NF-κB–Luc2 reporter. Spheroids were generated using a hanging-drop method in standard 96-well plates and matured for 8–10 days. Mature spheroids were subjected to acute 24 h glucolipotoxic challenge combining high glucose and palmitic acid and assessed for neutral lipid accumulation, NF-κB reporter activation (luciferase), and macrophage marker expression (qPCR). Results: Time-course characterization demonstrated progressive hepatocyte marker remodeling (albumin, alpha-fetoprotein, CYP3A4) and dynamic macrophage phenotype shifts (CD14, CD206, MARCO, TREM2). Acute glucolipotoxic challenge induced dose-dependent increases in neutral lipid accumulation and NF-κB reporter activation, accompanied by coordinated macrophage-associated transcriptional changes consistent with lipid-handling and tissue-remodeling programs. Post-challenge metabolic activity was retained under the selected stress conditions. As a proof-of-concept demonstration, three botanical extracts showed distinct attenuation profiles across the lipid and inflammatory endpoints. Conclusions: This 3D hepatocyte–macrophage co-culture model provides orthogonal readouts of steatosis and NF-κB-mediated inflammatory activation under glucolipotoxic stress, offering a reproducible, fit-for-purpose screening tool for investigating early glucolipotoxic hepatic responses and evaluating candidate compounds in a defined in vitro setting.

Fish oil (FO) is a rich source of omega-3 fatty acids (FA), which may be preferable FA for hepatic phosphatidylcholine (PC) synthesis via the actions of phosphatidylethanolamine N-methyltransferase. Our objective was to determine whether rumen-protected choline (RPC) feeding increased concentrations of circulating PC enriched with omega-3 FA in cows fed a bolus of rumen-protected FO. Eighteen Holstein cows (3.6 ± 2 [mean ± SD] lactations) were randomly assigned to 2 groups (n = 9/group) at −21 ± 3 d prepartum: unsupplemented (CON) or supplemented (RPC) with rumen-protected choline (0 or 60 g/d, respectively; 25% choline chloride; Ruprocol®; Vetagro S.p.A., Reggio Emilia, Italy) until d 35 postpartum. At d 27 ± 4 postpartum, all cows were provided with a gelatin capsule containing 100 g of lipid-microencapsulated FO as an esophageal bolus (36% FO; 10.2 g of omega-3 FA; Prototype 6; Vetagro S.p.A.). Cows were fed a total mixed ration and milked twice daily. Blood was sampled at 0, 10, and 24 h, relative to bolus delivery. Plasma was analyzed for choline and choline metabolites using LC/MS. Data were analyzed under a mixed model with the random effect of cow, and the fixed effects of treatment, hour, and their interaction. At h 0, plasma betaine concentrations were greater in RPC, relative to CON; however, plasma choline concentrations were not modified by treatment. Following FO bolus administration, circulating trimethylamine N-oxide and dimethylglycine (DMG) were increased by h 10. Cows supplemented with RPC tended to have greater plasma DMG and methionine, relative to CON. Although no changes were detected for plasma LPC-22:5, LPC-22:6, total LPC, PC-16:0/22:5 or PC-18:0/22:5, RPC group increased plasma LPC-20:5, PC-16:0/22:6, and PC-18:0/22:6, compared with CON. Plasma PC-18:0/20:5 and total PC concentrations were greater in RPC cows by h 24, relative to CON. We conclude that RPC feeding augmented the ability of dietary FO to increase plasma PC concentrations in early lactation cows.

Coccidiosis, caused by Eimeria parasites, is a major threat to global poultry production, and increasing restrictions on conventional anticoccidial drugs highlight the need for safer, more sustainable alternatives. Progress has been hindered by the lack of rapid, sensitive, and animal-sparing in vitro assays for quantifying parasite replication and drug efficacy. This study reports the development of a novel bioluminescent platform for anticoccidial screening based on a genetically modified Eimeria tenella line expressing NanoLuc luciferase (EtNluc). Parasite-associated bioluminescence enabled rapid and quantitative monitoring of intracellular development, allowing the tracking of different replication phases through schizont formation and merozoite release. Time course analysis showed minimal changes in relative light units (RLU) between 2 and 24 hours post infection (hpi), followed by a marked increase between 24 and 72 hpi, consistent with parasite replication. Among the tested multiplicities of infection (MOI), 4:1 exhibited the fastest growth, described by a linear model (slope = 2908 RLU/h, R2 = 0.84). A same-well repeated-measure analysis (2 and 72 hpi) confirmed the dose-dependent replication, with mean slopes of 2052.85, 765.07 and 523.63 RLU/h, respectively, supporting the selection of the MOI 4:1 for anticoccidial screening. These experimental conditions were used to evaluate the anticoccidial efficacy of commercial anticoccidial drugs (salinomycin and robenidine) and natural compounds (thyme and oregano essential oils, thymol, and carvacrol) under two experimental designs: short pre-incubation of sporozoites, and continuous exposure throughout intracellular development. Pre-incubation with commercial anticoccidials reduced invasion approximately to 65% for salinomycin and 44% for robenidine, whereas the essential oils and their bioactive constituents inhibited invasion by 30-55%, and reduced the replication slope to 33-60% of control values. Continuous exposure significantly impaired intracellular development for all treatments, reducing replication to 10-30% of controls, providing additional evidence that plant-derived compounds can complement commercial anticoccidials for integrated strategy for coccidiosis control in chickens. Overall, the EtNluc bioluminescent system provided a rapid, sensitive, and scalable method for quantifying E. tenella growth, suitable for in vitro anticoccidial screening, supporting the characterization of novel anticoccidial while reducing reliance on animal experimentation.

B vitamins, including niacin (vitamin B3), are synthesized by rumen microbes, but supplementation may provide additional benefits for ruminant health and productivity. Supplementing rumen-protected niacin (RPN) during the transition period can reduce lipolysis after calving and, consequently, may improve health and fertility of dairy cattle later in lactation. Our objective was to determine if supplementing RPN during the first 21 DIM would improve the health of dairy cows on a commercial dairy farm. We hypothesized that RPN would reduce mastitis, improve fertility, and reduce risk of leaving the herd during lactation. Holstein cows were blocked by parity and projected calving date, with treatments randomly assigned to cows within each block. Cows received RPN (n = 481; 26 g/d) through a supplement dispenser in the automated milk systems (AMS) in addition to their robot pellet, or the robot pellet only (CON; n = 593). Treatments were applied for the first 21 DIM, and cows were followed for the rest of their lactation. Milk yield, milk components (wk 1-3 of lactation), pre- and postpartum BCS, health records, and reproductive records were collected. Blood was collected from a random subset of 99 cows at 3 DIM and 97 cows at 10 DIM to assess plasma concentrations of niacin, metabolic biomarkers, and biomarkers of inflammatory status. Culling, proportion of cows pregnant, and mastitis risk were analyzed using Cox proportional hazard models. Mastitis incidence was analyzed with a linear mixed model and conception risk was analyzed using a chi 2 test. Supplementing RPN increased plasma nicotinamide concentration by 1,740 nM +/- 410.0 nM (SE; 50% increase), but it did not affect plasma nicotinic acid concentrations. Supplementing RPN reduced plasma insulin concentrations at 3 and 10 DIM across all parities. Circulating BHB and free fatty acid concentrations were greater for cows receiving RPN; the effect was greatest in cows in third or greater parity. Plasma haptoglobin was not affected by treatment. Rumen-protected niacin increased milk yield for first- and second-parity cows by wk 9 and 13 of lactation, respectively, and increased milk yield in those groups was sustained for the rest of lactation, resulting in 658 +/- 259.4 kg and 675 +/- 308.9 kg more milk for RPN-supplemented first- and second-parity cows. The risk of leaving the herd, mastitis incidence, and probability of pregnancy were not affected by RPN. Inquiries into the effects of RPN supplementation and its timing on metabolism are necessary to understand optimal supplementation strategies for RPN in dairy cattle.

The objective of this study was to test the effects of increasing levels of a microencapsulated blend of botanicals (MBB) against an F18+E. coli challenge on mucosa-associated microbiota, mucosal immunity, and morphology in the jejunum of nursery pigs. Thirty-two nursery pigs (6.8 ± 0.3 kg) were assigned to 4 dietary treatments in a randomized complete block design, with initial body weight and sex as blocks, and fed for 28 d in 3 phases (7/13/8 d, respectively). Four dietary treatments were the negative control (NC, receiving a corn-soy basal diet) and the 3 treatments (basal diet supplemented with 0.0, 0.1, and 0.2% of MBB). On d 7, pigs (except for NC) were orally inoculated with F18+E. coli (1.5 × 1010 CFU). On d 28, pigs were euthanized to collect jejunal tissue for histology and mucosa for jejunal mucosal immunity analysis. Growth performance was measured. Data were analyzed using SAS MIXED procedure and polynomial contrasts were conducted to evaluate linear and quadratic effects in challenged groups with SAS RSREG procedure. The 0.0% MBB decreased (P < 0.05) the relative abundance (RA) of Streptococcaceae in the jejunal mucosa compared with NC. Increasing levels of MBB quadratically changed (P < 0.05) the RA of Erysipelotrichaceae (max: 1.85 at 0.09% MBB) and Streptococcaceae (max: 2.35 at 0.08% MBB). The 0.0% MBB decreased (P < 0.05) the relative expression of TLR4 in the jejunum compared with the NC. Increasing levels of MBB both linearly and quadratically changed (P < 0.05) TLR4 (max: 0.9 at 0.07% MBB). The 0.0% MBB decreased (P < 0.05) TNF-α, IL-6, and IL-8 in the jejunum, whereas 0.0% MBB increased (P < 0.05) IgG compared with the NC. Increasing levels of MBB linearly increased (P < 0.05) IL-8 in the jejunum and quadratically changed (P < 0.05) ZO-1 in the jejunum (max: 1.4 at 0.08% MBB). The 0.0% MBB tended to decrease (P = 0.075) villus height in the jejunum compared with the NC. Increasing levels of MBB linearly increased (P < 0.05) villus height to crypt depth ratio in the jejunum. The 0.0% MBB decreased (P < 0.05) body weight on d 14 and gain-to-feed ratio from d 7 to 14 compared with the NC. Increasing levels of MBB both linearly and quadratically changed (P < 0.05) gain-to-feed ratio from d 7 to 20 (max: 0.9 at 0.10% MBB). In conclusion, F18+E. coli challenge negatively modulated the jejunal mucosa-associated microbiota and impaired intestinal morphology and growth performance of nursery pigs. Supplementation of MBB at a range of 0.07 to 0.10% provided the optimal mitigation from the negative impacts of F18+E. coli challenge on humoral immunity, intestinal integrity, jejunal morphology, and feed efficiency of nursery pigs.

L-lysine (L-Lys) is the first-limiting amino acid in swine nutrition, but free-form supplements exhibit poor intestinal absorption, reducing their bioavailability. This study aimed to enhance the gastric retention, controlled intestinal release, and systemic availability of L-Lys by optimizing solid lipid microcapsules (SLMs). SLMs were formulated using hydrogenated triglycerides (C16:0 or C18:1), free fatty acids, and varying emulsifier concentrations. Gastric retention and intestinal release were evaluated in vitro under simulated gastrointestinal conditions (a pepsin buffer at pH 5.0 for 2 h, followed by a pancreatin buffer at pH 6.5 for up to 8 h at 39 °C). SLMs with hydrogenated triglycerides showed significantly higher gastric retention (94-95%) than those with free fatty acids (48%). Specifically, C16:0 triglyceride-based SLMs achieved 74% intestinal release, which was enhanced to 90% with 1% emulsifier. This refined formulation was subsequently evaluated in vivo using weaned pigs (three groups; n = 4) fed a basal cornmeal diet. The treatments included a single oral administration of saline solution (placebo), free L-Lys (0.17 g/kg BW), or L-Lys SLMs (0.38 g/kg BW, equally providing L-Lys at 0.17 g/kg BW). The SLMs delayed the L-Lys plasma peak (T. max. 3-4 h vs. 1 h) and significantly increased the total L-Lys amount in the plasma over 24 h, demonstrating the enhanced relative bioavailability of encapsulated L-Lys.

The study aimed to evaluate the effects of increasing levels of a microencapsulated blend of botanicals (MBB) on the intestinal health and growth performance of nursery pigs challenged with F18(+)E. coli. Sixty-four nursery pigs (6.8 +/- 0.3 kg) were assigned to 4 dietary treatments in a randomized complete block design, with initial body weight and sex as blocks, and fed for 28 d in 3 phases. Treatments were a basal diet fed to pigs without F18(+)E. coli challenge (NC) and 3 levels of MBB (0.0%, 0.1%, and 0.2%) in pigs challenged with F18(+)E. coli. On day 7 of the study, pigs in the challenged group were orally inoculated with F18(+)E. coli (1.5 x 10(10) CFU). On days 7 and 21 post-challenge, pigs were euthanized to collect jejunal tissues and mucosa. Compared to the NC, 0.0% MBB increased (P < 0.05) relative abundance (RA) of Staphylococcus saprophyticus and reduced (P < 0.05) Streptococcus parasuis at days 7 and 21 post-challenge, respectively. Increasing levels of MBB decreased (linear: P < 0.05) RA of S. saprophyticus on day 7 post-challenge. Compared to the NC, 0.0% MBB increased (P < 0.05) jejunal NOD2 and IL-6 expression and decreased (P < 0.05) ZO-1 on day 7 post-challenge. Compared to the NC, 0.0% MBB decreased (P < 0.05) jejunal IL-6, IL-8, and TNF-alpha and increased (P < 0.05) IgG on day 21 post-challenge. Increasing levels of MBB increased OCLN (linear: P < 0.05) and ZO-1 (linear and quadratic: P < 0.05) on day 7 post-challenge and decreased toll-like receptor 4 (TLR4; linear and quadratic: P < 0.05). Compared to the NC, 0.0% MBB decreased (P < 0.05) Ki-67(+) on day 7 post-challenge. Increasing levels of MBB increased (linear: P < 0.05) Ki-67(+) on day 7 post-challenge and villus height (VH):CD on d 21 post-challenge. In the overall period, compared to the NC, 0.0% MBB decreased (P < 0.05) average daily gain. Increasing daily MBB intake linearly increased OCLN on day 7 and VH:CD on day 21, and reduced TLR4 and IL-8 on day 21 post-challenge, but exhibiting quadratic effects (P < 0.05) on ZO-1 (optimal at 0.12% of MBB), IgG (optimal at 0.14% of MBB), and G:F during days 7 to 20 and days 7 to 28 (optimal at 0.22% and 0.10% of MBB, respectively). In conclusion, F18(+)E. coli challenge negatively modulated the jejunal mucosal microbiota and reduced intestinal morphology and growth of nursery pigs. Supplementation of MBB at 0.10% to 0.14% provided optimal mitigation of the impacts of F18(+)E. coli challenge on humoral immunity, intestinal integrity, jejunal morphology, and feed efficiency of pigs.

Inducing inflammation in response to pathogen infection is known to be an energy-intensive process. An extended state of inflammation in production animals can be detrimental to performance parameters. Here, we compare two doses of a microencapsulated thymol-based feed additive blend and two different antibiotics in the context of a Salmonella Enterica subsp. Enterica serovar Enteritidis (SE) challenge. A total of 500, day-old, straight run chicks (Ross 708) were placed in floor pens (50 chicks/pen) and assigned to 5 groups with 2 replicates each. The groups were all fed with basal diets, without supplementation (Control) or supplemented with: tylosin at 25 g/MT (Tylosin); neomycin sulfate at 100 g/MT (Neomycin); or a microencapsulated thymol-based blend of botanicals at inclusions of 500 g/MT (Blend 500) or 1000 g/MT (Blend 1000). All the pens were orally challenged at day 4 with SE at 105 CFU/bird. At 7, 14, 21, and 35-days post hatch, ten animals from each pen were weighed and euthanized in order to culture enumerate SE in the ceca and determine SE prevalence in the liver. Gene based prevalence, gene expression, and kinome analysis were performed on jejunum samples collected at day 35. The higher dose of the tested botanical, Blend 1000, showed a statistically significant increase in final body weight relative to the other groups, indicating an energy benefit for this group. The Blend 1000 also showed a reduction in protein phosphorylation that corresponds to reduced inflammatory status that was unique compared to Blend 500 and the antibiotics. These birds also showed a clearance of orally inoculated SE demonstrating that reduced inflammation can benefit the broiler chicken in clearance of bacteria while maintaining growth.

Osteoarthritis (OA) is a prevalent joint disease in dogs. Botanicals (BOT) interacting with the endocannabinoid system (ECS) can reduce pain, inflammation, and other OA-related processes, and could become potential treatments for pets. This study investigates various BOT in vitro for their potential to reduce OA-like inflammation, oxidative stress, and apoptosis, and influence the expression of ECS enzymes or receptors. Capsicum oleoresin (10% capsaicinoids) at 100 ppm, cinnamaldehyde at 5 ppm, turmeric extract (95% curcuminoids) at 10 ppm, and eugenol at 10 ppm were selected for their documented ability to interact with the ECS and used as treatments. The efficacy of these BOT was assessed by pre-treating human chondrosarcoma cells (SW1353 - ATCC HTB-94) with BOT for 2 h before exposure to interleukin (IL)1β and tumor necrosis factor (TNF) α for 24 h. Following the challenge, the cells and supernatants were collected. Then, qPCR on selected markers and IL8 quantification with ELISA were conducted. Additionally, to assess antioxidant and anti-apoptotic capabilities, BOT-treated cells were exposed to menadione for 1 h to measure reactive oxygen species (ROS) and for 6 h to induce apoptosis and evaluate chondrocyte viability. Data were analyzed using one-way ANOVA with Tukey’s multiple comparisons (n = 3; P < 0.05) and reported in this work compared against the positive control. Under inflammatory conditions, SW1353 cells exhibited a significant increase in IL6 (234-fold; P = 0.0333) and IL8 (4,500-fold; P = 0.0395) expression, and MMP1 (13-fold, P = 0.0052), MMP3 (40-fold; P = 0.0474), and MMP13 (26-fold; P = 0.0260) expression. Among the different BOTs, only cinnamaldehyde effectively countered IL6 and IL8 upregulation, reducing their expression to 0 for IL6 and by 4,400-fold for IL8 (P = 0.0086). Additionally, it reduced MMP3 by 40-fold (P = 0.0242) and MMP13 by 24-fold (P = 0.0275), while eugenol significantly reduced MMP3 by 36-fold (P = 0.0250). Only cinnamaldehyde significantly decreased IL8 secretion by 86% (P < 0.0001), and all tested botanicals reduced chondrocyte apoptosis (P < 0.0001). Eugenol and turmeric extract exhibited significant antioxidant effects, reducing ROS levels by 70% compared with the positive control (P < 0.05). Concerning ECS response to the inflammatory challenge, MAGL levels increased by 4.2-fold (P = 0.0253), with cinnamaldehyde significantly reducing its mRNA expression by 4-fold (P = 0.0078). PPAR-γ expression showed a numerical decrease of 0.7-fold under inflammatory conditions, but cinnamaldehyde and capsicum oleoresin upregulated its mRNA expression by 2.3-fold (P = 0.0162) and 1.3-fold (P = 0.0006), respectively. Cinnamaldehyde also reduced DAGLα expression by 0.3-fold (P = 0.0079), and upregulated CBR2 and NRF2 mRNA expression by 7-fold (P < 0.0001) and 1.6-fold (P = 0.0295) respectively, while other botanicals had no effects. In conclusion, the tested BOTs displayed promising properties to mitigate OA in vitro, with cinnamaldehyde proving to be the most promising one. These findings suggest the potential of BOT active on ECS as a novel treatment for canine OA, prompting further in vivo research for comprehensive characterization.

The gustatory system is responsible for detecting and evaluating the palatability of the various chemicals present in food and beverages. Taste bud cells, located primarily on the tongue, communicate with the gustatory sensory neurons by means of neurochemical signals, transmitting taste information to the brain. It has also been found that the endocannabinoid system (ECS) may modulate food intake and palatability, and that taste bud cells express cannabinoid receptors. The purpose of this study was to investigate the expression of cannabinoid and cannabinoid-related receptors in the gustatory cells of the papillae vallatae and foliatae of ten piglets. Specific antibodies against the cannabinoid receptors (CB1R and CB2R), G protein-coupled receptor 55 (GPR55), transient receptor potential vanilloid 1 (TRPV1) and ankyrin 1 (TRPA1) were applied on cryosections of lingual tissue; the lingual tissue was also processed using Western blot analysis. Cannabinoid and cannabinoid-related receptors were found to be expressed in the taste bud cells and the surrounding epithelial cells. The extra-papillary epithelium also showed strong immunolabeling for these receptors. The results showed that these receptors were present in both the taste bud cells and the extra-gustatory epithelial cells, indicating their potential role in taste perception and chemesthesis. These findings contributed to understanding the complex interactions between cannabinoids and the gustatory system, highlighting the role of the ECS within taste perception and its potential use in animal production in order to enhance food intake.

Organic acids and botanicals have shown protective effects on gut barrier and against inflammation in broilers. However, their effects on intestinal digestive enzymes and nutrients transporters expression and functions have not been fully studied. The objective of this study was to understand how a microencapsulated blend of botanicals and organic acids affected intestinal enzyme activities and nutrient transporters expression and functions in broilers. A total of 288 birds were assigned to a commercial control diet or diet supplemented with 500 g/MT (metric ton) of the microencapsulated additive. Growth performance was recorded weekly. At d 21 and d 42, jejunum and ileum were isolated for enzyme (maltase, sucrase, and aminopeptidase) and transporter (SGLT1, GLUT2, GLUT1, EAAT3, B(0)AT1, and PepT1) analyses. Jejunum specific nutrients (glucose, alanine, and glutamate) transport activities were evaluated by Ussing chamber. Protein expression of nutrient transporters in small intestine were measured in mucosa and brush-border membrane (BBM) samples by western blot. Intestinal gene expression of the transporters was determined by RT-PCR. Statistical analysis was performed using Student's t-test comparing the supplemented diet to the control. The feed efficiency was significantly improved through the study period in the supplemented group (P <= 0.05). Significant changes of intestinal histology were shown in both jejunum (P <= 0.10) and ileum (P <= 0.05) after 21 d of treatment. At d21, jejunal maltase activity was upregulated (P <= 0.10). The Ussing chamber transport of glucose and alanine was increased, which was in line with increased gene expression (GLUT2, GLUT1, EAAT3, and B(0)AT1) (P <= 0.10 and P <= 0.05, respectively) and BBMV protein levels (B(0)AT1, P < 0.10). At d21, ileal sucrase and maltase activities were upregulated (P <= 0.05). Increased expressions of GLUT1, EAAT3, and B(0)AT1 were observed in both mRNA and protein levels (P <= 0.05). Similar pattern of changes was also shown at d42 of age. Our results suggest that feeding microencapsulated additives improves intestinal nutrient digestion and transporter expression and function in broilers, thereby enhancing feed efficiency.

IntroductionOsteoarthritis (OA) is a chronic degenerative joint disease characterized by the progressive degradation of articular cartilage, resulting in pain and reduced mobility. Turmeric (Curcuma longa L.) has been widely recognized for its anti-inflammatory and antioxidant properties, but the molecular mechanisms underlying its therapeutic effects remain inadequately explored. This study investigates the potential of turmeric oleoresin (TUR) to activate Cannabinoid Receptor 2 (CBR2) and its role in mediating anti-inflammatory and antioxidant effects in an in vitro OA model.Material and methodsMolecular docking and cAMP quantification assays were used to evaluate TUR’s agonistic activity on CBR2. Human chondrosarcoma cells (SW-1353) were treated with TUR under oxidative stress induced by menadione or inflammatory conditions simulated with IL-1β and TNF-α. The effects of TUR were assessed in the presence and absence of the CBR2 antagonist SR144528. Outcomes included changes in reactive oxygen species (ROS) production, inflammatory marker expression, oxidative defense markers and endocannabinoid system components and receptors.ResultsTUR was confirmed as a CBR2 agonist and significantly reduced ROS production, downregulated pro-inflammatory cytokines (IL-6, COX-2, metalloproteases), and suppressed signaling pathways such as NFKB1, ERK 1/2, and c-Myc. These effects were reversed upon CBR2 inhibition. TUR also enhanced HMOX-1 expression and modulated endocannabinoid-related enzymes, highlighting its impact on oxidative stress and the endocannabinoid system.DiscussionThese findings suggest that CBR2 activation is central to TUR’s anti-inflammatory and antioxidant effects. By modulating key pathways and endocannabinoid system components, TUR demonstrates potential as a novel therapeutic agent for OA management. Future studies could explore its clinical applications and further validate its molecular mechanisms in vivo.

This study examined the action of a blend of botanicals (BOT) against lipopolysaccharide (LPS)-induced inflammation on cultured hepatocytes and weaning piglets. In vitro studies examined HepG2 cells treated with BOT and challenged with Escherichiacoli LPS for 8 d. BOT treatment reduced IL-6 concentration in cell culture media across time (P < 0.05) and decreased pro-inflammatory cytokine expression on days 1 and 8 of experiment (TNF alpha, IL-1 beta; P < 0.05). BOT also increased the expression of antioxidant enzymes (GPX-2, SOD, CAT) on day 8 (P < 0.05), which was supported by lowered reactive oxygen species concentration after LPS challenge (P < 0.1). The in vivo study was conducted with 72 weaning pigs, allotted into 24 pens and divided into 3 groups: a negative control (CTR-, basal diet), a challenged control (CTR+) that received an intraperitoneal injection of E. coli O55:B5 LPS on days 14 and 16, and a challenged treated group which received a diet containing 1.5 g/kg of microencapsulated BOT (BOT+) for the whole duration of the study. Growth performance was determined weekly and, on days 21 (1 animal per pen) and 28 (remaining animals), pigs were sacrificed to collect liver and jejunal tissues. After the challenge, BOT+ pigs had increased BW on days 21 (P < 0.05) and 28 (P < 0.1) compared to CTR+. Similar improvements in average daily gain and FCR on days 14 to 21 (P < 0.05) and 21 to 28 (P < 0.1) were also seen in BOT+ group. In the liver, compared to CTR+ pigs, BOT+ pigs had downregulated expression of TLR-4, IL-6, IFN-gamma on day 21 (P < 0.05), and TLR-4, TNF-alpha, IL-8 on day 28 (P < 0.05). BOT+ also increased GPX-2 expression on days 21 and 28 (P < 0.05), while also upregulating SOD-1 and SOD-2 on day 21 (P < 0.05) and CAT on day 28 (P < 0.05) compared to CTR+. In the jejunum, BOT+ reduced inflammation by affecting cytokine expression (P < 0.05) and increasing the expression of tight-junction proteins, ZO-1 on day 21 and CLD-1 on day 28 (P < 0.05). Furthermore, BOT+ pigs had lower crypt depth on days 21 (P < 0.1) and 28 (P < 0.05), and increased villi-to-crypt ratio on days 21 and 28 (P < 0.05). By day 28, BOT+ intestinal measurements were restored to values similar to the CTR-. Finally, BOT+ also reduced mast cell activation on day 21 (P < 0.05) compared to CTR+. Considering all the findings, BOT controlled inflammatory activation and oxidative stress in liver cells, enhanced intestinal integrity, and as a result improved the growth performance of weaning piglets challenged with LPS. Piglets are particularly susceptible to stress due to the abrupt changes they face during weaning. These stressors cause a surge of oxidation and inflammation, particularly in the intestinal tract. Inflammation in the intestine causes a loss in its barrier function and facilitates the translocation of harmful compounds. Of particular concern is the translocation of lipopolysaccharide (LPS), which elicits an immune response in the liver, diverting energy from growth to inflammatory processes. Exposure to LPS also has the potential to have long-lasting detrimental effects on piglets' health. Research has identified the potential of many botanicals to minimize weaning stress through diverse modes of action. This study investigated the efficacy of a blend of botanicals (BOT) to help hepatocytes control inflammatory stress in vitro and to ameliorate the effects of an LPS challenge in piglets in vivo. Our in vitro and in vivo models successfully generated an inflammatory state. In vitro, BOT decreased inflammation and oxidation, and similar effects were seen in vivo, where BOT supplementation modulated the expression of cytokines in the liver and maintained intestinal integrity. These effects validate BOT ability to improve the performance of LPS-challenged piglets and support its utilization as a feed supplement to mitigate weaning stress.

Pharmacological doses of zinc oxide (ZnO) have been widely used in pig industry to control post-weaning diarrhea (PWD) symptoms exacerbated by enterotoxigenic Escherichia coli F4 infections. Because of environmental issues and regulatory restrictions, ZnO is no longer sustainable, and novel nutritional alternatives to manage PWD are urgently required. Botanicals represent a wide class of compounds employed in animal nutrition because of their diverse beneficial functions. The aim of this study was to investigate the in vitro protective action of a panel of essential oils and natural extracts on intestinal Caco-2 cells against an E. coli F4 infection. Moreover, we explored the potential mechanisms of action of all the botanicals compared to ZnO. Amongst the others, thyme essential oil, grape seed extract, and Capsicum oleoresin were the most effective in maintaining epithelial integrity and reducing bacterial translocation. Their mechanism of action was related to the modulation of cellular inflammatory response, the protection of tight junctions' expression and function, and the control of bacterial virulence, thus resembling the positive functions of ZnO. Moreover, despite their mild effects on the host side, ginger and tea tree essential oils provided promising results in the control of pathogen adhesion when employed during the challenge. These outcomes support the advantages of employing selected botanicals to manage E. coli F4 infections in vitro, therefore offering novel environmentally-friendly alternatives to pharmacological doses of ZnO capable to modulate host-pathogen interaction at different levels during PWD in pigs.

Previously, the supplementation of a microencapsulated blend of organic acids and botanicals improved the health and performance of broiler breeders under non-challenged conditions. This study aimed to determine if the microencapsulated blend impacted dysbiosis and necrotic enteritis (NE) in broiler breeders. Day-of-hatch chicks were assigned to non-challenge and challenge groups, provided a basal diet supplemented with 0 or 500 g/MT of the blend, and subjected to a laboratory model for NE. On d 20-21, jejunum/ileum content were collected for microbiome sequencing (n = 10; V4 region of 16S rRNA gene). The experiment was repeated (n = 3), and data were analyzed in QIIME2 and R. Alpha and beta diversity, core microbiome, and compositional differences were determined (significance at p ≤ 0.05; Q ≤ 0.05). There was no difference between richness and evenness of those fed diets containing 0 and 500 g/MT microencapsulated blend, but differences were seen between the non-challenged and challenged groups. Beta diversity of the 0 and 500 g/MT non-challenged groups differed, but no differences existed between the NE-challenged groups. The core microbiome of those fed 500 g/MT similarly consisted of Lactobacillus and Clostridiaceae. Furthermore, challenged birds fed diets containing 500 g/MT had a higher abundance of significantly different phyla, namely, Actinobacteriota, Bacteroidota, and Verrucomicrobiota, than the 0 g/MT challenged group. Dietary supplementation of a microencapsulated blend shifted the microbiome by supporting beneficial and core taxa.

Botanicals (BOTs) are well known for their anti-inflammatory and antioxidant activities. They have been widely used as feed additives to reduce inflammation and improve intestinal functions in agricultural animals. However, the effects of BOTs on chicken intestinal epithelial functions are not fully understood. The 3D apical-out chicken enteroids recapitulate the intestinal tissue, and allow convenient access to the luminal surface, thus serving as a suitable model for investigating gut functions. The aim of this study was to identify the roles of BOTs in protecting the intestinal epithelium in chicken enteroids under challenging conditions. Apical-out enteroids were isolated from the small intestines of 18 days-old chicken embryos. Lipopolysaccharide (LPS, 10 µg/mL) and menadione (400 µM) challenges were performed in the media with or without BOTs. Paracellular Fluorescein isothiocyanate–dextran 4kD (FD4) permeability, inflammatory cytokine gene expression, and reactive oxygen species (ROS) generation were analyzed post-BOTs and challenges treatments. Statistical analysis was performed using one-way ANOVA and post hoc multiple comparisons among treatments. The results showed that the LPS challenge for 24 h induced a 50% increase in FD4 permeability compared with nontreated control; thymol, thyme essential oil, and phenol-rich extract significantly (P < 0.02) reduced FD4 permeability by 25%, 41%, and 48% respectively, in comparison with LPS treatment. Moreover, the gene expression of inflammatory cytokines was upregulated, tight junction proteins and defensins were downregulated (P < 0.05) after 6 h of LPS treatment, while these BOTs treatments significantly restored the LPS-induced gene expression alterations (P < 0.05). Menadione oxidative challenge for 1 h significantly increased the ROS level compared with unchallenged control. Enteroids treated with thymol and thyme essential oils showed 30% reduced ROS levels, while the phenol-rich extract reduced them by 60%, in comparison with the challenged group (P < 0.0001). These data confirmed the role of BOTs in supporting the barrier function and reducing the disruptive effects of inflammation and oxidation in the chicken intestine.