Biology and Life Sciences

Abstract: Measurement of Mycobacterium avium subsp. paratuberculosis (MAP) load within macrophages using a mycobacterial growth inhibition assay (MGIA) is an accurate indicator of host immune control of MAP infection. Real-time fluorescent microscopy allows the continuous analysis of infected cells and the quantification of fluorescence intensity, which directly correlates with concentration of fluorescent bacteria. However, this method has not yet been evaluated for real-time quantification of free MAP bacilli or MAP-infected macrophages. The objective of this study was to develop an assay based on automated and real-time live-cell imaging (IncuCyte^®) for quantifying green fluorescence protein (GFP)-expressing MAP and to monitor MAP growth within infected macrophages. The fluorescence of serially diluted free MAP–GFP bacilli was quantified using the IncuCyte^® demonstrating that the fluorescence signal of 3 × 10⁷ CFU was significantly different from lowest MAP concentrations. Significant differences between uninfected and infected BOMAC cells (MOI; 1:10) in DMEM-F12, DMEM-F12 supplemented with fetal bovine serum (FBS), and RPMI-1640 supplemented with FBS were observed, indicating the suitability of these culture media for measuring MAP-GFP-host cell interaction in the IncuCyte^®. For the optimization of the MOI and cell density; bovine monocyte-derived macrophages (MDMs) and the BOMAC cells were seeded at different cell densities, infected with MAP-GFP at different MOIs, and after 2 h p.i. the fluorescence was quantified in the IncuCyte^® at different times p.i. Our results demonstrated that MAP phagocytosis is pathogen number and cell density dependent. Overall, the results showed that infection of 1 × 10⁶ cells at an MOI of 1:10 maintained a stable fluorescence signal of approximately 400 to 500 GCU⋅μm²/image" across 2 h to 4 days p.i.. In addition, good correlations between MAP-GFP (logCFUs) estimated in the BACTEC MGIT™ 960 system and fluorescence intensity measured in the IncuCyte^® were observed for free MAP-GFP bacilli and from MAP-GFP within infected BOMAC cells; r² = 0.79 and r² = 0.78, respectively. In conclusion, real-time fluorescent microscopy is a reliable method for quantifying fluorescent MAP and enables continuous monitoring of bacterial load within infected macrophages.

Abstract: Intramuscular fat (IMF) deposition is the primary economic driver of premium beef production, yet conventional phenotypic selection requires costly rearing to slaughter age. Although whole-genome sequencing has repeatedly flagged the KIRREL3 region on BTA29 as a candidate interval for meat quality in beef cattle, whether intronic polymorphisms within this gene are directly associated with carcass or meat-quality traits--and whether such marker effects remain stable across physiological stages--has not been validated. To address these gaps, we genotyped two intronic SNPs in KIRREL3 (g.30324329C>T, 63-bp site; g.30324473C>T, 203-bp site) in two age cohorts of Woking heifers (n = 226 at 36 months; n = 206 at 48 months), together with an independent validation cohort (n = 41) for fatty-acid profiling, meat-quality phenotyping, and multi-tissue expression analysis. Both loci were moderately polymorphic (PIC = 0.37) and in Hardy-Weinberg equilibrium. At 36 months, the TT genotype was associated with significantly higher dressing percentage, rib thickness, and meat grade than CC at both loci. By 48 months, associations at the 63-bp site persisted and the T allele showed complete dominance (CT and TT both exceeded CC in grade and rib thickness), whereas associations at the 203-bp site dissipated with age, showing no significant effect on intermuscular fat thickness or grade. In the validation cohort, palmitic acid (C16:0), linolenic acid (C18:3n3), and arachidonic acid (C20:4) followed a CC > TC >= TT trend (P < 0.05). Notably, CC animals displayed higher IMF and muscle KIRREL3 mRNA levels than TT animals, yet received lower meat grades--a pattern that awaits functional clarification. These findings identify KIRREL3 g.30324329C>T as a robust age-stable marker, providing a concrete target for early marker-assisted selection in Woking cattle.

Abstract: Background: Staphylococcus arlettae is a coagulase-negative staphylococcal species increasingly recognized as a reservoir of antimicrobial resistance determinants in animal, human, and environmental. Methods: In the present study, a multidrug-resistant S. arlettae isolate (BVC_SA31) isolated from bovine mastitis milk was characterized by whole-genome sequencing and comparative pangenomic analyses. Results: Antimicrobial susceptibility testing revealed resistance to penicillin, oxacillin, clindamycin, and erythromycin. Whole-genome sequencing of isolate generated a high-quality draft genome comprising of 36 contigs, 2,521 coding sequences, 10 rRNA genes and 57 tRNA genes. Species identity was confirmed by average nucleotide identity and tetranucleotide correlation analysis. Isolate was assigned to sequence type ST1203 and harbored antimicrobial resistance genes, including mph(C), blaARL-2, vanT, vanY, sdrM, sepA, and norC. Mobile genetic elements like insertion sequences (49), genomic islands (4), CRISPR loci (2), and plasmid-associated recombination systems (2) were identified. Comparative genomic analysis of 74 S. arlettae genomes revealed an open and highly diverse pangenome comprising 10,153 gene clusters, including 547 core genes, 1,024 soft-core genes, 1,267 shell genes, and 7,315 cloud genes. ANI-based clustering and codon usage analyses grouped the genomes into four distinct clusters, while core proteome phylogeny characterised two major global evolutionary lineages. Diverse repertoire of plasmid replicons, rep7a, rep7b, rep7c, rep10, rep13, rep15, rep19c, rep20, rep21, rep22, rep24, rep39, repUS10, repUS12, and repUS19 were identified. A total 39 antimicrobial resistance genes were identified, with msr(A) (46.62%), blaARL-2 (25.9%), mph(C) (19.24%), blaARL-1 (18.5%), mecA (8.88%), aph(2'')-Ii (7.4%), erm(C) (7.4%), lnu(A) (7.4%), tet(K) (7.4%), and dfrG (6.66%) being the most prevalent, confer resistance to macrolides, β-lactams, aminoglycosides, lincosamides, tetracyclines, and trimethoprim. Only two strains were found to harbour SSCmecA elements. Conclusions: In conclusion two global lineages of S. arlettae were identified and global antimicrobial resistance profiling of S. arlettae, highlighting its potential role as an emerging reservoir of antimicrobial resistance genes were identified.

Abstract: Germ cell formation is a fundamental process in sex determination, playing crucial roles in balancing oogonia and spermatogonia self-renewal and development. However, the regulatory genes fundamental to germ cell self-renewal and development remain to be elucidated. In this study, we constructed zebrafish lima1a knockout mutants. Lima1a is a homolog of human LIMA1, which is involved in cell motility, gene regulation, cellular metabolism, and the epithelial-to-mesenchymal transition (EMT). These knockout mutants were used in subsequent experiments to clarify the roles of lima1a in sexual differentiation and germ cell development. Compared with wild type (WT), lima1a^-/- zebrafish had smaller body sizes and smaller gonads. Nonetheless, lima1a^-/- zebrafish produced mature gametes and maintained a comparable sex ratio to their WT siblings. At a juvenile stage, lima1a^-/- females showed delayed and prolonged germ cell development, and this adversely affected follicle quality, subsequently impacting embryo development in adults. Additionally, adult male lima1a^-/- zebrafish exhibited defective spermatogenesis, and the ovary-to-testis transition in juveniles was delayed and prolonged. At 45 days post-fertilization (dpf), we observed that the expression levels of ovary-specific and testis-specific genes were significantly disrupted during the sexual development of lima1a^-/- zebrafish, suggesting that lima1a expression is crucial for the development and function of gonadal cells in ovaries and testes. These changes in gene expression also impacted the development of germ cells in lima1a^-/- zebrafish and caused delayed puberty onset. Furthermore, Vim, a marker for EMT, was upregulated in lima1a^-/- zebrafish gonadal cells, suggesting that lima1a maintains gonad differentiation and germ cell development by regulating the EMT process of gonadal cells. Collectively, these results reveal that lima1a is required for maintaining normal sexual development and the ovary-to-testis transition in juvenile zebrafish. Moreover, lima1a is required for gonadal germ cell development and maturation, and for embryo development.

Abstract: This study evaluated nano-iron sulfide (nFeS) as an iron source on the bioavailability, biocompatibility, and tolerance in broilers. Broilers were allocated to the five diets: an iron-deficient basal diet (ID), the basal diet with 80 mg Fe/kg as FeSO₄, or 20, 40, or 80 mg Fe/kg as nFeS. Results showed that nFeS treatments (40, 80 mg/kg) improved average daily gain and feed conversion rate compared to ID, showed comparable or even superior effects to FeSO₄ on growth performance during 1-21 days (p < 0.05). nFeS improved whole blood Hb content and PCV percentage at day 21, while increasing serum SOD and GSH-Px activities at day 42. nFeS (40, 80 mg/kg) had or tended to deposit higher iron content in liver, spleen, duodenum and serum than FeSO₄ groups. nFeS significantly increased breast meat lightness and redness compared with the ID (p < 0.05) and showed comparable effects on postmortem pH, drip loss, and shear force relative to FeSO₄. nFeS reshaped the microbial composition in the jejunum and cecum, particularly enriching the abundance of beneficial bacteria such as Lactobacillus in the jejunum. At the dose up to 400 mg/kg, nFeS revealed no negative effects in broilers. In conclusion, nFeS is an effective iron supplement with high bioavailability and comparability, and additional benefit in shaping intestinal microbiota.

Abstract:

Native Mexican turkeys (Meleagris gallopavo gallopavo) are an important genetic resource in family-based production systems exposed to marked regional and seasonal environmental variation. This study evaluated the effects of environmental gradients shape diet composition and hematological responses of native Mexican turkeys raised in family production systems. Fifty approximately six-month-old males were evaluated in a 5 × 2 factorial design including five physiographic regions and two seasons (rainy and dry). Crop content composition, estimated dietary nutrient composition, body weight, and hematological profile were analyzed using multivariate analysis of variance, principal component analysis (PCA), and multiple regression. Grains and energy-rich by-products predominated in the diet (58.2–76.2%), whereas the dry season increased dry plant fragments and crude fiber while reducing crude protein from 17.5 to 14.3% (p < 0.001). Body weight was greatest in the Neovolcanic Axis (5.20 kg) and lowest on the Coast (4.35 kg) (p < 0.001). Erythrocyte-related variables were higher in temperate, high-altitude regions, whereas leukocyte-related variables predominated in warmer regions (p < 0.05). PCA explained 71.3% of the total variation, identifying nutritional–productive and immunological–adaptive components. Physiographic region was the main determinant of diet composition and hematological responses, whereas the dry season intensified nutritional constraints and their effects on physiological adaptation.

Abstract: Evidence-based standards on environmental enrichment for lynxes under human care remains limited, despite their importance for the welfare of these wild cats and its recommendation by zoological associations. This study aimed to: (1) evaluate the scientific evidence available to guide practical decisions on lynx enrichment in controlled environment within an Evidence-Based Practice framework; and (2) examine enclosure enrichment structures use and social behaviour in a group of Eurasian lynxes. A total of seven relevant publications (3 on Lynx rufus) were identified, five of which through a scoping review. All studies reporting quantifiable outcomes indicated positive effects from at least some forms of environmental enrichment. We observed three Eurasian lynxes (Lynx lynx) housed at the Wildheart Animal Sanctuary, using the facility’s live-streaming webcams. Enclosure furnishing use by the lynxes was assessed from two-hundred opportunistically recorded instantaneous scans, while social behaviours were assessed from 200 minutes continuous recording. Lynxes used structural enrichment in more than half of the scan samples, and engaged in intra-specific affiliative behaviour. The use of the wooden turret was lower from sunrise to 12:59 pm than during other time periods (p< 0.001). Further species-specific research is needed to evaluate the effects of environmental enrichment on lynx behaviour and welfare.

Abstract: Stanniocalcin‑1 (STC‑1) is a multifunctional glycoprotein, yet its role in mitigating intestinal epithelial oxidative injury remains elusive. This study investigated the cytoprotective effects and underlying molecular mechanisms of STC‑1 in porcine intestinal epithelial cells (IPEC‑J2) subjected to tert‑butyl hydroperoxide (TBHP)‑induced oxidative stress. IPEC‑J2 cells were transfected with pcDNA3.1/STC‑1 prior to the TBHP challenge. STC‑1 overexpression markedly rescued IPEC‑J2 cells from TBHP‑induced cytotoxicity and apoptosis, as demonstrated by AO/PI dual-staining and a restored Bax/Bcl‑2 ratio. Concurrently, STC‑1 dramatically suppressed intracellular reactive oxygen species and mitochondrial superoxide accumulation while preserving the mitochondrial membrane potential. These physiological improvements were accompanied by enhanced total antioxidant capacity and upregulated activities of key antioxidant enzymes. Mechanistically, mRFP‑GFP‑LC3 reporter assays revealed STC‑1 enhanced autophagic processing and promoted Pink1/Parkin-mediated mitophagy to clear dysfunctional mitochondria. Furthermore, STC‑1 enhanced the AMPK–Nrf2/Sirt1 signaling axis, leading to Keap1 downregulation and the subsequent transcription of mitochondrial biogenesis markers (FoxO1, PGC‑1α, and TFAM) under stress conditions. Taken together, these findings demonstrate that STC‑1 safeguards porcine intestinal epithelial cells against oxidative injury by orchestrating a cooperative defense network that involves ROS scavenging, mitochondrial homeostasis, and antioxidant defense amplification via AMPK–Nrf2/Sirt1 pathway, presenting a potential therapeutic target for preventing stress-associated intestinal disorders in piglets.

Abstract:

Background/Objectives: Brazilian locally adapted sheep breeds represent valuable genetic resources. However, market appreciation of these breeds depends on genetic certification that ensures traceability, thereby qualifying their products as originating from sustainable production models. This study aimed to identify and evaluate the minimum set of highly informative SNPs for accurate and efficient breed assignment across five Brazilian sheep breeds. Methods: A total of 677 animal samples were used, the SNPs were selected from the Embrapa Multispecies 65K Illumina Infinium 1 chip, which contains 2,926 markers for Ovis aries. The dataset was randomly partitioned into training (80%) and testing (20%) sets. Markers were prioritized based on genetic differentiation using Pairwise Wright's fixation index (F_ST), implemented in the Toolbox for Ranking and Evaluation of SNPs (TRES), generating three nested reduced panels of 288, 192, and 96 SNPs. Finally, panel performance and preservation of population structure were evaluated using Random Forest classification, Principal Component Analysis (PCA), and ADMIXTURE. Results: The 96-SNP panel maintained high classification accuracy, comparable to larger subsets. This indicates that targeted marker selection is more effective than simply increasing marker density for breed assignment. Conclusions: While the development and analytical validation of a dedicated low-density genotyping assay remain necessary before routine implementation, the 96-SNP panel identified here provides a solid foundation for an efficient, cost-effective genomic tool. This panel will support breed certification, traceability, and the conservation of Brazilian locally adapted sheep genetic resources.

Abstract: Prioritizing cows for closer observation around calving is important for livestock management, but reliable same-day calving detection from behavior alone remains challenging. We examine whether daily 06:00–06:00 behavior summaries derived from ear-tag accelerometers can support strictly causal, calving-oriented triage in grazing cattle. Because birth annotations provide only daily-resolution supervision, we frame the task as weakly supervised day-level risk assessment rather than precise event timing. Therefore, we train a compact weakly supervised multilayer-perceptron daily-risk model using an exactly-one likelihood over a two-day candidate window comprising the day before birth and the recorded day of birth. The model uses current behavior summaries, deviations from cow-specific causal behavior baselines for the current and previous retained days, a history-gap variable, and missingness indicators. We evaluate the model using cow-grouped nested cross-validation on data from 134 calving cows and 4,891 cow-days. At the primary base operating point, pooled out-of-fold predictions show a measurable but modest signal, with F1 score 0.277, MCC 0.244, AUPRC 0.231, and candidate-window detection 47.0%. Comparator methods recover similar signals with different detection–burden trade-offs: a supervised positive-bag random forest improves F1 score and candidate-window detection, but increases non-candidate false-alert burden. A higher-threshold Alert state reduces the retained non-candidate false-alert rate to 0.0121, but lowers recall to 0.194. Applying the frozen deployment model to an independent non-calving cohort yields low daily Watch/Alert rates but non-negligible cow-level exposure. Overall, daily behavior summaries are better suited to lightweight herd-level risk stratification and human-in-the-loop monitoring than to reliable standalone calving detection.

Abstract: This study investigated the effects of apple, oak, and mesquite wood chip types on the physicochemical properties, VOC profiles, and sensory characteristics of brined smoked goat semimembranosus. Boneless muscles were brined in 1% NaCl and ran-domly assigned to a non-smoked control group or three wood chip-smoked groups. All smoked groups exhibited lower pH and reduced lipid oxidation compared to the con-trol (p < 0.05); TBARS values were significantly lower in all smoked groups than in the control (p < 0.05), indicating improved oxidative stability. Cooking loss and Warner–Bratzler shear force showed no significant differences among groups, indicating that fundamental textural properties were preserved. Notably, mesquite- and oak-smoked meats exhibited significantly higher abundances of key smoke-derived phenolic com-pounds — including guaiacol, phenol, o-cresol (2-methylphenol), and 2-methoxy-5-methylphenol — and sulfur-containing compounds such as carbon disul-fide and hexathiane, which correlated with reduced off-flavor scores and significant improvements in smoke flavor and overall acceptability compared to the control and apple-smoked groups (p < 0.05). These findings demonstrate that smoking goat meat with mesquite or oak wood chips is an effective processing strategy to mask the per-ception of species-specific off-flavor and enhance overall sensory acceptability.

Abstract: Bovine tuberculosis (bTB), caused by Mycobacterium bovis, remains an important constraint to water buffalo (Bubalus bubalis) productivity and a zoonotic risk within Pakistan's dairy sector. In this study, we performed a genome-wide association scan for single intradermal comparative cervical tuberculin (SICCT) reactor status in Nili-Ravi buffalo using the Axiom Buffalo array (90K), followed by a focused evaluation of the SLC11A1 (NRAMP1) candidate region. After quality control, 45,183 SNPs were retained for analysis, and a confirmed subset of 85 animals was used for the targeted study (50 SICCT-negative and 35 SICCT-positive). No marker achieved genome-wide significance in the full scan. However, within the SLC11A1 interval, the strongest nominal association was observed for AX-85139659 (odds ratio = 13.2, Fisher's P = 0.0039), followed by AX-85109412 (odds ratio = 0.09, P = 0.0092) and AX-85128873 (odds ratio = 0.14, P = 0.0457). The first two SNPs were located within a shared 27 kb haplotype block, with complete linkage disequilibrium (D′ = 1.00, r² = 0.76), suggesting that they are likely inherited together rather than representing independent signals. The identified SNPs in the SLC11A1 region provide preliminary evidence for genetic variation associated with bTB resistance in Nili-Ravi buffalo and warrant validation in larger populations.

Abstract: The loss of a companion animal can provoke grief whose intensity rivals human bereavement, yet its biological basis remains largely uncharted. This structured narrative review synthesizes neurobiological, endocrinological, immunological, and psychosocial evidence into a mechanistic framework offered not as established fact but as a source of testable hypotheses. We trace the substrates of the human-animal bond across six interacting systems and propose how disruption of each upon an animal's death may drive grief. Three carry comparatively robust analogical support (oxytocinergic signaling, hypothalamic-pituitary-adrenal [HPA] axis regulation, and neuroinflammatory activation) and three are more exploratory (dopaminergic, serotonergic, and endocannabinoid pathways). Integrating extracted study-level data into transparent prioritization heuristics, we identify the HPA axis, assessed with its coupled oxytocinergic partner, as the most defensible first target for a direct biomarker study, a conclusion robust across three methodologically independent analyses. No direct pet-loss evidence yet supports any proposal, and this gap is the review's central motivation. As applied context, we address euthanasia-related guilt, disenfranchised grief under East Asian norms (a tentative "double disenfranchisement" hypothesis we want tested), assessment instruments, evidence-graded therapies, a Korean tool-development roadmap, and One Health research priorities.

Abstract: Genotype-to-phenotype (G2P) research in farmed animals has entered a phase in which deep genome annotation, multi-omics and AI-enabled prediction can nominate variants, regulatory mechanisms and cellular pathways at scale, but causal validation remains a major bottleneck. In vitro cellular systems, from tractable primary cells to organoids and other advanced models, provide experimentally controlled contexts in which massively parallel reporter assays and CRISPR-based perturbations can test variant effects and define cellular phenotypes. Here, we examine how in vitro approaches can be deployed across host–pathogen interactions, genotype-by-environment responses, nutrition, adaptation and One Health research, and outline priorities for integrating in vivo, in vitro and in silico data into mechanistic G2P workflows.

Abstract: This study investigated whether rosemary extract (RE) supplementation selectively reshapes ruminal butyrate-producing microbiota and enhances functional milk properties in lactating dairy goats. Twenty-four goats were fed a control diet or a diet supplemented with 2.14 g/kg RE (n = 12) during a 42-day feeding trial. Rumen fermentation parameters, bacterial community composition, and milk quality indices - including bioactive proteins, amino acid and fatty acid profiles, and somatic cell count - were analyzed. RE supplementation significantly increased butyrate concentration and tended to elevate valeric acid. The relative abundances of fibrolytic bacteria (Ruminococcus albus and Fibrobacter succinogenes) and butyrate-producing taxa (i.e., Butyrivibrio) were significantly enriched (p < 0.05) in the RE group. Notably, dietary RE markedly elevated milk bioactive proteins, including lactoferrin, α-lactalbumin, and β-lactoglobulin (p < 0.05), and reduced somatic cell count (p < 0.05). These findings reveal a microbiota-mediated pathway through which RE enhances the functional quality of goat milk and support the potential of phytogenic feed additives.

Abstract: Turkish ambling horses represent an important component of Türkiye’s indigenous equine genetic resources. However, available morphometric information on these horses remains relatively limited. This study aimed to characterize morphometric traits of Turkish ambling horses by breeding origin, age, and sex, thereby providing baseline phenotypic data for future conservation and breeding programs. The findings provide novel insights into the phenotypic variation in Turkish ambling horses by integrating multiple breeding origins and evaluating the combined effects of breeding origin, age, and sex within a single analytical framework. A total of 102 Turkish ambling horses were evaluated, including indigenous horses raised in Bursa (n = 28), indigenous horses raised in Nazilli, Aydın Province (n = 65), and Afghan-origin ambling horses raised in Bursa (n = 9). Morphometric measurements of the head, body, and limbs were recorded, and the effects of breeding origin, age, and sex were analyzed using a general linear model. Significant differences among breeding origins were observed for several body and limb traits (p < 0.05), with Afghan horses exhibiting great-er body dimensions, particularly withers height, body length, chest depth, chest width, and heart girth, than the indigenous horse populations. Many body measurements showed a tendency to stabilize after four years of age, although some traits continued to vary among adult age groups. Males generally exhibited larger morphometric measurements than females. Correlation analysis revealed strong positive relationships among the principal body measurements, particularly between withers height and croup height (r = 0.84, p < 0.001), indicating a high degree of proportionality among major skeletal traits. Overall, this study offers one of the most comprehensive morphometric assessments of Turkish ambling horses from different breeding origins and establishes valuable baseline phenotypic data for indigenous Turkish horse populations. The findings contribute to the conservation and sustainable utilization of indigenous equine genetic resources and provide a scientific foundation for future phenotypic characterization, genetic studies, and breeding programs.

Abstract: Facial surface temperature can provide useful non-contact information for dairy-cow health, welfare, heat-stress, and reproductive-status monitoring. Infrared thermography can capture thermal information from facial regions such as the eyes, muzzle, nostrils, and ears, but infrared images alone often lack clear texture and anatomical boundaries, making reliable cow-head localisation difficult during mobile robotic inspection. Visible-light images provide richer structural cues but no temperature information. This study proposes YOLO11-AFE, a lightweight visible–thermal cow-head detection and heterogeneous-camera association framework for quadruped inspection robots. ADown was introduced for lightweight downsampling, C3k2_FE for adaptive feature enhancement, and SPPF_ECA for channel-aware multi-scale representation. An improved Hungarian matching algorithm was then used to establish one-to-one cow-head correspondence between synchronized visible-light and infrared pseudo-colour detections. YOLO11-AFE achieved Precision, mAP@0.5, and mAP@0.5:0.95 values of 97.51%, 96.26%, and 70.64% on the visible-light test subset, and 95.92%, 99.07%, and 91.41% on the infrared subset. Compared with YOLO11n, parameters and FLOPs were reduced by 17.37% and 18.17%. TensorRT deployment on Jetson Orin NX reached 36.71 FPS for the complete core pipeline. All 187 cow-head pairs were correctly associated, indicating that the framework provides an edge-deployable front end for future non-contact facial temperature monitoring.

Abstract: Canine anxiety is a widespread behavioral condition that compromises animal welfare and increases the risk of stress-related behavioral dysregulation. Severe anxiety disrupts cognitive processing, sleep regulation, and social functioning, often requiring pharmacological support to enable behavioral rehabilitation. This case report describes the clinical response of a 4-year-old Italian Volpino dog with chronic anxiety, hyperarousal, and stress-related dysregulation treated with a micellar CBD formulation (NegEnt). The treatment produced rapid improvements in sleep continuity, arousal modulation, social behavior, and impulse control without cognitive or motor impairment. Furthermore, a planned temporary discontinuation demonstrated the absence of withdrawal or rebound effects, followed by sustained long-term efficacy over a two-month maintenance period. These observations suggest that micellar CBD may facilitate a "window of cognitive plasticity" that supports behavioral therapy. Controlled studies are required to confirm these preliminary findings.

Abstract: Primary tracheal neoplasia is uncommon in domestic animals and humans, and most reported tracheal tumors are malignant. To the authors’ knowledge, this case represents the third confirmed tracheal leiomyoma reported in dogs. Similarly, in human medicine, tracheal leiomyomas have only been reported sporadically and account for approximately 1% of tracheal tumors. A 12-year-old spayed female Pitbull was presented as an emergency because of worsening recurrent respiratory signs that were refractory to medical treatment. On initial evaluation, reduced mobility of both arytenoid cartilages was observed, and left arytenoid lateralization was therefore performed. However, the patient continued to experience severe episodes of respiratory distress requiring orotracheal intubation. Subsequent bronchoscopy revealed an intraluminal tracheal mass partially obstructing the airway lumen. Because of this obstruction, an emergency temporary tracheostomy was performed caudal to the mass. Once the patient was clinically stabilized, computed tomography was performed to complete surgical planning. Surgical treatment consisted of segmental tracheal resection and end-to-end anastomosis, with removal of 24% of the total tracheal length. Histopathological examination confirmed the diagnosis of tracheal leiomyoma. Complete surgical excision was achieved, supporting a favorable prognosis.

Abstract: Fowl adenovirus serotype 11 (FAdV-11) is an important viral pathogen affecting both broiler and layer chickens and is the primary causative agent of inclusion body hepatitis (IBH), resulting in substantial economic losses through reduced meat and egg production worldwide. Despite its economic significance, no commercial vaccine is currently available specifically against FAdV-11. In the present study, to the best of our knowledge, the first immunoinformatics-based multi-epitope vaccine candidate against FAdV-11 was designed using conserved regions of the penton and fiber proteins. Conserved B-cell, cytotoxic T-lymphocyte (CTL), and helper T-lymphocyte (HTL) epitopes were identified, screened for antigenicity, allergenicity, toxicity, and immunogenicity, and assembled into a multi-epitope vaccine construct using appropriate linker peptides and a β-defensin adjuvant. The designed vaccine exhibited favorable physicochemical properties and was predicted to be highly antigenic, non-allergenic, and non-toxic. Secondary and tertiary structure prediction, refinement, and validation confirmed the structural stability and reliability of the vaccine construct. Molecular docking and molecular dynamics simulation analysis demonstrated stable interactions with immune-related Toll-like receptors (TLRs), while immune simulation predicted robust humoral and cellular immune responses with the establishment of long-term immunological memory. Codon optimization yielded a Codon Adaptation Index (CAI) of 0.982, and in silico cloning into the pET-28a(+) expression vector suggested efficient recombinant expression in Escherichia coli. Collectively, these findings indicate that the proposed vaccine represents a promising candidate for the prevention of FAdV-11-induced IBH in poultry. Nevertheless, comprehensive in vitro and in vivo studies are required to validate its immunogenicity, safety, and protective efficacy.