Journal of Dairy Science

Continuous video offers a dynamic view of dairy-cow behaviour, but its value for precision nutrition depends on alignment with physiological context. We developed a dairy digital-twin framework that fuses identity-associated behavioural records from an established video-analytics layer with body weight, milk production, milk fat, parity and days in milk. The 16-cow analytical cohort was monitored for up to 11 days in one tie-stall barn, yielding 153 cow-days after quality exclusions applied before model fitting. NRC (2001) expected dry matter intake provided a transparent physiological reference compatible with the daily records. In matched leave-one-cow-out analysis, adding video-derived feeding duration to body weight, fat-corrected milk and days in milk reduced RMSE from 2.015 to 1.763 kg DM/day, MAE from 1.267 to 1.159 kg DM/day and MAPE from 4.5% to 4.2%, while R{superscript 2} increased from 0.500 to 0.617. A secondary reduced index achieved RMSE 2.362 kg DM/day and MAPE 5.8% across held-out cows. Cow-level analysis delineated the operating domain: median per-cow MAPE was 4.15%, whereas the sole cow at 15 days in milk had MAPE 28.8%. Two independently recorded veterinary events were temporally concordant with unusual feeding trajectories, providing descriptive biological context. Because the endpoint was NRC-derived, these metrics quantify reference reconstruction rather than accuracy against observed intake. By converting continuous behavioural events into auditable cow-day states, the framework links physical animals to physiologically contextualized digital counterparts and establishes a scalable foundation for operator-focused dairy digital-twin decision support.

Insect proteins represent a promising and more sustainable alternative to soybean meal in swine nutrition. However, high dietary inclusion levels often result in reduced animal performance, indicating that factors other than crude protein concentration should be considered. This study evaluated the protein quality and degradability of feed-grade defatted black soldier fly (BSF) meal in comparison with a conventional organic protein core for post-weaning piglets. Protein content, amino acid composition, reactive lysine, Maillard reaction markers, buffering capacity, and protein hydrolysability were determined. Our results showed that defatted BSF meal exhibited a protein content and amino acid profile comparable to those of the protein core but contained substantially higher levels of free amino acids. Accounting for free amino acids reduced the estimated lysine damage from 18.4% to 10.4%, highlighting the importance of considering free amino acids when determining reactive lysine. In addition, BSF meal exhibited a higher buffering capacity and lower protein degradability than the protein core. These findings provide new insights into the nutritional limitations of feed-grade BSF meal and suggest that a more accurate assessment of reactive lysine, together with dietary strategies to reduce buffering capacity, may improve its utilization in post-weaning piglet diets.

Abstract The gayal (Bos frontalis) is an endangered semi-domesticated bovine species renowned for its high-quality beef. However, its semi-feral lifestyle, ongoing habitat fragmentation, and extensive genetic introgression from sympatric local cattle have led to dramatic population decline and severe erosion of purebred genetic integrity, posing substantial challenges to its conservation and utilization. To address the urgent demand for rapid, non-invasive, and field-compatible germplasm identification, we developed an integrated artificial intelligence (AI) framework that predicts genomic admixture composition from external morphological images. We constructed a comprehensive dataset comprising 6,245 morphological images and matched genomic sequences from 52 gayals maintained at the Yunnan Provincial Gayal Conservation Farms. Following a preliminary evaluation of nine deep learning models, five were incorporated into a anatomical segment-based multi-modal pipeline, among which Inception_V3 delivered the optimal overall performance. To enhance simultaneous extraction of local fine-grained features and global structural information, we further designed an innovative HybridInceptionViT model by integrating the multi-scale Inception module with the Vision Transformer (ViT) framework. This hybrid model significantly outperformed the baseline Inception_V3, boosting the accuracy of phenotype-derived prediction against genomic admixture estimate from 69.69% to 87.87% (absolute error <15%). This study establishes a practical, low-cost "phenotype-to-genotype" tool for rapid on-site gayal germplasm screening, offering a scalable strategy for the conservation and breeding management of endangered livestock, and holds broad application prospects for agricultural and livestock production systems.

Dairy-free foods must reproduce diverse functions of dairy, from foam stabilization in a latte to acid-gel structure in yogurt and melt-stretch behavior in mozzarella. Public consumer benchmarks rarely compare these categories under a common protocol. Here we analyze data from the NECTAR Taste of the Industry 2026 study, a blinded, randomized, in-person evaluation of commercial dairy-free products and dairy benchmarks. The full study included 2,183 consumers and 112 products across ten categories, with 3,220 product evaluations and 79,027 recorded survey responses, including 35,762 product-linked responses. The ten categories included barista milk, butter, cheddar, cream cheese, creamer, ice cream, fluid milk, mozzarella, sour cream, and Greek style yogurt. Within each category, we compare a dairy benchmark with the dairy-free product selected post hoc as the category leader. Participants rated overall liking, flavor, texture and mouthfeel, appearance, similarity to dairy, and purchase intent on seven-point scales and completed check-all-that-apply questions. In exploratory category-specific equivalence tests using a chosen margin of {+/-}0.05 points, barista milk, creamer, and fluid milk meet this margin. Mozzarella has the largest paired deficit of -1.46 points (unadjusted, p < 0.001), followed by Greek style yogurt and butter; all three remain significant after Holm correction across the ten primary comparisons. Descriptive penalty analysis associates off-flavor, artificial or chemical notes, stale or cardboard notes, bitter taste, and lingering aftertaste with the largest reductions in liking. Together, these results establish the first open sensory benchmark for dairy and dairy-free products, demonstrate that sensory parity is already achievable in several beverage categories, and identify the product categories and sensory attributes that offer the greatest opportunities for future innovation.

Micronutrient deficiencies are prevalent in Madagascar, where diets rely heavily on starchy staples and access to animal-source foods is limited. Small dried fish (SDF) are widely available, yet their nutritional value and health risks remain poorly documented. We combined market surveys, taxonomic identification, and micronutrient and heavy metal analyses of nine SDF types collected along National Road 7. The samples encompassed 33 fish families, were dominated by small pelagic species (Clupeidae and Engraulidae), and were appreciated by consumers. A daily portion (5 g for infants; 10 g for young children and women of childbearing age) contributed substantially to Recommended Nutrient Intakes (RNIs). Across samples and groups, SDF were rich (>30% of RNI) in selenium and, for infants and young children, in calcium. All samples were a source of (>15% of RNI), or rich in, phosphorus, whereas iron contributions were more variable but often substantial. Several samples exceeded 100% of RNIs for selenium, calcium, iron, or manganese in infants and young children, and some were also sources of magnesium and, less frequently, zinc. Vitamin A was absent from sun-dried samples but detected in a smoked freshwater type. Heavy metal concentrations varied markedly, and portions of several types led to estimated exposures to inorganic arsenic or cadmium exceeding reference values, whereas freshwater species and some pelagic types showed a more favorable nutrition-risk balance. Overall, SDF are affordable, nutrient-dense foods with strong potential to alleviate micronutrient deficiencies in Madagascar, while highlighting the need for type-specific guidance to balance nutritional benefits and contamination risks.

The exponential increase in the number of genotyped animals, combined with the availability of high-density SNP chips has introduced computational challenges for routine genomic evaluations, particularly during the construction of the genomic relationship matrix. Although higher-density SNP panels can facilitate the identification of causal mutations, their use substantially increases computational requirements without a proportional gain in genomic prediction performance. To optimize computational efficiency while maintaining accuracy of genomic predictions, this study compared five SNP selection strategies (i.e., random sampling, random sampling with inclusion of informative SNPs, linkage disequilibrium (LD)-based pruning, a Shannon entropy-based machine learning approach, and [[EQUATION]]-based prioritization) to develop reduced-density panels for Nellore cattle. Using high-density (HD) genotype data comprising 437,650 SNPs from 304,782 animals (after quality control) as reference, three reduced-density panels (25K, 45K, and 65K SNPs) panels were tested across five traits (i.e., Age at first calving, Stayability, Weaning weight, Yearling weight, Muscling) with diverse genetic architectures. Genomic estimated breeding values (GEBVs) derived from these reduced panels were compared to those obtained from the HD reference panel using Pearsons correlations, under both genomic best linear unbiased prediction (GBLUP) and single-step GBLUP (ssGBLUP) methods. In the GBLUP model, prediction accuracy generally improved with increased marker density. Random selection with and without the informative SNPs consistently yielded the highest accuracies, whereas the [[EQUATION]]-based approach showed the lowest agreement with the HD reference across all densities. In contrast, ssGBLUP demonstrated strong robustness to marker reduction, producing uniformly high correlations {approx}1.00) across all SNP densities and selection strategies. These findings indicate that optimized low-density SNP panels maintain prediction accuracy comparable to HD panels, offering a cost-effective tool for large-scale genomic evaluations.

The maned wolf (Chrysocyon brachyurus) is a near-threatened canid species that suffers from gastrointestinal (GI) disease under human care. While the cause remains poorly understood, recent studies report that altered gut microbiota is linked to GI disease in the domestic dog. The goal of this study was to describe the relationship between gut bacteria and environmental factors, GI health, short chain fatty acid (SCFA) peaks, and genetic relatedness in maned wolves. Fresh fecal samples were collected twice from wolves (first collection n = 29; second collection n = 25) housed in six facilities in the United States. Fecal alpha-1 proteinase inhibitor (FA1PI), a biomarker for GI health in canids, and SCFA peaks were quantified. Information on diet, housing group and history of GI disease or inflammation was provided by the zoological facility for each wolf. We characterized gut microbiota using 16S rRNA gene amplicon sequencing with qPCR abundance corrections. We found that dietary fiber gross energy (GE), protein GE, and number of daily produce items influenced gut microbiota. Additionally, there was a negative relationship between dietary fiber GE and Fusobacterium sp. abundance, corroborating previous findings linking fiber to altered gut microbiome composition in canids. Lastly, fiber GE was negatively correlated with FA1PI, connecting fiber to GI health in the maned wolf. Findings from this study suggest that increasing dietary fiber GE changes the gut microbiome and may improve GI health in the maned wolf, highlighting important management decisions that can be considered to maintain a healthy zoo-managed maned wolf population. IMPORTANCEFindings from this study are important because gastrointestinal disease is a common health concern for maned wolves under human care, and the causes are not well understood. The current study found that diet, especially the amount of fiber gross energy, can affect gut bacteria and may support better gastrointestinal health. Diet alterations could be a practical tool for zoos to support management of gastrointestinal health issues in maned wolves. These findings also show the importance of carefully managing diets for a near-threatened species, since better gastrointestinal health may improve overall health and well-being and support reproduction. More research is needed to determine the best amount and type of fiber, but our results provide foundational information for improving care and supporting healthy zoo-managed maned wolf populations.

Objective: Malnutrition and micronutrient deficiency are significant public health problems affecting the well-being of both the mother and her offspring. It is important to first quantify their burden in underserved communities and then to tackle this problem. The objective of this study was to assess the nutritional status, micronutrient deficiency profile, and associated determinants among pregnant women attending antenatal care clinics under PMSMA in selected government health facilities across three districts of Jharkhand, India. Design: The study employed a cross-sectional study design to assess the burden of malnutrition and micronutrient deficiency among pregnant women. Setting: The study was conducted in 4 health facilities in 3 districts of Jharkhand. The selected facilities were Rajendra Institute of Medical Sciences (RIMS), Ranchi; District Hospital (Sadar), Ranchi; District Hospital (Sadar), Godda; and Community Health Centre (Gamharia, Saraikela) under RHTC, Department of Community Medicine, Manipal Tata Medical College, Jamshedpur. The study was conducted during the period of September to December, 2022. Participant: Eligible pregnant women attending ANC clinics under PMSMA in the selected health facilities were enrolled until the required sample size of 977 was achieved. Pregnant women who were critically ill or those who presented with some emergency conditions were excluded from the study. Result: Based on BMI assessment, 38.6% of participants were malnourished, with 17.7% being underweight and 20.9% overweight or obese. Anaemia affected 69.3% of women, while clinical features suggestive of iron deficiency were observed among 21.1% of the participants. Vitamin A deficiency, iodine deficiency, and fluoride excess were also identified among a smaller proportion of women. Conclusion: The present study highlights a substantial burden of both malnutrition and micronutrient deficiency among pregnant women in Jharkhand and justifies the need for integrated maternal nutrition strategies during antenatal care.

BackgroundAlthough human milk (HM) confers important health benefits, how bioactive milk components (e.g., microbiota, oligosaccharides, and fatty acids) interact with infant genetics to influence childhood atopy remains poorly understood. ObjectiveWe investigated interactions between infant genomic susceptibility and exposure to maternal human milk components (HMCs) and assessed whether integrating these genetic and milk features improves prediction of childhood atopy. MethodsLeveraging infant genomic and maternal HMC data from the CHILD Cohort Study, we conducted gene-milk interaction analysis using linear regression models that integrated polygenic risk scores (PRS) of nursing infants with multiple HMC types. Gradient-boosting machines (GBMs) were used to evaluate predictive performance of HMCs and infant PRS for childhood atopy. ResultsChildhood atopy was associated with interactions between infant genomics (e.g., PRS associated with atopy) and exposure to specific human milk microbes (e.g., Abiotrophia, PBonf=0.005, {beta}=0.29), as well as networks of co-occurring HMCs (e.g., a module containing Bifidobacterium longum, 2-fucosyllactose, and eicosapentaenoic acid, P=0.009, {beta}=-12.3). A GBM integrating HMCs and infant PRS achieved the highest predictive performance for childhood atopy with an area under the curve (AUC) of 0.78, outperforming models based on individual HMC types or PRS alone (AUC range: 0.54-0.63). ConclusionIntegration of maternal HMC exposures with infant genomics reveals interaction effects that contribute to prediction of childhood atopy. Understanding how early-life exposures such as HMCs impact the health of children differently depending on their genomic profiles may facilitate the development of personalized intervention strategies to reduce the burden of these health outcomes during childhood. Key messagesO_LIInteractions between infant polygenic risk and exposure to human milk components are associated with childhood atopy. C_LIO_LINetworks of co-occurring human milk microbiota, oligosaccharides, and fatty acids may influence childhood atopy, with effects varying by infant genomic susceptibility. C_LIO_LIIntegration of human milk components with infant genomics improves prediction of childhood atopy compared with individual milk components or genomics alone. C_LI Capsule SummaryThis study demonstrates that interactions between infant polygenic risk and maternal milk components improve prediction of childhood atopy, highlighting opportunities for personalized early-life prevention strategies.

Cocoa (Theobroma cacao L.) is grown in Cote d'Ivoire (the world's leading producer), almost always in association with trees, yet these cocoa-based agroforestry systems (cocoa AFS) remain characterized largely descriptively, without a robust inferential framework or modern diversity indices. We analyse a survey database covering 474 farms and 103 localities across the three main cocoa-producing zones of Cote d'Ivoire, locally known as " loops " (East/South-East, Centre/Centre-West, South/South-West; surveys 2013-2016), applying a renewed ecological and statistical framework: Hill numbers (q = 0, 1, 2), individual-based Hurlbert rarefaction, multiplicative alpha/beta/gamma diversity partitioning, ordination (PCoA) and PERMANOVA on Bray-Curtis matrices, Ward's hierarchical clustering for a data-driven floristic typology, multivariate MANOVA, and linear mixed models. Across 99 adequately sampled localities (>10 stems), mean taxonomic richness and the Shannon index differed significantly among zones (MANOVA: Wilks' = 0.350; F8,180 = 15.5; p < 0.001), including after standardizing sampling effort by rarefaction (F = 8.72; p < 0.001). Floristic composition differed significantly by zone (PERMANOVA, pseudo-F = 2.85; p = 0.001), though with uneven within-group dispersion (PERMDISP, p = 0.024) that calls for caution in interpretation. The Centre/Centre-West zone showed the highest beta diversity (inter-farm floristic turnover = 16.4, versus 7.2 and 2.2 in the other two zones), revealing a highly heterogeneous mosaic of individual agroforestry choices rather than a homogeneous regional system. A typological classification based on floristic composition distinguishes four cocoa-AFS profiles, ranging from dense, diverse stands to impoverished systems with low densities of associated trees, partially but not fully overlapping with the zonal boundaries. At the scale of the three loops (n = 409 farms with both age and yield recorded), cocoa yield varied significantly by orchard age class (Kruskal-Wallis, H = 18.2; p < 0.001), following a bell-shaped profile peaking around 20-35 years, consistent with the intermediate-age yield optimum suggested qualitatively by the source study; this relationship, robust to log-transformation of yield and to the exclusion of extreme values, nonetheless remains of small magnitude (R2=1.5%) and confers no out-of-sample predictive power under cross-validation, illustrating the need to distinguish statistical significance from predictive utility. This reanalysis provides a reproducible and transferable statistical framework for characterizing West African cocoa AFS, and highlights the potential value of high beta diversity as a possible indicator of landscape resilience.

Protein engineering and precision fermentation provide an opportunity to increase the value of food proteins by improving their solubility, stability, functionality, or nutritional composition. Here, we use {beta}-lactoglobulin ({beta}LG) as a model protein to investigate how state-of-the-art computational protein design approaches affect these properties. First, the deep learning-based design tool ProteinMPNN was used to alter up to 20% of {beta}LG residues for increased stability. Second, the physics-based modeling platform PyRosetta was used to find positions in {beta}LG accommodating increased branched-chain amino acid (BCAA) content and up to 10 residues were simultaneously exchanged. Experimental characterisation of ProteinMPNN and stabilised BCAA-enriched variants showed similar secondary structure and oligomeric state as native {beta}LG. ProteinMPNN variants gave increased titers and increased thermal stability up to 15 {degrees}C, and this correlated with changes in the rate of surface pressure in droplet tensiometry. Stabilized BCAA-enriched mutants had altered acid solubility. Correlations between computationally derived biophysical metrics and experimental properties are presented and suggest some predictive power for surface hydrophobicity on protein yield.

Background: Many epidemiological studies rely on dietary exposures taken from baseline only. This limits our understanding of diet-disease associations because it requires assuming a level of temporal stability, either by individuals or dietary pattern composition. Objectives: This study aimed to evaluate these analytic assumptions of pattern structure consistency and baseline adherence using a cohort of US Black women with repeated measures of food frequency questionnaires (FFQ). Methods: Data from 6151 Black women aged 21-69 from the Black Women's Health Study with complete FFQ data in 1995, 2001, 2013, and 2021 were evaluated for temporal stability. Baseline dietary patterns were derived using an overfitted latent class model. Parameter estimates from the baseline model were then applied to subsequent waves to track individual transitions between existing patterns. Dietary patterns were also derived at each time point using an overfitted latent class model and assessed for changes in pattern composition over time. Results: Five baseline dietary patterns were identified in 1995. Only 18% of participants remained in the same baseline dietary pattern across all four time points, while all others transitioned to a different baseline-derived pattern. Dietary patterns derived independently at subsequent time points, yielded a different number of dietary patterns at each time point (2001: 6 patterns, 2013: 5 patterns, 2021: 4 patterns). Correlation strength of subsequent derived patterns and baseline patterns significantly weakened in strength after 2001 (40% pairings > 0.5), with no patterns correlated greater than 0.5 in 2021. Conclusion: Prospective studies that rely on baseline dietary exposure data cannot assume stability of pattern composition or individual pattern adherence over time, as it ignores changes in dietary habits and may bias our understanding of the diet-disease pathway.

Genomic signatures of selection can reveal loci underlying adaptation and disease resistance in livestock populations, but such analyses in water buffalo (Bubalus bubalis) have historically been constrained by the absence of a chromosome-level, species-native reference genome for SNP array data. We re-analyzed genotype data from 85 Nili-Ravi buffalo (Axiom Buffalo Genotyping 90K array, originally positioned using bovine (Bos taurus, UMD3.1) proxy coordinates, by performing a full coordinate liftover to the buffalo-native UOA_WB_1 assembly using an independently published SNP remapping resource. Following quality control (51,209 markers retained), haplotype phasing, and genome-wide integrated haplotype score (iHS) and Wrights Fst (case/control) selection scans, we evaluated 14 classical bovine-tuberculosis (bTB) candidate genes and identified six additional genes with putative immune function through an unbiased genome-wide screen. None of the 14 classical candidates (including SLC11A1, the Toll-like receptors, and IFNG) reached genome-wide significance in either scan. In contrast, six novel loci TNFSF18, IL2RB, TNFRSF19, IRF2, IL15, and CD28 showed significant iHS or Fst signals, four of which (TNFSF18, IL2RB, IL15, CD28) converge functionally on T-cell costimulation and cytokine receptor signaling (KEGG pathways map04660 and map04060, Bos taurus proxy annotation). Using extended haplotype homozygosity (EHH) decay, haplotype furcation structure, and per-marker haplotype counts as three independent lines of corroborating evidence, we classified these six genes into confidence tiers: TNFSF18 and IL2RB showed the strongest, most balanced support, while CD28 and IL15 signals were driven by very few haplotypes (3 and 5 of 30, respectively) and should be interpreted cautiously pending replication. These findings suggest that adaptive, cell-mediated immune signaling rather than the innate/macrophage-centred mechanisms emphasized by existing bTB candidate gene panels may be a more productive avenue for future selection studies in Nili-Ravi buffalo, while underscoring the value of buffalo-native coordinate systems for accurate genomic inference in this species.

Boiling raw milk before drinking is a common practice in Bangladesh, but its impact on residue levels of ciprofloxacin and enrofloxacin is not well known. This study looked at these antibiotics in 120 raw cow milk samples from 12 dairy areas near Dhaka, collected between November 2023 and March 2024. Each sample was split into two parts: one tested as raw milk and the other after boiling at home for 15 minutes. Residues were checked using matrix-matched HPLC-UV. Ciprofloxacin and enrofloxacin were found in 113 out of 120 samples (94.2%), and at least one of them was present in 117 samples (97.5%). We considered a level of 12.5 micrograms per liter or higher as detected. The average combined amount of these antibiotics dropped from 194.61 micrograms per kilogram in raw milk to 179.57 micrograms per kilogram after boiling, a decrease that was statistically significant (p < 0.001). However, the percentage of samples exceeding the EU maximum residue limit of 100 micrograms per kilogram only went down from 92.5% in raw milk to 90.0% after boiling, which was not statistically significant (p = 0.250). Using national milk consumption data as an estimate of intake, the hazard index for adults was 0.360 for raw milk and 0.332 for boiled. Calculated with a 10 kg body weight, these values were 2.159 and 1.992, respectively. Overall, boiling at home did reduce the levels of ciprofloxacin and enrofloxacin, but it usually did not bring high-residue samples below the EU limit.

Accurate tree volume estimation is central to forest management and carbon accounting. Allometric equations are widely used but limited in transferability across species, regions, and environmental conditions. Mobile Laser Scanning (MLS) offers a promising alternative through direct measurement of tree geometry; however, the influence of tree shape on MLS accuracy remains poorly understood. This study evaluated MLS-derived estimates of stem diameters, total tree height, and merchantable stem volume against destructive reference measurements from 176 trees spanning eight species (four hardwood, four softwood) in Wallonia, Belgium. A Zeb Horizon RT scanner was used; tree architectural descriptors extracted from the point cloud were tested for associations with measurement error. Across 7,824 stem diameter measurements, MLS achieved a mean error of 0.46 cm, with precision declining above 15 m. MLS-derived height outperformed Vertex IV clinometer measurements for hardwood species (RMSE% = 6.88 vs. 8.78) but performed slightly less well for softwoods (RMSE% = 7.36 vs. 6.14). QSM-based volume estimates systematically underestimated reference values, while taper-based reconstruction produced nearly unbiased estimates with an RMSE of 15.72%. Correlation analyses and PCA showed that tree architectural variables explained only a small fraction of MLS error variability. Diameter and height errors were largely independent of structural attributes, while volume errors showed moderate associations with tree size and crown density. These findings indicate that tree architecture is not a primary source of MLS measurement uncertainty. Future MLS-based forest inventory efforts should prioritize acquisition and processing optimization, as scanning conditions and forest structure appear more influential than tree shape.

The distribution of fat-soluble compounds (including antioxidants) in follicular fluid (FF) remains sparsely documented in relation to in vitro fertilization (IVF) outcomes and existing studies have reported diverging associations. This study aimed to describe plasma and FF concentrations of fat-soluble micronutrients in women undergoing IVF and to analyze their adjusted associations with ovarian function, embryo development and pregnancy outcomes. In 2021-2022, plasma and FF samples were collected from 82 women (first IVF cycle) at oocyte puncture, along with lifestyle data covering the three preceding months. Eleven compounds (two tocopherols, three xanthophylls, five carotenes and retinol) were quantified. All compounds were detected in both compartments (lowest in FF) except phytoene, undetectable in FF. Plasma and FF -tocopherol concentrations were positively associated with plasma estradiol levels before oocyte puncture (both p<0.01) while FF -carotene and lycopene were inversely associated with plasma progesterone concentrations (p=0.01 and 0.02, respectively). Plasma phytofluene and phytoene were positively associated with mature oocyte rate (p=0.03 and p=0.01, respectively), while FF retinol was negatively associated (p=0.03). Carotenes, tocopherols and retinol were inversely associated with later IVF outcomes: fertilization rate (p<0.001 for plasma g-tocopherol, 0.02 for FF retinol), top-quality embryo (p=0.02 for plasma phytofluene), biochemical pregnancy at day 7 post-embryo transfer (p=0.05 for plasma -tocopherol, 0.02 for plasma -carotene), clinical pregnancy (p=0.03 for plasma -tocopherol, 0.01 for plasma phytoene) and live birth (p=0.04 for plasma -tocopherol, 0.02 for plasma phytoene). Plasma and FF g-tocopherol were positively associated with embryo fragmentation (both p<0.05). Finally, among xanthophylls, only plasma {beta}-cryptoxanthin was positively associated with plasma progesterone concentrations (p=0.02). Our findings of heterogeneous associations between tocopherols, carotenes, retinol and IVF outcomes across the stages of IVF suggest a beneficial effect limited to early outcomes and support a complex and context-dependent role of these compounds in female reproduction. This manuscript has been submitted to PlosOne on August 19, 2026.

IntroductionPreeclampsia is characterized by a pro-inflammatory, anti-migratory and anti-angiogenic placental phenotype. Impaired spiral artery remodeling stemming from trophoblast dysfunction is a key pathogenic mechanism. Little is known about the processes that govern trophoblast function normally and in preeclampsia. In preeclampsia, placental Let-7b-5p is reduced. The objectives of this study were to determine the normal function of Let-7b-5p in human trophoblast cells, to examine whether the ssRNA sensors, Toll-like receptor (TLR) 7 and/or TLR8 are mediators of trophoblast Let-7b-5p function, and whether disruption of this pathway promotes a preeclampsia-like phenotype in the trophoblast. MethodsThe human first trimester trophoblast cell line, Sw.71, was transfected with a Let-7b- 5p mimic, a Let-7b-5p inhibitor, or scramble control. Cells were treated with or without the TLR7 inhibitor IRS661 or the TLR8 inhibitor CUCPT9a. Trophoblast migration was measured using a two-chamber assay and interactions with human endometrial endothelial cells (HEECs) was measured using a 3D matrigel model. Trophoblast anti-angiogenic sFlt-1 release was measured by ELISA and sFLT1 mRNA measured by RT-qPCR. ResultsTransfection of trophoblast cells with a Let-7b-5p mimic elevated migration through activation of TLR7 and TLR8, while in a TLR7-dependent manner, the Let-7b-5p mimic negatively regulated sFlt-1 production. Furthermore, inhibition of trophoblast Let-7b-5p reduced migration, elevated FLT1 mRNA expression and sFlt-1 release, and reduced trophoblast-endometrial endothelial cell interactions. ConclusionsThis study highlights a role for TLR7/TLR8-activating Let-7b-5p in promoting normal trophoblast function and endothelial interactions and that disruption in this miR-driven signaling pathway may be relevant to processes driving a pre-eclamptic placental phenotype. HighlightsTrophoblast migration is positively driven by Let-7b-5p activating TLR7 and TLR8 Let-7b-5p, via TLR7, negatively regulates trophoblast anti-angiogenic sFlt-1 production. Inhibition of trophoblast Let-7b-5p reduces trophoblast migration and normal interactions with endometrial endothelial cells, while sFlt-1 production is elevated. TLR7/TLR8-activating Let-7b-5p promotes normal trophoblast function and endothelial interactions and disruption in this miR-driven signaling pathway may promote a preeclamptic placental phenotype.

Introduction: Veterinary Professionals are at risk of contracting zoonoses, including potential emerging infections. Personal Protective Equipment (PPE) could reduce infection transmission risk, but use of it in the profession is low. Understanding Veterinary Professionals experiences with PPE could help identify facilitators and barriers, therefore informing a future strategy to improve usage. Methods: Two focus group discussion with veterinary nurses and two with veterinarians took place at a tertiary small animal teaching hospital. With an interpretivist epistemology, transcriptions were inductively coded and analysed using thematic analysis. Results: Veterinary Professionals frequently reported underusing PPE, despite significant zoonotic risk. Friction in interdisciplinary relationships negatively impacted veterinary professionals experiences with PPE: differing views on risk, policy and PPE, contrasting perceptions of each other, and challenges with communication impacted PPE decision-making. Barriers included an absence of initial recognition of risk, a staff culture of prioritising patient health over risk to self, friction in response to others PPE use or lack thereof, an overly complex policy, and a lack of cultural and institutional reaction to occupationally contracted zoonoses. Facilitators included effective inter and intradisciplinary communication and previous personal experience with serious zoonoses. Conclusion: Veterinary Professionals experiences with PPE, are shaped by social and professional factors, such as interdisciplinary friction, perceptions of self and others, of risk and policy. Recommendations are for the findings to be used locally to embed PPE use into everyday practice. At a national level, the United Kingdom Health Security Agency (UKHSA) should use the findings to evaluate current policies regarding PPE and zoonoses in veterinary practice and produce a simplified national guidance, in collaboration with veterinary professionals with lived experience. Further research into General Practice Veterinary Professionals experiences and the interdisciplinary social dynamics is recommended.

Advances in immunophenotyping of tumour-infiltrating lymphocytes (TILs) have improved our understanding of prognostic biomarkers and immune targets in human melanoma. However, whether the tumour-immune landscape in canine oral malignant melanoma (COMM) is concordant with that of human melanoma has not been properly defined. To address this gap, we developed a 16-colour spectral flow cytometry panel to characterise TIL phenotypic and functional profiles in COMM. Validation using mitogen-stimulated peripheral blood mononuclear cells from healthy dogs (n = 5) demonstrated robust identification of major T cell lineages, including regulatory T cells (Tregs) and memory subsets, and reliable evaluation of their activation and exhaustion status. COMM patients (n = 8) displayed a distinct protumour microenvironment, characterised by an increased proportion of Tregs, enrichment of tumour-specific exhausted-like T cells co-expressing programmed cell death protein 1 (PD-1) and tumour necrosis factor receptor 2 (TNFR2), and a reduction in cytotoxic CD8 and natural killer T (NKT) cell populations compared with tissue-resident (n = 4) and circulating (n = 8) lymphocytes. Analysis of additional solid tumours, including a mast cell tumour, nerve sheath tumour and adrenal cortical carcinoma, further supported the capability of the panel to identify similar patterns of immune dysregulation across diverse canine tumour landscapes. Collectively, this work describes the first detailed evaluation of canine TIL immunophenotyping using spectral flow cytometry and provides insights into the immunosuppressive mechanisms shaping the tumour microenvironment in COMM. These findings not only increase our understanding of canine tumour immunology but also identify potential immune targets and support ongoing comparative immuno-oncology efforts.

Robotic herding offers a promising off-animal approach to controlling the spatial distribution of grazing livestock. Nevertheless, the habituation of cattle to herding stimuli remains a critical obstacle to its practical use. This study presents the first field investigation of behavioral responses of grazing cattle to herding by a quadruped robot. We compared the herding efficacy of a drone and a quadruped robot, and tracked daily changes in responsiveness during continuous herding. Trials were conducted in a 1.1-ha pasture with Japanese Black breeding cows from June to October 2023. The quadruped robot elicited stronger avoidance responses with shorter latencies than the drone, and herding was more successful with the robot. During consecutive daily herding with the robot, behavioral responsiveness declined progressively over the initial 5 days, at a markedly slower rate than previously reported for drone herding. Following a 24-day interruption, responsiveness partially increased, but declined rapidly again over the subsequent two days. These results indicate that the quadruped robot constitutes a more persistent aversive stimulus than a drone for grazing cattle, although habituation management strategies (such as diversifying stimuli or combining sensory modalities) will be necessary for sustained control of grazing distribution.