Journal of Dairy Science

To investigate how the disturbances associated with a relocation to a bedded-pack barn, such as a housing system change, a milking system change and a social regrouping, impacts the behavior of lactating dairy cows, 38 cows from a total of 9 tie-stall or free-stall commercial farms were moved to a newly built bedded-pack barn on an enrollment basis, with a social regrouping occurring after 2 weeks. Scan sampling of video data was done to assess behavior expression in the pen, and live data was collected to assess milking reactivity and animal handling procedures. Results indicate that the cows adapted quickly to the relocation to the new housing system as there were no changes in the locations in the pen, the body positions or the behaviors of cows in time between arrival and regrouping. The social regrouping had a bigger impact with a decrease in 16% of the observed time spent lying and an increase of 9.7% of the observed time spent feeding. Cows also adapted quickly to the milking procedures with a rapid decrease in the occurrence of negative social interactions between cows at the parlor, and in needing less human-animal manipulations and less time to be brought to the parlor. The housing system of origin had a slight effect on behaviors with cows from tie-stalls spending 1.7 times more of the observed time lying than free-stall cows, and free-stall cows spending 1.6 times more of the observed time feeding than tie-stall cows. This study provides a better understanding of how dairy cows respond to disturbances and is encouraging for producers that need to make changes to their current housing system as cows were shown to be quickly adaptable to the challenges presented to them. SummaryDairy cows from cubicle systems were shown to adapt quickly after a relocation to a bedded pack barn, the first use of a milking parlor, and a social regrouping. This was supported by limited changes observed in their behaviors after the disturbances, and observed deviations were temporary and short-lived. Animal handling procedures also observed a quick improvement in time with the trips to the milking parlor needing 2x less time and 3.5x fewer physical contacts from handlers after 5 days. These results are encouraging to producers needing to make changes to their barns.

BackgroundFood intake is a complex trait in living organisms, where the genetics of food intake have been widely studied in humans, mice, Drosophila, cattle, pigs, chicken, and fish. In dairy cattle, intake of feed is highly linked to individuals energy balance, health, production, efficiency, and the environmental footprint of the individual to the society. Recent studies have provided solid evidence of the genetic variation of feed intake (FI) in dairy cattle population, but the genetic basis and molecular mechanism of dairy feed intake is still far from clear especially considering the lactation cycles of dairy cattle. This study aims to integrate stage-dependent genome-wide association (GWA) analyses, regional heritability mapping (RHM), and RNA-seq gene expression analyses to identify temporal functional variants associated with cattle dry matter intake (DMI) across multiple stages in lactation cycles. A total of 750,000 daily DMI records from 7,500 lactations of 2,300 cows were available with animals genotype and pedigree information. Total RNA-seq from blood were generated for 121 individuals in this population from 2 lactation stages. Data were split into multiple lactations stages for GWA, RHM, and transcriptomic analyses. ResultsStage-dependent GWAS and RHM identified 21 significant loci associated with DMI across multiple lactation stages. A total of 45 candidate genes were identified from GWA and RHM. Among all the 45 genes, six genes were later found significantly differently expressed between high and low feed intake animal groups using gene expression information from RNA-seq data. These genes show links to sugar and adipose metabolism, milk production, body weight, dopamine-reward pathways and immune functions. ConclusionsOur multi-omics analyses provide molecular evidence that the genetic basis of cattle DMI across lactation is not static. Temporal genomic variants associated with FI were identified with their transcriptomic patterns investigated, decoding the molecular mechanisms underlying DMI. Overall, the associated variants and candidate genes uncovered herein decoded genetic architecture of dairy feed intake on a temporal and multi-omics basis, enhancing the understanding of basic biology of dairy feed intake and informing breeding strategies aimed at improving dairy feed efficiency.

BackgroundHeat stress (HS) poses a major challenge to the dairy industry by reducing milk production, yet its cell type-specific effects in the bovine mammary gland remain incompletely defined. In this study, we recorded production traits and collected mammary biopsies from cows under thermoneutral (TN), HS, and pair-fed (PF) conditions. ResultsClinical measurements confirmed HS-induced physiological alterations. Compared with TN cows, HS cows exhibited reduced dry matter intake (DMI), milk yield, and yields of fat, protein, and lactose, along with increased water intake and milk urea nitrogen. The use of PF controls indicated that decreased DMI accounted for 45% of the milk-yield reduction, whereas direct HS effects accounted for the remaining reduction. We applied single-nucleus RNA-seq (snRNA-seq) on mammary biopsies to generate cell-resolved HS responses. We identified 14 distinct cell clusters, including epithelial, immune, and stromal populations. Under the TN condition, casein genes (e.g., CSN1S1, CSN2) were broadly expressed across luminal cells but were attenuated under HS, whereas luminal alveolar cells showed relative upregulation. Heat shock protein genes were strongly induced by HS, primarily in epithelial clusters. Gene-set enrichment analyses revealed increased ribosomal activities across HS-responsive clusters and enrichment of protein folding and metabolic pathways in luminal alveolar cells, suggesting elevated proteostasis demands under stress. Pseudotime analysis positioned luminal cells along a progenitor-to-secretory trajectory under TN, accompanied by increased casein gene expression, whereas under HS, mature luminal cells shifted toward a homeostasis regulatory state. Cell-cell communication analysis demonstrated HS-induced remodeling of interepithelial signaling, including altered ERBB4-mediated signaling from luminal hormone-sensing to alveolar lineages. Finally, transcription factor activity profiling highlighted cell type-specific HS-activated regulators and their downstream target genes. ConclusionsTogether, this cell type-resolved atlas delineates how HS alters bovine mammary epithelial function, developmental state, and intercellular crosstalk. These findings point to proteostasis pressure, disrupted signaling pathways, and rewired regulatory networks as mechanistic contributors to reduced lactational performance under HS, offering insights for improving heat resilience in dairy cattle.

Most common housing systems for dairy cows restrict their movement, which can influence welfare, gait, and hoof health of dairy cows. Outdoor access has been proposed as a management practice to offset these restrictions, but reported effects on cows locomotion vary and may not always be captured by traditional clinical assessments. In this study, we investigated gait and hoof through clinical (i.e., visual locomotion scoring and hoof lesion assessment) and subclinical (3D motion analysis, kinetic assessment, hoof infrared thermography and measuring claw conformation) methods to assess how limited provision of outdoor access affects non-lame cows housed in movement restricted environment. Thirty-six Holstein tie-stall cows were either given 1day/week (EX1) or 3days/week (EX3) of outdoor access (1h/day) during 5 consecutive weeks. Clinical and subclinical assessments of gait and hoof were performed before (Pre-trial), after 5 weeks of outing (Post-trial) and 8 weeks after outing (Follow-up). The results of this study revealed no clinical effect of outdoor access on cows locomotion score and hoof lesion prevalence. However, for subclinical assessment, both groups showed an increase in stride and stance time at Post-trial, with an increase in pressure applied by cows while standing in EX3 group and a reduction in coronary band temperature in both groups at Post-trial and Follow-up. Contact area and claw conformation changed after provision of outdoor access in both groups. This study illustrates that with the use of subclinical methods; we can reveal effects of outdoor access on gait and hoof health that might not be visible using the traditional methods.

Data from 80,713 first-calving cows (1984 to1989) of the Holstein, Mambi, and Siboney breeds, belonging to seven large dairy enterprises in Cuba and progenies of 1,297 sires, were analyzed. For each cow, the average across all lactations for at least 14 years after first calving was defined as individual productivity (PI), and the corresponding lifetime sum as accumulated productivity (PA); both traits were. Two genetic models were fitted: a classical Animal Model (M1) and a Sire maternal grandsire model (Sire MGS; M2), aimed at partitioning additive genetic variance into paternal and maternal-line components. Heritability estimates under model M1 were moderate (h2 {approx} 0.135 to 0.140), whereas M2 yielded higher values (h2 {approx} 0.158 to 0.170), reflecting increased additive variance due to a better connectedness across herds. Using estimated breeding values (EBV) for PI and PA, a global cow merit index (H1) was defined under M1. Under M2, a parental index (IM2) combining four standardized predictors (paternal and maternal-grandsire EBV for PI and PA) was constructed. Multiple regression of H1 on IM2 showed that the paternal and maternal-grandsire paths accounted for 73% and 27% of the variation, respectively, indicating a non-negligible maternal-line contribution. Model M2 provided the best overall fit according to information criteria and cross validation using two independent subsamples and the full population yielded correlations of 0.870 to 0.881, demonstrating strong predictive ability and stability of IM2 rankings. These results support the Sire MGS model as a structural extension of the Animal Model for breeding programs targeting lifetime productivity in tropical dairy cattle.

Introducing cognitive enrichment from an early age has the potential to enhance an animals capacity to learn both simple and complex tasks, promote neural plasticity, and support cognitive development. This is applicable for young cattle who are at a critical stage in their development and could benefit from the influence cognitive enrichment has on their behavioral expression. This study aims to explore the effects cognitive enrichment has on weaned dairy calves through analyzing behavioral measures of voluntary participation and short-term behavioral reactions to enrichment exposure. Our study involved a total of five pairs of weaned calves (n=8 treatment; n=2 control). The treatment groups were presented with three variations of a puzzle box, each equipped with unique challenges that offer different solutions (push, slide, pull). These boxes were provided to the calves twice daily over the span of nine days in an isolated corridor located behind their pen. We hypothesized that motivated calves would consistently engage with cognitive enrichment voluntarily over time and express directed natural behaviors, reflecting sustained participation across repeated trials. Results demonstrated that calves consistently visited the cognitive enrichment area across trials, with an average latency of 75.7 {+/-} 47.0s from the pen to the enrichment. Secondly, the calves spent a significant proportion of trial time within the enrichment area at 65% (870.1 {+/-} 21s). Lastly, all calves expressed a broad range of behaviors in line with their natural exploration within the enrichment area, while the puzzle box treatment groups expressed higher durations of behavioral expressions when compared to the control (F=11.7, p<0.0001). Combined, these results indicate the calves motivations to voluntarily participate in a cognitive challenge. While these are promising findings for cognitive enrichment and its applicability to dairy calves, further work is needed to understand broader parameters. Specifically, how can social dynamics influence enrichment interaction in groups, how can this type of enrichment be implemented on farms, and what are the long-term effects to providing cognitive enrichment in the early stages of development.

Epigenetics may play a crucial role in livestock adaptation to environmental challenges like heat stress. In recent years, a growing number of studies have investigated the epigenetic mechanisms underlying dairy cow adaptation to heat stress. However, there is still limited knowledge about the effects of heat stress on immune cells and immune-related phenotypes. Herein we aim to identify heat-stress induced DNA methylation variations on blood methylome potentially affecting regulatory regions and associated phenotypes. Blood samples were collected and peripheral blood mononuclear cell (PBMC) isolated from four cows before (D0) and after (D14) a 14-d heat stress challenge (cyclical THI 72-82) and, from four cows kept in thermoneutral conditions (THI 61-64). Heat-stressed cows had ad libitum access to diets supplemented with adequate levels of vitamin D and Ca (12,000 IU/kg of vitamin D and 0.73% Ca, respectively). To eliminate confounding effects due to differences in nutrient intake, cows maintained under thermoneutral conditions were pair-fed (PF) to their heat-stressed counterparts and received adequate concentrations of vitamin D and Ca as well. Reduced representation bisulphite sequencing (RRBS) was used to profile PBMCs methylome. Differential methylation analysis was performed using methylKit and DSS softwares ({Delta}meth [&ge;] 25%, adjusted p-value < 0.01), retaining only commonly detected differentially methylated cytosines (DMCs). A total of 2,908 DMCs were identified when comparing pre- and post-heat stress samples. After excluding 649 DMCs that were also detected under thermoneutral conditions, as these changes were likely associated with feed restriction inherent to the pair-feeding design rather than with heat stress per se, 2,259 heat stress-specific DMCs remained, predominantly hypomethylated. About half of the DMCs are annotated in intronic and intergenic regions; known to harbor regulatory elements. By intersecting the DMRs with publicly available functional annotation data, we observed hypomethylation on regulatory regions putatively affecting cows immune system. As an example, we identified a loss of methylation within an enhancer region of the MSN gene, which is involved in lymphocyte homeostasis, and a loss of methylation in the promoter region of MECP2, a well-established epigenetic regulator with a central role in chromatin organization and gene expression. These findings highlight the impact of heat stress on dairy cow immunity and provide new insights into its epigenetic regulation under environmental stress. Interpretative summaryThis study examined DNA methylation changes induced by heat stress in dairy cows to elucidate epigenetic mechanisms of thermal adaptation. Using RRBS on PBMCs, 2,259 heat stress-specific differentially methylated cytosines were identified, predominantly hypomethylated and enriched in regulatory regions. Functional annotation highlighted immune-related pathways, including hypomethylated regulatory regions near genes (e.g., MSN, ZBTB33, SLC25A5, GNAS, FAM3A, and MECP2) associated with immune function. These findings indicate that heat stress induces targeted epigenetic modifications potentially affecting immune regulation in dairy cows.

BackgroundThis study investigated rumen microbiome reconstitution and methane (CH4) emissions following a complete exchange of rumen contents between low- and high-CH4-yielding Norwegian Red dairy cows. Twenty cows were screened for CH4 yield, and two low and two high emitters were selected for rumen cannulation and content swap. Total rumen contents were swapped after complete evacuation and washing of both the rumen and omasum. Rumen samples were collected twice in weeks -1, 1, 3, and 7 for fermentation analysis, metagenomics, and metaproteomics, and at week 8 CH4 production was measured. ResultsPrior to the swap, low and high emitters produced 21.2 {+/-} 0.7 and 26.3 {+/-} 1.4 g CH4/kg dry-matter intake (DMI), respectively. Eight weeks after swap, CH4 yields were 12.7 {+/-} 0.3 and 28.9 {+/-} 0.3 g CH4/kg DMI, respectively, showing that the CH4 phenotype of each cow was maintained. Analysis of metagenome-derived 16S rRNA gene sequences showed that low emitters gradually re-established their original microbial community, whereas high emitters retained donor-like microbiota. Metaproteomic mapping suggested higher expression of Prevotella-associated succinate-propionate pathway enzymes in low emitters at week 7, though these differences were modest. ConclusionThese findings suggest that host factors influence CH4 output and microbial reconstitution, with low emitters restoring their native microbiome while high emitters retained a donor-associated community yet continued to emit high CH4. Results should be interpreted with caution given the small sample size (n = 2 per phenotype) and require confirmation in larger studies. ImportanceReducing enteric methane from cattle requires understanding whether the rumen microbiome or the host animal is the primary driver of methane output. We exchanged the entire rumen contents between low- and high-methane-yielding dairy cows and measured methane production alongside metagenomic and metaproteomic profiling over two months. Despite receiving each others microbiomes, each cows methane phenotype persisted--low emitters stayed low and high emitters stayed high. Microbiome reconstitution was asymmetric: low emitters restored their original microbial community, while high emitters retained the donor microbiota. Methanogen communities did not differ between phenotypes, pointing to host-level rather than microbial-level control of methane yield. These pilot findings suggest that breeding for favorable host traits may be essential for lasting methane reduction, and that microbiome transfer alone is unlikely to shift an animals methane phenotype. Larger studies are needed to confirm these observations.

Restlessness is a symptom of chronic boredom in humans and a behavioural phenomenon anecdotally described as a concern in bulls raised for fattening purposes, but it has so far not been addressed in research. The two studies presented in this paper aimed to gain first insights into restlessness in bulls. We operationally defined restlessness by the number of transitions between behaviours in a given time period, and quantified restlessness in bulls of different weight classes (300, 400, 500 kg) on farms keeping bulls on fully-slatted floors (n=8, Study 1) as well as across three different husbandry systems (fully-slatted floor (FS, n=4), straw-based (SB, n=4) and organic pasture (OP, n=3), Study 2). All farms were visited twice, and the behaviour of different individuals was continuously recorded for 15 minutes each between 9 a.m. and 5 p.m. (Study 1) and for 8 minutes each between 6 a.m. and 10 p.m. (Study 2). The effects of weight class and husbandry system were analysed using generalised linear mixed-effects models, and we ran a sequence analysis to cluster observations by the sequence, frequency, and duration of bulls behaviours in Study 1. Bulls kept in fully-slatted floor systems in Study 1 changed their behaviour on average 48.3 times per 10 minutes, with high variability both within and across farms. Weight class did not have a statistically supported effect on the number of transitions, and the sequence analysis revealed four clusters that differed in sequence and in the number of transitions. In Study 2, OP bulls showed fewer transitions than SB and FS bulls (X22 = 23.6, p < 0.001), while SB and FS bulls did not differ. While SB pens were more structured and offered more space per animal, both SB and FS systems can be characterised by monotony, which may explain the similar level of restlessness in both systems. Alternatively, or in addition, the high feeding intensity in SB and FS systems may have caused the higher number of transitions compared to the OP system, potentially elicited by subacute ruminal acidosis and/or laminitis and the resulting pain. However, these explanations are speculative and require systematic disentanglement in future studies. This study provides initial insights into restlessness in bulls and lays the groundwork for future research to identify the causes underlying restlessness and investigate its association with bull welfare.

Effective management of Bovine Respiratory Disease Complex (BRDC) requires timely, non-invasive diagnostic tools to protect calf health and welfare. Among early clinical signs, coughing stands out as both frequent and informative. To explore its potential for early BRDC detection, we deployed an artificial intelligence (AI)-driven acoustic monitoring system that recorded over 2,730 hours of audio during a 30-day period. Four experimental pens, each housing seven calves and stratified by infection status and antibiotic treatment, were equipped with a dedicated microphone to enable targeted acoustic surveillance. This configuration enabled pen-specific detection of cough events, which were subsequently classified using an AI HuBERT-based model trained on 1,045 labelled clips. The classifier achieved 92% accuracy. Temporal patterns in cough frequency aligned with infection dynamics, treatment responses, and circadian patterns. Notably, AI-detected coughs consistently preceded clinical scores by 1-2 days, confirming the systems sensitivity to early respiratory disorders. These findings support the use of acoustic surveillance as a valid, scalable, and autonomous tool for continuous monitoring and early warning of respiratory diseases in calves. ImplicationsThis study demonstrates that AI-powered acoustic monitoring enables real-time, non-invasive detection of coughs in calves for early warning of respiratory diseases, outperforming traditional veterinarian clinical scoring by 1-2 days. Its high accuracy and sensitivity to respiratory infection dynamics and treatment effects position it as a scalable tool for precision livestock farming.

Proteomic profiling provides a framework for describing breed-specific growth, physiological and adaptive mechanisms in livestock. This study compared the plasma proteomic profiles of Red Sokoto (RS) and West African Dwarf (WAD) goats, with emphasis on growth-related proteins. Plasma samples from 20 goats (RS = 10; WAD = 10) collected across three locations in southwestern Nigeria were analysed using liquid chromatography-mass spectrometry (LC-MS). A total of 66 plasma proteins were identified in RS goats and 59 in WAD goats, of which 14 were associated with growth regulation. Distinct breed-specific expression patterns were evident. RS goats exhibited higher abundance of fibronectin and calmodulin, indicating enhanced tissue remodelling, muscle development, and calcium-mediated signaling. In contrast, WAD goats showed relatively higher expression of key metabolic and endocrine regulators, including insulin, leptin, ghrelin, glucagon, adiponectin, epidermal growth factor, erythropoietin, and thrombopoietin, reflecting greater metabolic efficiency and adaptive resilience. Gene ontology enrichment analysis revealed marked functional divergence between breeds: RS goats demonstrated stronger enrichment of GO terms related to signal transduction efficiency, cell-matrix adhesion, calcium ion binding, and growth-related morphogenetic processes, whereas WAD goats showed enrichment of GO categories associated with energy metabolism, stress adaptation, catabolic regulation, and hematopoietic support. These findings indicate that breed differences in growth potential are driven more by pathway efficiency and functional integration than hormone abundance. Plasma proteomic and GO-based functional profiles highlight coordinated anabolic and structural growth regulation in RS goats and a resilience-oriented metabolic strategy in WAD goats, with important implications for breed-specific selection, conservation, and sustainable goat production systems.

Linkage disequilibrium (LD) and haplotype block structure govern the resolution and utility of genomic selection, marker-assisted selection, and genome-wide association studies (GWAS) in livestock. We performed a comprehensive genome-wide characterization of LD decay, haplotype block architecture, and population diversity across all 24 autosomes in Nili-Ravi buffalo (Bubalus bubalis; n = 85), using 43,543 post-quality-control SNPs. Mean genome-wide r2 was 0.124 (median 0.074) and mean D was 0.540 (median 0.481), with LD half-decay at {approx}70 kb. A total of 133 haplotype blocks encompassing 721 SNPs were identified (Gabriel et al., 2002). Haploview analysis of nine chromosomes harbouring bTB resistance candidate genes revealed contrasting selection signatures: directional selection at innate immune loci (IFNG, TLR1; H < 0.55) versus balancing selection at adaptive immune loci (BoLA-DRB3, SP110; H > 1.0). Critically, BBU15 Block 3 (28.6 kb; OR52E5/NCR1 locus, 47.16 Mb) showed a genome-wide significant integrated haplotype score (iHS; -log1 0 p = 5.408), directly co-localising with the published bTB susceptibility QTL (Bermingham et al., 2014). The TAA haplotype (frequency 53.3%) at this block represents a candidate resistance-associated haplotype for marker-assisted selection. These findings provide essential parameters for SNP panel design and bTB resistance breeding in South Asian buffalo.

BackgroundBlack soldier fly larvae (Hermetia illucens, BSFL) efficiently bio-convert organic waste into high-value protein, which has significant potential in domesticated animal feed formulations. BSFL growth and bioconversion potential can be enhanced through selective breeding, which requires accurate estimates of genetic parameters and knowledge of genotype-by-diet (G x D) interactions. However, comprehensive knowledge of G x D interactions is limited, and reports of genetic parameters are sparse across genetic strains and production environments globally. ResultsThis study estimated heritabilities, dominance effects and genetic correlations for BSFL growth traits and quantified G x D interactions. Phenotypes of 2,097 fifth-instar larvae reared on three diets were recorded, including larval body weight (LBW), length (LL), width (LW), and surface area (LSA). All larvae were genotyped using a custom 6K Allegro SNP panel. Genetic parameters and G x D interactions were estimated by fitting an additive-dominance model in ASReml-R. Heritabilities for growth traits were low across diets (0.05-0.14), with diet-specific estimates ranging from low to moderate (0.06-0.36). Dominance effects were significant across the traits (0.09-0.19), and genetic correlations were high among growth traits (>0.81), except between LW and LL (0.51). G x D interactions were moderate across diets (-0.04-0.49). ConclusionResults suggest that moderate to high genetic gain is achievable over a long-term breeding programme, given the genetic basis of growth traits and BSFs short generation interval (38-45 days). However, G x D interactions must be considered, either through combined or diet-specific selection strategies, and the significant dominance effects suggest heterosis could accelerate improvement.

Managing post-weaning diarrhoea (PWD) in piglets is difficult due to limits on antibiotics and zinc. Chitosan is emerging as a potential feed additive. We analysed a chito-oligosaccharide hydrochloride (COS-HCl), a low molecular weight (LMW) chitosan, and a medium molecular weight (MMW) chitosan, and assessed their effects on growth, faecal consistency, microbiota, and potential interference with enterotoxigenic Escherichia coli (ETEC). The three chitosans were characterised using {superscript 1}H-NMR, SEC-RI-MS, and SEC-RI-MALLS. COS-HCl had an Mw of 0.824 kDa; LMW and MMW showed Mw ranges of 14.4 kDa (0.3-30 kDa) and 116 kDa (15-600 kDa). Degrees of acetylation were 9.5%, 6.5%, and 15%. Two 42-day field studies evaluated average daily gain (ADG), faecal consistency, and microbiota. In the first trial, COS-HCl at 0.025-0.1% did not significantly affect ADG (-33 to - 12 g/d). In the second, LMW and MMW at 0.01% did not significantly change ADG (-7 and +3 g/d). Faecal consistency, ETEC shedding, and microbiota composition were similar to controls. An enzymatic HPLC-MS method enabled quantification of MMW chitosan in premix. Our results highlight the importance of advanced chitosan characterisation for precision nutrition and suggest that a threshold dosemay be needed to benefit growth and gut health in PWD management. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/714014v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@19c9e23org.highwire.dtl.DTLVardef@152461aorg.highwire.dtl.DTLVardef@7886e0org.highwire.dtl.DTLVardef@df0d9b_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundRecombination of parental haplotypes is a fundamental biological process that ensures proper segregation of homologous chromosomes and creates new combinations of alleles during meiosis. Crossover events are typically detected from large-scale pedigree-based genetic studies or linkage disequilibrium-based recombination maps, although these are generally limited to SNPs. Increasing amounts of long read sequencing and haplotype-resolved assemblies offer an alternative approach to examining recombination events at basepair resolution, albeit with much smaller sample sizes. ResultsHere, we analyse five high-quality genome assemblies from the Simmental cattle breed, including a newly assembled triobinned HiFi assembly of an Eringer x Simmental cross (N50 of 77 Mb and a k-mer quality value of 55.3). We integrate the five assemblies, of which two originate from maternal half-siblings, into a reference-free Simmental-specific pangenome. By considering path similarities in the pangenome, we were able to identify putative crossover events in the haplotypes of the half-siblings, as well as a greater number of events relative to the cousin due to an additional degree of generational separation. We validated the pangenome approach with phased SNPs called from linear alignments of maternal short read sequencing, with 23 of 30 chromosomes having the same recombination predictions. We identified 5 and 16.7 Mb of non-reference insertion sequences respectively shared or private to the half-siblings, enabling testing for recombination events beyond only SNP markers. We also identified four differentially methylated CpG clusters from the 5mC signal of HiFi reads which allowed us to narrow the window containing the putative recombination event from 35 to 20 Mb within the longest run of homozygosity. ConclusionStructural variants and methylation information identified from long read sequencing and genome assemblies may help identify recombination events in regions beyond those typically called from SNPs. Furthermore, while existing long read-based methylation calls can be noisy and report unrealistic intermediate methylation levels, 5mC methylation appears to be a promising avenue for distinguishing haplotypes in the absence of genomic variation.

Enteric methane emissions from ruminant livestock contribute to global warming, creating an urgent need for effective mitigation strategies that do not compromise animal productivity and welfare. Methanogenic archaea within the rumen microbiome drive enteric methane emissions. However, large-scale rumen-fluid sampling in commercial production systems is impractical, due to its invasive nature and the associated logistical challenges. This study hypothesised that rumination enables the capture of rumen microbial signals within the oral cavity and using oral microbiome profiles to provide a practical, non-invasive alternative method for proxy methane phenotyping in commercial production systems. To test the hypothesis, we estimated the oral microbiability, defined as the proportion of phenotypic variance in methane emissions explained by oral microbiome variation. Samples were collected from 209 animals across two trials in Queensland, Australia. Oral microbiome samples were obtained from all animals, with paired rumen samples in one trial, and methane emissions were measured using either the sulphur hexafluoride (SF6) tracer technique or the GreenFeed system. Microbial features were characterised using taxonomic and functional annotations, and microbiability was estimated using mixed linear models incorporating microbiome-based relationship matrices. Although the small sample size limited strong conclusions, the oral microbiability estimates reported in this study were comparable to those derived from rumen samples. Functional microbial profiles generally explained a greater proportion of methane variation than taxonomic profiles, suggesting that microbial function is more closely linked to methane production than community composition alone. However, these differences were not statistically significant due to large standard errors. These findings suggest that oral microbiome sampling potentially provides a practical, minimally invasive, scalable proxy method for methane emissions of individual cattle in grazing systems, where direct methane gas measurements are labour-intensive and difficult to implement. Integrating oral microbiome profiles in the existing breeding model with the host genetics, weight and environmental factors could provide a promising pathway for enabling selection for low emissions and advancing reduced emissions livestock farming under real-world production conditions. Lay summaryCattle produce methane as part of their normal digestion and this contributes to climate change. Reducing methane emission in grazing livestock systems is therefore important. However, measuring methane from individual grazing animals is difficult, costly, and often impractical under commercial conditions. The rumen microbiome has been used as a proxy for estimating methane emissions, but collecting rumen samples is invasive and impractical for large-scale use. Because rumination transfers material from the rumen to the mouth, we investigated whether microbes found in cattle mouths could also be used to estimate how much methane an individual animal produced. We suggest that mouth-swab sampling method can be an alternative to rumen fluid sampling because it was less invasive, relatively quick and practically applicable in commercial conditions. Importantly, the microbiome explained a meaningful proportion of the between-animal variation for methane emission. This suggests that collection of mouth swabs is a potentially scalable alternative proxy method to identify cattle that naturally produce less methane. Overall, our findings support the potential use of oral ruminant microbial information to improve breeding and management strategies aimed at reducing methane emissions while maintaining productive livestock systems. Teaser TextThis study demonstrates that collecting oral swabs from the mouths of grazing beef cattle could provide a scalable method to estimate individual methane emissions in commercial production systems, offering a practical alternative to invasive rumen sampling and complex gas measurement systems. These findings support the development of scalable breeding and management strategies for methane mitigation in large-scale livestock production systems.

Transcriptomic analyses are widely used to elucidate the molecular mechanisms driving gametogenesis and reproduction in fish, yet their accuracy depends heavily on appropriate normalization of gene expression data. Conventional approaches that rely on single or multiple reference genes are problematic during teleost oogenesis, as profound structural and physiological remodeling of the ovary challenges the assumption that commonly used reference transcripts remain stable. In this study, we assessed by qPCR the transcriptional variability of four widely used reference genes (actb, ef-1, gapdh, and 18S rRNA) throughout the oogenic cycle of the thicklip grey mullet (Chelon labrosus), using geNorm and NormFinder analyses, and we additionally evaluated total cDNA concentration as an alternative normalization factor. To examine the performance and interpretive consequences of each normalization strategy, we compared expression patterns of key steroidogenic genes (star, cyp19a1a, and cyp11b) normalized by individual reference genes, combinations of reference genes, or total cDNA concentration. All evaluated reference genes displayed notable transcriptional variability across oogenesis, confirming their limited suitability as sole internal controls. In contrast, normalization approaches integrating multiple reference genes and/or total cDNA concentration consistently provided greater stability and more reliable biological interpretation. These results support a refined and more robust normalization framework for transcriptional analyses in fish ovaries, particularly during stages of extensive tissue remodeling. Our findings demonstrate cDNA-based normalization is straightforward, rapid, and easy to implement across laboratories, providing a practical alternative for achieving accurate, reproducible transcript quantification in fish ovary studies.

Structural variants (SVs) are most effectively identified using long-read (LR) sequencing. However, long-read (LR) data remain limited, and sequenced samples often lack associated phenotypic information. To overcome this limitation, we combined pangenome-based (variation graph) and imputation approaches to enable large-scale SV association studies in the three main French dairy cattle breeds. A variation graph was constructed using 69,892 deletions, 89,900 insertions, and 17,402 duplications detected in 176 LR samples. We subsequently genotyped 939 samples for each SV in the panel by realigning their short read (SR) sequences to the graph. Validation analyses showed high genotype concordance rates for deletions (0.79) and insertions (0.79); however, the rates for duplications were low (0.14), leading to their exclusion from this study. Retained SVs were combined with single nucleotide variants (SNVs) and served as sequence-level imputation reference panel. From the SNP genotyping array data, we imputed SVs and SNVs for 11,902 Holstein, 3,753 Montbeliarde, and 3,053 Normande bulls. After quality control, more than 14 million SNVs and 40 thousand SVs were retained for within-breed genome-wide association analyses (GWAS) with daughter yield deviations for 13 traits related to milk production, udder health, fertility, and stature. The results of the GWAS demonstrated genetic architectures aligning with earlier discoveries and uncovered thirty-six unique significant associations between structural variants and traits. Conditional analysis revealed that ten of these SVs were the primary variants in the quantitative trait loci related to fat content, protein content, and stature.

BackgroundGene expression levels are affected by genetics and environmental effects. However, quantification of the influence of genetics and environmental effects on gene expression remains limited, especially in farm animals. Here, the relative influence of genetic and heat-related environmental variations on gene expression levels was investigated in pigs, using a backcross herd of diverse heat adaptation levels. Backcross animals were raised in either a tropical or temperate environment. Animals raised in temperate environment were subjected to an experimental heat stress at the end of their growth. ResultsWe identified 1,967 differentially expressed genes (DEGs) between pigs raised in the tropical (n = 181) and temperate (n = 180) facilities, and 472 DEGs throughout a 3 weeks experimental heat stress. Transcriptome-wide association (TWAS) study identified 139 associations between gene expression levels and thermoregulation/production traits. We detected 6,014 expression quantitative trait loci (eQTLs) associated with the expression level of 3,297 genes. Genetic variance was estimated to explain 36.3% of gene expression variance on average, and was the main source of variance for 27.7% of transcripts. Most eQTLs found are located in proximal regions (cis-eQTLs) and few within distal regions (trans-eQTLs) to their assigned genes. A trans-eQTL hotspot highlighted a hematopoietic mechanism driven by GPATCH8. An integration of GWAS and TWAS pointed to TMCO1 and ZNF184 as candidate genes for backfat thickness. ConclusionsThis study provides a better understanding of the impact of climate, heat stress and genetic influences on the pig whole blood transcriptome.

BackgroundGenomic prediction in livestock is predominantly based on additive models, even though dominance and other non-additive effects can contribute appreciably to phenotypic variance for fitness and fertility traits. Bayesian mixture models, such as Bayes R, have proven effective for modelling sparse, heterogeneous additive SNP effects, but most implementations do not explicitly accommodate dominance. In this study, we extended BayesR3 to jointly model additive and dominance marker effects within a unified Bayesian mixture framework, denoted BayesR3AD, and used this method to estimate additive and dominance effects for fertility and cow survival (longevity) in Holstein cattle. ResultsUsing real Holstein genotypes (227,942 animals, 74,626 SNPs), we simulated phenotypes with additive and dominance effects. When dominance was present in the simulated data, BayesR3AD improved prediction accuracy of genetic values by +0.1011 (0.6144 vs 0.5133; {approx}19.7% relative) compared with the additive-only BayesR3 model and recovered additive and dominance variance components without bias. Under purely additive simulations, dominance mixture components were effectively empty, confirming that the extended model shrinks unnecessary dominance effects toward zero. In real fertility data, including calving interval (63,378 records) and survival (68,514 records), BayesR3AD estimated small dominance variance ({approx}1-3% of total genetic variance). The model highlighted a very large additive loci at 57.82 Mb on BTA18 for both calving interval and survival. concordant with previous GWAS studies of Holstein fertility. Additionally, a large dominance effect was found at 44.37 Mb on BTA18 for calving interval implicating a heterozygote advantage that increases fertility. ConclusionsBayesR3AD provides a practical extension of BayesR3 that captures both additive and dominance contributions to genomic prediction. The method is robust, reverting effectively to the additive model when dominance is absent, while delivering accurate variance decomposition, and potential gains in prediction accuracy when dominance is present. Application to Holstein fertility traits demonstrates that dominance can be detected and quantified without compromising additive inference, supporting improved prediction of total genetic merit. While validated in cattle, BayesR3AD can be directly applied to other species to better model and predict traits related to fitness.