Harnessing Vaginal Probiotics for Enhanced Management of Uterine Disease and Reproductive Performance in Dairy Cows: A Conceptual Review.

Uterine disease in cattle impairs reproductive performance and profitability and increases antibiotic use and antimicrobial resistance. Thus, probiotics offer a promising alternative therapy. This review presents conceptual findings on the efficacy of probiotics in managing uterine diseases and fertility in cows. Probiotics containing Lactobacillus spp. and Bifidobacterium spp. individually or as composite formulations are known to improve fertility. Strategic intravaginal administration of these formulations would likely enhance uterine immunity, particularly during the postpartum period. While current findings on the benefits to uterine health are encouraging, there is still significant knowledge missing, including a lack of empirical information from large-scale field trials. This review underscores the need for evidence-based guidelines for probiotics, such as genomic selection of formulations, targeted delivery, or potential synergy with other interventions. Future research should address these gaps to maximize the potential of probiotics in managing uterine diseases and enhancing the reproductive health of dairy cattle.

Metabolomic and gut microbiome profiles across the spectrum of community-based COVID and non-COVID disease.

Whilst most individuals with SARS-CoV-2 infection have relatively mild disease, managed in the community, it was noted early in the pandemic that individuals with cardiovascular risk factors were more likely to experience severe acute disease, requiring hospitalisation. As the pandemic has progressed, increasing concern has also developed over long symptom duration in many individuals after SARS-CoV-2 infection, including among the majority who are managed acutely in the community. Risk factors for long symptom duration, including biological variables, are still poorly defined. Here, we examine post-illness metabolomic profiles, using nuclear magnetic resonance (Nightingale Health Oyj), and gut-microbiome profiles, using shotgun metagenomic sequencing (Illumina Inc), in 2561 community-dwelling participants with SARS-CoV-2. Illness duration ranged from asymptomatic (n = 307) to Post-COVID Syndrome (n = 180), and included participants with prolonged non-COVID-19 illnesses (n = 287). We also assess a pre-established metabolomic biomarker score, previously associated with hospitalisation for both acute pneumonia and severe acute COVID-19 illness, for its association with illness duration. We found an atherogenic-dyslipidaemic metabolic profile, including biomarkers such as fatty acids and cholesterol, was associated with longer duration of illness, both in individuals with and without SARS-CoV-2 infection. Greater values of a pre-existing metabolomic biomarker score also associated with longer duration of illness, regardless of SARS-CoV-2 infection. We found no association between illness duration and gut microbiome profiles in convalescence. This highlights the potential role of cardiometabolic dysfunction in relation to the experience of long duration symptoms after symptoms of acute infection, both COVID-19 as well as other illnesses.

Profiling post-COVID-19 condition across different variants of SARS-CoV-2: a prospective longitudinal study in unvaccinated wild-type, unvaccinated alpha-variant, and vaccinated delta-variant populations.

BACKGROUND: Self-reported symptom studies rapidly increased understanding of SARS-CoV-2 during the COVID-19 pandemic and enabled monitoring of long-term effects of COVID-19 outside hospital settings. Post-COVID-19 condition presents as heterogeneous profiles, which need characterisation to enable personalised patient care. We aimed to describe post-COVID-19 condition profiles by viral variant and vaccination status. METHODS: In this prospective longitudinal cohort study, we analysed data from UK-based adults (aged 18-100 years) who regularly provided health reports via the Covid Symptom Study smartphone app between March 24, 2020, and Dec 8, 2021. We included participants who reported feeling physically normal for at least 30 days before testing positive for SARS-CoV-2 who subsequently developed long COVID (ie, symptoms lasting longer than 28 days from the date of the initial positive test). We separately defined post-COVID-19 condition as symptoms that persisted for at least 84 days after the initial positive test. We did unsupervised clustering analysis of time-series data to identify distinct symptom profiles for vaccinated and unvaccinated people with post-COVID-19 condition after infection with the wild-type, alpha (B.1.1.7), or delta (B.1.617.2 and AY.x) variants of SARS-CoV-2. Clusters were then characterised on the basis of symptom prevalence, duration, demography, and previous comorbidities. We also used an additional testing sample with additional data from the Covid Symptom Study Biobank (collected between October, 2020, and April, 2021) to investigate the effects of the identified symptom clusters of post-COVID-19 condition on the lives of affected people. FINDINGS: We included 9804 people from the COVID Symptom Study with long COVID, 1513 (15%) of whom developed post-COVID-19 condition. Sample sizes were sufficient only for analyses of the unvaccinated wild-type, unvaccinated alpha variant, and vaccinated delta variant groups. We identified distinct profiles of symptoms for post-COVID-19 condition within and across variants: four endotypes were identified for infections due to the wild-type variant (in unvaccinated people), seven for the alpha variant (in unvaccinated people), and five for the delta variant (in vaccinated people). Across all variants, we identified a cardiorespiratory cluster of symptoms, a central neurological cluster, and a multi-organ systemic inflammatory cluster. These three main clusers were confirmed in a testing sample. Gastrointestinal symptoms clustered in no more than two specific phenotypes per viral variant. INTERPRETATION: Our unsupervised analysis identified different profiles of post-COVID-19 condition, characterised by differing symptom combinations, durations, and functional outcomes. Our classification could be useful for understanding the distinct mechanisms of post-COVID-19 condition, as well as for identification of subgroups of individuals who might be at risk of prolonged debilitation. FUNDING: UK Government Department of Health and Social Care, Chronic Disease Research Foundation, The Wellcome Trust, UK Engineering and Physical Sciences Research Council, UK Research and Innovation London Medical Imaging & Artificial Intelligence Centre for Value-Based Healthcare, UK National Institute for Health Research, UK Medical Research Council, British Heart Foundation, UK Alzheimer's Society, and ZOE.

Interrogating the Diversity of Vaginal, Endometrial, and Fecal Microbiomes in Healthy and Metritis Dairy Cattle.

The bovine genital tract harbors a dynamic microbiome. Genital tract microbial communities in healthy animals have been characterized using next-generation sequencing methods showing that microbe compositions differ between the vagina and uterus, more so during the postpartum period. Pre-calving fecal and vaginal, and endometrial swabs at the different postpartum intervals were collected from dairy cows. Microbiomes in these samples were determined based on bacterial 16S amplicon sequencing and compared between healthy (H; n = 10) control animals and cows that developed metritis (M; n = 10) within 21 days postpartum (DPP). Compared to healthy animals the pre-calving fecal and vaginal microbiomes of metritis animals were more abundant in sequences from the phylum Fusobacteria and the bacterial genera such as Escherichia-Shigella and Histophilus. In addition, compared to healthy animals, metritis cows harboured low microbial species diversity in the endometrium, as well as decreasing Proteobacteria and increasing Fusobacteria, Firmicutes, Actinobacteria, and Bacteroidetes abundances. The greatest taxonomic compositional deviations in endometrial microbial communities between the metritis and health cows were detected between 7 and 10 DPP. There was high taxonomic similarity detected between postpartum endometrial microbiomes and the prepartum vaginal and fecal microbiomes suggesting that colonization through bacteria ascending from the rectum and vagina to the uterine cavity might play a major role in establishing the endometrial microbiome postpartum. A deeper understanding of the establishment and dynamics of postpartum endometrial microbial communities in cows will thus provide crucial basic knowledge to guide the development of genital microbiome manipulation strategies for preventing uterine disease and improving fertility in dairy cows.

The effects of sustained fitness improvement on the gut microbiome: A longitudinal, repeated measures case-study approach.

The athlete gut microbiome differs from that of non-athletes in its composition and metabolic function. Short-term fitness improvement in sedentary adults does not replicate the microbiome characteristics of athletes. The objective of this study was to investigate whether sustained fitness improvement leads to pronounced alterations in the gut microbiome. This was achieved using a repeated-measures, case-study approach that examined the gut microbiome of two initially unfit volunteers undertaking progressive exercise training over a 6-month period. Samples were collected every two weeks, and microbiome, metabolome, diet, body composition, and cardiorespiratory fitness data were recorded. Training culminated in both participants completing their respective goals (a marathon or Olympic-distance triathlon) with improved body composition and fitness parameters. Increases in gut microbiota α-diversity occurred with sustained training and fluctuations occurred in response to training events (eg, injury, illness, and training peaks). Participants' BMI reduced during the study and was significantly associated with increased urinary measurements of N-methyl nicotinate and hippurate, and decreased phenylacetylglutamine. These results suggest that sustained fitness improvements support alterations to gut microbiota and physiologically-relevant metabolites. This study provides longitudinal analysis of the gut microbiome response to real-world events during progressive fitness training, including intercurrent illness and injury.

Four men in a boat: Ultra-endurance exercise alters the gut microbiome.

OBJECTIVES: Compositional and functional adaptions occur in the gut microbiome in response to habitual physical activity. The response of the gut microbiome to sustained, intense exercise in previously active individuals, however, is unknown. This study aimed to prospectively explore the gut microbiome response of four well-trained male athletes to prolonged, high intensity trans-oceanic rowing, describing changes in microbial diversity, abundance and metabolic capacity. DESIGN: A prospective, repeated-measures, within-subject report. METHODS: Serial stool samples were obtained from four male athletes for metagenomic whole-genome shotgun sequencing to record microbial community structure and relevant functional gene profiles before, during and after a continuous, unsupported 33-day, 5000 km transoceanic rowing race. Calorific intake and macronutrient composition were recorded by validated food frequency questionnaire and anthropometry was determined by body composition analysis and cardiorespiratory testing. RESULTS: Microbial diversity increased throughout the ultra-endurance event. Variations in taxonomic composition included increased abundance of butyrate producing species and species associated with improved metabolic health, including improved insulin sensitivity. The functional potential of bacterial species involved in specific amino and fatty acid biosynthesis also increased. Many of the adaptions in microbial community structure and metaproteomics persisted at three months follow up. CONCLUSIONS: These findings demonstrate that prolonged, intense exercise positively influences gut microbial diversity, increases the relative abundance of some bacterial species and up-regulates the metabolic potential of specific pathways expressing microbial gene products. These adaptions may play a compensatory role in controlling the physiological stress associated with sustained exertion as well as negating the deleterious consequences accompanying endurance exercise.

Moderate-intensity aerobic and resistance exercise is safe and favorably influences body composition in patients with quiescent Inflammatory Bowel Disease: a randomized controlled cross-over trial.

BACKGROUND: Overweight and metabolic problems now add to the burden of illness in patients with Inflammatory Bowel Disease. We aimed to determine if a program of aerobic and resistance exercise could safely achieve body composition changes in patients with Inflammatory Bowel Disease. METHODS: A randomized, cross-over trial of eight weeks combined aerobic and resistance training on body composition assessed by Dual Energy X-ray Absorptiometry was performed. Patients in clinical remission and physically inactive with a mean age of 25 ± 6.5 years and Body Mass Index of 28.9 ± 3.8 were recruited from a dedicated Inflammatory Bowel Disease clinic. Serum cytokines were quantified, and microbiota assessed using metagenomic sequencing. RESULTS: Improved physical fitness was demonstrated in the exercise group by increases in median estimated VO2max (Baseline: 43.41mls/kg/min; post-intervention: 46.01mls/kg/min; p = 0.03). Improvement in body composition was achieved by the intervention group (n = 13) with a median decrease of 2.1% body fat compared with a non-exercising group (n = 7) (0.1% increase; p = 0.022). Lean tissue mass increased by a median of 1.59 kg and fat mass decreased by a median of 1.52 kg in the exercising group. No patients experienced a deterioration in disease activity scores during the exercise intervention. No clinically significant alterations in the α- and ß-diversity of gut microbiota and associated metabolic pathways were evident. CONCLUSIONS: Moderate-intensity combined aerobic and resistance training is safe in physically unfit patients with quiescent Inflammatory Bowel Disease and can quickly achieve favourable body compositional changes without adverse effects. TRIAL REGISTRATION: The study was registered at ClinicalTrials.gov; Trial number: NCT02463916 .

A dual targeted ß-defensin and exome sequencing approach to identify, validate and functionally characterise genes associated with bull fertility.

Bovine fertility remains a critical issue underpinning the sustainability of the agricultural sector. Phenotypic records collected on >7,000 bulls used in artificial insemination (AI) were used to identify 160 reliable and divergently fertile bulls for a dual strategy of targeted sequencing (TS) of fertility-related ß-defensin genes and whole exome sequencing (WES). A haplotype spanning multiple ß-defensin genes and containing 94 SNPs was significantly associated with fertility and functional analysis confirmed that sperm from bulls possessing the haplotype showed significantly enhanced binding to oviductal epithelium. WES of all exons in the genome in 24 bulls of high and low fertility identified 484 additional SNPs significantly associated with fertility. After validation, the most significantly associated SNP was located in the FOXJ3 gene, a transcription factor which regulates sperm function in mice. This study represents the first comprehensive characterisation of genetic variation in bovine ß-defensin genes and functional analysis supports a role for ß-defensins in regulating bull sperm function. This first application of WES in AI bulls with divergent fertility phenotypes has identified a novel role for the transcription factor FOXJ3 in the regulation of bull fertility. Validated genetic variants associated with bull fertility could prove useful for improving reproductive outcomes in cattle.

The CD4(+) T cell methylome contributes to a distinct CD4(+) T cell transcriptional signature in Mycobacterium bovis-infected cattle.

We hypothesised that epigenetic regulation of CD4(+) T lymphocytes contributes to a shift toward a dysfunctional T cell phenotype which may impact on their ability to clear mycobacterial infection. Combined RNA-seq transcriptomic profiling and Reduced Representation Bisulfite Sequencing identified 193 significantly differentially expressed genes and 760 differentially methylated regions (DMRs), between CD4(+) T cells from M. bovis infected and healthy cattle. 196 DMRs were located within 10 kb of annotated genes, including GATA3 and RORC, both of which encode transcription factors that promote TH2 and TH17 T helper cell subsets respectively. Gene-specific DNA methylation and gene expression levels for the TNFRSF4 and Interferon-γ genes were significantly negatively correlated suggesting a regulatory relationship. Pathway analysis of DMRs identified enrichment of genes involved in the anti-proliferative TGF-ß signaling pathway and TGFB1 expression was significantly increased in peripheral blood leukocytes from TB-infected cattle. This first analysis of the bovine CD4(+) T cell methylome suggests that DNA methylation directly contributes to a distinct gene expression signature in CD4(+) T cells from cattle infected with M. bovis. Specific methylation changes proximal to key inflammatory gene loci may be critical to the emergence of a non-protective CD4(+) T cell response during mycobacterial infection in cattle.

Integrated analysis of the local and systemic changes preceding the development of post-partum cytological endometritis.

BACKGROUND: The regulation of endometrial inflammation has important consequences for the resumption of bovine fertility postpartum. All cows experience bacterial influx into the uterus after calving; however a significant proportion fail to clear infection leading to the development of cytological endometritis (CE) and compromised fertility. We hypothesised that early immunological changes could not only act as potential prognostic biomarkers for the subsequent development of disease but also shed light on the pathogenesis of endometritis in the postpartum dairy cow. METHODS: Endometrial biopsy RNA was extracted from 15 cows at 7 and 21 days postpartum (DPP), using the Qiagen RNeasy(®) Plus Mini kit and quality determined using an Agilent 2100 bioanalyser. Disease status was determined by histpathology based on inflammatory cell infiltrate. RNA-seq of both mRNA and miRNA libraries were performed on an Illumina® HiSeq(™) 2000. Paired reads were aligned to the bovine genome with Bowtie2 and differentially expressed genes were identified using EdgeR. Significantly over-represented Gene Ontology terms were identified using GO-seq, and pathway analysis was performed using KEGG. Quanititative real-time PCR was also performed for validation (ABI 7500 fast). Haematology was assessed using an automated ADVIA 2120 analyser. Serum proteins were evaluated by ELISA and metabolite analysis was performed using a Beckman Coulter AU 400 clinical analyser. Terminal-restriction fragment length polymorphism (T-RFLP) was used to obtain fingerprints of the microbial communities present. RESULTS: Next-generation sequencing from endometrial biopsies taken at 7 DPP identified significant induction of inflammatory gene expression in all cows. Despite the common inflammatory profile and enrichment of the Toll-like receptor and NFκB pathways, 73 genes and 31 miRNAs were significantly differentially expressed between healthy cows (HC, n = 9) and cows which subsequently developed CE at 7 DPP (n = 6, FDR < 0.1). While significant differential expression of 4197 genes in the transcriptome of healthy cows between 7 and 21 DPP showed the transition from a proinflammatory to tissue profliferation and repair, only 31 genes were differentially expressed in cows with CE (FDR < 0.1), indicating the arrest of such a transition. A link betwene the dysregulated inflammatory response and the composition of the uterine microbial communities was suggested by the presence of significant differences in uterine bacterial tRFLP profiles between HC and CE groups. Furthermore, inflammatory activity was not confined to the uterus; decreased circulating granulocytes and increased Acute Phase Protein (SAA and HP) expression levels were detected in plasma at 7 DPP in cows that developed CE. CONCLUSION: Our data suggests that the IL1 and IL17 inflammatory cascade activated early postpartum is resolved thereby restoring homeostasis in healthy cows by 21 DPP, but this transition fails to occur in cows which develop CE. Despite a common early inflammatory profile, elevated and differential expression of specific immune genes may identify cows at risk of prolonged inflammation and the development of CE postpartum.

MSTN genotypes in Thoroughbred horses influence skeletal muscle gene expression and racetrack performance.

Myostatin, encoded by the MSTN gene, is a member of the TGF-ß superfamily that regulates skeletal muscle development. A MSTN SNP significantly associated with Thoroughbred horse racing phenotypes has recently been identified as well as significant reductions in Thoroughbred skeletal muscle gene expression for three transcripts 400-1500 base pairs downstream of the MSTN gene following a period of training. Together, these findings indicate that MSTN genotypes may influence MSTN gene expression. To investigate this, MSTN mRNA expression was measured in biopsies from the middle gluteal muscle from 60 untrained yearling Thoroughbreds (C/C, n = 15; C/T, n = 28; T/T, n = 17) using two independent real-time qRT-PCR assays. MSTN gene expression was also evaluated in a subset (N = 33) of these animals using samples collected after a ten-month period of training. A significant association was observed between genotype and mRNA abundance for the untrained horses (assay I, P = 0.0237; assay II, P = 0.003559), with the C/C cohort having the highest MSTN mRNA levels, the T/T group the lowest levels and the C/T group intermediate levels. Following training, there was a significant decrease in MSTN mRNA (-3.35-fold; P = 6.9 × 10(-7) ), which was most apparent for the C/C cohort (-5.88-fold, P = 0.001). These data demonstrate the tight relationship between phenotype, genotype and gene expression at the MSTN gene in Thoroughbred racehorses.

A genome-wide SNP-association study confirms a sequence variant (g.66493737C>T) in the equine myostatin (MSTN) gene as the most powerful predictor of optimum racing distance for Thoroughbred racehorses.

BACKGROUND: Thoroughbred horses have been selected for traits contributing to speed and stamina for centuries. It is widely recognized that inherited variation in physical and physiological characteristics is responsible for variation in individual aptitude for race distance, and that muscle phenotypes in particular are important. RESULTS: A genome-wide SNP-association study for optimum racing distance was performed using the EquineSNP50 Bead Chip genotyping array in a cohort of n = 118 elite Thoroughbred racehorses divergent for race distance aptitude. In a cohort-based association test we evaluated genotypic variation at 40,977 SNPs between horses suited to short distance (≤ 8 f) and middle-long distance (> 8 f) races. The most significant SNP was located on chromosome 18: BIEC2-417495 ~690 kb from the gene encoding myostatin (MSTN) [P(unadj.) = 6.96 x 10⁻6]. Considering best race distance as a quantitative phenotype, a peak of association on chromosome 18 (chr18:65809482-67545806) comprising eight SNPs encompassing a 1.7 Mb region was observed. Again, similar to the cohort-based analysis, the most significant SNP was BIEC2-417495 (P(unadj.) = 1.61 x 10⁻9; P(Bonf.) = 6.58 x 10⁻5). In a candidate gene study we have previously reported a SNP (g.66493737C>T) in MSTN associated with best race distance in Thoroughbreds; however, its functional and genome-wide relevance were uncertain. Additional re-sequencing in the flanking regions of the MSTN gene revealed four novel 3' UTR SNPs and a 227 bp SINE insertion polymorphism in the 5' UTR promoter sequence. Linkage disequilibrium was highest between g.66493737C>T and BIEC2-417495 (r² = 0.86). CONCLUSIONS: Comparative association tests consistently demonstrated the g.66493737C>T SNP as the superior variant in the prediction of distance aptitude in racehorses (g.66493737C>T, P = 1.02 x 10⁻¹°; BIEC2-417495, P(unadj.) = 1.61 x 10⁻9). Functional investigations will be required to determine whether this polymorphism affects putative transcription-factor binding and gives rise to variation in gene and protein expression. Nonetheless, this study demonstrates that the g.66493737C>T SNP provides the most powerful genetic marker for prediction of race distance aptitude in Thoroughbreds.