Polymorphonuclear leukocytes (neutrophils) serve as critical indicators of innate immune function in dairy cattle. Accurate assessment of their functional capacities is essential for understanding the status of the innate immune system in health and disease or following immunomodulatory treatment. However, the lack of standardized protocols for evaluating PMN functional capacity in dairy cattle has led to inconsistencies in research methodologies and hindered cross-laboratory comparisons. To address this issue, we propose systematic, evidence-based methodologies for assessing PMN function in dairy cattle. It begins with establishing standardized protocols for blood sample collection, processing, storage, PMN isolation, and culture: protocols specifically designed to ensure PMN viability and generate reproducible outcomes across different laboratory settings. Building upon this foundation, we describe existing laboratory methodologies for assessing the 4 cardinal PMN functions: chemotaxis, phagocytosis, oxidative burst, and extracellular trap formation. We then elaborate on the advantages, limitations, and practical considerations of each approach by comparing natural PMN behavior in vivo with the corresponding in vitro assay. By establishing standardized methodological frameworks, this review aims to facilitate meaningful cross-laboratory comparisons and ensure that PMN functional measurements are accurate and reproducible, thereby allowing them to properly reflect the innate immune status across diverse experimental and laboratory settings.

We evaluated uterine bacterial growth patterns and their association with endometrial inflammation (EI) in equine samples collected using uterine swabs (US) or low-volume uterine lavage (UL). A database including 1,545 US and 2,066 UL with cytological and bacteriological results was retrospectively analysed. Endometrial inflammation was defined as ≥2 polymorphonuclear cells per high-power field, and bacterial culture was considered positive when aerobic growth occurred within 48 h, with the number of bacterial isolates per sample recorded (0, 1, 2, or ≥3). Data were analysed using generalized mixed-effects models including the number of isolates and bacterial species as fixed effects. The prevalence of EI was 4.8% for US and 36.6% for UL. For both techniques, EI prevalence was lower in samples with negative bacterial cultures (1.0 ± 0.3% for US and 10.7 ± 1.7% for UL) compared with samples yielding ≥1 bacterial isolate. In UL samples, isolation of a single bacterial species was associated with greater probability of EI (45.1 ± 2.6%) compared with samples yielding ≥3 isolates (33.3 ± 3.1%). For UL, the presence of Streptococcus sp. (β-haemolytic) and Staphylococcus aureus increased the probability of EI compared with their absence, whereas in US samples only Streptococcus sp. (β-haemolytic) increased EI probability. In conclusion, bacterial growth increased the likelihood of EI for both sampling techniques, with Streptococcus sp. (β-haemolytic) as the primary bacteria associated with evidence of EI. The integration of endometrial cytology with bacterial culture and pathogen identification improves interpretation of bacteriological findings and supports responsible antimicrobial use in mares.

Subclinical (SCE) and clinical endometritis (CE) are distinct manifestations of reproductive tract inflammatory disease in dairy cows. The development of both conditions stems from postpartum dysregulation of the inflammatory response or a shift in the composition of the uterine microbiome. To gain further insight into the host responses associated with these distinct conditions, we aimed to identify changes in the endometrial transcriptomic landscape in healthy postpartum dairy cows compared with those diagnosed with SCE or CE. Twenty-four multiparous Holstein cows were evaluated for uterine health status at 35 or 36 d postpartum using vaginal discharge scoring (Metricheck) and endometrial cytology (cytobrush). Based on these evaluations, cows were classified into 3 groups: healthy (n = 12; clear or no vaginal discharge and ≤5% endometrial PMN), SCE (n = 6; clear or no vaginal discharge and >5% PMN), and CE (n = 6; mucopurulent or worse discharge and >5% PMN). Endometrial samples collected via cytobrush were stored at -80°C and total RNA was isolated; RNA sequencing was performed using an Illumina NextSeq 500 platform, generating 75 bp single-end reads. Differentially expressed genes (DEG) were identified using DESeq2 with a significance threshold of P < 0.05 and |fold change| > 2. Pathway enrichment analyses were performed using the OmicShare platform to identify enriched biological pathways among the DEG. A total of 250 DEG were identified between healthy and SCE cows, 1,291 between healthy and CE cows, and 829 between SCE and CE cows. In SCE (as compared with healthy) cows, TNF, IL-17, NOD-like receptor signaling, and cytokine-cytokine receptor interaction pathways were upregulated, whereas the FoxO signaling pathway was downregulated. In CE compared with healthy cows, upregulated DEG were enriched in IL-17, TNF, chemokine, NOD-like receptor, NF-kappa B, and toll-like receptor signaling pathways, whereas downregulated DEG were enriched in PI3K-AKT, MAPK, AMPK, Wnt, PPAR, and metabolic pathways. In CE compared with SCE, upregulated DEG were enriched in NOD-like receptor, IL-17, chemokine, B cell receptor, and cytokine-cytokine receptor interaction pathways, and downregulated DEG were enriched in the metabolic pathways, fatty acid metabolism, insulin signaling pathway, and adipocytokine signaling pathway. These findings underscore that CE and SCE conditions involve an inflammatory event but likely arise from different mechanisms. The enrichment of immune signaling pathways in CE reflects a classic infectious response, whereas the metabolic and regulatory pathway alterations in SCE suggest a dysregulated inflammatory state linked to impaired resolution mechanisms. These results highlight the need for tailored prevention and treatment strategies, such as modulating immune regulation in SCE and targeting bacterial dysbiosis and tissue damage in CE cases.

Clinical endometritis (CE) is associated with bacterial pathogens while the same has not been proved about subclinical endometritis (SCE). We aimed to use shotgun metagenomic sequencing to investigate the associations between potentially unidentified pathogens and SCE. Uterine cytobrush samples from multiparous Holstein cows (n = 23) were taken at 21 days in milk (DIM) and sequenced via the Illumina shotgun platform. At 36 DIM, the cows were diagnosed as CE (n = 7), SCE (n = 7), or healthy (n = 9). We did not find differences in the alpha and beta diversity of bacteria and eukaryotes among the health groups. Relative abundance of typical pathogens i.e. Fusobacterium, Peptoniphilus, Peptostreptococcus, and Trueperella was greater in CE than healthy controls. We did not find evidence of eukaryotic or viral association in infection, yet, distinct patterns of bacterial co-occurrence were observed among pathogenic and non-pathogenic bacteria. In CE cows, Wnt/catenin pathway had lower abundance than SCE or healthy cows. Our findings support that CE is characterized by domination of pathogenic bacteria that intercorrelate, whereas SCE is not associated with bacterial colonization.

In brief:In vitro maturation is an essential tool in reproductive technologies, though its impact on oocyte quality remains a concern. This study shows that in vitro maturation alters gene expression and DNA methylation in bovine oocytes compared to in vivo matured oocytes, potentially compromising oocyte quality and developmental competence. Abstract:In vitro maturation of oocytes is a routine step in assisted reproduction but is associated with lower embryo development rates compared to oocyte maturation in vivo. We analyzed the genomic profiles of oocytes from the same cow, either matured in vivo or in vitro, using single-cell methylome and transcriptome sequencing, along with transcriptome analysis of corresponding cumulus cells. Both the transcriptome and methylome of the oocytes matured in vitro were altered. Notable changes included alterations in CpG islands associated with imprinted genes, including decreased methylation levels in MEST (PEG1), NNAT (both implicated in large offspring syndrome), and MIMT1. Transcriptomic analysis of their cumulus cells highlighted impaired mitochondrial function, hypoxia responses, and cell adhesion. Our findings highlight the extent to which the maturation environment can influence key epigenetic regulators and mRNA profiles that affect oocyte quality and subsequent developmental outcomes. The data provide a valuable resource for optimizing assisted reproductive technologies.

Following a significant increase in herd and farm sizes after the removal of milk quotas in Europe, the past 10 years have seen a slight yet steady decline in the population of cattle. This includes a reduction of approximately 5% in dairy and beef cattle. This trend is driven by various factors, such as changing market demands, economic shifts, and sustainability challenges in the livestock sector. Despite this, technological advancements in reproductive management have continued to enhance efficiency and sustainability, particularly in dairy production. The main areas of rapid development, which will continue to grow for improving fertility and management, include: i) genetic selection (including improved phenotypes for use in breeding programs), ii) nutritional management (including transition cow management), iii) control of infectious disease, iv) rapid diagnostics of reproductive health, v) development of more efficient ovulation/estrous synchronization protocols, vi) assisted reproductive management (and automated systems to improve reproductive management), vii) increased implementation of sexed semen and embryo transfer, viii) more efficient handling of substantial volumes of data, ix) routine implementation of artificial intelligence technology for rapid decision-making at the farm level, x) climate change and sustainable cattle production awareness, xi) new (reproductive) strategies to improve cattle welfare, and xii) improved management and technology implementation for male fertility. This review addresses the current status and future outlook of key factors that influence cattle herd health and reproductive performance, with a special focus on dairy cattle. These insights are expected to contribute to improved performance, health, and fertility of ruminants in the next 20 years.

In the present study, we examined the relationship between uterine involution, serum metabolites and calcium concentrations in clinically healthy postpartum dairy cows with or without subclinical endometritis (SCE). A cohort of 43 Holstein cows, sampled between 21 and 29 days postpartum, was selected based on the absence of clinical endometritis or other clinical postparturient diseases. Reproductive tract measurements included the diameters of the cervix and uterine horns, and blood sample analyses included serum total calcium, total cholesterol, triglycerides, non-esterified fatty acids, and β-hydroxybutyrate. Using the cytobrush, endometrial cytology samples were collected to assess the proportion of endometrial polymorphonuclear leukocytes (PMNs) at 21-29 days postpartum. Results indicated no association between SCE, defined as >5 % PMNs in endometrial smears, and either uterine dimensions or serum calcium levels. However, a general linear model, accounting for random effects of parity and days postpartum, demonstrated that lower serum calcium concentrations were associated with greater cervical and uterine horn diameters (P < 0.001), suggesting a linear dependence of uterine structural dimensions on calcium concentration. For all the other metabolites, no associations were found with cervical nor uterine horns diameters. This indicates blood calcium's role in supporting uterine involution during the late postpartum period. Interestingly, the PMN% in endometrial samples (characteristic of SCE) did not correlate with changes in uterine size, suggesting that other factors related to the periparturient period rather than serum calcium levels influence the development of SCE in the fourth week postpartum.

Previous studies in the horse highlight the potential benefit of prolonged in vitro maturation (IVM) (34 h) compared to short IVM (24 h) with or without prior oocyte holding, but little is known about the optimal IVM duration within this interval. To determine the effect of oocyte holding and duration of IVM ranged between 28 and 34 h on nuclear maturation, cleavage, blastocyst formation, and pregnancy rates, a retrospective study was performed in an equine clinical OPU-ICSI setting. The study included data of 2114 aspirated oocytes from 201 OPU-ICSI sessions. Duration of IVM was divided in three different time windows using quartiles, with 465 oocytes (22.0 %) between 28 and 30 h (first quartile), 1078 oocytes (51.0 %) >30 and 31.7 h (second and third quartiles), and 571 oocytes (27.0 %) >31.7 and 34 h (fourth quartile). Using logistic regression models, the effect of duration of IVM with and without holding was tested on nuclear maturation, cleavage, blastocyst, and pregnancy rates. The three IVM intervals did not show differences in nuclear maturation (respectively 64.5 +/- 0.48 %, 65.7 +/- 0.47 %, and 67.3 +/- 0.47 %), cleavage (respectively 59.7 +/- 0.49 %, 58.5 +/- 0.49 %, and 64.8 +/- 0.48 %), blastocyst (respectively 17.5 +/- 0.38 %, 19.0 +/- 0.39 %, and 20.8 +/- 0.41 %) nor pregnancy rates (respectively 65.4 +/- 0.49 %, 70.3 +/- 0.46 %, and 74.2 % +/- 0.44) (P >= 0.38). Oocyte holding prior to IVM did not affect the results either (P >= 0.15). In conclusion, oocyte holding and IVM duration between 28 and 34h do not significantly affect outcomes, allowing flexibility in the planning of clinical OPU-ICSI in horses.

Polymorphonuclear leukocytes are pivotal players in the innate immune response of dairy cows. Their functionality (phagocytosis [PC] and oxidative burst [OB]) and gene expression patterns, however, can be modulated by varying energetic conditions. Energetic fluctuations are particularly evident in periparturient dairy cows, when distinct levels of negative energy balance can trigger PMN dysfunction. This study aimed to assess how different energetic conditions affect PMN functionality and to examine their transcriptomic profile following an in vitro LPS challenge. Over 4 biological replicates, circulating PMN isolated from 4 pubertal nulliparous heifers were suspended in cell culture medium and supplemented with glucose (1.5 and 3 mM to mimic hypo- and normoglycemia, respectively) and with 1 mM nonesterified fatty acids (NEFA). Control PMN suspensions with no glucose or NEFA supplementation were incubated alongside. After 3 h of incubation under the aforementioned conditions, PMN were challenged with 50 μg/mL LPS for 1 h to mimic infection. At 0 and 3 h of incubation, the PMN viability (viable, apoptotic, or necrotic) and functionalities (PC and OB) were measured via flow cytometry. After the LPS challenge, the total RNA of PMN was extracted and sequenced on an Illumina platform. The effects of glucose and NEFA in the culture medium on PMN viability and functional parameters were fitted in mixed linear regression models, using the replicate as a random effect. The analyses of differentially expressed genes (DEG) were performed by edgeR. Regardless of NEFA, supplementation with 3 mM glucose increased PMN OB compared with PMN without glucose and NEFA supplementation. Transcriptomic analysis showed that PMN exposed to 3 mM glucose plus NEFA exhibited upregulation of oxidative e phosphorylation and reactive oxygen species pathways compared with 3 mM glucose alone (no-NEFA), suggesting that NEFA promotes an excessive PMN response. Under NEFA supplementation, PMN exposed to 1.5 mM glucose showed downregulation of energy metabolism and immune signaling pathways when compared with 3 mM glucose, indicating immune dysfunction related to metabolic stress (presence of NEFA) when glucose is low. These results provide insights into how different metabolic conditions during the transition period induce innate immune (dys)function in dairy cows.

Long noncoding RNAs (lncRNAs) can regulate gene expression by "sponging" microRNAs (miRNAs), reducing their inhibitory effects on mRNAs. However, this mechanism has been minimally investigated in preimplantation embryo development. In this study, we revisited existing RNA sequencing and small RNA sequencing data to investigate the role of lncRNAs in in vitro-produced bovine preimplantation embryos. Our findings revealed that although lncRNAs exhibit expression patterns similar to mRNAs, maternal lncRNAs degrade earlier than mRNAs during embryonic genome activation (EGA). Weighted gene co-expression network analysis identified 27 modules of mRNA and lncRNA, with enrichment analysis showing a significant negative correlation between the polycomb repressive complex pathway and blastocyst formation (R2 = -0.98). Additionally, bioinformatics analysis was used to predict and construct lncRNA-miRNA-mRNA networks, highlighting that lncRNAs bind more to miRNAs compared with mRNAs. Moreover, lncRNA-induced lncRNA-miRNA-mRNA axes participated in mRNA degradation and biogenesis around the EGA stage. These interactions became stronger after EGA, especially after the 16-cell stage. Overall, our study provides new insights into lncRNA-mediated regulatory networks during bovine preimplantation development.

Over 95% of Belgian Blue (BB) cows deliver via elective cesarean section (CS). The aim of this study was to determine the effect of elective CS on the incidence of subclinical endometritis (SCE) using cytobrush (CB) and low-volume lavage (LVL) techniques and evaluate the sensitivity of both techniques in detecting SCE. Uteri of BB cows (n = 100) were collected from the slaughterhouse. Cytobrush samples followed by LVL cytology samples were collected from all uterine horns (n = 200). The samples collected by CB was rolled on a microscope slide while LVL samples were centrifuged, and the pelleted cells were then scattered over a microscope slide. In total, three hundred nucleated cells were identified and the proportion of polymorphonuclear (PMN%) to endometrial cells was assessed. The cut-off for SCE was set at >= 1% PMN. To compare the CS horn with its CS-free counterpart, linear (PMN%) and logistic regression (SCE positive versus SCE negative) modeling was performed. The PMN% in CB (0.34 +/- 0.45%) samples was lower than in LVL (2.67 +/- 0.45%) samples (p < 0.0001), suggesting LVL is a more sensitive technique than CB. The CS horn had no effect on the PMN% (p = 0.99 for CB and p = 0.12 for LVL), and hence on the incidence of SCE (p = 0.18 for CB and p = 0.81 for LVL).

Reproductive tract inflammatory diseases (RTID) present significant health challenges in domestic animals, impacting welfare, fertility, and productivity. Traditionally, antibiotics have been the primary treatment for these conditions, however, the rise of antimicrobial resistance calls for alternative approaches. The microbiome of the female reproductive tract plays a vital role in maintaining reproductive health, and emerging evidence suggests that microbiome-based therapies, such as 'natural' or 'synthetic' microbiome transplantation, may offer sustainable solutions for RTID management. This review explores the composition and dynamics of the reproductive microbiome in both healthy and diseased states in cows, mares, sows, dogs, and cats. It also examines current treatments and the potential for microbiome-based interventions to replace or complement antibiotic therapies. Although research on microbiome-based therapies for preventing or treating RTID in domestic animals is virtually non-existent, vaginal and uterine microbiomes transplantation in mice and women show promise but require further investigation to evaluate their efficacy and safety across species with varying reproductive physiologies. Additionally, synthetic microbiome therapies present a controlled and reproducible alternative, though they face challenges in design, engraftment, and regulatory approval. The transition from antibiotic dependence to microbiome-based solutions marks a paradigm shift in veterinary medicine, but successful implementation demands a deeper understanding of host-microbiome interactions, rigorous safety protocols, and species-specific research.

Evaluating cumulus-oocyte complex (COC) morphology is commonly used to assess oocyte quality. However, clear guidelines on interpreting COC morphology data are lacking as this evaluation method is subjective. In the present study, individual in vitro embryo production was used, allowing follow-up of blastocyst formation for each COC. Images of immature COCs were presented to embryologists and two artificial intelligence (AI) models: deep neural network (DNN) and random forest classifier (RF). The aims were to (1) determine the most relevant morphological characteristics in distinguishing qualitative COCs, (2) review human-made predictions, and (3) build predictive AI models. Our experiments identified cumulus size as pivotal characteristic of COC quality, while embryologists assigned ooplasm morphology as most important. Inspection of COCs by the human eye showed significant limitations, as evidenced by their low predictive ability (balanced accuracy: 42.9%) and fair reliability. Our AI models outperformed the embryologists, yielding a balanced accuracy of 79.3% and 71.2% for DNN and RF, respectively. The first AI models that successfully predict developmental competence of immature bovine oocytes were created, outperforming embryologists and offering an objective perspective for COC morphology assessment. AI has emerged as a novel tool for oocyte appreciation, assisting decision-making in the embryology lab.

Introduction:We evaluated the impact of follicular fluid-derived small extracellular vesicles (FF-sEVs) supplementation during oocyte maturation in vitro on bovine embryo outcomes, comparing group and individual culture systems. Methods:Follicular fluid was aspirated from dominant follicles of four nulliparous Holstein heifers at 4.5 days post-ovulation. Small extracellular vesicles were isolated, characterized, and pooled to ensure balanced donor contribution. To confirm uptake, FF-sEVs were fluorescently labelled and co-cultured with cumulus-oocyte complexes (COCs) during in vitro maturation. Fluorescent labelling confirmed FF-sEVs internalization by oocytes and granulosa cells. Next, COCs were matured in vitro with FF-sEVs at varying concentrations (group system: 0, 5, 10, 25, 50 μg/mL; individual system: 0, 6.5, 12.5, 25 μg/mL), fertilized, and cultured. Blastocyst quality was assessed via differential-apoptotic staining. Results:In group culture, the control group exhibited higher day 8 blastocyst rates compared to 10, 25, and 50 μg/mL FF-sEVs groups, while 5 μg/mL FF-sEVs showed no difference. Blastocysts developed from oocytes matured in 25 and 50 μg/mL groups had reduced total cell numbers versus controls and groups matured in lower FF-sEVs concentrations. Conversely, individual maturation with 6.5 μg/mL FF-sEVs enhanced day 8 blastocyst rate, total cell counts, inner cell mass, and reduced apoptotic ratios compared to all other groups. Discussion and conclusion:We propose that intercellular communication in group cultures, potentially mediated by endogenous embryotropins (including sEVs), may mask FF-sEVs benefits. In individual systems, where such interactions are absent (or minimal), FF-sEVs significantly improved embryo competence. These findings underscore FF-sEVs as a promising tool to refine assisted reproductive technologies, contingent on culture conditions.

Abortions and perinatal mortalities (APM) substantially affect cattle industry efficiency. Various infectious and noninfectious factors have been associated with bovine APM worldwide. Infections are often considered pivotal due to their abortifacient potential, leading laboratories to primarily investigate relevant infectious agents for APM cases. Some infectious causes, such as Brucella abortus, have also a zoonotic impact, necessitating monitoring for both animal and human health. However, underreporting of bovine APM is a global issue, affecting early detection of infectious and zoonotic causes. Previous studies identified factors influencing case submission, but regional characteristics may affect results. In Belgium, farmers are obliged to report cases of APM within the context of a national brucellosis monitoring program. The inclusion criteria for this monitoring program cover abortions (gestation length of 42-260 d) and perinatal mortalities of (pre) mature calves following a gestation length of more than 260 d, which were stillborn or died within 48 h after birth. The objective of the present study was to describe the evolution in submission of APM cases within a mandatory abortion monitoring program in relation to subsidized initiatives in the northern part of Belgium. Based on the proportion of APM submissions versus the proportion of bovine reproductive females, an APM proportion (APMPR) was calculated, and factors at both animal and herd level that may influence this APMPR were explored by using linear models. This evaluation revealed that the APMPR increased with the introduction of an extensive analytical panel of abortifacient agents and a free on -farm sample collection from 0.44% to 0.94%. Additionally, an increase of the APMPR was associated with an outbreak of an emerging abortifacient pathogen (Schmallenberg virus; 1.23%), and the introduction of a mandatory eradica tion program for bovine viral diarrhea virus (BVDv; 1.20%). The APMPR was higher in beef compared with dairy cattle, and it was higher in winter compared with fall, spring, and summer. Smaller herds categorized in the first quartile had a higher APMPR compared with larger herds. Herds that submitted an APM in the previous year had a higher APMPR in the next year compared with herds without an APM submission. Finally, herds for which there was evidence of the presence of BVDv had a higher APMPR compared with herds without evidence of the presence of BVDv. In conclusion, the number of APM submissions increased after the introduction of a free on -farm sample collection and an extensive pathogen screening panel. Production type (beef), season (winter), smaller herd size, previous APM, and presence of BVDv seemed to have a positive effect on APMPR. However, even under mandatory circumstances, APM still seems to be underreported, since the APMPR was lower than the expected minimal rate of 2%. Therefore, further research is necessary to identify the drivers that convince farmers to submit APM cases to improve submission rates and ensure an efficient monitoring program for APM and eventually associated zoonotic pathogens.

Cumulus expansion is an important indicator of oocyte maturation and has been suggested to be indicative of greater oocyte developmental capacity. Although multiple methods have been described to assess cumulus expansion, none of them is considered a gold standard. Additionally, these methods are subjective and time-consuming. In this manuscript, the reliability of three cumulus expansion measurement methods was assessed, and a deep learning model was created to automatically perform the measurement. Cumulus expansion of 232 cumulus-oocyte complexes was evaluated by three independent observers using three methods: (1) measurement of the cumulus area, (2) measurement of three distances between the zona pellucida and outer cumulus, and (3) scoring cumulus expansion on a 5-point Likert scale. The reliability of the methods was calculated in terms of intraclass-correlation coefficients (ICC) for both inter- and intra-observer agreements. The area method resulted in the best overall inter-observer agreement with an ICC of 0.89 versus 0.54 and 0.30 for the 3-distance and scoring methods, respectively. Therefore, the area method served as the base to create a deep learning model, AI-xpansion, which reaches a human-level performance in terms of average rank, bias and variance. To evaluate the accuracy of the methods, the results of cumulus expansion calculations were linked to embryonic development. Cumulus expansion had increased significantly in oocytes that achieved successful embryo development when measured by AI-xpansion, the area- or 3-distance method, while this was not the case for the scoring method. Measuring the area is the most reliable method to manually evaluate cumulus expansion, whilst deep learning automatically performs the calculation with human-level precision and high accuracy and could therefore be a valuable prospective tool for embryologists.

BackgroundWithin the follicular fluid, extracellular vesicles (EVs) guide oocyte growth through their cargo microRNAs (miRNAs). Here, we investigated the role of EVs and their cargo miRNAs by linking the miRNAs found in EVs, derived from the fluid of an individual follicle, to the ability of its oocyte to become a blastocyst (competent) or not (non-competent).MethodsBovine antral follicles were dissected, categorized as small (2-4 mm) or large (5-8 mm) and the corresponding oocytes were subjected to individual maturation, fertilization and embryo culture to the blastocyst stage. Follicular fluid was pooled in 4 groups (4 replicates) based on follicle size and competence of the corresponding oocyte to produce a blastocyst. Follicular fluid-derived EVs were isolated, characterized, and subjected to miRNA-sequencing (Illumina Miseq) to assess differential expression (DE) in the 4 groups. Functional validation of the effect of miR-34c on embryo development was performed by supplementation of mimics and inhibitors during in vitro maturation (IVM).ResultsWe identified 16 DE miRNAs linked to oocyte competence when follicular size was not considered. Within the large and small follicles, 46 DE miRNAs were driving blastocyst formation in each group. Comparison of EVs from competent small and large follicles revealed 90 DE miRNAs. Cell regulation, cell differentiation, cell cycle, and metabolic process regulation were the most enriched pathways targeted by the DE miRNAs from competent oocytes. We identified bta-miR-34c as the most abundant in follicular fluid containing competent oocytes. Supplementation of miR-34c mimic and inhibitor during IVM did not affect embryo development. However, blastocyst quality, as evidenced by higher cell numbers, was significantly improved following oocyte IVM in the presence of miR-34c mimics, while miR-34c inhibitors resulted in the opposite effect.ConclusionThis study demonstrates the regulatory effect of miRNAs from follicular fluid-derived EVs on oocyte competence acquisition, providing a further basis for understanding the significance of miRNAs in oocyte maturation and embryonic development. Up-regulation of miR-34c in EVs from follicular fluid containing competent oocytes and the positive impact of miR-34c mimics added during IVM on the resulting blastocysts indicate its pivotal role in oocyte competence.

During the transition period, dairy cows exhibit heightened energy requirements to sustain fetal growth and lactogenesis. The mammary gland and the growing fetus increase their demand for glucose, leading to the mobilization of lipids to support the function of tissues that can use fatty acids as energy substrates. These physiological adaptations lead to negative energy balance, metabolic inflammation, and transient insulin resistance (IR), processes that are part of the normal homeorhetic adaptations related to parturition and subsequent lactation. Insulin resistance is characterized by a reduced biological response of insulin-sensitive tissues to normal physiological concentrations of insulin. Metabolic inflammation is characterized by a chronic, low-level inflammatory state that is strongly associated with metabolic disorders. The relationship between IR and metabolic inflammation in transitioning cows is intricate and mutually influential. On one hand, IR may play a role in the initiation of metabolic inflammation by promoting lipolysis in adipose tissue and increasing the release of free fatty acids. Metabolic inflammation, conversely, triggers inflammatory signaling pathways by pro-inflammatory cytokines, thereby leading to impaired insulin signaling. The interaction of these factors results in a harmful cycle in which IR and metabolic inflammation mutually reinforce each other. This article offers a comprehensive review of recent advancements in the research on IR, metabolic inflammation, and their intricate interrelationship. The text delves into multiple facets of physiological regulation, pathogenesis, and their consequent impacts.

Chronic endometritis (CE) in humans is asymptomatic inflammation of the endometrium, associated with poor reproductive outcomes. Similarly, asymptomatic endometrial inflammation in cows, termed subclinical endometritis (SCE), is associated with adverse reproductive outcomes. While the pathophysiology and treatment options for CE in humans remain poorly defined, the well-known financial implications of SCE in dairy cows have attracted intensive research. We performed a systematic review to determine potential areas of interest in human CE research, by analysing emergent themes that arose in studies of SCE in cows. A literature search for studies of subclinical endometritis in cows published between 1990 and November 2021 was performed across Embase, Medline, Scopus and CINAHL. Studies of symptomatic or clinical endometritis were excluded. A thematic analysis across two broad themes was explored: i) diagnostic methods of SCE and ii) pathophysiology of SCE. In total, 51 bovine studies were included. Twelve studies reported on diagnostic methodology. The primary emergent theme was the use of cytology for the diagnosis of SCE. Cytological analysis has a lower sensitivity than histopathology but is less invasive and more specific than alternative techniques such as ultrasound, vaginoscopy or metabolic markers. The subthemes related to pathophysiology were identified as the type of endometritis, metabolic stress, artificial insemination, infective causes and altered cellular pathways. Despite the lack of symptoms, cellular pathways of inflammation including NF-κB, MAPK and inflammasomes were found to be activated. The key themes related to the diagnosis and pathophysiology of SCE in cows identified in this systematic review highlight potential areas for future research into human CE. Lay summary Long-term or chronic endometritis (CE) is a condition associated with inflammation in the womb lining. People with CE commonly experience recurrent miscarriages and subfertility. The cause of CE is poorly understood, and as such there are no specific treatments. Comparatively, a form of CE (i.e. subclinical endometritis (SCE)) has been extensively researched in dairy cows due to its financial impact. A systematic review of cow studies was carried out and key themes around cause and diagnosis of SCE in cows were identified and analysed. In total, 44 studies were included. Six main themes around the way SCE is diagnosed were found, and examining cells (i.e. cytology) was found to be more sensitive and practical than other techniques. Six themes were also found for causes of SCE, notably difficult delivery, metabolic stress and infection. This study shows potential areas for future research into human SCE and provides insight into the causes of disease within humans.