The aim of this study was to explore alterations in plasma metabolites among mares afflicted with endometritis. Mares were divided into two groups, namely, the equine endometritis group (n = 8) and the healthy control group (n = 8), which included four pregnant and four non-pregnant mares, using a combination of clinical assessment and laboratory confirmation. Plasma samples from both groups of mares were analyzed through untargeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics. A total of 28 differentially abundant metabolites were identified by screening and identifying differentially abundant metabolites and analyzing the pathway enrichment of differentially. Ten metabolites were identified as potential biomarkers for the diagnosis of endometritis in mares. Among them, seven exhibited a decrease in the endometritis groups, including hexadecanedioic acid, oleoyl ethanolamide (OEA), [fahydroxy(18:0)]12_13-dihydroxy-9z-octa (12,13-diHOME), deoxycholic acid 3-glucuronide (DCA-3G), 2-oxindole, and (+/-)9-HPODE, and 13(S)-HOTRE. On the other hand, three metabolites, adenosine 5\'-monophosphate (AMP), 5-hydroxy-dl-tryptophan (5-HTP), and l-formylkynurenine, demonstrated an increase. These substances primarily participate in the metabolism of tryptophan and linolenic acid, as well as fat and energy. In conclusion, metabolomics revealed differentially abundant metabolite changes in patients with mare endometritis. These specific metabolites can be used as potential biomarkers for the non-invasive diagnosis of mare endometritis.

Peritoneal fluid lactate concentration is an important diagnostic tool in horses with abdominal pain. Information on peritoneal lactate concentrations is lacking following parturition in the mare.
To compare blood and peritoneal lactate concentrations in a population of mares within 36 h post-partum, report a normal reference range and identify any impact of retained foetal membranes (RFMs).
This is a retrospective study evaluating healthy mares from which blood and peritoneal samples had been obtained within 36 h of parturition. Exclusion criteria included signs of abdominal pain within this period. Data was interrogated for normality using a Shapiro-Wilk test. Wilcoxon signed-rank test and Bland-Altman analysis were used to compare blood and peritoneal lactate concentrations. Linear regression was used to compare age and breed data with peritoneal lactate concentrations. Significance was defined as p < 0.05.
Forty mares met the inclusion criteria. Mean age was 12.6 ± 4.1 years, and most mares were multiparous (65%). Peritoneal lactate ((1.2 (IQR = 0.9-1.6) mmol/L) was increased compared to blood lactate concentration (0.7 (IQR = 0-1.1)mmol/L; p < 0.001). Plasma total protein (TP) concentrations were 68 (IQR = 64-74) g/L and peritoneal protein concentrations 8 (IQR = 4-9.7) g/L. Six mares developed RFM. The median fold-increase in peritoneal lactate concentration compared to blood lactate concentration was 0.9 (IQR: 0.01-1.7; range: 0-2.5). The reference range for peritoneal fluid lactate concentration was 0-2.5 mmol/L.
Peritoneal lactate concentrations in healthy post-partum mares remained within the normal reference range and were not influenced by RFM or parturition. Increased peritoneal lactate in this group warrants further investigation.

The identification of typical behaviors in stage I of parturition, the opening phase, can be used to improve birth monitoring in the mare. Therefore, this study aimed to comprehensively analyze mare behavior during the opening phase. Real-time recordings of 66 births involving 56 warmblood mares were analyzed using camera footage. Behaviors such as increased locomotor activity, pawing with front hooves, lifting the tail, rolling, kicking with the hind legs toward the abdomen, and looking at the abdomen increased significantly (p < 0.001) in the four hours preceding parturition. Within the last hour of the observation period, a statistically significant change was observed for the duration of lying in the sternal and lateral position (p < 0.001). Significant correlations were observed between parity and the total number of repetitions of lying in the sternal position (p < 0.05). Furthermore, the birth process influenced the repetitions of lying in the lateral position (p < 0.05). These findings indicate distinct behavioral patterns during the opening phase of parturition, which were evident across the observed mares. Nonetheless, notable individual differences were also identified among the mares.

UNASSIGNED: A combined microbial and cytological examination of uterine samples is the main diagnostic method for endometritis in mares. This study aimed to describe a procedure for using the same uterine cytobrush (CB) for both bacteriological and cytological evaluation.
UNASSIGNED: The procedure consists of rolling the CB onto a sterilized glass slide immediately after collection and before the transfer into a sterile saline solution. In Experiment 1, a comparison between bacteriological results of the cotton swab (CS) and CB or pellet was made in 10 mares; in Experiment 2, bacteriological and cytological results were compared between different processing methods of CB in 28 mares; in other 6 mares, a CB was processed for cytology only, to investigate the reasons for the low cellularity of the pellet.
UNASSIGNED: The agreement between culture results from the CB and CS was evaluated, and a comparison between the cytological data obtained by different processing methods of CB was performed. The perfect agreement between the CB and CS microbiological results was found. The described procedure enables useful diagnostic smears for cytology. Moreover, the seeding of both the tip of CB and the saline solution used for the transport produced accurate bacteriological results.
UNASSIGNED: The protocol described in this study for the use of CB for both cytological and bacteriological analysis could be used for the diagnosis of endometritis. To maximize diagnostic sample quality, cytology slides must be prepared with meticulous care in the field to preserve cellular integrity and minimize artifacts.

Cumulus cell (CC) expansion is pivotal for oocyte maturation, during which CCs release factors that initiate paracrine signaling within the follicular fluid (FF). The FF is abundant in extracellular vesicles (EVs) that facilitate intercellular communication. Although bovine and murine EVs can control cumulus expansion, these effects have not been observed in equines. This study aimed to assess the impact of FF-derived EVs (ffEVs) on equine CC expansion, viability, and transcriptome. Cumulus-oocyte complexes (COCs) that underwent in vitro maturation (IVM) in the presence (200 µg protein/mL) or absence (control) of ffEVs were assessed for cumulus expansion and viability. CCs were isolated after 12 h of IVM, followed by RNA extraction, cDNA library generation, and subsequent transcriptome analysis using next-generation sequencing. Confocal microscopy images illustrated the internalization of labeled ffEVs by CCs. Supplementation with ffEVs significantly enhanced cumulus expansion in both compacted (Cp, p < 0.0001) and expanded (Ex, p < 0.05) COCs, while viability increased in Cp groups (p < 0.01), but decreased in Ex groups (p < 0.05), compared to the controls. Although transcriptome analysis revealed a subtle effect on CC RNA profiles, differentially expressed genes encompassed processes (e.g., MAPK and Wnt signaling) potentially crucial for cumulus properties and, consequently, oocyte maturation.

Determining whether endometrial microbial isolates are pathogens, contaminants, or even part of the \"normal\" microbiome is extremely complex, particularly given the absence of \"gold standard\" tests for endometritis. Population-level benchmarking and temporal monitoring can provide novel insights and a wider context to improve understanding. This study aimed to (i) estimate the prevalence of endometrial isolates from swabs of Thoroughbred broodmares in Newmarket, UK between 2014 and 2020; and (ii) evaluate the effects of year, mare age, and cytology findings on isolate prevalence. Generalised linear mixed models with a logit link, both null models and models using year of sampling, mare age, or cytology findings as predictors, were fitted to estimate isolate prevalence. Over the 7-year period, data were available from 18,996 endometrial-swab samples from 6050 mares on 290 premises. The overall isolate prevalence was 35.5% (95% confidence interval (CI) 33.0-37.9), and this varied significantly between years. The most prevalent isolates were β-hemolytic Streptococcus (17.9; 95% CI: 17-19) and E. coli (10.3%; 95% CI: 9.0-11.6). Isolate prevalence increased with mare age except for E. coli isolates, and with increasing category of cytology findings except for α-hemolytic Streptococcus isolates. The results provide novel estimates of isolate prevalence and highlight knowledge gaps around potential complexities in the interpretation of findings.

UNASSIGNED: In horse breeding, birth monitoring is an important factor in minimizing losses during parturition. Although different birth monitoring systems are available for this purpose, the current literature lacks systematic suitability analyses. This study aimed to address this gap in the literature. In order to achieve this, we examined a large number of foaling mares to assess the suitability of a transponder-based birth monitoring system attached to the vulva.
UNASSIGNED: Seventy warmblood mares were observed during foaling, and 86 foals were born during the foaling seasons of 2021 and 2022. Video surveillance in the foaling stable provided video recordings of births. This allowed the opportunity to assess the birth monitoring system\'s reporting accuracy. The exact times and reasons for each alarm were documented and the proportions of correctly detected births, false alarms, and unrecognized births were calculated.
UNASSIGNED: Overall, 96.5% of foalings were correctly detected using the birth monitoring system, with a sensitivity rate of 96% and a specificity rate of 91%. False alarms were primarily caused when a mare rubbed her tail against the stable walls.
UNASSIGNED: These data suggest that the tested transponder is well suited for monitoring the birth of mares. However, it is recommended that this method should be used in combination with other birth monitoring methods because not all births were detected correctly.

Introduction: Oocytes and follicular somatic cells within the ovarian follicle are altered during maturation and after exposure to culture in vitro. In the present study, we used a nontargeted metabolomics approach to assess changes in oocytes, cumulus cells, and granulosa cells from dominant, follicular-phase follicles in young and old mares. Methods: Samples were collected at three stages associated with oocyte maturation: (1) GV, germinal vesicle stage, prior to the induction of follicle/oocyte maturation in vivo; (2) MI, metaphase I, maturing, collected 24 h after induction of maturation in vivo; and (3) MIIC, metaphase II, mature with collection 24 h after induction of maturation in vivo plus 18 h of culture in vitro. Samples were analyzed using gas and liquid chromatography coupled to mass spectrometry only when all three stages of a specific cell type were obtained from the same mare. Results and Discussion: Significant differences in metabolite abundance were most often associated with MIIC, with some of the differences appearing to be linked to the final stage of maturation and others to exposure to culture medium. While differences occurred for many metabolite groups, some of the most notable were detected for energy and lipid metabolism and amino acid abundance. The study demonstrated that metabolomics has potential to aid in optimizing culture methods and evaluating cell culture additives to support differences in COCs associated with maternal factors.

Years of sire and dam selection based on their pedigree and athletic performance has resulted in a reduction in the reproductive capability of horses. Mare age is considered a major barrier to equine reproduction largely due to an increase in the age at which mares are typically bred following the end of their racing career. Nicotinamide adenine dinucleotide (NAD+) and its involvement in the activation of Sirtuins in fertility are an emerging field of study, with the role of NAD+ in oocyte maturation and embryo development becoming increasingly apparent. While assisted reproductive technologies in equine breeding programs are in their infancy compared to other livestock species such as cattle, there is much more to be learnt, from oocyte maturation to early embryo development and beyond in the mare, which are difficult to study given the complexities associated with mare fertility research. This review examines what is already known about the role of NAD+ and Sirtuins in fertility and discusses how NAD+-elevating agents may be used to activate Sirtuin proteins to improve equine breeding and embryo production programs both in vivo and in vitro.

Uterine diseases stand as the primary cause of infertility in mares; however, the diagnostic process often relies on obtaining endometrial biopsies and their hematoxylin-eosin staining. This review seeks to present the variability of uterine changes and their impact on fertility and underscore the utility of special stains, such as Masson trichrome, picrosirius red, elastica van Gieson, or periodic acid-Schiff, in enhancing diagnostic breadth. Connective tissue evaluation in the cervix is discussed, as it is subjected to cyclic changes and the impact on overall fertility. Vascular changes, particularly prevalent in multiparous mares, play a crucial role in adapting to physiological and pathological alterations, affecting early gestation and impeding placental development. Given that uterine vascular pathologies often involve fibrotic changes, connective tissue stains emerge as a valuable tool in this context. Moreover, equine endometriosis, predominantly associated with endometrial fibrosis, further highlights the relevance of special stains, suggesting their underutilization in the diagnostic process. Recognizing the subjective nature of diagnosing uterine pathologies and the need for additional diagnostic tools, we advocate for using dedicated stains in the histopathological evaluation of uterine samples. In conclusion, we encourage scientists and diagnosticians to embrace additional tools that enhance pathology visualization, enabling more reliable diagnoses concerning expected fertility.