In humans\' and experimental animals\' components of the somatotropic axis, such as growth hormone (GH) and insulin-like growth factor 1 (IGF-1) concentrations, decrease with advancing age. Although there is evidence regarding IGF-1, the effect of age on GH in mares, as well as the relationships between both parameters, have not yet been elucidated. On the other hand, although GH and IGF-1 are related to follicular development, it is unknown if they could be correlated with the circulating concentrations of ovarian steroids in mares, as occurs in other species. The hypothesis of this study was that both GH and IGF-1 could experience physiological changes with advancing age also in mares, and that both GH/IGF-1 could be correlated with oestradiol-17β (E2) and progesterone (P4), as recorded for other species. Hence, the objective of this study was to evaluate the concentrations of GH, IGF-1, E2, and P4 in mares, according to the different ages. Blood samples were drawn from 56 healthy cyclic Spanish Purebred mares belonging to four different age groups: 6-9 years, 10-13 years, 14-16 years and >16 years. Mares aged 6-9 years and 10-13 years showed higher GH concentrations (P < 0.05) than mares of 14-16 and >16 years; and mares aged 14-16 showed higher GH concentrations (P < 0.05) than >16 years (P < 0.05). Mares aged >16 years showed lower IGF-1 concentrations (P < 0.05) than mares of 6-9, 10-13 and 14-16 years (P < 0.05). The concentrations of E2 and P4 showed no significant differences among different age groups. Both GH and IGF-1 were not correlated with each other or with E2 and P4. The concentrations of E2 and P4 did not change with age. Advancing age leads to a decrease in the activity of the somatotropic axis in physiological cyclic mares, represented by a significant GH reduction, which, however, was ascribed for IGF-1 exclusively to mares over 16 years of age, without alterations in steroid hormone patterns.

OBJECTIVE: To describe the indications for and surgical technique of mastectomy of mares and to describe the outcome of 10 mares that underwent mastectomy in a retrospective case series.
METHODS: 10 mares having disease of one or both mammary glands.
METHODS: Medical records (1995 to 2022) from 2 university teaching hospitals were searched to identify mares that had undergone unilateral or bilateral mastectomy. Data regarding history, signalment, diagnostic tests, preoperative treatment, surgical procedure, and postoperative management were reviewed. Follow-up information was obtained by interviewing the owners by telephone.
RESULTS: One (n = 4 mares) or both mammary glands (6 mares) were excised for the following reasons: chronic bacterial mastitis (4), neoplasia (3), lymphangiectasia (1), pythiosis (1), and lymphoid hamartoma (1). None of the mares experienced intraoperative complications. The surgical site was closed primarily in 2 mares and left unsutured in 8 mares. Both sutured wounds developed a seroma, and 1 dehisced. The owners reported that the surgical wound, whether sutured or unsutured, was healed within 3 months. All mares returned to use for their intended purpose, but 3 mares were euthanized 2 to 4 years after surgery due to progression of disease. One mare drowned 1 year after discharge.
CONCLUSIONS: Mastectomy can be an effective treatment for mares suffering from disease of one or both mammary glands when the mare is refractory to medical treatment.

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.

The equine uterus is highly interrogated during estrus prior to breeding and establishing pregnancy. Many studies in mares have been performed during estrus under the influence of high estrogen concentrations, including the equine estrual microbiome. To date, it is unknown how the uterine microbiome of the mare is influenced by cyclicity; while, the equine vaginal microbiome is stable throughout the estrous cycle. We hypothesized that differences would exist between the equine endometrial microbiome of mares in estrus and anestrus. The aim of this study was two-fold: to characterize the resident endometrial microbiome of healthy mares during anestrus and to compare this with estrus. Double-guarded endometrial swabs were taken from healthy mares during estrus (n = 16) and in the following non-breeding season during anestrus (n = 8). Microbial population was identified using 16S rRNA sequencing. Our results suggest that the equine uterine microbiome in estrus has a low diversity and low richness, while during anestrus, a higher diversity and higher richness were seen compared to estrus. Despite this difference, both the estrus and anestrus endometrial microbiome were dominated by Proteobacteria, Firmicutes, and Bacteroidota. The composition of the microbial community between anestrus and estrus was significantly different. This may be explained by the difference in the composition of the endometrial immune milieu based on the stage of the cycle. Further research investigating the function of the equine endometrial microbiome and dynamics changes within the uterine environment is required.

Mesenchymal stromal cells (MSCs) have regenerative and immunomodulatory potential and may be used to treat injured tissues. Pregnancy has been associated with increased MSCs in the peripheral circulation in multiple species, but to date, there are no reports on this matter in horses. This study aimed to evaluate the effect of pregnancy on isolation efficiency and proliferation capacity of equine MSCs derived from the peripheral blood (PB) of mares. Venous blood samples were collected at the 11th month of gestation and 1 month after delivery from clinically healthy Arabian mares that presented normal pregnancies. Blood samples were processed for in vitro cellular culture and hormonal and metabolic profiles. MSCs were isolated and characterized by trilineage differentiation potential, immunophenotyping, analyzed by gene sequencing and proliferation assays. The isolation of peripheral blood mononuclear cells (PBMCs) of pregnant mares were associated with higher isolation efficiency and proliferative capacity of MSCs derived from peripheral blood (PB-MSCs) recovered pre-partum than those isolated post-partum. Although fetal gender, parity, 5α-reduced pregnanes, insulin, and cortisol were shown to affect cellular proliferation, individual factors and the small population studied must be considered. This study suggests that PB-MSCs from pregnant mares could be a valuable alternative source of MSCs for therapeutic purposes.

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.

To understand better the role that kisspeptin plays in regulating seasonal and estrous cycle changes in the mare, this study investigated the number, location and interactions between GnRH, kisspeptin and RFRP-3 neurons in the equine hypothalamus. Hypothalami were collected from mares during the non-breeding season, vernal transition and various stages of the breeding season. Fluorescent immunohistochemistry was used to label the neuropeptides of interest. GnRH cells were observed primarily in the arcuate nucleus (ARC), while very few labeled cells were identified in the pre-optic area (POA). Kisspeptin cells were identified primarily in the ARC, with a small number of cells observed dorsal to the ARC, surrounding the third ventricle (3V). The mean number of kisspeptin cells varied between animals and typically showed no pattern associated with season or stage of estrous cycle, but a seasonal difference was identified in the ARC population. Small numbers of RFRP-3 cells were observed in the ARC, ventromedial hypothalamus (VMH) and dorsomedial hypothalamus (DMH). The mean number of RFRP-3 cells appeared higher in pre-ovulatory animals compared to all other stages. The percentage of GnRH cell bodies with kisspeptin appositions did not change with season or stage of estrous cycle. The percentage of kisspeptin cells receiving inputs from RFRP-3 fibers did not vary with season or stage of estrous cycle. These interactions suggest the possibility of the presence of an ultra-short loop feedback system between these three peptides. The changes in RFRP-3 neurons suggest the possibility of a role in the regulation of reproduction in the horse, but it is unlikely to be as a gonadotropin inhibitory factor.