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Equine endometrosis is a degenerative and predominantly fibrotic condition resulting from progressive and irreversible multifactorial causes that influence the endometrium of mare. Tissue remodeling in the equine endometrium occurs as part of the pathogenesis of endometrosis, a process characterized by a shift in extracellular matrix (ECM) components. The relationship between matrix metalloproteinases and their specific inhibitors is crucial for the remodeling process. Collagen play a significant role in maintaining a healthy uterus and may promote fibrotic processes. The aim of this study was to quantify endometrial collagen deposition using picrosirius 25 red (PSR) staining, and to evaluate gene expression of collagen type 2 (COL-2) and 3 (COL-3), matrix metalloproteinases 1 (MMP-1) and 2 (MMP-2), their tissue inhibitor (TIMP-2), and tumor necrosis factor (TNF-α) in the endometrium of mares with different grades of fibrosis. The samples (n = 34) were classified into three categories based on the frequency and distribution of fibrosis-related changes in the endometrium: Category I (healthy endometrium, n = 12), Category II (moderate fibrosis, n = 12), and Category III (severe fibrosis, n = 10). Collagen quantification demonstrate a substantial proportional increase (P < 0.0001) in collagen deposition across Category I (11.72 ± 1.39 %), Category II (17.76 ± 1.29 %), and Category III (24.15 ± 1.87 %). In transcript evaluations, higher COL-2 expression was found in Category II than in mares classified as Category I or III. MMP-1 showed increased transcript expression in Category II compared to Category III endometrial samples. Higher expression of MMP-2 was detected in Category III than in Category I and II. TIMP-2 showed lower mRNA expression in Category III vs Category I and II. However, TNF-α gene expression was higher in Category II than in Categories I and III. This study demonstrates that endometrial evaluation using PSR can play an important role in routine analyses for the detection and objective quantification of collagen in endometrial tissues. Additionally, this study demonstrated through gene expression analysis that MMP-1 may be linked to physiological endometrial remodeling. In contrast, MMP-2 could be associated with fibrogenesis in the endometrium, which is regulated by the inhibitor TIMP-2. Furthermore, COL-2 and TNF-α could be considered as biological markers involved in the progression endometrosis in mares. As such, the results of this study may contribute to the development of future antifibrotic therapies that aim to delay or even reverse the pathological remodeling of the extracellular matrix in the uterus, in addition to optimizing the diagnosis and prognosis of endometrial fibrosis in mares. • Increased collagen accumulation in the uterine of mares with endometrosis. • Positive correlation between the animals' age and uterine collagen deposition. • Lower MMP-2 and higher TIMP-2 expression could be key for ECM reorganization. • TNF-α and COL-2 as possible markers of developing endometrosis. [ABSTRACT FROM AUTHOR]

Simple Summary: The present study aimed to detail the vaginal microbiome of mares on the post-delivery day under field conditions using the next-generation sequencing technique (NGS). This method allows for the determination of bacterial species composition at multiple levels and enables the assessment of biological diversity in the examined environment. It is believed that greater bacterial diversity is more beneficial for the physiology of the organ. The bacteria colonizing the vagina may include both saprophytic and potentially pathogenic microorganisms. Bacteria inhabiting the mucous membranes engage in constant dialogue with the host's immune system and are essential for maintaining balance on the mucous membranes. In the present study, various species of Corynebacterium spp. and Streptococcus spp. were identified, and their potential pathogenicity to the endometrium should be considered. The vaginal bacteria are critical for neonatal immunity, as well as for further infections and pathologies in foals and mares during the postpartum period. The vaginal microbiota was examined in six mares. Swabs were taken from the vaginal caudal wall within 12 h after natural delivery. Next-generation sequencing was used to analyze the composition of the vaginal microbiota and identify shared bacterial taxa. The post-delivery vaginal microbiome of mares included 18 identified families, 25 genera, and 33 species. The core families of the mares' microbiome comprised Leptotrichiaceae (21.72%), Peptostreptococcaceae/Tisserellales (15.54%), Corynebacteriaceae (13.32%), Aerococcaceae (10.84%), and Campylobacteraceae (4.26%). Shared bacterial species included Corynebacterium kutsheri, Campylobacter spp., Facklamia spp., and Oceanivirga uncultured bacteria. We found the presence of various Corynebacterium and Streptococcus species that could be involved in uterine disorders. Further research should focus on the possible pathogenicity of these bacteria for mares in the postpartum period and newborn foals. [ABSTRACT FROM AUTHOR]

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 (E 2) and progesterone (P 4), as recorded for other species. Hence, the objective of this study was to evaluate the concentrations of GH, IGF-1, E 2 , and P 4 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 E 2 and P 4 showed no significant differences among different age groups. Both GH and IGF-1 were not correlated with each other or with E 2 and P 4. The concentrations of E 2 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. • Age-related changes on GH, IGF-1, oestradiol-17β, and progesterone concentrations in mares were evaluated. • Mares aged 6–9 years and 10–13 years showed higher GH than mares of 14–16 and >16 years. • Mares aged 14–16 showed higher GH concentrations than >16 years. • Mares aged >16 years showed lower IGF-1 concentrations compared to all three age groups. • No correlations between GH, IGF-1, and ovarian steroids were observed. • Advancing age leads to a significant GH reduction which was ascribed for IGF-1 exclusively to mares >16 years [ABSTRACT FROM AUTHOR]

Background: Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. Results: Ultrasonographic monitoring and follicular fluid aspiration of preovulatory follicles in horses during the anovulatory (spring: non-breeding) and ovulatory (spring, summer, and fall: breeding) seasons and subsequent EV isolation and miRNA profiling identified significant variation in EV-miRNA cargo content. We identified 97 miRNAs with differential expression among the groups and specific clusters of miRNAs involved in the spring transition (miR-149, -200b, -206, -221, -328, and -615) and peak breeding period (including miR-143, -192, -451, -302b, -100, and let-7c). Bioinformatic analyses showed enrichments in various biological functions, e.g., transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Cluster analyses revealed distinct sets of significantly up- and down-regulated miRNAs associated with spring anovulatory (Cluster 1) and summer ovulation–the peak breeding season (Clusters 4 and 6). Conclusions: The findings from the current study shed light on the dynamics of FF-EV-coupled miRNAs in relation to equine ovulatory and anovulatory seasons, and their roles in understanding the mechanisms involved in seasonal shifts and ovulation during the breeding season warrant further investigation. [ABSTRACT FROM AUTHOR]

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. ANIMALS 10 mares having disease of one or both mammary glands. CLINICAL PRESENTATION 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 Iymphoid 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. CLINICAL RELEVANCE 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. [ABSTRACT FROM AUTHOR]

Background and Aim: In females of various species and experimental animals, iron (Fe) status in follicular fluid (FF) is associated with local physiological reproductive events related to follicle development, steroidogenesis, and oocyte maturation. However, these mechanisms remain unknown. This study aimed to determine and compare the intrafollicular and plasma concentrations of Fe, ferritin (Ferr), and transferrin (TRF) in cycling mares. Materials and Methods: Sixty ovaries were collected during the breeding season from 30 clinically normal mares raised for slaughterhouse meat production. Blood samples were collected before slaughter. Follicles were classified into three categories according to size: Small (20-30 mm; n = 20), medium (=31-40 mm; n = 20), and large (=41 mm; n = 20). The FF samples, after collection, were immediately taken to the laboratory for processing and were centrifuged, and the Fe and Ferr concentrations in the supernatant and plasma were determined by spectrophotometry. Results: Although intrafollicular Fe and Ferr were similar to plasma, TRF was significantly higher in FF than in systemic circulation (p < 0.05). Follicular development does not modify the status of Fe in the mare. Conclusions: Based on this evidence, it is possible that the acquisition of this molecule possibly originated from a local de novo source, whereas their diffusion through ultrafiltration does not play a relevant role. These results provide new scientific insights into the status of follicle Fe, suggesting its involvement in normal ovarian functions in mares. [ABSTRACT FROM AUTHOR]

Increased synthesis and deposition of collagen (COL) in the extracellular matrix (ECM) of equine endometrium contributes to endometrosis. Toll‐like receptors (TLRs) are transmembrane receptors involved in the innate immune response, recognized for their role in antigen recognition and previously associated with equine endometritis. The TLRs not only recognize pathogen‐associated molecular patterns but also regulate inflammations, fibrosis and cancer. The aim of this study was to explore the relationship between TLR expression at different stages of Kenney and Doig's (K‐D) grading and COL1 expression during the follicular phase of the oestrous cycle. Forty samples of endometrial tissues were collected post‐mortem from mares on the follicular phase of the oestrous cycle (10 samples of each K‐D category). Relative mRNA transcription of TLR‐2, TLR‐4 and COL1A2 genes was assessed using qPCR, and COL1 protein expression by Western blot analysis. The COL1A2 transcription increased in category IIB when compared to categories I, IIA and III endometria (p <.01). The relative protein abundance of COL1 showed no significant differences between endometrial categories (p >.05). As for the TLRs mRNA transcription, TLR‐2/‐4 transcripts increased in IIA when compared to the other K‐D endometria categories (p <.05). Our findings suggest that TLRs may be involved in the initiation of the endometrial inflammatory response. Additional studies are needed to explore TLRs' potential role as diagnostic markers for monitoring inflammation progression and fibrosis development, as well as their involvement in the mechanisms underlying fibrotic pathways. [ABSTRACT FROM AUTHOR]

The present study aimed to evaluate whether primed anoestrus mares are suitable recipients for embryos produced by intracytoplasmic sperm injection (ICSI). Anoestrus was confirmed in four mares and daily doses of oestradiol benzoate (6 mg in total) over 5 days were administered; after 3 days of rest, oral altrenogest was administered at 0.088 mg/kg and embryos (1 to 5 embryos per mare; 15 in total) were transferred 3.5 days after progesterone onset. Uterine lavage was conducted 48 h after transfer. The results revealed an 80% embryo recovery rate, and among the retrieved embryos, 67% showed evident intrauterine development. Hence, ICSI‐derived embryos can be successfully transferred to primed anoestrus mares, but more studies are required to ensure further embryo development and foaling. [ABSTRACT FROM AUTHOR]

Background and Aim: In females of various species and experimental animals, iron (Fe) status in follicular fluid (FF) is associated with local physiological reproductive events related to follicle development, steroidogenesis, and oocyte maturation. However, these mechanisms remain unknown. This study aimed to determine and compare the intrafollicular and plasma concentrations of Fe, ferritin (Ferr), and transferrin (TRF) in cycling mares. Materials and Methods: Sixty ovaries were collected during the breeding season from 30 clinically normal mares raised for slaughterhouse meat production. Blood samples were collected before slaughter. Follicles were classified into three categories according to size: Small (20–30 mm; n = 20), medium (≥31–40 mm; n = 20), and large (≥41 mm; n = 20). The FF samples, after collection, were immediately taken to the laboratory for processing and were centrifuged, and the Fe and Ferr concentrations in the supernatant and plasma were determined by spectrophotometry. Results: Although intrafollicular Fe and Ferr were similar to plasma, TRF was significantly higher in FF than in systemic circulation (p < 0.05). Follicular development does not modify the status of Fe in the mare. Conclusion: Based on this evidence, it is possible that the acquisition of this molecule possibly originated from a local de novo source, whereas their diffusion through ultrafiltration does not play a relevant role. These results provide new scientific insights into the status of follicle Fe, suggesting its involvement in normal ovarian functions in mares.

Abstract Background Ovarian follicular fluid (FF) is a dynamic environment that changes with the seasons, affecting follicle development, ovulation, and oocyte quality. Cells in the follicles release tiny particles called extracellular vesicles (EVs) containing vital regulatory molecules, such as microRNAs (miRNAs). These miRNAs are pivotal in facilitating communication within the follicles through diverse signaling and information transfer forms. EV-coupled miRNA signaling is implicated to be associated with ovarian function, follicle and oocyte growth and response to various environmental insults. Herein, we investigated how seasonal variations directly influence the ovulatory and anovulatory states of ovarian follicles and how are they associated with follicular fluid EV-coupled miRNA dynamics in horses. Results Ultrasonographic monitoring and follicular fluid aspiration of preovulatory follicles in horses during the anovulatory (spring: non-breeding) and ovulatory (spring, summer, and fall: breeding) seasons and subsequent EV isolation and miRNA profiling identified significant variation in EV-miRNA cargo content. We identified 97 miRNAs with differential expression among the groups and specific clusters of miRNAs involved in the spring transition (miR-149, -200b, -206, -221, -328, and -615) and peak breeding period (including miR-143, -192, -451, -302b, -100, and let-7c). Bioinformatic analyses showed enrichments in various biological functions, e.g., transcription factor activity, transcription and transcription regulation, nucleic acid binding, sequence-specific DNA binding, p53 signaling, and post-translational modifications. Cluster analyses revealed distinct sets of significantly up- and down-regulated miRNAs associated with spring anovulatory (Cluster 1) and summer ovulation–the peak breeding season (Clusters 4 and 6). Conclusions The findings from the current study shed light on the dynamics of FF-EV-coupled miRNAs in relation to equine ovulatory and anovulatory seasons, and their roles in understanding the mechanisms involved in seasonal shifts and ovulation during the breeding season warrant further investigation.

Bacterial endometritis is among the most common causes of subfertility in mares. It has a major economic impact on the equine breeding industry. The sensitivity of detecting uterine microbes using culture-based methods, irrespective of the sample collection method, double-guarded endometrial swab, endometrial biopsy, or uterine low-volume lavage (LVL), is low. Therefore, equine bacterial endometritis often goes undiagnosed. Sixteen individual mares were enrolled, and an endometrial sample was obtained using each method from all mares. After trimming, quality control and decontamination, 3824 amplicon sequence variants were detected in the dataset. We found using 16S rRNA sequencing that the equine uterus harbors a distinct resident microbiome during estrus. All three sampling methods used yielded similar results in composition as well as relative abundance at phyla (Proteobacteria, Firmicutes, and Bacteroidota) and genus (Klebsiella, Mycoplasma, and Aeromonas) levels. A significant difference was found in alpha diversity (Chao1) between LVL and endometrial biopsy, suggesting that LVL is superior at detecting the low-abundant (rare) taxa. These new data could pave the way for innovative treatment methods for endometrial disease and subfertility in mares. This, in turn, could lead to more judicious antimicrobial use in the equine breeding industry.

In the years 2022 and 2023 we investigated the prevalence of the ovulatory failure in 133 mares of different breed, 3 to 24 years old, brought to the Equine clinic of the University of Veterinary Medicine and Pharmacy (UVMP) in Košice. Our research involved the monitoring of the reproduction cycle in these mares with the aim to determine the prevalence of ovulatory failure during the breeding season, focusing on certain specific parameters that were considered sufficiently relevant with respect to the development of such failure. These parameters included environmental temperature and the amount of daylight the mares were exposed to, attributable to the months of the breeding season. In addition, we also evaluated the size of pre-ovulatory follicles and the influence of age of the investigated mares. During the mentioned years, one of the ovulatory failures occurred in 17 out of 133 mares (12.78 %). Examination by ultrasonography (USG) and the subsequent analysis of the occurrence of failures in relation to the investigated parameters allowed us to conclude that the length of daylight, age, and environmental temperature were the most important factors. Our observations indicated that the 15–16 h category of daylight presented the highest risk of development of cycle disorders with the most rapid increase observed for HAF. With respect to age, the highest occurrence of ovulatory failures was observed in the category of 15–24 years. The external temperature in the range of 18.1–23.0 °C affected the most the development of cycle disorders. Again, the effect was most evident with respect to HAF. This resulted in our recommendations for horse breeders that they should manage reproduction of their mares in a way so that the receptive phase of the cycle does not occur in the months during which the daylight length and environmental temperatures support ovulatory failure. We also recommend that if the owner′s wish is to keep in the breeding the mares older than 15 years, regular thorough gynaecological examination of these animals should be ensured, aimed at early diagnosis of ovulatory failure.

Assisted reproductive technologies (ARTs) are performed worldwide in the equine industry to produce genetically valuable foals. Among them, ovum pick up (OPU) combined with intra‐cytoplasmic sperm injection (ICSI) can now be more efficient than embryo transfer (ET) under optimal conditions. However, OPU is not a benign procedure for the mare and the process is costly. Improved efficiency is therefore in the interest of everyone, maximizing mare welfare and optimizing economics for the client. One of the key factors of success is the antral follicle count (AFC) at the time of OPU and subsequently the number of oocytes obtained. Variations in AFC are reported between individuals and between geographical areas. This leads to a significant increase in numbers of embryos produced per session in some countries compared to others, independent of the laboratory efficiency. This article revisits the basics of folliculogenesis involved in establishment of the antral follicle population and explores work in other species given the paucity of equine research in this area. The aim of the review is to elucidate interesting areas of further research that could generate essential information for clinicians and clients about the management and selection of the donor mare for OPU and potentially identify pharmacological targets for manipulation. [ABSTRACT FROM AUTHOR]

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. • Lack of reports onthe isolation of mesenchymal stromal cells (MSCs) from peripheral blood (PB-MSCs) in pregnant mares. • Pregnancy in Arabian mares was associated with higher isolation efficiency and proliferative capacity of PB-MSCs. • Fetal gender, parity, and levels of 5α-reduced pregnanes, insulin and cortisol were shown to affect cellular proliferation. • Pregnant mares could be a valuable, readily available source of MSCs for therapeutic purposes. [ABSTRACT FROM AUTHOR]

Objective: Leptospirosis is a worldwide zoonotic disease and well recognized infectious disease of horses. Equine leptospirosis is to cause the birth of weak foals, neonatal deaths and abortion after pregnancy period. Studies on leptospirosis in horses are generally investigations of urine samples and serological studies. Although leptospirosis causes reproductive disorders, genital sample studies that may be a source of infection have been ignored. The aims of this study were to study the prevalance of Leptospira by PCR using vaginal swab from apparently healthy horses. Methods: A total of 92 vaginal swab samples were collected and transferred to the bacterial diagnosis laboratory of Selcuk University Veterinary Faculty. The vulva and vagina were cleaned before swab samples were taken. All samples were stored in the refrigerator at -20°C and taken to the laboratory for processing. These samples were sent to the laboratory under cold chain conditions. DNA was extracted from suspicious samples and conventional PCR was used to detect Leptospira spp. Specific primers were selected and PCR was finalized for Leptospira spp. Results: As a result of the study, it was found that out of 92 mare's vaginal swab samples 7 (7.6%) were positive for Leptospira spp. This study is the first report from our country due to the detection of Leptospira spp DNA in asymptomatic mares. Conclusion: It revealed that the Leptospira PCR positive mare were not showing any signs and symptoms. When the results and observations were evaluated, it was thought that Leptospira PCR positive mares could play a role as a carrier in the transmission of leptospirosis. Our study is one of the rare studies on mares carrying the possible causative agent. Detection of leptospirosis by PCR can be considered as a reliable method for early detection of Leptospira shedding in asymptomatic animals. In addition, molecular studies from vaginal swab samples were observed as a rapid and definitive diagnostic option, considering the difficulty of isolation of the Leptospira agent and possible contaminations. According to the results of our study, it is recommended to reevaluate the control measures against the disease and to carry out molecular characterization and vaccination studies in risky areas. [ABSTRACT FROM AUTHOR]

Background: Transvaginal ultrasound‐guided aspiration (TUA) is used for post‐fixation twin reduction in mares. However, there is limited information regarding factors that influence pregnancy outcome after TUA. Objectives: To evaluate the effect of day of gestation on which TUA is performed, aspiration volume, puncture of the conceptus, medication administered before and after TUA, embryo location, mare age and parity and operator experience on pregnancy and foaling rates after TUA. Study design: Retrospective case series. Methods: Data were collected from case records of 464 TUAs performed by 14 operators in 422 mares diagnosed pregnant with dizygotic twins in two different facilities between 2010 and 2019. Pregnancy status was determined by ultrasonography at 5–7 days and 3–4 weeks after the TUA was performed. Subsequent pregnancy and foaling results were obtained by follow‐up communication. The effects of mare, gestation‐ and TUA‐related variables on pregnancy and foaling rates were analysed by the chi‐square‐test for homogeneity and Fisher's exact test and logistic regression. Results: TUA was performed between 21 and 82 days of gestation in unilaterally (267/359 [74.4%]) and bilaterally fixed (92/359 [25.6%]) twin pregnancies. A singleton pregnancy (218/381 [57.2%]), persistent twin pregnancy (60/381 [15.8%]), or the loss of both conceptuses (103/381 [27%]) was confirmed 5–7 days after TUA was performed. At 3–4 weeks post TUA 50.3% (163/324) of mares were diagnosed with a single viable pregnancy and 40.1% (127/317) went on to deliver a live single foal. TUA performed early in gestation (D 25–35) resulted in the birth of a live singleton foal in 49.3% (74/150) of mares. Main limitations: Missing retrospective data despite extensive follow‐up. Conclusion: This is the first large scale study to demonstrate that acceptable pregnancy and foaling rates can be achieved in mares diagnosed with twins when TUA is performed early in gestation (<40 days). [ABSTRACT FROM AUTHOR]

Background: Excessive inbreeding increases the probability of uncovering homozygous recessive genotypes and has been associated with an increased risk of retained placenta and lower semen quality. No genomic analysis has investigated the association between inbreeding levels and pregnancy loss. Objectives: To compare genetic inbreeding coefficients (F) of naturally occurring Thoroughbred Early Pregnancy Loss (EPLs), Mid and Late term Pregnancy Loss (MLPL) and Controls. The F value was hypothesised to be higher in cases of pregnancy loss (EPLs and MLPLs) than Controls. Study design: Observational case–control study. Methods: Allantochorion and fetal DNA from EPL (n = 37, gestation age 14–65 days), MLPL (n = 94, gestational age 70 days–24 h post parturition) and Controls (n = 58) were genotyped on the Axiom Equine 670K SNP Genotyping Array. Inbreeding coefficients using Runs of Homozygosity (FROH) were calculated using PLINK software. ROHs were split into size categories to investigate the recency of inbreeding. Results: MLPLs had significantly higher median number of ROH (188 interquartile range [IQR], 180.8–197.3), length of ROH (3.10, IQR 2.93–3.33), and total number of ROH (590.8, IQR 537.3–632.3), and FROH (0.26, IQR 0.24–0.28) when compared with the Controls and the EPLs (p < 0.05). There was no significant difference in any of the inbreeding indices between the EPLs and Controls. The MLPLs had a significantly higher proportion of long (>10 Mb) ROH (2.5%, IQR 1.6–3.6) than the Controls (1.7%, IQR 0.6–2.5), p = 0.001. No unique ROHs were found in the EPL or MLPL populations. Main limitations: SNP‐array data does not allow analysis of every base in the sequence. Conclusions: This first study of the effect of genomic inbreeding levels on pregnancy loss showed that inbreeding is a contributor to MLPL, but not EPL in the UK Thoroughbred population. Mating choices remain critical, because inbreeding may predispose to MLPL by increasing the risk of homozygosity for specific lethal allele(s). [ABSTRACT FROM AUTHOR]

Background: Despite being a large commercial breeding industry, there is little published data on the reproductive success of Standardbred mares. Objectives: To quantify the reproductive performance of Standardbred mares under artificial breeding systems in a commercial setting and determine the incidence of early embryonic and other pre‐partum losses. Study design: Retrospective cohort study. Methods: Data from four commercial farms were collected across four breeding years, and all mares were bred via artificial insemination. A total of 3995 mares contributed 7229 mare years. First‐cycle pregnancy rate (FCPR) and end of season pregnancy rate (SPR) were analysed in mixed‐effects logistic regression models. Time‐to‐conception interval was analysed in a Cox regression model. Results: The mean FCPR was 61.4% (confidence interval [CI] 60.3%–62.6%), the mean end of SPR was 84.7% (CI 83.8–85.5%), the mean live foal rate (FR) was 73.1% (CI 72.1%–74.2%). Mares located on‐farm were more probable to be pregnant in terms of both FCPR (odds ratio [OR] 1.168, CI 1.018–1.340, p = 0.026) and SPR (OR 2.026, CI 1.673–2.454, p < 0.001), mares inseminated with thawed–frozen semen were less probable to be pregnant in terms of FCPR (OR 0.598, CI 0.457–0.783, p < 0.001) and SPR (OR 0.479, CI 0.354–0.647, p < 0.001) compared with insemination with fresh‐extended semen. Older mares (14 years and older) were less probable to be pregnant in terms of FCPR (OR 0.795, CI 0.688–0.919, p = 0.002) and SPR (0.435, CI 0.352–0.538, p < 0.001) compared with young mares (3‐ to 8‐year old). Main limitations: Retrospective data relied on accurate record keeping of stud farms and no mare‐treatment or ovulation induction records were available. Live FRs relied mostly on annual foaling returns so fetal/foal deaths may be underrepresented. Conclusion: This study provides substantial baseline data on reproductive performance for Standardbred mares managed under a commercial artificial breeding system. [ABSTRACT FROM AUTHOR]

IntroductionProstaglandins (PG) are important regulators of the myometrial contractility in mammals. Endometrosis, a condition characterized by morphological changes in the equine endometrium, also affects endometrial secretory function. However, it remains unclear whether and how endometrosis affects myometrial function.MethodsThis study investigated: (i) mRNA transcription of genes encoding specific enzymes responsible for PG synthesis, such as prostaglandin—endoperoxide synthase (PTGS2), PGE2 synthase (PTGES), PGF2α synthase (PTGFS) and PG receptors: PGE2 receptors (PTGER1- 4), and PGF2α receptor (PTGFS) in equine myometrium and, (ii) the effects of PGE2 and PGF2α on myometrial contractile activity, during endometrosis in mares. The myometria used in experiments 1 and 2 were collected from mares in the mid-luteal (n = 23) and follicular (n = 20) phases of the estrous cycle, according to the histological classification of the endometrium (Kenney and Doig categories I, IIA, IIB, and III).ResultsIn experiment 1, changes in mRNA transcription of PG synthase or PG receptors in the myometrium during the course of endometrosis were determined using qPCR. During the mid-luteal phase, myometrial mRNA transcription of PTGES increased in mares with endometrial category IIB compared to category I. However, myometrial mRNA transcription of PTGER1 decreased during the progression of endometrosis compared to category I. During the follicular phase, mRNA transcription of PTGER1 and PTGER2 increased in mares with endometrial categories III or IIA, respectively. In addition, mRNA transcription of PTGFS increased in mares with endometrium category IIA compared to category I. In experiment 2, the force of myometrial contractions was measured using an isometric concentration transducer. In the follicular phase, PGE2 decreased the force of contractions in mares with endometrial categories IIA, IIB, and III compared to the respective control groups. Prostaglandin F2α increased the force of myometrial contractions in mares with category IIA endometrium, whereas it decreased in category IIB compared to the respective control groups.DiscussionWe concluded that in the progression of endometrosis there are changes in the myometrial transcription of mRNA encoding PG synthases and receptors, particularly PTGER1 and PTGER2. Mares with endometrosis had abnormal myometrial contractile responses to PG. These findings suggest that myometrial function may be compromised during the progression of endometrosis.

Background: 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. Objectives: 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). Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

A 9-year-old Thoroughbred mare with normal external genitalia and regular oestrus symptoms was gynecologically examined prior to insemination. This primary examination revealed the presence of a hypoplastic uterus and the lack of normal ovaries, and the mare was therefore subjected to more detailed diagnostics, including endocrinological, genetic, and clinical tests. Diagnostic imaging with the use of ultrasonography and endoscopy confirmed the underdevelopment of internal genitalia. Analysis of circulating sex hormones revealed very low concentrations of progesterone and oestradiol. Finally, cytogenetic analysis showed the presence of non-mosaic X trisomy (65,XXX), an aneuploidy of sex chromosomes that is rarely detected in horses. This finding was also confirmed by molecular methods, including highly sensitive droplet digital PCR (ddPCR) and microsatellite markers genotyping. Our study reveals the need for gynaecological and genetic evaluation of broodmares, even if their phenotype (including developed external genitalia and oestrus symptoms) shows no signs of potential abnormalities. [ABSTRACT FROM AUTHOR]

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. • Study investigated the number, location and interactions between GnRH, kisspeptin and RFRP-3 neurons in the hypothalamus of the mare. • Compared numbers across the estrous cycle and between seasons. [ABSTRACT FROM AUTHOR]

Infectious endometritis is considered one of the major causes of infertility and it can affect up to 60% of barren mares. It is characterized by the presence of one or more microorganisms in the reproductive tract and it is treated with the administration of antibiotics, ecbolic agents and uterine lavages. Ozone, thanks to its antimicrobial properties that are based on its high oxidative potential, could represent an effective alternative treatment for endometritis. The aim of this study was to test in vitro the bactericidal and fungicidal properties of different ozone formulations, either as gas (experiment 1) or dissolved in two liquid matrices (experiment 2), specifically distilled water or oil (Neozone 4000, Cosmoproject, Parma, Italy), onto 6 different species of microorganisms isolated from mares with clinical endometritis, namely Escherichia coli, Staphylococcus aureus, Streptococcus equi subsp. Zooepidemicus, Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans. In the first experiment, 3 clinical antibiotic-resistant strains per each species were exposed to different conditions: to O 2 O 3 gas mixtures (15 and 40 μg/ml for 1, 3 and 5 min), to 100 % O 2 or left untreated. The results showed a reduction of the microbial count of over 99,9% for every pathogen, time and concentration of O 2 O 3 gas mixtures tested. Furthermore, gaseous ozone showed both a time-dependant effect (5 vs 3 vs 1 min of exposure) and a concentration-dependant effect (40 vs 15 μg/ml) at 1 and 3 min, while after 5 min no differences were observed. In the second experiment, minimum inhibitory concentration (MIC), and minimum bactericidal/fungicidal concentration (MBC, MFC) of ozonated distilled water and ozonated oil were evaluated. Ozonated oil showed a bactericidal/fungicidal activity against all the strains tested (MIC range 12.5–25 % v/v , MBC/MFC range 12.5–50 % v/v) while ozonated distilled water didn't show an observable antimicrobial effect, discouraging its use as an antimicrobial agent for the treatment of endometritis. The results of this in vitro study indicate that both gaseous ozone and ozonated oil exerted remarkable antimicrobial activities and are promising alternative treatments for infectious endometritis, even when caused by antibiotic-resistant bacteria, and encourage further experiments in an effort to scale down or even prevent the use of antibiotics in equine reproduction. • Microorganisms isolated from mares with endometritis were tested in vitro. • O 2 O 3 gas mixture and ozonated oil showed a potent in vitro antimicrobial activity. • Ozonated distilled water was not effective in vitro as an antimicrobial agent. • O 2 O 3 gas mixture and ozonated oil were effective on antibiotic-resistant bacteria. [ABSTRACT FROM AUTHOR]

Simple Summary: Birth monitoring in the mare is crucial for preventing potential losses. To enhance the efficacy of birth monitoring and facilitate the development of monitoring programs rooted in behavioral parameters, we analyzed mare behavior during stage I of parturition. Our investigation revealed distinct behavioral patterns among mares, highlighting considerable variability among individuals. Interestingly, we observed correlations between parity, the birth process, and behavior during stage I of parturition, suggesting significant influences on the mare's behavior during this critical period. 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. [ABSTRACT FROM AUTHOR]

Background and Aim: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

The vaginal bacteria are critical for neonatal immunity, as well as for further infections and pathologies in foals and mares during the postpartum period. The vaginal microbiota was examined in six mares. Swabs were taken from the vaginal caudal wall within 12 h after natural delivery. Next-generation sequencing was used to analyze the composition of the vaginal microbiota and identify shared bacterial taxa. The post-delivery vaginal microbiome of mares included 18 identified families, 25 genera, and 33 species. The core families of the mares’ microbiome comprised Leptotrichiaceae (21.72%), Peptostreptococcaceae/Tisserellales (15.54%), Corynebacteriaceae (13.32%), Aerococcaceae (10.84%), and Campylobacteraceae (4.26%). Shared bacterial species included Corynebacterium kutsheri, Campylobacter spp., Facklamia spp., and Oceanivirga uncultured bacteria. We found the presence of various Corynebacterium and Streptococcus species that could be involved in uterine disorders. Further research should focus on the possible pathogenicity of these bacteria for mares in the postpartum period and newborn foals.

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. [ABSTRACT FROM AUTHOR]

Background and Aim: 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. Material and Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

Simple Summary: There is a lack of understanding as to whether the bacteria isolated from swabs of the surface of the lining of broodmares' uteri (endometrial swabs) are present due to infection or contamination, or whether they are, in fact, part of the normal flora. This study aimed to understand this further by investigating endometrial swabs taken from a population of Thoroughbred broodmares and submitted to a laboratory in the UK between 2014 and 2020; by describing bacterial prevalence (the estimated percentage of swabs from which bacteria are isolated); and by evaluating whether bacterial prevalence changed over time or between mares of different ages and with differing amounts of inflammation of the uterine lining. Over the 7-year period, 18,996 endometrial swabs were submitted from 6050 mares on 290 farms. The overall prevalence of bacteria was 35.5%. Beta-hemolytic Streptococcus (17.9%) and E. coli (10.3%) were the most prevalent types of bacteria. The prevalence of bacteria changed over time (between different years in which mares were sampled) and was also impacted by the mare age and by whether there was inflammation of the uterine lining at the time of sampling. Results from this study provide up-to-date prevalence estimates and suggest that the interpretation of Thoroughbred endometrial swab findings is complicated. 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. [ABSTRACT FROM AUTHOR]

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 tomass 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. [ABSTRACT FROM AUTHOR]

Simple Summary: The reproductive capacity of mares has suffered drastically as a result of years of selection based on athletic performance. The world of equine reproduction is now trying to catch up. Recent studies examining niacin deficiencies in women have indicated that nicotinamide adenine dinucleotide (NAD+) is vital for embryo and foetal development, with further animal models showing improvements to oocyte quality with the treatment of NAD+ precursors. Given the enormous benefits shown in these studies, these results show great promise in improving reproductive outcomes in mares. 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. [ABSTRACT FROM AUTHOR]

Equine endometrial degeneration is diagnosed by microscopy of an endometrial biopsy but it is uncertain if findings in a single biopsy represent the state of the entire endometrium. Previous studies have compared samples from multiple sites but conclusions are inconsistent. Further clarification is therefore needed. In this study, presence and characteristics of endometrial degeneration were compared in two full thickness specimens from the ventral base of both uterine horns, obtained post mortem from cyclic mares (n = 82). Hematoxylin and eosin-stained sections were blinded evaluated for periglandular fibrosis, glandular nests, dilated glands, glandular dilation, excessive lymphatic vessels, and lymphatic lacunae. Each section was also assigned to a modified Kenney and Doig categorization. Statistical analysis included multiple logistic regression models for fraction of mares with disagreement in the modified Kenney and Doig category and fraction of mares with disagreement for each parameter, multiple generalized linear regression models for fraction of parameters with disagreement, and equivalence tests for agreement of a cumulative score of degenerative parameters. Possible effect of age, mare type and estrous cycle stage were included in the analyses. For the Kenney and Doig categorization, 49% of the mares had good agreement, 46% had moderate agreement and 5% had poor agreement between the two biopsies. Disagreement in the Kenney and Doig categorization between the two biopsies increased with age (OR = 1.1, P = 0.009). Disagreement within each parameter ranged from 10% of mares (glandular nests), to 31% (periglandular fibroblasts) and 37% (lymphatic lacunae). Disagreement for changes in endometrial glands increased with age (OR = 1.15–1.16, P < 0.040). Several mares (33%) had disagreement in at least two parameters, and the number of parameters with disagreement increased with age (HR = 1.04, P = 0.010). When tested for equivalence, the cumulative scores for paired endometrial samples did not differ for mares younger than 18 years (P = 0.011). In conclusion, the degenerative changes were not uniformly distributed, revealed by a disagreement between paired endometrial samples for individual parameters and for the Kenney and Doig categorization, which increased with age. This suggests that conclusions based on a single biopsy, particularly in mares over 17 years of age, should be interpreted with caution, and evaluation of two biopsies should be considered. • Degenerative changes in the equine endometrium are not uniformly distributed. • Disagreement in Kenney & Doig categorization was observed in all age groups. • The risk of disagreement between paired endometrial samples increases with age. [ABSTRACT FROM AUTHOR]

Simple Summary: Uterine diseases are the leading cause of infertility in mares, causing increasing costs and losses in horses' breeding. Their current diagnosis is often supported by obtaining endometrial biopsies and hematoxylin–eosin staining, which is the basic staining used in histopathology. This review aims to show the variety of uterine changes affecting fertility and highlights the usefulness of special stains for connective tissue visualization—Masson trichrome, picrosirius red, elastica van Gieson, or periodic acid–Schiff—for a more comprehensive diagnosis. The fibrosis evaluation includes connective tissue changes in the cervix, the endometrium, and around/in the wall of blood vessels. Cervical connective tissue undergoes cyclic changes impacting fertility, whereas vascular changes, especially in multiparous mares, are crucial for adapting to physiological shifts, affecting early pregnancy and hindering placental development. Special stains are valuable for the identification of structural changes in the cervix and fibrosis in uterine blood vessels. Moreover, equine endometrosis, linked to fibrotic processes in the endometrium, emphasizes the need for wider use of special stains in diagnosis. Therefore, we advocate for special staining in reproductive tract evaluation due to its simplicity, accessibility, and effectiveness. We encourage scientists and diagnosticians to adopt additional tools for clearer pathology visualization, enabling reliable fertility predictions. 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. [ABSTRACT FROM AUTHOR]

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The aim of the present study was to develop a semi-quantitative urine pregnancy test for mares based on the Cuboni reaction and to verify the reliability of this test. The urine specimens were hydrolyzed by heating in the presence of hydrochloric acid. The resulting free estrogens were extracted from the urine matrix using toluene. Sulfuric acid was added to the toluene extract and the mixture was heated again. The lower layer in the test tube containing sulfuric acid was used for fluorescence measurements with excitation at 355 nm and measurement at 535 nm. The fluorometric Cuboni test revealed that the fluorescence counts in urine samples collected after the second trimester of gestation were significantly higher than those obtained from barren mares. The levels of estrogens, including equilin, estrone and estardiol-17ß exhibited a dose-dependent increase in fluorescence counts, whereas other steroids, such as progesterone, testosterone, and cortisol, did not affect fluorescence. Heat treatment of urine samples with hydrochloric acid significantly increased the fluorescence counts in those collected after the second trimester of gestation compared to non-pregnant samples, implying the presence of large amounts of conjugated estrogens in pregnant mare urine. Fluorescence counts in urine samples obtained during pregnancy showed a positive relationship with estrone concentrations as measured by enzyme immunoassay. The results of the present study showed that the fluorometric Cuboni test facilitates urine fluorescence counts depending on the urinary estrogen content and is capable of discriminating between pregnancy and non-pregnancy states beyond the second trimester of gestation in mares. [ABSTRACT FROM AUTHOR]

Abstract Background 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. Objectives 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). Methods 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

Resumo Este artigo trata das relações de parentesco tecidas por mulheres nas atividades cotidianas de mariscagem (coleta/captura de mariscos e crustáceos). O foco central da análise é a produção de parentes entre as casas e a maré, mediante as trocas cotidianas e ancestrais de um saber-fazer: a mariscagem. Com base na etnografia realizada entre as marisqueiras da ilha de Matarandiba (BA), procurou-se entender em que medida a maré, no seu sentido temporal, espacial e biossocial, pode compor a conjunção casa/corpo. Analisa-se como as relações de parentesco são tecidas a partir de uma circulação que acontece entre a casa e a maré por meio do ritmo da mariscagem. Propõe-se uma narrativa baseada na trajetória das comadres marisqueiras, focando principalmente nas relações afetivas enraizadas por elas no mangue.

Ensuring fast and efficient hemostasis is crucial for achieving optimal outcomes in laparoscopic ovariectomy surgery. This study compared the clinical outcomes of standing laparoscopic ovariectomy for medium-sized granulosa cell tumors (≤15 cm in size) using a 2-port LigaSure versus a 3-port bipolar electrode, focusing on operating time, mean blood loss, intraoperative and postoperative complications, and the duration of the prospective hospital stay. Twelve mares were divided into two groups: six underwent standing laparoscopic ovariectomy with LigaSure through a 2-port approach, while the remaining six underwent the standard 3-port procedure with the bipolar electrode. Our findings demonstrated that 2-port laparoscopic ovariectomy using LigaSure was not only technically feasible and safe but also offered several advantages, including shorter operating times, simplified procedures, decreased postoperative analgesic requirements, and improved cosmetic appearance of surgical wounds. Moreover, this technique proved to be a reliable method for achieving hemostasis of the mesovarium while also being technically straightforward, time-saving, and cost-effective. Overall, our study suggests that 2-port laparoscopic ovariectomy with LigaSure is a promising alternative to the standard 3-port approach. This approach not only benefits patients by potentially reducing postoperative discomfort and enhancing recovery but also provides advantages for surgeons in terms of efficiency and resource utilization.

Equine endometrosis is one of the major causes of equine infertility, often associated with signs of periglandular fibrosis occurring in the endometrium. As the oviduct is a key location of early embryo development, we hypothesized that disturbances in hormonal action may be associated with a loss of contact between mucosal cells in the uterus and oviducts. This study investigates the expression of intermediate filament proteins (vimentin and desmin), oestrogen receptor alpha (ERα), and progesterone receptor A (PRA) in the stroma and epithelia of the endometria and oviducts of healthy mares and mares with endometrosis. Endometrium samples were obtained from 56 mares, culled in a slaughterhouse, and were designated either healthy endometrium (n = 26) or endometrium with signs of moderate to severe endometrosis (n = 20). The phase of the ovarian cycle and the expression of vimentin, desmin, ERα, and PRA were compared between these groups. The expressions of both vimentin and desmin were higher in the endometrial stroma of the endometrosis group than in healthy endometria. The expression of ERα in the healthy endometrial stroma and luminal epithelium during the follicular phase was higher than in the luteal phase, but showed no differences in the endometrium of mares with endometrosis. PRA expression was significantly higher in the stoma oviduct during the follicular phase than in the luteal phase. It seems possible that mucosal cells may support the occurrence of moderate to severe endometrosis through their altered intermediate filament and sex steroid receptor interactions. In addition, the oviduct appears to be less sensitive to hormonal changes than the uterus. Changes in the expression of intermediate filament and steroid hormone proteins may be a leading factor contributing to the functional status of the oviduct in endometrosis.

The aim of the present study was to develop a semi-quantitative urine pregnancy test for mares based on the Cuboni reaction and to verify the reliability of this test. The urine specimens were hydrolyzed by heating in the presence of hydrochloric acid. The resulting free estrogens were extracted from the urine matrix using toluene. Sulfuric acid was added to the toluene extract and the mixture was heated again. The lower layer in the test tube containing sulfuric acid was used for fluorescence measurements with excitation at 355 nm and measurement at 535 nm. The fluorometric Cuboni test revealed that the fluorescence counts in urine samples collected after the second trimester of gestation were significantly higher than those obtained from barren mares. The levels of estrogens, including equilin, estrone and estardiol-17β exhibited a dose-dependent increase in fluorescence counts, whereas other steroids, such as progesterone, testosterone, and cortisol, did not affect fluorescence. Heat treatment of urine samples with hydrochloric acid significantly increased the fluorescence counts in those collected after the second trimester of gestation compared to non-pregnant samples, implying the presence of large amounts of conjugated estrogens in pregnant mare urine. Fluorescence counts in urine samples obtained during pregnancy showed a positive relationship with estrone concentrations as measured by enzyme immunoassay. The results of the present study showed that the fluorometric Cuboni test facilitates urine fluorescence counts depending on the urinary estrogen content and is capable of discriminating between pregnancy and non-pregnancy states beyond the second trimester of gestation in mares.

Background and Aim: 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. Materials and Methods: 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. Results: 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. Conclusion: 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.

The investigation of acute phase proteins in veterinary medicine has opened the doors towards the identification and use of new markers for a timely assessment of health status in both companion and food-producing animals. The aim of this paper is to review the literature available on the use of serum amyloid A (SAA), an acute phase protein, for the diagnosis and monitoring of reproductive disorders in animals. This review critically appraises the usefulness of such marker in clinical practice and summarizes the current state of knowledge. Recent advances in the diagnosis and monitoring of reproductive diseases are presented, highlighting where SAA evaluation may enhance early diagnostic tools for dogs, cats, cattle, and equines.

A cidade de Vigia de Nazaré, nordeste paraense, está situada em uma sub-região da Zona Costeira Amazônica, a Zona Costeira Paraense (ZCP), assentada nos níveis mais baixos dos terraços regionais e em planícies fluviomarinhas. Etapas da formação socioespacial da cidade evidenciam a problemática aqui analisada: a anexação de planícies fluviomarinhas ao contexto urbano, servindo para o assentamento espontâneo de diversas famílias, em sua maioria em situação de vulnerabilidade social. O objetivo é compreender, sob a perspectiva histórico-geográfica e da vulnerabilidade inerente, uma análise integrada dos processos físicos e sociais que expuseram e criaram no ambiente urbano espaços de vulnerabilidade ao risco hidrometeorológico. A análise dos dados revelou altos acumulados de precipitação anual para o contexto regional, com um período chuvoso bem definido, que duram de janeiro a maio (acima de 300 mm em média), posto em um ambiente sob influência de macromarés semidiurnas, podendo alcançar 4,5 m. Para o ano de 2022, foram verificados 60 registros de maré iguais ou acima de 4,2 m, nível de alerta local para coincidência com chuva. A ocupação recente deu-se em sua maior parte no nível morfológico de menor altimetria, a planície fluviomarinha urbanizada alcançou 1/3 do núcleo urbano. Este local, exposto à dinâmica do ambiente costeiro estuarino, exprime atualmente aspectos da degradação ambiental e alteração dos processos hidromorfodinâmicos.

Background and Aim: The adhesive progestin patch was investigated for estrus synchronization in mares because this method is convenient and safe in other species. This study aimed to compare the efficacy of a transdermal progestin patch versus oral altrenogest on estrus synchronization, preovulatory follicle development, and ovulation. Materials and Methods: Twenty-four broodmares were randomly divided into two groups. In Group 1, mares (n = 12) were fed 0.044 mg/kg altrenogest daily for 14 days. Group 2 (n = 12) mares were treated with adhesive transdermal progestin patches for 14 days. Mares were observed for estrus daily, from day 3 until day 7, after hormone withdrawal. Follicular development was determined by ultrasonography. The preovulatory follicle size and ovulation time after hormone treatments and ovulation rate were determined for both groups. Plasma progesterone levels were measured during the experimental periods and 2 days after hormone termination in both groups. Results: The results revealed that the transdermal progestin patch efficiently controlled follicular growth and estrus synchronization in mares. The percentage of mares exhibiting estrus was similar in the altrenogest (100.00%) and transdermal patch (91.67%) groups. Ovulation rates were equivalent with either altrenogest or progestin patch protocols (91.67% [11/12] vs. 83.33% [10/12]). In addition, the preovulatory follicle size was similar in mares treated with altrenogest and progestin patches. Conclusion: This study revealed that the transdermal progestin patch provides effective estrus synchronization and ovulation, similar to altrenogest treatment. However, the transdermal patch was more convenient with a shorter predictable ovulation time after estrus synchronization and should be considered as an alternative method for mares.

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.

Background and Aim: 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. Materials and Methods: 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. Results: 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. Conclusion: 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. [ABSTRACT FROM AUTHOR]

The application of trans-vaginal ovum pick up (OPU) and intracytoplasmic sperm injection (ICSI) is well established for commercial in vitro embryo production in horses. These assisted reproductive techniques are especially applied during the non-breeding season of the mare. However, little is known about how the health of the oocyte donor may affect the biochemical composition of the follicular fluid (FF) in small and medium-sized follicles routinely aspirated during OPU. This study aimed to investigate associations between systemic and FF concentrations of interleukin-6 (IL-6), total cholesterol, triglycerides, non-esterified fatty acids (NEFA), reactive oxygen metabolites (d-ROMs), biological antioxidant potential (BAP), and oxidative stress index (OSI) during the non-breeding season in mares. At the slaughterhouse, serum and FF of small (5–10 mm in diameter), medium (> 10–20 mm in diameter), and large (> 20–30 mm in diameter) follicles were sampled from 12 healthy mares. There was a strong positive association (P < 0.01) between the concentration of IL-6 in serum and those measured in small (r = 0.846), medium (r = 0.999), and large (r = 0.996) follicles. Serum concentrations of NEFA were positively correlated (P < 0.05) with those measured in small (r = 0.726), medium (r = 0.720), and large (r = 0.974) follicles. Values of total cholesterol and OSI in serum and medium follicles were significantly associated (r = 0.736 and r = 0.696, respectively). The serum concentrations of all lipid metabolites were markedly higher than those measured in FF of small- and medium-sized follicles. Values of IL-6 and OSI did not change significantly between serum and all follicle classes (P ≥ 0.05). To conclude, changes in the blood composition associated with inflammation, oxidative stress, and disturbed lipid metabolism of mares may lead to an inadequate oocyte microenvironment, which could affect oocyte quality and the success rate of OPU/ICSI programs. Further research should indicate whether these changes may ultimately affect in vitro oocyte developmental capacity and subsequent embryo quality. [ABSTRACT FROM AUTHOR]

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