Non-pregnant Mares Research Articles

Plasma Lipidomics Reveals Lipid Signatures of Early Pregnancy in Mares

Understanding the systemic biochemistry of early pregnancy in the mare is essential for developing new diagnostics and identifying causes for pregnancy loss. This study aimed to elucidate the dynamic lipidomic changes occurring during the initial stages of equine pregnancy, with a specific focus on days 7 and 14 post-ovulation. By analysing and comparing the plasma lipid profiles of pregnant and non-pregnant mares, the objective of this study was to identify potential biomarkers for pregnancy and gain insights into the biochemical adaptations essential for supporting maternal recognition of pregnancy and early embryonic development. Employing discovery lipidomics, we analysed plasma samples from pregnant and non-pregnant mares on days 7 and 14 post-conception using the SCIEX ZenoTOF 7600 system. This high-resolution mass spectrometry approach enabled us to comprehensively profile and compare the lipidomes across these critical early gestational timepoints. Our analysis revealed significant lipidomic alterations between pregnant and non-pregnant mares and between days 7 and 14 of pregnancy. Key findings include the upregulation of bile acids, sphingomyelins, phosphatidylinositols, and triglycerides in pregnant mares. These changes suggest enhanced lipid synthesis and mobilization, likely associated with the embryo’s nutritional requirements and the establishment of embryo–maternal interactions. There were significant differences in lipid metabolism between pregnant and non-pregnant mares, with a notable increase in the sterol lipid BA 24:1;O5 in pregnant mares as early as day 7 of gestation, suggesting it as a sensitive biomarker for early pregnancy detection. Notably, the transition from day 7 to day 14 in pregnant mares is characterized by a shift towards lipids indicative of membrane biosynthesis, signalling activity, and preparation for implantation. The study demonstrates the profound lipidomic shifts that occur in early equine pregnancy, highlighting the critical role of lipid metabolism in supporting embryonic development. These findings provide valuable insights into the metabolic adaptations during these period and potential biomarkers for early pregnancy detection in mares.

Untargeted Metabolomic Analysis Reveals Plasma Differences between Mares with Endometritis and Healthy Ones.

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.

Effect of age on amplitude of circulating catecholamine's change of healthy cyclic mares.

Catecholamines (CATs) are neurotransmitters and allostatic hormones whose plasma concentrations are physiologically modified in various species such as human, rats, mice and donkeys, with advancing age. However, currently these mechanisms are less well elucidated in horses and more specifically in mares. The hypothesis of this study was that, as in afore mentioned species, the CATs could experience physiological changes with advancing age. The objective of this study was to evaluate the concentrations of adrenaline (A), noradrenaline (NA), dopamine (DA), and serotonin (5-HT) in mares of different ages. Blood samples were drawn from 56 non-pregnant Spanish Purebred mares belonging to four different age groups: 6 to 9 years, 10 to 12 years, 13 to 16 years and > 16 years. The concentrations of A, NA, DA, and 5-HT were determined by competition EIA-Technical 3-CAt EIA, specifically validated for horses. Mares aged > 16 years showed lower A, DA, and 5-HT but higher NA concentrations than 6-9, 10-12, and 13-16 years (p < 0.05). Mares of 13-16 years showed lower A and higher NA than 6-9 and 10-12 years (p < 0.05). A and NA (r=-0.72; p < 0.05), and NA and 5-HT (r=-0.67; p < 0.05) were negatively correlated, and A and 5-HT (r = 0.74; p < 0.05) were positively correlated. Advanced age leads to a predominance of sympathetic nervous activity and lower serotonergic activity in non-pregnant mares.

Pregnancy rates according to follicle diameter and uterus edema in different age groups in two consecutive ovulations in Arabian mares

The study aimed to evaluate the pregnancy rates of mares in different age groups based on follicle diameter and uterine edema during two consecutive ovulations in three breeding seasons. Thirty Arabian mares were divided into two groups: below and equal to 10 years (Group A=54) and above 10 years old (Group B=36). Throughout the breeding season, the diameters of ovulatory follicles and uterine edema were recorded. After the first ovulation, when the follicle diameters of all mares in both Group A and Group B were analyzed independently of age, and compared between pregnant and non-pregnant mares, no differences were found (P&gt;0.05). The differences between the agerelated groups were insignificant (P&gt;0.05). Follicle diameters between pregnant and non-pregnant mares at the second ovulation were significantly different (P0.05). Differences between uterine edema values determined in mares that were pregnant and nonpregnant in two consecutive ovulations were found to be insignificant in terms of pregnancy (P&gt;0.05). As a result, although preovulatory diameter is not a reliable marker for pregnancy detection, it remains an important parameter for determining ovulation and artificial insemination timing. Uterine edema is only useful for determining the timing of insemination and is not associated with pregnancy. Moreover, since similar pregnancy rates were observed among different age groups, age alone is not a sufficient parameter for assessing reproductive efficiency in mares.

Hematological and biochemical reference values for the Brazilian Pony breed

The aim of this study was to characterize the hematological profile of the equine Brazilian Pony breed herds in northern of Rio de Janeiro state, Brazil. Blood samples were separated in categories: adult stallions, pregnant mares and non-pregnant mares. The red blood cells (RBC), packed cell volume (PCV), hemoglobin (Hgb), main corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), white blood cells (WBC) and differential count of basophils (Baso), eosinophils (Eos), band cells (Bands), segmented neutrophils (Neut), lymphocytes (Lym), monocytes (Mono), and platelets (Plat) were evaluated. The biochemical parameters of urea (Urea), alanine aminotransferase (ALP), aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), and creatinine (Creatinine) were measured in pregnant and non-pregnant mares. Analysis of variance was carried out with difference of hematological parameters between males and females. Also, analysis of variance was used to verify the effect of pregnancy on biochemical parameters. There was effect of sex on RBC, hemoglobin, VCM and MHCH, but PVC and MHC were not affected by sex. Higher means were observed in females. There was an effect of sex on WBC, eosinophils and neutrophils. There was effect of pregnancy on AST, but no effect on urea, GGT and Creatinine. According to the results obtained in this study, it can be concluded that the analysis of the blood count and the biochemical of the Brazilian Pony reveals specificities compared to other equine breeds. Thus, these results could be used as biomarkers for the evaluation of the Brazilian Pony breed in Rio de Janeiro.

Transcriptome identification of differential mammary genes of Kazakh horses during early pregnancy

Kazakh mares have attracted widespread attention with their outstanding lactation traits. Lactation is a complex dynamic process regulated by multiple factors. The extensive application of transcriptome sequencing technology enables researchers to further explore this biological issue. This study selected three pregnant and three non-pregnant Kazakh mares as the research subject. Their mammary glands were taken for transcriptome sequencing. The results show that there are 9 lncRNAs and 122 mRNAs differentially expressed between the two groups. GO enrichment analysis shows that there are 175 molecular functions, 59 cellular components, and 555 biological processes, including cellular hormone metabolic process, hormone catabolic process, and I-kappaB kinase/NF-kappaB signaling. KEGG enrichment analysis exhibits that these differential genes are mainly enriched in the NF-kappa B signaling pathway, steroid hormone biosynthesis, breast cancer, ECM-receptor interaction, and MAPK signaling pathway. WNT4, DPP4, and NFKBIA are key nodes regulating breast activation. Conclusions: Through the comparative analysis of the transcriptome data of mammary tissues of pregnant and non-pregnant mares, relevant differentially expressed genes are screened and analyzed. This study provides valuable fundamental data for investigating candidate genes related to the lactation regulation and mammogenesis of Kazakh horses.

Assessment of heavy metal and radioactive element levels in the ovarian follicular fluid of pregnant and non-pregnant Kyrgyz breed mares

Objective: In the current study, we aimed to ascertain the levels of heavy metals and radioactive elements in the ovarian follicular fluids of pregnant and non-pregnant Kyrgyz mares. Materials and Methods: To this end, follicular fluids were obtained from 49 Kyrgyz mares aged 3–5 years. The mares were in various stages of pregnancy (Group 1, n = 27) or were non-pregnant (Group 2, n = 22). We measured the levels of cadmium (Cd), lead (Pb), mercury (Hg), antimony (Sb), cobalt (Co), potassium (K), uranium (U), nickel (Ni), and thorium (Th) in follicular fluids using atomic emission mass spectrometry. Subsequently, the data were statistically analyzed according to whether the mare was pregnant or not, the pregnancy stage, the presence or absence of the corpus luteum in the ovaries, the corpus luteum’s diameter if present, and the presence or absence and diameter of follicles. Results: We found that K levels were higher in non-pregnant mares (0.00564 ± 0.000590 ppm, p = 0.009). Furthermore, Ni (0.01033 ± 0.001880 ppm, p = 0.07) and K (0.00563 ± 0.000498 ppm, p = 0.026) levels were higher in mares with a follicle diameter of 1–3 cm. However, these results did not change according to the month of pregnancy, number of follicles, presence or absence of the corpus luteum in the ovaries, number of corpora lutea, and their diameters. Conclusion: Thus, we concluded that heavy metal and radioactive element levels in the ovarian follicular fluids of Kyrgyz mares do not significantly change with pregnancy status, and there are limited detrimental effects of pollution on the follicular fluids.

Monthly and Pregnancy-Related Concentration of Cu and Zn in Serum of Mares in an Equine Breeding Herd

Copper (Cu) and zinc (Zn) are trace minerals with multiple biological functions, playing roles in fetal development and immune regulation. Despite their known significance, research on Cu and Zn administration and supplementation for pregnant mares is insufficient. This study aims to evaluate Cu and Zn serum concentrations monthly throughout the year and through pregnancy in Polish Konik mares. The study was conducted in 2020 on 36 mares from the Polish state stud farm, with 24 mares in the pregnant group and 12 mares in the non-pregnant group. Monthly blood samples were collected, and serum Cu and Zn concentrations were measured. The total Cu concentration in the serum of all mares was higher (p &lt; 0.0001) in August and September (13.98 ± 3.00 μmol/L) than in May, June, July, and November (11.04 ± 2.74 μmol/L). Similarly, the total Zn concentration was higher (p &lt; 0.0001) in August, September, October, November, and December (19.80 ± 9.72 μmol/L) than in May, June, and July (14.50 ± 6.94 μmol/L). No evidence of a linear relation between Cu and Zn concentrations was demonstrated (slopes difference p &lt; 0.001). No differences in Cu (p &gt; 0.05) or Zn (p &gt; 0.05) concentrations were found between pregnant (Cu: 12.08 ± 3.01 μmol/L; Zn: 18.02 ± 9.03 μmol/L) and non-pregnant (Cu: 11.23 ± 2.51 μmol/L; Zn: 18.59 ± 10.37 μmol/L) mares in any of the examined months of the year. In conclusion, the month of the year, rather than pregnancy, affects serum Cu and Zn concentrations.

Metabolic profile and body condition score of Crioulo horses finalists in the Freio de Ouro competition

The aim of this study was to assess the body condition score (BCS) and metabolic profile of finalists in the Freio de Ouro competition, stratified by sex. This study examined 84 Crioulo horses, comprising 43 non-pregnant mares and 41 stallions. Morphometric measurements, including BCS, cresty neck score (CNS), neck and heart girth, and assessments of fat depth at the neck and at the tail base, were conducted. Additional derived measurements such as the percentage of body fat (%BF), body mass index (BMI), fat mass (FM), fat-free mass (FFM), heart girth:height ratio (HG:HW), and neck circumference:height ratio (NC:HW) were calculated. A subgroup of 53 animals was selected for the evaluation of metabolic parameters, including triglycerides, cholesterol, low-density lipoprotein (LDL), high-density lipoprotein (HDL), lactate dehydrogenase (LDH), creatine kinase (CK), and adiponectin.In terms of BCS, 21% of participants were classified as overweight, with 49% having CNS≥3. Females exhibited greater fat deposition at the tail base and neck, along with higher %BF, FM, and HG:HW ratio, and lower NC:HWcompared to males. Among metabolic variables, females showed higher concentrations of LDH, LDL, CK, and lower concentrations of adiponectin than males. No differences were observed in morphometric measurements between participants that qualified for the final day of the competition and those which were disqualified. The study revealed that despite the rigorous physical demands and preparation required for the Freio de Ouro competition, a percentage of participants were overweight, displaying increased regional fat deposition in the neck. Although no metabolic changes related to increased adiposity were identified, the findings indicate a predisposition to the development of metabolic alterations. Additionally, females exhibited a greater accumulation of fat than males.

Effect of micronization and meal size of corn grain on glycemic response and in vitro hindgut acidosis potential in horses

This study aimed to 1) evaluate the interaction of corn grain micronization and starch levels per meal on equine plasma glucose, and 2) determine if micronization affects the risk of hindgut acidosis. Six mature (aged 6 to 10 years), healthy, non-pregnant mares (initial body weight [BW]: 301 to 463 kg) were used in a 2×3 factorial cross-over design. The treatments included two forms of corn grain (ground and micronized flaked) at three levels of starch (1, 1.5, and 2 g/kg BW per meal). The blood was sampled before and 30, 60, 90, 120, 180, 240, and 300 min after morning feeding and the glucose concentration in the plasma was determined. Small intestine and hindgut dry matter (DM) disappearances of ground and micronized corn were also compared using in vitro techniques. Micronized flaked corn grain showed three times more in vitro enzymatic DM disappearance (p < 0.001) compared with ground corn. Residues of in vitro enzymatic digestion of micronized flaked corn fermented 38.59 % faster than ground corn during in vitro hindgut incubation. The horses that consumed micronized flaked corn had higher post-prandial plasma glucose concentrations (p < 0.001). Increasing starch levels per meal from 1–2 g/kg BW resulted in higher plasma glucose concentrations (P = 0.005). However, no interaction of processing and starch meal size was found. Overall, processing the corn grain by micronization or increasing starch level per meal increased the plasma glucose concentrations, but the magnitude of the increases did not match that expected from in vitro studies.

Comparing Hematological and Biochemical Profiles of Pregnant and Non-pregnant Barb Mares Raised in Tiaret, Algeria

Background: The pure-bred Barb horse is a beloved breed from the Great Maghreb. Despite the breed’s prominence in Algeria, no gestational hematological or biochemical research has been done on this breed. Objectives: This study aimed to compare the hematological and biochemical parameters of pregnant and non-pregnant Barb mares in the first, second, and third trimesters of pregnancy. Methods: From 12 pregnant and 6 non-pregnant mares, 102 venous blood samples were taken, and their glucose (Glu), cholesterol (Cho), triglycerides (TG), total protein (TP), urea (Urea), Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), iron (Fer), calcium (Ca), phosphorus (P), and ferric reducing ability of plasma (FRAP) were assessed as biochemical variables. Also, red blood cells, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, white blood cells, and platelets were all measured as hematological variables. Results: The levels of ALP, ALT, GGT, and P decreased significantly throughout gestation, while Ca, TG, Fe, and Glu levels increased. AST concentrations decreased in the second and third trimesters, whereas Cho levels increased in the first and second trimesters. Urea levels increased significantly in the third trimester, and FRAP showed significant differences at different stages of pregnancy. Mean corpuscular hemoglobin concentration was significantly lower in the first and second trimesters, and hemoglobin values were significantly lower in the second trimester. The mean value of white blood cell count was slightly higher in late pregnancy, while platelet values significantly increased throughout all trimesters. Conclusion: The study provides valuable information on the changes in hematological and biochemical parameters during pregnancy in Barb mares. These findings can be used as a reference for future studies on the reproductive physiology of this breed.

Effects of pregnancy on electrocardiographic, vasovagal tonus index, and echocardiographic variables in horses.

Pregnancy affects maternal hemodynamics. The changes in autonomic nervous system activity for hemodynamics adaptation in pregnant horses are still unclear. Thus, this study aimed to examine the effect of pregnancy on electrocardiographic, vasovagal tonus index, and echocardiographic variables in horses. A total of 23 Thai native crossbred mares without any cardiac abnormalities were included in this study. The animals were assigned into two groups, a non-pregnant mare group (n =12) and a pregnant mare group (n = 11). Electrocardiogram recordings (paper speed = 25 mm/s and calibration = 10 mm/mV) were performed to obtain six limb leads (leads I, II, III, aVR, aVL, and aVF). The vasovagal tonus index (VVTI) was calculated to assess variability in heart rate over short periods using just 20 consecutive beats. Cardiac structure and function were evaluated by echocardiography. Heart rate, P wave duration, PR interval, QRS duration, QT interval, and T wave duration were significantly different between non-pregnant and pregnant horses (p < 0.05). Pregnant horses had significantly lower VVTI than non-pregnant (p < 0.05). There were no significant differences in cardiac structures including % interventricular septum (IVS), % left ventricular posterior wall (LVPW), IVS in diastole, left ventricular internal diameter at end-diastole, LVPW thickness at end-diastole, IVS in systole, left ventricular internal diameter at end-systole, LVPW thickness at end-systole, and left atrium/aortic roots ratio between the two groups. However, the pregnant horses had a significantly higher cardiac output and % ejection fraction than non-pregnant horses (p < 0.05). This study provided the first evidence that hemodynamic adaptations during pregnancy modified cardiac conduction, vasovagal tonus index, and echocardiographic variables in horses.

Longitudinal humoral immune response and maternal immunity in horses after a single live-attenuated vaccination against African horse sickness during the disease outbreak in Thailand.

African horse sickness (AHS) has become a newly emerging disease after an outbreak in northeastern Thailand in March 2020. Mass vaccination in horses with live-attenuated AHS virus (AHSV) vaccine is essential for AHS control and prevention. This study aimed to monitor the longitudinal humoral immune response before and after a single vaccination using a live-attenuated vaccine against AHS in stallions, mares, and pregnant mares, including maternal immunity in foals born from pregnant mares during the outbreak in Thailand. A total of 13 stallions and 23 non-pregnant and 21 pregnant mares were vaccinated with live-attenuated AHSV vaccines. Serum samples from selected horses were collected on the day of vaccination and 1, 6, 8, 9, 10, and 12-months post-vaccination. Furthermore, seven serum samples of foals born from vaccinated pregnant mares were collected on parturition date and 1, 3, and 6-months old. The antibody titer against AHS in all collected serum samples was evaluated using a commercial enzyme-linked immunosorbent assay kit. All data were analyzed for mean and standard deviation for each group of samples using a spreadsheet program. Antibody titers between times were analyzed using a one-way analysis of variance as repeated measurement, and antibody titers between horse groups were analyzed using a general linear model for statistically significant differences when p < 0.05. In stallion and non-pregnant mare groups, there were no statistically significant differences in antibody titers in all 6 time periods after vaccination. The antibody titer in the pregnant mare group showed a non-statistically significant difference between each gestation stage, except at 8 months post-vaccination. Furthermore, increasing antibody titers on days 1 and 3 after receiving colostrum in foals indicate the major role of transcolostral antibody transfer for AHS. This study demonstrated that a single AHS vaccination using a live-attenuated vaccine could stimulate high antibody titers sufficient for AHS control and prevention during the outbreak in Thailand. Similarly, the antibody response of vaccinated horses of both genders, including various stages of pregnant mares, was statistically not different.

Endocrine and metabolic profiles in pregnant and non-pregnant Criollo mares

Gestation leads to metabolic changes, prioritizing nutrients’ delivery to the developing fetus (Fowden et al. Equine Veterinary Journal. 1984; 16(4), 239-246). Insulin, adiponectin, and leptin are key metabolic hormones that have been associated with pregnancy success in several species (Coverdale et al. J Anim Sci. 2015; 93(7), 3261-3267.; Meikle et al. Anim. Reprod., v.15, (Suppl.1), p.899-911. 2018). Information on these hormones and their metabolites during gestation of horses is lacking, specifically in native breeds. Therefore, the objective of this study was to establish the endocrine-metabolic profile in pregnant and non-pregnant Criollo mares, a native breed that is popular in South America. Mares (n=56) were categorized as pregnant (n=41) or non-pregnant (n=15). Animals were bled between days 200 and 250 of gestation and the Body Condition Score (BCS) was determined (scale 1-9, Henneke et al. Equine Veterinary Journal. 1983; 15(4), 371-372). The controls were assayed at the same time samples were taken, during the winter, between June and August. Insulin was determined by immunoradiometric analysis, leptin and adiponectin by Radioimmunoassay and other biochemistry variables by spectrophotometer. Hematology variables were determined by hematologic counter. Variables were analyzed by ANOVA using a mixed procedure. The reproductive status did not affect theleptin (9.4±1.6 vs 9.0±1.0 ng/mL, P=0.804 in pregnant and non-pregnant mares respectively), adiponectin concentrations (18.9±2.7 vs 15.5±1.6 ng/mL, P=0.304 in pregnant and non-pregnant mares respectively), nor the BCS (7.3±0.2 vs 7.6±0.1, P=0.356 in pregnant and non-pregnant mares respectively). Insulin (18.2±2.9 vs 17.7±1.7 UI/mL, P=0.644 in pregnant and non-pregnant mares respectively) and glucose (87.0±2.6 vs 83.0±1.6 mg/dL, P=0.180 in pregnant and non-pregnant mares respectively) concentrations were not affected either. Cholesterol concentrations were not affected by the reproductive status, while triglyceride concentrations were higher in pregnant when compared to non-pregnant mares (31.0±1.9 vs 21.6±3.1 mg/dL, P=0.012). Urea and creatinine concentrations were also higher in pregnant than in non-pregnant mares (52.8±1.0 vs 46.7±1.6 mg/dL, P=0.002 and 1.25±0.03 vs 1.01±0.05 mg/dL, P=0.0002 respectively). Hepatic enzymes and white blood cell count were not affected by the reproductive status. This study shows higher concentrations of triglycerides, urea and creatinine in pregnant mares. These changes could be associated with the metabolic prioritization of glucose delivery to the developing fetus, which leads to a differential nutrient partitioning, as reflected in different concentrations of specific metabolites of the protein and lipid pathways.

Interleukin-1 beta treatment alters prostaglandin-related mRNA expression and cytokine abundance in equine endometrial culture

Interleukin-1β (IL-1β) is a proinflammatory cytokine that modulates the immunoinflammatory response and induces prostaglandin synthesis. IL-1β has a variety of roles in the endometrium during inflammation, decidualization, pregnancy, and post-partum. It can cause changes in endometrial gene expression and may affect cytokine, chemokine, and prostaglandin-related pathways. The relationship between IL-1β, prostaglandins, and downstream inflammatory responses in the equine uterine environment is not fully known. Our objective was to determine the effect that IL-1β has on prostaglandin-related gene expression, as well as cytokine and chemokine abundance, in endometrial cell culture. Light breed mares (n=8) were examined daily by transrectal ultrasound until ovulation. An endometrial biopsy was collected at day 11 post ovulation, and used for primary endometrial cell culture. Cells were cultured in DMEM with 10% FBS. At 80% confluence, cells were either not supplemented or supplemented with: 100 nM Progesterone (Prog); 1 nM Estradiol-17β (Est); or 100 nM Prog and 1 nM Est (P+E). After 24 hours, cells were treated with recombinant equine IL-1β at either 10 ng/mL (Low) or 1000 ng/mL (High), or 0.2% DMSO as an untreated control. Twenty-four hours post-treatment, supernatant was collected, and a bead-based multiplex assay was used to quantify concentration of twelve cytokines and chemokines. Total RNA was extracted from the cells and mRNA expression was evaluatedfor 10 prostaglandin-related genes, using the ΔCT method with ACTB as the reference gene. Data analysis was performed using JMP Pro 16, using a restricted maximum likelihood model with treatment and supplementation as factors, and mare as a random factor. Treatment with IL-1β High resulted in increased levels of CCL2, CCL5, and CCL11 (p<0.001) in cell culture supernatant, as well as increased mRNA expression of PTGS2 (p<0.02), regardless of supplementation. Cells supplemented with Prog or P+E and treated with IL-1β High had increased expression of IL1R1, IL1RAP, PTGS1, and PTGES compared to non-supplemented cells (p<0.05). IL-1β High treatment increased LiF expression in cells supplemented with Prog or P+E compared to untreated cells (p<0.05). IL-1β likely acts to modulate inflammatory pathways in the endometrium, altering chemokine levels and activating expression of PTGS2, leading to changes in downstream prostaglandin synthesis and other related pathways. Progesterone appears to increase expression of several target genes when compared to non-supplemented cells. Progesterone has been shown to be involved in the suppression of several inflammatory pathways, and investigation into the ovarian steroid regulation of genes and proteins in the equine endometrium is warranted. Further studies on the effect of IL-1β on molecular pathways and prostaglandin regulation could lead to a greater understanding of the uterine environment in the pregnant and non-pregnant mare.

Effect of mid-diestrus oxytocin treatment on early pregnancy in the mare

Progesterone supplementation in early pregnancy is commonplace in equine reproduction. Recently, there has been some controversy questioning the beneficial effects of routine progesterone administration and rising concerns of medication exposure as an occupational hazard for humans. Although premature luteal regression and resulting low plasma progesterone concentrations are rare in the mare, select cases benefit from luteal support. Duration of luteal function can be prolonged following oxytocin administration during mid-diestrus in the non-pregnant mare (Vanderwall.et.al. Journal of Equine Veterinary Science. 2012; 32:536-541). The aim of this field study was to investigate the effects of oxytocin administration on early pregnancy in the mare. Thoroughbred mares (n = 39; age 5 to 19 years) were enlisted and bred over 55 estrous cycles. Prior to natural breeding, uterine cultures were obtained, and mares were assigned to 1 of 2 groups: Oxytocin treatment (OT); oxytocin 60 units, intramuscularly, once daily, days 7-14 post-ovulation; or Control (CT); no treatment. Mare reproductive cycles were monitored by transrectal palpation and ultrasonography. Ovulation was documented and defined as day 0. Pregnancy diagnosis was performed by transrectal ultrasonography day 14 post-ovulation. If pregnancy was confirmed, a blood sample was collected and assayed for plasma progesterone concentration. Mares that did not conceive were administered prostaglandin F2αanalogue (cloprostenol; 250mcg once intramuscularly) at day 16 post-ovulation. These mares were rebred and treatment was determined by the stage in the study; therefore, some mares underwent the same treatment more than once. Subsequent pregnancy examinations were performed on days 28 and 45 of pregnancy. Data were analyzed using two-tailed Fisher's Exact tests for small counts, with significance defined at p < 0.05. First and per-cycle pregnancy rates for OT and CT were 90.9 and 91.3% and 68.2 and 55.1%, respectively. Median progesterone concentrations for OT were 6.9 and 7.0 ng/ml for CT. There were no differences between OT and CT groups for first (p = 0.165) or per cycle (p = 0.289) pregnancy rates, days 28 and 45 pregnancy rates (p = 1.0), or progesterone concentrations (p = 1.0). In this population of mares, oxytocin administration during mid-diestrus in early pregnancy did not induce a negative effect on pregnancy rate nor a positive effect on progesterone concentration. Whilst the study design did not assess the effect of OT on the inter-ovulatory interval (IOI), the authors’ hypothesize that the IOI would be increased, due to prolonged luteal function, in the non-pregnant OT mare if prostaglandin F2α analogue was not administered. Further investigation in this area may reveal potential beneficial effects of oxytocin administration in early pregnancy.

CfDNA of feto-placental origin is not detectable in plasma of pregnant mares by optimised sexing-qPCR.

Non-invasive prenatal testing is a cell-free-DNA (cfDNA)-based genetic screening, well-established in pregnant women, utilising cfDNA of feto-placental origin (cffDNA) isolated from maternal plasma (Christiaens et al, Prenatal Diagnosis. 2021;41:1316-1323). Fetal aneuploidy is described as causative for equine pregnancy loss (Shilton et al, Scientific Reports. 2020;10:13314), suggesting cffDNA-screening in mares is diagnostically warranted. The aims of the current study were to (i) optimise isolation methodology and examine presence of cffDNA in maternal plasma and (ii) develop a non-invasive cffDNA-based test to determine fetal sex in pregnant mares. Peripheral blood (10ml) from pregnant mares, carrying fetuses of confirmed sex (D70-80:n=4; last trimester:n=15), non-pregnant mares (n=2) and geldings (n=4) was collected into Streck®tubes. Samples were double-centrifuged within 24hrs, and supernatant transferred into Eppendorf® DNA-low-bind-tubes. CfDNA was isolated from multiple plasma aliquots per individual (QIAamp, ccfDNA-kit), applying two different temperatures (56°C, 21°C) for the magnetic-bead-binding step of the protocol. CfDNA was quantified by fluorometry (Qubit®fluorometer, 1XdsDNA-HS-assay). Control DNA was isolated from adult peripheral blood mononuclear cells (PBMC) and fetal tissue. CfDNA-isolation resulted in a median yield of 0.99ng/ml plasma (n=80 isolations) and did not differ (P>0.05) between aliquots of one individual or gestational stages but was significantly higher (P=0.0037) with an incubation temperature of 56°C (n=40), compared to 21°C (n=40). Quantification of cfDNA (n=6) by automatedelectrophoresis (cfDNA-Screentape, Agilent® TapeStation) was in alignment with fluorometry. A qPCR was optimized and validated for GAPDH and the Y-sequence TSPY utilizing 0.1ng and 0.2ng of DNA (indicated by cfDNA-concentrations, isolated above) from adult PBMC (n=41) and fetal tissue (n=6) of confirmed sex. A correct female / male classification was achieved for 95.1% (0.2ng) and 87.2% (0.1ng) of adult and 100% of fetal (0.1ng) DNA. Sensitivity (adult:0.2ng:100%; 0.1ng:100%), specificity (adult:0.2ng:100%; 0.1ng:95.2%) and PPV (adult:0.2ng:100%; 0.1ng:92.8%) were high. Next, qPCR accuracy was examined utilizing plasma cfDNA of non-pregnant mares (n=2) and geldings (n=4). Sensitivity, specificity and positive predictive value (PPV) for Y-chromosomal detection were 100%. In women, a minimum of 2-4% cffDNA is deemed diagnostic for prenatal testing. To determine TSPY detection at this ratio, male DNA was diluted in female DNA and TSPY accurately found at 4-10% but not 2%-dilutions. Finally, cfDNA from the plasma of pregnant mares (n=19) was analysed. All samples amplified GAPDH, however all clinically confirmed male pregnancies (6 / 19) were TSPY-negative. In conclusion, cfDNA was isolated from equine plasma and the yield improved by incubation at 56°C during the magnetic-bead-binding. A highly sensitive sexing-qPCR was validated for equine adult, fetal and cfDNA, but cffDNA was not detected in plasma of pregnant mares carrying male fetuses. CffDNA was not clearly observed by electrophoresis, suggesting isolation of cfDNA was of maternal but not feto-placental origin.

Global gene expression in equine endometrium around the time of maternal recognition of pregnancy

The conceptus-maternal communication during the preimplantation period, also known as maternal recognition of pregnancy (MRP), is crucial for the establishment and maintenance of pregnancy. This communication affects the endometrial transcriptome and proteome with subsequent reduction of endometrial pulsatile prostaglandin F2α secretion and the consequent extension of the corpus luteum's lifespan during early pregnancy in the mare. Also, this communication results in the preparation of the uterine environment for implantation. Any alteration of these processes will result in early embryonic death. Therefore, a better understanding of the normal endometrial transcriptomic changes around the time of MRP might be the key to understanding and preventing early embryonic loss. This study aimed to evaluate the endometrial transcriptome from Day 14 (Day 0 = ovulation) of pregnant and non-pregnant (i.e. diestrus) mares. RNA was isolated from pregnant (n=5) and non-pregnant (n=4) endometrial tissue samples. RNA-Seq was performed using Illumina NovaSeq6000 and differentially expressed genes (DEGs) were evaluated using DESeq.2 based upon an FDR<0.05. Our study identified 1193 DEGs (570 upregulated DEGs and 623 downregulated DEGs) in the pregnant endometrium as compared to the non-pregnant endometrium. In the current study, the pregnant endometrium was associated with downregulation (9.6-fold change) of mRNA forprostaglandin-endoperoxide synthase 2 (PTGS2, also known as cyclooxygenase-2 enzyme; COX2), which encodes for the rate-limiting enzyme in prostaglandin production. Gene ontology analysis of DEGs revealed that these genes were associated with relevant biological processes such as immune response (TNFAIP1, TNF, CXCR2, TNFSF15, IL15, TGFBR3, IL1B, TNFSF9, TLR8), regulation of trophoblast cell proliferation and migration (BOK, LIF, RBM46, TIMP1, CALR, GJA1), embryo growth and development (PTCH1, PTCH2R, DH10, GLI3, SHOX2, ZDBF2, EVX1, GREM2, KLF2, TWIST1), cell adhesion (COL28A1, EGFL6, ITGA3, FN1, NEDD9, LAMC2, RGMB, THBS4, RHOB, SELP, CHL1, CDHR1, MXRA8, FOLR3, NECTIN3), embryo implantation (MMP9 and LIF), placenta development (ADAM19, MAPK14, BIRC3), amino acids transportation (SLC1A1, SLC25A29, SLC36A2, SLC38A2, SLC7A5), steroid biosynthesis and metabolism (SRD5A1, HSD11B2, HSD17B7, CYP20A1), and progesterone receptors (PAQR5, and PGRMC1). Several of the DEGs identified in the current study are known to play a role in controlling endometrial receptivity to the conceptus in species other than the equine. These findings improve our understanding of the molecular changes taking place in equine endometrium around the time of MRP which might help us to understand and forestall early embryonic loss in the future.

Characterization of interferons in the equine conceptus and endometrium

Interferons (IFN) are involved in the maintenance and establishment of pregnancy in a variety of species. There are three types of IFNs, which include type I (IFN-α, β, ε, τ, and δ), type II (IFN-γ), and type III (IFN-λ1, λ2 and λ3). In the horse, IFNA, IFND and IFNO are expressed in the equine trophoblast, and IFNE is expressed in higher abundance in equine endometrium during the luteal phase. However, investigation of IFNs and their receptors during early equine pregnancy is still lacking. Our objective was to investigate the expression of IFN types I and II and their receptors, and other IFN related genes in equine conceptuses and endometrium during early pregnancy. Mares were examined daily by transrectal ultrasound when in estrus until the day (D) of ovulation (OV). Pregnant (P) group mares were bred using artificial insemination by a single fertile stallion. Conceptuses were detected and measured via transrectal ultrasound and collected via uterine lavage using 0.9% NaCl through a transcervical bivona catheter. Following conceptus retrieval, an endometrial biopsy was collected. Endometrial samples from nonpregnant (NP) mares were collected at D11 (NP11; n=6) and D13 (NP13; n=7), while endometrium and conceptuses were collected from pregnant mares at D11 (P11; n=7), D13 (P13; n=8), and D16 (P16; n=6). As the majority of nonpregnantmares would have undergone luteolysis, a D16 sample was not included in this study. Total RNA was isolated and evaluated for expression of interferon-related genes using Real-time PCR. The mean threshold cycle was determined and then normalized to the mean of the reference genes (GAPDH and ACTB) (ΔCT). A restricted maximum likelihood model was performed in JMP Pro 16 (Cary, NC) with mare as a random factor with significance set at P<0.05. There was no amplification for IFNB and IFND in the endometrium, and no amplification of IFNG, IFNB and IFNAR2 in the conceptus. In the endometrium, IFNAR1 and IFNGR1 had higher expression in P11 compared to P13 (P<0.02). Expression of endometrial IFNG mRNA was higher in P11 and P16 compared to P13 (P<0.004). There was no difference in the mRNA relative abundance of endometrial ISG15 and IFITM1 between groups or days. In the conceptus, IFNA, IFNO, and IFNGR2 expression were higher in D16 compared to other ages (P<0.05). D11 and D16 conceptuses had higher expression of IFNAR1 compared to D13 (P<0.0002), while IFNGR1 was higher in D16 compared to D11 (P<0.004). The role of interferons during maternal recognition of pregnancy in the horse is unknown. Unlike the pig, the horse embryo does not seem to express IFNG. Further investigation is underway.

Differential gene expression in the equine endometrium and conceptus from Day 11 to 16 post-ovulation

Mobility of the equine conceptus is essential for maternal recognition of pregnancy (MRP) and is regulated by the conceptus's own derived prostaglandin secretion. Our objective was to compare prostaglandin-related genes and other genes of interest during the expected MRP time. Light breed mares were examined daily by transrectal ultrasound when in estrus until the day (D) of ovulation. Mares in the pregnant (P) group were bred by artificial insemination with >500 million progressively motile normal sperm from one fertile stallion until D0. Conceptuses were detected and measured via transrectal ultrasound and collected via uterine lavage using 0.9% NaCl through a transcervical bivona catheter. Endometrium was collected via a transcervical biopsy punch following conceptus retrieval. Endometrium of non-pregnant (NP) mares was collected at: D11 (NP11; n=6) and D13 (NP13; n=7), while endometrium from the pregnant horn and conceptuses was collected at D11 (P11; n=7), D13 (P13; n=8), and D16 (P16; n=6). Total RNA from trophoblast and endometrial samples was isolated and evaluated for expression of PTGS2, PTGES, mPGES2, PTGER4, PTGER2, PTGIS, PTGIR, HPGD, SLCO2A1, CRTC2, NR3C1, CBR1, CREB, NF-Kβ, SPP1, IGFBP1, ESR1 and DNTTIP2 using Real-time PCR. Mean threshold cycle was determined and normalized to the reference gene (GAPDH and ACTB) (ΔCT). Statistical analyses were performed using JMP Pro 16, and data was ranked, transformed if necessary, followed by a restricted maximum likelihood model with mare as arandom factor to test differences between Groups and Day. Regarding endometrial mRNA, NP11 had higher expression for NF-Kβ, PTGIS, PTGIR and CBR1 compared to NP13 (p<0.007). ESR1, mPGES2, PTGIS, and CBR1 mRNA were higher in P11 compared to P16 (p<0.05), while CRTC2 was higher in P16 compared to P11 (p<0.06). Lastly, NF-Kβ, PTGIR and CBR1 were higher in P11 compared to P13 (p<0.01), but NF-Kβ P13 was lower compared to P16 (p<0.04). Regarding the conceptuses, CREB and PTGFR expressions were higher in D16 compared to D11 (p<0.003), while CRTC2, NR3C1, NF-Kβ and SPP1 were higher in D16 compared to D11 and 13 (p<0.02). D13 conceptuses had lower expression of HPGD compared to D11 and 16 (p<0.02). Lastly, D11 conceptuses had higher expression of mPGES2, PTGIS and PTGER4 compared to D13 and D16 (p<0.03). Higher expression of D11 PTGIS from both tissues may increase nutrient and blood flow to the endometrium in support of the early conceptus. The upregulation of endometrial NF-Kβ at D11 may be a result of endometrial receptivity through the regulation of inflammation, and the up-regulation of HPGD may function to regulate PGE2 during early pregnancy. Understanding embryonic and endometrial prostaglandin regulation during MRP may help to develop future therapies to ameliorate pregnancy loss in the horse.