Pregnant Mare Research Articles

Diagnostic and therapeutic insights into cystitis in a pregnant mare

Cystitis is relatively uncommon in horses and represents a significant health concern that can severely impact their ability to urinate and overall well-being. Clinical signs often include frequent urination accompanied by blood in the urine and a painful sensation during urination. To establish a definitive diagnosis, a comprehensive examination is imperative, which typically involves physical assessment, ultrasonography, cystoscopy, and rectal examination. Treatment is aimed at relieving bladder distention and preventing potential complications. In this report, a case of a six-year-old late-term pregnant mare was presented at Veterinary Clinics, with a history of voiding blood in urine. Further diagnostic evaluation confirmed cystitis, and appropriate therapeutic measures were taken, resulting in a successful recovery for the mare without complications.

Humoral immunity is not altered in overweight pregnant Crioulo mares

Both pregnancy and obesity can influence significant changes in the immune system. On this basis, the present study proposes to evaluate the humoral immune response of overweight pregnant mares in response to a commercial vaccine. Thirty pregnant Crioulo mares were separated according to body condition score (BCS) into overweight (BCS≥7/9) or lean-control (BCS= 5-6/9). In each group, the animals were subdivided into vaccinated and controls. The mares were vaccinated against EHV-1 in two doses spaced 21 days apart and had their blood collected monthly, for five months, for antibody evaluation. Both vaccinated groups had an increase in specific neutralizing antibodies after the vaccine. However, after the second dose, there was no increase in antibodies in any of the groups. Vaccinated overweight and lean-control mares did not differ at any time point. Therefore, this study demonstrated that obesity does not influence the humoral immune response in pregnant Crioulo mares.

Seasonal absence of supplementary corpora lutea in pregnant mares and the relationship with pregnancy loss

Two experiments were conducted in the Northern (UK) and Southern (Brazil) hemispheres to determine the effect of season (month of conception) on the development of supplementary CL (SCL) and the relationship with pregnancy loss. In experiment 1, 199 pregnancies were followed between Day 14 and term, to determine the number of SCL and pregnancy viability (Northern Hemisphere). From the 199 pregnancies, 178 were obtained from inseminations during the breeding season (March–September), while the rest, 21 pregnancies resulted from conceptions in the non-breeding season (October to February). Pregnancies conceived in the breeding season were more likely (P < 0.01) to have at least 1 SCL (75.8 %, 135/178) than pregnancies from the non-breeding season (33 %, 7/21). However, the pregnancy loss between Days 35 and 120 of pregnancy in mares with no SCL was similar (3.5 %, 2/57; P >0.1) than from mares with SCL (1.4 %, 2/142). In Experiment 2 (Southern Hemisphere), three groups of recipients were used based on their ovarian activity at the time of embryo transfer: Anestrus (n = 8), transitional (n = 7) and cyclic (n = 7) recipient mares. While all transitional and cyclic mares developed at least 1 SCL, only 50 % of anestrous recipients (4/8) developed SCL by 120 of gestation. In conclusion, the development of SCL in pregnant mares is influenced by the time of season of conception, therefore it appears to be regulated by the photoperiod and the endogenous seasonal variation in gonadotropin concentrations. Mares with no SCL were not at increased risk of pregnancy loss.

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.

Recombinant Salmonella enterica OmpX protein expression and its potential for serologically diagnosing Salmonella abortion in mares.

Salmonella abortion in mares is caused by Salmonella enterica subspecies enterica serovar abortus equi infection and is characterized by premature (abortion) or non-viable fetus birth. Although all horses are susceptible to infection, the condition is more often clinically manifested in pregnant mares, with most abortions recorded in young females. In addition, nonspecific clinical disease signs and poorly sensitive and effective bacteriological diagnostic methods hinder rapid and reliable infection diagnoses. Immunochemical methods such as enzyme-linked immunosorbent assay (ELISA) and immunochromatography assays can facilitate effective and rapid diagnoses. However, they require highly specific and active antigens and antibodies. This study aimed to generate a recombinant S. enterica outer membrane protein X (OmpX) and evaluate its suitability for serological diagnosis of Salmonella abortion in mares. Outer membrane protein X from the S. enterica antigen was synthesized de novo and expressed in Escherichia coli using the pET28 vector. Transformed E. coli cells were cultured under different conditions to detect recombinant OmpX (rOmpX) expression, and rOmpX purification and refolding were both conducted using metal affinity chromatography. Refolded and purified rOmpX was characterized by western blotting, liquid chromatography with tandem mass spectrometry, and ELISA. After optimized rOmpX expression, a 23 kDa molecular weight protein was identified. Amino acid sequence analysis using Mascot program suggested that these peptides were the OmpX protein from S. enterica. High specificity and diagnostic efficiency were recorded when rOmpX was used in ELISA against 89 serum samples from aborted and contact mares. Recombinant outer membrane protein, in comparison to the O antigen, demonstrated better diagnostic characteristics against sera from mares who aborted and contact horses.

Equine Placentitis in Mares Induces the Secretion of Pro-Inflammatory Cytokine eIL-1β and the Active Extracellular Matrix Metalloproteinase (MMP)-9.

Equine placentitis is characterized by infection and inflammation of the placenta. Different biomarkers associated with this inflammatory response have been evaluated in experimentally induced equine placentitis, but not in pregnant mares with spontaneous placentitis. The aim of the current study was to determine the concentration of eIL-1β and the activity of proMMP-2 and proMMP-9 in the serum of healthy mares and mares with placentitis on days 240 and 320 of gestation to explore whether these biomarkers are associated with equine maternal placentitis and/or with the birth of an infected or inviable foals. Serum samples were collected from sixteen pregnant English Thoroughbred mares, retrospectively classified as follows: (1) healthy mares with full-term gestation; and (2) mares with ultrasonographic signs of placentitis. The health of each foal was examined at birth, and it was decided to classify the cases into four groups: (1) healthy mares delivering a healthy foals (HM-HF, n = 6); (2) mares with USP delivering a healthy foal (USP-HF, n = 3); (3) mares with USP delivering a live septic foal (USP-LSeF, n = 4); and (4) mares with USP delivering a dead foal (USP-DF, n = 3). eIL-1β was quantified by ELISA, and proMMP-2 and proMMP-9 activity by gelatin zymography electrophoresis. In healthy mares, the serum concentrations of eIL-1β underwent a significant 16.5-fold increase from day 240 to day 320 of gestation. Although similar results were found in the mares with ultrasonographic signs of placentitis that delivered a healthy foal, those delivering a live septic or nonviable foal exhibited much higher concentrations of eIL-1β. proMMP-2 and proMMP-9 activity was not associated with maternal placentitis, foal infection, or death. Hence, the presence of placentitis severe enough to affect the health of the foal can be confirmed or discarded by determining the eIL-1β concentration in mares that have shown ultrasonographic signs of placentitis.

Measurement of Equine Placental Gonadotropin with the Antiserum Specific to Equine Pituitary Gonadotropin from Pregnant Mare Serum

Measurement of Equine Placental Gonadotropin with the Antiserum Specific to Equine Pituitary Gonadotropin from Pregnant Mare Serum

Use of deslorelin acetate to advance ovulation in goats for timed artificial insemination

The objective of this study was to determine the efficacy of deslorelin in advancing ovulation for timed artificial insemination (AI) protocols in goats. We hypothesized that deslorelin treatment advances the onset of ovulation and improves AI pregnancy rates. Does were synchronized using a 5-day CIDR (controlled internal drug releasing insert) protocol with prostaglandin treatment at CIDR insertion. For Experiment 1, does received 0.2 mg of intramuscular deslorelin (n = 9) or saline (control, n = 10) at CIDR removal. Serial blood collections and transrectal ultrasonography were performed to assess ovarian dynamics and identify ovulation. For Experiment 2, does received 0.2 mg of intramuscular deslorelin (n = 42), 5 ml of PMSG (pregnant mare serum gonadotropin)/hCG (human chorionic gonadotropin) (n = 42), or were left untreated (control, n = 42) at CIDR removal, and were subsequently bred by transcervical AI with fresh semen after 48 to 56 hours. Pregnancy diagnosis was performed at 50 and 90 days after AI. In Experiment 1, compared to control does, deslorelin-treated does had an increased (p &lt; 0.01) number of ovulations and increased (p &lt; 0.01) serum estradiol concentrations from 48 to 72 hours after CIDR removal. Serum progesterone concentrations did not differ between treatments. In Experiment 2, there was a main effect (p = 0.02) of treatment on pregnancy rates, with control does tending (p = 0.06) to have greater pregnancy rates than those treated with deslorelin or PMSG/hCG. Deslorelin treatment also resulted in decreased (p = 0.04) breeding season pregnancy rates and increased (p = 0.05) number of cycles to achieve pregnancy compared to control does. These results demonstrated that deslorelin not only has a super-ovulatory effect but also, at the dose given, can negatively impact does’ subsequent ovarian function and ability to achieve pregnancy.

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.

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.

Adaptive response of estrogen-iron axis in pregnant Purebred Spanish mares of different age.

The maintenance of iron (Fe) homeostasis is vital for the physiological function along life. In sexually mature humans and experimental animals, estrogens downregulate hepcidin (Hpc) expression, in order to improve the intestinal absorption and to mobilize Fe stores for maternal erythropoietic expansion and placental development. However, changes of these mechanisms related to regulation of Hpc on the availability of Fe during gestation with advancing age in mares, remain unknown. The objective of this study was to evaluate the interrelationships between serum Fe, Ferritin (Ferr) and Hpc with estrone (E1) and estradiol-17β (E2) concentrations in pregnant mares of different ages. Blood samples were taken from 40 pregnant Spanish Purebred mares belonging to 4 different age groups, 10 subjects for each group: 4-6 years, 7-9 years, 10-12 years, and > 12 years were used in this study. Fe concentrations of 4-6 and 7-9 years groups were higher (P < .01) than 10-12 and >12 years groups. Ferr concentrations of 4-6 years group were higher (P < .01) than other groups. Hpc concentrations increased and E1 decreased (P < .01) in > 12 years group compared to other age groups. E2 concentrations of 7-9, 10-12 and >12 years groups were higher (P < .01) than those of 4-6 years group; 7-9 years group had higher E2 concentrations (P < .01) than > 12 years group. Fe and Ferr were negatively correlated with Hpc (r = -0.81 and r = -0.67, respectively). E1 and E2 were negatively correlated with Fe (r = -0.23 and r = -0.11, respectively). E2 was positively correlated with Hpc (r = 0.78). In pregnant Spanish Purebred mare, the increase of estrogens, according to the more efficient iron status in response to Hpc inhibition and consequent mobilization of circulating and iron reserve, shows the existence of "estrogen-iron axis" in young mares. Nevertheless, these mechanisms are reversed in old mares, suggesting a less efficient iron metabolism with advancing age. It is hoped that new investigations are needed to understand in depth and clarify further the complex metabolic and hormonal mechanisms involved also in equine species.

Hematological indexes and iron status in pregnant mares.

During pregnancy, iron requirements are increased to meet optimal placental and fetal growth and the expansion of the maternal red-cell mass and to prevent complications related to the mother's iron deficiency anemia. Red-cell parameters and iron status provide consistent additional information for diagnosis of iron deficiency conditions. The aim of this study was to evaluate the serum iron status and its relation to hematological indexes in pregnant mares. Blood samples were taken from 31 Spanish Purebred mares over 11 months of pregnancy. Concentrations of iron (Fe), ferritin (Ferr), transferrin (T), and total iron-binding capacity (TIBC) increased significantly and unsaturated iron-binding capacity (UIBC) decreased as the pregnancy progressed without changes in red blood cell (RBC) count, hemoglobin (HB) concentration, packed cell volume (PCV), and transferrin saturation (TSAT). Fe and Ferr were positively correlated (). Fe and T () and Fe and TSAT () were positively correlated, and Fe and UIBC were negatively correlated (). T and TIBC were positively correlated (). Pregnancy in the Spanish Purebred mare is characterized by a progressive increase in Fe, Ferr, T, and TIBC and a decrease in UIBC without modification in hematological indexes. Hematological parameters and iron status seem to indicate a sufficiency for Fe transport and its related mobilization and utilization during gestation in Spanish Purebred mares.

The Effect of The Conditioned Medium from Human Embryonic Stem Cells on Mouse Oocytes In Vitro Maturation.

Some reports have indicated that conditioned medium from growing mouse embryonic stem cells (ESCs) provides a supportive condition for small follicles growing, oocyte maturation, and following embryo growth. The aim of this study is assessing in vitro maturation (IVM) and consequent in vitro fertilization (IVF) outcome of immature mouse oocytes using human embryonic stem cells conditioned medium (HESCM). In this experimental study, 240 germinal vesicle (GV) oocytes were took from NMRI female mice, aged 4-6 weeks, 48 hours before injection of 5 IU pregnant mare serum gonadotropin (PMSG). 120 GV oocytes without cumulus cells were cultured in each of the groups. 120 GV were cultured in HESCM as test groups and also 120 GV cultured in human embryonic stem cells medium (HESM) as control groups. After evaluating the metaphase II (MII) oocyte maturation rate at 8, 16 and 24 hours, the MII oocytes subsequently were fertilized in vitro and the two-cell embryo development rate was recorded at days 1, 2, and 3. Statistical analysis was performed by using the generalized estimating equations (GEE) method that calculated their rate ratio. Our data indicated there are significant differences between the maturation rates in HESCM and HESM (P=0.004), also the two-cell embryo development was significant between two culture media (P=0.00). Similar to some other studies, the secretome of the HESCM showed a significant impact on the IVM outcomes in mice.

In view of ovarian steroidogenesis and luteal construction to explore the effects of Bushen Huoxue recipe in mice of ovarian hyperstimulation

Ethnopharmacological relevanceBushen Huoxue recipe (BSHXR) is a widely used prescription medicine for treating gynecological diseases. We have previously found that BSHXR can improve the pregnancy outcome of controlled ovarian hyperstimulation (COH) mice by modulating the abnormal high level of progesterone. While the pharmacological mechanism of such therapeutic effect is not clear. Aim of the studyWe aimed to investigate the effects of BSHXR on the ovarian steroidogenesis and luteal function in mice undergoing COH. Materials and methodsA COH mouse model was established via an intraperitoneal injection of 0.4 IU/g pregnant mare serum gonadotropin (PMSG) and 1 IU/g human chorionic gonadotropin (HCG). The histological features of ovaries were observed using hematoxylin-eosin staining. The expression levels of FSHR, LHCGR, and key molecules in ovarian steroidogenesis, including CYP11A1, CYP17A1, CYP19A1, HSD3B1, and StAR, were examined via immunohistochemical staining, western blotting, and RT-qPCR. CD31, VEGFA, and FGF2 levels were assessed to evaluate ovarian vascularization. The protein and mRNA levels of ovarian ERK1/2, p-ERK1/2, MEK1/2, and p-MEK1/2 were also detected using western blotting, RT-qPCR, or immunofluorescence staining. ResultsCOH mice had a significantly increased volume and weight of the ovary and number of corpora lutea. In particular, COH exhibited a long-term influence on ovarian FSHR and LHCGR expression, disrupting the levels of CYP11A1, HSD3B1, and CYP17A1, causing poorer luteal angiogenesis. Compared with normal mice, the expression levels of ovarian VEGFA and FGF2 in COH mice were considerably lower on Day 1 after PMSG. On concomitant HCG treatment, both VEGFA and FGF2 expression surged dramatically on ED1 and then declined on ED4 and ED8. Moreover, the expression pattern of MEK1/2-ERK1/2 was almost consistent with that of VEGFA and FGF2. After treatment, BSHXR increased ovarian LHCGR, FSHR, CYP11A1, HSD3B1, and CYP17A1 levels, boosted luteal vascularization, and restored MEK1/2-ERK1/2 signaling in COH mice. ConclusionBSHXR restored the abnormally high progesterone level by regulating the CYP11A1 and HSD3B1 expression as well as promoted luteal angiogenesis, which was related with LHCGR-MEK1/2-ERK1/2-VEGFA/FGF2 signaling pathway in the ovary. This effect prevented the fluctuation of sex hormones in COH mice and benefited the outcome of pregnancy.

Small intestinal epiploic foramen entrapment in a seven-month pregnant Thoroughbred mare: a case report

A seven-month pregnant four-year-old Thoroughbred mare with colic weighing 600 kg was referred to the Jeju National University Equine Hospital. A physical examination and ultrasound suggested epiploic foramen entrapment. The patient underwent ileum resection and end-to-side jejunocecostomy. After surgery, the horse showed a consistent pain when introduced to feeding. A repeat laparotomy was performed to enlarge the stoma size with side-to-side jejunocecostomy. The repeat laparotomy was successful, and the mare gave birth uneventfully. This report describes a case of small intestinal epiploic foramen entrapment and the clinical outcomes of the 2 consecutive laparotomies a Thoroughbred mare in late gestation.

Estrogen-iron axis in cyclic mares: Effect of age

In woman and in animal models, estrogens are involved in iron (Fe) homeostasis supporting the hypothesis of the existence of an “estrogen-iron axis”. Since advancing age leads to a decrease in estrogen levels, the mechanisms of Fe regulation could be compromised. In cyclic and pregnant mares, to date, there is evidence linking the iron state with estrogens pattern. Then, the objective of this study was to determine the relationship among Fe, ferritin (Ferr), hepcidin (Hepc) and estradiol-17β (E2) in cyclic mares with advancing age. A total of 40 Spanish Purebred mares of different ranges of age was analyzed: 4–6 years (n = 10), 7–9 years (n = 10), 10–12 years (n = 10), and >12 years (n = 10). Blood samples were obtained on days −5, 0, +5 and + 16 of the cycle. Compared to mares of 4–6 years, serum Ferr was significantly higher (P < 0.01) and Fe significantly lower (P < 0.01) in mares >12 years of age. Hepc was significantly higher in mares >12 years (P < 0.01) than in those 7–9 years of age. E2 levels were higher in mares of 7–9 years (P < 0.01) than in 4–6 and >12 years of age. Fe and Ferr were negatively correlated with Hepc (r = −0.71 and r = −0.02, respectively). E2 was negatively correlated with Ferr and Hepc (r = −0.28 and r = −0.50, respectively), and positively with Fe (r = 0.31). There is a direct relationship between E2 and Fe metabolism, mediated by the inhibition of Hepc in Spanish Purebred mares. The reduction of E2 decreases the inhibitory effects on Hepc, increasing the levels of stored Fe and mobilizing less the free Fe in circulation. Based on the fact that ovarian estrogens participate in changes in the parameters indicative of iron status with age, the existence of an “estrogen-iron axis” in the mares'estrous cycle could be considered. Future studies are required to clarify these hormonal and metabolic interrelationships in the mare.

Metagenetic Analysis of the Pregnant Microbiome in Horses

Placentitis is the leading cause of infectious abortion in the horse. Additionally, it can result in weak and/or growth restricted offspring. While the etiology of ascending placentitis is well described in mares, less is known regarding the pathogenesis of other types, such as nocardioform placentitis. This study aims to identify the microbial communities in different body sites of the pregnant mare in early gestation to establish a core microbiome that may be perturbed in pathologic pregnancies such as placentitis. We hypothesize that the equine placenta harbors a distinct resident microbiome in early pregnancy when characterized by metagenetics and that there will be a disparity in bacterial communities from the oral, vaginal, and fecal microbiome. Samples were collected from the oral cavity, vagina, anus, and the allantoic portion of the allantochorion (“placenta”) from five pregnant mares between 96 and 120 days of gestation. The V4 region of the 16S rRNA gene was amplified for Illumina MiSeq sequencing to examine core bacterial communities present in the different body sites. Microbial community composition of the pregnant ponies by body site was significantly different (Bray–Curtis dissimilarity). The placenta was significantly different from the feces, oral cavity, and vagina. Alpha diversity measuring the Shannon diversity matrix was significant, with the body sites being a compounding variable, meaning there was a difference in richness and evenness in the different microbial communities. Feces had the greatest alpha diversity, while the oral cavity and placenta similarly had the least. In conclusion, metagenetics did reveal distinct community differences in the oral, fecal, vaginal, and placenta cavities of the horse. The equine placenta does show similarities in its microbial communities to the oral cavity. Further research needs to be completed to investigate how bacteria may be translocated to the placenta from these other body sites and how they contribute to the development of placentitis.

Detection of foaling using a tail-attached device with a thermistor and tri-axial accelerometer in pregnant mares.

It is desirable to attend to the mare at the time of foaling in order to assist fetal delivery and prevent complications. The early detection of the onset of labor is an important issue for the equine industry. The purpose of this study was to examine the applicability of a sensor for foaling detection using the data of surface temperature (ST), roll angle (rotation about the y-axis) and y-axis (long axis of the tail) acceleration which were collected from a multimodal device attached to the ventral tail base of the mare. The data were collected every 3 minutes in 17 pregnant mares. Roll angle differences from the reference values and the mare's posture (standing or recumbent) confirmed by video were compared and associated. Cohen's kappa coefficient was 0.99 when the threshold was set as ± 0.3 radian in roll angle differences. This result clearly showed that the sensor data can accurately distinguish between standing and recumbent postures. The hourly sensor data with a lower ST (LST < 35.5°C), a recumbent posture determined by the roll angle, and tail-raising (TR, decline of 200 mg or more from the reference value in y-axis acceleration) was significantly higher during the last hour prepartum than 2-120 hours before parturition (P < 0.01). The accuracy of foaling detection within one hour was verified using the following three indicators: LST; lying down (LD, change from standing to recumbent posture); and TR. When LST, LD and TR were individually examined, even though all indicators showed that sensitivity was 100%, the precision was 13.1%, 8.1% and 2.8%, respectively. When the data were combined as LST+LD, LST+TR, LD+TR and LST+LD+TR, detection of foaling improved, with precisions of 100%, 32.1%, 56.7% and 100%, respectively. In conclusion, the tail-attached multimodal device examined in this present study is useful for detecting foaling.

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.

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.