Transrectal ultrasonography of follicular dynamics and early pregnancy in mares: an on-site visual reference

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.

Background: During pregnancy, cardiovascular adaptations occur in order to meet maternal demands and adequate support for fetal development, but they are still unclear in the equine species, especially in the American Miniature horse breed. The main hemodynamic changes that occur during pregnancy are increased heart rate, cardiac output, and blood volume. It is necessary to use the reference values for the miniature breeds, in addition to the age range and reproductive condition, so the objective of this study was to evaluate the effect of pregnancy on electrocardiographic (ECG) parameters in mares and also of age in newborns foals of American Miniature horse breed. Materials, Methods & Results: Ten American Miniature Horse mares, between 7 and 19 years old (12.14 ± 4.05 years), 116.85 ± 24.09 kg of body weight were used in the experiment. All these mares were pluriparous, with 5.9 ± 3.5 of births, minimum of 2, and maximum of 12 foaling times. They were maintained in common social group in an indoor house stable with straw bed, with access to an outdoor paddock for several h per day. Twice a day they were fed with hay. Mineral supplements and water were available ad libitum. The ECG were obtained in 10 pregnant mares and in 10 neonates. All females were examined at a representative time of each trimester of pregnancy, at 30, 210, 300 days before and 21 days after delivery. Neonates were examined on different times: first 24 h after delivery, weekly on the 7th, 14th, 21st, 28th and 35th days of age. The ECG parameters considered were the heart rate and rhythm, duration and intervals (P, PR, QRS, QT, T) and amplitude (P, R, S, T) of ECG waves. The mean of pregnancy duration was 319.4 ± 4.97 days. Heart Rate (HR) varied between 51 to 59 bpm during pregnancy and 56 bpm on the 21st day after delivery. Comparing the clinical parameters of pregnant mares, non-pregnant mares and foal a difference was found for HR, Respiration Rate (RR) and Temperature (P = 0.001). All ECG parameters of pregnant, non-pregnant and foal mares showed a significant difference (P = 0.0001), except for the amplitude of the P1, P2 and R waves, which was similar for the 3 groups. The mean values of HR and amplitude of the positive T wave were higher in foals when compared to pregnant and non-pregnant mares, which in turn did not differ from each other. The mean duration of the P wave, PR segment, QRS complex, QT segment and T wave were the same among mares and greater than in foals. Comparing pregnant and non-pregnant mares, there was a difference in only 3 parameters: duration of the QTc segment, amplitude of the S wave and negative T wave. The non-pregnant mares had a longer duration of the QTc segment and a greater amplitude of the negative T wave, while the foals had a greater amplitude of the S wave.Discussion: Although this study did not measure HR at delivery time, it is worth to consider the action of the parasympathetic autonomic nervous system close to delivery, which reduces the HR of mares. This is because at delivery, sympathetic-adrenal activation should be avoided since the increase in sympathetic tone can cause uterine atony via B2 receptors. For this reason, it has been shown that, during physiological birth, horses are under parasympathetic control, and stress response mechanisms are not activated. It was conclude that the morphometry of the waves, intervals and ECG complexes, during the evolution of the reproductive state, the electrocardiographic aspects were generally associated with heart rate (PR and QT intervals). As for neonates, there was a discrepancy among the most of ECG parameters when compared to the adult female, regardless of reproductive status, clarifying the importance of ECG patterns not only for the breed, but also for the age group.

Dynamics of uterine diameter and endometrial morphology during the estrous cycle and early pregnancy in mares

Transrectal Doppler sonography of uterine blood flow during early pregnancy in mares.

Enrofloxacin may be an alternative antimicrobial for unresponsive cases of severe bacterial infections in pregnant mares. As pregnancy may affect drug bioavailability, distribution, metabolism and excretion, dose adjustment might be necessary. To determine the disposition of orally and intravenously administered enrofloxacin in pregnant and non-pregnant mares. Randomised cross-over experiment. Six light-breed, healthy pregnant mares (260days gestation) were given a single dose of either intravenous (5mg/kg bwt) or oral compounded (7.5mg/kg bwt) enrofloxacin, with the opposite dose administered after a 7-day washout. The protocol was repeated 45-60days post-partum, 15-30days after foals were weaned. Plasma samples were obtained via venepuncture at 0, 5, 10, 20, 30, 45, 60, 90min, and 2, 3, 4, 6, 8, 12, 24, 36, 48 and 72h after enrofloxacin administration. Enrofloxacin and ciprofloxacin concentrations were measured by LC-MS/MS. Concentration versus time data were analysed based on non-compartmental pharmacokinetics. Enrofloxacin AUC0-∞ was significantly higher in pregnant mares than non-pregnant mares after PO administration and tended to be higher after i.v. administration. Ciprofloxacin maximum plasma concentration (Cmax ) and concentration at 24h (C24h ) were higher, and half-life of the terminal phase (t½λz ) was longer in pregnant mares than non-pregnant mares after oral administration. Similarly, ciprofloxacin C24h was higher in pregnant mares with intravenous administration. Oral bioavailability did not differ based on pregnancy status. Only six healthy light breed mares were assessed. Disease or horse breed may affect the endpoints evaluated. A lack of established enrofloxacin AUC/MIC targets for equine pathogens limits pharmacokinetic-pharmacodynamic conclusions. The oral form of enrofloxacin was well absorbed, and oral bioavailability was comparable to previous studies. While differences in enrofloxacin and ciprofloxacin pharmacokinetics were seen between pregnant and non-pregnant mares, the recommended drug dose and dose intervals are appropriate for MIC <0.25µg/mL. Dosages may need to be adjusted for bacteria with a MIC >0.25µg/mL.

The objective of the present study was to examine if luteal blood flow (LBF) monitoring could be used as an additional prognostic tool for early pregnancy diagnosis, and we particularly focused on the differences in LBF between pregnant and nonpregnant mares. Furthermore, other possible developmental differences of corpus luteum (CL) between pregnant and nonpregnant mares were evaluated. The CL (n = 119) of 27 mares were monitored once daily in B- and Power-Doppler Mode on days 1, 2, 9, 12, and 16 after ovulation (day 0 = ovulation). The data were evaluated using the MIXED Linear Model with repeated measures, and parameters were estimated by the REML method. The course of LBF, area of CL, and pixel intensity differed in nonpregnant mares on a day-to-day basis in contrast to more stable values in pregnant mares. Further, the profiles of the courses were identical until day 9, but since day 12 the differences between pregnant and nonpregnant mares started to be prominent. The LBF, pixel intensity, and level of progesterone (P4) were similar in all mares until day 16, when smaller LBF, lower pixel intensity, and lower levels of P4 were found in nonpregnant mares (P = 0.04, P = 0.02, P &amp;lt; 0.05, respectively). In pregnant and nonpregnant mares the LBF was weakly (r = 0.29 in both) and pixel intensity strongly (r = 0.48 and 0.59, respectively) correlated to the levels of P4. LBF was strongly correlated to the area of CL in pregnant as well as nonpregnant mares (r = 0.72 and 0.64, respectively). In accordance with the results presented in our study we can state that LBF monitoring is not a suitable tool for early pregnancy diagnosis or prognosis as the differences between pregnant and nonpregnant mares are notable &amp;ndash; similarly to other indicators of CL status &amp;ndash; just after the onset of luteolysis (day 16) when embryo itself is detectable. &amp;nbsp;

The early pregnancy in mares - What do we still not know?

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

Absence of diurnal variation in serum progesterone concentrations in mares

Pregnancy diagnosis in the mare by immunoassay of estrone sulfate in serum and milk

Embryos, endometrial biopsies, and uterine lavage fluid were collected from pregnant and non-pregnant mares 14 days after ovulation. Embryos were cultured for 20.5 h with and without endometrial tissue from pregnant mares, and endometrial tissue was cultured alone. Endometrial content of PGF tended to be higher (P = 0.06) in non-pregnant than in pregnant mares, but the amount of PGF released from tissue during culture was similar for pregnant and non-pregnant mares. Lavage fluid from non-pregnant mares also tended (P = 0.08) to contain higher concentrations of PGF. Coincubation of embryos with endometrium from pregnant mares significantly (P = 0.01) lowered concentrations of PGF in medium. Tissue concentrations and release of PGE-2 and 6-keto-PGF-1 alpha were similar in endometrial samples from pregnant and non-pregnant mares and prostaglandin production was unaffected by the presence of an embryo during incubation. Horse embryos released all three prostaglandins during a 20.5-h incubation.

Maternal recognition of pregnancy (MRP) in the mare is an unknown process. In a non-pregnant mare on day 14 post-ovulation (PO), prostaglandin F2α (PGF) is secreted by the endometrium causing regression of the corpus luteum. Prior to day 14, MRP must occur in order to attenuate secretion of PGF. The embryo is mobile throughout the uterus due to uterine contractions from day of entry to day 14. It is unknown what signaling is occurring. Literature stated that infusing oil or placing a glass marble into the equine uterus prolongs luteal lifespan and that in non-pregnant mares, serum exosomes contain miRNA that are targeting the focal adhesion (FA) pathway. The hypothesis of this study is embryo contact with endometrium causes a change in abundance of focal adhesion molecules (FA) in the endometrium leading to decrease in PGF secretion. Mares (n = 3/day) were utilized in a cross-over design with each mare serving as a pregnant and non-pregnant (non-mated) control on days 9 and 11 PO. Mares were randomly assigned to collection day and endometrial samples and embryos were collected on the specified day. Biopsy samples were divided into five pieces, four for culture for 24 hours and one immediately snap frozen. Endometrial biopsies for culture were placed in an incubator with one of four treatments: [1] an embryo in contact on the luminal side of the endometrium, [2] beads in contact on the luminal side of the endometrium, [3] peanut oil in contact on the luminal side of the endometrium or [4] the endometrium by itself. Biopsies and culture medium were frozen for further analysis. RNA and protein were isolated from biopsies for PCR and Western blot analysis for FA. PGF assays were performed on culture medium to determine concentration of PGF. Statistics were performed using SAS (P ≤ 0.05 indicated significance). The presence of beads on day 9 impacted samples from pregnant mares more than non-pregnant mares and had very little impact on day 11. Presence of oil decreased FA in samples from pregnant mares on day 9. On day 11, oil decreased FA abundance in samples from non-pregnant mares. Embryo contact caused multiple changes in RNA and protein abundance in endometrium from both pregnant and non-pregnant mares. The PGF secretion after 24 hours with each treatment was also determined. On day 9, there was no change in PGF secretion compared to any treatments. On day 11, presence of peanut oil increased PGF secretion in samples from non-pregnant mares. In samples from non-pregnant mares, presence of an embryo decreased PGF secretion compared to control samples from non-pregnant mares. Results revealed that while beads and peanut oil may impact abundance of FA RNA and protein in endometrial samples, it does not appear to impact PGF secretion. Conversely, embryo contact for 24 hours with endometrium from a non-pregnant mare causes a decrease in PGF secretion. These results suggest that it is not just contact of any substance/object causing attenuation of PGF secretion, but the embryo itself is necessary to decrease PGF secretion.

Assessment of pregnancy in the late-gestation mare using digital infrared thermography

ABSTRACTBackground and Aim:Pregnancy induces significant anatomical and physiological changes, many of which are regulated by the autonomic nervous system (ANS). Heart rate variability (HRV) is a well-established non-invasive tool for assessing ANS activity. While changes in heart rate (HR) and HRV during the third-trimester of equine pregnancy are documented, there is limited understanding of cardiac autonomic adaptations during the early stages of gestation. This study aimed to compare HR and time-domain HRV parameters between healthy non-pregnant mares and those in the first and second trimesters of pregnancy.Materials and Methods:A total of 45 Thai native crossbred mares were enrolled and divided into three groups: Non-pregnant (n = 5), first-trimester pregnant (0–114 days; n = 18), and second-trimester pregnant (115–226 days; n = 22). All mares were clinically healthy and free from cardiac abnormalities. Electrocardiographic data were collected using a Holter electrocardiogram system over a 15 min period at rest, and HRV was analyzed using time-domain measures: Standard deviation of all NN intervals (SDNN), SDNN index, root mean square of successive differences, standard deviation of 5-min mean NN intervals, percentage of successive NN intervals >50 ms, and vasovagal tonus index (VVTI). Data were analyzed using Kruskal–Wallis and Mann–Whitney U-tests.Results:HR was significantly higher in first-trimester pregnant mares compared to non-pregnant mares (p < 0.05), and even higher in the second-trimester compared to the first (p < 0.05). However, there were no significant differences among the groups in any of the HRV parameters or VVTI.Conclusion:The findings indicate that cardiovascular adaptation during early pregnancy in mares is characterized by a progressive increase in HR, likely reflecting increased cardiac output to support fetal development. However, the lack of significant changes in time-domain HRV parameters and VVTI suggests that ANS balance is maintained during the first and second trimesters. These results provide valuable reference values for equine reproductive monitoring and contribute to a better understanding of physiological changes in early gestation.

Features of follicle-stimulating hormone–stimulated follicles in a sheep model: keys to elucidate embryo failure in assisted reproductive technique cycles