Luteolysis In Mares Research Articles

The Interaction Between Nodal, Hypoxia-Inducible Factor 1 Alpha, and Thrombospondin 1 Promotes Luteolysis in Equine Corpus Luteum.

The regulation of corpus luteus (CL) luteolysis is a complex process involving a myriad of factors. Previously, we have shown the involvement of Nodal in functional luteolysis in mares. Presently, we ask the extent of which Nodal mediation of luteolysis is done through regulation of angioregression. We demonstrated the interaction between Nodal and hypoxia-inducible factor 1 α (HIF1α) and thrombospondin 1/thrombospondin receptor (TSP1/CD36) systems, could mediate angioregression during luteolysis. First, we demonstrated the inhibitory effect of Nodal on the vascular marker platelet/endothelial cell adhesion molecule 1 (CD31). Also, treatment of mid CL explants with vascular endothelial growth factor A (VEGFA) showed a trend on activin-like kinase 7 (Alk7) protein inhibition. Next, Nodal was also shown to activate HIF1α and in vitro culture of mid CL explants under decreased oxygen level promoted Nodal expression and SMAD family member 3 (Smad3) phosphorylation. In another experiment, the crosstalk between Nodal and TSP1/CD36 was investigated. Indeed, Nodal increased the expression of the anti-angiogenic TSP1 and its receptor CD36 in mid CL explants. Finally, the supportive effect of prostaglandin F2α (PGF2α) on TSP1/CD36 was blocked by SB431542 (SB), a pharmacological inhibitor of Nodal signaling. Thus, we evidenced for the first time the in vitro interaction between Nodal and both HIF1α and TSP1 systems, two conserved pathways previously shown to be involved in vascular regression during luteolysis. Considering the given increased expression of Nodal in mid CL and its role on functional luteolysis, the current results suggest the additional involvement of Nodal in angioregression during luteolysis in the mare, particularly in the activation of HIF1α and TSP1/CD36.

Endogenous and exogenous effects of PGF2α during luteolysis in mares

An inhibitor of PGF2α biosynthesis (flunixin meglumine, FM) was used to study the role of endogenous PGF2α on the luteolytic effect of exogenous PGF2α in mares. A 2-h infusion of PGF2α at a constant rate (total dose, 0.1 mg) on Day 10 (ovulation = Day 0) was used to mimic the maximal concentrations of a spontaneous pulse of a PGF2α metabolite (PGFM). Treatment with FM (1.7 mg/kg) was done 1 h before and 5 h after the start of PGF2α infusion. In hourly blood samples beginning 1 h before the start of PGF2α infusion, progesterone decreased (P < 0.05) similarly by 5 h in each of the PGF2α and PGF2α+FM groups but not in the controls (n = 5). In a study of spontaneous luteolysis, the same FM dose was given every 6 h from Day 13 until Day 17 or earlier if CL regression was indicated by an 80% decrease in luteal blood-flow signals. Blood was sampled for progesterone assay each day and 8 h of hourly blood sampling was done each day to characterize PGFM concentrations and pulses. Progesterone (P4) was lower (P < 0.05) in controls than in an FM group (n = 7) by Day 15. Luteolysis (P4 < 1 ng/mL) ended on Days 14–19 in individual controls. In contrast, luteolysis did not end until after Day 20 in 4 of 7 FM-treated mares. In the three mares with completion of luteolysis before Day 20 in the FM group, the interval from beginning to end of luteolysis was longer (P < 0.02) (4.5 ± 0.6 days) than in the controls (3.0 ± 0.4 days). During 8-h sessions of hourly blood sampling on Day 14, concentration of PGFM was significantly lower in the FM group for the minimal, mean, and maximal per session. Pulses of PGFM were identified by a CV methodology on each day in 7 of 7 and 3 of 7 mares in the controls and FM group, respectively. The four FM-treated mares without a CV-identified pulse were the four mares in which luteolysis did not occur before Day 20. In mares with detected pulses, PGFM was lower at each nadir and at the peak (86% lower) in the FM group than in controls, but the interval between nadirs or base of a pulse was not different between groups. Hypothesis 1 that endogenous PGF plays a role in the luteolytic effect of exogenous PGF2α was not supported. Hypothesis 2 that an inhibitor of PGF2α biosynthesis prevented or minimized the prominence of PGFM pulses and increased the frequency of persistent CL was supported.

TGFB1 modulates in vitro secretory activity and viability of equine luteal cells

In the present report we describe the involvement of transforming growth factor B1 (TGF) in functional regression and structural luteolysis in the mare. Firstly, TGF and its receptors activin-like kinase (ALK) 5 and TGF receptor 2 were identified in corpus luteum (CL) steroidogenic, endothelial and fibroblast-like cells. Also, TGF and ALK5 protein expression were shown to be increased in Mid-, and Late-CL (p < 0.05). Subsequently, using an in vitro model with Mid-CL cells, we studied the role of TGF on secretory activity and cell viability. Cell treatment with TGF decreased progesterone (P4) and prostaglandin (PG) E2 concentrations in culture media (p < 0.05), and downregulated mRNA and protein of StAR, CYP11A1, cPGES and mPGES1 (p < 0.05). Conversely, TGF augmented PGF2a concentration in culture media, through PTGS2 and PGFS gene expression activation (p < 0.05). When cells were incubated with PGF2a, both TGF and ALK5 were upregulated (p < 0.05). Additionally, treatment with the pharmacological inhibitor of ALK5, ALK4 and ALK7 - SB431542 (SB) attenuated PGF2a functional and structural luteolytic actions. Indeed, SB blocked: (i) PGF2a inhibitory effect on StAR, CYP11A1, 3BHSD and mPGES1; (ii) PGF2a auto-amplification signal via PTGS2 and PGFS expression (p < 0.05); (iii) the PGF2a-induced BAX and FASL expression (p < 0.05). Finally, TGF decreased cell viability (p < 0.05) and promoted caspase 3 activity (p = 0.08) and the expression of pro-apoptotic FASL and BAX (p < 0.05). Our results suggest that TGF supports functional regression and structural luteolysis, and also confirm the importance of ALK5, ALK4 and ALK7 activation during PGF2a mediated luteolysis in mares.

Progesterone levels and days to luteolysis in mares treated with intrauterine fractionated coconut oil

Intrauterine plant oil infusion, including fractionated coconut oil, has been previously found to be a safe, inexpensive, and reversible method of prolonging the luteal phase in mares when administered on Day 10 of the estrous cycle. Our objective was to understand the uteroovarian response to the administration of fractionated coconut oil infusion in the uterus of diestrous mares. We hypothesized that intrauterine coconut oil administration on Day 10 would prolong luteal life span in a dose-dependent fashion and would result in higher serum progesterone levels than untreated mares at the expected time of luteolysis. Light-horse mares (n = 18) were examined using transrectal palpation and ultrasonography to determine if they had a normal interovulatory interval and were then examined daily in estrus until the day of ovulation (Day 0) and then every other day during an estrous cycle. Jugular blood was drawn on Day 11, Day 13, Day 15, and Day 17, centrifuged, and serum stored until assayed for progesterone (P4; Siemens Coat-a-Count Progesterone RIA, Los Angeles, CA, USA). Mares were randomly assigned to treatment and studied over one to two estrous cycles with a rest cycle after each treatment cycle. Groups were: control (n = 5), fractionated coconut oil 1.0 mL (Miglyol 810; Sasol Oil, Witten, Germany) infused in the uterus with an artificial insemination pipette on Day 10 (Group 1; n = 5) and fractionated coconut oil 0.5 mL infused in the uterus with an embryo transfer gun, on Day 10 (Group 2; n = 5). All statistical analyses were performed using analytical software (Stata SE, version 13.1, College Station, TX, USA) at P < 0.05. Data were first evaluated using the Shapiro–Wilk test for normality. Differences between groups in days to luteolysis (DTL) were examined using analysis of variance and Bonferroni, and the effect of day and treatment on P4 levels were examined using the Kruskal–Wallis and Dunn's all pairwise test. There was a significant difference in DTL between the groups (P = 0.0083), with fewer DTL in Group 1 compared to control (P = 0.011) and to Group 2 (P = 0.034). There was a significant effect of day (P < 0.0001) on P4 levels with Day 11 P4 levels higher than Day 15 and Day 17 and Day 13 P4 levels higher than Day 15 or Day 17 (P < 0.03). There was a significant effect of treatment group (P = 0.0098) on P4 levels with control and Group 2 levels higher than Group 1 mares (P = 0.0012, P = 0.0495, respectively). We concluded that intrauterine administration of 1 mL of fractionated coconut oil lowered P4 levels in diestrus in a dose-dependent fashion and did not prolonged the luteal phase of the mares.

138 Characterization of partial luteolysis in mares

Datawere analyzed by regression analysis and by a random effects mixedmodel. Therewere significant (P < 0.001) effects of time and assay method for the determination of progestins in the mixed model. Least squares means for progestins across the studied interval differed (P < 0.001) with P4 1⁄4 0.66 ng/mL; DHP 1⁄4 26.4 ng/ mL; ELISA progestins 1⁄4 47.9 ng/mL and mini-Vidas progestins 1⁄4 20.8 ng/mL (pooled SEM 1⁄4 0.66). These data confirm progestin determination by immunoassay during late gestation varies depending upon the assay used and the apparent cross reactivity to other progestins present in high concentrations during late gestation in the mare. More widespread application of LC-MS/MS for determination of progestins in the late pregnant mare will enhance the interpretation of progestin concentrations. Study was supported by the Albert G. Clay Endowment. Biom erieux provided the progestin immunoassays for the mini-Vidas.

Relationships among nitric oxide metabolites and pulses of a PGF2α metabolite during and after luteolysis in mares

Hourly circulating concentrations of a PGF2α metabolite (PGFM), progesterone (P4), and LH were obtained from a reported project, and concentrations of nitric oxide (NO) metabolites (NOMs; nitrates and nitrites) were determined in eight mares. Unlike the reported project, hormone concentrations were normalized to the peak of the first PGFM pulse of luteolysis (early luteolysis), second PGFM pulse (late luteolysis), and a pulse after luteolysis. The duration of luteolysis was 23.1 ± 1.0 hours, and the peak of the first and second PGFM pulses occurred 6.5 ± 0.9 and 14.8 ± 0.8 hours after the beginning of luteolysis. Concentration of P4 decreased progressively within and between the PGFM pulses Changes were not detected in LH concentration in association with the PGFM pulses. Concentration of NOMs was greater (P < 0.05) at the peak of the PGFM pulse during early luteolysis (88.8 ± 15.0 μg/mL) than during late luteolysis (58.8 ± 9.0 μg/mL). Concentration of NOMs began to decrease (P < 0.05) 4 hours before the peak of the PGFM pulse of early luteolysis. Concentration began to increase (P < 0.05) an hour after the peak of the PGFM pulse of late luteolysis. An NOM decrease and increase was not detected during the PGFM pulse after luteolysis. On a temporal basis, results indicated that NO either is not required for luteolysis in mares or has a role in or responds only during late luteolysis. A caveat is that the relative contribution of the CL versus other body tissues to circulating concentrations of NOMs in mares has not been determined.

Oxytocin induction of pulses of a prostaglandin metabolite and luteolysis in mares

A procedure for oxytocin (OT) administration on Day 13 postovulation was developed in mares for stimulation of a pulse of PGFM (a PGF2α metabolite) that mimics a natural PGFM pulse during luteolysis. Bolus treatment with each of five OT doses (1–10 IU/mare, n = 3) stimulated a burst of PGFM that was maximum in 4 minutes and was unlike a natural pulse. A 2-hour OT infusion of 1.25, 2.5, or 5 IU/100 kg (n = 4) induced a PGFM pulse similar to reported pulses; lower doses did not. The peak of an induced pulse (approximately 260–380 pg/mL) seemed similar to reported natural peaks (approximately 200–300 pg/mL), using the same assay system. The interval from nadir to nadir was 6.6 ± 0.2 hours. Percentage decrease in progesterone (P4) within 8 hours was greater (P < 0.05) for doses of 1.25, 2.5, or 5 IU/100 kg (43%–50%) than that for a vehicle group (11%). Treatment with flunixin meglumine (1.0 mg/kg), a cyclooxygenase inhibitor, decreased (P < 0.008) P4 concentration, but treatment 2 hours before the beginning of OT infusion (2.5 IU/100 kg) did not prevent the OT-induced PGFM pulses and the decrease in P4. In conclusion, a PGFM pulse was simulated by infusion of OT during 2 hours but not by a single OT bolus, and an OT-simulated PGFM pulse stimulated a decrease in P4 that was not prevented by a cyclooxygenase inhibitor. These are the first firm demonstrations that OT in mares as in other species has a role in luteolysis.

Expression of Aldo-keto Reductase 1C23 in the Equine Corpus Luteum in Different Luteal Phases

Regression of the corpus luteum (CL) is characterized by a decay in progesterone (P4) production (functional luteolysis) and disappearance of luteal tissues (structural luteolysis). In mares, structural luteolysis is thought to be caused by apoptosis of luteal cells, but functional luteolysis is poorly understood. 20α-hydroxysteroid dehydrogenase (20α-HSD) catabolizes P4 into its biologically inactive form, 20α-hydroxyprogesterone (20α-OHP). In mares, aldo-keto reductase (AKR) 1C23, which is a member of the AKR superfamily, has 20α-HSD activity. To clarify whether AKR1C23 is associated with functional luteolysis in mares, we investigated the expression of AKR1C23 in the CL in different luteal phases. The luteal P4 concentration and levels of 3β-hydroxysteroid dehydrogenase (3β-HSD) mRNA were higher in the mid luteal phase than in the late and regressed luteal phases (P<0.05), but the level of 3β-HSD protein was higher in the late luteal phase than in the regressed luteal phase (P<0.05). The luteal 20α-OHP concentration and the level of AKR1C23 mRNA were higher in the late luteal phase than in the early and mid luteal phases (P<0.05), and the level of AKR1C23 protein was also highest in the late luteal phase. Taken together, these findings suggest that metabolism of P4 by AKR1C23 is one of the processes contributing to functional luteolysis in mares.

Novel luteolytic mechanisms in equine corpora lutea: the role of Nodal signaling pathway and its interaction with Tumor necrosis factor alpha, Interferon gamma and Fas ligand

Novel luteolytic mechanisms in equine corpora lutea: the role of Nodal signaling pathway and its interaction with Tumor necrosis factor alpha, Interferon gamma and Fas ligand

Auto-amplification system in prostaglandin F2α production by endometrium for initiating and progressing luteolysis in mares

In many mammals, including the horse, prostaglandin F2a (PGF) secreted from the endometrium is the major luteolysin. When exogenous PGF is administered to induce luteolysis, the effective dose of PGF is lower in horses than in cattle. The reason why a low dose of PGF is successful in horses is not clear. We have hypothesized that a local autoamplification system of PGF production by the equine endometrium plays a role in inducing luteolysis following administration of a low dose of PGF. To test this hypothesis, we carried out the following in vivo and in vitro studies. In Experiment 1, mares (n1⁄46) at the mid-luteal stage were injected i.m. with 1.0 ml PGF analog (PGFa; cloprostenol 250 mg/ml) and blood samples from the jugular vein were collected frequently at fixed intervals. Progesterone and PGF metabolite (PGFM) concentrations in blood plasma were measured by enzyme immunoassay. Cloprostenol did not show a cross-reaction with PGFM in our assay system. Progesterone had decreased by 50% at 2 h and by about 90% at 24 h after PGFa administration. PGFM increased, showing a first peak at 45 min, then decreased and again increased to the level of the first peak at 16 h; levels exceeding that of the first peak were then maintained until 72 h after the PGFa administration. In Experiment 2, PGF receptor (PTGFR) mRNA expression in endometrial tissues collected from different stages of the estrous cycle was determined by real-time PCR. PTGFR mRNA expression was higher at the

Role of PGF2α in luteolysis based on inhibition of PGF2α synthesis in the mare

The effects of inhibition of PGF2α synthesis on luteolysis in mares and on the incidence of prolonged luteal activity were studied in controls and in a group treated with flunixin meglumine (FM), a PGF2α inhibitor (n = 6/group). The FM was given every 8 hours (1.0 mg/kg) on each of Days 14.0 to 16.7. Concentration (pg/mL) of PGF2α metabolite averaged over 8 hours of hourly blood sampling at the beginning of each day, was lower in the FM group than in the controls on Day 14 after ovulation (6.7 ± 1.3 vs. 13.8 ± 2.9, P < 0.05), Day 15 (15.0 ± 3.9 vs. 35.2 ± 10.4, P < 0.10), and Day 16 (21.9 ± 5.7 vs. 54.7 ± 11.4, P < 0.03). Concentration (ng/mL) of progesterone (P4) was greater in the FM group than in the controls on Day 14 (10.1 ± 0.9 vs. 7.7 ± 0.9, P < 0.08), Day 15 (9.2 ± 1.0 vs. 4.3 ± 1.0, P < 0.008), and Day 16 (5.6 ± 1.6 vs. 1.2 ± 0.4, P < 0.02). The interval from ovulation to the beginning of a decrease in P4 and to the end of luteolysis (P4 < 1 ng/mL) was each delayed (P < 0.03) by ∼1 day in the FM group. Intervals involving the luteal phase were long (statistical outliers, P < 0.05) in two mares in the FM group, indicating prolonged luteal activity. Results supported the hypotheses that (1) inhibition of PGF2α synthesis interferes with luteolysis in mares and (2) inhibition of PGF2α at the expected time of luteolysis may lead to prolonged luteal activity.

Diestrus administration of oxytocin prolongs luteal maintenance and reduces plasma PGFM concentrations and endometrial COX-2 expression in mares

The objectives were to: (1) evaluate the efficacy of varying intervals of oxytocin administration in preventing luteolysis in mares; (2) examine PGF2α release in mares experiencing prolonged diestrus secondary to oxytocin treatment; and (3) evaluate the endometrial expression of oxytocin receptor, estrogen receptor α, and prostaglandin synthesis enzymes after oxytocin administration. In experiment I, mares received oxytocin (60 IU, im) daily on Days 8 to 10, 8 to 12, or 8 to 14 postovulation, and control mares received sterile saline. Prolongation of diestrus was defined by elevation of serum progesterone >1.0 ng/mL through Day 30 postovulation. The proportion of mares experiencing prolonged cycles increased (P < 0.01) as the number of days of oxytocin administration increased. Oxytocin administration on Days 8 to 10, 8 to 12, and 8 to 14 prolonged luteal maintenance in 3/7, 4/7, and 6/7 mares respectively, compared with 0/7 control mares. Treated mares with prolonged diestrus had lower (P < 0.05) plasma PGFM concentrations at Day 16 than did mares with normal diestrus periods. In experiment II, endometrial biopsies from mares treated with oxytocin from Days 8 to 14 postovulation (N = 6) had reduced cyclooxygenase-2 expression (P < 0.05) compared with control mares (N = 6) as determined by quantitative reverse transcription polymerase chain reaction and immunohistochemical staining. Oxytocin administration prolonged luteal maintenance in mares, with an increasing number of mares responding to treatment as the number of days of oxytocin administration was increased beyond Day 8 postovulation. Luteal maintenance in mares was also associated with decreased plasma PGFM concentrations and reduced endometrial cyclooxygenase-2 expression.

Relationship Between Dose of Cloprostenol and Age of Corpus Luteum on the Luteolytic Response of Early Dioestrous Mares: A Field Study

The objective of this study was to establish and characterize the relationship between the dose of cloprostenol (37.5, 250, 500 and 750 μg) and the age of the early corpus luteum (CL) (80, 88, 96, 104 and 112 h) on the luteolytic response of mares. Behavioural oestrus and ultrasonographic signs of return to oestrus were considered as the occurrence of full luteolysis. A total of 298 mares were divided into groups according to dose of cloprostenol and CL age. There was an effect of dose of cloprostenol (p < 0.001) and age of the CL at the time of treatment (p < 0.001) on the percentage of mares with full luteolysis. The efficacy of 37.5 μg of d-cloprostenol was similar to that of 250 μg of d,l-cloprostenol (p > 0.05); and that of 500 similar to that of 750 μg (p > 0.05). The higher dose groups (500 and 750 μg) induced full luteolysis more frequently than the lower dose groups (37.5 and 250 μg) 96-104 h post-ovulation. There was no effect of CL age or cloprostenol dose on the interovulatory interval (p > 0.05). In conclusion, the effect of cloprostenol on the percentage of mares undergoing full luteolysis is dose-dependent. However, this effect is only evident in mares with CLs aged between 96 and 104 h. There is no advantage of administering more than 500 μg of d,l-cloprostenol (Estrumate(®)), to obtain a higher percentage of mares with full luteolysis in mares with CLs aged 80-112 h.

Hormone concentration changes temporally associated with the hour of transition from preluteolysis to luteolysis in mares

The temporal associations of cortisol, estradiol-17β, and oxytocin with pulses of PGFM at the common hour of transition between preluteolysis and luteolysis was studied in plasma from hourly blood samples in mares ( n = 8). The transitional hour was determined from progesterone concentrations and occurred between 2 PM and 2 AM in all mares. Pulses of PGFM were grouped into those occurring at the last pulse of preluteolysis (preluteolytic pulse), at the hour of transition (transitional), and during luteolysis (luteolytic). The preluteolytic PGFM pulse (45 ± 16 pg/ml at peak) and transitional pulse (42 ± 7 pg/ml) are reportedly less prominent than the first luteolytic pulse (193 ± 36 pg/ml). Cortisol increased ( P < 0.05) between −1 h and 0 h (peak) and then decreased ( P < 0.05) within the hours of the luteolytic PGFM pulse but did not change within the preluteolytic and transitional pulses. Estradiol increased ( P < 0.006) during −3 to 2 h of the luteolytic pulse but not for the other pulses. Oxytocin differed for the hours of the transitional PGFM pulse ( P < 0.02) and the luteolytic pulse ( P < 0.03) but did not differ significantly during the hours of the last preluteolytic pulse. Oxytocin increased ( P < 0.05) between −3 h and 0 h and then decreased ( P < 0.05) within each of the transitional and the luteolytic pulses. The oxytocin results are novel and support the hypothesis that on a temporal basis oxytocin in association with PGF2α accounts for the transition between preluteolysis and luteolysis within a single hour in mares, despite the small transitional PGFM pulse.

Concentrations of circulating hormones normalized to pulses of a prostaglandin F 2α metabolite during spontaneous luteolysis in mares

The temporal relationships between a pulse of 13,14-dihydro-15-keto-PGF 2α (PGFM) and the concentrations of circulating hormones during the luteolytic period were studied for 11 pulses in 11 mares (Equus caballus) using samples collected hourly. Mean PGFM pulses encompassed 4 h before to 4 h after the peak, and hormone data were normalized to the PGFM peak (Hour 0). Concentration of progesterone decreased (P < 0.05) between Hours –4 and –3 and continued to decrease linearly throughout the PGFM pulse. The concentrations of cortisol and prolactin increased (P < 0.004) during Hours –4 to 0 and decreased (P < 0.002) during Hours 0 to 4. Estradiol concentration increased (P < 0.02) during Hours –4 to 0 but did not change significantly after Hour 0. Concentrations of follicle-stimulating hormone and luteinizing hormone did not change significantly during the PGFM pulse, and the oxytocin results were equivocal. Percentage of corpus luteum area with color-Doppler signals of blood flow did not change significantly between Hours –4 and 0 and first began to decrease (P < 0.004) between Hours 0 and 2. Results demonstrated that concentrations of progesterone decreased linearly during a PGFM pulse, and cortisol, prolactin, and estradiol increased during the ascending portion of the pulse. The progesterone and gonadotropin results supported the hypothesis that the initial progesterone and gonadotropin increases that have been reported to occur in response to a single bolus luteolytic treatment with prostaglandin F 2α do not occur in response to the natural secretion of prostaglandin F 2α.

Progesterone responses to intravenous and intrauterine infusions of prostaglandin F2α in mares

The hypotheses were tested that prostaglandin F2alpha (PGF) travels from the uterus to the ovaries via a systemic route in mares, as opposed to a local route in ruminants, and that one pulse of PGF produces only partial luteolysis. Intravenous (i.v.) and intrauterine (i.u.) infusions of PGF were performed 8 days after ovulation at a constant rate for 2 h. Plasma concentrations of PGF were assessed by assay of 13,14-dihydro-15-keto-PGF2alpha (PGFM). Total doses administered were as follows: 0, 0.05, 0.1, 0.5 and 1.0 mg, i.v., PGF and 0 and 0.5 mg, i.u., PGF (n=4 mares per group). In addition, PGFM concentrations were determined for natural pulses from samples collected each hour during luteolysis (n=5). Progesterone was similarly reduced by 4 days after treatment in the 0.5 mg i.v., 0.5 mg i.u. and 0.0 mg i.u. groups. The area under the PGFM curve in the 0.1 mg i.v. group was similar to the area for natural PGFM pulses. Progesterone decreased to a similar concentration by 12 h in the 0.1, 0.5 and 1.0 mg i.v. groups, but thereafter was greater (P<0.05) in the 0.1 mg i.v. group. Progesterone concentrations reached <2 ng mL(-1) 6 days after treatment in the 0.05 and 0.1 mg i.v. groups and 2 days after treatment in the 0.5 and 1.0 mg i.v. groups. The results support the hypotheses of a systemic uteroluteal route for PGF transfer and that one pulse produces only partial luteolysis in mares.

Characterisation of pulses of 13,14-dihydro-15-keto-PGF2alpha (PGFM) and relationships between PGFM pulses and luteal blood flow before, during, and after luteolysis in mares

Blood collections for characterising 13,14-dihydro-15-keto-PGF2alpha (PGFM) pulses in mares and colour-Doppler examinations for estimating percentage of corpus luteum with blood-flow signals were done hourly for a 24-h session on Day 15 (ovulation = Day 0; n = 13 mares) or during 12-h sessions from Days 12 to 16 (n= 10 mares). Luteolysis was defined as extending from the beginning of a precipitous decrease in progesterone until progesterone was <2 ng mL(-1). Comparisons were made among preluteolysis, luteolysis, and postluteolysis. Greater prostaglandin F2alpha activity (mean PGFM concentration per session) occurred during luteolysis than during preluteolysis and postluteolysis. Statistically-detected PGFM pulses were smaller during preluteolysis with a highly variable interval from the last pulse to the beginning of luteolysis. Either two or three pulses were detected in each 24-h session during luteolysis and postluteolysis, after excluding three of eight sessions with no pulses during postluteolysis. Statistically, 17% of pulses during postluteolysis were prominent outliers. The nadir-to-nadir interval during a pulse (5 h), the peak-to-peak interval between pulses (9 h), and the resulting 4-h gap between pulses were similar during and after luteolysis. The decrease in progesterone encompassed the PGFM pulses, without a detectable fluctuation during a pulse. The percentage of corpus luteum with blood-flow signals did not change during the ascending portion of a PGFM pulse and decreased within 2 or 3 h after the peak, even during preluteolysis. Results indicated that a reported increase in luteal blood flow in heifers during the ascending portion of a PGFM pulse does not occur in mares.

Luteolysis, estrus induction, and clinical side effects in mares treated with a PDF2α analog, cloprostenol (Sinocrel 11-21)

Summary The objective of this study was to prove the effectiveness of cloprostenol, a synthetic prostaglandin analog for the induction of luteolysis in the mare, as well as to evaluate its clinical side effects. Twenty-five mares were treated on day 6 after ovulation with 0.075 mg cloprostenol. Blood samples were taken to determine plasma progesterone levels. Progesterone levels were below 1 ηg/mL at 42.2 ± 10.4 hours after treatment, which indicated that there was no longer a functional corpus luteum (CL). It was shown that in 96% (24/25) of the mares, the prostaglandin F2α (PGF2α) analog at the dose used had a luteolytic effect. All the mares showed estrous behavior within 5 days after application of the PGF2α analog. The prostaglandin treatment caused a significant increase ( P P > .05) in the heart rate and the respiratory rate between the treated group and the control group.

Changes in steady-state concentrations of messenger ribonucleic acids in luteal tissue during prostaglandin F2α induced luteolysis in mares

Transvaginal ultrasound-guided luteal biopsy was used to evaluate the effects of prostaglandin (PG)F2α on steady-state concentrations of mRNA for specific genes that may be involved in regression of the corpus luteum (CL). Eight days after ovulation (Hour 0), mares ( n = 8/group) were randomized into three groups: control (no treatment or biopsy), saline + biopsy (saline treatment at Hour 0 and luteal biopsy at Hour 12), or PGF2α + biopsy (5 mg PGF2α at Hour 0 and luteal biopsy at Hour 12). The effects of biopsy on CL were compared between the controls (no biopsy) and saline + biopsy group. At Hour 24 (12 h after biopsy) there was a decrease in circulating progesterone in saline group to 56% of pre-biopsy values, indicating an effect of biopsy on luteal function. Mean plasma progesterone concentrations were lower ( P < 0.001) at Hour 12 in the PG group compared to the other two groups. The relative concentrations of mRNA for different genes in luteal tissue at Hour 12 was quantified by real time PCR. Compared to saline-treated mares, treatment with PGF2α increased mRNA for cyclooxygenase-2 (Cox-2, 310%, P < 0.006), but decreased mRNA for LH receptor to 44% ( P < 0.05), steroidogenic acute regulatory protein to 22% ( P < 0.001), and aromatase to 43% ( P < 0.1) of controls. There was no difference in mRNA levels for PGF2α receptor between PG and saline-treated groups. Results indicated that luteal biopsy alters subsequent luteal function. However, the biopsy approach was effective for collecting CL tissue for demonstrating dynamic changes in steady-state levels of mRNAs during PGF2α-induced luteolysis. Increased Cox-2 mRNA concentrations suggested that exogenous PGF2α induced the synthesis of intraluteal PGF2α. Thus, the findings are consistent with the concept that an intraluteal autocrine loop augments the luteolytic effect of uterine PGF2α in mares.

Induction of luteolysis in mares by ultrasound-guided intraluteal treatment with PGF2alpha

To evaluate the technique of ultrasound-guided luteal injection in mares, PGF2alpha was administered under ultrasound guidance to horse mares (n = 7 to 9 per group) on Day 9 postovulation via either a systemic (im; zero, 0.01, 0.1, or 5 mg/dose) route or a local intraluteal (il; zero, 0.01 or 0.1 mg/dose) route. The luteolytic efficacy of each treatment was determined based on post-treatment decreases in progesterone concentration, interval to uterine edema (IE) and interovulatory interval (IOI). Local administration of PGF2alpha directly into the CL consistently induced luteolysis, at doses up to 50-fold lower than the lowest effective systemic dose. Significant decreases in IOI and IE occurred in mares treated with 5 mg PGF2alpha im or 0.1 mg PGF2alpha il, but did not occur in mares treated with 0.1 or 0.01 mg PGF2alpha im, 0.01 mg PGF il, vehicle il or vehicle im. Progesterone concentrations were reduced to less than 10% of pretreatment values by two days post treatment in mares treated with 5 mg PGF2alpha im or 0.1 mg PGF2alpha il. PGF2alpha doses of 0.1 mg im and 0.01 mg il were associated with smaller but significant progesterone decreases (to 66% and 46% of pre-treatment values, respectively) by two days post treatment. Progesterone values after administration of il vehicle did not differ from pre-treatment values by two days post treatment, but were significantly lower (53% of pre-treatment values) by four days post treatment. Intramuscular treatment with vehicle or 0.01 mg of PGF2alpha did not significantly reduce progesterone concentrations below pretreatment values. Overall, the minimum effective luteolytic dose of PGF2alpha given intraluteally was between 0.01 and 0.1 mg. Based on the results of this study, ultrasound-guided il injection appears to be a repeatable method for studying the direct effect of other chemicals on luteal function. However, the current procedure carries some risk, since three il injections were associated with ovarian abscesses.