Metagenomic characterization of the equine endometrial microbiome during anestrus.

The equine uterus is highly interrogated during estrus prior to breeding and establishing pregnancy. Many studies in mares have been performed during estrus under the influence of high estrogen concentrations, including the equine estrual microbiome. To date, it is unknown how the uterine microbiome of the mare is influenced by cyclicity; while, the equine vaginal microbiome is stable throughout the estrous cycle. We hypothesized that differences would exist between the equine endometrial microbiome of mares in estrus and anestrus. The aim of this study was two-fold: to characterize the resident endometrial microbiome of healthy mares during anestrus and to compare this with estrus. Double-guarded endometrial swabs were taken from healthy mares during estrus (n = 16) and in the following non-breeding season during anestrus (n = 8). Microbial population was identified using 16S rRNA sequencing. Our results suggest that the equine uterine microbiome in estrus has a low diversity and low richness, while during anestrus, a higher diversity and higher richness were seen compared to estrus. Despite this difference, both the estrus and anestrus endometrial microbiome were dominated by Proteobacteria, Firmicutes, and Bacteroidota. The composition of the microbial community between anestrus and estrus was significantly different. This may be explained by the difference in the composition of the endometrial immune milieu based on the stage of the cycle. Further research investigating the function of the equine endometrial microbiome and dynamics changes within the uterine environment is required.

Effect of Sampling Method on Detection of the Equine Uterine Microbiome during Estrus.

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

Breeding or assisted reproduction? Relevance of the horse model applied to the conservation of endangered equids.

Many wild equids are at present endangered in the wild. Concurrently, increased mechanization has pushed back the numbers of some old native horse breeds to levels that are no longer compatible with survival of the breed. Strong concerns arose in the last decade to preserve animal biodiversity, including that of rare horse breeds. Genome Resource Banking refers to the cryostorage of genetic material and is an approach for ex situ conservation, which should be applied in combination with in situ conservation programmes. In this review, we propose that, owing to the great reproductive similarity among the different members of the genus Equus, the domestic horse can be used to optimize cryopreservation and embryo production protocols for future application in wild equids. We will give this hypothesis a scientific underpinning by listing successful applications of epididymal sperm freezing, embryo freezing, intracytoplasmic sperm injection, oocyte vitrification and somatic cell nuclear transfer in domestic horses. Some ART fertilization methods may be performed with semen of very low quality or with oocytes obtained after the death of the mare.

Comparison of cell proliferation index in equine and caprine embryos using a modified BrdU incorporation assay.

The measurement of cell proliferation and cell viability using 5'bromo-2'deoxy-uridine (BrdU) labelling has been described in several cell types and species. The aim of this study was to adapt this technique to equine embryos and to compare the index of DNA replication (S-phase) between equine and caprine embryos. Seventeen equine embryos were recovered at day 6.5 post-ovulation and 20 caprine embryos were recovered at day 7 after the onset of estrus. Equine embryos were incubated during 1h at 39 degrees C in PBS containing 1mM of BrdU. Embryos were then treated in 0.05% trypsin during 15 min at 39 degrees C to permeabilise the capsule, and then embryos were rinsed in PBS containing 10% of foetal calf serum. After washing, embryos were immediately fixed in 2.5% paraformaldehyde with 0.3M NaOH during 15 min at ambient temperature. The S-phase was detected by immunocytochemistry technique. In caprine embryos, BrdU was visualised by the same technique but without the trypsin treatment. The percentage of cells (+/-S.E.M.) with BrdU incorporated into newly synthesised DNA strands was significantly higher in equine embryos (74+/-1) than in caprine (38+/-2). Our results demonstrated that BrdU incorporation assay can be used in equine embryos. This assay allows the determination of the proliferation index of live cells and could be used as an additional tool for evaluating the viability of embryos. The high percentage of cells incorporating BrdU during 1h of incubation with BrdU suggests that in comparison with the caprine embryos the cellular activity of proliferation is more intense in equine embryos and suggests that the cellular cycle is shorter in equine embryos.

Effects of gonadal steroids on the opioid regulation of LH and prolactin release in ovariectomized pony mares.

The aim of the present study was to investigate the role of ovarian steroids in the opioid regulation of LH and prolactin release in mares. Effects of the opioid antagonist naloxone on LH and prolactin secretion were determined in ovariectomized pony mares. The animals were pretreated with either progesterone (500 micrograms kg-1) or oestradiol benzoate (10 micrograms kg-1) for 8 days and subsequently with a combination of progesterone and oestradiol for an additional 8 days. Naloxone administration (0.5 mg kg-1 i.v.) resulted in a significant release of LH as well as prolactin in mares after pretreatment with either oestradiol benzoate or progesterone plus oestradiol benzoate (P < 0.05). No significant changes in LH and prolactin secretion were detected in progesterone-treated and non-steroid-treated ovariectomized mares. These results indicate that a prolonged oestrogen influence activates the opioid inhibition of LH and prolactin release in mares. In contrast to other species, progesterone alone does not activate a tonic opioid inhibition of LH and prolactin secretion, but modulates the effect of oestrogens. The opioid systems therefore seem to be regulated by a sequence of different steroid environments, as found during the oestrous cycle. The parallel increases in prolactin and LH secretion in mares may indicate a common regulatory pathway for these two hormones.

Induction of maternal behavior in non-parturient adoptive mares.

An attempt was made to elicit maternal behavior in non-parturient Welsh pony mares through a combination of hormonal treatment and vaginal-cervical stimulation (VCS). Lactation was induced in 16 nonpregnant, non-parturient mares via a combination of estradiol, progesterone and a dopamine antagonist (sulpiride). During the adoption trials, each lactating mare was confined behind a padded bar and a newborn foal was held near her head. Eight of the mares received two 3-min periods of VCS when the foster foal was introduced. Following VCS, the foal was released and its interactions with the adoptive mare observed until the acceptance criterion was met (i.e. the mare accepted the foal at the udder with no signs of aggression). The remaining eight adoptive mares were treated in the same manner but did not receive VCS. All 16 non-parturient mares eventually accepted and nursed their adopted foal. However, acceptance latencies were significantly shorter for mares in the VCS condition than for those without VCS, and did not differ between the VCS condition and a group of control mares with their biological offspring. In subsequent choice tests, both groups of foster mares (with/without VCS), like the control mares, displayed a preference for their 'own' foal. Once the non-parturient mares accepted their foster foal, their maternal behavior resembled that of control mothers. The positive effect of VCS on maternal acceptance may reflect a release of oxytocin triggered by this treatment.

Seasonality in mares.

In this review, we have attempted to summarize, based on recent data obtained in our laboratory and elsewhere, our current understanding of the regulatory mechanisms of seasonality and discuss the implications with regard to treatment strategies to advance the onset of cyclic reproductive activity in the early spring.

Fertility control using intrauterine devices: an alternative for population control in wild horses.

The purpose of this study was to develop a contraceptive method for feral horses. The feral horse population has increased significantly in recent years despite attempts to control numbers. As in most wild animal population control programs, contraceptive methods must be easy to apply, cause minimal disruption to the social structure and be fully reversible. In the present study, we tested the effectiveness of an intrauterine device (IUD) for fertility control in mares. Six mares were fitted with a silastic O-ring-shaped IUD on July 1 of Year 1. The IUD-treated mares were turned out with 12 nontreated mares and a fertile stallion in a large pasture until October 20 (112 d). None of the IUD-treated mares and all the nontreated mares became pregnant. The IUD-treated mares were maintained separately from the stallion during the winter. Following removal of the IUD on April 27 of Year 2, the mares were again introduced to the pasture with the stallion together with 6 nontreated mares. For the 6 mares previously treated with an IUD, the mean interval from introduction to the stallion to conception was 17.5 +/- 5 d or 1.3 cycles per pregnancy, and all mares produced a normal foal at term. Subsequently, 19 recorded mare breeding seasons resulted in 18 foals. Uterine cytology and histopathology indicate that the IUD causes mild chronic endometritis without permanent changes in the endometrium. We conclude that based on our observations, the O-ring-shaped IUD is an effective, safe and practical contraceptive method for mares.

Evidence for opioidergic inhibition of oxytocin release in periparturient mares.

In the horse mare, the onset of parturition is associated with an increase in oxytocin secretion, and it has been suggested that the onset of parturition may be triggered by endogenous oxytocin release. To test the hypothesis that oxytocin secretion is regulated by endogenous opioids in the periparturient period, we have 1) characterized oxytocin secretion in response to vaginocervical stimulation and 2) determined the effect of naloxone, an opioid antagonist, on oxytocin secretion induced by vaginocervical stimulation in prepartum mares and in postpartum mares at estrus and diestrus. During the last 2 months of pregnancy, the first diestrus and subsequent estrus post partum, a total of 66 vaginocervical stimulations were performed. Mares were pretreated with naloxone (0.5 mg/kg i.v.) or saline, administered 20 min before vaginocervical stimulation on subsequent days, using a randomized switchback design in which mares served as their own controls. Plasma was collected from 30 min before until 30 min after stimulation and was analyzed for oxytocin concentrations. Vaginocervical stimulation resulted in a significant increase in oxytocin secretion in all mares. Between Days 30 and 20 prepartum, the total amount of oxytocin secreted (calculated as area under the curve for 0 to 10 min after vaginocervical stimulation) was significantly greater in naloxone-treated than in saline-treated mares. From Day 20 prepartum until parturition, the differences between naloxone and saline-treated mares tended to decrease with approaching parturition, and were no longer statistically different. Peak plasma oxytocin concentrations were greater in naloxone-treated mares than in saline-treated mares during the entire prepartum period. During the postpartum period, total amount of oxytocin secreted following vaginocervical stimulation tended to be greater than during the prepartum period, and stimulated oxytocin secretion was significantly greater in naloxone-treated mares than in saline-treated mares. In conclusion, these data suggest that endogenous opioids suppress oxytocin secretion pre and post partum. It appears that opioid inhibition is not limited to the prepartum period, tends to decrease gradually towards parturition and is reinstated after foaling.

Induction of reproductive function in anestrous mares using a dopamine antagonist.

We investigated the role of dopamine in the regulation of seasonal reproductive activity in mares. Nine seasonal anestrous mares, maintained under a natural photoperiod, were treated daily with a dopamine D2 antagonist, [-]-sulpiride (200 mg/mare, im), beginning February 5 (day of year = 36) until the first ovulation of the year or for a maximum of 58. Nine untreated anestrous mares were maintained under the same conditions. The ovaries were examined by ultrasonography twice a week, and blood was collected three times a week for progesterone, LH, FSH and prolactin determinations. Mean day of first ovulation was significantly advanced for [-]-sulpiride-treated mares than control mares (mean day of year +/- SEM = 77.3 +/- 7.9 and 110.0 +/- 6.8, respectively; P < 0.01). Eight mares ovulated during [-]-sulpiride treatment while one mare failed to ovulate. Ovulation occurred 91 d after the start of treatment or on Day 127. All mares continued to have normal estrous cycles after the first ovulation. First cycle length and luteal progesterone concentrations did not differ between [-]-sulpiride-treated and control mares. Plasma prolactin concentrations were significantly increased at 2 and 9 h after [-]-sulpiride administration (P < 0.05), and had returned to basal levels by 24 h. At the time of the LH surge associated with the first ovulation, mean LH and FSH secretion was significantly higher in [-]-sulpiride-treated mares than in control mares (P < 0.05). These results suggest that dopamine plays a role in the control of reproductive seasonality in mares and exerts a tonic inhibition on reproductive activity during the anovulatory season.

Testosterone secretion during early pregnancy in mares.

We have characterized the testosterone secretion pattern during the first 80 d of pregnancy in mares and determined the sources that contribute to circulating testosterone levels during this period. Ten untreated, pregnant mares (Group 1), 10 altrenogest-treated, pregnant mares (Group 2), and 10 altrenogest-treated, pregnant mares in which the CL was eliminated by administration of PGF-2alpha on Day 16 (Group 3) were used in this study. Complete luteolysis occurred following PGF-2alpha administration in all mares in Group 3. Six of the 10 mares in Group 3 did not have an active CL until after Day 60 of pregnancy (Group 3a) and were included in the analysis. The remaining four mares developed a new CL on Days 32, 40, 43 and 49 of pregnancy and were excluded from analysis. Mares without a functional CL (Group 3a) had significantly lower testosterone concentrations than mares with a functional CL (Groups 1 and 2), during the period before equine chorionic gonadotropin (eCG) secretion. At the onset of eCG secretion, testosterone concentrations increased rapidly but the rate of increase decreased with time in mares with a functional CL (Groups 1 and 2). In mares without a functional CL (Group 3a), testosterone concentrations did not increase at the onset of eCG secretion but increased at a gradually increasing rate after Day 50. The lower testosterone concentration in mares without a functional CL before eCG secretion suggests that the CL contributes significantly to the circulating testosterone concentration during the period before eCG secretion. The close time relationship between the onset of eCG secretion and the increase in testosterone secretion in mares with a functional CL and the lack of a testosterone increase in pregnant mares without a functional CL suggest that the increase in testosterone secretion after Day 35 of pregnancy is the result of eCG-stimulated, luteal testosterone synthesis.

Expression of 3beta-hydroxysteroid dehydrogenase, cytochrome p450 17alpha-hydroxylase/17,20-lyase and cytochrome p450 aromatase enzymes in corpora lutea of diestrous and early pregnant mares.

In the pregnant mare, luteal estrogen production increases at the onset of equine chorionic gonadotropin (eCG) secretion by endometrial cups. In previous studies, we have demonstrated that eCG stimulates luteal androgen and estrogen production in pregnant mares. To further elucidate the regulation of steroidogenesis within the equine corpus luteum (CL) of pregnancy, we examined the expression of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), cytochrome P450 17alpha-hydroxylase/17,20 lyase (P450(17alpha)) and cytochrome P450 aromatase (P450(arom)) in luteal tissue samples collected during diestrus (Days 7 to 10) and pregnancy before (Days 29 to 35) and after (Days 42 to 45) the onset of eCG secretion. Immunoblot analyses revealed a single protein per enzyme with molecular weights of 48 kDa (3beta-HSD), 58 kDa (P450(17alpha)) and 56 kDa (P450(arom)). Steady-state levels of 3beta-HSD were lower in luteal tissue of diestrus than pregnancy, but expression did not change during pregnancy. Steady-state expression of P450(17alpha) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, P450(17alpha) expression was significantly higher after the onset of eCG secretion. Steady-state expression of P450(arom) in CL of diestrus was not significantly different from that of pregnancy. During pregnancy, luteal expression of P450(arom) was significantly lower after the onset of eCG secretion. These data support the hypotheses that eCG has a differential effect on the expression of luteal steroidogenic enzymes, that the eCG-induced increase in luteal estrogen production is the result of an increase in available aromatizable androgen due to an increase in P450(17alpha) expression and activity, and that increased luteal estrogen production is not due to an increase in aromatase expression.

Influence of exogenous progesterone on early embryonic development in the mare.

The influence of exogenous progesterone on the development of equine oviductal embryos was determined based upon the recovery of Day-7 uterine blastocysts from treated mares (n=13) that were given 450 mg progesterone daily between Days 0 and 6 and from untreated control mares (n=13). Daily administration of 450 mg progesterone in oil significantly (P<0.02) increased serum progesterone concentrations in the treated mares. There was no significant difference in the recovery rate of Day-7 embryos between treated and control mares (8/13 versus 6/13, respectively). Embryonic development, assessed by morphologic evaluation, embryo diameter, and number of cell nuclei was not significantly different for embryos from treated and from control mares. The results of this study indicate that administration of progesterone beginning on the day of ovulation does not affect the embryo recovery rate or embryonic development, based on evaluation of uterine blastocysts recovered at Day 7 after ovulation.

Persistence of the luteal phase following ovulation during altrenogest treatment in mares.

Two experiments were conducted to test the efficacy of altrenogest treatment in mares. The response to 15-d altrenogest treatment (Experiment 1) was characterized in 20 mares that were given 22 mg daily of altrenogest in oil (n = 10) or in gel (n = 10) from Day 10 to 25 after ovulation. In 17 mares, luteolysis occurred during altrenogest treatment (Day 17.7 +/- 0.5), while 2 mares retained their corpus luteum (CL), and 1 mare had a diestrous ovulation on Day 16, resulting in a prolonged luteal phase. Ten of the 17 mares in which the CL had spontaneously regressed returned to estrus after the end of treatment, and ovulated 5.7 +/- 0.8 d after the end of altrenogest treatment. Two of these 17 mares ovulated 2 and 3 d after the end of altrenogest treatment but ovulation was not accompanied by estrous behavior, and 5 mares ovulated during altrenogest treatment resulting in an interovulatory interval of 22.4 +/- 1.1 d (range: 20 to 25d). Five mares which ovulated during altrenogest treatment and 2 mares which ovulated during silent estrus after the end of altrenogest treatment failed to regress the CL around 14 d post ovulation, and had a prolonged luteal phase. In Experiment 2, the effect of altrenogest administered from luteolysis to ovulation on duration of the subsequent luteal period was analyzed. In 6 mares altrenogest was begun on Day 14 post ovulation and continued until the hCG-induced ovulation. The interval from ovulation during altrenogest treatment to spontaneous luteolysis was 45.6 +/- 2.4 d (range: 40 to 54d) in altrenogest-treated mares and was significantly longer than in 10 untreated control mares (14.5 +/- 0.3 d, range: 13 to 16d). The results suggest that the oil and gel altrenogest preparations are equally effective in modulating estrous behavior and time to estrus and ovulation. Altrenogest treatment started late in diestrus appears to result in a high incidence of ovulation during treatment and when luteolysis and ovulation occur during treatment; the subsequent luteal phase is frequently prolonged due to failure of regression of the CL.

Pure preovulatory follicular fluid promotes in vitro maturation of in vivo aspirated equine oocytes.

In the mare, rates of fertilization and development are low in oocytes matured in vitro, and a closer imitation of in vivo conditions during oocyte maturation might be beneficial. The aims of the present study were, therefore, to investigate whether (1) equine oocytes can be matured in vitro in pure equine preovulatory follicular fluid, (2) priming of the follicular fluid donor with crude equine gonadotrophins (CEG) before aspiration of preovulatory follicular fluid promotes the in vitro maturation rate, (3) the in vitro maturation rate differs between oocytes aspirated during estrus and those aspirated again 8 days after the initial follicular aspiration, and (4) high follicular concentrations of meiosis activating sterols (MAS) are beneficial for in vitro maturation of equine oocytes. During estrus, 19 pony mares were treated with 25 mg CEG. After 24 h (Al) and again after 8 days (A2), all follicles >4mm were aspirated and incubated individually for 30 h in the following culture media: standard culture medium (SM), preovulatory follicular fluid collected before CEG containing low MAS concentrations (FF1), preovulatory follicular fluid collected before CEG containing high MAS concentrations (FF2) or preovulatory follicular fluid collected 35 h after administration of CEG containing low MAS concentrations (FF3). Cumulus expansion rate was significantly affected by culture medium. The overall nuclear maturation rate was significantly higher for oocytes collected at A1 (67%) than for oocytes collected at A2 (30%). For oocytes collected at A1, the maturation rates were 71% (FF1), 61% (FF2), 79% (FF3) and 56% (SM). An electrophoretic protein analysis of the culture media revealed the presence of a 200-kDa protein in FF3. The results demonstrate that (1) equine oocytes can be matured during culture in pure equine preovulatory follicular fluid, (2) preovulatory follicular fluid collected after gonadotrophin-priming seems superior in supporting in vitro maturation than standard culture medium, (3) oocytes aspirated 8 days after a previous aspiration are less competent for in vitro maturation than oocytes recovered during the initial aspiration, and (4) the regulation of meiotic resumption during in vitro culture of equine oocytes might be related to the presence of a 200-kDa protein.

In vitro and in vivo comparison of Ham's F-10, Emcare holding solution and ViGro holding plus for the cooled storage of equine embryos.

Equine embryos have been successfully transferred after 24h cooled storage in Ham's F-10. The aim of this study was to compare the viability of equine embryos in vitro and in vivo after 6 and 24h cooled storage using three media and to examine the relationship between embryo size and viability after 24h cooled storage. In Experiment 1, the viability of embryos was evaluated using DAPI-staining after 0, 6 or 24h in Ham's F-10, 24h in Emcare embryo holding solution (EHS) or 24h in ViGro holding plus (VHP) (n=10/group). The mean number of dead cells was similar for embryos stored in Ham's F-10, EHS and VHP for 24h. Larger Day 7 embryos appear to withstand 24h cold storage better than small Day 7 embryos. The embryo quality for 24h cold storage was negatively correlated with size. In Experiment 2, 40 embryos were stored (n=20/group) either in Ham's F-10 or in EHS then transferred as pairs in recipient mares. Fifteen of the 20 recipient mares (75%) were pregnant. Out of 17 surviving embryos, 9 embryos (53%) were stored in Ham's F-10 and 8 (47%) in EHS. These results suggest that EHS and VHP offer a good alternative to Ham's F-10 for 24h cooled storage of equine embryos and that larger embryos may have a better viability after 24h of cooled storage than smaller embryos.

Pregnancy-specific elevations in fecal concentrations of estradiol, testosterone and progesterone in the domestic dog (Canis familiaris).

Estradiol (E2), testosterone (T) and progesterone (P4) concentrations were determined by enzyme-immunoassay in aqueous extracts of fecal samples obtained during anestrus, proestrus, estrus and metestrus of 11 nonpregnant and 11 pregnant bitches. Fecal hormone concentrations (ng/g) changed in relation to stage of cycle. Mean fecal steroid concentrations in 22 anestrous bitches and 3 ovariectomized bitches were low and similar for E2 (53 +/- 5 and 27 +/- 2), T (60 +/- 7 and 36 +/- 6), and P4 (62 +/- 6 and 86 +/- 15). Within 0 to 3 d of the ovulatory LH surge fecal E2 reached peak concentrations (301 +/- 38). The T peaks (281 +/- 41) were coincident or 1 to 3 d later. Fecal P4 was then elevated for approximately 2 m.o. Between Days 26 and 45 after ovulation, mean fecal P4 concentrations were higher (P < 0.05) in pregnant (401 +/- 60) than in nonpregnant bitches (164 +/- 23) and peak fecal P4 concentrations in individual animals were higher (P < 0.01) in pregnant (812 +/- 121) than in nonpregnant bitches (425 +/- 97). In the same period mean concentrations of E2 (117 +/- 13 vs 61 +/- 5) and T (102 +/- 10 vs 70 +/- 6) were also higher (P < or = 0.05) in pregnant than in nonpregnant bitches. Serum E2, T and P4 concentration were positively correlated (P = 0.1) with concentration in fecal samples obtained one day after serum collection. Although serial fecal ovarian steroid concentrations demonstrate the time course of ovulatory cycles, the diagnostic value of individual fecal samples appears limited. The ratios of peak to basal values were approximately 6, 5 and 7 for E2, T and P4, respectively, and were considerably lower than ratios of 12 to 50 previously reported for serum or plasma concentrations. The results demonstrate that there are pregnancy-specific increases in P4, E2 and T production reflected in fecal concentrations. While such increases are reflected in fecal samples, they are generally not evident in serum or plasma concentrations because of increased hemodilution, metabolism and clearance in pregnant bitches. The physiological stimulus for these increases, presumably ovarian in origin, or the potential role of prolactin is not known.

Urinary and plasma estrogen conjugates, estradiol and estrone concentrations in nonpregnant and early pregnant mares.

A direct radioimmunoassay for estrogen conjugates (EC) was applied to paired blood and urine samples collected from 20 mares and compared against estrone (E(1)) and estradiol-17beta (E(2)) to monitor changes in estrogen production during ovulatory cycles and early pregnancy. Blood samples were taken daily from five mares through two consecutive ovulations and from six mares at 6-h intervals starting 48 hours prior to ovulation and continuing after ovulation had occurred. Blood samples were also collected daily or three times per week from conception until Day 60 of pregnancy in nine pregnant mares. The mean urinary EC, plasma EC and plasma E(2) dynamics were parallel in nonpregnant mares, with a 3-fold increase in mean urinary EC concentrations from baseline to the ovulatory peak, a 1.8-fold increase in mean plasma EC concentrations and a 1.4-fold increase in mean plasma E(2) concentrations. In early pregnancy, a two-fold increase in mean plasma E(1) and EC concentrations occurred in concert with a five-fold rise in mean urinary EC concentrations, whereas plasma E(2) did not change. Following hydrolysis and chromatographic separation, E(1) and E(2) were identified as the hydrolytic products in the urine of nonpregnant and pregnant mares; however, an unidentified estrogen was the major hydrolytic product in nonpregnant mares and pregnant mares prior to Day 38 of pregnancy. The increased resolution of the EC profiles compared with the profiles of other estrogen components indicates that the determination of EC in urine or plasma provides a useful alternative method for monitoring reproductive events in mares.