Secretion of luteinizing hormone into pituitary venous effluent of the follicular and luteal phase mare: novel acceleration of episodic release during constant infusion of gonadotropin-releasing hormone.

We tested the hypothesis that continuous infusion of native GnRH into mares during the estrous cycle, at a dose of 100 µg/h, would elevate circulating concentrations of LH without disrupting the endogenous, episodic pattern of LH release. Ten cyclic mares were assigned to one of two groups (n = 5/group): (1) Control (saline) and (2) GnRH in saline (100 µg/h). On experimental day 0 (3 to 6 d after ovulation), osmotic pumps containing saline or GnRH were placed subcutaneously and connected to a jugular infusion catheter. Blood samples were collected from jugular catheters daily and at 5-min intervals from catheters placed in the intercavernous sinus (ICS) for 8 h on experimental day 4 (luteal phase; 7 to 10 d after ovulation), followed by an additional 6-h intensive sampling period 36 h after PGF(2α)-induced luteal regression (experimental day 6; follicular phase). Treatment with GnRH increased (P < 0.001) concentrations of LH by 3- to 4-fold in the peripheral circulation and 4- to 5-fold in the ICS. Continuous GnRH treatment accelerated (P < 0.01) the frequency of LH release and decreased the interepisodic interval during both luteal and follicular phases. Treatment with GnRH during the luteal phase eliminated the low-frequency, long-duration pattern of episodic LH release and converted it to a high-frequency, short-duration pattern reminiscent of the follicular phase. These observations appear to be unique to the horse. Further studies that exploit this experimental model are likely to reveal novel mechanisms regulating the control of gonadotrope function in this species.

Conceptus-mediated endometrial vascular changes during early pregnancy in mares: an anatomic, histomorphometric, and vascular endothelial growth factor receptor system immunolocalization and gene expression study.

This work examined how the conceptus modulates endometrial tissue remodeling and vascular development prior to implantation in mares. A macroscopic uterine examination was completed at day 21 of pregnancy. In situ morphology revealed that the endometrium involved in encroachment is restricted to the dorsal endometrium immediately overlying the yolk sac. The amount of stromal area occupied by blood vessels and the number of endometrial glands were increased during early pregnancy. Endometrial histomorphometry as well as the endometrial mRNA abundance and immunolocalization of VEGF, VEGFR1, VEGFR2, and Ki-67 was completed at days 14 and 21 of pregnancy, at day 10 of the estrous cycle, and during estrus. No obvious differences in VEGF and VEGFR1 protein localization were detected between pregnant and cycling mares but differential staining pattern for VEGFR2 and Ki-67 was observed. VEGFR2 localized to luminal and glandular epithelium of pregnant mares, while luminal epithelium was negative in cycling mares. Ki-67 staining was weak during the luteal phase but exhibited prominent luminal epithelium staining during estrus. In pregnant mares, all endometrial layers were Ki-67 positive. Quantitative RT-PCR revealed a greater abundance of VEGF mRNA during pregnancy. VEGFR2 transcript abundance was greatest in pregnant mares on day 21. This study supports the concept that the conceptus plays an active role in directing vasculogenesis within the uterus and thereby establishing hemotrophic nutrition that supports pregnancy after implantation.

Effects of estradiol-17beta administration on steady-state messenger ribonucleic acid (MRNA) encoding equine alpha and LH/CGbeta subunits in pituitaries of ovariectomized pony mares.

The process of sexual recrudescence in the springtime in mares is characterized by renewal of follicular growth and acquisition of steroidogenic competence. Concomitant with renewal of follicular steroidogenesis is re-establishment of LH biosynthesis and secretion. Research results from our laboratory indicate that increased estradiol and LH secretion occur in close temporal association before the first ovulation of the year. Therefore, the hypothesis tested in this experiment was that estrogen administration to ovariectomized pony mares during the equivalent time of early vernal transition would enhance LH biosynthesis as monitored by messenger ribonucleic acid (mRNA) encoding for the pituitary subunits of LH (alpha and LH/CGbeta). Mares were administered either sesame oil vehicle control, or estradiol (5 mg i.m. twice daily in sesame oil) for 3, 6 or 9 days, beginning on February 2. The pituitary glands were harvested, and examined for LH subunit mRNA by Northern Blot and slot blot analysis. There was a significant increase in LH secretion after 6 days of estradiol secretion compared with control vehicle administration. Similarly, there was a significant increase in both alpha and LH/CGbeta subunit mRNA when estradiol was administered for 9 days. These data indicate that estrogen stimulates LH subunit formation in mares during early equivalent vernal transition. These data do not, however, discriminate between a direct pituitary effect of estrogen, and a hypothalamic effect. Whether the surge of estradiol just prior to the first ovulation of the year is essential for the renewed biosynthesis of LH subunits cannot be determined from these data. However an important role of estrogen in the final stages of sexual recrudescence is indicated.

Effects of ovarian input on GnRH and LH secretion immediately postovulation in pony mares.

The potential involvement of ovarian factors in regulating GnRH and LH postovulation was studied in ovarian intact (Group 1; n=3) and ovariectomized (OVX; Group 2; n=3) mares (OVX within 12 hr of ovulation). Blood samples were collected every 10 min for 6 hr from jugular vein (JV) and intercavernous sinus (ICS) during estrus and on Day 8 postovulation for LH and GnRH analysis. Additionally, JV samples were collected twice daily (12-hr intervals) for 30 days for LH and progesterone (P4) analysis. A significant treatment x day effect (P<0.0001) describes declining plasma LH concentrations in intact mares, and regression analysis indicated that response curves were not parallel (P<0.001). Plasma LH concentrations remained elevated in OVX mares. LH increased further in OVX mares by Day 8 post-OVX (P<0.06), reflecting the increased (P<0.07) LH episode amplitude. GnRH decreased from estrus to Day 8 in both groups reflecting an effect of sampling period (P<0.03). GnRH episode amplitude declined (P<0.08) from estrus (62.8+/-3.1 pg/mL) to Day 8 (46.3+/-3.1 pg/mL) in OVX mares, but not in control mares (intact estrus, 36.5+/-6.4; intact Day 8, 37.5+/-7.3; OVX estrus, 62.8+/-3.1; OVX Day 8, 46.3+/-3.1 pg/mL). In conclusion, we propose that postovulatory LH decline requires ovarian feedback in mares, and that OVX alters GnRH secretory dynamics such that LH concentrations does not decline postovulation and, in fact, is further elevated with time after OVX.

Real-time monitoring of nitrile biotransformations by mid-infrared spectroscopy.

In this study mid-infrared spectroscopy was used to follow the enzyme kinetics involved in nitrile biocatalysis using whole cell suspensions of the bacterium Rhodococcus rhodochrous LL100-21. The bacteria were grown on acetonitrile to induce a two-step enzymatic pathway. Acetonitrile was biotransformed to acetamide by a nitrile hydratase enzyme and subsequently to acetic acid (carboxylate ion) by an amidase enzyme. The bacteria were also grown on benzonitrile to induce a one-step enzymatic pathway. Benzonitrile was biotransformed directly to benzoic acid (carboxylate ion) by a nitrilase enzyme. These reactions were followed by React IR using a silicon probe and gave excellent quantitative and qualitative real-time data of both nitrile biocatalytic reactions. This study has shown that this novel technique has potentially useful applications in biocatalysis.

The early fetal life of the equine conceptus.

This paper will discuss development of the equine conceptus, especially from the perspective of the maternal environment in which it develops and to which it has considerable influence.

Effect of oestradiol on LH secretion and pituitary responsiveness to GnRH in ovariectomized mares.

Long-term ovariectomized Pony mares were treated with oestradiol (0.2-5.0 mg; i.m.) at 12 h intervals for 10 days. Blood samples were collected by jugular venepuncture three times a day throughout the experiment and additional blood samples were collected at 15 min intervals for 12 h on days 0 and 10 (sampling periods 1 and 2, respectively). There were significant effects of oestradiol treatment (P < 0.05) and oestradiol treatment x day (P < 0.0001) on the mean LH concentrations each day. Regression analysis of LH time trends each day indicates that there is heterogeneity (P < 0.001) of regression due to the increasing LH concentrations in mares treated with oestradiol. These results indicate that oestradiol has a strong dose-related positive feedback on LH release in long term ovariectomized Pony mares. In Expt 2, pituitary sensitivity to GnRH in long term ovariectomized Pony mares after oestradiol administration was examined by administering a 100 mg bolus of GnRH (i.v.) twice a day on days 4 and 10 of a 10 day period of oestradiol administration. Blood samples were collected at 30 min intervals for 8 h on days 4 and 10 and once a day on the other days of treatment. The mean LH concentrations each day increased in oestradiol-treated mares over the 10 days of treatment (P < 0.002) and the pituitary responsiveness to GnRH also increased. These results indicate that oestradiol has a strong positive feedback effect on LH secretion by increasing the amplitude of the LH episode.

Influence and correction of temperature perturbations on NIR spectra during the monitoring of a polymorph conversion process prior to self-modelling mixture analysis.

The influence of temperature variations on the rank of a NIR dataset, has been investigated by comparing the results of principal component analysis (PCA) and evolving factor analysis (EFA), applied to two datasets measured at constant temperature and varying temperature. After temperature correction, the concentration profiles and spectra were obtained with PCA, SIMPLISMA and the orthogonal projection approach (OPA). The same resolution methods were used on the dataset measured at constant temperature.

Technical note: Application of the Box-Cox data transformation to animal science experiments.

In the use of ANOVA for hypothesis testing in animal science experiments, the assumption of homogeneity of errors often is violated because of scale effects and the nature of the measurements. We demonstrate a method for transforming data so that the assumptions of ANOVA are met (or violated to a lesser degree) and apply it in analysis of data from a physiology experiment. Our study examined whether melatonin implantation would affect progesterone secretion in cycling pony mares. Overall treatment variances were greater in the melatonin-treated group, and several common transformation procedures failed. Application of the Box-Cox transformation algorithm reduced the heterogeneity of error and permitted the assumption of equal variance to be met.

Effects of melatonin implants in pony mares. 2. Long-term effects.

The effects of melatonin implant treatment over a 4 wk period at the summer solstice on the transition into and out of the following anovulatory season were evaluated in ovary-intact and ovariectomized mares. Melatonin implants tended to delay the timing of the final ovulation of the breeding season (P = 0.0797) in the ovary-intact mares. Although the decline in LH secretion associated with the end of the breeding season was parallel between treatments and ovarian statuses, the rate of LH secretion, as expressed by its mathematical accumulation, was lower in ovariectomized, melatonin-treated mares than in ovariectomized, control mares suggesting that melatonin administration advanced the offset of the breeding season in ovariectomized mares (P = 0.0001). The first ovulation of the subsequent breeding season was significantly delayed in the melatonin-treated mares as compared with that of control mares (P = 0.0031). During reproductive recrudescence, the time of the onset of the increase in LH secretion was similar among all 4 groups but the patterns of LH secretion were different for each treatment and ovarian status combination (P = 0.0112). Mares with melatonin implants had a slower rate of increase in LH secretion than control mares (P = 0.0001), and ovariectomized mares had a faster rate of LH increase than intact mares (P = 0.0001). These results suggest that melatonin implants during the summer solstice can alter the annual reproductive rhythm in mares and support the concept that endocrine patterns of reproductive recrudescence are not entirely independent of the ovary.

Effects of melatonin implants in pony mares. 1. Acute effects.

The effects of melatonin implant treatment over a four week period on LH, estradiol (E2) and progesterone (P4) secretion during the breeding season were studied in ovary-intact and ovariectomized pony mares. Mares with melatonin implants had significantly higher daytime melatonin concentrations than mares with sharm implants (P = 0.0065). In ovariectomized mares, LH secretion did not differ between mares with melatonin and sham implants. In ovary-intact mares, melatonin implants altered the pattern of LH secretion (P = 0.0023) in such a way that an increase in LH secretion was observed during the periovulatory period. Estradiol and P4 secretion were unaffected by melatonin implants. These results suggest that constant administration of melatonin may enhance the secretion of LH during the periovulatory surge but does not adversely affect E2, P4 or basal LH secretion in mares during the breeding season.

The effect of pulsatile gonadotropin-releasing hormone and estradiol administration on luteinizing hormone and follicle-stimulating hormone concentrations in pituitary stalk-sectioned ovariectomized pony mares.

Hourly pulses of gonadotropin-releasing hormone (GnRH) or bi-daily injections of estradiol (E2) can increase luteinizing hormone (LH) secretion in ovariectomized, anestrous pony mares. However, the site (pituitary versus hypothalamus) of positive feedback of estradiol on gonadotropin secretion has not been described in mares. Thus, one of our objectives involved investigating the feedback of estradiol on the pituitary. The second objective consisted of determining if hourly pulses of GnRH could re-establish physiological LH and FSH concentrations after pituitary stalk-section (PSS), and the third objective was to describe the declining time trends of LH and FSH secretion after PSS. During summer months, ovariectomized pony mares were divided into three groups: Group 1 (control, n = 2), Group 2 (pulsatile GnRH (25 micrograms/hr), n = 3), and Group 3 (estradiol (5 mg/12 hr), n = 3). All mares were stalk-sectioned and treatment begun immediately after stalk-section. Blood samples were collected every 30 min for 8 h on the day before surgery (D0) and 5 d post surgery (D5) to facilitate the comparison of gonadotropin levels before and after pituitary stalk-section. Additionally, jugular blood samples were collected every 12 hr beginning the evening of surgery, allowing for evaluation of the gonadotropin secretory time trends over the 10 d of treatment. On Day 10, animals were euthanized to confirm pituitary stalk-section and to submit tissue for messenger RNA analysis (parallel study). Plasma samples were assayed for LH and FSH by RIA. Mean LH secretion decreased from Day 0 to Day 5 in Groups 1 and 3, whereas LH secretion tended (P < 0.08) to decrease in Group 2 mares. On Day 5, LH was higher (P < 0.01) in Group 2 (17.26 +/- 3.68 ng/ml: LSMEANS = SEM), than either Group 1 (2.65 +/- 4.64 ng/ml) or Group 3 (4.28 +/- 3.68 ng/ml). Group 1 did not differ from Group 3 on Day 5 (P < 0.40). Similarly, mean FSH levels decreased in all groups after surgery, yet Group 2 mares had significantly (P < 0.001) higher FSH concentrations (17.66 +/- 1.53 ng/ml) than Group 1 or Group 3 (8.34 +/- 1.84 and 7.69 +/- 1.63 ng/ml, respectively). Regression analysis of bi-daily LH and FSH levels indicated that the time trends were not parallel. These findings indicate: 1) Pituitary stalk-section lowered LH and FSH to undetectable levels within 5 d after surgery. 2) pulsatile administration of GnRH (25 micrograms/hr) maintained LH and FSH secretion, although concentrations tended to be lower than on Day 0, and 3) E2 did not stimulate LH or FSH secretion.

Comparative study between pony mares and ewes evaluating gonadotrophic response to administration of gonadotrophin-releasing hormone.

This study compared equine and ovine LH secretory responses to GnRH treatment. Dioestrous mares and ewes were challenged with continuous GnRH for 15 h. Mares that received constant GnRH (110 micrograms h-1) had sustained LH secretion (P < 0.01), whereas LH concentrations in ewes treated with continuous GnRH (25 micrograms h-1) initially increased, then declined and remained low, suggesting GnRH receptor desensitization or downregulation. In addition, progesterone-primed, ovariectomized mares and ewes were challenged with pulsatile or continuous GnRH for 5 days. Plasma LH concentrations were increased by day 5 in mares treated with pulsatile (25 micrograms pulse-1 h-1) and continuous (110 micrograms h-1) GnRH (P < 0.01). Furthermore, mean LH concentrations and time-response curves were not different. In contrast, ewes treated with continuous GnRH (2.5 micrograms h-1) demonstrated LH secretory patterns indicative of GnRH receptor downregulation on day 1 of treatment. LH concentrations in ewes treated with pulsatile GnRH (250 ng pulse-1 h-1) did not differ from controls. In conclusion, pony mares responded continuously to GnRH treatment (pulsatile and continuous), whereas ewes treated with continuous GnRH experienced reduced LH secretion. These findings suggest a unique hypothalamic-pituitary axis in pony mares.

Biochemical changes in the equine capsule following prostaglandin-induced pregnancy failure.

The equine embryonic capsule, an acellular covering that envelops the conceptus during the second and third weeks of pregnancy, is composed of mucin-like glycoproteins. Its structure is consistent with a dual role during early pregnancy: protection of the conceptus, and communication between the embryo and the mother. Loss of sialic acid from the capsular glycoproteins at day 16 correlates with the time of "fixation," or loss of conceptus mobility throughout the uterine horns. This study investigated how the structure of the capsule is linked to the maintenance of pregnancy. Six pregnancies, confirmed by ultrasound, were terminated by prostaglandin injection on day 14, prior to the time of embryo fixation. These "defective" conceptuses were collected at day 17, and the structure and molecular properties of their capsules were compared to those of day 17 conceptuses collected from 5 normal pregnancies. Defective capsules were not significantly different from normal capsules in terms of dry weight, amino acid composition, and content of neutral and amino sugars. However, defective capsules failed to show the loss of sialic acid normally occurring around the time of embryo fixation. Analysis of the capsular mucins following trypsin digestion was carried out by radioactive labeling with 3H on sialyl-oligosaccharides and 125I on tyrosine residues, followed by fast protein liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Differences in the trypsin fragmentation patterns indicated increased susceptibility of the defective capsules to proteolysis. We conclude that there is a temporal association between desialylation of the equine capsule and embryonic survival, and that failure to desialylate alters the properties of the capsule.

Relationship between endometrial oxytocin receptors and oxytocin-induced prostaglandin F2 alpha release during the oestrous cycle and early pregnancy in pony mares.

The effect of transcervical endometrial biopsy on the concentrations of plasma immunoreactive oxytocin and 15-keto-13,14-dihydro-prostaglandin F2 alpha (PGFM) was studied in 18 pony mares on days 8, 12 and 14 after ovulation, days 12 and 14 of early pregnancy and at oestrus. Five biopsy specimens were taken within 15 min and consecutive specimens from each mare were pooled two (A) and three (B) together for measurement of the number of oxytocin receptors. Blood samples were collected at intervals of 5 min for 15 min beginning just before the initial biopsy. Biopsy procedure elicited prompt oxytocin release in all mares. Pregnancy did not affect the response but day after ovulation had a significant influence on oxytocin release. The greatest increase in plasma oxytocin was observed on day 12 in both nonpregnant and pregnant mares and the lowest on day 8. The concentration of plasma PGFM rose linearly over the 15 min period in nonpregnant mares. This response increased progressively with time after ovulation and was greatest on day 14. There was no increase in circulating PGFM in pregnant mares. Endometrial oxytocin receptor concentration was lowest in mares at oestrus and highest in nonpregnant mares on day 14. Oxytocin receptor density in pregnant mares was similar to that in nonpregnant mares on day 12 but was significantly attenuated on day 14. The affinity of oxytocin receptors was lower in pregnant than in nonpregnant mares. Because of the positive correlation between PGF2 alpha release, endometrial oxytocin receptor density, and plasma oxytocin concentrations in nonpregnant mares, it is assumed that the release of PGF2 alpha was induced by oxytocin and was mediated by oxytocin receptors. Pregnancy-induced inhibition of PGF2 alpha release was not associated with suppression of oxytocin release or oxytocin receptor density. An embryo-derived factor is therefore the most likely cause for the suppression of PGF2 alpha release and interruption of the oxytocin-PGF2 alpha interaction in mares during early pregnancy.

Effect of beta-carotene administration on reproductive function of horse and pony mares.

The objective of this study was to determine whether supplemental beta-carotene would influence reproductive function in mares maintained on spring and summer pastures and to characterize plasma carotene concentrations during the estrous cycle. Carotene concentrations in plasma did not vary with day of estrous cycle (P = 0.7455). Mares receiving every other day injections of beta-carotene (400 mg; n = 4) or saline (10 ml; n = 4) during proestrus/estrus did not differ in plasma estradiol (E(2)) concentrations (P = 0.6313), follicle development (P = 0.8068), or plasma progesterone (P(4)) concentrations during the following diestrus (P = 0.4954). Moreover, no differences in plasma P(4) concentrations (P = 0.9047) were detected between mares receiving every other day injections of beta-carotene (400 mg; n = 4) or saline (10 ml; n = 4) during diestrus. However, administration of beta-carotene raised plasma carotene concentrations relative to controls when injected during proestrus/estrus (P = 0.0096) and diestrus (P = 0.0099). Pregnancy rates (P = 0.4900) and number of cycles required for pregnancy (P = 0.2880) were similar for mares administered injections of saline (10 ml; n = 37), beta-carotene (400 mg; n = 37), vitamin A (160,000 IU; n = 38), or vitamin A + beta-carotene (160,000 IU + 400 mg; n = 43), on the first or second day of estrus and on the day of breeding. Therefore, these results collectively suggest that supplemental beta-carotene does not affect the reproductive function of mares fed adequate dietary carotene. Whether supplemental beta-carotene would enhance reproductive function in mares on low carotene diets warrants further investigation.

MRI of healing osteochondritis dissecans fragment with absorbable pins.

We describe a patient who presented with an unstable osteochondritis dissecans (OD) fragment of the right knee demonstrated by MRI. He subsequently underwent surgical fixation with absorbable pins. Follow-up MRI showed the healing OD lesion without the artifact associated with metallic pins.

Pregnancy-associated endometrial expression of antileukoproteinase gene is correlated with epitheliochorial placentation.

Uterine expression of the mRNA encoding antileukoproteinase (ALP) is highest in pig uterus during mid- to late pregnancy, suggesting a stage of pregnancy-dependent role for this elastase/cathepsin G protease inhibitor in feto-maternal interactions. To examine a potential relationship between uterine synthesis of ALP and the type of placentation in mammalian species, the expression of ALP mRNA and/or protein in pregnant mares, cows, rats, and mice was evaluated. Genomic DNA and mRNA hybridization analyses were performed using a porcine ALP cDNA as probe. The concentration of ALP protein in reproductive tissues was determined by RIA using a polyclonal antibody raised against a synthetic peptide (ALP 16P) corresponding to amino acid residues 21-36 of the porcine ALP protein. A single ALP mRNA transcript of approximately 0.8 kb in length was detected in equine and bovine uterine tissues. The relative abundance of ALP mRNA in equine endometrium increased between days 125-170 (mid-pregnancy), and then decreased by day 215 of pregnancy. Similarly, the steady state levels of ALP mRNA in bovine endometrium and myometrium were higher during mid- to late than during early pregnancy. The levels of ALP mRNA in bovine fetal cotyledon were low and did not change significantly with stage of pregnancy. No hybridization was detected to pregnant rat endometrial tissues, although high stringency Southern blot analysis of porcine, bovine, and rat genomic DNAs using porcine ALP cDNA as probe predicted a high degree of nucleotide sequence homology in their respective ALP genes.(ABSTRACT TRUNCATED AT 250 WORDS)

A single gene encodes the beta-subunits of equine luteinizing hormone and chorionic gonadotropin.

Equine (e) CG and LH beta-subunits have identical amino acid sequences, including an extended carboxyl-terminal peptide (CTP). This suggests that unlike the corresponding human genes, the beta-subunits of eCG and eLH may be encoded by a single gene and share a common proximal promotor region. To explore this, we isolated and characterized the eLH/CG beta gene(s). Data from Southern analyses suggest that the eCG beta and eLH beta subunits are products of the same single copy gene (eLH/CG beta). Overlapping fragments of the eLH/CG beta gene and cDNA were amplified from equine genomic DNA and pituitary gland mRNA by the polymerase chain reaction, cloned, and sequenced. The eLH/CG beta gene spans less than 1.2 kilobase-pairs and has three exons that translate a CTP-containing polypeptide identical in sequence to that previously reported for the mature equine protein. There is, however, little amino acid homology shown between the CTP of human or equine CG beta subunit. In addition, unlike the human genes, the same TATAA-like element appears to be involved in directing initiation of transcription of the eLH/CG beta gene in placenta and anterior pituitary. Based upon these differences, we suggest that the CG beta genes evolved independently in humans and equids and that different mechanisms are involved in their patterns of placenta-specific expression.

Effects of steroid administration on pituitary luteinizing hormone and follicle-stimulating hormone in ovariectomized pony mares in the early spring: pituitary responsiveness to gonadotropin-releasing hormone and pituitary gonadotropin content.

These experiments tested the hypothesis that administration of steroid hormones to ovariectomized (OVX) mares during the vernal transition to the breeding season would influence LH and FSH secretion. Circulating gonadotropin concentrations, response to exogenous GnRH, and pituitary gonadotropin content were monitored. Experiments 1 and 2 were conducted, beginning 10 March, and 3 February, respectively, utilizing a total of 30 long-term OVX pony mares. In experiment 1, mares were administered vehicle (n = 5) or estradiol-17 beta (E2, n = 5, 5 mg/3 ml sesame oil), twice daily for 16 days. Blood samples were collected daily for assessment of circulating LH and FSH concentrations. On Day 10 of treatment, 400 micrograms GnRH were administered to all mares. LH increased significantly over days of treatment in the estradiol-treated group, but pituitary response to GnRH tended to be less than in control mares. Circulating FSH tended to decline over days of treatment in estradiol-treated mares, and the pituitary response to GnRH was significantly reduced. Pituitary LH, but not FSH, was increased on Day 16 of treatment with estradiol. In experiment 2, 20 OVX mares received, twice daily, vehicle (n = 5), E2, n = 5; 5 mg), progesterone (P4, n = 5; 100 mg), or progesterone plus estradiol (P4/E2, n = 5; 100 + 5 mg). Treatment continued for 14 days. GnRH (100 micrograms) challenges were administered on Days 6 and 13 of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)