A single dose of PGF2α does not consistently induce luteolysis in the equine CL until at least 5 days after ovulation, leading to the erroneous assumption that the early CL is refractory to the luteolytic effects of PGF2α. We hypothesized that serial administration of PGF2α in early diestrus would induce a return to estrus similar to mares treated with a single injection in mid-diestrus, and fertility of the induced estrus would not differ. The objectives of the study were to evaluate the effects of the 2 approaches as reflected by: (1) concentrations of plasma progesterone; (2) interovulatory and treatment-to-ovulation intervals; (3) the proportion of mares pregnant after artificial insemination. The study consisted of a balanced crossover design in which 10 reproductively normal Quarter Horse Mares were exposed to 2 treatments on 2 consecutive reproductive cycles. At detected ovulation (Day 0), mares were randomly allotted to 1 of 2 treatment groups: I, mid-diestrus treatment, administration of a single 10-mg dose of dinoprost tromethamine (PGF2α) im on Day 10; II, early diestrus treatment, administration of 10-mg PGF2α im twice daily on Days 0, 1, and 2 and once daily on Days 3 and 4. Mares in estrus and with a follicle 35 mm or greater in diameter were artificially inseminated with at least 2 billion motile sperm from a fertile stallion. Pregnancy was defined as detection of a growing embryonic vesicle on 2 consecutive examinations approximately 14 days after ovulation. Serial plasma samples were collected throughout the study period, and concentration of plasma progesterone was determined by RIA. A mixed-model ANOVA for repeated measures was used to analyze hormonal data. Interovulatory and treatment-to-ovulation intervals were compared by a paired t test and fertility by a McNemar chi-square analysis. All mares in group I underwent luteolysis after PGF2α administration denoted by mean (±SD) concentration of plasma progesterone of 0.25 ± 0.21 ng/mL detected 2 days after treatment. In group II, mean concentration of plasma progesterone remained below 1.0 ng/mL during treatment and until the onset of the next estrus. The mean interovulatory interval in group I was 18.5 ± 2.0 days compared with 13.1 ± 3.7 days in group II (P < 0.01). Treatment-to-ovulation intervals were 8.5 ± 2.0 days and 13.1 ± 3.7 days for groups I and II, respectively (P < 0.05). In both groups, 9 of 10 mares were pregnant (P = 1.0). Serial PGF2α administration beginning at ovulation consistently prevented luteal function in 10 of 10 mares in the present study without adversely affecting pregnancy rate of post-treatment cycles.

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 PGF2α-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.

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.

The equine embryo must signal its presence to the uterus for pregnancy to continue to term. Mobility of the conceptus throughout the uterus is crucial for its survival, and this action presumably permits the conceptus to transmit its antiluteolytic signal to the endometrium. Studies were completed to establish whether this unidentified antiluteolytic signal targets prostaglandin G/H synthase 2 (PGHS2), a rate limiting enzyme in converting arachidonic acid to prostaglandins (PGs). In the first study, quantitative RT-PCR was used to determine the relative abundance of PGHS2 mRNA in endometrium derived from estrous cyclic and pregnant mares on day 14 post-ovulation. PGHS2 mRNA abundance was substantially greater in endometrium from estrous cyclic mares. Additional studies were completed to better understand PGHS2 in equine endometrium. An estrogen and progesterone treatment regimen in ovariectomized mares was developed as a test model for detecting endometrial PGHS2 mRNA. Also, exposing endometrial explants to conceptus secretions (conditioned culture medium) decreased PGHS2 mRNA abundance whereas exposing explants to oxytocin increased PGHS2 mRNA abundance. Exposure to conceptus secretions also decreased PGF2alpha concentrations in explant-conditioned medium whereas oxytocin supplementation increased PGF2alpha concentrations in medium. These data support the hypothesis that PGHS2 is a target for the antiluteolytic signal produced by equine conceptuses during early pregnancy. Also, the endometrial explant culture system used for these studies can serve as a model for identifying and characterizing the maternal recognition of pregnancy factor in equids.

On Day 15 or 16 or pregnancy the mobile equine conceptus "fixates," i.e. lodges in the posterior segment of one uterine horn. Coincident with fixation, endometrium dorsal to the conceptus undergoes remarkable hypertrophic and hyperplastic changes which include increased endometrial gland size and numbers, and increased blood vessel size and numbers. We hypothesized that these localized changes, leading to endometrial encroachment onto the yolk sac area of the conceptus, are conceptus mediated, and involve alteration of Vascular Endothelial Growth Factor (VEGF) and its receptors (VEGFR-1 and VEGFR-2). To test the hypothesis, endometrial tissue samples were obtained from the endometrium dorsal to the conceptus or the contralateral horn in pregnant mares, and from equivalent sites in cycling mares. Samples were collected on Days 14, (n=5), and 21, (n=11) of pregnancy, and during estrus (n=6), and diestrus, (n=6). Samples were preserved for immunohistochemistry and for real time reverse transcription polymerase chain reaction (qPCR) analyzing expression of VEGF, VEGFR-1, and VEGFR-2. Additionally, Ki-67 was evaluated in endometrium by immunohistochemistry. Antisera were diluted as follows: anti-VEGF(147) polyclonal rabbit serum, 1:200 (sc-507; Santa Cruz Biotechnology, Inc), anti-VEGFR-1 polyclonal rabbit serum, 1:200 (Flt-1 C-17: sc-316; Santa Cruz Biotechnology, Inc.), anti-VEGFR-2 monoclonal mouse serum, 1:200 (Flk-1 A-3: sc-6251; Santa Cruz Biotechnology, Inc.), and anti-Ki-67 polyclonal rabbit serum, 1:1500 (NCL-Ki67p; Novocastra Laboratories Ltd.). Immunolocalization of VEGF and VEGFR-1 did not differ between pregnant and cyclic mares. Intense staining for VEGFR-2 was observed in all endometrial layers of pregnant mares, whereas the luminal epithelium of cycling mares exhibited only weak staining. Proliferation was evident by intense Ki-67 staining at the luminal epithelium of mares during estrus, but in diestrus, Ki-67staining was practically undetectable. During pregnancy, all endometrial layers stained positively for Ki-67, with more positive cells observed on Day 21 than on Day 14. There was no apparent difference in horn-side staining of any of the antigens, regardless of reproductive status. Expression of mRNA encoding VEGFR-1 was very low, and no differences were observed among reproductive statuses. In contrast, VEGF and VEGFR-2 exhibited significant (P < 0.03 and 0.005, respectively) differences among reproductive statuses, reflecting greater mRNA expression during pregnancy (Days 14 and 21, VEGF; and Day 21, VEGFR-2) compared with estrus or diestrus. Samples for qPCR were obtained only from endometrium ipsilateral to the conceptus in this experiment, so local versus intrauterine conceptus-mediated effects cannot be assessed. However, previous studies using color Doppler ultrasonography have shown a significant increase in endometrial blood flow primarily ipsilateral to the conceptus. Furthermore, morphometric analysis in the present study indicated significantly increased numbers and size of blood vessels, as well as endometrial glands in dorsal endometrium ipsilateral to the conceptus. These data provide strong support for the hypothesis that the equine conceptus directs the provision of its own nutrients through increased maternal endometrial vascularity and endometrial secretion, and suggests that the VEGF system plays a central role in this process. LAS is supported by a CAPES Scholarship from Brazil. (poster)

Polyclonal antisera and six distinct monoclonal antibodies (mAbs) were raised against constitutive cyclooxygenase (COX-1) purified from ram seminal vesicles. Immunoblotting experiments revealed that the polyclonal antisera and 4 of the mAbs strongly recognized human COX in platelet extracts. Different two-site immunometric assays of ram COX-1 were established using different combinations of mAbs. The assays were performed in 96-well microtiter plates coated with one mAb, with another mAb (covalently labeled with acetylcholinesterase (AChE)) as tracer. One combination (solid phase CX-101 + CX-105-AChE) exhibited the best sensitivity, with significant detection of concentrations as low as 23 pg/ml (0.3 fmol/ml of sheep COX-1). Unfortunately, this assay poorly cross-reacted with human COX-1 from platelet extracts. Another combination (solid phase CX-111 + CX-110-AChE) exhibited good recognition of human COX-1 but poor cross-reactivity with ram COX-1. Finally, purified anti-COX-1 IgG coated and CX-110-AChE were chosen as the best compromise since both good sensitivity (limit of detection, 113 pg/ml of ram COX-1) and significant cross-reactivity between COX-1 from both species were observed. In parallel, polyclonal antibodies were raised in rabbits against a peptide of 12 amino acids corresponding to the aminoterminal part of human COX-1. These polyclonal antibodies were affinity-purified and used in development of another two-site immunometric assay of COX-1 with CX-110-AChE as tracer. These two assays were used to analyze the COX-1 content of human platelets and cultured human umbilical vein cells (HUVEC). The results obtained with each assay were compared in terms of sensitivity and specificity. The validity of both assays was checked by analyzing platelets and HUVEC extracts previously fractionated by molecular sieve chromatography.

The objective of this study was to quantify serum progesterone levels, uterine features, and pregnancy rates in acyclic, embryo recipient mares using a bovine progesterone-releasing intravaginal device in a commercial embryo transfer (ET) program. The study included 73 recipient mares of unknown breed, aged 3–10 years, weighing 350–500 kg, and kept under an intensive management system on Tifton 85 (Cynodon spp.) pastures with water and mineral salt ad libitum. The horses were divided into two groups: a group with a progesterone-releasing intravaginal device (1 g progesterone, G-IVP4, n = 24) and a control group (G-iP4, n = 49) receiving an injection of 1,500 mg long-acting progesterone. Jugular blood was collected for the G-IVP4 group for subsequent progesterone measurement by radioimmunoassay on three occasions: Day 0 (D0), intravaginal device was placed; Day 5 (D5), day of the ET; and Day 9 (D9), day of pregnancy diagnosis. There was an increase (P < .0001) in serum progesterone levels on D5 and D9 compared with D0 (4.09 ± 0.81 and 6.45 ± 1.03 ng/mL vs. 0.71 ± 0.14 ng/mL). There were no differences among groups in the pregnancy rate (P > .05), with rates of 83.33% and 73.46% for G-IVP4 and G-iP4, respectively. In conclusion, the intravaginal route for absorption of 1 g of progesterone device increased the serum level of progesterone sufficiently to prepare the uterus of acyclic recipient mares for ET, and the conception rate was similar to the standard protocol using long-acting injectable progesterone.

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.

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 (α and LH/CGβ). 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 α and LH/CGβ 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.