Endometrial expression of estrogen and progesterone receptors in non-cyclic mares treated only with long-acting progesterone.

Administration of progesterone (P4) after estradiol is usually performed to prepare non-cyclic mares as embryo recipients. However, there are successful pregnancy reports after embryo transfer in non-cyclic mares treated only with progestins. The objective of this study was to evaluate endometrial gene expression and immunostaining for estrogen receptor alpha (ERα), beta (ERß) and progesterone receptor (PR) in seasonal anestrous mares treated with long acting P4 (LA P4). Endometrial tissue from eight seasonal anestrous mares was collected immediately before administration of 1.5 g of LA P4 and five days after. The receptors protein expression was evaluated by immunohistochemistry and the percentage of the immunostained area was determined by ImageJ software. Transcripts abundance for ERα, ERß and PR were determined by RT-qPCR. Blood samples were collected daily to measure plasma P4 concentrations. Protein expression for ERα was greater (p < 0.05) after LA P4 administration, although gene expression was not affected by treatment (p > 0.05). There was no difference for ERß protein expression (p = 0.07) and ERß gene expression was reduced (p < 0.05) after treatment. Gene and protein expression for PR was not altered (p > 0.05). In conclusion, endometrial PR and ERα expression patterns after LA P4 administration were similar to those previously found in protocols using estradiol prior to LA P4 to prepare non-cyclic mares as embryo recipients.

Administration of 2.5 mg of estradiol followed by 1,500 mg of progesterone to anovulatory mares promote similar uterine morphology, hormone concentrations and molecular dynamics to those observed in cyclic mares.

To test the hypothesis that the administration of 2.5 mg of estradiol benzoate (EB) followed by 1500 mg of long acting progesterone (LA P4) causes similar uterine changes and molecular dynamics in anovulatory mares to those observed in cyclic ones, we evaluated the changes of estrogen (ERα and ERß) and progesterone receptors (PR) in anestrous, transitional and cyclic mares by RT-qPCR and immunohistochemistry. In addition, we evaluated uterine edema, tonus and estrogens and progesterone plasma profile. Endometrial biopsies were taken from anestrous and transitional mares immediately before EB injection, 48 h after EB administration and five days after LA P4 was given. In cyclic mares, biopsies were collected at estrus and at five days after ovulation. Similar estrogen peaks were achieved after the injection of the single EB dose between treated and cyclic groups, as well as maximum uterine edema. Uterine tone was increased to diestrus levels after administration of 1500 mg of LA P4. Changes in relative abundance of transcripts for PR, ERα and ERß when progesterone stimulated endometrium was compared to estrogen stimulated endometrium were similar between cyclic and non-cyclic treated mares. However, apparent decreased PR in the endometrial glandular epithelium was not observed in non-cyclic mares five days after LA P4 administration as observed at five days after ovulation in cyclic mares. The protocol produced similar endometrial edema, uterine tonus and changes in relative abundance of PR, ERα and ERß transcripts to those observed in cyclic mares during late estrus and early diestrus, as well as similar estradiol and estrogen conjugate plasma concentrations.

Comparison of different regimens of estradiol benzoate treatments followed by long-acting progesterone to prepare noncycling mares as embryo recipients.

The present study evaluated the influence of different regimens of estradiol benzoate (EB) treatments followed by a single dose of long-acting progesterone (LA P4) on plasma estrogen and P4 concentrations in noncyclic mares prepared as embryo recipients. Twenty-one anestrous mares were distributed into three groups (n = 7 mares per group), according to the EB dose received (single dose of 2.5 mg, total of 5 mg in decreasing doses, and total of 10 mg in decreasing doses), which was followed by a single administration of 1500 mg of LA P4 in all groups. Mares were reevaluated during the ovulatory phase and seven of them became part of the cyclic nontreated control group. Ultrasonography was performed to monitor endometrial edema, and blood samples were collected to measure estradiol (E2), estrogen conjugate (EC), and P4 by RIA. Maximum uterine edema was achieved 24 hours after administration of EB in all treated groups. Maximum E2 concentrations were observed 24 hours after the first EB injection in treated groups and there were no differences (P > 0.05) among treatments. Maximum EC concentration was observed 24 hours after the single EB injection in the 2.5-mg group, whereas in the 5- and 10-mg groups EC peaks were observed 48 hours after the first EB administration. Maximum P4 concentrations were detected 24 hours after LA P4 injection, although higher P4 concentrations were observed in the group treated with 2.5 mg of EB than in that treated with 10 mg of EB (P < 0.05). Because P4 concentrations were reduced after administration of high doses of EB, we also measured 17α-hydroxyprogesterone (17-OH-P) to test the hypothesis that high concentrations of EB would accelerate the conversion of P4 to 17-OH-P. However, 17-OH-P concentrations paralleled P4 profile in all groups, irrespective of EB doses. In summary, the three EB treatment regimens induced similar E2 peaks, although the observation of EC peaks 24 hours after E2 peaks in the 5- and 10-mg groups indicate that an excess of E2 was given, which was converted into EC to be inactivated. Administration of 10 mg of EB reduced P4 concentrations 24 hours after LA P4 was given. We demonstrated that the mechanism by which this reduction occurred was not by an increase in P4 metabolism to 17α-OH-P. In conclusion, the use of 2.5 mg of EB followed by 1500 mg of LA P4 appears to be a more appropriate regimen to treat noncyclic mares, although additional studies are needed to verify embryo survival with this treatment dose.

Expression of receptors for ovarian steroids and prostaglandin E2 in the endometrium and myometrium of mares during estrus, diestrus and early pregnancy.

The objective of this study was to compare expression of estrogen receptor alpha (ER-α), ß (ER-ß), progesterone receptor (PR), as well as prostaglandin E2 type 2 (EP2) and 4 (EP4) receptors in the equine myometrium and endometrium during estrus, diestrus and early pregnancy. Tissues were collected during estrus, diestrus, and early pregnancy. Transcripts for ER-α (ESR1), ER-ß (ESR2), PR (PGR), EP2 (PTGER2) and EP4 (PTGER4) were quantified by qPCR. Immunohistochemistry was used to localize ER-α, ER-ß, PR, EP2 and EP4. Differences in transcript in endometrium and myometrium were compared by the ΔΔCT method. Expression for ESR1 (P<0.05) tended to be higher during estrus than diestrus in the endometrium (P=0.1) and myometrium (P=0.06). In addition, ESR1 expression was greater during estrus than pregnancy (P<0.05) in the endometrium and tended to be higher in estrus compared to pregnancy in the myometrium (P=0.1). Expression for PGR was greater (P<0.05) in the endometrium during estrus and diestrus than during pregnancy. In the myometrium, PGR expression was greater in estrus than pregnancy (P=0.05) and tended to be higher during diestrus in relation to pregnancy (P=0.07). There were no differences among reproductive stages in ESR2, PTGER2 and PTGER4 mRNA expression (P>0.05). Immunolabeling in the endometrium appeared to be more intense for ER-α during estrus than diestrus and pregnancy. In addition, immunostaining for PR during pregnancy appeared to be more intense in the stroma and less intense in glands and epithelium compared to estrus and diestrus. EP2 immunoreactivity appeared to be more intense during early pregnancy in both endometrium and myometrium, whereas weak immunolabeling for EP4 was noted across reproductive stages. This study demonstrates differential regulation of estrogen receptor (ER) and PR in the myometrium and endometrium during the reproductive cycle and pregnancy as well as abundant protein expression of EP2 in the endometrium and myometrium during early pregnancy in mares.

Supplementary corpora lutea monitoring allows progestin treatment interruption on day 70 of pregnancy in non-cyclic recipient mares.

The present study evaluated the effect of altrenogest treatment during 70 or 120 days of gestation on pregnancy maintenance in non-cyclic recipient mares and correlated the hormonal interruption findings with number, supplementary corpora lutea (SCL) formation period, and plasma progesterone (P4). Twenty five mares were used as recipients during anestrus, transitional or ovulatory phase and were assigned into groups according to altrenogest treatment period (70ALT, 120ALT or Control groups) or reproductive status at beginning of treatment (Anestrus, Transition or Cyclic/Control groups). Mares were evaluated by ultrasonography and quantification of plasma progesterone to monitor pregnancy status, SCL formation and P4 profile. After hormonal withdrawal, abortion was only observed on group 70ALT. The days of first SCL formation were similar (p=0.32) in the 70ALT and 120ALT groups and greater (p<0.01) than the Control group. In addition, the first SCL formation period occurred later during gestation in the anestrus group than in the transitional or cyclic mares. Progesterone synthesis in non-cyclic mares occurred in more advanced gestational period and showed lower concentration during the 120 days in relation to cyclic mares. It is suggested that progestin treatment interruption in non-cyclic recipient mares at 70 days of gestation allows pregnancy maintenance when SCL are observed and at 120 days enables maintenance in all recipient mares. In addition, the first SCL development period occurs in different gestational phases during pregnancy among anestrus, transitional and cyclic mares. This study improves the understanding of pregnancy physiology and enables progestins treatment interruption on day 70 of pregnancy in non-cyclic pregnant recipient mares.

Decreasing pH of mammary gland secretions is associated with parturition and is correlated with electrolyte concentrations in prefoaling mares.

The objectives of this study were to determine pH of the mammary gland secretions and the corresponding electrolyte concentrations in prefoaling mares. Pregnant mares (seven primiparous and seven multiparous) were monitored daily from 310-320 days of gestation until parturition. Prefoaling mammary gland secretions were collected, and pH was immediately determined with a pH meter and pH strip test. An aliquot of prefoaling mammary secretions was frozen and stored until further analyses. After parturition, samples from day -4 to 0 (day of foaling) were thawed and electrolyte concentrations (ie, Ca(2)(+), Mg(2)(+), Na(+), K(+) and Cl(-)) were determined with an automated analyser. Data were analysed via a mixed model with the mare as a random effect. Correlations were determined between pH and electrolyte concentrations by the Pearson product-moment for each pair. There was significant reduction in pH of mammary secretions on the day of foaling (P<0.0001), and most mares (11/14) with a pH ≤7 foaled within 24 hours. There was high correlation between the two pH methods (r=0.93). Additionally, there were significant (P<0.05) increases in Ca(2+) and K(+) concentrations, and significant decreases in Na(+) and Cl(-) concentrations from one day before to the day of foaling. The pH of mammary secretions was highly and significantly (P<0.001) correlated with Na(+) (r=0.87), Cl(-) (r=0.85), Ca(2+) (r=-0.88); and K(+)(r=-0.80) concentrations, and moderately correlated with Mg(2+) (r=-0.58). Daily evening pH measure of the mammary gland secretions can predict foaling in most mares.