Development of Porcine Accessory Sex Glands.

Accessory sex glands are recognized as targets of human disease and may have roles in reproductive success in livestock. The current experiments evaluated the influences of endogenous steroids on the development of porcine accessory sex glands, primarily in the neonatal period. When the aromatase inhibitor, letrozole, was used to inhibit the production of endogenous estrogens in the postnatal interval, growth of the seminal vesicles, prostate, and bulbourethral glands was stimulated. The weights of seminal vesicles, prostate, and bulbourethral glands approximately doubled at 6.5 weeks of age when the reduction in endogenous estrogens began at 1 week of age (p < 0.01). However, by 20 and 40 weeks of age, the weights of accessory sex glands were similar between the letrozole-treated boars and the vehicle-treated littermates indicating the growth stimulation was a transient effect when the treatment interval was short. The presence of both classical nuclear estrogen receptors and the G protein-coupled estrogen receptor in neonatal accessory sex glands indicated multiple signaling pathways might mediate the growth inhibition by endogenous estrogens. The absence of a detectable response when the classical estrogen receptors were blocked with fulvestrant (or when the androgen receptor was blocked with flutamide) suggests that endogenous estrogens act through the G protein-coupled estrogen receptor to inhibit the development of accessory sex glands during this neonatal to early juvenile interval.

Embryo-endometrial interaction associated with the location of the embryo during the mobility phase in mares.

Embryo-maternal crosstalk is essential to establish pregnancy, with the equine embryo moving throughout the uterus on days 9-15 (ovulation = day 0) as part of this interaction. We hypothesized that the presence of a mobile embryo induces local changes in the gene expression of the endometrium. On Day 12, the endometrial transcripts were compared among three groups: uterine horn with an embryo (P+, n = 7), without an embryo (P-, n = 7) in pregnant mares, and both uterine horns of nonbred mares (NB, n = 6). We identified 1,101 differentially expressed genes (DEGs) between P+ vs. NB and 1,229 DEGs between P- vs. NB. The genes upregulated in both P+ and P- relative to NB were involved in growth factor pathway and fatty acid activation, while downregulated genes were associated with oxytocin signaling pathway and estrogen receptor signaling. Comparing the transcriptome of P+ to that of P-, we found 59 DEGs, of which 30 genes had a higher expression in P+. These genes are associated with regulating vascular growth factors and the immune system, all known to be essential in early pregnancy. Overall, this study suggests that the mobile embryo influences the endometrial gene expression locally.

Anti-Müllerian hormone and inhibin-B concentrations vary cyclically in nonovulating queens within reference ranges established for determining gonadal status in cats.

OBJECTIVE: To define cyclic changes in anti-Müllerian hormone (AMH), inhibin-B, and progesterone concentrations and establish statistically valid, population-based clinical reference ranges in queens. ANIMALS: Cyclic queens (fertile, n = 6; infertile, 6) from an institutional breeding colony were blood sampled longitudinally, each for over 2 months, between November 2021 and February 2022, and residual serum samples from intact (n = 205) and ovariohysterectomized (49) queens from clinical submissions were used to establish reference ranges for intact and spayed females. METHODS: AMH and inhibin-B were measured using commercially available ELISAs, progesterone was measured using an in-house ELISA, and 90% CIs were calculated from these data. RESULTS: AMH and inhibin-B fluctuated in a highly correlated, cyclic pattern in 3 queens that did not ovulate immediately, whereas AMH declined as progesterone increased, indicative of ovulation, which occurred spontaneously early in the sampling period in 3 others; statistically valid reference ranges were established in intact and ovariohysterectomized females. CLINICAL RELEVANCE: Cyclic changes in hormone profiles were defined, providing relevant context for interpreting results in cases seeking to determine gonadal status (presence or absence of gonadal tissue) on the basis of established, population-based reference ranges reported here for cats for the first time.

Porcine-specific expression of the three functional CYP19 paralogs in early conceptus, placenta, and gonads.

In brief: Aromatase catalyzes the synthesis of estrogens and has been shown to have an important role during the establishment of pregnancy in the pig. This study confirmed the differential expression of the three aromatase isoforms. Abstract: Although three porcine aromatase isoforms have been identified, their gene expression profiles in reproduction are still poorly understood. Here, we identified by Sanger sequencing unique nucleotide signatures for the three paralogous copies of Cyp19 and analyzed by RT-PCR the occurrence of the Cyp19 and Cyp17a1 transcripts at different tissues and stages of conceptus and fetal-placental development. Cyp19a1 and Cyp19a3 expressions were detected in conceptuses and gonads, respectively. Cyp19a2 transcripts were identified on both the conceptuses and the placenta samples. Transcripts for Cyp17a1 were detected predominantly in conceptus and gonads. In the endometrium of day 21 pregnant females, as well as days 12 and 17 pseudopregnant females, we did not detect the expression of Cyp19a1, Cyp19a2, or Cyp19a3. In our study, we have demonstrated distinct transcriptional regulation for the three functional Cyp19 paralogs and a potential role for Cyp17a1 in controlling the secretion of estrogen from the conceptus and the placenta.

Case report: Androgen-secreting adrenocortical tumors in eight cats.

Urine marking, aggression, and other behavioral concerns are common reasons for cat owners to seek veterinary care. Empiric treatment for lower urinary tract disease or primary behavior disorders are commonly pursued, especially in those cases with normal routine laboratory evaluations. Herein, we report the clinicopathologic findings in eight sexually altered cats that were diagnosed with androgen-secreting adrenocortical tumors. Nearly all cats (n = 7) initially were evaluated for inappropriate urination and pungent urine, with additional behavioral concerns including aggression (n = 3) and excess vocalization (n = 4) commonly reported. Penile barbs (n = 5) were identified in all five male cats, and an enlarged clitoris was observed in one female cat. Testing of serum androgen concentrations revealed abnormally high androstenedione (n = 1) or testosterone (n = 7) concentrations. In the five cases with available adrenal tissue, histopathologic evaluation identified either an adrenocortical adenoma (n = 3) or adrenocortical carcinoma (n = 2). Hormonal abnormalities resolved and clinical signs improved in the four cats that underwent surgical adrenalectomy, with each of these cats surviving >1 year. However, clinical signs were minimally impacted with medical treatments, including one cat in which trilostane treatment failed to improve clinical signs or testosterone concentrations. This collection of cases underscores the importance of a detailed physical examination as well as the consideration of endocrine disturbances in cats undergoing evaluation for inappropriate urination or aggression. Furthermore, this report adds to the growing body of evidence that sex-hormone secreting adrenal tumors in cats may be an under-recognized syndrome.

Progesterone Analysis in Canine Breeding Management.

Progesterone is a worthwhile addition to the clinical assessment of cycle stage for breeding, elective cesarian delivery, and reproductive management in the bitch if reliably measured. Clinical decisions based on systemic progesterone concentrations also require the rapid return of results. Most commercially accessible analyses capable of returning results within a day still rely primarily on immunoassays of one kind or another. Point-of-care instruments utilizing similar technology have been developed more recently to enable results to be generated in-house. Repeated monitoring of progesterone on whatever platform can be useful if consistent collection and analysis protocols ensure acceptable precision, accuracy, and repeatability.

Pregnancy monitoring in mares: Ultrasonographic and endocrine approaches.

Methods to diagnose and monitor equine pregnancy continue to advance with improved instrumentation enabling the development of novel, non-invasive approaches to assess fetal well-being and viability using ultrasound and endocrine testing. From early embryonic loss to placentitis, that is typically encountered later in gestation, fetal viability and development as well as placental function can be evaluated using two fundamentally different, structural and functional, approaches. Ultrasound provides structural information on embryonic and fetal growth using such parameters as combined thickness of the uterus and placenta (CTUP), visual assessment of fetal fluids, activity, heart rate and multiple biometrics involving the fetal head and eyes, limbs and joints among many others, depending on the stage of gestation. Endocrine profiles that include progesterone and 5α-dihydroprogesterone, other metabolites, androgens and estrogens can be evaluated simultaneously using liquid chromatography-tandem mass spectrometry (LC-MS/MS) providing more functional information on fetal and placental competence and development. Endocrine information can be used in making clinical decisions including the need for progestin supplementation or when it can cease, and even estimating gestational stage in mares that cannot be easily palpated or scanned, as with mini-breeds or rancorous animals most notably. When used together, monitoring gestation by ultrasound and hormonal analysis provides unusual insight into feto-placental well-being and the progress of pregnancy, helping to identify problems needing therapeutic intervention.

Equine granulosa cell tumours among other ovarian conditions: Diagnostic challenges.

BACKGROUND: Granulosa cell tumours (GCT) are the most common ovarian tumours in mares. While the classical presentation may not represent diagnostic challenges, diagnosis is not easy in the early stages. OBJECTIVES: Illustrate the variability in the presentation and serum biomarkers associated with ovarian abnormalities in the mare. STUDY DESIGN: Retrospective case series. METHODS: Nonclassical cases of GCTs and other ovarian conditions were identified and behaviour, GCT endocrine results, palpation and ultrasonographic findings are described and the diagnostic value of each is discussed. RESULTS: Mares in this case series with GCTs had been presenting clinical signs ranging from no behavioural changes to behaviours including aggression, stallion-like and inability to work under saddle. Hormonal profiles of endocrinologically functional GCTs can be erratic and unpredictable. The clinical form and ultrasonographic appearance may also vary with time from an initially enlarged/anovulatory follicular structure that later develops a multicystic 'honeycomb' appearance. Mares with GCTs can also present with persistent anovulatory follicles or apparent luteal tissue that are unresponsive to treatment. If both ovaries are of relatively normal size and symmetry, but hormonal biomarkers are markedly increased (AMH >10 ng/mL, inhibin B and/or testosterone >100 pg/mL; 0.37 nmol/L), it is likely that a functional GCT is present. Still, it can be a challenge to decide which ovary to remove. Post-surgical endocrine testing can be helpful, especially if histopathology is not performed or a GCT is not found. MAIN LIMITATIONS: Cases limited to 14. CONCLUSIONS: Granulosa cell tumours present with a wide variety of clinical signs that do not fit what is commonly described as 'classic'. Only if AMH, testosterone and inhibin B concentrations are markedly increased, and there is an abnormally enlarged ovary, the diagnosis of a GCT is more confident. In the presence of normal size ovaries, normal hormonal biomarkers and abnormal behaviour, it is more likely that the ovaries are not involved.

Changes in testicular gene expression following reduced estradiol synthesis: A complex pathway to increased porcine Sertoli cell proliferation.

Porcine Sertoli cell number including number present at puberty is increased if testicular estradiol synthesis is reduced during the neonatal interval. Evaluating the changes in gene expression during the crucial interval of suppressed estradiol that leads to the increased Sertoli cell population will increase our understanding of Sertoli cell biology but this evaluation first required a more precise determination of the critical interval for treatment and timing of a detectable response. Previously, reduced testicular estrogens from 1 week of age were accompanied by increased Sertoli cell number at 6.5 weeks of age but the age at which Sertoli cell numbers were initially increased was unknown, one of the current objectives. Additional experiments were designed to further delineate the essential timing of treatment for the Sertoli cell response. Finally, changes in gene expression induced by the reduced estradiol synthesis were evaluated to elucidate molecular mechanisms. Experimental design typically consisted of one member of littermate pairs of boars treated with the aromatase inhibitor, letrozole, beginning at 1 week of age and the remaining member treated with canola oil vehicle. Weekly treatments continued through 5 weeks of age or tissue collection, whichever came first. Increases in Sertoli cell numbers were not detectable prior to 6.5 weeks of age and persistent treatment through 5 weeks of age was required to induce the increase in Sertoli cell numbers. This increase resulted from prolonging the first interval of Sertoli cell proliferation in the treated animals. Few genes exhibited dramatically altered transcription and similarities in pathway analysis or principal modified genes were quite limited in 2, 3, and 5-week-old boars. The critical timing and prolonged treatment required and the sequential changes in gene expression suggest a complex mechanism is involved in this model of increased proliferation of Sertoli cells.

Comparative analysis of steroids in cyclic and pregnant killer whales, beluga whales and bottlenose dolphins by liquid chromatography tandem mass spectrometry.

There exists a surprising diversity in the physiology and endocrinology of pregnancy among mammals in both the source (luteal/placental) and metabolism of progesterone. To evaluate the possible diversity of steroid metabolism within toothed cetaceans, we investigated 5α-reduced progesterone metabolites and androgens in cyclic (luteal phase) and pregnant captive killer whales, belugas and bottlenose dolphins (n = 5/species) bled longitudinally in early, mid- and late pregnancy (0.16, 0.50 and 0.85 fractions of 535, 464 and 380 gestation days, respectively). Mid-luteal samples were also collected. Serum was analyzed by liquid chromatography tandem-mass spectrometry as previously validated for (among others) progesterone, 20αOH-progesterone (20αOHP), 5α-dihydroprogesterone (DHP), several additional 5α-reduced metabolites and androgens (dehydroepiandrosterone, androstenedione and testosterone). The predominant mid-luteal pregnanes were: progesterone, belugas; progesterone and 20αOHP, dolphins; allopregnanolone (3α-DHP) and progesterone, killer whales. Progesterone was 2-4-fold higher in early pregnancy than mid-luteal samples but decreased thereafter. The predominant metabolite, 3ß,20α-dihydroprogesterone (3ß,20α-DHP; 40-80 ng/ml) was higher in mid- and late-than early gestation in all 3 species. Concentrations of 20αOHP and 3ß,20α-DHP were similar at mid-gestation but 20αOHP declined in late-gestation in killer whales, and 20αOHP was lower than 3ß,20α-DHP in belugas and dolphins throughout gestation. Other 5α-reduced metabolites, DHP, 3α-DHP and 20α-DHP, were far lower throughout pregnancy (<10 ng/ml). DHP and 3α-DHP decreased from early to mid-gestation in belugas, but changed little in killer whales and dolphins. These data suggest that progesterone metabolism is relatively conserved among these cetacean species. As in equine pregnancies, 3ß,20α-DHP is the major metabolite, increasing at the expense of progesterone as pregnancy progresses. Androstenedione and testosterone also increased detectably in mid- to late-gestation in these species. The tissue source remains unknown, but progesterone metabolism during gestation in these cetaceans is similar to horses and, together with androgens, may be reliable biomarkers of pregnancy.

Alteration of the mare's immune system by the synthetic progestin, altrenogest.

PROBLEM: Progestins are immunomodulatory in a variety of species. In the horse, the most commonly administered synthetic progestin is altrenogest (ALT), but its effect on the immune system of the non-pregnant mare is unknown. METHODS: Peripheral blood mononuclear cells (PBMCs) from diestrous mares were incubated with varying concentrations of progesterone (P4) or ALT to assess intracellular production of IFNγ and the expression of select cytokines. Additionally, ten mares received either ALT or VEH daily utilizing a switchback design beginning on the day of ovulation and continuing for 7 days. Circulating PBMCs and endometrial biopsies were obtained to assess the production and expression of the same cytokines. RESULTS: In vitro, both P4 and ALT caused a dose-dependent decrease in intracellular IFNγ in PBMCs. P4 caused a dose-dependent decrease in the expression of IFNγ, IL-10 and IL-4, while ALT caused an increase in the expression of IL-6 and IL-1ß in PBMCs. In vivo, ALT suppressed the intracellular levels of IFNγ in PBMCs on d6. While control mares experienced a decrease in IL-1ß expression from d0 to d6, ALT-treated mares did not. In the endometrium, ALT increased the expression of IL-1RN and IFNγ in comparison with VEH-treated mares. CONCLUSION: P4 and ALT appear to alter the immune system of the non-pregnant mare both systemically in addition to locally within the endometrium. Further research is necessary to determine the pathways through which this synthetic progestin functions on the immune system of the horse, and the consequences it may have.

Serum and tissue pregnanes and pregnenes after dexamethasone treatment of cows in late gestation.

Dexamethasone (DEX) initiates parturition by inducing progesterone withdrawal and affecting placental steroidogenesis, but the effects of DEX in fetal and maternal tissue steroid synthetic capacity remains poorly investigated. Blood was collected from cows at 270 days of gestation before DEX or saline (SAL) treatment, and blood and tissues were collected at slaughter 38 h later. Steroid concentrations were determined by liquid chromatography tandem mass spectrometry to detect multiple steroids including 5α-reduced pregnane metabolites of progesterone. The activities of 3ß-hydroxysteroid dehydrogenase (3ßHSD) in cotyledonary and luteal microsomes and mitochondria and cotyledonary microsomal 5α-reductase were assessed. Quantitative PCR was used to further assess transcripts encoding enzymes and factors supporting steroidogenesis in cotyledonary and luteal tissues. Serum progesterone, pregnenolone, 5α-dihydroprogesterone (DHP) and allopregnanolone (3αDHP) concentrations (all <5 ng/mL before treatment) decreased in cows after DEX. However, the 20α-hydroxylated metabolite of DHP, 20αDHP, was higher before treatment (≈100 ng/mL) than at slaughter but not affected by DEX. Serum, cotyledonary and luteal progesterone was lower in DEX- than SAL-treated cows. Progesterone was >100-fold higher in luteal than cotyledonary tissues, and serum and luteal concentrations were highly correlated in DEX-treated cows. 3ßHSD activity was >5-fold higher in luteal than cotyledonary tissue, microsomes had more 3ßHSD than mitochondria in luteal tissue but equal in cotyledonary sub-cellular fractions. DEX did not affect either luteal or cotyledonary 3ßHSD activity but luteal steroidogenic enzyme transcripts were lower in DEX-treated cows. DEX induced functional luteal regression and progesterone withdrawal before any changes in placental pregnene/pregnane synthesis and/or metabolism were detectable.

Anti-Müllerian hormone and ovarian aging in mares.

Anti-Müllerian hormone (AMH) is used as a marker of follicle population numbers and potential fertility in several species including horses but limited data exist across the lifespan. No one has decreased ovarian reserve experimentally to investigate whether a corresponding, quantitative decrease in AMH results. Concentrations of AMH across the lifespan were compiled from 1101 equine females sampled from birth to >33 years of age. Young and old mares (averaging 6 and 19 years) were hemi-ovariectomized and circulating AMH was assessed before and daily thereafter for 15 days. The remaining ovary was removed later and blood was drawn again before and after this second surgery for AMH determination. Polynomial regression analysis and analysis of mares grouped by 5-year intervals of age demonstrated AMH concentrations to be higher in mares aged 5-10 and 10-15 years than 0-5 years of age and lower in mares after 20 years of age. There was high variability in AMH concentrations among neonatal fillies, some of which had concentrations typical of males. Hemi-ovariectomy was followed by a decrease of AMH, almost exactly halving concentrations in intact mares. Concentrations of AMH had returned to intact levels in old mares before complete ovariectomy, as if exhibiting ovarian compensatory hypertrophy, but recovery of AMH was not evident in young mares. AMH may reflect ovarian senescence in mares after 20 years of age but is too variable to do so in the first two decades of life. The ovarian endocrine response to hemi-ovariectomy in mares appears to change with age.

Anti-Müllerian hormone profiling in prepubertal horses and its relationship with gonadal function.

Anti-Müllerian hormone (AMH) has gained increasing interest as a biomarker for assessment of gonadal activity. The ability to predict the ovarian follicular reserve of prepubertal female horses (fillies) or to identify stallions with testicular pathologies already during their prepubertal life has not been analyzed so far. Both would help to select fertile horses and reduce costs associated with keeping animals. The objectives of the present study were to (1) assess AMH, LH, FSH, progesterone (females) and testosterone (males) dynamics in prepubertal horses from birth onwards and (2) determine whether AMH concentrations detected in plasma of prepubertal female and male horses are correlated with postpubertal gonadal development. Warmblood foals (n = 30, 14 females, 10 normal males and 6 males with abnormal testicular development) born between February and May of two consecutive years (n = 28 in the first year and n = 2 the next year), were included in the study. Information on gestational length, parity of the dam and placental weight was collected for all foals. Blood samples for hormone analysis were collected from birth onwards every four weeks up to the age of one year. At two years, blood samples were collected on the day when antral follicle count (AFC) and total testicular volume (TTV) were assessed. AMH was detectable in the plasma of all animals from birth onwards and its concentration was significantly higher (P < .001) in males than in females, regardless of testicular development. In males, AMH and testosterone concentration were similar for all animals during the first year of life, regardless of testicular development. At two years, AMH concentration was higher (P < .05) in males with abnormal testicular development than in those with normal testes. In females, AMH concentration at two years was correlated with AMH concentration at birth (P < .05) and with AFC (P < .001). At birth, LH concentration was lower (P < .05) in stallions with abnormal testes (0.3 ±â€¯0.2 ng/ml) than in controls (0.6 ±â€¯0.2 ng/ml). A high negative correlation between AMH concentration and gestation length was observed in males during the first eight weeks of life (P < .01, r = -0.64 to -0.71). Elevated progesterone concentrations over 1 ng/ml were observed in several females starting with 20 weeks of age. This was paralleled by an increase in AMH concentration and was preceded by FSH and LH increases. In conclusion, AMH determination can be reliably used from two years onwards to identify stallions with abnormal testicular development, but it is inconclusive before puberty. In female horses, determination of AMH concentration at a prepubertal age allows for prediction of AMH and AFC after puberty. We suggest that premature luteinisation occurs before the onset of puberty in female horses and that LH secretion in the perinatal period is involved in testicular development and descent in the horse.

Endocrine and metabolic profile of peripubertal Standardbred colts.

The objectives of this study were to determine the concentrations of reproductive and metabolic hormones during the peripubertal period and to assess their relationship with testicular development and body fat deposition. Blood samples were collected from 23 healthy Standardbred colts every four weeks for twelve months. Colts were weighed monthly, and percent of body fat and testicular volume estimated by ultrasound. Onset of puberty was determined as the month when testosterone was two standard deviations above the previous mean. Plasma FSH, LH, leptin, estradiol-17ß, androstenedione, IGF-1, insulin, inhibin-A, and inhibin-B were analyzed for a seven month peripubertal period. Spring born Standardbred colts underwent puberty at 13 months of age; onset of puberty coincided with exponential testicular growth but did not coincide with an increase in cutaneous body fat deposition or leptin (p > 0.05). Plasma inhibin-B concentrations were significantly increased in the postpubertal period (p < 0.05), but no increase was seen in inhibin-A, androstenedione, FSH, LH, or estradiol-17ß. In conclusion, the rise in testosterone and subsequent onset of puberty coincides with rapid testicular growth but is not correlated with an increase in gonadotropins, IGF-1, cutaneous body fat or leptin in the horse.

Ovine placental steroid synthesis and metabolism in late gestation.

Steroid synthesis is required for pregnancy maintenance and for parturition, but comparatively little is known about the major metabolic routes that influence circulating concentrations. Dietary intake changes progesterone and estradiol concentrations in pregnant ewes but whether this reflects placental synthesis is unknown. Progesterone metabolism by 5alpha-reduction is a major metabolic route in other species and can influence the onset of parturition. Therefore, studies were conducted to (1) determine placental enzyme activity, progesterone, and estradiol measured by immunoassay in late gestation ewes on low-, moderate-, and high-nutritional planes, (2) to assess the significance of 5alpha-reduction of progesterone in determining progesterone concentrations in late gestation ewes (gestation day 145) given finasteride to inhibit 5alpha-reductase metabolism. In the second experiment, steroid profiles were examined comprehensively in blood and tissues by liquid chromatography tandem mass spectrometry for the first time in this species. Dietary intake altered progesterone and estradiol serum concentrations but without correlated changes in placental 3beta-hydroxysteroid dehydrogenase, 17alpha-hydroxylase/17,20-lyase cytochrome P450 or aromatase activity. 5alpha-reduced pregnane metabolites were identified in ewes at 145 days of gestation, but concentrations were lower than those of progesterone. Finasteride inhibited 5alpha-reduced progesterone metabolism but did not impact serum progesterone concentrations in these ewes. We conclude that (1) diet-induced changes in serum progesterone and estradiol concentrations are not likely a result of altered placental synthesis of sex steroid but most likely by their metabolism, and (2) metabolism by 5α-reduction is not a major determinant of systemic progesterone concentrations in late gestation ewes.

Steroidogenic enzyme activities in the pre- and post-parturient equine placenta.

Steroidogenic enzymes in placentas shape steroid hormone profiles in the maternal circulation of each mammalian species. These include 3ß-hydroxysteroid dehydrogenase/Δ5-4 isomerase (3ßHSD) and 17α-hydroxylase/17,20-lyase cytochrome P450 (P450c17) crucial for progesterone and androgen synthesis, respectively, as well as aromatase cytochrome P450 (P450arom) that converts Δ4-androgens to estrogens. 5α-reductase is another important enzyme in equine placentas because 5α-dihydroprogesterone (DHP) sustains pregnancy in the absence of progesterone in the second half of equine pregnancy. DHP and its metabolites decline dramatically days before foaling, but few studies have investigated placental enzyme activity before or at parturition in mares. Thus, key enzyme activities and transcript abundance were investigated in equine placentas at 300 days of gestation (GD300) and post-partum (term). Equine testis was used as a positive control for P450c17 activity. Substrates were incubated with microsomal preparations, together with enzyme inhibitors, and products were measured by liquid chromatography tandem mass spectrometry or radiometric methods (aromatase). Equine placenta expressed high levels of 3ßHSD, 5α-reductase and aromatase, and minimal P450c17 activity at GD300 compared with testis (600-fold higher). At foaling, 3ßHSD and aromatase activities and transcript abundance were unchanged but 5α-reductase (and P450c17) was no longer detectable (P < 0.05) and transcript was decreased. Trilostane inhibited 3ßHSD significantly more in testis than placenta, suggesting possible existence of different 3ßHSD isoforms. Equine placentas have significant capacity for steroid metabolism by 5α-reductase, 3ßHSD and aromatase but little for androgen synthesis lacking P450c17. Declining pre-partum 5α-reduced pregnane concentrations coincide with selective loss of placental 5α-reductase activity and expression at parturition in horses.

A comparison of progesterone assays for determination of peripheral pregnane concentrations in the late pregnant mare.

During the latter half of gestation in mares, there is a complex milieu of pregnanes in peripheral blood. Progesterone concentrations are often assessed by immunoassay during late gestation as a measure of pregnancy well-being; however, interpretation of results is complicated by the numerous cross-reacting pregnanes present in high concentrations during late gestation. Further, many mares are supplemented with an exogenous progestin, altrenogest, which may also cross-react with existing assays and further confound interpretation. The objectives of this study were: 1) to compare differences in pregnane concentrations determined with four immunoassays compared to LC-MS/MS and 2) to assess cross-reactivity observed with the same immunoassays, specifically considering pregnenolone (P5), progesterone (P4), 5α-dihydroprogesterone (DHP), allopregnanolone, and altrenogest. Blood samples from four healthy mares in late gestation were evaluated by immunoassay and by LC-MS/MS. Measured immuno-reactive progesterone (ir-progesterone) concentrations differed (p < 0.0001) between immunoassays, although results were highly correlated (r = 0.85-1.0; p < 0.001). Measured ir-progesterone concentrations by immunoassay were linearly associated (r2 = 0.68-0.76; p < 0.001) with concentrations of P5, P4, DHP, and allopregnanolone determined by LC-MS/MS. There was no detectable cross-reaction of altrenogest in any immunoassay, but varying degrees of cross-reactivity was observed with other pregnanes analyzed. These data confirm ir-progesterone concentrations during late gestation vary depending upon the assay used and the cross-reactivity to other pregnanes present in late gestation, although the synthetic progestin altrenogest did not affect the results of any immunoassay tested.

The dynamic steroid landscape of equine pregnancy mapped by mass spectrometry.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) allowed comprehensive analysis of various steroids detectable in plasma throughout equine gestation. Mares (n=9) were bled serially until they foaled. Certain steroids dominated the profile at different stages of gestation, clearly defining key physiological and developmental transitions. The period (weeks 6-20) coincident with equine chorionic gonadotropic (eCG) stimulation of primary corpora lutea and subsequent formation of secondary luteal structures was defined by increased progesterone, 17OH-progesterone and androstenedione, all Δ4 steroids. The 5α-reduced metabolite of progesterone, dihydroprogesterone (DHP) paralleled progesterone secretion at less than half the concentration until week 12 of gestation when progesterone began to decline but DHP concentrations continued to increase. DHP exceeded progesterone concentrations by week 16, clearly defining the luteo-placental shift in pregnane synthesis from primarily ovarian to primarily placental. The period corresponding to the growth of fetal gonads was defined by increasing dehydroepiandrosterone and pregnenolone (Δ5 steroids) concentrations from week 14, peaking at week 34 and declining to term. Metabolites of DHP (including allopregnanolone) dominated the steroid profile in late gestation, some exceeding DHP by weeks 13 or 14 and near term by almost tenfold. Thus Δ4 steroids dominated during ovarian stimulation by eCG, inversion of the ratio of progesterone: DHP (increasing 5α-pregnanes) marked the luteo-placental shift, Δ5 steroids defined fetal gonadal growth and 5α-reduced metabolites of DHP dominated the steroid profile in mid- to late-gestation. Comprehensive LC-MS/MS steroid analysis provides opportunities to better monitor the physiology and the progress of equine pregnancies, including fetal development.

COMPARISON OF TWO 4.7-MILLIGRAM TO ONE 9.4-MILLIGRAM DESLORELIN ACETATE IMPLANTS ON EGG PRODUCTION AND PLASMA PROGESTERONE CONCENTRATIONS IN JAPANESE QUAIL (COTURNIX COTURNIX JAPONICA).

Reproductive disease in captive avian species is common, and medical management is often chosen over surgical removal of the reproductive tract. In a previous study with Japanese quail, a single 4.7-mg deslorelin acetate implant reversibly decreased egg production in 6 out 10 birds for 70 days. The objective of the current study was to evaluate the effects of two 4.7-mg deslorelin acetate implants versus one 9.4-mg implant on egg production and plasma progesterone concentrations in Japanese quail ( Coturnix coturnix japonica). Following a 10-day period of consistent egg laying, 30 adult female Japanese quail were anesthetized and received two 4.7-mg deslorelin implants (n = 10), one 9.4-mg deslorelin implant (n = 10), or a single, identical placebo implant (n = 10) s.c. between the scapulae. Egg production was monitored daily, and plasma progesterone concentrations were measured on days 0, 14, 29, 120, 148, and 182 via enzyme-linked immunoassay. All birds were weighed periodically and euthanized at day 182, after which their reproductive tracts were evaluated at gross necropsy. Seven out of 10 birds treated with two 4.7-mg implants ceased egg laying 1 wk after implantation and remained nonovulatory for approximately 100 days. Cessation of egg laying for the 9.4-mg treatment group occurred in 7 out of 10 birds; onset was variable (weeks 5-12) and continued for the remainder of the study period. Plasma progesterone concentrations for deslorelin treatment groups were not significantly different compared to the placebo group at any time point. In conclusion, the two 4.7-mg and the one 9.4-mg implant treatments ceased egg laying in a similar number of birds, but the 9.4-mg implant had a slower onset of action and the effects on egg laying were inconsistent throughout the study period. Further studies evaluating use of deslorelin acetate in other avian species are needed.