Effect of 200 µg of gonadorelin hydrochloride at the first GnRH of a CIDR Synch program on ovulation rate and pregnancies per AI in Holstein heifers.

The initial ovulatory response during synchronization programs is often low in dairy heifers, largely due to follicular dynamics and hormonal dynamics. Specifically, the progesterone concentration (P4) at the time of the first GnRH treatment in a breeding program can influence the LH response, often resulting in a suboptimal ovulatory response. The objective of this study was to determine the effect of the highest label dose 200 µg (100 µg vs. 200 µg) of GnRH (50 µg gonadorelin hydrochloride per mL; Factrel®; Zoetis Inc. Madison, NJ) at the first GnRH of a 6-d CoSynch plus P4 device program on ovulatory response and pregnancy per AI (P/AI) in first service in Holstein heifers. A total of 1308 Holstein heifers were randomly allocated at the beginning of a 6-d CIDR-Synch program, Day 0, to receive either i.m. treatment of 100 µg (2CC, n = 655) or 200 µg (4CC, n = 653) of GnRH. Also, at Day 0, heifers received an intravaginal insert with 1.38 g of P4 (Eazi-Breed CIDR® Cattle Insert; Zoetis Inc., Madison, NJ). On Day 6, the insert was removed, and i.m. treatment of 25 mg of PGF2α (12.5 mg dinoprost tromethamine/mL; Lutalyse® HighCon Injection Zoetis) was administered. On Day 7, a second i.m. treatment of 25 mg of PGF2α was given, followed on Day 9 by concurrent i.m. treatment of 100 µg of GnRH and timed AI (TAI). A subset of 396 heifers had their ovaries scanned to evaluate ovulatory response, and blood samples were collected to measure the serum concentration of P4 at Day 0 and Day 6 of the study. The P4 concentrations at Day 0 were categorized as Low (≤3ng/mL) or High (>3ng/mL). The ovulatory response was greater for heifers receiving 4CC than 2CC at Day 0 (54.7% vs. 42.8%). The ovulatory response was greater for Low P4 than High P4 at Day 0 (54.3% vs. 37.8%). However, there was not an interaction between treatment and P4 concentrations (Low P4 2CC = 48.6% vs. High P4 2CC = 30.0%; Low P4 4CC = 60.0% vs. High P4 4CC = 45.5%). The ROC curve analysis indicates that P4 concentrations at Day 0 treatment could predict the ovulatory response, although the area under the curve was only 0.6. As expected, heifers that ovulated had increased P/AI (No = 55.6% vs. Yes = 67.7%); however, there was no effect of treatment on P/AI (2CC = 63.3% vs. 4CC = 59.6%), nor interactions between treatment and ovulation and treatment and P4 (HIGH vs LOW) for pregnancy outcomes. In summary, P4 concentration and increasing the dose of GnRH at Day 0 positively impacted ovulatory response in Holstein heifers. However, there was no interaction between treatment and P4 on ovulation and no subsequent impact of GnRH dose on P/AI.

Cystic ovarian disease in dairy cattle: Diagnostic accuracy when using B-mode and color Doppler ultrasound.

The most frequently reported definition of cystic ovarian disease in cattle is an abnormally persistent follicle (>7 to 10 d) with a diameter >25 mm. Discrimination between luteal and follicular ovarian cystic structures has traditionally been conducted by measuring the rim width of luteal tissue. The most common practice used in the field for diagnosis of cystic ovarian disease is examination by rectal palpation with or without the use of a B-mode ultrasound. Color Doppler ultrasound technology allows assessment of blood flow area measurements in the ovary, which has been proposed as a potential indirect measure for plasma progesterone (P4) concentrations. The objective of this study was to compare the diagnostic accuracy of differentiating luteal structures from follicular ovarian cysts using measures collected with B-mode and color Doppler transrectal ultrasonography. The definition of an ovarian cyst was a follicle greater than 20 mm in diameter in the absence of a corpus luteum that persisted for at least 10 d. A 3-mm luteal rim width was used to differentiate follicular and luteal cysts. A total of 36 cows were enrolled in the study during routine herd reproductive examination visits, with 26 and 10 having follicular and luteal cysts, respectively. Cows enrolled in the study were examined using a Mini-ExaPad mini ultrasound with color Doppler capabilities (IMV Imaging Ltd.). Blood samples were collected from each cow to measure P4 serum concentrations. History and signalment of each cow, including days in milk, lactation, times bred, days since last heat, milk composition, and somatic cell counts, were retrieved from an online database (DairyComp 305, Valley Agricultural Software). The accuracy of diagnosing follicular from luteal cysts based on luteal rim thickness was analyzed by receiver operating characteristic (ROC) curve using P4 as the gold standard, where P4 concentrations exceeding 1 ng/mL was defined as luteal, and all other structures with less P4 were considered follicular. Luteal rim and blood flow area were selected for further analysis because they presented the best ROC curves for differentiating cystic ovarian structures, with areas under the curve of 0.80 and 0.76, respectively. Luteal rim width of 3 mm was used as the cutoff standard in the study, resulting in sensitivity and specificity of 50% and 86%, respectively. Blood flow area of 0.19 cm2 was used as the cutoff standard in the study, resulting in sensitivity and specificity of 79% and 86%, respectively. When combining the use of luteal rim width and blood flow area to differentiate cystic ovarian structures, a parallel approach resulted in sensitivity and specificity of 73% and 93%, respectively, whereas an in-series approach resulted in sensitivity and specificity of 35% and 100%, respectively. In conclusion, the use of color Doppler ultrasonography when discriminating between luteal and follicular ovarian cysts in dairy cattle resulted in higher diagnostic accuracy compared with using B-mode ultrasonography alone.

The onset of puberty in colony-housed male and female titi monkeys (Plecturocebus cupreus): Possible effects of oxytocin treatment during peri-adolescent development.

Oxytocin has been used to treat neurodevelopmental conditions in adolescent patients but possible effects on reproductive development have not been well investigated. The effects of daily intra-nasal oxytocin treatment (12-18 months of age) on puberty and fertility were studied in colony-housed, male and female titi monkeys (Plecturocebus cupreus). Body weight, urinary conjugated pregnanes and estrogens (defining cyclicity) in females, and androgens and sperm in urine of in males, were measured from 1 to 3 years of age to detect puberty. Serum testosterone was also measured in males at 13, 23 and 33 months of age and hemi-castration at 3 years of age enabled assessment of testicular morphometry and oxytocin receptor expression. An oxytocin treatment*time interaction suggested a minor, transient suppression in weight gain after treatment ended. Note that females weighed 10% less across all ages. Oxytocin-treated females exhibited early, spurious ovulations but neither regular cyclicity (≈30 months) nor pregnancies were affected by treatment. Oxytocin did not affect the pubertal increase in urinary androgen or the first appearance of sperm, which occurred as early as 15 months of age. Treatment did delay the puberty-associated rise in serum testosterone in males. All males were pubertal by 22 months and all females by 32 months of age. Although no major male or female fertility outcome was observed, oxytocin demonstrated some physiological effects through a delay of testosterone secretion in males, induction of precocious ovulation in females, and a suppression of general weight gain for the months following treatment.

Effect of gonadotropin-releasing hormone administered at the time of artificial insemination for cows detected in estrus by conventional estrus detection or an automated activity-monitoring system.

The objectives of this study were to determine the effects of GnRH at the time of artificial insemination (AI) on ovulation, progesterone 7 d post-AI, and pregnancy in cows detected in estrus using traditional methods (tail chalk removal and mount acceptance visualization) or an automated activity-monitoring (AAM) system. We hypothesized that administration of GnRH at the time of AI would increase ovulation rate, plasma progesterone post-AI, and pregnancy per AI (P/AI) in cows detected in estrus. In experiment 1, Holstein cows (n = 398) were blocked by parity and randomly assigned to receive an injection of GnRH at the time of estrus detection/AI (GnRH, n = 197) or to remain untreated (control, n = 201) on 4 farms. The GnRH was administered as 100 µg of gonadorelin acetate. Ovarian structures and plasma progesterone were assessed in a subset of cows (GnRH, n = 52; control, n = 55) in experiment 1 at the time of AI and 7 d later. In experiment 2, a group of 409 cows in an AAM farm were enrolled as described for experiment 1 (GnRH, n = 207; control, n = 202). Data were categorized for parity (primiparous vs. multiparous), season (cool vs. warm), number of services (first vs. > first), DIM (>150 DIM vs. ≤150 DIM), and for AAM cows in experiment 2 for activity level (high: 90-100 index vs. low: 35-89 index). Pregnancy diagnosis was performed between 32 and 45 d post-AI (P1) and 60 to 115 d post-AI (P2). In experiment 1, there was no difference in plasma progesterone at day of estrus detection (control = 0.09 ng/mL vs. GnRH = 0.16 ng/mL), 7 d later (control = 2.03 ng/mL vs. GnRH = 2.18 ng/mL), and ovulation rate (GnRH = 83.2% vs. control = 77.9%) between treatments. There were no effects of GnRH in experiment 1 for P/AI at P1 (control = 43.3% vs. GnRH = 38.6%), P2 (control = 38.4% vs. GnRH = 34.5%), and for pregnancy loss (control = 9.8% vs. GnRH = 8.2%). In experiment 2, there were no effects of GnRH for P/AI at P1 (control = 39.6% vs. GnRH = 40.1%), P2 (control = 35.0% vs. GnRH = 37.4%), and for pregnancy loss (control = 9.5% vs. GnRH = 6.2%). There was a tendency for a parity effect on P/AI for P1, but not P2 or for pregnancy loss. High-activity cows had greater P/AI in P1 (low activity = 27.9% vs. high activity = 44.1%), P2 (low activity = 21.8% vs. high activity = 41.2%), and lower pregnancy loss (low activity = 20.7% vs. high activity = 5.1%), but there were no interactions between treatment and activity level. The current study did not support the use of GnRH at estrus detection to improve ovulatory response, progesterone 1 wk post-AI, and P/AI. More research is needed to investigate the relationship between GnRH at the time of AI and activity level in herds using AAM systems.

Steroidogenesis during prenatal testicular development in Spix's cavy Galea spixii.

Spix's cavy is a potentially good experimental model for research on reproductive biology and sexual development. The aim of the present study was to evaluate the ontogeny of the steroidogenic enzymes involved in testicular androgen synthesis during prenatal development. Testes were investigated on Days 25, 30, 40 and >50 of gestation. Immunohistochemistry and immunoblotting were used to establish the site and relative amount of androgenic enzymes, including 5α-reductase, cytosolic 17ß-hydroxysteroid dehydrogenase (17ß-HSDI) and mitochondrial microsomal 3ß-hydroxysteroid dehydrogenase (3ß-HSDII), throughout prenatal development. The testicular parenchyma began to organise on Day 25 of gestation, with the development of recognisable testicular cords. The mesonephros was established after Day 25 of gestation and the ducts differentiated to form the epididymis, as testicular cords were beginning to proliferate and the interstitium to organise by Day 30 of gestation, continuing thereafter. The androgen-synthesising enzymes 5α-reductase, 17ß-HSDI and 3ß-HSDII were evident in Leydig cells as they differentiated at all subsequent gestational ages studied. In addition, immunoblotting showed an increase in immunoreactivity for the enzymes at Days 30 and 40 of gestation (P<0.05) and a decrease at Day 50 of gestation (P<0.05). It is concluded that the increase in androgenic enzymes in Leydig cells coincides with the functional differentiation of the testes, and with the stabilisation and differentiation of mesonephric ducts forming the epididymis.

The steroid metabolome of pregnancy, insights into the maintenance of pregnancy and evolution of reproductive traits.

Modes of mammalian reproduction are diverse and not always conserved among related species. Progesterone is universally required to supports pregnancy but sites of synthesis and metabolic pathways vary widely. The steroid metabolome of mid-to late gestation was characterized, focusing on 5α-reduced pregnanes in species representing the Perissodactyla, Cetartiodactyla and Carnivora using mass spectrometry. Metabolomes and steroidogenic enzyme ortholog sequences were used in heirarchial analyses. Steroid metabolite profiles were similar within orders, whales within cetartiodactyls for instance, but with notable exceptions such as rhinoceros clustering with goats, and tapirs with pigs. Steroidogenic enzyme sequence clustering reflected expected evolutionary relationships but once again with exceptions. Human sequences (expected outgroups) clustered with perissodactyl CYP11A1, CYP17A1 and SRD5A1 gene orthologues, forming outgroups only for HSD17B1 and SRD5A2. Spotted hyena CYP19A1 clustered within the Perissodactyla, between rhinoceros and equid orthologues, whereas CYP17A1 clustered within the Carnivora. This variability highlights the random adoption of divergent physiological strategies as pregnancy evolved among genetically similar species.

New insights in equine steroidogenesis: an in-depth look at steroid signaling in the placenta.

Steroid production varies widely among species, with these differences becoming more pronounced during pregnancy. As a result, each species has its own distinct pattern of steroids, steroidogenic enzymes, receptors, and transporters to support its individual physiological requirements. Although the circulating steroid profile is well characterized during equine pregnancy, there is much yet to be explored regarding the factors that support steroidogenesis and steroid signaling. To obtain a holistic view of steroid-related transcripts, we sequenced chorioallantois (45 days, 4 months, 6 months, 10 months, 11 months, and post-partum) and endometrium (4 months, 6 months, 10 months, 11 months, and diestrus) throughout gestation, then looked in-depth at transcripts related to steroid synthesis, conjugation, transportation, and signaling. Key findings include: 1) differential expression of HSD17B isoforms among tissues (HSD17B1 high in the chorioallantois, while HSD17B2 is the dominant form in the endometrium) 2) a novel isoform with homology to SULT1A1 is the predominant sulfotransferase transcript in the chorioallantois; and 3) nuclear estrogen (ESR1, ESR2) and progesterone (PGR) expression is minimal to nonexistant in the chorioallantois and pregnant endometrium. Additionally, several hypotheses have been formed, including the possibility that the 45-day chorioallantois is able to synthesize steroids de novo from acetate and that horses utilize glucuronidation to clear estrogens from the endometrium during estrous, but not during pregnancy. In summary, these findings represent an in-depth look at equine steroid-related transcripts through gestation, providing novel hypotheses and future directions for equine endocrine research.

Steroid synthesis and metabolism in the equine placenta during placentitis.

Equine placentitis is associated with alterations in maternal peripheral steroid concentrations, which could negatively affect pregnancy outcome. This study aimed to elucidate the molecular mechanisms related to steroidogenesis and steroid-receptor signaling in the equine placenta during acute placentitis. Chorioallantois (CA) and endometrial (EN) samples were collected from mares with experimentally induced placentitis (n = 4) and un-inoculated gestationally age-matched mares (control group; n = 4). The mRNA expression of genes coding for steroidogenic enzymes (3ßHSD, CYP11A1, CYP17A1, CYP19A1, SRD5A1, and AKR1C23) was evaluated using qRT-PCR. The concentration of these enzyme-dependent steroids (P5, P4, 5αDHP, 3αDHP, 20αDHP, 3ß-20αDHP, 17OH-P, DHEA, A4, and estrone) was assessed using liquid chromatography-tandem mass spectrometry in both maternal circulation and placental tissue. Both SRD5A1 and AKR1C23, which encode for the key progesterone metabolizing enzymes, were downregulated (P < 0.05) in CA from the placentitis group compared to controls, and this downregulation was associated with a decline in tissue concentrations of 5αDHP (P < 0.05), 3αDHP (P < 0.05), and 3ß-20αDHP (P = 0.052). In the EN, AKR1C23 was also downregulated in the placentitis group compared to controls, and this downregulation was associated with a decline in EN concentrations of 3αDHP (P < 0.01) and 20αDHP (P < 0.05). Moreover, CA expression of CYP19A1 tended to be lower in the placentitis group, and this reduction was associated with lower (P = 0.057) concentrations of estrone in CA. Moreover, ESR1 (steroid receptors) gene expression was downregulated (P = 0.057) in CA from placentitis mares. In conclusion, acute equine placentitis is associated with a local withdrawal of progestins in the placenta and tended to be accompanied with estrogen withdrawals in CA.

Concentrations of sulphated estrone, estradiol and dehydroepiandrosterone measured by mass spectrometry in pregnant mares.

BACKGROUND: Few studies have provided a longitudinal analysis of systemic concentrations of conjugated oestrogens (and androgens) throughout pregnancy in mares, and those only using immunoassay. The use of liquid chromatography tandem mass spectrometry (LC-MS/MS) will provide more accurate concentrations of circulating conjugated steroids. OBJECTIVES: To characterise circulating concentrations of individual conjugated steroids throughout equine gestation by using LC-MS/MS. STUDY DESIGN: Longitudinal study and comparison of pregnant mares treated with vehicle or letrozole in late gestation. METHODS: Sulphated oestrogens and androgens were measured in mares throughout gestation and mares in late gestation (8-11 months) treated with vehicle or letrozole to inhibit oestrogen synthesis in late gestation. An analytical method was developed using LC-MS/MS to evaluate sulphated estrone, estradiol, testosterone and dehydroepiandrosterone (DHEAS) during equine gestation. RESULTS: Estrone sulphate concentrations peaked by week 26 at almost 60 µg/mL, 50-fold higher than have been reported in studies using immunoassays. An increase in DHEAS was detected from 7 to 9 weeks of gestation, but concentrations remained consistently low (if detected) for the remainder of gestation and testosterone sulphate was undetectable at any stage. Estradiol sulphate concentrations were highly correlated with estrone sulphate but were a fraction of their level. Concentrations of both oestrogen sulphates decreased from their peak to parturition. Letrozole inhibited estrone and estradiol sulphate concentrations at 9.25 and 10.5 months of gestation but, no increase in DHEAS was observed. MAIN LIMITATIONS: Limited number of mares sampled and available for analysis, lack of analysis of 5α-reduced and B-ring unsaturated steroids due to lack of available standards. CONCLUSIONS: Dependent on methods of extraction and chromatography, and the specificity of primary antisera, immunoassays may underestimate oestrogen conjugate concentrations in blood from pregnant mares and may detect androgen conjugates (neither testosterone sulphate nor DHEAS were detected here by LC-MS/MS) that probably peak coincident with oestrogen conjugates between 6 and 7 months of equine gestation.

Inhibin-A and Inhibin-B in stallions: Seasonal changes and changes after down-regulation of the hypothalamic-pituitary-gonadal axis.

The biological function of inhibin is mediated by two heterodimers, inhibin-A and inhibin-B. The relative importance of inhibin-A and -B in male reproductive function varies considerably across species with inhibin-B predominating in many species, whereas inhibin-A appears relatively more important in rams. Research reported to date in stallions has examined total or immunoreactive (ir) inhibin which does not distinguish the two heterodimers. Therefore, the objective of this study was to characterize changes in inhibin-A and inhibin-B concentrations in stallions: 1) across season for a period of one year, and 2) after downregulation of the hypothalamic-pituitary-gonadal (HPG) axis. In Study one, serum samples were obtained monthly from five stallions for a period of one year. Serum concentrations of inhibin-A, inhibin-B, testosterone and estrone sulfate were determined by ELISA. In Study two, stallions were treated with the GnRH antagonist, acyline (n = 4; 330 mg/kg acyline IM) or vehicle control (n = 4; vehicle alone) every five days for 50 days. Plasma concentrations of inhibin-A and -B were determined by ELISA at Days 0, 6, 12, 22, 37, 59, 80, 87 and 104 after initiation of acyline treatment. Testis volume was determined by ultrasonography at weekly intervals. In Study 1, both inhibin-A and inhibin-B showed seasonal changes in concentration with highest concentrations in increasing day length and lowest concentrations in short day lengths. Inhibin-B (overall mean 107.8 ±â€¯4.1 pg/mL) was present at 4.7-fold higher concentrations in serum than inhibin-A (overall mean 23.0 ±â€¯0.7 pg/mL). In Study 2, plasma concentrations of inhibin-B but not inhibin-A were significantly downregulated by administration of the GnRH antagonist, acyline. When the HPG axis was downregulated by acyline, testis volume was strongly correlated with inhibin-B (r = 0.73; P < 0.05) but not inhibin-A (r = 0.22; P = 0.20). In summary, inhibin-B appears to be the predominant form of inhibin in the stallion which undergoes seasonal regulation along with other reproductive parameters and is co-regulated with other endocrine parameters of the HPG axis.

5α-dihydroprogesterone concentrations and synthesis in non-pregnant mares.

In vivo and in vitro evidence indicates that the bioactive, 5α-reduced progesterone metabolite, 5α-dihydroprogesterone (DHP) is synthesized in the placenta, supporting equine pregnancy, but its appearance in early pregnancy argues for other sites of synthesis also. It remains unknown if DHP circulates at relevant concentrations in cyclic mares and, if so, does synthesis involve the non-pregnant uterus? Jugular blood was drawn daily from cyclic mares (n = 5). Additionally, ovariectomized mares (OVX) and geldings were administered progesterone (300 mg) intramuscularly. Blood was drawn before and after treatment. Incubations of whole equine blood and hepatic microsomes with progesterone were also investigated for evidence of DHP synthesis. Sample analysis for progesterone, DHP and other steroids employed validated liquid chromatography-tandem mass spectrometry methods. Progesterone and DHP appeared a day (d) after ovulation in cyclic mares, was increased significantly by d3, peaking from d5 to 10 and decreased from d13 to 17. DHP was 55.5 ± 3.2% of progesterone concentrations throughout the cycle and was highly correlated with it. DHP was detected immediately after progesterone administration to OVX mares and geldings, maintaining a relatively constant ratio with progesterone (47.2 ± 2.9 and 51.2 ± 2.7%, respectively). DHP was barely detectable in whole blood and hepatic microsome incubations. We conclude that DHP is a physiologically relevant progestogen in cyclic, non-pregnant mares, likely stimulating the uterus, and that it is synthesized peripherally from luteal progesterone but not in the liver or blood. The presence of DHP in pregnant perissodactyla as well as proboscidean species suggests horses may be a valuable model for reproductive endocrinology in other exotic taxa.

Effects of long-term, near-term, and real-time energy balance, and blood progesterone concentrations, on the pregnancy rate of contemporary dairy cows.

This study aimed to contribute to understanding the interface between reproductive and nutritional energetic physiology in contemporary dairy cattle. Multiparous Holstein cows (n = 32) between 70 and 180 days in milk were used in a study starting 10 d prior to the artificial insemination (AI) date and were estrous synchronized using a hormonal regimen. Fourteen cows were determined pregnant on day 39 post-AI. Coccygeal blood samples of all cows were collected on d -10 and -3 prior to AI to determine estrous cyclicity, as well as at AI and at 6, 13 and 20 d post-AI. Milk progesterone was measured 20 d post-AI, and body condition was scored (BCS; 1-5 scale) on days -10, 0, 13 and 27 relative to AI. Blood non-esterified fatty acid concentrations, measured on the same days as BCS, and changes of BCS from d -10 to AI were not predictive of pregnancy outcome. The BCS of cows on the day of AI was greater (P = 0.02) for pregnant cows with an approximate minimum BCS for a high probability of conception being 2.50. Serum progesterone concentrations of pregnant cows were greater (P < 0.05) on days 6, 13 and 20 post-AI, as was milk progesterone at day 20 post-AI (P < 0.01). Pregnant cows had greater (P = 0.02) net energy output (NEL), which is inconsistent with a common belief that low pregnancy rates in contemporary dairy cows are due to excessive milk production, but is consistent with published studies in this study area. The present research indicates that current low pregnancy rates in commercial high-producing multiparous dairy cattle may be partly due to breeding cows that have insufficient BCS to support pregnancy.

Inhibin-A and inhibin-B in cyclic and pregnant mares, and mares with granulosa-theca cell tumors: Physiological and diagnostic implications.

Studies in mares have examined serum inhibin concentrations using immuno-assays unable to distinguish dimeric inhibin-A from inhibin-B isoforms. Inhibin-A and inhibin-B immuno-assays were used to investigate concentrations in cyclic mares, young and old (6 vs 19 years old, respectively) mares following hemi-ovariectomy, mares during pregnancy and in mares with confirmed granulosa cell tumors (GCTs). Mares with inter-ovulatory intervals of 26 days had ovulatory peaks of inhibin-A averaging 80 pg/mL with a mid-cycle nadir of 5 pg/mL. Inhibin-A and inhibin-B concentrations were highly correlated (r = + 0.79, P < 0.01) though peak and nadir concentrations of inhibin-B were not significantly different. However, the ratio of inhibin-A to inhibin-B (A/B) changed significantly through the cycle, highest at ovulation and <1 (more inhibin-B than -A) at mid-cycle. Two mares with grossly extended inter-ovulatory intervals demonstrated mid-cycle inhibin-A (and inhibin-B) excursions suggestive of follicular waves. Follicle-stimulating hormone was negatively correlated with inhibin-A and -B concentrations in all 6 mares. Hemi-ovariectomy in young mares resulted in a significant decrease in inhibin-A and inhibin-B concentrations one day later (P < 0.05) but older mares did not, suggesting a possible extra-ovarian source(s) of these hormones. Both inhibin isoforms dropped to very low levels during pregnancy (P < 0.0001), inhibin-A (P < 0.0001) more rapidly than -B (P < 0.05), so that inhibin-B became the predominant measured form throughout most of gestation (P < 0.05). Mares with confirmed GCTs had elevated inhibin-B concentrations more reliably than inhibin-A but neither inhibin-A or -B was correlated with anti-Müllerian hormone concentrations. Collectively, concentrations of inhibin-A and -B were aligned with physiological events in healthy mares, though more pronounced cyclic changes were seen with inhibin-A. Inhibin-B concentrations were significantly associated with GCTs (P < 0.01), inhibin-A concentrations were not. While both inhibin-A and -B concentrations track physiological events such as cyclic follicular activity, only inhibin-B concentrations effectively signal ovarian neoplasia in mares.

Steroidogenesis during postnatal testicular development of Galea spixii.

The androgen/estrogen balance is essential for normal sexual development and reproduction in mammals. Studies performed herein investigated the potential for estrogen synthesis in cells of the testes of a hystricomorph rodent, Galea spixii The study characterized the expression of the key enzymes responsible for estrogen and androgen synthesis, cytochromes P450 aromatase (P450arom), 17α-hydroxylase/17,20-lyase (P450c17) respectively, as well as the redox partner NADPH cytochrome P450 oxido-reductase (CPR) required to support electron transfer and catalysis of these P450s, by immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR) analysis, throughout postnatal sexual development. Testes (immature, pre-pubertal, pubertal and post-pubertal) were collected, fixed for IHC (CYP19, CYP17 and CPR) and stored frozen for qPCR for the relevant gene transcripts (Cyp19a1 and Cyp17a1). Expression of P450c17 was significantly elevated at the pre-pubertal and pubertal stages. Based on IHC, P450c17 was expressed only in Leydig cell clusters. The expression of P450arom was detectable at all stages of sexual development of Galea spixii IHC data suggest that estrogen synthesis was not restricted to somatic cells (Leydig cells/Sertoli cells), but that germ cells may also be capable of converting androgens into estrogens, important for testicular function and spermatogenesis.

Multimodel precipitation responses to removal of U.S. sulfur dioxide emissions.

Emissions of aerosols and their precursors are declining due to policies enacted to protect human health, yet we currently lack a full understanding of the magnitude, spatiotemporal pattern, statistical significance, and physical mechanisms of precipitation responses to aerosol reductions. We quantify the global and regional precipitation responses to U.S. SO2 emission reductions using three fully coupled chemistry-climate models: Community Earth System Model version 1, Geophysical Fluid Dynamics Laboratory Coupled Model 3, and Goddard Institute for Space Studies ModelE2. We contrast 200 year (or longer) simulations in which anthropogenic U.S. sulfur dioxide (SO2) emissions are set to zero with present-day control simulations to assess the aerosol, cloud, and precipitation response to U.S. SO2 reductions. In all three models, reductions in aerosol optical depth up to 70% and cloud droplet number column concentration up to 60% occur over the eastern U.S. and extend over the Atlantic Ocean. Precipitation responses occur both locally and remotely, with the models consistently showing an increase in most regions considered. We find a northward shift of the tropical rain belt location of up to 0.35° latitude especially near the Sahel, where the rainy season length and intensity are significantly enhanced in two of the three models. This enhancement is the result of greater warming in the Northern versus Southern Hemispheres, which acts to shift the Intertropical Convergence Zone northward, delivering additional wet season rainfall to the Sahel. Two of our three models thus imply a previously unconsidered benefit of continued U.S. SO2 reductions for Sahel precipitation.

Progestin withdrawal at parturition in the mare.

Mammalian pregnancies need progestogenic support and birth requires progestin withdrawal. The absence of progesterone in pregnant mares, and the progestogenic bioactivity of 5α-dihydroprogesterone (DHP), led us to reexamine progestin withdrawal at foaling. Systemic pregnane concentrations (DHP, allopregnanolone, pregnenolone, 5α-pregnane-3ß, 20α-diol (3ß,20αDHP), 20α-hydroxy-5α-dihydroprogesterone (20αDHP)) and progesterone) were monitored in mares for 10days before foaling (n=7) by liquid chromatography-mass spectrometry. The biopotency of dominant metabolites was assessed using luciferase reporter assays. Stable transfected Chinese hamster ovarian cells expressing the equine progesterone receptor (ePGR) were transfected with an MMTV-luciferase expression plasmid responsive to steroid agonists. Cells were incubated with increasing concentrations (0-100nM) of progesterone, 20αDHP and 3α,20ßDHP. The concentrations of circulating pregnanes in periparturient mares were (highest to lowest) 3α,20ßDHP and 20αDHP (800-400ng/mL respectively), DHP and allopregnanolone (90 and 30ng/mL respectively), and pregnenolone and progesterone (4-2ng/mL). Concentrations of all measured pregnanes declined on average by 50% from prepartum peaks to the day before foaling. Maximum activation of the ePGR by progesterone occurred at 30nM; 20αDHP and 3α,20ßDHP were significantly less biopotent. At prepartum concentrations, both 20αDHP and 3α,20ßDHP exhibited significant ePGR activation. Progestogenic support of pregnancy declines from 3 to 5days before foaling. Prepartum peak concentrations indicate that DHP is the major progestin, but other pregnanes like 20αDHP are present in sufficient concentrations to play a physiological role in the absence of DHP. The authors conclude that progestin withdrawal associated with parturition in mares involves cessation of pregnane synthesis by the placenta.

Equine 5α-reductase activity and expression in epididymis.

The 5α-reductase enzymes play an important role during male sexual differentiation, and in pregnant females, especially equine species where maintenance relies on 5α-reduced progesterone, 5α-dihydroprogesterone (DHP). Epididymis expresses 5α-reductases but was not studied elaborately in horses. Epididymis from younger and older postpubertal stallions was divided into caput, corpus and cauda and examined for 5α-reductase activity and expression of type 1 and 2 isoforms by quantitative real-time polymerase chain reaction (qPCR). Metabolism of progesterone and testosterone to DHP and dihydrotestosterone (DHT), respectively, by epididymal microsomal protein was examined by thin-layer chromatography and verified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Relative inhibitory potencies of finasteride and dutasteride toward equine 5α-reductase activity were investigated. Pregnenolone was investigated as an additional potential substrate for 5α-reductase, suggested previously from in vivo studies in mares but never directly examined. No regional gradient of 5α-reductase expression was observed by either enzyme activity or transcript analysis. Results of PCR experiments suggested that type 1 isoform predominates in equine epididymis. Primers for the type 2 isoform were unable to amplify product from any samples examined. Progesterone and testosterone were readily reduced to DHP and DHT, and activity was effectively inhibited by both inhibitors. Using epididymis as an enzyme source, no experimental evidence was obtained supporting the notion that pregnenolone could be directly metabolized by equine 5α-reductases as has been suggested by previous investigators speculating on alternative metabolic pathways leading to DHP synthesis in placenta during equine pregnancies.

Review of the reproductive endocrinology of the pregnant and parturient mare.

Analytical advancements, especially methods using gas or liquid chromatography tandem mass spectrometry, have allowed more specific and reliable measurement of multiple steroid hormones in the plasma of mares throughout gestation and the periparturient period. Data such as these will form the central focus of this review. The comprehensive analyses possible with liquid chromatography tandem mass spectrometry illuminate the key physiological and developmental transitions that make equine gestation unique. Weeks 6 to 20 encompass endometrial cup formation and equine chorionic gonadotropic secretion that stimulates primary corpora lutea and induces formation of secondary luteal structures. The period is defined by increased progesterone, 17OH-progesterone, and androstenedione secretion, providing substrate feeding the rise in estrone sulfate that can be used as an aid in the diagnosis of pregnancy. The 5α-reduced metabolite of progesterone, dihydroprogesterone (DHP), parallels progesterone secretion during this period at less than half the concentration. After week 12, progesterone declines, but DHP concentrations continue to increase, exceeding progesterone by week 16, thereby defining the luteo-placental shift in pregnane synthesis from ovarian to primarily placental thereafter. The growth of fetal gonads begins around week 14 and is defined by increasing dehydroepiandrosterone, among other androgens, which fuels placental estrogen secretion, functioning as a true fetoplacental unit. Metabolites of DHP (including allopregnanolone) dominate in late gestation, some exceeding DHP by 10-fold near term. However, all major pregnanes decrease from 3 days before foaling, when fetal cortisol is reportedly rising. Though unique, equine pregnancy and parturition share many features in common with those seen in human pregnancy and birth.

Non-linear partitioning and organic volatility distributions of urban aerosols.

Gas-to-particle partitioning of organic aerosols (OA) is represented in most models by Raoult's law, and depends on the existing mass of particles into which organic gases can dissolve. This raises the possibility of non-linear response of particle-phase OA mass to the emissions of precursor volatile organic compounds (VOCs) that contribute to this partitioning mass. Implications for air quality management are evident: a strong non-linear dependence would suggest that reductions in VOC emission would have a more-than-proportionate benefit in lowering ambient OA concentrations. Chamber measurements on simple VOC mixtures generally confirm the non-linear scaling between OA and VOCs, usually stated as a mass-dependence of the measured OA yields. However, for realistic ambient conditions including urban settings, no single component dominates the composition of the organic particles, and deviations from linearity are presumed to be small. Here we re-examine the linearity question using volatility spectra from several sources: (1) chamber studies of selected aerosols, (2) volatility inferred for aerosols sampled in two megacities, Mexico City and Paris, and (3) an explicit chemistry model (GECKO-A). These few available volatility distributions suggest that urban OA may be only slightly super-linear, with most values of the normalized sensitivity exponent in the range 1.1-1.3, also substantially lower than seen in chambers for some specific aerosols. The rather low exponents suggest that OA concentrations in megacities are not an inevitable convergence of non-linear effects, but can be addressed (much like in smaller urban areas) by proportionate reductions in emissions.

The interrelationship between anti-Müllerian hormone, ovarian follicular populations and age in mares.

REASONS FOR PERFORMING STUDY: Anti-Müllerian hormone (AMH) is a granulosa-cell-derived glycoprotein, which plays an important inhibitory role during folliculogenesis. Concentrations of AMH are highly correlated with antral follicle counts (AFCs) in other species, which in turn are related to follicular reserve. Relatively little is known about AMH and AFC in the mare. OBJECTIVES: To determine plasma AMH concentrations and AFCs in mares of different ages, to measure the repeatability of AMH concentrations and AFCs within and across oestrous cycles and to assess the relationship between plasma AMH concentrations and AFCs with regard to mare age and follicle size. STUDY DESIGN: An observational study examining the relationship between AMH, AFC and age in 45 mares. METHODS: Young (3-8 years), middle-aged (9-18 years) and old mares (19-27 years) were examined by transrectal ultrasonography over 2 or 3 oestrous cycles. Plasma AMH concentrations and AFCs were determined, and antral follicles were classified by size into different groups. RESULTS: Plasma AMH concentrations varied widely between mares within similar age groups. Antral follicle counts were significantly lower in old mares than in young and middle-aged mares, and AMH concentrations were significantly lower in old than in middle-aged mares. A positive relationship was detected between AFC and AMH, and this relationship varied by mare age with a strong correlation in older mares (ρ = 0.86; P<0.0001), a moderate correlation in middle-aged mares (ρ = 0.60; P = 0.01) and no correlation in young mares (ρ = 0.40, P<0.4). The AMH concentrations were significantly related to the number of antral follicles between 6 and 20 mm in diameter, and the repeatability of AFCs and AMH concentrations was high within and between oestrous cycles. CONCLUSIONS: Our findings indicate that the relationship between AMH and AFC varies across age groups, and concentrations of AMH might be a better reflection of reproductive age than calendar age.