Seasonal and diel variation of hormone metabolites in European stonechats: on the importance of high signal-to-noise ratios in noninvasive hormone studies

Most vertebrates living in seasonal environments show seasonal reproductive cycles and diel rhythms. The rhythmicity in behavior and morphology is accompanied by diel and seasonal patterns of hormone secretions. In small animals, the investigation of diel patterns of hormones has been hampered because repeated blood sampling is difficult and may influence subsequent measurements. A possibility to avoid these caveats is to investigate excreted hormone metabolites instead. Here, we describe the diel excretion patterns of testosterone and corticosterone metabolites in a small captive songbird during 4 seasons: winter, early spring (Zugunruhe), summer, and autumn molt. Our approach is quite unique, because the diel patterns of steroids have rarely--if at all--been investigated in the same individuals within several seasons. Small birds should be ideal to investigate diel patterns of hormone metabolites, because they defecate frequently enough to establish a diel profile. However, concentration measurements of hormone metabolites rely on the assumptions that hormone metabolites are placed into droppings of similar mass (the "dropping amount assumption") and are excreted in constant time intervals (the "constant interval assumption"). These assumptions were clearly violated in our study, as the dropping mass per time interval and the defecation intervals varied depending on the time of day and season. We thus used the rate of hormone metabolite excretion as an alternative measure to concentration. Both testosterone and corticosterone metabolites showed diel and seasonal rhythmicity. Furthermore, the diel pattern varied depending on season. Concentration and rate measurements gave similar results when the differences between hormone metabolite levels were large-for example, when testosterone metabolites were compared across seasons. When the differences were more subtle, though, the 2 measures did not always correspond well, indicating that the violation of the 2 basic assumptions affected the comparability of concentration measurements. We conclude that diel and seasonal comparisons of hormone metabolites potentially give biologically meaningful results, especially when rates instead of concentrations are measured. However, such studies require awareness of the limitations and pitfalls of noninvasive hormone measurements, a carefully designed experiment, and very cautious interpretation of the data.     

Elevated plasma corticosterone decreases yolk testosterone and progesterone in chickens: linking maternal stress and hormone-mediated maternal effects

Despite considerable research on hormone-mediated maternal effects in birds, the underlying physiology remains poorly understood. This study investigated a potential regulation mechanism for differential accumulation of gonadal hormones in bird eggs. Across vertebrates, glucocorticoids can suppress reproduction by downregulating gonadal hormones. Using the chicken as a model species, we therefore tested whether elevated levels of plasma corticosterone in female birds influence the production of gonadal steroids by the ovarian follicles and thus the amount of reproductive hormones in the egg yolk. Adult laying hens of two different strains (ISA brown and white Leghorn) were implanted subcutaneously with corticosterone pellets that elevated plasma corticosterone concentrations over a period of nine days. Steroid hormones were subsequently quantified in plasma and yolk. Corticosterone-implanted hens of both strains had lower plasma progesterone and testosterone levels and their yolks contained less progesterone and testosterone. The treatment also reduced egg and yolk mass. Plasma estrogen concentrations decreased in white Leghorns only whereas in both strains yolk estrogens were unaffected. Our results demonstrate for the first time that maternal plasma corticosterone levels influence reproductive hormone concentrations in the yolk. Maternal corticosterone could therefore mediate environmentally induced changes in yolk gonadal hormone concentrations. In addition, stressful situations experienced by the bird mother might affect the offspring via reduced amounts of reproductive hormones present in the egg as well as available nutrients for the embryo.     

鸟粪对同里湿地公园土壤重金属及其形态的影响

为了解鸟粪对同里湿地公园土壤重金属全量及形态转化的影响,测定了公园内有鸟粪土壤、无鸟粪土壤、鸟粪沉积物的理化性质,Co、Cr、Cu、Ni、Zn全量及其形态分布,并进行统计分析。结果显示:同里湿地公园土壤及沉积物p H值平均含量达4.5,N、C、H、S和TP平均含量分别达到3.69、38.07、10.97、0.52、1.43g/kg,有鸟粪土壤中N、C、H和S含量显著高于无鸟粪土壤。Cu、Zn、Co平均含量呈现出鸟粪沉积物有鸟粪土壤无鸟粪土壤;Cr的平均含量表现为鸟粪沉积物无鸟粪土壤有鸟粪土壤;Ni呈现出有鸟粪土壤鸟粪沉积物无鸟粪土壤。总体上,鸟粪的进入增加了土壤Cu、Zn、Co、Ni含量。因此应及时清理土壤上覆鸟粪,降低对当地重金属污染风险。相对于无鸟粪土壤中不同重金属形态的分布,有鸟粪土壤中Co、Cr、Cu、Zn的活性态占全量的百分比均略有下降,非活性态呈上升趋势。鸟粪的加入,虽然会在一定程度上降低活性态重金属占总量的比例,但因总量和活性态含量均上升,向植物系统的迁移量会呈现增加趋势,因此对鸟粪施肥再利用应慎重。     

HPLC separation of corticosterone metabolites and its use in following corticosterone metabolism and transport in the isolated rat hepatocyte

Incubating isolated rat hepatocytes with tritium labelled and unlabelled corticosterone at 37 degrees C resulted in the rapid appearance of at least nine corticosterone metabolites. A quick, relatively easy, and quantitative high performance liquid chromatographic (HPLC) method was used to separate these metabolites and follow their rate of appearance both intra- and extracellularly. We found different intra- and extracellular amounts of each metabolite at a particular time and this suggested that some metabolites were more available for transport than others.     

Fecal Glucocorticoid Metabolites as Biomarkers in Equids: Assay Choice Matters

Free ranging animals are exposed to environmental, demographic, and ecological challenges over time, which can affect their health and fitness. Non-invasive biomarkers can provide insight into how animals cope with these challenges and assess the effectiveness of conservation management strategies. We evaluated how free ranging ponies (Equus ferus caballus) on the Carneddau Mountain range, North Wales respond to 2 stimuli: an acute stressor of an annual roundup event in November 2014, and spatial and temporal variation in ecological factors in 2018. We evaluated fecal glucocorticoid metabolites using 2 enzyme immunoassays (EIAs): an 11-oxoetiocholanolone EIA (measuring 11,17-dioxoandrostanes [11,17-DOAs]) and a corticosterone EIA. The former assay has been validated in equids, whereas there is limited evidence for the suitability of the latter. We used an additional parent testosterone EIA to measure fecal androgen metabolites in response to the ecological challenges. Following the roundup, the metabolite concentrations measured by the 2 glucocorticoid EIAs were not correlated. The 11,17-DOAs were elevated from the second day following the roundup and then slowly returned to pre-round levels over the next 2 weeks. In contrast, the metabolites measured by the corticosterone assay showed no response to the roundup. For the ecological data, all 3 assays detected a positive correlation between metabolites and social group size in males but not in females. The metabolite concentrations measured by the testosterone and corticosterone assays were highly correlated and were temporally associated with the onset of the breeding season, whereas the 11,17-DOAs were not. The co-variance of metabolites measured by the corticosterone and testosterone assays, and the lack of an acute response in the corticosterone assay to the roundup, suggests that metabolites detected by the corticosterone assay were not primarily associated with increased glucocorticoid production. We recommend using well-validated fecal biomarker assays of hypothalamus-pituitary-adrenal axis activity to evaluate and compare the effect of different management interventions and environmental change. © 2021 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society.     

Seeds in droppings of tropical fruit-eating birds: importance of considering seed composition

Summary The probability of interspecific seedling competition from bird droppings is high because fruit-eating birds, on average, deposit more than two species in a single dropping. Moreover, birds vary both in the number of plant species they deposit in a given dropping and in the seed composition of those droppings. In a preliminary experiment, I examined effects of interspecific seedling competition from seeds found in bird droppings. Certain plant species were competitively superior in pairwise growth experiments using six common second-growth species. The survival of certain shrubs depended on which species it was disseminated with in bird droppings. Rapid germination time may promote competitive superiority in some cases. Birds affect plant fitness not only because of their behavior following dispersal, but also because they deposit seeds in different densities and combinations.     

Urinary hormone analysis assists reproductive monitoring and sex identification of bell frogs (Litoria raniformis)

With the world currently facing a global amphibian extinction crisis, the development of techniques to help meet the needs of conservation managers and researchers studying the reproductive biology of amphibians is needed. Here, we developed enzyme immunoassays to measure estrone, testosterone, and progesterone hormone metabolites in the urine of Litoria raniformis, the southern bell frog. Concentrations of urinary estrone, testosterone, and progesterone increased during the breeding season for females (P < 0.05). Concentrations of urinary testosterone and progesterone increased for males during the breeding season compared with that for months where no reproductive behaviors were observed (P < 0.05). Furthermore, urinary estrone concentrations proved to be a reliable sexing tool for adult frogs, with no overlap between the sexes in 98% of cases, regardless of season. There was no difference in estrone (P = 0.204) or testosterone (P = 0.485) metabolite concentrations between samples taken immediately upon capture and those taken 12 to 24 h later from the same individual. Progesterone metabolite concentrations were lower on Day 2 than upon collection (P = 0.004). This is the first study to show that urinary hormone analysis can be a useful technique for reproductive monitoring in an amphibian. Additionally, hormone metabolite measures offer promise as sex identification tools for monomorphic species and for those whose secondary sex characteristics are visible only during the breeding season.     

Temperature effects on serum prolactin concentrations and activity of dopaminergic neurons in the infundibulum/pituitary stalk of calves

The effects of ambient temperature on serum concentrations of prolactin and neurochemical estimates of activity of dopaminergic neurons projecting to the infundibulum/pituitary stalk were investigated in Holstein bull calves. Accumulation of 3,4-dihydroxyphenylalanine (DOPA) in the infundibulum/pituitary stalk after intravenous injection of a decarboxylase inhibitor was used to estimate activity of these dopaminergic neurons. Increasing ambient temperature from 21 to 33 degrees C for 22 hr increased serum concentrations of prolactin and decreased activity of the dopaminergic neurons. Conversely, reducing ambient temperature from 22 degrees C to 11 degrees C decreased serum concentrations of prolactin and increased activity of these dopaminergic neurons. It is suggested that alterations in activity of dopaminergic neurons terminating in the infundibulum/pituitary stalk of bull calves may mediate acute temperature-induced changes in secretion of prolactin.     

Temperature stress can alter the photosynthetic efficiency and secondary metabolite concentrations in St. John's wort.

Temperature stress is known to cause many physiological, biochemical and molecular changes in plant metabolism and possibly alter the secondary metabolite production in plants. The hypothesis of the current study was that temperature stress can increase the secondary metabolite concentrations in St. John's wort. Plants were grown under controlled environments with artificial light using cool white fluorescent lamps and CO2 enrichment and 70-day-old plants were subjected for 15 days to different temperature treatments of 15, 20, 25, 30 and 35 degrees C before harvested. Major aim of the study was to increase the major secondary metabolites in St. John's wort by applying temperature stress and to evaluate the physiological status of the plant especially the photosynthetic efficiency and peroxidase activity of the leaf tissues exposed to different temperatures under precisely controlled environmental factors. Results revealed that relatively high (35 degrees C) or low (15 degrees C) temperatures reduced the photosynthetic efficiency of the leaves of St. John's wort plants and resulted in low CO2 assimilation. Net photosynthetic rates and the maximal quantum efficiency of PSII photochemistry of the dark adopted leaves (phi(p)max) decreased significantly in the leaves of plants grown under 35 or 15 degrees C temperature treatments. High temperature (35 degrees C) treatment increased the leaf total peroxidase activity and also increased the hypericin, pseudohypericin and hyperforin concentrations in the shoot tissues. These results provide the first indication that temperature is an important environmental factor to optimize the secondary metabolite production in St. John's wort and controlled environment technology can allow the precise application of such specific stresses.     

Anuran gender identification by fecal steroid analysis

This study tested the hypothesis that steroid hormone metabolites can be measured in anuran feces and their concentrations used to identify the sex of adults. Fecal samples from American toads, Bufo americanus, and boreal toads, B. boreas boreas, were extracted using ethyl acetate, and the concentrations of estradiol, progesterone and testosterone metabolites were measured by enzyme immunoassays with antibodies commonly used to evaluate steroid hormone concentrations in mammalian species. In American toads, mean testosterone metabolite concentrations (P<0.05) between males (224.3±15.5 ng/g feces) and females (80.7±10.6 ng/g), but estradiol and progesterone metabolite concentrations did not. In contrast, estradiol immunoreactivity differed (P<0.05) between male (19.0±1.8 ng/g) and female (48.3±6.3 ng/g) boreal toads. Progesterone and testosterone metabolite concentrations did not differ. Fecal hormone metabolite analysis offers a promising noninvasive approach to gender identification in anuran amphibians. However, the group of metabolites differentiating gender may not be consistent among species. Zoo Biol 0:1–12, 2005. © 2005 Wiley‐Liss, Inc.     

Testosterone and corticosterone during the breeding cycle of equatorial and European stonechats (Saxicola torquata axillaris and S. t. rubicola)

Northern-temperate male birds show seasonal changes in testosterone concentrations with a peak during the breeding season. Many tropical birds express much lower concentrations of testosterone with slight elevations during breeding. Here we describe testosterone and corticosterone concentrations of male stonechats from equatorial Kenya during different substages of breeding and molt. This tropical species has a short breeding season of approximately 3 months. We compare their hormone concentrations to previously published data of males of a northern-temperate relative, the European stonechat, also a seasonal breeder but with a breeding season of approximately 5 months. Equatorial stonechats show a pronounced peak of testosterone during the nest-building and laying stage. During all other stages, testosterone concentrations are low, similar to other year-round territorial tropical bird species. Corticosterone concentrations peak also during the nest-building and laying stage suggesting that this period of maximum female fecundity is a demanding period for the male. Equatorial stonechats have significantly lower concentrations of testosterone than European stonechats during all stages, except during the nest-building and laying stage. During this stage of maximum female fertility, testosterone levels tend to be higher in equatorial than in European stonechats. Our results suggest that equatorial stonechats belong to a group of tropical bird species that are characterized by a short breeding season and a brief high peak of testosterone during the female's fertile period. Such brief, but substantial peaks of testosterone may be common in tropical birds, but they may easily be missed if the exact breeding stage of individual birds is not known.     

Comparison of phospholipase production in shape Cryptococcus neoformans isolates from AIDS patients and bird droppings

Secreted phospholipase has been recently proposed as a virulence determinant in Cryptococcus neoformans as well as Candida albicans. This issue of cryptococcal phospholipase requires screening of phospholipase production in a larger number of isolates from clinical and environmental sources. In this study we examined phospholipase production in a total of 67 C. neoformans isolates from AIDS patients and bird droppings by using the egg-yolk plate method. Phenoloxidase activity, capsule size and growth at 37 °C were also measured in these strains in order to observe a possible relationship between phospholipase production of different C. neoformans strains and its virulence. Four of the 21 AIDS strains at 28 °C and 1 at 37 °C did not produce phospholipase, respectively. In contrast, 38 and 34 of the 46 bird dropping strains were negative for phospholipase production at 28, and 37 °C, respectively. Statistical analysis revealed a significant difference in phospholipase production, capsule size and growth ability at 37 °C, but not phenoloxidase activity, between the AIDS and the bird dropping strains. The highly prevalent distribution of phospholipase activity in the AIDS strains suggests a role of the enzyme in invading the host. This revised version was published online in June 2006 with corrections to the Cover Date.     

31P-NMR-studies on intracellular pH and metabolite concentrations in relation to the circadian rhythm, temperature and nutrition in Neurospora crassa.

Perfused mycelia of Neurospora crassa were analyzed in vivo with 31P-NMR. Both the cytoplasmatic and the vacuolar pH and the concentrations of phosphate metabolites were followed up to 30 h under constant conditions. No circadian changes were detected. However, slight changes in the nutrition or oxygen supply induced distinct changes in the intracellular pH and in the concentrations of metabolites. An increase of temperature from 21 to 43 degrees C lowered the intracellular pH and the metabolite concentrations. Changes in the perfusion rate affect the temperature responses, probably due to different availability of carbon sources and exhaustion of acid catabolites.     

Cool-night temperature induces spike emergence and affects photosynthetic efficiency and metabolizable carbohydrate and organic acid pools in <Emphasis Type="Italic">Phalaenopsis aphrodite</Emphasis>

After being acclimated to constant warm (28 degrees C day/28 degrees C night) and cool-night temperature (28 degrees C day/20 degrees C night) regimes in growth chambers for 2 weeks, the two groups of mature Phalaenopsis aphrodite subsp. formosana plants both clearly exhibited a diurnal oscillation of stomatal conductance, net CO(2) uptake rate, malate and starch levels, and the phosphoenolpyruvate carboxylase (EC 4.1.1.31) and NAD(+)-malic enzyme (EC 1.1.1.39) activities. Hence, P. aphrodite is an obligate crassulacean acid metabolism plant. Nevertheless, different night temperature greatly affected both the stomatal conductance and the contribution of ambient and respiratory CO(2) to the nocturnal accumulation of malate. However, the amounts of nocturnal accumulated malate and daily deposited starch appeared to have no significant difference between the two groups. These results demonstrate that P. ahrodite is congruent with the characteristics of CAM plants having great flexibility and plasticity in response to changes in environmental conditions. In addition, the formation of reproductive stem, viz. spike, was noticeably inhibited by a constant warm temperature, but induced by a fluctuating warm day and cool night condition. The relationship between the metabolic pool variation and spike induction of Phalaenopsis is also discussed.     

Penicillium persicinum, a new griseofulvin, chrysogine and roquefortine C producing species from Qinghai province, China

A unique Penicillium isolate from Chinese soil with terverticillate penicilli and ellipsoidal to cylindrical smooth-walled conidia, produces, in addition to the common metabolite ergosterol, copious amounts of an unknown peach-red pigment and the following secondary metabolites: griseofulvin, dechlorogriseofulvin, lichexanthone, roquefortine C, roquefortine D, chrysogine, 2-pyrovoylaminobenzamide, 2-acetyl-quinazolin-4(3H)-one. This isolate, CBS 111235, is described as Penicillium persicinum sp. nov., which belongs to subgenus Penicillium section Chrysogena but is morphologically similar to P. italicum. On the basis of the production of secondary metabolites it resembles P. griseofulvum and P. coprophilum. Sequence data using part of the beta-tubulin gene showed that it is phylogenetically related to P. chrysogenum and P. aethiopicum in section Chrysogena with which it shares both secondary metabolites and ability to grow at 37 degrees C.     

Biological Validations of Fecal Glucocorticoid,Testosterone, and Progesterone Metabolite Measurements in Captive Stumptail Macaques (<Emphasis Type="Italic">Macaca arctoides</Emphasis>)

Measuring hormone metabolites in fecal samples allows the noninvasive assessment of some steroid hormones in primates. However, noninvasive hormone assays need analytical and biological validation owing to variation in hormone metabolism and excretion between the sexes and across species. We aimed to validate the measurement of fecal glucocorticoid (fGC), testosterone (fT), and progesterone (fP) metabolites in 15 captive stumptail macaques (Macaca arctoides). We collected fecal samples before and after we induced a stress response by restraining and injecting the subjects with saline solution. We then measured hormone metabolites using a methanol extraction technique and ¹²⁵I radioimmunoassay kits. We analyzed the change in glucocorticoid production before and after the stressor, as well as sexual and social rank differences. For fT metabolite levels we investigated variation with sex, age, and social rank, and for fP metabolite levels, we tested for sexual and cycle phase differences. We found a significant increase in fGC metabolite levels 22–25 h poststressor in both sexes. The increase was greater in high-ranking than in low-ranking individuals. Levels of fT metabolites were higher in males than in females, correlated positively with rank only in males, and correlated negatively with age in both sexes. fP metabolite levels were higher in females than in males, and were higher during the luteal phase than in the follicular phase. These findings indicate that our assays reliably detected hormonal changes related to stress (fGC) and detected differences between social and sexual categories (fT, fP) in stumptail macaques.     

Stability of Avian Fecal Corticosterone Metabolite Levels in Frozen Avian Feces

ABSTRACT Fecal corticosterone metabolites are commonly used in avian ecology as a measure of response to stress. Recent research on mammals suggested that the manner in which samples are stored could be critical to alleviating any storage handling bias. Cross-reacting metabolites can increase glucocorticoid metabolites even after samples are frozen and, thus, result in an overestimation of hormone levels as the time increases between when samples were collected and when levels are measured. We examined effects of sample storage time on fecal corticosterone metabolites for 2 avian species across 165 days. We observed no change in fecal corticosterone metabolites across the sampling periods in either fulvous whistling-ducks (Dendrocygna bicolor) or white ibis (Eudocimus albus). Results suggest that avian fecal corticosterone metabolite levels do not change when samples are frozen for long periods of time and that there were no differences in the response between the 2 species we compared. This study demonstrated that avian fecal corticosterone samples are accurate even after freezing and, thus, studies that seek to address conservation questions may rely on these data. Studies of additional bird species are needed to generalize our findings to other avian taxa.     

The isolation and characterization of virulence factors of Cryptococcus spp. from saprophytic sources in the city of Ribeirão Preto, São Paulo, Brazil

Yeasts of the Cryptococcus genus are distributed in nature associated to animal and vegetal organic residues. Occasionally, species other than C. neoformans may be responsible for infectious diseases in human and animals. This study aims to determine the occurrence of Cryptococcus species in the atmosphere and bird droppings in the city of Ribeir?o Preto, S?o Paulo, Brazil, and to evaluate three virulence factors: capsule formation, growth at 37 degrees C and melanin production. We analyzed 86 environmental samples (54 droppings and 32 air). Of the 41 strains isolated, 15 were C. neoformans var. neoformans (12 droppings and 3 air), 15 C. albidus (12 droppings and 3 air), 9 C. laurentii (7 droppings and 2 air) and 2 C. uniguttulatus (from droppings). Capsules were produced by 93.3% of C. neoformans var. neoformans, 66.7% of C. albidus, 88.9% of C. laurentii and 50% (1/2) of C. uniguttulatus. All strains of C. neoformans, 20% of C. albidus and 44.4% of C. laurentii were able to grow at 37 degrees C. The melanin production on DOPA agar was verified in C. neoformans (93.3%), C. albidus (26.7%) and C. laurentii (66.7%). We concluded that different Cryptococcus species coexist in the same ecological niche and they are able to produce virulence factors.     

Social Status and Resistance to Disease in House Mice (Mus musculus): Status-related Modulation of Hormonal Responses in Relation to Immunity Costs in Different Social and Physical Environments

Unrelated and previously unfamiliar male mice were maintained in groups of six tor different periods and at different ambient temperatures. High- and low-ranking males differed in hormone-related resistance to a subsequent experimental infection with the blood protozoan Babesia microti and there were rank differences in the apparent modulation of immunodepressive hormones relative to an individual's internal, social and physical environment. Resistance to B. microti was influenced by serum concentrations of corticosterone and testosterone. Associations between aggressiveness and measures of immunocompetence and resistance were related to corticosterone concentration, but only among low rankers. Effects of testosterone on resistance were confined to high rankers but were not associated with aggressiveness, although high rankers maintained greater aggressiveness than low rankers in all experimental treatments. The effects of group duration and temperature on resistance can be interpreted in terms of changing hormone and antibody levels in relation to metabolic stress. The results emphasize the importance of interactions between underlying physiological changes in relationships between behaviour, immune function and disease.     

Biosynthesis and metabolism of testosterone by sertoli cell-enriched seminiferous tubules

Sertoli cell-enriched tubules isolated from rats which had been treated with 1,4-dimethyl sulfonyloxybutane were incubated with either [¹⁴C] progesterone or [¹⁴C] testosterone for 2 hours. Tubules of normal rats and fragments of Sertoli cell-enriched testes were incubated under the same conditions. Sertoli cell-enriched tubules converted progesterone to 20α-dihydroprogesterone, 17α-hydroxyprogesterone, androstenedione and testosterone. The major metabolite was 20α-dihydroprogesterone. The percentage conversion of progesterone into testosterone corresponded to a production of 10–20 ng testosterone. Sertoli cell-enriched tubules converted testosterone to dihydrotestosterone, androstenedione, 3α-androstanediol and 3β-androstanediol. Under our experimental conditions, dihydrotestosterone was the major 5α-reduced metabolite. Normal tubules converted progesterone and testosterone to the same metabolites as Sertoli cell-enriched tubules. Fragments of Sertoli cell-enriched testes were much more active than isolated tubules in metabolizing progesterone. They produced the same amounts of 5α-reduced metabolites of testosterone.