Steroid metabolism and validation of noninvasive endocrine monitoring in the African wild dog (Lycaon pictus)

The purpose of this study was to validate noninvasive endocrine monitoring techniques for African wild dogs (Lycaon pictus) and to establish physiological validity of these methods by evaluating longitudinal reproductive-endocrine profiles in captive individuals. To determine the primary excretory by-products of ovarian steroid metabolism, [¹⁴C]-progesterone and [³H]-estradiol were co-administered to a female and all excreta were collected for 80 hr postinjection. Radiolabel excretion peaked ≤ 18 hr postinfusion, and progesterone and estradiol metabolites were excreted in almost equivalent proportions in urine (39.7 and 41.1%, respectively) and feces (60.3 and 58.9%, respectively). Most of the urinary metabolites were conjugated (estradiol, 94.3 ± 0.3%; progesterone, 90.4 ± 0.5%), so that immunoassays for pregnanediol-3α-glucuronide (PdG) and estrogen conjugates (EC) were effective for assessing steroid metabolites. Two immunoreactive estrogens (estradiol and estrone) and at least one immunoreactive progesterone metabolite (3α-hydroxy-5α, pregnan-20-one) were detected in feces. Urine and fecal samples were collected (1–3 times per week) for 1.5 yr from one adult female and two adult males to assess longitudinal steroid metabolite excretion. Overall correlation of urinary PdG to matched, same-day fecal progesterone metabolites immunoreactivity was 0.38 (n = 71, P < 0.05). Similarly, urinary EC was correlated (P < 0.05) with same-day fecal estrogen immunoreactivity (r = 0.49, n = 71). During pregnancy and nonpregnant cycles, copulation occurred at the time of peak (or declining) estrogen metabolites and increasing progesterone metabolites concentrations. Estrus duration was 6–9 days and gestation lasted 69 days with parturition occurring coincident with a drop in progesterone metabolites. Males exhibited seasonal trends in fecal testosterone excretion with maximal concentrations from July to September coincident with peak mating activity. Although these limited longitudinal hormone profiles should be interpreted cautiously, noninvasive gonadal steroid monitoring suggests that: (1) both female and male wild dogs may exhibit reproductive seasonality in North America, (2) females are monoestrous, and (3) peak testicular activity occurs between August and October coincident with mating behavior. From a conservation perspective, noninvasive endocrine monitoring techniques should be useful for augmenting captive breeding programs, as well as for developing an improved understanding of the physiological mechanisms underlying reproductive suppression in response to social and ecological pressures. Zoo Biol 16:533–548, 1997. © 1997 Wiley-Liss, Inc.     

Serum and fecal steroid analysis of ovulation,pregnancy, and parturition in the black rhinoceros (Diceros bicornis)

Studies were conducted to determine: (1) if fecal hormone metabolite concentrations correlated with serum estrogen and progesterone concentrations, follicular activity and reproductive behavior in the black rhinoceros (Diceros bicornis) and (2) if threshold values of respective fecal metabolite concentrations correlated with pregnancy. Blood and fecal samples were collected, in conjunction with transrectal ultrasound and behavior observations, for an 18-month period from one black rhinoceros female. Subsequently, serial fecal samples were collected from 13 females in 10 zoos. Quantitative analysis of serum progesterone (P₄) and estradiol (E₂) was performed by radioimmunoassay (RIA): analysis of fecal estrogen metabolites (E) and fecal progesterone metabolites (P) were performed by enzyme immunoassay (EIA). Serum P₂ concentrations identified two luteal phase patterns and two nadirs which corresponded with behavioral estrus. Fecal E patterns indicated a sharp peak which corresponded with breeding. concentrations of fecal P illustrated identifiable nadirs and several peaks which corresponded to serum P₄ nadirs and luteal phases. Serum P₄ concentrations were not different between the luteal phase and pregnancy. Fecal P concentrations started to rise above luteal phase concentrations approximately 150 days postbreeding and remained elevated until immediately before parturition. Serum E₂ and fecal E concentrations rose and subsequently declined after parturition. In the fecal samples from seven pregnant females, fecal P concentrations were similarly elevated compared to six nonpregnant females. Results indicated that fecal steroid metabolites accurately reflected serum steroid hormone concentrations and that the measurement of P and E concentrations permitted the characterization of the estrous cycle, the diagnosis of pregnancy, and the onset of parturition. Zoo Biol 16:121–132, 1997. © 1997 Wiley-Liss, Inc.     

Non-invasive monitoring of ovarian function in several felid species by measurement of fecal estradiol-17β and progestins

An extraction and assay procedure to measure fecal estradiol-17β and progestin concentrations in several cat species was developed and validated for use for noninvasive monitoring of ovarian function. Fecal samples were collected over a range of 3–20 months from female tigers (three), lions (three), snow leopards (three), cheetahs (two), caracals (two), and domestic cats (five). Samples were extracted with 90% methanol, lipids removed with petroleum ether, and the estradiol and progestins in the methanol measured by radioimmunoassay (RIA). High Performance Liquid Chromatography (HPLC) fractionation and subsequent RIA of the fractions indicated that the estradiol-17β antiserum cross-reacted primarily with estradiol-17β in the feces of lions and tigers and was assumed to be specific for estradiol-17β in the feces of other species as well. However, there were several immunoreactive compounds, presumably progesterone metabolites, excreted in the feces which varied both quantitatively and qualitatively among species. The behavior of tigers, lions, cheetahs, and caracals was visually monitored during the collection period and frequency of sexual behaviors was positively correlated with increases in fecal estradiol in all species observed. The mean fecal estradiol-17β peaks were as follows: tigers, 128.0 ± 13.1; lions, 186.0 ± 14.8; snow leopards, 136.7 ± 15.9; cheetahs, 140.9 ± 9.0; caracals, 24.5 ± 4.0; and domestic cats 158.9 ± 19.3 ng/gm. Fecal progestin concentrations rose significantly (P < 0.001) only after breeding or during pregnancy and were as follows: tigers, 5.6 ± 0.6; lions, 1.9 ± 0.1; cheetahs, 8.4 ± 1.1; and caracals, 2.4 ± 0.4 μg/gm. Fecal progestins were elevated for one-half to two-thirds of the gestation length during presumed pseudopregnancy but remained elevated throughout successful pregnancies. These results suggest that ovarian function can be monitored noninvasively in the family Felidae by the measurement of fecal estradiol-17β and progestin concentrations. © 1995 Wiley-Liss, Inc.     

Comparison of different drying and storage methods on quantifiable concentrations of fecal steroids in the cheetah

Fecal steroid analysis is a powerful tool that can provide important information on the health, physiology, and reproductive status of nondomestic species. However, studying free‐ranging animals requires that feces be stored and transported from the collection site to the laboratory in a manner that prevents degradation or alteration of steroid metabolites. To determine the effects of different handling and storage methods on fecal steroids, 30 fresh fecal samples from five captive cheetahs were collected, thoroughly mixed, separated into aliquots, and processed (stored or dried) under different conditions. Concentrations of gonadal and adrenal steroid hormones were analyzed in feces stored frozen at –20°C or at room temperature in 95% ethanol. Both frozen and ethanol‐stored aliquots were desiccated using a lyophilizer, solar oven, or conventional oven. The steroid values from aliquots stored and desiccated using the different methods were compared to those obtained using the optimal storage method of freezing at –20°C and desiccating in a lyophilizer (control). Concentrations of corticoid, estrogen, progestagen, and androgen metabolites in fecal extracts were quantified by radioimmunoassay. Androgen metabolite concentrations were not significantly affected (P > 0.05) by storage or drying methods. Fecal samples stored at room temperature in ethanol and lyophilized also had steroid concentrations that did not differ (P > 0.05) from controls. However, the concentrations of corticoid and estrogen metabolites were significantly lower (P < 0.05), and progestagen metabolites were significantly higher (P < 0.05) in samples desiccated in solar and conventional ovens without regard to storage method. These results suggest that storage of fecal samples at room temperature in ethanol is the best alternative to freezing for subsequent analysis of steroid hormone concentrations. Differences in measured concentrations of hormones in oven‐desiccated samples could be due to hormone degradation or shifts in the immunodominant metabolite. Therefore, validation of storage and processing techniques should be included in the development of any new fecal steroid analysis methodology. Zoo Biol 21:215–222, 2002. © 2002 Wiley‐Liss, Inc.     

Excretion and metabolic fate of radiolabeled estradiol and testosterone in the cockatiel (Nymphicus hollandicus)

The metabolism and time courses for clearance of radiolabeled estradiol and testosterone were studied in the female cockatiel (Nymphicus hollandicus) using a simple technique of solubilizing dried fecal/urine matter in an aqueous solution. Carbon ¹⁴ radiolabeled estradiol and testosterone were injected intramuscularly and the urine and fecal matter collected for the pursuant 168 hr. The predominant radiolabel peak was found associated with the aqueous residue of the ether extracted aliquot for both hormones. High-performance liquid chromatographic (HPLC) separation of solubilized fecal/urine material collected during the first sampling interval (0–4 hr after injection) demonstrated that the majority of the excreted radiolabel was in the form of conjugated steroid metabolites in both the estradiol and testosterone injected birds. Subsequent hydrolysis of the aqueous residue of the ether extracted aliquots and HPLC characterized the estrogen and testosterone metabolites as being estrone/estradiol and a variety of androgen based moieties, respectively. By 24 hr postinjection, 79.4 and 79.1% of the original radiolabel was recovered in birds injected with estradiol and testosterone, respectively. These findings demonstrate that steroid hormone excretion in the cockatiel is a rapid and efficient process that is 79% complete by 24 hr and that the primary excretion products are conjugated metabolites. This study also supports the concept that fecal/urine collection is a practical and efficient method of monitoring sex steroid excretion and provides additional evidence that simple solubilization of fecal matter is a sufficient and efficient method for processing feces for subsequent metabolite measurements. Zoo Biol 16:505–518, 1997. © 1997 Wiley-Liss, Inc.     

Endocrine profiles during the estrous cycle and pregnancy in the Baird's tapir (Tapirus bairdii)

Serum samples were collected 1–3 times weekly from two Baird's tapirs (Tapirus bairdii) for 6 months in 1987–1988, and for more than 3 consecutive years beginning in 1989 to characterize hormone patterns during the estrous cycle and pregnancy. Based on serum progesterone concentrations, mean (±SEM) duration of the estrous cycle (n = 20) was 30.8 ± 2.6 days (range, 25–38 days) with a luteal phase length of 18.1 ± 0.4 days (range, 15–20 days). Mean peak serum progesterone concentrations during the luteal phase were 1.35 ± 0.16 ng/ml, and nadir concentrations were 0.19 ± 0.03 ng/ml during the interluteal period. Distinct surges of estradiol preceded luteal phase progesterone increases in most (14/20) cycles. Gestation length was 392 ± 4 days for three complete pregnancies. Mean serum progesterone concentrations increased throughout gestation and were 1.83 ± 0.13, 2.73 ± 0.13, and 4.30 ± 0.16 ng/ml during early, mid- and late gestation, respectively. Serum estradiol concentrations began to rise during mid-gestation, increasing dramatically during the last week of pregnancy. Patterns of serum estriol and estrone secretion during pregnancy were similar to that observed for estradiol. In contrast to progesterone and estrogens, serum cortisol concentrations were unchanged during pregnancy or parturition. Females resumed cycling 16.2 ± 2.0 days after parturition (n = 4) and, on two occasions, females became pregnant during the first postpartum estrus. These data suggest that the tapir cycles at approximately monthly intervals and that increases in serum progesterone are indicative of luteal activity. The interluteal period is relatively long, comprising approximately 40% of the estrous cycle. During gestation, progesterone concentrations are increased above luteal phase levels, and there is evidence of increased estrogen production during late gestation. The absence of increased cortisol secretion at the end of gestation suggests that this steroid does not play a major role in initiating parturition in this species. © 1994 Wiley-Liss, Inc.     

Use of group-specific antibodies to detect fecal progesterone metabolites during the estrous cycle of cows

Progesterone is metabolized to pregnanediones and hydroxylated pregnanes prior to its fecal excretion. Therefore, use of progesterone antibodies underestimates the actual amount of fecal metabolites. To improve the methodology of noninvasive fecal progesterone metabolite analysis, enzymeimmunoassays (EIA) using group-specific antibodies against 5-reduced 20-oxo-pregnane-C3-conjugates were developed. Fecal and milk samples were collected at 1- to 2-d intervals during the morning and evening milking throughout 1 estrous cycle in dairy cows (n = 12). Six immunoreactive metabolites were detected in the feces with high performance liquid chromatography (HPLC), eluting as 5α- and 5β-reduced pregnanes containing a 20-oxo-group (20-oxo-pregnanes). Fecal samples of 3 cows were analyzed by 3 EIAs using antibodies against 4-pregnene-6α-ol-3,20-dione 6HS:BSA (6HS-progesterone), 5α-pregnane-3β-ol-20-one 3HS:BSA and 5β-pregnane-3β-ol-20-one 3HS:BSA, respectively. The follicular and luteal phases were identifiable with each EIA. Luteal phase values and the differences between mean follicular (Days 0 to 2 and 19 to 21) and luteal phase (Days 10 to 16) values obtained with the 5-pregnane EIAs were 3- to 4-fold higher than with the 6HS-progesterone EIA. Since results with the former 2 EIAs were almost identical, the remaining samples were only analyzed by the EIA for 5β-pregnane-3α-ol-20-one. Fecal 20-oxo-pregnane concentrations were parallel to milk progesterone values, but had a lag time of about 0.5 d; the coefficient of correlation (P < 0.001) was 0.76 (y = 155.2 × + 37.2). Fecal 20-oxo-pregnane concentrations during the follicular and luteal phase were 39.5 ± 2.2 and 341 ± 15.2 ng/g feces, respectively. In conclusion, fecal 20-oxo-pregnanes are significantly correlated to milk progesterone concentrations. They consist of several metabolites and compared to a 6HS-progesterone antibody, their evaluation was improved using antibodies against 5-reduced pregnanes.     

Optimizing captive short-beaked echidna (Tachyglossus aculeatus) fecal sample identification and hormonal analysis

The objectives of this study were to develop a fecal marking protocol to distinguish male from female samples during the echidna breeding season and to determine if normalizing fecal progesterone metabolite data for inorganic content improves the detection of biologically relevant changes in metabolite concentrations. Over a period of 6 weeks, four echidnas were provided with green food coloring powder mixed into 20 g of their regular feed with the dose adjusted weekly by 0.05 g. The proportion of organic (feces) versus inorganic matter (sand) in the fecal samples of three echidnas was determined by combustion of organic matter. Hormonal data was then expressed as metabolite concentration per total dry mass (with sand) of extracted sample versus metabolite concentration per total mass of organic material (without sand). The optimal dose of food coloring powder was 0.30 g: this was excreted in the feces of all echidnas within 24 h of consumption with color present for two consecutive days. Correction for inorganic content (sand) did not significantly affect variability of fecal progesterone metabolite levels (mean CV ± SE with sand: 142.3 ± 13.3%; without sand: 127.0 ± 14.4%; W = 6, p = .2500), or the magnitude of change from basal to elevated fecal progesterone metabolite concentrations (mean ± SE with sand: 8.4 ± 1.7; without sand: 6.6 ± 0.5, W = 10, p = .1250). Furthermore, progesterone metabolite concentrations before and after correction for sand contamination correlated strongly (r = .92, p = < .001). These methods will facilitate future reproductive endocrinology studies of echidna and other myrmecophagous species.     

Correlation between serum and fecal concentrations of reproductive steroids throughout gestation in goats

Non-invasive techniques such as the measurement of fecal steroids are now widely used to monitor reproductive hormones in captive and free-ranging wild-life. These methods offer great advantages and deserve to be used in domestic animals. The aim of the present study was to determine the endocrine profile of dairy goats throughout pregnancy by the quantification of fecal progestins and estrogens and assess its correlation with serum concentrations. Blood and fecal samples were collected weekly from 11 adult, multiparous goats, from mating through pregnancy and 2 weeks post-partum. The extraction of estradiol and progesterone fecal metabolites was performed by dilution in ethanol. The radioimmunoassay (RIA) in solid phase was used to quantify serum 17beta-estradiol (estradiol) and progesterone, as well as their fecal metabolites. The mean concentrations of both fecal and serum estradiol started to increase between weeks 7 and 11, reached peak values near parturition and then decreased sharply (range: 19.8+/-5.8 ng/g of feces to 608.6+/-472.4 ng/g of feces and 0.007+/-0.005 ng/ml to 0.066+/-0.024 ng/ml). An increase in both fecal and blood progestagens occurred in the second week, mean concentrations remained greater until week 20, and then decreased in the last week of gestation and 2 weeks post-partum (range: 108.8+/-43.6 ng/g of feces to 3119.5+/-2076.9 ng/g of feces and 0.12+/-0.04 ng/ml to 13.10+/-4.29 ng/ml). The changes in blood and fecal hormone concentrations were analyzed and compared throughout gestation for each single goat, for each breed and for the whole group. Results indicated that matched values of serum and fecal hormone concentrations were correlated (r=0.79; p<0.001 for progesterone and r=0.84; p<0.001 for estradiol mean concentrations in the whole group). Regression analysis showed that logarithmic model allows significant prediction of serum from fecal concentrations with an R(2)=0.729 (y=0.013ln x-0.021) for estradiol and R(2)=0.788 (y=3.835ln x-18.543) for progesterone. Neither fecal nor serum concentrations were affected by the breed but a significant effect of the number of fetuses on progestin concentrations was found. Therefore, the profiles of progesterone and estradiol fecal metabolites reflect the serum concentrations of the same hormones in pregnant goats.     

Comparative analysis of gonadal and adrenal activity in the black and white rhinoceros in North America by noninvasive endocrine monitoring

Patterns of fecal reproductive steroid metabolites and adrenal corticoids were characterized for 12‐ to 24‐month periods in black (n = 10 male, 16 female) and white (n = 6 male, 13 female) rhinoceroses at 14 institutions. All black rhinoceros females exhibited at least some ovarian cyclicity on the basis of fecal progestogen analysis (range, 2–12 cycles/yr). However, cycles often were erratic, with many being shorter (<20 days; 18% of cycles) or longer (>32 days; 21%) than the average of 26.8 ± 0.5 days (n = 104 cycles). Five females exhibited periods of acyclicity of 2–10‐month duration that were unrelated to season. One complete and seven partial pregnancies were evaluated in the black rhinoceros. Fecal progestogens increased over luteal phase concentrations after 3 months of gestation. Females resumed cyclicity within 3 months postpartum, before calves were weaned (n = 5). Approximately half of white rhinoceros females (6 of 13) showed no evidence of ovarian cyclicity. Of the cycles observed, 5 were “short” (32.8 ± 1.2 days) and 24 were “long” (70.1 ± 1.6 days). Only two females cycled continuously throughout the study. One had both long (n = 9) and short (n = 2) cycles, whereas the other exhibited long cycles only (n = 5). Fecal estrogen excretion was variable, and profiles were not useful for characterizing follicular activity or diagnosing pregnancy in either species. Males of both species showed no evidence of seasonality on the basis of fecal androgen profiles. Androgen metabolite concentrations were higher (P < 0.05) in the black (27.6 ± 6.9 ng/g) than in the white (16.8 ± 3.1 ng/g) rhinoceros. An adrenocorticotropin hormone challenge in four black rhinoceros males demonstrated that the clearance rate of corticoid metabolites into feces was ～24 hours. Fecal corticoid concentrations did not differ between males and females, but overall means were higher in the black (41.8 ± 3.1 ng/g) than in the white (31.2 ± 1.7 ng/g) rhinoceros. In summary, fecal steroid analysis identified a number of differences in hormonal secretory dynamics between the black and white rhinoceros that may be related to differences in reproductive rates in captivity. Most black rhinoceros females exhibited some cyclic ovarian activity. In contrast, few white rhinoceroses demonstrated evidence of regular estrous cyclicity, and those females that were active had comparatively long cycles. Results also suggest that fecal corticoid concentrations reflect adrenal activity and may be species specific. Continued studies are needed to determine whether fecal corticoid measurements will be useful for understanding the cause of inconsistent gonadal activity in these two species. Because all but three (15.8%) of the white rhinoceroses evaluated in this study were less than 20 years of age compared to 73.1% (19 of 26) of the black rhinoceroses, the impact of age on reproductive and adrenal activity also needs to be evaluated further. Zoo Biol 20:463–486, 2001. © 2002 Wiley‐Liss, Inc.     

Differences in Fecal Androgen Patterns of Breeding and Nonbreeding Kori Bustards (Ardeotis kori)

To better understand breeding conditions to promote reproduction in captive kori bustards, fundamental endocrine studies measuring fecal androgen metabolites in male and female kori bustards were conducted. Feces collected weekly from males and females were analyzed for testosterone using enzyme‐linked immunoassay. Results from adult males (n = 5), adult females (n = 10), immature males (n = 10), and immature females (n = 10) revealed seasonally elevated testosterone concentrations in fertile, but not nonfertile adult males and females (P > 0.05). Adult females that were not maintained in a breeding group, or that did not produce eggs, did not demonstrate increases in testosterone compared to egg laying counterparts. In males, but not females, seasonal testosterone increases were accompanied by weight gain. Peaks in male fecal androgen metabolites ranged from 10‐ to 22‐fold higher than nonbreeding season (181.5 ± 19.1 vs. 17.0 ± 0.94 ng/g; P < 0.05). Mean breeding season values for adult males were 83.6 ± 6.1 ng/g vs. nonbreeding season values of 12.3 ± 0.73 ng/g (P < 0.05). In females, average breeding season testosterone concentrations were approximately 4‐fold higher than nonbreeding season (55.9 ± 6.0 vs. 14.5 ± 1.8 ng/g), with peaks 10‐ to 30‐fold higher. Results show that noninvasive fecal androgen metabolite analysis can provide a means of predicting fertility potential of male and female kori bustards and might be utilized to assess effects of modifying captive environments to promote reproduction in this species. Zoo Biol. 32:54‐62, 2013. © 2012 Wiley Periodicals, Inc.     

Hormonal and Behavioral Variation in Pied Tamarins Housed in Different Management Conditions

Pied tamarins are an endangered Amazonian primate that has limited breeding success in zoos. Unfortunately, little is known about their reproductive biology and adrenocortical activity. Objectives were: (1) determine if fecal hormones could be utilized to monitor gonadal and adrenocortical activity; (2) characterize male and female gonadal and adrenocortical hormones; and (3) determine if there were differences between adrenocortical activity and behavior in a nonbreeding, on‐exhibit (NB‐ON) pair compared to a breeding, off‐exhibit (B‐OFF) pair. Fecal samples were collected from four (two males; two females) individuals. Hormones were analyzed for fecal progesterone (FPM), androgen (FAM), and glucocorticoid (FGM) metabolites by enzyme immunoassay. Behavioral observations were conducted for 6 months. Data were collected on instantaneous behavior, location, and all occurrences of intraspecific behaviors. Fecal progesterone metabolites were validated by pregnancy (mean ± SE, pregnant: 28.47 ± 1.60 μg/g; nonpregnant: 8.63 ± 0.89 μg/g). Fecal androgen metabolites were higher (T = 31,971, P < 0.05) in the B‐OFF male (863.66 ± 46.30 μg/g) than the NB‐ON male (838.63 ± 60.70 μg/g). Fecal glucocorticoid metabolites were validated by response to veterinary procedure with elevated values (7.31 ± 1.48 μg/g) seven times the baseline (0.37 ± 0.04 μg/g) at 24‐hr postphysical. Females had higher baseline FGM than the males (P < 0.05). Baseline FGM were higher (P < 0.05) in the NB‐ON female (0.93 ± 0.03 μg/g) compared to the B‐OFF female (0.38 ± 0.02 μg/g). Similarly, the NB‐ON male's FGM baseline (0.71 ± 0.03 μg/g) were higher (P < 0.05) than the B‐OFF male (0.21 ± 0.01 μg/g). Behavioral data revealed stereotypical behaviors in the NB‐ON pair but no stereotypical behaviors in the B‐OFF pair. Fecal hormone monitoring and behavioral analysis may provide insight on the limited breeding success of pied tamarins in zoos. Zoo Biol. 32:299–306, 2013. © 2012 Wiley Periodicals, Inc.     

A physiological dose of estradiol with progesterone affects striatum biogenic amines

The acute effect of estradiol and progesterone on dopamine and serotonin metabolism in rat striatum was studied. One subcutaneous injection of 17 beta-estradiol (300 ng) and progesterone (150 micrograms) into intact male rats increased plasma levels of these steroids, while testosterone, corticosterone, and estrone remained unchanged. Dehydroepiandrosterone, androstane-3 beta, 17 beta-diol and dihydrotestosterone remained undetectably low. Prolactin decreased and androstane-3 alpha, 17 beta-diol, and 17-OH progesterone increased, but less than estradiol and progesterone. Peak levels of striatal dopamine, dihydroxyphenylacetic acid, and homovanillic acid were observed 15-45 min after steroid injection with a return to control values after 45-60 min, while serotonin and 5-hydroxyindoleacetic acid levels were slightly decreased. An injection of estradiol (70 ng) with progesterone (70 micrograms) to ovariectomized female rats left plasma prolactin levels unchanged, while striatum dopamine and serotonin as well as their metabolite concentrations peaked 15-60 min after steroid injection and returned to control values after 45-75 min. To allow for a better comparison of the action of these steroids, the effect of estradiol or progesterone alone and in combination on the brain of ovariectomized rats was compared in the same experiment. A similar increase in metabolites of dopamine levels was observed after these steroids alone or in combination, while dopamine levels were increased only after progesterone alone or in combination with estradiol. An injection of estradiol or progesterone to ovariectomized rats led to peak steroid concentrations at approximately the same time in the brain and plasma. In addition, plasma and brain steroid levels were significantly correlated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ovarian cycle approach by rectal temperature and fecal progesterone in a female killer whale,Orcinus orca

This study aimed to validate the measurements of body temperature and fecal progesterone concentrations as minimally invasive techniques for assessing ovarian cycle in a single sexually mature female killer whale. Rectal temperature data, fecal and blood samples were collected in the dorsal position using routine husbandry training on a voluntary basis. The correlations between rectal temperature and plasma progesterone concentration and between fecal and plasma progesterone concentrations were investigated. Fecal progesterone metabolites were identified by a combination of high‐performance liquid chromatography and enzyme immunoassay. Plasma progesterone concentrations (range: 0.2–18.6 ng/ml) and rectal temperature (range: 35.3–35.9°C) changed cyclically, and cycle lengths were an average (±SD) of 44.9±4.0 days (nine cycles) and 44.6±5.9 days (nine cycles), respectively. Rectal temperature positively correlated with the plasma progesterone concentrations (r=0.641, P<0.01). There was a visual trend for fecal progesterone profiles to be similar to circulating plasma progesterone profiles. Fecal immunoreactive progestagen analysis resulted in a marked immunoreactive peak of progesterone. The data from the single killer whale indicate that the measurement of rectal temperature is suitable for minimally invasive assessment of the estrous cycle and monitoring the fecal progesterone concentration is useful to assess ovarian luteal activity. Zoo Biol 30:285–295, 2011. © 2010 Wiley‐Liss, Inc.     

Reproductive endocrine patterns in captive female and male red wolves (Canis rufus) assessed by fecal and serum hormone analysis

Reproductive steroid profiles in female (n=13) and male (n=5) red wolves (Canis rufus) were characterized in fecal samples collected during the breeding season (December—May) and over a 1 year period, respectively. Blood samples from females (n=12) also were collected during the periovulatory period for luteinizing hormone (LH) and steroid analysis. High performance liquid chromatography (HPLC) of fecal extracts determined that estradiol and estrone constituted the major and minor forms, respectively, of fecal estrogen metabolites. Although native progesterone was present, pregnane metabolites predominated as the major forms of fecal progestins. HPLC analysis of fecal extracts from males revealed no native testosterone, but rather the predominance of more polar androgen metabolites. Based on hormone profiles and/or pup production, females were classified as pregnant (n=3), ovulatory‐nonpregnant (n=9), or acyclic (n=3). Longitudinal monitoring of females indicated no pregnancy‐specific differences in concentrations of either fecal progestagen or estrogen metabolites compared to ovulatory‐nonpregnant individuals; however, baseline progestagen concentrations were consistently elevated in acyclic females. There was good correspondence between serum and fecal steroid concentration during the periovulatory period. A rise in serum estrogens preceded the ovulatory LH surge which was then followed by a significant progesterone rise during the luteal phase. In males, changes in fecal androgen metabolite concentrations coincided with photoperiod fluctuations, increasing in late autumn and reaching peak concentrations during mid‐ to late winter just before the start of the breeding season. Collectively, these results serve as a database of ovarian and testicular endocrine events in this species, which can be utilized in population management and application of assisted reproductive technologies. Zoo Biol 21:321–335, 2002. © 2002 Wiley‐Liss, Inc.     

Low ambient temperature increases food intake and dropping production, leading to incorrect estimates of hormone metabolite concentrations in European stonechats

Non-invasive methods to measure steroid hormone metabolites in bird droppings or mammalian feces have become very popular. However, the accuracy of these measurements may be affected by many factors. Here, we use the stonechat (Saxicola torquata) as a passerine bird model to test whether differences in ambient temperature affect food intake and dropping production and whether these changes lead to measurement artefacts in hormone metabolite concentrations. In addition, we tested for diurnal patterns in hormone metabolites. We held European stonechats in climate chambers and subjected them to two different long-term ambient temperature regimes, +5 degrees C and +22 degrees C. As expected, food intake and dropping production was higher at +5 degrees C than at +22 degrees C. Plasma concentrations of corticosterone and testosterone did not differ between different ambient temperature regimes. However, corticosterone and testosterone metabolite concentrations (in ng/g) were significantly lower at +5 degrees C than at +22 degrees C. When we measured the rate of hormone metabolite excretion (in picogram per hour) instead of the concentration, there was no difference between treatment groups. Thus, the measurement of hormone metabolite concentrations can be flawed because, depending on the treatment, similar amounts of hormone metabolites can be excreted into very different amounts of droppings. In conclusion, hormone metabolite concentration measurements are sensitive to changes in ambient temperature and probably any other factor that alters metabolic rates. Any study involving systematic changes in metabolism--i.e., during molt, migration, hibernation, egg production, or seasonal comparisons--needs to take these caveats into account.     

Direct effect of the luteinizing hormone releasing hormone analog D-Trp6-Pro9-Net-LHRH on rat testicular steroidogenesis

The luteinizing hormone releasing hormone analog D-Trp⁶-Pro⁹-Net-LHRH (LHRH_a) inhibits rat testicular testosterone secretion. To determine whether LHRH_a decreases serum testosterone concentrations solely by inhibiting gonadotropin secretion or, in addition, by influencing directly testicular testosterone biosynthesis, we examined the effects of LHRH_a on the activities of 5 key testicular steroidogenic enzymes. Thirty hypophysectomized, hCG treated rats were given either LHRH_a (1 μg sc/day) or saline during 7 days. The LHRH_a treated animals exhibited a significant decrease of serum testosterone when compared to the control group (498 ± 37 ng/dl vs 2044 ± 105 ng/dl, mean ± SEM, P 〈0.001). 17-Hydroxyprogesterone serum levels were also decreased in the LHRH_a treated rats (61 ± 6 ng/dl vs 93 ± 7 ng/dl, P 〈0.005), while serum progesterone levels were similar in both groups of animals. These changes in steroid concentrations were associated with decreases in the musomal enzyme activities of 17-hydroxylase (37 ± 9 vs 654 ± 41 pmol/mg protein/min, P 〈0.001), 17, 20-desmolase (103 ± 9 vs 522 ± 47 pmol/mg protein/min, P 〈0.001), 3β-hydroxysteroid dehydrogenase (1.7 ± 0.02 vs 4.1 ± 0.1 nmol/mg protein/min, P 〈0.001), aromatase (95 ± 7 vs 228 ± 6 pmol/mg protein/ min, P 〈0.001) and 17-ketosteroid reductase (167 ± 9 vs 290 ± 18 pmol/mg protein/min, P 〈0.01) in the LHRH_a treated animals. These findings indicate that LHRH_a can inhibit directly rat testicular testosterone biosynthesis.     

Comparison of three protocols for superovulation of brown brocket deer (Mazama gouazoubira)

This study aims to evaluate the ovulation rate and the presence of functional corpora lutea after treatment by three different protocols designed to cause superovulation in brown brocket deer. Six female received an intravaginal device containing 0.33 g of progesterone (CIDR®) for 8 days, followed by 0.5 mg injection of estradiol benzoate at the time of insertion and 265 µg of cloprostenol at the time of removal. Afterwards, the hinds were divided into three groups (n = 2): Treatment A received injection of 600 IU eCG on Day 4 after CIDR® insertion; Treatment B received injection of 300 IU eCG at the same time; and Treatment C received injection of 250 IU FSH dissolved in PVP, also on Day 4 post‐insertion. The treatments were crossed over with 44–48 day intervals after CIDR® removal, such that all the deer were submitted to all three treatments. The mean ovulation rate (Treatment A = 3.40 ± 0.68, Treatment B = 1.40 ± 0.24, Treatment C = 0.80 ± 0.49), total ovarian stimulation (Treatment A = 4.80 ± 1.02, Treatment B = 1.80 ± 0.37, Treatment C = 1.40 ± 0.60), and mean CL diameter (Treatment A = 7.33 ± 0.76 mm, Treatment B = 3.94 ± 0.19 mm, Treatment C = 2.18 ± 0.49 mm) in Treatment A were significantly higher than the mean ovulation rates, total ovarian stimulation, and mean CL diameter in Treatments B and C. The mean fecal progesterone metabolites at the luteal phase in Treatment A (6,277.94±2,232.47 ng/g feces) was significantly different from Treatment C (1,374.82±401.77 ng/g feces). Thus, although fertility was not evaluated directly, Treatment A proved capable of induce superovulation in the species Mazama gouazoubira, presenting the greatest mean ovulation rates, with the formation of functional corpora lutea. lutea. Zoo Biol 31:642‐655, 2012. © 2011 Wiley Periodicals, Inc.     

Predictors of testosterone in zoo-managed male African elephants (Loxodonta Africana)

Reproductive complications for both male and female zoo-managed African elephants (Loxodonta africana) contribute to the rapidly declining population. In zoo-managed bull elephants, few studies have explored the potential physiological, physical, social, and environmental factors that influence bull fertility, particularly, androgen production. Testosterone is the essential steroid hormone for male sexual maturation and inadequate concentrations can be detrimental for spermatogenesis. Testosterone, fecal glucocorticoid metabolites, leptin, glucose, insulin, and triglycerides were analyzed from weekly fecal and blood serum samples taken over 6 months from six zoo-managed African elephant bulls (10–19 years of age). Testosterone levels were compared to endocrine factors, weekly social and environmental variables, daily musth signs, and body condition scores (BCS). The glucose-to-insulin ratio (G:I) was the only physiological biomarker found to be positively associated with testosterone. Predictive physical variables included Musth Score (+) and Moderate Exercise (+). Bulls with BCS signifying overweight (BCS 4) had lower testosterone (36.6 ± 1.6 ng/g fecal extraction [FE]) than bulls with healthy BCS 3; 51.2 ± 4.9 ng/g FE). Numerous social variables influenced testosterone concentrations, including Total Contact Day (+), Female Interaction Day (+), Indirect Contact Day (+), Indirect Contact Night (+) and Total No Contact (−). Both percentage of Time Outdoor and Time Mixed positively influenced testosterone, whereas testosterone decreased for percentage of Time Indoors. Each additional daily browse opportunity increased testosterone by approximately 7 ng/g FE. In managed care, the emphasis should be placed on optimizing these predictors of testosterone production to promote bull reproductive health.     

Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat (Prionailurus bengalensis euptilurus)

Although the Tsushima leopard cat (Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17β and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17β concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy.