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.     

Excretion of estrone,estradiol, and progesterone in the urine and feces of the female cotton-top tamarin (Saguinus oedipus oedipus)

The excretion of three gonadal steroids was studied in the urine and feces of female cotton-top tamarins (Saguinus oedipus oedipus). Each steroid, ¹⁴C-estrone, ¹⁴C-estradiol, and ¹⁴C-progesterone, was injected into a separate female cotton-top tamarin. Urine and feces were collected at 8 hr intervals for 5 days on the three tamarins. Samples were analyzed to determine the proportion of free and conjugated steroids. Steroid excretion patterns were determined by sequential ether extraction, enzyme hydrolysis, and chromatography. Labeled estrone was excreted in a slow and continuous manner into the urine (57%) and feces (43%) with 90% of the steroid conjugated. The nonconjugated form had an elution profile identical to ³H estrone, but the conjugated portion was not completely hydrolyzed by enzyme. Labeled estradiol was excreted primarily in the urine (87%) and was released rapidly. Over 90% of the injected ¹⁴C-estradiol was excreted in urine as a conjugate, of which 41% was converted to an estrone conjugate and the remaining 59% was excreted as a polar estradiol conjugate. Labeled progesterone was excreted primarily in the feces (95%), 61% of which was free steroid. Four to six individual peaks of radioactivity were found when using celite chromatography and high performance liquid chromatography (HPLC), indicating that progesterone is metabolized into several urinary and fecal metabolites. One of these peaks matched ³H-progesterone and others may be pregnanediols, pregnanetriols, and 17-hydroxyprogesterone. These steroidal excretion patterns help explain the atypical hormonal patterns seen during the tamarin ovarian cycle.     

Monitoring fecal samples for estrogen excretion across the ovarian cycle in Goeldi's monkey (Callimico goeldii)

In captive Goeldi's monkeys, estrogen concentration was determined in fecal samples collected from 4 cycling/unmated females and 4 postpartum/mated females in order to ascertain the potential of fecal estrogen monitoring for providing basic information about reproductive status in this endangered Amazonian monkey. Subjects were fed an omnivorous diet and first-void feces were collected in the home cage at 1–3-day intervals for 30–50 days from the cycling females, and at 6–14-day intervals around the estimated time of the postpartum ovulation in each of the 4 mated females. Estimates for the duration of ovarian cycles (22–26 days) and the timing of ovulation were based on cyclic profiles of either blood progesterone or urinary pregnanediol-3α-glucuronide. Fecal estrogen values were normalized using these plasma or urinary profiles. HPLC analysis of estrogen from postpartum fecal samples demonstrated the presence of unconjugated estrone and estradiol-17β (“unconjugated estrogen”). Unconjugated estrogen was extracted and its fecal concentration estimated via EIA. The correlation (r) between plasma estrone conjugates and fecal unconjugated estrogen across nonconception ovarian cycles was 0.65 and measurement of the latter generated cyclic profiles. A range of 4–36-ng unconjugated estrogen g^?1 feces was identified for follicular phases of nonconception cycles. Fecal unconjugated estrogen first exceeded the concentration range of the follicular phase 2–5 days after ovulation; the range was 49–402 ng g^?1 feces in samples collected during the remainder of these nonconception luteal phases. Luteal phase concentrations were on average 10-fold higher than follicular phase concentrations. Each of the 4 mated females conceived at its postpartum ovulation; concentrations of fecal unconjugated estrogen excreted by 3 of these females demonstrated a marked postovulatory increase. This study demonstrates that fecal unconjugated estrogen can be applied to monitor ovarian cyclicity in Goeldi's monkey. © 1994 Wiley-Liss, Inc.     

Diurnal variation on the excretion patterns of fecal steroids in common marmoset (Callithrix jacchus) females

The use of fecal steroid analysis to assess gonadal and adrenal function in primates has rapidly increased in recent years due to the ability to collect feces from nonhuman primates living in wild conditions. These techniques offer an exciting new potential for enhancing our knowledge of the endocrine status of free-living animals. Prior to using these techniques under field conditions, it is important to determine the diurnal variation of fecal excreted steroids for assessing possible time limitations on fecal collections. The following study investigates the diurnal frequency of defecation and patterns of steroid levels excreted in feces from four female common marmosets, Callithrix jacchus, living in a family group. These females represented three reproductive conditions: early pregnancy, ovarian cycling, and noncycling (postpubertal). Cortisol, estradiol, and progesterone were extracted and analyzed by enzyme immunoassay. Diurnal variations in steroid levels were found by ANOVA for cortisol and progesterone but not for estradiol. Significantly higher levels of cortisol were found in the afternoon, while the reverse was found for progesterone. All females showed the same pattern of steroid level change, except for cortisol in the pregnant female. Since all females defecated within the first hour after they awoke in the morning, this time was determined to be the most effective time to collect feces. The consistency of our findings reinforces the usefulness of this approach for studying reproductive and adrenocortical function in marmosets and also indicates that fecal collection should be limited to either morning or afternoon collections. Am. J. Primatol. 46:105–117, 1998. © 1998 Wiley-Liss, Inc.     

Excretion and measurement of estradiol and progesterone metabolites in the feces and urine of female squirrel monkeys (Saimiri sciureus)

The first objective of the present study was to determine the metabolic form and rate of excretion of ovarian hormone metabolites in the urine and feces of female squirrel monkeys injected with radiolabeled progesterone (Po) and estradiol. The major portion of the urinary metabolites of both hormones was excreted within 16-24 hr post-injection. Estrogen and Po isotopes in feces exhibited an excretion peak at 16 hr post-injection. The majority of recovered radiolabel of both hormones was excreted in feces. Chromatographic separation of fecal extractions indicated that the major estrogen metabolites in feces are in the free as opposed to the conjugated form. The radioactivity and immunoreactivity for estrone and estradiol (E(1) and E(2), respectively) in eluates of fecal samples subjected to celite co-chromatography indicated that both free E(1) and E(2) exist as excretion products in the feces of female squirrel monkeys. The major radioactive peaks for Po metabolites showed peaks in the elution profile at or very near the Po standard, and corresponded with the celite co-chromatography elution profile of Po standard when subjected to enzyme immunoassay (EIA). The second objective was to validate the application of EIA systems to measure fecal metabolites. Reproductive events of one female squirrel monkey across one annual reproductive cycle are described using the endocrine profile generated from fecal steroid assays. Examination of this profile confirmed that longitudinal fecal sampling and steroid hormone metabolite measurement in feces was not only feasible and practical, but accurately detected known reproductive events as well.     

Application of fecal steroid techniques to the reproductive endocrinology of female verreaux's Sifaka (Propithecus verreauxi)

Solid phase extraction, high performance liquid chromatography, and radioimmunoassay were used to test the validity of fecal steroid analysis for assessing ovarian function in sifaka (Propithecus verreauxi). Daily fecal samples were collected over a 4 month period from two cycling female sifaka, and single samples were collected from females during normal gestation and males while housed at the Duke University Primate Center. Tests of radioimmunoassay validity indicated that solid phase extraction and microradioimmunoassay techniques were reliable and accurate methods for quantifying ovarian steroids in sifaka feces. The progesterone (P₄) antibody specifically quantitated only P₄, while several estrogen metabolites made small contributions to immunoreactive measures of estradiol (E₂). A 1:10 dilution reduced these contributions to 3–15% of the estimated E₂ concentration. Although the spectral data suggested that E₂ was not the major metabolite present, it accounted for the majority of the immunoreactivity at normal assay dilutions. Fecal profiles of immunoreactive E₂ and P₄ in the conceptive female resembled serum profiles of other strepsirhines. E₂ and P₄ were elevated at the end of the conceptive cycle and were more markedly increased in late pregnancy in the two pregnant females. Mating behavior and indices of sexual interest were observed in conjunction with E₂ peaks, although not all peaks were accompanied by observations of sexual behavior. © 1995 Wiley-Liss, Inc.     

Steroid excretion during the ovarian cycle in captive and wild muriquis,Brachyteles arachnoides

Urine, feces, and copulation frequency were collected from two captive muriqui females, Brachyteles arachnoides, at the Centro de Primatologia do Rio de Janeiro following the resumption of postpartum ovarian cycles. Fecal steroid profiles from seven wild muriqui females at the Estação Biologica de Caratinga, Minas Gerais, Brazil, were compared to the captive females to determine the approximate patterns of steroid excretion relative to the urinary LH peak. Hormonal profiles from one of the captive female muriquis revealed a discrete urinary LH peak. For this female, fecal progesterone increased on the same day as the urinary LH peak, while fecal estradiol increased 6 days later and urinary steroids increased 5 days later. For both captive females, the onset of fecal progesterone increase was preceded by the onset of copulations, which occurred during at least a 5-day period. The complete fecal hormonal profiles of the one captive female for which continuous data were available were similar to those found in wild muriqui monkeys, with the onset of an increase in sustained progesterone levels occurring several days prior to the onset of sustained estradiol increase. These patterns suggest that fecal progesterone may be excreted rapidly in this species. The onset of sustained increase in fecal progesterone levels, together with the consistent delay in the onset of the sustained increase in estradiol, may provide the best indicators of the periovulatory period for muriqui females. Am. J. Primatol. 42:311–321, 1997. © 1997 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.     

Noninvasive analysis of fecal reproductive hormone metabolites in female veiled chameleons (Chamaeleo calyptratus) by enzyme immunoassay

The noninvasive technique of gonadal steroid metabolite measurement in feces for evaluation of reproductive activity has proven an effective and important tool for population management in various captive species, but has not yet been validated and used in reptile species. In this study, enzyme immunoassays (EIAs) were validated for the analysis of fecal samples from female veiled chameleons (Chamaeleo calyptratus) for estrogen (E2), testosterone (T), and progesterone (P) and their metabolites. High performance liquid chromatography and physiological methods (GnRH stimulation) were used for the validation of the assays. Biological events, such as skin color changes indicative of ovarian activity and oviposition, correlated with the cyclical pattern of E2, T and P metabolites in feces over a period of two reproductive cycles. This is the first study to report frequent longitudinal measurements of fecal hormone levels by EIA in a reptile species.     

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.     

Fecal steroid analysis of ovarian cycles in free-ranging baboons

This paper reports field and laboratory tests of serial sampling, solid phase extraction, and microradioimmunoassay methods for the collection, preservation, and analysis of fecal steroids. The field study was conducted in a troop of 87 yellow baboons (Papio cynocephalus) in the Tana River Primate Reserve, Kenya. Serial samples of four focal females and opportunistic sampling of 18 additional females over 22 days of sampling yielded a total of 62 samples, X = 3.1 ± 0.4/day, demonstrating the feasibility of regular field collection and extraction. Estradiol and progesterone concentrations in the field-extracted samples exhibited high recovery and statistically significant correlations (P < 0.05) with concentrations in the lab-extracted samples, suggesting that solid phase extraction could provide a useful alternative to freezing in sites where electricity or liquid nitrogen is not available. Tests of microradioimmunoassays demonstrated that these assays were sensitive, accurate, and precise when applied to the assay of fecal extracts, providing estimates of ovarian steroids that varied significantly with reproductive state. The demonstration that testosterone could be accurately and reliably assayed in fecal extracts suggests that these techniques also could be applied to the study of male reproductive function. Parallels between fecal profiles of cycling and pregnant baboons with patterns reported for serum steroids in baboons suggest that fecal steroids might be useful in distinguishing amenorrhea from early pregnancy in free-ranging baboons as well as in species lacking external indices of reproductive state. © 1995 Wiley-Liss, Inc.     

Measurement of urinary and fecal steroid metabolites during the ovarian cycle in captive and wild Japanese macaques, Macaca fuscata

We measured the concentration of steroid hormones from urine, feces, and blood samples of two captive Japanese macaques, Macaca fuscata, during nonconceptive ovarian cycles to compare the patterns of the excreted steroids with those of circulating steroids. Urine and feces were analyzed for estrone conjugates (E1C) and pregnanediol-3-glucronide (PdG) using enzyme immunoassays (EIAs), while plasma was analyzed for estradiol-17beta(E2), progesterone (P), and luteinizing hormone (LH) using radioimmunoassays (RIAs). Urinary and fecal E1C and PdG levels were approximately parallel to plasma E2 and P levels, respectively. The E1C profiles of daily urinary and fecal samples revealed a midcycle peak, followed by a sustained PdG increase lasting up to two weeks from the E1C peak. A fecal E1C peak was one day later than the urinary E1C peak. One of the captive females exhibited a discrete plasma LH peak, one indicator that ovulation has occurred, on the day following the urinary E1C peak, i.e., the same day of fecal E1C peak. We measured excreted steroids in nine wild females and determined the timing of ovulation by comparing fecal steroid profiles to those obtained in captive monkeys. Data from wild females indicated that eight of nine females conceived during their first ovulatory cycle of the sampling period, whereas the remaining female failed to conceive during the sampling period even though she ovulated. In the eight females that conceived, E1C increased again following the detected or estimated E1C peak, with levels comparable to the preovulatory peak levels, and sustained elevations of PdG for over 40 days. These data illustrate that the urinary and fecal profiles of ovarian steroid excretion obtained through the application of these noninvasive techniques provide an accurate approach for monitoring conceptive and nonconceptive ovarian cycle in captive and free-living Japanese macaques.     

Monitoring the reproductive status of female gorillas (Gorilla gorilla gorilla) by measuring the steroid hormones in fecal samples

The reproductive status of female gorillas (Gorilla gorilla gorilla) was estimated by measuring the sex steroid hormones in fecal samples instead of in blood samples. Fecal samples from female gorillas were used to examine the reliability of this non-invasive assay system, which included the extraction method for estradiol-17β (E₂) and progesterone (P) from fecal samples. Fecal samples from three female gorillas were collected daily for about 55 days, and fecal E₂ and P were assayed to clarify the fluctuation patterns of these steroids in the feces. Fecal sampling from one female was repeated for another 50-day period (starting 75 days after the end of the first observation period) and assayed to confirm if the menstrual cycle of this subject was ovulatory. Although fecal E₂ concentration measurements were quantitative by using this assay system, fecal P concentration measurements were semi-quantitative. Relative amounts of fecal P in fecal samples were estimated by using the values of B/B₀ (bound/total binding in the radioimmunoassay system). Two of the four fluctuation patterns of fecal hormones observed throughout the menstrual cycle for the three female gorillas were typical for normal ovulatory cycles. In the subject observed for two periods, one pattern was typical and the other atypical. The results show that this non-invasive method is simple and practical for monitoring the reproductive status of great apes as well as Old World monkeys.     

Seasonal changes in ovarian steroid hormone concentrations in the large hairy armadillo (Chaetophractus villosus) and the crying armadillo (Chaetophractus vellerosus)

Knowledge of armadillo reproductive physiology is essential for developing ex situ and in situ assisted reproductive techniques for propagating and/or controlling populations of these animals. The present study included assessment of fecal sex steroids by radioimmunoassay, determining reproductive status via monitoring ovarian activity (in the wild) and therefore reproductive status, in wild females of the large hairy armadillo (Chaetophractus villosus) and the crying armadillo (Chaetophractus vellerosus) in the southern hemisphere. Plasma and fresh fecal progesterone concentrations were not significantly correlated in either species. However, in both species, there was a significant positive correlation between plasma progesterone and dry fecal progesterone concentrations (r = 0.82, P < 0.05 and r = 0.60, P < 0.05, respectively). Dry fecal progesterone and estradiol concentrations were measured in one captive C. villosus (average baseline progesterone and estradiol concentrations 28.72 ± 11.75 ng/g dry feces and 3.04 ± 0.80 ng/g dry feces, respectively) and one captive C. vellerosus (average baseline progesterone and estradiol concentrations 14.05 ± 3.03 ng/g dry feces and 3.46 ± 1.20 ng/g dry feces, respectively) to detect hormonal peaks over 1 y; these occurred from late fall to early summer. Feces from wild C. villosus and C. vellerosus were also collected over 1 y to determine progesterone peaks, which occurred in winter and spring in both species (with no peaks during the summer or fall). Accordingly, C. villosus and C. vellerosus had a seasonal reproductive pattern. The significant correlations between dry fecal and plasma progesterone concentrations validated this method for monitoring reproductive status in these species.     

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.     

Behavioral and endocrine characteristics of the reproductive cycle in wild muriqui monkeys,Brachyteles arachnoides

The analysis of fecal ovarian steroids provides a powerful noninvasive method to obtain insights into ovulatory cycles, gestation length, and the timing of sexual interactions relative to the periovulatory period in wild primates. Techniques developed to collect and assay feces from free-ranging muriqui monkeys (Brachyteles arachnoides) for estradiol and progesterone yield the first explicit reproductive data on this species, and provide the first opportunity to evaluate the timing of observed copulations with muriqui ovarian cycles. Hormonal profiles from seven females indicate average cycle lengths of 21.0 ± 5.4 days (n=20). Females conceived after 3–6 ovulatory cycles. Gestation length averaged 216.4 ± 1.5 days for the five females for which conception cycles were sampled. Discrete copulation periods spanned an average of 2.1 ± 1.2 days (n=29), with intervals between these concentrated periods of copulations averaging 15.6 ± 6.7 days (n=20). There were no significant differences among females in cycle lengths, copulation period lengths, or copulation interval lengths. Ejaculation was visible following 71.8 ± 26.7% of copulations during the females' preovulatory periods. All females copulated outside the periovulatory period. The proportion of copulation days outside the periovulatory period was slightly greater (p=0.08) for primiparous females (64.8 ± 28.3%) than for multiparous females (28.7 ± 19.7%). Am. J. Primatol. 42:299–310, 1997. © 1997 Wiley-Liss, Inc.     

Excretion of radiolabeled estradiol metabolites in the slow loris (Nycticebus coucang)

The excretion pattern of estradiol was studied in the slow loris Nycticebus coucang) and the ring-tailed lemur (Lemur catta) in order to compare steroid excretion in two representative prosimian species. Daily urinary estrone conjugate measurements in the female loris provided little information when applied over prolonged periods. As a result of these negative data, a metabolic study was performed to determine if estrogen excretion patterns in the slow loris differed from those in the lemur, where urinary assays proved a useful tool in characterizing reproductive cycles. Radio-labeled estradiol was injected intravenously, and serial urine and fecal collections were analyzed for radiolabeled metabolites. The results of these studies demonstrate that more than 92% of the radiolabel was excreted in the feces of the loris, in contrast to only 16% excreted in the feces of the lemur.     

Fecal testosterone immunoreactivity as a non-invasive index of functional testosterone dynamics in male Japanese macaques (Macaca fuscata)

Validation of a simple method for the extraction and quantification of testosterone (T) from the excreta of male Japanese macaques (Macaca fuscata) is presented. Radioimmunoassay of paired fecal and serum samples collected from four intact sexually mature males during the breeding season provided profiles that were significantly correlated when samples were offset by approximately 48 hr. Additionally, no significant differences were observed in the pattern of temporal variation of T levels in serum and feces. Two castrated males were injected with radioinert T, and the patterns of excretion were observed by analysis of serial fecal and urine samples. Approximately 48 hr after the steroid was administered, a significant peak in the average fecal T levels was apparent. The injection event was also registered in the urine of both males, although qualitative differences were observed. These data suggest that measures of fecal T provide a reliable and non-invasive means of assessing gonadal function in this species. As the analysis of hormone levels in feces allows for frequent, stress-free sampling with minimal disruption, this method should be preferred in long-term orin situ applications requiring endocrine monitoring.     

Enzyme immunoassay of progesterone in the feces from beef cattle to monitor the ovarian cycle

The present study was undertaken to measure fecal progesterone concentration of beef cattle using antibody against authentic progesterone and to examine whether this method can monitor the ovarian cycle in beef cattle. Rectal fecal samples collected from 14 beef cattle were mixed with 6 ml of 100% methanol and shaken for 15 min. After centrifugation, supernatant was extracted with petroleum ether followed by an enzyme immunoassay (EIA) for progesterone. Specificity of the assay was examined by HPLC separation of fecal solution followed by the EIA in each fraction. The present assay identified only progesterone but not other metabolites in the feces sample that was extracted with petroleum ether. Sensitivity of the assay was estimated to be 0.0055 ng/ml (0.11 ng/g). Coefficient variations of intra- and inter-assay were 9.6-10.9% and 10.8-16.6%, respectively. Recovery rates ranged between 73 and 84%. Patterns in the fecal progesterone concentrations during the ovarian cycle were almost parallel to the plasma concentrations. A significant positive correlation was established between the fecal and plasma progesterone concentrations in individual animal (r=0.59-0.84, P<0.001, n=10) as well as pooled data (r=0.70, P<0.001, n=65). Fecal progesterone concentrations of day 0 (showing the nadir of concentration) of the ovarian cycle were less than 50 ng/g, which increased significantly toward day 9 (P<0.01). From days 14 to 18, there was significant reduction of fecal progesterone concentration (P<0.01). Ovarian cycles had at least 48 ng/g (mean=74 ng/g) of difference between minimum and maximum fecal progesterone concentrations. All cattle at days 9, 11 and 14 had higher fecal progesterone concentrations by more than 20 ng/g compared with day 0. These results suggest that the present EIA is suitable to measure the progesterone in cattle feces and can monitor ovarian cycle.     

川金丝猴粪尿中类固醇性激素抽提方法比较

本文旨在探讨川金丝猴粪尿中类固醇性激素的抽提方法, 实验比较了3 种尿液抽提溶剂和5 种粪便抽提方法的抽提回收率。结果显示, 以二氯甲烷为溶剂抽提雄猴尿液中的睾酮、雌猴尿液中的雌二醇和孕酮, 回收率分别为79.27%、73.39%和80.28%, 抽提效果较其它两种溶剂为好; 以乙醇加热法抽提雄猴粪便中的睾酮和雌二醇、雌猴粪便中的睾酮、雌二醇和孕酮, 回收率分别为1.33%、85.12%与67.41%、79.58%和77.71%, 抽提效果优于其它4 种方法,且该方法简便易于操作。实验表明, 以二氯甲烷和乙醇加热法分别抽提川金丝猴尿液和粪便中的类固醇性激素是较理想的抽提方法。