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.     

Measurement of fecal steroids in the black rhinoceros (Diceros bicornis) using group-specific enzyme immunoassays for 20-oxo-pregnanes

The metabolism and excretion of progesterone in different animal species results in several fecal 5-reduced progesterone metabolites (pregnanes), which in recent studies were quantified using progesterone antibodies. To increase the accuracy of fecal 20-oxo-pregnane evaluations in the black rhinoceros, enzyme immunoassays (EIA) using antibodies against 5α-pregnane-3β-ol-20-one 3HS:BSA (5α-20-one EIA) and 5β-pregnane-3α-ol-20-one 3HS:BSA (5β-20-one EIA) were developed. The assays showed high crossreactivities with pregnanes containing a 20-oxo group and are referred to as group-specific; results of these assays were compared with an EIA using an antibody against 6HS-progesterone (4-ene-20-one EIA). Fecal samples of both subspecies of the black rhinoceros (Diceros bicornis michaeli, n = 5, and Diceros bicornis minor, n = 1) during pregnancy were collected 1–3 times/week. HPLC separation showed three major immunoreactive fecal 20-oxo-pregnane peaks; their elution profiles and different crossreactivities in the three EIAs provided strong evidence that these peaks are 5α-pregnane-3, 20-dione, 5α-pregnane-3α-ol-20-one, and 5α-pregnane-3β-ol-20-one. Pregnane values in the pregnant animals continuously increased between months 3–7 and were significantly (P < 0.01) elevated above the levels of nonpregnant animals (0.2 μg/g) by week 11. During months 6–13 concentrations in the 5α-20-one and in the 5β-20-one EIA (5–11 μg/g) were significantly (P < 0.01) higher than in the 4-ene-20-one EIA (1.5–3 μg/g). In conclusion, the immunoreactive fecal 20-oxo-pregnane metabolites in the black rhinoceros are determined more accurately with antibodies against pregnane-20-one-C3 conjugates, as compared with a progesterone antibody. © 1996 Wiley-Liss, Inc.     

Patterns of fecal gonadal hormone metabolites in the maned wolf (Chrysocyon brachyurus)

Ex situ populations of maned wolves are not viable due to low reproductive efficiency. The objective of this study was to increase knowledge regarding the reproductive physiology of maned wolves to improve captive management. Fecal samples were collected 3-5 d/wk from 12 females of various reproductive age classes (young, prime breeding and aged) and reproductive histories (conceived and raised pups, conceived but lost pups, pseudo-pregnant and unpaired). Ovarian steroids were extracted from feces and assessed by enzyme immunoassay. Concentrations of estrogen metabolites gradually increased, beginning 2-5 d before breeding, and declined to baseline on the day of lordosis and copulation. Fecal progestin metabolite concentrations increased steadily during the periovulatory period, when sexual receptivity was observed, and remained elevated during pregnancy and pseudo-pregnancy. During the luteal phase, young and prime breeding-age females excreted larger amounts of progestins than those of older age classes. Furthermore, progestin concentrations were higher during the luteal phase of pregnant versus pseudo-pregnant bitches. Profiles of fecal progestin metabolites for three singleton females were unchanged throughout the breeding season, suggesting ovulation is induced in this species. However, this finding could be confounded by age, as these females were either young or aged.     

Fecal 20-oxo-pregnane concentrations in free-ranging African elephants (Loxodonta africana) treated with porcine zona pellucida vaccine

Because of overpopulation of African elephants in South Africa and the consequent threat to biodiversity, the need for a method of population control has become evident. In this regard, the potential use of the porcine zona pellucida (pZP) vaccine as an effective means for population control is explored. While potential effects of pZP treatment on social behavior of African elephants have been investigated, no examination of the influence of pZP vaccination on the endocrine correlates in treated females has been undertaken. In this study, ovarian activity of free-ranging, pZP-treated African elephant females was monitored noninvasively for 1 yr at Thornybush Private Nature Reserve, South Africa, by measuring fecal 5α-pregnan-3β-ol-20-on concentrations via enzyme immunoassay. A total of 719 fecal samples from 19 individuals were collected over the study period, averaging 38 samples collected per individual (minimum, maximum: 16, 52). Simultaneously, behavioral observations were made to record the occurrence of estrous behavior for comparison. Each elephant under study showed 5α-pregnan-3β-ol-20-on concentrations rising above baseline at some period during the study indicating luteal activity. Average 5α-pregnan-3β-ol-20-on concentrations were 1.61 ± 0.46 μg/g (mean ± SD). Within sampled females, 42.9% exhibited estrous cycles within the range reported for captive African elephants, 14.3% had irregular cycles, and 42.9% did not appear to be cycling. Average estrous cycle duration was 14.72 ± 0.85 wk. Estrous behavior coincided with the onset of the luteal phase and a subsequent rise in 5α-pregnan-3β-ol-20-on concentrations. Average 5α-pregnan-3β-ol-20-on levels positively correlated with rainfall. No association between average individual 5α-pregnan-3β-ol-20-on concentrations or cyclicity status with age or parity were detected. Earlier determination of efficacy was established via fecal hormone analysis with no pregnancies determined 22 mo post-treatment and onward. Results indicate the presence of ovarian activity amongst pZP-treated female African elephants in 2 yr after initial immunization. Further study should now be aimed toward investigating the long-term effects of pZP vaccination on the reproductive function of female African elephants.     

The effects of progesterone and its metabolites on the induction of sexual receptivity in rats

Estrogen-primed female rats were administered progesterone, 5 a-pregnan-3,20-dione, 5a-pregnan-3a-ol-20-one, 5a-pregnan-3β-ol-20-one, 4-pregnen-20a-ol-3-one, and 5a-pregnan-20a-ol-3-one in oil or oil alone and tested for the display of lordosis behavior 3 and 5 hr after progestin treatment. Progesterone was the most effective progestin in facilitating lordosis behavior. 5a-Pregnan- 3,20-dione and 5a-pregnan-3a-ol-20-one were partially effective, while the other progestins were no more effective than oil. The data suggest that the neuronal system which controls lordosis behavior is not entirely specific for progesterone.     

The impact of ecological variability on the reproductive endocrinology of wild female African elephants

Non-invasive endocrine methods enable investigation of the relationship between ecological variation and ovarian activity and how this impacts on demographic processes. The underlying physiological factors driving high variation in inter-calving intervals among multi-parous African elephants offer an interesting system for such an investigation. This study investigates the relationship between Normalized Differential Vegetation Index (NDVI), an ecosystem surrogate measure of primary productivity, and fecal progestin concentrations among wild female elephants. Matched fecal samples and behavioral data on reproductive activity were collected from 37 focal individuals during the two-year study. Linear mixed models were used to explore the relationship between fecal 5alpha-pregnane-3-ol-20-one concentrations and the independent variables of NDVI, calf sex, female age, gestation day, and time since last parturition. Among both non-pregnant and pregnant females, fecal 5alpha-pregnane-3-ol-20-one concentrations were significantly correlated with time-specific NDVI indicating a strong relationship between ecological conditions and endocrine activity regulating reproduction. In addition, the age of a female and time since her last parturition impacted hormone concentrations. These results indicate that the identification of an individual's reproductive status from a single hormone sample is possible, but difficult to achieve in practice since numerous independent factors, particularly season, impact fecal hormone concentrations. Regardless of season, however, fecal 5alpha-pregnane-3-ol-20-one concentrations below 1 microg/g were exclusively collected from non-pregnant females, which could be used as a threshold value to identify non-pregnant individuals. Collectively the information generated contributes to a better understanding of environmental regulation of reproductive endocrinology in wild elephant populations, information salient to the management and manipulation of population dynamics in this species.     

Detection and identification of plasma progesterone metabolites in the female Florida manatee (Trichechus manatus latirostris) using GC/MS/MS

Florida manatees (Trichechus manatus latirostris) have relatively low peripheral concentrations of progesterone (P4). The objective of this study was to determine if these relatively low P4 concentrations are associated with a high ratio of progestin metabolites and to document metabolite concentrations from individual blood samples obtained from manatees during diestrus or pregnancy. Metabolites known to exist in elephants—terrestrial manatee relatives—were targeted. These included 5α-reduced progestins (5α-pregnane-3,20-dione [5α-DHP] and 3α-hydroxy-5α-pregnan-20-one [5α-P3-OH]) and 17α-hydroxyprogesterone (17α-OHP), which occurs in Asian elephants. An additional, inactive metabolite, 20α-hydroxyprogesterone (20α-OHP), indicative of P4 overproduction, was also targeted. Progesterone itself was the predominant progestin detected in pregnant and nonpregnant manatee plasma (n = 10) using gas chromatography-mass spectrometry with tandem quadrupole detectors (GC/MS/MS). Progesterone concentrations in pregnant females varied from early (moderate to high) through mid and late (low) pregnancy. Progesterone concentrations ranged from low to high in nonpregnant, nonlactating females. The most commonly detected metabolite was 5α-P3-OH (n = 7), which occurred in pregnant (lower limit of detection [LLOD] to high) and nonpregnant (trace to high) females. The 5α-DHP metabolite was also detected in pregnant (LLOD to moderate) and nonpregnant (low) females. The 17α-OHP metabolite was not detected in any tested female. The 20α-OHP metabolite was detected in one nonpregnant, nonlactating, captive female (LLOD). Metabolites were most prevalent during early pregnancy, concurrent with maximum P4 concentrations. Based on their concentrations in peripheral circulation, we inferred that these metabolites may have, opposite to elephants, a limited physiologic role during luteal, pregnant, and nonpregnant phases in the manatee.     

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.     

An analysis of the metabolites of progesterone produced by isolated sertoli cells at the onset of gametogenesis

Sertoli cells isolated from 17 day old rats were maintained in culture and incubated with [¹⁴C]-progesterone for 20 h. The cells and media were extracted with ether/chloroform and the extracts chromatographed two-dimensionally on TLC and the radioactive metabolites visualized by autoradiography. Nine of the metabolites (constituting about 88% of total metabolite radioactivity) were identified by relative mobilities of the compounds and their derivatives in TLC and GC systems and by recrystallizations with authentic steroids as the following: 20α-hydroxypregn-4-en-3-one, 3α-hydroxy-5α-pregnan-20-one, 5α-pregnane3α,20α-diol, 17β-hydroxy-5α-androstan-3-one, 5α-pregnane-3,20-dione, 17-hydroxypregn-4-ene-3,20-dione, testosterone, 5α-androstane-3α,17β-diol and androst-4-ene-3,17-dione. Over 71% of the metabolite radioactivity was due to 20α-hydroxypregn-4-en-3-one, the major metabolite. 5α-reduced pregnanes constituted about 12% and C₁₉ steroids comprised about 2.9% of the radioactivity of the metabolites. Calculation of relative steroidogenic enzyme activities from initial reaction rates suggested the following activities in μunits/mg Sertoli cell protein: 20α-hydroxysteroid oxidoreductase (20α-HS0; 7.71), 5α-reductase (4.77), 3α-HS0 (3.57), 17α-hydroxylase (0.93), 17β-HS0 (0.34) and C₁₇-C₂₀ lyase (0.34). The relatively high rate of steroidogenic enzyme activities in the Sertoli cells of young rats may indicate that Sertoli cells are less dependent on Leydig cell steroidogenesis than has been assumed. Since nearly all the metabolites of progesterone and testosterone are now identified, it is possible to construct a picture of Sertoli cell steroidogenic activity.     

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.     

Evidence for steroid metabolism during the in vitro induction of maturation in oocytes of Rana pipiens

Oocytes of Rana pipiens exposed to exogenous progesterone in order to induce maturation have been observed to extensively metabolize this hormone. When progesterone was injected directly into the oocytes, they did not mature, but similar metabolism of progesterone occurred. The metabolites have been tentatively identified as the 5α-reduced derivatives, 5α-pregnanedione, 5α-pregnan-20α-ol-3-one, and 5α-pregnan-3β, 20α-diol, and the pathway of conversion has been examined. Samples of these steroids obtained from commercial sources and those extracted from progesterone-treated oocytes were effective in inducing maturation when added to the medium. Evidence is presented which suggests that steroid metabolism is not a prerequisite for maturation and that the metabolites like progesterone must interact with the oocyte surface to be effective.     

Metabolism of progesterone by chorionic cells of the early sheep conceptus invitro

Progesterone-4-¹⁴C was extensively metabolized during incubation with dispersed trophoblast prepared from chorionic membranes of the 21-day sheep conceptus. Of the metabolites formed, 17,20α-dihydroxypregn-4-en-3-one, 20α-hydroxypregn-4-en-3-one, 20(β-hydroxypregn-4-en-3-one, 5α-pregnane-3α,17,20α-triol, 5β-pregnane-3ga, 17,20α-triol, 5β-pregnane-3g,20α-diol, 3β-hydroxy-5α-pregnan-20-one, 3α-hydroxy-5β-pregnan-20-one, 20β-hydroxy-5β-pregnan-3-one, 5α-pregnane-3,20-dione and 5β-pregnane-3,20-dione were identified. These findings indicate that the sheep conceptus acquires extensive steroid metabolizing capability very early in pregnancy.     

Fecal steroid hormone analysis as an indicator of reproductive function in the cheetah

Techniques were developed and validated to measure fecal estrogen and progesterone concentrations of the female cheetah. Fecal samples were collected from seven mature females. Cheetahs were monitored before mating and continued until parturition. Four females had normal pregnancies, one conceived but the pregnancy resulted in spontaneous abortion, one was mated but apparently did not conceive and one was treated with gonadotropin-releasing hormone (GnRH) and human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Vaginal superficial cells increased with increasing estrogen concentrations. Peak estrogen occurred one day postcopulation. Increases in fecal progesterone concentrations, indicative of ovulation, occurred after copulation and hormonally induced ovulation. For the first time reproductive function can be monitored in the cheetah using noninvasive sample collection. © 1994 Wiley-Liss, Inc.     

Metabolism of progesterone by Dioscorea deltoidea suspension cultures

Dioscorea deltoidea plant tissue suspension cultures are capable of metabolizing progesterone to 5α-pregnan-3-β-ol-20-one and 5α-pregnan-3β,20β-diol. The latter product has not previously been reported as a metabolic product of progesterone by plant systems. Both transformation products are present as conjugates in this plant tissue culture.     

Biotransformation of progesterone by suspension cultures of Digitalis purpurea cultured cells

It has been shown that the cultured cells of Digitalis purpruea are capable of transforming progesterone (I) to 5α-pregnane-3,20-dione (II), 5α-pregnan-3β-ol-20-one (III), its glucoside (IV), 5α-pregnane-3β,20α-diol (V), its glucoside (VI), 5α-pregnane-3β,20β-diol (VII), its glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one (IX), its glucoside (X), Δ-pregnen-20β-ol-3-one (XI) and its glucoside (XII). 5α-Pregnan-3β-ol-20-one glucoside (IV), 5α-pregnane-3β,20α-diol glucoside (VI), 5α-pregnane-3β,20β-diol glucoside (VIII), Δ⁴-pregnen-20α-ol-3-one glucoside (X) and Δ⁴-pregnen-20β-ol-3-one glucoside (XII) have been found for the first time as new metabolises by plant tissue cultures. A scheme for the biotransformation of progesterone (I) has been proposed, and the reduction and glucosidation activities distinctly have been observed in these cultured cells.     

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.     

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.     

Evaluation of progesterone and 20-oxo-progestagens in the plasma of Asian (Elephas maximus) and African (Loxodonta africana) elephants

The corpus luteum of African elephants produces high amounts of 5α-reduced progesterone metabolites (5α-pregnane-3,20-dione and 5-α-pregnane-3α-ol-20-one), whereas progesterone itself is quantitatively less important, and plasma levels of progesterone during the estrous cycle in elephants are considerably lower than those of other mammals. The objective of this study was to compare the concentration of progesterone in plasma of Asian and African elephants as determined by specific progesterone assays with those of total immunoreactive progestagens containing a 20-oxo-group (20-oxo-P). These metabolites were determined by an enzyme immunoassay using an antibody against 5-α-pregnane-3β-ol-20-one, 3HS:BSA. Plasma of non-pregnant Asian (n = 4) and African (n = 4) elephants was collected at weekly intervals for periods of 8–15 months and at random intervals during pregnancy in one Asian elephant. High-performance liquid chromatography separation of plasma samples of both species demonstrated that in the 20-oxo-P assay, 5α-pregnane-3,20-dione makes up ˜60% of the total immunoreactive material. The progesterone and 20-oxo-P values during the estrous cycle showed a parallel pattern and were significantly correlated (P < 0.001; Asian: r = 0.80; y = 3.76 × –0.10; African: r = 0.75; y = 2.66 × –0.08). Progesterone and 20-oxo-P values in Asian and African elephants were <0.15 ng/ml during the follicular phase (weeks –4 to 0) of the estrous cycle; progesterone values during the luteal phase (weeks 2–9) were 0.60 ± 0.03 and 0.53 ± 0.03 ng/ml, and the 20-oxo-P values were 2.19 ± 0.16 and 1.48 ± 0.12 ng/ml, respectively. The 20-oxo-P values of the pregnant animal, although slightly higher, were comparable to those of non-pregnant elephants during the luteal phase. Total immunoreactive 20-oxo-P values are about three times higher than those of progesterone during the luteal phase, and 5α-pregnane-3,20-dione is the major immunoreactive 20-oxo-P in the plasma of Asian and African elephants. Zoo Biol 16:403–413, 1997. © 1997 Wiley-Liss, Inc.     

Monitoring hormones in urine and feces of captive bonobos (Pan paniscus)

Urinary and fecal hormones were analyzed on average every other day in 17 female bonobos kept at four US zoos (San Diego Zoo and Wild Animal Park, Milwaukee, Columbus, and Cincinnati). Ovarian cycle activity was monitored throughout the 15-month study period using estrogen and progesterone profiles and swelling charts. Behavioral data on sexual activity were also collected on a daily basis. Fecal and urinary samples were analyzed using high pressure liquid chromatography (HPLC), gas chromatography-mass spectrometry (CG-MS), and nanoelectrospray. Preliminary results indicate that in urine, both conjugated progestin and estrogen metabolites were abundant, while in fecal samples, free progestin metabolites from the 5a-pregnane series were found. Although traces of estrogen metabolites were detected in fecal samples, long-term monitoring of ovarian activity in our study yielded no meaningful estrogen profiles. In contrast, fecal progestin profiles, after adjusting for a one-day delay in excretion, closely matched the corresponding urinary progestin profiles. Using the identical antibody and tracer for both, fecal and urinary progestins, fecal samples yielded approximately ten times the relative amount of progestins compared to urinary progestins. Thus, when converted using a regression formula, fecal progestins may complete the picture obtained from urinary progestins, particularly in cases where the urine sample record is unavailable or incomplete. Evidence of the usefulness of urinary cortisol as a measure of stress is presented.     

Tracking the Ovarian Cycle in Black-and-Gold Howlers (<Emphasis Type="Italic">Alouatta caraya</Emphasis>) by Measuring Fecal Steroids and Observing Vaginal Bleeding

A better understanding of a species’ reproductive physiology can help conservation programs to manage primates in the wild and develop assisted reproductive technologies in captivity. We investigated whether measurements of fecal progestin and estrogen metabolites obtained by a radioimmunoassay could be used to monitor the ovarian cycle of Alouatta caraya. We also compared the occurrence of vaginal bleeding with the hormone profiles. We collected fecal samples from 3 adult and 1 subadult captive female over 5 mo and performed vaginal cytology for the adults. The interval between fecal progestin surges in the adult females was 19.11 ± 2.14 d (n = 18 cycles). Fecal progestin concentrations remained at basal values for 9.83 ± 2.21 d (n = 18) and rose to elevated values for 9.47 ± 0.72 d (n = 19). The subadult female showed basal levels of fecal estrogen and progestin concentrations throughout the study, suggesting that our hormone measurements are valid to monitor the ovarian cycle. Bleeding periods coincided with basal levels of fecal estrogens and progestin at intervals of 19.8 ± 0.9 d and lasted for 4.1 ± 1.0 d. Although we obtained these data from only 3 individuals, the results indicate that this species likely has a menstrual-type ovarian cycle. These data provide the first endocrine profile for the Alouatta caraya ovarian cycle and are similar to results obtained for other howler species. This similarity is important for comparative studies of howlers, allowing for a better understanding of their reproductive physiology and contributing to a critical information base for managing Alouatta species.