Urinary steroid evaluations to monitor ovarian function in exotic ungulates: III. Estrone sulfate and pregnanediol-3-glucuronide excretion in the Indian rhinoceros (Rhinoceros unicornis)

A study was undertaken to identify urinary estrogen and progesterone metabolites in the female Indian rhinoceros (Rhinoceros unicornis). Measurements of these metabolites were then used to monitor ovarian function and establish normal levels and patterns of steroid excretion during the estrous cycle and pregnancy. Urine samples were analyzed for estrone sulfate and pregnanediol-3-glucuronide (PDG) by direct radioimmunoassays. Both hormones produced discrete profiles reflecting ovarian activity in nonconceptive cycles. The estrous cycle was observed to be 48 days (range 39–64) with a mean follicular phase of 14.8 days (range 13–19), followed by a mean luteal phase of 19 days (range 17–21). Of the single gestation monitored, PDG levels rose above luteal phase levels by the third month after breeding and remained elevated throughout gestation. The combined estrogen and progesterone metabolite profiles present a complete evaluation of ovarian steriod production in the mature female Indian rhinoceros.     

Urinary steroid evaluations to monitor ovarian function in exotic ungulates: X. Pregnancy diagnosis in Perissodactyla

Enzymeimmunoassays (EIAs) for estrone conjugates (EC), pregnanediol-3-glucuronide (PDG), and C-19 and C-21 progesterone metabolites (C-19/C-21) were used to analyze urine samples from four nondomestic equid species, four tapir species, and two rhinoceros species in an attempt to identify if these assays could be used for diagnosing and monitoring pregnancy. The same urine samples were also analyzed for the presence of equine chorionic gonadotropin (eCG) activity, using a field dipstick test and a radioimmunoassay (RIA). The EC EIA was validated for three equid species and the Malayan tapir. Neither the PDG nor the C-19/C-21 EIAs were validated in any species evaluated. In equid species, the EC EIA demonstrated a specificity (the percentage of nonpregnant samples identified correctly) of 100% and a sensitivity (the percentage of pregnant samples identified correctly) of ≥ 88%. With the exception of the Grevy's zebra, the C-19/C-21 EIA showed a similar accuracy in identifying pregnant and nonpregnant equids. The PDG EIA was not sufficiently accurate to merit its use in equids or tapirs for pregnancy diagnosis. From the data collected, it appears analysis of a single urine by both the EC EIA and the C-19/C-21 EIA would be the best method of pregnancy detection during the last 2 trimesters of gestation, in equid species. In tapirs, the C-19/C-21 EIA was slightly more accurate for pregnancy diagnosis than the EC EIA. The C-19/C-21 EIA had a specificity of 93%, but a sensitivity of only 73% in tapir species. None of the EIAs evaluated demonstrated a sufficient specificity or sensitivity to be useful, as presently performed, for pregnancy diagnosis from a single sample in the black rhinoceros. The eCG dipstick used in this study did not prove a sufficiently reliable test for routine pregnancy in nondomestic equids. The eCG RIA results in the Przewalski's horses and the Hartman's mountain zebra were positive early in gestation, and indicate that gonadotropin analysis may be useful for pregnancy detection in these species. Only very low amounts of eCG activity was measured by the eCG RIA in the tapir and rhinoceros urine samples. © 1994 Wiley-Liss, Inc.     

The development and application of an enzyme immunoassay for urinary estrone conjugates

An enzyme immunoassay (EIA) for estrone conjugates is described and applied to urine samples from a female Indian rhinoceros, a female gorilla, and a female lion-tailed macaque. Concomitant measures of estrone conjugates in the same sample are compared to the values obtained with radioimmunoassay. High correlation coefficients for values obtained from each assay indicate that EIA measurements provide information that is comparable to values obtained by radioimmunoassay. EIA methods for urinary steroid conjugates can provide a practical tool to evaluate female reproductive status of zoo species without the need for a traditional endocrine laboratory.     

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.     

The detection of ovulation and early pregnancy in the baboon by direct measurement of conjugated steroids in urine

The pattern of excretion of urinary steroid metabolites in the olive baboon (Papio anubis) was examined during the menstrual cycle and in conception cycles in which embryos were surgically removed at intervals between day 11 and day 21 (day 0 = day of preovulatory estrogen peak). Conjugated estrone and pregnanediol-3α-glucuronide were measured in overnight urine samples by direct, nonextraction assays, and the levels were indexed by creatinine. Results showed that measurement of urinary conjugated estrone reflected preovulatory estrogen output and that pregnanediol-3α-glucuronide was an abundant urinary metabolite of progesterone. There was a defined postovulatory increase in the excretion of conjugated estrone during conception cycles in eight of ten animals. The timing of the increase ranged between day 13 and day 19 and was related to the appearance of elevated levels of urinary gonadotrophin. In four animals, increased estrogen excretion was first detected after the day of embryo removal, but this was most likely a response to chorionic gonadotrophin secreted before surgery. The findings demonstrate that measurement of conjugated estrone offers a rapid and practical approach for monitoring ovulation and implantation in the baboon by a single assay technique.     

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.     

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.     

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.     

Urinary and plasma gonadotropin concentrations in golden lion tamarins (Leontopithecus r. rosalia)

This paper describes the development and validation of a plasma and urinary gonadotropin immunoassay for golden lion tamarins (Leontopithecus rosalia), an endangered New World callitrichid primate. The assay is derived from a macaque chorionic gonadotropin assay and was validated for both plasma and urine samples in L. rosalia. Levels of immunoreactive LH/CG in lion tamarin urine were highly correlated (r = + 0.98) with gonadotropin bioactivity. Immunoreactive LH/CG levels were examined in two contexts: in the urine of adult females and in the plasma of adult males after administration of estrogen. Peaks of gonadotropin excretion were detected in samples collected from nonpregnant adult females. The peaks occurred immediately prior to cyclic elevations in urinary estrogen excretion. Plasma LH/CG concentration in males measured 24 and 48 hours after a single 50 μg injection of estradiol benzoate were significantly lower than levels at these time points measured after control treatment. Together, the results of this study point to the utility of the gonadotropin assay for monitoring reproductive function in both female and male lion tamarins.     

Estrogens in Plasma of the Hagfish,Myxine glutinosa (Cyclostomata)

Concentrations of estrogens in the plasma of Myxine glutinosa were measured in an attempt to determine whether different stages of reproduction are connected with certain levels of steroid concentration. After extraction with diethylether and column chromatography on Sephadex LH-20, the plasma was radioimmunoassayed for estrogens. Samples were subdivided into 9 groups according to the gonadal conditions of the animals. There appears to be a positive correlation in females between plasma estrogen concentration and egg size (taken as an indication of reproductive stage). After ovulation the level of plasma estrogens fell appreciably. Estrogen levels of most of the samples from male animals were also determined.     

Evidence for sulfatase and 17beta-hydroxysteroid dehydrogenase type 1 activities in equine epididymis and uterus

Our previous work showed that stallion testis produces high amounts of estrogens which are subsequently found in the ejaculate. These estrogens are mainly synthesized by testicular aromatase, and the major estrogen produced is estrone sulfate (E1S). The objective of this study was to investigate the potential role of E1S as a source of estrogens in the male and female horse reproductive tracts by determining whether both estrone sulfatase (Sulf) and 17beta-hydroxysteroid dehydrogenase type I (17beta-HSD1) activities were present in equine testes, epididymis and uterus. We assessed E1S bioconversion into estrone (E1) and estradiol (E2) in these tissues. Both Sulf and 17beta-HSD1 activities were well detected in the cauda epididymis and uterus. Additionally, Sulf activity was present in the distal corpus of the epididymis, and 17beta-HSDI in the proximal corpus. In contrast, aromatase gene expression, measured as an internal control of endogenous estrogen production, had high activity only in the testis. We found that seminal E1S of testicular origin can be metabolized to E2, especially in the cauda epididymis and uterus. Because E2 appears to play a major role in male and female reproduction, we propose that the bioconversion of seminal E1S could affect male and female fertility.     

Black rhinoceros (Diceros bicornis) in U.S. zoos: II. behavior,breeding success,and mortality in relation to housing facilities

The captive population of black rhinoceros (Diceros bicornis) is not self‐sustaining. The reasons for suboptimal reproduction and high mortality need to be investigated. This can only be achieved by cross‐institutional analyses of environments, behavior, and performance. In this study, we collected data on 23 zoos with black rhinoceros to compare zoo environments with reproductive success, mortality, and behavior. Institutional variation was characterized by enclosure area, percentage of walls around enclosure perimeter, percentage of public access along enclosure perimeter, climate, noise level, number of years zoo has maintained black rhinoceros, frequency of chlorine use, and number of male and female black rhinoceros at a zoo simultaneously. Birth and death rates for each institution were calculated from studbook records. We found that the breeding success of a zoo since 1973 correlated positively with enclosure area, and zoos with two or more females had a lower reproductive rate than zoos with only one female. Females residing during their pre‐reproductive years at a zoo with another reproductive female gave birth for the first time on average 3 years later than sole females. Mortality since 1973 correlated positively with percentage of public access. In Part I, we developed behavior profiles of 29.31 individual black rhinoceros from keeper ratings. Scores for males on the behavior trait Fear also correlated positively to percentage of public access, and we suggest that this aspect of black rhinoceros exhibits is a stressor for this species, especially the males. We found that different aspects of captive environments are associated with male and female black rhinoceros behavior. Male scores on the behavior trait dominant were higher in smaller enclosures, and female scores for a group of behaviors suggesting agitation (chasing/stereotypy/mouthing) were positively correlated with percentage of walls in their enclosure. These two behavior traits were found in Part I to be negatively correlated with the breeding success of an individual male or female. We re‐surveyed the behavior and husbandry of 29 black rhinoceros pairs in zoos 2 years after the original data were collected. The re‐survey confirmed that compatible black rhinoceros pairs are those with assertive females and submissive males, and that enclosure area and a low percentage of concrete walls around the enclosure are positive predictors of a pair's reproductive success. We conclude that temperament traits of individuals and characteristics of their captive environments both have an impact on a pair's breeding success. Our study demonstrates that cross‐institutional comparisons of zoo facilities, when integrated with behavioral assessments of individual animals, are a valuable tool for investigating potential causes of poor reproduction and well‐being in zoo animals. Zoo Biol 18:35–52, 1999. © 1999 Wiley‐Liss, Inc.     

Excretion of radiolabeled estradiol in the cat (Felis catus,L): A preliminary report

The time course and end products of estradiol metabolism were studied in the domestic cat, which has been chosen as a model for steroid metabolism studies in nondomestic felidae. Radiolabeled estradiol was injected intravenously into three adult female cats; one had a spontaneous estrus, one was induced with follicle-stimulating hormone, and one had been ovariohysterectomized; feces, urine, and blood were collected daily, and the radioactivity content was determined. Feces and urine contained 47 and 1% of the injected dose (0.33 μCi), respectively. Metabolites appeared earlier in the urine than in feces (d 1 vs d 2 postinjection), and excretion was completed on d 5; no radioactivity was detected in plasma 24 h postinjection. Estradiol metabolites were excreted as unconjugated estrogens (22%) and as conjugates hydrolyzable with β-glucuronidase and acid solvolysis (7 and 50%, respectively); the remaining 14% were not recoverable with any of the above methods. The major portion of the conjugates was estradiol-17β (64–80%) while 11–16% appeared as estrone. Endogenous cycles related to the spontaneous and induced ovarian activity were monitored by observation of estrous behavior, vaginal epithelium cornification, and plasma estradiol determination. The reproductive state of each animal had no effect on the time course or type of metabolite excreted. We found low proportions of injected radioactivity excreted in the urine and high residual levels remaining after hydrolysis and extraction in the feces. These findings suggest that although feces are an abundant source of estradiol metabolite in the cat, and probably in the exotic felidae, development of noninvasive methods for monitoring ovarian cycles in these species will depend on more efficient methods for urine hydrolysis, on the resolution of problems encountered in fecal steroid analysis, or on the identification of metabolites which may be measured directly in the urine without hydrolysis or extraction.     

Direct measurements of urinary estrone conjugates during the normal menstrual cycle of the gorilla (Gorilla gorilla)

A direct immunoassay for urinary estrone conjugates (estrone sulfate and estrone glucuronide) was used to assess the preovulatory estrogen rise in normal gorilla menstrual cycles. Immunoreactive estrone conjugates in samples concomitantly assessed for total estrogen immunoreactivity reflected similar profiles throughout the cycle; however, the speed and resolution of the direct assay for conjugates indicate this method to be more accurate in monitoring ovulation than the measurement of total immunoreactive estrogens. In a single conceptive ovarian cycle, urinary estrone conjugate continued to rise in the luteal phase, indicating that this test may also be useful for detecting early pregnancy. The application of this technique provides clear profile of ovarian function in gorillas as well as in other primate species.     

Urinary Progestogen and Estrogen Excretion During Pregnancy in Eulemur macaco flavifrons,E. rubriventer,and Hapalemur griseus occidentalis

Via a non-invasive approach, we aimed to provide comparative data on the presence and relative abundance of progestogen and estrogen metabolites excreted into the urine of Eulemur rubriventer, E. macaco flavifrons and Hapalemur griseus occidentalis and to characterize the patterns of progestogen and estrogen excretion during pregnancy. We found that estrone is the major urinary estrogen in Eulemur macaco flavifrons and Hapalemur griseus occidentalis, while 16-hydroxyestrone appeared to be the predominant estrogen in the urine of E. rubriventer. Estradiol-17ß was either absent (Hapalemur) or present in very low amounts. HPLC of progestogens had high levels of immunoreactivity in an assay against 5-pregnane-3-ol-20-one (5-P-3OH) in each species, though the nature of the progestogens measured by the 5-P-3OH assay differed among species. During pregnancy, 5-P-3OH levels remained relatively low in Eulemur rubriventer, whereas in E. macaco flavifrons and Hapalemur griseus occidentalis, a sustained elevation in levels occurred from mid-pregnancy until the final weeks before parturition. Estrogen excretion differed depending on the sex of the fetus in each species, with only females carrying male infants showing clearly elevated estrogen levels during the last 6–8 weeks of gestation. In conclusion, we demonstrate both species similarities and differences in the metabolism and urinary excretion patterns of reproductive hormones throughout gestation in the Lemuridae. The data not only extend our knowledge on the reproductive physiology of lemurs but also show that more studies on other lemur taxa are needed to provide a broader basis for interspecific comparison.     

Direct radioimmunoassay of specific urinary estrogen glucosiduronates in normal men and nonpregnant women.

Direct radioimmunoassay are described for the measurement of each of three specific estrogen glucosiduronates: estrone glucosiduronate, 17 beta-estradiol-17-glucosiduronate and estriol-16 alpha-glucosiduronate in urine. Each assay utilizes a specific antiserum prepared by complexing the carboxylic acid group of the appropriate glucosiduronate to the epsilon-amino group of lysine in bovine serum albumin or bovine thyroglobulin. The antisera showed little or no cross reactivity toward other estrogens that might be present in significant amounts in urine. These antisera were used for the direct assay of the conjugates in urine from normal men and nonpregnant women without prior extraction or chromatography. The values were similar to those obtained after extraction, chromatographic purification on DEAE-Sephadex and subsequent immunoassay; The following mean values +/- SE (microgram/g creatinine) were obtained: estrone glucosiduronate, male 10.1 +/- 0.6, follicular phase female 17.3+/- 1.6, luteal phase female 31.8 +/- 2.5; 17 beta-estradiol-17-glucosiduronate, male 1.7 +/- 0.3, follicular phase female 2.4 +/- 0.1, luteal phase female 4.2 +/- 0.4; estriol-16 alpha-glucosiduronate, male 1.8 +/- 0.2, follicular phase female 4.7 +/- 0.9, luteal phase female 10.0 +/- 1.6.     

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.     

Urinary and plasma estrogen conjugates, estradiol and estrone concentrations in nonpregnant and early pregnant mares

A direct radioimmunoassay for estrogen conjugates (EC) was applied to paired blood and urine samples collected from 20 mares and compared against estrone (E(1)) and estradiol-17beta (E(2)) to monitor changes in estrogen production during ovulatory cycles and early pregnancy. Blood samples were taken daily from five mares through two consecutive ovulations and from six mares at 6-h intervals starting 48 hours prior to ovulation and continuing after ovulation had occurred. Blood samples were also collected daily or three times per week from conception until Day 60 of pregnancy in nine pregnant mares. The mean urinary EC, plasma EC and plasma E(2) dynamics were parallel in nonpregnant mares, with a 3-fold increase in mean urinary EC concentrations from baseline to the ovulatory peak, a 1.8-fold increase in mean plasma EC concentrations and a 1.4-fold increase in mean plasma E(2) concentrations. In early pregnancy, a two-fold increase in mean plasma E(1) and EC concentrations occurred in concert with a five-fold rise in mean urinary EC concentrations, whereas plasma E(2) did not change. Following hydrolysis and chromatographic separation, E(1) and E(2) were identified as the hydrolytic products in the urine of nonpregnant and pregnant mares; however, an unidentified estrogen was the major hydrolytic product in nonpregnant mares and pregnant mares prior to Day 38 of pregnancy. The increased resolution of the EC profiles compared with the profiles of other estrogen components indicates that the determination of EC in urine or plasma provides a useful alternative method for monitoring reproductive events in mares.     

Menstrual cycle characterization and artificial insemination in the black mangabey (Cercocebus aterrimus)

Concentrations of immunoreactive estrone conjugates, pregnanediol-3-glucuronide, and luteinizing hormone were measured and indexed to creatinine in daily urine samples from three female black mangabeys (Cercocebus aterrimus). Daily observations of menstruation and perineal tumescence were recorded. The mean ± SEM lengths of the menstrual cycle [apparent cycle length of 26.0 ± 0.8 days determined by observation of intermenstrual intervals (n = 26); physiologic cycle length of 31.3 ± 5 days determined by urinary endocrine analysis (n = 4)], follicular phase [16.5 ± 4 days (n = 4)], and luteal phase [14.8 ± 1 day (n = 4)] were determined. The apparent cycle length is probably more accurate. Perineal tumescence began during or shortly after menstruation, increased concomitantly with increasing follicular phase conjugated estrone values, and reached maximal size in the periovulatory period. Ovulation was closely followed by a drop in conjugated estrone levels, an increase in urinary pregnanediol-3-glucuronide, and perineal detumescence. Peak concentrations of conjugated estrone and luteinizing hormone values were coincident. Pregnanediol-3-glucuronide accurately reflected luteal function in the black mangabey. Knowledge of the menstrual cycle parameters and their correlation to perineal tumescence was used to time artificial inseminations. Semen was obtained by rectal electroejaculation. Coagulum and extended semen, or trypsin-digested coagulum, were used for insemination. One insemination of trypsin-digested coagulum at the external os of the cervix resulted in a probable conception, follówed by apparent abortion after 3 weeks.     

Declining and low fecal corticoids are associated with distress, not acclimation to stress, during the translocation of African rhinoceros

Concentrations of adrenal steroid metabolites in feces are routinely used to assess the welfare of animals that are the subject of conservation efforts. The assumption that low and declining corticoid concentrations indicate the absence of stress and acclimation, respectively, is often made without experimental support or wild-animal comparisons, although intrinsic control of adrenal steroids might occur even under ongoing stress and distress. We adopted the capture and 11-week captivity of 18 black ( Diceros bicornis : 11 males, seven females) and 52 white ( Ceratotherium simum : 22 males, 30 females) rhinoceros as an experimental test of the relationship between corticoid concentrations and stress (translocation) and measured for suppressed gonad function as an indicator of distress – the biological cost of cumulative stressors. Fecal samples collected from the rectum at capture and during captivity were stored frozen and their corticoid, and androgen (in males) or progestin (in females), concentrations determined by radioimmunoassay. Corticoid profiles followed the expected pattern of being two to five times pre-capture levels (ng g⁻¹: black rhino: female 24.5±3.7, male 23.9±2.2; white rhino: female 16.3±1.6, male 12.3±2.4) for up to 17 days after capture and declined with time in captivity. Black rhinoceros and male white rhinoceros corticoids declined below pre-capture values and were associated with suppressed levels of androgens and progestins with increased time in captivity. Declining corticoids could not be interpreted as acclimation or the absence of stressors, without also measuring for distress in African rhinoceros. White rhinoceros female corticoid values remained elevated, although their gonad steroid levels were also suppressed. We discuss our findings for the management of rhinoceros in the wild and captivity.