Assessment of female reproductive status in captive-housed Hanuman langurs (Presbytis entellus) by measurement of urinary and fecal steroid excretion patterns.код для вставкиСкачать
American Journal of Primatology 37:27&284 (1995) RESEARCH ARTICLES Assessment of Female Reproductive Status in Captive-Housed Hanuman Langurs (Presbytis entellus) by Measurement of Urinary and Fecal Steroid Excretion Patterns M. HEISTERMANN', M. FINKE?, AND J.K. HODGES' 'Department of Reproductive Biology, German Primate Center, and 'Znstitute Anthropology, Uniuersity of Gottingen, Gottingen, Germany of The study reports on the use of urinary and fecal hormone measurements for monitoring female reproductive status in captive-housed Hanuman langurs (Presbytis entellus). Matched urine and fecal samples collected throughout '7 complete menstrual cycles of two females, and during part of one pregnancy in a third female were analyzed. Estrone conjugates (ElC) and immunoreactive pregnanediol glucuronide (PdG) in urine and immunoreactive estradiol (E2), progesterone (P41, pregnanediol (Pd) and 20ahydroxyprogesterone (20aOHP) in feces were measured by enzymeimmunoassay. E1C and PdG in urine were excreted in a cyclic pattern with E1C levels increasing 3- to 4-fold during the follicular phase t o reach preovulatory peak values 2 days before a defined rise in PdG concentrations. Cycle lengths ranged between 20 and 34 days comprising a variable follicular phase of 7-21 days and a more consistent luteal phase of 12-14 days. High pressure liquid chromatography (HPLC) analysis of fecal extracts confirmed the presence of all fecal hormones measured, but indicated large amounts of additional immunoreactivity in the three progestin assays. The patterns of excretion of fecal E2 and all three fecal progestins corresponded well with those of steroid metabolites in urine in showing a clear and well defined follicular phase E2 rise followed by a luteal phase progestin increase. Measurement of 20aOHP immunoreactivity revealed the most stable baseline and the highest follicular/luteal phase differential. Levels of all hormones were clearly elevated during pregnancy although urinary E1C and PdG showed a more pronounced increase compared to fecal metabolites. The results indicate that urinary and fecal hormone analysis can be applied to noninvasive monitoring of reproductive status in the Hanuman langur. o 1995 Wiley-Liss, Inc. Key words: Presbytis entellus, ovarian cycle, steroid excretion, urinary and fecal hormones, non-invasive methods Received for publication September 12, 1994; revision accepted February 2, 1995 Address reprint requests to Dr. Michael Heistermann, Department of Reproductive Biology, German Primate Center, Kellnemeg 4, 37077 Gottingen, Germany. 0 1995 Wiley-Liss, Inc. 276 I Heistermann et al. INTRODUCTION Field research on the Hanuman langur (Presbytis entellus) has focussed predominantly on questions regarding male and female reproductive strategies under different socioecological conditions [e.g., Hrdy, 1977; Winkler et al., 1984; Agoramorthy et al., 1988; Sommer & Rajpurohit, 19891. However, many questions concerning the mechanisms by which environmental pressures influence male and female reproduction have remained unanswered due to the difficulties in accurately determining female reproductive status in free-ranging animals. Reproductive assessment so far has been limited to observations of menstruation and sexual behavior [Sommer et al., 19921, measures which are both indirect and variable. Endocrine monitoring would enable a more direct assessment of female reproductive status, but information on the reproductive endocrinology of the Hanuman langur is limited and a reliable approach to noninvasive reproductive assessment suitable for application in the field has hitherto not been described. Although estrone and pregnanediol have been identified as the major urinary metabolites of estrogen and progesterone, respectively, during the ovarian cycle [Ramaswami, 19751, patterns of excretion were described only for estrone determined by chemical methods during two cycles [Shandilya et al., 19761. Furthermore, for the Hanuman langur, or any other species of Colobinae, no information exists on the measurement of hormones in feces or on the feasibility of applying this approach for monitoring reproductive status. The aims of the present study are therefore (i) to further define the endocrinology of the ovarian cycle in the Hanuman langur by the direct measurement of estrone conjugates and immunoreactive pregnanediol glucuronide in urine, and (ii) to establish and validate a method for the assessment of female reproductive status through the measurement of estrogen and progesterone metabolites in feces. METHODS Animals Three adult female Hanuman langurs of the Sri Lanka subspecies, Presbytis entellus thersites, aged 6.5-8 years were used in this study. Females were maintained together with an adult male and one subadult animal of each sex a t Krefeld Zoo, Germany. The animals were housed in an indoor enclosure with access to a seminatural outdoor island. A mixture of fruit and vegetables, supplemented with eggs, seeds, nuts and fresh leaves, were fed twice daily and water was available ad libitum. Sample Collection Through a total of seven ovarian cycles (3 and 4 cycles for each of two females) matched urine and fecal samples were collected 5-7 days a week and during the last two-thirds of one full-term pregnancy (including 5 weeks postpartum) in the third female. Samples were collected between 06.00 h and 10.00 h by positioning plastic sheets on the cage floor under the sleeping place of the animals, which were then observed until urination and defecation occurred. Only samples which were not cross-contaminated were collected (usually within 40 min of observation). In order to detect menstruation, urine samples were checked for the presence of blood by use of a stick test (Combur-9-Test, Boehringer Mannheim, Germany). Samples were frozen without preservatives at -20°C until analyzed. Sample Preparation and Hormone Assays Urine samples were analyzed for steroid conjugates by direct assays, without hydrolysis and extraction. Fecal samples (total amount collected) were lyophilized Steroid Excretion in Presbytis entellus I 277 and pulverized as described by Heistermann et al. [19931, and 0.05-0.10 g of the fecal powder was extracted with 5 ml 80% methanol by vortexing for 15 min. Following reduction of the extract to approximately 0.5 ml by evaporation under nitrogen, unconjugated steroids were extracted into diethylether (5 ml), and after evaporation to dryness, were reconstituted in 1 ml absolute methanol for assay. The overall mean recovery of 3H-progesterone used to monitor individual extraction efficiencies [see Heistermann et al., 19931 was 89.4 ? 0.3%(X +- S.E.M., n = 276 samples). Estrone conjugates (ElC) and immunoreactive pregnanediol-3-glucuronide (PdG) in urine samples were determined by direct microtiterplate enzymeimmunoassays as described by Heistermann and Hodges . Relative to estrone-3glucuronide (100%)significant cross-reactions in the E1C assay were only found for unconjugated estrone (71%)and estrone-3-sulfate (17%),with values < 1%for all other steroids tested. Cross-reactions in the PdG assay were PdG loo%, free pregnanediol22%, 20a-hydroxyprogesterone 32%,5a-pregnane-20a-ol-3-one 14%and < 1%for all other steroids tested, including androsterone and related C-19 steroids. Immunoreactive estradiol (E2) in fecal extracts was determined by microtiterplate enzymeimmunoassay using an antiserum raised in a rabbit against estradiol-6-carboxymethyloxime-(CMO)-BSA (donation from H. Meyer, Berlin). Estradiol-3-CMO coupled with peroxidase was used as a label. The antiserum showed the following cross-reactivities (at 50% binding) relative to estradiol (100%):estrone: 4.0%; estriol: 0.9%;estrone-3-glucuronide and estrone-3-sulfate: 0.6% and I 0.1% for all other steroids tested. In brief, diluted fecal extracts (50 p1 aliquots in duplicate) and E2 standards (50 p1, 0.49-125 pg per well) were combined with labelled E2 (50 pl) and antiserum (50 p1) and incubated at room temperature and in darkness for 4 h. After incubation, the plates were washed four times before peroxidase substrate solution (150 pl, containing 0.025% tetramethylbenzidine and 0.05% H,O,) was added, and the plates incubated in darkness for another 40 min. The enzyme reaction was stopped by the addition of 50 p12 mol/l H,SO, and absorbance measured at 450 nm on an automatic plate reader. Sensitivity of the assay was 0.9 pg per well. The antiserum for the measurement of immunoreactive 20a-hydroxyprogesterone (20aOHP) was raised in a rabbit against 20aOHP-20-CMO-BSA.Biotinylated 20aOHP (donation from E. Mostl, Vienna) in conjunction with peroxidaselabelled streptavidin 6-512, Sigma Chemie, Deisenhofen, Germany) was used as conjugate. The antiserum showed the following cross-reactivities (at 50% binding) relative to 20aOHP (100%):5a-pregnane-20a-ol-3-one: 25%; 5a-pregnane-20p-01%one: 1.9%; progesterone: 0.8%; pregnanediol: 0.3% and < 0.1% for all other steroids tested. In brief, diluted fecal extracts (50 p1) and 20aOHP standards (50 pl, 0.49-125 pg per well) were combined with labelled 20aOHP (50 p1>and antiserum (50 pl), and assayed in the same way as urinary PdG [Heistermann & Hodges, 19951. Sensitivity of the assay was 0.8 pg per well. Immunoreactive pregnanediol (Pd) and progesterone (P4) were measured by enzymeimmunoassays previously described by Heistermann et al. . To control for the variations in the volume and concentration of the voided urine, the creatinine content of each sample was determined by a creatinine analyzer (Beckmann Instruments, Brea, CA) using 1:20 diluted urine samples. Urinary hormone concentrations are expressed as masslmg creatinine (Cr). Validation of Urinary and Fecal Hormone Measurements Serial dilutions of urine and fecal extracts from samples of all reproductive stages gave displacement curves parallel to those obtained with the appropriate 278 I Heistermann et al. standards. Accuracy was assessed by determining the recovery of known amounts of pure steroid (five doses) added in quadruplicate to urine or to fecal powder before extraction. Mean & S.D. recovery values ranged from 89.3 2 3.3% for E2 to 118.7 k 9.6% for 20aOHP; recovery values for all other hormones were within these figures. Precision of all assays was assessed by replicate determinations of urine and fecal quality control pools. Intra-assay coefficients of variation ranged between 3.7% (ElC) and 11.2% (20aOHP), while interassay variability ranged between 9.6% (E2) and 19.5%(Pd). Specificity of fecal hormone measurements was assessed by high pressure liquid chromatography (HPLC) analysis of samples from the follicular and luteal phase of the ovarian cycle and mid- and late stages of pregnancy. Steroids were separated according to the method described by Heistermann et al. 119931. Coelution of immuno- and radioactivity of added tritiated Pd and P4 tracers confirmed the presence of substantial amounts of these hormones measured in the Pd and P4 assay, respectively, but indicated also the presence of additional immunoreactivity. The majority of immunoreactivity detected in the 20aOHP EIA did not coelute with authentic 20aOHP tracer, but was associated with a single peak with a polarity slightly less polar than that of 20aOHP. Thus, only minor amounts of 20aOHP appear to be present in feces and the 20aOHP measurements reported are predominantly of undetermined immunoreactivity. HPLC of fecal estrogens revealed that estrone ( E l ) was usually more abundant than estradiol (E2) but since there was no clear pattern of excretion of E l , measurement of E2 was used to generate cycle profiles. HPLC cochromatography indicated the measurement of E2 to be specific. Analysis of Data The follicular phase was defined a s the interval between the first day of menstruation until the day of the urinary E1C peak (day 01, whereas the luteal phase comprised the interval from the day after the E l C peak until the day before the next menstruation. A threshold value of three standard deviations above the mean of the preceding follicular phase values was calculated to assess timing of the first postovulatory increase in urinary and fecal progestin concentrations. An increase in concentrations above this threshold value was taken to indicate a statistically significant rise (P< 0.01) [Sachs, 1975; Jeffcoate, 19831. RESULTS The pattern of excretion of immunoreactive urinary E1C and PdG throughout two complete menstrual cycles in a n individual female is shown in Figure la. Although direct evidence for ovulation was not available, the profiles reveal a cyclic pattern of hormone excretion in which the presumed follicular and luteal components of the cycle can be clearly distinguished. Composite data for seven cycles, normalized to the day of the E1C peak, are shown in Figure 2a. Mean levels of E1C increased 3 to 4-fold during the follicular phase, from baseline levels of 130 ng/mg Cr to presumed preovulatory peak values of 450 ng/mg Cr. Thereafter, levels returned to those seen in the early follicular phase and there was no obvious secondary luteal phase increase. PdG levels were consistently low during the follicular phase (0.1-0.2 pg/mg Cr), but rose significantly 2 days after the E1C peak to reach maximum luteal phase values of 3 pg/mg Cr. Ten days after the E1C peak, mean PdG levels began to decline and had returned to baseline by the first or second day of menstruation. The pattern of excretion of E2 and 20aOHP immunoreactivity in feces throughout the menstrual cycle is shown individually in Figure l b and as com- 800 1 (4 600 - 400 - .ma W 10 0 20 30 40 50 n 400 4 - - 8 5 l .-I (b) c A ? a M 23. W PI X 0 0 10 20 30 40 50 0 Days of s a m p l e c o l l e c t i o n Fig. 1. Hormonal profiles of (a)estrone conjugates 1ElC) and immunoreactive pregnanediol glucuronide (PdG) in urine and (b)immunoreactive estradiol 1E2)and 20a-hydroxyprogesterone (20aOHP) in feces during two consecutive menstrual cycles in a Hanuman langur (female 2). The horizontal bars indicate the periods of menstruation. posite profiles in Figure 2b. Fecal measurements correspond well with their urinary counterparts in showing a clear and well defined follicular phase estrogen rise followed by a characteristic luteal phase progestin increase. Mean levels of fecal E2 showed a 5 to 6-fold increase during the follicular phase to reach a maximum value of 170 ng/g on the same day as the E1C peak in urine. In three of the seven cycles a delay of one day was seen between urinary and fecal progestin excretion, however, on average, the significant rise in both progestin measurements occurred 2 days after the urinary E1C peak. Maximum mean luteal phase levels of immunoreactive 20aOHP (7 pg/g) were approximately 30-fold higher than those measured in the follicular phase (0.2-0.3 pg/g). Although measurement of 20aOHP immunoreactivity revealed the most stable baseline and largest folliculadluteal phase differential, concentrations of immunoreactive pregnanediol and 280 / Heistermann et al. ,1 600 [ 4 1 (a) 400 2E Y 300 V 4 W A 0 200 1 d h 100 0 0 -10-6-6-4-2 0 2 4 6 6 10 12 14 16 h 250 ,I3 2M . d 200 c R 150 Po \ a" 100 W N W 50 A 0 -10-8-6-4-2 0 2 4 6 . . 8 10 12 14 16 Days before end after urinary EIC peak Fig. 2. Mean ( & S.E.M.) concentrations of (a)estrone conjugates (ElC) and immunoreactive pregnanediol glucuronide (PdG) in urine and (b) immunoreactive estradiol (E2) and 20a-hydroxyprogesterone (20aOHP) in feces representing 7 menstrual cycles in two female Hanuman langurs. Individual profiles were aligned to the day of the E1C peak in urine (day 0).The horizontal bar indicates the period of menstruation. progesterone were also markedly higher in the luteal compared with follicular phase (Table I), indicating that each measurement is potentially suitable for monitoring ovarian cyclicity. The lengths of individual menstrual cycles and their component phases are shown in Table 11. Cycle length in female 2 was stable at 23-24 days, whereas that of female 1varied between 20 and 34 days, essentially due to an extended follicular phase length (19 and 21 days) in two cycles. In the other cycles studied (n = 5), the follicular phase ranged between 7 and 10 days in length. The length of the luteal phase was between 12 and 14 days (n = 7). Urinary and fecal estrogen and progestin concentrations during the last twothirds of an individual full-term pregnancy and through the early postpartum period are illustrated in Table 111. Compared to levels in nonpregnant animals (see Figs. 1and 2 and Table I), mean concentrations of all five metabolites were clearly elevated by midpregnancy, although, with the exception of urinary ElC, there was a slight overlap of values. Mean concentrations of all hormones were highest during the last third of gestation, with estrogens (particular urinary E1C) showing the Steroid Excretion in Presbytis entellus I 281 TABLE I. Concentrations of Immunoreactive Fecal Progestins in the Follicular and Luteal Phases of the Ovarian Cycle of Two Hanuman Langurs Stage of cycle Fecal pragestin Follicular phase (days -6 to -Ub Luteal phase (days 5 to 10.9 k 0.9" 0.16 ? 0.01 0.06 ? 0.01 122.3 ? 9.9 5.1 t 0.3 0.3 k 0.03 Pregnanediol' 20cu-hydraxyprogesteronec Progesteroned "All values are expressed as mean ? S.E.M. in +gig dry weight bDay 0 = day of urinary E1C peak. 'Values calculated across 7 ovarian cycles. dValues calculated across 2 ovarian cycles. TABLE 11. Menstrual Cycle Characteristics in Two Hanuman Langurs* ~~ Female 1 Cycle lengtha Follicular phase lengthb Luteal phase length' Duration of menstruationd Female 2 Cl c2 c3 c4 c5 C6 c7 20 7 13 4 20 8 12 6 31 19 12 4 34 21 13 5 23 10 13 3 23 10 13 4 24 10 14 4 *All values are expressed in days. Yntermenstrual interval. bInterval between first day of menstruation and day of urinary E1C peak. 'Interval between first day after urinary E1C peak and day before menstruation. dAs detected by the presence of blood in urine. clearest increase in concentrations compared with levels in nonpregnant animals. Hormone concentrations declined to follicular phase values within 3 days (urinary metabolites) and 5 days (fecal metabolites) following parturition and remained low until the end of the sampling period. DISCUSSION The present study provides the first detailed account of the measurement of excreted steroid metabolites for noninvasive monitoring of female reproductive status in the Hanuman langur. The results show that measurements of estradiol and progesterone metabolites in both urine and feces are reliable methods for documenting ovarian cyclicity and pregnancy. Our results confirm and extend the findings of Shandilya et al.  and Ramaswami [19751 that estrone and pregnanediol are abundant steroid metabolites in the urine of cycling langurs. Although several studies have shown that androsterone is a major urinary progesterone metabolite in cercopithecines [e.g., Liskowski & Wolf, 1972; Shideler et al., 19931, it is unlikely that this metabolite contributed significantly to the PdG values reported here. In the first instance, cross-reactivity of androsterone in the PdG assay was < 0.1% and secondly, pregnanediol was reported to predominate over androsterone in both cycling and pregnant langurs [Ramaswami, 19751. The results of the present study furthermore demonstrate that both PdG and E1C are excreted in a cyclic pattern, with E1C rising from early follicular phase levels to a preovulatory peak followed by a significant rise in PdG during the luteal phase. The profiles of urinary E1C and 282 / Heistermann et al. TABLE 111. Concentrations of Urinary and Fecal Immunoreactive Steroids During Mid- and Late Pregnancy and the Postpartum Period in an Individual Hanuman LanPur Reproductive stage Steroid Urinary EICb Urinary PdGb Fecal E,' Fecal 20aOHPe Fecal Pd' Midpregnant (-140 to -110)" Late pregnant (-50 to -20)" Postpartum ( + S t 0 +35)" 3.2 k 0.2 16.4 1.3 0.5 5 0.06 11.0 2 0.9 242.1 ? 27.1 24.2 t 0.7 27.4 1.7 1.8 0.1 30.2 ? 1.8 563.8 t 35.6 0.02 0.002 0.10 & 0.01 0.01 0.002 0.15 2 0.02 10.2 2 0.7 * * * * * "Day of parturition = day 0. bValues are expressed a s mean 2 S.E.M. in pgimg creatinine. 'Values are expressed a s mean ? S.E.M. in pg/g dry weight. PdG are thus similar to those described for plasma estradiol and progesterone [Lohiya et al., 19881, respectively, indicating that in the Hanuman langur, as in a variety of other primate species [e.g., Eastman et al., 1984; Czekala et al., 1988; Shideler et al., 1990; Pryce et al., 1993; Heistermann & Hodges, 19941, the measurement of E1C and PdG in urine provides reliable information on both follicular development and corpus luteum function. Using the endocrine data to distinguish follicular and luteal components of the cycle, a consistent luteal phase length of 12-14 days was observed. In contrast, the length of the follicular phase showed marked variation (overall range 7-21 days) principally due to an extended follicular period in two of the four cycles of female 1. The reason for this degree of variation is not clear, since both prolonged cycles showed a late follicular phase E1C increase and a luteal phase PdG profile similar to those seen in all other cycles. Since, however, the majority of cycles (5/7) comprised a follicular phase of 7-10 days, we assume this to be more typical for the species and this is in agreement with estimates derived from limited laparoscopic examinations [David & Ramaswami, 19691 and measurements of plasma estradiol [Lohiya et al., 19881. Assuming a follicular phase of 7-10 days, the present data indicate an overall cycle length of 20-24 days, which corresponds well with mean intermenstrual intervals of 22-28 days previously reported for both captive and free-ranging animals [Lohiya et al., 1988; Sommer et al., 1992, for review]. Both estrone and estradiol could be measured in high amounts in feces with estrone usually being more abundant. In contrast to the cyclic pattern of estrone conjugates excreted into the urine, however, the pattern of unconjugated estrone in feces was found to be uninformative, while fecal estradiol corresponded well with the pattern of urinary E1C. While HPLC cochromatography confirmed the presence of substantial amounts of progesterone and pregnanediol in fecal extracts, the data additionally indicated a broad spectrum of metabolites in each of the three progestin assays used. This finding is not surprising and the presence of substantial amounts of immunoreactive fecal progestins has been demonstrated in a variety of other primate species [Ziegler et al., 1989; Heistermann et al., 1993; Shideler et al., 1994; Wasser et al., 19941. Although, quantitatively, greatest amounts were measured in the pregnanediol assay, measurement of fecal 20aOHP immunoreactivity resulted in the most stable baseline and the most pronounced luteal phase increase, suggesting that the metabolites detected in the 20aOHP assay might be more directly related to corpus luteum function. Identity of the compounds contributing to 20aOHP immunoreactivity, however, is not known. Steroid Excretion in Presbytis entellus I 283 Although the data of the present study are limited, they suggest that pregnancy in the Hanuman langur is characterized by the excretion of high levels of urinary and fecal estrogen and progestin metabolites. Both urinary hormones (particularly E1C) showed a more pronounced increase during gestation compared with the excretion of fecal metabolites. Assuming that the metabolism of estrogens and progestins does not change during pregnancy (HPLC profiles of nonpregnancy and pregnancy samples were similar), these results would suggest that during gestation, excretion of hormones via the urine is favored to the fecal route of excretion. Elevated levels of fecal steroids were nevertheless measured in pregnancy samples, indicating that fecal hormone analysis can be used for detecting mid- to late stages of gestation. However, the findings have to be confirmed, and since no samples from early stages of pregnancy were available, the usefulness of urinary and fecal hormone monitoring for early detection of pregnancy remains to be evaluated. Although further work is needed to examine the influence of diet on fecal hormone excretion in these primarily leaf-eating monkeys, as for various other primate species [e.g., Wasser et al., 1991; Strier & Ziegler, 1994; Shideler et al., 19941, the availability of methods for fecal hormone analysis provides new and interesting opportunities for combining behavioral and physiological studies on free-ranging Hanuman langurs. This may in turn lead to a better understanding of the mechanisms by which socioecological factors influence female reproduction. CONCLUSIONS 1. Excretion patterns of estrone conjugates and immunoreactive pregnanediol glucuronide in urine gave a reliable reflection of follicular development and corpus luteum function in the Hanuman langur. 2. Immunoreactive estradiol, progesterone, pregnanediol and 20a-hydroxyprogesterone in feces revealed cyclic patterns which showed a high correspondence with their urinary counterparts, indicating that fecal hormone analysis is a reliable alternative in monitoring ovarian function. 3. The approach of fecal hormone analysis provides a potentially useful tool for monitoring reproductive status in free-ranging langurs. ACKNOWLEDGMENTS We thank the Krefeld Zoo for providing us the opportunity to collect urine and feces from their Hanuman langurs. We also gratefully acknowledge the technical assistance in HPLC analysis provided by B. Bauer and the donations of antisera and enzyme label from H. Meyer and E. Mostl, respectively. The study was supported by a grant (Ho 1391/3-1)from the Deutsche Forschungsgemeinschaft to J.K. Hodges and M. Heistermann. REFERENCES Agoramoorthy, G.; Mohnot, S.M.; Sommer, V.; Srivastava, A. Abortions in free-ranging Hanuman langurs (Presbytis entel1us)-a male induced strategy? HUMAN EVOLUTION 3:297-308,1988, Czekala, N.M.; Roser, J.F.; Mortensen, R.B.; Reichard, T.; Lasley, B.L. Urinary hormone analysis as a diagnostic tool to evaluate ovarian function of female gorillas (Gorilla gorilla). JOURNAL OF REPRODUCTION AND FERTILITY 82:255-261, 1988. David, G.F.X.; Ramaswami, L.S. Studies on menstrual cycles and other related phenomena in the langur (Presbytis entellus entellus). FOLIA PRIMATOLOGICA 11: 300-316, 1969. Eastman, S.A.K.; Makawiti, D.W.; Collins, W.P.; Hodges, J.K. Pattern of excretion of urinary steroid metabolites during the ovarian cycle and pregnancy in the marmoset monkey. JOURNAL OF ENDOCRINOLOGY 102:19-26, 1984. Heistermann, M.; Hodges, J.K. Endocrine 284 I Heistermann et al. monitoring of the ovarian cycle and pregnancy in the saddle-back tamarin (Saguinus fuscicollis) by measurement of steroid conjugates in urine. AMERICAN JOURNAL OF PRIMATOLOGY, 35:117-127, 1995. Heistermann, M.; Tari, S.; Hodges, J.K. Measurement of faecal steroids for monitoring ovarian function in New World primates, Callitrzchidae. JOURNAL OF REPRODUCTION AND FERTILITY 99: 243-251, 1993. Hrdy, S.B. THE LANGURS OF ABU. FEMALE AND MALE STRATEGIES OF REPRODUCTION. Cambridge, Harvard University Press, 1977. Jeffcoate, S.L. Use of rapid hormone assays in the prediction of ovulation. Pp. 67-82 in OVULATION METHODS FOR ITS PREDICTION AND DETECTION. Chichester, John Wiley & Sons Ltd., 1983. Liskowski, L.; Wolf, R.C. Urinary excretion of progesterone metabolites in pregnant rhesus monkeys. PROCEEDINGS OF THE SOCIETY FOR EXPERIMENTAL BIOLOGICAL MEDICINE 139:1123-1 126, 1972. Lohiya, N.K.; Sharma, R.S.; Puri, C.P.; David, G.F.X.; Anand Kumar, T.C. Reproductive exocrine and endocrine profiles of female langur monkeys, Presbytis entellus. JOURNAL OF REPRODUCTION AND FERTILITY 82:485-492, 1988. Pryce, C.R.; Jurke, M.; Shaw, H.J.; Sandmeier, I.G.; Doebeli, M. Determination of ovarian cycle in Goeldiys monkey (Callirnico goeldii) via the measurement of steroids and peptides in plasma and urine. JOURNAL OF REPRODUCTION AND FERTILITY 99:427-435, 1993. Ramaswami, L.S. Some apsects of the reproductive biology of the langur monkey Presbytis entellus entellus Dufresne. PROCEEDINGS OF THE INDIAN NATIONAL SCIENCE ACADEMY 41:l30, 1975. Sachs, L. ed. ANGEWANDTE STATISTIK. Berlin, Springer-Verlag, 1975. Shandilya, L.N.; Ramaswami, L.S.; Shandilya, N. Oestrogen metabolites in urine during the menstrual cycle, pregnancy and puerperium in the Indian hanuman langur (Presbytisentellus entellus). JOURNAL OF REPRODUCTION AND FERTILITY 47: 7-11,1976. Shideler, S.E.; Munro, C.J.; Tell, L.; Owiti, G.; Laughlin, L.; Chatterton, Jr., R.; Lasley, B.L. The relationship of serum estradiol and progesterone concentrations to the enzyme immunoassay measurements of urinary estrone conjugates and immunoreactive pregnanediol-3-glucuronide in Macaca rnulatta. AMERICAN JOURNAL OF PRIMATOLOGY 22:113-122, 1990. Shideler, S.E.; Shackleton, C.H.L.; Moran, F.M.; Stauffer, P.; Lohstroh, P.N.; Lasley, B.L. Enzyme immunoassays for ovarian steroid metabolites in the urine of Macaca fascicularis. JOURNAL OF MEDICAL PRIMATOLOGY 22:301-312,1993. Shideler, S.E.; Savage, A.; Ortuno, A.M.; Moorman, E.A.; Lasley, B.L. Monitoring female reproductive function by measurement of fecal estrogen and progesterone metabolites in the white-faced saki (Pithecia pithecia). AMERICAN JOURNAL OF PRIMATOLOGY 32:95-108, 1994. Sommer, V.;Rajpurohit, L.S. Male reproductive success in harem troops of Hanuman langurs (Presbytis entellus). INTERNATIONAL JOURNAL OF PRIMATOLOGY 10:293-317, 1989. Sommer, V.;Srivastava, A.; Borries, C. Cycles, sexuality, and conception in freeranging langurs (Presbytis entellus). AMERICAN JOURNAL OF PRIMATOLOGY 28:l-27, 1992. Strier, K.B.; Ziegler, T.E. Insights into ovarian function in wild muriqui monkeys (Bruchyteles arachnoides). AMERICAN JOURNAL OF PRIMATOLOGY 32:31-40, 1994. Wasser, S.K.; Monfort, S.L.; Wildt, D.E. Rapid extraction of faecal steroids for measuring reproductive cyclicity and early pregnancy in free-ranging yellow baboons (Papio cynocephalus cynocephalus).JOURNAL OF REPRODUCTION AND FERTILITY 92:415-423,1991. Wasser, S.K.; Monfort, S.L.; Southers, J.; Wildt, D.E. Excretion rates and metabolites of oestradiol and progesterone in baboon (Papio cynocephulus cynocephalus) faeces. JOURNAL OF REPRODUCTION AND FERTILITY 101:213-220,1994, Winkler, P.;Loch, H.; Vogel, C. Life history of Hanuman langurs (Presbytis entellus)reproductive parameters, infant mortality, and troop development. FOLIA PRIMATOLOGICA 43:l-23,1984. Ziegler, T.E.; Sholl, S.A.; Scheffler, G.; Haggerty, M.A.; Lasley, B.L. Excretion of estrone, estradiol, and progesterone in the urine and feces of the female cotton-top tamarin (Saguinus oedipus oedipus). AMERICAN JOURNAL OF PRIMATOLOGY 17:185-195, 1989.