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S. K. Wasser, S. L. Monfort and D. E. Wildt

Summary. A rapid method was developed for extracting and assaying oestradiol and progesterone in faeces (n = 242) of female yellow baboons, free-living in Tanzania. Dose response studies generated slopes of 1·02 (r 2 = 0·99) for oestradiol and 1·09 (r 2 = 0·99) for progesterone, suggesting that this method accurately measured these steroids in faeces. Parallelism was proved by demonstrating that slopes produced from serially diluted samples were not different from those generated from standard curves (mean P value = 0·53 ± 0·17 for oestradiol and 0·44 ± 0·13 for progesterone). Faecal progesterone concentrations measured over several cycles in 2 females increased and decreased in correspondence to visual markers of the luteal phase (i.e. the period between sex-skin detumescence and menses), but the presumed preovulatory oestradiol peak was not observed consistently in all cycles. Progesterone profiles during early to midgestation in 3 females confirmed pregnancy by 25 days (14%) of gestation. Oestradiol profiles were more variable and were not indicative of pregnancy until 40 days (22%) of gestation. Radiolabel-infusion studies revealed that 32% of progesterone (n = 2) but only 11% of oestradiol (n = 2), was cleared through faeces. The latter findings may account for the greater variation observed in temporal oestradiol patterns during the baboon menstrual cycle and pregnancy. Compared with previous techniques, these new methods (i) save considerable time in assaying raw material and (ii) result in high extraction recoveries of faecal steroids ∼88% for oestradiol and 91% for progesterone). This approach may be particularly useful for studying physioloical function and endocrine–environmental interrelationships in free-living primate species.

Keywords: faeces; oestradiol; progesterone; free-ranging; baboon

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S. L. Monfort, N. P. Arthur and D. E. Wildt

Summary. Immunoreactive urinary oestrogen conjugates were assessed in daily urine samples (∼ 5 samples/week) collected from 8 Przewalski's mares maintained under semi-free-ranging pasture conditions. The relative percentage contributions of immunoreactive urinary oestrogens during different reproductive states (oestrus, luteal phase, early, mid- and late gestation) were determined using high-pressure liquid chromatography. In general, conjugated forms of oestrone (oestrone sulphate and oestrone glucuronide) were the major excreted immunoreactive oestrogens in non-pregnant and pregnant Przewalski's mares. Variations in urinary oestrogen conjugates indicated that the onset of oestrous cyclicity coincided with increasing daylengths, and the non-conception oestrous cycle was 24·1 ± 0·7 days (n = 17) in duration. Most copulations (29/35, 82·9%) were observed between Day −4 and Day +1 from the preovulatory oestrogen conjugates peak (Day 0). Based on known copulation dates, the mean gestation length was 48·6 ± 0·4 weeks (range 47·3–50·3 weeks). During pregnancy, urinary excretion of oestrogen conjugates increased ∼ 300-fold over levels in non-pregnant mares, reaching peak concentrations by Week +24 (51% of gestation). These results demonstrate that longitudinal reproductive events, including oestrous cyclicity and pregnancy, can be monitored precisely by evaluating urinary oestrogen conjugates in samples from Przewalski's mares maintained under semi-free-ranging conditions.

Keywords: Przewalski's horse; oestrogen; oestrous cycle; pregnancy; urinary steroids

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S. L. Monfort, C. Wemmer, T. H. Kepler, M. Bush, J. L. Brown and D. E. Wildt

Summary. Direct radioimmunoassays (RIA) for urinary oestrone conjugates and pregnanediol-3α-glucuronide (PdG) were used to study ovarian activity patterns and pregnancy in Eld's deer. In 2 does, urinary metabolite patterns were compared to temporal patterns of plasma LH, oestradiol-17β and progesterone. Preovulatory LH peaks occurred coincident with behavioural oestrus, and plasma progesterone secretion paralleled PdG excretion. Although plasma oestradiol-17β levels fluctuated between 5 and 10 μg/ml throughout the oestrous cycle, no preovulatory oestrogen surge was observed. Based on PdG excretion, non-conception oestrous cycles averaged 21·5 ± 2·1 days (±s.e.m., n = 65); however, 2 of 13 does exhibited prolonged oestrous cycles (30·1 ± 4·4 days; range 14–62 days, n = 14) characterized by sustained PdG excretion. Excluding these 2 females, the mean oestrous cycle was 18·5 ± 0·3 days (range 14·23 days, n = 51). Behavioural oestrus (12·24 h duration) was observed in 42 of 65 cycles (64·6%), and always corresponded with intercyclic troughs in PdG excretion (2·5 days duration). Mean gestation duration (n = 10) was 33·5 ± 0·4 weeks. PdG concentrations increased (P < 0·05) by Week –32 (3rd week of gestation), plateaued between Weeks −31 and −25, increased (P < 0·05) markedly by Week –22 and then rose steadily until parturition, declining (P < 0·05) rapidly thereafter. Mean excretion of oestrone conjugates remained low until Week −30, increased (P < 0·05) steadily to Week −24 (P < 0·05) and then returned to baseline by Week −17. Increased (P < 0·05) oestrone conjugates concentrations were detected again by Week −4 followed by a rapid increase to peak pregnancy levels by Week −1, declining (P < 0·05) precipitously after parturition. The results confirm that the Eld's deer is seasonally polyoestrous with onset (January–March) and cessation (August–October) of regular, cyclic ovarian activity coinciding with increasing and decreasing daylengths, respectively. Urinary PdG excretion accurately reflects cyclic ovarian activity and markedly elevated concentrations of this metabolite provide an accurate index of pregnancy. The simultaneous monitoring of oestrone conjugates appears useful for estimating the stage of pregnancy and predicting parturition onset.

Keywords: Eld's deer; oestrogen; progesterone; urinary steroids; LH; seasonality

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S. L. Monfort, K. D. Dahl, N. M. Czekala, L. Stevens, M. Bush and D. E. Wildt

Summary. Urinary excretion of oestrone conjugates, pregnanediol-3α-glucuronide (PdG) and 20α-hydroxypregn-4-en-3-one were measured from 8 weeks before oestrus to 2 weeks post partum and bioactive FSH was monitored during the periovulatory interval in a female giant panda. A biphasic urinary bioactive FSH excretory profile appeared to indicate a broad (∼ 10 day) follicular phase followed by a sharp preovulatory bioactive FSH surge coincident with an acute increase in urinary oestrone conjugates and behavioural oestrus. Weekly concentrations of urinary oestrone conjugates and PdG increased (P < 0·001) by Week 9 of gestation with 20α-hydroxypregn-4-en-3-one levels increasing 10–30-fold (P < 0·001) between Weeks 11 and 14. These observations indicate that the monoestrous giant panda does not appear to require a prolonged period of endogenous FSH release or multiple FSH peaks for ovarian priming and follicle selection to proceed normally. Furthermore, the delayed rise in urinary steroid excretion during the second half of gestation in the giant panda supports the concept of delayed implantation while the estimation of steroid conjugates in urine offers a non-invasive approach for monitoring pregnancy status in this endangered species.

Keywords: giant panda; oestrogen; progesterone; FSH; delayed implantation

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S. K. Wasser, S. L. Monfort, J. Southers and D. E. Wildt

Two unanaesthetized female yellow baboons (Papio cynocephalus cynocephalus) were infused (i.v.) with [3H]oestradiol and two with [3H]progesterone, early in the follicular phases of their cycles. One month later, the two females infused with [3H]oestradiol were simultaneously infused with [14C]progesterone and [3H]dehydroepiandrosterone. All urine and faeces were collected for 96 h after infusion. The proportion of steroid excreted in faeces (versus urine) was 10.0% for oestradiol and 40% for progesterone. Peak excretion in urine occurred 4.5 h after infusion. Peak excretion in faeces occurred an average of 36.4 h after infusion, with remarkable consistency between steroids. Eighty per cent of faecal oestradiol and progesterone metabolites were excreted as free (rather than conjugated) steroids. Simply boiling (20 min) the dried faecal sample in 90% ethanol proved to be the most rapid and efficient means of extracting these steroid metabolites. High pressure liquid chromatography and immunoreactivity studies revealed that oestradiol was excreted in faeces as oestradiol (36%), oestrone (44%) and a conjugated metabolite that co-eluted with oestrone sulfate (20%). Progesterone was excreted as eight different free forms, only a minor portion of which was progesterone, and what appeared to be a conjugated metabolite that co-eluted with pregnanediol-glucuronide (20%). The free progesterone metabolites were identified by gas-chromatography–mass-spectrometery as epimers of 5-pregnane-3-diol and 5-pregnane-3-ol-one. These data suggest that currently available immunoassays for free oestradiol and oestrone should adequately characterize faecal oestrogen profiles in baboons. However, high variability in crossreactivities of various progesterone antisera to progesterone metabolites in baboons makes antiserum selection a more serious concern in attempts to quantify faecal progestogen dynamics. All available data suggest considerable interspecific variability in faecal steroid metabolite excretion. This makes species-specific characterizations of the type reported here a necessary step for ensuring the accuracy of faecal endocrine monitoring.

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S. L. Monfort, J. L. Brown, T. C. Wood, C. Wemmer, A. Vargas, L. R. Williamson and D. E. Wildt

Plasma LH, FSH and testosterone were measured in blood samples collected via remote catheterization from six adult Eld's deer stags every 10 min for 8 h before and 2 h after GnRH (1 μg kg−1, i.v.) administration. Blood samples were collected within 2 weeks of the summer solstice (21 June), autumn equinox (22 September), winter solstice (21 December) and spring equinox (20 March). Marked seasonal variations in basal LH, FSH and testosterone concentrations were observed. From autumn, well-defined LH pulses were temporally associated with small, but detectable pulses in testosterone. During the winter transition into the breeding season, episodic LH pulses were also temporally associated with corresponding testosterone surges that lasted 2–3 h. High amplitude, low frequency testosterone surges were also observed during the spring, but often in the absence of detectable LH pulses. Basal LH and testosterone concentrations decreased during the summer and, although LH pulses were detected, associated testosterone pulses were absent. Only 37% of LH pulses occurred coincidentally with FSH pulses, and FSH pulses were generally less prominent. The increases in LH and FSH above basal concentrations after GnRH treatment were significant (P < 0.05) for all seasons. Increases in testosterone after GnRH treatment were greatest during the winter and spring, but testosterone also increased to a lesser extent during the autumn (P < 0.05). In contrast, testosterone concentrations were not different before and after GnRH treatment during summer. The net LH increase after GnRH treatment was similar (P > 0.05) for all seasons, whereas the proportional increase in LH was greatest (P < 0.05) during the summer and autumn when basal concentrations were lowest. Although the net FSH increase after GnRH treatment ranged only from 20 to 40 ng ml−1 during all seasons, the proportional rise in FSH after GnRH treatment tended to be highest during the summer and autumn when basal concentrations were lowest. Basal testosterone secretion and the net increase in testosterone after GnRH treatments were greatest during the spring rut (P < 0.05). Thus, in Eld's deer, it appears that pituitary gonadotrophs seasonally adjust their basal secretory 'set point', but the capacity to respond above basal concentrations is constrained to a maximum net increase in both LH and FSH, regardless of season. Because both basal LH and the net releasable pool of LH remain relatively constant, increased testosterone secretion after GnRH treatment probably represents increased testicular sensitivity to LH.

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S. K. Wasser, R. Thomas, P. P. Nair, C. Guidry, J. Southers, J. Lucas, D. E. Wildt and S. L. Monfort

A study was conducted in captive baboons to determine (i) the impact of cereal dietary fibre on faecal progestogen excretion, and (ii) whether means of controlling dietary effects could be identified. Blood was collected on 3 days per week and faeces on 5 days per week from four unanaesthetized cyclic female baboons, consecutively fed three diets of 5, 10 and 20% fibre for 90 days per diet. A 2 day lag time was detected before progesterone in the blood appeared in the faeces, regardless of diet (mean correlation was 0.62, P = 0.002). Increased dietary fibre had a negative effect on progestogen excretion (P < 0.004). Correspondence between blood and faecal progestogens was consistently greatest and the effect of dietary fibre least when faecal progestogens were expressed g−1 dry faeces. Several means of indexing faecal steroid excretion rates were examined including dehydroepiandrosterone (DHEA) and a number of byproducts of cholesterol metabolism. The cholesterol metabolite, cholestanone, was positively correlated with dietary fibre (r = 0.27; P < 0.04). Multiplying faecal progestogen concentration by the cholestanone g−1 dry faeces concentration increased the correlation between serum and cholestanone-indexed faecal progestogens (r = 0.78, P = 0.0001) compared with nonindexed progestogens (r = 0.71, P = 0.0001). We conclude that expressing faecal progestogens g−1 dry faeces may be sufficient and the most cost-effective method for controlling for most dietary effects when the objective is monitoring longitudinal endocrine status in baboons. However, it may be appropriate to express faecal progestogens by cholestanone concentrations when increased precision is needed to overcome the effects of profound variations in dietary fibre.

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J. L. Brown, M. Bush, C. Packer, A. E. Pusey, S. L. Monfort, S. J. O'Brien, D. L. Janssen and D. E. Wildt

Summary. Pituitary–gonadal function was examined in male lions free-ranging in the Serengeti Plains or geographically isolated in the Ngorongoro Crater of Tanzania. Lions were classified by age as adult (6·1–9·8 years), young adult (3·3–4·5 years) or prepubertal (1·4–1·6 years, Serengeti Plains only). Each animal was anaesthetized and then bled at 5-min intervals for 100 min before and 140 min after i.v. administration of saline or GnRH (1 μg/kg body weight). Basal serum LH and FSH concentrations were similar (P > 0·05) among age classes and between locations. In Serengeti Plains lions, net LH peak concentrations after GnRH were ∼ 25% greater (P < 0·05) in prepubertal than in either adult or young adult animals. GnRH-stimulated LH release was similar (P > 0·05) between adult and young adult lions, and these responses were similar (P > 0·05) to those measured in Ngorongoro Crater lions. Basal and GnRH-stimulated testosterone secretion was higher (P < 0·05) in adult than in young adult lions and lowest (P < 0·05) in prepubertal lions. Age-class differences in testosterone production were related directly to the concentrations of LH receptors in the testis (P < 0·05). Basal and GnRH-stimulated testosterone secretion and gonadotrophin receptor concentrations within age classes were similar (P > 0·05) between lions of the Serengeti Plains and Ngorongoro Crater. Lower motility and higher percentages of structurally abnormal spermatozoa were observed in electroejaculates of young adult compared to adult Serengeti Plains males (P < 0·05) and were associated with decreased steroidogenic activity. In contrast, there were no age-related differences in ejaculate characteristics of Ngorongoro Crater lions. Seminal quality in the Crater population was poor in adult and young adult animals and was unrelated to alterations in pituitary or testicular function. In summary, only seminal quality in adult male lions was affected by location, whereas age significantly affected both basal and GnRH-stimulated testosterone secretion and seminal quality (Serengeti Plains only) in sexually mature males. The striking seminal/endocrine differences among pride (breeding) males of different ages raises questions about the impact of age on individual reproductive performance in this species.

Keywords: lion; GnRH; LH; testosterone; receptors; testis; spermatozoa

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J. L. Brown, M. Bush, C. Packer, A. E. Pusey, S. L. Monfort, S. J. O'Brien, D. L. Janssen and D. E Wildt

Pituitary responses to gonadotrophin-releasing hormone (GnRH) and prolactin and steroid secretory profiles were examined in two populations of adult, female lions in the Serengeti (one outbred in the Serengeti Plains and one inbred in the Ngorongoro Crater) to determine whether reductions in genetic variability adversely affected endocrine function. GnRH-induced gonadotrophin secretion was also examined after adrenocorticotrophic hormone (ACTH) treatment to determine whether acute increases in serum cortisol altered pituitary function. Anaesthetized lions were administered (i) saline i.v. after 10 and 100 min of blood sampling; (ii) saline at 10 min and GnRH (1 μg kg−1 body weight) after 100 min; or (iii) ACTH (3 μg kg−1) at 10 min and GnRH after 100 min of sampling. Basal serum cortisol and basal and GnRH-induced gonadotrophin secretion were similar (P > 0.05) between females of the Ngorongoro Crater and Serengeti Plains. After ACTH, serum cortisol increased two-to threefold over baseline values and the response was unaffected (P > 0.05) by location. ACTH-induced increases in serum cortisol had no effect on subsequent basal or GnRH-stimulated luteinizing hormone (LH) or follicle-stimulating hormone (FSH) secretion. Overall mean serum progesterone concentrations ranged from 0.2 to 5.4 ng ml−1 with the exception of four females (two in the Serengeti and two in the Crater; progesterone range, 18.4–46.5 ng ml−1) that were presumed pregnant (three of these females were observed nursing cubs several weeks later). There were no differences (P > 0.05) between Serengeti and Crater lions in mean serum progesterone, oestradiol or prolactin concentrations, and hormone secretion was not influenced (P > 0.05) by GnRH or ACTH treatment. Although Ngorongoro Crater lions have decreased genetic variability, the reproductive–endocrine system of females appears functionally normal compared with outbred counterparts living in the Serengeti Plains. Furthermore, the acute rise in serum cortisol after ACTH administration in lions fails to alter subsequent GnRH-induced gonadotrophin release, suggesting that short-term changes in adrenal activity do not markedly affect pituitary responsiveness in this species.

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S. L. Monfort, J. L. Brown, M. Bush, T. C. Wood, C. Wemmer, A. Vargas, L. R. Williamson, R. J. Montali and D. E. Wildt

Blood samples, morphometric measurements and behavioural data were collected weekly for 52 weeks from six adult Eld's deer stags exposed to natural fluctuations in photoperiod (38°N latitude). Mean (± sem) serum LH concentrations reached peak values in the autumn (October), three months before FSH and testosterone concentrations reached peak values in early winter (January). Prolactin concentrations were inversely related (r = −0.733, P < 0.001) to LH and directly related to daylength, and maximal concentrations were observed during mid-summer (July) and minimal concentrations during early winter (January). The temporal pattern of circulating inhibin was positively correlated with FSH (r = 0.88, P < 0.001), but lagged behind the seasonal FSH increase by 1–3 weeks. Antler length, body weight and chest girth were maximal during pre-rut (December–January). Maximal scrotal circumference and combined testes volume were observed in mid-winter (February), whereas peak neck girth and behavioural aggression occurred 1–3 months later (March–May). On the basis of quarterly electroejaculation results, motile spermatozoa were produced in all seasons. However, the greatest number of motile spermatozoa per ejaculate was observed during the winter and spring, whereas the highest incidence of sperm pleiomorphisms (> 80%) was detected in the autumn. Histological assessments of the regressed testis (July) revealed fewer germ cells undergoing spermatogenesis and an increased incidence of degenerating and abnormal cell types. In summary, Eld's deer exhibit a circannual hypothalamic–pituitary–gonadal cycle with onset of pituitary activation occurring during the autumn and winter, whereas gonadal activity peaks during the winter and spring as daylengths are increasing. Marked circannual variations in circulating prolactin suggest that Eld's deer may use photoperiodic cues to modulate seasonal fertility; however, the existence of an endogenous seasonal rhythm operating independently of photoperiod cannot be excluded.