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.     

Breeding phenology in relation to NDVI variability in free-ranging African elephant

The phenology of reproduction is often correlated with resource availability and is hypothesized to be shaped by selective forces in order to maximize lifetime reproductive success. African elephants have the distinctive life history traits of a 22 month gestation and extended offspring investment, necessitating a long-term strategy of energy acquisition and reproductive expenditure to ensure successful offspring recruitment.
We investigated the relationship between the reproductive phenology of a wild elephant population and resource availability using remotely sensed Normalized Differential Vegetation Index (NDVI) data as a measure of time-specific primary productivity and hence forage quality.
The initiation of female elephants' 3+yr reproductive bout was dependent on conditions during the season of conception but timed so parturition occurred during the most likely periods of high primary productivity 22 months later. Thus, the probability of conception is linked to the stochastic variation in seasonal quality and the phenology of parturition is related to the predictable seasonality of primary productivity, indicating elephants integrate information on known current and expected future conditions when reproducing.
Juvenile mortality was not correlated with ecological variability, hence female fecundity rather than calf mortality appears to drive demographic processes in the study population.
Extreme climatic events, such as those associated with the El Niño-Southern-Oscillation (ENSO), acted to synchronize female fecundity in the population. This study suggests that the relationship between fecundity and ecological variability instigates the characteristic demographic fluctuations in elephant populations, rather than the mortality-driven fluctuations observed in many ungulate populations.     

Fecal steroid metabolites and reproductive monitoring in a female Tsushima leopard cat (Prionailurus bengalensis euptilurus)

Although the Tsushima leopard cat (Prionailurus bengalensis euptilurus) is one of the most endangered mammals in Japan, its reproductive physiology and endocrinology have been not elucidated. The objective was to establish the non-invasive monitoring of reproductive endocrinology in a female Tsushima leopard cat and to identify the types of fecal reproductive steroid metabolites in this species. Fecal concentrations of estrogen and progestin were determined by enzyme immunoassays, from 60 d before to 60 d after the last copulation, during three pregnancies. Fecal estrogen metabolite concentrations were increased before/around the mating period and after mid-pregnancy. Fecal progestin metabolite concentrations increased after the last copulation and remained high during pregnancy. The gestation period was 65.0 ± 0.6 d (mean ± SD). Fecal extracts were separated by high-performance liquid chromatography for identification of fecal metabolites. Fecal estrogens were identified as estradiol-17β and estrone. Fecal progestins during pregnancy contained 5α-reduced pregnanes: 5α-pregnan-3α-ol-20-one, 5α-pregnan-3β-ol-20-one and 5α-pregnan-3,20-dione, and nonmetabolized progesterone was barely detected in feces. In conclusion, measurement of fecal estrogen and progestin metabolites was effective for noninvasive reproductive monitoring in the Tsushima leopard cat. An immunoassay for fecal estradiol-17β concentrations seemed useful to monitor follicular activity, whereas an immunoassay with high cross reactivity for 5α-reduced pregnanes was useful to monitor ovarian luteal activity and pregnancy.     

The reproductive endocrinology and behavior of Vancouver Island marmot (Marmota vancouverensis)

The Vancouver Island marmot (Marmota vancouverensis; VIM) is one of North America's most endangered species with fewer than 150 individuals remaining in the wild. A captive breeding program was established across four facilities in Canada as an insurance population and source of animals for reintroduction to the wild. The purpose of this study was to gather information about the basic reproductive biology and behavior of this species, which is essential to improve captive breeding programs. Regular fecal samples were obtained from adult female (n = 14) and male (n = 10) marmots, 2 years of age and older, over 1-3 breeding seasons (2-3 months duration posthibernation) for steroid hormone analysis. Enzyme immunoassays were validated for quantifying fecal testosterone metabolite concentrations for males, and fecal estrogen and progesterone metabolite concentrations for females. Results indicated that fecal progesterone metabolite concentrations can be used to monitor ovulation and pregnancy. Behavioral monitoring through infrared video surveillance was conducted in four breeding pairs over a 2-year period (n = 7 behavioral profiles). Breeding behaviors correlated strongly with changes in reproductive endocrine profiles. A high frequency of play behavior or "wrestling" was observed in conjunction with breeding activity before an elevation in progesterone metabolite concentrations. Impending parturition was associated with increased aggression and exclusion of the male from the maternal nestbox as well as an increase in nesting activity. Observational data combined with hormonal analysis suggest that female VIMs are induced ovulators and that multiple breeding attempts may be required for ovulation and conception. Gestation appears to be approximately 34 days from peak breeding activity (32 days from estimated ovulation). Fecal testosterone concentrations suggest that testicular activity is seasonal with the reproductive activity occurring immediately posthibernation. Monitoring breeding behavior is a useful means of indicating estrus, conception and pregnancy, which can also be supported by the hormonal analysis of daily fecal samples of individual animals.     

Assessing reproductive profiles in female brown mouse lemurs (Microcebus rufus) from Ranomafana National Park,southeast Madagascar,using fecal hormone analysis

Studies on reproductive endocrinology in wild primate populations have greatly increased in the last decades owing to the development of noninvasive techniques that can be applied under field conditions. However, small‐bodied nocturnal species are not well represented on the long list of primates surveyed in the wild, and reproductive inferences regarding these animals in their natural habitats have not benefited from direct observations of hormonal changes. We collected fecal samples from female brown mouse lemurs (Microcebus rufus) in a southeastern rainforest of Madagascar in order to determine whether or not fecally excreted steroid levels show a consistent pattern of change during the reproductive season and are a useful complement to reproductive observations in wild‐trapped individuals. Initial data show variation in reproductive hormone levels before and after estrus and estimated day of parturition. Elevated levels of excreted estradiol (E₂) were observed around the time of estrus, whereas high levels of fecal progesterone (P) were seen during later stages of pregnancy and around parturition. A more complete picture of reproductive profiles in female mouse lemurs, and how they may change over the life span, can be obtained if hormone analyses are used to supplement field observations. Am. J. Primatol. 71:439–446, 2009. © 2009 Wiley‐Liss, Inc.     

Endocrine Correlates of Reproductive Status in Breeding and Nonbreeding Wild Female Moustached Tamarins

In callitrichid primates, reproduction is usually restricted to a single female per group. Reproductive rate is high and the occurrence of a postpartum estrus can lead to simultaneous lactation and pregnancy. In contrast, nonreproductive females often show ovarian inactivity. However, most studies on callitrichid reproductive physiology have been conducted in captivity, where conditions differ considerably from those in the wild, so that reproductive conditions may be strongly modified. Using fecal estrogen and progestogen measurements to monitor female reproductive status in 2 groups of wild moustached tamarins (Saguinus mystax), we examined 1) whether reproductive females in free-ranging groups also show postpartum estrus and 2) whether nonreproductive females demonstrate signs of ovarian activity. In both reproductive females, clear changes in the excretion pattern of progestogen and estrogen metabolites over time in combination with information on parturition dates allowed us to differentiate between pregnancy, a period of postpartum ovarian inactivity lasting for 54 and 64–82 days, and a period of ovarian activity before conception. Nonreproductive females demonstrated temporal fluctuations in hormone concentrations and absolute hormone levels that were similar to ones in the breeding females during the phase of ovarian activity. The results suggest that, in contrast to most captive female tamarins, reproductive females in wild groups of moustached tamarins do not have a postpartum estrus and that nonreproductive females show ovarian activity despite the presence of a breeding female. We therefore conclude that findings from captivity should be only carefully compared to the situation in the wild.     

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.     

Non-invasive faecal steroid monitoring of ovarian and adrenal activity in farmed blue fox (Alopex lagopus) females during late pregnancy, parturition and lactation onset

In the semi-domesticated blue fox, handling stress may influence reproductive performance and increase perinatal pup loss. Ovarian and adrenal steroids were analysed in faecal samples collected from mid-gestation through the first week of lactation in 40 female blue foxes to characterize hormone patterns during this important reproductive period. Daily faecal samples were collected from 40 foxes during 30 pregnancies, one late abortion and nine bred-matched non-pregnancies. Mean concentrations of faecal progestagens over the 10 days before birth were significantly higher in pregnant compared to non-pregnant females (51+/-1.50 microg/g versus 36+/-3.72 microg/g, respectively; P < 0.01). From 10 to 3 days before whelping, total faecal oestrogen concentrations also were higher (P < 0.01) in pregnant (1082+/-41.69 ng/g) than non-pregnant (628+/-72.43 ng/g) foxes, before declining to non-pregnant values (402+/-24.88 ng/g) after parturition. Overall mean faecal corticoid concentrations from 3 to 20 days before whelping differed between pregnant and non-pregnant foxes (128+/-3.11 ng/g versus 103+/-5.86 ng/g, respectively; P < 0.01). Furthermore, in pregnant foxes, corticoid excretion increased further from 2 days before to 3 days after whelping (216+/-13.71 ng/g; P < 0.01). Thereafter, corticoid concentrations were similar between pregnant and non-pregnant females (P > 0.05). In sum, the faecal steroid hormone patterns for oestrogens and progestagens were similar to those previously obtained by analyses of fox serum hormones, with both steroids being higher in pregnant than non-pregnant foxes at the end of gestation. The elevation in corticoid concentrations in pregnant females suggests that adrenal activation is involved in the initiation of parturition in the blue fox. Thus, faecal steroid analyses can be used to monitor ovarian activity during pregnancy and pseudopregnancy in farmed blue fox females.     

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.     

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.     

Testosterone and cortisol concentrations vary with reproductive status in wild female red deer

Although hormones are key regulators of many fitness and life history traits, the causes of individual level variation in hormones, particularly in wild systems, remain understudied. Whilst we know that androgen and glucocorticoid levels vary within and among individuals in mammalian populations, how this relates to key reproductive processes such as gestation and lactation, and their effects on a female''s measurable hormone levels are poorly understood in wild systems. Using fecal samples collected from females in a wild red deer population between 2001 and 2013, we explore how fecal androgen (FAM) and cortisol (FCM) metabolite concentrations change with age and season, and how individual differences relate to variation in reproductive state. Both FAM and FCM levels increase toward parturition, although this only affects FCM levels in older females. FCM levels are also higher when females suckle a male rather than a female calf, possibly due to the higher energetic costs of raising a son. This illustrates the importance of accounting for a female''s life history and current reproductive status, as well as temporal variation, when examining individual differences in hormone levels. We discuss these findings in relation to other studies of mammalian systems and in particular to the relatively scarce information on variation in natural levels of hormones in wild populations.     

Prediction of estrus cyclicity in Asian elephants (Elephas maximus) through estimation of fecal progesterone metabolite: development of an enzyme-linked immuno-sorbent assay

Asian elephants (Elephas maximus), prominent “flagship species”, are listed under the category of endangered species (EN - A2c, ver. 3.1; IUCN Red List 2009) and there is a need for their conservation. This requires understanding demographic and reproductive dynamics of the species. Monitoring reproductive status of any species is traditionally being carried out through invasive blood sampling and this is restrictive for large animals such as wild or semi-captive elephants due to legal, ethical, and practical reasons. Hence, there is a need for a non-invasive technique to assess reproductive cyclicity profiles of elephants, which will help in the species’ conservation strategies. In this study, we developed an indirect competitive enzyme linked immuno-sorbent assay (ELISA) to estimate the concentration of one of the progesterone-metabolites i.e., allopregnanolone (5α-P-3OH) in fecal samples of Asian elephants. We validated the assay which had a sensitivity of 0.25 μM at 90% binding with an EC₅₀ value of 1.37 μM. Using female elephants, kept under semi-captive conditions in the forest camps of Mudumalai Wildlife Sanctuary, Tamil Nadu and Bandipur National Park, Karnataka, India, we measured fecal progesterone-metabolite (5α-P-3OH) concentrations in six animals and showed their clear correlation with those of serum progesterone, measured by a standard radio-immuno assay. Statistical analyses using a Linear Mixed Effect model showed a positive correlation (P < 0.1) between the profiles of fecal 5α-P-3OH (range: 0.5-10 μg/g) and serum progesterone (range: 0.1-1.8 ng/mL). Therefore, our studies show, for the first time, that the fecal progesterone-metabolite assay could be exploited to predict estrus cyclicity and to potentially assess the reproductive status of captive and free-ranging female Asian elephants, thereby helping to plan their breeding strategy.     

Seasonal effects on the endocrine pattern of semi-captive female Asian elephants (Elephas maximus): timing of the anovulatory luteinizing hormone surge determines the length of the estrous cycle

Better breeding strategies for captive Asian elephants in range countries are needed to increase populations; this requires a thorough understanding of their reproductive physiology and factors affecting ovarian activity. Weekly blood samples were collected for 3.9 years from 22 semi-captive female Asian elephants in Thai elephant camps to characterize LH and progestin patterns throughout the estrous cycle. The duration of the estrous cycle was 14.6+/-0.2 weeks (mean+/-S.E.M.; n=71), with follicular and luteal phases of 6.1+/-0.2 and 8.5+/-0.2 weeks, respectively. Season had no significant effect on the overall length of the estrous cycle. However, follicular and luteal phase lengths varied among seasons and were negatively correlated (r=-0.658; P<0.01). During the follicular phase, the interval between the decrease in progestin concentrations to baseline and the anovulatory LH (anLH) surge varied in duration (average 25.9+/-2.0 days, range 7-41, n=23), and was longer in the rainy season (33.4+/-1.8 days, n=10) than in both the winter (22.2+/-4.5 days, n=5; P<0.05) and summer (18.9+/-2.6 days, n=8; P<0.05). By contrast, the interval between the anLH and ovulatory LH (ovLH) surge was more consistent (19.0+/-0.1 days, range 18-20, n=14). Thus, seasonal variation in estrous cycle characteristics were mediated by endocrine events during the early follicular phase, specifically related to timing of the anLH surge. Overall reproductive hormone patterns in Thai camp elephants were not markedly different from those in western zoos. However, this study was the first to more closely examine how timing of the LH surges impacted estrous cycle length in Asian elephants. These findings, and the ability to monitor reproductive hormones in range countries (and potentially in the field), should improve breeding management of captive and semi-wild elephants.     

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.     

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.     

Analyzing corticosterone metabolites in fecal samples of mice: a noninvasive technique to monitor stress hormones

In small animals like mice, the monitoring of endocrine functions over time is constrained seriously by the adverse effects of blood sampling. Therefore, noninvasive techniques to monitor, for example, stress hormones in these animals are highly demanded in laboratory as well as in field research. The aim of our study was to evaluate the biological relevance of a recently developed technique to monitor stress hormone metabolites in fecal samples of laboratory mice. In total, six experiments were performed using six male and six female mice each. Two adrenocorticotropic hormone (ACTH) challenge tests, two dexamethasone (Dex) suppression tests and two control experiments [investigating effects of the injection procedure itself and the diurnal variation (DV) of glucocorticoids (GCs), respectively] were conducted. The experiments clearly demonstrated that pharmacological stimulation and suppression of adrenocortical activity was reflected accurately by means of corticosterone metabolite (CM) measurements in the feces of males and females. Furthermore, the technique proved sensitive enough to detect dosage-dependent effects of the ACTH/Dex treatment and facilitated to reveal profound effects of the injection procedure itself. Even the naturally occurring DV of GCs could be monitored reliably. Thus, our results confirm that measurement of fecal CM with the recently established 5alpha-pregnane-3beta,11beta,21-triol-20-one enzyme immunoassay is a very powerful tool to monitor adrenocortical activity in laboratory mice. Since mice represent the vast majority of all rodents used for research worldwide and the number of transgenic and knockout mice utilized as animal models is still increasing, this noninvasive technique can open new perspectives in biomedical and behavioral science.     

Non-invasive assessment of the reproductive cycle in free-ranging female African elephants (Loxodonta africana) treated with a gonadotropin-releasing hormone (GnRH) vaccine for inducing anoestrus

ABSTRACT: BACKGROUND: In southern Africa, various options to manage elephant populations are being considered. Immunocontraception is considered to be the most ethically acceptable and logistically feasible method for control of smaller and confined populations. In this regard, the use of gonadotropin-releasing hormone (GnRH) vaccine has not been investigated in female elephants, although it has been reported to be safe and effective in several domestic and wildlife species. The aims of this study were to monitor the oestrous cycles of free-ranging African elephant cows using faecal progestagen metabolites and to evaluate the efficacy of a GnRH vaccine to induce anoestrus in treated cows. METHODS: Between May 2009 - June 2010, luteal activity of 12 elephant cows was monitored non-invasively using an enzyme immunoassay detecting faecal 5alpha-reduced pregnanes (faecal progestagen metabolites, FPM) on a private game reserve in South Africa. No bulls of breeding age were present on the reserve prior to and for the duration of the study. After a 3-month control period, 8 randomly-selected females were treated twice with 600 micrograms of GnRH vaccine (Improvac[REGISTERED SIGN], Pfizer Animal Health, Sandton, South Africa) 5-7 weeks apart. Four of these females had been treated previously with the porcine zona pellucida (pZP) vaccine for four years (2004-2007). RESULTS: All 12 monitored females (8 treated and 4 controls) showed signs of luteal activity as evidenced by FPM concentrations exceeding individual baseline values more than once. A total of 16 oestrous cycles could be identified in 8 cows with four of these within the 13 to 17 weeks range previously reported for captive African elephants. According to the FPM concentrations the GnRH vaccine was unable to induce anoestrus in the treated cows. Overall FPM levels in samples collected during the wet season (mean 4.03 micrograms/gram dry faeces) were significantly higher (P<0.002) than the dry season (mean 2.59 micrograms/gram dry faeces). CONCLUSIONS: The GnRH vaccination protocol failed to induce anoestrus in the treated female elephants. These results indicate that irregular oestrous cycles occur amongst free-ranging elephants and are not restricted to elephants in captivity. The relationship between ecological conditions and endocrine activity were confirmed. Free-ranging female elephants were observed to not cycle continuously throughout the year in the absence of adult bulls.     

Reproductive gonadal steroidogenic activity in the fishing cat (Prionailurus viverrinus) assessed by fecal steroid analyses

Non-invasive fecal steroid analyses were used to characterize gonadal activity in the fishing cat (Prionailurus viverrinus). Estrogen, progestagen and androgen metabolites were quantified in fecal samples collected for 12 months from four males and 10 females housed at seven North American zoological institutions. Male reproductive hormone concentrations did not vary (P>0.05) among season, and estrogen cycles were observed year-round in females and averaged (±SEM) 19.9±1.0 days. Mean peak estrogen concentration during estrus (460.0±72.6ng/g feces) was five-fold higher than baseline (87.3±14.0ng/g feces). Five of seven females (71.4%) housed alone or with another female demonstrated spontaneous luteal activity (apparent ovulation without copulation), with mean progestagen concentration (20.3±4.7μg/g feces), increasing nearly five-fold above baseline (4.1±0.8μg/g feces). The non-pregnant luteal phase averaged 32.9±2.5 days (n=13). One female delivered kittens 70 days after natural mating with fecal progestagen concentrations averaging 51.2±5.2μg/g feces. Two additional females were administered exogenous gonadotropins (150IU eCG; 100IU hCG), which caused hyper-elevated concentrations of fecal estrogen and progestagen (plus ovulation). Results indicate that: (1) male and female fishing cats managed in North American zoos are reproductively active year round; (2) 71.4% of females experienced spontaneous ovulation; and (3) females are responsive to exogenous gonadotropins for ovulation induction, but a regimen that produces a normative ovarian steroidogenic response needs to be identified.     

Impacts of the season and reproductive status on fecal reproductive and adrenocortical steroid metabolites in zoo Cuban crocodiles (Crocodylus rhombifer)

Conservation strategies for crocodilians often include captive breeding to create stable assurance populations. Evaluating adrenal and gonadal hormone patterns can provide animal managers with data to more effectively monitor animal welfare and reproductive status. This study evaluated the effects of season (breeding, nesting, or off), sex (male and female), and reproductive status of females (egg-laying/housed with a male or non-laying/housed solo) on concentrations of fecal glucocorticoid metabolite (FGM), fecal androgen metabolite (FAM), and fecal progestogen metabolite (FPM) in seven Cuban crocodiles, Crocodylus rhombifer, at the Smithsonian's National Zoological Park. Overall, seasonal changes in FGM and FPM concentrations were only observed in egg-laying females; FGM and FPM concentrations were both higher during the nesting season compared to the breeding and off seasons. Seasonal changes in FAM concentrations were only observed in males; males had higher FAM concentrations during the breeding and nesting seasons compared to the off season. Future studies investigating the use of fecal hormone metabolites in crocodilians are necessary to understand differences between individuals and species, to further elucidate the interactions between hormones and environmental factors, such as social housing, and to develop long-term datasets for the management of this species.     

Longitudinal fecal hormone analysis for monitoring reproductive activity in the female polar bear (Ursus maritimus)

The objective was to identify suitable enzyme immunoassays to monitor gonadal and placental function in the female polar bear. Immunoreactive progesterone, progesterone metabolite (PdG), estrogen, and androgen metabolite (T) concentrations were measured in fecal samples collected over 24 mo from captive female bears (N = 20). Whereas fecal extracts produced displacement curves parallel to the standard curve for each respective steroid, T and PdG more accurately reflected reproductive events. Concentrations of fecal T increased (P < 0.05) during the breeding season, and brief spikes were associated with estrus and mating. A postovulatory increase in PdG was not always detected, but sustained baseline T after mating appeared consistent with ovulation. Parturient bears excreted higher PdG concentrations (P < 0.05) during expected time of embryo implantation in Fall, and a late gestational rise in fecal T occurred 30 days prepartum. Many nonparturient bears also had a PdG rise in the Fall, suggesting they experienced either pregnancy loss or a pseudopregnancy. Differentiating pregnant and pseudopregnant states was not achieved using fecal PdG alone, but when combined with fecal T, comprehensive diagnoses could be made. Nonparturient bears demonstrated elevated (P < 0.05) fecal T during summer months, whereas parturient bears did not. In summary, noninvasive hormone monitoring techniques were established for the female polar bear. Although this study was directed at facilitating management and breeding efforts of captive polar bears, the methods could be applied to studies of reproductive function in wild populations.