Fecal Progesterone Metabolites Research Articles

Reproductive Seasonality, Estrous Cycle, Pregnancy, and the Recurrence of Postpartum Estrus Based on Long-Term Profiles of Fecal Sex Steroid Hormone Metabolites regarding Zoo-Housed Female Golden Takins (Budorcas taxicolor bedfordi).

This study investigates the non-invasive monitoring of the endocrine ovarian activities of captive female golden takins (Budorcas taxicolor bedfordi) based on long-term fecal sex steroid hormone metabolite dynamics. Fecal progesterone (P4) metabolite dynamics were monitored in nine females for 0.5-15 years between 2004 and 2022. Fecal estradiol-17β (E2) and estrone (E1) metabolites were measured during certain estrous cycles, and fecal E1 metabolite concentrations were measured during all gestation periods. The breeding season of the captive animals was mainly between May and December, and they were polyestrous animals whose breeding season begins during the long-day period. The onset of the breeding season occurred slightly earlier as age increased. The mean age (±SD) at puberty based on fecal P4 metabolite dynamics was 4.1 ± 2.9 years. The first conception ages ranged from 2.3-10.2 years. The mean estrous cycle period (±SEM) was 25.4 ± 1.1 days, and mounting and mating occurred in periods of low fecal P4 metabolite levels during the breeding season. The mean gestation period (±SD) from the estimated mating date to the calving date was 253.9 ± 5.7 days, and the fecal P4 metabolite distribution during pregnancy was bimodal. Fecal estrone metabolite levels gradually increased 21 weeks before delivery, peaked during the week of delivery, and then markedly decreased in the first week after delivery. Estrus resumed in the first April-August period after delivery (mean ± SD; 103.5 ± 40.9 days) or in May of the year after delivery (421.0 ± 16.5 days). This study revealed that the estrous cycle and pregnancy of female golden takins can be determined by fecal progesterone metabolite dynamics and that fecal estrone metabolite dynamics increases toward parturition and are useful for predicting the date of delivery. This endocrinological information is important for planned breeding efforts for the golden takins.

Evaluating the utility of harvester-collected samples for muskox (Ovibos moschatus) pregnancy diagnostics

Monitoring wildlife health in remote and vast regions of the Arctic can be difficult and expensive, but community-based monitoring programs can help alleviate these issues. Reliable bio-monitoring tools, compatible with sample types collected from remote regions, are needed to determine indicators of population health, such as reproductive rates. We sought to establish methods to diagnose pregnancy in muskoxen (Ovibos moschatus) using samples that are commonly collected in harvester-based sampling programs: blood on filter paper and feces. We first evaluated three pregnancy tests, pregnancy-associated glycoproteins (PAG) on filter paper blood samples and fecal progesterone and estrogen metabolites (FPM and FEM), using 12 captive female muskoxen of known pregnancy status. Pregnancy diagnostics using PAG were highly sensitive (100 %) and specific (100 %), with a diagnostic threshold of 19.4 percent positivity (%P) relative to the positive control of the test kit. FPM and FEM diagnostic accuracy differed by hormone and time period. FPM was most accurate in mid-gestation and FEM most accurate in late gestation (sensitivities and specificities of 100 %), but both were overall less accurate than the PAG test during gestation. PAG %P followed a biphasic pattern during gestation, FPM peaked in mid-gestation, and FEM peaked in late-gestation. Following the captive trial, we applied these tests to fecal samples and blood on filter paper samples from 159 adult female muskoxen that were submitted by harvesters through community-based monitoring programs in the Canadian Arctic. The observed patterns of PAG, FPM, and FEM in wild muskoxen throughout gestation were similar to captive muskoxen, though with lower levels of the respective biomarker, requiring different diagnostic thresholds for each test. This work with captive and wild muskoxen provides guidelines for pregnancy diagnostics in muskoxen throughout gestation using samples that can be easily collected by harvesters, allowing for improved monitoring of difficult to access populations through community-based approaches.

Non-Invasive Quantification of Faecal and Urine Reproductive Hormone Metabolites in the Naked Mole-Rat (Heterocephalus glaber).

The naked mole-rat (Heterocephalus glaber) occurs in colonies with a distinct dominance hierarchy, including one dominant, breeding female (the queen), 1-3 breeding males, and non-reproductive subordinates of both sexes that are reproductively suppressed while in the colony. To non-invasively evaluate reproductive capacity in the species, we first had to examine the suitability of enzyme immunoassays (EIAs) for determining progestogen and androgen metabolite concentrations in the naked mole-rat, using urine and faeces. A saline control and gonadotrophin-releasing hormone (GnRH) were administered to twelve (six males and six females) naked mole-rats which were previously identified as dispersers and housed singly. The results revealed that urine is possibly not an ideal matrix for progestogen and androgen metabolite quantification in naked mole-rats as no signal was detected in the matrix post GnRH administration. A 5α-Progesterone EIA and an Epiandrosterone EIA were identified as suitable for quantifying faecal progesterone metabolites (fPMs) and faecal androgen metabolites (fAMs) in males and females, respectively. The results suggest that there are individual variations in baseline fPM and fAM concentrations, and only two out of six females and no males exhibited an increase in fPM concentrations greater than 100% (-20% SD) post GnRH administration. Conversely, only four out of six females and three out of six males had an increase in fAM concentrations greater than 100% (-20% SD) following GnRH administration. These results imply that some naked mole-rat individuals have a reduced reproductive capacity even when they are separated from the queen.

Evaluation of minimally invasive estrus synchronization protocols in brown brocket deer (Subulo gouazoubira)

This study aimed to evaluate minimally invasive protocols for estrus synchronization in the brown brocket deer (Subulo gouazoubira). Females were submitted to Latin square design, in different treatments. All females received 0.25 mg of estradiol benzoate on the first day of treatment, concomitant with one of the following sources of progesterone: (1) DIP: an intravaginal progesterone releasing device for eight days, (2) MGA1x: once a day (in the morning) oral dose of 1 mg melengestrol acetate for eight days, (3) MGA2x: twice a day (morning and afternoon) oral doses of 0.5 mg of MGA for eight days, (4) P4LA: a single i.m. administration of 75 mg of long-acting progesterone (P4LA). Eight days after the beginning of each treatment, females received an i.m. administration of 265 µg of prostaglandin (PGF2α; cloprostenol). Treatment efficacy was evaluated by manifestation of behavioral estrus after treatment and concentration of fecal progesterone metabolites (FPM). The time to onset of estrus in treatment P4LA was significantly longer (180 ± 38.9 h) compared to DIP (63 ± 6.6 h), MGA1x (53 ± 14.4 h) and MGA2x (41 ± 10.1 h) (P = 0.008). According to individual baseline FPM and FPM concentration during the days after estrus, the corpus luteum formation was suggested in all females which responded to the treatments (93.75 %). Low synchrony, longer interval between PGF2α administration and onset of estrus suggest that the P4LA dose (75 mg) is too high and not effective for S. gouazoubira. DIP, MGA 1x and MGA 2x, were effective in estrus synchronization.

Assessment of a non-invasive approach to pregnancy diagnosis in gray whales through drone-based photogrammetry and faecal hormone analysis.

Knowledge of baleen whales' reproductive physiology is limited and requires long-term individual-based studies and innovative tools. We used 6 years of individual-level data on the Pacific Coast Feeding Group gray whales to evaluate the utility of faecal progesterone immunoassays and drone-based photogrammetry for pregnancy diagnosis. We explored the variability in faecal progesterone metabolites and body morphology relative to observed reproductive status and estimated the pregnancy probability for mature females of unknown reproductive status using normal mixture models. Individual females had higher faecal progesterone concentrations when pregnant than when presumed non-pregnant. Yet, at the population level, high overlap and variability in progesterone metabolite concentrations occurred between pregnant and non-pregnant groups, limiting this metric for accurate pregnancy diagnosis in gray whales. Alternatively, body width at 50% of the total body length (W50) correctly discriminated pregnant from non-pregnant females at individual and population levels, with high accuracy. Application of the model using W50 metric to mature females of unknown pregnancy status identified eight additional pregnancies with high confidence. Our findings highlight the utility of drone-based photogrammetry to non-invasively diagnose pregnancy in this group of gray whales, and the potential for improved data on reproductive rates for population management of baleen whales generally.

EVALUATING THE EFFICACY OF NONINVASIVE FECAL SAMPLING FOR PREGNANCY DETECTION IN ELK (CERVUS CANADENSIS).

Elk (Cervus canadensis) were reintroduced to Tennessee, USA in the early 2000s, with limited reproductive monitoring since initial release. We assessed the efficacy of noninvasive sampling for determining pregnancy using invasive (capture) and noninvasive (fecal collection in the field) techniques at the North Cumberland Wildlife Management Area (NCWMA), Tennessee. We captured 20 female elk 2019-2020, used pregnancy-specific protein B (PSPB) in blood to determine pregnancy and compared results to fecal progesterone metabolite (FPM) concentrations using two commercially available enzyme immunoassay (EIA) kits. Based on PSPB concentrations, 8/11 and 3/4 of captured adult elk (≥2.5 yr of age) were pregnant in 2019 and 2020, respectively; no 1.5-yr-old elk were pregnant (n=5). Using the progesterone EIA kit, FPM concentrations were x̄=192.84±38.63 ng/g (95% CI, 96.48-289.20) for nonpregnant and x̄=536.17±74.98 ng/g (95% CI, 375.97-696.36) for pregnant captured females. For the progesterone metabolite kit, FPM concentrations were x̄=188.16±43.39 ng/g (95% confidence interval [CI], 76.63-299.69) for nonpregnant and x̄=693.52±126.52 ng/g (95% CI, 407.31-979.72) for pregnant captured females. From February to May 2019, we collected 357 fecal samples in 65 areas across 489.62 km2 of the NCWMA. Using extracted DNA and analysis of 15 microsatellites, we identified 62 unique individuals from 128 female fecal samples collected on the landscape. We categorized females from landscape-collected feces as nonpregnant (35.5-40.3%; Metabolite-EIA kits), undetermined (1.6-6.5%; Metabolite-EIA kits), or pregnant (62.9-53.2%; Metabolite-EIA kits) based on a 95% CI of captured female FPM concentrations, giving an overall pregnancy rate of 53.2% using the recommended EIA kit. The pregnancy rate in sexually mature females may be higher, as it was not possible to distinguish age classes of landscape-collected fecal samples; therefore, some may have been from younger age classes not expected to be pregnant. Analysis of FPM may be useful at a population level to detect pregnancy.

Comparative analysis of gestation in three rhinoceros species (Diceros bicornis; Ceratotherium simum; Rhinoceros unicornis)

This comparative analysis evaluated endocrine profiles and gestation length data of captive pregnant black rhinoceros (Diceros bicornis), white rhinoceros (Ceratotherium simum), and greater one-horned (GOH) rhinoceros (Rhinoceros unicornis). Hormone profiles were collected over three decades as part of pregnancy diagnoses. After the third month of gestation, the luteo-placental shift in progesterone production in pregnant rhinoceroses causes a significant increase in the concentration of faecal progesterone metabolites. We defined a laboratory-specific value of 1000 ng/g faeces as a threshold for incipient feto-placental progesterone production. Using this value allowed a comparison between species and revealed significant individual differences within a species. The mean ± SEM gestation days for reaching the 1000 ng/g faeces threshold were 89.5 ± 2.9 (range 56–138 days; n = 39) in black, 96.0 ± 2.6 (58–138; n = 39) in white, and 117.8 ± 5.3 (74–173; n = 19) in GOH rhinoceroses. For the calculations of gestation length, we complemented our results from three decades of reproductive monitoring with data from the literature, resulting in about 70 values for each species. Gestation length in the black, the white and the GOH rhinoceros was 460.6 ± 1.5 (range: 436 – 486), 503.8 ± 1.3 (range: 480 – 525) and 480.5 ± 1.1 (range: 453 – 505) days, respectively. Daylight length significantly affected gestation length, while the sex of offspring had no effect. On average, pregnancies with parturitions in spring and summer were one week shorter than those in autumn and winter. Although rhinoceroses are non-seasonal breeders, most parturitions in captivity occur in autumn and winter. We also analysed preconception endocrine profiles in the white rhinoceros. Conceptions in this species occurred after oestrous cycles of approximately 35 days (n = 18), 70 days (n = 3), 15 days (n = 1), after periods of ovarian inactivity (n = 5), and during a foal heat within one month after stillbirth parturition (n = 1). In conclusion, this study provides a comprehensive overview of gestational parameters in three rhinoceros species.

Assessing reproduction in wolverines (Gulo gulo): Fecal progesterone levels in zoo and wild females

Wolverines (Gulo gulo) populations are under threat by increased human activities. A better understanding of their reproductive physiology would benefit research and monitoring of this species. The use of enzyme immunoassay (EIA) to measure fecal progesterone metabolites (FPM) is a powerful tool to understand wildlife reproductive physiology, and a non-invasive method for use in both zoo and wild populations. The main objective of this study was to determine reproductive status in female wolverines using FPM levels, first by monitoring of longitudinal changes in FPM in zoo housed females in breeding situations and second to compare those values to FPM measured in feces obtained from wild females observed in the field during reproductively active periods (Dec–Apr). Female reproductive physiology was assessed by measuring FPM in three zoo housed wolverines to determine species-specific levels arising from ovulation encompassing embryonic diapause and ovarian reactivation (resumption of ovarian luteal activity after diapause) during pregnancy or pseudopregnancy, which in turn enabled verification of reproductive status in 15 wild wolverines, monitored over three years. In the zoo females, FPM levels were sustained at basal levels if ovulation and/or breeding did not occur. After ovulation, both pregnancy or pseudopregnancy exhibited a bi-phasic pattern of FPM increases, with a 4.9-fold increase above baseline during embryonic diapause and a further 4.4-fold increase after ovarian reactivation. FPM levels during pregnancy and pseudopregnancy were indistinguishable, thus increases in FPM levels are only a confirmation of ovulation, and not diagnostic for pregnancy. In wild females, assessment of FPM levels in samples collected from December to February was able to predict a potentially pregnant/reproductively active female with levels above 8200 ng/g dried feces, or non-reproductive (nonovulatory) female during that period with levels below 1700 ng/g. Wild wolverine FPM levels indicative of pregnancy aligned with field observations of denning and/or lactation. FPM levels from samples collected from March and April were inconclusive as levels could be attributed to females being either postpartum or non-reproductive at that time. Timing of ovarian reactivation, and subsequent elevation of FPM, varied among individuals suggesting caution is warranted when assessing only one fecal sample per female. Understanding all factors that may impact reproductive success is essential for effective species conservation, particularly for species with low reproductive rates, like the wolverine. This study has demonstrated the potentially useful incorporation of non-invasive fecal hormone analysis for investigation of physiological parameters and reproductive status in wild wolverine females.

Understanding pregnancy-related hormones in female red pandas

Non-invasive techniques utilizing fecal samples to measure circulating steroid hormones have proven to effectively reflect the reproductive status of numerous species. Fecal progesterone metabolite (FPM) profiles have enhanced our understanding of the complicated reproductive strategies of many endangered species; however, this tool has been proven to not be optimal for pregnancy detection in the red panda (Ailurus fulgens). This study set out to investigate potential differences in FPM profiles (phases and lengths of hormone elevation) of seven female red pandas, both parturient and non-parturient, confirming FPM analysis is not a valuable indicator of pregnancy in this species. Secondly, examined whether a pregnancy related hormone, prostaglandin F2α and its metabolites (PGFM), can provide a reliable tool for confirming pregnancy in this species. Similarly, PGFM did not differentiate between parturient and non-parturient females until a significant peak the day of parturition (± 2 days). Interestingly, this is the first study to capture the PGFM surge associated with parturition, which may provide a tool for determining pregnancy in cases where live cubs were not observed.

Validation of non-invasive hormone analysis techniques to assist in the future identification of maternal roosts of ghost bats (Macroderma gigas)

The ghost bat (Macroderma gigas) is the largest microbat in Australia and occupies a large but contracting range. The ghost bat is listed as Vulnerable with a decreasing population trend by the IUCN Red List of Threatened Species. They occupy caves, disused mine adits and rock crevices as daytime roosts but distinguishing which are preferred as maternal roosts is challenging as sampling is difficult, and bats are easily disturbed while roosting. Identification of maternal roosts is a priority for conservation and management purposes, and therefore non-invasive hormone analysis was investigated as a potential tool for the future identification of the cave preferences of pregnant individuals. To validate fecal progesterone metabolite analysis techniques, fecal samples were collected from group housed female ghost bats at Perth Zoo between October and January, during the expected parturition period, over three years. Fecal samples were weighed (0.025 g), extracted with 2.5 ml of 80% methanol and analyzed for progesterone metabolite levels by enzyme-immunoassay. Significant elevations in fecal progesterone metabolite levels were detected in a subset of samples collected from co-housed females prior to parturition but not after, providing biological validation of the hormone analysis techniques. Over the three years, four pups were born with birth dates ranging from early November to late December. The mean fecal progesterone metabolite levels of non-pregnant females was significantly lower than those of pregnant females (206.5 ± 102.9 ng/g and 7003.6 ± 6078.0 ng/g respectively). These techniques can be used to monitor and evaluate the reproductive health of ghost bat populations over time and help identify factors influencing maternity roost fidelity. This data will provide valuable information for the conservation and management of the ghost bat and has the potential to be applied to other bat species.

Development of an enzyme immunoassay to measure urinary and faecal 5α-androst-16-en-3-one in pigs

Androstenone, a volatile steroid that possesses pheromonal activity, is responsible for boar taint, sexual interactions, and reproduction in pigs. A wide range of analytical methods has been developed to quantify and detect androstenone in adipose tissue and blood, which are invasive procedures. Therefore, the present study aimed to develop a non-invasive method to detect and quantify the androstenone. We produced group-specific polyclonal androstenone antibody to standardize and validate an enzyme immunoassay to measure faecal and urinary androstenone in Yorkshire boars and sows. Parallelism was performed to determine the immunoreactivity between faecal and urinary immunoreactive androstenone and respective antibody. In boars, urinary and faecal androstenone concentrations were higher on the day of mounting and copulation with sows. In sows, we also measured faecal progesterone metabolites to confirm the oestrus and mating. Faecal androstenone concentrations were peaked on the day of oestrus and mating in sows. Our results suggest that androstenone could be detected and quantified in faecal and urine samples of boars and sows.•Developed an enzyme immunoassay for measuring 5α-androst-16-en-3-one as a marker of boar taint and sex pheromone in urine and faeces of pigs•Detection of 5α-androst-16-en-3-one using a non-invasive method

The effects of animal transfers on the reproductive success of female white rhinoceroses (Ceratotherium simum simum) kept in European zoos

The Southern white rhinoceros (Ceratotherium simum simum) has been kept in European zoos since the 1960s. However, captive breeding success has been low, with social group composition, group size, and available space all playing a role. Female rhinoceroses that have never bred or not bred for a long time have a particularly increased risk of developing reproductive tract pathologies, often resulting in infertility at a young age. One management measure to stimulate breeding is to transfer non-reproducing animals to other zoos. This study evaluated the success of transfers of 4 – 28 years old white rhinoceroses between European zoos. We analyzed n = 90 (45 males and 45 females) transfers of white rhinoceroses between 1990 and 2018. Fecal progesterone metabolite levels were analyzed for a subset of female rhinoceroses. The success of a transfer was defined as a calf born within five years. The success rate after female transfers was 26.7%; however, when the age limit of transferred females is set at 18 years, the success rate was 44.4%. The success rate after a male transfer was 23.2%. In transferred females, 83% of births occurred within three years after a transfer. Births following the arrival of a new male were distributed over five years. After a male transfer, endocrine data were determined in 26 of 82 females affected by the transfer. Positive development of estrous cycle activity after the arrival of the new bull occurred in 13 females. In summary, the success of the transfers in terms of offspring birth and endocrine stimulation of cycle activity was lower than anticipated, and sometimes a considerable amount of time elapsed before a calf was born. Nonetheless, transfers are essential to promote breeding. The relatively low success of the transfers analyzed in this study relates to the partially advanced age of the white rhinoceroses studied. Transfers of juvenile or adolescent females currently conducted between European zoos reveal a better birth rate than the present study.

Investigating the utility of using fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas (Tachyglossus aculeatus)

This study demonstrates the utility of the analysis of fecal hormone metabolites as a reproductive management tool for captive short-beaked echidnas. Over three breeding seasons daily fecal samples were collected from female echidnas (n = 8) that were monitored continuously by video surveillance to confirm key reproductive events. Fecal progesterone metabolite concentrations were elevated above baseline values (448.0 ± 156.3 ng/g) during pregnancy and the luteal phase. However, compared to plasma progesterone the rise in fecal progesterone metabolite concentrations after copulation was delayed (3.3 ± 0.4 versus 8.3 ± 0.6 days, respectively), such that pregnancy was more reliably detected in its latter half when using fecal samples. Mating and oviposition were observed for 14 of the 19 pregnancies resulting in an estimated gestation of 16.7 ± 0.2 days (range 16.0–18.1 d). The estrogen enzyme-immunoassays tested (n = 3) in this study were not suitable for the fecal samples of the echidna. Fecal progesterone metabolites are an effective tool for confirming the timing and occurrence of estrous cycles in captive echidna colonies and can assist zookeepers in identifying possible causes of sub-optimal reproductive success without the unnecessary stress of repeated capture and anaesthesia for blood collection.

Optimizing captive short-beaked echidna (Tachyglossus aculeatus) fecal sample identification and hormonal analysis.

The objectives of this study were to develop a fecal marking protocol to distinguish male from female samples during the echidna breeding season and to determine if normalizing fecal progesterone metabolite data for inorganic content improves the detection of biologically relevant changes in metabolite concentrations. Over a period of 6 weeks, four echidnas were provided with green food coloring powder mixed into 20 g of their regular feed with the dose adjusted weekly by 0.05 g. The proportion of organic (feces) versus inorganic matter (sand) in the fecal samples of three echidnas was determined by combustion of organic matter. Hormonal data was then expressed as metabolite concentration per total dry mass (with sand) of extracted sample versus metabolite concentration per total mass of organic material (without sand). The optimal dose of food coloring powder was 0.30 g: this was excreted in the feces of all echidnas within 24 h of consumption with color present for two consecutive days. Correction for inorganic content (sand) did not significantly affect variability of fecal progesterone metabolite levels (mean CV ± SE with sand: 142.3 ± 13.3%; without sand: 127.0 ± 14.4%; W = 6, p = .2500), or the magnitude of change from basal to elevated fecal progesterone metabolite concentrations (mean ± SE with sand: 8.4 ± 1.7; without sand: 6.6 ± 0.5, W = 10, p = .1250). Furthermore, progesterone metabolite concentrations before and after correction for sand contamination correlated strongly (r = .92, p = < .001). These methods will facilitate future reproductive endocrinology studies of echidna and other myrmecophagous species.

Non-invasive assessment of fecal glucocorticoid, progesterone, and androgen metabolites and microbiome in free-ranging southern white rhinoceros (Ceratotherium simum simum) in South Africa

Increased poaching in northern South Africa has necessitated relocation of large numbers of southern white rhinoceros (Ceratotherium simum simum) to the Eastern Cape Province. The climate and grassland ecology of this province differ from that of northern South Africa which may impact the health of this species. This assessment of fecal steroid levels and microbiome in 10 free-ranging southern white rhinoceros in the Eastern Cape will provide insights into white rhinoceros physiology in this biome. Fecal steroid metabolites were analyzed using enzyme immunoassay (EIA) and ultra-performance convergence chromatography tandem mass spectrometry (UPC2-MS/MS). Fecal microbial composition was assessed via next generation sequencing. EIAs with antibodies raised against progesterone (P4; mouse monoclonal – CL425 clone), testosterone (T; rabbit polyclonal), corticosterone (B; sheep polyclonal) were utilized. Pregnant females had large quantities of fecal progesterone metabolites (FPMs) detected by CL425 EIA. Pregnant females also had native P4 and 11α-hydroxydihydroprogesterone (11αOHDHP4; 4-pregnen-11α-ol-3,20-dione) detected by UPC2-MS/MS but these concentrations were 1000-fold less than the concentrations of FPMs detected by the CL425 EIA. By contrast, non-pregnant females had FPM concentrations detected by CL425 EIA which were similar to native P4 and 11αOHDHP4 concentrations detected by UPC2-MS/MS. Mean fecal androgen metabolite (FAM) concentrations detected by the T EIA were similar between males and females. 11-ketoandrostenedione (11KA4) detected by UPC2-MS/MS was higher in females than males. However, there was no difference between males and females in the concentration of fecal glucocorticoid metabolites (FGMs) detected by the B EIA. Bacteroidia, followed by Clostridia, was the most abundant classes of fecal microbes. The unfiltered microbiome of females was more diverse than that of males. The core fecal microbiome of young rhinoceros had a higher observed species richness (Shannon diversity index, and Simpson diversity index) than that of old rhinoceros. In the alpha male, immobilization was associated with an increase in FGMs detected by 11-deoxycortisol (S) detected by UPC2-MS/MS coupled with decreased abundance of Spirochaetia. We detected substantially different FAM and FPM concentrations from those previously reported for both captive and wild white rhinoceros. Comparison of our UPC2-MS/MS and EIA results underscores the fact that most EIAs are highly cross reactive for many steroid metabolites. Our data also demonstrates a distinct effect of stress not only on FGMs but also on the fecal microbiome. This is the first non-invasive assessment of fecal steroid metabolites by UPC2-MS/MS and the fecal microbiome in wild white rhinoceros.

Use of a simplified non-invasive technic to monitor fecal progesterone metabolites and reproduction function in several zoo species: Efficacy of mini VIDAS® automate (bioMérieux)

Developing the zoos' ability to assess the reproductive status of the individuals they house is essential to improve the husbandry and management of these species. The use of non-invasive techniques such as fecal hormone analysis has been proven to be a simple and effective way to achieve this. Designed by bioMérieux, mini VIDAS® instrument is used in human and veterinary medicine to evaluate different endocrinological parameters, including serum or plasma progesterone. This study evaluates VIDAS® Progesterone (PRG) assay's efficacy to monitor fecal progestagens using a simple sample extraction protocol adapted to the zoo environment. We compared (1) VIDAS® PRG fecal profiles with established assays specifically designed for fecal progestagens analysis at the VetmedUni (Vienna, Austria) for okapis (Okapia johnstoni), greater one-horned rhinoceros (Rhinoceros unicornis), giraffes (Giraffa camelopardalis reticulata) and hippopotamus (Hippopotamus amphibius) (2) VIDAS® PRG fecal profiles with VIDAS® PRG serum profiles for African elephants (Loxodonta Africana), giant anteater (Myrmecophaga tridactyla) and white rhinoceros (Ceratotherium simum). Spearman mean correlations were: 0.6748 for African elephants (n = 2 animals), 0.7969 for giant anteater (n = 1 animal), 0.7926 for okapis (n = 2 animals), 0.6072 for greater one-horned rhinoceros (n = 4 animals), 0.6062 for giraffes (n = 4 animals) and 0.5740 for hippopotamus (n = 2 animals). Fecal progestagens analysis revealed estrous cycles in several species: 12.5 ± 0.5 weeks for African elephants (n = 2 cycles), 15.3 ± 1.1 days for okapis (n = 6 cycles), 44 ± 2.1 days for greater one-horned rhinoceros (n = 4 cycles) and 15.5 ± 0.5 days for giraffes (n = 4 cycles). We observed pregnancies in a giant anteater, an okapi and a hippopotamus. We observed a strong positive Spearman correlation (r > 0.60) for individuals exhibiting estrous cycles. These first results indicate that the mini VIDAS® can be used for monitoring of the reproductive status of non-domesticated species and can be a useful tool for the reproductive management through fecal progesterone analysis. A simple extraction protocol was suitable for sample preparation of fecal progesterone metabolite analysis. Further studies using a larger number of individuals per species at different reproductive stages could confirm the relevance of mini VIDAS® in the zoo community.

Variations, validations, degradations, and noninvasive determination of pregnancy using fecal steroid metabolites in free-ranging pronghorn

Pregnancy status is a key parameter used to assess reproductive performance of a species as it represents a starting point for measuring vital rates. Vital rates allow managers to determine trends in populations such as neonate survival and recruitment; two important factors in ungulate population growth rates. Techniques to determine pregnancy have generally involved capture and restraint of the animal to obtain blood samples for determining serum hormone levels. Non-invasive pregnancy assessment, via feces, eliminates any hazards between handler and animal, as well as removes handling-induced physiological biases. Using noninvasive fecal sampling, we conducted hormone validations, investigated pregnancy rates, and determined hormone degradation rates across five subpopulations of pronghorn (Antilocapra americana) in Idaho. Samples were collected during April-May of 2018 and 2019 from adult pronghorn of known sex and age class. Metabolites of testosterone, cortisol, 17β-estradiol, and progesterone were measured in fecal samples, and concentrations of estradiol and progesterone were examined for pregnancy determination. Average fecal progesterone metabolite (FPM) levels of pregnant females were more than double compared to levels of nonpregnant females. Fecal estrogen metabolite (FEM) levels did not differ during concurrent sampling. The largest difference in FPM levels between pregnant and nonpregnant females began on 28 April. Pregnancy determination sampling showed average FPM levels for all five subpopulations were significantly different than the nonpregnant female validation group. Nonetheless, pregnancy rates for some subpopulations lacked conclusive estimates due to early fecal sampling. Fecal glucocorticoid metabolites (FGM) levels significantly differed between pregnant females and male pronghorn, but did not differ from nonpregnant females. Degradation rates of FPM and FGM differed across days, with values for FPM from Day 1 being significantly different from all subsequent days, and after Day 9 for FGM, demonstrating the requirement of fresh samples to accurately measure hormone concentrations. We concluded that a noninvasive method to diagnosis pregnancy is possible in pronghorn via progesterone metabolites if fresh samples are collected during late gestation.

Non-Invasive Reproductive Hormone Monitoring in the Endangered Pygmy Hog (Porcula salvania).

Simple SummaryThe pygmy hog is one of the world’s rarest suids and classified as an endangered species. Efforts are being made to breed them in captivity and reintroduce them into the wild. In this study, we examined reproductive hormones in captive pygmy hogs using a non-invasive method by collecting 785 fecal samples from five females and two males for 12 months. High-pressure liquid chromatography was performed to examine the presence of immunoreactive progesterone and testosterone metabolites in the fecal samples. We standardized and validated enzyme immunoassays (EIA) for fecal progesterone and testosterone metabolites. Using progesterone EIA, we were able to detect pregnancies in four females and estimate the relevant gestation period. We also recorded 172 births from the captive breeding center and found strong seasonality patterns in births. In males, fecal testosterone metabolite concentrations were higher in the breeding season than in the non-breeding season as evidenced by elevated testosterone concentrations during breeding season. A significant difference in fecal progesterone metabolites concentration was observed between non-pregnant and pregnant females. This study can directly help in monitoring the reproductive status of reintroduced hogs both in the wild and in conservation breeding programs in India and elsewhere.The pygmy hog (Porcula salvania), until recently was classified as a critically endangered suid facing the threat of extinction due to habitat degradation. Efforts are being made to protect the pygmy hog from extinction and breed them in captivity under the Pygmy Hog Conservation Programme (PHCP). However, very little information is available on the reproductive physiology of pygmy hogs. Therefore, the present study aims to standardize enzyme immunoassays (EIAs) for monitoring pregnancy and reproductive status using progesterone and testosterone metabolites. A total of 785 fecal samples were collected from five females and two males over a period of one year from the PHCP Research and Breeding Centre, Guwahati, Assam. High-pressure liquid chromatography (HPLC) analysis revealed the presence of immunoreactive progesterone and testosterone metabolites in feces. Mating was observed in all five females, and four of them gave birth successfully. We were able to detect pregnancy using fecal progesterone metabolites. The mean gestation period, based on mating and parturition, was estimated to be 153.25 days from the four females studied. The breeding center recorded 172 births between 1996 and 2000 and found strong seasonal patterns in the birth rate, with most of the births occurring between May and June. In the males, fecal testosterone metabolites were significantly higher in the breeding season than in the non-breeding season. This is the first study on the subject and will help with future breeding programs in other captive breeding centers and with reproductive monitoring of reintroduced populations.

Linking socio-sexual and vocal behaviour with faecal progesterone and oestrogen metabolite levels in Southern white rhinoceros females.

Progesterone and oestrogen are the main gonadal steroid hormones that regulate the ovarian activity and induce the fertile oestrus period in females. The monitoring of this receptive phase is particularly decisive for captive breeding and is commonly based on the observation of female behavioural patterns that coincide with their hormonal oestrus. However, in the white rhinoceros (WR), a species that is well known for its impaired reproductive rate in captivity, the female behavioural and vocal indicators of receptivity have not been systematically investigated or linked to their hormonal states so far. In order to close this gap, we combined behavioural and acoustic recordings, conducted over an average period of 32days, with the analysis of faecal progesterone and oestrogen metabolite concentrations (fPM and fEM) in 27 adult Southern white rhinoceros (Ceratotherium simum simum; SWR) females from 10 European zoos. For eight of the study females, we were able to detect a receptive period indicated by their acceptance of sexual behaviour from the bulls. The comparison of behaviour and vocalization between receptive and non-receptive periods in these females demonstrated that particularly presenting and marking behaviour distinctly peaked during the receptive period, indicating the significance of olfactory signalling for female reproductive behaviour. Based on the analysis of fPM profiles, we were able to identify different reproductive states (cycling, non-cycling, pregnant) in 21 of 27 study females. In contrast, fEM profiles proved to be unsuitable for the detection of ovarian activity. For the majority (five of six females for which behavioural receptivity and endocrine cyclicity were determined), a coincidence of their receptive period and the hormonal oestrus, indicated by a nadir in fPM levels, could be detected. Conclusively, this study revealed a comprehensive behavioural repertoire that reflects the hormonal oestrus in SWR females and can therefore be reliably used for non-invasive ex situ reproduction monitoring.

Fecal progesterone and estrogen metabolite monitoring for cyclicity and pregnancy in southern tamandua (Tamandua tetradactyla).

Reproductive management of the southern tamandua (Tamandua tetradactyla) should include timed introductions for breeding to minimize aggression and pregnancy monitoring. Since serial blood sampling could cause unnecessary stress, and urinary progesterone metabolites are found in very low concentrations, this study sought to validate progesterone and estradiol enzyme immunoassays for measuring fecal progesterone metabolite (FPM) and fecal estrogen metabolite (FEM) concentrations in two females. Peaks in FEM concentrations coincided with breeding and conception, were 5-6 times higher than baseline concentrations, and were followed by clear luteal phases distinguished by FPM concentrations 5-6 times higher than baseline concentrations. FPM concentrations during the first 30-53 days of gestation overlapped with luteal phase concentrations, thereafter increasing to 8-25 times higher than baseline concentrations. FEM concentrations during the first 41-44 days of gestation remained near basal values for one female, whereas concentrations were 1.8 times higher than baseline for the second. FEM concentrations became elevated for the former by 44 days of gestation and increased further for the latter after 53 days, ultimately averaging four times higher than baseline for both females. The biphasic increase in FPM and FEM concentrations, follicular and luteal phase durations (follicular: 7 ± 1 days, luteal: 25 ± 1 days), total cycle length (41 ± 1 days), and gestation (161-165 days) documented in this study were consistent with previous reports from serum and urine analyses. Monitoring FPM and FEM is a reliable noninvasive method for tracking reproductive cycles and pregnancy in southern tamandua that overcomes the challenges associated with serum or urinary hormone analysis.