David C. Kersey

The use of noninvasive and minimally invasive methods in endocrinology for threatened mammalian species conservation.

Endocrinology is an indispensable tool in threatened species research. The study of endocrinology in threatened species not only advances knowledge of endocrine mechanism but also contributes to conservation efforts of studied species. To this end, endocrinology has been traditionally used to understand reproductive and adrenocortical endocrine axes by quantifying excreted steroid metabolites. From these studies a large body of knowledge was created that contributed to the field of endocrinology, aided conservation efforts, and created a template by which to validate and conduct this research for other species. In this regard noninvasive hormone monitoring has become a favored approach to study the basic endocrinology of wildlife species. Due to the increased understanding of endocrine physiology of threatened species, breeding rates of captive population have improved to levels allowing for reintroduction of species to restored natural ecosystems. Although these approaches are still employed, advances in biochemical, molecular, and genomic technologies are providing inroads to describe lesser known endocrine activity in threatened species. These new avenues of research will allow for growth of the field with greater depth and breadth. However, for all approaches to endocrinology, limitations on resources and access to animals will require innovation of current methodologies to permit broad application for use in threatened species research.

Parallel and seasonal changes in gonadal and adrenal hormones in male giant pandas (Ailuropoda melanoleuca)

Abstract The purpose of this study was to determine androgen and glucocorticoid (GC) hormonal patterns in male giant pandas (Ailuropoda melanoleuca) by monitoring gonadal and adrenal metabolites in feces. Initial validation experiments demonstrated comparable excretory patterns in urine versus feces for both androgen and GC measures. Matched urinary and fecal androgen and GC were correlated strongly with each other in a single male that was assessed over 2 years. A single pharmacological injection of adrenocorticotropic hormone caused a 15-fold GC increase in feces above baseline within 10 h, a peak at 12 h, and a return to baseline at 20 h, demonstrating the biological relationship between adrenal activation and GC excretion. Longitudinal androgen and GC excretory profiles in male giant pandas housed at North American (n  =  2) and Chinese (n  =  3) facilities were similar, with fecal androgens generally exceeding baseline coincident with the onset of the 5-month annual breeding season (January–June), after which values returned to nadir. Similarly, fecal GC excretion increased during the breeding season but was baseline thereafter. Fecal androgen and GC in a single male monitored through transition from subadult to sexual maturity also occurred in parallel. In this individual, basal fecal androgen and GC increased 88% and 66%, respectively, from 5 to 6 years of age. Collectively, these data demonstrate seasonal variations in gonadal activity in the giant panda by measuring androgen metabolites in feces, with elevations consistently occurring from January through June before a return to baseline for ∼4–6 months. Findings also reveal a similar temporal rise in adrenal GC patterns associated with breeding season onset, perhaps a mechanism to enhance metabolism, maximize body energy stores, and provide a competitive advantage in achieving mating opportunities. Examination of data from a single male suggests that the ability to produce these seasonal androgen and GC elevations is age dependent and occurs coincident with puberty.

Giant Pandas: Endocrinology of the giant panda and application of hormone technology to species management

INTRODUCTION Increasing breeding success in the giant panda requires a better understanding of its complex reproductive biology. We know that the female is typically mono-oestrus during a breeding season which occurs from February to May (within and outside China). Behavioural and physiological changes associated with pro-oestrus and oestrus last one to two weeks, during which the female exhibits proceptive behaviours, such as scent marking, to advertise her sexual receptivity (Lindburg et al ., 2001). During the peri-ovulatory interval, receptive behaviours (e.g. tail-up lordotic posture) climax with copulation generally occurring over a one- to three-day interval. Birthing occurs from June to October with a gestation of 85 to 185 days (Zhu et al ., 2001). This unusually wide gestation span is due to the phenomenon of delayed implantation, a varied interval before the conceptus implants in the uterus and begins foetal development. The driving force behind implantation in this species is unknown. The giant panda also experiences pseudopregnancy, whereby the female exhibits behavioural, physiological and hormonal changes similar to pregnancy. Behavioural and physiological cues associated with both pregnancy and pseudopregnancy include decreased appetite, nest-building and cradling behaviours, vulvar swelling and colouration, mammary gland enlargement and lethargy. Additionally, temporal and quantitative progesterone patterns (tracked by assessing urinary hormone by-products and progestins) are indistinguishable between pregnancy and pseudopregnancy. Therefore, no definitive test currently exists for identifying pregnant from pseudopregnant giant pandas.

The acute phase protein ceruloplasmin as a non-invasive marker of pseudopregnancy, pregnancy, and pregnancy loss in the giant panda.

After ovulation, non-pregnant female giant pandas experience pseudopregnancy. During pseudopregnancy, non-pregnant females exhibit physiological and behavioral changes similar to pregnancy. Monitoring hormonal patterns that are usually different in pregnant mammals are not effective at determining pregnancy status in many animals that undergo pseudopregnancy, including the giant panda. Therefore, a physiological test to distinguish between pregnancy and pseudopregnancy in pandas has eluded scientists for decades. We examined other potential markers of pregnancy and found that activity of the acute phase protein ceruloplasmin increases in urine of giant pandas in response to pregnancy. Results indicate that in term pregnancies, levels of active urinary ceruloplasmin were elevated the first week of pregnancy and remain elevated until 20–24 days prior to parturition, while no increase was observed during the luteal phase in known pseudopregnancies. Active ceruloplasmin also increased during ultrasound-confirmed lost pregnancies; however, the pattern was different compared to term pregnancies, particularly during the late luteal phase. In four out of the five additional reproductive cycles included in the current study where females were bred but no birth occurred, active ceruloplasmin in urine increased during the luteal phase. Similar to the known lost pregnancies, the temporal pattern of change in urinary ceruloplasmin during the luteal phase deviated from the term pregnancies suggesting that these cycles may have also been lost pregnancies. Among giant pandas in captivity, it has been presumed that there is a high rate of pregnancy loss and our results are the first to provide evidence supporting this notion.

Endocrine milieu of perioestrus in the giant panda (Ailuropoda melanoleuca), as determined by non-invasive hormone measures

The aim of the present study was to determine the efficacy of faecal hormonal measures for evaluating ovarian activity in a significant sized cohort of giant pandas during the perioestrual period. Faecal excretion of oestrogen and progestagen metabolites corresponded with urinary patterns and receptive behaviours. Longitudinal assessment of 10 females revealed that, on average, faecal oestrogen concentrations started to rise (P < 0.05) above baseline (baseline mean +/- s.e.m.; 64.7 +/- 6.6 ng g(-1)) 5 days before the preovulatory oestrogen peak (484.6 +/- 126.8 ng g(-1)), which was followed by a gradual descent over 4 days to nadir. Mean faecal progestagen metabolite concentrations increased approximately twofold above baseline (from 186.2 +/- 37.7 to 347.2 +/- 75.7 ng g(-1); P < 0.05) during the 20-day interval after the preovulatory oestrogen surge. Variability within and among females precluded the use of a threshold of oestrogen or progestagen metabolites to predict reproductive status, yet faeces collected 2-3 days per week provided sufficient data to recognise that an individual was in the perioestrual period. Finally, in females that were examined for at least 3 consecutive years, there was an 18-53 day variation in the onset and an 8-13 day variation in the duration of perioestrual behaviour from year to year. In summary, these findings indicate that gonadal hormone profiles associated with the period immediately before, during and after oestrus are accurately revealed by analysis of the fibrous faeces of the giant panda. This approach has potential value for providing point-in-time information on the reproductive status of free-living individuals.

Assessment of diurnal urinary cortisol excretion in Asian and African elephants using different endocrine methods.

Longitudinal urine samples were collected from Asian and African elephants to assess sample processing and immunoassay techniques for monitoring adrenal activity. Temporal profiles of urinary cortisol measured by RIA and EIA, with and without dichloromethane extraction, were similar; all correlation coefficients were >0.90. However, based on regression analyses, cortisol immunoactivity in extracted samples was only 72-81% of that of unextracted values. Within assay technique, RIA values were only 74-81% of EIA values. Collection of 24-hr urine samples demonstrated a clear diurnal pattern of glucocorticoid excretion, with the lowest concentrations observed just before midnight and peak concentrations occurring around 0600-0800 hr. These results indicate that elephants fit the pattern of a diurnal species, and that glucocorticoid production is affected by a sleep-wake cycle similar to that described for other terrestrial mammals. Cortisol can be measured in both extracted and unextracted urine using RIA and EIA methodologies. However, unexplained differences in quantitative results suggest there may be sample matrix effects and that data generated using different techniques may not be directly comparable or interchangeable.

Assessment of luteinizing hormone and prolactin immunoactivity in Asian and African elephant urine using assays validated for serum

Analysis of serum hormones is useful for timing artificial insemination (Luteinizing hormone) and diagnosing pregnancy (prolactin) in elephants. However, these tests require blood collection, which is not tolerated by all animals, and is impractical for field studies. Thus, developing a means to obtain these measures noninvasively could improve species management. Matched urine and serum was collected from Asian and African elephants daily throughout the follicular phase and after administration of a GnRH analogue for LH determination, and in pregnant and nonpregnant females for prolactin analyses using immunoassays validated for elephant serum. Despite identifying robust increases in circulating hormone concentrations, no concomitant changes in urinary LH or prolactin immunoactivity was detected. Concentration of samples by centrifugal filtration or ethanol precipitation did not increase the ability to measure biologically relevant changes in endogenous urinary LH or prolactin immunoactivity. Sample matrix interference was ruled out following sufficient recovery of exogenous LH or prolactin added to samples, except for samples concentrated >35-fold where some interference was suspected. These results suggest that elephants either do not excrete native LH or prolactin in urine, or concentrations are too low to be measured accurately by standard immunoassay techniques that are valid for serum analyses. Thus, it does not appear feasible or economically viable to use these noninvasive tests for ovulation detection or for pregnancy diagnosis in elephants.

Rising fecal glucocorticoid concentrations track reproductive activity in the female giant panda (Ailuropoda melanoleuca).

To better understand the adaptive significance of adrenal glucocorticoid (GC) variation in the giant panda, we assessed patterns of fecal GC excretion over time as well as during estrus, parturient and non-parturient luteal phases, lactation and acyclicity in 17 adult females. Fecal estrogen and GC patterns were positively correlated (P<0.05) in four of five periestrual females (r = 0.57-0.92). Among all reproductive states, fecal GC was highest (P<0.05) during periestrus (non-parturient, 495.9 ± 100.7 ng/g [mean ± SE]; parturient, 654.1 ± 10 6.5 ng/g; P>0.05). Concentrations of GC metabolites were lower (P<0.05) during the later stage of the luteal phase in non-parturient (334.8 ± 24.8 ng/g) compared to parturient (470.4 ± 54.0 ng/g) females. Although fecal GC concentrations in cyclic, non-parturient females did not differ (P>0.05) across all seasons, there were seasonal variations (P<0.05) in females that were acyclic and non-lactational. However, the overall lack of difference (P>0.05) in GC values between reproductively cyclic and acyclic females did not support the hypothesis that ovarian acyclicity is due to increased adrenal activity (related or unrelated to physiological stress). Furthermore, GCs may play an important role in the normal endocrine milieu associated with sexual receptivity and late pregnancy. These data demonstrate that both reproductive status and seasonal factors are important modulators of adrenal function in this endangered species.

The birth of a giant panda: Tracking the biological factors that successfully contribute to conception through to postnatal development.

Reproducing giant pandas (Ailuropoda melanoleuca) remains the most challenging aspect of managed care of this species. However, advancement in knowledge stemming from basic science research on the giant panda has facilitated a growth in the population. Here, we report the successful application of reproductive technologies, including noninvasive hormone monitoring, behavioral/morphometric observations, ultrasonographic evaluations, and acute phase protein assessment, in an individual female. By applying these approaches to one female, we report the practicality and usefulness of a multidisciplinary approach to reproductive care of the species. In addition, the utilization of various technologies across multiple physiological states also provided us an opportunity to record previously understudied events, such as maternal response to weaning and growth of a conceptus.

Use of urinary 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) concentrations to diagnose pregnancy and predict parturition in the giant panda (Ailuropoda melanolecua)

Pregnancy determination is difficult in the giant panda (Ailuropoda melanolecua), representing a challenge for ex situ conservation efforts. Research in other species experiencing pseudopregnancy indicates that urinary/fecal concentrations of 13,14, dihydro-15-keto-prostaglandin F2α (PGFM) can accurately determine pregnancy status. Our objective was to determine if urinary PGFM concentrations are associated with pregnancy status in the giant panda. Urinary PGFM concentrations were measured in female giant pandas (n = 4) throughout gestation (n = 6) and pseudopregnancy (n = 4) using a commercial enzyme immunoassay. Regardless of pregnancy status, PGFM excretion followed a predictable pattern: 1) baseline concentrations for 11–19 weeks following ovulation; 2) a modest, initial peak 14–36 days after the start of the secondary urinary progestagen rise; 3) a subsequent period of relatively low concentrations; and 4) a large, terminal peak at the end of the luteal phase. Pregnant profiles were distinguished by an earlier initial peak (P = 0.024), higher inter-peak concentrations (P < 0.001), and a larger terminal peak (P = 0.003) compared to pseudopregnancy profiles. Parturition occurred 23 to 25 days from the initial PGFM surge and within 24 hours of the start of the terminal increase. These pattern differences indicate that urinary PGFM monitoring can be used to predict pregnancy status and time parturition in the giant panda. Furthermore, this is the only species known to exhibit a significant PGFM increase during pseudopregnancy, suggesting a unique physiological mechanism for regulating the end of the luteal phase in the giant panda.