Comprehensive Breeding Techniques for the Giant Panda.

The dramatic growth of the captive giant panda (Ailuropoda melanoleuca) population exemplifies how the application of scientific findings to animal care and reproductive management can improve conservation breeding outcomes. Detailed behavioral studies of giant panda estrus, pregnancy and cub rearing have demonstrated the importance of husbandry management that supports natural reproductive behavior to enhance breeding success. Natural breeding has been valuably augmented by the development of assisted reproductive techniques founded through detailed studies of the reproductive physiology of the giant panda and outlining fundamental information about reproductive seasonality, male fertility and characterization of the estrous cycle. The resultant holistic understanding of giant panda reproduction has improved reproductive success in the captive population to such an extent that it is now self-sustaining and provides surplus animals for reintroduction. Despite these significant advances, there are knowledge gaps and remaining challenges to be addressed. Pregnancy detection remains the single biggest challenge when determining if natural mating or assisted breeding have been successful. Because pregnancy can only be determined in the few weeks prior to parturition, there are gaps in understanding and detecting delayed implantation and early embryonic loss. Additionally, dynamic management practices and standard of care for reproductive assistance needs to be developed. Only large breeding centers in China have the ability to promote normal reproductive behaviors and allow mate choice for the giant panda. These challenges need to be addressed in the near future in order to maintain a self-sustaining, genetically diverse and behaviorally competent captive population. This chapter documents the development of successful giant panda managed breeding programs by focusing on three key areas, (1) the development of science-driven reproductive techniques to improve fecundity in a species where the mating system was poorly understood, (2) how targeted research and adaptive management of social settings surrounding estrus and breeding improved reproductive success, and (3) insights and solutions to challenges faced across the program's history with future directions for research.

Reproductive activity in the peninsular pronghorn determined from excreted gonadal steroid metabolites.

Fecal hormone monitoring was employed to better define annual patterns of reproductive steroid metabolites from a breeding pair of peninsular pronghorn (Antilocapra americana peninsularis) maintained at the Los Angeles Zoo. Notably in the female, increased excretion of estrogen metabolites occurred during the breeding season (Jun-Aug), and a biphasic pattern in progestagen activity was measured during gestation. Of additional interest, a preterm increase in estrogen that continued for an additional 64 days post partum. Male androgen activity correlated with the female estrogen patterns, with a single successful copulation occurring during the breeding season; interestingly however, the male exhibited no reproductive behaviors during the female's preterm/post partum estrogen increase. These data are the first reproductive steroid profiles for the peninsular pronghorn and provide valuable insight that will aid efforts that link the species' reproductive physiology with conservation management.

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.

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.

Unique biphasic progestagen profile in parturient and non-parturient giant pandas (Ailuropoda melanoleuca) as determined by faecal hormone monitoring.

The luteal phase of the giant panda has been exclusively assessed by studying urinary hormone patterns in a very few individuals. To better understand hormonal dynamics of protracted progestagen excretion in this endangered species, we monitored hormonal metabolites in the fibrous faeces of multiple females in the USA and China. Giant pandas that were anoestrual during the breeding season excreted baseline progestagen throughout the year. In contrast, there were two distinctive periods when progestagen excretion increased in females that experienced behavioural oestrus, the first being modest, lasting for 61-122 days, and likely reflecting presumptive ovulation. This increase was far surpassed by a secondary rise in progestagen excretion associated with a rejuvenated luteal capacity or hormone production from an extra-gonadal source. The duration of this 'secondary' rise in progestagen excretion averaged approximately 45 days and terminated in a decline to baseline coincident with parturition or the end of a non-parturient luteal interval. Data revealed that, even with a complex, biphasic progestagen profile, the longitudinal patterns produced by giant pandas were relatively consistent among animals and across years within individuals. However, progestagen excretion patterns throughout this period could not be used to discriminate among non-pregnant, pregnant or pseudopregnant states.

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 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.

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.

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.

Use of fecal glucocorticoid and salivary cortisol concentrations as a measure of well-being of New York City carriage horses.

OBJECTIVE To use noninvasive approaches to assess stress in New York City (NYC) carriage horses during the course of their daily routine to determine whether use of these horses affected their well-being. DESIGN Prospective case control study. ANIMALS 13 (5 mares and 8 geldings) stabled working NYC carriage horses and 5 pastured (nonworking) NYC carriage horses (1 mare and 4 geldings). PROCEDURES Samples for determination of fecal glucocorticoid and salivary cortisol concentrations were collected on 3 successive days from 10, 8, and 9 working carriage horses during rest (time 1), preparation for work (time 2), and return to the stable (time 3) and at 1 hour after work (time 4). Infrared thermography (IRT) measurements were made to determine maximum temperature of the medial canthus at each time point. Fecal samples were also collected from 5 pastured carriage horses for determination of glucocorticoid concentrations. RESULTS No difference was found in mean ± SE fecal glucocorticoid concentrations between pastured (22.1 ± 9.8 ng/g) and working (19.5 ± 4.2 ng/g) carriage horses. A significant difference was found in salivary cortisol concentrations of working carriage horses between time 3 (0.96 ± 0.06 ng/mL) and time 4 (0.77 ± 0.07 ng/mL). The IRT measurement at time 2 (35.5 ± 0.64°C [95.9 ± 1.2°F]) was significantly lower than that at time 3 (36.2 ± 0.64°C [97.1 ± 1.2°F]). No other differences in IRT measurements were found. CONCLUSIONS AND CLINICAL RELEVANCE These working NYC carriage horses did not have physiologic responses indicative of a negative welfare status.

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.

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.