Polar Bear Signature Project The Lindner Center for Conservation and Research of Endangered Wildlife (CREW) at the Cincinnati Zoo & Botanical Garden is a global leader in research and conservation efforts with endangered wildlife species. Polar bears (Ursus maritimus) are under threat due to climate change but, despite the worldwide popularity of this iconic species, little is known about their unique physiology or how they will respond to a changing environment. Scientists at CREW are succeeding at integrating high tech scientific methods with innovative approaches to non-invasively study polar bear physiology, with a focus on reproduction. The techniques being developed are novel to the conservation and scientific communities and are likely to have broad application to other critically endangered populations that require monitoring without disruption. FemalesMale SeasonalitySexual MaturationARTContraceptionPublicationsCollaborations Monitoring the reproductive processes of females Scientists at CREW have been studying polar bear reproduction since 2008 and have monitored over 55 bears at 24 institutions. Fecal samples are collected 3-7 times/week and assayed for steroid hormone metabolite concentrations, specifically testosterone and progesterone, which are indicators of ovarian activity and pregnancy/pseudo-pregnancy, respectively. Results indicated that fecal steroid metabolites may be used to indicate ovulation has occurred, but cannot be used to distinguish pregnancy from pseudo-pregnancy. Although fecal steroid concentrations have provided valuable insight into which females are experiencing estrus and ovulating, it does not allow for the discernment between pregnancy and pseudopregnancy. A diagnostic pregnancy test not only would be useful in identifying pregnant females, it may also provide clues as to where the pregnancy process might be failing in so many of these females that fail to produce cubs. Additionally, the test may be useful in other species that experience pseudo-pregnancy as well, such as otters and red pandas. Because we have exhausted all known tests of fecal products that have been used to diagnose pregnancy in other species, it is evident that a novel avenue must be pursued. Fecal proteins Specific proteins may exhibit altered profiles in the feces of pregnant bears, but predicting appropriate candidate proteins to investigate is speculative at best. The objective of this study is to identify potential pregnancy biomarker proteins based on their increased abundance in the feces of pregnant polar bears compared to pseudo-pregnant females using two-dimensional in-gel electrophoresis (2D-DIGE) and mass spectrometry (MS). On average, 2200 proteins or protein fragments were present in each sample, but only five were found to be elevated significantly in the pregnant group. Of those five, transthyretin (TTR) was chosen as the first biomarker to explore further because it is produced by the placenta, so its association with the pregnant state is clear. Antibodies have been identified that detect the excreted form of the polar bear TTR protein and an enzyme-linked immunosorbent assay (ELISA) has been developed to quantify TTR concentrations in protein extracted from feces. The final step of the project (currently underway) involves comparing TTR excretion patterns among confirmed pregnant, pseudo-pregnant, and control bears. To do this, protein is being extracted from nearly 800 fecal samples to compare TTR concentrations among groups throughout the year and will be assayed for TTR concentrations. Not only would the validation of a pregnancy biomarker be useful in diagnosing pregnancy, but it may also provide insight into the prevalence and timing of pregnancy loss that may be occurring in females that breed but fail to produce cubs. Sniffer dog project (project completed) Over three years, a detection dog was trained to distinguish fecal samples originating from pregnant bears from all other samples. Although “Elvis” consistently signaled positively on novel samples from pregnancies on which he was trained, he did not signal positively on novel samples from novel pregnancies. We suspect that he has either memorized the scent signatures of the pregnancies on which he was trained and has not generalized his recognition to novel pregnancies or that the scent signature of the samples changes with time, impacting his analysis. However, because he is able to distinguish the pregnant (post-breeding) from the non-pregnant (pre-estrus) state in those pregnancies, testing is underway to determine the earliest pregnancy may be detectable. Fecal volatile organic compound (VOC) analysis (in process) In a project closely related to the sniffer dog project, volatile organic compound (VOC) analysis was performed on over 40 polar bear fecal samples to identify the chemicals present in the airspace above fecal samples. Of the 2705 different compounds identified, none was unique to pregnant bears or present in significantly higher concentrations in the pregnant versus pseudo-pregnant bears. Researchers at CREW are collaborating with analytical chemists to mine the complex dataset in an attempt to decipher the chemical signature associated with pregnancy. Monitoring male seasonality In an effort to determine the source of low reproductive success in polar bears in zoos, an important objective of CREW’s polar bear project was to characterize testosterone concentrations in males in zoos to verify that they are experiencing increases in testosterone during the breeding season. Furthermore, because captive bears generally live at lower latitudes and warmer climates relative to their wild cousins, they may provide insight into the ability of wild bears to adapt to a changing environment. In 2012, scientists at CREW completed a 3-yr study in which they monitored fecal testosterone concentrations in 14 adult male polar bears residing in 13 zoos ranging in location from Alaska to Arizona. Fecal samples were collected once per week year round, frozen, and shipped to CREW for analysis. Factors such as season, age, latitude, and the presence of females were evaluated to ascertain their effects on testosterone concentrations. Results showed that testosterone is significantly higher during the breeding season (Jan-May) compared to the rest of the year, similar to wild bears. Even males that did not breed or were not housed with females experienced seasonal testosterone increases, albeit lower concentrations than breeding males. Testosterone was lowest in the younger males, peaked when a male is ~10-18 years old, and then decreased with old age. Overall, latitude did not affect testosterone concentrations, although males at lower latitudes tended to exhibit lower testosterone during the summer months. It is unlikely that decreased testosterone in the summer would inhibit male fertility because the breeding season occurs at the end of the coldest months when testosterone concentrations were similar among all latitude groups. In conclusion, males in human care experience seasonal fluctuations in testosterone appropriate to the breeding season, comparable to their wild counterparts. There is no evidence that diminished seasonal cues or seasonal asynchrony between the sexes is responsible for low reproductive success in zoos. Results from this study do not reveal aberrations in testosterone production that would negatively affect the fertility of captive male polar bears. Monitoring sexual maturation in polar bears Polar bears in zoos generally are recommended for breeding starting around the age of five or six and then it may take a few years for them to conceive; however, recent DNA analysis of the wild population indicates that males as young as two or three may sire offspring and females as young as four can produce cubs. One of the aims of CREW’s Polar Bear Signature Project is to determine when juveniles become sexually mature; it is the first study to perform longitudinal fecal hormone monitoring on young polar bears throughout sexual maturation. With so few cubs born each year, it’s important that every sexual mature individual be paired for breeding to increase the chances of pregnancies and births. Because there are so few young individuals in the zoo population, compiling a complete dataset has been slow, however; the preliminary results are in: data indicate that males and females as young as three begin exhibiting seasonal changes in excretion patterns of steroid hormone metabolites, suggesting the onset of sexual maturation. Interestingly, one three-year-old female even exhibited a pseudo-pregnancy, despite the fact that breeding did not occur. Deciphering the fluctuations in hormone patterns associated with sexual maturity may allow for more appropriate breeding recommendations and will establish a knowledge-base of normal hormone patterns for juvenile polar bears. Since longitudinal sampling from wild individuals is logistically challenging, information learned from the zoo population may be applied to their wild cousins as well. Developing and Optimizing Assisted Reproductive Technologies (ART) in Polar Bears Semen collection and cryopreservation. Due to growing concern over reproductive failure in polar bears nationwide, CREW scientists are receiving requests from other zoological institutions to perform assisted reproductive procedures, such as artificial insemination (AI). A polar bear sperm bank is vital in supporting AI endeavors; however, the traditional method of semen collection from wildlife, electro-ejaculation, has been relatively unsuccessful in polar bears. Our compelling preliminary research has evaluated the use of a novel, minimally-invasive method of semen collection in this species. Although the mechanism is not well understood, there are reports of certain anesthetic drugs inducing ejaculation or causing semen to pool in the urethra in other species. One drug, medetomidine, seems to have this effect and also is already commonly used to anesthetize polar bears in zoos. To retrieve the sperm, a catheter is threaded into the urethra, a syringe is attached to the end of the catheter, and the catheter containing the sperm is slowly withdrawn. The procedure can be performed opportunistically at the time of a regularly scheduled physical examination and takes less than 5 minutes, so it doesn’t require a lengthy extension of anesthesia time. This new approach has resulted in an impressive 91% success rate and, for the first time, the creation of a polar bear sperm bank to store these valuable genetics is possible. A future goal is to perform semen collections on wild bears to preserve their genetics and also to investigate the impact of pollutants on the fertility of wild bears. Artificial Insemination (AI). Due to the shortage of males, potentially reproductively viable females are left without a male to breed. Consequently, the demand for developing ART is growing; however, little is known of the hormones orchestrating the intricacies of female reproduction in this species. Unlike humans and domestic species, information is non-existent regarding the use of exogenous hormones to overcome infertility in polar bears. Additionally, the need to determine the timing of hormones relative to performing an AI procedure is important to ensure that the insemination is performed around the time of ovulation. We have demonstrated successful ovulation induction in a polar bear using exogenous hormones; however, the results have been variable among individuals, so the precise hormones, dosages, and timing of administration relative to AI warrants further examination. Establishing a polar bear sperm bank and optimizing ART in this species will provide opportunities for conception to females that otherwise would not have a chance of pregnancy. Birth control that worked too well Unlike castration, which results in permanent sterility, hormonal contraceptives are considered to be temporary and reversible and, consequently, they play an integral role in managing animal population growth. Many polar bears in zoos were administered contraceptives in the early part of the 21st century due to lack of exhibit space and decreased need for additional zoo-born cubs. But more recently, the demand has risen and polar bears currently are in high demand. Contraceptive treatments were discontinued and nearly all females of reproductive age were paired for breeding; however, very few cubs are produced each year. From 2008 – 2016, CREW scientists monitored the fertility of ~30 mature female bears across North America, roughly half of which have been treated with contraceptives. Although the females that had been contracepted resumed normal breeding behaviors following treatment, only two (18%) gave birth. Both of these females had received just a single type of contraception whereas others had received different or multiple types over the years. In comparison, nearly 53% of the females that had never been contracepted produced cubs. A number of factors probably contribute to poor fertility, such as advanced maternal age and whether or not individuals had produced cubs prior to treatment, but it does appear that certain hormonal contraceptives have had unintended long term deleterious effects on the fertility of this species. Although it is unknown as to where, exactly, the pregnancy process is failing, CREW scientists are examining ways to overcome infertility to give these females a chance at motherhood. Recent peer-reviewed publications: Curry E, Safayi S, Meyerson R, Roth TL. (2015). Reproductive trends of polar bears in zoos (Ursus maritimus) in North American zoos: a historical analysis. Journal of Zoo and Aquarium Research 3:99-106. Curry E, Wyatt J, Sorel L, MacKinnon KM, Roth TL. (2014). Ovulation induction and artificial insemination of a polar bear in zoos (Ursus maritimus) using fresh semen. Journal of Zoo and Wildlife Medicine 45:645-649. Curry E, Roth TL, MacKinnon KM, Stoops MA. (2012). Factors influencing annual fecal testosterone metabolite profiles in male polar bears in zoos (Ursus maritimus). Reproduction in Domestic Animals 47:222-225. Stoops MA, MacKinnon KM, and Roth TL (2012). Longitudinal fecal hormone analysis for monitoring reproductive activity in the female polar bear (Ursus maritimus). Theriogenology 78: 1977-1986. Curry E, Stoops MA, Roth TL. (2012). Non-invasive detection of candidate pregnancy protein biomarkers in the feces of polar bears in zoos (Ursus maritimus). Theriogenology 78:308-314. Conference proceedings: Curry E, Roth TL, MacKinnon KM, Stoops MA. (2016). Monitoring the long-term effects of contraceptives on the fertility of polar bears in zoos (Ursus maritimus). International Congress on Canine and Feline Reproduction. Paris, France (June 22nd-25th). Curry E, Stoops MA, DeLorenzo CJ, MacKinnon KM, Roth TL. (2016). Using the ex situ population to advance the reproductive science of polar bears. 24th International Conference on Bear Research and Management. Anchorage, AK (June 12th-16th). Curry E and Roth TL. (2016). Poster presentation. A rapid, minimally-invasive method of collecting semen from polar bears. Proceedings of the International Embryo Transfer Society 42nd Annual Conference. Louisville, KY (January 23rd-January 26th). DeLorenzo C, Lynch B, Roth T, Petren K, Curry E. (2016). Poster presentation. Development of a non-invasive, fecal protein pregnancy test for polar bears. Proceedings of the International Embryo Transfer Society 42nd Annual Conference. Louisville, KY (January 23rd-January 26th). Curry E, Skogen M, Roth TL. (2014). Oral presentation. Evaluating the use of a detection dog and volatile organic compound analysis for non-invasive pregnancy diagnosis in the polar bear (Ursus maritimus). 23rd International Conference on Bear Research and Management. Thessaloniki, Greece (Oct 5th-11th). Curry E, Roth TL, MacKinnon KM, and Stoops MA. (2012). Platform presentation. Seasonal variation in fecal testosterone metabolite concentrations in male polar bears in zoos (Ursus maritimus). International Conference on Canine and Feline Reproduction. Whistler, BC (July 26th-29th). Roth TL, MacKinnon KM, and Stoops MA. (2011). Non-invasive fecal hormone monitoring for evaluating polar bear (Ursus maritimus) reproductive activity. Proceedings for the 20th International Conference on Bear Research & Management. Ottawa, ON (July 17th-23rd). Stoops MA, Vollmer L, and Roth TL. (2009). Faecal steroid analyses for monitoring reproductive function in polar bears (Ursus maritimus). Reproduction, Fertility and Development 21(1) 182-183. Roth TL, MacKinnon KM, and Stoops MA. (2009). Noninvasive fecal hormone monitoring for assessing reproductive activity and diagnosing pregnancy in the polar bear (Ursus maritimus). Proceedings for Advancing Bear Care 2009, Bear Care Group. San Francisco, CA. Collaborative Research Investigating the relationship between ambient conditions and thermoregulatory responses in polar bears (University of Guelph). By understanding links between thermoregulation demands and stress, adjustments for the environment of polar bears in zoos could be suggested to reduce temperature-associated stress thus improving the probability of successful breeding in captivity. Photogrammetry analysis of polar bear images (Purdue University, University of Wyoming, and Polar Bears International). As part of the Polar Bears International citizen science project in Churchill, Manitoba, Canada, researchers are using polar bears in zoos to optimize methodologies used to study the wild population. They are developing photographic methods to measure body condition to monitor long-term trends in morphometric changes related to alteration of environmental conditions. Fecal near infrared spectroscopy (NIRS) (Texas A&M). Dried fecal samples from pregnant and non-pregnant bears are scanned using a spectrometer and measurements of reflectance are recorded over the visible and near infrared ranges. If differences are found, this method could provide a non-invasive means of pregnancy diagnosis in this species. Exploring the metabolomic signature of pregnant polar bears (Cincinnati Children’s Hospital). Scientists at the Cincinnati Children’s Hospital Medical Center are characterizing the metabolites in polar bear fecal samples to determine if specific biomarkers or patterns are unique to pregnancy. Studies are underway to evaluate the metabolites in hundreds of samples. Polar bears (Ursus maritimus): connections between activity levels, temperature and day length (Miami University). The activity levels of zoo-housed bears at multiple facilities were assessed in relation to temperature and hours of daylight. Results of this study may help to define the conditions that polar bears in zoos should be housed in to enhance their health and behavior. Comparison of fecal and urinary steroid hormone excretion patterns in polar bears (SeaWorld San Diego). The aim of this research is to compare steroid excretion patterns in urine and feces collected from polar bears in zoos, especially those undergoing treatment for infertility. Characterization of various metabolite patterns will provide insight into the reproductive processes of this species. Evaluation of urinary and fecal hormone metabolites in pregnant polar bears (Memphis Zoo). This study aims to compare urinary and fecal steroid hormone excretion patterns in pregnant and non-pregnant polar bears. Annual fecal glucocorticoid metabolite concentrations in pregnant and pseudopregnant polar bears (Ursus maritimus) in North American zoos. (Brookfield Zoo) The goal of this study was to determine if glucocorticoid concentrations are higher in the feces of pseudo-pregnant polar bears versus pregnant bears. Patterns of diversity in the indigenous microbiota of mammals in human care (Stanford University) Researchers are investigating the various microbes present in the guts of many animals, including polar bears. Arctic Ambassador Center With CREW’s expanding involvement in polar bear research, conservation and education and the Zoo’s progress in going green, it was a natural next-step to formally name the Cincinnati Zoo & Botanical Garden one of Polar Bears International’s official Arctic Ambassador Centers. PBI Arctic Ambassador Centers are organizations endorsed by leading polar bear scientists and the AZA for actively engaging in saving polar bear habitat through greenhouse gas reductions within their organizations and communities. As such, CREW and the Zoo are committed to: 1) providing information about climate change to the local community on Zoo grounds, on the website and through special programs, 2) actively reducing our own carbon emissions and 3) maintaining our polar bears in an exhibit that meets all AZA standards. In return, we receive: 1) access to educational materials and biofacts from PBI, 2) support from PBI staff, 3) special opportunities for social networking and public relations and, perhaps most important, 4) the opportunity to engage in several unique PBI education programs.