Glucocorticoid Production, Activity Levels, And Personality Traits Of Fennec Foxes (Vulpes zerda) Managed For Different Roles In Zoos.

Fecal glucocorticoid metabolite (FGM) concentrations, activity, and personality were assessed for 35 fennec foxes (Vulpes zerda) to determine whether animals managed as ambassadors differed from exhibit or off-exhibit animals. A FGM assay, pedometer, and personality assessment tool were validated. Then, fecal samples and movement data were collected during winter and summer periods. Handling was recorded, and the personality of each fox was evaluated. Generalized linear mixed models assessed the relationships between FGM concentrations, activity, personality, handling, sex, season, rearing type, and role. FGM concentrations did not differ in relation to role or handling. Foxes were most active at night; the time of peak activity did not vary with role or handling. Foxes were more active in winter; males were more active than females, and ambassador foxes were more active than off-exhibit animals. Hand-reared foxes were more sociable, and, at one institution, ambassador foxes were more sociable than foxes in other roles. These results suggest that management for ambassador programs is not associated with changes in glucocorticoid production or circadian patterns but may increase activity and be associated with greater sociability.

Investigating Welfare Metrics for Snakes at the Saint Louis Zoo.

Modern herpetoculture has seen a rise in welfare-related habitat modifications, although ethologically-informed enclosure design and evidence-based husbandry are lacking. The diversity that exists within snakes complicates standardizing snake welfare assessment tools and evaluation techniques. Utilizing behavioral indicators in conjunction with physiological measures, such as fecal glucocorticoid metabolite concentrations, could aid in the validation of evidence-based metrics for evaluating snake welfare. We increased habitat cleaning, to identify behavioral or physiological indicators that might indicate heightened arousal in snakes as a response to the disturbance. While glucocorticoid metabolite concentrations increased significantly during a period of increased disturbance, this increase was not associated with a significant increase in tongue-flicking, a behavior previously associated with arousal in snakes. Locomotion behavior and the proportion of time spent exposed were also not affected by more frequent habitat cleaning. These results demonstrate the need to further investigate the behavioral and physiological responses of snakes to different aspects of animal care at a species and individual level. They also highlight the need to collect baseline behavioral and physiological data for animals, in order to make meaningful comparisons when evaluating changes in animal care.

DESLORELIN (SUPRELORIN®) USE IN NORTH AMERICAN AND EUROPEAN ZOOS AND AQUARIUMS: TAXONOMIC SCOPE, DOSING, AND EFFICACY.

The Association of Zoos and Aquariums Reproductive Management Center (RMC) in the US and the European Association of Zoos and Aquaria Reproductive Management Group (RMG) in Europe monitor efficacy of contraceptive products in participating institutions and use those results to inform contraceptive recommendations. This study used the joint RMC-RMG Contraception Database to analyze efficacy of deslorelin implants (Suprelorin®), a contraceptive used in a wide range of mammalian taxa. More recently its use has increased in birds and in some reptiles and fish. Deslorelin, a gonadotropin-releasing hormone (GnRH) agonist, stimulates the reproductive system before downregulating receptors on pituitary cells that produce hormones that stimulate gonadal steroids in both males (testosterone) and females (estradiol and progesterone), interrupting sperm production and ovulation, respectively. Nevertheless, it has been used mostly in females. Efficacy has been high in mammals, with failures resulting in offspring in only 1.3% of treated individuals and 0.5% of treatment bouts. The failure rate has been higher in birds, with 14.7% of individuals in 7.2% of bouts producing eggs, perhaps reflecting differences in avian GnRH molecules. Too few reptiles and fish have been treated for meaningful analysis. Although deslorelin appears very safe, a possible exception exists in carnivores, because the stimulatory phase can result in ovulation and subsequent sustained progesterone secretion that may cause endometrial pathology. However, the stimulatory phase can be prevented by treatment with megestrol acetate for 7 d before and 7 d after implant insertion. The two current formulations of Suprelorin are effective for minimums of 6 (4.7 mg) or 12 mo (9.4 mg). The data indicate that Suprelorin is an effective and safe contraceptive option for female mammals, although it may not be effective in males of some mammalian species. Further research is needed to ascertain its usefulness in nonmammalian taxa.

The past, present and future of hormonal contraceptive use in managed captive female tiger populations with a focus on the current use of deslorelin acetate.

Tigers (Panthera tigris spp.) are endangered in the wild; ensuring sustainable insurance populations requires careful planning within zoological collections. In captive situations, contraceptives are often used to control breeding and ensure genetically viable populations that contain manageable numbers of animals; reversible contraceptives are ideal because they offer flexibility for breeding management. Historically, synthetic progestins, such as melengestrol acetate implants, were used in female tigers, but these are associated with an increased risk of reproductive pathology and subsequent infertility. Recent management advice to ex-situ collections has been to transition to the use of gonadotropin-releasing hormone agonists, such as deslorelin acetate implants, which do not appear to have a similar risk of reproductive pathology but are associated with highly variable reversal times in exotic felids. Using data from 917 contraceptive records in female tigers captured by the Association of Zoos and Aquariums Reproductive Management Center and the European Association of Zoos and Aquaria Reproductive Management Group's joint Contraception Database and from supplementary surveys, this study reviews the changing use of contraceptives in captive female tigers. The aim was to describe the historical and current use of contraceptives and provide a comprehensive assessment on the use of deslorelin implants, including data on product protocols, efficacy, pathology, and reversibility. This study determined that current dose, frequency, reversibility, and anatomical placement sites of deslorelin implants are highly variable, indicating that specific, readily available, unified, evidence-based recommendations on the use of deslorelin would be useful for future contraceptive use in managed tiger populations.

HEMATOLOGY, PLASMA BIOCHEMISTRY, AND PLASMA PROTEIN ELECTROPHORESIS REFERENCE INTERVALS FOR BLUE IGUANAS (CYCLURA LEWISI) FROM GRAND CAYMAN ISLAND.

The blue iguana (Cyclura lewisi) is an endangered rock iguana species native to Grand Cayman, in the Cayman Islands. Health assessments were conducted on captive and free-roaming iguanas in 2001 and 2003-2014 and were performed in the summer wet season (June-July) of 2003-2004 and 2010-2014 and in the winter dry season (November-December) of 2001 and 2005-2009. Morphometric data were recorded from iguanas when blood samples were collected: 903 samples were collected and data from 890 samples from 775 iguanas were included. Samples were analyzed for hematology, plasma biochemistry, protein electrophoresis, mineral panels, 25-hydroxyvitamin D, and testosterone. Reference intervals were created for captive subadults, captive adults, and free-roaming adults when data were sufficient. Significant differences among these groups were described, as were differences on the basis of sex, season, and origin (captive vs free-roaming). In captive iguanas, most analytes were significantly different between subadults and adults, mature heterophils and copper were significantly higher in the dry season, zinc levels were significantly higher in the wet season, and cholesterol and triglycerides were significantly higher in adult females than adult males. Testosterone in adult males was significantly higher in the dry season. These results will aid in future health assessments and disease investigations in wild and captive populations of blue iguanas and are of comparative value for other Cyclura species that are free-roaming, captive, and, especially, in similar conservation release programs.

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

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

Estrous cyclicity and reproductive success are unaffected by translocation for the formation of new reproductive pairs in captive red wolves (Canis rufus).

This study investigated possible female-related causes for inconsistent success among reproductive pairs in the zoo-based red wolf (Canis rufus) population. Females (n = 13) at seven institutions were assessed for evidence of ovulation and normal reproductive cycles through the measurement of estradiol and progesterone metabolite excretion in feces. Fecal cortisol metabolites (FCM) were also measured. Factors potentially affecting FCM and/or estrous cyclicity were recorded, including exhibit status (on vs. off), reproductive history (proven vs. unproven), copulatory behaviors (ties observed: yes or no), pregnancy/parturition (pups or no pups produced), and translocation before the observed breeding season (yes or no). No differences were observed in baseline FCM between females housed on versus off-exhibit (p = .46) or between females producing pups and those who did not (p = .19). Baseline FCM were significantly lower among females observed in copulatory ties compared to females never observed in a tie (p = .04), and tended to be higher in females translocated before the breeding season compared to females in existing reproductive pairs (p = .11), and among historically unproven females compared to proven females (p = .11). All females evaluated had an endocrine profile indicative of ovulation and among the four females translocated to be paired with a new male before the breeding season, two had successful pregnancies, producing litters. Therefore, despite observed differences in baseline FCM among factors, estrous cyclicity and reproductive success are unaffected by translocation for the formation of new reproductive pairs in the zoo-based red wolf population.

Physical and Physiological Indicators of Welfare in Guinea Pigs (Cavia porcellus) Serving as Ambassador Animals.

Special encounters that allow contact between animals and guests are common in zoos and aquariums. Visitors to the Saint Louis Zoo may touch guinea pigs serving as ambassador animals. We evaluated two welfare indicators in ambassador guinea pigs by comparing glucocorticoid levels and body weights between periods when guinea pigs lived in a habitat accessible to the public and while off exhibit. Mean glucocorticoid levels did not differ between sexes or between on- and off-exhibit periods. There was significant individual variation, and females demonstrated greater variation than males. While on exhibit, glucocorticoid levels slightly but significantly increased in males and decreased in females. Moving guinea pigs between habitats only elicited a significant glucocorticoid response when females were moved off exhibit. Temporary closures of the exhibit had no effect on glucocorticoid levels in either sex. Analyses of the impact of handling rates on males found no impact on glucocorticoid levels. Guinea pigs' body weights were lower while on exhibit. We conclude that guinea pigs serving as ambassador animals at the Saint Louis Zoo demonstrate comparable physiological profiles while on and off exhibit and, when used in a rotational schedule, are a suitable species for animal encounters involving contact with the public.

Changes in human health parameters associated with an immersive exhibit experience at a zoological institution.

Zoological institutions often use immersive, naturalistic exhibits to create an inclusive atmosphere that is inviting for visitors while providing for the welfare of animals in their collections. In this study, we investigated physiological changes in salivary cortisol and blood pressure, as well as psychological changes among visitors before and after a walk through the River's Edge, an immersive, naturalistic exhibit at the Saint Louis Zoo. Study participants had a significant reduction in salivary cortisol and blood pressure after walking through the exhibit. Psychological assessments of mood found that most visitors felt happier, more energized, and less tense after the visit. Additionally, participants who spent more time in River's Edge, had visited River's Edge prior to the study, and had seen more exhibits at the Zoo prior to entering River's Edge experienced greater psychological and/or physiological benefits. We conclude that immersive, naturalistic exhibits in zoos can elicit positive changes in physiological and psychological measures of health and well-being and argue for a greater scientific focus on the role of zoos and other green spaces in human health.

Linkage between fecal androgen and glucocorticoid metabolites, spermaturia, body weight and onset of puberty in male African lions (Panthera leo).

There is limited physiological information on onset of puberty in male lions. The aim of this study was to use longitudinal non-invasive monitoring to: 1) assess changes in steroid metabolite excretory patterns as a function of age and body weight; 2) determine correlations between fecal androgen (FAM) and glucocorticoid (FGM) metabolite concentrations; and 3) confirm spermiogenesis non-invasively through urinalysis. Specifically, FAM and FGM metabolites were analyzed in samples collected twice weekly from 21 male lions at 17 institutions (0.9-16 years of age) for 3.8 months- 2.5 years to assess longitudinal hormone patterns. In addition, body weights were obtained approximately monthly from 10 individuals at five zoos (0.0-3.0 years), and urine was collected from six males at two facilities (1.2-6.3 years) and evaluated for the presence of spermatozoa. An increase in overall mean FAM occurred at 2.0 years of age, at which point concentrations remained similar throughout adulthood. The onset of puberty occurred earlier in captive-born males (<1.2 years of age) compared to wild-born counterparts (<2.5 years of age). Additionally, males in captivity gained an average of 7.3 kg/month compared to 3.9 kg/month for wild males over the first 2-2.5 years of age. Sperm (spermaturia) was observed in males as young as 1.2 years in captivity compared to 2.5 years in the wild (ejaculates). There was no difference in FAM or FGM concentrations with regards to age or season. Overall, this study demonstrates that: 1) captive male lions attain puberty at an earlier age than wild counterparts; 2) onset of puberty is influenced by body weight (growth rate); and 3) spermiogenesis can be confirmed via urinalysis. Knowledge about the linkage between body weight and onset of puberty could facilitate improved reproductive management of ex situ populations via mitigating the risk of unintended breedings in young animals.

Reactions of female cheetahs (Acinonyx jubatus) to urine volatiles from males of varying genetic distance.

Understanding the factors used by female cheetahs (Acinonyx jubatus) to make mate choice decisions could benefit zoo breeding programs, which currently assign mates based primarily on genetic distance. Because transporting animals between institutions is costly and can be stressful, females are often limited in the number of males available for mating. One solution would be to determine if an easily transported substance could be used to gauge interest by a female to a potential mate. Here, we investigate female interest in urine samples from males of different genetic distances. Twelve females at five institutions were offered scents from 17 males of varying genetic relatedness in a pair-wise choice paradigm. Behavioral responses of the females were recorded to determine preference. Results showed that females spent more time sniffing and in proximity to scents from the most distantly related males, but female response was not influenced by male urine testosterone concentration, female parity, age, or estrous cycling. Further research will be necessary to determine whether a female's interest in male urine translates to mate preference and acceptance before this technique can be applied to zoo breeding programs.

Red wolf (Canis rufus) sperm quality and quantity is affected by semen collection method, extender components, and post-thaw holding temperature.

Cryopreserving genetic resources is becoming increasingly important for species management. In the zoo-based red wolf (Canis rufus) population, inbreeding continues to increase in the absence of new founders. Through banking sperm, we preserve genetic diversity and create the ability to decrease inbreeding accumulation in the future. The quality and quantity of banked sperm can be affected by cryopreservation media and semen collection methods. This study's objectives were to further optimize semen extender used for red wolf sperm cryopreservation, investigate effects of post-thaw holding temperature, and to determine if urethral catheterization is an effective method for semen collection in this species. Semen collection via electroejaculation (EE) was performed on 39 adult red wolf males (ages 1 to 11) from 15 institutions. Urethral catheterization (UC) was attempted on a subset (n = 14) of those males, prior to EE. Thirteen different semen extenders were used for cryopreservation, which varied in osmolarity (HI or NORM), sugar source (glucose, fructose, or a combination), and cryoprotectant (glycerol or DMSO). Significant decreases in percent motility, forward progressive status (FPS), and acrosomal integrity were observed over time across all extenders (P < 0.0001). Among the extender components examined, post-thaw sperm motility and FPS were lower in DMSO versus glycerol based treatments (P < 0.005). Therefore, DMSO should be considered unsuitable as a cryoprotectant when freezing red wolf sperm. Effects of osmolarity and sugar source were minimal and temporally variable, however notably, a higher percentage of morphologically normal sperm were observed in the fructose-based extenders compared to glucose-based extenders post-thaw (P < 0.05). Additionally, post-thaw sperm motility and FPS declined more rapidly in samples maintained at 37 °C compared to samples held at room temperature (P < 0.05). Greater volumes of semen were collected using EE compared to UC (P = 0.041), and sperm samples collected using EE also had greater motility and FPS (P < 0.05). Additionally, though no gross morphological differences were observed, there were fewer sperm with intact acrosomes in the samples collected via UC (P = 0.0443). Thus, UC should not be considered sufficient for semen collection in red wolves when the desired fate of sperm is cryopreservation and/or AI. However, UC does provide an opportunity for a basic reproductive evaluation of a red wolf male.

Group management influences reproductive function of the male cheetah (Acinonyx jubatus).

Although the free-ranging cheetah is generally socially solitary, as many as 60% of males live in same-sex (usually sibling) coalitions. Under ex situ conditions, the cheetah experiences low reproductive success with only ~18% of males having ever produced young. Most male cheetahs (85%) are managed in captivity in coalitions, but with no data on the influence of social grouping on reproductive parameters. We examined the influence of singleton versus coalition management on various male cheetah physiological traits, including ejaculate quality and gonadal and adrenal hormone metabolite concentrations. We also assessed behaviour within coalitions for evidence of social hierarchy through initiation of interactions with group mates and relatedness to physiological traits. Ejaculate quality (including total motile and structurally normal spermatozoa per ejaculate) and androgen concentration profiles were higher (P<0.05) in coalition compared with singleton males. These results support the conclusion that testis function in the cheetah, specifically related to the development of normal, motile spermatozoa and androgen production, is influenced by management with same-sex conspecifics. The findings have implications for ex situ conservation breeding programs by suggesting that reproductive quality can be enhanced through group maintenance of cheetah males.

Serum Amyloid A Protein Concentration in Blood is Influenced by Genetic Differences in the Cheetah (Acinonyx jubatus).

Systemic amyloid A (AA) amyloidosis is a major cause of morbidity and mortality among captive cheetahs. The self-aggregating AA protein responsible for this disease is a byproduct of serum amyloid A (SAA) protein degradation. Transcriptional induction of the SAA1 gene is dependent on both C/EBPß and NF-κB cis-acting elements within the promoter region. In cheetahs, 2 alleles exist for a single guanine nucleotide deletion in the putative NF-κB binding site. In this study, a novel genotyping assay was developed to screen for the alleles. The results show that the SAA1A (-97delG) allele is associated with decreased SAA protein concentrations in the serum of captive cheetahs (n = 58), suggesting genetic differences at this locus may be affecting AA amyloidosis prevalence. However, there was no significant difference in the frequency of the SAA1A (-97delG) allele between individuals confirmed AA amyloidosis positive versus AA amyloidosis negative at the time of necropsy (n = 48). Thus, even though there is evidence that having more copies of the SAA1A (-97delG) allele results in a potentially protective decrease in serum concentrations of SAA protein in captive cheetahs, genotype is not associated with this disease within the North American population. These results suggest that other factors are playing a more significant role in the pathogenesis of AA amyloidosis among captive cheetahs.

Characterization of Ovarian Steroid Patterns in Female African Lions (Panthera leo), and the Effects of Contraception on Reproductive Function.

Because of poor reproduction after the lifting of an 8-year breeding moratorium, a biomedical survey of female lions in U.S. zoos was initiated in 2007. Fecal estrogen (FEM), progestagen (FPM) and glucocorticoid (FGM) metabolites were analyzed in samples collected 3-4 times per wk from 28 lions at 17 facilities (0.9-13.8 yr of age) for 4 mo-3.5 yr and body weights were obtained ~monthly from 17 animals at eight facilities (0.0-3.0 yr of age). Based on FEM, estrous cycle length averaged 17.5 ± 0.4 d in duration, with estrus lasting 4.4 ± 0.2 d. All but one female exhibited waves of estrogenic activity indicative of follicular activity; however, not all females expressed estrous behaviors (73%), suggesting silent estrus was common. Female lions experienced puberty earlier than expected; waves of estrogenic activity were observed as young as 1.1 yr of age, which may be related to a faster growth rate of captive vs. wild lions. Mean gestation length was 109.5 ± 1.0 d, whereas the non-pregnant luteal phase was less than half (46.0 ± 1.2 d). Non-mating induced increases in FPM were observed in 33% of females housed without a male, consistent with spontaneous ovulation. A number of study animals had been contracepted, and the return to cyclicity after treatment withdrawal, while variable, was ~4.0 yr and longer than the 1-yr expected efficacy, especially for those implanted with Suprelorin. For FGM, there were no differences in overall, baseline or peak mean concentrations among the age groups or across seasons, nor were there any relationships between reproductive parameters and FGM concentrations. Overall, results suggest that poor reproduction in lions after the breeding moratorium was not related to altered adrenal or ovarian steroid activity, but for some females may have been a consequence of individual institutions' management decisions.

Motile Sperm Output by Male Cheetahs (Acinonyx jubatus) Managed Ex Situ Is Influenced by Public Exposure and Number of Care-Givers.

The collective cheetah (Acinonyx jubatus) population in zoological institutions has never been self-sustaining because of challenges in natural reproduction. A retrospective analysis of North American zoo-breeding records has revealed that >90% of litters produced since 2003 occurred in facilities 'off-display' from the public. We examined seminal, endocrine, and behavioral traits of 29 adult male cheetahs that were: 1) managed in public exhibit or off-display facilities; 2) maintained by different numbers of cheetah-specific care-givers; and 3) living adjacent to varying numbers of adult conspecifics. Cheetahs housed off-display produced more total motile sperm/ejaculate (P = 0.04) than on-exhibit males. This finding was mirrored in our laboratory's historical records where two-fold more total motile sperm (P < 0.01) were measured in ejaculates from individuals with no public exposure (n = 43) compared to on-exhibit (n = 116) counterparts. Males at institutions with ≤3 care-givers also produced more total motile sperm/ejaculate (P < 0.03) and spent more time behaviorally active (P < 0.01) than at facilities using >3 care-givers. Exposure to high numbers of conspecifics within the same institution did not impact (P > 0.05) seminal traits, and presence of the public, care-giver number, or animals/facility had no influence (P > 0.05) on androgen or glucocorticoid excretion or other behavioral metrics. Findings indicate that male cheetahs are sensitive to general public exposure and too many care-givers, resulting in compromised motile sperm output/ejaculate with mechanism of action unrelated to altered androgen or glucocorticoid excretion.

INDUCTION OF CYTOKINE PRODUCTION IN CHEETAH (ACINONYX JUBATUS) PERIPHERAL BLOOD MONONUCLEAR CELLS AND VALIDATION OF FELINE-SPECIFIC CYTOKINE ASSAYS FOR ANALYSIS OF CHEETAH SERUM.

Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of cheetahs (Acinonyx jubatus ; n=3) and stimulated with lipopolysaccharides (LPS) to induce the production of proinflammatory cytokines TNF-α, IL-1ß, and IL-6 for establishment of cross-reactivity between these cheetah cytokines and feline-specific cytokine antibodies provided in commercially available Feline DuoSet® ELISA kits (R&D Systems, Inc., Minneapolis, Minnesota 55413, USA). This study found that feline-specific cytokine antibodies bind specifically to cheetah proinflammatory cytokines TNF-α, IL-1ß, and IL-6 from cell culture supernatants. The assays also revealed that cheetah PBMCs produce a measurable, cell concentration-dependent increase in proinflammatory cytokine production after LPS stimulation. To enable the use of these kits, which are designed for cell culture supernatants for analyzing cytokine concentrations in cheetah serum, percent recovery and parallelism of feline cytokine standards in cheetah serum were also evaluated. Cytokine concentrations in cheetah serum were approximated based on the use of domestic cat standards in the absence of cheetah standard material. In all cases (for cytokines TNF-α, IL-1ß, and IL-6), percent recovery increased as the serum sample dilution increased, though percent recovery varied between cytokines at a given dilution factor. A 1:2 dilution of serum resulted in approximately 45, 82, and 7% recovery of TNF-α, IL-1ß, and IL-6 standards, respectively. Adequate parallelism was observed across a large range of cytokine concentrations for TNF-α and IL-1ß; however, a significant departure from parallelism was observed between the IL-6 standard and the serum samples (P=0.004). Therefore, based on our results, the Feline DuoSet ELISA (R&D Systems, Inc.) kits are valid assays for the measurement of TNF-α and IL-1ß in cheetah serum but should not be used for accurate measurement of IL-6.