EVALUATION OF PLASMA CORTISOL AND LACTATE IN KOI (CYPRINUS CARPIO) EUTHANIZED BY IMMERSION IN TRICAINE METHANESULFONATE, CLOVE OIL, OR CARBON DIOXIDE GAS DISSOLVED IN WATER.

Little research has taken place on the effect of euthanasia methods on biophysical and biochemical changes at the time of euthanasia in fish. These changes are used in multiple species to determine stress levels before death. Koi (Cyprinus carpio) are an important fish species often used in laboratory research, kept in backyard ponds, and managed in zoological and aquarium collections. The current study evaluated euthanasia of koi by immersion in 0.5 g/L tricaine methanesulfonate (MS-222) (n = 10), 0.5 g/L clove oil (n = 8), 1 g/L clove oil (n = 10), and CO2 (n = 7) on time to cessation of opercular movement, plasma lactate levels, and plasma cortisol levels. CO2 had the longest mean time to cessation of opercular movement, and MS-222 had the shortest (mean CO2: 24.9 min, range 13.18-31.35 min; MS-222: 2.68 min, range 1.33-4.5 min). The difference was not significant between any of the groups for plasma cortisol or lactate levels. MS-222 demonstrated the highest cortisol levels, and CO2 had the lowest (mean CO2: 108.7 ng/ml, range 33.9-195.8 ng/ml; MS-222: 650.6 ng/ml, range 77.3-2374.9 ng/ml). Average lactate levels were highest for 1 g/L clove oil and lowest for 0.5 g/L clove oil (mean 0.5 g/L clove oil: 5.1 mmol/L, range 1.8-8.1 mmol/L; 1 g/L clove oil: 7.4 mmol/L, range 5.6-10.5 mmol/L).

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.

Noninvasive monitoring of steroid hormone production and activity of zoo-housed banteng (Bos javanicus).

This study describes patterns of steroid hormone production and activity for banteng (Bos javanicus), an endangered member of the Bovidae family. Using validated assays, concentrations of fecal progestagens, androgens, and glucocorticoids were quantified for four females and one male at the Saint Louis Zoo. A commercial activity monitor was also validated for assessing movement. The devices were then used to characterize activity in relation to season, reproductive status, and fecal steroid concentrations. General linear mixed models assessed differences in activity and steroid concentrations among individuals, in regards to reproductive status and season. Ovulatory cycle patterns, changes in activity around estrus and parturition, and events correlated with increased glucocorticoid production were also documented. Cycle lengths were 24.7 ± 0.4 days, and cycle lengths varied among individuals. Females cycled year-round, but luteal progestagen concentrations, along with glucocorticoids and male androgens, increased during the summer. Activity also increased in the summer. Progestagen concentrations were greater in pregnant females, and the gestation length of one pregnancy was 254 days. Pregnant females were less active overall, but activity increased the day before parturition. Activity was also greater preceding the onset of the luteal phase. The majority of glucocorticoid concentrations were in the range of baseline concentrations. However, a small number of elevated concentrations were correlated with husbandry and veterinary events. This study is the first to validate non-invasive methods for monitoring reproduction, welfare, and activity of banteng. Our results may contribute to the improved management of captive populations.

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.

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.

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.

Characterization of estrous cycles and pregnancy in Somali wild asses (Equus africanus somaliensis) through fecal hormone analyses.

Although reproduction in the domestic horse has been well described, less is known about reproduction in wild equids. This study describes endocrine patterns associated with estrous cycles and pregnancy for Somali wild asses (Equus africanus somaliensis), an endangered African equid. Fecal samples were collected three times per week for more than 2 years from five female Somali wild asses at the Saint Louis Zoo; progestagen and estrogen metabolites were quantified using commercially available immunoassays. Progestagen analysis indicated that cycle lengths were 27.2 ± 1.2 days and females cycled throughout the year. Progestagen levels during early pregnancy were low and not sustained above baseline until approximately 40 weeks prior to partition. Concentrations increased markedly around 16 weeks prior to delivery and peaked 2-3 weeks before birth. Fecal estrogen levels also increased significantly starting 40-45 weeks before parturition and reached their maximal value approximately 20 weeks prior to birth. Neither foal heat nor lactational suppression of estrus was observed, and females cycled within 45 days after delivery. These data are the first to describe the reproductive physiology of Somali wild asses. As the species faces increasing threats in the wild, this information may support conservation efforts by assisting with ex situ breeding programs.

Parent-offspring behavior of Jambu fruit doves (Ptilinopus jambu).

Fruit doves (Ptilinopus) constitute a genus of small to medium-sized, brightly colored arboreal birds, whose diets consist entirely of fruit. Little is known about the behavior of fruit doves because most species inhabit dense forests and are difficult to observe in the wild. This study describes the parental behavior of Jambu fruit dove pairs (Ptilinopus jambu) in a captive breeding program at the Saint Louis Zoo. Continuous video recordings were made of three pairs which raised a total of eight squabs over 2 years; daily rates of parental and squab behaviors were quantified. Overall, females were present at the nest, brooded their squabs, pecked, and attempted feedings more often than males. Parents also cared for their squabs at different times throughout the day. Males fed and brooded squabs during the middle of the day, while females fed throughout the day and brooded in the morning, evening, and overnight. Feeding rates were lower than those described for seed-eating doves (Columbinae), with hours between consecutive feedings, and squabs rarely begged before feeding events. Most squab behaviors involved initiating or terminating brooding and self-preening. These behaviors increased as squabs approached fledging, and coincided with a shift from full to partial brooding, and a decrease in parental allopreening. Older squabs also initiated feeding less frequently. Together, these data provide the first description of parental behavior in a Ptilinopus fruit dove. The results of this study may help improve captive breeding efforts, which are likely to become increasingly important for future conservation and reintroduction programs.

Reproductive behavior of the great hornbill (Buceros bicornis).

Great hornbills (Buceros bicornis) are a long-lived, monogamous species that forms strong pair-bonds, and mate compatibility is thought to be important for successful reproduction. Within AZA, great hornbills are listed as a red SSP. The population consists of a limited number of individuals that do not breed reliably, and improving reproduction is a top priority for the Coraciiformes TAG. To better understand mating behavior and evaluate mate compatibility, this study documented the behavior of pairs of great hornbills during and immediately after courtship. Using live observations, the study followed one female, an experienced and successful breeder, as she was paired with four successive males over 11 breeding seasons. Initially, males frequently vocalized, investigated the nest, and approached the female. As the female spent more time in the nest, these behaviors were replaced by regurgitation and food offering. The female was most often observed plastering and vocalizing. Behavioral differences between successful and unsuccessful pairs, possibly indicative of pair compatibility, included rates of approaching, billing, and biting. Numerous behaviors occurred more frequently during years that a chick hatched, including pseudoregurgitation, regurgitation, offering food items, and nest investigation. Males also spent more time in proximity to both the female and the nest during years that a chick hatched. Together, these results suggest that the amount of time pairs spend in proximity, the amount of time a male spends near the nest, and the frequency of certain behaviors may help evaluate compatibility and the likelihood of successful reproduction for pairs of great hornbills.

The effects of brood size on growth and steroid hormone concentrations in nestling eastern bluebirds (Sialia sialis).

Birds in multi-nestling broods often experience reduced growth and elevated nutritional and social stress as a result of competition for parental resources. While responses are often species-specific, experimentally increasing brood size has been shown to decrease growth and increase production of both testosterone and corticosterone in nestling passerines. To investigate the relationship between brood size, growth, and steroid hormone production in eastern bluebirds, we cross-fostered nestlings to small, medium, and large broods. Body mass, skeletal size, serum testosterone, and baseline serum corticosterone concentration were measured prior to fledging. Bluebird nestlings raised in large broods weighed less than nestlings in small and medium-sized broods and secreted elevated concentrations of testosterone. Nestling corticosterone concentrations did not vary in response to brood size or body condition, but concentrations were higher in males compared to females. Our results suggest that nestling bluebirds experiencing nutritional and social stress increase testosterone production. Elevated concentrations may enhance begging and competitive abilities in smaller young. Higher concentrations of corticosterone in males may be related to sex-biased provisioning. Further experimental work is needed to ascertain the effects of elevated testosterone concentrations in nestling bluebirds, as well as the causes and consequences of elevated corticosterone for male nestlings.

Changes in plasma hormone levels correlate with fledging in nestling Leach's storm-petrels.

Leach's storm-petrels accumulate large amounts of body mass throughout the nestling period. Approximately 4days before fledging, nestlings weigh 50-100% more than adults. In order to shed this excess mass, nestlings engage in behavioral anorexia, and leave the burrow when they are light enough to fly. During this prefledging period, we measured several plasma hormones (corticosterone, thyroxine, and testosterone) to determine whether the behavioral changes associated with fledging are correlated with endocrine changes. In several species, including petrels, corticosterone levels are known to increase near fledging. Reduced food consumption has been shown to elevate corticosterone levels and decrease thyroxine levels in nestling birds. In nestling storm-petrels, levels of both corticosterone and thyroxine increased. Storm-petrels were found to secrete measurable levels of testosterone, but levels did not change during the prefledging period. Increased corticosterone levels might be part of an endocrine signal that initiates changes in feeding behavior, or may result from reduced food intake. Elevated thyroxine levels may be related to metabolic changes involved in mass loss. Future experimental work is needed to ascertain whether the described endocrine changes are responsible for, or result from, prefledging changes in petrel feeding behavior.

Egg size and yolk steroids vary across the laying order in cockatiel clutches: a strategy for reinforcing brood hierarchies?

When a female bird begins incubation before clutch completion, the nestlings hatch sequentially, and a size hierarchy forms within the brood. This size hierarchy may be minimized or exacerbated through differential allocation of resources to eggs across the laying order. In this study, we characterize intra-clutch variation in cockatiel clutches by measuring egg mass, yolk mass, and concentrations of yolk testosterone, androstenedione, and corticosterone. Cockatiels are a long-lived member of the Psittaciformes. Because asynchronous hatching may reduce sibling competition and allow for extended development periods in long-lived birds, we predicted that female cockatiels would allocate maternal resources in a way that would reinforce the brood size hierarchy. Significant within-clutch differences in egg size and steroid concentrations were observed. Eggs at the end of the laying sequence were smaller and had significantly smaller yolks than eggs early in the laying order. Fifth-laid eggs, as well as first-laid eggs, contained significantly lower concentrations of testosterone than eggs in other positions of the laying sequence. No differences in yolk androstenedione concentration were observed. Yolk corticosterone concentrations increased linearly with laying order. Together, these patterns might reinforce the brood size hierarchy created by asynchronous hatching.

A simplified method for extracting androgens from avian egg yolks.

Female birds deposit significant amounts of steroid hormones into the yolks of their eggs. Studies have demonstrated that these hormones, particularly androgens, affect nestling growth and development. In order to measure androgen concentrations in avian egg yolks, most authors follow the extraction methods outlined by Schwabl (1993. Proc. Nat. Acad. Sci. USA 90:11446-11450). We describe a simplified method for extracting androgens from avian egg yolks. Our method, which has been validated through recovery and linearity experiments, consists of a single ethanol precipitation that produces substantially higher recoveries than those reported by Schwabl (1993. Proc. Nat. Acad. Sci. USA 90:11446-11450). Zoo Biol 28:137-143, 2009.