North American Zoological Institutions Research Articles

Non-invasive assessment of fecal glucocorticoid and androgen metabolites in the pygmy hippopotamus (Choeropsis liberiensis)

The pygmy hippopotamus (Choeropsis liberiensis) is an endangered species endemic to the Upper Guinea Forest ecosystem in West Africa. We have limited information concerning the species’ reproduction and well-being under managed care. We therefore developed non-invasive methods for characterizing gonadal androgen and adrenal hormone profiles in pygmy hippos using fecal samples collected from 12 males and 12 females housed in North American zoological institutions. We aimed to: 1) identify and validate enzyme immunoassays (EIAs) for measuring metabolites of corticosteroids and testosterone in feces; and 2) test whether gonadal activity is correlated with previous breeding history, season or type of housing. For glucocorticoids, several EIAs for measuring metabolites were investigated. A group-specific EIA exhibiting cross-reactivity with 11,17-dioxoandrostane (DOA) metabolites of cortisol most clearly reflected adrenocortical activity in response to pharmocological challenge with adrenocorticotropic hormone (ACTH) in both males and females. However, day-to-day concentrations of this metabolite in the feces of pygmy hippos that did not undergo ACTH challenge were near the detection limits of the assay, making this EIA impractical for assessing glucocorticoid activity in this species. Another group-specific EIA, exhibiting cross-reactivity with 5α-pregnane-3β,11β,21-triol-20-one, produced biologically relevant data and evidence of an appropriate response to pharmacological challenge with exogenous ACTH. The testosterone metabolite assay C196 (Arbor Assays, Ann Arbor, Michigan, USA) also produced biologically coherent data: adult males exhibited the highest mean androgen metabolite concentrations (477 ng/g), followed by adult females (259 ng/g) and juvenile males (160 ng/g). Proven breeding males had higher, but not significantly different, mean concentrations (472 ng/g) to unproven males (352 ng/g; P = 0.400). Similarly, adult males housed outdoors year-round in subtropical climates exhibited higher, but not statistically different mean concentrations (554 ng/g) to males in temperate climates that were housed indoors at least part of the year (412 ng/g; P = 0.208). There were, however, significant differences in mean concentrations among seasons for adult males, with higher values in spring (546 ng/g) and summer (542 ng/g) than in autumn (426 ng/g) and winter (388 ng/g, P = 0.003). In conclusion, we identified EIAs for the measurement of fecal metabolites of androgens and glucocorticoids that can be used for further studies to monitor gonadal activity in male pygmy hippos and adrenocortical activity in both sexes. We also identified a seasonal trend in male gonadal activity in this species under managed care in North America. Finally, our findings highlight an important consideration when using non-invasive methods for evaluating fecal cortisol metabolites: ACTH used for pharmacological validation of an EIA does not necessarily equate to biological relevance.

CAUSES OF MORTALITY AND COMORBIDITIES IN CAIMAN LIZARDS (DRACAENA GUIANENSIS) AT SIX ZOOLOGICAL INSTITUTIONS.

An understanding of the main causes of mortality in caiman lizards (Dracaena guianensis) under managed care is imperative to promote optimal husbandry, health, and welfare. A retrospective review of morbidity and mortality in caiman lizards from North American zoological institutions between 2005 and 2020 was conducted. Postmortem data, including gross necropsy and histopathology findings, were available for 32 caiman lizards (n = 12 subadults, n = 20 adults) from six zoological institutions. Necropsy reports were evaluated to collect general demographic data, categorize cause of death (accident/trauma, congenital/genetic, degenerative/geriatric, infectious, deposition disease, neoplastic, unknown, and multifactorial), and assess common comorbidities. Infectious disease was the most common cause of mortality in adult lizards (8/20; 40%) with amoebiasis and bacterial etiologies being overrepresented. Demise due to traumatic/accidental injury was the second most common cause of death in adult lizards (3/20;15%) and included blunt force trauma or suspected drowning. Infectious disease (4/12; 33.3%) and trauma/accidental injury (4/12; 33.3%) were also the most common causes of death in subadults. The most common comorbidities or other incidental findings identified during necropsy included trematode parasitism (15/32; 46.9%), arteriosclerosis (11/32; 34.4%), and adrenocortical hyperplasia (6/32; 18.8%). This retrospective review suggests that management practices to prevent and control infectious diseases and mitigate traumatic injury play a pivotal role in the long-term care and survival of caiman lizards in managed care.

RETROSPECTIVE ANALYSIS OF MORTALITY CAUSES IN MANAGED KIRK'S DIK-DIKS (MADOQUA KIRKII) IN NORTH AMERICA FROM 1988 to 2019.

Kirk's dik-diks (Madoqua kirkii) are the most common dik-dik species managed in North American zoological institutions, but their numbers are declining at a concerning rate, with less than 40 individuals currently housed in accredited institutions. This retrospective study reports the causes of mortality in Kirk's dik-diks in North American zoological institutions from 1988 to 2019. Out of 15 institutions accredited by the Association of Zoos and Aquariums (AZA) currently housing Kirk's dik-diks, nine contributed to this study (60% participation). Eighty-four necropsy records were reviewed to determine the primary affected body system and etiological cause of death across and within age categories. Neonatal death (prior to 1 mon of age) was most common (38.1%), followed by death in adults (29.8%), geriatric animals (19.0%), and juveniles (13.1%). As a whole population, causes of death by body system were multisystemic (47.6%), musculoskeletal (15.5%), respiratory (8.3%), and digestive (8.3%). Neonatal complications accounted for 50.0% of all deaths in animals prior to 1 mon of age. In juveniles, a nutritional cause of death was most common (27.3%) and significantly higher compared to measures of this cause within other age categories. In adults, metabolic etiologies and trauma each accounted for 28.0% of deaths. Degenerative etiologies were most common in geriatric individuals, representing 31.3% of the deaths. Death from infectious disease was found across all ages, representing 11.9% of all mortalities. Results from this study provide a baseline reference for this species and may aid clinicians in decision-making as it relates to the medical care and management of this species during different life stages.

Phenylbutazone pharmacokinetics in southern white rhinoceros (Ceratotherium simum simum) after oral administration.

Southern white rhinoceros (Ceratotherium simum simum) frequently develop painful conditions, such as traumatic injuries or osteoarthritis, necessitating the administration of pain-relieving medications. One of the preferred treatments is the nonsteroidal anti-inflammatory drug phenylbutazone because of the availability of oral formulations and the familiarity of its use in horses. For the main study, a single oral dose of phenylbutazone at 2mg/kg was administered to healthy adult rhinoceros (n=33) housed at six North American zoological institutions. Each rhinoceros had up to four blood samples collected under voluntary behavioural restraint at up to four predetermined time points (0, 1, 1.5, 2, 3, 4, 6, 8, 10, 24, 30 and 48h). Drug analysis was performed by high-performance liquid chromatography. The population pharmacokinetic parameters were calculated with nonlinear mixed-effects modelling, and analysis showed a peak concentration (CMAX ) of 3.8µg/ml at 1.8h and an elimination half-life of 9h. The concentrations achieved were similar to what has been reported for horses and were within the half maximal effective concentration for horses for at least 10h. A multi-dose trial in five rhinoceros receiving 2mg/kg orally once daily for five days found mild accumulation at a predicted factor of 1.2. This study represents the first pharmacokinetic data of phenylbutazone in any rhinoceros species.

Nutrient Composition of Locally Available Browses Consumed by Matschie’s Tree Kangaroos (Dendrolagus matchiei) in Six North American Zoological Facilities

Locally collected browses (n=17 spp.) consumed by Matschie’s tree kangaroos (Dendrolagus matschiei) in 6 North American zoological institutions were analyzed for comparison with native plants eaten by this species in Papua New Guinea to evaluate dietary suitability. Primary nutrients including crude protein and fat, fiber, starch, non-fiber carbohydrates, and ash were determined using standard analytical methods for forages. Macrominerals calcium (Ca), phosphorus (P), magnesium (Mg), potassium (K), sodium (Na) and sulfur (S), as well as trace elements copper (Cu), iron (Fe), manganese (Mn), molybdenum (Mo) and zinc (Zn) were quantified in leaf (n=18), flower (n=1), twig (n=9) and bark (n=6) samples. Tannin content was estimated through the bovine serum albumin methodology. On a dry matter basis (DMB) , foods averaged (± SD) moderate protein (12 ± 5%) and soluble carbohydrate (27 ± 12%) content, along with low starch (1 ± 1%) and crude fat (3 ± 2%) values, and moderate to high values in fiber fractions (neutral detergent fiber 49 ± 15%, acid detergent fiber 33 ± 13%, lignin 11 ± 5%). Macromineral concentrations (Ca 2.2 ± 1.6%, P 0.2 ± 0.1%, Mg 0.3 ± 0.2%, K 1.5 ± 0.6%, Na 0.03 ± 0.04%, S 1.2 ± 1%) and select trace minerals were within anticipated ranges (Cu 11 ± 5 mg/kg, Mo 1 ± 1 mg/kg and Zn 33 ± 18 mg/kg); exceptions Fe (122 ± 11 mg/kg) and Mn (51 ± 81 mg/kg) were considered on the high end of dietary adequacy for most herbivores. Leaves differed significantly from woody parts for all proximate nutrients, as well as K, S, Fe, and tannin content. Consumed in a 50:50 DMB ratio, locally available browses provide similar nutrient profiles as plants eaten by free-living tree kangaroos. Combined data provide information useful in establishing nutrient targets for dietary development, leading to improved health, welfare, and feeding management of tree kangaroo populations under human care.

Diagnosing cardiovascular disease in western lowland gorillas (Gorilla gorilla gorilla) with brain natriuretic peptide.

Cardiovascular disease is a leading cause of death in zoo-housed great apes, accounting for 41% of adult gorilla death in North American zoological institutions. Obtaining a timely and accurate diagnosis of cardiovascular disease in gorillas is challenging, relying on echocardiography which generally requires anesthetic medications that may confound findings and can cause severe side effects in cardiovascularly compromised animals. The measurement of brain natriuretic peptide (BNP) has emerged as a modality of interest in the diagnosis, prognosis and treatment of human patients with heart failure. This study evaluated records for 116 zoo-housed gorillas to determine relationships of BNP with cardiovascular disease. Elevations of BNP levels correlated with the presence of visible echocardiographic abnormalities, as well as reported clinical signs in affected gorillas. Levels of BNP greater 150 pb/mL should alert the clinician to the presence of myocardial strain and volume overload, warranting medical evaluation and intervention.

Development of a 12-plex of new microsatellite markers using a novel universal primer method to evaluate the genetic diversity of jaguars (Panthera onca) from North American zoological institutions

Maintenance of genetic diversity in both zoo-managed and wild animals is critical to population health. Jaguars are considered Near Threatened throughout their geographic range, and are Endangered in the United States. Genetic diversity of zoo-managed jaguar populations in North America using microsatellite markers has not been reported. To evaluate genetic diversity in jaguars housed in North American zoological institutions compared to wild and other zoo jaguar populations, we created a 12-plex of ten tetranucleotide (n = 10) and two pentanucleotide (n = 2) autosomal microsatellite loci that have not previously been described for use in jaguars, using a universal primer approach which minimizes cost and DNA quantity requirements. A subset of jaguars from North American zoological institutions (n = 35, approximately 28% of population) were genotyped using DNA extracted from whole blood. Microsatellite loci were polymorphic with a range of 3–10 alleles (average 6.42 alleles per locus), and expected heterozygosity (He) between 0.47 and 0.85. Overall population He was 0.73. Probability of identity of individuals (PI) and siblings (PIsibs) were 6.7 × 10− 13 and 2.1 × 10− 5, respectively, confirming that this panel can be used reliably to identify individuals. Using this novel multiplex microsatellite panel, we concluded that jaguars from the North American zoo population have high genetic diversity, comparable to reported wild and other zoo jaguar populations.

Nutrient evaluation of a pork by-product and its use as environmental enrichment for managed large exotic cats.

North American zoological institutions typically feed ground raw meat diets to large exotic cats. These diets typically are nutritionally complete, but lack physical properties characteristic of whole prey. Lack of mastication and prey manipulation may contribute to behavioral and health challenges. Pork by-products may provide environmental enrichment to mitigate these challenges. The objectives of this study were to evaluate a pig head for nutritional composition and to determine if a pig head was biologically relevant environmental enrichment for managed large exotic cats. Pig heads consisted of: DM: 48.5%; OM: 60.7%; CP: 38.4%; fat: 22.0%; CF: 13.5%; TDF: 3.4%; GE: 4.1 kcal/g DM. Five individually housed exotic cats (Panthera tigris tigris, Panthera tigris altaica (n = 2), Panthera tigris jacksoni, Panthera leo) were observed in 2-h blocks, 24-h before pig head introduction (Baseline), at time of pig head introduction (Enrichment) and immediately after the pig head was removed (Post Enrichment) via instantaneous scan sampling for 4 consecutive weeks. Active behaviors were 55.7% higher on Enrichment compared to Baseline days, and 26.4% higher compared to Post Enrichment days (p<0.0001). Active behaviors were 39.8% higher on Post Enrichment compared to Baseline days (p<0.0001). Total active behaviors were highest (p<0.0001) in week 3 and lowest (p<0.0001) in week 4 with differences as high as 64.5% seen among weeks. In conclusion, pig heads have potential to provide nutrient dense enrichment to large exotic cats, and employing a pig head as environmental enrichment increased active behaviors and did not lose novelty.

Sources of morbidity in lorises and pottos in North American zoos: A retrospective review, 1980-2010.

Delineating patterns of morbidity can reveal management practices in need of reassessment to improve individual welfare, as well as population health and sustainability. We reviewed medical records from 38 North American zoological institutions for 276 slender lorises, slow lorises, and pottos born between January 1, 1980 and December 31, 2010. This sample included animals identified as 116 Nycticebus pygmaeus, 84 N. coucang, 48 Loris tardigradus tardigradus, 6 L.t. nordicus (now classified as L. lydekkerianus nordicus), and 22 Perodicticus potto. Taxonomy for lorises and pottos is developing, and two of these populations (N. coucang and P. potto) likely included hybrids and/or multiple species. Our focus was to examine trends based on species and age. Across all species, whole body disease events, abnormalities of bloodwork, and dental diseases were the most common sources of morbidity. Other major sources of morbidity varied by species and included trauma, respiratory disease, and ocular disease. A recent upsurge in research has informed feeding practices for slow lorises living in human care, and a similar, evidence-based approach is needed to improve diets for other species. Given the prevalence of trauma in this sample, social needs and reproductive management practices are also important areas for further investigation. Species-level health trends reveal risk factors for individual welfare that can guide husbandry practices in zoos, as well as in sanctuaries caring for the influx of lorises and pottos rescued from the growing wildlife trade.

EVALUATION OF POTENTIAL RISK FACTORS ASSOCIATED WITH CATARACT IN CAPTIVE MACARONI (EUDYPTES CHRYSOLOPHUS) AND ROCKHOPPER PENGUINS (EUDYPTES CHRYSOCOME).

Complete ophthalmic examinations were performed on 160 Macaroni penguins ( Eudyptes chrysolophus ) and 90 Rockhopper penguins ( Eudyptes chrysocome ) at eight North American zoological institutions. Cataract prevalence in the Macaroni population was 46.5% (n = 74) of penguins and 42.3% (135/319) of eyes. Cataract prevalence in the Rockhopper population was 45.5% (n = 40) of penguins and 40.6% (73/180) of eyes. The mean age of Macaroni penguins without ocular disease was 7.4 ± 5.8 yr, while that of Rockhoppers was 9.8 ± 6.4 yr. Risk factors for cataract were examined through husbandry surveys completed by each institution and by evaluation of light intensity and ultraviolet (UV) light measurements acquired in each penguin exhibit. Risk factors associated with cataract in Macaroni penguins included age, dietary smelt, hand-feeding, and fluorescent exhibit lighting. Risk factors associated with cataract in Rockhopper penguins included age, dietary capelin, increasing population density, and increasing length of minimum photoperiod. Factors associated with decreased odds of cataract in Macaroni penguins included saltwater pool, monitoring of water quality for salinity, pH, and alkalinity; use of water additives; presence of pool filtration and sterilization systems; use of metal halide lightbulbs; increasing light intensity; and UV spectrum lighting. Factors associated with decreased odds of cataract in Rockhoppers included dietary herring and krill, increasing exhibit land area, pool temperature monitoring, increasing maximum photoperiod, and increasing minimum UV light.

Histologic and Immunohistochemical Characterization of Pheochromocytomas in 20 Clouded Leopards ( Neofelis nebulosa).

Clouded leopards in North American zoological institutions have a high frequency of pheochromocytomas and were identified in 32 of 70 (45%) animals necropsied. Archival sections of adrenal gland from 20 adult clouded leopards with unilateral or bilateral pheochromocytomas collected between 1984 and 2011 were examined by light microscopy and immunohistochemistry, and case demographics were reviewed. Affected leopards were older than 10 years of age (mean, 16 years; range, 11-19 years), and males were overrepresented (12 males, 8 females). Pedigree analysis yielded no evidence for heritability. Five clouded leopards had bilateral neoplasms. Pheochromocytoma was the cause of death due to invasion of the caudal vena cava and fatal hemorrhage in 4 cases. Most pheochromocytomas were well-demarcated, nodular, and expansile masses composed of cords and packets of neoplastic polygonal cells. Five pheochromocytomas had vascular invasion, of which 4 resulted in hemorrhage that was the cause of death. One of the latter pheochromocytomas also had pulmonary metastasis. Ultrastructurally, neoplastic cells had cytoplasmic structures consistent with both norepinephrine- and epinephrine-containing granules. In all cases, neoplasms were immunohistochemically positive for chromogranin A, protein gene product 9.5, and synaptophysin. A subset of neoplasms evaluated by tissue microarray were positive for met-enkephalin and β-endorphin and negative for melan-A. Histologically, 7 of 20 (35%) clouded leopards with pheochromocytomas had retinal detachment, retinal degeneration, or intramyocardial muscular arteriosclerosis, suggestive of hypertension. Pheochromocytomas can cause mortality and may be a source of clinically significant hypertension in clouded leopards. These neoplasms share similar histologic, immunohistochemical, and ultrastructural characteristics with those of other species.

Evaluation of captive gibbons (Hylobates spp., Nomascus spp., Symphalangus spp.) in North American Zoological Institutions for Gibbon Ape Leukemia Virus (GALV).

This study evaluated 79 captive gibbons (Hylobates, Nomascus, and Symphalangus spp.) within 30 North American zoological institutions for evidence of exposure to and possible infection with gibbon ape leukemia virus (GALV). Enzyme-linked immunosorbent assays (ELISAs) on gibbon serum samples revealed the presence of antibodies against GALV antigens in 28% of animals, indicating previous exposure or possibly protective immunity to GALV. Virus detection in gibbon blood or serum using polymerase chain reaction (PCR) or co-culture of gibbon peripheral blood mononuclear cells with human cells was negative for all samples submitted. The majority (19/27, 70%) of animals with reported health conditions were clinically healthy at the time of sample collection. Historically accrued clinical data were used to assess association of diseases in gibbons antibody positive for GALV. The results suggest captive gibbons could mount an immune response to GALV and show no evidence of infection. There was no association with neoplastic conditions in seropositive animals. The potential role of gibbons as a reservoir for GALV and the role of GALV as an epizoonotic-zoonotic agent or as a contributor to gibbon ape morbidity and mortality are not substantiated by the study findings.

Hormonal and behavioral patterns of reproduction in female hylobatids

Ovarian cyclicity, reproductive behavior, and gestation length were characterized in female hylobatids using non-invasive fecal steroid analyses and behavioral data. Progestagen metabolites were quantified in fecal samples collected for 3 months from seven females housed at seven North American zoological institutions. Mean (±SEM) ovarian cycle length was 23.1±1.5 days (seven females; 22 cycles) and was similar across individuals and species (range: 15.3–27.3 days). Gestation length in white-cheeked gibbons was 191±7.0 days (n=2), and female-initiated reproductive behavior occurred throughout the ovarian cycle for each individual. This was the first study of its kind to use fecal hormone metabolite analysis in combination with behavioral observations to characterize female reproductive traits for various hylobatid species. These results contribute to our general knowledge of the basic biology of hylobatids, highlight the importance of evaluating both hormonal and behavioral information, and assist the management and breeding of zoo-housed populations of these endangered primates to support overall conservation efforts.

Reproductive gonadal steroidogenic activity in the fishing cat ( Prionailurus viverrinus) assessed by fecal steroid analyses

Non-invasive fecal steroid analyses were used to characterize gonadal activity in the fishing cat ( Prionailurus viverrinus). Estrogen, progestagen and androgen metabolites were quantified in fecal samples collected for 12 months from four males and 10 females housed at seven North American zoological institutions. Male reproductive hormone concentrations did not vary ( P > 0.05) among season, and estrogen cycles were observed year-round in females and averaged (±SEM) 19.9 ± 1.0 days. Mean peak estrogen concentration during estrus (460.0 ± 72.6 ng/g feces) was five-fold higher than baseline (87.3 ± 14.0 ng/g feces). Five of seven females (71.4%) housed alone or with another female demonstrated spontaneous luteal activity (apparent ovulation without copulation), with mean progestagen concentration (20.3 ± 4.7 μg/g feces), increasing nearly five-fold above baseline (4.1 ± 0.8 μg/g feces). The non-pregnant luteal phase averaged 32.9 ± 2.5 days ( n = 13). One female delivered kittens 70 days after natural mating with fecal progestagen concentrations averaging 51.2 ± 5.2 μg/g feces. Two additional females were administered exogenous gonadotropins (150 IU eCG; 100 IU hCG), which caused hyper-elevated concentrations of fecal estrogen and progestagen (plus ovulation). Results indicate that: (1) male and female fishing cats managed in North American zoos are reproductively active year round; (2) 71.4% of females experienced spontaneous ovulation; and (3) females are responsive to exogenous gonadotropins for ovulation induction, but a regimen that produces a normative ovarian steroidogenic response needs to be identified.

The effectiveness of indigestible markers for identifying individual animal feces and their prevalence of use in North American zoos

Techniques for analyzing hormone metabolites in animal excreta have created many opportunities for noninvasive monitoring of health, reproduction, and welfare in zoo animals, but can be difficult to implement when individual samples are not readily identifiable in animal groups. A common approach to this problem is to feed animals an indigestible marker that subsequently appears in feces, but there has been little systematic research on the use of such "fecal markers." First, we used an online survey to assess the prevalence of fecal marker use in North American zoological institutions. Second, we conducted a series of experimental tests utilizing commonly employed fecal markers in a variety of typical zoo taxa to determine the: (1) effectiveness of several markers to accurately distinguish samples in a variety of species, (2) minimum quantity of marker needed for detection, and (3) length of time between ingestion and detection in the feces. The majority of the 45 institutions that completed the survey reported using fecal markers with their collections. The survey also revealed that the most frequently used markers are seeds/grains and food colorants, with the former generally used in Carnivora and the latter in Primates. Our experimental data confirmed the success of these taxa/marker combinations and also revealed that food colorants function as markers in a variety of avian, reptilian, and mammalian species. Our data describe successful fecal markers for a wide variety of zoo taxa and should, therefore, be useful for zoological managers and researchers needing to employ fecal markers in future investigations.

Analysis of nutrient concentrations in the diet, serum, and urine of giraffe from surveyed North American zoological institutions

The objectives of the present research were to conduct a survey to investigate the health history and feeding practices of giraffe in captivity in North America and to obtain samples of hay, concentrate, browse, urine, and serum to compare across zoos, possible factors relating to the development of urolithiasis. Forty-one out of 98 institutions contacted responded, representing 218 giraffe. All responding zoos fed concentrate and alfalfa hay was the primary forage. Sixty-five percent of zoos fed browse and 43 different species of browse were listed. Six zoos reported a history of urolithiasis, seven reported wasting syndrome, and 10 reported sudden death. The median daily amount (as fed) of concentrate and hay offered were 5.45 kg (range of 2.73-9.55 kg) and 6.82 kg (range of 2.53-12.50 kg), respectively. The concentrate:hay ratio of the offered diet ranged from 0.22 to 3.47 with a median value of 0.79. Forty-three percent of the institutions offered a ratio greater than 1:1. Samples of concentrate and hay (six zoos), serum (five zoos), and urine (seven zoos) were obtained for chemical analyses. Analyzed nutrient content of the consumed diet, measured by weighing feed and orts for three consecutive days, met recommendations for giraffe, but was excessive for crude protein and P. Concentrate:hay and serum P were positively correlated (r=0.72; P<0.05). High dietary P content and a high level of concentrate relative to hay may be contributing factors to urolith formation and warrant further investigation.

199 IN VITRO EMBRYO PRODUCTION IN THE CLOUDED LEOPARD (NEOFELIS NEBULOSA)

The clouded leopard, a spotted, mid-sized cat native to southeast Asia, is classified as Endangered by the U.S. Fish and Wildlife Service. The Species Survival Plan (SSP) program has designated that clouded leopards in North American zoological institutions are a research population with the focus of enhancing management and developing assisted reproductive techniques. In this study, we examined (1) ovarian response to exogenous gonadotropin stimulation, (2) in vitro production of embryos by IVF and intracytoplasmic sperm injection (ICSI), and (3) in vivo developmental ability of in vitro-derived embryos. Eight females at Audubon Center for Research of Endangered Species (ACRES) in New Orleans, LA, were used as oocyte donors and embryo recipients. During non-estrual, but otherwise unknown, stages of the estrous cycle, females 4 to 10 years old at our first treatment were administered a total of 15 (n = 8) or 20 (n = 7) IU of porcine FSH (i.m; Sioux Biochemical Co., Sioux City, IA) daily for 4 days. On Day 5, 10 (n = 8) or 15 (n = 7) IU of porcine LH (i.m; Sioux Biochemical Co.) was given and laparoscopic oocyte retrieval (LOR) was done 24 h later. One, 2, or 3 LOR were performed on 3, 3, and 2 females, respectively (total n LOR = 15), 10 (67%) of which were done on females that were 8 to 12 years of age. A total of 176 pre-ovulatory oocytes (mean = 14.7; range = 3-31) were recovered from 12 LOR performed on 6 females. Two females, a 5-year-old and an 11-year-old, did not respond to gonadotropin treatment. Of 5 females with ≥2 LOR, an average of 16.2 and 14.6 oocytes were recovered at the first and second LOR, respectively. Luteal tissue was present on the ovaries at 3 of 4 LOR performed during January-April as compared with 0 of 11 LOR performed from June-December. Semen was obtained by electroejaculation of 3 males (6, 9, and 11 years of age) and used after cryopreservation [TEST-yolk (media prepared with egg yolk, TES, and Tris) + 6% glycerol] or after storage at 4°C for 24 h (cooled). In vitro production, cryopreservation, and transfer of embryos were done as described by Pope et al. (2006 Theriogenology 66, 1518-1524). Cleavage frequency was 43% overall, 55% (64/117) after IVF, and 20% after ICSI with frozen (10/43) or cooled (2/16) sperm. After IVF with frozen sperm, cleavage rate was 63% (48/76) v. 39% (16/41) with cooled sperm. On Days 5, 57, 26, and 17% of embryos were morula (&gt; 16 cells), early morula (≈16 cells) and pre-morula (&lt; 16 cells), respectively. Forty-four Day 5 embryos were cryopreserved at a slow, controlled rate, 24 of which were thawed and transferred by laparotomy to the uteri of 3 Day 5 gonadotropin-treated recipients (7-9 embryos/female). Similarly, 28 Day 5 fresh embryos were auto-transferred to 3 Day 5 recipients (5-15 embryos/female). Most embryos transferred were morulae (83%) produced by IVF using frozen sperm (71%). None of the recipients established pregnancies, a failure that, undoubtedly, was multifaceted. Age may have been a factor because most recipients were 8 to 11 years old. Nonetheless, we have (1) shown that clouded leopard females are responsive to exogenous gonadotropins, (2) produced embryos in vitro, both by IVF and ICSI, using both cryopreserved and cooled sperm, and (3) provided further evidence of seasonality in ovarian activity.

Captive reproduction of the Jamaican iguana (Cyclura collei)

The Jamaican iguana (Cyclura collei) was considered to be extinct until its rediscovery in 1990 in the Hellshire Hills region of Jamaica. Between 1994 and 1996, several wild caught juveniles were placed in six North American zoological institutions including the Indianapolis Zoo. The intent was to establish a self-sustaining captive population outside of Jamaica in order to serve as an ancillary population should the wild numbers decline. Several environmental parameters such as temperature, humidity, photoperiod and diet were manipulated to encourage reproduction in captivity. In 2006, two clutches of eggs were deposited by two separate females for a total of 35 eggs. Twenty-six eggs were fertile and 22 hatched after a 76-83-day incubation at 30.3-30.8 degrees C (86.5-86.7 degrees F). The average weight of the neonates was 26.4 g and the average total length was 21.7 cm. This successful reproduction of C. collei constitutes the first North American hatching for this species. Environmental parameters, incubation techniques and neonate morphometry can serve as a baseline for further propagation of the species. This represents a positive milestone for the continuing conservation of this critically endangered species.