HEMATOLOGY AND BLOOD CHEMISTRY VALUES IN CUBAN CROCODILES (CROCODYLUS RHOMBIFER) HOUSED AT THE ZAPATA SWAMP CROCODILE FARM, CUBA.

We report hematology and biochemistry reference intervals (RI) for the critically endangered Cuban crocodile (Crocodylus rhombifer). In November 2019, we sampled 43 adult crocodiles (6 male, 37 female) under human care at the Zapata Swamp Crocodile Farm in Matanzas, Cuba. These crocodiles are part of a breeding program for the species registered by the Convention on International Trade in Endangered Species (CITES). Visual health evaluations were performed immediately after manual restraint, and blood was collected from the postoccipital sinus. We performed packed cell volume (PCV), total solids (TS), complete blood counts (CBC), and biochemistry profiles for each crocodile on the day of sampling. Mean PCV (n = 42) was 21.1 ± 5.0% and TS (n = 42) 7.3 ± 1.2 mg/dl, respectively. Absolute white blood cell (WBC) (n = 40) was 9.6 ± 5.7 ｘ 109/L. Similar to other crocodilian species, the dominant leukocyte was lymphocytes (70.7 ± 10.4%), followed by heterophils (18.7 ± 9.7%). Two of the crocodiles had a high heterophil:lymphocyte ratio (0.87 and 0.74), although on visual exam they were both considered healthy. The range of creatine kinase was 41-1,482 U/L, and the higher values may be a reflection of muscle exertion at time of handling. Limitations to the study included skewed sex ratios and high lipemia and hemolysis in most samples collected. These are the first reference intervals reported for this species, including the first descriptions of WBC morphology. These data are valuable for the management of animals at the Zapata Swamp Crocodile Farm, for comparison with free-living Cuban crocodiles in Cuba, and for comparison with those managed under human care outside of Cuba.

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.

Crocodilian perivitelline membrane-bound sperm detection.

Advanced reproductive technologies (ART's) are often employed with various taxa to enhance captive breeding programs and maintain genetic diversity. Perivitelline membrane-bound (PVM-bound) sperm detection has previously been demonstrated in avian and chelonian species as a useful technique for breeding management. In the absence of embryotic development within an egg, this technique can detect the presence of sperm trapped on the oocyte membrane confirming breeding, male reproductive status, and pair compatibility. PVM-bound sperm were successfully detected in three clutches of Cuban crocodile (Crocodylus rhombifer) eggs at the Smithsonian's National Zoological Park (NZP) for the first time in any crocodilian species. PVM-bound sperm were detected in fresh and incubated C. rhombifer eggs, as well as eggs that were developing (banded) and those that were not (not banded). The results of this study showed significant differences in average sperm densities per egg between clutches (p = 0.001). Additionally, there was not a significant difference within clutches between eggs that banded and those that did not band (Clutch A, p = 0.505; Clutch B, p = 0.665; Clutch C, p = 0.266). The results of this study demonstrate the necessity to microscopically examine eggs that do not develop (do not band), to determine if sperm is present, which can help animal managers problem solve reproductive shortcomings. PVM-bound sperm detection could be a useful technique in assessing crocodilian breeding programs, as well as have potential uses in studies assessing sperm storage, artificial insemination, and artificial incubation.

Age, fertility and reproductive behavior in cuban crocodiles, Crocodylus rhombifer, at the Smithsonian's National Zoological Park.

The Smithsonian's National Zoological Park (NZP) has a long history with Cuban crocodiles, Crocodylus rhombifer, beginning in 1900's when the first animals arrived at the NZP. Today, the Zoo has two breeding groups of C. rhombifer and has observed and analyzed reproductive behavior and fertility rates over the last three years. Fertility rates were determined initially by observing the formation of an opaque band that forms on the shell of a fertile egg, called banding. The fertility rates by banding were later compared to the observation made after opening the eggs to verify fertility. In addition to tracking fertility, nesting and agonistic behavior were also observed. Several notable observations were documented over the same period. These included a male predating a nesting female's eggs, increased aggression between two females housed together, the continued development of a partially banded egg, and the discovery of 19 additional egg shells post oviposition by both females in the enclosure. Here we discuss the nest phenology, fertility and behavior of the five exhibited C. rhombifer at the Smithsonian's National Zoological Park over a 3-year period. Zoo Biol. 34:278-284, 2015. © 2015 Wiley Periodicals Inc.

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.

Crocodylian conservation and evolution insights from an anatomical and histological examination of phalli from male false gharial (Tomistoma schlegelii).

As wild population threats for the endangered false gharial (Tomistoma schlegelii) persist, conservation breeding programs, including developing semen collection techniques for subsequent artificial insemination, are becoming important species conservation measures. Developing reproductive biology understanding of a species is important to developing best practices and hopefully maximizing reproductive successes. However, information on crocodylians functional copulatory anatomy in general is lacking. To that end, zoological facilities and conservation centres have the exceptional opportunity to contribute new understandings that may not otherwise be attainable regarding crocodylian reproductive anatomy, particularly during routine physical examinations or post-mortem necropsies. Therefore, to better understand T. schlegelii reproductive biology, to contribute knowledge in support of zoo breeding conservation efforts and to contribute to what is known overall about crocodylian reproduction, we investigated phallic anatomy of adult male Tomistoma from two zoological populations, the St. Louis Zoo, USA and Sungai Dusun Wildlife Reserve, Peninsular Malaysia. Here, we present the gross anatomical features and histological analysis of underlying tissue-level details in pursuit of a better understanding of copulatory function and associated gamete transfer mechanisms. While much of the overall Tomistoma phallic morphology and inferred function corresponds to that of other crocodylian species and speaks to conserved aspects of functional anatomy across taxa, species-specific aspects of glans and glans tip morphology are also identified. These novelties are discussed in a general function and overall broader evolutionary contexts.

Parthenogenesis in a captive Asian water dragon (Physignathus cocincinus) identified with novel microsatellites.

Reptiles show varying degrees of facultative parthenogenesis. Here we use genetic methods to determine that an isolated, captive female Asian water dragon produced at least nine offspring via parthenogenesis. We identified microsatellites for the species from shotgun genomic sequences, selected and optimized primer sets, and tested all of the offspring for a set of seven microsatellites that were heterozygous in the mother. We verified that the seven loci showed high levels of polymorphism in four wild Asian water dragons from Vietnam. In all cases, the offspring (unhatched, but developed eggs, or hatched young) had only a single allele at each locus, and contained only alleles present in the mother's genotype (i.e., were homozygous or hemizygous). The probability that our findings resulted from the female mating with one or more males is extremely small, indicating that the offspring were derived from a single female gamete (either alone or via duplication and/or fusion) and implicating parthenogenesis. This is the first documented case of parthenogenesis in the Squamate family Agamidae.

Influence of extracellular environment on the motility and structural properties of spermatozoa collected from hormonally stimulated Panamanian Golden Frog (Atelopus zeteki).

A better understanding of the factors influencing the biology of amphibian spermatozoa after release from the testis is a prerequisite to the development of sperm preservation methods. The objective of the study was to determine the effect of extracellular conditions (exposure to water and different temperatures) over time on the sperm motility and structural properties (including morphology and DNA integrity) collected from hormonally stimulated Atelopus zeteki. Following intraperitoneal injection of gonadotropin-releasing hormone agonist (des-Gly10, D-Ala6, Pro-NHEt9 GnRH; 4 µg/g of body weight), human chorionic gonadotropin (hCG, 10 IU/gbw), or Amphiplex™ (0.4 µg/gbw GnRH-A + 10 µg/gbw metoclopramide hydrochloride), spermic urine samples from 27 males were collected and analyzed for sperm motility, morphology and DNA integrity while maintained at room temperature (23 °C), 4 °C, or diluted in water (hypo-osmotic environment) over a period of 46 min post-collection. Percentages of sperm motility and forward progressive motility remained high (>60%) when spermic urine was kept at room temperature or at 4 °C for 46 min regardless of the hormonal stimulation method. Dilution in water at room temperature greatly reduced the percentage of motile spermatozoa and forward progression (<50%) as well as DNA integrity (32.8% of intact cells) after 23 min while morphology did not differ (30.4% of normal cells), regardless of the hormone stimulation. This is the first systematic study on the effect of extracellular environment over time on A. zeteki sperm quality. This will contribute to the development of sperm handling protocols and reproductive technologies for this and other endangered Atelopus species.

Effects of hormonal stimulation on the concentration and quality of excreted spermatozoa in the critically endangered Panamanian golden frog (Atelopus zeteki).

Knowledge of basic gamete biology is critical to better protect and propagate endangered amphibian species and also to develop reproductive technologies combined with germplasm cryopreservation. The objectives of the study were to test different hormonal stimulations and then characterize the quantity and quality of Panamanian golden frog (Atelopus zeteki) spermatozoa. Following intraperitoneal injection of the gonadotropin-releasing hormone agonist (des-Gly10, D-Ala6, Pro-NHEt9--GnRH 1, 2 or 4 µg/g of body weight), human chorionic gonadotropin (hCG; 5 or 10 IU/gbw), or Amphiplex™ (0.4 µg/gbw GnRH-A + 10 µg/gbw metoclopramide hydrochloride), spermic urine samples from 29 males were collected at different time points (from 0.5 to 24 h post-injection) to analyze the concentration, motility, and morphology of the spermatozoa. Peak of sperm concentration was observed at 3.5 h post injection for all hormonal treatments. Amphiplex™ led to the highest sperm concentrations (4.45 ± 0.07 × 106 cells/mL) followed by 4 µg/gbw GnRH-A (2.65 ± 0.21 × 106 cells/mL). Other stimulation protocols and doses induced sperm production, but at lower levels (ranging from 1.34 to 1.70 × 106 cells/mL). More than 60% of spermatozoa were motile following all treatments but the highest motility (>90%) was obtained from the 10 IU/gbw hCG treatment. Spermic urine samples obtained with all hormone treatments had higher pH (ranging from 7.1 to 7.8) than the urine alone (6.7-6.8). Spermatozoa were filiform and elongated with an apical acrosome, a mitochondrial sheath, a small midpiece and a long tail with an undulating membrane. More than 80% of cells were morphologically normal and 50-70% had intact DNA. These sperm characteristics were not influenced by hormonal treatments. This first comprehensive characterization of sperm samples following optimized hormonal stimulations in A. zeteki lays the foundation for more fundamental studies, reproductive technologies, and future preservation strategies.

Reduced immune function predicts disease susceptibility in frogs infected with a deadly fungal pathogen.

The relationship between amphibian immune function and disease susceptibility is of primary concern given current worldwide declines linked to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). We experimentally infected lowland leopard frogs (Lithobates yavapaiensis) with Bd to test the hypothesis that infection causes physiological stress and stimulates humoral and cell-mediated immune function in the blood. We measured body mass, the ratio of circulating neutrophils to lymphocytes (a known indicator of physiological stress) and plasma bacterial killing ability (BKA; a measure of innate immune function). In early exposure (1-15 days post-infection), stress was elevated in Bd-positive vs. Bd-negative frogs, whereas other metrics were similar between the groups. At later stages (29-55 days post-infection), stress was increased in Bd-positive frogs with signs of chytridiomycosis compared with both Bd-positive frogs without disease signs and uninfected control frogs, which were similar to each other. Infection decreased growth during the same period, demonstrating that sustained resistance to Bd is energetically costly. Importantly, BKA was lower in Bd-positive frogs with disease than in those without signs of chytridiomycosis. However, neither group differed from Bd-negative control frogs. The low BKA values in dying frogs compared with infected individuals without disease signs suggests that complement activity might signify different immunogenetic backgrounds or gene-by-environment interactions between the host, Bd and abiotic factors. We conclude that protein complement activity might be a useful predictor of Bd susceptibility and might help to explain differential disease outcomes in natural amphibian populations.

Clinicians' Real World Perceptions of Pre-Nephrectomy Diagnostic Biopsy Performance as a Driver of Reduction in Unnecessary Surgeries in Renal Tumors.

Operative removal of oncocytomas is generally unnecessary, but not infrequent in the context of renal masses. The infrequent use of pre-nephrectomy biopsies is a function of historical limitations of histopathological differential diagnosis in this setting. Assessment of clinicians' receptiveness to a novel molecular diagnostic approach to this challenge was undertaken by means of a survey vehicle administered to 102 practicing urologists and pathologists who met inclusion criteria related to their actual clinical activity. Survey results supported the previously reported observations on misdiagnosis with urologists' reported rates of 25% inconclusive results, and an additional 17% disagree with the final surgical diagnosis. The self-reported rate of 9% for pre-operative biopsies was comparable to prior reports, but 39% of urologists who are not currently performing pre-operative biopsies expressed interest in introducing them into their practice for this purpose with an improved diagnostic. Almost all urologists (94%) felt it important not to resect benign oncocytomas and 62% indicated they would use a test which improved the ability to sub-type renal tumors pre-operatively. The level of performance benchmark of the unidentified prototypic microRNA-based diagnostic as reported previously in the literature was deemed sufficient to change care in these cases by 73%. Overall they predicted a 38% rate of biopsies and resulting increases in decisions to forgo nephrectomy or to perform only partial nephrectomy. Pathologists also expressed support for the use of this technology in the context of inadequate specimens and for improved sub-typing of these tumors in inconclusive cases.

Using operant conditioning and desensitization to facilitate veterinary care with captive reptiles.

In addition to being a large component of most zoological collections, reptile species are becoming more popular as family pets. Reptiles have the cognitive ability to be trained to facilitate daily husbandry and veterinary care. Desensitization and operant conditioning can alleviate some of the behavioral and physiological challenges of treating these species. A survey of reptile training programs at zoos in the United States and worldwide reveals that there are many successful training programs to facilitate veterinary care and minimize stress to the animal. Many of the techniques being used to train reptiles in zoological settings are transferable to the exotic pet clinician.