Breathing Air and Living Underwater: Molecular Evolution of Genes Related to Antioxidant Response in Cetaceans and Pinnipeds.

Cetaceans and pinnipeds are lineages of mammals that have independently returned to the aquatic environment, acquiring varying degrees of dependence on it while sharing adaptations for underwater living. Here, we focused on one critical adaptation from both groups, their ability to withstand the ischemia and reperfusion experienced during apnea diving, which can lead to the production of reactive oxygen species (ROS) and subsequent oxidative damage. Previous studies have shown that cetaceans and pinnipeds possess efficient antioxidant enzymes that protect against ROS. In this study, we investigated the molecular evolution of key antioxidant enzyme genes (CAT, GPX3, GSR, PRDX1, PRDX3, and SOD1) and the ROS-producing gene XDH, in cetaceans and pinnipeds lineages. We used the ratio of non-synonymous (dN) to synonymous (dS) substitutions as a measure to identify signatures of adaptive molecular evolution in these genes within and between the two lineages. Additionally, we performed protein modeling and variant impact analyzes to assess the functional consequences of observed mutations. Our findings revealed distinct selective regimes between aquatic and terrestrial mammals in five of the examined genes, including divergences within cetacean and pinniped lineages, between ancestral and recent lineages and between crowns groups. We identified specific sites under positive selection unique to Cetacea and Pinnipedia, with one site showing evidence of convergent evolution in species known for their long and deep-diving capacities. Notably, many sites under adaptive selection exhibited radical changes in amino acid properties, with some being damaging mutations in human variations, but with no apparent detrimental impacts on aquatic mammals. In conclusion, our study provides insights into the adaptive changes that have occurred in the antioxidant systems of aquatic mammals throughout their evolutionary history. We observed both distinctive features within each group of Cetacea and Pinnipedia and instances of convergence. These findings highlight the dynamic nature of the antioxidant system in response to challenges of the aquatic environment and provide a foundation for further investigations into the molecular mechanisms underlying these adaptations.

Osteological profiling of femoral diaphysis and neck in aquatic, semiaquatic, and terrestrial carnivores and rodents: effects of body size and locomotor habits.

The increased limb bone density documented previously for aquatic tetrapods has been proposed to be an adaptation to overcome buoyancy during swimming and diving. It can be achieved by increasing the amount of bone deposition or by reducing the amount of bone resorption, leading to cortical thickening, loss of medullary cavity, and compaction of trabecular bone. The present study examined the effects of locomotor habit, body size, and phylogeny on the densitometric, cross-sectional, and biomechanical traits of femoral diaphysis and neck in terrestrial, semiaquatic, and aquatic carnivores, and in terrestrial and semiaquatic rodents (12 species) by using peripheral quantitative computed tomography, three-point bending, and femoral neck loading tests. Groupwise differences were analyzed with the univariate generalized linear model and the multivariate linear discriminant analysis supplemented with hierarchical clustering. While none of the individual features could separate the lifestyles or species adequately, the combinations of multiple features produced very good or excellent classifications and clusterings. In the phocid seals, the aquatic niche allowed for lower femoral bone mineral densities than expected based on the body mass alone. The semiaquatic mammals mostly had high bone mineral densities compared to the terrestrial species, which could be considered an adaptation to overcome buoyancy during swimming and shallow diving. Generally, it seems that different osteological properties at the levels of mineral density and biomechanics could be compatible with the adaptation to aquatic, semiaquatic, or terrestrial niches.

Establishment and Characterization of a Primary Fibroblast Cell Culture from the Amazonian Manatee (Trichechus inunguis).

The vulnerable status of the Amazon manatee, Trichechus inunguis, indicates the need to seek measures to guarantee its conservation. In this context, the cultivation of cells in vitro is a strategy that should at least guarantee the preservation of their genetic material. Thus, we established for the first time a primary culture of Amazonian manatee fibroblasts (TINsf) from a skin biopsy of a young male. Karyotypic analysis of the 3rd, 7th, and 12th passages confirmed the taxonomic identity of the species T. inunguis (2n = 56/NF = 92) and indicated that this culture presents genomic stability. Gene and protein expression of vimentin at the 13th passage show the predominant presence of fibroblasts in TINsf. To test the cell line's responsiveness to materials and demonstrate a possible application of this culture, it was exposed to andiroba seed oil (ASO), and its viability and proliferative capacity were evaluated. ASO demonstrated toxic effects at the highest concentrations and longest exposure times tested, reproducing results observed in human cultures, indicating the applicability of TINsf in toxicological and biotechnological studies. After cryopreservation, the TINsf line maintained its proliferative potential, indicating the establishment of a new culture available for future studies.

Regiospecific analysis of lipidome in the brain from mammals of land and aquatic habitats-by liquid chromatography-mass spectrometry.

The brain is a complex organ demonstrated by the occurrence of specific types of functional lipids. Despite some studies focusing on providing the animal brain lipid signature, there are limited studies focusing on the comprehensive and regiospecific characterization of multiple animal brain lipidome. Herein we characterized about 294 lipid molecular species from six different lipid classes in different portions of the brain after fixation from mammals of different habitats, fully-aquatic (n = 6), semi-aquatic (n = 6), and terrestrial (n = 4), using liquid chromatography-mass spectrometry. The untargeted brain lipid profiling revealed a significant difference in total lipid levels between fully-aquatic, semi-aquatic, and terrestrial mammals. The polyunsaturated fatty acids and cholesterol esters are abundant in brain tissue of semi-aquatic followed by fully-aquatic mammals whereas phosphatidylethanolamines are profoundly high in terrestrial species. The regiospecific analysis revealed a predominance of sphingolipids in all the groups but no significant differences were observed between the different portions of the brain such as the cerebellum, cortex, pons, spinal cord, and thalamus. Interestingly the multivariate analysis showed almost the same lipid compositions in the spinal cord and thalamus of terrestrial mammals. Overall, this is the first report to compare the comprehensive brain-lipidome among different mammalian groups inhabiting three distinct habitats. These results indicate that the brain lipid composition is specific to the animal habitat.

Pseudogenization of the Hair-Related Genes PADI3 and S100A3 in Cetaceans and Hippopotamus amphibius.

Hair-related genes in mammals play important roles in the development and maintenance of hair and other keratinous structures in mammals. The peptidyl arginine deiminase 3 (PADI3) gene encodes an enzyme that catalyzes the conversion of arginine residues to citrulline. The S100 calcium binding protein A3 (S100A3) gene encodes a protein that is highly expressed in the hair cuticle and contains arginine residues that are converted to citrullines by PADI enzymes. In this study, we investigated the pseudogenization events of PADI3 and S100A3 in cetaceans and Hippopotamus amphibius. We found that PADI3 underwent three independent pseudogenization events during cetacean evolution, in baleen whales, toothed cetaceans other than Physeter catodon, and P. catodon. Notably, the entire PADI3 gene is absent in the baleen whales. Pseudogenization of S100A3 occurred independently in cetaceans and H. amphibius. Interestingly, we found that in cetaceans S100A3 underwent pseudogenization before PADI3, suggesting that differential selection pressures were acting on the two genes. Our findings provide valuable insights into the molecular evolution of these genes in cetaceans and hippopotamuses, highlighting their importance for understanding the evolution of hair-related genes.

Vomeronasal Receptors Associated with Circulating Estrogen Processing Chemosensory Cues in Semi-Aquatic Mammals.

In numerous animals, one essential chemosensory organ that detects chemical signals is the vomeronasal organ (VNO), which is involved in species-specific behaviors, including social and sexual behaviors. The purpose of this study is to investigate the mechanism underlying the processing of chemosensory cues in semi-aquatic mammals using muskrats as the animal model. Muskrat (Ondatra zibethicus) has a sensitive VNO system that activates seasonal breeding behaviors through receiving specific substances, including pheromones and hormones. Vomeronasal organ receptor type 1 (V1R) and type 2 (V2R) and estrogen receptor α and ß (ERα and ERß) were found in sensory epithelial cells, non-sensory epithelial cells and lamina propria cells of the female muskrats' VNO. V2R and ERα mRNA levels in the VNO during the breeding period declined sharply, in comparison to those during the non-breeding period, while V1R and ERß mRNA levels were detected reversely. Additionally, transcriptomic study in the VNO identified that differently expressed genes might be related to estrogen signal and metabolic pathways. These findings suggested that the seasonal structural and functional changes in the VNO of female muskrats with different reproductive status and estrogen was regulated through binding to ERα and ERß in the female muskrats' VNO.

Hemotropic Mycoplasma spp. in Aquatic Mammals, Amazon Basin, Brazil.

Hemotropic Mycoplasma spp. (hemoplasmas) are uncultivable bacteria that infect mammals, including humans. We detected a potentially novel hemoplasma species in blood samples from wild river dolphins in the Amazon River Basin, Brazil. Further investigation could determine pathogenicity and zoonotic potential of the detected hemoplasma.

Taste receptors in aquatic mammals: Potential role of solitary chemosensory cells in immune responses.

The sense of taste is associated with the evaluation of food and other environmental parameters such as salinity. In aquatic mammals, anatomic and behavioral evidence of the use of taste varies by species and genomic analysis of taste receptors indicates an overall reduction and, in some cases, complete loss of intact bitter and sweet taste receptors. However, the receptors used by taste buds in the oral cavity are found on cells in other areas of the body and play an important role in immune responses. In the respiratory tract, an example of such cells is solitary chemosensory cells (SCCs) which have bitter and sweet taste receptors. The bitter receptors detect chemicals given off by pathogens and initiate an innate immune response. Although many aquatic mammals may not have a role for taste in the assessment of food, they likely would benefit from the added protection that SCCs provide, especially considering respiratory diseases are a problem for many aquatic mammals. While evidence indicates that some species do not possess functional bitter receptors for taste, many do have intact bitter receptor genes and it is important for researchers to be aware of all roles for these receptors in homeostasis. Through a better understanding of the anatomy and physiology of aquatic mammal's respiratory systems, better treatment and management is possible.

Microbiome Composition and Function in Aquatic Vertebrates: Small Organisms Making Big Impacts on Aquatic Animal Health.

Aquatic ecosystems are under increasing stress from global anthropogenic and natural changes, including climate change, eutrophication, ocean acidification, and pollution. In this critical review, we synthesize research on the microbiota of aquatic vertebrates and discuss the impact of emerging stressors on aquatic microbial communities using two case studies, that of toxic cyanobacteria and microplastics. Most studies to date are focused on host-associated microbiomes of individual organisms, however, few studies take an integrative approach to examine aquatic vertebrate microbiomes by considering both host-associated and free-living microbiota within an ecosystem. We highlight what is known about microbiota in aquatic ecosystems, with a focus on the interface between water, fish, and marine mammals. Though microbiomes in water vary with geography, temperature, depth, and other factors, core microbial functions such as primary production, nitrogen cycling, and nutrient metabolism are often conserved across aquatic environments. We outline knowledge on the composition and function of tissue-specific microbiomes in fish and marine mammals and discuss the environmental factors influencing their structure. The microbiota of aquatic mammals and fish are highly unique to species and a delicate balance between respiratory, skin, and gastrointestinal microbiota exists within the host. In aquatic vertebrates, water conditions and ecological niche are driving factors behind microbial composition and function. We also generate a comprehensive catalog of marine mammal and fish microbial genera, revealing commonalities in composition and function among aquatic species, and discuss the potential use of microbiomes as indicators of health and ecological status of aquatic ecosystems. We also discuss the importance of a focus on the functional relevance of microbial communities in relation to organism physiology and their ability to overcome stressors related to global change. Understanding the dynamic relationship between aquatic microbiota and the animals they colonize is critical for monitoring water quality and population health.

Factors influencing exposure of North American river otter (Lontra canadensis) and American mink (Neovison vison) to mercury relative to a large-scale reservoir in northern British Columbia, Canada.

Although reservoir creation increases fish mercury (Hg) concentrations, little information exists on its effects on Hg concentrations in aquatic mammals. River otters (Lontra canadensis) and American mink (Neovison vison) are two aquatic mammals that have been used as model species for assessing Hg bioaccumulation in aquatic systems. We assessed Hg and selenium (Se) concentrations in these two species within and outside of the Williston Reservoir (Peace-Williston (PW) watershed) in northern British Columbia (BC) and used these data to investigate potential explanatory factors (i.e., watershed, gender, trophic level (δ15N), and regional geology) influencing Hg concentrations. Hg concentrations in otter and mink inhabiting the Mackenzie watershed (outside the PW) were significantly lower than other watersheds in Northern BC. The general trend was the same for both species; the Peace-Williston having the highest and Mackenzie having the lowest Hg concentrations. For mink, the Peace-Williston watershed, higher trophic levels, and higher proportions of igneous/metamorphic bedrock were all significant influences on higher Hg concentrations (logistic regression). Higher trophic levels or proportions of of igneous/metamorphic bedrock, however, were not directly associated with the PW watershed suggesting there may be an impoundment effect. Baseline data on natural Hg inputs before planned anthropogenic changes occur is a critical first step to aiding interpretations of Hg-related effects on wildlife populations and their related ecosystems.

Survey of Brucella spp. and Leptospira spp. antibodies in cetaceans and manatees of the Amazon basin and Atlantic Ocean, Brazil.

Aquatic mammals can act as sentinels of emerging and resurging pathogens in the environment. Brucella spp. and Leptospira spp. are 2 zoonotic pathogens relevant to aquatic mammals, and their detection can be used to assess pathogen exposure. In this study, serum from 84 individuals-63 cetaceans (families Iniidae, n = 37; Delphinidae, n = 22; and Kogiidae, n = 4) and 21 West Indian manatees Trichechus manatus-was tested by the Rose Bengal Test (RBT) and a commercial competitive enzyme-linked immunosorbent assay (c-ELISA) for detecting Brucella spp. antibodies, and the microscopic agglutination test (MAT) for screening Leptospira spp. exposure. Overall, 4.8% (3/63) of cetaceans were positive by RBT and 15.9% (10/63) by c-ELISA for Brucella spp. Serum from 8 c-ELISA positive cetaceans (with available serum) was further tested via serum agglutination test (SAT) and 1 individual was positive. c-ELISA was more sensitive than RBT. Exposure to Brucella spp. was found in 5 cetacean species: Clymene dolphin Stenella clymene, short-finned pilot whale Globicephala macrorhynchus, pygmy killer whale Feresa attenuata, melon-headed whale Peponocephala electra and Atlantic bottlenose dolphin Tursiops truncatus in the Atlantic Ocean, Brazil, expanding the range of known Brucella seropositive aquatic hosts. No evidence of Brucella spp. exposure was found in Iniidae and Kogiidae odontocetes and manatees. Antibodies against Leptospira spp. were not detected in cetaceans and sirenians by MAT. These results contribute to the evaluation of different Brucella spp. serological methods in cetaceans and manatees and highlight the epidemiology of zoonotic pathogens in aquatic mammals of the southwestern Atlantic Ocean and the Amazon basin.

Social relationships and death-related behaviour in aquatic mammals: a systematic review.

Some aquatic mammals appear to care for their dead, whereas others abandon their live offspring when conditions are unfavourable. This incredible variety in behaviours suggests the importance of comparing and contrasting mechanisms driving death-related behaviours among these species. We reviewed 106 cases of aquatic mammals (81 cetaceans and 25 non-cetaceans) reacting to a death event, and extrapolated 'participant' (age class, sex, relationship and decomposition) and 'social' characteristics (escorting, calf dependence, alloparental care, herding and dispersal patterns) from published and unpublished literature. A multiple correspondence analysis (MCA) was performed to explore the relationships between these characteristics and death-related behaviours, with species clustered based on MCA scores. Results showed that both cetaceans and non-cetaceans react to death but in different ways. Non-cetaceans, characterized by a short maternal investment, were observed to protect the dead (defending it from external attacks), while cetaceans spent much longer with their offspring and display carrying (hauling, spinning, mouthing with the carcass and diving with it) and breathing-related (lifting and sinking the carcass) activities with the dead generally in association with other conspecifics. Our work emphasizes the need of increased documentation of death-related cases around the world to improve our understanding of aquatic mammals and their responses to death.This article is part of the theme issue 'Evolutionary thanatology: impacts of the dead on the living in humans and other animals'.

Eye Optics in Semiaquatic Mammals for Aerial and Aquatic Vision.

Based on anatomical measurements of refractive structures in the eye, the positions of focused images were computed for several groups of semiaquatic mammals: rodents, a nonpinniped semiaquatic carnivore (the sea otter), and pinniped carnivores (seals, sea lions, and the walrus). In semiaquatic rodents, eye optics enable emmetropia in the air but cause substantial hypermetropia in the water. In semiaquatic carnivores, there are several mechanisms for amphibious vision that focus images on the retina in both air and water. These mechanisms include the potential for a substantial change in the lens shape of sea otters and the presence of the corneal emmetropic window in pinnipeds. The results suggest that several groups of mammals that independently adapted to aquatic environments vary in how their visual systems adapted to aquatic vision.

Transition to an Aquatic Habitat Permitted the Repeated Loss of the Pleiotropic KLK8 Gene in Mammals.

Kallikrein related peptidase 8 (KLK8; also called neuropsin) is a serine protease that plays distinct roles in the skin and hippocampus. In the skin, KLK8 influences keratinocyte proliferation and desquamation, and activates antimicrobial peptides in sweat. In the hippocampus, KLK8 affects memory acquisition. Here, we examined the evolution of KLK8 in mammals and discovered that, out of 70 placental mammals, KLK8 is exclusively lost in three independent fully-aquatic lineages, comprising dolphin, killer whale, minke whale, and manatee. In addition, while the sperm whale has an intact KLK8 reading frame, the gene evolves neutrally in this species. We suggest that the distinct functions of KLK8 likely became obsolete in the aquatic environment, leading to the subsequent loss of KLK8 in several fully-aquatic mammalian lineages. First, the cetacean and manatee skin lacks sweat glands as an adaptation to the aquatic environment, which likely made the epidermal function of KLK8 obsolete. Second, cetaceans and manatees exhibit a proportionally small hippocampus, which may have rendered the hippocampal functions of KLK8 obsolete. Together, our results shed light on the genomic changes that correlate with skin and neuroanatomical differences of aquatic mammals, and show that even pleiotropic genes can be lost during evolution if an environmental change nullifies the need for the different functions of such genes.

Cryptosporidium spp. and Giardia sp. in aquatic mammals in northern and northeastern Brazil.

Cryptosporidium and Giardia are protozoans that can infect humans and wild and domestic animals. Due to the growing importance of diseases caused by protozoan parasites in aquatic species, we aimed to evaluate the frequency of infection by Cryptosporidium spp. and Giardia sp. in aquatic and marine mammals in the northern and northeastern regions of Brazil. We collected 553 fecal samples from 15 species of wild-ranging and captive aquatic mammals in northern and northeastern Brazil. All samples were analyzed by the Kinyoun technique for identification of Cryptosporidium spp. oocysts. Giardia sp. cysts were identified by means of the centrifugal-flotation technique in zinc sulfate solution. Subsequently, all samples were submitted for direct immunofluorescence testing. The overall frequency of infection was 15.55% (86/553) for Cryptosporidium spp. and 9.04% (50/553) for Giardia sp. The presence of Cryptosporidium spp. was detected in samples from 5 species: neotropical river otter Lontra longicaudis (15.28%), giant otter Pteronura brasiliensis (41.66%), Guiana dolphin Sotalia guianensis (9.67%), Amazonian manatee Trichechus inunguis (16.03%), and Antillean manatee T. manatus (13.79%). Giardia sp. was identified in L. longicaudis (9.23%), P. brasiliensis (29.16%), pygmy sperm whale Kogia breviceps (100%), dwarf sperm whale K. sima (25%), S. guianensis (9.67%), T. inunguis (3.81%), and T. manatus (10.34%). This is the first report of Cryptosporidium spp. in L. longicaudis, P. brasiliensis, and S. guianensis, while the occurrence of Giardia sp., in addition to the 2 otter species, was also identified in manatees, thus extending the number of hosts susceptible to these parasitic agents.

Vibrio parahaemolyticus- and V. alginolyticus-associated meningo-encephalitis in a bottlenose dolphin (Tursiops truncatus) from the Adriatic coast of Italy.

A case of Vibrio parahaemolyticus- and V. alginolyticus-associated meningo-encephalitis in a bottlenose dolphin (Tursiops truncatus) found stranded along the Adriatic coast of Italy in 2016 is herein reported, along with a minireview on V. parahaemolyticus and V. alginolyticus infections in aquatic mammals. Macroscopically, two abscesses were found in the dolphin's forebrain, along with an extensive, bilateral, parasitic broncho-pneumonia. Histologically, a suppurative-to-pyogranulomatous meningo-encephalitis involved the brain but not the cerebellum. Microbiological investigations yielded isolation of V. parahaemolyticus and V. alginolyticus from the aforementioned abscesses and from the brain parenchyma, respectively, with simultaneous recovery of Shewanella algae from the heart and of Photobacterium damselae from a blowhole swab. Although V. parahaemolyticus and V. alginolyticus, which are widely distributed across marine ecosystems worldwide, likely played a role in the development of the suppurative meningo-encephalitis in this dolphin, we are not aware of previous isolations of any of these two bacteria neither from cetacean brain lesions, nor from abscesses in aquatic mammals.

TREATMENT OF PULMONICOLA COCHLEOTREMA INFECTION WITH IVERMECTIN-PRAZIQUANTEL COMBINATION IN AN ANTILLEAN MANATEE (TRICHECHUS MANATUS MANATUS).

The aim of this study was to report the use of an oral combination of ivermectin plus praziquantel in the treatment of a Pulmonicola cochleotrema in an Antillean manatee ( Trichechus manatus manatus). A female manatee was found exhibiting respiratory changes and the presence of parasites in the nares. Based on clinical manifestations presented by the manatee, a symptomatic therapeutic protocol was employed, which included an anthelmintic treatment using a combination of ivermectin plus praziquantel. The parasites retrieved were identified as P. cochleotrema. The fourth day after the onset of the therapeutic protocol, the clinical signs declined and on the seventh day posttreatment no clinical signs were observed. This is the first time a therapeutic protocol of ivermectin plus praziquantel has been used in the treatment of P. cochleotrema in manatees.

Morphology of the eyeball from the Humpback whale (Megaptera novaeangliae).

Aquatic mammals underwent morphological and physiological adaptations due to the transition from terrestrial to aquatic environment. One of the morphological changes regards their vision since cetaceans' eyes are able to withstand mechanical, chemical, osmotic, and optical water conditions. Due to insufficient information about these animals, especially regarding their sense organs, this study aimed to describe the morphology of the Humpback whale (Megaptera novaeangliae) eyeball. Three newborn females, stranded dead on the coast of Sergipe and Bahia, Brazil, were used. Samples were fixed in a 10% formalin solution, dissected, photographed, collected, and evaluated through light and electron microscopy techniques. The Humpback whale sclera was thick and had an irregular surface with mechanoreceptors in its lamina propria. Lens was dense, transparent, and ellipsoidal, consisting of three layers, and the vascularized choroid contains melanocytes, mechanoreceptors, and a fibrous tapetum lucidum. The Humpback whale eyeball is similar to other cetaceans and suggests an adaptation to diving and migration, contributing to the perception of differences in temperature, pressure, and lighting.

Urinary parameters of Trichechus inunguis (Mammalia, Sirenia): reference values for the Amazonian Manatee

The Amazonian manatee, Trichechus inunguis (Natterer 1883) is endemic to the Amazon Basin and is currently considered a vulnerable species. In order to establish normality ranges of urinary parameters to help monitor the health of this species in captivity, chemical urinalyses were performed on twelve males and nine females of various age groups. Urine was collected once a month for twelve months in the tanks just after being drained, by placing stainless steel containers under the genital slit of females and applying abdominal massages to males in order to stimulate urination. Quantitative data of glucose, urea, creatinine, uric acid and amylase levels were obtained using colorimetric spectrophotometry. Dip strips were also useful for routine analyses, despite only providing qualitative results. Normal range to glucose levels, regardless of sex or age class, was 3.0 to 3.6 mg.dL-1, coinciding with qualitative values of glucose measured by dip strips. Statistical differences observed in some parameter levels suggest that some urine parameters analysed must take into consideration the sex and the age class of the animal studied, being these differences less remarkable in creatinine and amylase levels. To this last one, statistical difference was detected only in the calve's urine (7.0 to 11.5 mg.dL-1) compared to other age classes samples (4.1 to 5.3 mg.dL-1). The results presented here may be used as comparative data in future research on urinalysis in related species.

O peixe-boi da Amazônia, Trichechus inunguis (Natterer 1883) é endêmico da Bacia Amazônica e é considerado atualmente como espécie vulnerável. Com o objetivo de estabelecer valores de normalidade para os parâmetros urinários com vistas a ajudar no monitoramento da saúde desta espécie em cativeiro, foi realizada urinálise química em doze machos e nove fêmeas de várias classes etárias. A urina era coletada uma vez por mês, durante doze meses, nos tanques, logo após esvaziamento, colocando-se coletores de aço inoxidável sob o orifício genital das fêmeas e aplicando massagem abdominal nos machos para estimular a micção. Dados quantitativos dos níveis de glicose, ureia, creatinina, ácido úrico e amilase foram obtidos por meio de espectrofotometria colorimétrica. O uso de tiras reativas também foi útil para análises de rotina, apesar de somente fornecerem resultados qualitativos. A faixa de normalidade para os níveis de glicose, independente de classe etária e sexo foi de 3,0 a 3,6 mg.dL-1, coincidindo com os valores qualitativos identificados pelas tiras reativas. Diferenças estatísticas observadas nos níveis de alguns parâmetros analisados sugerem que estes devem levar em consideração o sexo e a classe etária do animal estudado. Estas diferenças foram menos marcantes nos níveis de creatinina e de amilase, sendo que para esta última foi detectada diferença estatística apenas nas urinas de filhotes lactentes (7,0 a 11,5 mg.dL-1), em comparação às amostras das demais classes etárias (4,1 a 5,3 mg.dL-1). Os resultados aqui apresentados podem ser usados como dados comparativos em futuras pesquisas sobre urinálise em espécies relacionadas.