Study of inorganic elements in different organs and tissues of Amazonian manatee (Trichechus inunguis) from Brazil.

Multielement concentrations (P, S, Cl, K, Ca, Cr, Mn, Fe, Ni, Cu, Zn, Rb, and Rh) and total mercury (T-Hg) were analyzed in different organs and tissues of Amazonian manatee (Trichechus inunguis). Samples of 27 T. inunguis specimens, maintained in the collection of the Amazonian Center for the Research and Preservation of Aquatic Mammals, were used, situated in an area highly impacted by gold mining in the northern region of the Brazilian Amazon. Samples of aquatic plants used as food by the animals were also analyzed. The elements S, Cl, K, Cr, and Mn accumulated mainly in the musculature, while Fe and Cu were more concentrated in the liver. Trace elements, such as rubidium (Rb) and rhodium (Rh), not previously reported in the organs of animals of the family Trichechidae, were also identified. The averages for T-Hg in the skin, muscle, encephalon, liver, kidney, and lung samples were, respectively, 0.1540 ± 0.1332, 0.0593 ± 0.1044, 0.0517 ± 0.0467, 0.0486 ± 0.0543, 0.0237 ± 0.0336, and 0.0013 ± 0.0032 µg.g-1. The values obtained for the vibrissae samples were below the limit of quantification, which allows for the conclusion that this tissue cannot be used as a contamination marker. It was observed that even when kept in a conservation breeding site, these animals were exposed to non-essential trace elements. Differences in the accumulation of elements were observed between the different organs and tissues analyzed. The presence of contaminants in animals that live in a preservation center, even at low levels, deserves attention.

A hybrid swarm of manatees along the Guianas coastline, a peculiar environment under the influence of the Amazon River plume.

The West Indian (Trichechus manatus) and Amazonian (T. inunguis) manatees have a sympatric occurrence at the mouth of the Amazon River. A result of this interspecific encounter is the occurrence of hybrids, which are frequently found along the coasts of Amapá state in Brazil, French Guiana and Guyana. Here we present new genetic evidence indicating the occurrence of a hybrid swarm along the Guianas Shield coastline, which is an interspecific hybrid zone that also separates T. manatus populations located east (Brazil) and west (Caribbean, Gulf of Mexico, Florida and Antilles). In addition, we suggest that this hybrid population occupies a peculiar mangrove-rich environment under strong influence of the Amazon River plume, which requires an independent management and should be considered a special conservation area.

Acoustical and anatomical determination of sound production and transmission in West Indian (Trichechus manatus) and Amazonian (T. inunguis) manatees.

West Indian (Trichechus manatus) and Amazonian (T. inunguis) manatees are vocal mammals, with most sounds produced for communication between mothers and calves. While their hearing and vocalizations have been well studied, the actual mechanism of sound production is unknown. Acoustical recordings and anatomical examination were used to determine the source of sound generation. Recordings were performed on live captive manatees from Puerto Rico, Cuba and Colombia (T. manatus) and from Peru (T. inunguis) to determine focal points of sound production. The manatees were recorded using two directional hydrophones placed on the throat and nasal region and an Edirol-R44 digital recorder. The average sound intensity level was analyzed to evaluate the sound source with a T test: paired two sample for means. Anatomical examinations were conducted on six T. manatus carcasses from Florida and Puerto Rico. During necropsies, the larynx, trachea, and nasal areas were dissected, with particular focus on identifying musculature and soft tissues capable of vibrating or constricting the airway. From the recordings we found that the acoustical intensity was significant (P < 0.0001) for both the individuals and the pooled manatees in the ventral throat region compared to the nasal region. From the dissection we found two raised areas of tissue in the lateral walls of the manatee's laryngeal lumen that are consistent with mammalian vocal folds. They oppose each other and may be able to regulate airflow between them when they are adducted or abducted by muscular control of arytenoid cartilages. Acoustic and anatomical evidence taken together suggest vocal folds as the mechanism for sound production in manatees.

First attempt to monitor luteinizing hormone and reproductive steroids in urine samples of the Amazonian manatee (Trichechus inunguis).

The aims of this study were to validate an enzyme immunoassay (EIA) for the measurement of luteinizing hormone (LH) in urine samples of Amazonian manatees (Trichechus inunguis; Mammalia: Sirenia) and to monitor urinary LH and reproductive steroids during the ovarian cycle in this species. Urine samples were collected from two captive males following a hormonal challenge with a gonadotropin-releasing hormone (GnRH) analogue. The urinary LH results from hormonal challenge were compared with urinary androgens for the purpose of EIA validation. Furthermore, urine samples were collected daily, over a 12-wk period, from two captive adult females, for 2 consecutive yr. The urinary LH pattern from females was compared with the patterns of urinary progestagens and estrogen conjugates throughout the ovarian cycle. An LH peak was observed in both male Amazonian manatees after the hormonal challenge, occurring prior to or together with peak androgen levels. In the females, the ovarian cycle ranged from 40 to 48 days (mean of 43.7 days). Two distinct peaks of estrogen conjugates were observed across all cycles analyzed, and the urinary LH peaks observed were accompanied by peaks of urinary estrogen conjugates. The EIA was validated as a method for the quantification of urinary LH from Amazonian manatees, as it was able to detect variations in the levels of LH in urine samples. These results suggest that T. inunguis exhibits a peculiar hormonal pattern during the ovarian cycle. Therefore, further studies are desirable and necessary to clarify the relationship between this hormonal pattern and morphological changes, as well as mating behavior, in Amazonian manatee.

Endocrine monitoring of the ovarian cycle in captive female Amazonian manatees (Trichechus inunguis).

The Amazonian manatee (Trichechus inunguis; Mammalia: Sirenia), a threatened aquatic mammal endemic to the Amazon basin, is the only sirenian that lives exclusively in fresh water. Information about the reproductive endocrinology of the Amazonian manatee is scarce; therefore, the aim of this study was to monitor salivary progesterone and estradiol patterns during the ovarian cycle in T. inunguis. Salivary samples were collected daily during a 12-week period of two consecutive years from two captive adult females. The salivary estradiol and progesterone were measured by enzyme immunoassay. The results were analyzed in an iterative process of excluding values that were higher than the mean plus 2 standard deviations until the basal values were determined. The interval between two peaks of salivary estradiol followed by a rise of progesterone was considered as one complete cycle for the calculation of the cycle length. We observed only three complete cycles in all samples analyzed. The cycle length ranged from 42 to 48 days (mean of 44.67 days). We also observed two distinct salivary estradiol peaks during all cycles analyzed, with the first peak occurring before the rise in salivary progesterone and the second occurred followed by a return to basal progesterone levels. This is the first in-depth study of the ovarian cycle in Amazonian manatees. Our results demonstrate that salivary samples can be a useful tool in the endocrine monitoring of this species and suggest that T. inunguis shows a peculiar hormonal pattern during the ovarian cycle, a finding that may have physiological and ecological significance in the reproductive strategy of these animals.

Tempo de passagem de duas dietas no trato gastrointestinal do peixe-boi da Amazônia Trichechus inunguis (Natterer, 1883) em cativeiro / Transit time of two diets in the gastrointestinal tract of the Amazonian manatee Trichechus inunguis (Natterer, 1883) in captivity

The objective of this study was to determine the transit time of two diets in the digestive tract of the Amazonian manatee in captivity. We tested separately two different diets: one composed exclusively of grass of the genus Brachiaria (experimental diet - ED- A 1) and the other composed of grass Brachiaria added with small portions of extruded pellets for horses (experimental diet ED- A 2). Two healthy adult manatees were selected and isolated from the rest and underwent a period of food acclimation with the experimental diets for 15 days. After this period, the experimental diet was marked with a plastic colored tape of 10-cm length and given to the animals. The manatees were monitored at intervals of 1 hour and all fecal material was collected until recovery of the markers. The mean transit time of ED - A1 was 123h57min , about 5.16 days and ED - A 2 was 125h04min or 5.21 days. There was no statistical difference (P <0.05) between the transit time of the two diets provided. The transit time observed (approximately 5 days) has also been reported by other authors for this species and is considered a strategy to increase the absorption time of food nutrients. Despite the small sample size, the results suggest that the use of pellets in the diet of the Amazonian manatee did not affect the transit time of the grass. Therefore, it is reasonable to believe that the introduction of concentrated food (pellets) does not affect the efficiency of the animal to digest and absorb food properly.

Com o objetivo de testar o tempo de passagem do alimento no trato digestório de peixes-bois da Amazônia em cativeiro, foram testadas separadamente duas dietas distintas. Uma composta exclusivamente de capim do gênero Brachiaria (dieta experimental - DE 1) e outra de capim do gênero Brachiaria acrescentado de pequenas porções de ração extrusada para eqüinos (dieta experimental - DE 2). Foram selecionados do plantel do INPA dois animais adultos machos sadios, os quais foram isolados dos demais e submetidos a um período de aclimatação às dietas experimentais por 15 dias. Após este período, as dietas foram marcadas com uma fita plástica de 10 cm e fornecidas aos animais que foram monitorados em intervalos de uma hora. Todo material fecal foi coletado até a recuperação dos marcadores plásticos. A média do tempo de passagem da DE 1 foi de 123h57min, cerca de 5,15 dias e da DE 2 foi de 125h04min ou 5,21 dias. Não houve diferença estatística (P<0,05) entre as dietas fornecidas. O tempo de passagem observado (aproximadamente 5 dias) coincide com o relatado por outros autores para a espécie, sendo esse tempo considerado uma estratégia para aumentar o tempo de absorção nutricional dos alimentos. Apesar do número reduzido de amostras, os resultados sugerem que o uso da ração na alimentação não interfere no tempo de passagem do capim pelo trato digestório do peixe-boi. Com isso, sugere-se que a introdução de alimento concentrado (ração) não afeta a eficiência na digestão e absorção correta do alimento.

Phylogeography and sex-biased dispersal across riverine manatee populations (Trichechus inunguis and Trichechus manatus) in South America.

Phylogeographic patterns and sex-biased dispersal were studied in riverine populations of West Indian (Trichechus manatus) and Amazonian manatees (T. inunguis) in South America, using 410bp D-loop (Control Region, Mitochondrial DNA) sequences and 15 nuclear microsatellite loci. This multi-locus approach was key to disentangle complex patterns of gene flow among populations. D-loop analyses revealed population structuring among all Colombian rivers for T. manatus, while microsatellite data suggested no structure. Two main populations of T. inunguis separating the Colombian and Peruvian Amazon were supported by analysis of the D-loop and microsatellite data. Overall, we provide molecular evidence for differences in dispersal patterns between sexes, demonstrating male-biased gene flow dispersal in riverine manatees. These results are in contrast with previously reported levels of population structure shown by microsatellite data in marine manatee populations, revealing low habitat restrictions to gene flow in riverine habitats, and more significant dispersal limitations for males in marine environments.

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 mgxdL-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 mgxdL-1) compared to other age classes samples (4.1 to 5.3 mgxdL-1). The results presented here may be used as comparative data in future research on urinalysis in related species.

Urinary parameters of Trichechus inunguis (Mammalia, Sirenia): reference values for the Amazonian Manatee / Parâmetros urinários de Trichechus Inunguis (Mammalia, Sirenia): valores-referência para o peixe-boi da Amazônia

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.

Placentation in the Amazonian manatee (Trichechus inunguis).

Evidence from several sources supports a close phylogenetic relationship between elephants and sirenians. To explore whether this was reflected in similar placentation, we examined eight delivered placentae from the Amazonian manatee using light microscopy and immunohistochemistry. In addition, the fetal placental circulation was described by scanning electron microscopy of vessel casts. The manatee placenta was zonary and endotheliochorial, like that of the elephant. The interhaemal barrier comprised maternal endothelium, cytotrophoblasts and fetal endothelium. We found columnar trophoblast beneath the chorionic plate and lining lacunae in this region, but there was no trace in the term placenta of haemophagous activity. The gross anatomy of the cord and fetal membranes was consistent with previous descriptions and included a four-chambered allantoic sac, as also found in the elephant and other afrotherians. Connective tissue septae descended from the chorionic plate and carried blood vessels to the labyrinth, where they gave rise to a dense capillary network. This appeared to drain into shorter vessels near the chorionic plate. The maternal vasculature could not be examined in the same detail, but maternal capillaries ran rather straight and roughly parallel to the fetal ones. Overall, there is a close resemblance in placentation between the manatee and the elephant.