O objetivo deste estudo foi verificar a viabilidade da dosagem de andrógenos em amostras de saliva, secreção lacrimal, urina e fezes de peixe-boi da Amazônia realizando um desafio hormonal. Dois peixes-bois amazônicos adultos machos (A-1 e A-2) foram submetidos a um protocolo de experimentação de doze dias (D-1 a D10). No D0 os animais receberam uma injeção intramuscular de GnRH exógeno. Amostras de saliva, secreção lacrimal, urina e fezes foram coletadas diariamente (entre 08h00 e 09h00) e mantidas a -20°C até o ensaio. As amostras de fezes foram liofilizadas, extraídas com metanol 80% e diluídas em tampão antes do radioimunoensaio (RIE). As amostras de urina sofreram hidrólise ácida e foram diluídas em soro bovino depletado. As amostras de saliva e secreção lacrimal foram dosadas sem etapa de extração, porém, o ensaio foi adaptado para aumentar a sensibilidade do teste. Os ensaios hormonais foram realizados utilizando um conjunto comercial de RIE para testosterona total. Um pico de andrógenos (mediana+2DI) somente foi observado nas amostras de saliva, urina e fezes de ambos os animais. Porém, os picos de andrógenos fecais ocorreram depois (cinco dias) dos picos de andrógenos urinários e salivares. Este intervalo está correlacionado com o longo tempo de passagem da digesta pelo trato gastrointestinal na espécie. Os picos salivares e urinários ocorreram muito próximos, provavelmente com poucas horas de intervalo. Esses resultados demonstram que as concentrações de andrógenos em amostras de saliva, urina ou fezes refletem consistentemente os eventos fisiológicos e são ferramentas de grande utilidade no monitoramento reprodutivo de peixes-bois da Amazônia. / The aim of this study was to verify the viability of androgens measurement in saliva, lacrimal secretion, urine and fecal samples of Amazonian manatee by carrying out hormonal challenge. Two adult male manatees (A-1 and A-2) were submitted to an experimentation protocol of twelve days (D-1 to D10). On D0 the animals received an intramuscular injection of GnRH-analogue. Salivary, lacrimal, urinary and fecal samples were collected daily (between 08h00 and 09h00) and frozen at -20°C until assayed. Fecal samples were lyophilized, extracted with 80% methanol and diluted in buffer before the radioimmunoassay (RIA). Urine samples underwent acid hydrolysis and diluted in depleted bovine serum. Salivary and lacrimal samples were assayed without extraction step, but, the assay was adapted to improve the sensibility. Hormonal assays were carried out with a commercial testosterone RIA kit. An androgen peak (>median+2IQR) was observed only in salivary, urinary and fecal samples of both animals. However, the fecal androgens peaks occurred later than urinary and salivary androgens peaks. These intervals are correlated with the long digesta passage time in this species. The salivary and urinary peaks were very close, probably with few hours of interval. These results show that androgens concentrations in saliva, urine or feces samples reflect reliably physiological events and are powerful tool for reproductive monitoring of Amazonian manatees.

Os objetivos deste estudo foram determinar os níveis hormonais urinários e salivares durante o ciclo estral em peixes-bois da Amazônia, e verificar a existência de variação nos padrões dos hormônios reprodutivos presentes na saliva e na urina durante dois trimestres diferentes do ano. Foram utilizados 7 animais (4 machos e 3 fêmeas) adultos, alojados no LMA/INPA. As coletas ocorreram durante dois anos em dois trimestres diferentes do ano (Trimestre I e Trimestre II), onde, durante 12 semanas de cada trimestre foram colhidas amostras de saliva dos machos e urina e saliva das fêmeas. A testosterona salivar nos machos foi dosada por radioimunoensaio usando conjunto diagnóstico comercial. Os estrógenos, progestinas e LH urinários e progesterona e estradiol salivares nas fêmeas foram dosados por enzimaimunoensaio. O ciclo estral foi estimado em 39,67&plusmn;1,15 dias e 44&plusmn;2,00 dias para as matrizes urinária e salivar, respectivamente, com a presença peculiar de dois picos de estrógenos concomitantes com picos de LH urinário antes da elevação das progestinas, característica anteriormente observada somente para elefantes. Nos machos, a testosterona salivar apresentou um pico no final do Trimestre II (35,91&plusmn;5,64 pg/mL; P<0,05), sugerindo uma antecipação dos machos de T. inunguis à época reprodutiva das fêmeas. Para as fêmeas, os esteroides urinários e salivares e o LH urinário apresentaram valores significativamente maiores na maioria dos meses do Trimestre I quando comparado ao Trimestre II (P<0,05), e as fêmeas, em grande parte, apresentaram padrão hormonal cíclico no Trimestre I e padrão acíclico no Trimestre II, sugerindo, assim, a existência de sazonalidade reprodutiva na espécie mesmo em condições de cativeiro. Desta forma, foi possível concluir que as características peculiares do ciclo estral do peixe-boi da Amazônia podem ter importância biológica na estratégia reprodutiva da espécie, como ocorrem em elefantes; que aparentemente T. inunguis apresenta sazonalidade reprodutiva, e esta seria sincronizada por outro fator ambiental que não a disponibilidade de alimento; e que é possível utilizar tanto a matriz urinária quanto a salivar no monitoramento endócrino-reprodutivo de peixe-boi da Amazônia, sendo ferramentas importantes para estudos da biologia reprodutiva da espécie. / The aims of this study were to define urinary and salivary hormone levels during estrous cycle in Amazonian manatees; and to verify the existence of differences on urinary and salivary reproductive hormones during two different trimesters of the year. Seven adult animals (4 males and 3 females), kept at LMA/INPA were analyzed. Salivary samples from males and urinary and salivary samples from females were collected during 12 weeks in two different trimesters, during two consecutive years. Salivary testosterone from males was measured by radioimmunoassay kit. Urinary estrogens progestins and LH and salivary estradiol and progesterone from females were measured by enzyme immunoassay. The estrous cycle showed duration of 39.67&plusmn;1.15 days and 44.00&plusmn;2.00 days for urinary and salivary matrices, respectively, and showed two estrogens peaks accompanied by peaks of urinary LH before the rise of progestins. This hormonal pattern was previously reported only in elephants. The males showed a salivary testosterone peak at the end of Trimester II (35.91&plusmn;5.64 pg/ml; P<0.05), this fact suggest a male anticipation to the female reproductive season. The females showed high values of urinary and salivary steroids and urinary LH during most of months of the Trimester I when compared with the Trimester II (P<0.05), and the females usually showed hormonal cyclic pattern during Trimester I and acyclic pattern during Trimester II, suggesting the existence of reproductive seasonality even in captive conditions. Therefore, I conclude that the peculiar characteristics of Amazonian manatees estrous cycle may have biological importance for mating strategies, as in elephants; T. inunguis apparently shows reproductive seasonality, and it is not synchronized by food availability; it is possible to use as saliva as urine to monitor the reproductive physiology of Amazonian manatee, being important tools in studies about reproductive biology for this species.

O objetivo deste estudo foi verificar a viabilidade da dosagem de andrógenos em amostras de saliva, secreção lacrimal, urina e fezes de peixe-boi da Amazônia realizando um desafio hormonal. Dois peixes-bois amazônicos adultos machos (A-1 e A-2) foram submetidos a um protocolo de experimentação de doze dias (D-1 a D10). No D0 os animais receberam uma injeção intramuscular de GnRH exógeno. Amostras de saliva, secreção lacrimal, urina e fezes foram coletadas diariamente (entre 08h00 e 09h00) e mantidas a -20°C até o ensaio. As amostras de fezes foram liofilizadas, extraídas com metanol 80% e diluídas em tampão antes do radioimunoensaio (RIE). As amostras de urina sofreram hidrólise ácida e foram diluídas em soro bovino depletado. As amostras de saliva e secreção lacrimal foram dosadas sem etapa de extração, porém, o ensaio foi adaptado para aumentar a sensibilidade do teste. Os ensaios hormonais foram realizados utilizando um conjunto comercial de RIE para testosterona total. Um pico de andrógenos (mediana+2DI) somente foi observado nas amostras de saliva, urina e fezes de ambos os animais. Porém, os picos de andrógenos fecais ocorreram depois (cinco dias) dos picos de andrógenos urinários e salivares. Este intervalo está correlacionado com o longo tempo de passagem da digesta pelo trato gastrointestinal na espécie. Os picos salivares e urinários ocorreram muito próximos, provavelmente com poucas horas de intervalo. Esses resultados demonstram que as concentrações de andrógenos em amostras de saliva, urina ou fezes refletem consistentemente os eventos fisiológicos e são ferramentas de grande utilidade no monitoramento reprodutivo de peixes-bois da Amazônia. / The aim of this study was to verify the viability of androgens measurement in saliva, lacrimal secretion, urine and fecal samples of Amazonian manatee by carrying out hormonal challenge. Two adult male manatees (A-1 and A-2) were submitted to an experimentation protocol of twelve days (D-1 to D10). On D0 the animals received an intramuscular injection of GnRH-analogue. Salivary, lacrimal, urinary and fecal samples were collected daily (between 08h00 and 09h00) and frozen at -20°C until assayed. Fecal samples were lyophilized, extracted with 80% methanol and diluted in buffer before the radioimmunoassay (RIA). Urine samples underwent acid hydrolysis and diluted in depleted bovine serum. Salivary and lacrimal samples were assayed without extraction step, but, the assay was adapted to improve the sensibility. Hormonal assays were carried out with a commercial testosterone RIA kit. An androgen peak (>median+2IQR) was observed only in salivary, urinary and fecal samples of both animals. However, the fecal androgens peaks occurred later than urinary and salivary androgens peaks. These intervals are correlated with the long digesta passage time in this species. The salivary and urinary peaks were very close, probably with few hours of interval. These results show that androgens concentrations in saliva, urine or feces samples reflect reliably physiological events and are powerful tool for reproductive monitoring of Amazonian manatees.

Florida manatees (Trichechus manatus latirostris) live in the shallow, often turbid inland and coastal waters of the southeastern United States. Since their vision is poor (Bauer et al., 2003), other senses probably guide orientation. Previous studies have found that manatees can hear over 40 kHz (Gerstein et al., 1999) and have the capacity for rapid auditory temporal processing (Mann et al., 2005). However, it is not known if manatees have the ability to localize underwater sounds. Two Florida manatees were trained to identify underwater sound source locations using a four-choice discrimination paradigm. Three broad-band signals ( 0.2 - 20, 6 - 20, and 0.2 2kHz) were tested at four durations (3,000, 1,000, 500, and 200ms) and two tonal signals (4 and 16kHz) were tested with a 3,000ms duration. A total of 1,008 test trials were analyzed per subject. Both manatees learned the task easily, and could localize all of the test signals at a performance rate well above the 25% chance level. Within all of the broad-band conditions, performance accuracy ranged from 93% - 79% for Buffett, and 93% - 51% for Hugh. Broad-band signal duration did not have an effect on performance accuracy with Buffett who ranged from 89% to 87%, but did with Hugh who ranged from 87% - 58%. Broad-band frequency type did not have an effect on performance accuracy with Buffett who averaged 90%, 86%, and 89%, but may have with Hugh who averaged from 76%, 68%, and 65% at the 0.2 20, 6 20, and 0.2 2 kHz conditions. Both animals performed above chance levels with the pure tone signals, but at a much lower accuracy rate with Hugh at 49% and 32% and Buffett at 44% and 33% with the 4 kHz and 16 kHz conditions. Results from this experiment provide information about the manatees ability to localize different types of sounds in a controlled environment. This knowledge is important for understanding how manatees detect and localize noise generated from conspecifics and boat engines and contributes to making competent conservation management decisions about these endangered marine mammals.

Submitted by (edna.saturno@ufrpe.br) on 2016-10-13T12:47:32Z No. of bitstreams: 1 Joao Carlos Gomes Borges.pdf: 2198512 bytes, checksum: de1f2ccf01e9496dcc35acd9a6874feb (MD5) / Made available in DSpace on 2016-10-13T12:47:32Z (GMT). No. of bitstreams: 1 Joao Carlos Gomes Borges.pdf: 2198512 bytes, checksum: de1f2ccf01e9496dcc35acd9a6874feb (MD5) Previous issue date: 2007-02-12 / The aim of the present study was to verify the occurrence of oocysts of Cryptosporidium spp. in Antillean manatees (Trichechus manatus) and Amazonian manatees (Trichechus inunguis) from Brazil, and also evaluate the hydric transmission of the coccidium and the influence between this pathogen and the staff involved directly in captive animal care and management. Fecal samples were collected from 34 Antillean manatees, 12 Amazonian manatees and also 21 employees involved in the management activities. Water samples, used to the manatees consumption and management were also collected. The fecal samples were processed by sedimentation in formol-ether and Kinyoun technique. Before the water samples were processed, this was submitted to a filtration process in membrane (diameter of 47 mm, porosity of 3 μm) under negative pressure. At the end of the analyzing operations, the positive samples were submitted to the Direct Immunoflorescence Test. The results obtained by light and fluorescence microscopy analysis showed alcohol-acid-resistant structures compatible with oocyst of Cryptosporidium spp. in 25% (34/136) and 4,30% (05/115) of thematerial proceeding from Antillean manatees and Amazonian manatees, respectively; 23,80% (10/42) of the material obtained from the employees; and 66,67% (04/06) of the water samples analyzed. The results showed the possibility of Antillean manatees and Amazonian manatees could become infected with Cryptosporidium spp., and participate in the epidemiology of this waterborne disease by eliminate of viable oocyst in water environment wich can infect man and a wide range of domestic and wild animals. The hand of the frequency of Cryptosporidium spp. in staff involved directly in captive animal care and management could be the way to prevent the infections the manatee’s species. / O objetivo neste trabalho foi verificar a ocorrência de oocistos de Cryptosporidium spp. em peixes-boi marinhos (Trichechus manatus) e peixes-boi amazônicos (Trichechus inunguis) no Brasil, além de avaliar a transmissão hídrica deste coccídio e a sua influência com os funcionários diretamente envolvidos no manejo dos animais mantidos em cativeiro. Para tanto foram coletadas amostras fecais provenientes de 34 peixes-boi marinhos, 12 peixes-boi amazônicos e 21 funcionários envolvidos em atividades de manejo, bem como amostras de água superficial destinada a manutenção e consumo dos animais. As amostras fecais foram processadas através da sedimentação pelo formoléter e coradas pela técnica de Kinyoun. No que concerne as amostras de água, estas foram submetidas ao processo de filtração em membranas (47 mm de diâmetro e 3 μm de porosidade) sob pressão negativa. No final das análises, as amostras positivas foram submetidas ao Teste de Imunofluorescência Direta. Os resultados obtidos namicroscopia de luz e fluorescência revelaram oocistos de Cryptosporidium spp. em 25% (34/136) e 4,30% (05/115), sendo inerentes respectivamente aos peixes-boi marinhos e amazônicos, 23,80% (10/42) do material proveniente dos funcionários e 66,67% (04/06) das análises de água. Os resultados finais apontam a possibilidade dos peixes-boi marinhos e peixes-boi amazônicos infectarem-se com Cryptosporidium spp., e participar da epidemiologia das doenças veiculadas pela água, através da eliminação de oocistos viáveis ao ambiente que podem infectar o homem e um grande número de animais domésticos e silvestres. O controle da freqüência de infecções de Cryptosporidium spp. nos funcionários diretamente envolvidos no manejo e cuidados dos animais mantidos em cativeiro pode ser um dos caminhos para prevenir a infecção nas espécies de peixes-boi.

Os objetivos deste estudo foram determinar os níveis hormonais urinários e salivares durante o ciclo estral em peixes-bois da Amazônia, e verificar a existência de variação nos padrões dos hormônios reprodutivos presentes na saliva e na urina durante dois trimestres diferentes do ano. Foram utilizados 7 animais (4 machos e 3 fêmeas) adultos, alojados no LMA/INPA. As coletas ocorreram durante dois anos em dois trimestres diferentes do ano (Trimestre I e Trimestre II), onde, durante 12 semanas de cada trimestre foram colhidas amostras de saliva dos machos e urina e saliva das fêmeas. A testosterona salivar nos machos foi dosada por radioimunoensaio usando conjunto diagnóstico comercial. Os estrógenos, progestinas e LH urinários e progesterona e estradiol salivares nas fêmeas foram dosados por enzimaimunoensaio. O ciclo estral foi estimado em 39,67&plusmn;1,15 dias e 44&plusmn;2,00 dias para as matrizes urinária e salivar, respectivamente, com a presença peculiar de dois picos de estrógenos concomitantes com picos de LH urinário antes da elevação das progestinas, característica anteriormente observada somente para elefantes. Nos machos, a testosterona salivar apresentou um pico no final do Trimestre II (35,91&plusmn;5,64 pg/mL; P<0,05), sugerindo uma antecipação dos machos de T. inunguis à época reprodutiva das fêmeas. Para as fêmeas, os esteroides urinários e salivares e o LH urinário apresentaram valores significativamente maiores na maioria dos meses do Trimestre I quando comparado ao Trimestre II (P<0,05), e as fêmeas, em grande parte, apresentaram padrão hormonal cíclico no Trimestre I e padrão acíclico no Trimestre II, sugerindo, assim, a existência de sazonalidade reprodutiva na espécie mesmo em condições de cativeiro. Desta forma, foi possível concluir que as características peculiares do ciclo estral do peixe-boi da Amazônia podem ter importância biológica na estratégia reprodutiva da espécie, como ocorrem em elefantes; que aparentemente T. inunguis apresenta sazonalidade reprodutiva, e esta seria sincronizada por outro fator ambiental que não a disponibilidade de alimento; e que é possível utilizar tanto a matriz urinária quanto a salivar no monitoramento endócrino-reprodutivo de peixe-boi da Amazônia, sendo ferramentas importantes para estudos da biologia reprodutiva da espécie. / The aims of this study were to define urinary and salivary hormone levels during estrous cycle in Amazonian manatees; and to verify the existence of differences on urinary and salivary reproductive hormones during two different trimesters of the year. Seven adult animals (4 males and 3 females), kept at LMA/INPA were analyzed. Salivary samples from males and urinary and salivary samples from females were collected during 12 weeks in two different trimesters, during two consecutive years. Salivary testosterone from males was measured by radioimmunoassay kit. Urinary estrogens progestins and LH and salivary estradiol and progesterone from females were measured by enzyme immunoassay. The estrous cycle showed duration of 39.67&plusmn;1.15 days and 44.00&plusmn;2.00 days for urinary and salivary matrices, respectively, and showed two estrogens peaks accompanied by peaks of urinary LH before the rise of progestins. This hormonal pattern was previously reported only in elephants. The males showed a salivary testosterone peak at the end of Trimester II (35.91&plusmn;5.64 pg/ml; P<0.05), this fact suggest a male anticipation to the female reproductive season. The females showed high values of urinary and salivary steroids and urinary LH during most of months of the Trimester I when compared with the Trimester II (P<0.05), and the females usually showed hormonal cyclic pattern during Trimester I and acyclic pattern during Trimester II, suggesting the existence of reproductive seasonality even in captive conditions. Therefore, I conclude that the peculiar characteristics of Amazonian manatees estrous cycle may have biological importance for mating strategies, as in elephants; T. inunguis apparently shows reproductive seasonality, and it is not synchronized by food availability; it is possible to use as saliva as urine to monitor the reproductive physiology of Amazonian manatee, being important tools in studies about reproductive biology for this species.

Submitted by Amanda Silva (amanda.osilva2@ufpe.br) on 2015-03-05T13:59:07Z No. of bitstreams: 2 TESE Fábia de Oliveira Luna.pdf: 6070926 bytes, checksum: 5c0233b17f6af6c3a2f420f1d6eb3248 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-03-05T13:59:07Z (GMT). No. of bitstreams: 2 TESE Fábia de Oliveira Luna.pdf: 6070926 bytes, checksum: 5c0233b17f6af6c3a2f420f1d6eb3248 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013 / The West Indian manatee (Trichechus manatus) and the Amazonian manatee (Trichechus inunguis) belong to the mammalian Order Sirenia, classified respectively as critically endangered and as vulnerable in Brazil. Both species have been hunted commercially since the sixteenth century, resulting in a drastic reduction or elimination of manatees in some places. This hunting pressure may have caused isolation of the populations resulting in geographic genetic isolation, with a reduction of their genetic diversity. The objective of this study is to understand the genetic diversity and population structure of the West Indian manatee (T. manatus) in Brazil; verify the existence of hybridization between the manatees T. manatus and T. inungus; define the degree of relatedness of individuals in captivity; and provide strategies for the conservation of the species. In northeastern Brazil, a large number of dependent calves strand on beaches. Many of these calves have been rescued alive and rehabilitated for future release. Previously, the identification of release sites did not take into account the genetic issues. Through this study, it has been possible to identify suitable release sites based on phylogenetic traits of the manatees. The genetic structure of the population was studied using nuclear DNA in the program STRUCTURE, and comparing FST and RST values. On the Northeast coast one population was identified, which is subdivided in two subpopulations. A different genetic population was found in the Northwest of the Amazon River estuary (extreme north of Brazil, Guyana and Venezuelan). Manatees from the Estuary of the Amazon River grouped with Amazonian manatees from the Santarém region to form a third population. A total of four manatee Management Units were identified along the Brazilian coast, which should be treated separately for management and conservation needs. Using a combination of molecular markers (mtDNA and nDNA) and cytogenetic analyses the existence of hybridization between T. manatus and T inunguis was confirmed, although this does not appear to be a frequent occurrence. Some of the dependent calves rescued, and other individuals transferred from irregular captive conditions to the CMA/ICMBio, were kept together and breeding occurred. Maintaining related manatees together can lead to inbreeding. It was possible 29 to establish the degree of relatedness between individuals kept at CMA/ICMBio and verify the extistence of inbreeding. To avoid inbreeding, hybridization, and overpopulation in captivity, it is necessary that males and females be separated immediately, thereby preventing reproduction ex-situ. The present study allows us to establish actions for the conservation of the West Indian manatee. Actions that should be prioritized include: protection and restoration of habitat with the creation and implementation of specific protected areas; creation and maintenance of travel corridors that allow gene flow; reduction and elimination of anthropogenic pressures on the species; stopping pointless reproduction in captivity; designing further studies in the north of Brazil to better understand the hybridization and implement the action plan.

The Florida manatee (Trichechus manatus latirostris), a subspecies of the West Indian manatee, is one of the most endangered marine mammals in United States waters. The Florida manatee is the only manatee that ranges into subtropical and temperate regions. During the winter months manatees adopt a “refuging strategy” where they aggregate at warm-water sources immediately following decreases in the ambient water temperature to below 20° C (68° F) in order to avoid cold stress syndrome (CSS). During the winter manatees aggregate in warm water refuges, including natural warm water springs and the effluent discharges of power plants. The purpose of this study was to determine the number of manatees that aggregate and utilize the waters of the Florida Power and Light (FPL) Plant in Port Everglades (PPE), Florida, its effluent canal, and the surrounding Intracoastal Waterway during the winter months. This study documents the importance of Port Everglades as a wintering refuge for the Florida manatee. This study analyzed the inverse relationship between the number of manatees present at a warm water effluent and water temperature. In this study data were collected over five manatee winter seasons (between 15 November and 31 March) from 1999-2004, from both boat-based and land-based surveys monitoring the presence of manatees in the effluent canal from the FPL electricity generating facility in Port Everglades, FL and the Intracoastal Waterway. Findings indicated that there was an inverse relationship between the number of manatees present and water temperature, where more manatees were present in cooler months, and fewer in warmer months. This study also analyzed the parameters of Catch per Unit Effort (CPUE) as well as heating degree-days and their effect on and relationship to the number of manatees present. The higher the heating degree-days number, the more severe, or cold, winter this indicates. The year with the highest heating degree-days, 24.98, was the 2002-2003 season, which was also the season with the highest number of manatees observed, 393, and the highest CPUE, 10.62 manatees/day.