Gestational follow-up in ewes - placentome size and fecal progesterone metabolite levels

Background: Transabdominal ultrasonography in sheep allows real-time diagnosis and monitoring of pregnancy as well as measurement of the size of placentomes, which are structures that connect the fetal and maternal portions, that increase as pregnancy progresses. Progesterone is involved in the maintenance of gestation, with high levels noted during pregnancy. In this context, it is hypothesized that measurements of placentome and progesterone could help in monitoring pregnancy in sheep. Therefore, the main objectives of this study included evaluating whether placentome size and fecal progesterone levels are associated with gestational age in ewes and whether measurement of the placentome and progesterone could be used as an estimate of gestational age. Materials, Methods & Results: A total of 63 pregnant adult (between 2 and 5 years of age) Santa Inês ewes were monitored in the city of Boa Vista, Roraima. All ewes were kept in the paddock with Panicum maximun cv. tanzania, mineral salt, and water ad libitum, supplemented with 200 g/animal/day of soybean residue. Ewes were subjected to ultrasonography every 21 days from day 42 of pregancy until term to monitor pregnancy. Ultrasonography was performed using a linear probe to measure the placentome, including length × width in centimeters. Also every 21 days, fecal samples were collected for measurement of progesterone level. Feces were collected directly from the rectal ampulla, stored individually in plastic bags, and frozen at -20ºC until processing. In the laboratory, these feces samples were freeze-dried and subjected to hormonal extraction with 80% methanol. Then, the levels of fecal progesterone metabolites were measured by enzyme immunoassay. Data was tabulated and submitted to statistical analyses, including descriptive stastistic, correlation, KruskalWallis test and Tukey's test. The levels of fecal progesterone metabolites increased significantly between the 3rd and 4th months of pregnancy, with a decrease close to delivery. Placentome size increased significantly between the 2nd and 3rd months of pregnancy. Placentome measurements did not correlate with progesterone levels and showed a weak correlation with gestational age. Therefore, we concluded that the measurements of placentome size and a single evaluation of fecal progesterone metabolites were not considered efficient methods for estimating gestational age. Discussion: Measurement of progesterone level in fecal samples is a non-invasive hormone monitoring method that provides values like that of blood levels and is less stressful for the animals at the time of sample collection. The wide physiological variation makes it difficult or even useless to use fecal progesterone, when is measured only once per animal, as an indicator of the presence of pregnancy. Despite this limitation, serial sampling is useful for monitoring of hormones during the pregnancy period and is an important tool for research purposes. Placentome size measurements correlated weakly with gestational age and did not correlate with progesterone level. Thus, the presence of placentomes is indicative of the presence of pregnancy but was considered inefficient for determining gestational age in sheep.

Evaluation of the use of dry blood spots for progesterone determination in ewes / Avaliação do uso de manchas secas de sangue para dosagem de progesterona em ovinos

The use of dried blood spots on filter paper has been shown to be a practical alternative in several studies with humans and animals, enabling a simple means of storing and transporting viable blood samples for various laboratory analyses. However, its applicability in the measurement of progesterone in animals is scarce. Thus, the objective of this study was to evaluate the feasibility of using dried blood spots for the measurement of progesterone in sheep. In total, 38 blood samples from 6 sheep were dripped onto filter paper, and the remainder of each sample was separated into serum. The progesterone levels in the serum samples and in the dry drops were measured by enzyme immunoassay and subsequently correlated. The levels of progesterone in the serum and dry spots showed a high correlation between the matrices (R2 = 0.9694). In conclusion, this study demonstrated the feasibility of using samples of dried sheep blood spots for the measurement of progesterone, and the storage and transport technique can be applied in the field.

O uso de manchas secas de sangue em papel filtro tem se demonstrado uma alternativa prática em vários estudos com humanos e animais, possibilitando um meio simples de armazenamento e transporte de amostras de sangue viáveis para várias análises laboratoriais. Entretanto, sua aplicabilidade na dosagem de progesterona em animais é escassa. Deste modo, o objetivo deste estudo foi avaliar a viabilidade do uso de manchas secas de sangue para a dosagem de progesterona em ovinos. Assim, 38 amostras de sangue de 6 ovelhas foram gotejadas em papel filtro e o restante de cada amostra foi separado o soro. Os níveis de progesterona nas amostras de soro e nas gotas secas foram mensurados por enzimaimunoensaio e posteriormente correlacionados. Os níveis de progesterona no soro e nas manchas secas apresentaram uma alta correlação entre as matrizes (R2=0,9694). Em conclusão, este estudo demonstrou a viabilidade do uso de amostras de manchas secas de sangue de ovino para a dosagem de progesterona, podendo a técnica de armazenamento e transporte ser aplicada a campo.

Utilização de medidas osteológicas para estimativa de comprimento corporal em peixe-boi da Amazônia / Using osteological measurements to estimate body length in Amazonian manatees

O comprimento corporal é um importante parâmetro em estudos morfológicos, ecológicos e comportamentais de uma espécie e contribui para o entendimento da condição corporal de um indivíduo. Este parâmetro é essencial para estratégias de conservação e manejo, informando estudos que avaliam taxas de crescimento, maturidade física e classificação dos indivíduos em grupos de idade, promovendo uma melhor precisão aos parâmetros biológicos de uma espécie. O objetivo desse trabalho foi identificar preditores de comprimento corporal para o peixe-boi da Amazônia (Trichechus inunguis) utilizando caracteres métricos de material osteológico. Onze medidas lineares do crânio, mandíbula, escápula e úmero foram coletadas de 41 esqueletos de peixe-boi da Amazônia de diferentes classes de idade (filhote, juvenil e adulto). Os dados foram analisados por regressão linear simples. O comprimento côndilobasal foi o melhor preditor de comprimento corporal para a espécie (R = 0,943), entretanto, todos os ossos avaliados mostraram pelo menos um caractere com capacidade de predizer o comprimento corporal (R2 > 0,9). Os resultados deste estudo são úteis para inferir o comprimento corporal de peixes-boi da Amazônia a partir de ossos depositados em museus e coleções biológicas, expandindo o potencial informativo destes materiais. (AU)

Using osteological measurements to estimate body length in Amazonian manatees / Utilização de medidas osteológicas para estimativa de comprimento corporal em peixe-boi da Amazônia

Body length is an important parameter in morphological, ecological and behavioral studies of a species and contributes to the understanding of the body condition of individuals. This parameter is essential for conservation and management strategies by informing studies evaluating growth rates, physical maturity and classification of individuals into age groups, promoting better accuracy for the biological parameters of the species. The aim of this study was to identify predictors of body length for Amazonian manatees (Trichechus inunguis) using metric characters of osteological materials. Eleven linear measurements of skull, jaw, scapulae and humeri were collected from 41 Amazonian manatee skeletons of different age classes (calf, juvenile and adult). Data were analyzed by simple linear regression. The condylobasal length was the best predictor of body length for the species (R = 0.943), however, all bones evaluated showed at least one measurement with the capacity to predict body size (R2 > 0.9). Results of this study are useful for inferring body length of Amazonian manatees using bones deposited in museums and biological collections, expanding the informative potential of these materials.(AU)

O comprimento corporal é um importante parâmetro em estudos morfológicos, ecológicos e comportamentais de uma espécie e contribui para o entendimento da condição corporal de um indivíduo. Este parâmetro é essencial para estratégias de conservação e manejo, informando estudos que avaliam taxas de crescimento, maturidade física e classificação dos indivíduos em grupos de idade, promovendo uma melhor precisão aos parâmetros biológicos de uma espécie. O objetivo desse trabalho foi identificar preditores de comprimento corporal para o peixe-boi da Amazônia (Trichechus inunguis) utilizando caracteres métricos de material osteológico. Onze medidas lineares do crânio, mandíbula, escápula e úmero foram coletadas de 41 esqueletos de peixe-boi da Amazônia de diferentes classes de idade (filhote, juvenil e adulto). Os dados foram analisados por regressão linear simples. O comprimento côndilobasal foi o melhor preditor de comprimento corporal para a espécie (R = 0,943), entretanto, todos os ossos avaliados mostraram pelo menos um caractere com capacidade de predizer o comprimento corporal (R2 > 0,9). Os resultados deste estudo são úteis para inferir o comprimento corporal de peixes-boi da Amazônia a partir de ossos depositados em museus e coleções biológicas, expandindo o potencial informativo destes materiais.(AU)

Análise histológica do segmento sexual renal de Suaçuboia / Histological analysis of the renal sexual segment of Suaçuboia

Corallus hortulanus is a snake with wide geographical distribution and valid ecological importance. However, information about their reproductive biology is scarce and most of the data obtained is from snakes in captivity. Thus, the work aimed to describe the histological aspects of the sexual segment of the kidney (SSK), a specialized part of the kidneys. Therefore, histological analysis and measurement of SSK were performed from 12 specimens of Corallus hortulanus collected in different months of the year. The histological aspects of the SSK observed were similar to those described for other species of snakes and the tubular segments had an average diameter of 76.8 ± 25.6 µm. However, a great variation was observed in the morphological aspects (hypertrophy and presence and disposition of granules) and in the diameter of the tubular segments among the individuals analyzed, apparently related to reproductive seasonality. The results obtained in this study contribute to a greater understanding of the reproductive aspects of C. hortulanus and the possible seasonal variations should be better investigated.

Análise histológica do segmento sexual renal de Suaçuboia / Histological analysis of the renal sexual segment of Suaçuboia

Corallus hortulanus is a snake with wide geographical distribution and valid ecological importance. However, information about their reproductive biology is scarce and most of the data obtained is from snakes in captivity. Thus, the work aimed to describe the histological aspects of the sexual segment of the kidney (SSK), a specialized part of the kidneys. Therefore, histological analysis and measurement of SSK were performed from 12 specimens of Corallus hortulanus collected in different months of the year. The histological aspects of the SSK observed were similar to those described for other species of snakes and the tubular segments had an average diameter of 76.8 ± 25.6 µm. However, a great variation was observed in the morphological aspects (hypertrophy and presence and disposition of granules) and in the diameter of the tubular segments among the individuals analyzed, apparently related to reproductive seasonality. The results obtained in this study contribute to a greater understanding of the reproductive aspects of C. hortulanus and the possible seasonal variations should be better investigated.(AU)

Monitoring of progesterone and estrone fecal metabolites throughout gestation in ewes / Avaliação dos metabólitos fecais de progesterona e estrona em ovelhas durante a gestação

The aim of this study was to monitor the progesterone and fecal estrone metabolites throughout gestation in ewes correlating with the serum levels of these steroid hormones. Therefore, fecal and serum samples were collected from 5 weeks before mating and gestation until two weeks postparturition. Serum levels of progesterone and estrone and their fecal metabolites were measured by enzyme immunoassay. Serum and fecal hormonal patterns showed a significant correlation for both hormones (R = 0.8572, P < 0.001 for progesterone and R = 0.5893, P < 0.001 for estrone). The fecal progesterone metabolite levels showed significant increasing values among the three thirds of pregnancies, consistent with the serum levels and with the literature. Additionally, the prepartum peak of estrone in the fecal matrix was identified but without observation in the serum matrix due to the blood collection interval used. Therefore, this study demonstrated the viability of progesterone and estrone monitoring throughout gestation using fecal samples, making noninvasive longitudinal endocrine monitoring throughout gestation possible in this species.(AU)

O objetivo deste estudo foi monitorar os níveis de metabólitos fecais de progesterona e estrona ao longo da gestação em ovelhas, correlacionando-os com os níveis séricos desses hormônios esteroides. Assim, amostras de fezes e sangue foram colhidas de cinco ovelhas no período pré-cobertura e durante a gestação, até duas semanas após o parto. Os níveis séricos de progesterona e estrona e de seus metabólitos fecais foram mensurados por enzimaimunoensaio. Os perfis hormonais séricos e fecais apresentaram correlação positiva significativa para os dois hormônios (R = 0,8572, P < 0,001 para progesterona e R = 0,5893, P < 0,001 para estrona). Os níveis de metabólitos fecais de progesterona apresentaram valores significativamente crescente entre os terços da gestação, corroborando com os níveis séricos e com os relatos da literatura. Adicionalmente, foi possível evidenciar o pico pré-parto de estrona na matriz fecal, porém sem registro na matriz sérica, provavelmente devido ao intervalo de coletas aplicado. Deste modo, este estudo demonstrou a viabilidade do monitoramento dos níveis de progesterona e estrona durante a gestação em ovinos utilizando amostras fecais, possibilitando monitoramento endócrino longitudinal não invasivo durante a gestação nessa espécie.(AU)

Monitoring of progesterone and estrone fecal metabolites throughout gestation in ewes / Avaliação dos metabólitos fecais de progesterona e estrona em ovelhas durante a gestação

The aim of this study was to monitor the progesterone and fecal estrone metabolites throughout gestation in ewes correlating with the serum levels of these steroid hormones. Therefore, fecal and serum samples were collected from 5 weeks before mating and gestation until two weeks postparturition. Serum levels of progesterone and estrone and their fecal metabolites were measured by enzyme immunoassay. Serum and fecal hormonal patterns showed a significant correlation for both hormones (R = 0.8572, P < 0.001 for progesterone and R = 0.5893, P < 0.001 for estrone). The fecal progesterone metabolite levels showed significant increasing values among the three thirds of pregnancies, consistent with the serum levels and with the literature. Additionally, the prepartum peak of estrone in the fecal matrix was identified but without observation in the serum matrix due to the blood collection interval used. Therefore, this study demonstrated the viability of progesterone and estrone monitoring throughout gestation using fecal samples, making noninvasive longitudinal endocrine monitoring throughout gestation possible in this species.

O objetivo deste estudo foi monitorar os níveis de metabólitos fecais de progesterona e estrona ao longo da gestação em ovelhas, correlacionando-os com os níveis séricos desses hormônios esteroides. Assim, amostras de fezes e sangue foram colhidas de cinco ovelhas no período pré-cobertura e durante a gestação, até duas semanas após o parto. Os níveis séricos de progesterona e estrona e de seus metabólitos fecais foram mensurados por enzimaimunoensaio. Os perfis hormonais séricos e fecais apresentaram correlação positiva significativa para os dois hormônios (R = 0,8572, P < 0,001 para progesterona e R = 0,5893, P < 0,001 para estrona). Os níveis de metabólitos fecais de progesterona apresentaram valores significativamente crescente entre os terços da gestação, corroborando com os níveis séricos e com os relatos da literatura. Adicionalmente, foi possível evidenciar o pico pré-parto de estrona na matriz fecal, porém sem registro na matriz sérica, provavelmente devido ao intervalo de coletas aplicado. Deste modo, este estudo demonstrou a viabilidade do monitoramento dos níveis de progesterona e estrona durante a gestação em ovinos utilizando amostras fecais, possibilitando monitoramento endócrino longitudinal não invasivo durante a gestação nessa espécie.

Ferramentas da reprodução na conservação dos mamíferos aquáticos da Amazônia / Reproductive tools in the conservation of Amazonian aquatic mammals

O peixe-boi da Amazônia (Trichechus inunguis) e o boto-vermelho (Inia geoffrensis) são os mamíferos aquáticos da Amazônia com maior risco de extinção. Deste modo, esta revisão apresenta os avanços no conhecimento sobre a biologia reprodutiva destas espécies, bem como as lacunas no conhecimento existentes, podendo estas ser, de grande importância, para futura aplicação de biotecnologias como aliada na conservação destas espécies.

The Amazonian manatee (Trichechus inunguis) and the Amazon river dolphin (Inia geoffrensis) are the most threatened aquatic mammals of the Amazon. Therefore, this review highlights the advances on knowledge about reproductive biology of these species, as well as the information gaps, which is of great importance to the future use of biotechnologies as a tool for the conservation of those species.

Ferramentas da reprodução na conservação dos mamíferos aquáticos da Amazônia / Reproductive tools in the conservation of Amazonian aquatic mammals

O peixe-boi da Amazônia (Trichechus inunguis) e o boto-vermelho (Inia geoffrensis) são os mamíferos aquáticos da Amazônia com maior risco de extinção. Deste modo, esta revisão apresenta os avanços no conhecimento sobre a biologia reprodutiva destas espécies, bem como as lacunas no conhecimento existentes, podendo estas ser, de grande importância, para futura aplicação de biotecnologias como aliada na conservação destas espécies.(AU)

The Amazonian manatee (Trichechus inunguis) and the Amazon river dolphin (Inia geoffrensis) are the most threatened aquatic mammals of the Amazon. Therefore, this review highlights the advances on knowledge about reproductive biology of these species, as well as the information gaps, which is of great importance to the future use of biotechnologies as a tool for the conservation of those species.(AU)

Changes in the blood parameters of the Amazonian manatee (Trichechus inunguis) after long-distance transportation / Alterações nos padrões sanguíneos do peixe-boi da Amazônia (Trichechus inunguis) após transporte de longa distância

In this study we report the hematological, biochemical and hormonal parameters in a juvenile male Amazonian manatee measured before transport, immediately after transport, and during adaptation to a new facility. The animal was transported from Manaus, Amazonas State, Brazil, to São Paulo, São Paulo State, Brazil, (2,733 km) within 6 hours. Among all blood parameters analyzed, we observed obvious neutrophilia, lymphopenia, and increases in the neutrophil/lymphocyte ratio and serum glucose and aspartate aminotransferase (AST) levels, but these parameters subsequently returned to normal. These results suggest that transport and changes in the environment are temporary stressful events for Amazonian manatees. We, therefore, recommend monitoring the hematological and biochemical parameters before and after translocation to minimize the effects of handling stressors in this species.

Neste estudo, são relatados os parâmetros hematológicos, bioquímicos e hormonais em um peixe-boi da Amazônia macho jovem, medidos antes do transporte, imediatamente depois, e durante a sua adaptação a um novo recinto. O animal foi transportado de Manaus, Estado do Amazonas, Brasil, para São Paulo, Estado de São Paulo, Brasil, (2.733 km) durante 6h de viagem. Entre os parâmetros sanguíneos analisados, foi observada clara neutrofilia, linfopenia e aumento na relação neutrófilo/linfócito e nos níveis de glicose e aspartato aminotransferase (AST), mas estes parâmetros posteriormente voltaram aos valores normais. Estes resultados sugerem que o transporte e as mudanças no ambiente são eventos estressantes para o peixe-boi da Amazônia. Desta forma, é recomendável o monitoramento dos parâmetros hematológicos e bioquímicos antes e após a translocação para minimizar os efeitos de agentes estressores nesta espécie.

Fisiologia reprodutiva do peixe-boi da Amazônia (Trichechus inunguis) em cativeiro: ciclicidade ovariana e padrões hormonais em machos e fêmeas em dois trimestres diferentes do ano / Reproductive physiology of captive Amazonian manatee (Trichechus inuguis): ovarian cyclicity and hormonal patterns in males and females during two different trimesters of the year

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. [...] 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±1.15 days and 44.00±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±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

Morphological analysis of testes from post-hatch chicks submitted to temperature variation during incubation / Análise morfológica dos testículos de pintainhos recém-eclodidos submetidos à variação de temperatura durante a incubação

This study aimed to evaluate the effects of temperature variation during incubation on testicular morphology of post-hatch chicks. We utilized 60 eggs incubated under different temperatures: Group 1 37.5°C; Group 2 39.5°C; and Group 3 34°C. Chicks were weighted and the testes were histologically analyzed. All eggs from Group 3 showed embryonic death. There were no significant differences in body weight and testicular morphology between Groups 1 and 2; however, there was a difference in the diameter of the seminiferous tubules (p < 0.001). The increase in temperature to 39.5°C during incubation causes a decrease in diameter of the seminiferous tubules. (AU)

Este estudo objetivou avaliar os efeitos da variação de temperatura durante a incubação na morfologia testicular de pintainhos recém-eclodidos. Foram utilizados 60 ovos incubados em diferentes temperaturas: Grupo 1 37,5°C; Grupo 2 39,5°C; e Grupo 3 34°C. Os pintainhos foram pesados e seus testículos analisados histologicamente. Todos os ovos do Grupo 3 apresentaram morte embrionária. Não houve diferenças significativas no peso e na morfologia testicular entre os Grupos 1 e 2, porém, houve diferença no diâmetro dos túbulos seminíferos (p < 0,001). A elevação da temperatura para 39,5°C durante a incubação causa a redução do diâmetro dos túbulos seminíferos.(AU)

Morphological analysis of testes from post-hatch chicks submitted to temperature variation during incubation / Análise morfológica dos testículos de pintainhos recém-eclodidos submetidos à variação de temperatura durante a incubação

This study aimed to evaluate the effects of temperature variation during incubation on testicular morphology of post-hatch chicks. We utilized 60 eggs incubated under different temperatures: Group 1 ­ 37.5°C; Group 2 ­ 39.5°C; and Group 3 ­ 34°C. Chicks were weighted and the testes were histologically analyzed. All eggs from Group 3 showed embryonic death. There were no significant differences in body weight and testicular morphology between Groups 1 and 2; however, there was a difference in the diameter of the seminiferous tubules (p < 0.001). The increase in temperature to 39.5°C during incubation causes a decrease in diameter of the seminiferous tubules.

Este estudo objetivou avaliar os efeitos da variação de temperatura durante a incubação na morfologia testicular de pintainhos recém-eclodidos. Foram utilizados 60 ovos incubados em diferentes temperaturas: Grupo 1 ­ 37,5°C; Grupo 2 ­ 39,5°C; e Grupo 3 ­ 34°C. Os pintainhos foram pesados e seus testículos analisados histologicamente. Todos os ovos do Grupo 3 apresentaram morte embrionária. Não houve diferenças significativas no peso e na morfologia testicular entre os Grupos 1 e 2, porém, houve diferença no diâmetro dos túbulos seminíferos (p < 0,001). A elevação da temperatura para 39,5°C durante a incubação causa a redução do diâmetro dos túbulos seminíferos.

Noninvasive monitoring of androgens in male Amazonian manatee (Trichechus inunguis): biologic validation.

The Amazonian manatee (Trichechus inunguis) is endemic in the Amazonian basin and is the only exclusively fresh water sirenian. Historically hunted on a large scale, this species is now considered endangered, and studies on the reproductive physiology are critical for the improvement of reproductive management of captive and wild populations of manatees. The aim of this study was to verify the viability of androgen measurement in saliva, lacrimal, urine, and fecal samples of the Amazonian manatee by conducting a hormone challenge. Two adult male manatees (A-1 and A-2) were submitted to an experimentation protocol of 12 day (D1 to D10). On D0, the animals received an intramuscular injection of gonadotropin-releasing hormone (GnRH)-analogue. Salivary, lacrimal, urinary, and fecal samples were collected daily (between 0800 hours and 0900 hours) and frozen at -20 degrees 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 were diluted in depleted bovine serum. Salivary and lacrimal samples were assayed without the extraction step. Hormonal assays were conducted with a commercial testosterone RIA kit. An androgen peak (> median + 2 interquartile range [IQR]) was observed in all matrices of both animals, although it was less prominent in the lacrimal samples of A-2. However, the fecal androgen peak (A-1 peak = 293.78 ng/g dry feces, median [IQR] = 143.58 [32.38] ng/g dry feces; A-2 peak = 686.72 ng/g dry feces, median [IQR] = 243.82 [193.16] ng/g dry feces) occurred later than urinary (A-1 peak = 648.16 ng/mg creatinine [Cr], median [IQR] = 23.88 [30.44] ng/mg Cr; A-2 peak = 370.44 ng/mg Cr, median [IQR] = 113.87 [117.73] ng/mg Cr) and salivary (A-1 peak = 678.89 pg/ml, median [IQR] = 103.69 [119.86] pg/ml; A-2 peak = 733.71 pg/ml, median [IQR] = 262.92 [211.44] pg/ml) androgen peaks. These intervals appear to be correlated with the long digesta passage time in this species. The salivary and urinary peaks were closely associated. These results demonstrate that androgen concentrations in saliva, urine, or feces samples reflect reliably physiologic events and are a powerful tool for noninvasive reproductive monitoring of Amazonian manatees.