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African painted dogs (Lycaon pictus, APD) are highly endangered, with fewer than 7000 remaining in nature. Captive breeding programs can preserve a genetically diverse population and provide a source of individuals for reintroductions. However, most programs are initiated from few founders and suffer from low genetic diversity and inbreeding. The aims of this study were to use molecular markers to assess genetic variation, inbreeding, and relatedness among APDs in the North American captive population, to use these data to realign studbook records, and to compare these data to wild populations and to the European captive population to facilitate the development of a global management plan. We sequenced mitochondrial and major histocompatibility (MHC) class II loci and genotyped 14 microsatellite loci from 109 APDs from 34 institutions in North America. We identified three likely studbook errors and resolved ten cases of uncertain paternity. Overall, microsatellite heterozygosity was higher than reported in Europe, but effective population size estimates were lower. Mitochondrial sequence variation was extremely limited, and there were fewer MHC haplotypes than in Europe or the wild. Although the population did not show evidence of significant inbreeding overall, several individuals shared high relatedness values, which should be incorporated into future breeding programs.

Dissertação de Mestrado apresentada no ISPA – Instituto Universitário para obtenção de grau de Mestre na especialidade de Biologia Marinha e Conservação Com o tempo, o homem tem vindo a tomar cada vez mais consciência da grande importância da proteção, preservação e recuperação do meio ambiente, assim como das espécies ameaçadas. Aliadas à missão de conservação estão várias entidades que, diariamente, trabalham com intuito de manter populações saudáveis e autossustentáveis. Este estudo apresenta os resultados da análise demográfica e genética da população do Leão-marinho-da-califórnia [Zalophus californianus (Lesson, 1828)], espécie gerida no âmbito de Programa ex situ da EAZA (EEP). Realizou-se um questionário que foi enviado a diversas instituições, com o objetivo de criar um Plano de Gestão a Longo Prazo para a espécie, através de um bom controlo do tamanho da sua população. Os dados desta população encontram-se registados no seu Studbook desde 1991, dados estes que são essenciais para monitorizar e gerir o estabelecimento e o crescimento de populações mantidas em cativeiro, facilitando o desenvolvimento de uma população viável e ajudando a maximizar a diversidade genética e a estabilidade demográfica. Através da atualização e correção de erros do Studbook foi possível aumentar a percentagem de pedigree conhecido. Para a análise demográfica e genética da população utilizou-se o software PMx. Os resultados mostram que a população do EEP se encontra em crescimento, demostrando parâmetros demográficos positivos, tais como: taxa instantânea de crescimento (r =0,039); taxa de crescimento populacional (λ = 1,040) e; taxa líquida de reprodução (Ro = 1,605). Relativamente aos parâmetros genéticos, a diversidade genética da população é alta e tem potencial para aumentar, o que indica um bom estado genético. Over time, man has become increasingly aware of the great importance of protecting, preserving, and recovering the environment, as well as threatened species. Allied to the conservation mission are several entities that work daily in order to maintain healthy and self-sustainable populations. This study presents the results of the demographic and genetic analysis of the population of the California Sea Lion [Zalophus californianus (Lesson, 1828)], a species managed under the EAZA Ex situ Programme (EEP). A questionnaire was prepared and sent to several institutions, with the aim of creating a long-term management plan for the species, through good control over the size of its population. This population's data has been recorded in a Studbook since 1991. This data is essential to monitor and manage the establishment and growth of populations kept in captivity, facilitating the development of a viable population, and helping to maximize genetic diversity and demographic stability. By updating and correcting Studbook errors, it was possible to increase the percentage of pedigree known. For the demographic and genetic analysis of the population, the PMx software was used. The results show that the population of the EEP is growing, showing positive demographic parameters, such as: instantaneous growth rate (r = 0.039); population growth rate (λ = 1,040); net reproductive rate (Ro = 1.605). Regarding genetic parameters, the genetic diversity of the population is high and has the potential to increase, which indicates a good genetic status.

El objetivo principal de ésta tesis fue realizar un análisis demográfico, genético y reproductivo de la población de jaguares (Panthera onca) cautivos en Colombia. En el análisis demográfico se realizó la construcción del Studbook Regional para Jaguares (Panthera onca) (n=38), se obtuvo el censo de los animales que han estado presentes en las colecciones zoológicas de Colombia desde el año de 1970 hasta el año 2009. Se estableció la distribución de los ejemplares vivos en la actualidad por clases de edades. Se obtuvieron las características demográficas para las poblaciones actuales y poblaciones modelos de jaguares machos y hembras; también se construyó la tabla de vida para machos y hembras. En el análisis genético, a partir de los registros, se calcularon las estadísticas poblacionales. Se encontró que en la población se encuentran 2 nuevos posibles fundadores potenciales (n=2). Se realizaron las proyecciones reproductivas para un tiempo de 5, 10, 15 y 20 años con el fin de incrementar la pobalción de 25 individuos a 40. Se realizó la selección de apareamientos empleando el valor de la media de parentesco MK, y la ubicación geográfica. Se realizó una propuesta de meta poblacional para los jaguares en Colombia de conservar el 90% de la diversidad genética durante un periodo de 100 años, teniendo en cuenta las siguientes variables: Tiempo generacional (T =12), Tasa de crecimiento poblacional (λ= 1.02), Tamaño poblacional actual (N =25), Número efectivo poblacional actual (Ne =6.5), Relación Ne/N (0.26), Diversidad genética actual (GD =0.78), Población máxima permitida (n=40), Población requerida para alcanzar metas (n=37), FGE reclutado por cada nuevo fundador (FGE =0.4). El plan de manejo poblacional para el jaguar en parques zoológicos de Colombia fue propuesto estableciendo metas de manejo poblacional para los siguientes 100 años. También se realizó un análisis genético de la población de jaguares cautivos en parques zoológicos de Colombia mediante el análisis tanto de registros del Studbook Regional para Jaguares como de muestras de ADN obtenidas de 20 animales vivos (n=20), con el cual se construyó el pedigrí para la población empleando 9 microsatélites. Las paternidades y maternidades fueron asignadas por el programa CERVUS y las relaciones de parentesco fueron obtenidas por el programa KINSHIP. El análisis del Studbook se realizó por medio del programa PM2000 en donde se obtuvo el valor de la diversidad genética (GD) actual de la población (GD=0.7832), el valor de diversidad genética potencial (GDp=0.9113), valor génico (GV=0.7846), el valor del equivalente del genoma fundador actual (FGE=2.31) y potencial (FGEp=5.64), el valor del genoma fundador sobreviviente actual (FGS=3.14) y potencial (FGSp=5.64), el coeficiente medio de consanguinidad (F=0.0179), el número efectivo poblacional (Ne=5.26), al igual que la relación (Ne/N =0.2667) y la media de parentesco MK (Mean KINSHIP) para cada individuo. En el análisis de los microsatélites se calcularon las frecuencias alélicas por cada microsatélite y se encontró un promedio de alelos por locus de 5.67. El promedio de la heterocigocidad observada general fue de 0,687 y el de la heterocigocidad esperada 0,684. Se encontró que todos los locus se encuentran en equilibrio Hardy–Weinberg. Para esta población se encontró un exceso de heterocigotos en los locus FCA075, FCA043, FCA736, FCA220 y FCA391 y exceso de homocigotos solo para los locus FCA 441, FCA 008, FCA 224 mediante los estimados de Fis de Weir y Cockerham's (W and C), y Robertson y Hill's (R and H). El promedio de Fis para la población fue (Fis W and C= -0,0157) y (Fis R and H=-0,0109) indicando que la población se encuentra en exogamia. No se encontraron diferencias significativas para la prueba de desequilibrio por ligamiento. Los análisis de verosimilitud obtenidos en CERVUS resultaron con asignaciones de la maternidad y de paternidad. Los asignamientos se realizaron según el logaritmo natural de la tasa de verosimilitud (LOD). Se obtuvieron las probabilidades de relación y parentesco asignadas por KINSHIP de las simulaciones y se encontraron relaciones significativas (p0.05) en 6 parejas de jaguares, (p0.01) para 3 parejas y (p0.001) para 11 parejas. Con este estudio se logró establecer la relación entre individuos y se logró construir el pedigrí de la población de jaguares cautivos en parques zoológicos de Colombia como base fundamental para el establecimiento de un plan de conservación ex situ para el Jaguar en parques zoológicos de Colombia. Por último se realizó la caracterización reproductiva de un grupo de jaguares cautivos (n=6) en las instalaciones de la Fundación Zoológico Santa Cruz en Cundinamarca, Colombia, mediante un análisis longitudinal no invasivo de estradiol y progesterona en hembras y testosterona en machos. En un periodo de un 1 año dividido en cuatro épocas climatológicas las hembras confinadas en solitario fueron evaluadas con el objetivo de caracterizar la actividad ovárica y determinar la presentación de ovulaciones espontáneas. En los machos la actividad testicular. En las hembras se realizó un segundo seguimiento después de la aplicación de un protocolo convencional de gonadotropinas. La hormonas fueron extraídas de muestras de materia fecal de tres hembras (n=3) y dos machos (n=2). La medición de estradiol se realizó por medio de radioinmunoanálisis (RIA) y la medición de progesterona y testosterona por medio de ensayo por inmunoabsorción ligado a enzimas (ELISA). El modelo lineal de regresión de efectos mixtos encontró diferencias significativas en las concentraciones de estadiol en las diferentes estaciones (Chi square = 15.97, DF = 3, p 0.01). Las comparaciones de medias estacionales se realizó mediante el método de la diferencia significativa honesta de Tukey (DSH), revelando diferencias significativas entre las estaciones: Sequía 1 vs. Lluvias 2 (p 0.01), Lluvias 1 vs. Lluvias 2 (p 0.05), y Sequía 2 vs. Lluvias 2 (p 0.05). En las tres jaguares hembras se encontraron elevaciones de progesterona compatibles con ovulaciones expontáneas y el modelo lineal de regresión de efectos mixtos también encontró diferencias significativas en las concentraciones de progesterona en las diferentes estaciones (Chi square = 28.56, DF = 3, p 0.01). El DSH mostró diferencias significativas solo entre las estaciones: Sequía 2 vs.Lluvias 2 (p 0.01). La estación con el nivel promedio más bajo fue lluvias 2 (Octubre, Noviembre, y Diciembre). Durante esta estación periodos de anestro fueron registrados entre 31 y 83 días. Las tres hembra mostraron picos de estradiol después de la administración de gonadotropina coriónica equina (eCG). El análisis longitudinal no invasivo para los machos (Macho 1 y Macho 2) no mostró diferencias significativas en el curso del año de análisis. El análisis seminal de los tres machos adultos (n = 3) fue realizado por medio de electroeyaculación bajo anestesia general (Macho 1 and 2) y por lavado epididmal post mortem (Macho 3). La concentración media de espermatozoides encontrada fue 5.7 x 106 ± 1.1 x 106 espermatozoides/ml. La motilidad progresiva promedio fue de 80 %. El porcentaje de espermatozoides normales obtenidos por electro eyaculación + desviación estandar (DS) fue 80 ± 2.8%, y las anormalidades más frecuentemente encontradas fueron defectos de cabeza (7 ± 1.4%). El fluido obtenido en el lavado epididimal mostró 35 ± 1.4% de espermatozoides normales, y la anormalidad más frecuente fueron las gotas citoplasmáticas distales (39 ± 11.3%). Abstract. The objective of this thesis was to analyze the demographic, genetic and reproductive status of captive jaguars in Colombia. In the demographic analysis the Regional Studbook for Jaguars (Panthera onca) (n=38) was constructed using the software SPARKS. The studbook data were analyzed by the software population management 2000 (PM2000). The census of the animals that had been present in the zoological collections of Colombia from the year of 1970 until the year 2009 was obtained. The distribution of the living animals by age classes was established and the demographic characteristics were obtained for the current population and model population of male and female jaguars, as well as the life table for males and females. The reproductive projections were obtained for 5, 10, 15 and 20 years in order to increase the captive population from 25 individuals to 40. The selection of matings was based on the Mean KINSHIP (MK) value, and the geographical location. It is presented the population goals for the jaguars in Colombia to conserve 90% of the genetic diversity during a 100 year period, for the variables: Generational Time (T =12), populational growth rate (λ = 1.02), current populational size (N =25), current populational effective Number (Ne =6.5), Relationship (Ne/N =0.26), current genetic Diversity (GD =0.78), allowed maximum Population (n=40), required Population to reach goals (n=37), FGE recruited by each new founder (FGE =0.4). The population management plan for the Jaguar (Panthera onca) in Colombia was proposed for the Colombian Zoological Parks establishing population management goals for the next 100 years. Also, a genetic analysis for the captive population of jaguars (Panthera onca) was done in zoological parks of Colombia based upon the Colombian Regional Studbook for Jaguars and from DNA obtained form 20 living animals (n=20) in order to establish the pedigree for the captive population using 9 microsatellites. The assignments for paternities and maternities were done with for the program CERVUS and the relationship between animals were established with the KINSHIP program. The analysis of the Studbook was done with the PM2000 program generating the following values: genetic diversity for the population (GD=0.7832), potential genetic diversity (GD=0.9113), genic value (GV=0.7846), current founder genome equivalent (FGE=2.31) and potential p(FGE=5.64), current and potential founder genome survivor (FGS=3.14; pFGS=5.64 respectively). Other values calculated were, mean coefficient of consanguinity (F=0.0179), population effective number (Ne=5.26) and the Mean KINSHIP (MK) for each individual. With the microsatellites analysis, the allelic frequencies were calculated for each locus with an average of alleles per loci of 5.67. The averages of the observed and expected heterozigocity were 0.687 and 0.684 respectively. It was found that all loci are in Hardy-Weinberg equilibrium. For this population an excess of heterozygous was found for the locus FCA075, FCA043, FCA736, FCA220 and FCA391 and an excess of homozygous for the locus FCA 441, FCA 008, FCA 224 by Fis of Weir and Cockerham's (W and C), and Robertson and Hill's (R and H). The average of Fis for the population was (Fis W and C = -0.0157) and (Fis R and H=-0.0109) indicating that the population is in exogamy. No significant differences were found for the linkage disequilibrium test. The likelihood analyses obtained in CERVUS assigned maternities and paternities. The assignments were done according to the value Pair Top LOD. The relationship probabilities were obtained by KINSHIP. In the simulations, a significant relationship (p 0.05) were found in 6 pairs of jaguars, (p 0.01) in three pairs and (p 0.001) for 11 pairs. With this study the relationships between animals was established and the pedigree of the captive population of jaguars was built in order to develop an ex situ conservation plan for the species in the Colombian zoological parks. In the last part of the study a reproductive analysis of a captive group of jaguars (n = 6) at the Santacruz Zoological Foundation in Cundinamarca, Colombia, was conducted by performing a longitudinal, noninvasive, hormonal analysis of estradiol and progestogens in females and of androgens in males. During four seasons, female jaguars confined in solitary were evaluated for ovarian activity and spontaneous ovulation, male jaguars for testicular activity. A second hormonal follow-up was conducted in the females after administration of gonadotropins. Hormones were extracted from fecal samples of three females (n = 3) and two males (n = 2). Estradiol measurements were obtained by radioimmunoassay (RIA) and progestogens by enzyme immunoassay (EIA). The linear mixed-effect regression (LMER) showed that there was a significant effect of seasons in the concentrations of estradiol (Chi square = 15.97, DF = 3, p 0.01). Post hoc comparisons of all pairs of seasonal means were conducted according to Tukey’s honest significant difference (HSD), revealing significant differences between seasons: Dry 1 vs. Rains 2 (p 0.01), Rains 1 vs. Rains 2 (p 0.05), and Dry 2 vs. Rains 2 (p 0.05). Elevations of progestogens compatible with spontaneous ovulation occurred in three jaguars and the LMER showed that there was also a significant effect of seasons (Chi square = 28.56, DF = 3, p 0.01). Post hoc comparisons showed significant differences only between seasons: Dry 2 vs. Rains 2 (p 0.01). The season with the lowest average concentration was Rains 2 (October, November, and December). During this season, periods of anestrous were registered that lasted between 31 and 83 days. The three females presented estradiol peaks after the administration of eCG. A noninvasive longitudinal analysis for androgens was also made (Males 1 and 2) over the course of one year, and no significant differences were found between the different seasons. A seminal analysis of three adult male jaguars (Panthera onca) (n = 3) was also performed after electro-ejaculation under general anesthesia (Male 1 and 2) and by a postmortem epididymal wash (Male 3). The mean concentration of spermatozoids was 5.7 x 106 ± 1.1 x 106 spermatozoa/ml. The progressive motility + SD averaged 80 %. The percentage of normal spermatozoids obtained by electro-ejaculation was 80 ± 2.8%, and the abnormalities found more frequently were head defects (7 ± 1.4%). The seminal fluid obtained by epididymal flush contained 35 ± 1.4% normal spermatozoids, and the most frequent abnormalities found corresponded to distal cytoplasmic droplets (39 ± 11.3%). Doctorado

Genealogical records of animals (studbook) are created to avoid reproduction between closely related individuals, which could cause inbreeding, particularly for such endangered species as the Panthera onca (Linnaeus, 1758). Jaguar is the largest felid in the Americas and is considered an important ecological key species. In Mexico, wild jaguar populations have been significantly reduced in recent decades, and population decline typically accompany decreases in genetic variation. There is no current census of captive jaguars in Mexico, and zoos do not follow a standardized protocol in breeding programs based on genetic studies. Here, we emphasise the importance of maintaining an adequate level of genetic variation and propose the implementation of standardised studbooks for jaguars in Mexico, mainly to avoid inbreeding. In addition, achieving the aims of studbook registration would provide a population genetic characterisation that could serve as a basis for ex situ conservation programmes.

Financiadora de Estudos e Projetos Species kept in captivity are of great importance for in situ conservation of natural populations. In order to fulfill this role, they need to be properly managed with the aim to avoid the consequences of a captive environment, such as: loss of genetic diversity, inbreeding depression, accumulation of deleterious alleles and genetic adaptation to captivity (the first three due to the small number of breeding stock). Thus, with the aim to test founder effects in captive ―populations‖, we used as study model the spectacled bears from Brazilian zoos. This ―population‖ consists of twenty-one bears found in eight zoos along the country, which are originated from just twelve founding individuals. To test a possible founder effect, we analyzed twelve heterologous microsatellite loci for all the specimens of the species in Brazil. Results for the number of alleles and allelic richness obtained through the rarefaction method showed that spectacled bears in captivity have reduced levels of allelic richness in relation to three natural populations (Colombia, Ecuador and Venezuela) studied by other authors; suggesting that bears from Brazilian zoos suffered the consequences of a founding effect, as they have only a fraction of the gene pool of the ancestral population. From the information present in the studbook was possible to verify that there are two families of spectacled bears, and according to the individuals´s multilocus genotypes, we found alleles that are present in a same family and absent in the other. Regarding the kinship, there was a discrepancy in the results with the molecular estimators through the softwares ML-Relate and Coancestry and the information given by the institutions. The information obtained in this work are fundamental to the ex situ conservation program in Brazil with the objective of maintain as high as possible the genetic diversity for future reintroduction plans of the species in nature. Espécies mantidas em cativeiro são de grande importância na conservação in situ de populações naturais. A fim de que possam desempenhar este papel é necessário que sejam devidamente manejadas, para que as consequências que o cativeiro traz sejam evitadas, como por exemplo: perda de diversidade genética, depressão endogâmica, acúmulo de alelos deletérios e adaptação genética ao cativeiro (as três primeiras devido ao pequeno número de espécimes dos plantéis). No Brasil, uma espécie de mamífero que vem sendo criada em cativeiro desde 1970 e que passou por um efeito fundador, é o urso de óculos (Tremarctos ornatus). O plantel existente hoje totaliza o número de vinte e um ursos, encontrados em oito zoológicos do país, o qual se originou de apenas doze indivíduos fundadores. Para avaliar as consequências de um conhecido efeito fundador, analisamos doze locos heterólogos de microssatélites para todos os exemplares da espécie nos zoos do país, através de amostragem não-invasiva. Resultados referentes ao número de alelos e à riqueza alélica obtida por meio do método de rarefação mostraram que os ursos de óculos de cativeiro possuem valores reduzidos de riqueza alélica em relação a três populações naturais (Colômbia, Equador e Venezuela) estudadas por outros autores; sugerindo que os ursos dos zoológicos brasileiros sofreram as consequências de um efeito fundador, pois possuem apenas uma fração do pool gênico da população ancestral. A partir das informações presentes no studbook foi possível verificar que existem duas famílias de ursos de óculos, e de acordo com os genótipos multilocos dos indivíduos, encontramos alelos que estão presentes em uma mesma família e ausentes na outra. Em relação ao parentesco, houve uma discrepância nos resultados encontrados com os estimadores moleculares através dos softwares ML-Relate e Coancestry e o histórico cedido pelas instituições. As informações obtidas em nosso trabalho, portanto, são fundamentais para que os ursos encontrados nos zoológicos do Brasil possam participar na formação de casais em futuros programas de reprodução ex situ com o propósito de serem utilizados em planos de reintrodução da espécie na natureza.