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Universidade Federal Rural Do Semi-Árido Programa De Pós-Graduação Em Ciência Animal Universidade Federal Rural do Semi-Árido Programa de Pós-Graduação em Ciência Animal ANA LIZA PAZ SOUZA OTIMIZAÇÃO DE PROTOCOLOS DE CRIOPRESERVAÇÃO DO SÊMEN DE CATETOS (Pecari tajacu LINNEAUS, 1758) MOSSORÓ-RN 2015 ANA LIZA PAZ SOUZA Otimização de protocolos de criopreservação do sêmen de catetos (Pecari tajacu Linneaus, 1758) Tese apresentada ao Programa de Pós Graduação em Ciência Animal da Universidade Federal Rural do Semi- Árido de Mossoró, para a obtenção do título de Doutora em Ciência Animal. Departamento: Ciências Animais Área de Concentração: Morfofisiologia e Biotecnologia Animal Orientador: Prof. Dr. Alexandre Rodrigues Silva Mossoró-RN 2015 Dados Internacionais de Catalogação na Publicação (CIP) Biblioteca Central Orlando Teixeira (BCOT) Setor de Informação e Referência S725o Souza, Ana Liza Paz. Otimização de protocolos de criopreservação do sêmen de catetos (Pecari tajacu LINNEAUS, 1758) / Ana Liza Paz Souza. - Mossoró, 2015. 145f: il. Orientador: Dr. Alexandre Rodrigues Silva Tese (Doutorado em Ciência Animal) - Universidade Federal Rural do Semi-Árido. 1. Cateto. 2. Aloe vera. 3. Criopreservação. 4. Dodecil Sulfato de Sódio. 5. Lipoproteína de Baixa Densidade. I. Título. RN/UFERSA/BCOT CDD 636.9734 Bibliotecária: Vanessa de Oliveira Pessoa CRB-15/453 FOLHA DE AVALIAÇÃO Nome do Autor: Ana Liza Paz Souza Título: Otimização de protocolos de criopreservação do sêmen de catetos (Pecari tajacu Linneaus, 1758) Tese apresentada ao Programa de Pós Graduação em Ciência Animal da Universidade Federal Rural do Semi- Árido de Mossoró, para a obtenção do título de Doutora em Ciência Animal. Data: 10/02/2015 Banca Examinadora: Mossoró-RN 2015 Dedicatória, Ao único que é digno de receber toda honra e toda gloria! Porque Dele, por Ele, para Ele são todas as coisas! Meu Deus AGRADECIMENTOS À minha mãezinha, Maria Auxiliadora Ferreira Paz, minha estrutura, mulher forte, que não mediu esforços para me dá o bem mais precioso, o estudo. Te amo muito mamãe! Aos meus irmãos Artur Luiz Ferreira Paz e André Levi Ferreira Paz, presentes de Deus na minha vida. A meu esposo Manoel Nilson Batista Filho, pela amizade, confiança, paciência, força e amor que tem me dado, meu porto seguro. A minha pequena florzinha, Naila Cecília, que mesmo ainda dentro do meu ventre já me traz uma sensação única e inexplicável. A minha cunhada e amiga Gleiziely Melo por compartilhar comigo as minhas aflições e por me presentear com minha sobrinha que tanto amo, Ana Lavínia. A minha família adotiva Santiago de Azevedo, por estarem sempre presentes na minha vida. Ao meu orientador Alexandre Rodrigues Silva, por me orientar com tamanha dedicação, acreditou em mim e pacientemente me ensinou além do que há em livros, me ensinou a ser perseverante, a ter autoconfiança e me incentivou a fazer a diferença. A quem muito admiro. Aos meus colegas de pesquisa do laboratório de conservação de germoplasma animal que muito me ajudaram; Gabriela Lima, Gislayne Peixoto, Thibério Castelo, Andreia Silva, Lívia Campos, Camila Praxedes, Luana Bezerra, Artur Brilhante, Artur Lago. Em especial ás minhas amigas, irmãs Gabriela Lima e Gislayne Peixoto que estavam sempre ao meu lado em qualquer situação. Ao Professor Dr. Moacir Franco de Oliveira por está sempre disponível a me ajudar disponibilizando o seu tempo nas minhas coletas. Aos funcionários do CEMAS, Seu Almeida pela coragem e força de vontade a me ajudar. Ao Professor Dr. Marc Henry pelos conhecimentos e ajuda na extração da lipoproteína de baixa densidade. Ao Programa de Pós Graduação em Ciência Animal da Faculdade de Medicina Veterinária da UFERSA e a todos os seus professores e funcionários. As minhas três inseparáveis amigas Polyanna Dantas, Maria Marília e Jussara Figueiredo que mesmo de longe acompanharam mais essa batalha. A CAPES, pelo apoio financeiro. A todos que torceram por mim. Muito Obrigada! RESUMO Souza, A L P. OTIMIZAÇÃO DE PROTOCOLOS DE CRIOPRESERVAÇÃO DO SÊMEN DE CATETOS (Pecari tajacu LINNEAUS, 1758). Universidade Federal Rural do Semi-Arido, UFERSA - Mossoró, RN 2015. A presente tese teve como objetivo aperfeiçoar protocolos de criopreservação de sêmen de catetos (Pecari tajacu). Para tanto, foram utilizados animais adultos oriundos do Centro de Multiplicação de Animais Silvestres (CEMAS) da Universidade Federal Rural do Semi-Árido (UFERSA), cujo sêmen foi coletado por eletroejaculação e imediatamente avaliado. O primeiro experimento objetivou avaliar o efeito da lipoproteína de baixa densidade (LDL) purificada, em relação à gema de ovo (G) e determinar a concentração ideal (20%, 10% e 5%) para criopreservação de sêmen em catetos. Após a análise dos dados verificou-se que a LDL (20%) pode ser utilizada em substituição à gema de ovo, tendo sido verificados 36.4±5.3% e 21.8±6.9 de motilidade espermática total após a descongelação, respectivamente. No segundo experimento, avaliou-se a ação do gel de Aloe vera (AV) tanto na refrigeração quanto na congelação do sêmen de catetos, em diferentes concentrações (20%, 10%, 5%) ou associado à gema de ovo (10%:10%). Verificou-se que o gel de Aloe vera (20%) se mostrou eficiente para a refrigeração a 5°C, mantendo a longevidade espermática por até 48 horas de avaliação apresentando uma motilidade total de 54.2±21.3%; quanto ao processo de criopreservação, verificou-se que o uso de Aloe vera a 20% pode substituir a gema de ovo, porém a associação desta com a gema promoveram a melhor preservação da viabilidade (32.7±2.3%), resposta osmótica (37.8±4.2%) e integridade de membrana (40.2±3.1). No terceiro experimento verificou-se a ação da adição (0,5% e 1,0%) do detergente a base de dodecil sulfato de sódio (SDS), Equex®STM, no sêmen de catetos. Entretanto, observou-se que este, em nenhuma concentração proposta, foi eficiente em aumentar a longevidade da célula espérmática de catetos além dos 15 minutos, conforme previamente descrito para a espécie. Os experimentos realizados foram suficientes a responder as hipóteses propostas, sendo úteis para o aperfeiçoamento de protocolos de criopreservação de sêmen de catetos (Pecari tajacu). Palavra Chave: Cateto, Criopreservação de sêmen, Lipoproteína de baixa Densidade, Dodecil Sulfato de Sódio, Gel de Aloe vera. ABSTRACT Souza, A L P. CRYOPRESERVATION PROTOCOLS OPTIMIZATION OF SEMEN COLLARED PECCARIES (Pecari tajacu LINNEAUS, 1976). Federal Rural University of the Semi-Arid, Mossoró, RN 2015. The present thesis had as objective optimized peccaries semen cryopreservation protocols (Pecari tajacu). Therefore, adult animals were used originated from the Multiplication Center of Wild Animals (CEMAS) of the Federal Rural University of the Semi-Arid (UFERSA), which semen was collected by electroejaculation and immediately evaluated. The first experiment aimed at evaluating the effect of low- density lipoprotein (LDL) purified in relation to egg yolk (G) and defines the ideal concentration (20%, 10% and 5%) for semen cryopreservation in peccaries. After the data analysis it was found that LDL (20%) can be used to replace egg yolk having been verified 36.4 ± 5.3 and 21.8 ± 6.9% of the total sperm motility after thawing, respectively. The second experiment evaluated the action of Aloe vera gel (AV) both refrigeration and in the peccaries of semen freezing in different concentrations (20%, 10%, 5%) or associated with egg yolk (10%:10%). It was found that Aloe vera gel (20%) proved to be efficient cooling at 5 ° C, maintaining the longevity of sperm for up to 48 hours of total motility assessment having a 54.2 ± 21.3%; as the cryopreservation process, it was found that the use of Aloe vera 20% can replace egg yolk, but its association with gem promoted better preservation of viability (32.7 ± 2.3%), osmotic response (37.8 ± 4.2%) and membrane integrity (40.2 ± 3.1). In the third experiment evaluated the addition of the action (0.5% and 1.0%) of base detergent sodium dodecyl sulfate (SDS), Equex®STM, the peccaries of semen. However, it was observed that, at any concentration proposal was effective in increasing the longevity of sperm cell peccaries over 15 minutes as previously described for the species. The experiments have been sufficient to answer the hypotheses and are useful for the improvement of semen cryopreservation protocols of collared peccary (Pecari tajacu). Keyword: Peccaries, semen cryopreservation, low density lipoprotein, Sodium dodecyl sulfate, Aloe vera. LISTA DE FIGURAS Figura 1: Espécie Pecari tajacu.....................................................................................24 Figura 2: Testículo de catetos (Pecari tajacu) demonstrando a bolsa testicular despigmentada e sem pelos grosseiros............................................................................26 Figura 3: Testículo de cateto (Pecari tajacu) dissecado demonstrando a fáscia espermática fibrosa..........................................................................................................27 Figura 4: Imagem histológica do testículo de cateto (pecari tajacu).............................28 Figura 5: Pênis do cateto (Pecari tajacu) em forma de espiral......................................29 Figura 6: Diagrama da ação da lipoproteína de baixa densidade...................................46 Artigo 3 Innovative use of Aloe Vera for collared peccary (Pecari tajacu) semen conservation Figure 1: Values (means ± SEM) for sperm viability, morphology and osmotic response in collared peccaries (Pecari tajacu) chilled semen (n=5) diluted in Tris plus egg yolk (20%) or Aloe vera extract (20%), storage during 48h at 5 ºC. 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