Nutrient absorption is important in all stages of life. As the diet of an animal changes from birth on, morphological and biochemical adaptation can be anticipated in order to accommodate changing demands. The main focus of the present study was to examine the relationship between age and diet on the potential for peptide transport via PepT1 in the gastrointestinal tract of lambs and to relate changes of peptide transport capability to morphological changes. A 2x4 factorial arrangement of treatments was used with 32 crossbred lambs. Four blocks were created based upon gender, birth type (single or twin), birth weight, and birth date. Lambs were randomly allotted at birth to receive or not to receive a creep diet. All lambs were allowed to nurse. Sampling times of 2, 4, 6, or 8 wk were randomly allotted to lambs. Samples for RNA extraction and histological evaluation were taken from the dorsal rumen, ventral rumen, omasum, duodenum, jejunum, and ileum. Villi were about 7% shorter (P < 0.09) in lambs receiving creep feed. Papillary height and width increased linearly (P < 0.001 and P < 0.0001, respectively) with age. Total and keratinized epithelial cells in the stomach decreased (P < 0.03 and P < 0.004, respectively) with age and were fewer (P < 0.0002 and P < 0.0001, respectively) in lambs receiving creep feed. Creep feeding appears to have slightly altered the mucosal structure of the small intestine and it was advantageous in that it stimulated papillary growth and thus predisposed the rumen for the introduction of feed into the diet. A 2.8 kb oPepT1 mRNA was present in all tissues studied by 2 wk, and age did not significantly influence the abundance of oPepT1 mRNA in the small intestine or stomach. In the small intestine, abundance of oPepT1 mRNA was greatest (P < 0.0007) in the jejunum. In the stomach, abundance of oPepT1 mRNA was greatest (P < 0.01) in the dorsal rumen. In the stomach, particularly in the rumen, a greater abundance of oPepT1 mRNA was observed in lambs not receiving the creep diet. It seems likely that a stimulus for development is coming from the non-luminal direction, possibly blood-borne, and may be involved in the ontogenesis of oPepT1. Peptide transport appears to be a physiologically important process in the young lamb and the rumen appears to be involved in the transport of peptides, particularly in nursing lambs. / Master of Science

Embryonic stem cells (ESCs) are pluripotent cells derived from the early embryo and are able to differentiate into cells belonging to the three germ layers. They are a valuable tool in research and for clinical use, but their applications are limited by ethical and technical issues. In 2006 a breakthrough report described the generation of induced pluripotent stem cells (iPSCs). IPSCs are ESC-like cells generated from somatic cells by forcing the ectopic expression of specific transcription factors. This circumvents the ethical issues about the use of embryos in research and provides multiple opportunities to understand the mechanisms behind pluripotency. The aim of this project was to generate sheep iPSCs and characterise them. In order to learn the technique I initially repeated the original iPSC methodology: the putative mouse iPSCs I have generated display a morphology typical of ESCs, characterised by a high nuclear to cytoplasmic ratio, and form colonies with neat edges and smooth domes. These cells are positive to Nanog, a marker of pluripotency, and can give rise to cells belonging to the mesodermal and the ectodermal lineages when differentiated in vitro. Since the main aim of the thesis was the derivation of sheep pluripotent cells, once established the protocol in mouse, I then moved to the generation of ovine iPSC colonies. The cells I have generated have a morphology similar to that of mouse ESCs, express markers of pluripotency such as alkaline phosphatase and Nanog and can differentiate in vitro and in vivo into cells belonging to the three germ layers. Additionally, these ovine iPSCs can contribute to live born chimeric lambs, although at low level.

Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever (MCF) in susceptible ruminants. Through an unknown mechanism, presence of the virus leads to proliferation of NK-like T cells that are not targetrestricted by the MHC class molecules. These host cells cause the symptoms found in MCF; fever, swollen lymph nodes, and necrotic lesions of the nasal, conjunctival, and oral mucosa, which usually leads to death of the host. MicroRNAs (miRNAs) are ~22 nt RNA molecules expressed by eukaryotes and viruses that regulate genes post-transcriptionally. Viral miRNAs have been found to regulate cellular genes to control the cell cycle and have a role in pathogenesis. It was hypothesised that OvHV-2 expresses miRNAs and these play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 encodes miRNAs. Bioinformatic analysis was conducted on deep sequencing data from RNA of OvHV-2- immortalised T cells. Candidate miRNAs were selected if they adhered to miRNA secondary structure. 46 candidate miRNAs were found, with three clusters on the minus strand; one at the 5’ end and the other two in a 9.3 kb region that contains no predicted open reading frames. The 8 most abundant candidates were successfully validated by northern hybridisation for small RNAs. The majority of the predicted targets for the 8 validated OvHV-2 miRNAs were from the OvHV-2 genome. This study adds OvHV-2 to the list of herpesviruses that encode miRNAs and provides another tool for studying the pathogenesis of MCF.

Orientador: Luiz Claudio Nogueira Mendes / Coorientador: Marcelo Vasconcelos Meireles / Banca: Alex Akira Nakamura / Banca: Fernanda Bovino / Resumo: A infecção patogênica por Eimeria spp., denominada eimeriose ou coccidiose, tem sido indicada como responsável por mortes, queda no desenvolvimento e baixa produtividade em criações animais, ocorrendo principalmente em indivíduos jovens devido à imaturidade do sistema imunológico. Quando são submetidos a um sistema intensivo de produção, como em situação de confinamento, cordeiros podem se tornar ainda mais susceptíveis às infecções por espécies de Eimeria, devido à alta concentração de indivíduos, que potencializa a dispersão do parasito, e ao estresse associado à criação. No estado de São Paulo, já foram identificadas dez espécies de Eimeria em estudos prévios e, dentre elas, E. ovinoidalis foi considerada a mais patogênica para ovinos. Dessa forma, o objetivo deste trabalho foi descrever as espécies do gênero Eimeria Schneider, 1875 que ocorreram em um confinamento de cordeiros, bem como as dinâmicas da eliminação de oocistos no ambiente e correlação com o ganho de peso médio diário (GMD) durante nove semanas. Cento e quatro cordeiros de diversas raças e cruzas, com aproximadamente 60 dias de vida, foram confinados e submetidos a pesagens, avaliações clínicas e coprológicas periódicas. Amostras de fezes que continham mais de 500 oocistos de Eimeria por gramas de fezes (OoPG) foram separadas para esporulação, posteriormente determinando-se as espécies às quais os oocistos pertenciam. Dentre os 677 oocistos avaliados, as seguintes espécies foram identificadas: E. parva Kotla... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Pathogenic infection caused by Eimeria spp., denominated eimeriosis or coccidiosis, has been pointed out as responsible for deaths, decreased development and low productivity for livestock animals, affecting mainly young individuals due to immature immunological system. When submitted to an intensive breeding system, as a feedlot, lambs can become even more susceptible to Eimeria infections, due to high concentration of animals, which potentializes parasite dispersion, and to stress generated by farming conditions. In São Paulo State, ten Eimeria species were identified in previous studies and, among them, E. ovinoidalis, was considered the most pathogenic for ovine. So, this study aimed to describe species of the Eimeria Schneider, 1875 that occurred in a lamb feedlot, as well as the dynamics of oocyst output in the environment and its correlation with daily weight gain (DWG) during nine weeks. One hundred and four lambs of various breeds and crossbreeds, at approximately 60 days old, were placed in a feedlot and submitted to periodic weighing, clinical and coprological evaluations. Fecal samples that had more than 500 Eimeria spp. oocysts per gram (OPG) were separated for sporulation, and oocysts were identified at species level. Among the 677 evaluated oocysts, the following species were identified: E. parva Kotlan, Moscy & Vajda, 1929, E. crandallis Honess, 1942, E. ovinoidalis McDougald, 1979, E. weybridgensis Norton, Joyner & Catchpole 1974, E. bakuensis Musaev, 1970 (s... (Complete abstract click electronic access below) / Mestre

Os agonistas alfa 2 adrenérgicos são sedativos empregados na rotina clínica de ruminantes, e têm, entre outras, a vantagem de possuir antagonistas específicos, que aumentam a segurança no uso destes medicamentos. Tendo em vista a escassez de estudos acerca dos parâmetros hemodinâmicos e respiratórios em ovinos em posição quadrupedal e submetidos à sedação com xilazina ou dexmedetomidina com posterior reversão pelo atipamezole, realizou-se o presente estudo. Para tanto, foram utilizados 12 ovinos, machos, com idade entre um e dois anos, peso médio de 37,7kg, distribuídos em dois grupos de seis animais que foram submetidos a dois tratamentos distintos com média de três semanas de intervalo, em estudo do tipo prospectivo, encoberto e aleatório, sendo designados como Grupo XILA (cloridrato de xilazina 0,2 mg/kg IM) e Grupo DEX (cloridrato de dexmedetomidina 15 &micro;g/kg IM). Para a instalação do cateter de artéria pulmonar os animais foram anestesiados com auxílio de máscara facial com isofluorano em oxigênio, e na sequência foram intubados e mantidos sob anestesia com o mesmo agente até o fim da instrumentação. Após a recuperação anestésica foram avaliados os parâmetros hemodinâmicos, hemogasométricos e respiratórios durante os primeiros 60 minutos com os animais em posição quadrupedal, para obtenção dos parâmetros basais. Finalizada esta avaliação, os animais foram submetidos aos protocolos de sedação segundo o seu grupo. Os parâmetros foram verificados e registrados aos cinco (S5), 15 (S15) e 30 minutos (S30) após sedação, e, após a aplicação de 30 &micro;g/kg IM de atipamezole aos cinco (R5) e 15 minutos (R15). Foram avaliados frequência e ritmo cardíaco, pressão arterial sistêmica, parâmetros hemodinâmicos, frequência respiratória, hemogasometria arterial e venosa mista, temperatura central, glicemia e grau de sedação. Os índices ventilatórios e hemodinâmicos foram calculados. O período de latência em XILA e DEX foram 3,9 e 5,2 minutos. A xilazina promoveu sedação mais intensa, com diferença significativa entre os grupos aos 5 e 15min após sedação (momentos S5 e S15). Após a sedação, observou-se redução significativa da frequência cardíaca nos dois grupos, que refletiu no débito cardíaco, índice cardíaco e pressão arterial média. Houve elevação não significativa da resistência vascular sistêmica em ambos os grupos, mas que após o atipamezole ficou significativamente mais baixa no grupo XILA. A xilazina causou taquipneia, que foi inibida após o atipamezole. Não houve alterações clinicamente importantes nos valores de PaCO2, PaO2, SaO2 e nem nos índices de ventilação, indicando que não ocorreu hipoxemia, hipercapnia ou hipoventilação durante a sedação. A glicemia elevou-se de maneira significativa nos dois grupos mantendo-se elevada mesmo após o antagonista. Após administração do atipamezole os animais levaram 10,0 e 11,7 minutos para ficarem em posição quadrupedal e 19,3 e 30 minutos para reversão dos efeitos da xilazina e da dexmedetomidina, respectivamente. Tanto a xilazina como a dexmedetomidina promoveram sedação segura, com poucos efeitos hemodinâmicos e cardiorrespiratórios, sugerindo que a administração pela via intramuscular seja adequada para sedação de ovinos com xilazina ou dexmedetomidina / Adrenergic alpha 2 agonists are sedatives used in ruminants clinical routine and have, among others, the advantage of specific antagonists which increase the safety of these drugs. Given the scarcity of studies on the hemodynamic and respiratory parameters in sheep in standing position and undergoing sedation with xylazine or dexmedetomidine with subsequent reversal by atipamezol, this study was performed. For this purpose were used 12 male sheep, aged between one and two years, average weight 37,7kg. Sheep were divided into two groups of six animals that were submitted to two different treatments with a mean interval of three weeks, in a study of prospective hidden and random type, being designated as XILA Group (xylazine hydrochloride 0.2 mg/kg) and DEX Group (dexmedetomidine hydrochloride 15 &micro;g/kg) given by intramuscular route. For the installation of pulmonary artery catheter, the animals were anesthetized with facial mask with isofluorane in oxygen, and were intubated and maintained under anesthesia with the same agent until the end of instrumentation. After recovery of anesthesia, we evaluated the hemodynamic, blood gas and respiratory parameters during the first 60 minutes with animals in standing position, to obtain the baseline parameters. Completed this evaluation, the animals were submitted to sedation protocols according to their group. The parameters were checked and registered to 5 (S5), 15 (S15) and 30 minutes (S30) after sedation, and after application of 30 &micro;g/kg atipamezole by intramuscular route to 5 (R5) and 15 minutes (R15). Frequency and heart rate, systemic blood pressure, hemodynamic parameters, respiratory rate, arterial and mixed venous blood gas analysis, core temperature, blood glucose and degree of sedation were evaluated. Ventilatory and hemodynamic indices were calculated. The onset period in XILA and DEX were 3.9 and 5.2 minutes. Xylazine promoted more intense sedation, with a significant difference between the groups at 5 and 15 minutes after alpha 2 administration (S5 and S15). After sedation, significant decrease in heart rate was observed in both groups, which reflected in cardiac output, cardiac index and mean arterial pressure. There was no significant increase in systemic vascular resistance in both groups, but after atipamezol was significantly lower in the XILA group. Xylazine caused tachypnea, which was inhibited after atipamezol. There were no clinically significant changes in PaCO2, PaO2, SaO2 nor in the ventilation indices, indicating that there was no hypoxemia, hypercapnia or hypoventilation during sedation period. Blood glucose rose significantly in both groups, remained higher even after antagonist. After administration of atipamezol sheep needed 10.0 and 11.7 minutes to remain in standing position and 19.3 and 30 minutes to reverse the effects of dexmedetomidine and xylazine, respectively. Xylazine and dexmedetomidine promoted safe sedation with few hemodynamic and cardiorespiratory effects, suggesting that the intramuscularly route is suitable for sedation of sheep with xylazine or dexmedetomidine

O presente trabalho objetivou avaliar a capacidade de um sal mineral rico em molibdênio (Mo) em prevenir a intoxicação cúprica acumulativa (ICA), analisando variáveis clínicas, sangüíneas e os teores de cobre (Cu) e Mo hepático. Foram utilizados 25 ovinos da raça Ilede-France, aleatoriamente distribuídos em cinco grupos de cinco animais de cada, sendo que o grupo 1 recebia dieta contendo 80% volumoso e 20% concentrado, os grupos 2 e 3 recebiam 50% volumoso e 50% concentrado, e os grupos 4 e 5 recebiam a mesma dieta dos grupos 2 e 3 com a adição diária de 150 mg de sulfato de Cu até o término do experimento (150 d). Os grupos 1, 3 e 5 recebiam sal mineral contendo 300 ppm de Mo. Foram realizadas três biópsias hepáticas para determinação da concentração de Cu, Mo e Zn neste órgão e três ensaios de retenção aparente de Cu e Mo (0 d, 75 d, 150 d). Quinzenalmente, foi realizado exame clínico e coleta de amostras de sangue e urina. Três ovinos do grupo 4 e um do grupo 5 manifestaram ICA. Não existiu diferença entre a freqüência de mortalidade entre os grupos (P = 0,56). Os teores de Cu hepático nos ovinos com ICA (2450 ppm) foram superiores aos que não intoxicaram (1518 ppm). Quanto maior a ingestão de Mo na dieta menor foi o acúmulo de Cu hepático ao término do experimento (r = -0,72). Ovinos com ICA aumentaram a concentração de Zn hepático nas fases finais da intoxicação. A presença de altas atividades de GGT (>56,5 U/L) e de AST (>120 U/L) indicaram de maneira efetiva a presença de destacado acúmulo de Cu nos tecidos hepáticos (1000 ppm). / The aim of this project is to evaluate the capacity of a molybdenum-rich mineral salt in the prevention of cumulative cooper poisoning (CCP) in sheep, through clinical and blood exams and hepatic copper and molybdenum concentrations. Twenty five Ile-de-France sheep were randomly distributed into five groups of five animals each, where group 1 received a 80% forrage and 20% concentrate diet, groups 2 and 3 received a 50% forrage and 50% concentrate diet, and groups 4 and 5 received the same diet as groups 2 and 3 with a daily supplementation of 150mg of copper sulphate until the end of the experiment (150 d). Groups 1, 3 and 5 received a mineral salt with 300 ppm of molybdenum. For three times during the experiment (day zero, 45th and 105th day) a liver biopsy was carried out to evaluate the degree of copper accumulation in this organ. Clinical examination was followed every two weeks, as far as blood and urine samples were withdrawn. Three sheep from group 4 and one sheep from group 5 presented clinical picture of CCP. There was no difference in the frequency of mortality between groups 4 and 5 (P = 0.56). The liver copper concentration was higher in sheep with CCP (2450 ppm) compared to sheep that not presented CCP (1518 ppm). The higher the ingestion of molybdenum in the diet the lower the liver copper concentration at the end of the experiment (r = -0.72). Sheep with CCP had higher liver zinc concentration. The presence of high activity of GGT (> 56.5 U/L) and AST (>120 U/L) indicated effectively the high accumulation of copper in liver (> 1000 ppm).

O presente trabalho teve como objetivo avaliar os efeitos de diferentes condições nutricionais (suplementação e não suplementação) na atividade cíclica reprodutiva (ACR) em ovelhas das raças Santa Inês (SI), Romney Marsh (RM) e Suffolk (SU) mantidas sob pastejo, e submetidas ao efeito macho, durante época de anestro sazonal sob fotoperíodo natural no Estado de São Paulo. Avaliou-se também os efeitos destes tratamentos nas concentrações plasmáticas de melatonina (MEL) e na duração (horas) da secreção noturna deste hormônio. Todos animais foram mantidos sob pastejo, sendo que metade de cada raça recebeu suplementação e a outra não, caracterizando os tratamentos de suplementação e não suplementação, respectivamente. O tratamento de suplementação forneceu de 100 a 110% dos requerimentos protéicos (PB) e de 130 a 140% dos requerimentos energéticos (NDT), enquanto que o tratamento de não suplementação forneceu de 60 a 70% de PB e 100% de NDT. Os tratamentos nutricionais iniciaram 21 dias antes da introdução dos machos, e continuaram por todo o período de permanência dos mesmos (45 dias). Para o efeito macho, foram utilizados três machos (rufiões), impregnados com tinta na região prepucial para detecção do estro, os quais permaneceram isolados das fêmeas por um período de 60 dias. Colheitas de sangue foram realizadas antes (a cada 3 dias) e após (a cada 2 dias) a introdução dos machos, para dosagem de progesterona plasmática, a fim de monitorar a ACR das fêmeas. Foram também realizadas colheitas de sangue para dosagem de MEL plasmática: antes do início do experimento, antes da introdução dos machos e no final do experimento, em período de 24 horas, a cada 2 (noite) ou 4 (dia) horas. Os parâmetros avaliados foram: ganho de peso, ACR após introdução dos machos nos animais que se encontravam em anestro, dias para retorno da ACR, concentrações plasmáticas de MEL, duração da secreção de MEL. Estes parâmetros foram analisados utilizando o procedimento GLM no SAS, seguido pelo teste de ukey para estabelecer a comparação entre médias. O tratamento nutricional teve efeito no ganho de peso (P<0,001), no número de dias para retorno da ACR (P<0,1), nas concentrações de MEL (P<0,1) e na duração da secreção deste hormônio (P<0,1). Animais suplementados apresentaram em média, maior ganho de peso, maiores concentrações de MEL plasmática, e maior duração da secreção deste hormônio. Diferenças raciais foram encontradas para ganho de peso, retorno da ACR e concentrações plasmáticas de MEL. Fêmeas SI, perderam peso, enquanto que as RM mantiveram e as SU ganharam peso durante todo o período experimental, independente do tratamento nutricional. Todas as fêmeas SI retornaram à ACR em média 5 dias após a introdução dos machos, já as fêmeas SU suplementadas não retornaram à ACR, enquanto que as não suplementadas retornaram em 23 dias. Nenhuma ovelha RM retornou à ACR. Assim, conclui-se que para a raça SI só efeito macho é eficiente em ativar o retorno da ACR, e para a raça SU, deve-se aliar esta prática com um bom manejo nutricional, e para a RM, o efeito macho mesmo associado à nutrição não são capazes de amenizar a estacionalidade da ACR. E por fim, a nutrição afeta a duração da secreção de MEL. / This study evaluated the effects of different nutritional conditions (suplementation and no suplementation) in reproductive cyclic activity (RCA) for Santa Inês (SI), Romney Marsh (RM) and Suffolk (SU) breeds, in pasture, submitted to the male effect, during seasonal anoestrus, under natural photoperiod in São Paulo State. It was also evaluated the effects of nutritional treatments for plasmatic melatonin (MEL) concentration and for the duration of MEL secretion. All ewes were mainted in pasture, receiving food supplementation or not, distinguishing the supplementation or not supplementation treatments. The supplementation treatment supplied 100-110% of protein requeriments (CP) and 130-140% of energy requeriments (TDN). The no supplementation treatment supplied 60-70% CP and 100% TDN requeriments. The nutritional treatments were started 21 days before the rams introduction and lasted throughout the permanence period (45 days). To the male effect, it used three vasectomized rams impregnated with ink at the prepucial area in order to facilitate the estrus verification, isolated from the females for 60 days. Blood samples were collected before (every 3 days) and after (every 2 days) the rams introduction to determine de plasmatic progesterone to follow up the ewes reproductive cyclic activity (RCA). Blood samples were also collected to determine the plasmatic MEL dosage: before the beginning of the experiment, before the rams introduction and in the end of the experiment, during 24 hours, every 2 dark hours or 4 light hours. There was evaluated the following parameters: weight gain, RCA after male introduction for the ewes in anoestrus, days to RCA return, MEL concentration and MEL secretion duration. The parameters were analyzed by analisis of variance using the GLM (General Linear Procedure) of the Statistical System (SAS) software. Tukey test was used to compare means. The nutritional treatment resulted efficient for weight gain (P<0,001), days to return RCA (P<0,1), MEL concentration (P<0,1) and the duration of its hormone secretion (P<0,1). Supplemented ewes showed higher weight gain, higher MEL plasmatic concentration and higher hormone secretion duration in average. There was verified breed differences for weight gain, RCA return and MEL plasmatic concentration. SI ewes lost weight in average, RM ewes maintained it and SU ewes gained weight during the whole experimental period not taking into account the nutritional treatment. SI ewes had, in average, their RCA returned 5 days after the rams introduction not taking into account the nutritional treatment. However, SU ewes presented different behaviours due treatments: the ones that received supplementation did not return the RCA while, the non supplemented ones returned the RCA within 23 days. RM ewes did not return RCA no matter which treatment had received. Therefore, one may conclude that for SI ewes the male effect is enough for the RCA return. For SU breed one should combine this practice to good nutritional management. And for RM neither the male effect nor the nutrition are capable to diminish the RCA seasonally. Finally, one may conclude that the nutrition affects the amount and duration of MEL secretion.

Para estudo de diferentes períodos de protocolos de indução do estro e de um novo dispositivo de progesterona para pequenos ruminantes (DPR - Tecnopec, Brasil) foram realizados dois experimentos em ovelhas da raça Texel durante a contraestação reprodutiva. Experimento 1: com o objetivo de comparar a eficiência de protocolos de indução do estro em períodos de nove versus doze dias e com a utilização de dispositivos novos versus usados através das taxas de gestação e manifestação de estro, foram utilizados 160 ovelhas Texel. Os animais foram divididos aleatoriamente em 4 grupos: protocolo de 9 dias com a utilização de um dispositivo feito em silicone novo (G9N, n=91), protocolo de 9 dias com a utilização de dispositivo usado (G9U, n=25), protocolo de 12 dias com a utilização de dispositivo novo (G12N, n=26) e protocolo de 12 dias com a utilização de dispositivo usado (G12U, n=18). Os dispositivos foram colocados no dia zero (D0) e retirados no dia nove (D9) nos grupos G9N e G9U e doze dias após (D12) nos grupos G12N e G12U. Junto com a aplicação de 0,125mg de cloprostenol (Estron, Agener, Brasil) e 500UI de eCG (Folligon®, Intervet, Netherland). Experimento 2: com o objetivo de comparar o Dispositivo Pequenos Ruminantes (DPR - Tecnopec, Brasil) versus esponja impregnada com medroxiprogesterona Progespon® (MSD, Brasil) no protocolo de 9 dias foram utilizadas 152 ovelhas Texel. Os animais foram divididos aleatoriamente em 2 grupos: protocolo de 9 dias com a utilização da esponja Progespon® (GP, n=61) e protocolo de 9 dias com a utilização do Dispositivo Pequenos Ruminantes (GDPR, n=91). O protocolo utilizado foi semelhante ao G9N do experimento 1 exceto pelo tipo de dispositivo utilizado. No experimento 1 e 2 as variáveis analisadas foram: taxas de manifestação de estro, concepção e prenhez e dispersão no intervalo de cio. O exame de ultrassonografia para detecção de prenhez (Chisson, KYLUMAX) foi realizado com 30 e 60 dias da retirada do dispositivo. O delineamento utilizado foi inteiramente casualizado e os dados analisados através do programa SAS System for Windows (SAS, 2000). Como resultados observou-se que não houve diferenças entre os protocolos de 9 e 12 dias, assim como entre os dois tipos de dispositivos em relação a taxa de manifestação de estro, concepção e prenhez. Houve maior taxa de manifestação de estro (P<0,05)) do dispositivo novo se comparado com o usado. Foi observado menor (P<0,05) dispersão na ocorrência de estro no protocolo de 9 dias utilizando o dispositivo novo. Conclui-se que os protocolos de 9 e 12 dias e ambos dispositivos são semelhantes quanto a eficácia de indução do estro em ovelhas Texel durante a contraestação reprodutiva e que o dispositivo novo apresenta maior taxa na manifestação de estro em comparação com o dispositivo usado. / Two experiments were conducted to study different periods of estrus induction and study of a new progesterone device for small ruminants (DPR - Tecnopec, Brazil) protocols in Texel ewes during the non-breeding season. Experiment 1: 160 Texel ewes were used in order to compare the efficiency of estrus induction protocols with the nine versus twelve days periods and the use of new versus used devices through the rates of estrus manifestation and pregnancy. The animals were randomly divided into 4 groups: 9 days protocol with the use of the new device (G9N, n = 91) and 9 days protocol with the use of used devices (G9U, n = 25) 12 days protocol with the use of the new device (G12N, n = 26) and 12 days protocol with use of used device (G12U, n = 18). The devices were placed on day zero (D0) and removed on day nine (D9) in the G9N and G9U groups and twelve days after (D12) in the groups G12U and G12N, together with the use of 0.125 mg of cloprostenol (Estron, Agener, Brazil) and 500 IU of eCG (Folligon®, Intervet, Netherland). Experiment 2: 152 Texel ewes were used in order to compare the Small Ruminants Device (DPR - Tecnopec, Brazil) versus Sponge impregnated with medroxyprogesterone Progespon® (MSD, Brazil) in the 9 days protocol. The animals were randomly divided into 2 groups: 9 days protocol using sponge Progespon® (GP, n = 61) and 9 days protocol using Small Ruminants Device (GDPR, n = 91). The protocol used was similar to that G9N of experiment 1 except by the type of device used. 312 sheep were used in the period of September to November 2012. The considered methodologies in experiment 1 were: rates of estrus manifestation, conception and pregnancy and dispersion in the heat interval. The ultrasound examination for detection of pregnancy (Chisson, KYLUMAX) was performed at 30 and 60 days after D9 and D12. The experimental design was completely randomized and analyzed using the SAS System for Windows (SAS, 2000) program. As results it was shown no differences between the protocols 9 and 12 days, as well as between the two types of devices in relation to the estrus manifestation, conception and pregnancy rate. There was a higher estrus manifestation rate (P <0.05) of the new device compared with the used one. It was observed lower (P <0.05) dispersion in the occurrence of estrus in the 9 days protocol. We conclude that the protocols 9 and 12 days and both devices are similar in efficacy to induce estrus in Texel ewes during the non-breeding season and the new device has the highest rate of estrus manifestation compared to the used device.

This research was undertaken in order to design techniques and processes that enable the manufacture of effective antivenoms. To prepare a broad specificity anti venom for European vipers from chicken yolk it was first necessary to develop a simple effective method to extract avian immunoglobulin (lgY). A specific fluoroimmunoassay was developed to monitor IgY recovery and serum IgY levels in immunised hens. The most promising extraction methods from the literature were compared using a triglyceride kit to monitor lipoprotein removed and SDS-PAGE and ELISA to monitor purity and activity respectively. Caprylic acid followed by ammonium sulphate proved the best method. Unfortunately only low levels of specific IgY were achieved and it was necessary to include an affinity purification step to demonstrate their effectiveness in an EDso test. Pepsin, papain and trypsin all produced Fab' fragments from IgY but only pepsin digested the resultant Fc fragments. Pepsin could also digest other proteins in egg yolk, thereby avoiding the need to salt fractionate IgY prior to its digestion with a consequent improvement in the recovery of Fab'. A small scale affinity purification (SSAP) assay was developed, characterised and used to determine specific antibody levels in ovine antisera. Small doses (l5IAg) of venom produced significant specific levels but larger doses produced a better response and were used to produce anti venom. Binding studies with SSAP demonstrated a high concentration of specific antibodies in V.latastei antisera that bind to components in the venoms of other European vipers. A specific ovine F(ab')2-based V.latastei antivenom approximately twice as potent as the anti venom used currently in Spain was prepared from the ovine antisera. Evidence is presented that SSAP should supersede manual ELISA for assessing specific antibody levels in antisera. No major gain in recovery and purity resulted from processing whole blood rather than serum for preparing antivenom.

Para estudo de diferentes períodos de protocolos de indução do estro e de um novo dispositivo de progesterona para pequenos ruminantes (DPR - Tecnopec, Brasil) foram realizados dois experimentos em ovelhas da raça Texel durante a contraestação reprodutiva. Experimento 1: com o objetivo de comparar a eficiência de protocolos de indução do estro em períodos de nove versus doze dias e com a utilização de dispositivos novos versus usados através das taxas de gestação e manifestação de estro, foram utilizados 160 ovelhas Texel. Os animais foram divididos aleatoriamente em 4 grupos: protocolo de 9 dias com a utilização de um dispositivo feito em silicone novo (G9N, n=91), protocolo de 9 dias com a utilização de dispositivo usado (G9U, n=25), protocolo de 12 dias com a utilização de dispositivo novo (G12N, n=26) e protocolo de 12 dias com a utilização de dispositivo usado (G12U, n=18). Os dispositivos foram colocados no dia zero (D0) e retirados no dia nove (D9) nos grupos G9N e G9U e doze dias após (D12) nos grupos G12N e G12U. Junto com a aplicação de 0,125mg de cloprostenol (Estron, Agener, Brasil) e 500UI de eCG (Folligon®, Intervet, Netherland). Experimento 2: com o objetivo de comparar o Dispositivo Pequenos Ruminantes (DPR - Tecnopec, Brasil) versus esponja impregnada com medroxiprogesterona Progespon® (MSD, Brasil) no protocolo de 9 dias foram utilizadas 152 ovelhas Texel. Os animais foram divididos aleatoriamente em 2 grupos: protocolo de 9 dias com a utilização da esponja Progespon® (GP, n=61) e protocolo de 9 dias com a utilização do Dispositivo Pequenos Ruminantes (GDPR, n=91). O protocolo utilizado foi semelhante ao G9N do experimento 1 exceto pelo tipo de dispositivo utilizado. No experimento 1 e 2 as variáveis analisadas foram: taxas de manifestação de estro, concepção e prenhez e dispersão no intervalo de cio. O exame de ultrassonografia para detecção de prenhez (Chisson, KYLUMAX) foi realizado com 30 e 60 dias da retirada do dispositivo. O delineamento utilizado foi inteiramente casualizado e os dados analisados através do programa SAS System for Windows (SAS, 2000). Como resultados observou-se que não houve diferenças entre os protocolos de 9 e 12 dias, assim como entre os dois tipos de dispositivos em relação a taxa de manifestação de estro, concepção e prenhez. Houve maior taxa de manifestação de estro (P<0,05)) do dispositivo novo se comparado com o usado. Foi observado menor (P<0,05) dispersão na ocorrência de estro no protocolo de 9 dias utilizando o dispositivo novo. Conclui-se que os protocolos de 9 e 12 dias e ambos dispositivos são semelhantes quanto a eficácia de indução do estro em ovelhas Texel durante a contraestação reprodutiva e que o dispositivo novo apresenta maior taxa na manifestação de estro em comparação com o dispositivo usado. / Two experiments were conducted to study different periods of estrus induction and study of a new progesterone device for small ruminants (DPR - Tecnopec, Brazil) protocols in Texel ewes during the non-breeding season. Experiment 1: 160 Texel ewes were used in order to compare the efficiency of estrus induction protocols with the nine versus twelve days periods and the use of new versus used devices through the rates of estrus manifestation and pregnancy. The animals were randomly divided into 4 groups: 9 days protocol with the use of the new device (G9N, n = 91) and 9 days protocol with the use of used devices (G9U, n = 25) 12 days protocol with the use of the new device (G12N, n = 26) and 12 days protocol with use of used device (G12U, n = 18). The devices were placed on day zero (D0) and removed on day nine (D9) in the G9N and G9U groups and twelve days after (D12) in the groups G12U and G12N, together with the use of 0.125 mg of cloprostenol (Estron, Agener, Brazil) and 500 IU of eCG (Folligon®, Intervet, Netherland). Experiment 2: 152 Texel ewes were used in order to compare the Small Ruminants Device (DPR - Tecnopec, Brazil) versus Sponge impregnated with medroxyprogesterone Progespon® (MSD, Brazil) in the 9 days protocol. The animals were randomly divided into 2 groups: 9 days protocol using sponge Progespon® (GP, n = 61) and 9 days protocol using Small Ruminants Device (GDPR, n = 91). The protocol used was similar to that G9N of experiment 1 except by the type of device used. 312 sheep were used in the period of September to November 2012. The considered methodologies in experiment 1 were: rates of estrus manifestation, conception and pregnancy and dispersion in the heat interval. The ultrasound examination for detection of pregnancy (Chisson, KYLUMAX) was performed at 30 and 60 days after D9 and D12. The experimental design was completely randomized and analyzed using the SAS System for Windows (SAS, 2000) program. As results it was shown no differences between the protocols 9 and 12 days, as well as between the two types of devices in relation to the estrus manifestation, conception and pregnancy rate. There was a higher estrus manifestation rate (P <0.05) of the new device compared with the used one. It was observed lower (P <0.05) dispersion in the occurrence of estrus in the 9 days protocol. We conclude that the protocols 9 and 12 days and both devices are similar in efficacy to induce estrus in Texel ewes during the non-breeding season and the new device has the highest rate of estrus manifestation compared to the used device.