As Espécies Reativas de Oxigênio (EROs) conferem proteção aos seres vivos contra os contínuos ataques de microrganismos, além de serem responsáveis por diversos eventos fisiológicos. No entanto um desequilíbrio entre a produção das EROs e os agentes antioxidantes resulta diversos processos patológicos nos mais variados sistemas em seres humanos e em animais. Durante a gestação ocorrem alterações do metabolismo que podem levar a um aumento de subprodutos da oxidação. Assim como a gestação, o nascimento também impõe um período com alta demanda energética, alta tensão de oxigênio e, por consequência, determina um momento crítico na vida do neonato, por ser exigida uma rápida adaptação da condição hipóxica (intra útero) para hiperóxica (extra útero). Desta forma, o objetivo desta dissertação foi verificar o estado oxidativo das éguas no terço final da gestação, no periparto e no pós-parto levando em consideração o efeito da paridade, bem como a condição oxidativa de neonatos durante os primeiros sete dias de vida. Como indicadores de oxidação foram mensurados os níveis de TBARS e oxidação de proteína. Foi quantificado o ferro total e como parâmetros antioxidantes, foram medidas as atividades das enzimas GPx e SOD, e as concentrações de bilirrubina. Nos potros verificamos que a SOD não apresentou diferença significativa no periodo analisado. As concentrações de bilirrubina foram mais baixas no primeiro tempo avaliado, e tanto a bilirrubina total quanto a indireta elevaram-se às 12 horas e então caíram entre as 72h e 168h. Já a GPx demonstrou aumento da sua atividade nos tempos 12 e 72h quando comparada ao tempo 0h. Verificou-se altos níveis de TBARS no primeiro momento pós-nascimento, uma conseguinte diminuição às 12h seguida de estabilização nos demais tempos. Já a avaliação do período gestacional das éguas indicou um efeito de interação entre paridade e tempo gestacional apenas para o ferro total. SOD e oxidação proteica não apresentaram alterações significantes no período estudado. Tanto a GPx quanto as TBARS apresentaram efeito de tempo, com evidente alteração entre o parto, apresentando aumento e o pós-parto apresentando diminuição de atividade e das concentrações dessas variáveis. Concluímos que em potros, a peroxidação lipídica ao nascimento apresentou-se alta sugerindo um balanço pró-oxidativo durante tal período, o que poderia caracterizar um aumento nos níveis de EROs com finalidade de completar importantes eventos fisiológicos. Quanto a bilirrubina indireta e a GPx podemos sugerir que frente aos altos níveis da peroxidação lipídica houve um estímulo para ativação dos sistemas antioxidantes que envolvem essas biomoléculas e que as duas tenham agido concomitantemente visando equilibrar os níveis de EROs. Com relação às éguas, apontamos que a paridade não tem influencia sobre o estabelecimento da homeostase oxidativa em éguas e que no momento do parto as mesmas passam por um desbalanço oxidativo transiente. Ou seja, o desbalanço oxidativo faz parte tanto do momento do parto quanto da primeira semana de vida dos potros, possivelmente desempenhando um papel fisiológico em abas categorias. / Reactive Oxygen Species (ROS) produced protect living beings against the continuous attacks of microorganisms, as well as being responsible for several physiological events, so oxidative homeostasis is a premise. However, an imbalance between the production of ROS and the antioxidant agents causes several pathological processes in the most varied systems in humans and animals. During pregnancy it is suggested that alterations of the metabolism take place, consequently the increase of by-products of the oxidation during the gestational period. Like gestation, the birth also imposes a period with high energy demand, high oxygen tension and, consequently, it determines a critical moment in the life of the newborn because it is required a rapid adaptation of the same to the change of hypoxic condition (intra uterus) To hyperoxic (extra uterus). The aim of this dissertation was to verify the oxidative status of mares in the final third of gestation, peripartum and postpartum, taking into account the parity effect, as well as the oxidative condition of neonates during the first seven days of life. As oxidation indicators, the levels of TBARS and protein oxidation were measured. Total iron was quantified and as antioxidant parameters, activities of GPx and SOD enzymes, and bilirubin concentrations were measured. In the foals we verified that the SOD showed no significant difference in the analyzed time. Bilirubin concentrations were lower in the first time evaluated, and both total and indirect bilirubin increased at 12 hours and then fell between 72h and 168h. On the other hand, GPx showed an increase in its activity in times 12 and 72h when compared to time 0h. There were high levels of TBARS at the first post-birth moment, a consequent decrease at 12h, followed by stabilization at the other times. The results with mares indicated interaction effect between parity and gestational time only for total iron. SOD and protein oxidation did not present significant alterations in the studied period. Both GPx and TBARS presented a time effect, with an evident alteration between childbirth and postpartum, and there was an increase and decrease, respectively, in the activity and concentrations of these variables. We conclude that in foals, lipid peroxidation at birth was high suggesting a pro-oxidative balance during such period, which could characterize an increase in the levels of ROS in order to complete important physiological events. Regarding indirect bilirubin and GPx, we can suggest that in the face of the high levels of lipid peroxidation there was a stimulus for the activation of the antioxidant systems that involve these biomolecules and that the two have acted concomitantly in order to maintain the high levels of EROs at non detrimental levels. Regarding mares, we pointed out that parity has no influence on the establishment of oxidative homeostasis in mares and that at the time of delivery they undergo transient oxidative imbalance. That is, oxidative imbalance is part of both the calving moment and the first week of life of foals, possibly playing a physiological role in categories.

Com a utilização de 18 éguas, adultas, sem raça definida, com peso médio de 521±56 Kg, em delineamento inteiramente casualisado com três tratamentos e seis repetições por tratamento com medidas repetidas no tempo. Os objetivos deste trabalho foi verificar os efeitos da suplementação dietética com ácidos graxos ômegas 3 e ômega 6 sobre a composição do colostro e leite de éguas e transferência imunitária para os potros. Diferenças significativas encontradas foram na qualidade do leite das éguas onde foi observado efeito de tempo (semanas) sobre as variáveis de gordura, proteína, lactose e sólidos totais. Para a variável sólidos totais observou-se efeito do tratamento, semana e da inclusão de óleo na dieta. Observou-se nos potros uma atividade linfoproliferativa, no grupo óleo de soja semelhante ao encontrado no grupo controle, em ambos os períodos analisados. O grupo óleo de linhaça apresentou atividade linfoproliferativa maior do que a encontrada nos dois outros grupos no dia sete. No dia trinta, o grupo de linhaça continuou a apresentar uma maior atividade proliferativa, no entanto sem diferença estatística. A suplementação fonte de óleo aumentou a energia dietética disponível e a resposta proliferativa das éguas do dia sete. Observou-se que a suplementação com óleo de linhaça promoveu um aumento das concentrações dos isótopos IgGa e IgGb no colostro. A concentração média de IgGa no grupo óleo de linhaça foi quase 4 vezes superior aquela encontrada no colostro das éguas do grupo controle e quase 3 vezes superior aquela encontrada no grupo suplementado com óleo de soja com uma significância de. Assim concluímos que o ômega 3 melhorou a posta linfoproliferativa e aumentou a concentração de IgGb, porem não alterou os componentes do leite. / With the use of 18 mares, adult mongrel with a mean weight of 521 ± 56 kg in completely randomized design with three treatments and six replicates per treatment with repeated measures. The objectives of this study was to determine the effects of dietary supplementation with fatty acids omega 3 and omega 6 on the composition of colostrum and milk of mares and foals for immune transfer. Significant differences were found in milk quality mares where was no effect of time (weeks) on the variables of fat, protein, lactose and total solids. For variable total solids was observed effect of treatment week and oil inclusion in the diet. It was observed in foals lymphoproliferative activity in the soybean oil group similar to that found in the control group in both periods analyzed. The group linseed oil lymphoproliferative showed activity greater than that found in the other two groups on day seven. On the thirtieth day, the flaxseed group continued to show a higher proliferative activity, however no statistical difference. The source of oil supplementation increased dietary energy available and the proliferative response of mares of day seven. It was observed that supplementation with flaxseed oil promoted increases in concentrations of isotopes and IgGa IgGb in colostrum. The average concentration of IgGa group in linseed oil was almost 4 times higher than that found in colostrum of mares in the control group and almost 3 times higher than that found in the group supplemented with soybean oil with a significance. Thus we conclude that omega 3 put lymphoproliferative improved and increased the concentration of Ig, but it does not change the components of milk.

<i>Rhodococcus equi</i> (<i>R. equi</i>) is an intracellular, gram-positive coccobacillus that causes pneumonia in foals aged 2 to 4 months. Neonatal foals are susceptible to <i>R. equi</i> infection probably due to inefficient Toll-like receptor (TLR)-2 signaling and inability to produce interferon gamma. One of the reasons for inefficient receptor signaling and recognition of <i>R. equi</i> by the foals immune system may be the inefficient sequestration of TLRs in lipid rafts, which act as signaling platforms. However, there are no protocols to isolate lipid rafts from equine cells and, therefore, no data on the association of TLRs with the lipid rafts in the lung cells of normal and infected foals. Because of the clinical importance of the disease, there is considerable interest in developing effective prophylactic methods, which in turn requires a better understanding of fundamental immunology of the foals. In this study, I have examined the effect of <i>R. equi</i> vaccination on the lung inflammation induced following challenge with <i>R. equi</i>. I also developed a protocol to isolate lipid rafts from broncho-alveolar lavage (BAL) cells and investigated the association of lipid rafts with TLRs.<p> In the first study, 15 mixed breed draft-type foals up to 7 weeks of age were studied. The foals were divided into control (n=7) and a vaccinated (n=8). The control foals were given 10 mL phosphate buffered saline intramuscularly while the vaccinated group was vaccinated on day 0 of the study followed by a booster on day 14. All the foals were challenged with <i>R. equi</i> (5x106 cells/mL into the dorso-caudal region of the right lung lobe). BAL was performed on day 14, 28 and 35 and all the foals were euthanized on day 49 of the study.<p> The study design did not leave any non-infected foal at the end of the experiment. Therefore, lung samples were obtained from two untreated control (non-vaccinated non-infected) foals from the Department of Veterinary Pathology, University of Saskatchewan were used. The data showed similar levels of lung inflammation in both the control and vaccinated foal groups based on BAL cytology, gross pathology and histopathology. Gross and histopathological studies indicated that both control and vaccinated foals developed granulomatous lesions. Immunohistology showed increased expression of TLR4, TLR2 and TNF alpha in alveolar septa and in some cases in the vascular endothelium and airway epithelium in the lungs of both groups compared to the untreated control foals. Western blots showed increased expression of TLR2 but not TLR4 in the lung extracts from both the vaccinated and the control foals. Vaccinated foals showed higher concentrations of TNF alpha(p=0.0219) in their BAL on day 28 but lower concentrations of IL-10 (p=0.0172) in their lung extracts collected on day 49 compared to the controls. There were no differences in IFN gamma and protein concentrations between the two groups.<p> To understand the role of lipid rafts in TLR4 and TLR2 signaling, I developed an efficient and simpler protocol to isolate lipid rafts from BAL cells of foals and confirmed their identity by localizing Flotillin-1 and GM-1 (fractions 6-9), which are lipid raft markers, and transferrin receptor (fractions 1-4) which is present in non-lipid raft fractions. Lung macrophages from naïve foals lacked sequestration of Flotillin-1 and GM-1 in the higher fractions compared to the vaccinated foals. Further, the data showed that while TLR4 and TLR2 were localized in most of the fractions (1-9) in control foal BAL collected on day 14 and 28, the TLR4 and TLR2 association was restricted to fractions 6-9 in the lipid rafts isolated from BAL cells of vaccinated foals. These data suggest that BAL cells of neonatal foals may not have effective signaling machinery because of lack of association of TLR2 and TLR4 with lipid rafts.<p> Taken together, the data show similar levels of lung inflammation in the control and vaccinated foals upon infection with <i>R. equi</i>. The vaccination, however, appeared to have some effect on the immunohistologic expression of TLR2, TLR4 and TNFalpha in the lung tissues, and increased association of TLR2 and TLR4 with the lipid raft fractions. Based on the higher expression of TNF alpha and lower expression of IL-10, the vaccinated foals may be more competent to mount an immune response against <i>R. equi</i>.

Graduation date: 1993

<i>Rhodococcus equi</i> (<i>R. equi</i>) is an intracellular, gram-positive coccobacillus that causes pneumonia in foals aged 2 to 4 months. Neonatal foals are susceptible to <i>R. equi</i> infection probably due to inefficient Toll-like receptor (TLR)-2 signaling and inability to produce interferon gamma. One of the reasons for inefficient receptor signaling and recognition of <i>R. equi</i> by the foals immune system may be the inefficient sequestration of TLRs in lipid rafts, which act as signaling platforms. However, there are no protocols to isolate lipid rafts from equine cells and, therefore, no data on the association of TLRs with the lipid rafts in the lung cells of normal and infected foals. Because of the clinical importance of the disease, there is considerable interest in developing effective prophylactic methods, which in turn requires a better understanding of fundamental immunology of the foals. In this study, I have examined the effect of <i>R. equi</i> vaccination on the lung inflammation induced following challenge with <i>R. equi</i>. I also developed a protocol to isolate lipid rafts from broncho-alveolar lavage (BAL) cells and investigated the association of lipid rafts with TLRs.<p> In the first study, 15 mixed breed draft-type foals up to 7 weeks of age were studied. The foals were divided into control (n=7) and a vaccinated (n=8). The control foals were given 10 mL phosphate buffered saline intramuscularly while the vaccinated group was vaccinated on day 0 of the study followed by a booster on day 14. All the foals were challenged with <i>R. equi</i> (5x106 cells/mL into the dorso-caudal region of the right lung lobe). BAL was performed on day 14, 28 and 35 and all the foals were euthanized on day 49 of the study.<p> The study design did not leave any non-infected foal at the end of the experiment. Therefore, lung samples were obtained from two untreated control (non-vaccinated non-infected) foals from the Department of Veterinary Pathology, University of Saskatchewan were used. The data showed similar levels of lung inflammation in both the control and vaccinated foal groups based on BAL cytology, gross pathology and histopathology. Gross and histopathological studies indicated that both control and vaccinated foals developed granulomatous lesions. Immunohistology showed increased expression of TLR4, TLR2 and TNF alpha in alveolar septa and in some cases in the vascular endothelium and airway epithelium in the lungs of both groups compared to the untreated control foals. Western blots showed increased expression of TLR2 but not TLR4 in the lung extracts from both the vaccinated and the control foals. Vaccinated foals showed higher concentrations of TNF alpha(p=0.0219) in their BAL on day 28 but lower concentrations of IL-10 (p=0.0172) in their lung extracts collected on day 49 compared to the controls. There were no differences in IFN gamma and protein concentrations between the two groups.<p> To understand the role of lipid rafts in TLR4 and TLR2 signaling, I developed an efficient and simpler protocol to isolate lipid rafts from BAL cells of foals and confirmed their identity by localizing Flotillin-1 and GM-1 (fractions 6-9), which are lipid raft markers, and transferrin receptor (fractions 1-4) which is present in non-lipid raft fractions. Lung macrophages from naïve foals lacked sequestration of Flotillin-1 and GM-1 in the higher fractions compared to the vaccinated foals. Further, the data showed that while TLR4 and TLR2 were localized in most of the fractions (1-9) in control foal BAL collected on day 14 and 28, the TLR4 and TLR2 association was restricted to fractions 6-9 in the lipid rafts isolated from BAL cells of vaccinated foals. These data suggest that BAL cells of neonatal foals may not have effective signaling machinery because of lack of association of TLR2 and TLR4 with lipid rafts.<p> Taken together, the data show similar levels of lung inflammation in the control and vaccinated foals upon infection with <i>R. equi</i>. The vaccination, however, appeared to have some effect on the immunohistologic expression of TLR2, TLR4 and TNFalpha in the lung tissues, and increased association of TLR2 and TLR4 with the lipid raft fractions. Based on the higher expression of TNF alpha and lower expression of IL-10, the vaccinated foals may be more competent to mount an immune response against <i>R. equi</i>.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2002. / Härtill 5 uppsatser.

A infecção por Rhodococcus equi é a causa mais comum de pneumonia em potros durante os primeiros 6 meses de idade e as medidas utilizadas no controle da infecção são freqüentemente ineficientes. Os protocolos de tratamento são longos, de alto custo e nem sempre eficazes. A prevenção da rodococose através da imunização de potros com vacina eficaz e segura tem sido um desafio. Estudos recentes se baseiam na utilização da proteína A associada a virulência (VapA), que tem sido descrita como importante indutora da imunidade contra R. equi. Este estudo avaliou a resposta clínica e imunitária desenvolvida por potros imunizados com cepa atenuada de S. enterica Typhimurium capaz de expressar o antígeno VapA (grupo teste) ou apenas S. enterica Typhimurium sem o gene vapA (grupo controle), antes e após o desafio experimental. Duas etapas experimentais foram estabelecidas de acordo com a via de imunização: intranasal ou oral em até 12 horas após o nascimento e aos 14 dias de idade. Os animais do grupo teste e do grupo controle foram desafiados experimentalmente com cepa virulenta de R. equi 28 dias após o nascimento. Para acompanhar o desenvolvimento dos sinais clínicos, foram realizados exames clínicos, hemogramas e exames complementares de imagem. O padrão de resposta imune desenvolvida foi avaliada através da dosagem de munoglobulinas, mensuração da expressão de citocinas, ensaio de proliferação de linfócitos, isolamento de R. equi e perfil citológico do LTB. As manifestações clínicas foram observadas de maneira menos intensa nos animais do grupo teste da segunda etapa experimental e óbitos decorrentes da rodococose ocorreram apenas nos animais do grupo controle (2/3). Os animais do grupo teste apresentaram uma resposta de IgGb mais intensa quando comparada aos animais do grupo controle, porém não foi observado diferença estatística. A linfoproliferação e a expressão de citocinas de padrão Th1 foram superiores nos animais do grupo teste. Em contraste, os animais do grupo controle apresentaram uma resposta caracterizada pela maior expressão da citocina IL-4. A produção de IgA local foi mais significativa nos animais imunizados com Salmonella carreando vapA. Não foram observados efeitos tóxicos graves resultantes da imunização em nenhuma das etapas experimentais. A vacinação de potros neonatos com S. enterica Typhimurium expressando VapA foi considerada segura, capaz de modular eficientemente a resposta imune e aparentemente capaz de proteger contra a infecção experimental com R. equi. / Rhodoccocus equi is the major cause of pneumonia in foals during the first six months and control measures are frequently ineffective. Treatment protocols are long, expensive and do not always produce good results. Rhodococcosis prevention through immunization of foals using a safe and efficient vaccine is still a challenge. Recent studies are based on the use of the virulence associated protein A (vapA) which has been described as an important inducer of immunity against R. equi. The present study evaluated the clinical and immune response of foals vaccinated with an attenuated strain of S. enterica Typhimurium expressing VapA antigen (test group) or S. enterica Typhimurium without the vapA gene (control group), previous to and following experimental challenge. Two experimental phases were established according to the immunization route: intranasal or oral vaccination up to 12 hrs following birth and at 14 days of age. The experimental and control groups were challenge on day 28 with a virulent stain of R. equi. Clinical examination, complete hemograms and image complementary exams were used to evaluate the development of clinical signs. Immune response patterns were evaluated though immunoglobulin dosage, cytokine expression, lymphocyte proliferation essays, isolation of R. equi and cytological profiles of TBW. Clinical manifestation was less intense in the test group during the second experimental phase, and death occurred only in the control group (2/3) and was due to R. equi pneumonia. The test group produced a more intense IgGb response when compared to controls however no statistical difference was observed. Lymphoproliferation and Th1 cytokine expression were higher in the test group. In contrast, controls produced an IL-4 response. Local IgA was significantly higher in animals immunized with Salmonella carrying vapA. Immunization protocols produced no severe toxic effect. The vaccination of neonatal foals with S. enterica Typhimurium expressing VapA was considered safe, produced efficient modulation of the immune response and is apparently able to protect against experimental R.equi infection.

Infecções pulmonares de potros jovens por Rhodococcus equi resultam em grave pneumonia levando à morte um grande número de animais todos os anos. Até o momento não há nenhuma vacina aceita globalmente para a prevenção da rodococose equina. O único tratamento preconizado é baseado em antibioticoterapia, porém os protocolos clínicos são longos, de alto custo, com efeitos colaterais e tem favorecido a seleção de cepas resistentes aos antibióticos. Diversas estratégias no desenvolvimento de uma vacina segura e eficiente contra a rodococose foram propostas, porém, não induziram um efeito protetor considerável. O principal fator de virulência do R. equi descrito e amplamente estudado é a proteína vapA, no entanto, outras proteínas localizadas na ilha de patogenicidade estão presentes em amostras de R. equi virulento extraídos de animais infectados. Trabalhos recentes têm demonstrado a presença do gene vapG em todas as cepas virulentas e este gene é altamente expresso quando a bactéria reside no interior de macrófagos, tornando-o um possível alvo vacinal. Nosso grupo já possui experiência prévia no uso de linhagem de Salmonella enterica Typhimurium atenuada carreando a proteína vapA. Baseado nos resultados positivos obtidos, foi construída uma linhagem atenuada de S. enterica Typhimurium 3987 expressando a proteína vapG. A administração desta linhagem em camundongos foi capaz de induzir proteção contra R. equi virulento. Os resultados observados foram a colonização e persistência da Salmonella nos órgãos alvo, a redução da carga bacteriana de R. equi, e a indução de um perfil imune protetor semelhante ao observado em animais adultos resistentes. Observou-se o aumento da produção de IL-12p70 e IFN-, alem da presença de níveis aumentados de IL-4 e a redução dos níveis de TNF-. Observou-se também o aumento na subpopulação de células T auxiliares (CD4+) com perfil de memória, além de aumento na população de linfócitos B totais quando comparado aos grupos controles. Este conjunto de resultados indica que a imunização com Salmonella enterica Typhimurium expressando a proteína vapG gera uma resposta imune celular eficiente tornando esta linhagem uma possível candidata a vetor vacinal. Alem disso, sugere que outros antígenos do R. equi podem ser / Pulmonary infections in young foals by Rhodococcus equi result in severe pneumonia, leading to death a large number of animals every year. There is no globally accepted vaccine for the prevention of equine rhodococosis so far. To date, the only acceptable treatment is based on antibiotics, but the clinical protocols are long lasting, expensive, having side effects and favoring the emergence of drug resistant strains. Several strategies for developing a safe and effective vaccine against rhodococosis have been proposed, however, none has induced a significant protective effect. The main virulence factor of R. equi described and extensively studied is the protein vapA, although other proteins encoded by genes in pathogenicity islands are detected in samples of virulent R. equi, isolated from infected animals. Recent works have demonstrated that the vapG gene is present in all virulent strains, and that this gene is highly expressed when bacteria reside within macrophages, making it a potential vaccine target. Our group has already been working with an attenuated Salmonella enterica Typhimurium strain expressing the VapA protein. Even though our results were very promising, we decided to construct a second vaccine strain expressing the VapG protein. Based on the positive results, it was constructed an attenuated strain of S. enterica Typhimurium 3987 expressing the vapG protein. Interestingly, the VapG-expressing strain induced protection against virulent R. equi in mouse model of infection. We could observe colonization and persistence of Salmonella vaccine cells in target organs, with reduction of bacterial loads of R. equi and induction of a protective immune profile similar to that seen in resistant adult animals. We could also observe increased production of IL-12p70 and IFN- in addition to the presence of increased levels of IL-4 and reduced levels of TNF-. Moreover, we detected an increase in the subpopulation of T helper cells (CD4), with a profile of memory, as well as in the population of B lymphocytes, when compared to control groups. This set of results shows that immunization with Salmonella enterica Typhimurium expressing the VapG protein raises an efficient cellular immune response, making this strain a potential candidate for vaccine vector. Furthermore, this work suggests that other R. equi antigens may be taken into account for vaccine construction, besides the VapA protein utilized in the majority of studies.

Com a utilização de 18 éguas, adultas, sem raça definida, com peso médio de 521±56 Kg, em delineamento inteiramente casualisado com três tratamentos e seis repetições por tratamento com medidas repetidas no tempo. Os objetivos deste trabalho foi verificar os efeitos da suplementação dietética com ácidos graxos ômegas 3 e ômega 6 sobre a composição do colostro e leite de éguas e transferência imunitária para os potros. Diferenças significativas encontradas foram na qualidade do leite das éguas onde foi observado efeito de tempo (semanas) sobre as variáveis de gordura, proteína, lactose e sólidos totais. Para a variável sólidos totais observou-se efeito do tratamento, semana e da inclusão de óleo na dieta. Observou-se nos potros uma atividade linfoproliferativa, no grupo óleo de soja semelhante ao encontrado no grupo controle, em ambos os períodos analisados. O grupo óleo de linhaça apresentou atividade linfoproliferativa maior do que a encontrada nos dois outros grupos no dia sete. No dia trinta, o grupo de linhaça continuou a apresentar uma maior atividade proliferativa, no entanto sem diferença estatística. A suplementação fonte de óleo aumentou a energia dietética disponível e a resposta proliferativa das éguas do dia sete. Observou-se que a suplementação com óleo de linhaça promoveu um aumento das concentrações dos isótopos IgGa e IgGb no colostro. A concentração média de IgGa no grupo óleo de linhaça foi quase 4 vezes superior aquela encontrada no colostro das éguas do grupo controle e quase 3 vezes superior aquela encontrada no grupo suplementado com óleo de soja com uma significância de. Assim concluímos que o ômega 3 melhorou a posta linfoproliferativa e aumentou a concentração de IgGb, porem não alterou os componentes do leite. / With the use of 18 mares, adult mongrel with a mean weight of 521 ± 56 kg in completely randomized design with three treatments and six replicates per treatment with repeated measures. The objectives of this study was to determine the effects of dietary supplementation with fatty acids omega 3 and omega 6 on the composition of colostrum and milk of mares and foals for immune transfer. Significant differences were found in milk quality mares where was no effect of time (weeks) on the variables of fat, protein, lactose and total solids. For variable total solids was observed effect of treatment week and oil inclusion in the diet. It was observed in foals lymphoproliferative activity in the soybean oil group similar to that found in the control group in both periods analyzed. The group linseed oil lymphoproliferative showed activity greater than that found in the other two groups on day seven. On the thirtieth day, the flaxseed group continued to show a higher proliferative activity, however no statistical difference. The source of oil supplementation increased dietary energy available and the proliferative response of mares of day seven. It was observed that supplementation with flaxseed oil promoted increases in concentrations of isotopes and IgGa IgGb in colostrum. The average concentration of IgGa group in linseed oil was almost 4 times higher than that found in colostrum of mares in the control group and almost 3 times higher than that found in the group supplemented with soybean oil with a significance. Thus we conclude that omega 3 put lymphoproliferative improved and increased the concentration of Ig, but it does not change the components of milk.

Group size affects individual and collective vigilance levels of prey species. As individual vigilance decreases with increasing group size, the indirect risk of predation to each individual and the group as a whole will also decrease (dilution and many-eyes effect) which may have a decreasing effect on stress responses on group level as well. Where predation risk is low, other factors like group size might influence stress-related glucocorticoid output in prey species. I test the relationship between group size and individual and collective vigilance levels in a plains zebra (Equus burchelli) population living in an environment with low adult predation risk due to the absence of lions (Panthera leo) in the Dinokeng Game Reserve (DGR). I also test for an effect of season (wet vs. dry) on these levels. Vigilance levels are expected to be higher in summer than winter, due to a seasonal influence on susceptibility to predation or the ease of predator detection. Finally, I test if the presence of foals in a band influences individual and collective vigilance levels. Mothers are expected to increase their vigilance levels since foals are most susceptible to predation by smaller predators in the reserve. I also aimed to specifically investigate how group size affects adrenocortical endocrine activity in male and female individuals of plains zebra bands living in an environment with low predation risk. With an increase in group size, and individual vigilance expecting to decrease, the indirect risk of predation to each individual and the group as a whole will also decrease (due to both dilution and many-eyes effect) which may decrease stress on group level as well. Affiliative social interactions between females could also have the effect of improving fitness and reducing stress levels in this species. We therefore evaluated the reliability of different enzyme-immunoassays (EIA) for monitoring glucocorticoid metabolite levels in plains zebra faeces by conducting an adrenocorticotropic hormone (ACTH) challenge test and associated translocation event. Individual vigilance decreased with group size, confirming a classic group size effect; while collective vigilance remained constant meaning individuals could reduce their own scanning and rely on scanning by other group members. Individual vigilance differs between seasons, with higher levels recorded in summer, but no seasonal effect is found on rate of scanning and collective vigilance. Interestingly, bands with foals have a lower level of individual vigilance than bands without foals, which may be due to added nutritional stress on mothers during lactation. A constant collective vigilance level means mothers may be able to increase foraging time without increasing predation risk. I successfully identified two group-specific EIAs as suitable for assessing adrenocortical endocrine activity in male and female plains zebra, but gender-specific differences in response to ACTH administration and translocation were detected. In winter, bands of free-ranging plains zebra differ significantly in terms of faecal glucocorticoid metabolite (FGM) output, but due to the fact that the bands tested also differed in size as well as composition, the potential band size effect were analysed for males and females separately. Males in bands of greater size exhibit higher FGM levels than males in small bands; explained by the females’ need for protection, resource competition and reproduction pressure. FGM levels in females did not differ between bands of different sizes, indicating that potential changes in vigilance behaviour per se might not alter FGM output in a low-predation risk environment. This study confirms firstly, the classic group size effect on vigilance in plains zebra in this low predation risk environment and secondly, that group size and sex should be considered when looking at FGM levels in this social species. / Dissertation (MSc)--University of Pretoria, 2012. / Zoology and Entomology / unrestricted