Secondary plant metabolites have recently been gaining interest in livestock production systems following the ban of in-feed antibiotics within the European Union. The rise in antimicrobial resistance found in pathogenic and non-pathogenic bacteria has lead to increased interest in the research community regarding the use of phythochemicals as an alternative to antibiotics. The purpose of this research was to evaluate the impact of including phytochemicals in a livestock production system. Specifically, a high tannin-containing forage, sainfoin (Onobrychis viciifolia), was evaluated in vitro for its antimicrobial effect on Escherichia coli. We determined that phytochemicals alone are not as inhibitory as synthetic antibiotics. Thus, the use of combination therapy to deter the development of antimicrobial resistance was evaluated. A myriad of plant compounds were screened for their synergistic interactions with ciprofloxacin. Geraniol, an essential oil, was identified to possess good antimicrobial activity and synergistic interactions with ciprofloxacin. Therefore the effect of long term exposure to both ciprofloxacin and geraniol were examined. Results demonstrated that once an antimicrobial concentration threshold was reached, resistance to ciprofloxacin increased markedly in the presence of both geraniol and ciprofloxacin. Finally, an in vivo trial was conducted in which forty steers were fed sainfoin or alfalfa over a 9-week period to evaluate its ability to reduce E. coli shedding and its impact on gut microbiota in the context of popular theoretical ecology concepts. Results from the in vivo study indicate that sainfoin was able to promote a slight decrease in generic E. coli shedding which could be maintained throughout the trial. Using high-throughput sequencing, the effect of sainfoin on the microbial ecosystem of the ruminant gut was evaluated. Sainfoin induced a significant shift in the microbial community structure of the rumen and to a lesser extent in the hindgut. Using ecology theories, a hypothesis was formulated regarding the mechanisms that mediate the development of tolerance and the fundamental ecological processes controlling microbial population shifts. Understanding how the gut ecosystem functions and predicting its behaviour in the presence of various fluctuating environmental conditions will enable more efficient manipulation of the rumen and promote best management practices in livestock production.

Secondary plant metabolites have recently been gaining interest in livestock production systems following the ban of in-feed antibiotics within the European Union. The rise in antimicrobial resistance found in pathogenic and non-pathogenic bacteria has lead to increased interest in the research community regarding the use of phythochemicals as an alternative to antibiotics. The purpose of this research was to evaluate the impact of including phytochemicals in a livestock production system. Specifically, a high tannin-containing forage, sainfoin (Onobrychis viciifolia), was evaluated in vitro for its antimicrobial effect on Escherichia coli. We determined that phytochemicals alone are not as inhibitory as synthetic antibiotics. Thus, the use of combination therapy to deter the development of antimicrobial resistance was evaluated. A myriad of plant compounds were screened for their synergistic interactions with ciprofloxacin. Geraniol, an essential oil, was identified to possess good antimicrobial activity and synergistic interactions with ciprofloxacin. Therefore the effect of long term exposure to both ciprofloxacin and geraniol were examined. Results demonstrated that once an antimicrobial concentration threshold was reached, resistance to ciprofloxacin increased markedly in the presence of both geraniol and ciprofloxacin. Finally, an in vivo trial was conducted in which forty steers were fed sainfoin or alfalfa over a 9-week period to evaluate its ability to reduce E. coli shedding and its impact on gut microbiota in the context of popular theoretical ecology concepts. Results from the in vivo study indicate that sainfoin was able to promote a slight decrease in generic E. coli shedding which could be maintained throughout the trial. Using high-throughput sequencing, the effect of sainfoin on the microbial ecosystem of the ruminant gut was evaluated. Sainfoin induced a significant shift in the microbial community structure of the rumen and to a lesser extent in the hindgut. Using ecology theories, a hypothesis was formulated regarding the mechanisms that mediate the development of tolerance and the fundamental ecological processes controlling microbial population shifts. Understanding how the gut ecosystem functions and predicting its behaviour in the presence of various fluctuating environmental conditions will enable more efficient manipulation of the rumen and promote best management practices in livestock production.

Submitted by Socorro Pontes (socorrop@ufersa.edu.br) on 2017-06-21T13:36:52Z No. of bitstreams: 1 TalytaLN_TESE.pdf: 1505855 bytes, checksum: f450d9b1a9eb7f3e1dc8fddf45518523 (MD5) / Approved for entry into archive by Vanessa Christiane (referencia@ufersa.edu.br) on 2017-06-22T11:39:09Z (GMT) No. of bitstreams: 1 TalytaLN_TESE.pdf: 1505855 bytes, checksum: f450d9b1a9eb7f3e1dc8fddf45518523 (MD5) / Approved for entry into archive by Vanessa Christiane (referencia@ufersa.edu.br) on 2017-06-22T11:41:55Z (GMT) No. of bitstreams: 1 TalytaLN_TESE.pdf: 1505855 bytes, checksum: f450d9b1a9eb7f3e1dc8fddf45518523 (MD5) / Made available in DSpace on 2017-06-22T11:42:26Z (GMT). No. of bitstreams: 1 TalytaLN_TESE.pdf: 1505855 bytes, checksum: f450d9b1a9eb7f3e1dc8fddf45518523 (MD5) Previous issue date: 2017-04-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Tramadol is an atypical opioid analgesic widely used in humans and in the routine of the small animal clinic. Due to the lack of studies that determine its pharmacokinetic and pharmacodynamic characteristics, its use in production animals is restricted. The objective of this research was to evaluate the clinical, familial and bioavailability effects of tramadol in goats. The study was divided into two phases: the first one evaluated the clinical effects of tramadol at doses of 2 and 4 mg / kg intravenously; In the second stage, the bioavailability and pharmacokinetic parameters of tramadol and O-desmethyltramadol were evaluated after intravenous and intramuscular administration at a dose of 4 mg / kg. Opioid has been shown to be safe and effective in the control of orchiectomy pain and should be given every 6 and 8 hours at doses of 2 and 4 mg / kg. The intramuscular opioid has bioavailability of 62%. The data from this research provide information on tramadol and its metabolites, and serve as the basis for future studies involving the drug and the plasm / O tramadol é um analgésico opioide atípico amplamente utilizado em humanos e na rotina da clínica de pequenos animais. Devido ausência de estudos que determinam as características farmacocinéticas e farmacodinâmicas, sua utilização em animais de produção é restrita. Objetivou-se com esta pesquisa avaliar os efeitos clínicos, farmacocinética e biodisponibilidade do tramadol em caprinos. O estudo foi dividido em duas fases: na primeira, realizou-se avaliação dos efeitos clínicos do tramadol nas doses de 2 e 4 mg/kg intravenoso em caprinos submetidos a orquiectomia e na segunda etapa, realizou-se avaliação da biodisponibilidade e dos parâmetros farmacocinéticos do tramadol e O-desmetiltramadol após administração intravenosa e intramuscular em caprinos, na dose de 4 mg/kg. O opioide mostrou-se seguro e eficaz no controle da dor da orquiectomia, devendo ser administrado a cada 6 e 8 horas, nas doses de 2 e 4 mg/kg. O opioide intramuscular tem biodisponibilidade de 62%. Os dados desta pesquisa fornecem informações relevantes a respeito do tramadol e seus metabólitos e servem de base para estudos futuros envolvendo o fármaco e a matriz biológica / 2017-06-21

A emissão de gases de efeito estufa (GEE) é uma das principais causas do aquecimento global, sendo uma problemática mundial das últimas décadas. O dióxido de carbono (CO2), o metano (CH4) e oxido nitroso (N2O) são os principais GEE e os ruminantes são uns dos maiores contribuintes com a produção desses gases no mundo, devido ao processo digestivo de fermentação entérica. Na busca de estratégias para diminuir as emissões de metano e melhorar a produtividade animal, aditivos alimentares têm sido utilizados nas dietas dos animais. Assim, o objetivo deste trabalho foi avaliar a inclusão de aditivos alimentares sobre a produção de metano determinada pela técnica do gás traçador SF6, assim como, o consumo de matéria seca, a dinâmica ruminal e a contagem total e diferencial de protozoários do rúmen. Seis vacas não-gestantes e não-lactantes, com peso vivo médio de 784 ± 87 kg e canuladas no rúmen, foram distribuídas a uma das três dietas experimentais, seguindo-se delineamento experimental em quadrado latino 3x3 replicado (n= 18 unidades experimentais), sendo os tratamentos: 1) Controle (CON): Dieta basal sem inclusão de aditivo; 2) Monensina (MON): Dieta com adição de 300 mg de monensina sódica por animal por dia; 3) Tanino (TAN): Dieta com adição de 68 g de extrato de tanino condensado de Acácia-negra (Acacia mearnsii) por animal por dia. Os alimentos foram fornecidos duas vezes ao dia, na forma de ração completa. Cada período experimental foi constituído por 21 dias, sendo 10 dias de adaptação às respectivas dietas. A partir do dia 16 até o dia 21 foram coletados os dados de consumo de matéria seca e de produção de metano, sendo este último determinado a cada 24 horas, pela técnica do gás traçador de SF6. No dia 21, coletou-se conteúdo ruminal para determinação de protozoários. Quanto à dinâmica ruminal, foi realizado o esvaziamento ruminal nos dias 10 (3 horas após alimentação matinal) e 11 (imediatamente antes da alimentação matinal) de cada período experimental. Os resultados foram analisados através do procedimento MIXED onde o modelo incluiu o efeito de tratamento como fator fixo e os efeitos de animal dentro de quadrado, quadrado e período como fatores aleatórios. Não houve diferenças significativas (P>0,05) entre os tratamentos para as variáveis do consumo de matéria seca, como também, para os parâmetros de dinâmica ruminal (P>0,05) (matéria seca do conteúdo ruminal, massa líquida, massa sólida, massa total, assim como taxa de desaparecimento). A emissão de metano (expressa em g/d, g/kg PV, g/kg PV0,75 ou Mcal/Ani/d) com o tratamento com monensina foi menor em relação ao tratamento controle. Para a contagem total e diferencial de protozoários foi verificado efeito de aditivo para a subfamília Diplodiniinae, sendo que, o tratamento com monensina diminuiu em 27,5% a contagem desta subfamília em relação ao tratamento com tanino. Referente ao gênero Isotricha, foi observado que os tratamentos com monensina ou com tanino diminuíram em 31 e 30% respectivamente, este gênero em relação ao tratamento controle. A adição de monensina (17 mg/kg de MSI) revela-se uma alternativa para reduzir as perdas energéticas geradas na produção de metano, assim como também na redução de protozoários, que albergam microrganismos metanogênicos. Em relação ao tanino (0,4% na dieta) acredita-se que com doses mais elevadas na dieta possa resultar numa redução da emissão de metano / The emission of greenhouse gases (GHG) is a major cause of global warming, being a worldwide concern in recent decades. Carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O) are the main greenhouse gases and ruminants are one of the major contributors to the production of these gases around the world, due to the enteric fermentation process. In the search for strategies to reduce methane emissions and to improve animal productivity, food additives have been used in animal diets lately. Thus, the aim of this trial was to assess the inclusion of food additives on methane emissions in cattle, using the sulfur hexafluoride (SF6) tracer technique, as well as on dry matter intake, rumen dynamics and total and differential counts of ruminal protozoa. Six non-pregnant and non-lactating rumen-cannulated cows (784 ± 87 kg) were assigned to a replicated 3x3 Latin square (18 experimental units). Treatments were: 1) Control (CON) basal diet with no additive inclusion; 2) Monensin (MON) addition of 300 mg of sodium monensin per animal per day, 3) Tannin (TAN) addition of 68 g of concentrated extract of condensed tannin (Acacia mearnsii) per animal per day. The animals were fed total mixed ration twice daily. Each experimental period consisted of 21 days the first 10 days were used for diet adaptation. From day 16 up to 21, data about dry matter intake and methane production were collected, the latter done every 24 hours using the sulfur hexafluoride (SF6) tracer technique. On the day 21, ruminal content was sampled for protozoa determination. Regarding the rumen dynamics, the rumen was emptied on days 10 (3 hours post-morning feeding) and 11 (right before morning feeding) of each experimental period. The results were analyzed by MIXED procedure; the model included the effect of treatment as fixed factor and the effects of period, square and animal within square as random factors. There were no significant differences (P>0.05) among treatments for dry mater intake variables, nor for ruminal dynamics parameters (ruminal content dry matter, liquid mass, solid mass, total mass or disappearance rate). Methane emission (expressed in g/day, g/kg LW, g/kg LW0.75 or Mcal/Ani/day) was lower for the group receiving monensin compared to the control group. For total and differential counts of protozoa, the additives affected the Diplodiniinae subfamily, i.e. monensin decreased the count of this subfamily by 27.5%, compared to tannin. Regarding the Isotricha genus, treatments with monensin or tannin decreased it by 31 and 30% respectively, compared to the control treatment. The addition of monensin (17 mg/kg DMI) revealed to be an alternative to reduce the energy lost by methane production, as well as to decrease the protozoa, which host methanogen microorganisms. Regarding tannin (0.4% in the diet), it is believed that higher doses in the diet can lead to a reduction in methane emission

A intensificação do sistema de produção da carne ovina é influenciada por algumas técnicas de manejo como a castração e o sistema de confinamento, tendo como objetivo produto de melhor qualidade sensorial e nutricional desejada pelo consumidor e maior rentabilidade para o produtor. Portanto, objetivou-se com este trabalho avaliar desempenho, características de carcaça e da carne, qualidade sensorial e perfil de ácidos graxos da carne de cordeiros não castrados e castrados abatidos em diferentes períodos de confinamento. Foram utilizados 48 machos Dorper x Santa Inês com 32,3 ± 5,04 kg de peso corporal e 104 dias de idade no início do experimento. Os animais foram alojados dois por baia de acordo com o peso inicial (bloco) e após 14 dias de adaptação ao local foram alimentados com uma dieta com 75% grão de milho inteiro, 20% de pelete protéico mineral e 5% de feno de capim coast cross. Os animais foram desmamados aos 90 dias de idade e após a adaptação vinte e quatro animais foram castrados com o burdizzo aos 113 dias de idade. A alimentação e as sobras foram pesadas diariamente para determinações de matéria seca e eficiência alimentar. Os animais foram pesados no início do experimento e a cada 14 dias. Foram perdidas duas parcelas experimentais. Os animais foram abatidos após 36 ou 78 dias de confinamento (metade de cada sexo) para determinação das características da carcaça e da carne e foram colhidas amostras do m. Longissimus dorsi para análise do perfil de ácidos graxos e análise sensorial com painel treinado. Em vista dos resultados obtidos para cordeiros jovens desse tipo de cruzamento e alimentados com esta dieta, os animais não castrados tiveram melhor desempenho, características de carcaça e de carne que os castrados, já na qualidade sensorial poucos resultados foram significativos enquanto que no perfil de ácidos graxos houve efeitos e interações entre a condição sexual e o tempo de confinamento, indicando que animais castrados abatidos aos 36 dias tiveram um carne mais magra, com maior porcentagem de n-3 e relação AGP:AGS. / The intensification of the production system of sheep meat is influenced by some management techniques such as castration and confinement system, aiming to produce better sensory and nutritional quality desired by consumers and more profitable for the producer. Therefore, the aim of this study was to evaluate performance, carcass characteristics and meat sensory quality and fatty acid profile of lamb meat from non-castrated and castrated slaughtered at different periods of confinement. We used 48 male Dorper x St. Agnes with 32.3 ± 5.04 kg body weight and 104 days old at the start of the experiment. The animals were housed two per cage according to the initial weight (block) and after 14 days of adaptation to site were fed a diet containing 75% whole grain corn, 20% protein pellet mineral and 5% grass hay Coast cross. The animals were weaned at 90 days of age and after adjustment twenty-four animals were castrated with burdizzo to 113 days of age. The food and leftovers were weighed daily for determinations of dry matter and feed efficiency. The animals were weighed at baseline and every 14 days. We lost two experimental plots. The animals were killed after 36 or 78 days of confinement (half of each sex) to determine the characteristics of the carcass and meat and samples were taken from m. Longissimus for analysis of fatty acid profile and sensory evaluation with trained panel. In view of the results obtained for young lambs this type of intersection and fed this diet, the animals were not castrated best performance, carcass characteristics and meat castrated, have sensory quality were few significant results while in acid profile acids were effects and interactions between the sexual condition and the time of confinement, indicating that castrated animals slaughtered at 36 days had a more lean meat, with higher n-3 and relationship AGP: AGS.

Fescue toxicosis is problematic for ruminant livestock, causing weight loss and low productivity when fed endophyte-infected forages. Complete underlying mechanisms of toxicosis are unknown therefore; the objective of the study was to determine if ruminally dosed ergot alkaloids impact rumen motility. Cannulated steers were pair-fed a forage diet and ruminally dosed with endophyte-free (E-) or endophyte-infected (E+) tall fescue seed. An 8-h period of rumen motility collection began 4-h after feeding by monitoring pressure change via a wireless telemetry and transducer system. In experiment 1, steers were paired by weight and assigned to E- or E+ treatment. Overall, E+ steers had more frequent contractions. On d 7 - 9, both treatments had lower frequencies and E- steers had greater amplitude of contractions, which corresponded with decreased DM intake. In experiment 2 steers remained in pair, but switched treatment. During the 57 d E+ steers received titrated levels of ergovaline + ergovalinine. There was no difference between treatments for frequency or amplitude of contractions, but increasing dosage, decreased frequency (d 1 - 44) and amplitude, coinciding with lower DM intakes. Alteration in rumen motility associated with changes in intake may be responsible for the decreased productivity in ruminants consuming E+ forages.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13338号 / 論農博第2881号 / 新制||農||1079(附属図書館) / 学位論文||R2||N5245(農学部図書室) / (主査)教授 廣岡 博之, 教授 松井 徹, 准教授 熊谷 元 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Les hydrocarbures aromatiques polycycliques (HAP) sont des polluants organiques persistants (POP) issus d’une combustion incomplète de matière organique, d’origine naturelle (incendies de forêt) et anthropique (chauffage au gaz, trafic routier, industrie…). La consommation par les animaux d’élevage de couverts végétaux et sols contaminés en HAP, couplée à une forte lipophilie de ces derniers, constitue un risque potentiel en terme de contamination des produits animaux (lait, viande, œufs…). Dans ce travail, nous nous sommes intéressés à l’évaluation de l’exposition du ruminant laitier aux HAP au travers de l’utilisation de biomarqueurs métaboliques d’exposition orale aux HAP. Nous avons tout d’abord testé l’aptitude du 1-hydroxypyrène dans l’urine et/ou le lait à être utilisé comme biomarqueur métabolique spécifique d’exposition orale et subchronique (7 jours, 40 jours) de la chèvre au mélange : phénanthrene, pyrène, benzo(a)pyrene. Ceci en utilisant de l’huile comme vecteur de contamination sur la plage 0.04-50 mg/jour de chacun des 3 HAP. Les résultats démontrent entre autres que (i) le 1-hydroxypyrène est excrété de manière proportionnelle au niveau d’exposition sur toute la plage d’exposition testée dans le lait (taux de transfert stable de l’ordre de 1% dans le lait et 10% dans l’urine) ; (ii) qu’un plateau d’excrétion est obtenu au plus tard 10 jours après le début de l’exposition. La seconde partie de ce travail a permis de démontrer le potentiel de l’activité ethoxyresorufin-o-deethylase (EROD) dans les lymphocyte périphérique sanguins (LPS) à être utilisé comme un biomarqueur d’exposition orale aspécifique du ruminant laitier aux POP CYP450 inducteurs, dont certains HAP. Des cinétiques d’induction EROD lymphocytaire par exposition orale aux HAP ont ainsi pu être modélisées par un modèle de type logistique sur 40 jours d’exposition suivis de 10 jours post-exposition. Une ébauche de courbe dose-réponse de type Michaelis-Menten a par ailleurs pu être déterminée, autorisant plusieurs commentaires relatifs au métabolisme des HAP par le ruminant laitier. Une dernière étude cinétique, menée chez le rat en mode subchronique sur 32 jours, a finalement permis, entre autres, de mettre en évidence une bonne corrélation entre les activités EROD dans les lymphocytes sanguins périphériques, le foie et le tissu cérébral. L’ensemble des résultats obtenus démontre la pertinence de l’usage combiné de l’activité EROD lymphocytaire et du 1-OH pyrène dans le lait ou l’urine comme outils d’évaluation pratiques et accessibles du risque lié à l’ingestion de POP par les ruminants laitiers / Polycyclic aromatic hydrocarbons (PAH) are persistent organic pollutants (POP) produced during the incomplete burning of organic materials. Their production can be of natural origin (forest fire) or anthropic origin (gaz heating, vehicular traffic, industry). The ingestion of PAH contaminated vegetal covers or soils by farm animals, coupled with the high lipophily of these PAH, therefore represents a potential hazard in terms of contamination of animal products (milk, meat, eggs…). In the present work, we focused on the evaluation of the dairy ruminant exposure to PAH through the use of metabolic biomarkers of exposure. At first we tested the potential of 1-hydroxypyrene excreted in urine or milk to be used as a metabolic and specific biomarker of subchronic (7 to 40 days) and oral exposure of the goat to a ternary mixture consisting of phenanthrene, pyrene and benzo(a)pyrene. Each PAH was solubilized in oil to reach contamination levels in the range 0.04-50 mg/day. Results demonstrate that (i) 1-hydroxypyrene excretion in milk and urine is proportional to the level of exposure all along the tested exposure range (stable transfer rates of 1-OH pyrene: about 1% in milk and 10 % in urine); (ii) excretion of 1-OH pyrene reached a plateau at the latest 10 days after the beginning of exposure. In the second part of this work, it was demonstrated that the ethoxyresorufin-o-deethylase (EROD) activity, when measured in peripheral blood lymphocytes (PBL), can be used as a convenient and non-specific biomarker of oral and chronic exposure of dairy ruminant to CYP 450 inducting POP, such as many PAH. Induction kinetic of EROD activity PBL could be fitted with a logistic-like model over 40 days of exposure followed by 10 days post-exposure. An approximate dose/response curve could be fitted using a Michaelis-Menten-like model, allowing for several comments about the metabolism of PAH in dairy ruminant. A final kinetic study, which was run on rats under subchronic conditions (32 days), next to other results, showed a good correlation between EROD activities in PBL, liver and brain. Achieved results demonstrate the relevance of the combined use of the EROD activity in PBL and of the 1-OH pyrene in milk or urine as convenient and cost-limited tools for risk assessment in terms of PAH and more generally POP ingestion by dairy ruminants

Le facteur nutritionnel affecte de façon significative la sécrétion et la composition des constituants du lait qui conditionnent sa qualité nutritionnelle. Dans la glande mammaire, ces processus font intervenir de nombreux gènes dont l’expression est modulée par l’alimentation, cependant les mécanismes de régulation sous-jacents ne sont pas connus. Les microARNs (miARN) sont des petits ARN non codants qui se lient sur leurs ARNm cibles pour en réguler l’expression. Ils ouvrent donc des pistes d’investigation pour la compréhension de ces mécanismes. La première partie de mon travail de thèse a consisté à obtenir une meilleure connaissance des miARN exprimés dans la glande mammaire, notamment en dressant les miRNomes de référence par séquençage haut débit chez la souris, la vache et la chèvre. Ensuite, pour la première fois, l’impact de la nutrition sur l’expression des miARN mammaires a été étudié. Deux modèles ruminants, un modèle dit « extrême » et un modèle de supplémentation lipidique proche des conditions d’élevage, ont permis d’identifier 30 et 2 miARN, respectivement, dont l’expression est nutrirégulée. L’analyse in silico des cibles des miARN nutrirégulés a révélé un rôle potentiel de ceux-ci dans le métabolisme des lipides. Certaines des cibles sont effectivement différentiellement exprimées dans ces modèles, parmi celles-ci certains gènes sont essentiels pour la lactation tels que ESR1. Enfin, une étude pilote de la fonction de trois miARN nutrigulés a été initiée in vitro dans des cellules épithéliales mammaires bovines. Ces travaux permettent donc d’apporter des premiers éléments pour la compréhension de la régulation de l’expression des gènes en réponse à la nutrition et de l’impact des miARN sur la lactation. / Nutrition significantly affects the secretion and the composition of milk which determine its nutritional quality. In the mammary gland, regulation of these processes involves numerous genes which expression can be affected by nutrition. However, their regulations remain unclear. MicroRNAs (miRNA) are small non coding RNA which can bind mRNAs and regulate their expression of target genes. Consequently, they offer opportunities to understand the regulation of gene expression in response to nutrition. The first step of my PhD aimed to obtain a better knowledge of miRNA expressed in the mammary gland. Mammary miRNome were established from the lactating mouse, cow and goat using high-throughput sequencing. Later, the effect of nutrition on the expression of miRNA in the mammary gland was analyzed for the first time. Two models in ruminants, a food deprivation (“extreme” model) and a lipid supplementation (model similar to breeding conditions) highlighted 30 and 2 nutriregulated miRNA, respectively. The analysis of nutriregulated miRNA’s predicted targets, in silico, revealed their potential role in lipid metabolism. Some of those target genes have been previously identified as differently expressed in the same conditions and could thus be involved in the regulation of the expression of genes essential for the mammary gland function, such as ESR1. Finally, three nutriregulated miRNA were selected and used in a preliminary study of their functions in vitro in bovine mammary epithelial cells. These works bring first evidences in understanding the nutritional regulation of gene expression in the mammary gland as well as the role of miRNA in lactation.

Peste des petits ruminants virus (PPRV) belongs to the family paramyxoviridae which comprises non segmented negative sense RNA viruses including measles and rinderpest virus. PPRV is the causative agent of peste des petits rumaninats disease (also known as sheep or goat plague disease) in small ruminants. The viral genome contains a non segmented negative sense RNA encapsidated by viral encoded nucleocapsid protein (N-RNA). Viral transcription is carried out by the virus encoded RNA dependent RNA polymerase complex represented by the large protein L and phosphoprotein P. Viral transcription begins at the 3’ end of the genome synthesising all the viral transcripts (3’-N-P-M-F-HN-L-5’). A remarkable feature common to all members of Paramyxoviridae family is the gradient of transcription from 3’ end to the 5’ end due to attenuation of polymerase transcription at each gene junction. The objectives of the present study are characterization of peste des petits ruminants virus transcription and the associated activities required for post transcriptional modification of viral mRNA. In addition, an attempt has been made to develop in vitro transcription with heterologous combination of PPRV and RPV polymerase proteins. The first reaction in capping involves removal of γ-phosphate from triphosphate ended precursor mRNA by RNA triphosphatase. The domain having RNA triphosphatase activity within the L protein has been identified and expressed independently in E. coli. The details of the objectives are presented below. 1. Development of in vitro transcription system for PPRV mRNA synthesis In order to develop an in vitro transcription reconstitution system for PPRV, the viral RNP complex comprising large (L), phospho (P) and N protein encapsidating viral genomic RNA was purified from virus infected Vero cells. The in vitro transcription reconstituted system with RNP complex was able to synthesise all the viral mRNA as analysed by RT-PCR. As a control, total RNA from virus infected cells was isolated and analysed by RT-PCR. In order to refine the in vitro transcription system, separately expressed recombinant polymerase complex was used to reconstitute transcriptional activity in vitro. For this,viral genomic RNA (N-RNA) was purified from PPRV infected cells using CsCl density gradient centrifugation. The recombinant baculovirus for PPRV P protein was earlier generated in the lab. A recombinant baculovirus harbouring the L gene of PPRV was generated in the present study (described in part one). The viral RNA polymerase consisting of L-P complex was expressed in Sf21 insect cells and partially purified by ultra centrifugation on 5-20% glycerol gradient. Glycerol gradient fraction containing the L-P complex was found to be active in the in vitro transcription reconstitution system. Further quantitation of transcripts made in vitro and in infected cells has been carried out by real time PCR. Notably, the gradient of polarity of transcription of viral mRNA observed in vitro with the partially purified recombinant L-P complex was similar to the gradient observed in infected cells. Host proteins have been shown to modulate the transcription of many paramyxoviruses. In order to test the role of host factors, uninfected cell lysate of Vero cells was added to the in vitro transcription reaction and the transcript level was measured by real time PCR. The result showed an increase in the transcription by addition of host proteins suggesting the involvement of host factors in viral transcription. Further, the newly developed in vitro reconstitution system was used to test if recombinant L and P proteins of RPV can functionally replace PPRV L and P protein in the in vitro transcription complementation assay. The result presented in part one indicates that the L or P protein of PPRV can be replaced by RPV L and P protein in heterologous transcription reconstitution system ,with a reduced efficiency. However, the homologous polymerase complex of RPV failed to recognise the N-RNA genomic template of PPRV. 2. RNA triphosphatase activity of PPRV L protein and identification of RNA triphosphatase domain Post transcriptional modification of mRNA such as capping and methylation determines the translatability of viral mRNA by cellular ribosome. In negative sense RNA viruses, synthesis of viral mRNA is carried out by the viral encoded RNA polymerase in the host cell cytoplasm. Since the host capping and methylation machinery is localized to the nucleus, viruses should either encode their own mRNA modification enzymes or adopt alternative methods as has been reported for orthomyxoviruses (cap snatching) and picornaviruses (presence of IRES element). In order to test, if PPRV RNA polymerase possesses any of the capping activities, the RNP complex containing the viral N-RNA and RNA polymerase (L-P) were purified from virion. Using the purified RNP complex, the first activity required for mRNA capping, RNA triphosphatase was tested and the results are described in part two. RNP complex purified from virion showed both RNA triphosphatase (RTPase) activity. The RNA triphosphatase from viruses, fungi and other eukaryotes have been classified into two groups, metal dependent and metal independent. The cleavage of the γ-phosphate from triphosphate ended precursor mRNA by L protein of PPRV was found to be metal dependent. So, by the metal dependency of the RTPase reaction, PPRV L protein was assigned to the metal dependent RTPase tunnel family. One of the key features of metal dependent RTPase group members is the ability to hydrolyse γ-β phosphoanhydride bond of NTPs. PPRV L protein associated with RNP complex also was also able to cleave γ-β phosphoanhydride bond of NTPs. Owing to the large size of L protein (240 KDa), it is conceivable that the L protein functions in a modular fashion for different activities pertaining to mRNA synthesis and post transcriptional modification. Sequence comparison of L proteins from different morbilliviruses revealed the presence of three conserved domains namely domain I (aa 1-606), domain II (aa 650-1694) and domain III (aa 1717-2183). Domain II has the catalytic motif for viral RNA dependent RNA polymerase. Multiple sequence alignment of PPRV L protein with known RNA triphosphatases predicted a two hundred amino acid long region on L protein comprising the C terminus of domain II and N terminus of DIII as a possible candidate for RNA triphosphatase domain. The above predicted domain was cloned and expressed in E. coli. The ability of the purified recombinant RTPase domain to cleave γ-β phosphoanhydride bond of RNA was tested. The results described in part two suggest that the predicted RTPase domain has RNA triphosphatase activity. In addition to RNA triphosphatase, the RTPase domain also has the NTPase activity. The RNA triphosphatase of DNA viruses, yeasts and other fungi have three motifs essential for enzyme activity. Motif A and motif C are rich in glutamate and are involved in metal binding. Motif B is rich in basic amino acids and forms the centre for catalysis. The glutamate residue (E1647) of motif A of PPRV L protein RTPase domain was converted to alanine and the loss of RTPase activity was assessed. The results summarised in appendix 1 shows that the E1647A mutant has reduced RNA triphosphatase and NTPase activity.