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Instituto De Ganadería De Montaña (IGM) Instituto de Ganadería de Montaña (IGM) EFECTO DE LA ADICIÓN DE LÍPIDOS A LA DIETA DE OVEJAS LECHERAS SOBRE LOS MICROORGANISMOS IMPLICADOS EN EL METABOLISMO RUMINAL DE LOS ÁCIDOS GRASOS Y LA REGULACIÓN NUTRICIONAL DE LA LIPOGÉNESIS MAMARIA Tamara Castro Carrera León, Diciembre 2014 EFECTO DE LA ADICIÓN DE LÍPIDOS A LA DIETA DE OVEJAS LECHERAS SOBRE LOS MICROORGANISMOS IMPLICADOS EN EL METABOLISMO RUMINAL DE LOS ÁCIDOS GRASOS Y LA REGULACIÓN NUTRICIONAL DE LA LIPOGÉNESIS MAMARIA EFFECT OF THE ADDITION OF LIPIDS TO THE DIET OF DAIRY EWES ON THE MICROORGANISMS INVOLVED IN RUMEN FATTY ACID METABOLISM AND THE NUTRITIONAL REGULATION OF MAMMARY LIPOGENESIS Memoria de Tesis Doctoral presentada por Tamara Castro Carrera dirigida por Dr. Álvaro Belenguer Ferrando y Dr. Pablo Gutiérrez Toral para acceder al grado de Doctor, dentro del programa de Doctorado “Medicina, Sanidad y Producción Animal y Ciencia de los Alimentos” de la Universidad de León. León, diciembre de 2014 La autora de esta memoria ha disfrutado de una ayuda del Programa JAE Predoc para la formación de personal investigador del Consejo Superior de Investigaciones Científicas (CSIC) cofinanciada por el Fondo Social Europeo. El Ministerio de Economía y Competitividad (MINECO), a través del Proyecto AGL2011-23700, ha aportado la mayor parte de la financiación necesaria para llevar a cabo los trabajos que componen esta memoria. La Junta de Castilla y León también ha contribuido gracias al Proyecto CSI023U13. Deseo expresar mi agradecimiento a todas las personas e instituciones que han puesto a nuestra disposición sus conocimientos y los medios necesarios para la realización de este trabajo. CONTENIDOS ÍNDICE Página CONTENIDOS .................................................................................................................... i Índice ............................................................................................................................. iii Índice en inglés .......................................................................................................... vii Abreviaturas ..................................................................................................................xi Abreviaturas en inglés ................................................................................................xi RESUMEN ........................................................................................................................... 1 RESUMEN EN INGLÉS .................................................................................................... 9 INTRODUCCIÓN ............................................................................................................ 15 REVISIÓN BIBLIOGRÁFICA ....................................................................................... 23 1. Mecanismos responsables de la composición de ácidos grasos de la leche de los rumiantes .................................................................................... 25 1.1. Origen metabólico de los lípidos de la leche ................................................. 25 1.2. Efecto de la alimentación sobre el perfil de ácidos grasos de la leche ........ 27 2. Microbiología del metabolismo de los lípidos en el rumen ........................... 29 2.1. Ecosistema microbiano ruminal ...................................................................... 29 2.1.1. Bacterias........................................................................................................ 30 2.1.2. Protozoos ...................................................................................................... 33 2.1.3. Hongos ......................................................................................................... 34 2.1.4. Arqueas ........................................................................................................ 35 2.1.5. Bacteriófagos ................................................................................................ 35 2.2. Procesos metabólicos y microorganismos implicados ................................. 36 2.2.1. Lipólisis ........................................................................................................ 36 2.2.2. Biohidrogenación ........................................................................................ 38 2.2.3. Hidratación .................................................................................................. 42 2.2.4. Efecto de la alimentación ........................................................................... 44 2.3. Técnicas de estudio de la comunidad microbiana ruminal ......................... 46 2.3.1. Técnicas tradicionales de cultivo .............................................................. 47 2.3.2. Técnicas basadas en biología molecular .................................................. 48 2.3.2.1. Técnicas de identificación de la huella genética ............................... 50 2.3.2.2. Técnicas basadas en la secuenciación ................................................ 53 2.3.2.3. Técnicas cuantitativas .......................................................................... 56 iv Contenidos 3. Metabolismo de los lípidos en los tejidos del animal en lactación............... 60 3.1. Tejido secretor mamario ................................................................................... 60 3.1.1. Síntesis de ácidos grasos de novo ............................................................. 61 3.1.2. Captación de los ácidos grasos circulantes en el plasma ...................... 63 3.1.3. Elongación de los ácidos grasos ............................................................... 65 3.1.4. Desaturación de los ácidos grasos ............................................................ 65 3.1.5. Esterificación de los ácidos grasos ........................................................... 67 3.1.6. Síntesis y secreción del glóbulo graso ...................................................... 68 3.1.7. Factores de transcripción implicados en la regulación nutricional de la lipogénesis mamaria ............................................................................. 69 3.2. Tejido adiposo ................................................................................................... 72 3.3. Técnicas de estudio de la expresión de los genes ......................................... 74 3.3.1. PCR cuantitativa con transcriptasa inversa ............................................ 75 3.3.2. Microarrays de ADN ................................................................................... 76 4. Referencias ............................................................................................................... 77 PLANTEAMIENTO DE LA TESIS ............................................................................... 97 PRUEBA I – Cambios en la población ruminal de Butyrivibrio en ovejas lecheras alimentadas con una dieta suplementada con aceite de girasol y microalgas marinas ........................................................................................................ 101 Resumen ..................................................................................................................... 103 1. Introducción .......................................................................................................... 104 2. Material y métodos ............................................................................................... 104 3. Resultados y discusión ........................................................................................ 106 4. Conclusión ............................................................................................................. 108 5. Referencias ............................................................................................................. 108 PRUEBA II – Utilización de la pirosecuenciación 454 y el polimorfismo de la longitud de los fragmentos terminales de restricción para evaluar la comunidad bacteriana ruminal en ovejas lecheras alimentadas con microalgas marinas ........................................................................................................ 111 Resumen ..................................................................................................................... 113 1. Introducción .......................................................................................................... 114 2. Material y métodos ............................................................................................... 115 2.1. Animales, dietas y diseño experimental ...................................................... 115 2.2. Medidas, recogida de muestras y análisis ................................................... 116 2.3. Extracción de ADN y cuantificación ............................................................ 116 2.4. Análisis mediante T-RFLP ............................................................................. 116 Contenidos v 2.5. Pirosecuenciación y análisis de secuencias .................................................. 117 2.6. Análisis estadísticos ........................................................................................ 118 3. Resultados .............................................................................................................. 119 3.1. Análisis de la comunidad bacteriana mediante T-RFLP ............................ 119 3.2. Análisis de la comunidad bacteriana mediante pirosecuenciación .......... 123 4. Discusión ...............................................................................................................
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