Influencia de la dieta de los corderos sobre el sistema inmune de los mismos

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Anastas a s Palmas g üello He n r ector, D DE LA S Universit a : Sanidad A Tít u E LA DIE E MA IN M e a presen t io Argüell o de Gran C n ríquez Anexo II S PALM A a rio de Sa n Animal u lo de la T TA DE L M UNE DE tada por o Henríque z C anaria, a L o A S DE G R n idad Ani m T esis OS COR D LOS MI S Don Lor e z 21 de oct u o renzo E n R AN CA N m al y Seg u D EROS S S MOS” e nzo Enri q u bre de 20 El Doct o rique He r N ARIA g uridad S OBRE E L q ue Hern á 13 o rando, r nández C L á ndez C astellan o o ANASTASIO ARGÜELLO HENRÍQUEZ, PROFESOR TITULAR DE UNIVERSIDAD EN EL DEPARTAMENTO DE PATOLOGÍA ANIMAL, PRODUCCIÓN ANIMAL, BROMATOLOGÍA Y TECNOLOGÍA DE LOS ALIMENTOS DE LA FACULTAD DE VETERINARIA DE LA UNIVERSIDAD DE LAS PALMAS DE GRAN CANARIA INFORMA: Que Don Lorenzo Enrique Hernández Castellano, Licenciado en Veterinaria, ha realizado bajo mi dirección y asesoramiento el presente trabajo titulado “INFLUENCIA DE LA DIETA DE LOS CORDEROS SOBRE EL SISTEMA INMUNE DE LOS MISMOS” considerando que reúne las condiciones y calidad científica para optar al título de Doctor por la Universidad de Las Palmas de Gran Canaria. Las Palmas de Gran Canaria, a 21 de octubre de 2013 Fdo. Anastasio Argüello Henríquez IN F UNI V F LUEN C E L Las V ERSIDA D F A C IA DE L SIST E Lore n Palmas d D DE LA S A CULTA D TESI LA DI E E MA IN M n zo Enriq u d e Gran C S PALM A D DE VE T S DOCT O E TA DE M UNE D u e Herná n C anaria, a A S DE G R T ERINA R O RAL LOS C O D E LOS n dez Cas t a 21 de oc R AN CA N R IA O RDER O MISM O t ellano t ubre de 2 N ARIA O S SO B O S 2 013 B RE Agradecimientos Al D for m grac i b ue n pers o man o A la punt o dura n y pa s topi c llam a Al D labo r por t grac i A A n de p o que d pens a adie s A Is a sie mp escu c pasa d me r e sino resp e vez “abr i peri o Ag ra D r. Anasta s m arme para i as tambié n n o es salir f o na, ¿Quié n o s un baño y Dra. Noe m o s, comas, e n te este pe r s ábamos h o c )” o “mi g ar te al 109 3 D r. Juan F r atorio de f t ransmitir t i as por tod a n tonio, co m o rtada de T d ará ese fa m a bas que i b s tradas a ra s a bel y a J o m pre dejaba c harme y a c d o juntos, t ecuerde co n la pasión e ctivos pad r que puede n i gado” y p o o do de tiem p ra decimie n s io Ar g üell empezar u n n por hacer m f uera y co n n sino iba a y una coci n m í Castro N e spacios y m r iodo. Grac i o ras hablan d g en Berebe r 3 o visitar e l F rancisco f orma inge n t u pasión p a s tus leccio m pañero de T esis … ¡y m oso cabl e b as a libra r s carse cont r o sué (tambi é para “lue c onsejarme , t anto dentr o n stantemen t que m e h r es. A Isab e n hablar c o o rque creo q p o tan cort o n to s  o Henríqu e n duro ca m m e abrir u n n ocer otras irse con to d n a para un c o N avarro. M m ayúscula s as por esas d o de tema s r ”. Sé que l despacho 2 Capote Á l n iosa (¿qui é or la gana d n es…. ¡¡¡y reparto de p lo que me q e submari n r te de mí, r a ese cable é n conocid o g o despué s , por no no m o como fue r t e que es S e h a transmit i e l y Pedro p o nmigo. A q ue nunca m o . e z. Muchís i m ino, que s i n poco m á culturas!. A da su famil olegio?. ¡ M M uchas gra c s , sino que t tardes en l a s como “m i como “di r 2 1 para co n l varez, un é n sino iba d ería, base dos piedra s p apas, de c a queda/le q u n o que co m te recomie ). ¡¡¡MU o como Jes ú s mañana d m brar la “i n r a del labor a e govia y n o i do por la p or siempr e Mar y Ca m e han di c i mas graci a i n duda m e s mis “hor A parte de u ia a Etiopí a M uchas grac i c ias por to d t e debo tod o a s que baja b s 3 euros d r ectora pos t n tarte cualq u palmero c a llamarm e fundamen t s !!!!! a rrera, de m u eda por a g m unica A m ndo que v a CHAS GR A ú s de la Fe ) d entro de n finidazz” d a torio. A é l o “Madrizz ” ciencia. M e tener una p rmen y P e c ho tantas v a s por dar m e rece la pe n i zontes”, c o u n gran dir e a para cons t i as por tod o d o Noemí; o el esfuerz b a “un mo m e Binter”, “ t iz a ”, siem p u ier cosa. ¡ M c apaz de r e e Ovis arie s al de nues t m áster, de d o g uantar!. R e m érica con a yas mand a A CIAS PO R ) . A ella le / un mes” c e buenos m l le/la/lo ag r ” la verdad e M uchas gra c p alabra am a e dro por h v eces la pal a m e la oport u n a recorre r . o mo dices e ctor, eres u t rui r con su o Tacho! a ti te deb o z o que has e m ento” a bu “ Mangarín ( p re estarás M uchas Gr a e bautizar a s ?). Much a t ro trabajo. o ctorado, d e e cuerda qu e Europa, a s a ndo a tus R TODO!!! / la/lo agra d c ualquier c m omentos q u r adezco no e ra capital d c ias tambi é a ble y grac i h acerme se n abra “cari ñ u nidad de Muchas tú: ¡Qué u na gran s propias o no solo e mpleado scar algo ( trending ahí para a cias! todo el s gracias Muchas e edición e siempre s í que si ballenas ezco que o sa para u e hemos solo que d el reino, é n a sus i osa cada n tir muy o” en un A Davinia, mi abogada particular durante la tesis y la organización hecha persona. Muchas gracias por tu colaboración. Quiero que sepas que pasarás a la historia como la chica que quiso, pero nunca consiguió, que me sacara la Ikea Family Mastercard para contratar un seguro médico un 10% más barato y así ahorrar en la operación de miopía. Ahhhh…. y que la cria del camello es el guelde y no el guelfo . A Alexandr, el cual siempre está disponible para echar una mano a la respuesta de: ¡lo que mande patrón!. De él recuerdo muchas frases graciosas, como la de:¡Aquí hay poder! (mientras con dos dedos se señalaba el hombro con los galones). ¡Muchas gracias patrón! A Vanessa, la verdad, nunca llegué a pensar que una persona que me cayó tan mal en el primer año de carrera, pudiera llegar a ser una chica tan buena y simpática. La verdad es que tu llegada marcó un antes y un después y no solo por el ruido de tus tacones. A mi visviquense favorito Aridany, muchísimas gracias por haber dado ese “wonderful” toque “ArtAttack” al laboratorio. Gracias a ti sé que el croché y las otras manualidades no son lo mío. Muchas gracias por la ayuda prestada, sobre todo en la última paridera. A Diego, una de las personas más trabajadoras que conozco. Es la única persona que puede venderte un colchoncito viscoelástico con espuma HR de alta densidad, supervisar un campo de lechugas, hacerse el segundo máster y echarte una mano en el laboratorio en el mismo día. Muchas gracias por todo Diego, siempre serás el “Self-suckling man”. A la futura veterinaria Paula Paola, muchas gracias por estar siempre dispuesta a echarme una mano en lo que sea, desde hacer un ELISA, una chito o un complemento hasta darle el biberón a los corderos, tomar muestras de sangre u ordenar el laboratorio. Gracias Paolilla!! A los ayudantes de granja (Juan, Luis, Serafín y Bernardo) y a Ventura. A ellos les debo los diversos temas discutidos entre las 8:00-8:30, acompañados siempre de una buena taza de café. Muchas gracias por “meter a viaje” a las ovejas y sus cuellos en la ordeñadora. A la unidad de Enfermedades Infecciosas, sobre todo a Fátima, la cual ha sido durante estos años mi almacén de reactivos de emergencia. Muchas gracias a todos por la ayuda. A mi ovejero favorito, Don Antonio Juan Manuel Alonso Ruiz. Muchas gracias por colaborar con parte de los animales de esta Tesis. Además, tengo que agradecerle las tardes que he pasado aprendiendo de su compañía, charlando de temas tan variados que van desde las autovacunas, los “tetones” y la “potra” de la ubre de las ovejas, pasando por la D.O. del Queso de Flor de Guía y finalizando en la genética y descendencia de su toro Galeote. A Toni, Perico, Alejandro y compañía. Muchas gracias por acordarse de mí para cualquier evento lúdico-festivo, pese a que en la mayoría de las ocasiones me es imposible ir. A Juan Ramón (the best Couch Potato) tengo que agradecerle su pasión por los idiomas, la cual me ha ayudado mucho en las estancias. Muchas han sido, además, las horas que he pasado junto a su familia (María, Paula, Sara y sus padres Fernando y Toñi). Muchas gracias a los alumnos de veterinaria que han colaborado en la lactancia de los corderos usados en esta Tesis, ya que sin ellos estos experimentos no hubieran sido posibles. Al Ministerio de Educación y a su programa de Formación del Profesorado Universitario (FPU) por la financiación recibida para la realización de esta Tesis doctoral. A mi madre y a mi padre les agradezco lo que soy. A mi madre le agradezco que use siempre “sus poderes de madre” para saber cómo me siento sin tener que decírselo, porque como dice ella: “madre solo hay una”. Le agradezco mucho su gestión de la economía del hogar, ya que pese a que a veces no había dinero para lujos, siempre hubo dinero para un libro, un curso o cualquier “cosa del estudio”, por eso sé que mi educación no hubiera sido la misma sin ella. A mi padre le agradezco ese gran esfuerzo que ha hecho trabajando toda su vida para darme la educación que él no pudo tener. Además me enseñó a trabajar, aunque nunca llegaré a su nivel, ya que como dice él, “este hombro ha acarreado más sacos de papas que lentejas dan por un duro” o esas famosas cajas de agua hechas madera “que pesaba más la caja que las botellas de agua”. A mis hermanos María del Cristo y Carmelo Javier (que sé que a mamá le hace ilusión que salgan los nombres enteros), mi primo Enrique Emmanuel, mis tíos, mis abuelos y todos mis familiares que están o han estado conmigo. Gracias a todos porque sé que pase lo que pase, puedo contar siempre con ellos para cualquier cosa. A María. Muchísimas gracias por acompañarme en la distancia, aunque a veces la diferencia horaria fuera de 7 horas. La verdad es que no sé qué me depara el futuro, pero de lo que estoy completamente seguro es que quiero y deseo que sigas formando parte de mi vida durante muchísimo tiempo. Introducción Introducción 7 1. Generalidades Son varios los estudios que han demostrado la relación existente entre la supervivencia de los rumiantes neonatos y un correcto encalostrado (Argüello et al., 2004b; Castro et al., 2005b; Castro et al., 2009; Castro et al., 2011). De hecho, se ha descrito que el calostro se basa en una mezcla de diversos componentes tales como grasa, lactosa, vitaminas y minerales que presentan un alto valor nutricional (Ontsouka et al., 2003). Además, y a pesar de la clara importancia nutricional que posee el calostro, éste contiene una potente mezcla de proteínas que participan, de manera activa, en la protección del neonato contra diversos patógenos y otros cambios ambientales producidos tras el parto (Bendixen et al., 2011). Por otro lado, la proteómica es campo de la ciencia que se encarga de estudiar los proteomas, entendiendo estos últimos como grandes y complejos conjuntos de proteínas expresadas procedentes de un tejido, órgano, organismo o fluido, entre otras. Pese a que el estudio de la proteómica es un campo relativamente nuevo, es uno de los que más rápido se han desarrollado en los últimos años, recibiendo un gran reconocimiento internacional y siendo una herramienta usada de forma amplia y diversa tanto en la investigación animal como en la humana (Penque, 2009; Eckersall et al., 2012). Dentro de esta área de trabajo se incluyen por ejemplo, la transformación de músculo a carne (Paredi et al., 2012) o su uso en la industria láctea (Roncada et al., 2012). En cuanto a los estudios proteómicos del calostro, existen diversos autores que han usado la proteómica para describir por ejemplo, los cambios que se producen en el perfil proteico durante la transición entre calostro y leche (Reinhardt and Lippolis, 2008; Stelwagen et al., 2009; Nissen et al., 2012) o los procesos proteolíticos que se producen en el estómago de los lechones después de la ingesta de calostro (Danielsen et al., 2011). 2. Definición y funciones del calostro en los rumiantes domésticos El calostro se define como la primera secreción formada en la glándula mamaria de las hembras de los mamíferos unas semanas antes del parto. En cuanto a las funciones del calostro, cabe destacar que el calostro es la primera fuente de energía para Introducción 8 los rumiantes neonatos, estimula la eliminación de los meconios y por último y más importante, participa de forma activa en la transferencia de inmunidad pasiva desde la madre al neonato, protegiendo a éste frente a infecciones durante los primero días de vida (Kramer et al., 2001). 2.1. El calostro como fuente de energía En cuanto a la importancia del calostro como fuente de energía, éste participa de forma activa en el incremento de la temperatura corporal de los neonatos. Así, Vermorel et al. (1983) describieron que la temperatura corporal de terneros mantenidos a 10°C aumentaba un 18 % y un 9 % en la primera y segunda hora tras la administración del calostro. Debido a lo anteriormente descrito y tras observar que una de las principales causas de mortalidad durante las primeras horas de vida en los corderos era la hipotermia, Eales et al. (1982) propusieron realizar un adecuado encalostrado como medida preventiva para evitar el descenso de temperatura de estos animales. En este mismo sentido, Hamadeh et al. (2000) afirmaron que la ingesta temprana de calostro es primordial para la inducción de la termogénesis en corderos neonatos. 2.2. Expulsión de los meconios Si bien es cierto que esta función puede parecer menos importante que la anterior, el calostro juega un papel fundamental como estimulador del peristaltismo intestinal en los rumiantes neonatos debido a su alto contenido en sales de magnesio. Son varios autores (García de Jalón et al., 1990; Barza et al., 1993) los que describen que, esta función es de vital importancia para favorecer la expulsión del meconio, evitando así la colonización bacteriana de la mucosa intestinal. 2.3. Establecimiento del vínculo materno-filial Los patrones de comportamiento que desarrollan los corderos neonatos durante los primeros momentos de vida tienen como objetivo fundamental el establecimiento del vínculo materno-filial, durante el periodo sensible de la madre, y a realizar lo antes posible el primer amamantamiento (Val-Laillet et al., 2004). El citado vínculo se instaura después del parto y tiene lugar en un periodo muy específico y breve de tiempo (Ramirez et al., 1997). Introducción 9 En corderos, el consumo de calostro produce una mayor actividad y vigorosidad sobre los mismos, viéndose favorecido el reconocimiento materno (Nowak et al., 1989). Por el contrario, Gonzalez and Goddard (1998) encontraron que el aporte de una dosis extra de calostro comercial no mejoraba el establecimiento del vínculo materno-filial. Estos autores describen como la existencia de un reflejo de saciedad por repleción estomacal podría reducir las posibilidades de que se produzcan mejoras en la citada relación entre la madre y el cordero. 2.4. El calostro como elemento esencial para la transferencia de inmunidad pasiva Por otro lado, y tal y como ya ha sido descrito anteriormente, la complejidad de la placenta epiteliocorial (vacas y búfalos; Wildman et al., 2006; Padua et al., 2010) o sinepitelicorial (pequeños rumiantes; Wooding et al., 1986) hace que la transferencia de inmunoglobulinas a través de la placenta no sea posible, al contrario de lo que ocurre en otras especies con una placenta hemocorial (humanos y ratones; Chucri et al., 2010). Por esto mismo, los rumiantes neonatos son considerados agammaglobulinémicos (terneros y búfalos) o hipogammaglobulinémicos (corderos y cabritos) al nacimiento (Argüello et al., 2004b; Castro et al., 2005b; Castro et al., 2009; Moreno-Indias et al., 2012c), siendo extremadamente importante la ingesta de calostro en estas especies, ya que de ello depende la absorción de proteínas esenciales para conseguir una correcta transferencia de inmunidad pasiva, y por consiguiente disminuir los porcentajes de mortalidad perinatal (Stelwagen et al., 2009; Castro et al., 2011; Danielsen et al., 2011). En cuanto a la absorción de proteínas calostrales, las inmunoglobulinas son las proteínas que han sido estudiadas en profundidad. Sin embargo, cada vez se está teniendo en mayor consideración la importancia del resto de proteínas presentes en el calostro en el desarrollo de una correcta transferencia de inmunidad pasiva (Smith and Foster, 2007). No obstante, y a pesar de la funciones previamente descritas, el calostro es también importante desde el punto de vista nutricional, ya que provee al animal de una serie de elementos nutritivos tales como grasa, proteína, lactosa, minerales o vitaminas que son fundamentales en este período tan temprano de la vida (Ontsouka et al., 2003). Por otro lado, el calostro contiene otro tipo de sustancias tales como agentes antiinflamatorios y antimicrobianos (Stelwagen et al., 2009), factores de crecimiento que controlan el Introducción 10 desarrollo gastrointestinal temprano (Purup et al., 2007), citoquinas, enzimas y otros muchos péptidos (Koldovsky, 1980; Blum and Hammon, 2000), tal y como puede ser observado en la figura 1. Por último, es necesario remarcar que muchos de los péptidos bioactivos presentes en el suero y en la membrana de los glóbulos grasos, tales como las inmunoglobulinas, la lactoferrina o los factores de crecimiento, están presentes en una mayor concentración en calostro que en leche, reflejando una vez más las importancia del calostro en la salud y estado inmune de los rumiantes neonatos (Pakkanen and Aalto, 1997; Scammel, 2001). Figura 1. Visión esquemática de algunos de los principales componentes del calostro y de la leche que participan en la defensa del rumiante neonato.(Adaptado de Wheeler et al., 2007). MFGM: Membrana del glóbulo graso. Introducción 11 La correcta absorción de estos componentes, se lleva a cabo gracias a la acción combinada de la baja actividad proteolítica presente en el tracto gastrointestinal de los rumiantes neonatos (Guilloteau et al., 1983), la presencia de α1antitripsina presente en el calostro (Jensen, 1978; Pallavicini et al., 1984; Ramos et al., 2010) y el gran porcentaje de enterocitos en estado de apoptosis (Castro-Alonso et al., 2008). Sin embargo, estas condiciones especiales para absorber las proteínas de forma intacta van desapareciendo durante las primeras 48 horas de vida, por lo que es de vital importancia alimentar a los rumiantes con calostro durante este periodo (Bush and Staley, 1980; Moore et al., 2005) con el objetivo de que estos animales adquieran unos adecuados niveles iniciales de inmunoglobulinas y otros componentes proteicos en el plasma sanguíneo (Quigley et al., 2000; Christley et al., 2003). La falta de un correcto encalostrado y por lo tanto de una correcta transferencia de inmunidad pasiva, lleva consigo que los rumiantes neonatos sean considerablemente más susceptibles a enfermedades infecciosas, incrementándose los porcentajes de mortalidad a estas edades (Ahmad et al., 2000; da Nobrega et al., 2005; Nowak and Poindron, 2006). Por todo lo anteriormente descrito, la fase de encalostrado es considerada fundamental para mantener el bienestar de los rumiantes neonatos. 3.Componentes mayoritarios del calostro relacionados con el sistema inmune 3.1. Inmunoglobulinas Las inmunoglobulinas son proteínas de un alto peso molecular, pertenecientes al grupo de las glicoproteínas. Estas pueden clasificarse de diversas formas en base a su movilidad electroforética, su peso molecular y su estructura antigénica. Estas proteínas son producidas por las células plasmáticas y los linfocitos B, siendo responsables de la inmunidad humoral. Como principales funciones biológicas de las inmunoglobulinas figuran la activación de la vía clásica del sistema de complemento y la formación de los complejos antígeno-anticuerpo. Introducción 12 De los diferentes isotipos de inmunoglobulinas existentes en los mamíferos (IgG, IgM, IgA, IgD, IgE), la IgG es la que se encuentra en mayor concentración en el plasma sanguíneo, desempeñando el papel más importante en cuanto a los mecanismos de defensa mediados por anticuerpos se refiere (Tizard, 1992). Esta inmunoglobulina presenta un peso molecular de 180 kDa y contiene cuatro cadenas polipeptídicas enlazadas y unidas mediante puentes disulfuro. Debido a este bajo peso molecular y a su estructura simple, constituye, junto con la IgD, el grupo de las inmunoglobulinas con menor peso molecular, lo que facilita la salida de estas inmunoglobulinas del torrente circulatorio con el fin de alcanzar rápidamente los espacios tisulares y las superficies corporales. Específicamente, dentro de la IgG se pueden distinguir dos subisotipos (IgG1 e IgG2), los cuales son diferenciables mediante el uso de métodos electroforéticos (Meckenzie, 1970; Butler et al., 1971; Kehoe, 1971). Además la presencia de estos dos isotipos en el calostro se encuentra regulada de forma diferente, ya que mientras que la IgG1 presenta un tipo de transporte selectivo, la IgG2 lo realiza por medio de la difusión pasiva (Lascelles, 1979). Sin embargo, la mayoría de la IgG presente en el calostro es IgG1, la cual proviene del torrente sanguíneo. Así, Castro et al. (2006) encontraron que la concentración de IgG sanguínea, en cabras de raza Majorera, descendía un 38,70% durante los dos últimos meses de gestación. En el caso de los rumiantes, la IgG es la que aparece con una mayor concentración en sangre y calostro, pudiendo llegar a representar entre el 80 y 90% del total de las inmunoglobulinas presentes en los mismos (Klaus et al., 1969; Neubauer and Schone, 1979; Lacetera et al., 1996). Figura 2. Oveja en el último estadío de la gestación. Introducción 13 En ganado bovino la IgG1 supone el 50% de las inmunoglobulinas del suero sanguíneo, siendo la más predominante en leche (Tizard, 1992). En un estudio realizado por Ciupersescu (1977) en corderos, se estudió como la IgG2 representa escasamente el 1% de las inmunoglobulinas presentes en la sangre durante las primeras etapas de la vida. Siguiendo esta misma línea, Lascelles (1979) encontró que la concentración de IgG1 en el calostro de vaca y oveja era de 48,24 y 94,10 mg/ml, respectivamente; mientras que la concentración de IgG2 en el calostro de estas mismas especies era de 3,98 y 2,50 mg/ml. Por otro lado, el segundo subtipo de inmunoglobulina más importante en el torrente sanguíneo es la IgM, ya que tras la IgG, es la que más concentrada está en el suero sanguíneo de la mayoría de los mamíferos. La IgM es la inmunoglobulina de mayor peso molecular (900 kDa) y se encuentra constituida por la unión de 5 monómeros estabilizados mediante puentes disulfuro. Contiene además un pequeño péptido rico en cisteína, llamado cadena J, la cual, al igual que los monómeros, se encuentra unida mediante puentes disulfuro. Debido a su gran tamaño, estas inmunoglobulinas quedan retenidas en el torrente sanguíneo y no participan en la defensa extravascular. Sin embargo, pueden difundir a través de los epitelios gracias a la cadena J, ya que puede unirse a los receptores presentes en la superficie de las células. En el calostro, a diferencia de la IgG, la IgM se produce a nivel local (Lascelles, 1979) . 3.2. Chitotriosidasa La Chitotriosidasa (ChT) es una enzima secretada por los macrófagos en su último estadío de diferenciación. La función de esta enzima es la de hidrolizar la quitina de la pared celular de hongos y nematodos. Han sido varios los autores que han descrito como la ChT juega un papel importante durante la respuesta inmune (Di Rosa et al., 2005; Malaguarnera et al., 2005) y los procesos inflamatorios (Di Rosa et al., 2006; Malaguarnera et al., 2006). Por otro lado, la actividad de esta enzima se encuentra muy elevada en el suero plasmático de pacientes que padecen enfermedades como la malaria, la enfermedad de Gaucher tipo 1 o en alteraciones hematológicas (Barone et al., 1999; Barone et al., 2003). En este sentido, Musumeci et al. (2005) observaron como la actividad de la ChT en el calostro y suero sanguíneo de mujeres africanas era significativamente mayor que en las mujeres caucásicas (1230 nmol/ml/hora y 293 Introducción 14 nmol/ml/hora, mujeres africanas y caucásicas, respectivamente). En este estudio, no se encontró una correlación entre la actividad de la ChT del calostro y el suero sanguíneo en ninguno de los dos grupos estudiados, lo cual llevó a estos autores a sugerir que la actividad de la ChT es debida a una secreción local por la activación de los macrófagos. Estudios similares fueron realizados en cabras de raza Majorera por Argüello et al. (2008b), encontrando que en el calostro de estos animales la actividad de esta enzima era de 3912 nmol/ml/hora en el momento del parto y de 465 nmol/ml/hora el día 4 después del parto. En otro estudio realizado con la misma raza, Moreno-Indias et al. (2012c) observaron valores de hasta 9431 nmol/ml/hora, justo después del parto, llegando a 3553 nmol/ml/hora a las 10 horas tras el parto. Si se realiza una comparación entre especies, se observa que la actividad de la ChT descrita en el calostro de cabra es mayor que la descrita en la especie humana debido probablemente a que las cabras presentan un mayor contacto con parásitos y hongos. En cuanto a la actividad de la ChT descrita en cabritos (Argüello et al., 2008b), se han observado valores de 2664 nmol/ml/hora al nacimiento y de 9231 nmol/ml/hora a los 49 días de vida, concluyendo que la actividad de esta enzima en torrente sanguíneo aumenta con la edad. Esto puede ser explicado, tal y como se ha descrito anteriormente, por la progresiva activación de los macrófagos con la edad de los animales. En cambio, Moreno-Indias et al. (2012b) no encontraron diferencias en la actividad de la ChT en los primeros 35 días de vida de los animales. Asimismo, Rodríguez et al. (2009) no encontraron diferencias en la actividad de la citada enzima durante los 5 primeros días de vida de cabritos alimentados con distintas concentraciones de calostro caprino liofilizado. Esto refuerza la idea de que la ChT no es absorbida a nivel intestinal y puede jugar un rol importante en la defensa local en el lumen intestinal (Argüello et al., 2008b). Sin embargo, Wold and Adlerberth (2000) sugieren que la ChT debe ser destruida o inactivada antes de llegar al intestino, al igual que ocurre con otras proteínas de la leche materna. 3.3. Sistema de Complemento El Sistema de Complemento es un componente central del sistema inmune innato, además de interceder en los mecanismos de respuesta mediante anticuerpos. Tiene tres actividades fisiológicas principales: defensa frente a infecciones bacterianas, Introducción 21 Tabla 1. Procedimientos de centrifugación para separar las MFGM. Especie Centrifugación Referencia Velocidad(g) Tiempo (min.) Temp. (ºC) Vaca 5000 15 4 (Zhang et al., 2011) Vaca 15000 15 10 (Fong et al., 2008) Vaca 10000 15 4 (Reinhardt and Lippolis, 2008) Humano* 1500 20 25 (Patton and Huston, 1986) Humano 4000 30 4 (Lonnerdal and Forsum, 1985) Oveja* 1500 20 25 (Martini et al., 2013) *Adición necesaria de 5 gr de Sacarosa/100 ml Después de este proceso, el calostro desnatado debe de ser dividido en las dos últimas fracciones. En la bibliografía encontramos multitud de métodos para separar las caseínas del suero de calostro, sin embargo, todos ellos coinciden en el uso de la ultracentrifugación, la precipitación por acidificación del calostro o la combinación de ambos. Algunas de las técnicas más usadas se encuentran en la tabla 2. Tabla 2. Procedimientos para la separación de las caseínas y del suero de calostro. Especie Centrifugación Acidificación Referencia Velocidad (g) Tiempo (min.) Temp. (ºC) Vaca 16500 30 4 C2H4O2 (1N) (Golinelli et al., 2011) Vaca C2H4O2 (30% v/v, pH 4,6) (Mier et al., 2008; Zhang et al., 2011) Vaca 15000 15 10 HCl (1M, pH 4,6) (Fong et al., 2008) Vaca* 44000 30 4 (Boehmer et al., 2008) Vaca 100000 60 4 (Le et al., 2010) Humano* 13000 30 4 HCl (1M, pH 4,6) + 60 mmol CaCl2 (Liao et al., 2011) Humano* 189000 60 4 HCl (1M, pH 4,3) + 60 mmol CaCl2 (Kunz and Lonnerdal, 1989) Vaca 1500 10 5 C2H4O2 (1N) + CH3COONa (1N) (Fox, 2003; Jensen et al., 2012) Vaca 100000 60 4 (Jensen et al., 2012) Humano 10000 15 20 HCl (1M, pH 4,6) (Lonnerdal and Forsum, 1985) *La centrifugación fue repetida dos veces Introducción 22 6.2. Proteínas mayoritarias presentes en el calostro El primer grupo de proteínas que pueden ser aisladas del calostro o de la leche, son las localizadas en la MFGM, la cual envuelve al glóbulo de grasa (Lonnerdal and Forsum, 1985). Durante los últimos años, la mayoría de los estudios en la MFGM han sido realizados en vacas lecheras (Mondy and Keenan, 1993; Mather, 2000; Ye et al., 2002; Fong et al., 2007; Bianchi et al., 2009), sin embargo, existe un rápido crecimiento del interés por la MFGM de otras especies lecheras. Debido a este motivo, algunos de los estudios mas recientes sobre la MFGM han sido realizados en otras especies, tales como cabras (Cebo et al., 2010), ovejas (Addis et al., 2011; Pisanu et al., 2011) o búfalas (D'Ambrosio et al., 2008). Tal y como ha sido descrito por Mather (2000) son 7 las principales proteínas mayoritarias presentes en la MFGM, siendo 2 de ellas las que más relación poseen con la inmunidad. Estas proteínas son la mucin-1 (MUC-1) y la xanthine dehydrogenase/oxidase (XDH/XO). Figura 4. Principales componentes de la MFGM. www.charriau.com Introducción 23 MUC-1 es una proteína glicosilada presente en la superficie apical de las células epiteliales de muchos órganos, tales como pulmones, estómago o intestino (Hollingsworth and Swanson, 2004). Esta proteína ha sido aislada en vacas (Patton et al., 1995), donde ésta se encuentra en una baja concentración (40 mg/l) si es comparada con la encontrada en la MFGM de humanos (729-805 mg/l) (Peterson et al., 1998) . Esta proteína también fue aislada en cabras (Campana et al., 1992) y pese a que no existe información en la bibliografía sobre su concentración en calostro, se ha descrito que la MUC-1 caprina presenta una reacción cruzada con la bovina, pero no con la humana. Esta proteína puede jugar un papel importante en la protección de superficies expuestas frente a daños físicos o a infecciones microbianas (Schroten et al., 1992; Patton et al., 1995; Peterson et al., 1998). Además, la MUC-1 podría participar en la reacción inmune de los neonatos, debido a la capacidad de esta proteína de unirse y secuestrar a los microorganismos patógenos en el tracto gastrointestinal (Schroten et al., 1992; Peterson et al., 1998). Con respecto a la XDH/XO, ésta es principalmente sintetizada en la glándula mamaria (Bruder et al., 1983; Parks and Granger, 1986) y alcanza su máxima concentración durante el último periodo de la gestación e inmediatamente después del parto (Kurosaki et al., 1996). Esta proteína ha sido aislada de la MFGM de vacas, cabras y ovejas, aunque aún no han sido establecidas las concentraciones de esta proteína en dichas especies. Sin embargo, basándose en muestras de leche, se ha descrito que la XDH/XO ovina y bovina tienen una concentración similar (Mondy and Keenan, 1993; Ye et al., 2002; Pisanu et al., 2011). La función de esta proteína no está definida claramente, pero se ha sugerido que podría desempeñar un papel estructural y funcional en la formación de la MFGM (Ishii et al., 1995), además de ser uno de los componentes antibacterianos del calostro y de la leche, proporcionando una fuente de H2O2 para la lactoperoxidasa (Bjorck and Claesson, 1979). Por último, esta proteína podría actuar como inmunomodulador, produciendo una exacerbación de la respuesta inflamatoria o induciendo la expresión de genes que codifican para diferentes componentes tales como proteínas de adhesión, receptores celulares y otros componentes de la respuesta inmune (Mather, 2000). Introducción 24 El segundo grupo de proteínas que puede ser obtenido del calostro, son las denominadas proteínas del suero. Estableciendo el suero de calostro bovino como referencia, ha sido estudiado que este contiene más de 200 tipos de proteínas, de las cuales la β -lactoglobulina, α -lactalbumina, albúmina sérica bovina (BSA), inmunoglobulinas y la lactoferrina se encuentran en una mayor concentración (Sgarbieri, 2004; Korhonen and Pihlanto, 2007; Kawecka and Radko, 2011; Roncada et al., 2012). De este grupo de proteínas, las inmunoglobulinas juegan el papel más importante en cuanto a la función inmune se refiere, sin embargo existen otras proteínas como la α –lactalbumina y la lactoferrina que poseen otras funciones inmunes importantes (Korhonen and Pihlanto, 2007; Tang et al., 2011). En cuanto a la α –lactalbumina, ésta ha sido asociada a la regulación de la producción de lactosa (Kleinberg et al., 1983), sin embargo ha sido descrito que esta proteína también juega un papel importante como inmunomodulador en el calostro y la leche humana y de vaca (Montagne et al., 2000; Korhonen and Pihlanto, 2007; Marnila and Korhonen, 2011). Además son varios los estudios que describen la presencia de esta proteína en calostro (Kawecka and Radko, 2011; Nissen et al., 2012). .Ha sido observado que la concentración de esta proteína en calostro no difiere significativamente entre cabras, vacas y ovejas (2,77 mg/ml, 2 mg/ml y 2,3 mg/ml, respectivamente) (Perez et al., 1990; Levieux et al., 2002), mostrando que la concentración de esta proteína en calostro es 1,5 veces mayor que la encontrada en leche. La lactoferrina, es otra de las proteínas mayoritarias del suero de calostro, siendo muy importante la acción de ésta durante las infecciones. Así, Konuspayeva et al. (2008) estudiaron las diferencias de concentración en calostro de varias especies tales como camella (5,1 mg/ml), vaca (0,84 mg/ml), cabra (3,09 mg/ml), oveja (1,56 mg/ml) o búfala (2,1 mg/ml). Resultados similares fueron observados por Nissen et al. (2012) en calostro de vaca usando 2DE-LC-MS/MS y ELISA. En cuanto a su función biológica primaria, la lactoferrina es una proteína transportadora de los iones hierro. Asimismo, la lactoferrina tiene una amplia variedad de funciones biológicas, muchas de las cuales no parecen estar relacionadas con su capacidad de transportar iones de hierro (Brock, 2002), así, la lactoferrina participa, por ejemplo, como un factor indiscutible en la Introducción 25 inmunidad innata, siendo sintetizada por el epitelio de la glándula mamaria (Sánchez et al., 1992; Adlerova et al., 2008), proveyendo actividad antimicrobiana (bactericida y fungicida) a la glándula mamaria y también al recién nacido (Bellamy et al., 1992). Por último, debido a que la concentración de esta proteína incrementa durante las reacciones inflamatorias e infecciones virales, ésta ha sido clasificada como una proteína inflamatoria de fase agua (Kanyshkova et al., 2001). Con respecto a la última de las fracciones que podemos obtener del calostro, las caseínas pueden ser clasificadas en 4 subtipos (αs1, αs2, β and κ), siendo además responsables de algunas funciones biológicas importantes, tales como el transporte de iones (Ca, PO4, Fe, Zn, Cu), el de actuar como inmunomoduladores y por último el ser precursores de péptidos bioactivos (Korhonen and Pihlanto, 2007). En cuanto a esta última característica de las caseínas, ha sido descrito que los fragmentos proteolíticos de las caseínas poseen funciones antimicrobianas (Lahov and Regelson, 1996), sugiriendo que las proteasas presentes en el calostro podrían participar en la propia inmunidad del individuo. Estos péptidos bioactivos están despertando el interés de los investigadores, ya que podrían ser usados como posibles fuentes naturales de alimento que reporten un beneficio directo a la salud de los seres humanos (Gauthier et al., 2006), debido probablemente a que estimulan el sistema inmune innato en la glándula mamaria y pueden prevenir las infecciones de esta durante el periodo de secado (Silanikove et al., 2005). 7.Proteínas minoritarias en calostro Pese a la gran importancia de las proteínas mayoritarias del calostro anteriormente descritas, existe un creciente interés en aumentar el conocimiento general de las proteínas minoritarias presentes en el mismo, asi como su relación con los procesos relacionados con la salud de humanos y animales (Yamada et al., 2002; Reinhardt and Lippolis, 2008; Golinelli et al., 2011). Poniendo como ejemplo el proteoma del calostro humano, un total de 151 proteínas han sido identificadas, siendo 83 de ellas nunca descritas en leche o calostro humano (Palmer et al., 2006). Probablemente muchas de estas proteínas pueden estar relacionadas con importantes Introducción 26 funciones en el recién nacido, tales como la regulación del crecimiento, el transporte de nutrientes o lo que es más importante, la TIP. En los rumiantes, la identificación y el conocimiento de la función de las proteínas presentes en el suero de calostro y la MFGM están aún incompletos y por lo tanto muchas de las funciones de estas proteínas no ha sido aún descritas (Fong et al., 2008; Reinhardt and Lippolis, 2008; Rusu et al., 2009). Las técnicas bioquímicas, principalmente los métodos basados en los geles 2DE, han sido las más usadas para la identificación de las proteínas minoritarias del calostro (Yamada et al., 2002; O'Donnell et al., 2004). Sin embargo, actualmente estas técnicas están empezando a ser reemplazadas por otras técnicas como el iTRAQ o el Label-free, ya que confieren varias ventajas en comparación con las técnicas basadas en los geles 2DE, siendo capaces de identificar un mayor número de proteínas, usando una menor cantidad de muestra y obteniendo unos resultados más reproducibles. Sin embargo, debido al elevado coste del equipamiento, de los instrumentos y los reactivos, estas técnicas no se encuentran disponibles en la mayoría de los laboratorios. 7.1. Técnicas comúnmente usadas para identificar proteínas minoritarias Los procedimientos tales como inmunoabsorción (Murakami et al., 1998; Yamada et al., 2002; Palmer et al., 2006), isoelectro enfoque (IEF) (Zuo and Speicher, 2002), affinity tagging (Holland et al., 2006), y semi-coupled anionand cationexchange chromatography (Fong et al., 2008) han sido usados como herramientas para eliminar las proteínas mayoritarias e incrementar la abundancia relativa de éstas en análisis proteómicos de muestras complejas como el calostro. A pesar de las técnicas descritas en la tabla 2 para eliminar las caseínas de las muestras de suero de calostro, es necesario además depleccionar las proteínas mayoritarias (principalmente inmunoglobulinas), ya que estas enmascaran a las proteínas minoritarias en los análisis proteómicos (Yamada et al., 2002). Este efecto adverso de las inmunoglobulinas en los estudios proteómicos de proteínas minoritarias se produce porque éstas están localizadas en la misma región del gel 2DE que muchas de las proteínas minoritarias, tales como la transferrina, plasmina, lipoproteína lipasa y fosfatasa alcalina, entre otras, que quedan enmascaradas por esta Introducción 27 proteína mayoritaria (Fong et al., 2008). Por este motivo es necesario eliminar las proteínas mayoritarias del calostro y así poder analizar los cambios en el perfil proteómico de las proteínas minoritarias. Actualmente existen varios kits capaces de eliminar o reducir la presencia de proteínas mayoritarias en el calostro. Así, Golinelli et al. (2011) usaron y compararon la eficacia del kit Albumin and IgG removal (GE Healthcare Life Sciences, UK) y del kit Vivaspin 500 ultrafiltration cartridge (GE Healthcare Life Sciences, UK) con una membrana de 100 kDa de peso molecular en la depleción de las proteínas mayoritarias del calostro. Estos autores observaron como el kit Albumin and IgG removal fue ineficaz a la hora de eliminar la IgG del suero de calostro bovino, probablemente porque estos kits están compuestos por una agarosa que contiene anticuerpos frente a la IgG de humano, lo que hace que debido a la falta de reacción cruzada entre la IgG humana y bovina, estas últimas no hayan sido captadas por los anticuerpos frente a las IgG de humano. Sin embargo, estos autores también describen que el tratamiento del suero de calostro con el kit Vivaspin 500 eliminó la mayoría de la IgG e IgA, mejorando la detección de proteínas minoritarias en las muestras. Otro kit usado comúnmente para la depleción de las proteínas mayoritarias es el ProteoMiner (Bio-Rad, USA), el cual se basa en una exclusiva librería de hexapéptidos con la propiedad de unirse con la misma capacidad a proteínas de diferente abundancia (Bandhakavi et al., 2011). Por lo tanto, cuando una muestra que contiene una mezcla compleja de proteínas es expuesta a estos hexapéptidos, cada uno de éstos se unen a las proteínas mayoritarias y por lo tanto se saturan rápidamente, permaneciendo la gran mayoría de las proteínas mayoritarias en suspensión y sin unir a estos hexapéptidos. En el caso de las proteínas minoritarias, estas no llegan a saturar a sus hexapéptidos y por lo tanto todas las proteínas minoritarias son captadas por estos ligandos. Como resultado del tratamiento con este kit, tendremos que pese a que las proteínas mayoritarias siguen estando presentes, estas se encuentran en una proporción mucho más reducida, permitiendo así un incremento relativo del porcentaje de las proteínas minoritarias (Boschetti and Righetti, 2008; D’Amato et al., 2009). Cabe destacar, que pese a que este kit fue creado específicamente para muestras de plasma y suero, ha sido también comprobada su eficacia en otro tipo de muestras tales como orina (Castagna et al., Introducción 28 2005), bilis (Guerrier et al., 2007), plaquetas, glóbulos rojos y clara de huevo (Guerrier et al., 2008). Con respecto a las muestras de leche y calostro, Liao et al. (2011) usaron el kit Proteominer para depleciones en leche y calostro de humano, mostrando que la presencia de muchas proteínas minoritarias incrementó y que a su vez la presencia de proteínas mayoritarias decreció considerablemente. Sin embargo, estos autores describieron que este kit tiene un efecto muy limitado a la hora de eliminar las caseínas restantes en el suero de calostro y leche. De la misma forma, este kit fue usado para estudiar el proteoma de la leche de vaca, obteniendo un incremento considerable de las proteínas minoritarias (D’Amato et al., 2009). Es necesario remarcar que pese a que este kit es capaz de producir un incremento en la presencia de proteínas minoritarias, las proteínas mayoritarias presentes en el calostro y leche tales como la lactoferrina o la αlactalbúmina, además de las caseínas, son aún detectadas tras el tratamiento con este kit. Sin embargo, a pesar de que se ha demostrado la efectividad de este kit en la depleción de proteínas mayoritarias, es necesario resaltar el efecto que produce este kit en estudios proteómicos cuantitativos usando iTRAQ, en los cuales se mide la abundancia relativa de cada proteína (Bandhakavi et al., 2011). Tal y como describieron Mouton-Barbosa et al. (2010) después del tratamiento de las muestras con este kit, las proteínas mayoritarias no mantiene su verdadera cantidad relativa después del tratamiento, ya que saturan los ligandos de la librería de hexapéptidos, sin embargo las Figura 5: Presentación comercial del kit Proteominer (Bio-Rad, USA) y del kit Albumin and IgG removal (GE Healthcare Life Sciences, UK). Introducción 29 proteínas minoritarias, al no saturar los ligandos, mantienen su concentración original en la muestra. Por lo tanto, y tomando la información descrita anteriormente, el kit de depleción Proteominer es una excelente herramienta a la hora de descubrir las partes más profundas del proteoma de multitud de muestras compuestas por una gran mezcla de proteínas de diferente concentración. Sin embargo, algunas consideraciones deben de tenerse cuando este kit es usado en estudios proteómicos cuantitativos. 7.2.Función de algunas proteínas minoritarias y su relación con el sistema inmune Es muy importante entender como las proteínas minoritarias del calostro pueden mejorar o modificar la respuesta inmune ya sea a nivel de la madre o a nivel del recién nacido, por lo que el estudio de estas proteínas por separado es de extrema importancia. Una de estas proteínas minoritarias del calostro es la plasmina. Esta proteína es una enzima fibrinolítica que juega un papel muy importante en la disolución de los coágulos de fibrina, previniendo así procesos de trombosis (Ogiwara et al., 2010). A pesar de su función en el torrente sanguíneo, esta proteína ha sido identificada en calostro y leche (Dupont et al., 1998; Le et al., 2010). Tal y como fue descrito por Dupont et al. (1998), el calostro de vaca posee una concentración en plasmina 10 veces superior (0,49 µg/ml) a la que se encuentra en leche (0,04 µg/ml). Además Rebucci et al. (2005) encontraron que tanto la plasmina descrita en la leche de vaca, oveja y cabra es idéntica a la encontrada en sangre. Asimismo, otros autores han observado que la plasmina tiene una función inmune, ya que contribuye a la migración de los neutrófilos hasta el punto de infección (Renckens et al., 2006). Siguiendo esta línea, Theodorou et al. (2010) observaron como la concentración de plasmina se incrementaba en sangre y leche tras una mastitis aguda en ovejas lactantes. Otra de las proteínas minoritarias presentes en el calostro es la serum amyloid A (SAA). Esta proteína se encuentra principalmente en complejos con lipoproteínas, existiendo varias isoformas que varían en concentración en función de la especie (Uhlar and Whitehead, 1999). La SAA es una de las proteínas más conservadas dentro de los Introducción 30 mamíferos, confirmando la premisa de que esta proteína tiene una función básica y esencial en el sistema inmune, formando parte de la fase aguda de la inflamación (Eckersall et al., 2006; Pyorala et al., 2011). Esta proteína ha sido también identificada en el calostro de varias especies tales como humana (Kumon et al., 2011) yegua, vaca y oveja (McDonald et al., 2001; Le et al., 2010). Así mismo, se ha descrito que la concentración de SAA circulante en plasma sanguíneo se incrementa 1000 veces entre las 24 y 48 horas tras la infección o inflamación, partiendo de niveles basales de 5-8 mg/ml (Faty et al., 2012), mostrando así la importancia de esta proteína en los procesos inflamatorios. Además, una variante de esta proteína de fase agua, la SAA-3, ha sido descrita su expresión en células de la glándula mamaria en respuesta a patógenos, estando presente en la leche durante las mastitis (McDonald et al., 2001) y en el calostro (McDonald et al., 2001; Larson et al., 2005), sugiriendo el importante papel de esta proteína en la reacción inmune. Así, McDonald et al. (2001) encontraron que la concentración de la SAA-3 es mucho mayor en calostro que en leche (267,45 µg/ml y 2,63 µg/ml, respectivamente). Estos autores además describieron que la concentración de esta proteína en calostro de ovejas es menor que la encontrada en vacas (62,83 µg/ml y 267,45 µg/ml, respectivamente). De manera general, la proteína SAA desempeña un gran número de acciones proinflamatorias, tales como quimiotáctico de los neutrófilos, monocitos y linfocitos T, causando la infiltración de los leucocitos y promoviendo la adhesión de los neutrófilos a la células endoteliales (Badolato et al., 1994; Xu et al., 1995; Su et al., 1999), estimulando a la liberación de citoquinas (Furlaneto and Campa, 2000; He et al., 2003) y metaloproteínasas (Lee et al., 2005b) por parte de los neutrófilos y los monocitos. De acuerdo a lo descrito por He et al. (2009), estas características de la SAA sugieren que tiene una función clave no solo en el establecimiento, sino también en el mantenimiento de la inflamación. Otra interesante proteína minoritaria presente en el calostro es el fibrinógeno, precursor de la fibrina, el componente mayoritario de los trombos sanguíneos. Sin embargo esta proteína tiene también una función defensiva, ya que ha sido demostrado que la concentración de fibrinógeno se incrementa durante los procesos de inflamación aguda (Tamzali et al., 2001; Ganheim et al., 2003). Además, Yamada et al. (2002) Introducción 37 Bruder, G., H. W. Heid, E. D. Jarasch, and I. H. Mather. 1983. Immunological Identification and Determination of Xanthine-Oxidase in Cells and Tissues. Differentiation 23(3):218-225. Bush, L. J. and T. E. Staley. 1980. Absorption of Colostral Immunoglobulins in Newborn Calves. Journal of Dairy Science 63(4):672-680. Butler, J. E., A. J. Winter, and G. G. Wagner. 1971. Symposium: bovine immune system. J Dairy Sci 54(9):1309-1314. Campana, W. M., R. V. Josephson, and S. Patton. 1992. Presence and GeneticPolymorphism of an Epithelial Mucin in Milk of the Goat (Capra-Hircus). Comp Biochem Phys B 103(1):261-266. Castagna, A., D. Cecconi, L. Sennels, J. Rappsilber, L. Guerrier, F. Fortis, E. Boschetti, L. Lomas, and P. G. 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Characterization of protein components of natural and heat-treated milk fat globule membranes. Int. Dairy J. 12(4):393-402. Zaiou, M. and R. Gallo. 2002. Cathelicidins, essential gene-encoded mammalian antibiotics. J Mol Med 80(9):549-561. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415(6870):389-395. Introducción 56 Zhang, L. Y., J. Q. Wang, Y. X. Yang, D. P. Bu, S. S. Li, and L. Y. Zhou. 2011. Comparative Proteomic Analysis of Changes in the Bovine Whey Proteome during the Transition from Colostrum to Milk. Asian Austral J Anim 24(2):272278. Zuo, X. and D. W. Speicher. 2002. Comprehensive analysis of complex proteomes using microscale solution isoelectrofocusing prior to narrow pH range twodimensional electrophoresis. Proteomics 2(1):58-68 Objetivos Objetivos 59 Capítulo 1 Revisar el importante papel que juega el calostro en la transferencia de inmunidad pasiva, especialmente las proteínas mayoritarias presentes en el mismo. Además, también se detallará como las proteínas minoritarias presentes en éste pueden intervenir en procesos inmunes, dado que estos procesos tiene una gran relevancia no solo en la supervivencia del recién nacido, sino en el incremento de la protección frente a agentes infecciosos. Capítulo 2 Determinar la evolución del peso vivo, así como la concentración de IgG e IgM y la actividad del Sistema de Complemento y la Chitotriosidasa durante los primeros 5 días de vida de los corderos criados debidos al sistema de lactancia (natural vs. artificial), la fuente de calostro (cabra vs. oveja) y el tiempo transcurrido hasta la primera toma de calostro (2 vs. 14 h después del nacimiento). Capítulo 3 Analizar el efecto de la dieta de los corderos (leche de oveja, lactorremplazante y leche en polvo entera para consumo humano) sobre la evolución del peso vivo, la concentración de IgG e IgM y la actividad del Sistema de Complemento y la Chitotriosidasa durante la lactancia y el destete. Objetivos 60 Capítulo 4 Investigar el peso vivo y el estado inmune de corderos criados con un sistema de lactancia natural y corderos criados en un sistema de lactancia artificial y encalostrados con dos cantidades de IgG diferentes (4 g de IgG/ kg de peso vivo y 8 g de IgG/ kg de peso vivo). Capítulo 5 Analizar las proteínas minoritarias presentes en el plasma sanguíneo de corderos debido a la ingesta de calostro durante las primeras 14 horas de vida, contribuyendo así a incrementar el conocimiento de la importancia del calostro en la transferencia de inmunidad pasiva y el desarrollo del sistema inmune de los corderos. Capítulo 6 Describir el proteoma del calostro ovino y el plasma de cordero, así como hacer una cuantificación relativa de cómo la ingesta de calostro puede modificar el proteoma del plasma de los corderos recién nacidos. Objectives Chapter 1 69 colostrum intake and the absorption of colostrum proteins play an essential role in PIT and ultimately on newborn survival rates (Stelwagen et al., 2009; Castro et al., 2011; Danielsen et al., 2011). Such protein absorption is mainly based on immunoglobulins (IgG, IgM and IgA, mainly), however it has been also described the importance of non-immunoglobulin proteins absorption from colostrum to the newborn ruminants blood in order to ensure a correct PIT (Smith and Foster, 2007). In spite of this primary role, colostrum is also important to the newborn animal as the first source of nutrition, supplying essential nutrients such as fat, proteins, lactose and minerals at this early stage of life (Ontsouka et al., 2003). Finally, colostrum also contains other substances such as antimicrobial and anti-inflammatory agents (Stelwagen et al., 2009), as well as growth factors that control early gastrointestinal development (Purup et al., 2007), cytokines, enzymes and numerous other peptides (Koldovsky, 1980; Blum and Hammon, 2000) as shown in figure 1. Many of the bioactive whey and milk fat globule membrane (MFGM) proteins, notably immunoglobulins, lactoferrin and growth factors, are present in colostrum in higher concentration than in milk, reflecting the importance of colostrum in the health of newborn ruminants (Pakkanen and Aalto, 1997; Scammel, 2001). Figure 1. Schematic representation of the major known proteins involved in the host defence system in milk and colostrum. (Adapted from Wheeler et al. (Wheeler et al., 2007)). MFGM: Milk Fat Globule Membrane. Capítulo 1 70 The absorption of these colostrum components is favoured by a low proteolytic activity in the gastrointestinal tract of newborn animals (Guilloteau et al., 1983) and also by trypsin inhibitors present in colostrum (Jensen, 1978; Pallavicini et al., 1984; Ramos et al., 2010). However, these special conditions for intact colostrum proteins absorption decrease along the first 48 hours after birth, so it is crucial to feed newborn ruminants with colostrum during this period (Bush and Staley, 1980; Moore et al., 2005), in order for them to acquire an adequate initial concentration of serum Ig’s (Quigley et al., 2000; Christley et al., 2003). In addition, it has been described that newborn ruminants that are not fed with colostrum during the first hours of life are more susceptible to diseases, considerably increasing mortality rates (Ahmad et al., 2000; da Nobrega et al., 2005; Nowak and Poindron, 2006). For all these reasons, colostrum intake is very important to ensure the survival of newborn ruminants. 3. PROTEOMICS AS A TOOL TO ANALYZE AND IDENTIFY PROTEINS FROM COLOSTRUM Colostrum is a complex body fluid produced by the mammary gland of pregnant mammals. Colostrum composition is regulated by hormones (estradiol and progresterone) during colostrogenesis (Castro et al., 2011) that may be defined as the prepartum transfer of components, mainly immunoglobulins, from maternal bloodstream into mammary secretions during a short period (Barrington and Parish, 2001). This process ceases abruptly immediately before parturition (Brandon et al., 1971). There are several factors that affect colostrogenesis, such as species, breed, age, nutrition, litter size, length of dry period and health status (Csapo et al., 1994; Awadeh et al., 1998a; Maunsell et al., 1998). Moreover, colostrum components are secreted by different mechanisms (Patton and Jensen, 1975), so there are proteins directly produced in the mammary gland or transferred from the bloodstream (or both). For example, while IgG is transported to the mammary gland from the blood stream, part of the IgA is synthesized within the mammary gland by plasma cells which had migrated into the gland (Wheeler et al., 2007), as shown in figure 1 . Proteomics is a powerful tool that can simultaneously analyse several hundred proteins in complex mixtures; however, because of the wide range of protein concentrations and subcellular locations, there is no general protocol for the separation of the complete colostrum proteome. Accordingly, and in order to increase the coverage of the colostrum proteome, it is necessary to study the different components separately. Some of these techniques are based in the pre-fractionation of the colostrum sample in order to Chapter 1 71 reduce complexity before proteomics analysis(Gagnaire et al., 2009). These previous steps are in accordance with the different properties of the proteins, namely charge, size or hydrophobicity. The gel-based approach has been the most used method for decades, and is still widely used, being the two-dimensional gel electrophoresis (2DE) method commonly employed in quantitative proteomics (Wu et al., 2006). However, it has been also described a clear interest on liquid chromatography separations (Danielsen et al., 2011), which have several distinct advantages in comparison with the 2DE studies. Finally, mass spectrometry (MS) has increasingly become the method of choice for analysis of complex protein samples. MALDI TOF/TOF MS, for instance is a robust, sensitive and relatively inexpensive. Moreover, this method has produced much of the protein identifications data reported in the literature(Webster and Oxley, 2012). However, there are other techniques, such as isobaric tag for relative and absolute quantitation (iTRAQ) that allows studying deeply the proteome of complex samples with higher accuracy. Several proteome studies have characterized the components of colostrum in different species such as bovine and human (Reinhardt and Lippolis, 2008; Stelwagen et al., 2009; Agarwal et al., 2011). Nevertheless, little is known about colostrum and the process that takes place in the newborn, namely degradation, proteolytic protection, internalization and retention in the intestinal tissue. Moreover, it is necessary to study how this primary food source passes through the gastrointestinal tract of the suckling neonate and how the above-mentioned factors affect the different colostrum components. 4. MANAGEMENT EFFECTS ON COLOSTRUM AND NEWBORN RUMINANTS As described by Castro et al. (Castro et al., 2011) the management during the last weeks of pregnancy is extraordinarily important, because it is in this period that colostrum is secreted. There are other factors such as litter size and number of lactation that affect final colostrum composition. Moreover, colostrum management in the first hours of life is crucial for the survival of the newborn ruminant. However, in spite of the importance of these factors in the final composition and on colostrum protein absorption by newborn ruminants, no proteomics studies have been published on these effects. Capítulo 1 72 4.1 Nutrition during gestation The relation between maternal nutrition during late pregnancy and mammary gland development, colostrum composition and volume produced after partum has long been described (Mellor and Murray, 1985). In fact, dairy animals that receive a balanced feed produce more colostrum than underfed animals. Regarding immune components present in colostrum, nutrition management may vary the final concentration of some immune components. Accordingly, Awadeh et al. (Awadeh et al., 1998b) described how cows supplemented during late pregnancy with selenium increased their IgG concentration in colostrum. On contrast, Swanson et al. (Swanson et al., 2008) did not observe any effect in sheep supplemented during the late pregnancy neither in the colostrum yield nor in IgG concentration. 4.2 Litter size and number of lactations Litter size is a very controversial effect. Csapó et al. (Csapó et al., 1994) found that colostrum IgG concentration was higher in twin Hungarian White goats and Hungarian Merino sheep than in singles. However, Argüello et al. (Argüello et al., 2006) did not find differences in colostrum IgG concentration between Majorera goats with different litter sizes. There has been some controversy on the issue of the number of lactations. While it has been shown to increase IgG concentration in bovine (Oyeniyi and Hunter, 1978) and caprine colostrum (Ha et al., 1986), Dos Santos et al. (Santos, 1994) and Argüello et al. (Argüello et al., 2006) did not observe differences in the colostrum IgG concentration due to the number of lactations in Saanen and Majorera goats, respectively . 4.3 First colostrum intake by newborn ruminants As described in section 2, newborn ruminants can absorb large molecules. Nevertheless and considering colostrum, this special ability decreases along the first 48 hours after birth. It is important to notice that the first colostrum ingestion is very important to acquire a correct PIT, although other aspects such as colostrum IgG concentration or volume of ingested colostrum also take high relevance in PIT. As reported (Chigerwe et al., 2009) Holstein calves fed with 3 litres of colostrum between the first 4 hours after birth acquire an adequate PIT. However, the same author observed that calves that took their first Chapter 1 73 colostrum intake between 4 and 12 hours after birth also had an adequate PIT, although the latter animals were fed with a high quality colostrum (IgG concentration > 100µg/µL). 5. COLOSTRUM FRACTIONS AND HIGH ABUNDANT PROTEINS 5.1 Colostrum fractions As described in Section 3, there is no adequate protocol for the separation of whole colostrum. It is therefore necessary to separate different fractions. Colostrum proteins are divided into three main groups according to the fraction where they are found: caseins, whey proteins and proteins from the MFGM, as summarized in figure 2. Figure 2. Schematic procedure for the separation of the three main groups of colostrums proteins. Two fractions can be obtained upon centrifugation: Milk Fat Globule Membrane (MFGM) and skimmed colostrum. The skimmed colostrum can be treated with ultracentrifugation or acidification (or both) to obtain the last last fractions: Caseins and Whey proteins. These three fractions can be isolated in two steps. The first step is based in the centrifugation of the colostrum in order to separate the fat layer (MFGM) and the skimmed colostrum that includes caseins and whey proteins. Several centrifugation protocols may be found in the literature as summarized in table 1, although some of them have only been tested in milk samples. Ultracentrifugation Acidification (pH 4.3-4.6) Centrifugation Colostrum Skimmed colostrum Insoluble fraction (Caseins) Soluble fraction (Whey proteins) Fat (MFGM) Capítulo 1 74 Table 1. Centrifugation procedures for isolation of MFGM. Specie Centrifugation Reference Speed (g) Time (min.) Temp. (ºC) Cow 5,000 15 4 (Zhang et al., 2011) Cow 15,000 15 10 (Fong et al., 2008) Cow 10,000 15 4 (Reinhardt and Lippolis, 2008) Human* 1,500 20 25 (Patton and Huston, 1986) Human 4,000 30 4 (Lonnerdal and Forsum, 1985) Ewe* 1,500 20 25 (Martini et al., 2013) *With addition of 5g of Sucrose/100mL of milk After this process, the resulting skimmed colostrum needs to be divided in the two last fractions (whey proteins and caseins). In the literature, several methods may be found to isolate caseins from the skimmed colostrum or milk sample; however, all of them have in common the use of ultracentrifugation, acid precipitation or the combination of both methods. Some of the most used techniques are shown in table 2. Table 2. Procedures to isolate caseins and whey proteins from milk and colostrum. Species Centrifugation Acid precipitation Reference Speed (g) Time (min.) Temp. (ºC) Cow 16,500 30 4 C2H4O2 (1N) (Golinelli et al., 2011) Cow C2H4O2 (30% v/v, pH 4.6) (Mier et al., 2008; Zhang et al., 2011) Cow 15,000 15 10 HCl (1M, pH 4.6) (Fong et al., 2008) Cow* 44,000 30 4 (Boehmer et al., 2008) Cow 100,000 60 4 (Le et al., 2010) Human* 13,000 30 4 HCl (1M, pH 4.6) + 60 mmol CaCl2 (Liao et al., 2011) Human* 189,000 60 4 HCl (1M, pH 4.3) + 60 mmol CaCl2 (Kunz and Lonnerdal, 1989) Cow 1,500 10 5 C2H4O2 (1N) + CH3COONa (1N) (Fox, 2003; Jensen et al., 2012) Cow 100,000 60 4 (Jensen et al., 2012) Human 10,000 15 20 HCl (1M, pH 4.6) (Lonnerdal and Forsum, 1985) *Centrifugation repeated twice Chapter 1 75 5.2. High abundant proteins As described above, one of the three groups of proteins that can be found in colostrum and milk, are located in a complex membrane, called milk fat globule membrane (MFGM) that surrounds triacylglycerols droplets (Lonnerdal and Forsum, 1985).Traditionally, most of the research on MFGM has been focused in dairy cattle (Mondy and Keenan, 1993; Mather, 2000; Ye et al., 2002; Fong et al., 2007; Bianchi et al., 2009), however the interest in different aspects of non-bovine milk is fast growing. For this reason, some recent studies on MFGM of other species, such as goats (Cebo et al., 2010), sheep (Addis et al., 2011; Pisanu et al., 2011) or water buffaloes (D'Ambrosio et al., 2008) may be found in the literature. As described (Mather, 2000), there are 7 HAP in the MFGM. Two of the most important proteins related to immunity are mucin-1 (MUC-1) and xanthine dehydrogenase/oxidase (XDH/XO). MUC-1 is a glicycoprotein that is present in the apical surface of epithelial cells from different organs, such as lungs, stomach, intestine and others (Hollingsworth and Swanson, 2004). This protein has been isolated in cattle (Patton et al., 1995) where it’s concentration is lower (40 mg/ ) by comparison to human MFGM (729-805 mg/L) (Peterson et al., 1998). This protein was isolated in goats (Campana et al., 1992), albeit no information seems to be available on its concentration levels. Nevertheless, it has been demonstrated that it has cross-reactivity with cow MUC-1 and no cross-reactivity with human MUC-1. Regarding its biological function, this protein may play an important role in the protection of exposed surfaces from physical damage and invasive pathogenic microorganisms (Schroten et al., 1992; Patton et al., 1995; Peterson et al., 1998). In addition, MUC-1 may participate in the immune reaction in the suckling neonate because of its capacity of binding and sequestering pathogenic microorganisms within the gut lumen (Schroten et al., 1992; Peterson et al., 1998). Concerning XDH/XO, it is essentially synthesized in the mammary gland (Bruder et al., 1983; Parks and Granger, 1986) and it reaches maximum values during late pregnancy and immediately after parturition (Kurosaki et al., 1996). This protein has been isolated from MFGM in cows, goats and sheep, although differences between them have so far not been analysed in colostrum. However, using milk samples, it has been described that XDH/XO from sheep and cow MFGM have similar concentrations (Mondy and Keenan, 1993; Ye et al., 2002; Pisanu et al., 2011). The function of this protein is not completely defined, but it has been suggested that it may play structural and functional roles in the formation of the MFGM Capítulo 1 76 (Ishii et al., 1995) and also as having antibacterial properties in colostrum and milk, providing a source of H2O2 for lactoperoxidase (Bjorck and Claesson, 1979). Finally, this protein could act as an immunomodulator either causing tissue damage and exacerbation of the inflammatory response or inducing expression of genes encoding, for example, adhesive proteins, cell receptors, and components of the immune system (Mather, 2000). Another group of proteins that can be found in colostrum are whey proteins. Setting bovine milk as reference, whey contains more than 200 different proteins, with β -lactoglobulin, α -lactalbumin, bovine serum albumin (BSA), immunoglobulins and lactoferrin being the major constituents (Sgarbieri, 2004; Korhonen and Pihlanto, 2007; Kawecka and Radko, 2011; Roncada et al., 2012). From this group of proteins, immunoglobulins play the most important role in the innate immune transfer, however there are other proteins with important immune functions, particularly α –lactalbumin and lactoferrin (Korhonen and Pihlanto, 2007; Tang et al., 2011). Conventionally, α –lactalbumin has been associated to the regulation of lactose production (Kleinberg et al., 1983), however it has been recently described that this protein plays also a role as an immunomodulator in human and bovine colostrum and milk (Montagne et al., 2000; Korhonen and Pihlanto, 2007; Marnila and Korhonen, 2011). The concentration of this protein in colostrum does not differ significantly between goats, cows and sheep (2.77 mg/mL, 2 mg/mL and 2.3 mg/mL, respectively) (Perez et al., 1990; Levieux et al., 2002), showing that the concentration of this protein is approximately 1.5 times higher than those found in milk. The other important HAP in the immune response against infections is lactoferrin. Konuspayeva et al. (Konuspayeva et al., 2008) studied the differences of this protein concentration in colostrum from several mammals such as camels (5.1 mg/mL), cows (0.84 mg/mL), goats (3.09 mg/mL), sheep (1.56 mg/mL) or water buffaloes (2.1 mg/mL). Similar results were found by Nissen et al. (Nissen et al., 2012) in bovine colostrum, using 2DE-LC-MS/MS and quantitative ELISA. The main function of lactoferrin is the binding and transport of iron ions. Additionally, lactoferrin has a wide variety of biological functions, many of which do not appear to be connected with its iron binding ability (Brock, 2002). Lactoferrin works as an innate immune factor synthesized by the mammary epithelium (Sánchez et al., 1992; Adlerova et al., 2008), that provides antimicrobial activity (bactericide and fungicide) to the mammary Chapter 1 77 gland and also to the newborn (Bellamy et al., 1992). Due to the increase in its concentration during most inflammatory reactions and some viral infections, it has been classified as an acute-phase protein (Kanyshkova et al., 2001). With reference to casein, there are four main types (αs1, αs2, β and κ), that are responsible for important biological functions such as ion carriers (Calcium, Fosfate, Iron, Zinc, Cupper), bioactive peptide precursors and immunomodulators (Korhonen and Pihlanto, 2007). It was also demonstrated that the casein proteolytic fragments have an antimicrobial activity (Lahov and Regelson, 1996), suggesting that proteases may also play a role in the host defence. In addition, peptides derived from caseins are receiving much attention as possible sources of natural bioactivity with health benefits for humans (Gauthier et al., 2006), probably because they stimulate the innate immune system within the mammary gland and prevent udder infections during the dry phase (Silanikove et al., 2005). 6. LOW ABUNDANT PROTEINS FROM COLOSTRUM In addition to the high importance of HAP in colostrum, there is a growing interest in LAP in order to increase the general knowledge about their biological role in animal and human health (Yamada et al., 2002; Reinhardt and Lippolis, 2008; Golinelli et al., 2011). In humans for example, a total of 151 proteins were recently identified, albeit the fact that 83 of them had never been previously described in human colostrum or milk (Palmer et al., 2006). Probably, many of this newly identified proteins could be involved in important colostrum roles such as regulation of growth, nutrient transport and what it is more important, PIT. In ruminants, knowledge on the identity and function of the LAP constituents of whey and MFGM is still incomplete, although many of these proteins may have uncharacterized biological properties (Fong et al., 2008; Reinhardt and Lippolis, 2008; Rusu et al., 2009). Biochemical techniques, namely 2DE gel methods have been one of the most common techniques used to identify LAP in both milk and colostrum (Yamada et al., 2002; O'Donnell et al., 2004). However, these techniques are starting to be replaced by other such as iTRAQ or label-free methodologies that confer several advantages in comparison to 2DE gel methods. These new techniques are able to identify a higher number of proteins using less amount of sample and producing more reproducible results. On the other hand, the main disadvantage of these techniques is the expensive cost of the equipment, instruments and reagents, not frequently available in most laboratories. Capítulo 1 78 6.1. Most common techniques used to identify low abundant proteins Approaches such as immunoabsorption (Murakami et al., 1998; Yamada et al., 2002; Palmer et al., 2006), isoelectric focusing (IEF) (Zuo and Speicher, 2002), affinity tagging (Holland et al., 2006) , and semicoupled anionand cation-exchange chromatography (Fong et al., 2008) have been used as a tool to remove HAP and increase the relative abundant of LAP in proteomic analysis of complex samples such as colostrum. Despite of the removal of caseins by procedures such as those described in table 2, the large amount of immunoglobulins and high abundant proteins in, for example, ruminant whey is particularly difficult for LAP proteomic analysis (Yamada et al., 2002). As a consequence of their high abundant in colostrum, similar isoelectric point and molecular weight, immunoglobulins are located in the same region of the 2DE-gel where most of the LAP, such as transferrin, plasmin, lipoprotein lipase, alkaline phosphatase, and others yet to be identified are also found (Fong et al., 2008). For this reason, it is necessary to remove HAP (namely immunoglobulins) from colostrum as they can mask low abundant proteins. Presently, several commercial kits are available to remove immunoglobulins. Golinalli et al. (Golinelli et al., 2011) used and compared the removal efficacy of whey HAP proteins from colostrum using the albumin and IgG removal kit (GE Healthcare Life Sciences, UK) and the Vivaspin 500 ultrafiltration cartridge (GE Healthcare Life Sciences, UK) with a membrane of a molecular-mass cut-off of 100 kDa. These authors observed that the Albumin and IgG removal kit was ineffective in removing IgG from the bovine colostrum whey. It is known that the albumin and IgG removal kit contains agarose-immobilized anti-IgG against human proteins. The failure of these antibodies to capture the IgG present in bovine colostrum whey could be ascribed to a lack of cross-reactivity of the antibodies to the bovine proteins. However, these authors described that the treatment of colostrum whey with Vivaspin 500 removed most of the IgG and IgA, rendering possible the detection of LAP. Another common depletion technique used to deplete high abundant proteins is the ProteoMiner kit (BioRad, USA) that consists of a unique library of hexapeptides, which bind proteins of different abundance with same capacity (Bandhakavi et al., 2011). Consequently, when a complex protein mixture is exposed to a ligand library, each bead with affinity to an abundant protein will rapidly become saturated and therefore the vast majority of the same protein will remain unbound. In contrast, low abundant proteins Chapter 1 85 [16]Wildman, D. 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Vet Immunol Immunop 2001, 83 (3–4), 203-211. Capítulo 1 92 93 Capítulo 2 Chapter 2 Chapter 2 101 Colostrum feeding period 149 At birth, animals were randomly divided in five different groups. The natural rearing 150 (NR) group was composed by 20 lambs who fed colostrum directly from their dams. 151 The other 40 lambs were removed from their dams, dried, weighed and ear tagged and 152 then divided into 4 blocks (10 lambs each) in accordance with the method of colostrum 153 feeding and without contact with the dam. Lambs blocks were allocated into rooms 154 equipped with central heating at a room temperature of 20ºC and providing at least 0.3 155 m2 floor space per lamb. Goat Colostrum 2 h (GC2) and Goat Colostrum 14 h (GC14) 156 groups received a goat colostrum pool that was previously pasteurized at 63ºC for 30 157 minutes according to Trujillo et al. (2007). Sheep Colostrum 2 h (SC2) and Sheep 158 Colostrum 14 h (SC14) groups received a sheep colostrum pool that was pasteurized by 159 the procedure previously described. Lambs from GC2 and SC2 were bottle-fed 160 colostrum at 2 h, 14 h and 24 h after birth and GC14 and SC14 lambs were bottle-fed 161 colostrum at 14 h and 24 h after birth. Because there are no references concerning the 162 amount/concentration of colostrum that should be given to lambs in order to achieve a 163 correct PIT, by the end of the colostrum feeding period all the artificial rearing groups 164 (GC2, GC14, SC2 and SC14) received a total colostrum amount equivalent to 4 g of 165 IgG/kg of BW, which is the amount recommended by Castro et al. (2005b) for goat 166 kids. 167 168 The goat and sheep colostrum IgG concentration (41.32 and 64.37 mg/mL, respectively) 169 was determined using a commercial ELISA kit (Bethyl laboratories, Montgomery, TX, 170 USA). 171 172 Capítulo 2 102 The NR group was raised with dams and had free access to dam colostrum/milk until 173 end of the experiment. After colostrum period, artificial rearing groups (GC2, GC14, 174 SC2 and SC14) received a commercial milk replacer at 16% (w/w; MR group, 175 Bacilactol Corderos y Cabritos, Saprogal, La Coruña, Spain; 95.5% DM, 23.6% CP and 176 22.7% ether extract, air-dry powder basis). These groups were fed ad libitum (37ºC), 177 using nipple buckets twice a day (8:00 am and 05:00 pm). 178 179 BW recording and sample collection 180 All experimental animals were weighed before each blood extraction and data was 181 expressed in kg (MOBBA, Barcelona, Spain; accuracy, 5g). Blood samples were taken 182 before morning feeding from the jugular vein in 2.5 ml K-EDTA tubes. Subsequently, 183 blood was centrifuge at 2190 x g during 5 minutes at 4º C (Hettich-Zentrifugen, 184 Universal 32 R, Germany), storing the obtained plasma at -80° C until analysis. In the 185 colostrum and milk feeding period, blood samples were taken at 2 h after birth (labeled 186 as sample 0) and then at 1, 2, 3, 4, 5 d after birth. 187 188 To determine plasma IgG and IgM concentration commercial ELISA kits (Bethyl 189 Laboratories, Montgomery, TX, USA) were used, setting a purified sheep IgG and IgM 190 as a standard reference. Results were expressed as mg of immunoglobulin/mL of 191 plasma. 192 Chitotriosidase activity was measured following the procedure described previously by 193 Argüello et al. (2008a) in goat blood plasma. In this procedure, 1 µL of undiluted blood 194 plasma with 100 µL of a solution containing 22 mM artificial substrate (4195 methylumbelliferyl-d-N, N′, N″ triacetylchitotriose) in 0.5 M citrate phosphate buffer 196 (pH 5.2) was incubated for 15 min at 37°C. The reaction was stopped with 5 mL of 0.5 197 Chapter 2 103 M Na2CO3-NaHCO3 buffer (pH 10.7). Fluorescence was measured using a fluorimeter 198 (Perkin Elmer, Norwalk, CT) at 365 nm of excitation and 450 nm of emission. The ChT 199 activity was expressed as nanomoles of substrate hydrolyzed/mL/h. 200 201 Complement System activity (TCA and ACA) was measured by the hemolytic rate 202 according to a novel technique described by Moreno-Indias et al. (2012b) in goat kid 203 blood plasma. In this technique a DGHB++ buffer [Hepes Gelatin Veronal Buffer with 204 Ca++ and Mg++: 5 mM HEPES, 71 mM NaCl, 0.15 mM CaCl2, 0.5 mM MgCl2, 2.5% 205 (w/v) glucose, 0.1% (w/v) gelatin, pH 7.4] was used to measure total Complement 206 System activity, and DGHB-Mg-EGTA buffer [4.2 mM Hepes, 59 mM NaCl, 7.0 mM 207 MgCl2, 2.08% (w/v) glucose, 0.08% (w/v) gelatin, 10 mM EGTA, pH 7.4] to measure 208 the alternative pathway. For total Complement System activity, rabbit red blood cells 209 and lamb plasma were diluted to 5% in DGHB++; 100 µL of each was then mixed in a 210 microtiter plate and incubated at 37°C for 1 h. Cells were removed by centrifugation 211 (2500  g, 5 min, 4ºC), and supernatant absorbance was measured at 405 nm using a 212 micro-plate reader. Complete hemolysis was achieved by mixing the cells with distilled 213 water (100 µL), and spontaneous lysis was produced by mixing the diluted rabbit red 214 blood cells with DGHB++. Complement-induced hemolysis of rabbit red blood cells by 215 the test sera was calculated using the formula: [(A405 sample − A405 spontaneous 216 lysis) / (A405 complete hemolysis − A405 spontaneous lysis)]  100. The same protocol 217 was performed with DGHB-Mg-EGTA buffer to measure the alternative pathway. 218 219 220 221 Capítulo 2 104 Statistical analyses 222 Statistical analyses were performed using SAS, Version 9.00 (SAS Institute Inc., Cary, 223 NC). The SAS PROC MIXED procedure for repeated measurements was used to 224 evaluate the effect of rearing system (natural vs. artificial) colostrum source (goat vs. 225 sheep) and timing of the first colostrum intake (2 vs. 14 h after birth) on IgG and IgM 226 concentration, ChT and Complement System activity and BW of lambs from birth to 5 d 227 after birth. A Bonferroni’s test was used to evaluate differences between groups. 228 229 RESULTS AND DISCUSSION 230 Results relating to BW, IgG and IgM concentration, ChT activity, TCA and ACA 231 during the first 5 d after birth are shown in Table 1. NR was the only group that 232 increased the BW after birth; however weights for groups reared under artificial 233 conditions decreased from partum to 4 d after birth, with a trend of increasing weight at 234 5 d. It was observed that there were no BW differences between the animals at birth (0 235 d), although at 4 d and 5 d the NR group was heavier than the others (GC2, GC14, SC2 236 and SC14), with no BW differences being found between these four artificial rearing 237 groups. According to Lanza et al. (2006), factors associated with an increased suckling 238 frequency could be related to the differences between NR and the other groups. As 239 observed by Rodríguez et al. (2008), lambs that are reared under restricted conditions 240 (twice ad libitum daily) presented a lower BW gain rate (253 g/d) than animals that 241 were raised with their dams (307 g/d). In addition, Argüello et al. (2004a) observed that 242 goat kids reared under a natural rearing system had a higher weight than those from an 243 artificial rearing system. In contrast of these findings, Napolitano et al. (2002) did not 244 find BW gain rate differences between lambs reared under natural or artificial 245 conditions (180 g/d and 170 g/d, respectively). It is important to remark that not only 246 Chapter 2 105 neither the colostrum source (goat vs. sheep), nor the delay of the colostrum intake (2 247 vs.14 h after birth) showed effects on lamb growth during the colostrum period. 248 249 Focusing on the IgG concentration, at birth (0 d) all the animals had a basal IgG 250 concentration in their blood plasma, probably from maternal origin through the 251 placenta, as it has been referenced by Castro et al. (2011). These values increased 252 during the first 24 h after birth, although the highest concentration was obtained in the 253 NR group (16.79 mg/mL), probably because the colostrum intake were not restricted in 254 these animals and a higher amount of IgG could be absorbed. However, IgG 255 concentration in the artificial rearing groups (GC2, GC14, SC2 and SC14) remained 256 constant in the following days (2, 3, 4 and 5 d after birth). No differences were observed 257 between artificial rearing groups in this period, probably because all these animals 258 received the same amount of IgG in relation to their BW at birth. Several authors have 259 observed how an increase of the total amount of IgG present in colostrum intake 260 increase the IgG present in newborn ruminant blood not only in lambs (Halliday and 261 Williams, 1979) but also in calves (Muller and Ellinger, 1981; Stott and Fellah, 1983) 262 and goat kids (Castro et al., 2005b; Rodríguez et al., 2009). In addition Firat et al. 263 (2003) reported differences in the milk feeding period between lambs raised under 264 natural and artificial conditions, describing that lambs raised under artificial rearing 265 system had generally lower plasma IgG levels than lambs raised under natural 266 conditions. Results observed in this study show that no differences in IgG concentration 267 were found to be due to the colostrum source (goat vs. sheep) or the colostrum intake 268 delay (2 vs. 14 h). 269 270 Capítulo 2 106 No animals from any of the groups presented detectable IgM in the blood at birth (0 d). 271 Groups studied showed an increase in IgM concentration during the first days after 272 birth; however this concentration tended to gradually decrease in the following days in 273 groups NR, SC2 and SC14. When groups were compared, the largest differences were 274 perceived between the NR group and the artificial rearing groups (GC2, GC14, SC2 and 275 GC14), showing that NR group presented the highest IgM concentration from 1 d to 5 d. 276 These differences between natural and artificial rearing groups during this period could 277 be produced because the artificial rearing groups received a restricted amount of 278 colostrum in comparison with those who were reared under natural conditions. The 279 same effect was observed in newborn goat kids by Rodríguez et al. (2009) who 280 observed that animals fed with a higher IgM amount in colostrum presented a higher 281 IgM concentration on blood. Moreover, Stott and Fellah (1983) observed a quadratic 282 relationship between the amount of IgM in colostrum and plasma IgM concentration in 283 calves. According to the results of this study, no differences were observed due to the 284 colostrum source (goat vs. sheep) and colostrum intake time (2 h vs. 14 h after birth) 285 when artificial rearing groups were analyzed in this period. 286 287 The ChT activity was found to be similar during the first 4 d after birth, increasing in 288 GC14 and SC14 at the end of this period, although no differences were observed 289 between groups in the whole period. The evolution of this enzyme activity has not been 290 previously described in sheep, although Argüello et al. (2008a) described higher values 291 in goat kids at birth (2664 nmol/mL/h) and if this is compared with results of the present 292 study (1017.22, 804.68, 897.46, 946.33 and 1019.69 nmol/mL/h in NR, GC2, GC14, 293 SC2 and SC14, respectively), it demonstrates an increase of this enzyme activity in goat 294 kids blood at 21 days of life (6000 nmol/mL/h). In accordance with these findings, 295 Chapter 2 107 Rodríguez et al. (2009) did not find any differences in blood ChT activity of goat kids 296 that were fed with colostrum at different IgG concentrations. As tends to happen with 297 other proteins from breast milk, ChT may be inactivated or destroyed before arriving in 298 the intestine (Wold and Adlerberth, 2000), so the primary role of ChT from colostrum 299 must be to protect the intestinal lumen of the newborn, increasing the activity of this 300 enzyme when the animal becomes older by the progressively macrophages activation 301 (Argüello et al., 2008a). The present results suggest that neither rearing method (natural 302 vs. artificial) nor colostrum source (goat vs. sheep) nor colostrum intake time (2 h vs.14 303 h after birth) seemed to affect ChT activity in newborn lambs. 304 305 Complement System activity (TCA and ACA) was not detectable at birth (0 d) in any of 306 the studied groups, becoming detectable from 1 d after birth and reaching the maximum 307 value at the end of this period (5 d). NR group obtained the highest Complement System 308 activity (TCA and ACA) from 2 d (20.56% and 15.05%, TCA and ACA, respectively) 309 to 5 d (42.43% and 36.26% of TCA and ACA, respectively). In general, no differences 310 in Complement System activity were found between artificial rearing groups. In 311 accordance with these findings, Eckblad et al. (1981a) suggested that Complement 312 System components in colostrum may play an important role in the development of the 313 Complement System activity in newborn animals through gut absorption, so a higher 314 amount of colostrum intake may produce an earlier development of the Complement 315 System activity, as happened in the NR group. Similarly, Castro et al. (2008) showed 316 the importance of dam milk in the earlier Complement System activation compared with 317 milk replacer feeding. Results of the current study indicate that neither the colostrum 318 source (goat vs. sheep) nor the colostrum intake delay (2 h vs. 14 h after birth) affect the 319 Complement System development. 320 Capítulo 2 108 CONCLUSION 321 In this study, lambs from the natural rearing system showed in general, higher immune 322 condition and BW than animals reared under the different artificial system during the 323 first days after birth. However, further studies will be necessary in order to achieve a 324 similar initial IgG concentration in the artificial rearing groups compared to the natural 325 ones. 326 327 These findings may improve the management systems in place in lamb farms as it 328 shows that it is not necessary to colostrum feed immediately after birth and, also, that 329 goat colostrum could be used to bottle fed newborn lambs. 330 331 ACKNOWLEDGMENTS 332 Author L.E. Hernández-Castellano, acknowledges financial support from the 333 Formación del Profesorado Universitario (FPU) program (Ministry of Education, 334 Madrid, Spain). Authors acknowledge Ms. Heather Briggs for her help in the grammar 335 revision of this manuscript. 336 337 REFERENCES 338 Ahmad, R., A. Khan, M. T. Javed, and I. Hussain. 2000. The level of immunoglobulins in relation 339 to neonatal Lamb mortality in Pak-Karakul sheep. Vet Arhiv 70(3):129-139. 340 Argüello, A., N. Castro, M. Batista, I. Moreno-Indias, A. Morales-DelaNuez, D. 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Journal of Dairy Science 66(6):1319-1328. 438 Trujillo, A. J., N. Castro, J. M. Quevedo, A. Arguello, J. Capote, and B. Guamis. 2007. Effect of 439 heat and high-pressure treatments on microbiological quality and immunoglobulin G stability 440 of caprine colostrum. Journal of Dairy Science 90(2):833-839. 441 Winter, A. 2011. Bovine neonatal pancytopenia, and anaemia in lambs caused by feeding cow 442 colostrum. Veterinary Record 168(3):84-84. 443 Winter, A. C. and M. J. Clarkson. 1992. Farm investigations of anemia in labms caused by 444 feeding cow colostrum. Veterinary Record 131(10):213-216. 445 Wold, A. E. and I. Adlerberth. 2000. Breast feeding and the intestinal microflora of the infant - 446 Implications for protection against infectious diseases. Pages 77-93 in Short and Long Term 447 Chapter 3 117 ABSTRACT 48 There are several factors that can affect the lamb BW and immune parameters before 49 weaning, such as rearing system or milk source. For this reason the aim of this 50 experiment was to evaluate the effect of diet (sheep milk-NR vs. milk replacer-MR vs. 51 whole powdered cow milk-CM) on the BW and immune parameters during milk 52 feeding period and weaning. In this study 60 lambs were randomly divided according to 53 treatment (NR, MR and CM). Blood plasma was used to measure the immunoglobulin 54 concentration (IgG and IgM), Chitotriosidase activity and Complement System activity 55 (Total and Alternative pathways, TCA and ACA, respectively). Results showed that 56 lambs reared with NR presented higher BW, IgG, IgM, TCA and ACA than animals 57 reared with MR or CM at 3 d and 5 d. However, during the weaning, these differences 58 disappeared. At the end of this period, animals reared with MR and CM showed higher 59 BW than lambs reared with NR (15.28, 16.89 and 17.66 kg in NR, MR and CM groups, 60 respectively, P<0.05). Moreover, MR and CM groups showed higher IgM 61 concentrations at the end of this period than NR group (1.05, 1.90 and 1.60 mg/mL in 62 NR, MR and CM, respectively, P<0.05). The findings showed in this study, may 63 improve the management in sheep farms, reducing the expenses of the artificial rearing 64 systems and, consequently, increasing the economic benefits of the sheep producers. 65 66 Keywords: Chitotriosidase, Immunity, lamb, whole powdered cow milk, weaning 67 68 69 70 71 72 Capítulo 3 118 INTRODUCTION 73 Newborn ruminants have three critical periods related to their immune system in the 74 first month of life: colostrum intake, milk feeding, and weaning. The management in 75 these periods affects the final animal performances (Marsico et al., 1993; Massimini et 76 al., 2007; Mastellone et al., 2011). 77 78 Nowadays, there is an increasing number of high production dairy farms, in which 79 artificial rearing is chosen in order to increase the amount of sheep milk available for 80 processing (cheese, yogurt) (Demiroren et al., 1995; Napolitano et al., 2008) and 81 simplifying management (Emsen et al., 2004). In this system, lambs are separated from 82 dams at early age (0–2 d) and then, they are fed with a milk replacer. The milk feeding 83 period is an important stage in the newborn lamb because milk is the only energy source 84 for these animals. Furthermore, in order to achieve an optimum performance, it has been 85 recommended to feed lambs with milk replacers formulated specifically for them 86 (Frederiksen, 1980), which are mainly based on cow milk, cereals and vegetable fats 87 (Bañón et al., 2006). However, milk replacer for lambs is usually considerably more 88 expensive than high quality calf milk replacer or even whole powdered cow milk for 89 human consumption (in some regions). 90 91 Weaning is a critical phase in domestic ruminant production, mainly because any 92 change produced in feeding strategies (frequency and composition) can be perceived as 93 a stressor in preweaning ruminants. Weaning is well known to increase susceptibility to 94 a variety of infectious diseases in ruminants due to the attenuation of the immune 95 system under high stress conditions, such as diet changes (Sowinska et al., 2001; 96 Hickery, 2003). For this reason, there is an increasing interest in finding effective 97 Chapter 3 119 dietary stress reducers and immune enhancers that may improve the disease resistance 98 in weaning ruminants (Kwon et al., 2011). 99 100 The immunoglobulin plasma concentration (IgG and IgM, mainly) is probably the most 101 important humoral immune parameter, being deeply studied in lambs (Klobasa and 102 Werhahn, 1989; Mukkur et al., 1998; Hashemi et al., 2008); however, there are other 103 immune components, such as the Chitotriosidase (ChT) activity and the Complement 104 System activity, that play a fundamental role in the innate immune response, acting as a 105 part of the host defense in newborn ruminants. Chitotriosidase is predominantly a 106 secretory protein that is able to hydrolyze chitin in the cell wall of fungi and nematodes 107 (Barone et al., 1999). This enzyme is a functional chitinase with high homology to 108 chitinases that belong to family of 18 glycosyl hydrolases. Chitotriosidase has an 109 important relation with the host defense and for this reason it has been investigated in 110 humans (Musumeci et al., 2005) and goats (Argüello et al., 2008a; Hernández111 Castellano et al., 2011; Moreno-Indias et al., 2012c), being this enzyme has never been 112 described in sheep or lambs . 113 114 The Complement System activity (Total (TCA) and Alternative (ACA) pathways) is 115 involved in specific and nonspecific immunity, playing an important role in defense 116 mechanisms against infectious microorganisms (Rodríguez et al., 2009), being one of 117 the first immune barriers to pathogens (Petrova and Mehta, 2007). The Complement 118 System in mammals has been well described, particularly in humans and mice, but it has 119 been also examined in ruminants, such as cows and goats (Mayilyan et al., 2008; 120 Moreno-Indias et al., 2012a; Moreno-Indias et al., 2012c). However, there are few 121 studies about the Complement System activity in sheep and lambs. 122 Capítulo 3 120 The aim of the present study was to analyze the effect of diet (sheep milk-NR, milk 123 replacer-MR and whole powdered cow milk-CM) on the BW evolution, the IgG and 124 IgM blood concentration and the ChT and Complement System activity during milk 125 feeding period and weaning. 126 127 MATERIAL AND METHODS 128 The present study was performed in the Department of Animal Science of the 129 Universidad de Las Palmas de Gran Canaria in Canary Islands (Spain) on 60 lambs (30 130 males and 30 females) of Canary breed. Animal procedures were approved by the 131 Ethical Committee of the University. 132 133 Colostrum Period 134 At birth, animals were randomly divided in three different groups according to diets 135 (NR, MR and CM). The NR group was composed of 20 lambs that were fed colostrum 136 directly from their dams. The other 40 lambs were artificial reared without dam contact. 137 The MR and CM animals received a pool of fresh sheep colostrum that was previously 138 pasteurized at 63ºC for 30 minutes according to Trujillo et al. (2007). Because no 139 recommendation about the requirements of colostrum IgG/BW in artificially reared 140 lambs was found in the literature, lambs received 4g of IgG/Kg of BW during the 141 colostrum period (0-2 d after birth), according to recommend concentration for goat 142 kids (Castro et al., 2005a). Sheep colostrum IgG concentration was determined using a 143 commercial ELISA kit (Bethyl laboratories, Montgomery, TX, USA), using purified 144 sheep IgG (Bethyl laboratories, Montgomery, TX, USA) for the standard curve. 145 146 147 Chapter 3 121 Milk Feeding Period 148 The NR group was raised with unlimited access to their dams until weaning period. 149 Ewes were not milked during this period. After colostrum period, the other 40 lambs 150 were randomly allotted into two artificial rearing groups: one of them received a 151 commercial milk replacer at 16% (w/w; MR group, Bacilactol Corderos y Cabritos, 152 Saprogal, La Coruña, Spain; 95.5% DM, 23.6% CP and 22.7% ether extract, air-dry 153 powder basis), and the other one received whole powdered cow milk for human 154 consumption at 16% (w/w; CM group, whole powdered milk, Arla foods, Denmark; 155 97% DM, 27% CP, 28% ether extract, air-dry powder basis). Both groups were fed ad 156 libitum (37ºC), using nipple buckets twice a day (8:00 a.m. and 05:00 p.m.). 157 158 Weaning Period 159 Animals started the weaning period when they reached 10 Kg of BW. During the 160 weaning period (30 d) the three studied groups had free access to starter feed (18% CP 161 and 3.4% ether extract), alfalfa hay and water. The NR lambs were removed from dams 162 and placed in a pen. Ewes were milked during the whole weaning period once daily 163 (09:00 a.m.). During the first wk, lambs had access to ewes twice daily (10:00 a.m. after 164 milking, and 05:00 p.m.), reducing this access to once a day (05:00 p.m.) during the 165 second wk, and thereafter, lambs had no more access to ewes. The MR and CM groups 166 were fed with half a liter of the selected diet twice a day during the first wk (10:00 a.m. 167 and 05:00 p.m.), reducing to once daily at the second wk (05:00 p.m.). During the rest 168 of the period animals received neither cow milk nor milk replacer. 169 170 171 172 Capítulo 3 122 BW and sample collection 173 During the milk feeding period, blood samples were taken at 3 d, 5 d and 20 d after 174 birth. Another sample was collected when animals reached 10 Kg of BW. During the 175 weaning period, samples were obtained every 5 days until the end of the experiment. 176 Animals were weighed before blood extraction (MOBBA, Barcelona, Spain; accuracy, 177 5g). Blood samples were taken before feeding from the jugular vein in 2.5 mL tubes 178 with EDTA. After that, blood was centrifuged at 2190 g for 5 minutes at 4ºC (Hettich179 Zentrifugen, Universal 32 R, Germany), storing the plasma at -80° C until analysis. 180 181 To determine plasma IgG and IgM concentrations, a commercial ELISA kit (Bethyl 182 Laboratories, Montgomery, TX, USA) was used, setting a purified sheep IgG and IgM 183 as a standard reference. Results were expressed in mg of immunoglogulin/mL of 184 plasma. 185 186 Chitotriosidase activity was measured following the procedure described by Argüello et 187 al. (2008a). In this procedure, 1 µL of undiluted blood plasma with 100 µL of a solution 188 containing 22 mM artificial substrate (4-methylumbelliferyl-d-N, N′, N″ 189 triacetylchitotriose) in 0.5 M citrate phosphate buffer (pH 5.2) was incubated for 15 min 190 at 37°C. The reaction was stopped with 5 mL of 0.5 M Na2CO3-NaHCO3 buffer (pH 191 10.7). Fluorescence was measured using a fluorimeter (Perkin Elmer, Norwalk, CT) set 192 to an exictation at 365 nm and emission at 450 nm. The ChT activity was expressed as 193 nanomoles of substrate hydrolyzed per milliliter per hour. 194 195 Complement System activity (TCA and ACA) was measured using a novel technique 196 described by Moreno-Indias et al. (2012b). A DGHB++ buffer [Hepes Gelatin Veronal 197 Chapter 3 123 Buffer with Ca++ and Mg++: 5 mM HEPES, 71 mM NaCl, 0.15 mM CaCl2, 0.5 mM 198 MgCl2, 2.5% (w/v) glucose, 0.1% (w/v) gelatin, pH 7.4] was used to measure the total 199 Complement System activity, and DGHB-Mg-EGTA buffer [4.2 mM Hepes, 59 mM 200 NaCl, 7.0 mM MgCl2, 2.08% (w/v) glucose, 0.08% (w/v) gelatin, 10 mM EGTA, pH 201 7.4] was used to measure the alternative pathway. For total Complement System 202 activity, rabbit red blood cells and lamb plasma were both diluted to 5% in DGHB++; 203 100 µL of each was then mixed in a microtiter plate and incubated at 37°C for 1 h. Cells 204 were removed by centrifugation (2500  g, 5 min, 4ºC), and supernatant absorbance was 205 measured at 405 nm using a microplate reader. Complete hemolysis was achieved by 206 mixing the cells with distilled water (100 µL), and spontaneous lysis was produced by 207 mixing the diluted rabbit red blood cells with DGHB++. Complement-induced hemolysis 208 of rabbit red blood cells by the test sera was calculated using the formula: [(A405 209 sample − A405 spontaneous lysis) / (A405 complete hemolysis − A405 spontaneous 210 lysis)]  100. The same protocol was performed with DGHB-Mg-EGTA buffer to 211 measure the alternative pathway. 212 213 Statistical analyses 214 Statistical analyses were performed using SAS, Version 9.00 (SAS Institute Inc., Cary, 215 NC). The SAS PROC MIXED procedure for repeated measures was used to evaluate 216 the effect of the milk source (NR, MR, CM) on BW evolution, IgG and IgM 217 concentration, ChT and Complement System activity during milk feeding and weaning 218 period. A Tukey’s test was used to evaluate differences between groups. 219 220 221 222 Capítulo 3 124 RESULTS AND DISCUSSION 223 Table 1 shows the results of BW, IgG and IgM concentration, ChT activity, TCA and 224 ACA during the milk feeding period. In reference to BW evolution, all the studied 225 groups increased their initial BW at 3 d after birth; although differences between groups 226 where observed. Sheep milk group showed the highest BW at 3 d, 5 d and 20 d after 227 birth (4.95, 5.43, 8.96 kg, respectively). Due to the similar BW at birth between groups 228 (4.15, 4.35 and 4.46 kg in NR, MR and CM groups, respectively), differences between 229 NR and the other two groups could be related to factors associated with the increase of 230 suckling frequency in animals reared in the NR group (Lanza et al., 2006). As it has 231 been studied by Rodríguez et al. (2008), lambs that are reared under restricted 232 conditions presented lower ADG (253 g/d) than animals that were raised with their 233 dams (307 g/d). Baumrucker and Blum (1993) found that dam’s milk has a growth 234 promoter that is not present in milk replacers, which could explain the higher BW in NR 235 lambs. In agreement with these findings, Argüello et al. (2004a) obtain heavier goat kids 236 in a natural rearing system than goat kids fed with milk replacer. However, when MR 237 and CM groups were compared, no differences due to the diet were observed during this 238 period. 239 240 Focusing on the IgG concentrations, all animals decreased the IgG concentrations in 241 blood from 3 d until 5 d after birth. In the NR group, IgG concentrations also decreased 242 at 20 d (6.47 mg/mL) and at the end of the period (5.22 mg/mL). On the other hand, IgG 243 concentrations in MR and CM groups remained similar at 20 d (3.15 and 4.44 mg/mL in 244 MR and CM groups, respectively), but decreased at the end of the milk feeding period 245 (2.78 and 2.88 g/mL in MR and CM groups, respectively). The highest IgG 246 concentrations were obtained by the NR lambs throughout the studied period, probably 247 Chapter 3 125 because of the unrestricted amount of colostrum ingested. In agreement with these 248 results, Altiner et al. (2005) described that lambs reared with their dams obtained higher 249 IgG concentrations in blood than lambs reared with milk replacer. Nevertheless, Firat et 250 al. (2003) found no differences in the milk feeding period, between lambs that received 251 a similar colostrum intake and lambs raised under natural and artificial conditions, 252 obtaining higher IgG concentrations than the results showed in this study. On the other 253 hand, comparing both artificial rearing groups (MR and CM), show that no differences 254 were observed in IgG concentrations during the whole studied period. To the best of our 255 knowledge, no references about the immune evolution due to the different milk sources 256 used in artificial rearing has been found in lambs, moreover, Moreno-Indias et al. 257 (2012b) did not find any difference in IgG concentration between goat kids artificial 258 reared with fresh goat milk or cow milk. These results support the idea that there were 259 no differences between lambs reared with MR or CM on blood IgG concentrations, 260 although higher IgG values were obtained in the NR group, probably produced by the 261 higher colostrum intake of this last group. 262 263 The NR group obtained the highest IgM concentration at 3 d after birth (1.32 mg/mL), 264 decreasing at 5 d and 20 d and remaining constant until the end of the milk feeding 265 period (0.98, 0.71 and 0.78 mg/mL, respectively). A different evolution was observed in 266 MR and CM groups, whereas the IgM concentration decreased at 5 d (0.41 mg/mL in 267 both MR and CM) and kept constant at 20 d (0.50 and 0.39 mg/mL, respectively), 268 increasing and obtaining the highest value at the end of the milk feeding period (0.82 269 mg/mL and 0.65 mg/mL in MR and CM, respectively). According to findings observed 270 by Nonnecke et al. (2012) in colostrum-fed and colostrum-deprived calves, animals fed 271 with colostrum showed a rapid increase of serum IgG and IgM concentrations after 272 Capítulo 3 126 colostrum intake, decreasing these concentrations with age. The colostrum-deprived 273 calves, in contrast, had very low or undetectable serum IgG and IgM concentrations, 274 followed by an age-related increase of IgG and IgM concentrations, suggesting 275 endogenous production of these immunoglobulins. Although in the present study this 276 evolution was only observed in IgM concentrations, it has been described that IgM is 277 the first immunoglobulin produced by the organism (Ehrenstein and Notley, 2010). The 278 NR group registered higher IgM concentrations at 5 d (0.98 mg/mL) and at 20 d (0.71 279 mg/mL) than the MR and CM groups at the same time. As it was described before, the 280 unrestricted access of the NR group to colostrum could produce a higher IgM 281 concentration in blood. At the end of this period, no differences were observed between 282 groups. No differences between MR and CM were detected during the whole milk 283 feeding period. Moreno-Indias et al. (2012b) did not observed differences, due to the 284 milk source (Goat milk and cow milk) in goat kids reared under an artificial rearing 285 system from partum to 35 d of life. In this study, no differences in blood IgM 286 concentrations were observed between NR, MR and CM diets at the end of this period, 287 although NR group showed higher IgM concentrations at 5 d than the other two groups. 288 289 Chitotriosidase activity remained constant from 3 d after birth to the end of this period 290 in the three studied groups. Moreover, even a punctual exception was found at 20 d; no 291 differences were found between different groups in this period. The ChT activity is 292 related to the immune status of animals; however there are no studies that have 293 described this enzyme in sheep. In goat kids similar values to these results were 294 reported by Rodríguez et al. (2009), who similarly demonstrated that ChT activity in 295 goat kids does not vary with age during the first 5 d of life. A similar evolution was also 296 described by Moreno-Indias et al. (2012b), although higher values than present results 297 Chapter 3 133 Mastellone, V., G. Massimini, M. E. Pero, L. Cortese, D. Piantedosi, P. Lombardi, D. Britti, and L. 475 Avallone. 2011. Effects of Passive Transfer Status on Growth Performance in Buffalo Calves. 476 Asian Austral J Anim 24(7):952-956. 477 Matos-Gomes, N., M. Katsurayama, F. H. Makimoto, L. L. O. Santana, E. Paredes-Garcia, M. A. 478 D. Becker, and M. C. Dos-Santos. 2010. Psychological Stress and Its Influence on Salivary Flow 479 Rate, Total Protein Concentration and IgA, IgG and IgM Titers. Neuroimmunomodulation 480 17(6):396-404. 481 Mayilyan, K. R., Y. H. Kang, A. W. Dodds, and R. B. Sim. 2008. The Complement System in 482 Innate Immunity. Pages 219-236 in Innate Immunity of Plants, Animals, and Humans. Vol. 21. 483 H. Heine, ed. Springer Berlin Heidelberg. 484 Moreno-Indias, I., A. W. Dodds, A. Arguello, N. Castro, and R. B. Sim. 2012a. The complement 485 system of the goat: Haemolytic assays and isolation of major proteins. Bmc Vet Res 8. 486 Moreno-Indias, I., A. Morales-delaNuez, L. E. Hernandez-Castellano, D. Sanchez-Macias, J. 487 Capote, N. Castro, and A. Arguello. 2012b. Docosahexaenoic acid in the goat kid diet: Effects on 488 immune system and meat quality. Journal of animal science 90(11):3729-3738. 489 Moreno-Indias, I., D. Sanchez-Macias, N. Castro, A. Morales-delaNuez, L. E. Hernandez490 Castellano, J. Capote, and A. Arguello. 2012c. Chemical composition and immune status of 491 dairy goat colostrum fractions during the first 10 h after partum. Small Ruminant Research 492 103(2-3):220-224. 493 Mukkur, T. K. S., K. H. Walker, and G. H. McDowell. 1998. Passive immunisation of neonatal 494 lambs via colostrum and milk of ewes previously immunised with live attenuated Salmonella 495 typhimurium protects neonatal lambs from experimental salmonellosis. Comp. Immunol. 496 Microbiol. Infect. Dis. 21(4):327-336. 497 Musumeci, M., L. Malaguarnera, J. Simpore, R. Barone, M. Whalen, and S. Musumeci. 2005. 498 Chitotriosidase activity in colostrum from African and Caucasian women. Clin Chem Lab Med 499 43(2):198-201. 500 Napolitano, F., G. F. Cifuni, C. Pacelli, A. M. Riviezzi, and A. Girolami. 2002. Effect of artificial 501 rearing on lamb welfare and meat quality. Meat Science 60(3):307-315. 502 Napolitano, F., C. Pacelli, A. Girolami, and A. Braghieri. 2008. Effect of information about 503 animal welfare on consumer willingness to pay for yogurt. Journal of Dairy Science 91(3):910504 917. 505 Nonnecke, B. J., W. R. Waters, J. P. Goff, and M. R. Foote. 2012. Adaptive immunity in the 506 colostrum-deprived calf: Response to early vaccination with Mycobacterium bovis strain bacille 507 Calmette Guerin and ovalbumin. Journal of Dairy Science 95(1):221-239. 508 Oswald, I. P., F. Lantier, and G. Bourgy. 1990. Classical and Alternative Pathway Hemolytic 509 Activities of Ovine Complement - Variations with Age and Sex. Vet Immunol Immunop 510 24(3):259-266. 511 Petrova, A. and R. Mehta. 2007. Dysfunction of innate immunity and associated pathology in 512 neonates. Indian J Pediatr 74(2):185-191. 513 Rhind, S. M., H. W. Reid, S. R. McMillen, and G. Palmarini. 1998. The role of cortisol and beta514 endorphin in the response of the immune system to weaning in lambs. Anim Sci 66:397-402. 515 Rodríguez, A. B., R. Landa, R. Bodas, N. Prieto, A. R. Mantecon, and F. J. Giraldez. 2008. Carcass 516 and meat quality of Assaf milk fed lambs: Effect of rearing system and sex. Meat Science 517 80(2):225-230. 518 Rodríguez, C., N. Castro, J. Capote, A. Morales-delaNuez, I. Moreno-Indias, D. Sanchez-Macias, 519 and A. Arguello. 2009. Effect of colostrum immunoglobulin concentration on immunity in 520 Majorera goat kids. Journal of Dairy Science 92(4):1696-1701. 521 Sowinska, J., H. Brzostowski, Z. Tanski, and K. Czaja. 2001. The weaning stress response in 522 lambs of different age. Czech J Anim Sci 46(11):465-468. 523 Capítulo 3 134 Trujillo, A. J., N. Castro, J. M. Quevedo, A. Arguello, J. Capote, and B. Guamis. 2007. Effect of 524 heat and high-pressure treatments on microbiological quality and immunoglobulin G stability 525 of caprine colostrum. Journal of Dairy Science 90(2):833-839. 526 527 Chapter 3 135 Capítulo 3 136 Capítulo 4 Chapter 4 Cápitulo 4 139 Running head: Colostrum importance on lamb immune status 1 2 The importance of colostrum period management on the weight and immune 3 parameters in lambs: from birth to weaning. 4 L. E. Hernández-Castellanoa, A. Suárez-Trujilloa, D. Martell-Jaizmea, G. Cugnob, N. 5 Castroa and A. Argüelloa. 6 aDepartment of Animal Science, Universidad de Las Palmas de Gran Canaria, 35413 7 Arucas, Gran Canaria, Spain. 8 bpreguntar a Graziano 9 10 11 12 13 14 15 16 17 18 19 Chapter 4 140 ABSTRACT 20 The aim of this study was to investigate the BW and immune status of lambs reared 21 under natural conditions and lambs reared under artificial conditions fed with two 22 different colostrum amounts (4 g/kg of BW and 8 g/kg of BW of IgG). In this study 60 23 lambs were randomly divided according to treatment. Twenty lambs remained with their 24 dams (NR group). Forty lambs were removed from their lambs at birth and were bottle 25 fed with 4 g/kg of BW (C4 group) or 8 g/kg of BW of IgG (C8 group). Blood plasma 26 samples and BW recording were done before feeding. Blood plasma was used to 27 determine the immunoglobulin concentration (IgG and IgM) and the Complement 28 system activity (Total and Alternative pathways). Lambs from NR group showed higher 29 BW than C4 and C8 groups during milk feeding period, while those groups had higher 30 BW than NR group at the end of weaning period (15.28, 16.89 and 16.95 kg in NR, C4 31 and C8 groups, respectively, P<0.05). With reference to immune parameters, C8 and 32 NR groups had higher plasma IgG and IgM concentration than C4 group during milk 33 feeding period (P<0.05). Addtionally, C4 and C8 groups showed a similar IgG 34 concentration and higher IgM concentration than NR group at the end of weaning 35 period (P<0.05). Complement system activity was higher in NR group than in C4 and 36 C8 groups during the first 3 days after birth (P<0.05). As a conclusion, lambs fed with 37 the amount of colostrum equivalent to 8g of IgG/ kg of BW showed similar immune 38 parameters than lambs from NR group getting a better BW at the end of the weaning 39 period. 40 41 Keywords: Immune, lamb, colostrum, weaning, artificial rearing. 42 Cápitulo 4 141 INTRODUCTION 43 The relation between colostrum and the survival of newborn ruminants has been long 44 characterized (Argüello et al., 2004b; Castro et al., 2005b; Castro et al., 2009; Castro et 45 al., 2011). In fact, colostrum contains a complex mixture of proteins that actively 46 participate in the protection of the dam and the neonate (Passive Inmune Transfer; PIT) 47 against pathogens and other post-partum environmental challenges (Bendixen et al., 48 2011). Moreover, colostrum contains diverse components, such as fat, lactose, vitamins 49 or minerals that have a high nutritional importance (Ontsouka et al., 2003). 50 In particular, colostrum plays an important role in newborn lambs, because they are 51 born hypo gammaglobulinemic, due to the complexity of the synepitheliochorial 52 ruminant placenta which does not allow a sufficient transfer of immunoglobulins from 53 the dam to the foetus (Castro et al., 2009). Additionally, it has been described that lambs 54 which are not fed with colostrum in the first hours of life are more susceptible to 55 diseases and mortality (Ahmad et al., 2000; da Nobrega et al., 2005; Nowak and 56 Poindron, 2006). 57 Nowadays, there are an increasing number of high production dairy farms, where lambs 58 are reared under an artificial feeding system. In those cases, lambs are bottle-fed with 59 colostrum and milk replacer in order to increase the amount of milk available for 60 processing (cheese, yogurt) (Demiroren et al., 1995; Napolitano et al., 2008), 61 simplifying the animal management (Emsen et al., 2004). 62 It has been described by several authors (Castro et al., 2005b; Morales-delaNuez et al., 63 2009; Moreno-Indias et al., 2012b) that goat kids need to be fed with an amount of 64 colostrum equivalent to 4 gr of IgG/kg of BW, divided into 3 meals before 48 h after 65 Chapter 4 142 birth, in order to make an appropriate PIT. However, no references about the amount of 66 colostrum required by newborn lambs reared under artificial conditions have been 67 found. This knowledge has a high relevance on the survival of the lambs during these 68 first days of life, because it is essential to reach an adequate initial immunoglobulin 69 concentration on blood (O'Doherty and Crosby, 1997; Quigley et al., 2000; Christley et 70 al., 2003). 71 The aim of this study was to investigate the BW and immune status of lambs reared 72 under natural conditions and lambs reared under artificial conditions fed with two 73 different colostrum amounts (4 g IgG/ kg of BW and 8 g IgG/ kg of BW). 74 75 MATERIAL AND METHODS 76 The present study was performed in the Department of Animal Science of the 77 Universidad de Las Palmas de Gran Canaria in the Canary Islands (Spain) on 60 lambs 78 (30 males and 30 females) from Canary breed. Animal procedures were approved by the 79 Ethical Committee of the University. 80 81 Colostrum and Feeding Period 82 At birth, animals were randomly divided into three different groups. The natural rearing 83 (NR) group was composed of 20 lambs which sucked colostrum directly from their 84 dams. The other 40 lambs were removed from their dams and then divided in 2 blocks 85 (20 lambs each) according to colostrum feeding treatment (4 g of IgG/ kg of BW and 8 86 g of IgG/kg of BW) without dam contact. Each room had central heating conferring a 87 room temperature of 20ºC (approximately) and providing at least 0.3 m2 floor space per 88 lamb. Both artificial rearing groups (C4 and C8) received a sheep colostrum pool that 89