UNIVERSIDAD POLITÉCNICA DE MADRID ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS INFLUENCE OF NUTRITIONAL AND MANAGEMENT PRACTICES ON PRODUCTIVE PERFORMANCE AND EGG QUALITY OF BROWN EGG EGG-LAYING HENS INFLUENCIA DE FACTORES NUTRICIONALES Y DE MANEJO SOBRE LA PRODUCTIVIDAD Y CALIDAD DEL HUEVO EN GALLINAS PONEDORAS RUBIAS TESIS DOCTORAL Adriano Pérez Bonilla INGENIERO AGRÓNOMO 2012 DEPARTAMENTO DE PRODUCCIÓN ANIMAL ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS INFLUENCE OF NUTRITIONAL AND MANAGEMENT PRACTICES ON PRODUCTIVE PERFORMANCE AND EGG QUALITY OF BROWN EGG EGG-LAYING HENS INFLUENCIA DE FACTORES NUTRICIONALES Y DE MANEJO SOBRE LA PRODUCTIVIDAD Y CALIDAD DEL HUEVO EN GALLINAS PONEDORAS RUBIAS Adriano Pérez Bonilla INGENIERO AGRÓNOMO DIRECTOR DE TESIS Gonzalo González Mateos Dr. INGENIERO AGRÓNOMO 2 “Lo importante es no dejar de hacerse preguntas” “Una enorme cantidad de experimentos no pueden probar definitivamente que tengo razón, pero un solo experimento puede probar que estoy equivocado” “Se debe hacer todo tan sencillo como sea posible, pero no más sencillo” Albert Einstein “La ciencia es la progresiva aproximación del hombre al mundo real” Max Planck “El saber te hará libre” Anónimo 3 A mis padres, hermanos, abuelos, tios y a Clara 4 AGRADECIMIENTOS ¡Por fin!, después de mucho tiempo de estudio y trabajo a las espaldas llego a la culminación de todo mi esfuerzo tanto profesional como personal, por ello, a estas alturas de la “película” no me gustaría dejarme a nadie en el tintero. En primer lugar agradecer a mis padres la educación recibida a lo largo de mi vida, educarme en la filosofía del esfuerzo, del tesón, del compañerismo, de la solidaridad, del “no todo vale”, en fin, gracias por todos los logros que he conseguido gracias a vosotros, vuestro cariño, comprensión, aguante y esfuerzo. A mis hermanos David e Israel, porque sin su apoyo y motivación hasta el infinito hubiera sido imposible acabar esta tesis entre otras muchas cosas. Especial mención a mi piticli, mi pequeña, mi niña, mi muro de las lamentaciones, mi psicóloga, mi profesora de estadística, de Excel, de Word, de Power Point, de SAS.. Clara, sabes que sin ti no lo hubiera consegido. Gracias amor!. A la dirección de mi empresa, Camar Agroalimentaria por permitirme la realización de esta Tesis y confiar en mí a lo largo de todo este período, sin duda, de una de las cosas de las que me siento más orgulloso es ver plasmado mi trabajo en el día a día y sentir que contribuyo de forma importante en la mejora del negocio. Muchas gracias. 5 A Gonzalo, simplemente decirte que te estaré eternamente agradecido por todo el trabajo que hemos desarrollado juntos estos últimos años y sobre todo al enorme esfuerzo que se que ha supuesto las correcciones, mirar datos, etc, mientras estabas de punta a punta del mundo. Me siento un privilegiado al trabajar con una persona con tanto prestigio y con tanta capacidad de trabajo. A todo el personal de Camar Agroalimentaria y en especial a “mis granjeros”, Félix, Ángel y Mario; y a los auxiliares de laboratorio Gianina y Eugenia por todo el trabajo bien hecho, por todos esos fines de semana, por toda esa cantidad de días de puesta, peso de huevo, contar gallinas, pesar gallinas…etc, por todos esos detalles de manejo en nave y laboratorio, sin vosotros esto no hubiera sido posible. Mil gracias. Al mis compañeros del equipo de trabajo de Gonzalo (Mohamed, Martina, Julio, Lourdes, Carine, Maziar, Sara, Beatriz, Pilar, Sergio, Vahid, Hissam ….) por el escrupuloso trabajo realizado en nave y laboratorio. Os debo mucho. A mi compañero y amigo Samuel Novoa, por su inestimable ayuda en las naves, por ser tan trabajador, tan buen profesional y ser un amigo en momentos difíciles. A la granja experimental de Nutreco (Poultry Research Center), mi segunda familia, y en especial a Pedro Pérez de Ayala y Rosa Rocha por permitirme “asaltar” la fábrica y a Marcos, Paco y Borja por su excelente trabajo en la fabricación de los piensos. Gracias igualmente a Ángel Fernández por resolverme las 1000 dudas sobre los métodos de análisis de laboratorio y al grupo de 6 investigadores de avicultura (Ángela y Jon) por resolverme dudas “existenciales” y a mi antigua “jefa” Anabel por su apoyo incondicional. A todos mis profesores de la rama de producción animal, tanto de la E.U.I.T. Agrícolas como de la E.T.S.I. Agrónomos por formarme como persona y como profesional a lo largo de estos 14 años, por “moldearme”, por generarme esta inquietud interior sobre nuestra responsabilidad y compromiso con la sociedad. A los editores de la sección de Metabolismo y Nutrición del Poultry Science (Robert Elkin, Enric Esteve y Markus Rudehustcord) y a los revisores de los artículos, por sus inestimables correcciones y comentarios en el desarrollo de los artículos. MUCHAS GRACIAS A TODOS LO CONSEGUIMOS!!!! Adriano Pérez Bonilla 7 INDEX RESUMEN .................................................................................................................... 19 ABSTRACT .................................................................................................................. 25 CHAPTER 1: LITERATURE REVIEW AND OBJECTIVES ............................... 30 1. LITERATURE REVIEW................................................................................................ 31 1.1. Introduction ...................................................................................................... 31 1.2. Effect of the main cereal of the diet on hen productivity and egg quality........ 34 1.3. Effect of supplemented fat of the diet on hen productivity and egg quality ..... 36 1.4. Effect of linoleic acid of the diet on hen productivity and egg quality............. 38 1.5. Effect of protein and aminoacids content of the diet on hen productivity and egg quality ............................................................................................................... 39 1.6. Effect of energy content of the diet on hen productivity and egg quality ......... 42 1.7. Effect of initial body weight at the onset of lay on hen productivity and egg quality ...................................................................................................................... 44 2. OBJECTIVES ............................................................................................................. 46 3. REFERENCES ............................................................................................................ 47 CHAPTER 2: EFFECTS OF THE MAIN CEREAL AND TYPE OF FAT OF THE DIET ON PRODUCTIVE PERFORMANCE AND EGG QUALITY OF BROWNEGG LAYING HENS FROM 22 TO 54 WEEKS OF AGE (TRIAL 1) ................. 59 1. INTRODUCTION ........................................................................................................ 60 2. MATERIAL AND METHODS ........................................................................................ 62 2.1. Husbandry, Feeding Program, and Experimental Diets.................................. 62 2.2. Laboratory Analyses ........................................................................................ 64 8 2.3. Productive Performance and Egg Quality ....................................................... 65 2.4. Statistical Analysis ........................................................................................... 66 3. RESULTS .................................................................................................................. 67 3.1. Laboratory analysis.......................................................................................... 67 3.2. Productive performance ................................................................................... 67 3.3. Egg quality ....................................................................................................... 68 4. DISCUSSION ............................................................................................................. 68 4.1. Productive performance ................................................................................... 68 4.2. Egg quality ....................................................................................................... 71 5. CONCLUSIONS .......................................................................................................... 72 6. REFERENCES ............................................................................................................ 81 CHAPTER 3: EFFECT OF CRUDE PROTEIN AND FAT CONTENT OF THE DIET ON PRODUCTIVE PERFORMANCE AND EGG QUALITY TRAITS OF BROWN EGG-LAYING HENS WITH DIFFERENT INITIAL BODY WEIGHT (TRIAL 2) ...................................................................................................................... 87 1. INTRODUCTION ........................................................................................................ 88 2. MATERIALS AND METHODS ...................................................................................... 89 2.1. Husbandry, Feeding Program, and Experimental Diets.................................. 89 2.2. Analytical Evaluation of Ingredients and Feeds .............................................. 90 2.3. Productive performance and egg quality ......................................................... 91 2.4. Statistical analysis ............................................................................................ 92 3. RESULTS .................................................................................................................. 92 4. DISCUSSION ............................................................................................................. 93 5. REFERENCES .......................................................................................................... 103 9 CHAPTER 4: EFFECTS OF ENERGY CONCENTRATION OF THE DIET ON PRODUCTIVE PERFORMANCE AND EGG QUALITY OF BROWN EGGLAYING HENS DIFFERING IN INITIAL BODY WEIGHT (TRIAL 3)........... 108 1. INTRODUCTION ...................................................................................................... 109 2. MATERIALS AND METHODS .................................................................................... 110 2.1.Husbandry, Diets, and Experimental Design .................................................. 110 2.2. Laboratory Analysis ....................................................................................... 111 2.3. Productive Performance and Egg Quality ..................................................... 112 2.4. Statistical Analysis ......................................................................................... 113 3. RESULTS ................................................................................................................ 113 3.1. Productive Performance ................................................................................ 113 3.2. Egg Quality .................................................................................................... 114 4. DISCUSSION ........................................................................................................... 115 4.1. Productive Performance ................................................................................ 115 4.1.1. AMEn Concentration of the Diet .......................................................... 115 4.1.2. Initial Body Weight ............................................................................... 118 4.2. Egg Quality .................................................................................................... 119 4.2.1. AMEn Concentration of the Diet .......................................................... 119 4.2.2. Initial Body Weight ............................................................................... 121 5. REFERENCES .......................................................................................................... 122 CHAPTER 5: GENERAL DISCUSSION AND CONCLUSIONS ........................ 133 1. GENERAL DISCUSSION ............................................................................................ 134 1.1. Productive performance in egg-laying hens .................................................. 134 1.1.1. Effect of the main cereal of the diet ...................................................... 134 10 1.1.2. Effect of source, fatty acid profile, and level of supplemented fat of the diet ....................................................................................................................... 135 1.1.3. Effect of linoleic acid of the diet ........................................................... 137 1.1.4. Effect of energy content of the diet ....................................................... 138 1.1.5. Effect of initial body weight of the hens ............................................... 141 1.2. Egg quality in brown egg-laying hens............................................................ 143 1.2.1. Effect of the main cereal of the diet ...................................................... 143 1.2.2. Effect of source, fatty acid profile, and level of supplemented fat of the diet ....................................................................................................................... 144 1.2.3. Effect of linoleic acid of the diet ........................................................... 146 1.2.4. Effect of energy content of the diet ....................................................... 147 1.2.5. Effect of initial body weight of the hens ............................................... 149 ANNEX I: RESUMEN EN ESPAÑOL..................................................................... 151 1. INTRODUCCIÓN ...................................................................................................... 152 2. REVISIÓN BIBLIOGRÁFICA ...................................................................................... 155 3. OBJETIVOS DE LA TESIS DOCTORAL ....................................................................... 169 EXPERIMENTO 1. EFECTOS DEL CEREAL PRINCIPAL Y EL TIPO DE GRASA EN LA DIETA SOBRE LOS PARÁMETROS PRODUCTIVOS Y LA CALIDAD DE HUEVO EN GALLINAS PONEDORAS RUBIAS EN EL PERIOD 22-54 SEMANAS DE VIDA ......................................... 170 1. MATERIAL Y METODOS .......................................................................................... 171 1.1. Crianza, Programa de Alimentación y Dietas Experimentales ..................... 171 1.2. Análisis de Laboratorio.................................................................................. 173 1.3. Variables Productivas y Calidad de Huevo ................................................... 174 1.4. Análisis Estadístico ........................................................................................ 175 2. RESULTADOS.......................................................................................................... 176 11 2.1. Variables Productivas .................................................................................... 176 2.2. Calidad del Huevo .......................................................................................... 177 3. DISCUSIÓN ............................................................................................................. 177 3.1. Variables Productivas .................................................................................... 177 3.2. Calidad de Huevo ........................................................................................... 179 4. CONCLUSIONES ...................................................................................................... 180 EXPERIMENTO 2. EFECTOS DEL NIVEL DE PROTEÍNA BRUTA Y EL CONTENIDO DE GRASA EN LA DIETA SOBRE LOS PARÁMETROS PRODUCTIVOS Y LA CALIDAD DEL HUEVO EN GALLINAS PONEDORAS RUBIAS CON DISTINTOS PESOS VIVOS ......................................................... 181 1. MATERIAL Y MÉTODOS .......................................................................................... 182 1.1. Crianza, Programa de Alimentación y Dietas Experimentales ..................... 182 1.2. Análisis de Laboratorio.................................................................................. 183 1.3. Variables Productivas y Calidad de Huevo ................................................... 184 1.4. Análisis Estadístico ........................................................................................ 185 2. RESULTADOS.......................................................................................................... 185 3. DISCUSIÓN ............................................................................................................. 186 4. CONCLUSIONES ...................................................................................................... 191 EXPERIMENTO 3. EFECTOS DE LA CONCENTRACIÓN ENERGETICA DE LA DIETA SOBRE LOS PÁRAMETROS PRODUCTIVOS Y LA CALIDAD DE HUEVO EN GALLINAS PONEDORAS RUBIAS CON DISTINTOS PESOS VIVOS.......................................................................................... 192 1. MATERIAL Y MÉTODOS .......................................................................................... 193 1.1. Crianza, Dietas y Diseño Experimental ......................................................... 193 1.2. Análisis de Laboratorio.................................................................................. 195 1.3. Productive Performance and Egg Quality ..................................................... 195 1.4. Statistical Analysis ......................................................................................... 196 12 2. RESULTADOS.......................................................................................................... 197 2.1. Variables Productivas .................................................................................... 197 2.2. Calidad de Huevo ........................................................................................... 198 3. DISCUSIÓN ............................................................................................................. 198 3.1. Variables Productivas .................................................................................... 198 4. CONCLUSIONES ...................................................................................................... 204 CONCLUSIONES GENERALES E IMPLICACIONES DE LA TESIS DOCTORAL........................ 206 REFERENCES .................................................................................................................. 210 13 ABBREVIATIONS LIST 14 ABBREVIATION LIST ºC: degree Celsius; grado centígrado *: P<0.05 **: P<0.01 ***: P<0.001 %: percentage; porcentaje AA: amino acid; aminoácido ADFI: average daily feed intake AMEn: nitrogen-corrected apparent metabolizable energy Arg: arginine; arginina AVO: acidulated vegetable oil soapstocks; oleina vegetal BW: body weight BWG: body weight gain C18:2: linoleic acid; ácido linoleico Ca: calcium; calcio cm: centimeter; centímetro cm2:centimeter square; centímetro cuadrado CF: crude fiber; fibra bruta CMD: consumo medio diario CP: crude protein; protein bruta Cys: cysteine, cisteina DM: dry matter; material seca d: day; día EE: ether extract; extracto etéreo EMAn: energía metabolizable aparente corregida en nitrógeno. 15 EnE: energy efficiency; eficiencia energética et al.: and others; y colaboradores FA: fatty acid; ácido graso FCR: feed conversion ratio FEDNA: Fundación Española para el Eesarrollo de la Nutrición Animal FI: feed intake g:gram GE: gross energy; energía bruta GLM: general lineal model GMD: geometric mean diameter; diámetro geometric medio GSD: geometric standard desviation; desviación estándart geométrica h: hour; hora HU: haugh unit; unidades haugh IC: índice de conversión Ile: isoleucine; isoleucina IU: international unit(s); unidades internacionales kcal: kilocalorie; kilocaloría kg: kilogram; kilogramo L: linear effect, efecto lineal LNL: linoleic acid; acido linoleico Lys: lysine, lisina m: meter; metro m2: square meter; metro cuadrado mEq: miliequivalent, miliequivalentes Met: methionine; metionina mg: miligram; miligramo 16 mm: milimeter; milímetro N: normal n: number of replicates per treatment; numero de réplicas por tratamiento nm: nanometer; nanometro NRC: National Research Council NS: not significant difference (P> 0.10); diferencia no significativa (P> 0,10) NSP: nonstarch polysaccharides; polisacáridos no amiláceos P: probability; probabilidad P: phosphorus; fósforo ppm: parts per million, partes por millon PUFA: polyunsaturated fatty acid PV: peso vivo Q: quadratic effect, efecto cuadrático SAS: Statistical Analysis Systems SBO: Soy bean oil; aceite de soja SCWL: Single Comb White Leghorn; gallinas Leghorn SD: standard deviation sem: semana SEM: standard error of the mean SFA: saturated fatty acid SFAT: supplemental fat Thr: threonine Trp: tryptophan, triptófano TSAA: total sufur amino acids; aminoácidos azufrados totales Val: valine; valina vs.: versus 17 µ: average; media µm: micrometer, micrometros wk: week XL: extra large eggs; huevos de tamaño XL (>73 g) 18 Resumen RESUMEN 19 Resumen Resumen El objetivo general de esta Tesis Doctoral fue estudiar la influencia de diversos factores nutricionales y de manejo sobre la productividad y la calidad del huevo en gallinas ponedoras comerciales rubias. Los factores estudiados fueron: 1) Cereal principal y tipo de grasa en la dieta; 2) Nivel de proteína bruta y grasa en la dieta; 3) Nivel energético de la dieta; 4) Peso vivo al inicio del período de puesta. En el experimento 1, la influencia del cereal principal en la dieta y el tipo de grasa suplementada en la dieta sobre los parámetros productivos y la calidad del huevo fue estudiado en 756 gallinas rubias de la estirpe Lohmann desde la sem 22 hasta las 54 de vida. El experimento se realizó mediante un diseño completamente al azar con 9 tratamientos ordenados factorialmente, con 3 cereales bases (maíz, trigo blando y cebada) y 3 tipos de grasa que variaban en su contenido en ácido linoléico (aceite de soja, oleína vegetal mezcla y manteca). Todas las dietas satisfacian las recomendaciones nutricionales para gallinas ponedoras rubias según el NRC (1994) y FEDNA (2008). La unidad experimental fue la jaula para todas las variables. Cada tratamiento fue replicado 4 veces, y la unidad experimental estuvo formada por 21 gallinas alojadas en grupos de 7. Las dietas fueron formuladas con un contenido nutritivo similar, excepto para el ácido linoléico, que varió en función del tipo de cereal y grasa utilizado. Así, dependiendo de la combinación de estos elementos el contenido de este ácido graso varió desde un 0.8% (dieta trigo-manteca) a un 3.4% (dieta maíz-aceite de soja). Este rango de ácido linoléico permitió estimar el nivel mínimo de este nutriente en el pienso que permite maximizar el peso del huevo. Los parámetros productivos y la calidad del huevo se controlaron cada 28 días y el peso de las aves se midió individualmente al inicio y al final del experimento con el objetivo de estudiar la variación en el peso vivo de los animales. No se observaron interacciones entre el tipo de cereal y grasa en la dieta para ninguna de las variables 20 Resumen productivas estudiadas. Los tratamientos experimentales no afectaron a las principales variables productivas (porcentaje de puesta, peso del huevo y masa de huevo). Sin embargo, la ganancia de peso fue mayor en gallinas alimentadas con maíz o trigo que las gallinas alimentadas con cebada (243 vs. 238 vs. 202 g, respectivamente; P< 0.05). En el mismo sentido, las gallinas alimentadas con manteca obtuvieron una mayor ganancia de peso que las gallinas alimentadas con aceite de soja u oleína vegetal (251 vs. 221 vs. 210 g, respectivamente; P< 0.05). En cuanto a las variables estudiadas en relación con la calidad del huevo, ninguna de las variables estudiadas se vio afectada por el tratamiento experimental, salvo la pigmentación de la yema. Así, las gallinas alimentadas con maíz como cereal principal obtuvieron una mayor puntuación en relación con la escala de color que las gallinas alimentadas con trigo y con cebada (9.0 vs. 8.3 vs. 8.3, respectivamente; P< 0.001). La pigmentación de la yema también se vio afectada por el tipo de grasa en la dieta, así, las gallinas alimentadas con manteca obtuvieron una mayor puntuación de color en relación con la escala de color que las gallinas alimentadas con aceite de soja u oleína vegetal (8.9 vs. 8.5 vs. 8.2, respectivamente; P< 0.001). La influencia del contenido en ácido linoléico respecto al peso de huevo y masa de huevo fue mayor a medida que el contenido de dicho ácido graso se redujo en la dieta. Así, la influencia de la dieta en los radios peso de huevo/g linoléico ingerido y masa de huevo/g linoléico ingerido fue significativamente mayor a medida que el contenido en dicho ácido graso disminuyo en la dieta (P< 0.001). Los resultados del ensayo indican que las gallinas ponedoras rubias no necesitan más de un 1.0% de ácido linoléico en la dieta para maximizar la producción y el tamaño del huevo. Además, se pudo concluir que los 3 cereales y las 3 grasas utilizadas pueden sustituirse en la dieta sin ningún perjuicio productivo o referente a la calidad del huevo siempre que los requerimientos de los animales sean cubiertos. 21 Resumen En el experimento 2, la influencia del nivel de proteína bruta y el contenido de grasa de la dieta sobre los parámetros productivos y la calidad del huevo fue estudiado en 672 gallinas ponedoras rubias de la estirpe Lohmann entre las sem 22 y 50 de vida. El experimento fue conducido mediante un diseño completamente al azar con 8 tratamientos ordenados factorialmente con 4 dietas y 2 pesos vivos distintos al inicio de puesta (1592 vs. 1860g). Tres de esas dietas diferían en el contenido de proteína bruta (16.5%, 17.5% y 18.5%) y tenían un contenido en grasa añadida de 1.8%. La cuarta dieta tenía el nivel proteico más elevado (18.5%) pero fue suplementada con 3.6% de grasa añadida en vez de 1.8%. Cada tratamiento fue replicado 4 veces y la unidad experimental consistió en 21 gallinas alojadas dentro de grupos de 7 animales en 3 jaulas contiguas. Todas las dietas fueron isocalóricas (2750 kcal EMAn/kg) y cubrieron las recomendaciones en aminoácidos para gallinas ponedoras rubias (Arg, Ile, Lys, Met, Thr, Trp, TSAA y Val) según el NRC (1994) y FEDNA (2008). Los efectos de los tratamientos sobre las variables productivas y la calidad de huevo fueron estudiados cada 28 días. La dieta no afecto a ninguna de las variables productivas estudiadas a lo largo del período productivo. Sin embargo, el peso inicial origino que las gallinas pesadas consumieran más (120.6 vs. 113.9 g; P< 0.001), obtuvieran un porcentaje de puesta mayor (92.5 vs. 89.8%; P< 0.01) y un peso del huevo mayor (64.9 vs. 62.4 g; P< 0.001) que las gallinas ligeras. El peso inicial de las gallinas no afecto al IC por kg de huevo ni a la mortalidad, sin embargo, la ganancia de peso fue mayor (289 vs. 233 g; P< 0.01) y el IC por docena de huevos fue mejor (1.52 vs. 1.57; P< 0.01) en las gallinas ligeras que en las gallinas pesadas. En cuanto a la calidad del huevo, la dieta no influyó sobre ninguna de las variables estudiadas. Los resultados del ensayo muestran que las gallinas ponedoras rubias, independientemente de su peso vivo al inicio de la puesta, no necesitan una cantidad de proteína bruta superior a 16.5% para maximizar la producción, asegurando que las dietas 22 Resumen cubren los requerimientos en AA indispensables. Asimismo, se puedo concluir que las gallinas con un peso más elevado al inicio de puesta producen más masa de huevo que las gallinas con un peso más bajo debido a que las primeras producen más cantidad de huevos y más pesados. Sin embargo, ambos grupos de peso obtuvieron el mismo IC por kg de huevo y las gallinas más livianas en peso obtuvieron un mejor IC por docena de huevo que las pesadas. En el experimento 3 la influencia de la concentración energética sobre los parámetros productivos y la calidad del huevo fue estudiada en 520 gallinas ponedoras rubias de la estirpe Hy-Line en el período 24-59 sem de vida. Se utilizaron 8 tratamientos ordenados factorialmente con 4 dietas que variaron en el contenido energético (2650, 2750, 2850 y 2950 kcal EMAn/kg) y 2 pesos vivos distintos al inicio del período de puesta (1733 vs. 1606g). Cada tratamiento fue replicado 5 veces y la unidad experimental consistió en una jaula con 13 aves. Todas las dietas se diseñaron para que tuvieran una concentración nutritiva similar por unidad energética. Las variables productivas y de calidad de huevo se estudiaron mediante controles cada 28 días desde el inicio del experimento. No se observaron interacciones entre el nivel energético y el peso inicial del ave para ninguna de las variables estudiadas. Un incremento en la concentración energética de la dieta incrementó la producción de huevos (88.8 % vs. 91.2 % vs. 92.7 % vs. 90.5 %), masa de huevo (56.1 g/d vs. 58.1 g/d vs. 58.8 g/d vs. 58.1 g/d), y eficiencia energética (5.42 vs. 5.39 vs. 5.38 vs. 5.58 kcal EMA/g huevo) de forma lineal y cuadrática (P< 0.05) y afectó significativamente a la ganancia de peso (255 g vs. 300 g vs. 325 g vs. 359 g; P<0.05) . Sin embargo, un incremento en la concentración energética provocó un descenso lineal en el consumo de los animales (115 g vs. 114 g vs. 111 g vs. 110 g; P< 0.001) y un descenso lineal y cuadrático en el IC por kg de huevo (2.05 vs. 1.96 vs. 1.89 vs. 1.89; P< 0.01). En cuanto a la calidad del huevo, un incremento en el contenido energético de la dieta provocó una reducción en la calidad del albumen de 23 Resumen forma lineal en forma de reducción de Unidades Haugh (88.4 vs. 87.8 vs. 86.3 vs. 84.7; P< 0.001), asimismo el incremento de energía redujo de forma lineal la proporción relativa de cáscara en el huevo (9.7 vs. 9.6 vs. 9.6 vs. 9.5; P< 0.001). Sin embargo, el incremento energético propició un incremento lineal en la pigmentación de la yema del huevo (7.4 vs. 7.4 vs. 7.6 vs. 7.9; P< 0.001). El peso vivo al inicio de la prueba afecto a las variables productivas y a la calidad del huevo. Así, los huevos procedentes de gallinas pesadas al inicio de puesta tuvieron una mayor proporción de yema (25.7 % vs. 25.3 %; P< 0.001) y menor de albumen (64.7 vs. 65.0; P< 0.01) y cáscara (9.5 vs. 9.6; P< 0.05) respecto de los huevos procedentes de gallinas ligeras. Consecuentemente, el ratio yema:albumen fue mayor (0.40 vs. 0.39; P< 0.001) para las gallinas pesadas. Según los resultados del experimento se pudo concluir que las actuales gallinas ponedoras rubias responden con incrementos en la producción y en la masa del huevo a incrementos en la concentración energética hasta un límite que se sitúa en 2850 kcal EMAn/kg. Asimismo, los resultados obtenidos entre los 2 grupos de peso al inicio de puesta demostraron que las gallinas pesadas al inicio de puesta tienen un mayor consumo y producen huevos más pesados, con el consecuente aumento de la masa del huevo respecto de gallinas más ligeras. Sin embargo, el IC por kg de huevo fue el mismo en ambos grupos de gallinas y el IC por docena de huevo fue mejor en las gallinas ligeras. Asimismo, la eficiencia energética fue mejor en las gallinas ligeras. 24 Abstract ABSTRACT 25 Abstract Abstract The general aim of this PhD Thesis was to study the influence of different nutritional factors and management on the productivity and egg quality of comercial Brown laying hens. The factor studied were: 1) The effect of the main cereal and type of fat of the diet; 2) The effect of crude protein and fat content of the diet; 3) The effect of energy concentration of the diet; 4) The effect of initial body weight of the hens at the onset of lay period. In experiment 1, the influence of the main cereal and type of supplemental fat in the diet on productive performance and egg quality of the eggs was studied in 756 Lohmann brown-egg laying hens from 22 to 54 wk of age. The experiment was conducted as a completely randomized design with 9 treatments arranged factorially with 3 cereals (dented corn, soft wheat, and barley) and 3 types of fat (soy oil, acidulated vegetable soapstocks, and lard). Each treatment was replicated 4 times (21 hens per replicate). All diets were formulated according to NRC (1994) and FEDNA (2008) to have similar nutrient content except for linoleic acid that ranged from 0.8 (wheat-lard diet) to 3.4% (corn-soy bean oil) depending on the combination of cereal and fat source used. This approach will allow to estimate the minimum level of linoleic acid in the diets that maximizes egg weight. Productive performance and egg quality traits were recorded every 28 d and BW of the hens was measured individually at the beginning and at the end of the experiment. No significant interactions between main factors were detected for any of the variables studied. Egg production, egg weight, and egg mass were not affected by dietary treatment. Body weight gain was higher (243 vs. 238 vs. 202 g; P<0.05) for hens fed corn or wheat than for hens fed barley and also for hens fed lard than for hens fed soy oil or acidulated vegetable soapstocks (251 vs. 221 vs. 210 g; P< 0.05). Egg quality was not influenced by dietary treatment except for yolk color that was greater (9.0 vs. 8.3 vs. 26 Abstract 8.3; P< 0.001) for hens fed corn than for hens fed wheat or barley and for hens fed lard than for hens fed soy oil or acidulated vegetable soapstocks (8.9 vs. 8.5 vs. 8.2, respectivamente; P< 0.001). The influence of linoleic acid on egg weight and egg mass was higher when the fatty acid was reduced in the diet. Thus, the influence of the diet in egg weight/g linoleic acid intake and egg mass/g linolec acid intake was higher when the amount of this fatty acid decreased in the diet (P< 0.001). It is concluded that brown egg laying hens do not need more than 1.0% of linoleic acid in the diet (1.16 g/hen/d) to maximize egg production and egg size. The 3 cereals and the 3 fat sources tested can replace each other in the diet provided that the linoleic acid requirements to maximize egg size are met. In experiment 2, the influence of CP and fat content of the diet on performance and egg quality traits was studied in 672 Lohmann brown egg-laying hens from 22 to 50 wk of age. The experiment was conducted as a completely randomized design with 8 treatments arranged factorially with 4 diets and 2 initial BW of the hens (1,592 vs. 1,860 g). Three of these diets differed in the CP content (16.5, 17.5, and 18.5%) and included 1.8% added fat. The fourth diet had also 18.5% CP but was supplemented with 3.6% fat instead of 1.8% fat. Each treatment was replicated 4 times and the experimental unit consisted of 21 hens allocated in groups of 7 in 3 adjacent cages. All diets were isocaloric (2,750 kcal AME/kg) and met the recommendations of brown egg-laying hens for digestible Arg, Ile, Lys, Met, Thr, Trp, TSAA, and Val. Productive performance and egg quality were recorded by replicate every 28-d. For the entire experimental period, diet did not affect any of the productive performance traits studied but the heavier hens had higher ADFI (120.6 vs. 113.9g; P< 0.001), egg production (92.5 vs. 89.8%; P< 0.01), and egg weight (64.9 vs. 62.4g; P< 0.001) than the lighter hens. Initial BW did not affect feed conversion per kilogram of eggs or hen mortality but BW gain was higher (289 vs. 233g; P< 0.01) and FCR per dozen of eggs was better (1.52 vs. 1.57; P< 0.01) for the lighter 27 Abstract than for the heavier hens. None of the egg quality variables studied was affected by dietary treatment or initial BW of the hens. It is concluded that brown egg-laying hens, irrespective of their initial BW, do not need more than 16.5% CP to maximize egg production provided that the diet meet the requirements for key indispensable amino acids. Heavier hens produce more eggs that are larger than lighter hens but feed efficiency per kilogram of eggs is not affected. In experiment 3, the influence of AMEn concentration of the diet on productive performance and egg quality traits was studied in 520 Hy-Line brown egg-laying hens differing in initial BW from 24 to 59 wks of age. There were 8 treatments arranged factorially with 4 diets varying in energy content (2,650, 2,750, 2,850, and 2,950 kcal AMEn/kg) and 2 initial BW of the hens (1,733 vs. 1,606 g). Each treatment was replicated 5 times (13 hens per replicate) and all diets had similar nutrient content per unit of energy. No interactions between energy content of the diet and initial BW of the hens were detected for any trait. An increase in energy concentration of the diet increased (linear, P< 0.05; quadratic P< 0.05) egg production (88.8 % vs. 91.2 % vs. 92.7 % vs. 90.5 %), egg mass (56.1 g/d vs. 58.1 g/d vs. 58.8 g/d vs. 58.1 g/d), energy efficiency (5.42 vs. 5.39 vs. 5.38 vs. 5.58 kcal AMEn/g of egg), and BW gain (255 g vs. 300 g vs. 325 g vs. 359 g; P<0.05) but decreased ADFI (115 g vs. 114 g vs. 111 g vs. 110 g; P< linear, P< 0.001) and FCR per kg of eggs (2.05 vs. 1.96 vs. 1.89 vs. 1.89; linear, P< 0.01; quadratic P< 0.01). An increase in energy content of the diet reduced Haugh units (88.4 vs. 87.8 vs. 86.3 vs. 84.7; P< 0.01) and the proportion of shell in the egg (9.7 vs. 9.6 vs. 9.6 vs. 9.5; P< 0.001). Feed intake (114.6 vs. 111.1 g/hen per day), AMEn intake (321 vs. 311 kcal/hen per day), egg weight (64.2 vs. 63.0 g), and egg mass (58.5 vs. 57.0 g) were higher for the heavier than for the lighter hens (P<0.01) but FCR per kg of eggs and energy efficiency were not affected. Eggs from the heavier hens had higher proportion of yolk (25.7 % vs. 25.3 %; P< 0.001) and lower of albumen (64.7 vs. 65.0; P< 0.01) and 28 Abstract shell (9.5 vs. 9.6; P< 0.05) than eggs from the lighter hens. Consequently, the yolk to albumen ratio was higher (0.40 vs. 0.39; P< 0.001) for the heavier hens. It is concluded that brown egg-laying hens respond with increases in egg production and egg mass, to increases in AMEn concentration of the diet up to 2,850 kcal/kg. Heavy hens had higher feed intake and produced heavier eggs and more egg mass than light hens. However, energy efficiency was better for the lighter hens. 29 Chapter 1. Literature review and objectives CHAPTER 1: Literature review and objectives 30 Chapter 1. Literature review and objectives 1. Literature review 1.1. Introduction Global egg production and trade have shown a remarkable and dynamic growth during the last 40 years. From 1970 to 2009, egg production increased faster than production of beef and veal or pig meat. In 1970 World egg production accounted for about 19,540 million tons with the 4 major producer countries being USA, Russia (URSS at this time), Japan and China. In 2009 the production of eggs reached levels of 62,8 million tons with China, USA, India and Japan as the leader countries (FAOSTAT, 2011). Egg production growth between 1960 and 2007 was very fast in Asia, especially in China, medium to slow with continuous upwards in Africa and South America and slow in Europe and Oceania. In 2007, Asia production accounted for 38 million tons, Africa for 2.3 million tons and South America for 3.4 million tons. Because of management and logistic problems, relatively few eggs are traded internationally. In 2008, world exports of egg reached a value of 4,083 million US dollars, a growth of about 17.7% as compared with that of 2004. Major exporter countries, were The Netherlands, China, Spain and Poland. The total importation of eggs in 2008 reached a value of 3,846 million dollars, an increase of 15.3% as compared with that of 2004. The largest importers of eggs in 2008 were Germany, The Netherlands, France and China. According to FAOSTAT (2011), egg consumption per person per year improved steadily from 2000 to 2007. The global average consumption increased from 8.1 kg in 2001 to almost 8.6 kg in 2007. Egg consumption in Asia grew at a faster rate than in the remaining areas of the world. By 2007, Asia reached a record of 8.8 kg of egg consumption per person. Newest studies were not done but FAO data indicate that in 2010 the average yearly egg consumption in the world was above 9.2 kg per person. 31 Chapter 1. Literature review and objectives According to MARM (2010) there were 44 million laying hens destined for egg production in Spain in 2009. These hens were housed in a total of 1,370 registered layer houses. Approximately, 95.7% of the birds were housed in cages, 2.4% were free range and 1.7% were on floor. Organic production occupies only 0.1% of the census. These numbers are expected to change in the new future because of the new Europe-Union legislation on behalf of animal welfare. In 2004, Spain recorded the highest level of egg production (1.13 x 103M dozen of eggs). The highest production of eggs occurred in Castilla-La Mancha (32%), followed by Castilla y Leon (17%), Valencia (9%), and Cataluña (8%). Export market is very important for the Spanish egg industry. Egg production covered local demand and allows exporting a large proportion (around 23% of the total production in 2008 and 2009). The main destination of exports is the European Union, with France being the first importer country (41%) followed by Germany (14%), United Kingdom (12%), The Netherlands and Portugal (11% each) (MARM, 2010). Between 2000 and 2009, egg consumption decreased from 17.5 to 11.3 kg per person per year (a general decrease of about 36%). However, this decrease in egg consumption was not linear; it suffered a fluctuation in 2004 and a sharply decrease thereafter. The economic success of the egg industry depends on egg mass produced by each hen during the whole lay period. This objective is influenced by the length of the laying period but also by the number of eggs produced and the size of these eggs. Also, the percentage of marketable eggs and the relative cost of raw materials are important factors to be considered to reach the economic objectives. Egg loss produced from farm to consumer because of egg handling accounts for 5 to 7% of all eggs laid (Roland, 1988). Most of these losses are related to poor shell quality of eggs produced at the end of the production cycle. In addition, the external and internal quality of the eggs is to be taken into account to reduce the incidence of rejected eggs and improve selling prize. Egg rate depends mainly on genetics but health status together with management and feeding 32 Chapter 1. Literature review and objectives practices of pullets and hens are contributing factors. Thus, the economic success of a laying hen operation requires a curve with a sharp, high peak of production at the start of the laying cycle and a good persistency throughout the entire egg-laying cycle. It is widely accepted that high peak productions are positively related with egg mass production. Egg size has important connotations related to the success of egg operation in countries such as Spain in which consumers show preferences for large egg. In these countries, under these circumstances, producers tend to increase the duration of the egglaying cycle because egg size increases with age of the hens. The objective of poultry nutritionists is to formulate diets that maximize performance variables including egg production and egg size early in the production cycle and reduce shell problems at the end of the production cycle. In order to meet these objectives, nutritionists need to play with level of nutrient requirements (energy concentration, crude protein and AA level), use of raw materials (type of cereal and fat) and obtain a high uniformity and the target body weight (BW) of the pullets at the onset of the laying cycle, to reach the optimum performance of the hens. Productive performance and optimal quality of the eggs produced are the two main factors succeed in the egg industry and to meet both targets depends partially on the good nutritional management of the birds. Thus, it is important to check effects of the nutritional variables used in the lay period to improve productive performance on the throughout external (percentage of unmarketable eggs: broken, dirty, shell-less eggs) and the internal (albumen height, yolk color, and the different proportion of yolk and albumen) quality of the eggs. In the current research we investigated the effects of key nutritional components or productive performance and egg quality of commercial brown egg-laying hens during the whole laying cycle. The factors studied were: 1) The effect of main cereal and type of 33 Chapter 1. Literature review and objectives fat in the diet, 2) The effect of crude protein level and added fat in the diet, 3) The effect of energy level in the diet, 4) The effect of initial BW of the hens at the onset of lay period on performance and egg quality variables. 1.2. Effect of the main cereal of the diet on hen productivity and egg quality Cereals are rich in starch and are the most widely used ingredients as energy sources in poultry feeds. In addition, cereals provide also part of the crude protein (CP) and AA require by the birds. Starch utilization by poultry depends on the cereal used, because cereals differing in the nature and the structure of their starch fraction. Moreover, starch digestion depends on factors such as soluble cell-wall polysaccharide content, nature of starch granule, presence of anti-nutritional factors in the grain, and the digestive capacity of the animal (Classen, 1996). Many studies have been conducted to ascertain the nutritive value of the different starch present in nature but the prediction of its nutritive value and utilization by laying hens has not been elucidated yet. The most common cereals produced in Spain and used in poultry diets are corn (Zea mays L.) wheat (Triticum L.) and barley (Hordeum vulgare L.). Corn has less CP (7.5% vs. 10.2% vs. 9.6%) and crude fiber (2.3% vs. 2.6% vs. 4.7%) and more starch (63.3% vs. 60.2% vs. 53%), ether extract (3.6% vs. 1.6% vs. 1.8%), linoleic acid (1.81% vs. 0.64% vs. 0.71%), and AMEn (3,280 vs. 3,100vs. 2,800 kcal/kg) than wheat or barley (Fundación Española Desarrollo Nutrición Animal, 2010). In addition, the chemical composition and nutritive value of corn is quite uniform compared with that of wheat and barley, but the contents vary depending on factors such as cultivar, agronomic practices, weather conditions, length of storage period, feed form, and type of bird (Pirgozliev et al., 2003; GutiérrezÁlamo et al., 2008; Frikha et al., 2011). Under commercial conditions, many egg producers formulate diets for laying hens with a minimum of corn to ensure a high feed intake and to maximize egg size early in the production cycle specially when the pullets 34 Chapter 1. Literature review and objectives have a early lay stimulation. The reasons for this practice are unknown but might be related to the more uniform nutritive value of corn and the better structure of the feed when coarse corn is included in the diet (Frikha et al., 2009). Also, corn has more linoleic acid (LNL) content than wheat and barley, and an increase in LNL content of the diet might result in an improvement in egg weight, especially in young hens under hot climate conditions in which feed intake is low (Jensen et al., 1958; Scragg et al., 1987; Grobas et al., 1999a). On the other hand, wheat and barley contain a high and variable amount of nonstarch polysaccharides (NSP) including arabinoxylans, and β-glucans, which are known to increase digesta viscosity and reduce productive performance in poultry (Lázaro et al., 2003, García et al., 2008). Thus, the level of inclusion of wheat and barley in poultry diets depends on many factors such as, the species considered, age, and nutrient profile, including AMEn, CP, and NSP. Several reports have compared corn, wheat, and barley in the diet on productive performance of laying hens, broilers, and pullets. In general, these studies suggest that wheat and barley are a good alternative to corn in these species. In laying hens, Craig and Goodman (1993), Lázaro et al. (2003), Liebert et al. (2005), and Safaa et al. (2009) have reported similar hen productivity when the 3 cereals were compared and with wheat and barley diets supplemented with exogenous enzymes. In contrast, Coon et al. (1988) compared corn and barley and reported higher ADFI and poorer FCR in hens fed the enzyme-supplemented barley diets than in corn fed. In broilers, Mathlouthi et al. (2002) reported similar performance when 60% of the corn was substituted by a combination of 40% wheat and 20% barley. Also, Ruiz et al. (1987) reported similar BWG and FCR in broilers fed mash when corn was substituted by wheat. However, Crouch et al. (1997) compared corn and two varieties of wheat at 40% of inclusion in mash diets for broilers and found that BWG and FCR were impaired with one of the two wheats. In pullets, Frikha et al. (2009) reported higher BWG in pullets fed corn than in pullets fed wheat, both diets being supplemented with 35 Chapter 1. Literature review and objectives enzymes. The reasons for these discrepancies are unknown but might be related to the use with different enzyme complex and the estimated value in the increase of the energy. The information available on the effects of the main cereal of the diet on eggquality is scarce. In general, the inclusion of barley and wheat increased the incidence of dirty eggs compared with the inclusion of corn (Francesch et al., 1995). Similarly, Lázaro et al. (2003) found that the substitution of corn by wheat affected the percentage of dirty egg quality of Single Comb White Leghorn (SCWL) hens from 20 to 44 wk of age. However, Jamroz et al. (2001) reported similar egg quality from hens fed wheat of barley diets supplemented with enzymes. Moreover, Çiftci et al. (2003) and Safaa et al. (2009) reported that the substitution of corn by wheat in enzyme supplemented diets did not affect the percentage of dirty eggs in SCWL or brown egg-laying hens, respectively. 1.3. Effect of supplemented fat of the diet on hen productivity and egg quality Fats are used in poultry to increase the energy content of the diets. The inclusion of fat in the diet usually results in an increase in egg size. Fat inclusion resulted often in higher energy intake, increased BW gain and egg weight (Grobas et al., 2001; Bouvarel et al., 2010), probably because of improved palatability with less dust formation (ISA Brown, 2011). Also, supplemental fat has been shown to reduce rate of feed passage, facilitating the contact between digesta and enzymes, improving digestibility and utilization of other nutrients such as the lipid and carbohydrate fractions of dietary ingredients (Mateos and Sell, 1980b, 1981). Whitehead et al. (1993) studied the effect of supplemented fat on egg weight and concluded that maize oil weight more compared with others sources of fat such as fish oil (long chain polyunsaturated fatty acids (FA), coconut oil (shorter chain saturated FA) or tallow (medium to long chain length saturated FA). Probably, readily absorbable unsaturated FA of corn oil improves the increased egg. Grobas et al. (2001) studied the 36 Chapter 1. Literature review and objectives effect of 4 different sources of supplemented fat on egg weight and reported that eggs were heavier when hens were fed diets supplemented with soy oil than when supplemented with linseed oil, olive oil, or tallow. On the other hand, supplemental fat might reduce egg shell quality, especially in old hens. Atteh and Leeson (1983, 1984, 1985) studied the effect of FA profile on performance and mineral metabolism of egglaying hens and broilers and reported that fat and some minerals can interfere together, leading to the formation of insoluble soaps responsible of the decrease in absorption of both FA and minerals. Furthermore, they reported that soap formation was higher with saturated (palmitic and stearic acids) that with unsaturated FA and that an increase in the Ca content of the diet increased of soap formation. Many studies have shown that a reduction in supplemental fat (SFAT) significantly decrease egg size (Keshavarz and Nakajima, 1995; Grobas et al., 1999a,b; Bohnsack et al., 2002; Sohail et al., 2003). Grobas et al. (2001) reported that SFAT improved egg weight and egg mass output in both SCWL hens and brown egg-laying hens throughout the production cycle. The same authors, Grobas et al. (1999b) compared isonutritive diets for brown egg-laying hens differing in fat content (0 and 4 %) from 22 to 65 wk of age and observed that SFAT improved productive performance and egg size but that FCR was not affected. In this research, the improvement in egg rate observed occurred from 38 to 61 wk of age whereas the beneficial effects on egg weight were more noticeable from 22 to 57 wk of age. Whitehead (1981) showed that supplementation of the diets with 4 or 30 g fat/kg significantly increased egg weight. Whitehead et al. (1993) compared 5 inclusion levels of fat (0, 10, 20, 40, and 60 g/kg diet) and concluded that, with the exception of fish oil, which hindered productive performance when included at the level of 20 g/kg, maize oil, tallow, and coconut oil perform well till 40 g/kg of fat inclusion. Furthermore, Grobas et al. (1999b) showed that supplementation of the diet with 40 g fat/kg increased egg weight as compared with a non supplemented diet. 37 Chapter 1. Literature review and objectives However, the authors showed that further increases from 5 to 10% of fat supplementation did not have any positive effect on egg weight (Grobas et al., 2001). Added fat increased both yolk and albumen weights, but in some researches the improvement was proportionally greater for the albumen than for the yolk (Grobas et al., 1999b). Whitehead (1995) hypothesized that the beneficial effect of SFAT on albumen weight was due to the influence of certain unsaturated FA on the production of oestrogen which is the main responsible for albumen secretion Regarding egg quality traits, Grobas et al. (1999a) observed that the increase in egg weight with SFAT was accompanied by a similar increase (3.5%) in yolk and albumen weights. The mechanism by which SFAT increases egg size is uncertain. Whitehead et al. (1991) suggested that SFAT increased yolk weight by stimulating lipid deposition and albumen weight by stimulating oestrogen secretion which controls protein synthesis in the oviduct. Parsons et al. (1993) reported that a reduction in SFAT from 6 to 2% of the diet reduced the proportion of large and above eggs in SCWL. Same results have been reported by Bohnsack et al. (2002) with similar type of diets. Haugh units were not affected by SFAT (Usayran et al., 2001; Grobas et al., 2001). Previous research has shown that SFAT exerts a favorable effect on egg weight beyond that attributable to an increase in LNL content of the diet (Shannon and Whitehead, 1974; Sell et al., 1987; Keshavarz, 1995; Grobas et al., 1999a). 1.4. Effect of linoleic acid of the diet on hen productivity and egg quality The effect and requirement of LNL to maximize hen productivity and egg size is subject of debate. Under commercial conditions, many guides for feeding laying hens (H& N International, 2008; Lohmann, 2010) recommend increasing the level of LNL in the diet to at least 1.8% to maximize egg size. In contrast, Shannon and Whitehead (1974) and Whitehead (1984) recommended 1.0% LNL in the diet whereas Scragg et al. (1987) 38 Chapter 1. Literature review and objectives recommended up to 2% dietary LNL to increase egg size in brown-egg laying hens. Ribeiro et al. (2997) reported higher egg weight in broiler breeder hens fed 1.90 % LNL diets than in hens fed 1.50 % LNL diets. In all these studies, other productive variables studied, such as egg production, egg mass, and FI, were not affected by LNL content of the diet. Grobas et al.(1999b) studying the effect of LNL level of brown egg-laying from 22 to 65 wk of age reported that a reduction from 1.65 to 1.15% in the level of LNL did not affect performance variables. These authors concluded that the LNL requirement of brown laying hens for maximal productivity from 22 to 65 wk of age is not greater than 1.15 % of the diet. In fact, Grobas et al. (1999c) reported that 0.79 % LNL tended to reduce egg weight with respect to 1.03 or 2.23 % LNL in brown hens from 20 to 32 wk of age but hen-day production, egg mass, FI, FCR, and BW were not affected by LNL. In respect of egg quality traits, Grobas et al. (1999c) reported that LNL levels ranged from 0.79 to 2.73% in diets for brown egg-laying hens did not affect the percentage of marketable eggs and the percentage of broken and dirty eggs, Haugh units, or the proportion of egg components. March and McMillan (1990) and Whitehead et al. (1993) indicated that LNL supplementation to diets deficient in this essential FA increased yolk weight, probably through an improvement in the mechanism by which lipoproteins are synthesized or taken up by the developing ova. 1.5. Effect of protein and aminoacids content of the diet on hen productivity and egg quality The ideal protein can be defined as the exact AA balance, with no deficiencies or excess, required for maintenance and production. It maximizes the effective use of dietary protein and can substantially reduce production costs, increase farm profitability and minimize nitrogen excretion. The CP level and level of AA have an important role in the egg size. Thus, the daily requirements of an egg-laying hen are 2-4 g for maintenance 39 Chapter 1. Literature review and objectives and 10-13 g for egg production. In the peak period, the hens needs at a least of 17 g of balance CP to express the maximal genetic potential (Summers, 1986). Diets for laying hens are formulated to meet the requirements for those indispensable AA that may limit egg production, namely Lys, Met, Thr, and TSAA. According to NRC (1994) diets based on corn and soybean meal with 15.0% CP can satisty the AA requirements of brown egglaying hens consuming 110g of feed per day. However, several commercial guidelines for laying hens (Lohmann, 2010; ISA Brown, 2011) recommend CP levels varying from 17.4 to 18.2%. Is accepted that egg size increased with increases in CP (Hawes and Kling, 1993; Hussein et al., 1996; Bouvarel et al., 2010), especially at the onset of lay period (Parsons et al., 1993) whereas others (Summers and Leeson, 1983) reported no benefits on egg production and egg weight with dietary CP above NRC (1994) requirement (16.5%). Keshavarz and Nakajima (1995) reported that the increased in egg weight with the increase of CP was due to an increase in albumen proportion. In contrast, after peaking the hens tend to overconsume and increased the fat proportion in the body (Proudfoot et al., 1988). Thus, is a good practice to reduce the percentage of CP in the diet throughout the lay period to improve the efficiency (Harms, 1986). Summers (1986) reported at the end of the lay period an extra energetic cost in oxidation process to eliminate the excess of nitrogen, producing liquid faeces, extre large eggs and poor egg quality. However, some authors (Pilbrow and Morris, 1974; Wethli and Morris, 1978; Huyghebaert et al., 1991; Joly, 1995) recommended maintain the CP level at the end of the lay period according to the poor efficiency in the use of AA by old hens than in young hens. Ballam (1985) reported that the AA requirements were higher to optimize the egg weight than to optimize the egg production. This author estimated a 10% increase in Met and Lys to improve the egg weight without any effect on egg production. Also, Summers et al. (1991) reported that a deficiency in CP level in the diet affected more in the egg 40 Chapter 1. Literature review and objectives size than in egg production. However, Morris and Gous (1988) showed in disagreement because of the different coefficient of variation of both variables, thus, the coefficient of variation for egg production and egg weight were 0.20 and 0.10 respectively. Therefore, low difference in egg weight could be statistically different but this effect could not be showed in egg production. These authors in a review of the CP and AA requirements in laying hens reported similar reductions in egg weight and egg production with a reduction of 10% in the level of CP in the diet, but, if the reduction is more severe the reduction is higher in egg production than egg weight. Schutte et al. (1994) reviewed a serie of experiment about TSAA and Met requirements. Thus, Roland et al. (1992) recommended high levels of TSSA at the beginning of the lay period whereas Summers and Leeson (1993) and Klien and Hawes (1990) did not observed any improve on the performance. Lys is the second limitant AA in practical feeds (March and Biely, 1963; Sell and Hodgson, 1966). Thus, Joly (1995) reported that a deficient amount of lys in the diet decreased egg mass (reducing a 65% of egg production and a 35% in egg weight). Moreover, Nathanael and Sell (1980) reported that egg weight increased quadratically with the increase of lys level. In contrast, Harms and Ivey (1993) and Prochaska et al. (1996) did not dettect any effect on performance with increases in lys level. Is possible that in the research of Nathanael and Sell (1980) other AA was limitant. Egg quality including percentage of dirty eggs, albumen height, and eggshell traits are affected by CP levels. Thus, Fariborz et a. (2007) compared isoenergetic diets containing 16.3 or 17.8% CP and reported that albumen height, shell thickness, and shell strength were not affected by the CP content of the diet. However, Hammershoj and Kjaer (1999) reported that Haugh units (HU) declined as the level of CP of the diet increased from 13.7% to 17.9% 41 Chapter 1. Literature review and objectives 1.6. Effect of energy content of the diet on hen productivity and egg quality The analyze of energy balance is the usually form to calculate the amount of FI and the animal production (De Blas, 1991). This author, in a series of researches, estimated 107,8 kcal AME/kg0,75 for maintenance energy requirements (from 90 to 120 kcal/kg0,75) and 8,39 kcal AME/g for increases of BW and 1,94-2,25 kcal AME/g for production energy requirements. Thus, a egg-laying hen with a BW of 2.0 kg, with a BWG of 0.8 g/d with a egg mass of 58 g/d, needs 300-320 kcal of AME per day. Hens eat to satisfy their energy requirements and therefore an increase in the energy content of the diet should decrease ADFI proportionally (Hill et al., 1956). Bouvarel et al. (2010) reviewed a series of experiments conducted in laying hens during the last 20 years and reported that as an average, a 10% increase in AMEn content of the diet reduced FI by only 5.5%. Changes in energy concentration of the diet have resulted in contrasting results in respect to productive performance (Harms et al., 2000). In laying hens, Grobas et al. (1999c) reported that increasing the AMEn content of the diet from 2,680 to 2,810 kcal/kg (a 4.8% increase) decreased feed intake by the same proportion (a 5.0% decrease) but that egg production and egg mass were not affected. Similarly, Peguri et al. (1991) reported a 5% decrease in FI but similar egg production when the AMEn of the diet was increased from 2,700 to 2,910 kcal/kg (a 8% increase). In contrast, Joly and Bougon (1997) reported in brown egg-laying hens from 19 to 68 wk of age a 1.3% increase in egg production and a 4.5% increase in egg mass as the energy content of the diet increased from 2,200 to 2,700 kcal AMEn/kg. Most of published trials about the effect of energy level in the diet reported an improve in egg weight with the increase in the energy concentration of the diet (De Groote, 1972; Walker et al., 1991). The hens tend to maintain its energy intake modifying the FI (Leeson et al., 1973; Newcombe and Summers, 1985), overconsuming energy in 42 Chapter 1. Literature review and objectives high energy diets (Morris, 1968; De Groote, 1972; Walker et al., 1991). Thus, the excess of nutrients improve the egg weight (De Groote, 1972; McDonald, 1984; Leclerq, 1986; Walker y col., 1991). According to these authors the egg weight improved from 0.10 to 0.20% per each 100 Kcal. Bouvarel et al. (2010) analyzed data from 11 experiments conducted for the last 20 years and reported that egg weight increased 0.96 g per each 100 kcal of increase in dietary AMEn. The reasons for the discrepancies among authors in relation to the effects of an increase in energy content of the diet on egg weight are not apparent but might be related with the level of fat and the LNL content of the control diet. The effect of energy levels on egg production showed different results, thus, while Mathlouthi et al. (2002) reported in SCWL hens that egg production increased as the AMEn of the diet increased from 2,650 to 2,750 kcal/kg, Grobas et al., (1999c) in brown hens fed diets varying from 2,680 to 2,810 kcal AMEn/kg, Harms et al. (2000) in brownand SCWL hens fed diets varying in AMEn from 2,500 to 3,100 kcal/kg , and Jalal et al. (2006, 2007) in SCWL hens fed diets varying from 2,800 to 2,900 kcal AMEn/kg did not detect any significant difference in egg production with changes in the energy content of the diet. In commercial flocks, is a common practice increasing the energy concentration of the diet at the onset of lay period, especially, when the pullets have not a homogeneous BW or when the pullets have a low BW at the beginning of lay period. Thus, some authors reported that in hot climate like Spain, the increase of energy concentration of the diet improve the performance especially in light hens (Kling and Hawes, 1990; Daghir, 1995). The reasons for the discrepancies among authors in respect to the variation in egg quality values with increases in AMEn of the diet are not apparent but might be related with the different use of basal diets and fats. Grobas et al. (1999a) reported that the increase in energy concentration of the diet did not affect the percentage of dirty, broken, or shell less throughout the laying period. Some authors reported effects with energy 43 Chapter 1. Literature review and objectives increases on albumen quality. Zimmermann and Andrews (1987) and Junqueira et al. (2006) reported that the increase in energy concentration of the diet did not affect the HU. However, Wu et al. (2005) reported a decrease in HU when the AMEn of the diets was increased from 2,720 to 2,960 kcal/kg. The reasons for the discrepancies among authors in respect to the variation in HU values with increases in AMEn of the diet are not apparent. Xanthophylls, the main pigment source responsible for egg yolk color, are highly soluble in fat. Gunawardana et al. (2008) reported higher yolk pigmentation in SCWL hens fed a diet with 5.0% added fat than in hens fed a control diet without any added fat. Also, Lázaro et al. (2003) reported higher yolk pigmentation in SCWL hens fed high AMEn diets. Also, when fat is used to increase the energy concentration of the diet the proportion of shell in the egg might be affected. Junqueira et al. (2006) reported a linear decrease in egg shell proportion as the AMEn increased from 2,850 to 3,050 kcal/kg in brown egg-laying hens from 76 to 84 wk of age. However, Gunawardana et al. (2008) did not find any effect of energy content of the diet on egg shell proportion in SCWL fed diets varying in AMEn content from 2,750 to 3,050 kcal/kg. 1.7. Effect of initial body weight at the onset of lay on hen productivity and egg quality Spanish consumers have a preference for heavy eggs for which they are willing to pay an extra price. In consequence, egg producers need to obtain a high percentage of large and extra large eggs. The amount of large eggs is a challenge for the first part of the laying cycle. Thus, increase the percentage of large eggs early in the lay production cycle, increase feed intake and BW of pullets and avoid poor uniformity of the flock at the beginning of the egg production cycle, especially under hot weather conditions is a challenge (Frikha et al., 2009). 44 Chapter 1. Literature review and objectives The information available about the effects of initial body weight at the onset of lay period in brown hens on productive performance and egg quality is very scarce. Body weight at the onset of egg production is a major factor influencing hen productivity. Egg weight throughout the production cycle is largely determined by the initial BW of the hen (Harms et al., 1982; Leeson and Summers, 1987). Heavier hens at the onset of the laying period consumed more feed and produced bigger eggs throughout the egg-cycle than lighter hens (Summers and Leeson, 1983; El Zubeir and Mohammed, 1993). Bish et al. (1985) reported that heavy SCWL hens (1,377 g) produced heavier eggs than medium (1,256 g) and light (1,131 g). In addition, heavier hens produced more eggs but had similar FCR per kg of eggs than lighter hens, confirming the results of Keshavarz (1995). This author, reported a 1.4 g difference in egg weight between light (1,151 g) and heavy (1,333 g) SCWL hens from 18 to 62 wk of age. The information available about the effects of initial body weight on egg quality is very limited. In general, is accepted that eggs from the heavy hens are heavier than light hens. Also, heavy hens had higher proportion of yolk and lower of albumen than eggs from the light hens. Probably, heavy hens produce heavier yolks than lighter hens, because of their higher feed intake that results in eggs with higher proportion of yolk (Leeson and Summers, 2005). 45 Chapter 1. Literature review and objectives 2. Objectives The general aim of this Doctoral Thesis was to study the influence of nutritional factors that might affect the productivity and egg quality of commercial brown laying hens. Thus, to reach these goals three trials were carried out in the period 2009-2011 under commercial management conditions. The effects of type of cereal and fat (trial 1), crude protein level and initial body weight of the hen (trial 2) and energy level and initial body weight of the hen (trial 3) were carried out. Also, the second objective was study the influence of management, and nutritional factor and the use of some raw material usefully in the nutrition of the hens conducing to a reduction in productive cost. In experiment 1, a study was carried out from 22 to 54 wk of age in 756 brown egg-laying hens (Lohmann) to study the effect of 3 main cereals (Corn, Wheat, and Barley), and 3 types of fat (Soy oil, Acidulated Soapstocks, and Lard) in the diet on productive performance and egg quality. As a result of the interaction between cereal and fat used the effect of different levels of linoleic acid was obtained and its effects were measured. (Chapter 2). In experiment 2, a study was carried out to study the effect of 3 protein levels (18.5%, 17.5%, and 16.5% of CP) and 2 levels of added fat (3.6% and 1.8%) on performance and egg quality of brown egg-laying hens (Lohmann). The design was carried out with 3 diets that differing in the level of CP (18.5%, 17.5%, and 16.5%) with only 1.8% of added fat, and a fourth diet contained 18.5% CP but 3.6% of added fat. This model was carried out in 2 groups of hens (heavy and light) differing in BW respect to the target value (Lohmann guide at the age of starting experiment). (Chapter 3) In experiment 3, a study was conducted to study the effect of 4 energy levels (2,950, 2,850, 2,750, and 2,650 kcal AMEn/kg) in 2 groups of Hy Line brown hens (heavy and light) differing in BW respect to the target value (Hy Line guide at the age of starting 46 Chapter 1. Literature review and objectives experiment). The diets was isonutritive per kcal. Productive performance and egg quality was measured throughout lay period from 22 to 59 wk of age. (Chapter 4). 3. References Atteh, J. O., S. Leeson, and R. J. Julian. 1983. Effects of dietary levels and types of fat on performance and mineral metabolism of broiler chicks. Poult. Sci. 62:2403-2411. Atteh, J. O., and S. Leeson. 1984. Effects of dietary saturated or unsaturated fatty acids and calcium levels on performance and mineral metabolism of broiler chicks. Poult. Sci. 63:2252-2260. Atteh, J. O., and S. Leeson. 1985. Response of laying hens to dietary saturated and unsaturated fatty acids in the presence of varying dietary calcium levels. Poult. Sci. 64:520-528. Ballam, G. C. 1985. Integrating nutrition and management to affect egg size. Poul. Tribune. 91:40-44. Bish, C. L., Beane, W. L., Rugzler, P. L. and Cherry, J. A. 1985. Body weight influence on egg production. Poult. Sci. 64:2259-2262. Bohnsack, C. R., R. H. Harms, W. D. Merkel, and G. B. Russell. 2002. Performance of commercial layers when fed diets with four levels of corn oil or poultry fat. J. Appl. Poult. Res. 11:68-76. Bouvarel, I., Y. Nys, M. Panheleux, and P. Lescoat. 2010. Comment l’alimentation des poules influence la qualité des oeufs. INRA Prod. Anim. 23:167-182. Çiftci, I., E. Yenice, and H. Eleroglu. 2003. Use of triticale alone and in combination with wheat or maize: effects of diet type and enzyme supplementation on hen 47 Chapter 1. Literature review and objectives performance, egg quality, organ weights, intestinal viscosity and digestive system characteristics. Anim. Feed Sci. Technol. 105:149–161. Classen H. L. 1996. Cereal grain starch and exogenous enzymes in poultry diets. Anim. Feed Sci. Technol. 62:21-27. Coon, C. N., I. Obi, and M. L. Hamre. 1988. Use of barley in laying hens diets. Poult. Sci. 67:1306-1313. Craig, L.W. and T. Goodman. 1993. Utilization of feed enzymes in laying hen rations. J. Appl. Poult. Res. 2:68-74. Crouch, A. N., J. L. Grimes, P. R. Ferket, and L. N. Thomas. 1997. Enzyme supplementation to enhance wheat utilization in starter diets for broilers and turkeys. J. Appl. Poult. Res. 6:147-154. Daghir, N. J. 1995. Replacement pullet and layer feeding and management in hot climate. Poultry Production in Hot Climates. Eds.: Daghir, N.J. CAB International, Cambridge. UK. pp.: 219-253 De Blas, C. 1991. Necesidades energéticas de las gallinas ponedoras. Nutrición y alimentación de gallinas ponedoras. Eds.: De Blas, C. y Mateos, G.G. MAPA. Madrid. AEDOS. Barcelona. Mundi-Prensa. Madrid. pp.:145-159. De Groote, G. 1972. A marginal income, and cost analysis of the effect of nutrient density on the performance of white leghorn hens in battery cages. Br. Poult. Sci.13:503-520. El Zubeir, E.A., and O.A. Mohammed. 1993. Dietary protein and energy effects on reproductive characteristics of commercial egg type pullets reared in arid hot climate. Anim. Feed Sci. Technol. 41:161-165. 48 Chapter 1. Literature review and objectives FAOSTAT. 2011. Food and Agriculture Organization, production and trade. faostat.fao.org. Visited July 2011. Fariborz, K., M. Faraji, and S. K. Dehkordi. 2007. Effects of reduced-protein diets at constant total sulfur amino acids: lysine ratio on pullet development and subsequent laying hen performance. Am. J. Anim. Vet. Sci. 2:89-92. Frikha, M., H. M. Safaa, M. P. Serrano, X. Arbe, and G. G. Mateos. 2009. Influence of the main cereal and feed form of the diet on performance and digestive tract of brown-egg laying pullets. Poult. Sci. 88:994–1002. Frikha, M., H. M. Safaa, M. P. Serrano, E. Jiménez-Moreno, and G. G. Mateos. 2011. Influence of the main cereal of the diet and particle size of the cereal on peroductive performance and digestive traits of brown-egg laying pullets. Anim. Feed. Sci. Technol. 164:106-115. Francesch, M., A.M. Pérez-Vendrell, E. Esteve-García, and J. Brufau. (1995). Enzyme supplementation of a barley and sunflower-based diet on laying hen performance. J. Appl. Poult. Res. 4:32-40. Fundación Española Desarrollo Nutrición Animal. 2010. Normas FEDNA para la Formulación de Piensos Compuestos. C. de Blas, G. G. Mateos, and P. G. Rebollar, ed. Fund. Esp. Desarro. Nutr. Anim., Madrid, Spain. García, M., R. Lázaro, M. A. Latorre, M. I. Gracia, and G. G. Mateos 2008. Influence of enzyme supplementation and heat processing of barley on digestive traits and productive performance of broilers. Poult. Sci. 87:940-948. 49 Chapter 1. Literature review and objectives Grobas, S., G. G. Mateos, and J. Mendez. 1999a. Influence of dietary linoleic acid on productive and egg weight of eggs and egg components in young brown hens. J. Appl. Poult. Res. 8:177-184. Grobas, S., J. Mendez, C. De Blas, and G. G. Mateos. 1999b. Influence of dietary energy, supplemental fat and linoleic acid concentration on performance of laying hens at two ages. Br. Poult. Sci. 40:681-687. Grobas, S., J. Mendez, C. De Blas, and G. G. Mateos. 1999c. Laying hen productivity as affected by energy, supplemental fat, and linoleic acid concentration of the diet. Poult. Sci. 78:1542-1551. Grobas, S., J. Méndez, R. Lázaro, C. de Blas, and G. G. Mateos. 2001. Influence of source and percentage of fat added to diet on performance on fatty acid composition of egg yolks of two strains of laying hens. Poult. Sci. 80:1171-1179. Gunawardana, P., D. A. Roland Sr., and M.M. Bryant. 2008. Effect of energy and protein on performance, egg components, egg solids, egg quality, and profits in molted Hy-line W-36 hens. J. Appl. Poult. Res. 17:432-439. Gutiérrez-Álamo, A., P. Pérez de Ayala, M. W. A. Verstegen, L.A. Den Hartog, and M. J. Villamide. 2008. Variability in wheat: factors affecting its nutritional value. World’s Poult. Sci. J. 64:20–39. H & N International. 2008. Brown Nick Management Guide. H & N International, Cuxhaven, Germany. Hammershoj, M. and J. B. Kjaer. 1999. Phase feeding for laying hens: effect of protein and essential amino acids on egg quality and production. Acta Agric. Scand., Sect. A, Animal Sci. 49:31-41. 50 Chapter 1. Literature review and objectives Harms, R. H., P. T. Costa, and R. D. Miles. 1982. Daily feed intake and performance of laying hens grouped according to their body weight. Poult. Sci. 61: 1021-1024. Harms, R. H. 1986. Should phase feeding be used for laying hens?. Feedstuffs 58:18. Harms, R. H. and Ivey, F. J. 1993. Performance of commercial laying hens fed various supplemental amino acids in a corn-soybean meal diet. J. Appl. Poult. Res. 2: 273282. Harms, R.H., G. B. Russell, and D. R. Sloan. 2000. Performance of four strains of commercial layers with major changes in dietary energy. J. Appl. Poult. Res. 9:535-541. Hawes, R. O. and Kling, L. J. 1993. The efficacy of using prelay and early-lay rations for brown-egg pullets. Poul. Sci. 72:1641-1649. Hill, F. W., D. L. Anderson, and L. M. Dansky. 1956. Studies of the energy requirements of chickens 3. The effect of dietary energy level on the rate and gross efficiency of egg production. Poult. Sci. 35:54-59. Hussein, A. S., Cantor, A. H., Pescatore, A. J. and Johnson, T. H. 1996. Effect of dietary protein and energy levels on pullet development. Poult. Sci. 75:973-978. Huyghebaert, G., De Groote, G., Butler, E. A. and Morris, T. R. 1991. Optimun isoleucine requirement of laying hens and the effect of age. Br. Poult. Sci. 32: 471-481. Isa Brown. 2011. Nutrition Management Guide. Institut de Selection Animale. B.V, Boxmeer, The Netherlands. 51 Chapter 1. Literature review and objectives Jalal, M.A., S. E. Scheideler, and D. Marx. 2006. Effect of bird cage space and dietary matabolizable energy level on production parameters in laying hens. Poult. Sci. 8:5306-5311. Jalal, M.A., S. E. Scheideler, and E. M. Pierson. 2007. Strain response of laying hen to varying dietary energy levels with and without avizyme supplementation. J. Appl. Poult. Res. 16: 289-295. Jamroz, D., J. Skokupinska, J. Orda, A. Wiliczkiewicz, and A. M. Klunter. 2001. Use of wheat, barley or triticale in feed for laying hens supplemented with carbohydrates derived from Trichoderma longibrachiatum. J. Appl. Anim. Res. 19:255-263. Jensen, L. S., J. B. Allred, R. E. Fry, and J. McGinnis. 1958. Evidence for an unidentified factor necessary for maximum egg weight in chickens. J.Nutri. 65:219-223. Joly, P. (1995) Puesta al día de las necesidades de aminoácidos de las gallinas ponedoras. XXXII Symposium de avicultura. Sección española de la Asociación Mundial de Avicultura Científica (WPSA). Barcelona. Spain. pp.: 67-81. Joly, P., and M. Bougon. 1997. Influence du niveau énergétique sur les performances des pondeuses à oeufs roux et évolution de l´ingéré en fonction de l`âge. 2ême Journée de la Recherche Avicole. Vol. 2:115-120. Junqueira, O.M., A. C. de Laurentiz, R. da Silva Filardi, E. A. Rodrigues, and E. M. Casartelli. 2006. Effects of energy and protein levels on egg quality and performance of laying hens at early second production cycle. J. Appl. Poult. Res. 15:110-115. 52 Chapter 1. Literature review and objectives Keshavarz, K., and S. Nakajima. 1995. The effect of dietary manipulations of energy, protein, and fat during the growing and laying periods on early egg weight and egg components. Poult. Sci. 74:50-61. Keshavarz, K. 1995. Further investigations on the effect of dietary manipulations of nutrients on early egg weight. Poult. Sci. 74:62-74. Kling, L.J. and Hawes, R. O. 1990. Effect of fat, protein, and methionine concentrations on egg size and production in early matured brown-egg-type pullets. Poult. Sci. 69:1943-1949. Lázaro, R., M. García, M. J. Araníbar, and G.G. Mateos. 2003. Effect of enzyme addition to wheat-, barley- and rye-based diets on nutrient digestibility and performance of laying hens. Br. Poult. Sci. 44:256-265. Leeson, S., Lewis, D. and Shrimpton, D. H. 1973. Multiple linear regression equations for the prediction of food intake in the laying fowl. Br. Poult. Sci. 14: 595-608. Leeson, S., and J. D. Summers. 1987. Effect of immature body weight on laying performance. Poult. Sci. 66:1924-1928. Leeson, S., and J. D. Summers. 2005. Comercial Poultry Nutrition, 3rd Edition. Nottingham University Press. Nottingham, UK. Liebert, F., J. K. Htoo, and A. Sünder. 2005. Performance and nutrient utilization of laying hens fed low-phosphorus corn-soybean diets supplemented with microbial phytase. Poult. Sci. 84:1576-1583. Lohmann. 2010. Management Guide for Lohmann Brown-Classic. Lohmann Tierzucht. GMBH. Cuxhaven, Germany. 53 Chapter 1. Literature review and objectives March, B. E. and Biely, J. 1963. Control of egg size in chickens through dietary amino acid balance. Nature. 200:702-703. March, B. E. and McMillan, C. 1990. Linoleic acid as a mediator of egg size. Poult. Sci. 69:634-639. MARM, 2010. Ministerio de Medio Ambiente y Medio Rural y Marino. El sector de la avicultura de puesta en cifras. Principales indicadores económicos en 2009. Subdirección general de productos ganaderos, Madrid, Spain. Mateos, G. G. and J. L. Sell. 1980b. Influence of graded levels of fat on utilization of pure carbohydrate by the laying hen. J. Nutr. 110:1894-903. Mateos, G. G. and. J. L. Sell. 1981. Nature of the extrametabolic effect of supplemental fat used in semipurified diets for laying hens. Poult. Sci. 60:1925-1930. Mathlouthi, N., M. Larbier, M. A. Mohamed, and M. Lessire. 2002. Performance of laying hens fed wheat, wheat-barley or wheat-barley-wheat bran based diets supplemented with xylanase. Can. J. Anim. Sci. 82:193-199. Morris, T. R. 1968. The effect of dietary energy level on the voluntary calorie intake of laying hens. Br. Poul. Sci. 9:285-295. Morris, T. R. and Gous, R. M. 1988. Partitioning of the response to protein between egg number and egg weight. Br. Poul. Sci. 29:93-99. Nathanael, A. S. and Sell, J. L. (1980) Quantitative measurements of the lysine requirement of the laying hen. Poult. Sci. 59:594-597. 54 Chapter 1. Literature review and objectives Newcombe, M. and Summers J. D. 1985. Effects on increasing cellulose in diets fed as crumble or mash on the food intake and weight gains of broilers and Leghorn chicks. Br. Poult. Sci. 26:35-42. NRC. 1994. Nutrient Requirements of Poultry. 9th ed. Natl. Acad. Press, Washington, DC. Parsons, C. M., Koelkebeck, K. W., Zhang, Y., Wang, X. and Leeper, W. 1993. Effect of dietary protein and added fat levels on performance of young laying hens. J. Appl. Anim. Res. 2:214-220. Peguri, A., and C. Coon. 1991. Effect of temperature and dietary energy on layer performance. Poult. Sci. 70:126-138. Pilbrow, P. W. and Morris, T. R. 1974. Comparison of lysine requirements amongst eight stocks of laying fowl. Br. Poult. Sci. 15:51-73. Pirgozliev, V. R., C. L. Birch, S. P. Rose, P. S. Kettlewell, and M. R. Bedford. 2003. Chemical composition and the nutritive quality of different wheat cultivars for broiler chickens. Br. Poult. Sci. 44:464–475. Prochaska, J. F., Carey, J. B. and Shafer, D. J. 1996. The effect of L-lysine intake on egg component yield and composition in laying hens. Poult. Sci. 75:1268-1277. Proudfoot, F.G., Hulan, H. W. and McRae, K. B. 1988. Performance comparisons of phased protein dietary regimens fed to commercial Leghorns during the laying period. Poult. Sci. 67:1447-1454. Ribeiro, B.R.C., L.J.C. Lara, N.C. Baiao, C.A.A. Lopez, M.A. Fiuza, S.V. Conçado, and G.M.M. Silva. 2007. Effect of linoleic acid level in the diet on the weight, 55 Chapter 1. Literature review and objectives composition and eclodibility of broiler breeders eggs. Arq. Bras. Med. Vet. Zootec. 59: 789-796. Ruiz, N., J. E. Marion, R. D. Miles, and R. B. Barnett. 1987. Nutritive value of new cultivars of triticale and wheat for broiler chick diets. Poult. Sci. 66:90–97. Roland, D. A., Kao, D. K., Bryant, M. M. and Self, J. (1992) Econometric nutrition: Maximizing profits in commercial Leghorn by optimizing feeding method, total sulfur amino acid intake and environmental temperature. Degussa Technical Symposium. Degussa Corp., Indianapolis, IN. pp.:47-57 Roland, D. A., Sr. 1988. Research note: eggshell problems: Estimates of incidence and economic impact. Poult. Sci. 67:1801-1803. Safaa, H. M., E. Jiménez-Moreno, D. G. Valencia, M. Frikha, M. P. Serrano and G. G. Mateos 2009. Effect of main cereal of the diet and particle size of the cereal on productive performance and egg quality of brown egg-laying hens in early phase of production. Poult. Sci. 88:608-614. Schutte, J. B., De Jong, J. and Bertram, H. L. 1994. Requirements of the laying hen for sulfur amino acids. Poult. Sci. 73:274-280. Scragg, R. H., Loga, N. B. and Geddes, N. 1987. Response of egg weight to the inclusion of various fats in layer diets. Br. Poul. Sci. 28:15-21. Sell, J. L. and Hodgson, G. C. 1966. Wheat-soybean meal rations for laying hens. Poult. Sci. 45:247-253. Sell, J. L., Angel, C. R. and Escribano, F. 1987. Influence of supplemental fat on weights of egg and yolks during early egg production. Poult. Sci. 66:1807-1812. 56 Chapter 1. Literature review and objectives Shannon, D. W. F. and Whitehead, C .C. 1974. Lack of response in egg weight or output to increasing levels of linoleic acid in practical layers’ diets. J. Sci. Food Agric. 25:553-561. Sohail, S. S., M. M. Bryant, and D. A. Roland, Sr. 2003. Influence of dietary fat on economic returns of commercial Leghorns. J. Appl. Poult. Res. 12:356–361. Summers, J. D. 1986. Feeding light hibrids in lay. Poultry International. 25:22-26. Summers, J. D., Atkinson, J. L. and Spratt, D. 1991. Supplementation of low protein diet in an attempt to optimize egg mass output. Can. J. Anim. Sci. 71:211-220 Summers, J.D. and S. Leeson. 1983. Factors influencing early egg size. Poult. Sci. 62:1155-1159. Usayran, N., M. T. Farran, H. H. O. 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Effects of the main cereal and nd type of fat of the diet CHAPTER 2: Effects ffects of the main cereal and type of fat of the diet on productive performance and egg quality of brown-egg egg laying hens from 22 to 54 weeks of age (Experiment 1) PUBLISHED IN: POULTRY SCIENCE 90:2801-2810 90:2801 doi:10.3382/ps.2011-01503 59 Chapter 2. Effects of the main cereal and type of fat of the diet 1. Introduction Corn (Zea mays L.) and soft wheat (Triticum aestivum L.) are two cereals commonly used as energy sources in poultry diets.In some countries, barley (Hordeum vulgare L.) is also an attractive commercial alternative.Corn has less protein and dietary fiber and more starch, fat, AMEn, and linoleic acid (LNL) content than wheat or barley. In addition, the chemical composition and nutritive value of corn is quite uniform compared to that of wheat and barley, but all grain contents vary depending on factors such as cultivar, agronomic practices, weather conditions, and length of the storage period (Pirgozliev et al., 2003; Gutiérrez-Alamo et al., 2008; Frikha et al. 2011). In commercial practice, many egg producers formulate diets for laying hens with at least 20-30% corn to insure high feed intake and maximize egg size early in the production cycle, in spite of the frequent higher relative cost of corn. The reasons for this practice is unknown but might be related to the more uniform nutritive value of corn and the better feed structure when coarse corn is included in the diet (Frikha et al., 2009). Also, corn has more LNL content than wheat and barley, and an increase in LNL content of the diet might result in an improvement in egg weight, especially in young pullets under hot climate conditions in which FI is low (Jensen et al., 1958; Scragg et al., 1987; Grobas et al., 1999a). In addition, wheat and barley contain a high and variable amount of non starch polisaccharides (NSP) that is known to increase digesta viscosity and reduce productive performance in poultry (Lázaro et al., 2003, Garcia et al., 2008). The effect of the main cereal of the diet on egg quality has not been studied in detail. In general, the inclusion of barley and wheat increased the incidence of dirty eggs as compared with the inclusion of corn (Francesch et al., 1995) but the problem was reduced to levels similar to that of corn when diets were supplemented with enzymes (Lázaro et al., 2003). 60 Chapter 2. Effects of the main cereal and type of fat of the diet Fats are used in poultry to increase the energy content of the diets. The inclusion of fat resulted often in lower feed consumption but higher energy intake, egg weight, and BW gain (Grobas et al., 2001; Bouvarel et al., 2010), probably because of improved palatability with less dust formation (ISA Brown, 2011). Also, supplemental fat has been shown to reduce rate of feed passage, facilitating the contact between digesta and enzymes and improving digestibility and utilization of other nutrients such as the lipid and carbohydrate fractions of dietary ingredients (Mateos and Sell, 1980b, 1981). Soybean oil (SBO), acidulated vegetable oil soapstocks (AVO), a by-product of the oil industry for human consumption, and lard, are the most commonly used fat sources in laying hen diets in Spain. Soybean oil contains more LNL and AMEn than AVO and lard (Fundación Española Desarrollo Nutrición Animal, 2003). In addition, the LNL and AMEn content of AVO may be more variable than that of SBO or lard because they depend on the characteristics of the original oils and the refining process used. Scragg et al. (1987) reported increases in egg weight with increases in dietary levels of LNL of up to 2.0%. Moreover, some commercial guidelines on management and nutrition of laying hens (H & N International, 2008; Bábolna Tetra, 2009; Lohman, 2010) recommend dietary LNL levels higher than 1.8% (2.0 g/hen/d) to optimize egg production and insure a rapid increase in egg size at the beginning of the laying period. However, Shutze et al. (1959), Grobas et al. (1999b,c), and Safaa et al. (2009) reported no benefits with levels of LNL in the diet above 1.0-1.15%. The hypothesis of this research was that when the diet is supplemented with enzymes, wheat and barley can substitute corn as the main cereal of the diet without any negative effect on performance or egg quality. Similarly, the 3 fat sources tested could be used indistinctly as a source of dietary energy without any loss of performance or egg quality. Also, we hypothesize that egg weight of hens fed diets containing 4.3% 61 Chapter 2. Effects of the main cereal and type of fat of the diet supplemental fat could be maximized with levels of LNL in the diet of approximately 1.0% (1.16 g/hen/d). The aim of this study was to determine the influence of the main cereal of the diet (corn, wheat, and barley) and type of supplemental fat (soy oil, acidulated vegetable soapstocks, and lard) on productive performance and egg quality of brown laying hens from 22 to 54 wks of age. 2. Material and methods 2.1. Husbandry, Feeding Program, and Experimental Diets All experimental procedures were approved by the animal Ethics Committee of the Universidad Politécnica de Madrid and were in compliance with the Spanish guidelines for the care and use of animals in research (Boletín Oficial del Estado, 2005). In total, 756 20-wk-old Lohmann Brown laying hens were obtained from a commercial flock (El Canto Agroalimentaria S.L, Toledo, Spain). From 20 to 22 wks of age all hens were fed a common corn-soybean meal diet. At 22 wks, the hens were weighed individually and placed at random in cages (7 hens) provided with an open trough feeder and 2 nipple drinkers in an environmentally controlled barn. Each treatment was replicated 4 times and the experimental unit consisted of 3 adjacent cages (21 hens) (600 x 575 mm; General Ganadera S.A, Valencia, Spain). The ambient temperature in the barn varied according to the month considered (22 ± 3 ºC in March, first month of the trial and 28 ± 3 ºC in July, last month of the trial). The light program was constant and consisted of 16 h light per day. The cereals used were grown in Spain and were obtained from commercial sources; the SBO was supplied by Bunge Ibérica S.A. (Barcelona, Spain), the AVO by Oleínas y Grasas S.L. (Tarragona, Spain), and the lard from Ibergrasa S.A. (Madrid, 62 Chapter 2. Effects of the main cereal and type of fat of the diet Spain). The AVO was a commercial mixture of vegetable soapstocks and was composed primarily of by-products of the palm oil and soy oil refinery industry. Two different batches of cereals and lipid sources were used during the trial: one batch for the first four 28-d periods and a second batch for the last four 28-d periods of the experiment. The calculated and determined composition of the cereals and fat sources used are shown in Tables 1 and 2, respectively. The experiment was conducted as a completely randomized design with 9 diets organized factorially with 3 main cereals and 3 sources of supplemental fat. Barley and wheat were included in their respective diets in substitution of 45 percentage units of corn. The ingredient composition of these diet was adjusted to insure that all had similar, AMEn and indispensible AA content (Fundacion Española Desarrollo Nutricion Animal, 2003). However, no attempt was made to equalize the LNL content of these diets. Within each cereal series of diets, the 3 fat sources were introduced (wt:wt) at a level of 4.3% and no attempt was made to maintain constant their AMEn and LNL contents. Thus, diets based on SBO contained slightly more AME (2.750, 2.727, and 2.730 Kcal/kg, respectively) and more LNL than diets based on AVO or lard (Table 3). Because of the experimental design, the levels of LNL varied from 0.8 to 3.4% depending on the combination of cereal and fat source used, with the lower values reported for diets based on wheat and lard and the higher for diets based on SBO and corn. The LNL content of diets based on wheat or barley and supplemented with lard, was lower than 1.1%, the value recommended by NRC (1994) for brown-egg laying hens consuming 110 g feed/d.Moreover, most of the experimental diets had lower levels of LNL than currently used by the industry (1.7 to 2.0%). Otherwise, all the diets met or exceeded the nutrient requirements for brown egg-laying hens (Fundacion Española Desarrollo Nutricion Animal, 2008). A commercial enzyme complex that included β-glucanase and xylanase activity (Endofeed, GNC Bioferm Inc., Saskatoon, SK, Canada), was included at the dose 63 Chapter 2. Effects of the main cereal and type of fat of the diet recommended by the supplier in all diets to insure maximal nutrient utilization. Also, a commercial additive (Lucanmix, BASF Ibérica, Tarragona, Spain) based on canthaxantin and ester of β-apo-8-carotenoic was included in fixed amounts in all the diets to satisfy consumer preference for egg yolk pigmentation. The ingredient composition and the calculated nutritive value of the experimental diets are presented in Tables 3 and 4, respectively. The cereal fraction of the feeds was ground using a hammer mill provided with a 7.5 mm screen. 2.2. Laboratory Analyses Representative samples of cereals and diets were ground in a laboratory mill (Model Z-I, Retsch Stuttgart, Germany) provided with a 1-mm screen and analyzed for moisture by the oven-drying (method 930.01), total ash using a muffle furnace (method 942.05), nitrogen by combustion (method 990.03) using a LECO analyzer (Model FP-528, LECO, St. Joseph, MI), starch by the α-amylase glucosidase (method 996.11), CF by sequential extraction with diluted acid and alkali (method 962.09), and Ca and P by spectrophotometry (methods 968.08 and 965.17) as described by AOAC International (2000). Neutral and acid detergent fiber of the cereals were determined sequentially as described by Van Soest et al. (1991) and expressed on an ash-free basis. Ether extract was determined by Soxhlet analysis after 3 N HCl acid hydrolysis (Boletín Oficial del Estado, 1995), and gross energy using an isoperibol bomb calorimeter (Model 356, Parr Instrument Company, Moline, IL). The fatty acid content of cereals and fat sources were determined by gas-liquid chromatography (GC-14B, Shimadzu, Kyoto, Japan) as indicated by Grobas et al. (1999b). Quality traits of the fat sources, including insoluble impurities (method 926.12), moisture (method 984.20), and unsaponifiable material (method 933.08) were determined as indicated by AOAC International (2000). Non eluted material, which reflects the indigestible portion of fat in the sample, was 64 Chapter 2. Effects of the main cereal and type of fat of the diet determined by gas chromatography (method 977.17) as indicated by AOAC International (2000). The acid value, which measures the amount of KOH in mg needed for neutralizing the free fatty acids present in 1 g of the fat sample, was determined by method Cd-3d-63 of AOCS (1998). The initial peroxide index (method 16) was determined as indicated by Boletín Oficial del Estado (1995). All these analyses were conducted in duplicate samples. The geometric mean diameter of cereals (Table 1) and diets (Table 4) for each of the two periods considered were determined in triplicate in 100 g samples using a Retsch shaker (Retsch, Stuttgart, Germany) provided with 8 sieves ranging in mesh from 5,000 to 40 µm according to the methodology outlined by ASAE (1995). 2.3. Productive Performance and Egg Quality Eggs were collected daily and egg weight was measured in all eggs produced the last 2 days of each of the eight 28-d periods. Also, feed intakewas measured by replicate by period and cumulative and mortality was recorded as produced. From these data, hen-day egg production, egg weight, egg mass, ADFI, and feed conversion ratio (FCR) per kilogram and per dozen of eggs, were calculated by period and cumulatively. Also, the ratio (g/g) between average egg mass or egg weight and LNL intake by treatment were calculated. In addition, all the hens were weighed individually at the beginning and at the end of the experiment, and the BW gain per replicate was calculated. The number of clean, dirty, broken, shell-less, and double-yolked eggs was recorded daily by replicate. An egg was considered as dirty when a spot of any kind or size was detected on the shell as evaluated by two independent observers blind to treatment. In addition, all the eggs used for egg weight determination (produced the last 2-d of each 28-d period), were graded as described by the European Council Directive (2006). The categories recorded were extra large (>73 g), large (73 to 63 g), medium (63 65 Chapter 2. Effects of the main cereal and type of fat of the diet to 53 g), and small (< 53 g). Also, shell density and internal quality (Haugh unit and yolk color by Roche color fan) were measured in 10 eggs per replicate chosen at random from eggs produced the last day of each 28-d period. A multitester equipment (QCM System, Technical Services and Supplies, Dunnington, York, UK) was used. In addition, shell thickness of 5 eggs collected at random from eggs produced the last day of each 28-d period from each replicate was measured with a digital micrometer (model IT-014UT, Mitotuyo, Kawasaki, Japan). The average of 3 measurements was used to estimate shell thickness of each egg. At the end of the second experimental period the proportion of albumen and yolk were determined in the 10 eggs per replicate used for egg quality measurements as indicated by Safaa et al. (2008). Also, the pH value of the yolk and albumen fractions was measured in these eggs using a pH meter (Accumet 910, Kent City, MI) as indicated by Shang et al. (2004). 2.4. Statistical Analysis The experimental design was completely randomized with 9 treatments arranged factorially and the main effects (type of cereal and of supplemental fat) and its interactions were analyzed by ANOVA using the GLM procedure of SAS Institute (1990). No significant interactions between main effects were observed for any of the traits studied and therefore, the interaction was removed from the model. The homogeneity of the variance of the data for all traits was tested by the Levene´s Test (Hovtest option of the GLM procedure). All performance and egg quality data were homogeneous, except for mortality; therefore, mortality data were analyzed after arcsin transformation. When the effects of cereal and fat source were significant, the Tukey test was used to make pairwise comparisons to separate treatment means. Results in tables are presented as means, and differences were considered significant at P < 0.05. 66 Chapter 2. Effects of the main cereal and type of fat of the diet 3. Results 3.1. Laboratory analysis The determined chemical composition of cereals (Table 1) and lipid sources (Table 2) were similar to expected values (Fundación Española Desarrollo Nutrición Animal, 2003). The LNL content of the 2 batches of fats used was 58.6 and 55.7% for SBO, 23.9 and 21.6% for AVO, and 8.4 and 8.0% for lard. Gross energy was lowest for AVO, consistent with its higher moisture, insoluble impurities, and non-eluted material content as compared with SBO or lard. The geometric mean diameter of the 2 batches of cereals and diets used was higher for barley than for corn with that of wheat being intermediate (Tables 1 and 4, respectively). 3.2. Productive performance No interactions between cereal and supplemental fat on performance of laying hens were detected and therefore, only the P-values for main effects are presented (Table 5). Also, dietary treatment had no significant effects on productive performance in any of the periods considered and therefore, only cumulative effects are presented.For the entire experimental period,egg production (92.9, 91.5, and 92.1 % for corn, wheat, and barley, respectively) and egg weight (64.5, 63.6, and 64.1 g for corn, wheat, and barley, respectively) were not affected by type of cereal. Consequently, egg mass did not differ among treatments (59.9, 58.2, and 59.1 g for hens fed corn, wheat, or barley, respectively). In contrast, hens fed wheat and corn had higher BW gain than hens fed barley (243, 238, and 202 g, respectively; P< 0.05).Source of fat did not affect any of the productive traits studied except for BW gain that was higher for hens fed lard than for hens fed SBO or AVO (251, 221, and 210 g, respectively; P< 0.05). Cumulatively, average mortality was 7.5% and was not affected by dietary treatment. 67 Chapter 2. Effects of the main cereal and type of fat of the diet The ratio between g of egg weight or g of egg mass produced and g of LNL intake according to treatment is shown in Table 6. The ratio was lowest for hens fed SO, irrespective of the main cereal of the diet and highest for hens fed lard and wheat, followed by hens fed lard and barley, and lard and corn (P < 0.001). 3.3. Egg quality For the entire experimental period the percentage of dirty, broken, shell-less, and doubleyolked eggs, as well as Haugh units, and shell thickness and density of the eggs were not affected by diet (Table 7).Yolk pigmentation was higher (P< 0.001) for hens fed corn than for hens fed wheat or barley. Also, yolk eggs from hens fed lard were better pigmented (P< 0.001) than yolks from hens fed SBO or AVO. At 30 wks of age, dietary treatment did not affect percentage of yolk or albumen weight, or the pH of these two egg fractions (Table 8). 4. Discussion 4.1. Productive performance For the entire experimental period, ADFI, egg production, egg weight, and FCR were similar for the 3 cereals, results that agree with previous reports (Craig and Goodman., 1993; Lázaro et al., 2003; Safaa et al., 2009) that have shown that when wheat and barley diets were supplemented with exogenous enzymes, laying hen productivity was not affected by the main cereal of the diet. Moreover, Mathlouthi et al. (2002) reported similar performance in broilers when 60% of corn was substituted by a combination of 40% wheat and 20% barley supplemented with enzymes. Also, Ruiz et al. (1987) reported similar BW gain and FCR for diets based on corn than for diets based on wheat in broilers from 1 to 21 d of age. In contrast, Coon et al. (1988) reported higher ADFI and poorer FCR in hens fed enzyme supplemented barley diets than in hens fed corn diets. In 68 Chapter 2. Effects of the main cereal and type of fat of the diet the current experiment, BW gain was greater in hens fed corn or wheat than in hens fed barley, results that agree with data of Berg et al. (1959) who reported higher BW gain for hens fed corn than for hens fed barley. In contrast, Frikha et al. (2009) reported higher BW gain in pullets fed corn than in pullets fed wheat, both diets being supplemented with enzymes. In general, the information available indicated that when the diets are supplemented with enzymes, wheat and barley can substitute for corn in poultry diets, in agreement with the data of the current experiment. For the entire experimental period, the substitution of SBO by AVO or lard in the diet did not affect any of the productive performance traits studied except for BW gain that was higher for hens fed lard than for hens fed SBO or AVO. We are not aware of any published paper reporting on the effects of these 3 supplemental fats on BW gain of the hens. In the current research, a higher percentage of the energy intake of the hens fed lard was diverted to BW gain rather than to energy deposition in eggs was compared with hens fed lard than when fed SBO or AVO. Vila and Esteve-Garcia (1996) and Sanz et al. (1999, 2000) reported that broilers fed diets supplemented with tallow or lard had higher abdominal fat deposition than broilers fed isoenergetic diets based on more unsaturated vegetable oils, consistent with the results of the current experiment. The effect of dietary LNL on egg size is a subject of debate. Under commercial conditions, many commercial guides for feeding laying hens (H&N International, 2009; Lohmann, 2010) recommend to increase the level of LNL in the diet to at least 1.8% (2.0 g/hen/d) to maximize egg size. Moreover, Scragg et al. (1987) recommended up to 2% dietary LNL to increase egg size in brown-egg laying hens and Ribeiro et al. (2007) reported higher egg weight in broiler breeder hens fed 1.9% LNL diets than in hens fed 1.5% LNL diets. However, data from Jensen et al. (1958), Shutze et al., (1959), and Grobas et al. (1999a, b) do not support current feeding practices of using 1.8% LNL in the diet to maximize egg size. The reasons for the discrepancies between practical 69 Chapter 2. Effects of the main cereal and type of fat of the diet nutritionists and researchers in respect to LNL requirements to maximize egg size are not known but might be related to the composition of the diets used. For example, under practical conditions, the increase in LNL content of the diet is achieved by increasing the level of fat and thus, the effects on LNL level and fat inclusion are confounded. In this respect, Grobas et al. (1999b) suggested that laying hens require no more than 1.15% LNL in the diet (1.33 g/hen/d) for maximal egg weight and that when this minimal amount of LNL is met, an increase in supplemental fat, irrespective of its LNL content, might result in further increase in egg size. In addition, Mateos and Sell (1980a) demonstrated in laying hens that supplemental fat increases the utilization of other components of the diets such as the carbohydrate fraction (Mateos and Sell, 1980a). Consequently, the increase in egg size observed with the use of high levels of dietary LNL might be a result of the increase in fat content rather than LNL level “per se”. In the current experiment, the lower egg weight was observed for hens fed wheat and lard (62.8 g) and the larger for hens fed corn and SBO or AVO (64.9 and 65.0 g, respectively) although no significant differences were detected (P> 0.05) . The LNL content of the diets based on wheat and lard (0.8%) was below values recommended by most researchers (Jensen et al., 1958; Shutze et al., 1959). In fact, the NRC (1994) recommends a minimum of LNL in the diet of 1.0% (1.10 g/hen/d) in brown-egg laying hens. Probably, the low LNL content of the wheat and lard diets was insufficient to maximize egg weight. It is of interest to notice the differences in the ratio between egg size (g) or egg mass produced (g/d) and LNL intake (g/d) by the hens fed the different diets. This ratio (Table 7) was 2.5 times higher for hens fed the lard diets than for hens fed the SBO diets, with hens fed the AVO diets being intermediate. The information provided suggests that brown-egg laying hens can use indistinctly any of the 3 cereals or lipid sources tested as energy sources without any 70 Chapter 2. Effects of the main cereal and type of fat of the diet effect on performance provided that the amount of dietary LNL is maintained around 0.91.0% (1.04 -1.16 g/hen/d). 4.2. Egg quality Type of cereal did not affect any of the egg quality traits studied, except for egg yolk pigmentation that was increased when corn was used. The beneficial effect on yolk pigmentation observed with corn feeding was expected because all the diets, independent of the cereal used, were supplemented with the same amount of exogenous pigment source. The information available on the effects of the main cereal of the diet on egg quality traits, other than yolk pigmentation, is scarce. Jamroz et al. (2001) reported similar egg quality from hens fed wheat or barley diets supplemented with enzymes. On the other hand, Francesch et al. (1995) have reported a higher incidence of dirty eggs in hens fed barley than in hens fed corn. The only report available comparing egg quality in hens fed high levels of these 3 cereals is that of Lázaro et al. (2003). In this report, the inclusion of barley and wheat increased the percentage of dirty eggs as compared with the inclusion of corn. However, no other quality traits (shell weight, percentage of shell-less, and Haugh units) were affected by the main cereal of the diet. Moreover, when enzymes were added to the wheat and barley diets, the incidence of dirty eggs decreased to levels similar to those found for the corn diet. Çiftci et al. (2003) and Safaa et al. (2009) reported that the substitution of corn by wheat in enzyme supplemented diets, did not affect the percentage of dirty eggs in Single Comb White Leghorn and brown egg-laying hens, respectively, in agreement with the results of the current trial. In the current experiment, Roche color fan values of the yolks were greater with lardthan with SBO- or AVO supplemented diets. We have not found any report in the literature comparing the influence of type of fat on yolk pigmentation. The fatty acid profile of lard was more saturated than that of the SBO or AVO and thus, dietary 71 Chapter 2. Effects of the main cereal and type of fat of the diet pigments were probably more stable in the presence of lard than in the presence of more unsaturated fat sources, both in the feed and in the gastrointestinal tract. Cereal source did not affect the pH of yolk and albumen in fresh eggs. Woodward et al. (1987) reported that yolk rupture strength of 50-wk-old hens decreased with the age of the eggs but that the decline was more rapid for egg yolks from birds fed a corn diet than from birds fed a wheat diet. Scheideler et al. (2010) reported that supplementation of the diet with 250 IU vitamin E/kg improved vitelline membrane strength of fresh eggs. These authors reported that vitamin E supplementation increased yolk pH and reduced albumen pH and that these changes in pH were responsible for the beneficial effects observed on vitelline membrane strength. In the current experiment, no effects of cereal or fat source on albumen and yolk pH were detected. Thus, neither cereal type nor supplemental fat source was expected to affect this aspect of egg quality. 5. Conclusions We conclude that corn, wheat, and barley can be used successfully in laying hen diets to levels of up to 45% provided that the diets supplied a minimal amount of linoleic acid. Also, soybean oil, acidulated vegetable soapstocks, and lard can be used as a source of energy in the diet without any significant effect on performance or egg quality provided that the diets met the requirements in linoleic acid of the hens. Under practical feeding conditions, the requirements of hens for linoleic acid to maximize egg size is lower than recommended by most practical nutritionists and guidelines for laying hens. Therefore, current practices of formulating commercial diets with at least 1.8% linoleic acid are not justified. 72 Table 1. Chemical composition of the experimental cereals (% as fed basis, unless otherwise indicated). Calculated analysis AMEn (kcal/kg) Determined analysis2 DM CP Ether extract Linoleic acid Starch Ash Neutral detergent fiber Acid detergent fiber Crude fiber GMD3, µm GSD4 , µm 1 Barley Wheat Corn 2,800 3,150 3,260 22-38 wks 38-54 wks 90.6 89.8 11.2 9.6 1.9 2.0 0.8 0.9 53.1 52.0 2.5 2.3 17.4 18.7 4.8 4.7 4.1 4.7 927 950 ±2.43 ±2.41 22-38 wks 38-54 wks 88.8 88.9 11.1 9.7 1.8 2.0 0.7 0.8 60.9 61.2 1.5 1.8 12.8 13.9 3.3 3.4 2.7 3.3 859 885 ±2.46 ±2.61 22-38 wks 38-54 wks 86.5 86.4 7.2 7.0 3.4 3.5 2.1 2.1 63.3 64.5 1.4 1.2 8.5 8.4 1.9 1.9 1.6 1.7 777 788 ±2.59 ±2.60 1 According to Fundación Española Desarrollo Nutrición Animal (2003). Analyzed in triplicate. 3 Geometric mean diameter. 4 Log normal geometric SD. 2 Table 2. Chemical analyses of the experimental fats (% as fed basis, unless otherwise indicated) Calculated analysis SBO1 AVO2 Lard 9.000 22-38 wks 38-54 wks 8.450 22-38 wks 38-54 wks 8.550 22-38 wks 38-54 wks 3 AMEn (kcal/kg) Determined analysis4 Gross energy (kcal/kg) 9,315 9,374 9,030 9,190 9,395 Fatty acid profile(g/100g fatty acids) Myristic acid 0.3 0.1 1.0 0.5 1.3 Palmitic acid 10.0 10.1 15.7 21.2 23.1 Palmitoleic acid 0.2 0.2 2.1 0.9 3.1 Margaric acid 0.2 0.08 0.2 0.1 0.3 Estearic acid 4.5 3.7 5.1 3.5 11.6 21.0 23.1 47.1 47.6 48.8 Oleic acid 5 Linoleic acid 58.6 55.7 23.9 21.6 8.4 Linolenic acid 5.0 5.6 1.5 2.1 0.5 Other fatty acids 0.2 1.4 3.4 2.5 2.9 0.04 0.03 2.7 1.6 0.8 Moisture Non eluted material 1.5 0.1 15.2 6.9 3.1 Acid value 1.2 0.2 52.2 48.0 0.1 Initial peroxide index (meq/kg) 2.0 2.3 1.1 0.8 4.0 Insoluble impurities 0.1 0.04 0.3 0.3 0.2 1 Soybean oil. 2 Acidulated vegetable soapstocks. 3 According to Fundación Española Desarrollo Nutrición Animal (2003). 4 Analyzed in duplicate. 5 Calculated values in diet formulation were 56, 23 and 8.5% for SBO, AVO, and lard, respectively. 9,433 1.2 23.0 2.6 0.3 10.4 51.4 8.0 0.6 2.5 0.02 0.1 0.5 2.1 0.1 74 Table 3. Ingredient composition of the experimental diets (%, as-fed basis) Ingredient 1 SBO Barley AVO2 Lard SBO Wheat AVO Lard SBO Corn AVO Lard Corn 15.6 15.6 15.6 10.2 10.2 10.2 51.4 51.4 51.4 Wheat ---45.0 45.0 45.0 ---Barley 45.0 45.0 45.0 ------SBO 4.3 --4.3 --4.3 --AVO -4.3 --4.3 --4.3 -Lard --4.3 --4.3 --4.3 Soya bean meal, 47% 23.3 23.3 23.3 17.4 17.4 17.4 22.0 22.0 22.0 Sunflower meal, 32% 0.6 0.6 0.6 10.9 10.9 10.9 10.1 10.1 10.1 Methionine-OH, 88% 0.15 0.15 0.15 0.12 0.12 0.12 0.12 0.12 0.12 L-Lys-HCL, 78% ---0.02 0.02 0.02 ---Sepiolite3 0.32 0.32 0.32 1.40 1.40 1.40 1.33 1.33 1.33 Monocalcium phosphate 0.98 0.98 0.98 0.91 0.91 0.91 1.1 1.1 1.1 Calcium carbonate 9.0 9.0 9.0 9.0 9.0 9.0 8.9 8.9 8.9 Sodium chloride 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.35 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 0.40 Vitamin and mineral premix4 1 Soybean oil. (Supplied by Bunge Ibérica S.A., Barcelona, Spain). 2 Acidulated of vegetable soapstocks(Supplied by Oleínas y Grasas S.L., Tarragona, Spain). 3 Complex magnesium silicate clay used as inert material (Manuel Riesgo S.A., Madrid, Spain). 4 Supplied per kg of diet: vitamin A (trans-retinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 2,000 IU; vitamin E (DL-α-tocopheryl acetate), 10 mg; vitamin B1, 1 mg; vitamin B2, 4 mg; vitamin B6, 1 mg; vitamin 12 (cyanocobalamin), 15 mg; vitamin K3, 2.5 mg; choline (choline chloride), 150 mg; nicotinic acid, 25 mg; pantothenic acid (D-calcium pantothenate), 7,5 mg; folic acid 0,10, mg; manganese (MnO), 70 mg; zinc (ZnO), 50 mg; iron (FeSO4 H2O), 30 mg; copper (CuSO4 5H2O), 5 mg; iodine [Ca(IO3)2], 0,5 mg; selenium (Na2SeO3), 0,3 mg; canthaxantin; 2,4 g; ester of β-apo-8-carotenoic, 1,7 g (Lucanmix yellow/red, BASF, Tarragona, Spain), [(Endo-1.3(4) -β-glucanase (EC 3.2.1.6), 150 IU/g; Endo-1.4-β-xylanase (EC 3.2.1.8) 105 IU/g; (Endofeed, GNC Bioferm, Saskatchewan, SK, Canada), Natuphos, (BASF Española, S.A, Tarragona, Spain), 6-phytase (EC 3.1.3.26), 300 FTU)]. 75 Table 4. Chemical composition of the experimental diets (%, as-fed basis unless otherwise indicated) SBO1 Barley AVO2 Lard SBO Wheat AVO Lard SBO Corn AVO Calculated analysis3 AMEn (kcal/kg) 2,750 2,727 2,730 2,750 2,727 2,730 2,750 2,727 Digestible Lys 0.76 0.76 0.76 0.69 0.69 0.69 0.74 0.74 Digestible Met 0.37 0.37 0.37 0.38 0.38 0.38 0.39 0.39 Digestible Tre 0.53 0.53 0.53 0.51 0.51 0.51 0.56 0.56 Digestible Trp 0.18 0.18 0.18 0.18 0.18 0.18 0.18 0.18 Linoleic acid 3.1 1.8 0.9 3.0 1.7 0.8 3.4 2.1 Ca 3.8 3.8 3.8 3.8 3.8 3.8 3.8 3.8 Total P 0.68 0.68 0.68 0.68 0.68 0.68 0.73 0.73 Available P 0.45 0.45 0.45 0.45 0.45 0.45 0.45 0.45 4 Determined analysis GE (kcal/kg) 3,683 3,718 3,773 3,686 3,660 3,714 3,664 3,635 DM 89.8 89.9 89.3 90.0 89.9 89.8 89.9 89.9 CP 18.2 17.7 18.1 18.6 18.2 18.4 18.1 18.0 Ether extract 6.1 5.7 5.8 6.2 5.8 5.9 6.1 5.8 Total ash 12.4 12.9 12.5 12.7 12.7 12.6 12.5 12.7 GMD5, µm 934 923 965 870 886 863 757 745 5 ±2.38 ±2.34 ±2.36 ±2.40 ±2.33 ±2.33 ±2.36 ±2.38 GSD , µm GMD6, µm 909 888 878 832 828 830 778 822 GSD6, µm ±2.34 ±2.31 ±2.26 ±2.28 ±2.24 ±2.29 ±2.25 ±2.21 1 Soybean oil (Supplied by Bunge Ibérica S.A., Barcelona, Spain). 2 Acidulated vegetable soapstocks (Supplied by Oleínas y Grasas S.L., Tarragona, Spain). 3 According to Fundación Española Desarrollo Nutrición Animal (2003). 4 Analyzed in triplicate. Data correspond to the average of feeds supplied from 22 to 38 wks and for 38 to 54 wks of age. 5 Geometric mean diameter and Log normal geometric SD. Data correspond to diets fed from 22 to 38 wks of age. 6 Data correspond to diets fed from 38 to 54 wks of age Lard 2,730 0.74 0.39 0.56 0.18 1.2 3.8 0.73 0.45 3,666 89.0 17.5 5.9 12.3 753 ±2.41 800 ±2.21 76 Table 5. Influence of the main effects of cereal and fat source of the diet on performance of laying hens from 22 to 54 wks of age Cereal Fat Cereal Barley Wheat Corn Fat SBO1 AVO2 Lard Egg production Egg weight Egg mass Feed intake FCR FCR BWgain Mortality (%) 92.1 91.5 92.9 (g) 64.1 63.6 64.5 (g/d) 59.1 58.2 59.9 (g/d) 115.3 115.4 117.3 (kg/kg) 1.95 1.98 1.96 (kg/dozen) 1.52 1.53 1.55 (g) 202b 243a 238a (%) 7.4 6.4 8.9 91.7 92.6 92.2 1.10 64.3 64.5 63.5 0.26 58.9 59.7 58.5 0.78 221b 210b 251a 9.71 6.1 7.1 9.4 1.85 115.6 115.8 116.6 1.11 Probability NS NS 1.96 1.94 1.99 0.017 1.53 1.55 1.53 0.016 SEM3 Effect4 Cereal NS NS NS NS NS Fat NS NS NS NS NS 1 Soybean oil. 2 Acidulated vegetable soapstocks. 3 Standard error of the mean (12 replicates of 21 hens each per treatment). 4 The interaction between main cereal of the diet and source of supplemental fat was not significant (P >0.05). a, b Means within cereal or fat source with different superscripts are significantly different (P <0.05) . * P ≤ 0.05. * * NS NS 77 Table 6. Influence of diet on the ratios between egg weight (g) and egg mass (g/d) produced and g of daily linoleic acid (LNL) intake Cereal Corn Barley Wheat Corn Barley Wheat Corn Barley Wheat SEM3 1 SBO SBO SBO AVO2 AVO AVO Lard Lard Lard Egg weight:g LNL intake 16.26g 18.24g 17.81g 26.41f 31.05e 32.91d 45.65c 64.12b 71.48a 0.333 Probability *** *** *** Egg mass:g LNL intake 15.09f 16.58f 15.88f 24.33e 28.91d 29.30d 42.33c 59.17b 63.07a 0.769 Cereal *** Fat *** Cereal*fat *** 1 Soybean oil. 2 Acidulated vegetable soapstocks. 3 Standard error of the mean (4 replicates of 21 hens each per treatment). *** P ≤ 0.001 a, g Means within a column with different superscripts are significantly different (P <0.05). 78 Table 7. Influence of the main effects of cereal and fat source of the diet on egg quality variables from 22 to 54 wks of age Cereal Fat Cereal Barley Wheat Corn Fat SBO2 AVO3 Lard Dirty eggs % 2.9 3.5 3.4 Broken eggs % 0.95 1.20 1.17 Shell-less eggs % 0.24 0.15 0.13 Double yolked % 0.15 0.14 0.17 Shell thickness mm 0.372 0.372 0.369 3.5 2.9 3.3 0.26 1.10 1.21 1.00 0.19 0.21 0.18 0.14 0.05 0.14 0.19 0.14 0.03 Probability NS NS 0.370 0.371 0.373 0.013 Shell density mg/cm2 77.0 77.0 76.9 Haugh units RCF1 88.0 87.0 86.3 8.3b 8.3b 9.0a 77.4 76.6 76.9 1.99 86.8 87.6 86.9 2.82 8.5b 8.2b 8.9a 0.40 NS NS *** *** SEM4 Effect5 Cereal NS NS NS NS NS NS NS NS NS Fat NS 1 Roche color fan. 2 Soybean oil. 3 Acidulated vegetable soapstock. 4 Standard error of the mean (12 replicates of 21 hens each per treatment). 5 The interaction between main cereal of the diet and source of supplemental fat was not significant (P >0.05). *** P ≤ 0.05. a, b Means within cereal or fat source with different superscripts are significantly different (P <0.05). 79 Table 8. Influence of the main effects of cereal and fat sources of the diet on percentage of yolk and albumen and pH at 30 wks of age Albumen weight Yolk Albumen Yolk weight Cereal Fat (%) (%) pH pH Cereal Fat SEM Barley 25.2 61.6 6.2 8.9 Wheat 25.4 61.3 6.3 8.9 Corn 24.9 62.1 6.2 9.0 Soy oil 24.7 62.2 6.2 8.9 Soapstocks 25.5 61.6 6.2 8.9 Lard 25.4 61.3 6.2 8.9 0.02 0.05 1 Effects 0.94 2 0.97 Probability Cereal NS NS NS NS Fat NS NS NS NS 1 Standard error of the mean (12 replicates of 21 hens each per treatment) The interaction between main cereal of the diet and source of supplemental fat was not significant (P >0.05) NS, not significant (P > 0.05). 2 80 Chapter 2. Effects of the main cereal and type of fat of the diet 6. References ASAE. 1995. Method of determining and expressing fineness of feed materials by sieving. Pages 461-462 in Agriculture Engineers Yearbook of Standards. American Society of Agriculture Engineers. Standard S319.2. Am. Soc. Agric. Eng., St. Joseph, MO. 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Effects of the main cereal and type of fat of the diet Lázaro, R., M. García, M. J. Araníbar, and G. G. Mateos. 2003. Effect of enzyme addition to wheat, barley and rye based diets on nutrient digestibility and performance of laying hens. Br. Poult. Sci. 44:256-265. Lohmann, 2010. Management Guide for Lohmann Brown-Classic. Lohmann Tierzucht. GMBH. Cuxhaven, Germany. Mateos, G. G. and. J. L. Sell. 1980a. Influence of graded levels of fat on utilization of pure carbohydrate by the laying hen. J. Nutr. 110:1894-1903. Mateos, G. G. and. J. L. Sell. 1980b. Influence of carbohydrate and supplemental fat source on the metabolizable energy of the diet. Poult. Sci. 59:2129-2135. Mateos, G. G. and. J. L. Sell. 1981. Nature of the extrametabolic effect of supplemental fat used in semipurified diets for laying hens. Poult. Sci. 60:1925-1930. Mathlouthi, N., S. Mallet, L. Saulnier, B. Quemener, and M. Larbier. 2002. Effect of xylanase and β-glucanase addition on performance, nutrient digestibility and physico-chemical conditions in the small intestine contents and caecal microflora of broiler chickens fed a wheat and barley-based diet. Anim. Res. 51:395–406. NRC. 1994. Nutrient Requirements of Poultry. 9 threv. ed. Natl. Acad. Press, Washington, DC. Pirgozliev, V. R., C. L. Birch, S. P. Rose, P. S. Kettlewell, and M. R. Bedford. 2003. Chemical composition and the nutritive quality of different wheat cultivars for broiler chickens. Br. Poult. Sci. 44:464–475. Ribeiro, B.R.C., L.J.C. Lara, N.C. Baiao, C.A.A. Lopez, M.A. Fiuza, S.V. Conçado, and G.M.M. Silva. 2007. Effect of linoleic acid level in the diet on the weight, composition and eclodibility of broiler breeders eggs. Arq. Bras. Med. Vet. Zootec. 59: 789-796. Ruiz, N., J. E. Marion, R. D. Miles, and R. B. Barnett. 1987. Nutritive value of new cultivars of triticale and wheat for broiler chick diets. Poult. Sci. 66:90–97. 84 Chapter 2. Effects of the main cereal and type of fat of the diet SAS Institute. 1990. SAS STATs User's Guide: Version 6, 4th ed. SAS Inst. Inc., Cary, NC. Safaa, H. M., M. P. Serrano, D. G. Valencia, M. Frikha, E. Jiménez-Moreno, and G. G. Mateos 2008. Productive performance and egg quality of brown egg-laying hens in late phase of production as influenced by level and source of calcium in the diet. Poult. Sci. 87:2043-2051. Safaa, H. M., E. Jiménez-Moreno, D. G. Valencia, M. Frikha, M. P. Serrano and G. G. Mateos 2009. Effect of main cereal of the diet and particle size of the cereal on productive performance and egg quality of brown egg-laying hens in early phase of production. Poult. Sci. 88:608-614. Sanz, M., A. Flores, and C.J. Lopez-Bote. 1999. Effect of fatty acid saturation in broiler diets on abdominal fat and breast muscle fatty acid composition and susceptibility to lipid oxidation. Poult. Sci. 78:378-382. Sanz, M., A. Flores, P. Perez de Ayala, and C.J. Lopez-Bote. 2000. Higher lipid accumulation in broilers fed saturated fats than in those fed unsaturated fats. Br. Poult. Sci. 40:95.101. Scheideler, S.E., P. Weber, and D. Monsalve. 2010. Supplemental Vitamin E and selenium effects on egg production and egg deposition of α-tocopherol and selenium. J. Appl. Poult. Res. 19:354-360. Scragg, R. H., N. B. Logan, and N. Geddes. 1987. Response of egg weight to the inclusion of various fats in layer diets. Br. Poult. Sci. 28:15-21. Shang, X. G., F. L. Wang, D. F. Li, J.D. Yin, and J. Y. Li. 2004. Effects of dietary conjugated linoleic acid on the productivity of laying hens and egg quality during refrigerated storage. Poult. Sci. 83:1688-1695. Shutze, J.V., L. S. Jensen, and J. McGinnis. 1959. Further studies on unidentified nutritional factors affecting egg size. Poult. Sci. 38:1247-1252. 85 Chapter 2. Effects of the main cereal and type of fat of the diet Van Soest, P.J., J.B. Robertson, and A. Lewis. 1991. Methods for dietary fiber, neutral detergent fiber, and nonsarch polysaccharides in relation to a nimal nutrition. J. Dairy Sci. 74:3583-3597. Vila, B., and E. Esteve-Garcia. 1996. Studies on acid oils and fatty acids for chickens. 1. Influence of age, rate of inclusion and degree of saturation on fat digestibility and metabolisable energy of acid oils. Br. Poult. Sci. 37:105-117. Woodward, S. A., R. C. Fluck, M. D. Ouart, and D.M. Janky. 1987. Influence of Florida 301 wheat in laying hen diets on egg yolk rupture strength. Poult. Sci. 66:14141416. 86 Chapter 3. Effect of crude protein and fat content of the diet CHAPTER 3: Effect ffect of crude protein and fat content of diet on productive performance and egg quality traits of brown egg-laying laying hens with different initial body weight (Experiment 2) PUBLISHED IN: POULTRY SCIENCE 91:1400-1405 91:1400 doi:10.3382/ps.2011-01917 87 Chapter 3. Effect of crude protein and fat content of the diet 1. Introduction Diets for laying hens are formulated to meet the requirements for those indispensable amino acid (AA) that may limit egg production, namely Lys, Met, Thr, and TSAA. According to NRC (1994), diets based on corn and soybean meal with 16.5% CP satisfy the AA requirements of brown egg-laying hens consuming 110 g feed per day. However, in many countries it is a common practice to formulate diets with CP levels in excess of 16.5%. In fact, several commercial guidelines for laying hens (H & N International, 2008; Lohmann, 2010; Isabrown, 2011) recommend CP levels varying from 17.4% to 18.2% (19.1 to 20.0 g CP/hen/d) for the first part of the production cycle. The reasons behind this practice are unknown but might be related to the interest to maximize egg size and reduce the possibility of a non-conventional indispensable AA (i.e., Arg, Ile, Val) limiting egg weight and hen production. In this respect, Bouvarel et al. (2010) reported that egg size was a function of the amount of CP ingested and that per each extra g of CP ingested, egg weight increased by 1.3 g. However, an excess of CP in the diet increases nitrogen load to the environment (Summers, 1993; Blair et al., 1999; Roberts et al., 2007a; Latshaw and Zhao, 2011), might impair feed efficiency (Vieira et al., 2004), and often results in increased feed cost. Supplemental fat may play an important role in improving productive performance and egg size of laying hens at the beginning of the egg production cycle (Grobas et al., 1999b; Pérez-Bonilla et al., 2011). The effects of supplemental fat on egg size depend on level and type of fat used as well as on the linoleic acid (LNL) content of the diet (Jensen et al., 1958; Shutze et al., 1962; Grobas et al., 1999c; Bohnsack et al., 2002). In addition, supplemental fat might improve the digestibility of other components of the diet (Mateos and Sell, 1980a, b) as well as egg mass production and feed efficiency (Grobas et al., 1999a; Bouvarel at al., 2010). 88 Chapter 3. Effect of crude protein and fat content of the diet Body weight at the onset of egg production is a major factor influencing hen productivity. Egg weight throughout the production cycle is largely determined by the initial BW of the hen (Harms et al., 1982; Leeson and Summers, 1987). Therefore, heavier birds at the onset of egg production will produce larger eggs during the whole laying cycle than small birds (Summers and Leeson, 1983; El Zubeir and Mohammed, 1993). Because of genetic improvement, pullets reach sexual maturity at younger ages than traditionally and consequently, BW is reduced at this stage. The authors have not found any research conducted in the last years comparing productive performance in brown egg-laying hens varying in initial BW. The objectives of this research were to determine the effects of increased dietary CP from 16.5% to 18.5% and fat content from 1.8 to 3.6% of otherwise isonutritive diets, on productive performance and egg quality of Lohmann Brown egglaying hens differing in initial BW from 22 to 50 wk of age. 2. Materials and methods 2.1. Husbandry, Feeding Program, and Experimental Diets All experimental procedures were approved by the animal Ethics Committee of the Universidad Politécnica de Madrid and were in compliance with the Spanish guidelines for the care and use of animals in research (Boletín Oficial del Estado, 2007). In total, 672 Lohmann Brown hens were obtained at 20 wk of age from a commercial flock (El Canto Agroalimentaria S.L, Toledo, Spain), weighed individually, and classified as light (1,592 ± 75 g) or heavy (1,860 ± 86 g). These BW compared with an expected value of 1,640 ± 57 g for similar type of birds at same age (Lohmann, 2010). Within each BW group, hens were randomly distributed into 16 replicates formed by 21 birds housed in groups of 7 in 3 adjacent cages (600 × 575 mm; General Ganadera S.A, Valencia, Spain) provided with an open trough feeder and 2 nipple drinkers. For 2 wk 89 Chapter 3. Effect of crude protein and fat content of the diet prior to the beginning of the experiment (20-22 wk of age), the hens were fed a common commercial diet based on corn and soybean meal. Room temperature was recorded daily throughout the experiment with the minimum average value (20 ± 3ºC) recorded in March (beginning of the experiment) and the maximum (27 ± 3ºC) recorded in July. The lighting program consisted of 16 h light per day throughout the experiment. All the diets were isocaloric (2,750 kcal AMEn/kg) and had similar digestible TSAA content. The main difference among the first 3 diets was the CP content (16.5, 17.5, and 18.5%, respectively). The last diet had also 18.5% CP content but included 3.6% fat rather than 1.8% fat. Adjustments in the ingredient compositionwere made to maintain constant the nutritive value of all the diets. Because of the experimental design, diets with the higher CP content had also more digestible Arg, Ile, Lys, Thr, and Val content than diets with the lower CP content, but in all cases the level of all these AA were higher than recommended by NRC (1994) and Fundacion Española Desarrollo Nutricion Animal (2008)for brown laying hens. The cereal portion of these diets was ground using a hammer mill provided with a 7.5 mm screen. 2.2. Analytical Evaluation of Ingredients and Feeds Representative samples of the diets were ground using a laboratory mill (Model Z-I, Retsch Stuttgart, Germany) provided with a 1-mm screen and analyzed for moisture by oven-drying (method 930.01),total ash using a muffle furnace (method942.05), nitrogen by combustion (method 990.03) using a LECO analyzer (Model FP-528, LECO, St. Joseph, MI) and Ca and P by spectrophotometry (methods 968.08 and 965.17)as described by AOAC International (2000). Ether extract was determined by Soxhlet analysis (method 4b) after 3N HCl acid hydrolysis as indicated inBoletín Oficial del Estado (1995) and GE was determined using an isoperibol calorimeter bomb (Model 1356, Parr Instrument Company, Moline, IL). The geometric mean diameter of the diets 90 Chapter 3. Effect of crude protein and fat content of the diet was determined in triplicate in 100 g samples using a Retsch shaker (Retsch, Stuttgart, Germany) provided with 8 sieves ranging in mesh from 5,000 to 40 µm according to the methodology outlined by ASAE (1995). The ingredient composition and the calculated and determined chemical analyses of the diets are presented in Table 1. 2.3. Productive performance and egg quality Eggs were collected daily and egg weight was measured in all eggs produced the last 2 days of each of the seven 28-d periods. Feed intake was controlled by replicate every 28d period, and mortality was recorded as produced. From these data, ADFI, egg production, egg weight, egg mass, and feed conversion ratio (FCR) per kilogram and per dozen of eggs were calculated by period and cumulatively. In addition, all the hens were weighed individually at the beginning of the experiment and at the end of each of the seven 28-d laying periods, and the BW gain (BWG) per replicate was calculated. The number of dirty, broken, and shell-less eggs was recorded daily by replicate. An egg was considered dirty when a spot of any kind or size was detected on the shell, as evaluated by two independent observers blind to treatment. Egg quality was measured in 10 eggs collected randomly from each replicate the last day of each 28-d period. Eggs were individually weighed and broken, and albumen and shell quality variables were determined using an egg multitester equipment (QCM-System, Technical Services and Supplies, Dunnington, York, UK). Albumen height was measured using an electronic height gauge (QCH-System, TSS). Yolk color was determined with the Roche Color Fan (QCC-System, TSS) as indicated by Vuilleumier et al. (1969). Shell density (mg/cm2) was calculated as the weight of the dry shell divided by the surface area using the QCM System equipment (Technical Services and Supplies, Dunnington, York, UK). In addition, shell thickness was measured in 5 eggs per replicate chosen at random at the end of each 28-d period using a digital micrometer (model IT-014UT, Mitotuyo, 91 Chapter 3. Effect of crude protein and fat content of the diet Kawasaki, Japan). The average of three measurements taken at the centre of the egg was used forfurther evaluation. 2.4. Statistical analysis The experimental design was completely randomized with 8 treatments arranged factorially and the main effects (diet and initial BW of the pullets) and the interaction were analyzed by ANOVA using the GLM procedure of SAS Institute (1990). No significant interactions between main effects were observed for any of the traits studied and therefore, the interaction was removed from the model.The homogeneity of the variance of the data for all traits was tested using the Levene´s Test (Hovtest option of GLM procedures). All data on performance and egg quality traits were homogeneous, except for mortality and therefore, mortality data were analysed after arcsin transformation. When the effects of diet and BW of the hens were significant, a Tukey test was used to make pairwise comparisons to separate treatment means. Results in tables are presented as means, and differences were considered significant at P < 0.05. 3. Results No significant interactions between dietary treatment and period were detected for any of the variables studied and therefore, only cumulative data are discussed. Mortality was considered normal (4.9%) and not related to treatment. Diet did not affect any of the productive performance traits studied. However, ADFI (120.6 vs. 113.9 g; P< 0.001), egg production (92.5 vs. 89.8%; P< 0.01), egg weight (64.9 vs. 62.4 g; P< 0.001), and egg mass (60.0 vs. 56.1 g; P< 0.001) were higher for the heavier than for the lighter hens (Table 2). The FCR per kilogram of eggs was not affected by initial BW of the hens but FCR per dozen of eggs was better for the lighter than for the heavier hens (1.52 vs. 1.57; P< 0.01). Also, cumulative BWG was higher for the lighter than for the heavier hens (289 92 Chapter 3. Effect of crude protein and fat content of the diet vs. 233 g; P< 0.01). Data by period on the main effects of initial BW of the hens onADFI, egg production, egg weight, FCR, and BWG of the hens are shown in Figure 1. Feed intake and egg weight were higher (P< 0.001) for the heavier than for the lighter hens in all periods. Body weight gain was higher (P < 0.001) for the lighter than for the heavier hens from 22 to 26 wk of age but no significant differences were found after this period. Incidence of dirty eggs, percentage of broken and shell-less eggs, albumen quality, shell density, and shell thickness were not affected by diet or by the initial BW of the hens. However, yolk pigmentation was greater (P< 0.01) in eggs from hens fed the 1.8% fat diets than in eggs from hens fed the 3.6% fat diet (Table 3). 4. Discussion Crude protein content of the diet did not affect any of the productive performance traits studied, results that are consistent with NRC (1994) recommendations, indicating that the requirements for all indispensable AA are satisfied when the ADFI of the hens was 110 g of a diet with 16.5% CP (corresponding to a CP intake of 18.1 g CP/d). In fact, in the current study, the average CP intake of the hens fed the 16.5% CP diets was in excess of NRC (1994) recommendations (18.6 g/d for the light pullets and 19.8 g/d for the heavy pullets, corresponding to a feed intake of 112.6 and 119.7 g/hen/d, respectively). Kling et al. (1985) compared in two different experiments with brown egg-laying hens from housing to 66 wk of age, 4 diets organized factorially with 2 levels of Met (0.3% vs. 0.4%) and 2 levels of CP (17% and 19%) and observed that an increase in the CP content of the diet did not affect hen performance in any of the two experiments. Similarly, Junqueira et al. (2006) reported that increasing the CP of the diet of molted brown egglaying hens from 16 to 20%, while maintaining constant the AMEn concentration and the Met content, did not affect performance.Also, Summers and Leeson (1983) reported similar performance and egg size of Single Comb White Leghorn (SCWL) hens from 20 93 Chapter 3. Effect of crude protein and fat content of the diet to 32 wk of age fed dietsbalanced for AMEn and Met that contained 17% or 22% CP.In contrast, Roberts et al. (2007b) reported that a decrease in CP content (19.8% to 19.1% from 23 to 31 wk, 18.1% to 17.1% from 32 to 44 wk, and 16.9% to 15.8% from 45 to 58 wk of age) of isoenergetic diets with similar indispensable AA profile, reduced egg production, egg mass, and feed efficiency of SCWL.However, egg weight, ADFI, and BWG were not affected by a decrease in CP content. Keshavarz (1995), using isocaloric diets organized as 3 × 3 factorial, reported that an increase in CP content (17, 19, and 21%) of diets with increased levels of Met (0.34, 0.38, and 0.42%) did not affect egg production, egg weight, ADFI, or BWG of SCWL hens from 18 to 38 wk of age. However, it was observed that from 26 to 34 wk of age egg weight increased when hens were fed the 21% CP diets. The results of the current experiment support that indispensable AA intake rather than nitrogen intake “per se” modulates egg size and hen performance. Moreover, the data indicate than an excess of CP intake did not impaired feed efficiency. In contrast, Vieira et al. (2004) reported that feed efficiency was impaired in broilers from 14 to 35 d of age when the level of CP of diets balanced for indispensable AA, was increased from 20.5% to 26%. The effects of supplemental fat on ADFI, egg production, and egg weight are a subject of debate. Usually, an increase in energy concentration of the diet is accompanied by an increase in supplemental fat and in LNL content (Grobas et al., 2001; Frikha et al., 2009). Consequently, the 3 effects (AMEn concentration, level of supplemental fat, and LNL content) are confounded and can not be separated under most practical feeding conditions. In the current experiment, an increase in supplemental fat from 1.8 to 3.6% in isocaloric diets in which LNL was in excess of hen requirements (1.9 to 2.5%) did not affect egg weight or productive performance of the hens, in agreement with the report of Grobas et al. (1999c). Keshavarz (1995) compared in SCWL from 18 to 38 wk of age isocaloric diets based on corn or barley that included 3 sources of fat (tallow, blended fat, 94 Chapter 3. Effect of crude protein and fat content of the diet or corn oil) at 2 levels of supplementation (2% and 4%) with an unsupplemental control diet.The author reported no difference in egg weight, egg production, or BWG among treatments, consistent with the results of the current trial. Keshavarz and Nakajima (1995) reported that the inclusion of 4% of a blended animal and vegetable fat in isocaloric diets for SCWL from 18 to 34 wk of age increased egg weight and BWG as compared with a control non-fat supplemented diet. However, in this research egg production was reduced with fat supplementation. Bohnsack et al. (2002) compared diets supplemented with 2, 4, and 6% of either corn oil or poultry fat to SCWL hens from 26 to 38 wk of age and reported that hens fed 4 or 6% fat produced heavier eggs than those fed 0 or 2% fat, but that egg production was not affected. However, in this research the concentration in AMEn of the diets increased with the level of supplemental fat and therefore, the effects of AMEn concentration of the diet and level of supplemental fat were confounded. Grobas et al. (1999b) reported that the supplementation of isonutritive diets for Isa Brown hens with 4% of different mixtures of acid oil soapstocks and animal fat, while maintaining constant the LNL level at 1.15%, increased egg weight in young hens (22 to 26 wk of age) but not in older hens (74 to 78 wk of age). Safaa et al. (2008) reported that an increase in added fat from 1.1 to 3.0% while maintaining constant the AMEn concentration (2,700 kcal/kg) of the diets improved egg production, egg weight, and FCR per kilogram of eggs in Hy-Line Brown egg-laying hens from 59 to 70 wk of age. However, no effects were observed in a second experiment using similar type of diets in Lohmann Brown hens of similar age. Pullets that were heavier at the onset of egg production ate more feed and produced heavier eggs than pullets that were lighter. However, FCR per kilogram of eggs was not affected and in fact, FCR per dozen of eggs was better for the lighter pullets. The authors have not found any recent report on the effects of initial BW of brown egg-laying pullets on egg production. In the current experiment, ADFI increased 2.7 g and egg weight 0.93 95 Chapter 3. Effect of crude protein and fat content of the diet g per each 100 g increase in initial BW of the pullets. Similar results have been reported by Harms et al. (1982) in two separate experiments with SCWL hens from 31 to 47 wk of age. In a first experiment, these authors reported an increase of 0.88 g in egg weight per each 100 g of extra initial BW in hens varying in BW from 1,411 g to 1,546 g whereas in the second experiment, conducted with hens weighing 1,546 g and 1,684 g, the increase was of 1.6 g. In both experiments egg production was similar and feed efficiency was poorer for the heavier hens. Summers and Leeson (1983) compared egg performance of SCWL pullets sorted by BW at 18 wk of age into 4 groups (1,107, 1,205, 1,281, and 1,383 g). They observed that from 19 to 25 wk of age, the heavier pullets laid more eggs than the lighter pullets and that per each 100 g of extra initial BW, egg weight increased by 0.9 g. Moreover, egg production decreased with the reduction in BW but FCR per kilogram of eggs was not affected. Similarly, Leeson and Summers (1987) distributed SCWL pullets into 3 BW groups; 997, 1,100, and 1,226 g at 15 wk of age in experiment 1 and 1,308, 1,411, and 1,564 g BW at 19 wk of age in experiment 2. The authors reported that ADFI increased 3.2 g (experiment 1) and 3.6 g (experiment 2) per each 100 g of extra BW from housing to 67 wk of age, values that were slightly higher than the value of 2.7 g observed in the current experiment. Keshavarz (1995) compared the performance of light (1,151 g) and heavy (1,333 g) SCWL pullets from 18 to 62 wk of age and reported similar egg production but a 2 g increase in feed intake and a 1.4 g increase in egg weight per each 100 g of extra initial BW. Furthermore, the author reported that cumulative BWG from 17 to 62 wk was 24.2% higher for the lighter than for the heavier hens, consistent with the value of 20.8% reported in the current experiment. Egg quality, including percentage of dirty eggs, albumen height, and egg shell traits were not affected by dietary treatment or by the initial BW of the hens. The data agree with results of Junqueira et al. (2006) who observed that an increase in CP content of the diet did not affect Haugh units or egg shell thickness. Similar results have been reported for 96 Chapter 3. Effect of crude protein and fat content of the diet egg shell quality by Wolford and Tanaka (1970). Also, Fariborz et al. (2007) comparing isoenergetic diets containing 16.3 or 17.8% CP reported that albumen height, shell thickness, and shell strength were not affected by the CP content of the diet. In fact, Williams (1992) indicated that hen strain and age were the most important factors affecting albumen quality and that nutrition did not have a great impact on this variable. However, Hammershoj and Kjaer (1999) reported that Haugh units declined as the level of CP of the diet increased from 13.7 to 17.9%. The authors did not give any explanation for their finding. Supplementation of the high CP diet with 3.6% fat did not affect any of the shell and albumen quality traits studied. In broilers, Atteh et al. (1983) indicated that the inclusion of saturated fats in the diet increased the formation of soaps between fatty acids and the Ca salts, resulting in lower Ca retention. In the current experiment, no effects of extra fat supplementation of the diet on shell quality were observed, data that agree with results of Safaa et al. (2008) who reported similar egg shell quality in late phase of production in hens fed diets with 1.1 or 3% supplemental fat (soy oil or palm oil). Probably, the amount of Ca soaps present at the small intestine level in the hens of the current experiment was limited, because the fat used was unsaturated and the soaps formed may dissociate at the pH values encountered in this section of the gastrointestinal tract. Yolk pigmentation was lower in eggs from hens fed the diet containing 3.6% fat than in hens fed the diets containing 1.8% fat, a difference that was expected because corn was included only in the 1.8% fat supplemented diets. It is concluded that increasing the levels of CP of the diet from 16.5 to 18.5% and of fat from 1.8 to 3.6% does not affect performance or egg quality in hens from 22 to 50 wk of age, irrespective of the initial BW of the pullets. Egg production, egg weight, and ADFI are higher in heavier than in lighter hens but egg quality is not affected by the initial BW of the hens. Moreover, FCR expressed as kilogram of feed per kilogram of 97 Chapter 3. Effect of crude protein and fat content of the diet eggs is not affected by the initial BW of the pullets and in fact, FCR per dozen of eggs improves with the lighter BW. Cumulative BWG is higher for the light BW group than for the heavy BW group. Thus, the practice of increasing the CP content of the diet over NRC (1994) recommendations to maximize egg weight and hen productivity is not justified in any of the two groups of hens. In general, overall hen productivity is improved when heavier hens are used, but the economical advantage of this practice might depend on price difference between egg weight grades as well as on relative cost of feed ingredients. 98 Table 1. Ingredient and chemical composition of the experimental diets (% as fed basis unless otherwise stated) Crudeprotein, % 18.5 18.5 17.5 16.5 Fat, % 3.6 1.8 1.8 1.8 Ingredient Corn 35.50 35.00 34.45 Wheat 30.00 20.00 20.00 20.00 Barley 30.99 3.60 6.70 9.80 Soybean meal, 47%CP 24.30 28.00 24.70 21.40 Sunflower meal, 32%CP 0.24 0.26 0.93 1.66 Soybean oil 3.60 1.80 1.80 1.80 DL -Methionine,99% 0.15 0.12 0.15 0.17 Monocalcium phosphate 1.00 1.00 1.00 1.00 Calcium carbonate 9.00 9.00 9.00 9.00 Sodium chloride 0.32 0.32 0.32 0.32 Vitamin and mineral premix1 0.40 0.40 0.40 0.40 Calculated analysis2 AMEn(Kcal/Kg) 2,750 2,750 2,750 2,750 CP 18.5 18.5 17.5 16.5 Digestible Arg 1.07 1.12 1.05 0.98 Digestible Ile 0.68 0.72 0.67 0.62 Digestible Lys 0.81 0.87 0.80 0.73 Digestible Met 0.39 0.39 0.40 0.41 Digestible Met+ Cys 0.67 0.67 0.67 0.67 Digestible Thr 0.57 0.62 0.58 0.54 Digestible Trp 0.20 0.20 0.19 0.18 Digestible Val 0.78 0.82 0.77 0.72 CF 3.3 2.7 2.9 3.0 EE 5.2 4.0 4.0 4.0 Linoleic acid 2.5 1.9 1.9 1.9 Total ash 11.2 11.2 11.1 11.0 Ca 3.80 3.80 3.80 3.80 Digestible P 0.36 0.34 0.33 0.33 Determined analysis3 GE (Kcal/kg) 3,721 3,594 3,591 3,543 CP 18.7 18.9 17.6 16.7 EE 4.9 3.4 3.3 3.3 DM 91.9 91.0 91.0 90.9 Total ash 12.2 13.1 12.9 12.1 Particle size GMD, µm4 938 874 871 830 GSD5 ± 2.24 ± 2.29 ± 2.34 ±2.33 1 Supplied per kilogram of diet: vitamin A (trans-retinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 2,000 IU; vitamin E (DL-α-tocopheryl acetate), 10 mg; vitamin B1, 1 mg; vitamin B2, 4 mg; vitamin B6, 1 mg; vitamin 12 (cyanocobalamin), 15 mg; vitamin K3, 2.5 mg; choline (choline chloride), 150 mg; nicotinic acid, 25 mg; pantothenic acid (D-calcium pantothenate), 7,5 mg; folic acid, 0.1 mg; manganese (MnO), 70 mg; zinc (ZnO), 50 mg; iron (FeSO4 H2O), 30 mg; copper (CuSO4 5H2O), 5 mg; iodine [Ca(IO3)2], 0,5 mg; selenium (Na2SeO3), 0,3 mg; canthaxantin; 2,4 g; ester of β-apo-8-carotenoic, 1,7 g (Lucanmix yellow/red, BASF, Tarragona, Spain), [(Endo-1.3(4) -β-glucanase (EC 3.2.1.6), 150 IU/g; Endo-1.4-β-xylanase (EC 3.2.1.8) 105 IU/g; (Endofeed, GNC Bioferm, Saskatchewan, SK, Canada), Natuphos, (BASF Española, S.A, Tarragona, Spain), 6-phytase (EC 3.1.3.26), 300 FTU)]. 2 According to Fundación Española Desarrollo Nutrición Animal (2010). 3 Analyzed in triplicate. 4 Geometric mean diameter. 5 Geometric standard desviation ═ log normal SD. 99 Table 2. Influence of CP and fat content of the diet and initial BW ofthe hens on performance from 22 to 50 wk of age CP Fat % % 18.5 3.6 18.5 1.8 17.5 1.8 16.5 1.8 Diet 18.5 18.5 17.5 16.5 3.6 1.8 1.8 1.8 SEM2 Initial1 BW Egg production Egg weight Egg mass Feed intake FCR FCR BWG Mortality % g g/d g/d kg/kg kg/dozen g % High Low High 93.6 88.6 91.6 65.2 62.9 65.2 61.0 55.8 59.7 122.4 113.2 121.2 2.01 2.03 2.03 1.58 1.53 1.59 241 290 233 2.4 3.6 4.8 Low 91.4 61.9 56.6 115.0 2.03 1.51 275 3.6 High Low High Low 92.4 90.3 92.3 89.1 64.9 62.5 64.3 62.4 60.0 56.4 59.3 55.6 119.2 114.8 119.7 112.6 1.99 2.04 2.02 2.03 1.55 1.53 1.56 1.52 224 332 234 260 8.3 5.9 5.9 4.8 91.1 91.5 91.3 90.7 64.1 63.6 63.7 63.3 58.4 58.2 58.2 57.4 117.8 118.1 117.0 116.1 2.02 2.03 2.01 2.02 1.56 1.55 1.54 1.54 266 254 278 247 3.0 4.2 7.1 5.3 0.02 0.02 2.01 2.03 1.57a 1.52b 0.01 0.01 15.0 1.21 NS NS NS ** NS ** NS NS 0.99 Initial BW High Low SEM3 92.5a 89.8b 0.70 0.34 64.9a 62.4b 0.24 0.65 60.0a 56.1b 0.46 0.80 120.6a 113.9b 0.56 21.2 233b 289a 1.71 5.3 4.5 4 Effect Probability Diet NS NS NS Initial BW ** *** *** 1 The initial BW±SD was 1.860 ± 86g and 1.592 ± 75g, for the heavy and light hens, respectively. 2 Standard error of the mean (8 replicates of 21 hens each per treatment). 3 Standard error of the mean (16 replicates of 21 hens each per treatment). 4 The interaction between type of feed and initial BW of the hens was not significant (P> 0.05). NS: not significant; ** P < 0.01; *** P < 0.001 a, b Means within a column with different superscripts are significantly different(P<0.05). NS *** 100 Table 3. Influence of CP and fat content of the diet and BW of the hens on egg quality from 22 to 50 wk of age Dirty Broken Shell-less Shell CP Fat Initial1 eggs eggs eggs density BW % % % % % mg/cm2 18.5 3.6 High 2.8 1.2 0.18 76.7 Low 2.6 1.1 0.10 81.2 18.5 1.8 High 3.3 0.9 0.15 78.5 Low 2.8 1.0 0.21 79.1 17.5 1.8 High 3.3 1.0 0.09 80.9 Low 2.6 0.6 0.19 74.8 16.5 1.8 High 2.6 0.8 0.11 79.8 Low 3.2 0.9 0.49 78.3 Diet 18.5 3.6 2.7 1.1 0.14 78.9 18.5 1.8 3.0 1.0 0.18 78.8 17.5 1.8 2.9 0.8 0.14 77.8 16.5 1.8 2.9 0.8 0.30 79.0 3 SEM 0.32 0.20 0.07 2.92 Initial BW High Low SEM4 3.0 2.8 0.23 1.0 0.9 0.14 0.13 0.25 0.05 Probability Effect5 Diet NS NS NS Initial BW NS NS NS 1 The initial BW±SD was 1.860 ± 86g and 1.592 ± 75g, for the heavy and light hens, respectively. 2 Roche color fan. 3 Standard error of the mean (8 replicates of 21 hens each per treatment). 4 Standard error of the mean (16 replicates of 21 hens each per treatment). 5 The interaction between type of feed and initial BW of the hens was not significant (P> 0.05). NS: not significant; ** P < 0.01 a, b Means within a column with different superscripts are significantly different(P<0.05). Shell thickness mm 0.365 0.374 0.374 0.371 0.371 0.371 0.374 0.377 Haugh Units RCF2 84.8 83.9 84.2 87.4 82.7 84.0 84.5 83.5 7.5 7.3 8.5 8.3 8.5 8.8 8.6 8.8 0.369 0.372 0.371 0.375 0.004 84.4 85.8 83.3 84.0 1.63 7.4b 8.4a 8.6a 8.6a 0.30 78.9 78.3 2.07 0.371 0.373 0.003 84.1 84.7 1.16 8.3 8.3 0.23 NS NS NS NS NS NS ** NS 101 *** *** *** 120 *** *** *** 115 110 105 100 22-26 A 26-30 30-34 34-38 38-42 42-46 46-50 Egg production (%) *** 125 96 NS * * † 94 * † 92 90 NS 88 86 84 B Age (wk) 22-26 26-30 30-34 34-38 38-42 42-46 46-50 67 66 65 64 63 62 61 60 59 58 C 200 *** *** *** *** *** *** *** 22-26 26-30 30-34 34-38 38-42 42-46 46-50 Age (wk) *** Feed conversion (kg/kg) Egg weight (g) Age (wk) 2,15 NS 2,10 NS 2,05 NS NS NS NS 2,00 NS 1,95 1,90 1,85 1,80 D 22-26 26-30 30-34 34-38 38-42 42-46 46-50 Age (wk) Heavy hens (Initial BW = 1,860 ± 86g) 150 BW gain (g) Feed intake (g/d) Figure 1. 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Roberts, S.A., H. Xin, B. J. Kerr, J. R. Russell, and K. Bregendahl. 2007b. Effects of dietary fiber and reduced crude protein on nitrogen balance and egg production in laying hens. Poult. Sci. 86:1716-1725. Safaa, H. M., M. P. Serrano, D. G. Valencia, X. Arbe, E. Jiménez-Moreno, R. Lazaro, and G. G. Mateos. 2008. Effects of the levels of methionine, linoleic acid, and added fatin the diet on productive performance and egg qualityof brown laying hens in the late phase of production. Poult. Sci. 87:1595-1602. 106 Chapter 3. Effects of crude protein and fat content of the diet SAS Institute. 1990. SAS STATs User's Guide: Version 6, 4th ed. SAS Inst. Inc., Cary, NC. Shutze, J. V., L. S. Jensen, and J. McGinnis. 1962. Accelerated increase in egg weight of young pullets fed diets supplemented with corn oil. Poult. Sci. 41:1846-1851. Summers, J. D. and S. Leeson. 1983. Factors influencing early egg size. Poult. Sci. 62:1155-1159. Summers, J. D. 1993. Reducing nitrogen excretion of the laying hen by feeding lower crude protein diets. Poult. Sci. 72:1473-1478. Vieira, S. L., A. Lemme, G. B. Goldenberg, and I. Brugalli. 2004. Responses of growing broilers to diet with increased sulfur amino acids to lysine ratios at two dietary protein levels. Poult. Sci. 83:1307-1313. Vuilleumier, J. P., 1969. The Roche yolk colour fan – An instrument for measuring yolk colour. Poult. Sci. 48:767-779. Williams, K. C. 1992. Some factors affecting albumen quality with particular reference to Haugh unit score. World´s Poult. Sci. J. 48:5-16. Wolford, J. H. and K. Tanaka. 1970. Factors influencing egg shell quality- A review. World´s Poult. Sci. J. 26:763-780. 107 Chapter 4. Effect of energy concentration of the diet CHAPTER 4: Effects of energy concentration of the diet on productive performance and egg quality of brown egg-laying laying hens differing in initial body weight (Experiment 3) ACCEPTED FOR PUBLISHED IN: POULTRY SCIENCE TBC: 1-11 doi:10.3382/ps.201 doi:10.3382/ps.2012-02526 108 Chapter 4. Effect of energy concentration of the diet 1. Introduction A major problem affecting egg production and egg weight of modern strains of laying hens is the reduced feed intake (FI) often observed at the onset of egg production (Summers and Leeson, 1983). A low FI results in hens that do not reach the standard BW at this stage of growth which in turn reduces egg size during the whole egg laying cycle (Harms et al., 1982; Leeson and Summers, 1987). Changes in energy concentration of the diet have resulted in contrasting results in respect to energy intake, feed efficiency, and productive performance of the hens (Harms et al., 2000). Grobas et al. (1999c) reported that a 4.8% increase in the AMEn content (from 2,680 to 2,810 kcal/kg) of diets with a constant AME: Lys ratio decreased feed intake by 5.0% without affecting egg production or egg mass. In contrast, Peguri et al. (1991) reported an increase in egg production of 0.30% and a decrease in FI of 2.75 g per each 100 kcal increased in diets ranged from 2,700 to 2910 kcal AMEn/kg. Also, Valkonen et al. (2008) reported an increase in egg production as the AMEn content of the diet increased from 2,340 to 2,630 kcal/kg. These data suggest that an increase in energy content might be more beneficial when low density energy diets are used. Hens adjust FI to satisfy their energy requirements (Hill et al., 1956). Consequently, an increase in energy concentration of the diet should reduce FI proportionally. An increase in energy is usually obtained by adding fat to the diet and supplemental fat might improve the utilization of other components of the diet (Mateos and Sell, 1980a,b). Consequently, and increase in the AMEn content of the diet might improve nutrient utilization and egg size (Grobas et al., 1999b). The influence of energy concentration of the diet on egg quality traits has not been studied in detail. Junqueira et al. (2006) did not detect any difference in HU or eggshell quality in brown egg-laying hens fed diets varying in AMEn content from 2,850 to 3,050 kcal/kg. 109 Chapter 4. Effect of energy concentration of the diet On the other hand, Gunawardana et al. (2008) did not detect any difference in the proportion of yolk and albumen in eggs in SCWL hens fed diets varying in AMEn content from 2,750 to 3,055 kcal/kg. In contrast, Wu et al. (2005) reported that yolk weight increased and haugh units (HU) decreased as the AMEn of the diet increased from 2,720 to 2,955 kcal/kg. The authors have not found any information on the effects of energy concentration of the diet on egg quality of hens differing in initial BW. The hypothesis of the current research was that an increase in AMEn concentration of the diet could improve energy intake and productive performance of the hens and that the beneficial effects could be more pronounced for the light than for the heavy hens. The objectives of the present research were to study the effects of energy concentration of the diet on productive performance and egg quality of brown egg-laying hens differing in initial BW from 24 to 59 wk of age. 2. Materials and methods 2.1.Husbandry, Diets, and Experimental Design All experimental procedures were approved by the animal Ethics Committee of the Universidad Politécnica de Madrid and were in compliance with the Spanish guidelines for the care and use of animals in research (Boletín Oficial del Estado, 2007). Hy-Line Brown laying hens (n=520) were obtained from a commercial flock (Camar Agroalimentaria S.L, Toledo, Spain) and housed in a total environmentally controlled barn. The hens were housed in groups of 13 in enriched cages (635 x 1,200 mm; Facco S.A., Padova, Italy) equipped with an open trough feeder and 2 nipple drinkers. They were weighed individually at 21 wk of age and sorted into 2 groups according to BW; heavy (1,733 ± 48g) and light (1,606 ± 39g). These values differed from the target weight 110 Chapter 4. Effect of energy concentration of the diet of 1,685± 35g recommended by the supplier (Hy-Line International, 2011). Room temperature was recorded daily throughout the experiment with a minimum average value of 19 ± 3ºC recorded in January (start of the experiment) and a maximum of 28 ± 3ºC recorded in July. The light program was constant and consisted of 16 h light per day. From 21 to 24 wks of age all hens were fed a common corn-soybean meal diet (2,750 kcal AME/kg, 17.5% CP, and 0.39% Met). From 24 (start of the experiment) to 59 wk of age hens were fed one of 4 diets that varied in AMEn from 2,650 to 2,950 kcal/kg but had similar nutrient content per unit of energy. For the manufacturing of the feeds the two extreme diets were formulated (Fundacion Española Desarrollo Nutricion Animal, 2010) and the intermediate feeds were obtained by judicious mixing of these two summit diets in adequate proportions. All the diets met or exceeded the nutrient requirements for brown egg-laying hens (Fundacion Española Desarrollo Nutricion Animal, 2008). An enzyme complex that included β-glucanase and xylanase activity (Endofeed, GNC Bioferm Inc., Saskatoon, SK, Canada), was included at the dose recommended by the manufacturer in all diets to insure maximal nutrient utilization. Also, a commercial pigment mixture based on canthaxantin and the ester of β-apo-8-carotenoic (Miavit Nutrición Animal S.L., Tarragona, Spain) was included in fixed amounts in these feeds. The ingredient composition and the calculated and determined nutrient content of the experimental diets are presented in Table 1. The experimental design was completely randomized with 8 treatments arranged factorially with 4 energy levels (2,650, 2,750, 2,850, and 2950 kcal AMEn/kg) and 2 initial BW of the hens (1,733 vs. 1,606 g). Each treatment was replicated 5 times and the experimental unit was a cage with 13 hens. 2.2. Laboratory Analysis 111 Chapter 4. Effect of energy concentration of the diet Representative samples of the diets were ground in a laboratory mill (Model Z-I, Retsch Stuttgart, Germany) provided with a 1-mm screen and analyzed for moisture by ovendrying (method 930.01), total ash using a muffle furnace (method 942.05), and nitrogen by combustion (method 990.03) using a LECO analyzer (Model FP-528, LECO, St. Joseph, MI), as described by AOAC International (2000). Ether extract was determined by Soxhlet analysis after 3 N HCl acid hydrolysis (Boletín Oficial del Estado, 1995) and gross energy using an isoperibol bomb calorimeter (Model 356, Parr Instrument Company, Moline, IL). The geometric mean diameter of the diets were determined in triplicate in 100 g samples using a Retsch shaker (Retsch, Stuttgart, Germany) provided with 8 sieves ranging in mesh from 5,000 to 40 µm (Table 1) according to the methodology outlined by ASAE (1995). 2.3. Productive Performance and Egg Quality All eggs produced were collected daily and egg weight was measured in all eggs laid during the last 2 days of each of the nine 28-d periods. Feed intake was measured by replicate every 28-d and mortality was recorded as produced. All the hens were weighed individually at the beginning and at the end of each experimental period. From these data, hen-day egg production, egg weight, egg mass, ADFI, daily energy intake, feed conversion ratio (FCR) per kilogram and per dozen of eggs, energy efficiency expressed as kilocalories of AMEn per gram of egg, and BW gain were calculated by period and cumulatively. The number of dirty, broken, and shell-less eggs were recorded daily by replicate. An egg was considered as dirty when a spot of any kind or size was detected on the shell as evaluated by two independent observers blind to treatment. Haugh units and yolk pigmentation were measured per replicate in 10 eggs chosen at random from eggs produced the last day at 39, 48, 55, and 59 wk of age, using a Multitester equipment 112 Chapter 4. Effect of energy concentration of the diet (QCM System, Technical Services and Supplies, Dunnington, York, UK) as indicated by Pérez-Bonilla et al. (2011). Proportion of shell, albumen, and yolk of the eggs, and the yolk to albumen ratio were determined per replicate in the same 10 eggs collected for egg quality measurements. The yolk and the shell were separated from the albumen using a paper tissue to remove any adhered material (Safaa et al., 2008). Albumen weight was calculated by difference between egg weight and the weights of the yolk and the shell. 2.4. Statistical Analysis The experiment was conducted as a completely randomized design with 8 treatments arranged factorially and main effects (energy level and initial BW) and its interactions were analyzed by ANOVA using the GLM procedure of SAS Institute (1990). Normal distribution of residuals and variance homogeneity of the data was tested by UNIVARIATE procedure and the Levene´s Test, respectively. Mortality data were analyzed by GENMOD procedure of SAS Institute (1990), using a binomial distribution. The link function was logit transformation (ln(µ/1-µ)). When the effects of energy and initial BW were significant, the Tukey test was used to make pairwise comparisons to separate treatment means. In addition, polinomial contrasts were performed using the REG procedure of SAS Institute (1990) to study the linear (L) and quadratic (Q) effects of dietary energy on the different traits studied. Results in tables are presented as means and differences were considered significant at P < 0.05. 3. Results 3.1. Productive Performance No interactions between energy content of the diet and initial BW of the hens were detected for any of the traits studied and therefore, only main effects are presented. For the entire experimental period, egg production (88.8, 91.2, 92.7, and 90.5 %; L, P< 0.01; 113 Chapter 4. Effect of energy concentration of the diet Q, P< 0.01), egg mass (56.1, 58.1, 58.8, and 58.1 g/d; L, P< 0.01; Q, P< 0.01), AMEn intake (304, 313, 316, and 324 kcal/hen per day; L, P< 0.001), energy efficiency (5.42, 5.39, 5.38, and 5.58 kcal AMEn/g egg; L, P< 0.001; Q, P< 0.001), and BW gain (255, 300, 325, and 359 g; L, P< 0.001) increased as the AMEn content of the diet increased from 2,650 to 2,950 kcal/kg (Table 2). However, ADFI (114.8, 114.0, 111.0, and 110.0 g; L, P< 0.001), FCR per kilogram (2.05, 1.96, 1.89, and 1.89 kg/kg; L, P< 0.001; Q, P< 0.01) and per dozen of eggs (1.54, 1.48, 1.42, and 1.44 kg/dozen; L, P< 0.01; Q, P< 0.01) decreased as the energy content of the diet increased (Table 2). Egg weight and mortality rate were not affected by diet. The effects of energy concentration of the diet on ADFI, egg production, egg weight, and BW gain by each of the nine 28-d period are shown in Figure 1. Initial BW of the hens affected most productive variables studied, including egg weight, egg mass, ADFI, and FCR per dozen of eggs. For the entire experimental period, heavier hens had higher ADFI (113.9 vs. 111.0 g; P< 0.001) and AME intake (321 vs. 311 kcal/hen per day; P < 0.001) and produced more egg mass (58.5 vs. 57.0 g; P <0.01) and bigger eggs (64.2 vs. 63.0 g; P< 0.01) than lighter hens. However, egg production, FCR per kilogram of eggs, energy efficiency, BW gain, and mortality rate were not affected by the initial BW of the hens. The effects of initial BW of the hen on ADFI, egg production, egg weight, and BW gain by each of the nine 28-d period are shown in Figure 2. 3.2. Egg Quality Diet did not affect percentage of dirty, broken, and shell-less egg, or the proportion of yolk and albumen in the egg (Table 3). However, HU (L, P< 0.001) and shell weight decreased (L, P< 0.001) and yolk pigmentation increased (L, P< 0.001) as the energy concentration of the diet increased (Table 3). Initial BW of the hens did not affect 114 Chapter 4. Effect of energy concentration of the diet percentage of dirty eggs, broken eggs, or shell-less. The proportion of yolk was higher (P< 0.001) and that of albumen lower (P< 0.01) for heavy than for the light hens. Consequently, the yolk to albumen ratio was higher (P< 0.001) for the heavier than for the lighter hens (Table 3). The effects of energy content of the diet and initial BW of the hens on HU, proportion of shell in the egg, and yolk to albumen ratio in the different periods studied are shown in Figure 3 and 4, respectively. 4. Discussion 4.1. Productive Performance 4.1.1. AMEn Concentration of the Diet Hens eat to satisfy energy requirements and therefore an increase in the energy content of the diet should decreased ADFI proportionally (Hill et al., 1956). However, in the current research, a 11% in the energy content of the diet (from 2,650 to 2,950 kcal AMEn/kg) decreased FI by only 4% resulting in an increase in energy intake of 7%. These results agree with data of Bouvarel et al. (2010) who reviewed a series of experiments conducted in laying hens during the last 20 years and reported that as an average, a 10% increase in AMEn content of the diet reduced feed intake by only 5.5%. Moreover, Keshavarz (1998) reported in SCWL from 18 to 66 wk of age that a 8% increase in the AMEn concentration of the diet (from 2,815 to 3,035 kcal/kg) increased energy intake by 9%. The data indicate that laying hens tend to overconsume energy when the AMEn of the diet is increased. Probably, the inclusion of extra-amounts of fat to those diets might improve palatability resulting in higher than expected feed consumption. Egg production increased as the AMEn concentration of the diet increased from 2,650 to 2,850 kcal/kg, but a further increase to 2,950 kcal/kg did not result in further improvements. Similarly, Mathlouthi et al. (2002) reported in SCWL hens that egg 115 Chapter 4. Effect of energy concentration of the diet production increased as the AMEn of the diet increased from 2,650 to 2,750 kcal/kg. In contrast, Grobas et al., (1999c) in brown hens fed diets varying from 2,680 to 2,810 kcal AMEn/kg, Harms et al. (2000) in brown-and SCWL hens fed diets varying in AMEn from 2,500 to 3,100 kcal/kg , and Jalal et al. (2006, 2007) in SCWL hens fed diets varying from 2,800 to 2,900 kcal AMEn/kg, and, did not detect any significant difference in egg production with changes in the energy content of the diet. Egg weight was not affected by energy concentration of the diet, consistent with data of Grobas et al. (1999b), Ciftci et al. (2003), and Valkonen et al. (2008). However, Harms et al. (2000) and Wu et al. (2005, 2007b) reported that egg weight increased linearly with increases in dietary energy. Bouvarel et al. (2010) analyzed data from experiments conducted for the last 20 years and reported that egg weight increased 0.96 g per each 100 kcal of increase in dietary AMEn. The reasons for the discrepancies among authors in relation to the effects of an increase in energy content of the diet on egg weight are not apparent but might be related with the linoleic acid (LNL) and fat contents of the control diet. When the energy concentration of the diet increases, there is usually a concomitant increase in both LNL and fat content. If the control diet had a LNL content below hen requirements, an increase in AMEn will result in higher intakes of this nutrient and in increases in egg size. In the current study, the LNL content of the control diet was 1.35%, probably above hen requirements for maximizing egg weight (Jensen et al., 1958; Shutze et al., 1962; Irandoust et al., 2012). In addition, the level of added fat used for increase the the energy content of the diet was increased from 0.92 to 6.02% and Grobas et al. (1999c) reported that an increase in fat content of the diet resulted in increases in egg weight. In this respect, Grobas et al. (1999b) suggested that laying hens require no more than 1.15% LNL in the diet (1.33 g/hen per day) for maximal egg weight and that when this minimal amount of LNL was met, an increase in supplemental fat, irrespective of its LNL content, resulted in further increases in egg size. 116 Chapter 4. Effect of energy concentration of the diet In the current research, egg mass increased as the AMEn of the diet increased from 2,650 to 2,750 kcal/kg but a further increase to 2,850 or 2,950 kcal/kg did not result in any further improvement. These results agree with data of Keshavarz (1998) who reported similar egg mass in SCWL hens fed diets with 2,820 or 3,040 kcal AMEn/kg. Also, Wu et al. (2005) reported that an increase in the AME of the diet from 2,720 to 2,960 kcal/kg did not affect egg mass in SCWL hens from 21 to 36 wk of age. In contrast, Joly and Bougon (1997) reported an increase in egg mass of 4.5% as the AMEn of the diet increased from 2,200 to 2,700 kcal/kg in brown hens from 19 to 68 week of age. Probably, an increase in the energy concentration of the diet might be more effective in improving egg mass production, when the diets were low than when the diets were high in energy content. Feed conversion ratio improved as the energy content of the diet increased, in agreement with most published reports (Grobas et al., 1999a,b; Wu et al., 2005). In contrast, Keshavarz (1998) reported no differences in feed efficiency in SCWL hens from 18 to 66 wk of age fed diets with 2,820 or 3,040 kcal AMEn/kg. Similarly, Valkonen et al. (2008) reported no differences in energy efficiency between two feeding programs in SCWL hens from 41 to 73 wk of age that consisted in two set of diets differing in 230 kcal in the 3 periods considered (2,380 vs. 2,610 kcal AMEn/kg as an average) . In the current research, hens fed the higher energy diet (2,950 kcal AMEn/kg) had lower FI but higher energy intake than hens fed the other diets but the excess of energy was derived to increases in BW gain rather than to improvements in egg mass production. Consequently, the efficiency of converting feed energy to egg mass was hindered when the high energy diet (2,950 kcal AMEn/kg) was used. On the other hand, hens fed the low energy diet (2,650 kcal AMEn/kg) consume less energy than hens fed the other diets. Probably the amount of energy consumed by the hens fed the low energy diet was less than precised for optimal egg production, which resulted in reduced egg mass. It seems possible that 117 Chapter 4. Effect of energy concentration of the diet hens fed the low energy diets were not able to increase feed intake to satisfy their energy requirements because of limited GIT capacity. Daily BWG increased 0.11 g/hen per day per each 100 kcal increase in AMEn concentration of the diet a value that is below the 0.20 g reported by Grobas et al. (1999c) in brown laying-hens from 22 to 65 wk of age fed diets varying in AMEn content from 2,680 to 2,810 kcal/kg and the 0.45 g reported by Harms et al. (2000) in SCWL from 36 to 44 wk of age fed diets with 2,520 to 3,080 kcal AMEn/kg. In contrast, Keshavarz (1998) reported an increase in BW of the hens of 0.014 in SCWL hens fed diets varying in AMEn content from 2,820 to 3,040 kcal/kg from 20 to 66 wk of age. Modern brown egg-laying hens might respond to increases in energy content of the diets with moderate increases in BW gain, with higher increases when high energy diets were used. The results of the current trial suggest that modern brown-egg laying hens might not regulate precisely feed intake according to energy requirements, when extreme AMEn concentrations of the diet are used. Hens fed high AMEn diets (i.e., 2,950 kcal/kg) tended to overconsume energy with a positive effect on BWG but not in egg mass production whereas hens fed low AMEn diets (i.e., 2,650 kcal/kg) tended to underconsume energy, with a negative impact on egg mass production. 4.1.2. Initial Body Weight The information available on the effects of initial BW of brown egg-laying hens on productive performance is very limited. Heavier hens at the onset of the laying period ate more feed and produced bigger eggs throughout the cycle than lighter hens (Summers and Leeson, 1983; El Zubeir and Mohammed, 1993). Bish et al. (1985) reported that heavy SCWL hens (1,377 g) produced heavier eggs than medium (1,256 g) and light (1,131 g) hens, results that are consistent with the findings of the current research. In addition, heavier hens produced more eggs but had similar FCR per kilogram of eggs than lighter 118 Chapter 4. Effect of energy concentration of the diet hens, confirming the results of Keshavarz (1995) and Pérez-Bonilla et al. (2012). Also, egg weight increased significantly with increases in initial BW of the hens. Keshavarz (1995) reported a 1.4 g difference in egg weight between light (1,151 g) and heavy (1,333 g) SCWL hens from 18 to 62 wk of age. Similarly, Pérez-Bonilla et al. (2012) reported that egg weight was 2.5 g higher in heavy (1,860 g) than in light (1,592 g) brown layinghens from 22 to 50 wk of age. One of the hypothesis of the present research was that light hens could show higher increases in energy intake and BW gain with increases in energy concentration of the diet than heavy hens. However, no interactions between initial BW and AMEn concentration were observed for any of the traits studied. In fact, the BW of the heavy and light hens were 18 and 19% higher at 50 wk of age than at 24 wk of age. These results indicate that modern brown egg-laying hens respond similarly to increases in energy content of the diet, irrespective of BW at the onset of egg production, data that are consistent with the report of Pérez-Bonilla et al. (2012) who observed increases inBW with age of 18 and 12% in heavy and light hens, respectively. 4.2. Egg Quality 4.2.1. AMEn Concentration of the Diet Energy concentration of the diet did not affect the percentage of dirty, broken, or shell less eggs throughout the laying period, consistent with data of Grobas et al. (1999a). However, albumen quality decreased linearly with increases in AME concentration of the diet, in disagreement with data of Zimmermann and Andrews (1987) and Junqueira et al. (2006). Moreover, Wu et al. (2005) reported a decrease in HU when the AMEn of the diets was increased from 2,720 to 2960 kcal/kg. The reasons for the discrepancies among authors in respect to the variation in HU values with increases in AMEn of the diet are not apparent. In the experiment of Wu et al. (2005), diets were not balanced for CP and 119 Chapter 4. Effect of energy concentration of the diet AA content with increases in energy content, and the authors suggested that the decrease in HU observed was possibly due to the lower AA intake of the hens fed the high energy diets. However, in the current experiment HU decreased with increases in AMEn concentration in spite of all diets having similar CP and AA content per unit of energy. The main differences with respect to ingredient composition of the diets in the current experiment were that the high energy diets had more fat and wheat and less barley than the low energy diets. But Grobas et al. (1999a,b) and Safaa et al. (2008) did not observe any effect of supplemental fat on HU of the eggs and Lázaro et al. (2003) and PérezBonilla et al. (2011) did not observe any effect of the main cereal of the diet on albumen quality. Yolk pigmentation increased linearly with increases in energy concentration of the diet, in spite of all diets having similar levels of corn and pigmenting additives. Xanthophylls, the main pigment source responsible for egg yolk color, are highly soluble in fat. As we increased the energy concentration of the diet, the level of fat increased, favoring the absorption of xanthophylls in the gastro intestinal tract of the hen. Similar data have been reported by Lázaro et al. (2003) in SCWL hens fed high AMEn diets. Also, Gunawardana et al. (2008) observed higher yolk pigmentation in SCWL hens fed a diet with 5% added fat than in hens fed a control diet without any added fat. The proportion of shell in the egg decreased linearly as the energy content of the diet increased in agreement with the results of Junqueira et al. (2006) who reported a linear decrease (Y = 13.252 – 0.0016x, R2 = 0.57) in egg shell proportion as the AMEn increased from 2,850 to 3,050 kcal/kg in brown hens from 76 to 84 wk of age. However, Gunawardana et al. (2008) did not find any effect of energy content of the diet on egg shell proportion in SCWL using 4 added energy levels (from 0 to 238 kcal AMEn/kd). The level of dietary fat increased the energy content of the diet increased fat, might form soaps with the Ca salts present in the feed, resulting in a reduction in Ca retention and in 120 Chapter 4. Effect of energy concentration of the diet the relative weight of the shell (Atteh and Leeson; 1983b, 1984). In contrast, Safaa et al. (2008) in brown laying-hens reported similar egg shell quality in late phase of production in hens fed diets that included 1.1 or 3% of a 40:60 mixture of soy oil and palm oil. Probably the proportion of saturated fatty acids in the lipid fraction might affect soap formation and final quality of the shell. 4.2.2. Initial Body Weight The percentage of dirty, broken, and shell-less eggs, and the HU and yolk pigmentation of the eggs were not affected by the initial BW of the hens, in agreement with data of Pérez-Bonilla et al. (2012). However, eggs from the heavy hens had higher proportion of yolk and lower of albumen than eggs from the light hens. Consequently, yolk to albumen ratio was higher for the heavier than for the lighter hens. The authors have not found any published report on the effects of initial BW of the hens on egg quality or on the proportion of egg components to compare with the data of the current research. Probably, heavy hens produce heavier yolks than lighter hens because of their higher feed intake, resulting in eggs with higher proportion of yolk (Leeson and Summers, 2005). In summary, an increase in energy content of the diets from 2,650 to 2,950 kcal AMEn/kg affected performance and egg quality of the hens. Hens fed the higher energy diet (2,950 kcal AMEn/kg) had higher energy intake than hens fed the 2,750 and 2,850 kcal AME/kg diets but the energy excess was derived to increases in BW gain rather than to improvement in egg production. On the other hand, hens fed the 2,650 kcal AME/kg diet had a reduced energy intake below requirements for optimal productive performance. An increase in energy concentration of the diet reduced HU and the proportion of shell in the egg but did not affect yolk to albumen ratio. Heavy hens had higher feed intake and produced more mass of eggs than light hens but energy efficiency was not affected. An increase in the energy content of the diet increased BW of the hens but the response was 121 Chapter 4. Effect of energy concentration of the diet similar for all hens irrespective of the initial BW. Heavy hens had higher yolk to albumen ratio than lighter hens. Productive performance was higher for heavier than for lighter hens but the economical advantage of increasing BW of the hens at the start of the laying cycle might depend on price difference between egg weight grades as well as on relative cost of feed ingredients. 5. References AOAC International. 2000. Official Methods of Analysis. 17th ed. AOAC Int., Gaithersburg, MD. ASAE. 1995. Method of determining and expressing fineness of feed materials by sieving. 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Ley 32/2007 de 7 de Noviembre para el cuidado de los animales, en su explotación, transporte, experimentación y sacrificio. BOE 268:45914-45920. 122 Chapter 4. Effect of energy concentration of the diet Bouvarel, I., Y. Nys, M. Panheleux, and P. Lescoat. 2010. Comment l´alimentation des poules influence la qualité des oeufs. INRA Prod. Anim. 23: 167-182. Çiftci, I., E. Yenice, D. Gökҫeyrek, and E. Öztürk. 2003. Effects of energy level and enzyme supplementation in wheat-base layer diets on hen performance and egg quality. Acta Agric. Scand. Sect. A Anim. Sci. 53:113-119. El Zubeir, E. A., and O. A. Mohammed. 1993. Dietary protein and energy effects on reproductive characteristics of commercial egg type pullets reared in arid hot climate. Anim. Feed Sci. Technol. 41:161-165. Fundación Española Desarrollo Nutrición Animal. 2008. Necesidades Nutricionales para Avicultura: Pollos de Carne y Aves de Puesta. R. Lázaroand G. G. Mateos, ed. Fund. Esp. Desarro. Nutr. Anim., Madrid, Spain Fundación Española Desarrollo Nutrición Animal. 2010. Normas FEDNA para la Formulación de Piensos Compuestos. C. de Blas, G. G. Mateos, and P. G. Rebollar, 3ª ed. Fund. Esp. Desarro. Nutr. Anim., Madrid, Spain. Grobas, S., G. G. Mateos, and J. Mendez. 1999a. Influence of dietary linoleic acid on production and weight of eggs and egg components in young brown hens. J. Appl. Poult. Res. 8:177-184. Grobas, S., J. Mendez, C. De Blas, and G. G. Mateos. 1999b. Influence of dietary energy, supplemental fat and linoleic acid concentration on performance of laying hens at two ages. Br. Poult. Sci. 40:681-687. Grobas, S., J. Mendez, C. De Blas, and G. G. Mateos. 1999c. Laying hen productivity as affected by energy, supplemental fat, and linoleic acid concentration of the diet. Poult. Sci. 78:1542–1551. Gunawardana, P., D. A. Roland Sr., and M.M. Bryant. 2008. Effect of energy and protein on performance, egg components, egg solids, egg quality, and profits in molted Hyline W-36 hens. J. Appl. Poult. Res. 17:432-439. 123 Chapter 4. Effect of energy concentration of the diet Harms, R. H., P. T. Costa, and R. D. Miles. 1982. Daily feed intake and performance of laying hens grouped according to their body weight. Poult. Sci. 61: 1021-1024. Harms, R.H., G. B. Russell, and D. R. Sloan. 2000. Performance of four strains of commercial layers with major changes in dietary energy. J. Appl. Poult. Res. 9:535541. Hill, F. W., D. L. Anderson, and L. M. Dansky. 1956. Studies of the energy requirements of chickens 3. The effect of dietary energy level on the rate and gross efficiency of egg production. Poult. Sci. 35:54-59. Hy-Line International, 2011. Hy-Line Variety Brown, Commercial Management Guide 2009-2011. Hy-Line International, West Des Moines, IA, USA. Irandoust, H., A. H. Samie, H. R. Rahmani, M. A. Edriss, and G. G. Mateos. 2012. Influence of source of fat and supplementation of the diet with vitamin E and C on performance and egg quality of laying hens from forty four to fifty six weeks of age. Anim. Feed Sci. Technol. (In-Press). Jalal, M.A., S. E. Scheideler, and D. Marx. 2006. Effect of bird cage space and dietary matabolizable energy level on production parameters in laying hens. Poult. Sci. 8:5306-5311. Jalal, M.A., S. E. Scheideler, and E. M. Pierson. 2007. Strain response of laying hen to varying dietary energy levels with and without avizyme supplementation. J. Appl. Poult. Res. 16: 289-295. Jensen, L. S., J. B. Allred, R. E. Fry, and J. McGinnis. 1958. Evidence for an unidentified factor necessary for maximum egg weight in chickens. J. Nutr. 65:219-223. Joly, P., and M. Bougon. 1997. Influence du niveau énergétique sur les performances des pondeuses à oeufs roux et évolution de l´ingéré en fonction de l`âge. 2ême journée de la Recherche Avicole. 1997.2:115-120. 124 Chapter 4. Effect of energy concentration of the diet Junqueira, O.M., A. C. de Laurentiz, R. da Silva Filardi, E. A. Rodrigues, and E. M. Casartelli. 2006. Effects of energy and protein levels on egg quality and performance of laying hens at early second production cycle. J. Appl. Poult. Res. 15:110-115. Keshavarz, K. 1995. Further investigations on the effect of dietary manipulations of nutrients on early egg weight. Poult. Sci. 74:62-74. Keshavarz, K. 1998. The effect of light regimen, floor space, and energy and protein levels during the growing period on body weight and early egg size. Poult. Sci. 77:12661279. Lázaro, R., M. García, M. J. Araníbar, and G. G. Mateos. 2003. Effect of enzyme addition to wheat, barley and rye based diets on nutrient digestibility and performance of laying hens. Br. Poult. Sci. 44:256-265. Leeson, S., and J. D. Summers. 1987. Effect of immature body weight on laying performance. Poult. Sci. 66:1924-1928. Leeson, S., and J. D. Summers. 2005. Comercial Poultry Nutrition, 3rd Edition. Nottingham University Press. Mateos, G. G. and. J. L. Sell. 1980a. Influence of graded levels of fat on utilization of pure carbohydrate by the laying hen. J. Nutr. 110:1894-1903. Mateos, G. G. and. J. L. Sell. 1980b. Influence of carbohydrate and supplemental fat source on the metabolizable energy of the diet. Poult. Sci. 59:2129-2135. Mathlouthi, N., M. Larbier, M. A. Mohamed, and M. Lessire. 2002. Performance of laying hens fed wheat, wheat-barley or wheat-barley-wheat bran based diets supplemented with xylanase. Can. J. Anim. Sci. 82:193-199. Peguri, A., and C. Coon. 1991. Effect of temperature and dietary energy on layer performance. Poult. Sci. 70:126-138. 125 Chapter 4. Effect of energy concentration of the diet Pérez-Bonilla, A., M. Frikha, S. Mirzaie, J. Garcia, and G. G. Mateos. 2011. Effects of the main cereal and type of fat of the diet on productive performance and egg quality of brown-egg laying hens from 22 to 54 weeks of age. Poult. Sci. 90:2801-2810. Pérez-Bonilla, A., C. Jabbour, M. Frikha, S. Mirzaie, J. Garcia, and G. G. Mateos. 2012. Effect of crude protein and fat content of the diet on productive performance and egg quality traits of brown egg-laying hens with different initial body weight. Poult.Sci. 91:1400-1405. Safaa, H. M., M. P. Serrano, D. G. Valencia, M. Frikha, E. Jiménez-Moreno, and G. G. Mateos. 2008. Productive performance and egg quality of brown egg-laying hens in late phase of production as influenced by level and source of calcium in the diet. Poult. Sci. 87:2043-2051. SAS Institute, 1990. SAS STATs User’s Guide.Version 6, 4th ed. SAS Institute Inc., Cary, NC. Shutze, J. V., L. S. Jensen, and J. McGinnis. 1962. Accelerated increase in egg weight of young pullets fed diets supplemented with corn oil. Poult. Sci. 41:1846-1851. Summers, J. D. and S. Leeson. 1983. Factors influencing early egg size. Poult. Sci. 62:1155-1159. Valkonen , E., E. Venalainen, L. Rossow, J. Valaja. 2008. Effects of dietary energy content on the performance of laying hens in furnished and conventional cages. Poult. Sci. 87:844-852. Vuilleumier, J.P., 1969. The Roche yolk colur fan – An instrument for measuring yolk colour. Poult. Sci. 48:767-779. Wu, G., M. M. Bryant, R. A. Voitle, and D.A. Roland Sr. 2005. Effect of dietary energy on performance and egg composition of Bovans white and Dekalb white hens during phase I. Poult. Sci. 84:1610-1615. 126 Chapter 4. Effect of energy concentration of the diet Wu, G., P. Gunawardana, M. M. Bryant, R. A. Voitle, D.A Roland Sr. 2007b. Effect of molting method and dietary energy on postmolt performance, egg components, egg solid, and egg quality in Bovans white and Dekalb white hens during second cycle phases two and three. Poult. Sci. 86:869-876. Zimmermann, N. G., and D. K. Andrews. 1987. Comparison of several induced molting methods on subsequent performance of Single Comb White Leghorn hens. Poult. Sci. 66: 408-417. 127 Table 1. Ingredient composition and calculated and determinated analyses of the experimental diets (% as-feed bases, unless stated otherwise). AMEn (kcalAME/kg) Ingredient Corn Wheat Barley Soya bean meal, 47% CP Soybeanoil DL-Methionine, 99% Monocalciumphosphate Limestone1 Sodium chloride Vitamin and mineral premix2 Calculated analysis3 AMEn(kcal AME/ kg) CP Ether extract Digestible Met Digestible Met+Cys Digestible Lys Digestible Thr DigestibleTrp Digestible Val Linoleicacid Total Ca Total P Available P Determined analysis4 Gross energy (kcal/ kg) DM CP Ether extract Total ash GMD5, µm GSD5, µm 2,650 2,750 2,850 2,950 6.60 27.15 33.19 21.37 0.92 0.14 0.86 8.97 0.30 0.50 6.60 34.02 22.24 23.35 2.61 0.15 0.95 9.27 0.31 0.50 6.60 41.10 10.95 25.39 4.34 0.17 1.04 9.59 0.32 0.50 6.60 47.96 27.37 6.02 0.18 1.13 9.91 0.33 0.50 2,650 16.9 2.7 0.39 0.78 0.81 0.58 0.17 0.02 1.35 3.66 0.68 0.44 2,750 17.5 4.3 0.41 0.82 0.86 0.61 0.18 0.02 2.23 3.80 0.69 0.46 2,850 18.1 6.0 0.43 0.86 0.90 0.64 0.19 0.02 3.13 3.94 0.71 0.48 2,950 18.8 7.6 0.45 0.90 0.95 0.67 0.20 0.02 4.01 4.08 0.73 0.49 3,561 91.5 16.9 2.6 12.3 990 ±2.25 3,657 91.7 17.6 4.1 12.3 945 ±2.23 3,776 91.6 18.2 5.7 12.5 971 ±2.20 3,824 91.9 18.4 7.0 12.6 1,020 ±2.02 1 50% of the calcium carbonatewas supplied coarsely ground (3/6 mm screen). Supplied per kilogram of diet: vitamin A (trans-retinyl acetate), 10,000 IU; vitamin D3 (cholecalciferol), 2,000 IU; vitamin E (DL-α-tocopheryl acetate), 10 mg; vitamin B1, 1 mg; vitamin B2, 4 mg; vitamin B6, 1 mg; vitamin 12 (cyanocobalamin), 15 mg; vitamin K3, 2.5 mg; choline (choline chloride), 150 mg; nicotinic acid, 25 mg; pantothenic acid (D-calcium pantothenate), 7.5 mg; folic acid, 0.10 mg; manganese (MnO), 70 mg; zinc (ZnO), 50 mg; iron (FeSO4 H2O), 30 mg; copper (CuSO4 5H2O), 5 mg; iodine [Ca(IO3)2], 0,5 mg; selenium (Na2SeO3), 0,3 mg; canthaxantin; 2,4 g; ester of β-apo-8-carotenoic, 1,7 g (Lucanmix yellow/red, Basf, Tarragona, Spain), [(Endo-1.3(4)β-glucanase (EC 3.2.1.6), 150 IU/g; Endo-1.4-β-xylanase (EC 3.2.1.8), 105 IU/g; (Endofeed, GNC Bioferm, Saskatchewan, SK, Canada), Natuphos 5000 [300 FTU/ Kg of 6-phytase (EC 3.1.3.26), Basf Española, S.A, Tarragona, Spain]. 3 According to Fundación Española Desarrollo Nutrición Animal (2010). 4 Analyzed in triplicate. 5 Geometric mean diameter and log normal geometric SD. 2 128 Table 2. Influence of the AMEn of the diet and initial BW of the laying hens on productive performancefrom 24 to 59 wks of age1 Feed AMEn Initial Eggproduction Eggweight Eggmass FCR AME intake intake FCR2(kg/kg) (kcal AME/kg) BW (%) (g) (g/d) (kg/dozen) (kcal/hen/d) (g/hen/d) 2,650 2,750 2,850 2,950 SEM6 Main effects AMEn BW gain (g) Mortality4 (%) High5 Low5 High Low High Low High Low 88.8 88.9 91.7 90.8 93.2 92.2 91.0 90.1 0.84 63.5 62.8 64.5 62.9 64.1 62.8 64.6 63.7 0.39 56.4 55.8 59.1 57.1 59.8 57.9 58.8 57.4 0.65 115.9 113.6 115.4 112.5 113.0 108.9 111.1 108.9 0.98 2.06 2.05 1.95 1.98 1.90 1.88 1.89 1.89 0.015 1.55 1.52 1.49 1.47 1.44 1.40 1.45 1.43 0.014 307 301 317 309 322 310 328 321 2.7 5.45 5.40 5.37 5.42 5.39 5.36 5.58 5.60 0.043 2,650 2,750 2,850 2,950 88.8c 91.2ab 92.7a 90.5bc 0.59 63.1 63.7 63.5 64.1 0.27 56.1b 58.1a 58.8a 58.1a 0.46 114.8a 114.0a 111.0b 110.0b 0.69 2.05a 1.96b 1.89c 1.89c 0.010 1.54a 1.48b 1.42c 1.44c 0.010 304c 313b 316b 324a 1.9 5.42b 5.39b 5.38b 5.58a 0.030 255c 300bc 325ab 359a 17.3 1.5 3.1 0.8 4.6 64.2a 63.0b 0.19 58.5a 57.0b 0.32 113.9a 111.0b 0.49 1.95 1.49a 1.95 1.46b 0.008 0.007 Probability *** *** NS *** 319a 310b 1.4 5.45 5.44 0.021 313 307 12.2 1.9 3.1 *** *** *** NS ** NS NS NS L*** NS L*** Q*** L*** NS NS NS SEM7 Initial BW EnE3 (kcal AME/ g egg) High Low 91.2 90.5 SEM8 0.42 Effect9 AMEn *** NS ** *** Initial BW NS *** ** *** Contrasts AMEn linear L*** NS L** L*** L*** AMEn quadratic Q*** NS Q** NS Q** a,b,c Means with different superscript within each main effect are significantly different (P < 0.05). 1 Data presented correspond to the means of 9 periods of 28-d each. 2 FCR = Feed conversion ratio. 3 EnE = Energy efficiency. 4 Analyzed by GENMOD procedure. 5 Initial BW±SD of 1,733 ± 48g and 1,606 ± 39g, for the heavy and light hens, respectively. 6 SEM (5 replicates of 13 hens each per treatment). 7 SEM (10 replicates of 13 hens each per treatment). 8 SEM (20 replicates of 13 hens each per treatment). 9 The interaction between AMEn and initial BW was not significant (P > 0.05). *P < 0.05; **P< 0.01; ***P< 0.001. L** Q** 272 239 284 316 331 320 365 352 24.5 1.5 1.5 3.1 3.1 0.0 1.5 3.1 6.1 129 Table 3. Influence of the AMEn of the diet and initial BW of the laying hens on egg quality from 24 to 59 wks of age1 Relative weight of egg (%) AMEn (kcal AME/kg) 2,650 2,750 2,850 2,950 SEM4 Main effects Energy Initial BW Dirty eggs (%) Broken eggs (%) Shell-less eggs (%) Haugh units High3 Low3 High Low High Low High Low 5.23 6,84 5.12 5.65 5.36 5.10 5.88 6.13 0.618 0.93 1.62 1.74 1.55 1.67 1.24 1.97 1.69 0.379 0.09 0.13 0.19 0.25 0.06 0.06 0.10 0.13 0.060 88.7 88.2 87.2 88.4 85.0 87.6 84.8 84.5 2.26 2,650 2,750 2,850 2,950 6.03 5.38 5.23 6.01 0.437 1.27 1.65 1.46 1.83 0.268 0.11 0.22 0.06 0.12 0.040 88.4a 87.8a 86.3b 84.7c 1.60 High Low 5.40 5.93 0.309 1.58 1.53 0.190 0.11 0.14 0.030 86.4 87.2 1.13 SEM5 Initial BW Yolk pigmentation2 Shell Yolk Albumen Yolk to albumen ratio 7.3 7.5 7.5 7.3 7.6 7.7 8.0 7.8 0.40 9.7 9.7 9.6 9.6 9.5 9.7 9.4 9.5 0.20 25.8 25.3 25.4 25.5 25.8 25.4 26.0 25.3 0.44 64.5 65.0 65.0 64.9 64.7 64.9 64.6 65.2 0.52 0.403 0.390 0.393 0.394 0.400 0.393 0.404 0.389 0.0101 7.4c 7.4bc 7.6ab 7.9a 0.28 9.7a 9.6a 9.6a 9.5b 0.13 25.5 25.4 25.6 25.6 0.31 64.7 64.9 64.8 64.9 0.38 0.396 0.393 0.396 0.397 0.0072 7.6 7.6 0.20 Probability *** ** NS NS 9.5b 9.6a 0.09 25.7b 25.3a 0.22 64.7b 65.0a 0.26 0.400a 0.392b 0.0051 ** * NS *** NS ** NS *** L*** NS NS NS NS NS NS NS SEM6 Effect7 AMEn NS NS NS Initial BW NS NS NS Contrasts8 AMEn linear NS NS NS L*** L*** AMEn quadratic NS NS NS NS NS a,b,c Means with different superscript within each main effect are significantly different (P < 0.05). 1 Data presented correspond to the average value of 4 measurements (39, 48, 55, and 59 wk of age). 2 Measured using the DSM color fan according to Vuilleumier (1969). 3 Initial BW±SD were 1,733 ± 48g and 1,606 ± 39g, for the heavy and light hens, respectively. 4 SEM (5 replicates of 13 hens each per treatment). 5 SEM (10 replicates of 13 hens each per treatment). 6 SEM (20 replicates of 13 hens each per treatment). 7 The interaction between AMEn and initial BW was not significant (P > 0.05). NS: not significant *P < 0.05; **P< 0.01; ***P< 0.001. 130 Figure 1: Effect of AMEn concentration of the diet (kcal/kg) on egg production (A), egg weight (B), feed intake (C), and BW gain (D) from 24 to 59 wk of age. 98 ** NS NS NS * * 92 * 67 ** 94 ** *** 90 88 Egg weight (g) Egg production (%) 96 SEM: 0.27 P > 0.10 69 SEM: 0.59 P < 0.01 65 NS NS NS * NS NS NS 63 NS 86 61 84 82 28-31 2,650 120 * 32-35 36-39 2,750 40-43 44-47 2,850 48-51 52-55 *** *** *** *** 114 24-27 28-31 32-35 36-39 40-43 44-47 48-51 52-55 56-59 2,650 SEM: 0.98 P < 0.001 118 Feed intake (g/hen/d) B 2,950 kcal/kg * 116 56-59 100 2,750 2,850 SEM: 17.3 P < 0.01 *** NS *** 112 20 106 -20 24-27 28-31 32-35 36-39 40-43 44-47 48-51 52-55 56-59 C NS NS NS 110 0 NS NS 40 108 24-27 28-31 32-35 36-39 40-43 44-47 48-51 52-55 56-59 D 2,650 2,750 2,850 2,950 kcal/kg NS NS 60 * 2,950 kcal/kg 80 *** BW gain (g) A 59 24-27 2,650 2,750 2,850 2,950 kcal/kg 131 Figure 2: Effect of initial BW of the hens on egg production (A), egg weight (B), feed intake (C), and BW gain (D) from 24 to 59 wk of age. 96 NS NS ** 65 NS 92 ** SEM: 0.42 P > 0.10 * NS NS NS 90 NS 88 Egg weight (g) Egg production (%) 94 67 NS 86 ** NS ** ** NS ** 63 ** 61 84 82 A * 24-27 59 28-31 NS 32-35 36-39 40-43 44-47 48-51 Heavy hens (1,733 ± 48g) 120 SEM: 12.2 B NS Heavy hens (1.733 ±48g) 100 * *** BW gain (g) 116 114 112 C110 D 108 106 24-27 28-31 32-35 36-39 40-43 44-47 48-51 52-55 56-59 Light hens (1,606 ± 39g) 60 NS NS NS NS 80 Heavy hens (1,733 ± 48g) NS Light hens (1.606 ±39g) NS * 118 Feed intake (g/hen/d) 24-27 28-31 32-35 36-39 40-43 44-47 48-51 52-55P > 56-59 0.10 Light hens (1,606 ± 39g) *** *** ** ** SEM: 0.49 52-55P < 56-59 0.001 NS NS 40 20 0 -20 24-27 28-31 32-35 36-39 40-43 44-47 48-51 52-55 56-59 Heavy hens (1,733 ± 48g) Light hens (1,606 ± 39g) 132 Chapter 5. General discussion and conclusions CHAPTER 5: General discussion and conclusions 133 Chapter 5. General discussion and conclusions 1. General discussion 1.1. Productive performance in egg-laying hens 1.1.1. Effect of the main cereal of the diet In trial 1, from 22 to 54 weeks of age, important variables like egg production, egg weight, egg mass, FI, and FCR per kilogram and per dozen of egg and mortality did not differ independent of the cereal used in agreement with previous studies (Craig and Goodman., 1993). As a result of experimental design and in order to avoid the effects of NSP of the diet that included wheat and barley, diets were supplemented with exogenous enzymes. This fact produced that laying hen productivity was not affected by the use of wheat and barley in agreement with Lázaro et al. (2003) who reported in SCWL hens (Hy-line W-77) located individually that cereals with soluble fiber like wheat and barley can replace corn successfully in laying hen diets. Also, the same author reported that viscosity due to NSP was reduced via enzyme supplementation improved digestibility of nutrients and hen performance. Also, Safaa et al. (2009) reported no difference in Lohman brown hens fed with two diets that contained 50% of dented corn and 50% of hard wheat. It is noticeable to comment that in both studies (Lazaro et al., 2003 and Safaa et al., 2009) was used the same commercial enzyme complex that contained β-glucanase (EC 3.2.1.6.) andxylanase activity (Endofeed, GNC Bioferm Inc., Saskatoon, Saskatchewan, Canada). In contrast of the current research, Coon et al. (1988) reported higher FI and poorer FCR per kilogram in hens fed enzyme-supplemented barley diets than in hens fed corn diets. The only effect founded in the current research related with the use of different cereal was the BW gain. Thus, BW gain was greater in hens fed corn or wheat than in hens fed barley, which were results agree with data of Berg (1959) who reported higher BW gain for hens fed corn than for hens fed barley. 134 Chapter 5. General discussion and conclusions 1.1.2. Effect of source, fatty acid profile, and level of supplemented fat of the diet In trial 1, the substitution of SBO by AVO or lard in the diet did not affect any of the productive performance traits studied except to BW gain of the birds. Thus, BW gain of hens fed lard was higher than hens fed SBO or AVO confirming that more energy consumed by the hens fed lard was diverted to BW gain rather than to energy deposition in eggs as compared with hens fed SBO or AVO. In broilers, Vila and Esteve-García (1996) reported that broilers fed diets supplemented with tallow or lard had higher abdominal fat deposition than broilers fed isoenergetic diets based on more unsaturated vegetable oils, which is consistent with the results of the current experiment. In contrast, Grobas et al. (2001) in brown hens did not find this effect in BW gain of the hens using 2 types of unsaturated fat (soy oil and linseed oil) and 2 types of monounsaturated fat (tallow and olive oil) respect to control diet without added fat. Although no significant difference were found throughout experiment for all performance variables except to BW gain, the lower egg weight was observed for hens fed lard (saturated fat), and the larger EW was observed for hens fed SBO or AVO (unsaturated fat) confirming previous data of Grobas et al. (2001). This authors reported tha thens fed with tallow and olive oil improve EW (3 g as average) respect to hens fed the monounsaturated fat (tallow or olive oil). This result was expected by the authors because the LNL content of the monounsaturated diets were less than 1% which has been shown to be insufficient to maximize egg size (Grobas et al., 1999c). The effects of supplemental fat on performance of hens were a subject of debate in trial 2. The increase of added fat from 1.8 to 3.6% of soy oil in the diet did not affect any performance variables throughout experiment 2 in agreement with the finding of Usayran et al. (2001) who reported in SCWL hens from no difference among diets with 0 or 4% of added fat from 28 wk of age to peak of lay. In contrast, Mateos and Sell (1981) 135 Chapter 5. General discussion and conclusions indicated that SFAT might benefit hen productivity by slowing down the rate of food passage, allowing more time for the contact of enzymes and dietary components which in turn might result in an improvement in performance variables, especially in EW. Parsons et al. (1993) reported in SCWL hens from 21 to 40 wk of age an improved in egg production and EW using diets with added fat (from 2 to 6%) respect to diets without added fat. Also, Safaa et al. (2008) reported that egg production (77.0 vs. 79.3%), EW (64.9 vs. 66.3 g), egg mass (49.8 vs. 52.5%), and FCR (2.36 vs. 2.26) improved with the increase in SFAT from 1.1 to 3.0%. Moreover, Grobas et al. (1999b) reported in brown hens from 22 to 65 wk of age an improvement in egg production (88.0 vs. 89.8%), EW (64.1 vs. 65.3 g) and egg mass (56.4 vs. 58.6 g/d), but FCR (2.08 vs. 2.06) was not affected in diet with 0 or 4.0%. Improves in productive performance by the use of added fat have been reported by several authors (Jensen et al. 1983; Bohnsack et al. 2002; Sohail et al. 2003). The reasons for the discrepancies among authors are not know but might be related to the type of diet and the confusing effect between added fat, AMEn, and LNL content. Due to experimental design, to increase added fat was accompanied by an increase in energy concentration and LNL content of the diet (1.9 vs. 2.5%, respectively). Grobas et al. (1999b,c) reported in brown hens from 22 to 74 wks of age that an increase in the dietary SFAT with LNL held constant at 1.15% increase food consumption and energy intake and this increase in energy intake resulted in an additional increase in BW and in an improvement in egg production and EW. Also, the same authors reported that an interaction between age an SFAT. Thus, EW increased with SFAT in young but not in old hens. 136 Chapter 5. General discussion and conclusions 1.1.3. Effect of linoleic acid of the diet The effect of LNL content of the diet was studied in trial 1 as a consequence of experimental design. Thus, due to the interaction of the main cereal of the diet and the type of fat 9 levels of LNL were obtained (from 3.4% to 0.8, for corn-SBO and wheatlard respectively). Opposite to the initial hypothesis about the expected effect of LNL on performance variables, especially, the improve of EW, there were not significant different between diets on productive variables, thus the level of LNL did not affect the performance results, specially the EW. In spite of this results, and although no significant differences were detected, the lower EW was observed for hens fed wheat and lard (62.8 g), and the larger EW was observed for hens fed corn and SBO or AVO (64.9 and 65.0 g, respectively). Probably, the low LNL content of the wheat and lard, below values recommended by most researchers (Jensen et al., 1958; Shutze et al., 1959), was insufficient to maximize EW. Also, was interested to notice the difference in the ratio between egg size or egg mass produced and LNL intake by the hens fed the different diets. The ratio was 2.5 times higher for hens fed the lard diets than for hens fed the SBO diets, with hens fed the AVO diets being intermediate. The information provided in the current study about the minimum level of LNL to maintain the performance variables was around 0.9-1.0% whereas under commercial conditions, many commercial guides for feeding laying hens (H & N international, 2008; Lohmann, 2010) recommend increasing the level of LNL in the diet to at least 1.8% (2.0 g/hen per d) to maximize egg size. Also, in experimental conditions Scragg et al. (1987) recommended up to 2.0% dietary LNL to increase egg size in brown egg-laying hens. However, Shutze et al. (1959), and Grobas et al. (1999a,b) do not support current feeding practices of using 1.8% LNL in the diet to maximize egg size in agreement with the current study. In fact, Grobas et al. (1999b) in brown hens from 22 to 74 wk of age reported no differences in EW among diets differing 137 Chapter 5. General discussion and conclusions in LNL content from 1.10 to 1.60%.This authors reported that once the LNL requirement of hens is fulfilled with 1.15% of the diet, the SFAT increased EW independently of the LNL content of the diet. In the trial 3, the design of diets provoked that when the energy concentration of the diet increases, there is usually a concomitant increase in both fat and LNL content. Thus, the level of added fat used in the diets, increased from 0.92 to 6.02% as the energy content of the diet increased from 2,650 to 2,950 and the level of LNL increased from 1.35 to 4.0% respectively. In spite of this, the increase in the energy concentration did not affect the EW among diets in disagreement with the results of Grobas et al. (1999c) who reported brown hens from 22 to 65 wk of age that an increase in added fat from 0 to 4% content of the diet resulted in increases in EW. Grobas et al. (1999b) suggested that laying hens require no more than 1.15% LNL in the diet to maximize EW and that when this minimal amount of LNL was met, an increase in supplemental fat resulted in further increases in egg size, irrespective of its LNL content. Therefore, the effects of increasing the energy content of the diet on EW might depend on the fat and LNL contents of the basal diet. 1.1.4. Effect of energy content of the diet The influence of energy content of the diet was studied in experimental 3. Thus, important variables like FI, EW, FCR, and BW of the hens were affected by treatment. Thus, in this research, an increase in the energy content of the diet from 2,650 to 2,950 kcal AMEn/kg (an 11% increase) decreased FI by 4% resulting in an increase in energy intake of 7% in agreement with the results of Bouvarel et al. (2010) who reviewed a series of experiments conducted in laying hens during the last 20 years and reported that as an average, a 10% increase in AMEn content of the diet resulted in a reduction in feed intake of only 5.5% in agreement with the results of the current experiment. However, 138 Chapter 5. General discussion and conclusions Keshavarz (1998) reported in SCWL from 18 to 66 wk of age that an increase in the AMEn concentration of the diet from 2,815 to 3,035 kcal/kg (an 8% increase) resulted in a 9% increase in energy intake.The data indicate that laying hens do not regulate precisely FI according to requirements and tended to overconsume energy as the AMEn of the diet increases. Egg production increased as the AMEn concentration of the diet increased from 2,650to 2,850 kcal/kg but an increase to 2,950 kcal/kg did not result in any further improvement. Similarly, Mathlouthi et al. (2002) reported in SCWL hens that egg production increased as the AMEn of the diet increased from 2,650 to 2,750 kcal/kg. In contrast, Grobas et al., (1999c) in brown hens fed diets varying from 2,680 to 2,810 kcal AMEn/kg, Harms et al. (2000) in brown-and SCWL hens fed diets varying in AMEn from 2,500 to 3,100 kcal/kg, and Jalal et al. (2006, 2007) in SCWL hens fed diets varying from 2,800 to 2,900 kcal AMEn/kg did not detect any significant difference in egg production with changes in the energy content of the diet. These data support the hypothesis that an excess in energy intake caused by changes in diet composition, results primarily in increases in BW gain rather than in further increases in egg mass production.Egg weight was not affected by energy concentration of the diet, consistent with data of Grobas et al. (1999b), Ciftci et al. (2003), and Valkonen et al. (2008). However, Harms et al. (2000) and Wu et al. (2005, 2007b) reported that EW increased linearly with increases in dietary energy. Bouvarel et al. (2010) analyzed data from 11experiments conducted for the last 20 years and reported that EW increased 0.96 g per each 10 kcal of extra energy intake per day. The reasons for the discrepancies among authors in relation to the effects of an increase in energy content of the diet on EW are not apparent but might be related with the level of fat and the linoleic acid (LNL) content of the experimental diets. Feed conversion ratio improved as the energy content of the diet increased, in agreement with most published reports (Grobas et al., 1999a,b;Wu et al., 2005).Hens eat feed to satisfy their energy requirements and therefore, high AMEn 139 Chapter 5. General discussion and conclusions diets results in better FCR. Moreover, supplemental fat has been shown to reduce rate of feed passage, facilitating the contact between digesta and enzymes and improving digestibility and utilization of other nutrients such as the lipid and carbohydrate fractions of the diet (Mateos and Sell, 1980b, 1981).In contrast, Keshavarz (1998) reported no differences in feed efficiency in SCWL hens from 18 to 66 wk of age fed diets with 2,820 or 3,040 kcal AMEn/kg. Similarly, Valkonen et al. (2008) reported no differences in energy efficiency in SCWL hens from 41 to 73 wk of age fed very low (2,380 kcal AMEn/kg as an average) or low (2,610 kcal AMEn/kg as an average) energy diets. In the current research, hens fed the higher energy diet (2,950 kcal AMEn/kg) had lower FI but higher energy intake than hens fed the other diets but the excess of energy was derived to increases in BW gain rather than to improvements in egg mass production. Consequently, the efficiency of converting feed energy to egg mass was hindered when the high energy diet was used. On the other hand, hens fed the low energy diet (2,650 kcalAMEn/kg) consumed less energy than hens fed the other diets. Probably because of physical limitation of the gastro intestinal tract (GIT), the amount of energy consumed by the hens fed the low energy diet was below requirements for optimal egg production, resulting in reduced egg mass. Body weight gain increased 0.11 g/hen per day per each 100 kcal increase in AMEn concentration of the diet, a value that was below the 0.20 g reported by Grobas et al. (1999c) in brown egg-laying hens from 22 to 65 wk of age fed diets varying in AMEn content from 2,680 to 2,810 kcal/kg, and the 0.45 g reported by Harms et al. (2000) in SCWL from 36 to 44 wk of age fed diets with 2,520 to 3,080 kcal AMEn/kg. In contrast, Keshavarz (1998) reported an increase in BW of the hens from 20 to 66 wk of age of only 0.014, in SCWL hens fed diets varying in AMEn content from 2,820 to 3,040 kcal/kg. Modern brown egg-laying hens might respond to increases in energy content of the diets with increases in BW gain with effects being more noticeable when high energy diet sare used. 140 Chapter 5. General discussion and conclusions The results suggest that modern brown-egg laying hens might not regulate accurately feed intake according to energy requirements when very high or low energy diets are used. Hens fed high AMEn diets (i.e., 2,950 kcal/kg in the current trial) tended to overconsume energy with a positive effect on BWG but not in egg mass production. On the other hand, hens fed low AMEn diets (i.e., 2,650 kcal/kg in the current trial) tended to underconsume energy, with a negative impact on egg mass production. 1.1.5. Effect of initial body weight of the hens The influence of initial body weight of the hens at the onset of lay period was subject of debate in trials 2 and 3. Nowadays, laying pullets are coming into production and peaking several weeks earlier than was the case a few years ago. Although there may be some debate as to the contribution of genetics, management, and nutrition in bringing about this change, most researchers agree that earlier maturing pullets usually result in more profitable layers (Leeson and Summers, 2005). One of the problems with earlier maturing pullets, and a factor that continues to keep them from being readily accepted by the industry is a higher percentage of small eggs. In spite of this, the potential studies that could be realized with an earlier maturing pullet and hens at the onset of lay cycle continues to generate research interest. The influence of initial body weight of the hens was studied in trial 2 in 672 Lohmann Brown hens which were weighed individually and classified as light (1,592 ± 75 g) or heavy (1,860 ± 86 g) compared with a target BW of 1,640 ± 57 g. Thus, throughout experimental period ADFI (P< 0.001), egg production (P< 0.001), EW (P< 0.001), and egg mass (P< 0.001) were higher for the heavier than for the lighter hens. In trial 3 the influence of two groups differing in initial BW was studied in 520 Hy-Line brown hens. Hens were weighed individually and classified as light (1,606 ± 39 g) and heavy (1,733 ± 48g) with a target BW of 1,685± 35g for hens of this age. Like the 141 Chapter 5. General discussion and conclusions trial 2, heavy hens obtained higher FI (P< 0.001), EW (P< 0.001), egg mass (P< 0.01) and FCR per dozen of eggs whereas egg production and FCR per kilogram of egg was not different in this case between both groups of hens. In both studies data are in agreement with Harms et al. (1982) who reported in three groups of SCWL divided as light (range from 1,301 to 1,500 g), medium (range from 1,501 to 1,580 g), and heavy (range from 1,581 to 1,780 g), higher FI, egg production, EW, and egg mass (P< 0.05) in heavy hens than in light hens. Also, Summers and Leeson (1983) reported in SCWL hens divided in four groups of weight at 18 wks of age as very light (1,107 g), light (1,205 g), medium (1,281 g), and heavy (1,383 g); higher egg production, FI, and EW (P< 0.05) from 19 to 25 wks. Moreover, Bish et al. (1985) reported in SCWL hens divided into three body weight categories as light (1,131 g), medium (1,256 g), and heavy (1,377 g) at 20 wks of age no significant different in egg production but the FI (P< 0.05), FCR per kilogram and per dozen of egg (P< 0.05) was higher in the heavy hens than lighter hens with the medium being intermediate. The result of Bish et al. (1985) is partially agreed with the results of trial 2 and 3. It is noticeable to comment the different range of weight between trials. In trial 2 the difference between both groups was 268 g as average, while in trial 3 this difference was 127 g as average. Consequently, the differences in performance variables were more consistent in trial 2 than in trial 3. Thus, in trial 2 the severe range of BW resulted in light hens obtained higher BW throughout the laying cycle respect to heavy hens whereas this effect was not observed in trial 3. In spite of this, the increase in FI and BW of the hens per each 100 g increased was similar. Thus, in trial 2 FI increased 2.7 g and EW 0.93 g, and in trial 3 FI increased 2.4 g and EW 0.94 g per each 100 g increase in initial BW of the hens. The results of trial 2 and 3 are consistent with Harms et al. (1982) who reported that the BW increased in all groups of weight but the increase was higher in the light hens 142 Chapter 5. General discussion and conclusions group. In contrast, Summers and Leeson (1983) reported in four SCWL hens weight groups from 19 to 25 wks of age that body weight appeared to be the main factor controlling EW for the young pullet and all four groups of pullets gained a similar amount of BW from 19 to 25 wks of age, results that are in disagreement with both trials 2 and 3. 1.2. Egg quality in brown egg-laying hens 1.2.1. Effect of the main cereal of the diet The effects of the main cereal on egg quality were studied in trial 1. The initial hypothesis about the use of this three cereal was that barley and wheat in high proportion could increase the percentage of dirty eggs. Also it was though that the high percentage of wheat will disturb the quality of albumen with a reduction in albumen quality especially in summer when FI is low. Opposite of the initial hypothesis, the type of cereal did not affect any of the egg quality traits studied (dirty, broken, shell-less, double yolked egg, and HU) except for egg yolk colour, which was increased when corn was used. This effect was expected because all of the diets, independent of the cereal used, were supplemented with the same amount of exogenous pigment source. The available information about the effect of the main cereal in the diet on egg quality is scarce. Thus, the only report available the last year was that of Lázaro et al. (2003a) who compared in SCWL from 20 to 44 wk of age this 3 cereals in the diet. In this experiment, the inclusion of barley and wheat increased the percentage of dirty eggs as compared with the inclusion of corn in disagreement with the current research. However, no other egg quality variables were affected by the inclusion of corn. This author reported in SCWL that wheat and barley supplemented with enzymes improves digestibility and could replace corn successfully in laying hens diets. Thus, when enzymes were added to the wheat and barley diets, the incidence of dirty eggs decreased to levels similar to those found for the 143 Chapter 5. General discussion and conclusions corn diet in agreement with the current study. In fact, Safaa et al. (2009) reported that substitution of corn by wheat in enzyme supplemented diets did not affect the percentage of dirty eggs in brown hens in agreement with the results of the current trial. Moreover, Mathlouthi et al. (2003), Lázaro et al. (2003b, 2004), and García et al. (2008) reported that enzymes reduce intestinal viscosity in poultry and thus improve the consistency of the excreta. In contrast, Francesch et al. (1995) reported a higher incidence of dirty eggs in hens fed barley than in hens fed corn. Shell thickness and shell density was not affected by type of cereal in agreement with results of Çiftci et al. (2003) who reported in SCWL from 27 to 43 wk of age that egg shell thickness were not affected when 30% of corn was substituted by wheat. Our result is strongly consistent because the corn substituted in the current study was 45%. According with the initial hypothesis of the study, the effect of type of cereal on albumen quality was studied with the albumen pH measurement. Thus, no effects of cereal on albumen pH were detected. Woodward et al. (1987) reported that yolk rupture strength of 50-wk-old hens decreased with the age of the eggs, but that the decline was more rapid for egg yolks from birds fed a corn diet that from birds fed a wheat diet. 1.2.2. Effect of source, fatty acid profile, and level of supplemented fat of the diet In trial 1, the type of fat and its effects of egg quality traits were studied. The information available about the effect of this three fats is scarce. The initial hypothesis about the use of this three fats were that AVO could increase the percentage of dirty eggs and also it was though that the high percentage of this fat included in the diet might provoked problems in egg shell quality. In this context, Lard was the fat source that could be used in mixtures with AVO to avoid problems. Soybean oil was the control fat used in this experiment. In spite of the initial hypothesis, the type of fat did not affect any of the egg quality variables studied including dirty, broken, shell-less, and double-yolked eggs. 144 Chapter 5. General discussion and conclusions Also, shell characteristics like shell thickness, shell density and HU did not affected by treatment. This results are in agreement with Fraga-Benitez et al. (1987) who reported in SCWL hens no effect in shell thickness using 0, 2, 4, 6, 8, or 10% of nonacidulated sunflower soapstock for 168 d of lay cycle. Also, Pardío et al. (2005) reported in SCWL hens from 20 to 35 wk of age that increases in soybean soapstock from 25 to 100% in the diet did not affect shell thickness respect to control diet including SBO. This authors reported that HU was not affected by treatment in agreement with current study. Moreover, Safaa et al. (2008) did not find any significant effect in shell quality and HU in brown hens from 56 to 68 wk of age using palm oil and soy bean oil in the diet. However, Grobas et al. (2001) using four types of fat (Tallow, olive oil, soy oil, and linseed oil) respect to the control diet without fat reported higher shell thickness in soy oil and olive oil respect to tallow and linseed oil. The same authors did not find effect in HU. The yolk color was the only egg quality variable affected by treatment. Thus, the color fan values of the yolks were greater with lard than with SBO or AVO supplemented diets. The fatty acid profile of lard was more saturated than that of the SBO or AVO, an thus, dietary pigments were probably more stable in the presence of lard than in the presence of more unsaturated fat sources, both in the feed and in the gastrointestinal tract, result that is in disagreement with Grobas et al. (2001) who reported in brown hens similar yolk color among 4 different fat sources (Tallow, olive oil, soy oil, and linseed oil). The effect of SFAT on egg quality traits was studied in trial 2 and trial 3. Thus, in trial 2 supplementation of the high CP diet with 3.6% fat did not affect any of the shell and albumen quality traits studied. In broilers, Atteh et al. (1983) indicated that the inclusion of saturated fats in the diet increased the formation of soaps between fatty acids and the Ca salts, resulting in lower Ca retention. In the current experiment, no effects of extra fat supplementation of the diet on shell quality were observed, data that agree with results of Safaa et al. (2008) who reported similar egg shell quality in late phase of 145 Chapter 5. General discussion and conclusions production in hens fed diets with 1.1 or 3% supplemental fat (soy oil or palm oil). Probably, the amount of Ca soaps present at the small intestine level in the hens of the current experiment was limited, because the fat used was unsaturated and the soaps formed may dissociate at the pH values encountered in this section of the gastrointestinal tract. Yolk pigmentation was lower in eggs from hens fed the diet containing 3.6% fat than in hens fed the diets containing 1.8% fat, a difference that was expected because corn was included only in the 1.8% fat supplemented diets. In trial 3, the level of dietary fat increased from 2.6 to 7.0% as the energy content of the diet increased and the free added fat might form soaps with the Ca salts present in the feed, resulting in a reduction in Ca retention and in the relative weight of the shell (Atteh and Leeson, 1983, 1984). In contrast, Safaa et al. (2008) did not find any difference in hes fed diets with 1.1 or ·3% of supplemental fat. Probably, the ratio saturated:unsaturated fatty acids in the lipid fraction of the diet, might affect soap formation, fat digestibility, and final deposition of Ca in the egg shell. 1.2.3. Effect of linoleic acid of the diet Due to experimental design of the diets the effect of LNL was studied in trial 1, 2 and 3. Thus, in trial 1 the LNL content varied among diets depending of the type and fat used in the diet, thus, the level of LNL ranged between 0.8% (wheat-lard diet) and 3.4% (cornSBO). In trial 2, the effect of 3.6 or 1.8% of added fat in the diet provoked the variation in the LNL content (from 2.5% to 1.9% for the 3.6% and 1.8% of added fat, respectively). In trial 3, the increase of AMEn of the diet with increases in the fat content increased the LNL level from 1.35% to 4.01% for the 2,650 and 2,950 kcal/kg diet, respectively. The level of LNL did not affect any of the variables studied in trial 1 and trial 2 in agreement with Safaa et al. (2008) who reported in brown hens that reducing the LNL 146 Chapter 5. General discussion and conclusions content of the diet from 1.6 to 1.12% did not affect egg quality or proportion of egg components, which agrees with Grobas et al. (1999c), who reported that LNL levels ranging from 0.79 to 2.73% in diets for brown hens did not affect the percentage of broken and dirty eggs, HU, or the proportion of egg components. In trial 3 the increase in LNL content of the diet did not affect any of the egg quality variables. The effects on HU, yolk pigmentation, and shell proportion of the egg in this trial might be related with the SFAT in agreement with Grobas et al. (1999b) who reported that once the LNL requirement of hen is fulfilled (1.15% of the diet or less), SFAT increased egg weight independently of LNL and AMEn content of the diet and might was the origin of differences in shell, yolk, and albumen proportion, and yolk to albumen ratio. March and McMillan (1990) and Whitehead et al. (1993) indicated that LNL supplementation to diets deficient in this essential fatty acid increased yolk weight, probably through an improvement in the mechanism by which lipoproteins are synthesized or taken up by the developing ova. In the present study, the increase in LNL content of the diet did not increase yolk weight, indicating that LNL was not limiting egg size (including the level of 0.8% in the wheat-lard diet of trial 1). 1.2.4. Effect of energy content of the diet The effect of energy on egg quality traits was studied in trial 3. Energy concentration of the diet did not affect the percentage of dirty, broken, or shell less eggs throughout the laying period, consistent with data of Grobas et al. (1999a). However, albumen quality decreased linearly with increases in AME concentration of the diet, in disagreement with data of Zimmermann and Andrews (1987) and Wu et al. (2005). The reasons for the discrepancies are not apparent but in the experiment of Wu et al. (2005) diets were not balanced for CP and AA content with increases in energy content, and the authors 147 Chapter 5. General discussion and conclusions suggested that the decrease in HU observed was possibly due to the lower AA intake of the hens fed the high energy diets. However, in the current experiment HU decreased with increases in AMEn concentration, in spite of all diets having similar CP and AA content per unit of energy. Also, Valkonen et al. (2008) reported in SCWL hens no significant differences in HU comparing diets from 2,340 to 2,630 kcal AME/kg from 36 to 68 wk of age. Moreover, Gunawardana et al. (2008) did not observe any difference in albumen quality comparing diets from 2,750 to 3,053 kcal AMEn/kg. Some authors have been reported that the nutrition of the hens do not appear to have any great effect of albumen quality (Wells, 1968; Naber, 1979) whereas other authors reported that ingredient composition might affect albumen quality (Mateos and Puchal, 1982). In this respect, the main differences in ingredient composition of the 4 diets used in trial 3 were that the high energy diets had more fat and wheat and less barley than the low energy diets. However, Grobas et al. (1999a,b) and Safaa et al. (2008) did not observe any effect of supplemental fat on HU of the eggs, and Lázaro et al. (2003) did not observe any effect of the main cereal of the diet on albumen quality. Yolk pigmentation increased linearly with increases in energy concentration of the diet, in spite of all diets having similar levels of corn and pigmenting additives. Xanthophylls, the main pigment source responsible for egg yolk color, are highly soluble in fat. As we increased the energy concentration of the diet, the level of fat increased, favoring the absorption of xanthophylls in the gastro intestinal tract of the hen in agreement with Lázaro et al. (2003). The proportion of shell in the egg decreased linearly as the energy content of the diet increased, in agreement with the results of Junqueira et al. (2006) who reported in brown egg-laying hens from 76 to 84 wk of age, a linear decrease in egg shell proportion as the AMEn increased from 2,850 to 3,050 kcal/kg. However, Gunawardana et al. (2008) did not find any effect of energy content of the diet on egg shell proportion in 148 Chapter 5. General discussion and conclusions SCWL fed diets varying in AMEn content from 2,750 to 3,050 kcal/kg. The level of dietary fat increased as the energy content of the diet increased and the free added fat might form soaps with the Ca salts present in the feed, resulting in a reduction in Ca retention and in the relative weight of the shell (Atteh and Leeson, 1983, 1984). In contrast, Safaa et al. (2008) reported similar egg shell quality in the late phase of the production cycle of brown egg-laying hens fed diets that included 1.1% or 3% of supplemental fat (soy oil and palm oil). Probably, the ratio saturated:unsaturated fatty acids in the lipid fraction of the diet, might affect soap formation, fat digestibility, and final deposition of Ca in the egg shell. 1.2.5. Effect of initial body weight of the hens The effect of initial BW of the hens on egg quality variables was subject of debate in trial 2 and 3. In general, the information available about the effect of initial BW of the hens on egg quality variables in very limited. In trial 2, the hens differing in 2 groups as heavy (1,860 ± 86 g) and light (1,592 ± 75 g) did not show any difference in egg quality traits throughout lay period. In trial 3, the hens differing in 2 groups as heavy (1,733 ± 48g) and light (1,606 ± 39g) the percentage of dirty, broken, and shell-less eggs, and the albumen quality and yolk pigmentation of the eggs were not affected by the initial BW of the hens, in agreement with data of trial 2. However, in trial 3, eggs from the heavy hens had higher proportion of yolk and lower of albumen than eggs from the light hens. Consequently, yolk to albumen ratio was higher for the heavier than for the lighter hens. The authors have not found any published report on the effects of initial BW of the hens on egg quality or on the proportion of egg components to compare with the data of the current research. Probably, heavy hens produce heavier yolks than lighter hens, because of their higher feed intake, which may result in eggs with higher proportion of yolk (Leeson and Summers, 2005). 149 Annex 1: Resumen en Español UNIVERSIDAD POLITÉCNICA DE MADRID DEPARTAMENTO DE PRODUCCIÓN ANIMAL ESCUELA TÉCNICA SUPERIOR DE INGENIEROS AGRÓNOMOS El amplio resumen en lengua castellana que se presenta a continuación se presenta para cumplir con uno de los requisitos necesarios para la presentación de la Tesis Doctoral en Inglés y poder optar así al título de Doctor Ingeniero Agrónomo. EL DOCTORANDO Adriano Pérez Bonilla INGENIERO AGRÓNOMO VºBº DIRECTOR DE TESIS Gonzalo González Mateos Dr. INGENIERO AGRÓNOMO 150 Annex 1: Resumen en Español ANNEX I: Resumen esumen en Español 151 Annex 1: Resumen en Español 1. Introducción La producción global de huevos y su comercialización han mostrado un remarcable y dinámico crecimiento durante los últimos 40 años. Desde el año 1970 hasta el año 2009 la producción de huevo se incremento de una manera más rápida que la producción de carne de bovino como de porcino. En 1970 la producción mundial de huevo contabilizaba 19.540 millones de toneladas, situándose USA, Rusia, Japón y China como los 4 principales países productores. En 2009 la producción de huevo alcanzó niveles de 62,8 millones de toneladas siendo China, USA, India y Japón los países que lideran la producción (FAOSTAT, 2011). La producción de huevos creció entre los años 1960 y 2007 de manera consistente y rápida en Asia, especialmente en China. En el mismo sentido, África y América del Sur obtuvieron crecimientos continuos, sin embargo en Europa y Oceanía dichos incrementos tuvieron una menor importancia cuantitativa (FAOSTAT, 2011). En el año 2007, la producción asiática contabilizó 38 millones de toneladas de huevo, 2.3 millones de toneladas en África y 3.4 millones de toneladas en América del Sur. Debido al manejo y problemas relacionados con la logística, es importante los huevos que son comercializados internacionalmente. De hecho, en el año 2008 las exportaciones mundiales de huevo llegaron a valores de 4.083 millones de dólares, un crecimiento de un 17.7% comparado con datos del año 2004 (FAOSTAT, 2011). Los países exportadores de huevo sin Holanda, China, España y Polonia. La importación total de huevos en 2008 supuso un valor de 3.850 millones de dólares, un incremento del 15.3% comparado con datos del año 2004 (FAOSTAT, 2011). Los principales países importadores de hubo son Alemania, Holanda, Francia y China. Según datos del FAOSTAT (2011), el consume per capita de huevo mejoró en el período comprendido entre el año 2000 y el 2007. La media global se incrementó desde 152 Annex 1: Resumen en Español 8.1 kg hasta 8.6 kg por persona y año en 2007. Particularmente incipiente es el crecimiento del consumo per cápita en Asia (8.8 kg de huevo por persona y año). Estudios llevados a cabo por la FAO indican que en el año 2010 el consumo medio mundial se situó en 9.2 kg por persona y año (un incremento de un 7% respecto al año 2007). Según el MARM (2010) el número total de ponedoras en España se situó en 44 millones de aves. Este censo representó un total de 1370 explotaciones. Aproximadamente, 95.7% de las ponedoras presentes en España se alojaron en jaulas, 4.2% supusieron las ponedoras explotadas en suelo (camperas y aviario) y sólo u 0.1% fueron ponedoras explotadas bajo el régimen de producción biológica u orgánica. La legislación referente al bienestar animal ha producido variaciones en estos. La producción española obtuvo en el año 2004 el mejor dato productivo de su historia (1.13 x 103 millones de docenas). De esta producción Castilla-La Mancha (32%), Castilla y León (17%), Valencia (9%) y Cataluña (8%) representaron más del 60% de la producción global española. En general la avicultura de puesta española tiene un marcado carácter exportador, de hecho, del total de la producción española, aproximadamente un 23% del total de la producción fue utilizado para la exportación. Los principales países destinatarios de esta mercancía se sitúan dentro de la Unión Europea con Francia (41%), Alemania (14%), Reino Unido (12%), Holanda (11%) y Portugal (11%) (MARM, 2010). En cuanto a la tendencia en el consumo de huevo, en el período comprendido entre el año 2000 y 2009, el consumo de huevo descendió desde 17.5 kg por persona y año a 11.3 kg por persona y año (un descenso de un 36%). El éxito económico de toda industria dedicada a la producción de huevos depende de la masa de huevo producida por cada ave alojada en la instalación a lo largo de todo el período de puesta. Este objetivo depende de la duración del período de puesta así como del número de huevos producidos y el tamaño de los mismos. Secundariamente, el 153 Annex 1: Resumen en Español porcentaje de huevos comercializables y el coste relativo de las materias primas son importantes factores que han de ser considerados para alcanzar estos objetivos económicos. La obtención del mayor número de huevos comercializables, está relacionado con la calidad externa e interna del huevo, reduciéndose la incidencia de huevos rechazados antes de su comercialización. Unos datos productivos óptimos dependen principalmente de la capacidad genética del animal unido a un óptimo estatus sanitario. En este sentido, el manejo de la nutrición y alimentación tanto en pollitas como en ponedoras son factores que contribuyen a la mejora de dichos parámetros productivos. Así, el éxito económico de las explotaciones de gallinas ponedoras requiere una curva de producción óptima, con una persistencia alta a lo largo de todo el período de puesta y un pico de puesta máximo, acorde con la genética del ave. Es generalmente aceptado que un pico de producción elevado está positivamente relacionado con una masa de huevo por ave alojada elevada. El tamaño del huevo tiene unas importantes connotaciones relacionadas con el éxito económico de toda explotación de ponedoras, especialmente en países como España donde el consumidor prefiere huevos con un peso mayor para el consumo. Consecuentemente, bajo estas circunstancias, los productores tienden a incrementar la duración del período de puesta debido a que las gallinas incrementan el peso del huevo a medida que incrementan su edad. El objetivo del nutricionista es formular dietas que maximicen las variables productivas (porcentaje de puesta y peso del huevo), desde los primeros estadios productivos y reduciendo los problemas que aparecen al final del ciclo productivo. Para cumplir con estos objeticos, el nutricionista necesita jugar con los niveles de nutrientes, requerimientos energéticos, proteicos y aminoacídicos, así como el uso de distintas materias primas que tienen una importancia clave en cuanto al coste de las fórmulas (cereales y grasas). Secundariamente, el nutricionista necesita obtener lotes 154 Annex 1: Resumen en Español con una alta uniformidad y peso vivo adecuados a la estirpe al inicio del período de puesta. Unas variables productivas y una calidad del huevo óptima son los 2 principales factores que tienen que producirse en una industria de avicultura de puesta. Para alcanzar estos 2 objetivos, un buen manejo nutricional de las aves es clave. Es importante, por tanto, comprobar los efectos del manejo nutricional de las aves a lo largo del periodo de puesta mediante la comprobación sistemática de la calidad externa del huevo (porcentaje de huevos rotos, sucios y fárfaras) e interna del huevo (altura de albumen, color de yema, y proporciones de yema y albumen). En general, según datos de Roland et al., (1988) las pérdidas desde las granjas hasta el consumidor final se situó entre un 5-7% para el total de la producción. La mayoría de esas pérdidas están relacionadas con una calidad pobre de la cáscara especialmente al final del período de puesta. En el presente trabajo de investigación se estudiaron los efectos de componentes nutricionales clave tanto para las variables productivas como para la calidad del huevo en gallinas ponedoras rubias a lo largo de todo el período experimental. Los factores estudiados fueron: 1) El efecto del cereal principal y el tipo de grasa en la dieta, 2) El efecto del nivel de proteína bruta y grasa añadida en la dieta, 3) El efecto del nivel energético in la dieta, 4) El efecto del peso inicial de las gallinas al inicio del período de puesta, sobre las variables productivas y la calidad del huevo.. 2. Revisión bibliográfica Los cereales son ricos en almidón y son comúnmente utilizados como fuentes energéticas en raciones para avicultura. Además, los cereales suministran parte de proteína bruta y aminoácidos requeridos por las aves. La utilización del almidón por las aves depende principalmente del cereal utilizado ya que difieren en la naturaleza y estructura. La digestión del almidón depende de factores como el contenido de la pared celular, la 155 Annex 1: Resumen en Español naturaleza granular del almidón y la presencia de factores antinutritivos en el grano, así como la capacidad digestiva del animal (Classen, 1996). Numerosos estudios han sido realizados para obtener el valor nutricional de diferentes almidones provenientes de cereales en gallinas ponedoras. Sin embargo, el valor nutricional de dichos almidones no ha sido ajustado todavía. Los principales cereals producidos en España y utilizados en dietas para ponedoras son el Maíz (Zea mays L.), Trigo (Triticum L.) y Cebada (Hordeum vulgare L.). El Maíz tiene menos proteína bruta (7.5% vs. 10.2% vs. 9.6%) y fibra bruta (2.3% vs. 2.6% vs. 4.7%). Sin embargo, tiene más almidón (63.3% vs. 60.2% vs. 53%), grasa bruta (3.6% vs. 1.6% vs. 1.8%), ácido linoléico (1.81% vs. 0.64% vs. 0.71%) y energía (3,280 vs. 3,100 vs. 2,800 kcal/kg) que el trigo y la cebada. (Fundación Española Desarrollo Nutrición Animal, 2010). La composición química y el valor nutricional de maíz is bastante uniforme comparada con la del trigo y la cebada, sin embargo, dichas características dependen principalmente de factores como el cultivar de origen, las prácticas agronómicas, las condiciones climáticas, el tiempo de almacenaje, las características físicas del grano y en último caso el tipo de ave (Pirgozliev et al., 2003; Gutiérrez-Álamo et al., 2008; Frikha et al., 2011). En condiciones prácticas, muchos productores de huevo formulan dietas para ponedoras con una mínima cantidad de maíz con el objetivo de asegurar altos consumos, maximizando de esta manera el tamaño del huevo sobre todo en las primeras fases del ciclo de puesta, momento especialmente importante cuando una estimualción temprana es utilizada en las pollitas. En general, las razones de esta práctica son desconocidas pero quizá estén relacionadas con la mayor uniformidad del valor nutricional del maíz y la obtención de una mejor estructura del pienso al utilizar una molienda grosera en dicho cereal (Frikha et al., 2009). Asimismo, el maíz tiene una mayor concetración de ácido linoleico (LNL) en comparación con el trigo y la cebada, con lo cual, el mayor contenido 156 Annex 1: Resumen en Español de LNL en la dieta quizá resulte en incrementos en el peso del huevo sobre todo en aves jóvenes bajo condiciones climáticas que propicien bajos consumos (Jensen et al., 1958; Scragg et al., 1987; Grobas et al., 1999a). Por otro lado, el trigo y la cebada contienen altas cantidades de factores antinutritivos como son los polisacáridos no amiláceos en forma de arabinoxilanos y βglucanos, los cuales, incrementan la viscosidad de la digesta y producen una disminución en los rendimientos productivos de los animales (Lázaro et al., 2003, García et al., 2008). Por ello, el nivel de inclusión del trigo y la cebada en dietas para aves depende de varios factores como son la especie, la edad de los animales y el perfil de nutrientes incluyendo la energía, el contenido en proteína y los polisacáridos no amiláceos. Diversos estudios se han llevado a cabo con el objetivo de comparar estos tres cereales (maíz, trigo y cebada) y su efecto en las variables productivas de ponedoras, broilers y pollitas. En general, estos estudios sugieren que el trigo y la cebada son una buena alternativa al maíz en dietas para estas 3 especies. En ponedoras, Craig y Goodman. (1993), Lázaro et al. (2003), Liebert et al. (2005), y Safaa et al. (2009) han obtenido resultados productivos similares comparando el maíz con el trigo y la cebada suplementadas con enzimas exógenas (xilanasas y β-glucanasas) mientras que otros autores como es el caso de Coon et al. (1988) obtuvieron mayores consumes y peor IC en animales alimentados con cebada suplementada con enzima, respesto de los animals alimentados con maíz. En broiler, Mathlouthi et al. (2002) obtuvo parametros productivos similares cuando un 60% de maíz en la dieta fue sustituido por una combinación de 40% de trigo y 20% de cebada. En el mismo sentido, Ruiz et al. (1987) obtuvo una ganancia diara e IC similar en broilers alimentados en harina cuando el maíz fue sustituido por el trigo. Sin embargo, contrariamente a estos resultados Crouch et al. (1997) al comparar el maíz con dos variedades de trigo en un 40% de inclusión en dietas en harina, obtuvo mejor ganancia media diaria e IC en animales alimentados con una de las dos variedades de trigo. En 157 Annex 1: Resumen en Español pollitas, Frikha et al. (2009) obtuvo una mayor ganancia media diaria en pollitas alimentadas con maíz que las que fueron alimentadas con trigo, siendo ambas dietas suplementadas con enzimas. Las razones de estas discrepancias son desconocidas pero quizá estén relacionadas con el diseño de las dietas basales. La infomación disponible sobre los efectos de la utilización de distintos cereales en la dieta sobre la calidad del huevo es escasa. En general, la inclusión de cebada y trigo ha sido asociada a un aumento en la incidencia de huevos sucios en comparación con el maíz (Francesch et al., 1995). En este sentido, Lázaro et al. (2003) al sustituir maíz por trigo en dietas de gallinas ponedoras blancas en el período 20-44 sem de vida obtuvo un mayor porcentaje de huevos sucios en los animales alimentados con trigo respecto de los alimentados con maíz. Sin embargo, otros autores (Jamroz et al., 2001; Çiftci et al., 2003; Safaa et al., 2009) informan que dietas basadas en trigo y cebadas suplementadas con enzimas obtuvieron los mismos resultados en cuanto al porcentaje de huevos sucios que los animales alimentados con maíz. Las grasas son utilizadas en avicultura con el objetivo de incrementar la energía de las dietas. La inclusión de grasa en la dieta normalmente esta asociada a un incremento en la peso del huevo. La utilización de grasas produce a menudo incrementos en la energía ingerida por el ave, asi como incrementos en el peso vivo y el peo del huevo (Grobas et al., 2001; Bouvarel et al., 2010), probablemente debido a una mejora en la palatabilidad del pienso como consecuencia de una menor formación de finos (ISA Brown, 2011). La suplementación de grasa ha demostrado tener un efecto positivo en la reducción del tránsito digestivo, facilitando el contacto de la digesta con las enzimas digestivas, mejorando la digestibilidad y utilización de otros componentes de la dieta como son los carbohidratos (Mateos y Sell, 1980b, 1981). Whitehead et al. (1993) estudiaron los efectos de la grasa suplementada en la dieta sobre el peso del huevo concluyendo que los animales alimentados con aceite de maíz obtuvieron un mayor peso 158 Annex 1: Resumen en Español de huevo respecto de los animales alimentados con grasa poliensaturada de cadena larga (aceite de pescado), saturadas de cadena media y larga (Sebo) y saturada de cadena corta (aceite de cacahuete). La razón para la mejora del aceite de maíz respecto del resto de grasas fuera un perfil de ácidos grasos más apropiado (poliinsaturado de cadena medialarga) para el incremento del peso del huevo. Grobas et al. (2001) estudiaron el efecto de cuatro grasas suplementadas en la dieta sobre el peso del huevo, y reporta que las gallinas alimentadas con aceite de soja obtuvieron huevos con un peso mayor que las gallinas alimentadas con aceite de linaza, aceite de oliva o sebo. Un factor a tener en cuenta en el manejo de las grasas en la dieta es el que comentan Atteh and Leeson (1983, 1984, 1985) al estudiar el efecto del perfil de los ácidos grasos sobre el metabolismo mineral de la ponedora y el broiler. Estos autores reportan que la grasa y algunos minerales en la dieta pueden interferirse mutuamente, llegándose a la formación de sopas o jabones insolubes responsables del descenso en la absorción tanto de los ácidos grasos presentes en la grasa como de los minerales. Según estos autores, dicho efecto tendría una mayor relevancia dependiendo de la presencia de ácidos grasos saturados (palmítico y estárico) en la dieta. Diversos estudios han mostrado que la reducción en la grasa añadida al pienso produce una disminución en el tamaño del huevo (Keshavarz y Nakajima, 1995; Grobas et al., 1999a,b; Bohnsack et al., 2002; Sohail et al., 2003). Así, Grobas et al. (2001) reportan que la grasa suplementada en la dieta mejora el peso del huevo y la masa de huevo exportada tanto en gallinas blancas como en gallinas rubias a lo largo de todo el período de puesta. El mismo autor Grobas et al. (1999b) al comparar dietas isonutritivas que diferían en el contenido de grasa añadida desde 0 a 4% en gallinas rubias en el período 22-65 sem, observan que la grasa añadida mejora las rendimientos productivos y el peso del huevo. Sin embargo el mismo autor (Grobas et al., 2001) no obtuvo nuevos incrementos en el peso del huevo al aumentar la inclusión de grasa en la dieta desde 5% a 10%. Whitehead et al. (1993) utilizando 5 niveles de inclusion de grasa (0, 10, 20, 40, 159 Annex 1: Resumen en Español and 60 g/kg dieta) y 4 tipos de grasas (aceite de maíz, aceite de cacahuete, sebo y aceite de pescado) observó que a excepción del aceite de pescado, el cual mejoraba los rendimientos productivos hasta niveles de 20 g/kg de dieta, el resto de grasas obtuvieron buenos resultados productivos hasta niveles de 40 g/kg dieta. Los principales efectos de la grasa añadida al pienso sobre la calidad del huevo están relacionados con la proporción relativa tanto de yema como de albumen. Grobas et al. (1999a) observaron que el incremento en el peso del huevo, como consecuencia de la inclusión de grasa añadida al pienso, incremento tanto el porcentaje de yema como de albumen en una cantidad cercana al 3.5%. Posteriormente, dichos autores (Grobas et al., 1999b) observaron incrementos tanto en la proporción de yema como de albumen pero el incremento fue proporcionalmente mayor en el albumen. Whitehead et al. (1991) observaron que la grasa añadida incrementó el peso de la yema debido a la estimulación en la deposición lipídica y el peso del albumen mediante la estimulación estrogénica. Posteriormente, Whitehead (1995) reportó que los efectos beneficiosos de la grasa añadida al pienso sobre la proporción de albumen son debidos a la influencia de ciertos ácidos grasos insaturados en la producción estrogénica, la cual, es la principal responsable de la secreción de albumen. Sin embargo, (Usayran et al., 2001; Grobas et al., 2001) no observaron ningún efecto en las unidades haugh con la grasa añadida al pienso. En cuanto al efecto de la grasa añadida al pienso sobre el resto de variables relacionadas con la calidad del huevo destacar que Parsons et al. (1993) observarón que una reducción en la grasa añadida al pienso desde un 6% a un 2% redujo la proporción de huevos de gramaje L y XL en gallinas blancas al igual que Bohnsack et al. (2002). Trabajos experimentales previos han mostrado que la grasa añadida tiene un efecto positivo sobre el peso del huevo, debido entre otras cosas, a un incremento en LNL de la dieta a medida que la grasa se incrementa en la dieta (Shannon and Whitehead, 1974; Sell et al., 1987; Keshavarz, 1995; Grobas et al., 1999a). De este modo, el efecto y 160 Annex 1: Resumen en Español necesidades de LNL para maximizar la productividad de las ponedoras y el peso del huevo es objeto de debata. En condiciones comerciales, muchas guías de manejo de gallinas ponedoras (H& N International, 2008; Lohmann, 2010) recomiendan incrementar el nivel de LNL en la dieta con un mínimo de 1.8% con el objetivo de maximizar le peso del huevo. Sin embargo, Shannon and Whitehead (1974) y Whitehead (1984) recomendaron niveles de 1.0% en la dieta mientras Scragg et al. (1987) recomendó niveles por encima de un 2.0% para maximizar el peso del huevo en gallinas rubias. Ribeiro et al. (1997) observaron un mayor peso del huevo en reproductoras pesadas alimentadas con un nivel de LNL de 1.9% respecto de gallinas alimentadas con niveles de un 1.5%. Grobas et al. (1999b) estudió el efecto del nivel de LNL en gallinas ponedoras rubias en el período 22-65 sem de vida y observó que la reducción desde 1.65% hasta 1.15% en el nivel de LNL no afectó a las variables productivas. Estos autores concluyeron que las necesidades en LNL en gallinas ponedoras rubias con el objetivo de maximizar la productividad en el período 22-65 sem no es mayor que 1.15%. Asimismo, Grobas et al. (1999c) observaron que niveles de LNL de 0.79% tendió a reducir el peso del huevo respecto de niveles de 1.03 o 2.23% en gallinas ponedoras rubias en el período 20-32 sem de vida, sin embargo, el resto de variables productivas no se vieron afectadas por el tratamiento. Respecto de la influencia del nivel de LNL sobre los parámetros de calidad del huevo, Grobas et al. (1999c) observaron que el nivel de LNL no afectó al porcentaje de huevos comercializables, porcentaje de huevos rotos, sucios, unidades haugh, o la proporción de yema o albumen. March y McMillan (1990) y Whitehead et al. (1993) indicaron que la suplementación con LNL en dietas deficitarias en este ácido graso esencial, incremento el peso de la yema, probablemente mediante una mejora en el mecanismo de síntesis de lipoproteínas llevado a cabo en el oviducto. 161 Annex 1: Resumen en Español El concepto de perfil de proteína ideal puede ser definido como el balance exacto de AA, sin deficiencias ni excesos, necesarios para el mantenimiento y la producción del ave. Maximizar la efectividad en el uso de proteína conlleva el reducir las emisiones de nitrógeno, reducir los costes de producción e incrementar el beneficio de la explotación. El nivel de proteína bruta y el nivel de AA tienen un importante rol en el tamaño del huevo. Así, las necesidades de una gallina ponedora son entre 2-4 g para su mantenimiento y entre 10-13 g para la producción de huevo. En el período de puesta, las gallinas necesitan al menos 17 g de proteína bruta para expresar su máximo potencial genético (Summers, 1986). Las dietas para gallinas ponedoras están formuladas para cubrir las necesidades para los AA limitantes en la producción de huevo como son la Lys, Met, Thr y TSSA. Según el NRC (1994) las dietas basadas en maíz y harina se soja con un 15.0% de proteína bruta pueden satisfacer las necesidades en AA en gallinas ponedoras que consumo de media diaria 110 g. Sin embargo, las actuales guías de manejo comercial de ponedoras (Lohmann, 2010; ISA Brown, 2011) recomiendan niveles de proteína bruta que varían entre 17.4 %-18.2 %. Existen discrepancias sobre el efecto del nivel de proteína bruta y el peso del huevo. En general es aceptado que el peso del huevo se incrementa a medida que el nivel de proteína bruta es incrementado (Hawes y Kling, 1993; Hussein et al., 1996; Bouvarel et al., 2010) especialmente al comienzo del período de puesta (Parsons et al., 1993). Sin embargo, otros autores no observarón beneficios en los parámetros productivos utilizando niveles por encima de los recomendados por el NRC (1994) de 16.5%. Keshavarz y Nakajima (1995) observaron que el incremento en el peso del huevo con el incremento en el nivel de proteína bruta fue debido a un incremento en la proporción de albumen. Sin embargo, después del pico de puesta las gallinas tendieron a consumir más viéndose aumentado la proporción de grasa corporal en estos animales (Proudfoot et al., 1988). Por ello, es una buena práctica reducir el porcentaje de proteína en la dieta a lo largo del 162 Annex 1: Resumen en Español período de puesta con el objetivo de mejorar la eficacia nutricional (Harms, 1986). Summers (1986) observó al final del ciclo de puesta la existencia de un coste energético extra producido como consecuencia de los procesos oxidativos para eliminar el exceso de nitrógeno, produciéndose heces líquidas, huevos de mayor tamaño y una calidad de cáscara pobre. Como contraste, diversos autores (Pilbrow y Morris, 1974; Wethli y Morris, 1978; Huyghebaert et al., 1991; Joly, 1995) recomiendan mantener un nivel de proteína al final del período de puesta debido a la pobre eficiencia en el uso de los AA por parte de las gallinas viejas. Ballam (1985) observó que las necesidades en aminoácidos fueron mayores para optimizar el peso del huevo que para optimizar el porcentaje de puesta de los animales. Este autor estimó que se puedía utilizar un incremento del 10% en Met y Lys para incrementar el peso del huevo sin ningún efecto en el porcentaje de puesta. En el mismo sentido, Summers et al. (1991) observaron que una deficiencia en el nivel de proteína bruta de la dieta afectó de manera más importante al tamaño del huevo que a la producción de huevo. Sin embargo, Morris y Gous (1988) se mostraron en desacuerdo con estos resultados ya que observaron que los coeficientes de variación para ambas variables (peso del huevo y porcentaje de puesta) eran distintos, concretamente 0.20 y 0.10 respectivamente; con lo cual, pequeñas diferencias en el peso del huevo darían diferencias significativas mientras que las mismas diferencias en el caso del porcentaje de puesta no serían significativamente distintas. Estos mismos autores realizaron una revisión de las necesidades en proteína bruta y AA en gallinas ponedoras, observando reducciones similares en el peso del huevo y en la producción de huevo con reducciones de un 10% en el nivel de proteína bruta de la dieta, sin embargo, con reducciones mayores la reducción fue mas severa en la puesta que en el peso del huevo. Schutte y col. (1994) realizaron una revision sobre una serie de experimentos basados en las necesidades de AA azufrados y Met. Así, Roland et al. (1992) 163 Annex 1: Resumen en Español recomiendan altos niveles de AA azufrados al comienzo del periodo de puesta, mientras que Summers y Leeson (1993) y Klien y Hawes (1990) no observaron ninguna mejora en los parámetros productivos. El segundo aminoácido limitante en las dietas es la Lys (March y Biely, 1963; Sell y Hodgson, 1966). Joly (1995) observó que una deficiencia en la cantidad de lys en la dieta producía una disminución en la masa de huevo, debido a una reducción en la producción de huevo y en peso de huevo de 65% y 35% respectivamente. En el mismo sentido, Nathanael y Sell (1980) observaron que el peso del huevo se incrementó de forma cuadrática con el incremento del nivel de lys en la dieta. Contrariamente, Harms y Ivey (1993) y Prochaska et al. (1996) no detectaron ningun efecto sobre las variables productivas al incrementar el nivel de lys en la dieta. Es posible que en el estudio de Nathanael y Sell (1980) otro AA fuera limitante. En general es aceptado que la calidad del huevo, incluyendo el porcentaje de huevos sucios, la altura de albumen y la calidad de cáscara son afectados por el contenido de proteína en la dieta. En este sentido, Hammershoj y Kjaer (1999) observaron un empeoramiento en las unidades haugh a medida que el nivel de proteina bruta se incrementó desde 13.7% hasta 17.9%. Sin embargo, Fariborz et a. (2007) observaron en dietas isoenergéticas que diferían en el contenido de protein bruta (16.3 vs. 17.8%) que la altura de albumen, espesor de cáscara y resistencia a la fractura de la cáscara no fueron afectadas por el nivel de proteína en la dieta. El análisis del balance energético es la manera de calcular el consumo de alimento diario y la producción diaria en el ave (De Blas, 1991). Este autor, analizó una serie de experimentos estimando unas necesidades medias de mantenimiento de 107,8 kcal EMA/kg0,75 con unos márgenes desde 90 a 120 kcal EMA/kg0,75, 8,39 kcal EMA/g para incrementos en el PV y 1,94-2,25 kcal EMA/g para cubrir las necesidad energéticas de producción. Así, una gallina ponedora con un PV de 2.0 kg con una GMD de 0.8 g/d, con una massa exportada de huevo de 58 g/d, necesita entre 300-320 kcal de EMA por día. 164 Annex 1: Resumen en Español Las gallinas comen para satisfacer sus necesidades energéticas, consecuentemente, un incremento en el nivel energético de la dieta debería disminuir el consumo proporcionalmente (Hill et al., 1956). Bouvarel et al. (2010) revisaron una serie de experimentos llevados a cabo en gallinas ponedoras en los últimos 20 años llegando a la conclusion que, como media, un incremento del 10% en el contenido energetico de la dieta reduce sólo un 5.5% el consumo de los animales. Los cambios en la concentración energética de la dieta han producido resultados contradictorios respecto a la productividad de las aves (Harms et al., 2000). Grobas et al. (1999c) observaron que un incremento en la EMAn desde 2,680 a 2,810 kcal/kg (un incremento de un 4.8%) disminuyó el consumo en la misma proporción (un 5.0%) pero la producción de huevos y la masa exportada por animal no fue afectada por el tratamiento. En el mismo sentido, Peguri et al. (1991) observaron un descenso de un 5% en el consumo cuando el nivel energético de la dieta fue incrementado desde 2,700 a 2,910 kcal/kg ( un incremento de un 8%). Sin embargo, Joly y Bougon (1997) observarón en gallinas rubias en el período 19-68 sem de vida un incremento de un 1.3% en la puesta y un 4.5% en la masa de huevo a medida que el contenido energético se incremento desde 2,200 a 2,700 kcal de EMAn. Diversos trabajos publicados sobre el efecto del nivel energético de la dieta informan que el incremento en el nivel de EMA en la dieta conlleva un incremento en el peso del huevo (De Groote, 1972; Walker et al., 1991). En general, las gallinas tienden a mantener su ingesta de energía modificando el consumo (Leeson et al., 1973; Newcombe y Summers, 1985), sobreconsumiendo energía en dietas altas en energía (Morris, 1968; De Groote, 1972; Walker et al., 1991) y con ello este exceso de nutrientes produce el incremento en el peso del huevo (De Groote, 1972; McDonald, 1984; Leclerq, 1986; Walker y col., 1991). Según estos autores el peso del huevo mejora en unos márgenes entre 0.10% - 0.21% por cada 100 kcal. Bouvarel et al. (2010) analizando datos de 11 trabajos experimentales llevados a cabo en los últimos 20 años informó que el peso del 165 Annex 1: Resumen en Español hubo se incremento en 0.96 g por cada 100 kcal de incremento en la energía de la dieta. Las razones de las discrepancias entre autores en relación con los efectos de incrementar el contenido energético de la dieta sobre el peso del huevo no son claros pero quizá estén relacionados con el nivel de grasa y LNL en la dieta control. Los efectos del nivel energetico de la dieta sobre las variables productivas en ponedoras muestran diferentes resultados. Mathlouthi et al. (2002) observó en gallinas blancas que la producción de huevo aumentaba a medida que el contenido energético de la dieta aumentó desde 2,650 a 2,750 kcal EMA/kg, mientras, Grobas et al., (1999c) en gallinas rubias observó el mismo efecto utilizando dietas desde 2,680 a 2,810 kcal EMAn/kg. Sin embargo, Jalal et al. (2006, 2007) en gallinas blancas utilizando dietas que variaron desde 2,800 a 2,900 kcal EMA/kg no detectaron ningún efecto en la producción de las aves. En general, en condiciones prácticas es una práctica común incrementar el nivel energético de la dieta al inicio del período de puesta, especialmente, cuando las pollitas recibidas en la nave de puesta no tienen un peso adecuado ni homogéneo. Así, algunos autores observan que en climas cálidos el incremento de la concentración energética de la dieta mejora los parámetros productivos especialmente en gallinas ligeras (Kling y Hawes, 1990; Daghir, 1995). Respecto al efecto del nivel energético sobre la calidad del huevo, existen discrepancias entre autores. Así, Grobas et al. (1999a) observaron que el incremento en el contenido energético de la dieta no afecto al porcenaje de huevos sucios, rotos o fárfararas a lo largo del período de puesta. Algunos autores han observado que el nivel energético de la dieta tiene influencia en la calidad del albumen. Así, Wu et al. (2005) observaron que un incremento en el contenido energético de la dieta desde 2,720 a 2,960 kcal EMA/kg oiriginó un descenso en las unidades haugh. Sin embargo, Zimmermann y Andrews (1987) y Junqueira et al. (2006) no observaron ningun efecto en la calidad del albumen cuando el contenido energético de la dieta fue incrementado. 166 Annex 1: Resumen en Español Las xantofilas, la principal fuente de pigmentación de la yema de huevo son solubles principalmente en la grasa. Gunawardana et al. (2008) observaron una mayor pigmentación de la yema en gallinas blancas alimentadas con un 5.0% de grasa añadida respect de gallinas alimentadas con una dieta control sin grasa añadida. Lázaro et al. (2003) observaron una mayor pigmentación de yema en gallinas blancas con dietas que contenían una mayor concentración energética. Además, cuando la grasa es utilizada para incrementar el nivel energético de la dieta la proporción de cáscara presente en el huevo puede verse afectada. Junqueira et al. (2006) observaron en gallinas ponedoras rubias un descenso lineal en la proporción de cáscara a medida que el nivel energético de la dieta se incremento desde 2,850 a 3,050 kcal EMA/kg en el período 76-84 sem de vida. Contrariamente, Gunawardana et al. (2008) no observaron ningun efecto al aumentar en nivel energético de la dieta desde 2,750 a 2,050 kcal EMA/kg sobre la proporción de cáscara. El consumidor español tiene preferencia por huevos con mayor gramaje, por los cuales paga un mayor precio en el Mercado. En consecuencia, los productores de huevo necesitan obtener un alto porcentaje de huevos de tamaños L y XL. La cantidad de huevos L en la primera parte del ciclo de puesta es un reto. Así, el incremento del porcentaje de huevos de mayor tamaño, el consumo diario, el peso vivo y una adecuada uniformidad del lote de pollitas que inicia el período de puesta bajo condiciones de clima calido como es el caso de España, es un reto a alcanzar (Frikha et al., 2009). La información disponible sobre los efectos del peso vivo inicial al comienzo del período de puesta sobre los parámetros productivos y la calidad del huevo es muy escasa. El peso vivo al inicio del período de puesta es el principal factor que influye en la productividad de las gallinas ponedoras. El peso del huevo a lo largo del ciclo de puesta esta fuertemente influenciado por el peso vivo inicial del ave al inicio de puesta (Harms et al., 1982; Leeson and Summers, 1987). Las gallinas que inicial el ciclo de puesta con 167 Annex 1: Resumen en Español un peso por encima del estándar racial, tienen consumos mayores y producen huevos de mayor gramaje a lo largo del ciclo de puesta respecto de gallinas que inician la puesta con un peso inferior al estándar racial (Summers and Leeson, 1983; El Zubeir and Mohammed, 1993). Bish et al. (1985) observó que gallinas blancas pesadas (1,377 g) producían huevos más pesados que gallinas de peso medio (1,256 g) o gallinas ligeras (1,131 g). Además, las gallinas pesadas produjeron mayor masa de huevo pero tuvieron un IC por kg similar a las gallinas ligeras, confirmando los resultados de Keshavarz (1995) el cual observó diferencias en el peso del huevo de 1.4 g entre 2 grupos de ponedoras clasificadas como pesadas (1,333 g) o ligeras (1.151 g) en el período productivo 18-62 sem de vida. La información disponible sobre los efectos del peso vivo inicial sobre la calidad del huevo es muy escasa. En general, es aceptado que los huevos procedentes de gallinas más pesadas (por encima del estándar racial), son más pesados que los procedentes de gallinas ligeras (por debajo del estándar racial). Asimismo, las gallinas pesadas poseen una mayor proporción de yema y menor de albumen que las gallinas ligeras. Probablemente, las gallinas pesadas producen yemas de mayor tamaño ya que su consumo diario es mayor y como consecuencia el tamaño del huevo es mayor (Leeson and Summers, 2005) 168 Annex 1: Resumen en Español 3. Objetivos de la Tesis Doctoral El objetivo principal de la presente Tesis Doctoral fue estudiar la influencia de diversos factores que afectan tanto a la productividad como a la calidad del huevo en gallinas ponedoras rubias. Así, para alcanzar estos objetivos, se llevaron a cabo 3 experimentos en el período 2009-2011 bajo condiciones comerciales de manejo. El efecto del tipo de cereal principal y el tipo de grasa en la dita (experimento 1), el nivel de proteína bruta en la dieta y el peso vivo inicial de la gallina al inicio de puesta (experimento 2), y el nivel de energía en la dieta y el peso vivo inicial de la gallina al inicio de puesa (experimento 3) fueron diseñados bajo los anteriores objetivos. Secundariamente, se estudio la influencia del manejo sobre los factores nutricionales y el uso de materias primas clave en la nutricion de ponedoras con el objetivo de reducir los costes de producción a nivel práctico. 169 Annex 1: Resumen en Español Efectos del cereal principal y el tipo de grasa en la dieta sobre los parámetros productivos y la calidad calid del huevo en gallinas ponedoras rubias en el periodoo 22-54 semanas de vida (Experimento 1) PUBLICADO EN: POULTRY SCIENCE 90:2801-2810 90:2801 doi:10.3382/ps.2011 doi:10.3382/ps.2011-01503 170 Annex 1: Resumen en Español La hipotesis del presente experimento fue que cuando una dieta basada en trigo y cebada es suplementada con enzimas, ambos cereales pueden sustituir al maiz sin ningun efecto negativo sobre las variables productivas o la calidad del huevo. De forma similar, se podría utilizar indistintamente, oleína vegetal o manteca en sustitución de aceite de soja sin ningun efecto negativo en los parámetros productivos o en la calidad de huevo. El peso del huevo en gallinas alimentadas con dietas que contenían 4.3% de grasa añadida al pienso podrían maximizarse con niveles de ácido linoléico en la dieta de 1.0% aproximadamente, suponiendo un consumo medio de los animales de 116 g/gallina y dia y una ingesta de 1.16 g LNL/gallina/dia. El objetivo de este experimento fue estudiar la influencia del cereal principal en la dieta (maíz, trigo y cebada) y el tipo de grasa añadida al pienso (aceite de soja, oleína vegetal y manteca) sobre los parámetros productivos y a calidad de huevo en gallinas ponedoras rubias en el período 22-54 semanas de vida. 1. Material y metodos 1.1. Crianza, Programa de Alimentación y Dietas Experimentales Todos los procedimientos experimentales realizados fueron aprobados por el comite de ética de la Universidad Politécnica de Madrid y estuvo acorde con la Guía Española para el cuidado y el uso de animales en experimentación (Boletín Oficial del Estado, 2005). En total, 756 gallinas ponedoras rubias de la estirpe Lohmann fueron obtenidas de un lote comercial (El Canto Agroalimentaria S.L, Toledo, Spain). Desde la semana 20 a la 22 de vida todos los animales comieron un pienso pre-experimental común basado en maíz-harina de soja. En la semana 22 de vida todas las gallinas fueron pesadas individualmente y alojadas al azar en grupos de 7 animales en jaulas provistas de un comedero y 2 bebederos de tetina en una nave bajo control automatizado ambiental. Cada tratamiento fue replicado 4 veces y la unidad experimental consistió en 3 jaulas (600 x 171 Annex 1: Resumen en Español 575 mm; General Ganadera S.A, Valencia, Spain) adjacentes (21 gallinas). La temperatura ambiental en la nave varió acorde con el mes considerado (22 ± 3 ºC en Marzo, primer mes de la prueba y 28 ± 3 ºC en Julio, último més de la prueba). El programa de luz fue constante y consistió en 16 horas de luz al día. Los cereales utilizados en la prueba provenían de proveedores comerciales. Las grasas (aceite de soja, oleína vegetal y manteca) fueron suministradas por Bunge Ibérica S.A. (Barcelona, Spain), Oleínas y Grasas S.L. (Tarragona, Spain), e Ibergrasa S.A. (Madrid, Spain) respectivamente. La oleína vegetal fue una mezcla comercial compuesta principalmente por subproductos de la industria del refino del aceite de palma y soja. Dos lotes de cereales y grasas fueron utilizados durante el experimento: el primer lote para los primeros 4 períodos de 28 dias y el segundo lote para los últimos 4 períodos de 28 dias del experimento. El experimento fue realizado mediante un diseño completamente al azar con 9 dietas organizadas factorialmente con 3 cereales principales en la dieta y 3 tipos de grasas. La cebada y el trigo fueron incluidos en sus respectivas dietas en sustitución de un 45% de maíz. La composición de las dietas fue ajustado para asegurar que tuvieran una EMAn y contenido en AA similar según (Fundacion Española Desarrollo Nutricion Animal, 2003). Sin embargo, no se intento igualar el nivel de LNL de dichas dietas. La principal variación en las 3 dietas que contenían el mismo tipo de cereal fue que al incluir un mismo nivel de grasa (4.3%) tanto la EMAn como el LNL varió acorde a esta inclusión. Así, las dietas basadas en aceite de soja contuvieron una mayor proporción de energía y más LNL que las dietas basadas en oleína vegetal o manteca. Debido al diseño experimental, el nivel de LNL de la dieta varió desde 0.8 to 3.4% dependiendo de la combinación del cereal y la grasa utilizada, así el valor mas bajo de LNL fue la dieta que contenía trigo y manteca y el mas alto fue la dieta basada en aceite de soja y maíz. El contenido en LNL de la dieta basada en trigo o cebada suplementados con manteca fue 172 Annex 1: Resumen en Español más baja que 1.1%, siendo este nivel el recomendado por el NRC (1994) para gallinas ponedores rubias que consumen 110 g. Además, la mayoría de las dietas diseñadas tuvieron niveles inferiores de LNL que los utilizados actualmente por la industria (1.7%2.0%). Sin embargo, todas las dietas cubrieron o excedieron las necesidades en nutrientes recomendadas por Fundacion Española Desarrollo Nutricion Animal (2008). Se utilizó un complejo enzimático commercial, el cual, incluía β-glucanasas y xylanasas (Endofeed, GNC Bioferm Inc., Saskatoon, SK, Canada), y fue utilizado en la dosis recomendada por el fabricante. Asimismo, se utilizó un pigmentante sintético basado en canthaxantina y ésteres de carotenoides (β-apo-8-carotenoide) que fueron incluidos en cantidades fijas en todas las dietas. Todas las dietas fueron molidas con un molino de martillos a través de una criba de 7.5 mm. 1.2. Análisis de Laboratorio Muestras representativas de cereales y piensos fueron molidas en el laboratorio con un molino de martillos (Model Z-I, Retsch Stuttgart, Germany) provisto de un tamiz de 1mm. Posteriormente, se analizó humedad mediante estufa (método 930.01), cenizas totals mediante mufla (método 942.05), nitrógeno por combustión-Dumas (método 990.03) utilizando un analizador LECO (Modelo FP-528, LECO, St. Joseph, MI), almidón mediante la medida de la α-amilasa glucosidasa (metodo 996.11), fibra bruta mediante extracción con dilución ácido-básica (método 962.09), Ca y P mediante espectrofotometría (métodos 968.08 y 965.17) según describe la AOAC Internacional (2000). La fibra neutro detergente y ácido detergente fue determinada de forma secuencial según describe Van Soest et al. (1991). El extracto etéreo fue determinado mediante Soxhlet después de una hidrólisis ácida según describe el Boletín Oficial del Estado (1995) y la energía bruta fue analizada mediante bomba calorimétrica adiabática Modelo 356, Parr Instrument Company, Moline, IL). El contenido en ácidos grasos de los 173 Annex 1: Resumen en Español cereales y grasas fue determinado mediante cromatografía líquida de gases (GC-14B, Shimadzu, Kyoto, Japan) según la metodología descrita por Grobas et al. (1999b). La calidad de las grasas, incluyendo impurezas insolubles (método 926.12), humedad (método 984.20) y material insaponificable (método 933.08) fueron determinados según describe la AOAC International (2000). La materia no elucible, la cual refleja la fracción indigestible de la grasa fue determinada mediante cromatografía de gases (método 977.17) según indica la AOAC International (2000). La acidez oleíca, la cual mide la cantidad de KOH en mg necesarios para neutralizar los ácidos grasos libres presentes en 1 g de grasa fue determinado mediante el método Cd-3d-63 de la AOCS (1998). El índice de peróxidos (método 16) fue determinado según indica el Boletín oficial del Estado (1995). Todos los análisis fueron realizados en muestras por duplicado. El tamaño medio de partícula de los cereales y las dietas de cada lote fabricado, fue determinado por triplicado en muestras de 100 g utilizando un tamizador Retsch (Retsch, Stuttgart, Germany) provisto de 8 tamices con una luz de malla que oscilo entre 5,000 y 40 µm según la metología descrita por ASAE (1995). 1.3. Variables Productivas y Calidad de Huevo Los huevos fueron recogidos diáriamente y el peso del huevo fue medido en todos los huevos producidos 2 días antes de cada control de 28 días. El consumo fue medido por réplica, obteniendo consumos por período y acumulado. La mortalidad fue recogida según se produjo a lo largo de la prueba. Mediante estos registros, el porcentaje de puesta, peso de huevo, masa de huevo, consumo medio diario, e IC por kg y por docena de huevos fue calculado por período y acumulado. El ratio (g/g) entre la masa de huevo o peso de huevo y LNL ingerido por tratamiento fue calculado para todo el período experimental. Asimismo, todas las gallinas fueron pesadas individualmente al inicio y al final de la prueba obteniéndose la ganancia de peso vivo por réplica. 174 Annex 1: Resumen en Español El número de huevos limpios, sucios, rotos, fárfaras y dobles yemas fue recogido diariamente por réplica. Un huevo fue considerado como sucio cuando cualquier tipo de material o mancha fue detectado en la cáscara mediante la evalución de 2 observadores independientes. Todos los huevos utilizados para el cálculo del gramaje fueron clasificados según la European Council Directive (2006) como extra large (> 73 g), large (73-63 g), medium (63-53 g) y small (< 53 g). La densidad de cáscara y la calidad interna del huevo (Unidades Haugh y color de yema) fueron medidas en 10 huevos por réplica, elegidos al azar, el último día de cada período de 28 días. Un equipo multitest QCM System, Technical Services and Supplies, Dunnington, York, UK) fue utilizado para tal fin. De los 10 huevos anteriores, en 5 de ellos, fue medido el espesor de cáscara mediante un micrómetro digital (modelo IT-014UT, Mitotuyo, Kawasaki, Japan) utilizando la media de 3 medidas. Por último, al final del segundo período experimental se midió la proporción de albumen y yema en 10 huevos de cada réplica según la metodología descrita por Safaa et al. (2008). En estos mismos huevos se midió el pH de ambas fracciones mediante un pHmetro (Accumet 910, Kent City, MI) según la metodología descrita por Shang et al. (2004). 1.4. Análisis Estadístico El diseño experimental se baso en un modelo completamente al azar con 9 tratamientos organizados factorialmente. Los efectos principales (tipo de cereal y grasa) y sus interacciones fueron analizados mediante un análisis de varianza utilizando el procedimiento GLM de SAS (SAS Institute, 1990). No se observaron interacciones significativas entre el tipo de cereal y grasa, como consecuencia la interacción fue eliminada del modelo. La homogeneidad de varianzas de los datos de todas las variables fue analizada mediante el Test de Levene (Opción Hovtest del procedimiengo GLM). Todas las variables productivas y de calidad de huevo fueron homogéneas, excepto para 175 Annex 1: Resumen en Español la mortalidad. De este modo, la mortalidad fue analizada después de realizar una transformación logarítmica (arcoseno). Cuando los efectos del cereal y la grasa fueron significativos, se utilizó el Test de Tukey como medida de separción y comparación de medias. Una diferencia fue considerada significativa cuando P< 0.05. 2. Resultados 2.1. Variables Productivas No se detectaron interacciones entre el tipo de cereal grasa a lo largo del período experimental. Los tratamientos no tuvieron efecto sobre las variables productivas en ninguno de los períodos considerados. Teniendo el cuenta el período global, el porcentaje de puesta (92.9, 91.5 y 92.1 % para el máiz, trigo y cebada, respectivamente) y el peso del huevo (64.5, 63.6 y 64.1 g para el máiz, trigo y cebada, respectivamente) no fueron afectados por el tipo de cereal. Consecuentemente, la masa de huevo no fue distinta entre tratamientos (59.9, 58.2 y 59.1 g para el máiz, trigo y cebada, respectivamente). Sin embargo, las gallinas alimentadas con trigo y maíz tuvieron una mayor ganancia de peso que las gallinas alimentadas con cebada (243, 238 y 202 g, respectivamente; P< 0.05). La fuente de grasa no afecto a ninguna de las variables productivas estudiadas, excepto para la ganancia de peso, que fue mayor en las gallinas que consumieron manteca respecto de las gallinas que consumieron aceite de soja u oleína vegetal (251, 221 y 210 g, respectivamente; P< 0.05). La mortalidad media del experimento fue 7.5% y no fue afectada por el tratamiento. En cuanto al ratio entre los g de peso de huevo o g de masa de huevo producidos por g de LNL ingerido, fue menor en gallinas alimentadas con aceite de soja, independientemente del cereal utilizado. Sin embargo dicho ratio fue mayor en gallinas alimentadas con manteca y trigo, seguidas de las alimentadas con manteca-cebada, y manteca-máiz (P < 0.001). 176 Annex 1: Resumen en Español 2.2. Calidad del Huevo Para el periodo global, el porcentaje de huevos sucios, rotos, fárfaras y dobles yemas, así como las unidades haugh y el espesor de cáscara y densidad de cáscara no fueron afectados por la dieta. El color de yema fue mayor (P< 0.001) en huevos de gallinas alimentadas con maíz que en huevos de gallinas alimentadas con trigo o cebada. Asimismo, los huevos de gallinas alimentadas con manteca obtuvieron una pigmentación de huevo mayor (P< 0.001) que los huevos de gallinas alimentadas con aceite de soja u oleína vegetal. En la semana 30 de vida de los animales, la dieta no influyó sobre el porcentaje de yema o albumen, así como el pH de ambas fracciones. 3. Discusión 3.1. Variables Productivas Para el periodo global, el consumo medio diario, el porcentaje de puesta, el peso del heuvo y el IC fue similar para los 3 tipos de cereales, resultados que están en concordancia con experimentos previos (Craig and Goodman., 1993; Lázaro et al., 2003; Safaa et al., 2009) los cuales mostraron que cuando dietas basadas en trigo y cebada son suplementadas con enzimas, la productividad de la ponedora no se ve afectada. Mathlouthi et al. (2002) en broilers, observó que cuando el 60% del maíz en la dieta era suplementado con una combinación de 40% de trigo y 20 de cebada suplementados con enzimas, las variables productivas eran similares. Sin embargo Coon et al. (1988) observaron mayor consumo y peor IC en gallinas alimentadas con dietas basadas en cebada que gallinas que se alimentaron con dietas basadas en maíz. En el presente trabajo, la ganancia de peso fue mayor en gallinas alimentadas con maíz y trigo que las gallinas alimentadas con cebada, resultados que están acorde con los presentados por Berg et al. (1959) el cual, observó una mayor ganancia de peso en 177 Annex 1: Resumen en Español gallinas alimentadas con maíz que en gallinas alimentadas con cebada. En el mismo sentido, Frikha et al. (2009) observó una mayor ganancia de peso en pollitas alimentadas con maíz respecto a las pollitas alimentadas con dietas basadas en trigo y cebada suplementadas con enzimas. En el periodo global la sustitución de aceite de soja por oleína vegetal o manteca no afectó a ninguna de las variables estudiadas excepto para la ganancia media diaria que fue mayor para las gallinas alimentadas con aceite de soja u oleína vegetal. Los autores del presente trabajo no han encontrado ninguna publicación comparando el efecto de estas 3 grasas sobre la ganancia de peso en gallinas. En el presente trabajo, un alto porcentaje de la energía ingerida por las ponedoras que consumieron manteca fue derivado a incrementar la ganancia de peso en vez de depositarse en el huevo en comparación con las gallinas que consumieron aceite de soja u oleína vegetal. Vila y Esteve-García (1996) y Sanz et al. (1999, 2000) observaron que los pollos alimentados con dietas que contenían sebo o manteca tenían una mayor deposición de grasa abdominal respecto de los pollos que consumieron piensos basados en grasas vegetales insaturadas, resultados que son consistentes con los obtenidos en el presente trabajo. Los efectos del LNL sobre el tamaño del huevo fue objeto de debate en el presente trabajo. Bajo condiciones comerciales, muchas guías de manejo de ponedoras (H&N International, 2009; Lohmann, 2010) recomiendan incrementar el nivel de LNL en la dieta, al menos un 1.8% con el objetivo de incrementar el peso del huevo. Scragg et al. (1987) recomendaron niveles por encima de un 2.0% para incrementar el peso del huevo en gallinas rubias. Ribeiro et al. (2007) observaron mayores pesos de huevo en gallinas reproductoras pesadas cuando fueron alimentadas con dietas que tenían niveles de 1.9% de LNL respecto de dietas que contenían 1.5%. Sin embargo, Jensen et al. (1958), Shutze et al., (1959) y Grobas et al. (1999a,b) no apoyan estas prácticas en el uso de niveles de 1.8% de LNL. Las razones de las discrepancias entre nutricionistas a nivel práctico e 178 Annex 1: Resumen en Español investigadores residen en la composición de las dietas utilizadas. Por ejemplo, bajo condiciones comerciales, el incremento del nivel de LNL en la dieta es alcanzado mediante el incremento de grasa en la dieta. De este modo los efectos del nivel de LNL y la inclusión de grasa están confundidos. En este sentido, Grobas et al. (1999b) sugirieron que las gallinas ponedoras no requerían mas de un 1.15% de LNL en la dieta para maximizar el peso del huevo cuando; y que cuando una cantidad mínima de LNL es satisfecha en la dieta, un incremento en la grasa añadida al pienso resulta en nuevos incrementos de peso de huevo. 3.2. Calidad de Huevo El tipo de cereal no afectó a ninguna de las variables estudiadas excepto para el color de yema que fue mayor en los huevos de gallinas que consumieron maíz. El efecto beneficioso de la pigmentación de la yema mediante la utilización de maíz era esperada ya que independientemente del cereal utilizado todas las dietas fueron suplementadas con la misma cantidad de pigmentante sintético. La información disponible sobre los efectos del tipo de cereal en la dieta sobre la calidad del huevo es escasa. Jamroz et al. (2001) observó una calidad de huevo similar en gallinas alimentadas con trigo o maíz, ambas dietas suplementadas con enzimas. Çiftci et al. (2003) y Safaa et al. (2009) observaron que la sustitución de maíz por trigo suplementado con enzimas no afectó al porcentaje de huevos sucios en gallinas leghorn blancas o en ponedoras rubias, respectivamente. Sin embargo, Francesch et al. (1995) observó una mayor incidencia de huevos sucios en gallinas alimentadas con cebada respecto a gallinas alimentadas con maíz.. 179 Annex 1: Resumen en Español 4. Conclusiones × El Maíz, trigo y cebada pueden utilizarse de forma exitosa en dietas para ponedoras en niveles de un 45% asegurándose una cantidad mínima de ácido linoléico. × El aceite de soja, oleína vegetal o manteca pueden ser utilizadas como fuente de energía en la dieta sin ningún tipo de efecto perjudicial. × Bajo condiciones prácticas los requerimientos de las gallinas ponedoras con el objetivo de maximizar el peso del huevo son menores que las recomendaciones de la mayoría de guías de manejo comerciales. × Las actuales prácticas en el manejo de la nutrición de la gallina ponedora comercial de disponer, al menos, de un nivel de ácido linoléico de 1.8% o mayor no están justificadas. 180 Annex 1: Resumen en Español Efectos del nivel de proteína bruta y el contenido de grasa en la dieta sobre los parámetros productivos y la calidad del huevo en gallinas ponedoras rubias con distintos pesos vivos (Experimento 2) PUBLICADO EN: POULTRY SCIENCE 91:1400-1406 91:1400 doi:10.3382/ps.2011 doi:10.3382/ps.2011-01917 181 Annex 1: Resumen en Español La hipótesis del presente trabajo fue que en gallinas que comienzan el ciclo de puesta con un peso por debajo del estándar racial, sería recomendable utilizar niveles altos de proteína bruta en la dieta y AA con el objetivo de optimizar las variables productivas de estos animales. Asimismo, el aumento del nivel de grasa en la dieta conllevaría una recuperación del peso de los animales y como consecuencia una mejora en las variables productivas. El aumento del nivel de proteína y grasa no tendría ningún efecto negativo sobre la calida del huevo. Por ello el objetivo del presente trabajo experimental fue determinar los efectos del incremento de proteína bruta de la dieta desde 16.5% hasta 18.5% y del contenido en grasa de la dieta desde 1.8% hasta 3.6% en dietas isonutritivas sobre los parámetros productivos y la calidad del huevo en el período 22-50 semanas de vida. 1. Material y métodos 1.1. Crianza, Programa de Alimentación y Dietas Experimentales Todos los procedimientos experimentales realizados fueron aprobados por el comite de ética de la Universidad Politécnica de Madrid y estuvo acorde con la Guía Española para el cuidado y el uso de animales en experimentación (Boletín Oficial del Estado, 2007). En total 672 gallinas rubias de la estirpe Lohmann que fueron obtenidas de un lote comercial (El Canto Agroalimentaria S.L, Toledo, Spain), fueron pesadas individualmente y clasificadas como ligeras (1,592 ± 75 g) o pesadas (1,860 ± 86 g) respecto de un peso vivo esperado según la guía de manejo de 1,640 ± 57 g (Lohmann, 2010). Dentro de cada grupo de peso, las gallinas fueron distribuidas al azar dentro de 16 réplicas. Cada réplica estuvo compuesta por 21 gallinas (7 gallinas en 3 jaulas adyacentes) (600 × 575 mm; General Ganadera S.A, Valencia, España). Las 2 semanas anteriores al comienzo del ensayo (20-22 sem) las gallinas fueron alimentadas con un 182 Annex 1: Resumen en Español pienso común basado en maíz y harina de soja. La temperatura de la instalación fue recogida diariamente a lo largo del experimento con una mínima temperatura recogida en marzo (20 ± 3ºC , comienzo del experimento) y una máxima temperatura recogida en Julio (27 ± 3ºC). El programa de luz consistió en 16 horas de luz y 8 de escuridad a lo largo de todo el período experimental. Todas las dietas fueron isoenergéticas (2,750 kcal EMAn/kg) y tuvieron una cantidad similar de AA azufrados. La principal diferencia entre las 3 primeras dietas utilizadas fue el contenido en proteína bruta (16.5%, 17.5% y 18.5%, respectivamente). La última dieta contenía un nivel de proteína bruta de 18.5% pero incluyó un 3.6% de grasa añadida en vez de 1.8% de grasa que incluyeron las otras 3. Ajustes en la composición de ingredientes en la dieta fue realizado para mantener constante el valor nutritivo de todas las dietas. Como consecuencia del diseño experimental, las dietas con un contenido mayor de proteína bruta contenían mayor cantidad de AA, pero en cualquier caso cubrieron las necesidades recomendadas por el NRC (1994) y por la Fundacion Española Desarrollo Nutricion Animal (2008) para gallinas rubias. Todas las dietas fueron molidas con un molino de martillo utilizando un tamaño de criba de 7.5 mm. 1.2. Análisis de Laboratorio Muestras representativas de cereales y piensos fueron molidas en el laboratorio con un molino de martillos (Model Z-I, Retsch Stuttgart, Germany) provisto de un tamiz de 1mm. Posteriormente, se analizó humedad mediante estufa (método 930.01), cenizas totales mediante mufla (método 942.05), nitrógeno por combustión-Dumas (método 990.03) utilizando un analizador LECO (Modelo FP-528, LECO, St. Joseph, MI), Ca y P mediante espectrofotometría (métodos 968.08 y 965.17) según describe la AOAC Internacional (2000). El extracto etéreo fue determinado mediante Soxhlet después de una hidrólisis ácida según describe el Boletín Oficial del Estado (1995) y la energía bruta 183 Annex 1: Resumen en Español fue analizada mediante bomba calorimétrica adiabática Modelo 1356, Parr Instrument Company, Moline, IL). Todos los análisis fueron realizados en muestras por duplicado. El tamaño medio de partícula de los cereales y las dietas de cada lote fabricado, fue determinado por triplicado en muestras de 100 g utilizando un tamizador Retsch (Retsch, Stuttgart, Germany) provisto de 8 tamices con una luz de malla que oscilo entre 5,000 y 40 µm según la metología descrita por ASAE (1995). 1.3. Variables Productivas y Calidad de Huevo Los huevos fueron recogidos diáriamente y el peso del huevo fue medido en todos los huevos producidos 2 días antes de cada control de 28 días. El consumo fue medido por réplica cada 28 días, obteniendo consumos por período y acumulado. La mortalidad fue recogida según se produjo a lo largo de la prueba. Mediante estos registros, el porcentaje de puesta, peso de huevo, masa de huevo, consumo medio diario, e IC por kg y por docena de huevos fue calculado por período y acumulado. Asimismo, todas las gallinas fueron pesadas individualmente al inicio y al final de la prueba obteniéndose la ganancia de peso vivo por réplica. El número de huevos limpios, sucios, rotos, fárfaras y dobles yemas fue recogido diariamente por réplica. Un huevo fue considerado como sucio cuando cualquier tipo de material inerte o mancha fue detectado en la cáscara mediante la evalución de 2 observadores independientes. La calidad del huevo fué medida en 10 huevos por réplica, elegidos al azar, el último día de cada período de 28 días. Cada huevo fue individualmente pesado, y en cada uno de ellos se analizó la calidad del albumen y de la cáscara de cáscara mediante un equipo multitest QCM System, Technical Services and Supplies, Dunnington, York, UK). La densidad de cáscara se calculó como el peso de la cáscara en seco dividido por la superficie de la misma y el espesor de cáscara fue medido mediante un micrómetro digital (modelo IT-014UT, Mitotuyo, Kawasaki, Japan) 184 Annex 1: Resumen en Español utilizando la media de 3 medidas tomadas en la zona medial del huevo. El color de yema se midió mediante escala Roche según la metodología descrita por Vuilleumier et al. (1969). 1.4. Análisis Estadístico El diseño experimental se baso en un modelo completamente al azar con 9 tratamientos organizados factorialmente. Los efectos principales (tipo de cereal y grasa) y sus interacciones fueron analizados mediante un análisis de varianza utilizando el procedimiento GLM de SAS (SAS Institute, 1990). No se observaron interacciones significativas entre el tipo de cereal y grasa, como consecuencia la interacción fue eliminada del modelo. La homogeneidad de varianzas de los datos de todas las variables fue analizada mediante el Test de Levene (Opción Hovtest del procedimiengo GLM). Todas las variables productivas y de calidad de huevo fueron homogéneas, excepto para la mortalidad. De este modo, la mortalidad fue analizada después de realizar una transformación logarítmica (arcoseno). Cuando los efectos del cereal y la grasa fueron significativos, se utilizó el Test de Tukey como medida de separción y comparación de medias. Una diferencia fue considerada significativa cuando P< 0.05. 2. Resultados No fueron dietectadas interaciones entre los tratamientos a lo largo del período experimental. La mortalidad fue considerada normal (4.9%) y no relacionada con el tratamiento. Las dietas experimentales no afectaron a ninguna de las variables productivas estudiadas. Sin embargo, el consumo (120.6 vs 113.9; P< 0.001), el porcentaje de puesta (92.5 vs. 89.8%; P< 0.01), el peso del huevo (64.9 vs. 62.4; P< 0.001) y la masa de huevo (60.0 vs. 56.1; P< 0.001) fueron mayores en las gallinas pesadas respecto de las gallinas ligeras. El IC por kg de huevo no fue afectado por el peso 185 Annex 1: Resumen en Español vivo inicial de las gallinas pero el IC por docena de huevo fue mejor en las gallinas ligeras respecto de las gallinas pesadas (1.52 vs. 1.57; P< 0.01). La ganancia media diaria fue mayor en las gallinas ligeras respecto a las pesadas (289 vs. 233 g; P< 0.01). A lo largo del período experimental las gallinas pesadas tuvieron un mayor consumo y peso del huevo que las gallinas ligeras (P < 0.001). La incidencia de huevos sucios, rotos, fárfaras, calidad del albumen, densidad de cáscara y espesor de cáscara no fueron afectados por el tratamiento experimental o el peso inicial de las gallinas. Sin embargo, el color de yema fue mayor (P< 0.01) en huevos de gallinas que comieron las dietas que tenían 1.8% de grasa respecto de las que comieron el pienso que contenía 3.6% de grasa. 3. Discusión El porcentaje de protein bruta de la dieta no afecto a ninguna de las variables productivas estudiadas, resultados que son consistentes con las recomendaciones del NRC (1994) las cuales indican que las necesidades en AA indispensables son cubiertas cuando a gallinas que consumen 110g se les aporte un pienso con 16.5% de proteina bruta. De hecho, en el presente estudio la media de proteína ingerida por las gallinas en el pienso que contenía 16.5% de proteína bruta estuvo por encima de las recomendaciones del NRC (1994) (18.6 g/d para las gallinas ligeras y 19.8 g/d para las gallinas pesadas, que corresponden a consumos de 112.6 y 119.7 g/gallina /día, respectivamente). Los resultados del presente trabajo están deacuerdo con los datos de Kling et al. (1985). Dichos autores compararon en ponedoras rubias desde el inicio de puesta hasta la sem 66 de vida, dos niveles de Met (0.30% vs. 0.40%) y 2 niveles de proteína bruta (17.0% vs. 19.0%) y observaron que un incremento en el contenido de proteína de la dieta no afectó a ninguna de las variables productivas estudiadas. De forma similar, Junqueira et al. (2006) observaron que un incremento del nivel de proteína de la dieta en gallinas rubias mudadas desde un 16% a un 20% manteniendo constante la EMAn y el nivel de Met no afectó a ninguna de las 186 Annex 1: Resumen en Español variables productivas estudiadas. Summers y Leeson (1983) obtuvieron parámetros productivos similares en gallinas blancas alimentadas con niveles de proteína de 17% o 22% y balanceadas en EMAn y Met desde la semana 20 a la 32 de vida. Sin embargo, Roberts et al. (2007b) observaron en gallinas blancas que un descenso en el contenido de proteína bruta desde 19.8% a 19.1% en el período 45-58 sem de vida en dietas isoenergéticas con un perfil de AA similar, redujo la puesta, la masa de huevo y empeoró el IC. Keshavarz (1995) observarón que un incremento en el nivelde proteína de la dieta desde 17% hasta 21%, con incrementos en el nivel de Met desde 0.34 hata 0.42% no afectó a la puesta, peso del huevo, consumo, o ganancia de peso en gallinas blancas en el período 18-38 semanas de vida. Los resultados del presente experimento apoyan el razonamiento por el cual la ingesta de AA en vez del nitrógeno “per se” es la responsable de modular el tamaño del huevo y las variables productivas de las gallinas. Los efectos de la grasa añadida sobre el consumo, puesta y peso del huevo fueron objeto de debate. Normalmente, un incremento en la concentración energética del pienso va acompañada de in incremento en la grasa añadida al pienso y en el contenido en LNL (Grobas et al., 2001; Frikha et al., 2009). Consecuentemente, los 3 efectos (concentración energética, nivel de grasa añadida y nivel de LNL) están confundidos y no pueden ser separados en la mayoría de las situaciones prácticas en cámpo. En el presente trabajo, un incremento de la grasa añadida al pienso desde 1.8% a 3.6% en dietas isocalóricas, en las cuales, el nivel de LNL estaba por encima de las necesidades (desde 1.9% a 2.5%) no afectó a ninguna de las variables productivas estudiadas. Estos resultados están en concordancia con los observados por Grobas et al. (1999c). Keshavarz (1995) comparó 2 niveles de grasa añadida al pienso (2% y 4%) frente a una dieta sin grasa añadida en gallinas blancas en el período 18-38 semanas de vida, y observó que las dietas no afectaron a variables productivas claves como el peso del huevo, la puesta o la ganancia de peso de los animales. Sin embargo, Keshavarz y Nakajima (1995) observaron que la 187 Annex 1: Resumen en Español inclusión de un 4% de grasa animal y vegetal en dietas isocalóricas en gallinas blancas, incremento el peso del huevo y la ganancia media diaria comparado con la dieta control sin grasa añadida. Bohnsack et al. (2002) observaron en gallinas blancas en el período 2638 sem que los animales que comieron piensos que contenían un 4 y un 6% de grasa añadida tuvieron un mayor peso de huevo que las dietas que no contenían grasa o la que contenía un 2%, sin embargo el resto de variables productivas no se vieron afectadas. Sin embargo hay que señalar que en este estudio la concentración de EMAn de las dietas se incremento con la grasa añadida el pienso, con lo cual, los efectos de la concentración energética y en nivel de grasa añadida están confundidos. Grobas et al. (1999b) observaron en gallinas rubias Isa Brown que la suplementación de la dieta con un 4% de grasa (diferente mezclas de oleína vegetal y grasa animal) manteniendo constante el nivel de LNL en un 1.15%, incrementaba el peso del huevo en gallinas jóvenes (sem 22-26 de vida) pero no en gallinas viejas (sem 74-78 de vida). Safaa et al. (2008) observaron que cuando un incremento en grasa añadida al pienso desde 1.1% a 3.0% manteniendo constante la concentración energética (2,700 kcal EMA/kg) de la dieta, mejoró el porcentaje de puesta, el peso del huevo y el IC por kg de huevo en gallinas rubias estirpe Hy-Line en el período productivo 59-70 sem. Las pollitas que empezaron el ciclo de puesta con un peso por encima del estándar racial consumieron mas pienso y produjeron huevos más pesados que las pollitas que empezaron el ciclo de puesta por debajo del estándar racial. Sin embargo el IC por kg de huevo no fue afectado, de hecho, el IC por docena de huevo fue mejor para las pollitas que empezaron el ciclo de puesta con el peso mas bajo. Los autores del presente trabajo no han encontrado ningún artículo científico reciente sobre los efectos del peso vivo inicial de las pollitas sobre los parámetros productivos. En el presente experimento, el consumo se incremento en 2.7 g y el peso del huevo en 0.93 g por cada 100 g de incremento de peso vivo en las pollitas. Resultados similares fueron observados por 188 Annex 1: Resumen en Español Harms et al. (1982) en 2 experimentos con gallinas blancas desde la sem 31 a la 47 de vida. En el primer experimento, estos autores observaron un incremento de 0.88 g en el peso del huevo por cada 100 g de incremento de peso en gallinas que variaron el peso desde 1,411 g hasta 1,546 g, sin embargo en el segundo experimento, con un rango de peso de 1,546 g y 1,684, el incremento fue de 1.6 g. En ambos experimentos la producción de huevo fue similar y el IC fue peros en gallinas pesadas. Summers y Leeson (1983) compararon las variables productivas en pollias blancas divididas por peso vivo a las 18 sem de vida en 4 grupos (1,107 g, 1,205 g, 1,281 g y 1,383 g). Estos autores observaron que en el período 19-25 sem, las gallinas pesadas obtuvieron mejores porcentajes de puesta que las gallinas ligeras y que por cada 100 g de peso extra el peso del huevo se incremento en 0.9 g, sin embargo el IC por kg de huevo no se vió afectado. De manera similar, Leeson y Summers (1987) distribuyeron pollitas blancas en 3 grupos de peso (997 g, 1,100 g y 1,226 g) en la sem 15 de vida en el experimento 1 y (1,308 g, 1,411 g, y 1,564 g) en la sem 19 en el experimento 2. Los autores observaron que el consumo se incrementó 3.2 g y 3.6 g por cada 100 g de incremento de peso en el experimento 1 y 2 desde el inicio de puesta hasta la sem 67, respectivamente. Keshavarz (1995) observó porcentajes de puesta similares en gallinas blancas en el período 18-62 sem de vida divididas en 2 grupos de peso, ligeras (1,151g) y pesadas (1,333 g), sin embargo, el consumo se incrementó en 2 g y el peso del huevo en 1.4 g por cada 100 g de incremento de peso en los animales. Además, estos autores observaron un incremento en la ganancia de peso en dicho período de 24.2% mayor en gallinas ligeras respecto de las gallinas pesadas, acorde con lo observado en el presente experimento (20.8%). La calidad del huevo, incluyendo porcentaje de huevos sucios, altura de albumen y calidad de cáscar no fueron afectados por la dieta o el peso inicial de las gallinas. Los datos del presente experimento están en concordancia con los presentados por Junqueira et al. (2006) el cual, observó que el incremento del nivel de proteína bruta en la dieta no 189 Annex 1: Resumen en Español agecto a las unidades haugh o la calidad de cáscara. Otros autores confirman estos resultados (Wolford and Tanaka., 1970; Fariborz et al., 2007). De hecho, Williams et a., (1992) indicó que la genética de la gallina y la edad de la misma eran los factores mas importantes que afectan a la calidad del abúmen, y que la nutricion no tiene un gran impacto en esta variable. Sin embargo, Hammershoj y Kjaer (1999) observaron que las unidades haugh descendieron a medida que el nivel de proteína se incremento de 13.7 a 17.9%. La suplementación de la dieta con un nivel alto en proteína con un 3.6% de grasa añadido no afectó a ninguna de las variables relacionadas con la calidad del huevo. En el presente experimento no hubo ningún en la calidad de cáscara relacionado con el incremento de grasa en el pienso, resultados que están en concordancia con los obtenidos por Safaa et al. (2008), los cuales, observaron que la calidad de la cáscara en la última fase de producción en gallinas rubias suplementadas con 1.1% o 3.0% de grasa. En broilers, Atteh et al. (1983) indicó que la inclusión de grasa saturada a la dieta incrementa la formación se jabones entre los ácidos grasos y las sales cálcicas, disminuyendo la retención de calcio. Probablemente, la cantidad de jabones presentes en el intestino delgado en las gallinas del presente ensayo fueran escasos, ya que la grasa utlizada fue una grasa insaturada (aceite de soja), potenciándose la disociación de estos jabones con el pH del tracto gastrointestinal. La pigmentación fue menor en los huevos procedentes de gallinas alimentadas con la dieta que contenía un 3.6% de grasa en relación con el resto de dietas que contenía un 1.8%. Esta diferencia era esperada ya que el maíz fue incluido sólo en las dietas que contenían un 1.8% de grasa añadida. 190 Annex 1: Resumen en Español 4. Conclusiones × Incrementar el nivel de proteína bruta en el pienso desde 16.5% a 18.5% y de grasa añadida al pienso de 1.8% a 3.6% no afectó ninguna de las variables productivas estudiadas o la calidad del huevo en el período productivo 22-50 sem independientemente del peso de la gallina al inicio del ciclo de puesta. × Variables productivas importantes en el rendimiento de la explotación como fueron el porcentaje de puesta, el peso del huevo y el consumo fueron mayores en gallinas pesadas respecto de las gallinas ligeras. Asimismo, las variables relacionadas con la calidad del huevo no se vieron afectadas por el peso inicial de las gallinas al comienzo del ciclo de puesta. × El IC por kg de huevo no fue afectado por el peso inicial de las gallinas, de hecho el IC por docena de huevo fue mejor en las gallinas ligeras respecto de las gallinas pesadas. Ese dato puede indicar que el manejo y gestión de los lotes debe monitorizarse o modularse dependiendo del objetivo comercial de una empresa. Así, el uso de gallinas pesadas o ligeras dependerá de si producimos huevos a granel o estuchados, así como el destino final de la producción, ya que el cliente nacional requiere un tamaño más grande de huevo, sin embargo, si nuestro objetivo es la importación requeriremos un menor gramaje de huevo. × A nivel de campo, la utilización del incremento del nivel de proteína bruta por encima de las recomendaciones del NRC (1994) con el objetivo de maximizar el peso del huevo no está justificada in ninguno de los 2 grupos de gallinas. × En general, las gallinas que inicial el ciclo de puesta con un peso superior al estándar racial poseen unos mejores resultados productivos, pero la ventaja de estos animales depende de la diferencia en el peso del huevo respecto de animales con menor peso, así como del coste relativo de las materias primas. 191 Annex 1: Resumen en Español Efectos de la concentración energetica energetic de la dieta sobre los párametros productivos y la calidad del de huevo vo en gallinas ponedoras rubias con distintos pesos vivos (Experimento 3) ACEPTADO PARA PUBLICAR EN: POULTRY SCIENCE TBC: 1-11 doi:10.3382/ps.201 doi:10.3382/ps.2012-02526 192 Annex 1: Resumen en Español La hipótesis del presente trabajo fué que un incremento en la concentración energetica de la dieta podría incrementar la ingesta caloric de las gallinas mejorando los parámetros productivos. Dicho efecto podría ser más pronunciado en gallinas ligeras que inician el ciclo de puesta con un peso por debajo del estándar racial respecto de gallinas más pesadas. El objetivo del presente estudio fue estudiar los efectos de la concentración energetica de la dieta sobre los parámetros productivos y la calidad del huevo en gallinas ponedoras rubias que tenían 2 pesos distintos al inicio del ciclo de puesta, a lo largo del período productivo entre la semana 24 y 59 de vida. 1. Material y Métodos 1.1. Crianza, Dietas y Diseño Experimental Todos los procedimientos experimentales realizados fueron aprobados por el comite de ética de la Universidad Politécnica de Madrid y estuvo acorde con la Guía Española para el cuidado y el uso de animales en experimentación (Boletín Oficial del Estado, 2007). En total 520 gallinas rubias de la estirpe Hy-Line fueron obtenidas de un lote comercial (Camar Agroalimentaria S.L, Toledo, Spain) y fueron pesadas en la sem 21 de vida individualmente y clasificadas como ligeras (1,606 ± 39 g) o pesadas (1,733 ± 48g) respecto de un peso vivo esperado según la guía de manejo de 1,685 ± 35g (Hy-Line International, 2011). Dentro de cada grupo de peso, las gallinas fueron distribuidas al azar dentro de 16 réplicas. Cada réplica estuvo compuesta por 13 gallinas alojadas en jaulas enriquecidas provistas de un comedero y 2 bebederos de cazoleta (635 x 1,200 mm; Facco S.A., Padova, Italia). Las 2 semanas anteriores al comienzo del ensayo (20-22 sem) las gallinas fueron alimentadas con un pienso común basado en maíz y harina de soja. La temperatura de la instalación fue recogida diariamente a lo largo del experimento con una mínima temperatura recogida en enerro (19 ± 3ºC , comienzo del experimento) y 193 Annex 1: Resumen en Español una máxima temperatura recogida en Julio (28 ± 3ºC). El programa de luz consistió en 16 horas de luz y 8 de escuridad a lo largo de todo el período experimental. Todas las dietas fueron isoenergéticas (2,750 kcal EMAn/kg) y tuvieron una cantidad similar de AA azufrados. La principal diferencia entre las 3 primeras dietas utilizadas fue el contenido en proteína bruta (16.5%, 17.5% y 18.5%, respectivamente). La última dieta contenía un nivel de proteína bruta de 18.5% pero incluyó un 3.6% de grasa añadida en vez de 1.8% de grasa que incluyeron las otras 3. Ajustes en la composición de ingredientes en la dieta fue realizado para mantener constante el valor nutritivo de todas las dietas. Como consecuencia del diseño experimental, las dietas con un contenido mayor de proteína bruta contenían mayor cantidad de AA, pero en cualquier caso cubrieron las necesidades recomendadas por el NRC (1994) y por la Fundacion Española Desarrollo Nutricion Animal (2008) para gallinas rubias. Todas las dietas fueron molidas con un molino de martillo utilizando un tamaño de criba de 7.5 mm. Desde la sem 21 hasta la 24 de vida todas las gallinas consumieron un pienso común basado en maíz y harina de soja (2,750 kcal EMAn, 17.5% de proteína bruta y 0.39% de Metionina). Desde la sem 24 de vida (comienzo del experimento) hasta la sem 59 de vida las gallinas fueron alimentadas con 4 dietas que variaron en el contenido energético desde 2,650 hasta 2,950 kcal/kg pero todas tuvieron una cantidad equivalente de nutrientes por unidad energética. Para la fabricación de las 4 dietas experimentales, se formularon las 2 dietas extremas (2,650 y 2,950 kcal/kg), con lo cual, las dietas intermedias fueron obtenidas mezclando cantidades adecuadas de las 2 anteriores. Todas las dietas cubrieron las necesidades nutricionales recomendadas por la Fundacion Española Desarrollo Nutricion Animal (2008). Se utilizó un complejo enzimático commercial, el cual, incluía β-glucanasas y xylanasas (Endofeed, GNC Bioferm Inc., Saskatoon, SK, Canada), y fue utilizado en la dosis recomendada por el fabricante. Asimismo, se utilizó un pigmentante sintético basado en canthaxantina y ésteres de 194 Annex 1: Resumen en Español carotenoides (β-apo-8-carotenoide) que fueron incluidos en cantidades fijas en todas las dietas. El diseño experimental fue completamente al azar con 8 tratamientos ordenados factorialmente con 4 niveles de energia (2,650, 2,750, 2,850, y 2950 kcal EMA/kg) y 2 pesos de gallinas (1,733 vs. 1,606 g). Cada tratamiento fue replicado 5 veces y la unidad experimental fue una jaula con 13 gallinas. 1.2. Análisis de Laboratorio Muestras representativas de cereales y piensos fueron molidas en el laboratorio con un molino de martillos (Model Z-I, Retsch Stuttgart, Germany) provisto de un tamiz de 1mm. Posteriormente, se analizó humedad mediante estufa (método 930.01), cenizas totales mediante mufla (método 942.05), nitrógeno por combustión-Dumas (método 990.03) utilizando un analizador LECO (Modelo FP-528, LECO, St. Joseph, MI), Ca y P mediante espectrofotometría (métodos 968.08 y 965.17) según describe la AOAC Internacional (2000). El extracto etéreo fue determinado mediante Soxhlet después de una hidrólisis ácida según describe el Boletín Oficial del Estado (1995) y la energía bruta fue analizada mediante bomba calorimétrica adiabática Modelo 1356, Parr Instrument Company, Moline, IL). Todos los análisis fueron realizados en muestras por duplicado. El tamaño medio de partícula de los cereales y las dietas de cada lote fabricado, fue determinado por triplicado en muestras de 100 g utilizando un tamizador Retsch (Retsch, Stuttgart, Germany) provisto de 8 tamices con una luz de malla que oscilo entre 5,000 y 40 µm según la metología descrita por ASAE (1995). 1.3. Productive Performance and Egg Quality Los huevos fueron recogidos diáriamente y el peso del huevo fue medido en todos los huevos producidos 2 días antes de cada control de 28 días. El consumo fue medido por 195 Annex 1: Resumen en Español réplica cada 28 días, obteniéndose consumos por período y acumulado. La mortalidad fue recogida según se produjo a lo largo de la prueba. Mediante estos registros, el porcentaje de puesta, peso de huevo, masa de huevo, consumo medio diario, IC por kg y por docena de huevos, y eficiencia energética expresado como las calorías de EMAn por g de huevo fue calculado por período y acumulado. Asimismo, todas las gallinas fueron pesadas individualmente al inicio y al final de la prueba obteniéndose la ganancia de peso vivo por réplica. El número de huevos limpios, sucios, rotos, fárfaras y dobles yemas fue recogido diariamente por réplica. Un huevo fue considerado como sucio cuando cualquier tipo de material inerte o mancha fue detectado en la cáscara mediante la evalución de 2 observadores independientes. Las Unidades haugh y el color de yema fue medido por réplica en 10 huevos elegidos al azar el último día de la sem 39, 48, 55 y 59 de vida mediante equipo multitest (QCM System, Technical Services and Supplies, Dunnington, York, UK) según metodología descrita por Pérez-Bonilla et al. (2011). La proporción de cascara, albumen, y yema de los huevos, así como el ratio yema:albumen fue determinado por réplica en los mismos 10 huevos recogidos para las medidas de calidad de huevo. La yema y la cáscara fueron separados del albumen utilizando un papel secante, eliminando cualquier tipo de material adherido según metología descrita por Saraa et al. (2008). El peso del albumen fue calculado por diferencia entre el peso del huevo y los pesos de la yema y la cáscara. 1.4. Statistical Analysis El diseño experimental se baso en un modelo completamente al azar con 8 tratamientos organizados factorialmente. Los efectos principales (nivel de energía y peso inicial) y sus interacciones fueron analizados mediante un análisis de varianza utilizando el procedimiento GLM de SAS (SAS Institute, 1990). Se testó mediante el procedimiento 196 Annex 1: Resumen en Español UNIVARIATE y Test de Levene la distribución normal de los residuos y la homogeneidad de varianzas. La mortalidad fue analizada mediante el procedimiento GENMOD de SAS (SAS Institute, 1990), utilizando una distribución binomial. La función link utilizada para el análisis fue la función logit (ln(µ/1-µ)) siendo µ la media del tratamiento.Cuando los efectos del cereal y la grasa fueron significativos, se utilizó el Test de Tukey como medida de separción y comparación de medias. Además, contrastes polinomiales fueron diseñados utilizando el procedimiento REG de SAS (SAS Institute, 1990) para estudiar el efecto linal (L) y cuadrático (Q) de la concentración energética de la dieta. Una diferencia fue considerada significativa cuando P< 0.05. 2. Resultados 2.1. Variables Productivas No se observaron interacciones entre el contenido energético de la dieta y el peso inicial de las gallinas para ninguna de las variables estudiadas a lo largo del período experimental. Para el período global considerado, el porcentaje de puesta (88.8, 91.2, 92.7 y 90.5%; L, P< 0.01; Q, P< 0.01), la masa de huevo (56.1, 58.1, 58.8 y 58.1 g/d; L, P< 0.01; Q, P< 0.01), energía ingerida (304, 313, 316 y 324 kcal/gallina y día; L, P< 0.001), eficiencia energética (5.42, 5.39, 5.38 y 5.58 kcal AMEn/g egg; L, P< 0.001; Q, P< 0.001), y ganancia media diaria (255, 300, 325 y 359 g; L, P< 0.001) se incrementaron a medida que el contenido energético de la dieta se incremento desde 2,650 hasta 2,950 kcal/kg. Sin embargo, el consumo (114.8, 114.0, 111.0 y 110.0 g; L, P< 0.001), IC por kg de huevo (2.05, 1.96, 1.89 y 1.89 kg/kg; L, P< 0.001; Q, P< 0.01) y por docena de huevo (1.54, 1.48, 1.42 y 1.44 kg/docena; L, P< 0.01; Q, P< 0.01) disminuyo a medida que el contenido energético de la dieta se incrementó. El peso del huevo y la mortalidad no fueron afectados por la dieta. 197 Annex 1: Resumen en Español El peso inicial de las gallinas afectó a varias variables productivas, incluyendo peso del huevo, masa del huevo, consumo e IC por docena de huevo. Para el periodo experimal global, las gallinas pesadas consumieron más pienso (113.9 vs. 111.0 g; P< 0.001) ingirieron más energía (321 vs. 311 kcal/gallina por día; P < 0.001), tuvieron mayor masa de huevo (58.5 vs. 57.0 g; P <0.01) y mayor peso de huevo (64.2 vs. 63.0 g; P< 0.01) que las gallinas ligeras. Sin embargo, el porcentaje de puesta, el IC por kg de huevo, la eficiencia energética, la ganancia de peso y la mortalidad no fueron afectadas por el peso inicial de las gallinas. 2.2. Calidad de Huevo La dieta no afectó al porcentaje de huevos sucios, rotos, fárfaras o la proporción de yema y albumen en el huevo. Sin embargo, las unidades haugh (L, P< 0.001) y la cáscara de huevo (L, P< 0.001) disminuyó y la pigmentación de la yema aumentó (L, P< 0.001) a medida que la concentración energética se incrementó. El peso inicial de las gallinas no afecto al porcentaje de huevos sucios, rotos o fárfaras. La proporción de yema fue mayor (P< 0.001) y la de albumen fue menor (P< 0.01) en las gallinas pesadas respecto de las ligeras. Consecuentemente, el ratio yema:albumen fue mayor (P< 0.001) para las gallinas pesadas. 3. Discusión 3.1. Variables Productivas Las gallinas comen para satisfacer sus necesidades energéticas, de tal manera que un incremento en el nivel energético de la dieta disminuye proporcionalmente el consumo (Hill et al., 1956). Sin embargo, en el presente experimento, un incremento de un 11% en la energía de la dieta (desde 2,650 hasta 2,950 kcal EMAn/kg) disminuyó el consumo, pero solamente un 4%, obteniéndose un incremento en la energía ingerida de un 7%. 198 Annex 1: Resumen en Español Estos resultados coinciden con los obtenidos por Bouvarel et al. (2010), la cual, observó en una serie de experimentos llevados a cabo en los últimos 20 años, que como media, un incremento de un 10% en la EMAn redujo el consumo pero sólo un 5.5%. Keshavarz (1998) en gallinas blancas observaron en el período 18-66 sem de vida que un incremento de un 8% en la EMAn de la dieta desde 2,815 hasta 3,035 kcal/kg incrementó la energía ingerida en un 9%. Los datos indican que las gallinas ponedoras tienden a consumir energía extra cuando la EMAn de la dieta es incrementada. Probablemente, la inclusión de cantidades extra de grasa pueda mejorar la palatabilidad del pienso produciéndose un mayor consumo. El porcentaje de puesta se incrementó a medida que la concentración energética de la dieta se incrementó desde 2,650 hasta 2,850 kcal/kg, pero nuevos incrementos energéticos hasta 2,950 kcal/kg no resultaron en nuevas mejoras. Mathlouthi et al. (2002) observaron en gallinas blancas que la producción se incrementó a medida que el nivel energético de la dieta se incrementó desde 2,650 hasta 2,750 kcal/kg. Sin embargo, Grobas et al. (1999c) en gallinas rubias alimentadas con dietas que variaron desde 2,680 hasta 2,810 kcal/kg, Harms et al. (2000) en gallinas blancas y rubias alimentadas con dietas que variaron desde 2,500 hasta 3,100 kcal/kg, y Jalal et al. (2006, 2007) en gallinas blancas alimentadas con dietas que variaron desde 2,800 hasta 2,900 kcal/kg, no detectaron ninguna diferencia significativa en producción de huevo con cambios en el contenido energético de la dieta. El peso del huevo no fue afectado por el incremento en la concentración energética deacuerdo con los datos observados de Grobas et al. (1999b), Çiftci et al. (2003), y Valkonen et al. (2008). Sin embargo, Harms et al. (2000) y Wu et al. (2005, 2007b) observaron que el peso del huevo aumentó de forma lineal con el incremento en el nivel energético de la dieta. Las razones de estas discrepancias entre autores no son aparentes pero quizá estén relacionadas con el nivel de LNL y contenido de grasa de la dieta control. Cuando la concentración energética de la 199 Annex 1: Resumen en Español dieta se incrementa, hay un incremento tanto en el nivel de LNL como en el contenido de grasa. En el presente experimento, el contenido de LNL de la dieta control fue 1.35%, probablemente suficiente para maximizar el peso del huevo (Jensen et al., 1958; Shutze et al., 1962; Irandoust et al., 2012). Además, el nivel de grasa añadida utilizada para incrementar el nivel energético se incrementó desde 0.92 hasta 6.02%. Grobas et al. (1999c) observó que un incremento en la grasa añadida de la dieta incrementó el peso del huevo. En el presente experimento la masa de huevo se incrementó a medida que la concentración energética de la dieta aumentó desde 2,650 hasta 2,750 kcal/kg, sin embargo nuevos incrementos hasta 2,850 o 2,950 kcal/kg no originaron nuevas mejoras. Estos resultados siguen la tendencia de los observados por Keshavarz (1998), el cual, obtuvo una masa de huevo similar en gallinas ponedoras blancas alimentadas con dietas que variaron en su concentración energética desde 2,820 hasta 3,040 kcal EMAn/kg. Sin embargo, Joly y Bougon (1997) observaron un incremento en la masa de huevo de 4.5% a medida que la concentración energética del pienso se incrementó desde 2,200 hasta 2,700 kcal/kg en gallinas rubias en el período productivo 19-68 sem de vida. Probablemente, el incremento en la concentración energética del pienso sea mas efectiva mejorando la masa del huevo cuando las dietas utilizadas en los ensayos posean un nivel energético mas bajo. El IC por kg de pienso mejoró a medida que el nivel enegético de la dieta se incrementó, en consonancia con los datos publicados por diversos autores (Grobas et al., 1999a,b; Wu et al., 2005). Sin embargo, Keshavarz (1998) no obtuvo diferencias in gallinas blancas en el período 18-66 sem de vida alimentadas con dietas que variaron desde 2,820 hasta 3,040 kcal EMAn/kg. En el presente trabajo, las gallinas alimentadas con la dieta alta en energía (2,950 kcal EMAn/kg) tuvieron un menor consumo pero una mayor ingesta energética que las gallinas que comieron el resto de dietas, sin embargo, el exceso de energía fue derivado a incrementar el peso vivo en vez de mejorar la producción. Consecuentemente, la eficiencia de convertir la energía del 200 Annex 1: Resumen en Español alimento en masa de huevo fue mejorada cuando se utilizó la dieta alta en energía (2,950 kcal EMAn/kg). Sin embargo, las gallinas que consumieron la dieta baja en energía (2,650 kcal EMAn/kg) consumieron menos energía que las del resto de dietas. Probablemente la cantidad de energía consumida por las gallinas de la dieta baja en energía fue menor que el necesario para optimizar la producción, obteniéndose una menor masa de huevo. Es posible que las gallinas alimentadas con la dieta baja en energía no pudieran incrementar su consumo para cubrir sus necesidades energéticas debido a limitaciones en la capacidad del tracto gastrointestinal. La ganancia media diaria se incrementó 0.11 g/gallina y dia por cada 100 kcal de aumentó en la concentración energética, un valor que es menor que los 0.20 g observados por Grobas et al. (1999c) en gallinas rubias en el período 22-65 sem de vida con dietas que contenían 2,680 y 2,810 kcal/kg; y los 0.45 g observados por Harms et al. (2000) en gallinas leghorn blancas en el período 36-44 sem de vida con dietas que contenían 2,520 o 3,080 kcal/kg. Sin embargo, Keshavarz (1998) obstuvo un incremento en el peso vivo en gallinas blancas de 0.014 utilizando dietas con 2,820 o 2,040 kcal/kg en el período 20-66 sem de vida. Las actuales gallinas ponedoras quizá respondan a incrementos en la concentración energética de la dieta con incrementos moderados en el peso vivo, o con grandes incrementos cuando se utilizan dietas con una alta concentración energética. Los resultados del presente trabajo sugieren que las gallinas ponedoras modernas quizá no regulen de una forma precisa su consumo con el objetivo de cubrir sus necesidades energéticas cuando dietas con una concentración energética extrema (alta o baja) son utilizadas. Las gallinas que consumieron la dieta alta en energía (2,950 kcal/kg) tendieron a sobreconsumir energía con un efecto positivo sobre la ganancia de peso de los animales, pero no se observaron mejoras en la masa del huevo, mientras que las gallinas que consumieron la dieta baja en energía (2,650 kcal/kg) tendieron a reducir su consumo energético con un impacto negativo sobre la masa de huevo. 201 Annex 1: Resumen en Español La información disponible sobre los efectos del peso vivo inicial de las gallinas sobre las variables productivas son muy limitados. Las gallinas pesadas al inicio del ciclo de puesta consumieron más y produjeron huevos mas grandes a lo largo de todo el ciclo de puesta respecto de las gallinas ligeras (Summers y Leeson, 1983; El Zubeir y Mohammed, 1993). Bish et al. (1985) observó que gallinas blancas pesadas (1,377 g) producían huevos más pesados que gallinas medianas (1,256 g) y ligeras (1,131 g), resultados que son consistentes con los obtenidos en el presente experimento. Además las gallinas pesadas produjeron más huevos pero el IC por kg de huevo fue similar que el de las gallinas ligeras, confirmando los resultados de Keshavarz (1995). El peso del huevo se incrementó significativamente con incrementos en el peso vivo inicial de las gallinas. Keshavarz (1995) observó una diferencia de 1.4 g entre gallinas ligeras con un peso de 1,151 g y gallinas pesadas con un peso de 1,333 g en el período 18-62 sem de vida. 3.2. Calidad de Huevo La concentración energética de la dieta no afectó al porcentaje de huevos sucios, rotos o fárfaras a lo largo del período de puesta, consistentes con los datos obtenidos por Grobas et al. (1999a). Sin embargo, la calidad del albumen disminuyó con el incremento en la concentración energética de la dieta. Wu et al. (2005) obseraron una disminución en las unidades haugh cuando la dieta se incremento de 2,720 a 2,960 kcal/kg. Las raones de las discrepancias entre autores respecto de la variación en las unidades haugh con incremento en la concentración energética de la dieta no son claros. En el experimento de Wu et al. (2005) las dietas no eran isonutritivas, así los autores sugieren que el descenso en las unidades haugh podrían originiarse debido a la menor ingesta de AA por parte d las gallinas que comieron la dieta alta en energía. Sin embargo, en el presente estudio el descenso en las unidades haugh ocurrió siendo todas las dietas isonutritivas por unidad energética. En este sentido, puede ser que las diferencias residieran en las materias primas 202 Annex 1: Resumen en Español utilizadas. La principal diferencia en la composición de las dietas fue que la dieta alta en energía tenía más trigo y menos cebada que las dietas con menos energía. Sin embargo, Lázaro et al. (2003) no observó ningún efecto del tipo de cereal sobre la calidad del albumen. La pigmentación de la yema se incrementó linalmente con el incremento de la concentración energética de la dieta a pesar de que todas las dietas tuvieron niveles similares de maíz y pigmentante sintético. Las Xantofilas, el principal pigmentante responsable del color de la yema, es soluble en grasa. A medida que incrementamos la concentración energética de la dieta, el nivel de grasa fue incrementado favoreciendo la absorción de xantofilas en el tracto gastrointestinal de la gallina. Similares resultados han sido observados por Lázaro et al. (2003) y Gunawardana et al. (2008). La proporción de cascara en el huevo disminuyo linalmente con el incremento de la concentración energética de la dieta in concordancia con los resultados de Junqueira et al. (2006) el cual observó un descenso lineal en la proporción de cáscara a medida que se incremento el contenido energético de la dieta desde 2,850 hasta 3,050 kcal/kg en gallinas rubias en el período 76-84 sem de vida. Sin embargo, Gunawardana et al. (2008) no observó ningún efecto en la proporción de cáscara al utilizar 4 niveles de grasa añadida al pienso (aumentando desde 0 hasta 238 kcal EMAn/kg). El nivel de grasa que incrementa la concentración energética del pienso quizá pueda formar jabones con las sales cálcicas presentes en el alimento, produciéndose una reducción en la retención de calcio y en el peso relativo de la cáscara (Atteh and Leeson; 1983b, 1984). Sin embargo, Safaa et al. (2008) obtuvieron una calidad de cáscara similar en la última fase de producción en gallinas rubias alimentadas con dietas que incluyeron 1.1 o 3.0% de una mezcla de aceite de soja y aceite de palma. Probablemente la proporción de ácidos grasos saturados en la fracción lipídica quizá propició la formación de jabones. El porcentaje de huevos sucios, rotos y fárfaras, unidades haugh y color de yema no fueron afectados por el peso inicial de las gallinas. Sin embargo, los huevos de las 203 Annex 1: Resumen en Español gallinas pesadas tuvieron una mayor proporción de yema y menor de albumen que las gallinas ligeras. Consecuentemente, el ratio yema:albumen fue mayor para las gallinas pesadas que para las gallinas ligeras. Los autores no han encontrado ningún artículo científico publicado sobre los efectos del peso vivo inicial de las gallinas sobre la calidad del huevo o la proporción de los distintos componentes del huevo. Probablemente, las gallinas pesadas producen yemas más pesadas que las gallinas ligeras debido a que su consumo es mayor, obteniéndose huevos con mayor proporción de yema (Leeson and Summers, 2005). 4. Conclusiones × Un incremento en el contenido energético de las dieta desde 2,650 hasta 2,950 kcal EMAn/kg afectó a las variables productivas y la calidad del huevo a lo largo del período de puesta × Las gallinas que consumieron la dieta alta en energía (2,950 kcal EMAn/kg) tuvieron una mayor ingesta de energía que las gallinas que consumieron las dietas con 2,750 y 2,850 kcal EMAn/kg, respectivamente, pero el exceso de energía ingerida fue derivado a incrementar el peso vivo en vez de mejorar la productividad de las gallinas. × Las gallinas que consumieron la dieta baja en energía (2,650 kcal EMAn/kg) tuvieron un consumo energético por debajo de sus necesidades con el objetivo ve maximizar los resultados productivos. × Un incremento en la concentrción energética de la dieta disminuyó la calidad del albumen y la proporción de cáscara en el huevo, pero no afecto al ratio yema:albumen. × Las gallinas pesadas tuvieron un mayor consumo y mayor masa de huevo que las gallinas ligeras, pero la eficiencia energética de ambos grupos no fue afectada por la concentración energética. 204 Annex 1: Resumen en Español × Un incremento en la concentración energética de la dieta incrementó el peso vivo de las gallinas, pero la respuesta fue similar para todas las gallinas independientemente de su peso al inicio del ciclo de puesta. × Las gallinas pesadas tuvieron un ratio yema:albumen mayor que las gallinas. × Los datos productivos fueron mejores en gallinas pesadas que en gallinas ligeras pero la ventaja económica de incrementar el peso vivo de las gallinas al inicio del ciclo de puesta quizá dependa de los precios de los distintos tamaños de huevo así como del coste relativo de las materias primas. 205 Annex 1: Resumen en Español Conclusiones generales e Implicaciones de la Tesis Doctoral 206 Annex 1: Resumen en Español Conclusiones Generales e Implicaciones × El Maíz, trigo y cebada pueden utilizarse de forma exitosa en dietas para ponedoras en niveles de un 45% asegurándose una cantidad mínima de ácido linoléico sin afectar a los parámetros productivos o a la calidad del huevo. El aceite de soja, oleína vegetal o manteca pueden ser utilizadas como fuente de energía en la dieta sin ningún tipo de efecto perjudicial sobre los parámetros productivos o a la calidad del huevo. × Incrementar el nivel de proteína bruta en el pienso desde 16.5% a 18.5% y de grasa añadida al pienso de 1.8% a 3.6% no afectó a ninguna de las variables productivas estudiadas o la calidad del huevo. × Un incremento en el contenido energético de las dieta desde 2,650 hasta 2,950 kcal EMAn/kg afectó a las variables productivas y la calidad del huevo a lo largo del período de puesta. × En general, las gallinas que inician el ciclo de puesta con un peso superior al estándar racial poseen unos mejores resultados productivos, pero la ventaja de estos animales depende de la diferencia en el peso del huevo respecto de animales con menor peso, así como del coste relativo de las materias primas. × Bajo condiciones prácticas los requerimientos de las gallinas ponedoras con el objetivo de maximizar el peso del huevo son menores que las recomendaciones de la mayoría de guías de manejo comerciales. Las actuales prácticas en el manejo de la nutrición de la gallina ponedora comercial de disponer, al menos, de un nivel de ácido linoléico de 1.8% o mayor no están justificadas. × Variables productivas importantes en el rendimiento de la explotación como fueron el porcentaje de puesta, el peso del huevo y el consumo fueron mayores en gallinas pesadas que en las gallinas ligeras. Las variables relacionadas con la calidad del huevo no se vieron afectadas por el peso inicial de las gallinas al comienzo del ciclo de puesta. 207 Annex 1: Resumen en Español × El IC por kg de huevo no fue afectado por el peso inicial de las gallinas, de hecho el IC por docena de huevo fue mejor en las gallinas ligeras respecto de las gallinas pesadas. Ese dato puede indicar que el manejo y gestión de los lotes debe monitorizarse o modularse dependiendo del objetivo comercial de una empresa. Así, el uso de gallinas pesadas o ligeras dependerá de si producimos huevos a granel o estuchados, así como el destino final de la producción, ya que el cliente nacional requiere un tamaño más grande de huevo, sin embargo, si nuestro objetivo es la importación requeriremos un menor gramaje de huevo. × A nivel de campo, la utilización del incremento del nivel de proteína bruta por encima de las recomendaciones del NRC (1994) con el objetivo de maximizar el peso del huevo no está justificada in ninguno de los 2 grupos de gallinas. × Las gallinas que consumieron la dieta alta en energía (2,950 kcal EMAn/kg) tuvieron una mayor ingesta de energía que las gallinas que consumieron las dietas con 2,750 y 2,850 kcal EMAn/kg, respectivamente, pero el exceso de energía ingerida fue derivado a incrementar el peso vivo en vez de mejorar la productividad de las gallinas. × Las gallinas que consumieron la dieta baja en energía (2,650 kcal EMAn/kg) tuvieron un consumo energético por debajo de sus necesidades con el objetivo ve maximizar los resultados productivos. × Un incremento en la concentración energética de la dieta disminuyó la calidad del albumen y la proporción de cáscara en el huevo, pero no afecto al ratio yema:albumen. × Las gallinas pesadas tuvieron un mayor consumo y mayor masa de huevo que las gallinas ligeras, pero la eficiencia energética de ambos grupos no fue afectada por la concentración energética. × Un incremento en la concentración energética de la dieta incrementó el peso vivo de las gallinas, pero la respuesta fue similar para todas las gallinas independientemente de su peso al inicio del ciclo de puesta. 208 Annex 1: Resumen en Español × Las gallinas pesadas tuvieron un ratio yema:albumen mayor que las gallinas. 209 Annex 1: Resumen en Español Referencias Atteh, J. 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