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MASTER THESIS Effect of fiber inclusion in the rearing diets and energy concentration of the laying diets on productive performance and egg quality of brown egg-laying hens from 18 to 46 weeks of age OMAR BOUALI Animal Production Engineer 2013 The supervisors: Dr. Gonzalo GONZALES MATEOS & Dra. Rosa Lázaro García راقولا و ةنيكسلا اوملعت و ،ملعلا اوملعت باطخلا نب رمع ************** Acquire knowledge, and learn tranquility and dignity Omar ibn al-Khattab AGRADECIMIENTOS El presente trabajo de investigación fue realizado bajo la supervisión de Dr Gonzalo Gonzáles Mateos y Dra Rosa Lázaro Garcia. A quienes me gustaría expresar mi más profundo agradecimiento, por hacer posible la realización de este estudio. Además, de agradecer su paciencia, tiempo y dedicación que tuvieron para que esto saliera de manera exitosa. Gracias por su apoyo por ser parte de la columna vertebral de mi tesis de Master. A Dios por protegerme durante todo mi camino y darme fuerzas para superar obstáculos y dificultades a lo largo de toda mi vida. A la dirección de la empresa, Camar Agroalimentaria por permitirme la realización de esta Tesis, y en especial, a Medín de Vega y Jorge Herrera por confiar en mí a lo largo de todo este periodo A la dirección de IAM Zaragoza, Dr. Ignacio Romagoza, Dr. José Antonio Guada y Dr. Armando de Occon por su disponibilidad continúa para ayudarme A Dr. Carlos de de Blas Beorlegui y Dr. Javier García por haber compartir conmigo sus conocimientos y sus oportunos consejos A mi padre Mounir, que siempre lo he sentido presente en mi vida. Y sé que está orgulloso de la persona en la cual me he convertido. A mi madre Aicha, que con su demostración de una madre ejemplar me ha enseñado a no desfallecer ni rendirme ante nada y siempre perseverar a través de sus sabios consejos. A mi tio Omar, quien con sus consejos ha sabido guiarme para culminar mi carrera profesional A mis hermanos Mokhtar, Nissaf y Manel quienes con su ayuda, cariño y comprensión han sido parte fundamental de mi vida. A Manel, por acompañarme durante todo este arduo camino y compartir conmigo alegrías y fracasos. A mis amigos, especialmente Adriano, Lourdes, Mohamed, Husham y Rodrigo que siempre me han prestado un gran apoyo moral y humano, necesarios en los momentos difíciles de este trabajo. A los compañeros y becarios del Departamento, quiero agradeceros el buen trato, el apoyo y la ayuda que siempre me habéis dispensado y sobre todo, esa especial amabilidad y esos momentos inolvidables de alegría, cariño y buen humor durante el transcurso de los años de mi estancia. Por último a los miembros del tribunal de mi tesis por aceptar formar parte de mi tribunal y ajustar sus agendas A todos, muchas gracias; OMAR BOUALI INDEX Index I INDEX OF CONTENTS Page CHAPTER 1: ABSTRACT, RESUMEN, RESUME 1 CHAPTER 2: LITERATURE REVIEW AND OBJECTIVES 13 CHAPTER 3: THE EXPERIMENT: EFFECT OF FIBER INCLUSION IN THE REARING DIETS AND ENERGY CONCENTRATION OF THE LAYING DIETS ON PRODUCTIVE PERFORMANCE AND EGG QUALITY OF BROWN EGG-LAYING HENS FROM 17 TO 46 WEEKS OF AGE. 45 Abbreviation keys IX ABBREVIATION KEYS ºC: degree Celsius %: percentage *: P<0.05 **: P<0.01 ***: P<0.001 AA: amino acid ADFI: average daily feed intake AMEn: nitrogen-corrected apparent metabolizable energy Arg: arginine BW: body weight BWG: body weight gain Ca: calcium CF: crude fiber cm: centimeter cm2: square centimeter CP: crude protein Cys: cysteine d: day; día DM: dry matter; materia seca EE: ether extract et al.: and others FCR: feed conversion ratio FEDNA: Fundación Española para el Desarrollo de la Nutrición Animal; Spanish Foundation for the Development of Animal Nutrition Abbreviation keys X g: gram, gramo GIT: gastro intestinal tract GIT: gastrointestinal tract GLM: general lineal model GMD: geometric mean diameter GSD: geometric standard deviation h: hour; hora HCl: hydrochloric acid IC: índice de conversión Ile: isoleucine IU: international unit kcal: kilocalorie kg: kilogram Lys: lysine M: milliones m: meter; metro m2: square meter Met: methionine mg: miligram min: minute mm: milimeter N: nitrogen n: number of replicates per treatment NRC: National Research Council NS: non significant difference (P> 0.10) Abbreviation keys XI NSP: non starch polysaccharides P: phosphorus P: probability ppm: parts per million RL: relative length (cm/kg body weight) RW: relative weight (g/kg body weight) SAS: Statistical Analysis Systems SCWL: Single Comb White Leghorn SD: standard deviation SEM: standard error of the mean SI: small intestine T: tons Thr: threonine Trp: tryptophan Val: valine vs.: versus wk: week μm: micrometer CHAPTER 1: ABSTRACT, RESUMEN, RÉSUMÉ ABSTRACT Chapter 1: Abstract 1 ABSTRACT A common commercial practice used to enhance feed intake in young hens at the onset of the egg laying cycle consists in increasing the fiber content of the rearing diets. The inclusion of fiber in these diets might help in the adaptation of the gastrointestinal tract (GIT) of the bird for higher consumption during the laying phase, which may result in improved hen productivity. The experimental consisted in 12 treatments with 6 rearing diets (0-17wk) and 2 laying diets (18-46wk) in a factorial arrangement. Two of the rearing diets had similar nutrient content and were based on barley or corn. The other 4 diets formed a 2x2 factorial with 2 fiber sources (straw and sugar beet pulp) included at 2 or 4% at expense of in the corn diet. In the laying phase, half of the pullets of these 6 dietary treatments were fed diets with 2,650 or 2,750 kcal AMEn/kg but with similar nutrient content per unit of energy. No interactions between rearing and laying phase diets were found. Type of diet during the rearing did not affect laying hen productivity or the relative weight (% BW) of the GIT, the relative length (cm/kg BW) of the hens or of the small intestine, or the pH of the gizzard contents. Hens fed the high energy diet during the laying period ate less feed (P<0.001) and had better FCR (P<0.01) and higher BW gain (P<0.05) than hens fed the low energy diet. Egg quality was not affected by dietary treatment except for the dirty eggs that was higher (P<0.001) for the low energy diet. We conclude that neither the main cereal nor the inclusion of a fiber source in the rearing phase diets affected GIT development or laying hen production at 46 wk of age. Also, the use of high energy diets during the laying phase reduced ADFI and improved FCR but did not affect egg production or egg weight. Key words: energy, pullet, straw, sugar beet pulp Chapter 1: Résumé 9 RÉSUMÉ Une des pratiques utilisées pour améliorer la prise alimentaire chez les jeunes poules au début du cycle de ponte consiste à augmenter la teneur en fibres de l'alimentation des poulettes. L'inclusion de la fibre dans ces régimes pourrait aider à l'adaptation de l'appareil digestif de la poule pour avoir une consommation plus élevée au cours de la période de ponte, ce qui peut améliorer sa productivité ultérieurement. Le dispositif expérimental était complètement aléatoire avec 12 traitements avec 6 régimes utilisés durant la phase d'élevage des poulettes (0-17semaines) et 2 régimes utilisés durant la phase d'élevage des poules pondeuses (17-46semaines) suivant un arrangement factoriel. Deux des régimes alimentaires des poulettes étaient basés sur l'orge ou du maïs , et les autres quatre régimes restants ont formé un factoriel 2x2 avec deux sources de fibres ( la paille et la pulpe de betterave à sucre) incluses à des niveaux de 2 et 4% dans le régime alimentaire basé sur le maïs. Durant la période de ponte, la moitié des poulettes de ces 6 traitements alimentaires ont reçu une alimentation à 2.650 ou à 2.750 kcal EMAn / kg avec une même teneur en éléments nutritifs par unité de EMAn pour 2.650 ou 2.750 kcal AMEn / kg. Il n´y a pas d´interactions observées entre les régimes alimentaires utilisés durant la phase des poulettes et ceux qui ont été utilisés durant la phase de ponte. Le type d'alimentation distribué aux poulettes n'a pas affecté la productivité des poules pondeuses et même le poids relatif (% Poids vif) du tube digestif, le pH du contenu du gésier, ou la longueur des poules (cm / kg de poids vif) ou de l'intestin grêle. Les poules nourries avec le régime contenant un niveau élevé d´énergie durant la période de ponte ont présenté une baisse de consommation alimentaire (P <0,001), et par contre un meilleur indice de consommation (P <0,01) et un gain de poids plus élevé (P <0,05) que celui des poules nourries avec un régime Chapter 1: Résumé 10 alimentaire contenant un niveau faible d´énergie. La qualité des œufs n'a pas été affectée par les traitements alimentaires pratiqués, sauf qu´ il y ait une augmentation du pourcentage des œufs sales (P <0,001) affectée par la diète qui contient un niveau faible d´énergie. Nous concluons que ni le type de céréale utilisé, ni l'inclusion des sources de fibres dans le régime alimentaire des poulettes n’ont affecté le développement du tube digestif à 46 semaines d'âge ou même la production d´œufs. En outre, l'utilisation des régimes alimentaires avec un niveau élevé en énergie au cours de la période de ponte réduit la consommation alimentaire et améliore l´indice de consommation, mais il n'y a pas d'incidence que ce soit sur la production d'œufs ou sur le poids des œufs euxmêmes. Mots clés: énergie, la paille, la pulpe de betterave á sucre, poulette CHAPTER 2: LITERATURE REVIEW AND OBJECTIVES Chapter 2: Literature review and objectives 13 1. General comments The present Master Thesis has been carried out thanks to a CDTI research project (Ministerio de Ciencia e Innovacion, Madrid) with Camar Agroalimentaria. S. L, a leading egg production company placed in Toledo (Spain). The objectives of the egg production industry to attain optimal performance and adequate economic output of a given flock of laying hens.Thus;  The economic success of the egg industry depends on total egg mass produced by hen during the whole lay period. The total egg mass production per hen is influenced not only by the length of the laying period but also by the persistency of the egg production curve, the number of eggs produced, and the size of these eggs.  The economic success of a laying hen operation requires a high peak of egg production of adequate size at the start of the laying cycle and a good persistency throughout the laying period. It is widely accepted that a high peak of production is positively related with egg mass production. Chapter 2: Literature review and objectives 14 2. Literature review 2.1. Introduction Spain, with around 37 M of industrial laying hens in 2012 is one of the leader egg producer countries in EU. This production corresponds to 12.3% of the total census of the EU-27 (European Commission, 2012). Total egg production in Spain is around 795,000 t per year (FAOSTAT, 2012a). Moreover, in 2011, Spain exported 92,000 t, mostly to other European countries, especially Germany (FAOSTAT, 2012b). Egg consumption in Spain is estimated to be over 220 eggs per person per year, equivalent to 14 kg of egg (FAOSTAT, 2012a). The use of non-enriched cages for egg production has been banned in the EU-27 in January 2012 and consequently, Spanish producers had to reconvert their facilities to meet the legislation which has caused a redaction of egg production. At present time, laying hens are housed primarily in enriched cages (93.5%) with a small percentage of hens housed on floor (4%) or under free range (2.6%) conditions (REGA, 2012). The profitability of the egg industry depends of 3 keys factors; number of eggs per hen housed, egg size, and percentage of eggs that reach the table of the final consumer. One of the most critical points to improve egg rate and egg quality is proper management and feeding and nutrition practices of the pullets during the rearing phase. Adequate body weight (BW) at sexual maturity of pullets and uniformity are positively correlated with high peak production, persistency of egg production curve, and proportion of large eggs (Summers and Leeson, 1994). Adequate feed intake (FI) during the rearing phase will result in pullets with a well-developed gastrointestinal tract (GIT) that will allow the birds to meet their nutritional requirements, especially in the critical period of the onset of laying. However, the information available on nutrient requirements and the influence of dietary fiber on growth and development of the GIT is very limited in Chapter 2: Literature review and objectives 15 pullets. More information is needed to help the nutritionists to formulate diets that maximize FI and BW gain (BWG) and at the same time allows proper development of the GIT of the pullets and an adequate uniformity of the flock. 2.1. Utilization of fiber in poultry diets The dietary fiber (DF) has typically been an undesirable component of diets. However, two main factors have increased recently the interest of dietary fiber inclusion in diets for chicks. The first one is the need for using non-traditional ingredients with larger concentrations of fiber and the second one is a growing public concern regarding the use of in-feed antimicrobials for livestock production. The concern is because of the potential transmission of antibiotic resistance from animals to humans (Witte et al., 2002). Therefore, the interest in dietary alternatives to promote or maintain health in chicks has grown. Some authors have indicated that DF is the major source of energy to support microbial populations in the gastrointestinal tract (Bach Knudsen et al., 1991; Jørgensen et al., 1996). The chemical properties of DF, as well as its fermentation in the intestine, have an important role in keeping the balance between communities of commensals and pathogens (Montagne et al, 2003). Also, the intestinal microbiota has the capacity to modulate the host’s immune response (Bauer et al., 2006). Thus, the source and concentration of DF in the diets of chicks represent an opportunity to manipulate the susceptibility and capacity to recover from enteric diseases. On other hand, DF might help to protect the mucosa of the GIT, reducing the incidence of ulcers and colitis, as well as chronic inflammation of the digestive mucosa (Mateos et al., 2012). Chapter 2: Literature review and objectives 16 2.2.1. Definition of dietary fiber The DF was originally defined as ""the skeletal remains of plant cells in the diet, which are resistant to hydrolysis by the digestive enzymes of man" (Trowell, 1974). This excluded the polysaccharides added to the diet such as food additives (e.g. plant gums, modified cellulose) and the definition was later expanded to include "all polysaccharides and lignin, which are not digested by the endogenous secretions of the human digestive tract (Trowell et al., 1976). Despite extensive research over the last quarter of the 20th century, the definition of DF has been continuously debated and no universal agreement has yet been reached (Cummings et al., 1997; DeVries et al., 1999). However, currently most researchers are using either a physiological or a chemical definition. According to the physiological definition, DF is "the dietary components resistant to degradation by mammalian enzymes", while chemically it is "the sum of non-starch polysaccharides (NSP) and lignin" (Theander et al., 1994). The DF is predominantly found in plant cell walls and consists of a complex mixture of NSP that are associated with a number of other, non-carbohydrate components (McDougall et al., 1996). It is now known that DF exhibits a range of physical properties that act in concert with the chemical properties to determine the physiological effects of the feed. In the analysis of DF, it is common to characterize DF in accordance with its solubility. Soluble NSP (s-NSP) tend to impair digestion and absorption of nutrients in the fore-gut, whereas the negative effects of insoluble NSP (i-NSP) are less pronounced. The i-NSP primarily act in the large intestine where, increase faecal bulk, dilute colonic contents and decrease transit time. Chapter 2: Literature review and objectives 17 2.2.2. Physicochemical properties of fiber The DF exhibits a range of physical properties that act in concert with the chemical properties to determine the physiological effects in animals. The physicochemical properties that are more commonly studied include: particle size and bulk volume, hydration properties and the viscosity, lipid adsorption capacity, surface area and porosity characteristics, and ion exchange capacity (Guillon and Champ, 2000, 2002). 2.2.2.1. Particle size and bulk volume Particle influences a number of events occurring in the GIT, such as transit time, fermentation capability, and bulk of the fecal contents (Guillon and Champ, 2000). Particle size depends on the nature of cell walls present in the ingredients and the degree of processing. Auffret et al. (1994) indicated that grinding not only reduces particle size but also modifies the structure of the fiber. Particle size of the fiber component may vary during transit throughout the GIT as a result of chewing, grinding in the gizzard and bacterial degradation in the large intestine. In addition, some components involved in the cohesiveness of the fiber matrix may be solubilized during the digestive process. Consequently, particle size of the fiber before ingestion is not necessarily relevant to assess the potential action and benefits of fiber on transit time. The analysis of particle size distribution is typically carried out by dry sieving through a series of sieves with decreasing mesh size as described by American Society of Agriculture Engineers (1995). The measurement of particle size in wet form may be more relevant when comparing the bulk volume of fiber in the GIT than the measurements in dry form. In any case, the method and system utilized for the determination of particle size of fiber sources should be always indicated. Chapter 2: Literature review and objectives 24 benefits of the enzymes should be attributed to their capacity to reduce the viscosity of the digesta, or their capacity to release nutrients encapsulated in the fiber matrix of the grain cell walls, or both (Bedford, 2002). 2.2.4.2. The insoluble fraction of dietary fiber As previously mentioned i-NSP share some physical features with s-NSP, but the negative effects exerted by i-NSP are much less severe. For example, i-NSP may display some water-holding capacity, but their viscosity-inducing properties are relatively low (Smits and Annison, 1996). Although i-NSP are less rapidly fermented by the microflora, they are not inert, and may indeed interact with the constituents of the digesta. In theory, these effects may partially be mediated by the intestinal microflora as i-NSP tend to favour certain bacterial species (Baurhoo et al., 2007), some of which display a capacity to deconjugate bile salts (Guban et al., 2006). Further, depending on the pH, some NSP may associate with cations (Smits and Annison, 1996). Although certain part of fiber may improve mineral retention in broiler chickens (Ortiz et al., 2009), the effects of fiber on different minerals are ambiguous (van der Aar et al., 1983). Overall, there is a scarcity of in vivo experiments on the effects of i-NSP on the uptake of nutritionally important minerals for poultry like calcium. In contrast, the suggestion that i-NSP may stimulate the digestion of macronutrients and benefit bird welfare has gained increasing attention in the scientific community. The majority of hypothesized explanations for these observations relate to the effects of i-NSP on gastrointestinal function. Whereas water-soluble particles dissolve in the intestinal fluids and rapidly pass the anterior digestive tract, i-NSP are retained for a longer period of time in the crop and gizzard (Ferrando et al., 1987; Vergara et al., 1989). Increased retention time of digesta in the anterior digestive tract may alleviate problems with over-consumption of feed in broilers (Svihus et al., 2010), probably as feed intake is Chapter 2: Literature review and objectives 25 partially controlled by distension-sensitive receptors in this section of the digestive tract (Duke, 1988). Numerous studies have demonstrated that i-NSP stimulate gizzard development (Riddell, 1976; Hetland et al., 2003, 2005; Jiménez-Moreno et al., 2010) and reduce the occurrence of spontaneous gizzard erosion and ulceration (Kaldhusdal et al., 2012). The notion that gizzard development is important to digestion derives from several experiments. For example, gizzard size is correlated with the efficiency of mechanical degradation of raw potato starch granules (Rogel et al., 1987), and broiler chickens selected for high utilization of a wheat-based diet displayed increased gizzard weights (de Verdal et al., 2010). The effects of i-NSP on gizzard function are not restricted to the control of feed intake and the digesta particle size, but probably also involve the regulation of digesta flow throughout the digestive tract. Ingestion of i-NSP increases bile acid content and amylase activity in digesta, hypothetically via an increased gastroduodenal reflux (Hetland et al., 2003). An increased frequency of small intestinal reflux has been suggested to facilitate the digestion of nutrients (Duke, 1997). In addition to the aforementioned effects of i-NSP on gizzard grinding capacity, the elevated contents of bile acids in digesta reported by Hetland et al. (2003) may help explaining the increased digestibilities of fat in young broiler chickens fed i-NSP from soybean hulls and oats (Jiménez-Moreno et al., 2009). When considering the effects of i-NSP on nutrient utilization, the importance of particle size should be emphasized. Since digesta must pass through the gastroduodenal junction of the gizzard, larger particles are retained for a longer time and consequently, stimulate gizzard function more than fine particles do (Jiménez-Moreno et al., 2010). Chapter 2: Literature review and objectives 26 2.3. Energy level in poultry diet Hens eat to satisfy their energy requirements and therefore an increase in the energy content of the diet should decrease FI proportionally (Hill et al., 1956). 2.3.1. Energy content of laying hen diets 2.3.1.1. Effects on productive performance Bouvarel et al. (2010) reviewed a serie of experiments conducted with laying hens during the last 20 years. They reported that as an average, a 10% of 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 with respect to productive performance (Harms et al., 2000). In laying hens, Grobas et al. (1999c) have reported that increasing the AMEn content of the diet from 2,680 to 2,810 kcal/kg a (4.8% of increase) decreased feed intake by the same proportion a (5.0% of decrease) but egg production and egg mass were not affected. Similarly, Peguri et al. (1991) reported a 5% of decrease in FI but similar egg production when AMEn of the diet was increased from 2,700 to 2,910 kcal/kg a (8% of increase). In contrast, Joly and Bougon (1997) reported a 1.3% of increase in egg production and 4.5% of increase in egg mass as the energy content of the diet increased from 2,200 to 2,700 kcal AMEn/kg in brown laying hens diets from 19 to 68 wk of age. Most of published trials which study the effect of energy level of diet reported an improvement in egg weight (EW) with increasing levels of energy (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), over consuming energy in high energy diets (De Groote, 1972; Walker et al., 1991). Thus, the excess of nutrients improve the EW (De Groote, 1972; Walker y col., 1991). According to these authors, Chapter 2: Literature review and objectives 27 EW 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 EW 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 energy level on EW are not apparent but might be related with the level of fat and the linoleic acid (LNL) content of diets. The effects of energy level on egg production are variable. Thus, while Mathlouthi et al. (2002) reported with Single Comb White Leghorn (SCWL) hens that egg production increased as the AMEn of diet increased from 2,650 to 2,750 kcal/kg, Grobas et al., (1999c) with brown hens fed diets varying from 2,680 to 2,810 kcal AMEn/kg, Harms et al. (2000) with 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. In commercial flocks, increasing the energy content of diet at the onset of laying period is quite common, especially when the pullets have not an homogeneous BW or when the pullets have a low BW at the beginning of laying period. Thus, some authors have reported that in places with hot climate like Spain, the increase of energy concentration of the diet can improve the performance especially in light hens (Kling and Hawes, 1990; Daghir, 1995). 2.3.1.2. Effects on egg quality The reasons for the discrepancies among authors in respect to the variation in egg quality results with AMEn of diet are not apparent, but might be related to 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. Zimmermann and Andrews (1987) and Junqueira et al. Chapter 2: Literature review and objectives 28 (2006) reported that the increase in energy concentration of the diet did not affect the haugh units (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. On the other hand, 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. These results can be related to a better absorption of Xanthophylls, the main pigment source responsible for egg yolk color, which are soluble in fat. Also, when fat is used to increase the energy concentration of the diet, the proportion of shell in the egg could be affected due to fat can modify calcium absorption, basically in broilers. However, this effect is not clear in pullets or laying hens. 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. 2.3.2. Fat content of laying hen diets 2.3.2.1. Effects on productive performance Fats are used in poultry feeds to increase the energy content of diets. Fat inclusion usually results in higher energy intake, BW gain and EW (Grobas et al., 2001; Bouvarel et al., 2010), probably due a better 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, 1980). Whitehead et al. (1993) studied the effect of supplemented fat Chapter 2: Literature review and objectives 29 on EW and concluded that eggs produced with maize oil were heavier than those produced 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 EW. Grobas et al. (2001) studied the effect of 4 different sources of supplemented fat on EW 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. Atteh and Leeson (1983, 1984,1985) studied the effect of FA profile on performance and mineral metabolism of laying hens and broilers, and reported that fat and some minerals like calcium 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 an increase in the Ca content of the diet increased the soap formation. Many studies have shown that a reduction in supplemental fat (SFAT) decreases egg size (Keshavarz and Nakajima, 1995; Grobas et al., 1999a,b; Sohail et al., 2003). Grobas et al. (2001) reported that SFAT improved EW and egg mass output in both, SCWL hens and brown laying hens throughout the production cycle. Grobas et al. (1999b) compared isonutritive diets for brown 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, although 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 EW were more noticeable from 22 to 57 wk of age. Whitehead (1981) showed that supplementation of diets with 0.4 or 3 % of fat significantly increased EW. Whitehead et al. (1993) compared 5 inclusion levels of fat (0, 1, 2, 4, and Chapter 2: Literature review and objectives 30 6 %) and concluded that, with the exception of fish oil, which hindered productive performance when included at 2 %, maize oil, tallow, and coconut oil perform well till 4 % of inclusion. Furthermore, Grobas et al. (1999b) showed that supplementation of the diet with 4 % increased EW as compared with a non-supplemented diet. However, these authors showed that further increases from 5 to 10% of fat supplementation did not have any positive effect on EW (Grobas et al., 2001). 2.3.2.2. Effects on egg quality Added fat increased, both yolk and albumen weights, but in some studies 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 oestrogens which are the main responsible for albumen secretion Regarding egg quality traits, Grobas et al. (1999a) observed that the increase in EW 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 (Grobas et al., 2001; Usayran et al., 2001). Previous research has shown that SFAT exerts a favorable effect on EW beyond that attributable to an increase in LNL content of the diet (Sell et al., 1987; Grobas et al., 1999a). Chapter 2: Literature review and objectives 31 3. Objectives The general aim of this Master Thesis was to study the influence of nutritional factors during the rearing and laying phases that might affect the productivity and egg quality of commercial brown egg-laying hens. With this objective, six experimental diets for the rearing phase varying in the main cereal (Barley or corn) and with different level of inclusion of two fiber sources straw and sugar beet pulp, and two diets with different level of energy for the laying phase diets to study: -The effects of diets composition during the rearing and laying phases in eggs quality during the laying phase -The effects of diets composition during the rearing and laying phases of the development of the GIT of the hens at 48 wk of age - The effects of inclusion of different sources of fiber in the rearing diets and its effect on productive performance during the laying cycle. -Potential interaction between the characteristics of the rearing phase diet and the laying hen diet on performance of the hens during the laying cycle. Chapter 2: Literature review and objectives 33 4. Reference American Society of Agriculture Engineers. 1995. Method of determining and expressing fineness of feed materials by sieving. Pages 461-462 in Agriculture Engineers Yearbook of Standards, ASAE standard S319.2. St. Joseph, MO. Apajalahti, J., A. Kettunen, and H. Graham. 2004. Characteristics of the gastrointestinal microbial communities, with special reference to the chicken. World's Poult. Sci. Jr. 60:223-232. 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. 62:24032411. 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. Auffret, A., M. C. Ralet, F. Guillon, J. L. Barry, and J. F. Thibault. 1994. Effect of grinding and experimental conditions on the measurement of hydration properties of dietary fibers. Lebensmittel-Wissenschaft und –Tech 27:166-172. Bach Knudsen, K. E., and I. Hansen. 1991. Gastrointestinal implications in pigs of wheat and oat fractions. 1. Digestibility and bulking properties of polysaccharides and other major constituents. B. Jr. Nutr. 65:217-232. Bauer, E., B. A. Williams, H. Smidt, M. W. A. Verstegen, and R. Mosenthin. 2006. Influence of the gastrointestinal microbiota on development of the immune system in young animals. Curr. Issues Intest. Microbiol. 7:35-52. Baurhoo, B., L. Phillip, and C.A. Ruiz-Feria. 2007. Effects of purified lignin and mannan oligosaccharides on iintestinal integrity and microbial populations in the ceca and litter of broiler chickens. Poult. Sci. 86:1070-1078. Bedford, M.R. 2002. The role of carbohydrases in feedstuff digestion. In: McNab, J.M., Boorman, K. N. (Ed.) Poult. Fds.Sup. 319-336. Wallingford, Oxon, the UK: CAB International. Björnhag, G. 1989. Transport of water and food particles through the avian ceca and colon. Jr. Exp. Zoo. 252:32-37. Chapter 2: Literature review and objectives 40 Summers, J. D., and S.Leeson. 1994. Laying hen performance as influenced by protein intake to sixteen weeks of age and body weight at point of lay. Poult. Sci. 73:495-501. Svihus, B. and H. Hetland. 2001. Ileal starch digestibility in growing broiler chickens fed on a wheat-based diet is improved by mash feeding, dilution with cellulose or whole wheat inclusion. Br. Poult. Sci. 42:633-637. Svihus, B., A. Sacranie, V. Denstadli, and M. Choct. 2010. Nutrient utilization and functionality of the anterior digestive tract caused by intermittent feeding and inclusion of whole wheat in diets for broiler chickens. Poult. Sci. 89:2617-2625. Theander,O., P. Aman, E. Westerlund, and H. Graham. 1994. Enzymatic1chemical analysis of dietary fber. J. AOAC Inter. 77:703-709. Teirlynck, E., L. Bjerrum, V. Eeckhaut, G. Huygebaert, F. Pasmans, F. Haesebrouck, J. Dewulf, R. Ducatelle, and F. Van Immerseel. 2009. The cereal type in feed influences gut wall morphology and intestinal immune cell infiltration in broiler chickens. B. Jr. Nutr .102:1453-1461. Thibault,J.-F., M. Lahaye, and F. Guillon.1992. Physico-chemical properties of food plant cell walls. In: Schweizer,T.F.,Edwards,C.A. (Eds.),Dietary fbre: A Component of Food: Nutritional Function in Health and Disease. Springer, London,21-39. Trowell, H., 1974. Definition of fiber. Lancet 503 Trowell,H., D. A. T. Southgate,T. M. S. Wolever, A. R. Leeds, M. A. Gassull,and D. J. A. Jenkins. 1976. Dietary fbre redefned. Lancet 967. Usayran, N., M. T. Farran, H. H. O. Awadallah, I. R. Al-Hawi, R. J. Asmar, and V. M. Ashkarian. 2001. Effects of added dietary fat and phosphorus on the performance and egg quality of laying hens subjected to a constant high environmental temperature. Poult. Sci. 80:1695–1701. Van der Aar, P.J., G. C. Fahey, S. C. Ricke, S. E. Allen, and L. L. Berger. 1983. Effects of dietary fibers on mineral status of chicks. Jr. Nutr. 113:653-661. Vergara, P., C. Ferrando, M. Jiménez, E. Fernández, and E. Goñalons. 1989. Factors determining gastrointestinal transit time of several markers in the domestic fowl. Expe. Phys. 74:867-874. Viveros, A., A. Brenes, M. Pizarro, and M. Castaño.1994. Effect of enzyme supplementation of a diet based on barley, and autoclave treatment, on apparent Chapter 2: Literature review and objectives 41 digestibility, growth performance and gut morphology of broilers. Anim. Feed. Sci. Technol. 48:237-251. Walker, A. W., S. A. Tucker, and N. J. Lynn. 1991. Effects of nutrient density and fat content on the performance of laying hens. Br. Poult. Sci. 32:1138-1139. Wang, G., C. Ekstrand, and J. Svedberg. 1998. Wet litter and perches as risk factors for the development of foot pad dermatitis in floor-housed hens. Br. Poult. Sci. 39:191-197. Whitehead, C. C. 1981. The response of egg weight to the inclusion of different amounts of vegetable oil and linoleic acid in the diet of laying hens. Br. Poult. Sci. 22:525532. Whitehead, C. C., A. S. Bowman, and G. D. Griffin. 1991. The effects of dietary fat and bird age on the weights of eggs and egg components in the laying hen. Br. Poult. Sci. 32:565-574. Whitehead, C. C., A. S. Bowman, and G. D. Griffin. 1993. Regulation of plasma oestrogen by dietary fats in the laying hen: relationship with egg weight. Br.Poult. Sci. 34:999-1010. Whitehead, C. C. 1995. Plasma oestrogen and the regulation of egg weight in laying hens by dietary fats. Anim. Feed. Sci. Technol. 53:91-98. Witte, W., I. Klare, and G. Werner. 2002. Molecular ecological studies on spread of antibiotic resistance genes. Anim. Biotechnol. 13:57-70. 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. Wu, Y.B., V. Ravindran, D. G. Thomas, M. J. Birtles, and W. H. Hendriks. 2004. Influence of phytase and xylanase, individually or in combination, on performance, apparent metabolisable energy, digestive tract measurements and gut morphology in broilers fed wheat-based diets containing adequate level of phosphorus. Br. Poult. Sci. 45:76-84. 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. CHAPTER 3: THE EXPERIMENT EFFECT OF FIBER INCLUSION IN THE REARING DIETS AND ENERGY CONCENTRATION OF THE LAYING DIETS ON PRODUCTIVE PERFORMANCE AND EGG QUALITY OF BROWN EGG-LAYING HENS FROM 18 TO 46 WEEKS OF AGE. Chapter 3: The experiment 45 1. Introduction Egg production and egg quality depends on multiple factors, including nutrition during the rearing and laying phases. Grains are the main ingredients of poultry feeds and birds are well equipped to utilize diets with high starch contents (Hetland et al., 2003). Most research conducted with poultry have shown similar productive performance in hens fed diets based on corn or barley, provided that the diets were supplemented with an enzyme complex with adequate β-glucanase and xylanase activity (Lázaro et al. 2003; Safaa et al, 2009; Pérez Bonilla et al, 2011). Fiber is an important component of poultry diets but fiber cannot be digested by the bird, although it might affect gastrointestinal tract (GIT) development and gizzard function (González-Alvarado et al., 2010). Traditionally, dietary fiber has been considered as a diluent of the nutrient content of the feed with negative impact on palatability and nutrient digestibility (Mateos et al., 2002, 2012). The increased availability of vegetable sources with high fiber content, such as DDGS from the ethanol industry, has increased the interest to study the effects of dietary fiber on poultry performance. Recent studies have shown that the inclusion of adequate amounts of certain types of fiber in the feed improves the adaptation of the GIT to existing production systems and might reduce digestive disorders in poultry, improving bird performance (Jiménez-Moreno et al., 2009, 2013 a, b) It is a common commercial practice to increase the fiber content of the feeds during the last part of the rearing phase to enhance the ability of the pullet to increase voluntary feed intake at the start of the laying period. However, the scientific information available on the benefit of this practice is very limited. The hypothesis of this study was that changes in the development of the GIT during the rearing phase, a consequence of including fibrous ingredients in the diet, could improve subsequent hen Chapter 3: The experiment 46 production and that the benefits could be more pronounced in hens fed during the laying phase low energy diets than in hens fed high energy diets. 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, 2013). In total, 480 Lohmann Brown egg-laying pullets were fed during the 3 periods (1 to 5 wk, 5 to 10 wk, and 10 to 17 wk of age) of the rearing phase one of 6 diets that consisted in 2 isonutritve diets based on barley or corn, and 4 extra diets that included 2 or 4% of either cereal straw or sugar beet pulp at the expense of the corn diet. The ingredient composition and nutritive value of the diets and the management of the pullets during the rearing phase have been reported elsewhere (Guzman et al., 2013). At 17 wk of age, the pullets were moved from the rearing house to the laying hen facility where they were housed in groups of 10 in enriched cages (0,64 m × 1,2 m; Facco S.A., Padova, Italy) equipped with an open trough feeder and 2 nipple drinkers. At arrival to the laying hen facility, hens received a common diet that included an antibiotic (Tylosin, 200 ppm/Elanco valquimica, Madrid, Spain) for 7 d and then, their respective experimental laying diets to 46 wk of age. Half of the pullets of each previous rearing group received a low (LE; 2,650 kcal AMEn/kg) or a high (HE; 2,750 kcal AMEn/kg) energy diet. The two laying diets had similar nutrient content per unit of energy and therefore, the AMEn: indispensable AA ratio did not vary between the 2 diets (Table 1). Diets were formulated according to Fundacion Española Desarrollo Nutrición Animal (2010) and met or exceeded the nutrient requirements for brown-egg laying hens Chapter 3: The experiment 47 according to Fundacion Española Desarrollo Nutrición Animal (2008). No commingling was practiced and therefore, all the hens within each laying cage belonged to the same pullet replicate. Pullets and hens were vaccinated and managed according to commercial practices (Lohmann, 2012). The light program during the laying phase consisted of 16 h of continuous light per day throughout the experiment. The temperatures inside the barn were recorded daily an varied from 28 ±3ºC in June (first period of the experiment) to 19 ± 3ºC in January (last period of the experiment). The experimental design was completely randomized with 12 treatments arranged as a 6x2 factorial with 6 diets used during the rearing phase and 2 diets used during the laying phase as main effects. Each of the 12 treatments was replicated 4 times and the experimental unit during the laying period was an enriched cage with 10 hens. 2. 2. Laboratory Analysis The nutritive values of the diets used during the rearing phase have been reported elsewhere (Guzmán et al., 2013). Representative samples of the laying phase diets were ground in a laboratory mill (Model Z-I, Retsch Stuttgart, Germany) equipped with a 0.5-mm screen and analyzed for moisture by oven-drying (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 3N HCl acid hydrolysis (Boletín Oficial del Estado, 1995) and gross energy in an isoperibol bomb calorimeter (Model 356, Parr Instrument Company, Moline, IL). The amino acids (AA) content of the diets were determined by chromatography (Hewlett-Packard 1100, Waldbronn, Germany) as described by De Coca-Sinova et al (2008). The geometric mean diameter (GMD) of the diets was determined in triplicate in 100 g samples using Chapter 3: The experiment 48 a Retsch shaker (Retsch, Stuttgart, Germany) equipped with 8 sieves ranging in mesh from 5,000 to 40 µm according to the methodology outlined by the ASAE (1995). The determined analyses and the GMD of the diets are shown in (Table 2). 2. 3. Productive Performance and Egg Quality Feed intake and egg production were recorded by replicate for each of the seven 28 d periods and for the entire experimental period (18 to 46 wk of age). All the eggs produced the last 2 days of each week were weighed by replicate and the average value of the 4 wk was used to estimate average egg weight for each of the 7 experimental periods. In addition, hens were weighed by replicate at the start of the experiment and at the end of each of the 7 experimental periods. Mortality was recorded and weighed as produced. From these data, egg production, average daily feed intake (ADFI), egg mass, feed conversion ratio (FCR) per kilogram of eggs and per dozen of eggs, and BW gain were calculated by period and cumulatively. In addition, the number of dirty, broken, and shell-less 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 (Lázaro et al., 2003). Egg quality, including Haugh units, yolk color, shell strength, shell thickness, and shell color were measured in 6 eggs collected randomly from each replicate the last day of each of the 7 experimental periods. Haugh units and yolk color (Roche color fan) were measured using a Multitester equipment (QCM System, Technical Services and Supplies, Dunnington,York, UK) as indicated by Pérez Bonilla et al., (2012). Shell color was measured using a Minolta colorimeter (Chroma Meter Model CR-200, Minolta Corp., Ramsey, NJ) and the Hunter color values, L* (lightness), a* (green to red), and b* (blue to yellow) values were recorded (Hunter and Harold, 1987). Egg shell strength was evaluated applying increased pressure to the broad pole of the egg using a press meter, and expressed in kg/cm2. Shell thickness was measured at the two pole ends and Chapter 3: The experiment 49 at the middle section of the egg using a digital micrometer (QCM System) and the average of the 3 measurements of each of the 6 eggs was used for further analyses. 2. 4. Digestive Traits At 46 wk of age, after the corresponding productive performance control, 2 birds per replicate were randomly selected, weighed individually, and euthanized by CO2 inhalation. The digestive tract (from the end of the crop to the cloaca, including the digestive contents, spleen, liver, and pancreas) was removed aseptically and weighed. Then, the liver, and the full proventriculus and full gizzard were excised and weighed. Data on organ weight were expressed relative (%) to BW. Then, the gizzard was emptied from any digesta, cleaned, dried with desiccant paper, and weighed again. The fresh digesta content of the gizzard was measured as the difference between the full and the empty organ weight and expressed relative to the weight (%) of the full organ. In addition, the pH of the gizzard digesta was measured in these hens using a digital pH meter fitted with a fine tip glass electrode (model 507, Crison Instruments S.A., Barcelona, Spain) as indicated by Jiménez-Moreno et al. (2009) (Table5). No attempt was made to measure the fresh digesta content of the proventriculus. The length of the hens, measured from the tip of the beak to the end of the longest phalanx in extended birds, and the length of the duodenum (from gizzard to pancreo-biliary ducts), jejunum (from pancreo-biliary ducts to Meckel’s diverticulum), ileum (from Meckel’s diverticulum to ileo-cecal junction), and the two ceca (from the ostium to the tip of the right and left ceca) were measured on a glass surface using a flexible tape with a precision of 1 mm (Table 6). Tarsus length and tarsus diameter (measured in the middle point of the bone) were also determined using a digital caliper. All these measurements were expressed in cm/kg BW. All traits were measured in duplicate and the average value of the two measurements was used for further statistical analysis. Chapter 3: The experiment 56 kcal/kg. The authors have not found any report in the literature comparing the effects of energy concentration of the diet on the size and the length of the different organs of the GIT to compare with the results of current experiment. Probably, a change in the energy level of the diet of 100 kcal AMEn/kg is not an important factor that could affect GIT traits. Frikha et al. (2009, 2011) and González-Alvarado et al. ( 2007, 2008) have shown that other factors such as feed form, main cereal of the diet of egg-laying pullets, and inclusion of fiber in the diet might affect GIT development in poultry. 5. Conclusion Type of cereal or the inclusion of 2 to 4% of a fiber source in the rearing diets resulted in similar feed intake and hen performance during the laying phase. No interactions between the characteristics of the rearing diets and the energy content of the laying diets were detected for any laying trait. Therefore, the effects on an increase in the level of fiber in the rearing diets on the development and function of the gastrointestinal tract disappear with time and have no effects on subsequent hen performance. An increase in the energy content of the laying diets reduces feed intake, improves feed efficiency, and increases BW gain but did not affect any of the other production variables studied. Chapter 3: The experiment 57 Table 1. Ingredient composition and calculated analysis (% as fed bases, unless otherwise indicated) of the experimental diets. AMEn (kcal/kg) Low3 High4 Ingredient Wheat 40.0 40.0 Corn 20.0 17.0 Soybean meal, 47% CP 17.1 18.0 Sunflower meal, 34% CP 10.3 11.0 Sunflower oil soapstocks 1.45 3.39 Dicalcium phosphate 1.08 1.18 Calcium carbonate 8.24 8.50 Sodium chloride 0.28 0.30 DL-methionine, 99% 0.11 0.13 Sepiolite 0.94 - Vitamin and mineral premix1 0.50 0.50 Calculated analysis2 Dry matter 89.4 89.8 EMAn, Kcal/kg 2,650 2,750 Crude protein 17.6 18.2 Linoleic acid 1.7 2.8 Crude fiber 4.4 4.6 Neutral detergent fiber 11.1 11.4 Digestible amino acids Arg 1.05 1.09 Ile 0.63 0.65 Lys 0.68 0.71 Met 0.37 0.40 Met+cys 0.63 0.66 Thr 0.53 0.55 Trp 0.18 0.19 Val 0.72 0.75 Total ash 12.4 12.9 Ca 3.59 3.72 Total phosphorus 0.61 0.64 Digestible phosphorus 0.29 0.31 1Provided the following (per kilogram of diet): vitamin A (trans-retinyl acetate), 6,000 IU; vitamin D3 (cholecalciferol), 1,200 IU; vitamin E (all-ractocopherol-acetate), 5 mg; vitamin K3 (bisulphate menadione complex), 1.5 mg; riboflavin, 3.5 mg; betaine, 67.5 mg; thiamin (thiamine-mononitrate), 1 mg; vitamin B12 (cyanocobalamin), 15 µg; Se (Na2SeO3), 0.1 mg; I (KI), 1.9 mg; Cu (CuSO4・5H2O), 4 mg; Fe (FeCO3), 18 mg; Mn (MnO), 66 mg; and Zn (ZnO), 2According to Fundación Española Desarrollo Nutrición Animal (2010). 3Low energy: 2,650 kcal AMEn/kg 4High energy: 2,750 kcal AMEn/kg Chapter 3: The experiment 58 Table 2. Determined analysis1and particle size distribution2 of the experimental laying phase diets (% as fed basis, unless otherwise indicated) AMEn (kcalAMEn/kg) Low3 High4 Composition Gross energy, kcal/kg 3,848 3,872 Dry matter 94.2 93.8 Ether extract 5.0 5.2 CP 17.5 18.5 Arg 1.16 1.18 Ile 0.72 0.73 Lys 0.80 0.81 Met 0.41 0.43 Met+cys 0.72 0.74 Thr 0.63 0.64 Trp 0.22 0.22 Val 0.83 0.85 Total ash 13.7 14.2 Particle size >2,500 11.80 12.77 1,250 36.31 36.93 630 29.21 28.99 315 17.91 15.11 160 4.77 6.18 <80 0.01 0.03 GMD5 ± GSD6 1,112± 2.06 1,132± 2.10 1Analyzed in triplicate 2Sieve diameter, µm.The percentage of particles smaller than 40 µm and bigger than 2500 µmwere negligible for all diets. 3Low energy: 2,650 kcal AMEn/kg 4High energy: 2,750 kcal AMEn/kg 5Geometric mean diameter 6GSD = Log normal SD. Chapter 3: The experiment 59 Table 3. Influence of source and level of fiber of the rearing phase diets and AMEn of the laying phase diets on productive performance of the hens (18 to 46wk of age) Rearing phase Laying phase Egg production (%) Egg weight (g) Egg mass (g/d) Feed intake (g/d) FCR3 (kg/kg) FCR (kg/dozen) BW gain (g) Cereal Fiber source Inclusion level, % AME, kcal/kg Barley 0 2,650 94.5 60.7 57.4 114.9 2.04 1.48 335 2,750 94.0 61.3 57.7 111.4 1.97 1.44 354 Corn 0 2,650 94.0 62.4 58.7 115.8 2.01 1.50 371 2,750 93.7 62.0 58.2 111.8 1.96 1.45 381 Straw 2 2,650 95.1 61.0 58.1 114.5 2.00 1.47 322 2,750 93.4 62.3 58.3 111.6 1.95 1.46 378 4 2,650 93.8 62.1 58.4 114.8 2.00 1.49 327 2,750 94.1 60.9 57.4 112.6 2.01 1.46 392 SBP1 2 2,650 90.7 62.7 56.9 113.4 2.04 1.53 337 2,750 92.7 62.7 58.2 110.5 1.94 1.45 336 4 2,650 94.0 61.1 57.5 114.8 2.03 1.49 342 2,750 90.1 62.1 56.1 109.6 2.00 1.49 407 Main effects Rearing phase diets Fiber source Straw 94.1a 61.6 58.0 113.4 1.99 1.47 355 SBP 91.9b 62.2 57.2 112.1 2.00 1.49 356 Inclusion level, % 0 93.9 62.2 58.4 113.8 1.98 1.48 376 2 93.0 62.2 57.9 112.5 1.98 1.48 343 4 93.0 61.5 57.3 113.0 2.01 1.48 367 Laying phase diets, AMEn/kg 2,650 kcal 93.7 61.7 57.8 114.7a 2.02 1.49a 339b 2,750 kcal 93.0 61.9 57.6 111.3b 1.97 1.46b 375a SEM2 1.20 0.56 0.83 1.29 0.027 0.021 25.3 Probability General model4 0.160 0.118 0.653 0.024 0.102 0.239 0.302 Main effects Fiber source during rearing phase 0.030 0.223 0.186 0.240 0.660 0.201 0.964 Fiber level during rearing phase 0.734 0.266 0.320 0.619 0.270 0.943 0.259 Energy level during laying phase 0.369 0.512 0.713 <.0001 0.002 0.008 0.016 Contrast Straw ,0 vs 2 and 4% 0.814 0.223 0.565 0.757 0.683 0.644 0.353 Straw, 2 vs 4% 0.832 0.756 0.706 0.666 0.344 0.553 0.725 SBP, 0 vs 2 and 4% 0.068 0.922 0.077 0.206 0.433 0.505 0.374 SBP, 2 vs 4% 0.719 0.255 0.673 0.843 0.936 0.612 0.435 Straw linear 0.921 0.220 0.505 0.951 0.361 0.913 0.513 SBP linear 0.142 0.289 0.053 0.228 0.184 0.584 0.964 1 Sugar Beet palp 2 Standard error of the mean (4 replicates of 10 hens each per treatment) 3 Feed conversion ratio 4All of the interactions studied between level and source of fiber, and energy level were not significant (p>0.10) a, b Means with different superscripts are significantly different Chapter 3: The experiment 60 Table 4. Influence of source and level of fiber of the rearing phase diets and AMEn of the laying phase on egg quality from 18 to 46 wk of age Rearing phase Laying phase Dirty eggs % Broken eggs % Shell-less eggs % Haugh Units RCF1 Shell strength kg/cm2 Shell thickness mm Shell color Cereal Fiber source Inclusion level, % AME, kcal/kg L2 a3 b4 Barley 0 2,650 2.2 0.65 0.10 91.4 10.2 5.2 0.381 62.1 15.2 32.4 2,750 1.6 0.67 0.34 89.9 10.2 5.0 0.390 62.0 15.1 32.5 Corn 0 2,650 2.7 0.64 0.32 90.2 10.3 5.1 0.375 62.4 15.0 32.3 2,750 1.8 0.66 0.22 91.3 10.2 5.0 0.381 61.7 15.2 31.9 Straw 2 2,650 2.4 0.47 0.24 91.1 10.2 5.0 0.384 60.4 16.3 33.0 2,750 1.6 0.89 0.20 91.0 10.1 5.1 0.385 61.9 15.1 32.1 4 2,650 2.8 0.44 0.18 90.9 10.3 5.2 0.384 61.3 15.4 32.2 2,750 1.7 0.73 0.20 90.7 10.1 5.2 0.378 61.9 15.1 32.3 SBP 2 2,650 2.2 0.83 0.33 91.3 10.4 4.9 0.380 60.9 15.7 32.4 2,750 2.0 0.33 0.13 90.7 10.2 5.2 0.384 61.6 15.3 32.0 4 2,650 2.4 0.32 0.19 90.3 10.3 5.3 0.384 61.4 15.4 32.4 2,750 1.3 0.43 0.28 90.7 10.3 5.2 0.383 61.0 15.8 32.2 Main effects Rearing phase diets Fiber source Straw 2.1 0.63 0.21 90.9 10.2 5.1 0.383 61.4 15.5 32.4 SBP 2.0 0.48 0.24 90.8 10.3 5.1 0.383 61.2 15.5 32.2 Inclusion level, % 0 2.3 0.65 0.27 90.8 10.2 5.1 0.378 62.05 15.1 32.1 2 2.0 0.63 0.23 91.0 10.2 5.0 0.383 61.2 15.6 32.4 4 2.0 0.48 0.21 90.7 10.2 5.2 0.382 61.4 15.4 32.3 Laying phase diets, AMEn/kg 2,650 kcal 2.5a 0.56 0.23 90.9 10.3 5.1 0.381 61.4 15.5 32.5 2,750 kcal 1.7b 0.62 0.23 90.7 10.2 5.1 0.383 61.7 15.3 32.2 SEM5 0.40 0.142 0.080 0.70 0.14 0.20 0.005 0.53 0.36 0.34 Probability General model6 0.188 0.108 0.494 0.927 0.952 0.940 0.761 0.361 0.392 0.706 Main effect Fiber source during rearing phase 0.596 0.217 0.597 0.744 0.232 0.891 0.986 0.712 0.743 0.442 Fiber level during rearing phase 0.855 0.328 0.692 0.717 0.996 0.365 0.361 0.197 0.327 0.593 Energy level during laying phase <0.001 0.508 0.996 0.692 0.334 0.916 0.454 0.402 0.295 0.137 Contrast Straw, 0 vs 2 and 4% 0.767 0.895 0.372 0.788 0.644 0.680 0.234 0.142 0.267 0.227 Straw, 2 vs 4% 0.564 0.532 0.726 0.733 0.901 0.446 0.522 0.387 0.252 0.380 SBP, 0 vs 2 and 4% 0.471 0.211 0.623 0.994 0.572 0.752 0.240 0.081 0.172 0.593 SBP, 2 vs 4% 0.537 0.186 0.950 0.468 0.978 0.314 0.704 0.882 0.834 0.802 Straw linear 0.970 0.646 0.326 0.952 0.746 0.478 0.486 0.394 0.691 0.545 SBP linear 0.310 0.064 0.681 0.727 0.649 0.455 0.241 0.112 0.191 0.561 1 Roche color fan. 2L* value means lighter color; a higher 3a* value means a redder color; a higher 4b* value means a more yellow color 5Standard error of the mean (4 replicates of 10 hens each per treatment) 6All of the interactions studied between level and source of fiber, and energy level were not significant (p>0.10) Chapter 3: The experiment 61 Table 5. Influence of source and level of fiber of the rearing phase diets and AMEn of the laying phase diets on relative weight (% BW) of the digestive organs and on gizzard content (% organ weight) of hens at46 wk of age Rearing phase Laying phase BW (g) GIT1 (%) Liver (%) Proventriculus2 (%) Gizzard Cereal Fiber source Inclusion level, % AME, kcal/kg RW2 (%) Weight2 (g) Content (%) pH Barley 0 2,650 1910 12.7 2.65 0.55 2.73 52.15 35.04 3.87 2,750 1799 12.0 2.70 0.56 2.60 46.87 33.46 3.87 Corn 0 2,650 1941 12.0 2.49 0.51 2.74 53.01 35.48 4.10 2,750 1802 12.3 2.78 0.51 2.58 46.73 33.83 4.05 Straw 2 2,650 1843 12.1 2.63 0.53 2.79 51.53 32.97 3.97 2,750 1776 12.9 2.83 0.58 2.82 50.05 32.16 4.04 4 2,650 1904 12.6 2.56 0.60 2.98 56.33 32.40 4.12 2,750 1851 12.6 2.57 0.53 2.72 50.30 30.52 3.87 SBP 2 2,650 1819 13.0 2.74 0.54 2.70 49.02 31.75 4.21 2,750 1862 12.5 2.57 0.56 2.69 50.35 30.01 4.13 4 2,650 1747 12.8 2.58 0.56 2.74 47.76 31.89 4.00 2,750 1874 12.7 2.73 0.62 2.83 52.88 33.03 4.11 Main effects Rearing phase diets Fiber source Straw 1843 12.5 2.65 0.56 2.83 52.05 32.01 4.00 SBP 1826 12.7 2.66 0.57 2.74 50.00 31.67 4.11 Inclusion level, % 0 1872 12.1 2.64 0.51 2.66 49.87 34.65 4.08 2 1825 12.6 2.69 0.55 2.75 50.24 31.73 4.09 4 1844 12.6 2.61 0.58 2.82 51.82 31.96 4.02 Laying phase diets, AMEn/kg 2,650 kcal 1861 12.5 2.61 0.55 2.78 51.63 33.25 4.04 2,750 kcal 1827 12.5 2.70 0.56 2.71 49.53 32.17 4.01 SEM3 65.3 0.36 0.097 0.051 0.138 3.150 1.870 0.140 Probability General model4 0.646 0.605 0.368 0.942 0.824 0.639 0.676 0.760 Main effect Fiber source during rearing phase 0.717 0.420 0.911 0.721 0.344 0.356 0.791 0.247 Fiber level during rearing phase 0.727 0.196 0.503 0.288 0.344 0.683 0.261 0.793 Energy level during laying phase 0.368 0.898 0.118 0.731 0.345 0.239 0.304 0.667 Contrast Straw, 0 vs. 2and4% 0.625 0.199 0.922 0.849 0.144 0.432 0.153 0.516 Straw, 2 vs. 4% 0.304 0.883 0.116 0.259 0.731 0.430 0.536 0.963 SBP, 0 vs. 2and4% 0.426 0.057 0.850 0.160 0.481 0.962 0.101 0.760 SBP, 2 vs. 45 0.653 0.993 0.966 0.465 0.518 0.841 0.380 0.390 Straw linear 0.925 0.242 0.460 0.308 0.168 0.281 0.144 0.576 SBP linear 0.358 0.104 0.882 0.135 0.370 0.887 0.344 0.873 1Gastrointestinal tract full 2All of digestive organ expressed are full 3Standard error of the mean (4 replicates of 10 hens each per treatment) 4All of the interactions studied between level and source of fiber, and energy level were not significant (p>0.10) Chapter 3: The experiment 62 Table 6. Influence of source and level of fiber of the rearing phase diets and AMEn of the laying phase diet on the relative length (L, cm/kg BW) of the hen and of the tarsus, small intestine (SI) and cecum of hens at 46 wks of age Rearing phase Laying phase Relative length, cm/kg BW3 Cereal Fiber source Inclusion level, % AME, kcal/kg Hen Tarsus Tarsus width Duodenum Jejunum Ileum SI1 Cecum Barley 0 2,650 35.0 5.3 0.67 14.1 41.4 37.7 86.1 12.5 2,750 36.8 5.6 0.72 13.9 41.5 37.6 86.1 11.3 Corn 0 2,650 33.6 5.3 0.67 14.0 40.2 36.3 85.2 12.1 2,750 36.0 5.5 0.71 13.8 42.1 38.6 87.5 11.8 Straw 2 2,650 36.0 5.5 0.69 13.1 40.4 37.2 84.1 13.3 2,750 36.8 5.6 0.73 14.0 43.5 38.5 89.0 12.4 4 2,650 35.2 5.4 0.68 13.7 41.4 38.8 87.1 12.2 2,750 35.4 5.5 0.71 15.4 41.9 38.5 88.0 12.7 SBP 2 2,650 36.2 5.5 0.73 13.7 44.0 39.1 89.9 11.7 2,750 35.3 5.4 0.67 13.4 41.5 37.0 85.2 12.4 4 2,650 36.9 5.6 0.75 14.8 44.3 41.5 90.8 12.3 2,750 34.9 5.5 0.68 12.9 42.7 37.1 86.2 12.9 Main effects Rearing phase diets Fiber source Straw 35.9 5.5 0.70 14.1 41.8 38.2 87.1 12.7 SBP 35.8 5.5 0.71 13.7 43.1 38.7 88.1 12.3 Fiber level, % 0 34.8 5.4 0.69 13.9 41.1 37.5 86.4 12.0 2 36.1 5.5 0.70 13.6 42.4 37.9 87.1 12.4 4 35.6 5.5 0.71 14.2 42.6 39.0 88.0 12.5 Laying phase diets, AMEn/kg 2,650 kcal 35.5 5.4 0.70 13.9 42.0 38.4 87.2 12.4 2,750 kcal 35.9 5.5 0.70 13.9 42.2 37.9 87.0 12.3 SEM2 1.23 0.17 0.024 0.79 1.58 1.66 3.05 0.91 Probability General model4 0.796 0.897 0.218 0.705 0.732 0.765 0.926 0.960 Main effect Fiber source during rearing phase 0.961 0.943 0.771 0.543 0.242 0.734 0.644 0.567 Fiber level during rearing phase 0.494 0.725 0.738 0.527 0.551 0.540 0.792 0.756 Energy level during laying phase 0.552 0.448 0.691 0.975 0.769 0.543 0.915 0.823 Contrast Straw, 0 vs. 2 and 4 0.313 0.479 0.550 0.763 0.624 0.589 0.782 0.376 Straw, 2 vs. 4 0.362 0.546 0.565 0.197 0.833 0.632 0.741 0.630 SBP, 0 vs. 2 and 4 0.875 0.441 0.435 0.725 0.145 0.404 0.511 0.657 SBP, 2 vs. 4 0.333 0.645 0.395 0.823 0.643 0.449 0.753 0.498 Straw Linear 0.679 0.761 0.804 0.366 0.144 0.483 0.693 0.615 SBP Linear 0.367 0.382 0.226 0.986 0.754 0.276 0.480 0.491 1Small intestine: was evaluated from data from the duodenum, jejunum and ileum 2Standard error of the mean (4 replicates of 10 hens each per treatment) 3The BW of hen was indicated in table5 4All of the interactions studied between level and source of fiber, and energy level were not significant (p>0.10) Chapter 3: The experiment 63 Figure 1. Effect of fiber source of the rearing phase diets on egg production (A), egg weight (B), feed intake (C), feed conversion ratio (D) and BW gain (E) from 18 to 46 wk of age. Chapter 3: The experiment 64 Figure 2. Effect of level of fiber of the rearing phase diets on egg production (A), egg weight (B), feed intake (C), feed conversion ratio (D) and BW gain (E) from 18 to 46 wk of age. Chapter 3: The experiment 65 Figure 3. Effect of AMEn concentration of the laying phase diet (kcal/kg) on egg production (A), egg weight (B), feed intake (C), feed conversion ratio (D) and BW gain (E) from 18 to 46wk of age. Chapter 3: The experiment 73 Anejo Ingredient composition of the rearing phase diets (Guzmán et al., 2013) Table:Ingredient composition (% as fed basis, unless otherwise indicated) of the experimental diets 0-5 week 5-10 week 10-17 week Barley Corn Barley Corn Barley Corn 0% 2% 4% 0% 2% 4% 0% 2% 4% Barley 35.0 - - - 45.0 5.0 4.9 4.8 50.0 25.0 24.5 24.0 Corn - 40.0 39.3 38.5 - 40.0 39.2 38.4 - 40.0 39.2 38.4 Wheat 24.0 18.0 17.6 17.3 26.7 15.0 14.7 14.4 19.3 1.1 1.08 1.06 Soybean meal (47% CP) 32.1 35.0 34.3 33.6 23.6 25.2 24.7 24.2 18.0 18.8 18.4 18.0 Wheat bran - - - - - 10.1 9.9 9.7 7.3 10.2 10.0 9.8 Fiber sourcea - - 2.0 4.0 - - 2.0 4.0 - - 2.0 4.0 Poultry fat 4.6 2.65 2.54 2.44 1.03 1.04 1.02 0.98 1.02 0.5 0.49 0.48 Monocalcium phosphate 1.96 2.05 2.01 1.95 1.16 1.35 1.32 1.3 1.97 1.96 1.93 1,89 Calcium carbonate 1.29 1.26 1.22 1.20 1.43 1.28 1.26 1.23 1.46 1.5 1.47 1.45 Sodium chloride 0.34 0.36 0.35 0.34 0.35 0.33 0.32 0.31 0.35 0.35 0.34 0.34 DL-methionine (99%) 0.21 0.18 0.18 0.17 0.17 0.16 0.15 0.15 0.1 0.08 0.08 0.07 L-Lys HCl (78%) - - - - 0.05 0.04 0.03 0.03 - - L-Thr (98%) - - - - 0.01 - - - - 0.01 0.01 0.01 Vitamin and mineral premixb 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 a The fiber sources used were sugar beet pulp and straw, depending on experimental treatment. b Provided the following (per kilogram of diet): vitamin A (trans-retinyl acetate),6,000 IU; vitamin D3 (cholecalciferol), 1,200 IU; vitamin E (all-rac-tocopherolacetate), 5 mg; vitamin K3 (bisulphatemenadione complex), 1.5 mg; riboflavin, 3.5 mg; betaine, 67.5 mg; thiamin (thiamine-mononitrate) ,1 mg; vitamin B12 (cyanocobalamin), 15 µg; Se (Na2SeO3), 0.1 mg; I (KI), 1.9 mg; Cu (CuSO4・5H2O), 4 mg; Fe (FeCO3), 18 mg; Mn (MnO), 66 mg; and Zn (ZnO), 37 mg. 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