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Universidad de Zaragoza INTERNATIONAL CENTER FOR ADVANCED MEDITERRANEAN AGRONOMIC STUDIES MEDITERRANEAN AGRONIMIC INSTITUTE OF ZARAGOZA EFFECT OF LEVEL OF DIETARY SOLUBLE FIBRE AND THREONINE ON DIGESTION AND GROWTH PERFORMANCE IN POST-WEANING RABBITS Cecilia Alexandra CASTILLO MARTINEZ This work has been carried out at the Deparment of Animal Production, Escuela Técnica Superior de Ingenieros Agrónomos, Universidad Politécnica de Madrid, under the supervision of Dr. Rosa CARABAÑO LUENGO and Dr. Javier GARCÍA ALONSO. Thesis presented as a requirement for aspire to academic degree de Máster of Science en Nutrición animal: ACKNOWLEDGEMENT I wish to express my sincere gratitude to Mediterranean Agronomic Institute of Zaragoza and SENESCYT for giving me the opportunity to study the Specialization and Master of Science. By extension I’d like to offer my thanks to the Department of Animal Production of Escuela Técnica Superior de Ingenieros Agrónomos - Universidad Politécnica de Madrid that made this research possible I would like to express to thank to my supervisors Professor Rosa Carabaño and Javier García for giving me the opportunity to work with them, for their guidance, advice throughout the research project I would like to thank especially to my parents Julio y Lucia, my brother Roberto and my sister Diana, have given me their unequivocal support throughout during. Edgar, thank you for your support, love and companionship at all. I want to show my gratitude to my entire friend from Ecuador who has encouraged me with their best wishes, to all my friends in Spain for all their supports, kindness, friendship and making my stage very agreeable. Cecilia Alexanda CASTILLO MARTINEZ Junio, 17 del 2013 i ABSTRACT The aim of this work was to study the effect of soluble fibre and threonine deficiency on digestion and performance after weaning in rabbits. Four diets in a 2 x 2 factorial arrangement were used with two level of soluble fibre (89 vs.119 g/kg) and two level of threonine (5 vs. 6.4 g/kg). The effect of soluble fibre was studied substituting alfalfa hay (100 g/kg) by sugar beet pulp (126 g/kg), and the level of threonine was obtained supplemented L-threonine (0 vs. 1.42 g/kg). In the first experiment, 128 mixed-sex weanling rabbits of 35-d of age were used to determine the apparent faecal digestibility of DM and CP from 42 to 46 d (12/diet), the apparent ileal digestibility of DM and CP (at 46 d. 20/diet) and mucosa morphology (10/diet). In the second experiment, 140 weanling 25 d old rabbits of both sexes were used to determine the apparent faecal (from 32 to 35 d. 11/diet) and ileal (at 35 d. 20/diet) digestibility of DM, CP, TDF, starch and mucin concentration, mucosa morphology (10/diet) and growth traits (35/diet). In both experiments rabbits were fed with the experimental diets for 10 days and then they were slaughtered. In the first experiment rabbits fed with the high level of soluble fibre showed higher relative weight of total digestive tract and caecum and a lower caecal pH than those fed with a low level (P ≤ 0.032), with no effect on growth traits. The increase of soluble fibre level in the diet led to a higher villous height/crypt depth ratio (P < 0.001) and increased the number of goblet cells per villi (P<0.001). Threonine level did not affect any trait. In the second experiment, mortality decreased with the high level of soluble fibre (P = 0.002), and tended to be reduced with threonine level (P = 0.091). The increase of soluble fibre level also increased the villous height/crypth depth ratio (P < 0.001), the number of goblet cells (P = 0.008) and the ileal and faecal flow of mucins (P ≤ 0.033). However, no effect of threonine level on these traits was detected, but a positive effect on ileal starch digestibility in low soluble fibre diet was found (P < 0.001). The ileal and faecal digestibility of TDF increased with soluble fibre level (P ≤ 0.065), with no effect on organ weights. In conclusion, the increase of soluble fibre improved intestinal mucosa integrity and mucin secretion leading to a better health status of the rabbits when the sanitary conditions worsened. Mucosa barrier traits were not affected by dietary threonine level although in poor sanitary conditions a low threonine level impaired rabbit health suggesting a limiting status of this amino acid. ii RESUMEN El objetivo de este trabajo fue estudiar el efecto de un pienso deficitario en fibra soluble y treonina sobre la digestión y rendimiento en conejos post destete. Para ello se formularon cuatro piensos experimentales con un arreglo factorial 2 x 2 usando dos niveles fibra soluble (89 vs.119 g/kg MS) y dos niveles de treonina (5 vs. 6.4 g/kg MS). El efecto de la fibra soluble fue estudiado mediante la sustitución de heno de alfalfa (100 g/kg) por pulpa de remolacha (126 g/kg), y el nivel de treonina suplementando Ltreonina (0 vs. 1.42g/kg). En el primer experimento, se utilizaron 128 gazapos destetados a los 35 días de edad, y se determinó la digestibilidad fecal aparente de la MS y proteína bruta (PB) desde los 42 a los 46 días (12/pienso) y la morfología de la mucosa (10/pienso). En el segundo experimento, se utilizaron 140 gazapos destetados a los 25 días de edad, y se determinó la digestibilidad fecal aparente (desde los 32 a 35 días de edad) e ileal (a los 35 días de edad) de la MS, PB, fibra dietética total y almidón. Además, se cuantificó la concentración de mucinas, la morfología de la mucosa (10/pienso) y los rendimientos productivos (35/pienso). Los piensos experimentales fueron suministrados durante 10 días, tras los cuales los animales fueron sacrificados. En el primer experimento, los animales que recibieron los piensos con un mayor nivel de fibra soluble mostraron un mayor peso relativo del tracto gastrointestinal y del ciego, y un menor pH cecal en comparación con aquellos animales alimentados con los piensos con un bajo nivel de fibra soluble (P ≤ 0.032), sin observarse efecto alguno en el rendimiento productivo. Un mayor nivel de fibra soluble en la dieta condujo a un incremento del ratio longitud de villi/profundidad de cripta (P<0.001) y del número de células caliciformes por villi (P<0.001). Los niveles de treonina no afectaron a ninguno de estos parámetros. En el segundo experimento, un mayor nivel de fibra soluble redujo la mortalidad (P = 0.002) y un nivel adecuado de treonina también tendió a disminuirla (P = 0.091). Un mayor nivel de fibra soluble incrementó el ratio longitud de villi/profundidad de cripta (P < 0.001), el número de células caliciformes (P = 0.008) y el flujo ileal y fecal de mucinas (P ≤ 0.033). El nivel de treonina no afectó a ninguna de estas mediciones, sin embargo hubo un efecto positivo en la digestibilidad ileal del almidón en las dietas con bajo nivel fibra soluble (P < 0.001). Un mayor nivel de fibra soluble en la dieta incrementó la digestibilidad ileal y fecal de la FDT (P ≤ 0.065), sin afectar el peso de los órganos. En conclusión, el incremento del nivel de fibra soluble mejora la integridad de la mucosa intestinal y la secreción de mucinas, mejorando el iii estado sanitario de los animales cuando las condiciones sanitarias empeoraron. El nivel de treonina no afectó a ninguna característica de la barrera intestinal, si bien cuando las condiciones sanitarias son deficientes un nivel bajo de treonina deterioró el estado de salud de los animales, sugiriendo una limitación de este aminoácido. iv RESUMÉ L’objectif de ce travail était d'étudier l'effet des fibres solubles et la carence en thréonine sur la digestion et la performance après le sevrage chez les lapins. Quatre régimes ont été utilisés dans un arrangement factoriel de 2 x 2, avec deux niveaux de fibres solubles (89 vs.119 g/kg) et deux niveaux de thréonine (5 vs. 6,4 g/kg). L'effet des fibres solubles a été étudié en substituant la paille de luzerne (100 g/kg) par la pulpe de betterave sucrière (126 g/kg), et le niveau de la thréonine a été obtenu en supplémentant la ration par la L-thréonine (0 vs 1,42 g/kg). Dans la première expérience, 128 lapereaux, de deux sexes, sevrés et de 35 jours d'âge ont été utilisés pour déterminer la digestibilité fécale apparente de MS et PB entre le 42ème et 46ème jour (12/ration), la digestibilité iléale apparente de MS et BP (au 46ème jour, 20/ration) et la morphologie de la muqueuse (10/ration). Dans la deuxième expérience, 140 lapereaux sevrés de 25 jours d’âge et des deux sexes ont été utilisés pour déterminer la digestibilité fécale apparente (entre 32 à 35 jours, 11/ration) et iléale (à 35 jours. 20/ration) de la MS, PB, FDT, la concentration de l'amidon et de la mucine, la morphologie de la muqueuse (10/ration) et les paramètres de croissance (35/ration). Dans les deux expériences, les lapereaux ont été nourris avec les rations expérimentales pendant 10 jours, puis ils ont été abattus. Dans la première expérience, les lapins alimentés par un niveau élevé de fibre soluble ont montré poids relatif plus élevé de l'appareil digestif total et de caecum, en plus, un pH caecal plus bas que ceux nourris avec un faible niveau de fibre soluble (P≤ 0,032), sans effet sur les paramètres de croissance. L'augmentation du niveau de fibres solubles dans l'alimentation a conduit à la formation de villosité plus longue y un ratio de profondeur/hauteur des cryptes supérieur (P<0,001) et augmenté le nombre de cellules caliciformes par villosités (P<0,001). Le niveau de thréonine n’a eu aucune incidence sur les paramètres. Dans la deuxième expérience, la mortalité a diminué avec le niveau élevé de la fibre soluble (P=0,002), et tend à être réduit avec le niveau thréonine (P = 0,091). L'augmentation du niveau de fibres solubles a également augmenté le rapport hauteur/profondeur des cryptes des villosités (P<0,001), le nombre de cellules à mucus (P = 0,008) et le flux iléal et fécal des mucines (P≤0,033). Cependant, aucun effet du niveau de la thréonine sur ces caractéristiques de croissance a été détectée, mais un effet positif sur la digestibilité iléal de l'amidon dans le ration alimentaire de faible contenu en fibres solubles a été trouvé (P<0,001). La digestibilité iléale et fécale de FDT augmentait avec 1 I. INTRODUCTION AND OBJETIVES Recent studies have been focused on optimizing the post-weaning feed to improve digestive health in weaning rabbits. Total dietary fibre is the main component in the feed of rabbits accounting for 35 and 50% of the diet. Its importance is related to the influence exerted on the rate of passage of digesta and its function as substrate for microbiota, which in turn affect and regulate rabbit growth performance and digestive health. Insoluble fibre is the most important total dietary fibre fraction. It has been extensively studied and their chemical and physical characteristics play an important role in the digestive physiology of the rabbit and it is essential to avoid digestive disturbances (Gidenne et al., 2010). In contrast, soluble fibre has been scarcely studied in rabbit nutrition. It comprises non-starch and non-NDF polysaccharides (Hall, 2003). It is a minor, heterogeneous and highly degradable fraction of the total dietary fibre (Trocino et al., 2013a). Most diets in rabbits include sugar beet pulp (SBP) to increase the level of soluble fibre. The level of soluble fibre recommended is around 12% in the diet in the growth period (Trocino et al., 2013a). In a context of epizootic rabbit enteropathy, the inclusion of soluble fibre in the diet has been shown to have positive effects on intestinal health, which has been associated to an improvement of the intestinal mucosa integrity and the modulation of intestinal microbiota (Gómez-Conde et al., 2007, 2009). Another important effect of the inclusion of soluble fibre in the diet is its positive effect on mucin secretion. Studies carried out in rats by Satchithanandam et al. (1990) found that supplementation with 5% citrus fibre in a purified diet increased the mucin secretion in the stomach, small intestine and colon. In rabbits, the inclusion of sugar beet pulp and pectin as source of soluble fibre increase the number of goblet cells, the mucin concentration in the ileum and mucin flow to the caecum (Abad, 2011; El Abed et al., 2011b). Mucins are important components of the mucus layer. They are secreted by globet cells and play an important role in the prevention of chemical, mechanical, enzymatic damage of intestinal mucosa and bacterial adhesion in the mucosa (Deplancke and Gaskins, 2001). Mucins contain on average 20% of protein that is particularly rich in 2 threonine, serine and proline, representing 42% of amino acids composition of intestinal mucin in rabbits (Roberton et al., 1989). The increase of mucin production might modify the threonine requirements. The aim of this work was to study the effect of soluble fibre and threonine deficient diets on rabbits digestion and performance. To prove this effect four diets were formulated combining 2 levels of soluble fibre and threonine (according to requirements vs. deficient) (de Blas and Mateos, 2010; Trocino et al., 2013a) OBJETIVES: Identify the effect of soluble fibre, threonine and the possible interaction between the level of soluble fibre and threonine on: • Growth performance in the post weaning period. • Digestibility of nutrients (ileal and faecal digestibility). • Mucin production. • Morphology of the intestinal mucosa (villus height, crypt depth and goblet cells number). 3 II. LITERATURE REVIEW 2.1. Definition of dietary fibre Dietary fibre (DF) has been defined as the "sum of lignin and all polysaccharides that are not digested by enzymes secreted by the digestive system of man" (Trowell et al., 1976) and chemically defined as the set of non-starch polysaccharides and lignin that belong to the cell wall (Theander et al., 1997). It has been difficult to establish a precise definition of dietary fibre due to complex physical structure, chemical composition and high number of constituents. Recently, the Codex Alimentarius Commission (2009) defined dietary fibre as the carbohydrate polymers with ten or more monomeric units, lignin and other associated constituents, which are not hydrolyzed by the endogenous enzymes in the small intestine of humans and exert some specific physiology effects in the digestive tract. 2.2. Characterization and quantification of dietary fibre In general, dietary fibre is constituted by a series of components distributed in the plant cell wall and in the cytoplasm, according to a chemical structure that results in specific physical properties (Gidenne et al., 2010. See Figure 1). Plant cell wall is composed of microfibrils of cellulose forming a strong framework that gives rigidity to the plant. The microfibrils are embedded in a matrix composed by a lignin network cemented with another matrix of polysaccharides and glycoproteins (Cápita, 1996). Other polymers that comprise the cell wall are hemicellulose and pectin substances. The main cytoplasm components are storage carbohydrates like fructan, mannans, oligosaccharides and resistant starch and their proportion, as other components, depends on the particular plant. Full details of the fibre components of the main raw materials used in animal feed are described by Selvendran (1984), Bach Knudsen (1997), Gidenne et al. (2010) and Hall (2003). The most common definitions to refer to the animal physiology are insoluble and soluble fibre. The first one is comprised mainly by cellulose hemicelluloses and lignin, represents the greatest proportion of total dietary fibre (TDF). Soluble fibre (SF) represents the lowest proportion of TDF, that comprises the non-starch and non-NDF polysaccharides, the main components that are considered 4 part of the soluble fibre fraction are pectin substances, (1 → 3) (1 → 4)-β-glucans, fructans and gums (Hall, 2003). Figure 1: Schematic representation of the cell wall of the plant and its main components. (Gidenne et al., 2010). Due to the heterogeneity in the three-dimensional matrix of plant cell walls there is not available a methodology to accurately determine the dietary fibre of a feedstuff. According to the concept of dietary fibre, it could only be determined by the digestive balance in the animal. An indirect estimation of dietary fibre may be performed by different methodologies reviewed by (Bach Knudsen) 2001 and Mertens (2003) in which non-fibrous constituents are extracted well solubilized with chemical solutions, hydrolyzed enzymatically or by combining both procedures, once isolated the residue of fibre can be measured gravimetrically or chemically, leading to three methods: chemical-gravimetric, enzymatic-gravimetric and enzymatic-chemical methods. The most widely method used for determining TDF is the enzymatic gravimetric, this method simulate the digestion when incubating the samples with enzymes (amylases, proteases and amyloglucosidase). The TDF can divide into soluble (SDF) e insoluble fibre (IDF). Ethanol is used to precipitate and recover the indigestible water soluble polysaccharides (Prosky et al., 1992; AOAC, 2000). An alternative measure of soluble fiber is that of neutral detergent-soluble fiber, or those non-starch non-NDF polysaccharides soluble in neutral detergent plus heat-stable, α-amylase. (Hall et al., 1997, 1999). 5 In order to determine the component of cell wall matrix and estimate its major subcomponents (cellulose, hemicelluloses and lignin) the sequential analyses of detergent system: neutral detergent fibre (NDF), acid detergent fibre (ADF) and acid detergent lignin (ADL) were developed by Van Soest (1963). Modifications with heat stable α-amylase (aNDF) have been proposed to extent this application to grains, concentrated feed, also this fraction has been corrected by ash and reported in organic matter (aNDFom) (Mertens et al., 2002). In order to compare with another method for insoluble fibre, this fraction also need to be corrected for crude protein (CP) (aNDFom - cp) (Abad et al., 2013). Another alternative to estimate insoluble fibre is the in vitro indigestibility dry matter (ivDMi). Ramos et al. (1992) adapted the enzymatic in vitro method developed by Boisen (1991) for pigs to estimate the nutritive value of rabbit feeds. The aim of this method is to remove the digestible fractions of diet by using enzymes in three steps, the main part of indigestible fraction of the diet is dietary fibre. In order to compare with another method for insoluble fibre, the ivDMi2 and 3 steps need to be corrected for ash and crude protein (Abad et al., 2013). Soluble fibre can be estimated by difference using three procedures: TDF–IDF (SDFIDF), TDF–ivDMi2 (SDFivDMi2), and TDF – aNDFom-cp (SDFaNDFom-cp). (Abad et al., 2013). The quantification of soluble fibre depends on the method used to determine it, showing SDFaNDFom-cp the highest value between the three procedures (Abad et al., 2013). This method is used in most studies related on the role of soluble fibre in growing rabbits (Trocino et al., 2013a). The differences observed between these three procedures might be accounted for the different analytical conditions used in extraction procedures (mainly temperature, but also pH and reagents) to remove starch and protein in each method (Marlett et al., 1989). This is especially important in pectin rich feedstuffs, like SBP, where the use of boiling NDF solution (containing EDTA, a chelating agent of calcium bound in pectin complexes) led to solubilization of a higher amount of substances than for ivDMi2. While the use of SDFIDF showed intermediate values between SDFaNDFom-cp and SDFivDMi2 (Abad et al., 2013). Dietary fibre the major fraction of rabbit diets, where it accounts for 40 – 50% of the total diet. The levels of fibre in complete experimental feeds used for the growing rabbit are shown in Table 1. 6 Table 1: Levels of fibre (g kg−1 dry matter) in complete experimental feeds used for the growing rabbit (n = 111) (Villamide et al., 2009). Average Minimum Maximum aNDFom 368 248 443 ADFom 169 135 284 ADL 56 27 195 Hemicellulose 172 59 251 Cellulose 140 42 220 *SF 77 18 147 Other feed constituents Starch 176 82 324 Crude protein 166 122 244 Sugars 53 31 163 Ether extract 32 10 71 aNDFom: neutral detergent fibre assayed with a heat stable amylase and expressed exclusive of residual ash; ADF: acid detergent fibre; ADL: acid detergent lignin; CP: crude protein; EE: ether extract. *SF: determined by (Trocino et al., 2013a) using 18 experimental diets, the soluble fibre was calculated by difference between TDF–NDF, or as neutral detergent soluble fibre (NDSF, Hall et al., 1997), the pectin content of the diets given in the paper. 2.3. Effect of dietary fibre on digestion and gut barrier health 2.3.1. Fibre digestion Dietary fibre can be digested only through microbial fermentation in the digestive tract (de Blas et al. 1999). Some components of dietary fibre are degraded prior to entering the caecum of rabbit by the pectinase activity, the main fibrolytic enzyme present in the stomach, small intestine and cecum, and it is explained by caecotrophy (Marounek et al. 1995). One of the most important constituents of the pectins are uronic acid, that showed a relative high ileal digestibility (20 to 52%) (Gidenne, 1992; Carabaño et al., 2001). Moreover the high digestibility of soluble fibre in the ileum can be explained by the existence of a wide type of ileal microbiota (Gómez-Conde et al., 2007, 2009). On the other hand, glucose and xylose the major monomers from insoluble fibre in most fibre sources, showed a much lower ileal digestibility (Gidenne, 1992; Carabaño et al. 2001). Ileal digestibility coefficient of aNDFom-cp and SF (TDF – aNDFom-cp) increased with the level of soluble fibre, with increasing the proportions of SBP and apple pulp (Abad et al., 2012). Despite the low digestibility of the insoluble fibre, supplying insoluble dietary fibre to growing rabbits is essential to avoid digestive disturbances, as it warrants an adequate rate of passage (Gidenne et al., 2010). 7 The main site for dietary fibre fermentation seem to be the caecum, because of the higher microbial activity of the digesta and longer retention time in this segment, although it is mainly fermented the soluble fibre and short sized fibre particles (Gidenne et al., 2010). The ileal and caecal microbial population secretes enzymes capable of hydrolyzing the main components of dietary fibre. Greater enzymatic activity for degrading pectins and hemicelluloses than for degrading cellulose has been detected in several studies (Marounek et al., 1995; Gidenne et al., 2000, 2002). The source of fibre has a significant effect on the enzymatic activity (pectinolytic and cellulolytic) being higher in SBP diets than in alfalfa and wheat bran diets (Falcao e Cunha et al., 2004). It agrees with the increases in the digestion efficiency of fibre and in the microbial activity with the level of pectin substances in the diet Garcíaet al. (2000), such as accurs when SBP is introduced in them (Trocino et al., 2013a). These results are parallel to the faecal digestibility of the corresponding dietary fibre constituents in rabbits. Hemicelluloses show a higher digestibility than cellulose (46 vs. 27%). (Gidenne et al., 2010). Although the relative contents of digestible hemicellulose and cellulose in the diet might vary depending on the source of the fibre used (Trocino et al., 2013a). Lignin and cutin are considered almost totally undegradable and might limit the digestibility of hemicellulose in a great extent than that of cellulose (Gidenne et al., 2010). Therefore, increasing the levels of sugar beet pulp as source of soluble fibre with low lignified (insoluble fibre) and with a high hemicellulose to cellulose ratio has often been associated with an increase NDF faecal digestibility (See Figure 2), improving digestibility and energy value. In recent studies the apparent faecal digestibility of soluble fibre has been determined showing a range from 69.7 to 95.1% and a mean of 84.9%. (Abad. 2011, 2012; Trocino et al., 2010, 2011, 2013b). In all the cases the soluble fibre was determined by the difference between (TDF - aNDFom-cp). The inclusion of SBP as source soluble fibre in the diets, is associated with an increase of the relative weight of total digestive tract, stomach, caecum, as well as an increase of the volatile fatty acid concentration, especially acetate and propionate, and a reduction of caecal pH (Fraga et al., 1991; García et al., 1993; Carabaño et al., 1997; García et al., 2002b; Falcao e Cunha et al., 2004: Gómez-Conde et al., 2009; Trocino et al., 2011). This effect might be due to insoluble fraction of SBP rather than to the soluble fraction (El Abed et al., 2011a). 8 Figure 2: Effect of level of inclusion of soluble fibre on NDF faecal digestibility (Abad et al., 2011, 2012; Trocino et al., 2010, 2011, 2013b). 2.3.2. Effect of dietary fibre on gut barrier health The intestinal barrier plays a key role in the protection of rabbits and other animals against pathogens because it prevents the colonization and the translocation of bacteria and toxins. (Carabaño et al., 2008). Intestinal barrier begins in the intestinal lumen through the acidification and continues with the protection mechanisms of the epithelium by a mucus layer secreted by goblet and paneth cells. This mucus protects the lining of mechanical chemical or enzymatic damage and bacterial adhesion (Deplancke and Gaskins, 2001). Once this protection is lost and bacteria and toxins are in contact with the epithelium, the mucosal immune system linked to the mucosa is put into operation, first non-specifically and then developing tolerance mechanisms (Montage et al., 2004). Several criteria have been proposed to characterize mucosal integrity in poultry and pigs. Some of the most frequently used parameters are: enzyme activity in the lumen, brush border enzyme activity, mucosa morphology, others barrier function trait and microbial activity (Van der Kils and Jansman, 2002). In non-ruminants, level and source of dietary fibre affect intestinal mucosa (Montagne et al., 2003). In rabbits, both insoluble and soluble fibre plays an important role in morphology of intestinal mucosa. The inclusion of soluble fibre in the diet in substitution of insoluble fibre, improves the structure (villous height/crypt depth) and functionality (greatest sucrose activities) of mucosa and the immune response (See 9 Figure 3, Gómez-Conde et al., 2007). This study suggested that the soluble fibre has a protective effect upon the mucosa that favors an immune response. This positive effect of soluble fibre on the intestinal mucosa may also be related with the modulation of caecal microbiota (Gómez-Conde et al., 2007, 2009), reducing the frequency of detection of several potential pathogens as Clostridium perfringens and Campylobacter spp. These results suggest a positive influence of soluble fibre on intestinal health. Likewise pectin compared highly lignified fibre improved morphology of the jejunal mucosa and the activity of intestinal cells (Chiou et al., 1994). Similarly, rabbits fed with SBP showed higher villi height / crypt depth ratio than those fed with sunflowers hulls and straw (El Abed et al., 2011b). These authors also found that the animals fed with the soluble fraction of SBP (pectin) or with the insoluble fraction of SBP, showed intermediate values between sunflowers hulls/straw and SBP diet. Therefore, it seems that the beneficial effect on intestinal morphology is an additive effect of both soluble and insoluble fibre fractions of the SBP. However, these changes in gut mucosa morphology might be age-dependent as they were lower in animals at 45 d of age (Àlvarez et al., 2007) or not observed in older rabbits (51-56 d of age) (Trocino et al., 2010, 2011; Xiccato et al., 2011). The sampling site (jejunum vs. ileum), time after weaning and health status of the animals may also contribute to these differences (Trocino et al., 2013a). Figure 3: Effect of fibre source and soluble fibre levels on the morphology of the jejunal mucosa in 35-d-old rabbits (Gómez-Conde et al., 2007). P= 0.001 P= 0.001 a 16 of alfalfa hay with soya-bean protein concentrate in starter diets for young rabbits did not affect jejunal morphology (Chamorro et al., 2007). There is scarce information about the effect of the protein on the mucus layer especially in rabbits. Studies performed in calves showed that both the level and source of protein influenced the production of mucin. When dietary crude protein supplied by skim milk powder rose from 14 to 278 g of CP/kg DMI, the flow of mucin protein increased at the duodenum (+300%) (Montage et al., 2000). The flow of mucin protein also, increased by 70% at the duodenum and at the jejunum when protein from skim milk powder was partially replaced by soybean protein concentrate and hydrolyzed soybean protein isolate. When it is replaced by potato protein concentrate, the mucin flow also increased at the duodenum (+24%) and at the ileum (+52%) (Montage et al., 2000). In pigs the study conducted by Piel et al. (2007) showed that both protein and fibre types affected mucin secretion. Diets with high indigestible protein and fibre increased mucin secretion by 46% in relation to other more digestible diets. 2.5. Importance of nitrogen endogenous losses in rabbit The classical definition of endogenous nitrogenous comes from Mitchell (1924), according to his definition, endogenous nitrogen is the nitrogen found in chyme or faeces when a nitrogen-free diet has been fed. Endogenous secretions come from various sources including saliva, pancreatic secretions, and bile, sloughed off epithelial cells, serum albumin and mucin (Nyachoti et al., 1996). In pigs, this fraction when passing into the small intestine can be hydrolyzed and absorbed before reaching terminal ileum while into large intestine can be metabolized by local microbes like the mucin, or excreted through feces (Souffrant et al., 1993). Endogenous losses have been divided into basal and the specific endogenous losses, the first one are directly related to the dry matter intake (Hess and Sève, 1999), and the specific endogenous losses are induced by specific composition and characteristics of the diet (Boisen and Moughan, 1996). In this sense, García et al. (2004) and Villamide et al. (2013) found a lineal relationship between feed intake and endogenous ileal protein flow, in rabbits using diets with highly digestibility casein. The endogenous ileal flow of N increased with the feed intake around 6 mg of N/g DMI (Villamide et al., 2013). 17 Endogenous nitrogen accounts for 50 -75% of the total ileal flow of nitrogen in the digesta of rabbits, the remaining percentage correspond to the indigestible nitrogen linked to NDF and to the small presence microbial nitrogen (García et al., 2005; Villamide et al., 2013). According to these studies the level of endogenous nitrogen losses in rabbits is greater to that found in pigs (5.45 g/kg DMI vs. 2.02 g/kg DMI) (Jansman et al., 2002; Villamide et al., 2013) using the same methodology of determination (casein based diet). The major reason could be the higher amount of fibre in rabbit diets respect to other non-ruminant species, 270-330 vs. 30-80 g NDF/kg DM in rabbits and pigs diets, respectively (García et al., 2005). The endogenous flow of amino acids determined from rabbits fed a casein basal diet acids varied from 5.053 g/kg DMI for glutamic acid to 0.38 g/kg DMI for methionine, being 2.21 g/kg DMI for threonine (García et al., 2004. Table 3). Table 3: The amino acid composition (g/16 g N) of endogenous ileal protein of rabbits fed a casein basal diet. Amino acid composition (g/16gN) Endogenous flow of amino acid (g/kg DMI) García et al. (2004) Llorente et al. (2006) García et al. (2004) Villamide et al. (2013) Essential amino acids Arginine 4.63 3.60 1.85 1.23 Cystine 3.11 2.70 1.24 0.94 Histidine 1.53 1.30 0.61 0.44 Isoleucine 3.72 3.80 1.49 1.30 Leucine 4.77 4.30 1.90 1.47 Lysine 3.76 3.60 1.50 1.21 Methionine 0.96 0.70 0.38 0.27 Phenylalanine 2.09 4.10 0.83 1.40 Threonine 5.53 5.60 2.21 1.89 Valine 5.64 5.10 2.25 1.72 Nonessential amino acids Alanine 3.56 3.40 1.41 1.15 Aspartic acid 7.53 7.20 3.01 2.46 Glutamic acid 12.7 12.5 5.05 4.24 Glycine 5.18 8.00 2.07 2.76 Proline 5.41 4.70 2.16 1.59 Serine 6.45 5.80 2.57 1.96 Tyrosine 2.07 3.50 0.83 1.18 The ileal endogenous N flow comes from different origins that condition the endogenous amino acid profile that seems to be rather constant. The predominant endogenous amino acids found in the ileal digesta are glutamic acid, aspartic acid, 18 threonine, serine and glycine. (García et al., 2004; Llorente et al., 2006. Table 3). The concentration of threonine is high in the ileal endogenous losses 5.53 g per 16 g N compared to lysine and methionine 3.76 g and 0.96 g per 16 g N, respectively. It implied a higher ileal flow of threonine compared lysine and methionine. This important flow of threonine can be explained by the mucins content that are rich in threonine (Roberton et al., 1989). The specific endogenous losses are related to the characteristics of the diet. The presence of fibre and anti-nutritional factors can double its production in pigs (Boisen and Moughan, 1996). Some studies have determined the true ileal digestibility of protein and amino acids from raw materials frequently used in rabbits feeding. These studies suggest that correction the ileal endogenous mainly affected the ileal digestibility of those amino acids in the major proportion in the endogenous substance flow, as threonine, and/or minority amino acid total amino acid content of the feedstuff (Table 4. García et al., 2005). Table 4: Apparent ileal (AI) and true ileal (TI) digestibility of raw material. Raw Material Autor AID TID TID - AID CP THR CP THR CP THR Maize Llorente et al. (2007) 49.4 15.8 78.2 62.0 28.8 46.2 Wheat Llorente et al. (2007) 67.0 44.0 89.1 84.3 22.1 40.3 Wheat bran García et al. (2005) 52.9 43.9 69.8 74.4 16.9 30.5 Barley grain García et al. (2005) 61.9 45.7 79.6 72.5 17.7 26.8 Gluten feed Llorente et al. (2007) 65.5 49.8 78.1 71.5 12.6 21.7 Peas Llorente et al. (2006) 76.1 63.3 86.3 84.6 10.2 21.3 Alfalfa hay García et al. (2005) 59.0 56.2 74.2 75.2 15.2 19.0 Sunflower 28% Llorente et al. (2006) 76.1 74.1 88.3 90.4 12.2 16.3 Sunflower 38% Llorente et al. (2006) 80.4 76.3 89.0 88.8 8.60 12.5 Soybean tosted Llorente et al. (2006) 82.3 76.1 93.7 87.9 11.4 11.8 Sunflower 36% García et al. (2005) 80.7 73.8 86.1 84.6 5.40 10.8 Soybean meal Llorente et al. (2006) 86.7 81.4 93.6 91.3 6.90 9.90 One of the most important constituents of endogenous losses are mucins, as mentioned in Section 2.3. Mucins are secreted by specialized cells called and paneth goblet cells and have an important role in the intestinal barrier. The most remarkable property of mucin is its ability to form a gel, a viscoelastic semisolid material that adheres to the epithelial surface and provides a physical barrier between the underlying cell surface and lumen (Bansil et al., 1995). 19 The mucin are glycoproteins that are constituted by oligosaccharide chains of 5– 15 monomers, exhibit moderate branching and are attached to the protein core by Oglycosidic bonds to the hydroxyl side chains of serine, threonine and proline and arranged in a “bottle brush” configuration around the protein core (Bansil et al. 1995). Mantle and Thakore (1988) determined the carbohydrate profile of purified rabbit intestinal and colon mucins (Table 5). Whereas Roberton et al. (1989) using different techniques of purification of mucin in different species, determined protein content in different species around 20% of dry weight, and similar features in their amino acid profile (Table 6). The major amino acid residues (serine, threonine and proline) tend to comprise 40-50 mol/100mol. A recent study conducted by Romero et al. (2011), determined a protein concentration of 25% in crude mucin of ileal digesta in rabbits using different type of grinding of barley and dehydrated alfalfa, although these authors did not purify intestinal mucin from other proteins. Table 5: Carbohydrate profiles of purified rabbit intestinal and colonic mucins (mol/100mol) (Mantle and Thakore, 1988). Upper small intestine Mid small intestine Distal small intestine Proximal colon Carbohydrates Fucosa 9.5 9.0 9.9 10.3 Mannose 2.1 2.0 1.6 1.8 Galactose 21.6 21.9 22.7 23.7 N-acetylglucosamine 21.9 21.1 21.1 21.1 N-acetylgalactosamine 28.4 29.8 29.7 20.2 Sialic acid 16.4 16 15.0 22.9 Sulphate 5.9 2.0 6.8 5.3 Table 6: Amino acid composition of purified mucins (mol/100mol) (Roberton et al., 1989). Mucin of small intestine Mucin of colon mucin Rabbit Human Pig Rat Rabbit Human Serine + Proline + Threonine 42.3 45.5 52.3 48.7 42.3 55 Aspactic acid + Glutamic acid 14.8 13.5 8.8 13.1 16.3 12.3 Glycine + Valine + Alanine 20.9 17.6 16.6 14.7 18.8 15.9 Leucine + Isoleusine 8.2 10.5 7.4 8.3 8.4 8.2 Rest of amino acid 13.7 12.9 14.9 15.2 14.2 8.6 Protein (% of dry weight) 18.6 14 17.9 13.4 17.4 — 20 The carbohydrate structures found on mucin macromolecules are extraordinarily diverse, providing a vast array of potential binding sites for both commensal and pathogenic organisms. The attachment of microbes to intestinal mucins depends on many factors such as the composition and quantity of mucins, intestinal motility and rate of intestinal fluid flow. Increased mucin secretion depends on many factors one of them is the inclusion of fibre and protein in the diet, as previously commented. (Deplancke and Gaskins, 2001). 2.6. Requirements and function of threonine for growing rabbits After lysine and methionine, threonine is the next most limiting amino acid in swine and poultry diets, while for rabbit diets is the second and sometimes the first (de Blas et al., 2000). The raw materials used in diets for rabbits have a strong deficit of threonine that might require to be incorporated into the diet as synthetic threonine (Lthreonine) (Colin and Ghezal-Triki, 2001). The quantitative essential amino acid requirement of the growing rabbit, estimated, using weight gain as the response criterion, indicated a threonine requirement of 0.50% of the diet (Adamson and Fisher, 1973). An approach to balance the supply of amino acids is to use the concept of ideal protein. This method was used in growing rabbits by Moughan et al. (1988) and consists in supplying a dietary protein with an amino acid pattern similar to that of the amino acid of the whole body. Body the lysine concentration is 6.12 g/16gN and threonine concentration 3.94 g /16gN, accounting for 0.64 of lysine. This amino acid pattern can be regarded as an approximated ideal balance of dietary amino acids. Also, de Blas et al. (1998) supplementing a basal diet with L-threonine determined a minimal dietary concentration of 6.0 crude and 4.0 g/kg digestible threonine to maximize the performance of growing rabbits (See Table 7). A level of dietary threonine below or above (0.30% - 0.80%, in the diet respectively) of the optimum (0.50%) shows negative impact on feed intake and weight gain (Adamson and Fisher, 1973). Studies carried out in pigs have demonstrated that 60% of dietary threonine is retained on the first-pass metabolism (Stoll et al., 1998). Once taken up by the mucosal cells, threonine may have different metabolic fates, including oxidation. Threonine is 21 catabolized either by threonine dehydratase to NH4+ and 2-ketobutyrate, which is irreversibly converted to CO2, or by threonine dehydrogenase to form 2-amino-3ketobutyrate, which is mainly converted to glycine and acetyl-coenzyme A (Ballevre et al., 1990). It appears that most threonine used by the intestine is for mucosal and secretory protein synthesis because threonine oxidation represents only 2–9% of the total threonine utilized (Schaart et al., 2005). Table 7: Threonine requirement expressed in units total and faecal digestible in the growing-finishing rabbits according to different authors. Thr total Thr/lys total Thr dig Thr dig/Lys dig Adamson and Fisher (1973) 0.50 0.71 _ _ Davidson and Spreadbury (1975) 0.58 0.62 _ _ Colin and Ghezal-Triki (2001) 0.60 _ _ _ de Blas et al., (1998) 0.60 0.67 0.40 0.67 de Blas and Mateos (2010) 0.62 0.85 0.43 0.75 Thr total: Threonine total, Thr/lys total: ratio threonine/lysine total; Thr dig: Digestible threonine; Thr dig/Lys dig: ratio threonine/lysine digestible. In pigs, deficit of threonine below those recommended levels (-30%) reduced of villus height and crypt depth compared to the control diet in the ileum section whereas proximal and distal in the jejunum no differences were found, this low level of threonine did not affect growth performance nor growth of the intestine (Hamard et al. 2007). The presence of threonine is essential for the production of mucin in rat and piglets (Faure et al., 2005; Law et al., 2007). Threonine deficient diets affected negatively the quantity as well as the characteristics of mucins. This decrease in mucin production is directly related with a severely lower numbers of goblet cells (Faure et al., 2005). Other research shows, that the deficit or excess of threonine in the diet (0.37 - 1.11%, true ileal digestible threonine (TIDT) respectively) reduced the synthesis of intestinal mucosal protein and mucins compared with pigs fed 0.74 % TIDT according current NRC requirements (Wang et al., 2007). These results are consistent with later studies, which observed reduction in the levels of acidomucins and sulfomucins, respectively, in the ileum and duodenum of weanling pigs fed either a low or high level of dietary threonine (Wang et al., 2010). The mucins are continuously synthesized and there is a continuous and irreversible loss of threonine (Van Der Schoor et al., 2002). In rabbits through caecotrophy process, threonine can be recycled form microbial protein that can contribute to 21% of the total threonine intake (Nicodemus et al. 1999). 22 All these studies have been carried out in pigs, whereas there is scarce information on rabbits. Recent studies in pigs suggest that there may be some interaction between soluble fibre and threonine. The results of several studies (Zhu et al., 2005; Myrie et al., 2006) showed that soluble non-starch polysaccharides increased endogenous losses of amino acids especially threonine and affected the utilization of this amino acid for protein deposition and growth. However, Święch et al. (2012) showed that 8% inclusion pectin in the diet did not affect the number of goblet cell producing acidic and neutral mucins mid-jejunum and ileum. However, these authors found a the positive effect of pectin on the number of goblet cell containing neutral mucins in crypts of the ileum, that suggested an increase of the rate of mucosal protein synthesis particularly of threonine rich mucin protein. In this study the inclusion of pectin did not produce evident changes in threonine metabolism. Additionally there was a reduction of total tract protein digestibility and therefore the nitrogen retention was decreased with supplementation. 23 III. MATERIAL AND METHODS 3.1. Experimental diets Four diets in a 2 x 2 factorial arrangement were used with two level of soluble fibre (89 vs.119 g/kg) and two level of threonine (Thr) (5 vs. 6.4 g/kg). The effect of soluble fibre was studied substituting alfalfa hay (100 g/kg) by sugar beet pulp (126 g/kg), the level of Thr was obtained supplemented L-threonine (0 vs. 1.42 g/kg). Diets were formulated according to the concept of ideal protein, thus faecal digestible lysine levels were 0.57% in all diets in order to be a sub-limiting aminoacid according to recent lysine requirements (Carabaño et al., unpublished). In the diets supplemented with threonine the ratio digestibility threonine/lysine was 0.75, whereas diets not supplemented with threonine the ratio was 0.58. It resulted in two diets with low soluble fibre without or with Thr supplementation (LSF/LThr and LSF/HThr), and two diets with high soluble fibre without or with Thr supplementation (HSF/LThr and HSF/HThr). Diets contained similar concentrations of CP and NDF (162 and 326 g/kg DM respectively). To determine the apparent ileal digestibility, 5 g of DM/kg of alfalfa hay labeled with Yb2O3 was included in all diets. The diets were formulated to meet the nutrients requirement suggested by de Blas and Mateos (2010), except for soluble fibre and threonine. Ingredients and chemical composition of the experimental diets are showed in Table 8. 3.2. Experiment 1. 3.2.1. Animal and housing This study was approved by the Committee of Ethics of the Departamento de Producción Animal of the Universidad Politécnica de Madrid. All animals were handled according to the principles of animal care published by Spanish Royal Decree 1201/2005 (BOE, 2005). Crossbred (New Zealand White × Californian) healthy rabbits mixed-sex were used in the experiments. The first experiment was carried out in the facilities of NUTRECO. Rabbits were housed in metabolism cages measuring 40 × 51 × 32 cm to collect their faeces in the faecal digestibility trial, while animals used to determine ileal digestibility were housed in pairs. Housing conditions were controlled during the whole experimental period as follows: a 12-h light-dark cycle was established and temperature conditions were maintained between 18 and 23°C by 24 heating systems combined with continues forced ventilation. Rabbits had ad libitum access to feed and water. 3.2.2. Determination of ileal and faecal digestibility Fourty-eight mixed-sex weanling rabbits of 35-d of age and a body weight (BW) of 831 ± 90g (mean ± standard deviation) were blocked by litter and randomly assigned to the 4 experiment diets (12 rabbits/diet). After 7-d of adaptation to the diets, feed intake was recorded and the faeces collected for 3 days consecutive to determine the apparent faecal digestibility of DM and CP. A second group of eighty weanling rabbits of both sexes, 35-d of age and a BW of 833 ± 116 g (mean ± standard deviation) were blocked by litter and randomly assigned to the four experimental diets (20 rabbits/diet) to determine the apparent ileal digestibility of DM and CP. Feed intake, growth rate, feed efficiency, mortality and morbidity were recorded up to 46-d of age. The symptoms of morbidity taken into consideration were: aqueous diarrhoea, mucus in faeces and relative low body weight as well as sharp reduction of feed intake and once slaughtered distended intestinal segments by gas and liquid and compacted caecal content. At 46-d animals with 1506 ± 171 g (mean ± standard deviation) were slaughtered by CO2 inhalation between 19:00 and 21:00 h to minimize the influence of caecotrophy. The whole gastrointestinal tract was weighed, the stomach and caecum were extracted and weighed with their content and the caecal pH was measured. The caudal 20 cm of the ileum in each case was then excised, emptied and the digesta were frozen in dry ice. For the chemical analysis of the ileal content, samples were freeze-dried and grounded. Due to the small amounts of sample, ileal content from 2 rabbits for each treatment were pooled (10 pool/treatment). The ileal digestibility of DM and CP was determined by the dilution technique using ytterbium as a marker according Goméz-Conde et al. (2007). 3.1.4. Determination of mucosa morphology To study mucosa morphology 6 cm sample to the measurement of intestinal mucosa morphology was excised from the middle part of the jejunum of rabbits (10/treatment) and placed into a 10% neutral buffered formaldehyde solution (pH 7.2 to 7.4). 25 3.3. Experiment 2 3.3.1. Animal and housing The second experiment was carried out in the facilities of ETS de Ingenieros Agrónomos de la Universidad Politécnica de Madrid, following the same standards as in the first experiment. The animals were caged in individual flat-deck cages measuring 60 × 25 × 32 cm high, while those used for faecal digestibility were caged in metabolism cages measuring 40 × 51 × 32 cm that allowed the faeces collection. Housing conditions were controlled during the whole experimental period as follows: a 12-h light-dark cycle was established and temperature conditions were maintained between 18 and 23°C by heating systems combined with continues forced ventilation. Rabbits had ad libitum access to feed and water. 3.3.2. Determination of ileal and faecal digestibility One hundred and forty weanling 25 d old rabbits of both sexes, and live weight of 355 ± 44 g (mean ± standard deviation) were blocked by litter and randomly assigned to the 4 experimental diets (35 rabbits/diet). After 7-d, feed intake was recorded and the faeces collected for 3 consecutive days to determinate from 32 to 35 d of age the apparent faecal digestibility of DM, CP, TDF and starch according to Gómez-Conde et al. (2011). Growth rate, feed efficiency, mortality and morbidity were recorded up to 35-d. Symptoms of morbidity were: aqueous diarrhoea, mucus in faeces and relative low body weight as well as sharp reduction of feed intake and once slaughtered distended intestinal segments by gas and liquid and compacted caecal content. At 35-d animals with a live weight of 764 ± 89 g (mean ± sd) were slaughtered by CO2 inhalation between 19:00 and 21:00 h to minimize the influence of cecotrophy, to determine the apparent ileal digestibility of DM, CP, TDF and starch. The same measurements were made than in experiment 1 (weight of digestive organs and caecal pH) and ileal digesta was collected. Due to the small amount of sample, ileal content from 3 to 5 rabbits for each treatment were mixed resulting in 8 pool/diet, in which were determined Yb and CP to calculate ileal digestibility of DM and CP. A sample of 0.5 g from each pool was taken to constitute a unique pool of 4 g per treatment, in which were determined starch, TDF and crude mucin. The values obtained were used to calculate ileal starch and TDF digestibility in combination with the Yb content of the original pools. The ileal flow of DM, CP, starch, TDF and mucin was calculated by 32 33 V. DISCUSSION The inclusion of sugar beet pulp to increase the dietary soluble fibre in post weaning diets have demonstrated to exert a positive effect on intestinal health of rabbits (Trocino et al., 2013a) probably mediated through changes the intestinal microbiota and in the mucosa integrity, that seems to increase the endogenous substances, and specifically the mucin secretion (El Abed et al., 2011b, 2013). This situation might increase the threonine requirements as observed in rats (Faure et al., 2005). In our first experiment, there was no mortality in the post weaning period (35-46 d), the growth performance showed relatively high figures, taking into account the sublimiting faecal digestible lysine content of diets, and there was no effect of soluble fibre inclusion on these traits nor in feed intake, feed efficiency and in the ileal and faecal DM and CP digestibility. However, the inclusion of soluble fibre increased the relative weight of digestive tract and caecum, in spite of the lack of effect on feed intake. These effects are more related with the insoluble fraction than with the soluble fraction of sugar beet pulp (El Abed et al., 2011a). The increase of soluble fibre also decreased caecal pH, suggesting a higher fermentation activity in the caecum as previously described (Gómez-Conde et al., 2009; El Abed et al., 2011a). Our results also confirm a positive effect of the inclusion of soluble fibre on the mucosa integrity, improving the morphology and increased the number of goblet cells in the jejunal mucosa as observed previously (Gómez-Conde et al., 2007; El Abed et al., 2011b, 2013). It might suggest a higher intestinal mucin production (Abad, 2011; El Abed et al., 2011b), although in this experiment it was not measured, which might increase the threonine requirements of the rabbits. However, feeding rabbits under their threonine requirements had no effect on growth performance, mucosa morphology and number of goblet cells, and did not interact with level of soluble fibre. These results are similar than those reported in piglets by Hamard et al. (2007) that did not found any effect of a threonine deficit on growth performance and goblet cells number, although they found a negative effect of a threonine deficit on the villous height/crypth depth ratio. Our results contrast with the negative effect of soluble fibre when pigs were fed a threonine limiting diet on protein deposition (Zhu et al., 2005), although these authors provoked higher differences in soluble fibre among diets than in the present work (0 vs. 12%), or with the positive effect of optimal threonine levels on intestinal mucosal barrier of neonate and weanling 34 pigs (Law et al., 2007; Wang et al., 2010). It would suggest that threonine requirements just after weaning might be lower than those proposed for the average growing period, which would be in accordance with the results obtained by de Blas et al. (1998). It might be accounted for the relative higher demand of amino acids at weaning for growth than for maintenance, the main destination for threonine, circumstance also observed in pigs (NRC, 2012), even when the type of diet (high in soluble fibre) might have enhanced threonine requirements. On the other hand, this work used recommendations obtained 15 years ago (de Blas et al., 1998) when rabbit growth capacity was lower (around 20%) than that of the rabbits used in this work. These recommendations seems to meet current rabbit requirements which might be explained by a higher efficiency of the utilization of amino acids for growth retention and maintenance in the current hybrids as suggested by García-Palomares et al. (2006). The second experiment were carried out in a farm with periodic outbreaks of epizootic rabbit enteropathy, and the incidence of mortality in the post weaning period (25-35 d) was significant. The reduction of level of soluble fibre increased the mortality rate confirming its positive effect described by Trocino et al. (2013a). In this study, also the reduction of threonine level under the recommended value tended to impair mortality incidence, which might indicate its limiting content in low threonine diets, in contrast with the results of the first experiment especially with poor sanitary conditions. However, the mortality rate was not recorded in the whole fattening period and we must be carefully in the interpretation of these results. The better health status in rabbits fed high soluble fibre diets accounts for their higher feed intake and the trend to have a higher growth rate. The increase of soluble fibre enlarged the length of the villous, increased the villous height/crypth depth ratio leading to an improvement of ileal starch digestibility, confirming again the already reported positive effect of soluble fibre on mucosa morphology (Gómez-Conde et al., 2007). Furthermore, soluble fibre inclusion also increased the number of goblet cells per villi, and the ileal and faecal flow of intestinal mucins suggesting that its effect on the intestinal mucus layer might be one of the preventive mechanism exerted by soluble fibre, similar to that observed in rats (Ito et al., 2009). In fact, mucin secretion seems to be accompanied by a pool of antimicrobial substances, mainly secreted by Paneth cells in the crypth, encharged to avoid the mucosa colonization (McGuckin et al., 2011). The low of intestinal mucin in the faeces indicated that they are extensively fermented in the caecum (96 %) and might 35 be another factor influencing the profile of caecal microbiota. In spite of the strong effect of soluble fibre on goblet cells and mucin production in this experiment, or the positive trend observed for threonine content on mortality, no effect of threonine level was reported on mucosa morphology or mucin production, in contrast with previous results in rats and pigs (Faure et al., 2005; Wang et al., 2007). However, dietary threonine improved ileal starch digestibility in low soluble fibre diets with no effect in high soluble fibre diets. This result might indicate that supplementation of threonine in low soluble fibre diets would enhance mucosa functionality (Gómez-Conde et al., 2007). Level of threonine did not modify ileal CP digestibility but faecal CP digestibility decreased for high threonine diets. It could be explained through a more important presence of endogenous substances in faeces, although faecal mucin content was not affected by threonine, or by a higher presence of microorganisms. The increase of soluble fibre tended to improve the ileal TDF digestibility which is a similar result than those reported by Abad et al. (2012). It is accounted for the increase of both soluble fibre and insoluble fermentable fibre when sugar beet pulp is used for increasing the level of dietary soluble fibre (Abad, 2011). This effect might be related to the changes observed in ileal microbiota profile when dietary soluble fibre is increased (Gómez-Conde et al., 2007; El Abed et al., 2013), although in this study the TDF flow to the caecum was not modified by soluble fibre in contrast to the flow of mucin or that of starch, that tended to be higher for low soluble fibre diets, especially for that with low threonine diet content (LFS/LThr). Faecal digestibility of TDF and NDF also improved with soluble fibre in agreement with Abad et al. (2013) and Trocino et al. (2013a), with no effect of threonine, as expected. Surprisingly, the diet high in both soluble fibre and threonine showed the lowest faecal starch digestibility. It might be attributed to a higher presence of microorganisms that contain minor but appreciable starch content. Microbial protein accounts for 40% of the total CP content in the hard feces (Carabaño et al., 2000), and α-linked glucose starch glucose) content in bacteria was around 2.4% DM (Merry and McAllan, 1983). In contrast with our first experiment, soluble fibre did not influence relative weight of organs that according to the younger rabbits used were higher in the second experiment. Anyway, caecal pH followed the same trend and tended to be acidified with the increase of soluble fibre which is in agreement with the higher faecal digestibility of TDF and NDF reported. 36 37 VI. CONCLUSIONS The levels of inclusion of soluble fibre and threonine in the diet did not affect the growth performance rabbits, which, might indicate that the inclusion of soluble fibre did not affect threonine requirements for growth. The increase of soluble fibre improved intestinal mucosa integrity and mucin secretion leading to a better health status of the rabbits when the sanitary conditions worsened. Mucosa barrier traits were not affected by dietary threonine level suggesting that diets met threonine requirements. However, in poor sanitary conditions a low dietary threonine level impaired rabbit health status suggesting a limiting status of this amino acid, although no clear effects on mucosa integrity were detected. Further, researches on this topic are warranted to clarify these effects. 38 39 REFERENCES AACC., 2001. The definition of dietary fibre report of the dietary fibre definition. Committee Cereal Foods World. 46, 112-126. Abad, R., 2011. 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Journal of Animal Science. 83, 1044-1053. 51 Table 8: Ingredients and chemical composition of experimental diets L-SF/ L-Thr L-SF/ H-Thr H-SF/ L-Thr H-SF /H-Thr Soluble fibre, g/kg 89 89 119 119 Threonine1, g/kg 5 6.4 5 6.4 Ingredientes, g/kg Wheat 135.6 134.2 100 98.6 Barley meal 78.7 78.7 79.6 79.6 Sunflower meal 79.0 79 97.2 97.2 Alfalfa hay 295.0 295 195 195 Sugar beet pulp — — 126 126 Wheat bran 300.0 300 300 300 Cereal straw 72.4 72.4 70 70 Soy bean oil 18.9 18.9 10 10 Alfalfa hay – Yb 5.0 5.0 5.0 5.0 L-Lysine-HCl 2.2 2.2 2.3 2.3 L-Treonine — 1.42 — 1.42 DL-methionine 0.5 0.5 1.4 1.4 Sodium chloride 4.0 4.0 4.0 4.0 Calcium carbonate 5.7 5.7 6.5 6.5 Vitamin and mineral premix2 3.0 3.0 3.0 3.0 Analyzed composition, g/kg DM Dry matter 919 927 918 924 Ash 65.9 67.1 64.1 63.6 Crude protein 164 165 161 158 Starch 238 240 219 196 Total dietary fibre 415 403 448 454 Insoluble fibre aNDFom 366 354 379 373 aNDFom-cp 326 313 336 330 Acid detergent fibre 173 172 182 177 Acid detergent lignin 34.5 37.3 34.2 33.5 ivDMi23 356 349 380 393 ivDMi3 298 296 295 301 Soluble fibre TDF-aFNDmo-cp 88 90.0 113 125 TDF-ivDMi2 58.5 53.5 68.5 60.6 CP-TDF, % 37.2 36.6 38.8 39.6 CP-NDF, % 24.5 24.8 27.5 28.0 Calculated composition1, g/kg Lysine 7.0 7.0 7.2 7.2 Methionine 2.8 2.8 3.7 3.7 Threonine 5.0 6.4 5.0 6.4 Calcium 8.0 8.0 8.0 8.0 Phosphorus 5.2 5.2 5.1 5.1 Sodium 2.0 2.0 2.1 2.1 Calculated faecal digestible content4, g/kg Lysine 5.7 5.7 5.7 5.7 Threonine 3.0 4.4 3.0 4.4 1 Calculated value using Tables (Fedna 2010) 2 Provided by Trouw Nutrition. Vitamin and mineral premix composition (per kg of diet); mg/kg: MgO: 240 mg; S: 240 mg; Mg as Mg2O3: 20 mg; ZnO: 60 mg; Cu as CuSO45H2O: 18 mg; I as KI: 1.10 mg; Co as 2CoCO3.3Co(OH)2 H2O: 0.30 mg; Se as Na2SeO3: 0.05 mg; Fe as FeCO3: 78 mg; vitamin A: 9999.9 UI; vitamin D3: 1080 UI; vitamin E like of acetate dl-alpha-tocopherol: 36 UI; vitamin K: 1 mg; vitamin B1: 2 mg; vitamin B2: 6 mg; vitamin B6: 2 mg; vitamin B12: 10 mg; niacin: 50 mg; calcium pantothenate: 20 mg; folic acid: 5 mg; pantothenic acid: 18.4 mg; Biotin: 60 mg; E771 diclazuril 0.5g/100g (clinacox 0.5%): 1 mg; E320 butylhydroxyanisole (BHA): 0.12 mg; E321 butylhydroxytoluene (BHT): 1.32 mg; E324 ethoxyquin: 0.19 mg. 3ivDMi2; ivDMi3: in-vitro indigestibility of dry matter 2 and 3 steps, respectively. 4Estimated from García et al. (2005) and Villamide et al. (2013). 52 Table 9: Effect of level of soluble fibre and threonine on growth traits in rabbits from 35 until 46 d old rabbits (Exp. 1) Experimental diets P-value L-SF / L-Thr L-SF / H-Thr H-SF / L-Thr H-SF / H-Thr SEM1 Soluble fibre Threonine Soluble fibre x Threonine BW 35d Soluble fibre, g/kg 89 89 119 119 Threonine, g/kg 5 6.4 5 6.4 ADG, g/d 55.9 55.7 58.1 55.1 1.22 0.51 0.21 0.27 ˂.0001 ADFI, g/d 98.8 102 106 101 2.66 0.27 0.69 0.17 ˂.0001 Feed efficiency, g/g 0.544 0.546 0.564 0.571 0.025 0.12 0.75 0.86 0.32 1n=10 cages (2 rabbits /cage). ADG: average daily gain. ADFI: average daily feed intake. Table 10: Effect of level of soluble fibre and threonine on digestive traits of 46 d old rabbits (Exp. 1) Experimental diets P-value L-SF / L-Thr L-SF / H-Thr H-SF / L-Thr H-SF / H-Thr SEM1 Soluble fibre Threonine Soluble fibre x Treonine Soluble fibre, g/kg 89 89 119 119 Threonine, g/kg 5 6.4 5 6.4 Live weight 46 d2, g 1495 1501 1531 1495 15.6 0.33 0.33 0.17 Weight of organs (%LW) Digestive tract 22.2 21.5 22.6 22.8 0.45 0.032 0.52 0.27 Stomach 6.08 5.89 6.35 6.09 0.19 0.22 0.24 0.86 Caecum 7.66 7.15 7.94 8.55 0.31 0.009 0.87 0.075 Caecal pH 5.68 5.64 5.53 5.58 0.035 0.002 0.78 0.12 1n=20; 2For the statistical analysis of live weight at 46 d was used weaning weight at 35 d as covariate (P<.0001). 53 Table 11: Effect of level of soluble fibre and threonine on apparent ileal in rabbits at 46 d old and faecal digestibility in rabbits from 42 to 46 d old (Exp. 1) Experimental diets P-value L-SF/ L-SF/ H-SF/ H-SF/ SEM1 Soluble fibre Threonine Soluble fibre x Threonine L-Thr H-Thr L-Thr H-Thr Soluble fibre, g/kg 89 89 119 119 Threonine, g/kg 5 6.4 5 6.4 ADFI 42-46, g/d 63.8 62.1 60.9 63.0 1.2 0.40 0.84 0.12 Apparent ileal digestibility,% Dry matter 44.2 — 45.4 — 2.36 0.77 — — Crude protein 45.6 — 45.1 — 5.90 0.77 — — Apparent faecal digestibility,% Dry matter 63.8 62.1 60.9 63 1.2 0.42 0.83 0.12 Crude protein 74.5 75.4 74.1 74.6 1.12 0.62 0.54 0.84 1n = 12 for apparent faecal digestibility; n = 10 for apparent ileal digestibility. ADFI: average daily feed intake. Table 12: Effects of level of soluble fibre and threonine on the morphology of the jejunal mucosa in 46 d old rabbits (Exp. 1) Experimental Diet P-value L-SF/ L-Thr L-SF/ H-Thr H-SF/ L-Thr H-SF/ H-Thr SEM1 Soluble fibre Threonine Soluble fibre x Threonine Soluble fibre, g/kg 89 89 119 119 Threonine, g/kg 5 6.4 5 6.4 Villous height, µm 409 401 529 539 25.9 <.0001 0.96 0.72 Crypt depth, µm 115 113 106 109 3.13 0.061 0.91 0.49 Villous height/crypt depth 3.60 3.55 4.90 5.07 0.30 <.0001 0.95 0.86 Nº Globet cell/villi 12.3 14.3 19.2 17.4 1.20 <0.001 0.92 0.13 1n=10. 54 Table 13: Effect of level of soluble fibre and threonine on the growth traits and mortality in rabbits from 25 until 35 d (Exp. 2) Experimental diet P-value L-SF/ L-Thr L-SF/ H-Thr H-SF/ L-Thr H-SF/ H-Thr SEM1 Soluble fibre Threonine Soluble fibre x Threonine Weight 25 d Litter Soluble fibre, g/kg 89 89 119 119 Threonine, g/kg 5 6.4 5 6.4 n 27 27 30 31 ADG, g/d 35.1 36.1 37.2 38.1 1.3 0.10 0.43 0.95 <0.001 0.080 ADFI, g/d 52.3 51.9 55.1 61.4 2.13 0.004 0.16 0.11 0.058 0.12 Feed efficiency, g/g 0.674 0.689 0.686 0.637 0.018 0.25 0.34 0.070 0.041 0.96 Mortality,% 17.1 14.3 5.71 0.00 — 0.002 0.091 0.14 — — Morbidity,% 5.71 8.57 8.57 11.4 — 0.54 0.54 0.93 — — 1n= 35 for mortality and morbidity trial. ADG: average daily gain. ADFI: average daily feed intake. Table 14: Effect of level of soluble fibre and threonine on digestive traits of 35 d old rabbits (Exp. 2) Experimental diets P-value L-SF/ L-Thr L-SF/ H-Thr H-SF/ L-Thr H-SF/ H-Thr SEM1 Soluble fibre Threonine Soluble fibre x Threonine Soluble fibre, g/kg 89 89 119 1119 Threonine, g/kg 5 6.4 5 6.4 Live weight 35 d2, g 743 759 785 769 19.5 0.06 0.99 0.21 Weight of organs (%LW) Digestive tract 27.8 28.9 28.5 27.6 0.92 0.81 0.91 0.29 Stomach 7.47 7.45 7.53 7.59 0.29 0.75 0.94 0.88 Caecum 9.36 9.48 9.26 9.4 0.29 0.73 0.67 0.98 Caecal pH 5.41 5.43 5.34 5.33 0.05 0.10 0.91 0.77 1n=20; 2For the statistical analysis of live weight at 35 d was used weaning weight at 25 d as covariate (P<.0001). 55 Table 15: Effect of level of soluble fibre and threonine on apparent ileal in rabbits at 35 d old and faecal digestibility in rabbits from 32 to 35 d old (Exp. 2) Experimental diet P-value L-SF/ L-Thr L-SF/ H-Thr H-SF/ L-Thr H-SF/ H-Thr SEM1 Soluble fibre Threonine Soluble fibre x Threonine Soluble fibre, g/kg 89 89 119 119 Threonine, g/kg 5 6.4 5 6.4 ADFI 32-35 d, g/d 52.9 52.6 56.7 60.8 2.12 0.008 0.39 0.31 Apparent ileal digestibility,% Dry matter 45.6 45.9 44.5 44.9 3.11 0.73 0.89 0.99 Crude protein 50.0 49.3 46.4 47.1 4.75 0.53 0.99 0.88 Starch 89.1c 91.2b 92.6a 92.2ab 0.51 <0.001 0.013 <0.001 TDF 5.14 5.22 13.7 15.7 5.01 0.065 0.84 0.85 Apparent faecal digestibility,% Dry matter 66.4 66.0 66.3 65.5 1.10 0.65 0.37 0.81 Crude protein 81.8 80.9 81.7 79.6 0.62 0.27 0.021 0.34 Starch 98.9a 98.9a 98.4ab 98.3b 0.027 <0.001 0.001 0.021 TDF2 33.2 31.2 37.7 39.9 1.78 0.003 0.79 0.34 aNDFom-cp 24.3 22.1 27.4 26.5 1.62 0.035 0.36 0.68 3Soluble fibre 69.7 70.8 74.2 76.3 3.48 0.18 0.64 0.88 1n = 11 for apparent faecal digestibility and n = 8 for apparent ileal digestibility. ADFI: average daily feed intake. a,b,c Mean values in the same row with a different superscript differ, P <0.05. 2 TDF: correted by mucin 3 SF: calculated by the difference (TDF-aNDFom-cp) 56 Table 16: Effect of level of soluble fibre and threonine on ileal flow in 35 d old rabbits (Exp. 2) Experimental diet P-value L-SF/ L-Thr L-SF/ H-Thr H-SF/ L-Thr H-SF/ H-Thr SEM1 Soluble fibre Threonine Soluble fibre x Threonine Soluble fibre, g/kg 89 89 119 119 Threonine, g/kg 5 6.4 5 6.4 Ileal flow, g/d Dry matter 28.9 28.6 31.3 33.8 2.11 0.11 0.65 0.54 Crude protein 4.43 4.40 4.85 5.10 0.42 0.21 0.82 0.74 Starch 1.38 1.12 0.91 0.93 0.12 <0.001 0.16 0.083 TDF 20.9 20.3 21.8 23.9 1.65 0.18 0.64 0.41 1n = 8 for dry matter and protein ileal flow; n=1 for starch and TDF (total dietary fibre) ileal flow. Table 17: Effect of level of soluble fibre and threonine on mucin concentration in ileal digesta and faeces in 35 d old rabbits (Exp. 2) Experimental diet P-value L-SF/ L-Thr L-SF/ H-Thr H-SF/ L-Thr H-SF/ H-Thr SEM1 Soluble fibre Threonine Soluble fibre x Threonine Soluble fibre, g/kg 89 89 119 119 Threonine, g/kg 5 6.4 5 6.4 Crude mucin, % Ileal digesta 5.06 5.12 6.38 6.79 — — — — Faeces 0.25 0.27 0.31 0.29 0.024 0.15 0.95 0.48 Crude protein of mucin, % Ileal digesta 31.7 32.4 31.5 28.5 — — — — Faeces 24.7 25.0 25.5 26.5 — — — — Mucin flow, g/d Ileum 1.47 1.46 2.00 2.25 0.143 <0.001 0.39 0.38 Faeces 0.056 0.063 0.083 0.075 0.008 0.033 0.95 0.41 1n= 1 for ileal crude mucin concentration; n = 10 for faecal crude mucin %; n= 8 for ileal flow of mucin and n= 10 for faecal flow of mucin; n=1 for crude protein% of ileal and faeces mucin. 57 Table 18: Effects of level of soluble fibre and threonine on the morphology of the jejunal mucosa in 35 d old rabbits (Exp. 2) Experimental Diet P-value L-SF/ L-Thr L-SF/ H-Thr H-SF/ L-Thr H-SF/ H-Thr SEM1 Soluble fibre Threonine Soluble fibre x Threonine Soluble fibre, g/kg 89 89 119 119 Threonine, g/kg 5 6.4 5 6.4 Villous height, µm 422 417 558 548 26.2 <.0001 0.78 0.92 Crypt depth, µm 102 101 102 97 2.80 0.59 0.34 0.43 Villous height/crypt depth 4.10 4.19 5.43 5.60 0.18 <.0001 0.47 0.83 Nº Goblet cell/villi 12.6 13.8 15.7 14.6 0.70 0.0085 0.96 0.11 1n=10.