The role of digestible fibre, starch and protein on health status and perfromance in diets for growing rabbits

Introduction and objectives Since 1996, the spread-out of Epizootic Rabbit Enteropathy (ERE) in European rabbit breeding stock has remarkably increased mortality and losses and impaired feed conversion, due to the large number of morbid animals. The lack of identification of an etiological agent responsible for the syndrome and the attribution of a multifactorial character to it, as well as the limitations imposed by EU legislation on the use of antibiotics, have stimulated research on feeding and management strategies capable of preventing or at least limiting the impact of enteropathy. In particular, studies in the past few years intended to specify the nutritional requirements in rabbits during weaning and post-weaning, in relation to the possibility of reducing the damages caused by enteropathy. Among the various nutrients, the most digestible fibre fractions (DF: pectins and hemicelluloses) seem to favour the health status of the animals, in addition to enhance caecal the fermentative activity and feed efficiency (Gidenne and García, 2006). By increasing soluble fibre level in starter weaning diets, C. perfringens and other opportunistic pathogens at caecal level were reduced, followed by the mortality caused by diarrhoea (Soler et al., 2003; Gómez-Conde et al., 2007). By keeping a constant ADF content and replacing starch and protein by digestible fibre (Perez et al., 2000; Gidenne et al., 2001; Marguenda et al., 2006), digestive disorders decreased and rabbit health status improved. Reducing insoluble fibre (Gutiérrez et al., 2002; Alvarez et al., 2007) and increasing the more soluble fractions (García-Ruiz et al., 1997; Carabaño et al., 2008) had also positive effects on intestinal mucosa integrity. Few are yet known nowadays on the interaction between digestible fibre and protein. Lack (18%) may favour digestive disorders and mortality, modifying caecal fermentative activity and microflora composition (Maertens e De Groote, 1988; Lebas, 1989; Carabaño et al., 2008, 2009). Reducing protein level from 18 to 16% (in diets with the same ileal digestibility) significantly reduced the presence of Clostridium perfringens and mortality by epizootic rabbit enteropathy (Chamorro et al., 2007), while further reduction from 16 to 14% did not have any effect on mortality, though reducing anaerobic bacteria at ileum (García-Palomares et al., 2006a, 2006b; Carabaño et al., 2009). The use of less digestible protein sources may also increase the nitrogen flux at caecal level and favour the development of pathogens such as E. coli and Clostridia spp, thus impairing animal health and performance (Gutiérrez et al., 2003; Chamorro et al., 2005, 2007; Carabaño et al., 2008, 2009). Given the late attention of the mass media and consumers on the use of not genetically modified raw materials, a certain interest exists in evaluating protein sources alternative to soybean, which is certainly not modified biotechnologically. Among these, sunflower meal is widely used in rabbit nutrition, usually combined with soybean meal. The use of diets based only on sunflower meal demands a careful evaluation of the aminoacidic equilibrium. The experimental activities realized in the frame of the present PhD thesis followed the general objectives of defining rabbit nutritional needs during post-weaning and fattening with special regards to the different fibrous fractions, mostly the more digestible (pectins, hemicelluloses), the starch content in relation to fibre, as well as the dietary protein supply with the aim of 1) improving rabbits intestinal conditions 2) reducing the incidence and severity of digestive pathologies 3) increasing feed efficiency, and 4) guaranteeing high growth performance and final meat quality. The objectives of the present thesis were pursued along the following four studies. Experiment 1 Digestible fibre level and substitution of soybean with sunflower meal in diets for growing rabbits. At 34 d, 216 rabbits (837±48 g LW) of both genders from a hybrid line were divided in six experimental groups of 36 units and fed ad libitum with six isoproteic diets (15.9% CP) formulated according to a 2 x 3 factorial design, with two protein sources (soybean vs. sunflower meal) and three levels of DF to ADF ratio (1.0, 1.1 and 1.2). The apparent digestibility of nutrients and the digestible energy (DE) concentration of the diets were determined in vivo (Perez et al., 1995). Caecal contents and intestinal mucosa for villi and crypts measurements were sampled at 56 d of age. Faeces and feed samples were analysed according to A.O.A.C methods (2000) and the European harmonized procedures EGRAN (2001). The content of Total Dietary Fibre (TDF) was determined through a gravimetric-enzymatic procedure that requires the treatment with α-amylase, protease and aminoglucosidase (Megazyme Int. Ireland Ltd., Wicklow, Ireland). The content of digestible fibre (DF) was calculated as the difference between the TDF and the ADF content, thus theoretically including, pectins and hemicelluloses. Volatile fatty acid concentration was measured on the supernatant by gas chromatography using the method of Osl (1988). At 76 d of age, rabbits were slaughtered in a commercial slaughterhouse and carcasses were dissected at the Department according to the international scientific protocols (Blasco et al., 1993). The pH (Xiccato et al., 1994) and the colour (CIE, 1976) of longissimus lumborum and biceps femoris were measured. The data were analyzed by ANOVA using the GLM procedure (SAS Inst. Inc., Cary, NC) and considering the effects of DF to ADF ratio and of the protein source. The effects of gender was also included in the model to analyze variability of data of growth performance, slaughter results, carcass and meat traits. Mortality, morbidity and sanitary risk were analyzed by the CATMOD procedure of SAS. The digestibility of dry matter, gross energy, crude fibre and fibrous fractions significantly increased with DF to ADF ratio, due to the higher inclusion of beet pulp and the lower inclusion of alfalfa. The digestibility and nutritive value of the diets increases with DF to ADF ratio not only for the higher presence of digestible fibre constituents, but also for the increased digestibility (9-10 points) of all fibre fractions: ADF digestibility increased from 14.6 to 25.6%; hemicelluloses digestibility from 40.3 to 49.1%; pectins digestibility from 85.0 to 93.8% (P<0.01). This increment may be associated with the lower grade of lignification and complexity of cell walls and to the higher susceptibility of structural carbohydrates to the action of digestive enzymes, animal or bacterial ones. Protein source had a more limited effect on the digestibility of nutrients. In the sunflower diets, crude protein digestibility tended to decrease (P=0.06), consistently with the lower digestibility of crude protein in sunflower meal rather than soybean meal. Ether extract and hemicelluloses digestibility were higher (P<0.001) in sunflower diets than in soybean diets, while ADF and pectins were less digestible. Nutritive value of diets was moderate and increased by DF to ADF ratio (from about 9-10 MJ/kg) regardless to the protein source and follows the current recommendations for rabbit nutrition during post-weaning. During the trial, sanitary problems were limited and unaffected by the feeding treatment. Nor intestinal mucosa traits or caecal fermentation activity were influenced by feeding treatments, while pH of caecal content was significantly reduced by increasing the DF/ADF ratio (P=0.04). By increasing DF/ADF ratio and the nutritive value of the diets, daily growth rate, live weight of rabbits and, consequently, slaughter results did not vary, while feed consumption tended to decrease, mainly in the first period of the trial, from 34 to 55 days of age, and feed conversion linearly improved (from 3.55 to 3.42 and 3.30 in the whole trial; P<0.001). Neither growth performance nor slaughter results and meat traits were affected by protein source. Experiment 2 Digestible fibre/starch ratio and protein level for feeding growing rabbits. At 32 d, 246 rabbits (837±48 g LW) of both genders from a hybrid line were divided in six experimental groups of 36 units and fed ad libitum with six diets formulated with a constant ADF level (18%), but differing in protein levels (15 vs. 17%) and DF/starch ratio (0.8, 1.5 and 2.8), according to a bi-factorial arrangement. The in vivo digestibility trial (on 60 animals from 52 to 56 d of age), the sampling of caecal content and intestinal mucosa (on 36 rabbits at 55 d), histological analyses of gut mucosa and chemical analyses of experimental diets, faeces and caecal content as well as slaughter and dissection procedure and meat quality analyses were performed according to the methodologies detailed in the first experiment. The data were analyzed by ANOVA using the GLM procedure and considering the effects of DF/starch ratio and protein level. Sex effects were also included in the model to analyze variability of data of growth performance, slaughter results, carcass and meat traits. Increasing dietary protein significantly improved dry matter (about +1 point) and nutrient digestibility. The digestible energy content varied little depending on dietary protein, however, and, therefore, DP/DE ratio was higher in diets at 16% CP than those at 15% CP. In both cases, protein supplies satisfy requirements of growing rabbits (De Blas and Mateos, 1998). Increasing DF/starch ratio, starch apparent digestibility significantly decreased (although to a low extent in absolute value), while greatly increased fibre and fibrous fractions digestibility. Dry matter and gross energy digestibility did not change significantly, however. The nutritive value of diets, that is the digestible energy (DE) content, did not change with DF/starch ratio, since energy supplied by DF was similar to that provided by starch within constant crude fibre and NDF levels. Since hemicelluloses did not change appreciably, this result may be largely ascribable to the complete digestibility of pectins (100%) and, to a lesser extent, to the general increase in digestibility of all fibrous fractions, also the less digestible ones (e.g. ADF). Even though mortality and morbidity were low during the trial, a significant effect of dietary treatment was detected. Mortality and sanitary risk tended (P=0.10-0.11) to increase in rabbits fed with the highest protein content diets. Lower DF/starch ratio diets provoke acute ERE and increased mortality (10 vs. 1.4 and 1.4%, P=0.04), though affected animals recovered faster, so the sanitary risk did not differ among the three groups. Rabbits fed the diet with the lowest DF/starch and highest protein level showed significantly higher mortality (17.1 vs. 1.5%) and sanitary risk (20.0 vs. 8.1%) compared to the other groups. The protein level did not affect neither filling of single compartment nor caecal fermentation activity, while crypt depth (P=0.02) and villi/crypt ratio (P=0.11) decreased with low protein diets. Increasing DF/starch ratio did not modify any morphometric variable, while increased the total amount of volatile fatty acids, even though at a non-significant level, and rose (P=0.02) the acetate proportion, main fermentation product, while reduced valerate proportion (P<0.01). Growth performance was satisfying for the slaughter age and the genetic type used, but not significantly affected by the experimental treatments, which only modified minor traits of carcasses and meat quality. Experiment 3 Reducing dietary protein and increasing digestible fibre/starch ratio in diets for growing rabbits. At 29 d, 282 rabbits (596±58 g LW) of both genders from a hybrid line were divided in six experimental groups of 47 units and fed ad libitum with six diets formulated with similar content of ADF (18%) and differing in crude protein level (15.0, 15.5, 17.0% CP) and DF/starch ratio (1.1, 2.0), according to a bi-factorial arrangement. Two in vivo digestibility trials were performed on 60 animals (10 per diet) from 35 to 39 d and from 56 to 60 d of age; at 38 d caecal content and jejunum mucosa were sampled from 36 rabbits (6 animals per group). Digestibility trials, caecal and gut mucosa sampling, histological analysis and chemical analyses of experimental diets, faeces and caecal contents were performed according to the methodologies previously described. At 78 d of age, rabbits were slaughtered and carcasses dissected according to the procedures described above. Nitrogen balance was also calculated on data recorded individually estimating the body N content at the various ages using the formula proposed by Szendro et al. (1998). The same data were statistically analysed in order to evaluate the effect of dietary factors on nitrogen retention, intake and excretion. The data were analyzed by ANOVA using the GLM procedure and considering the effects of protein level, DF/starch ratio as well as their interaction. The effects of sex was also included in the model to analyze variability of data of growth performance, slaughter results, carcass and meat traits. The digestibility coefficients of nutrients measured in the two digestibility trials were analyzed by a three-way ANOVA using the GLM procedure and considering the effects of age, protein level, DF/starch ratio as well as their interactions. Increasing the protein level significantly increased nutrients digestibility, and thus the nutritive value, but decreased ether extract and starch digestive utilization. The partial replacement of alfalfa by soybean and sunflower meal and the increase in barley and dried beet pulp maintained a constant digestible fibre level and decreased the less digestible fibrous fractions, thus reducing the feed transit and increasing the digestibility. Nutrient digestive efficiency significantly improved by increasing DF/starch ratio. The reduction of alfalfa, the increase of barley and dried beet pulps, at constant digestible fibre content, may explain the increase in digestibility due to the slower feed transit caused by the reduction of less digestible fibre (mainly hemicelluloses) and the increase of pectins content. Dry matter and gross energy digestibility did not change with the age of animals. Digestive utilization of crude protein significantly decreased (from 79.2 to 75.7%, P<0.001), while digestibility of starch (from 96.4 to 97.9%, P<0.001), crude fibre (from 16.7 to 22.2%, P<0.001) and of fibrous fractions (TDF, NDF, ADF, hemicelluloses) increased, except for DF. In the first period of growth (29-50 d of age), reducing the dietary protein below 15.5% significantly reduced (P<0.001) daily weight gain (from 53.1 and 55.1 g/d in diets with 15.5% and 17% protein to 49.3 g/d in diets with 14% protein) and live weight at 50 d of age (1712 and 1755 vs. 1631 g), without affecting feed intake. In the second period (50-78 d) performance was not influenced by protein level. In the overall period, rabbits fed low-protein diets showed lower growth performance than those fed high-protein diets; while those fed medium-protein diets did not differ significantly from the other groups. Feed conversion varied according to the growth rate and significantly improved in the post-weaning and whole period by increasing protein, while was not influenced in the second period. When increasing dietary protein, despite stimulating nitrogen retention, total nitrogen excretion increased from 80.4 to 95.0 and 115.0 g, corresponding to 1.64, 1.94 and 2.35 g N excreted per day. When referring to high-protein diets (=100), nitrogen excretion was reduced by 17 and 30% by decreasing the protein level in medium- and low-protein diets, while retained N decreased only by 3 and 6%, respectively. The N excretion was reduced more in the first rather than in the second period of growth. By increasing DF/starch ratio, daily growth rate during post-weaning was significantly stimulated, feed intake reduced and as a consequence feed conversion improved. From 50 days of age until slaughter, growth was similar, but the effects on feed intake and conversion remained evident and significant. As a consequence, higher DF/starch ratio increased live weight at 50 days of age, even though the differences were no longer significant at the end of the trial, reducing feed intake and conversion (P<0.001) in the whole experiment. Increasing DF/starch ratio also reduced N excretion by 5.5%, but without effecting body N retention that improved by 2%. Health status was satisfactory in all groups and not influenced by feeding treatments. By increasing the protein content from 14 to 17.0%, VFA production increased and caecal pH decreased, while ammonia did not change. Higher inclusion of barley and dried beet pulps, at constant DF content, in diets with higher protein content may explain the more intense caecal fermentative activity. By increasing DF and reducing starch, intestinal transit got slower and gut incidence tended to increase, mainly due to caecal filling. Caecal fermentative activity increased together with VFA production (from 64.0 to 78.4 mmol/l; P<0.001) reducing pH (from 5.87 to 5.71) and ammonia production (from 4.7 to 2.3 mmol/l; P<0.001). Jejunum mucosa morphometry was not influenced by dietary protein or digestible fibre level. Protein effect on live and slaughter weight reflected changes in all correlated variables: increasing dietary protein content significantly increased live weight and carcass weight and tended (P=0.09) to improve slaughter dressing percentage. Other carcass and meat characteristics were not affected. Regarding growth performance, DF/starch ratio slightly affected slaughter and carcass traits, with small variations (P<0.10) of dissectible fat and hind leg muscle/bone ratio. Experiment 4 Starch and pectin levels in diets for growing rabbit: effects on health status, digestive physiology, growth performance and carcass and meat quality. At 34 d, 240 rabbits (827±26 g LW) of both genders from a hybrid line were divided in six experimental groups of 40 units and fed ad libitum with six diets formulated according to a bi-factorial design 2 x 3, with three levels of starch (5, 10 and 15%) and two levels of pectins (5 and 10%). Health status was controlled daily to detect promptly the occurrence of diseases, especially digestive problems. The in vivo digestibility trial on 60 animals from 52 to 56 d of age, the sampling of caecal content and intestinal mucosa on 36 rabbits at 51 d, histological analyses of gut mucosa and chemical analyses of experimental diets, faeces and caecal content were performed according to the methodologies previously described. At 75 d of age, rabbits were slaughtered and carcasses dissected following procedures described above. The data were analyzed by ANOVA using the GLM procedure and considering the effects of starch and pectins level, as well as their interaction. Sex effects were also included in the model to analyze variability of data of growth performance, slaughter results, carcass and meat traits. Increasing dietary starch improved dry matter and nutrient digestibility (from 54.7% of low-starch diets to 62.9% of high-starch diets). In high-pectins diets crude fibre (8.1 to 26.4%) and all fibre fractions digestibility was higher. Extreme pectin/starch ratio diets (0.5 vs. 2.0) showed very similar digestibility and nutritive values (DE: 9.5 MJ/kg). Diet digestibility was maintained at high level by replacing barley meal with dried beet pulp, without changing the inclusion rate of alfalfa meal. The digestibility of fibre fractions changed according to the dietary pectin level: within diets at 5% pectins, digestibility of crude fibre changed in a restricted range (from 7.2% to 9.6%, P>0.05), while significant differences were found for fibre fractions; within diets at 10% pectins, digestibility increased for crude fibre (23.5 to 30.3%), NDF (34.3 to 45.1%), ADF (25.7 to 32.1%) and hemicelluloses (45.4 to 60.8%), without significant differences for pectins digestibility. Increasing dietary starch significantly increased final live weight at 75 d and daily growth and reduced feed intake with a corresponding improvement in feed conversion (3.52 to 3.31 and 3.13, P<0.001) in the whole period. During post-weaning, daily weight gain was higher (P<0.001) and feed intake tended to be lower with high-starch diets (P=0.06), while in the second period daily growth was similar, but feed intake was significantly reduced (172 to 159 g/d; P<0.001) probably due to a better chemiostatic regulation of appetite. Dietary pectins level significantly affected growth performance: increasing concentrations stimulated daily weight gain (especially during post-weaning), reduced feed intake and improved feed conversion (3.59 vs. 3.05; P<0.001). Final live weight was higher in animals fed diets containing 10% pectins compared to those fed diets with 5% pectins, both at 55 days of age and at the end of the trial. Health status was good for all rabbits (only two animals died) and caecal fermentation activity changed little with dietary treatment: N-ammonia production was higher in the caecum of animals fed low-starch diets, likely because of the higher protein intake (DP/DE: 12.9, 12.2 and 11.6 g/MJ respectively for the diets with 5, 10, and 15% starch). With increasing dietary starch, total VFA production did not change, while the increase of acetate (P=0.12) and the reduction of propionate (P<0.01) were unexpected. The chemical characteristics of the diets may explain the result obtained in this experiment: 1) even the highest level of starch used (about 16%, average value of diets L1 and H1) was compatible w

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