Effect of cellobiose and dietary soluble fibre on fermentation, growth performance and health in rabbits

Resumen

The present thesis constitutes a step forward in advancing the knowledge of the supplementation of cellobiose in the drinking water of rabbits and its possible relationship with soluble fibre level in the diet. There is a positive effect of soluble fibre on rabbit digestive health and therefore on the reduction of mortality in weaning rabbits. Nevertheless, this does not seem to end the problem faced by producers. This thesis aims: 1) to determine the adequate dose of cellobiose and its potential interaction with the dietary soluble fibre level, 2) to characterize the ileal and caecal fermentative profile of the cellobiose when combined with different levels of soluble fibre and 3) to evaluate the influence of cellobiose and soluble fibre supplementation on the in vitro caecal fermentation of cellobiose and other carbohydrate-rich feedstuffs. These objectives were developed in six experiments. In experiment one, the aim of the study was to examine whether the combination of dietary soluble fibre and cellobiose exerts a synergistic effect on growth performance, health status, fermentation traits and immune response in rabbits. Six treatments in a 3 × 2 factorial arrangement were used: 3 cellobiose concentrations in drinking water (0.0, 7.5 and 15.0 g/L) × 2 dietary levels of soluble fibre (84.0 and 130 g/kg DM, for the low soluble fibre (LSF) and high soluble fibre (HSF) diets, respectively). A total of 264 young rabbits (35/treatment) were weaned at 34 d of age and had ad libitum access to feed and water. At 46 d of age the other 9 rabbits/treatment were slaughtered and ileal and caecal digesta collected to analyze VFA profile and the immune response in the appendix mucosa. At 48 d of age the cellobiose supplementation was withdrawn and the experimental diets were replaced by a standard commercial diet until 61 d of age. From 34 to 48 d of age there was a linear increase of mortality with the level of cellobiose in the HSF group (0 vs. 17.1%; P = 0.003). In contrast, a quadratic effect of cellobiose level on mortality was found in the LSF group, the rabbits offered 7.5-cellobiose showing the lowest mortality (5.7 vs. 21.4%; P = 0.030). Cellobiose level had a quadratic effect on ADFI, ADG and G:F in this period (P ≤ 0.047), with the 7.5-cellobiose groups having the best growth performance. In contrast, only minor changes on these traits were observed from 48 d of age onwards. Cellobiose level influenced quadratically the ileal VFA concentrations (P = 0.014), showing the maximal value the 7.5-cellobiose groups. In rabbits fed 7.5-cellobiose-LSF a change of acetate to propionate, butyrate and valerate was found in the ileum (P ≤ 0.070). Increasing cellobiose levels reduced linearly caecal VFA concentrations in HSF fed rabbits, but no effect was detected in LSF groups (P = 0.033). The level of soluble fibre increased VFA concentrations in both the ileum (by 22%; P < 0.001) and the caecum (by 11%; P = 0.005). The relative gene expression of IL-6, IL-10, and TNF-α, iNOS, MUC-1 and toll like receptors (TLR-2 and TLR-4) in the appendix increased linear and quadratically with increasing levels of cellobiose (P ≤ 0.063). In conclusion, in rabbits fed LSF diets a dose of 7.5 g cellobiose/L drinking water would be recommended, whereas levels of cellobiose supplementation beyond 7.5 g/L should be avoided in rabbits fed HSF diets. The second, third and fourth experiments were performed to evaluate the effect of supplement cellobiose (≤ 7.0 g/L) on performance of growing rabbits. In experiment 2, six treatments in a 3 × 2 factorial arrangement were used: 3 cellobiose concentrations in drinking water (0.0, 3.5, and 7.0 g/L) × 2 feeds differing in the soluble fibre level (71.9 vs. 130 g/kg DM, for (LSF) and (HSF) diets, respectively). A total of 192 young rabbits (32/treatment) were weaned at 26 d of age and had ad libitum access to feed and water. For experiment 3, the same cellobiose concentrations from experiment 2 were evaluated in rabbits fed with a LSF diet (71.9 g/kg DM) and for this propose, 186 young rabbits (62/treatment) weaned at 26 d of age were used. In experiment 4, four treatments in a 2 × 2 factorial arrangement were structured: 2 cellobiose concentrations in drinking water during the pre-weaning period × 2 cellobiose concentrations in drinking water during the post-weaning period (0.0 and 7.0 g/L in both periods). For the pre-weaning period, 20 litters (10 kits/litter) were used. Each litter was divided at 22 d of age in two groups (5 kits/group) until 29 d of age. From each group 4 kits/litter were weaned at 29 d of age and assigned at random 0.0 or 7.0 g of cellobiose/L. Cellobiose supplementation affected feed intake and feed efficiency in all experiments (P ≤ 0.045). In experiment 2, supplemented 3.50 g of cellobiose/L increased (P = 0.017) weight gain compared with the others cellobiose concentrations (0.0 and 7.0 g of cellobiose/L), resulting in greater weight at 56 d of age (P = 0.017). Results from 26 to 40 d of age in experiment 3 differed from experiment 2, as the increment of cellobiose concentration tended to decrease feed efficiency (P = 0.098). Despite this, from 40 to 50 d of age (experiment 3) increment the cellobiose concentration increased the feed efficiency (P = 0.009). In experiment 4, from 22 to 29 d of age (pre-weaning period), cellobiose supplementation decreased (P = 0.042) the feed intake. After weaning, from 29 to 43 d rabbits supplemented with cellobiose, increased (P ≤ 0.001) the weight gain and the feed efficiency (51.7 vs. 42.4 g/d and 0.457 vs 0.385 g/g, respectively). The increment of cellobiose concentration tended to reduce the mortality in rabbits fed with LSF diets (experiments 2 and 3; P ≤ 0.098). Contrary, in experiment 4 supplemented the animals with cellobiose in both periods tended to increment the mortality (P = 0.087). In conclusion, supplement moderates concentrations of cellobiose (≤ 7.0 g/L) would be recommended for rabbits fed with LSF diets. In experiment 5, the influence of substrate pre-digestion and donors’ diet on in vitro caecal fermentation of different substrates in rabbits was investigated. Eight hybrid rabbits were fed two experimental diets containing either low (LSF; 84.0 g/kg DM) or high soluble fibre (HSF; 130 g/kg DM) levels. In vitro incubations were conducted using batch cultures with soft faeces as inoculum and four fibrous or fibre-derived, low-starch and low-protein substrates: D-cellobiose (CEL), sugar beet pectin (PEC), sugar beet pulp (SBP) and wheat straw (WS). Substrates in half of the cultures were subjected to a 2-step pepsin/pancreatin in vitro digestion without filtration, and the whole residue (soluble, insoluble and added enzymes) was incubated at 40ºC. Gas production was measured until 144 h, and volatile fatty acid (VFA) production at 24 h incubation was determined. Cultures without substrate (blanks) were included to correct gas production values for gas released from endogenous substrates and added enzymes. Pre-digestion had no influence on in vitro gas production kinetic of WS, and only reduced the time before gas production begins (lag time; by 31%; P = 0.042) for SBP, but for both substrates the pre-digestion decreased the molar proportion of acetate (by 9%; P ≤ 0.003) and increased those of propionate and butyrate (P ≤ 0.014). For CEL, the pre-digestion increased the gas and total VFA production (by 30 and 114%), shortened the lag time (by 32%), and only when it was combined with LSF inoculum 38 percentage units of acetate were replaced by butyrate (P ≤ 0.039). Treatments had a minor influence on in vitro fermentation traits of SBP. The results showed that the pre-digestion process influenced the in vitro caecal fermentation in rabbits, but the effects were influenced by donors’ diet and the incubated substrate. A pre-digestion of substrate is recommended before conducting in vitro caecal fermentations. The level of soluble fibre in the donors’ diet also influenced the in vitro caecal fermentation but its effect depended on the type of substrate. In the last experiment, the in vitro caecal fermentation of five substrates low in starch and protein content [D-(+)-glucose (GLU), D-cellobiose (CEL), sugar beet pectin (PEC), sugar beet pulp (SBP) and wheat straw (WS)] was investigated using as inoculum soft faeces from rabbits receiving different levels of cellobiose and soluble fibre. Twenty four rabbits were supplemented three levels of cellobiose in the drinking water (0.0, 7.5 and 15.0 g/L) and fed two experimental diets containing either low (LSF; 84.0 g/kg DM) or high soluble fibre (HSF; 130 g/kg DM) levels. All substrates were subjected to a two-step pepsin/pancreatin in vitro pre-digestion, and the whole residue was used as substrate for the in vitro incubations. Gas production was measured until 144 h, and volatile fatty acid (VFA) production was determined at 24 h incubation. Experimental treatments did not affect SBP fermentation and had only a subtle influence on fermentation of WS and GLU. In contrast, fermentation of CEL was markedly affected, and cellobiose supplementation × donors’ diet interactions were detected for most gas production parameters. Both, the fractional gas production (k) and maximal gas production rates were linearly increased (P ≤ 0.025) and the initial delay in the onset of gas production (Lag) linearly decreased (P < 0.001) by cellobiose supplementation with the HSF inoculum, with no differences (P > 0.05) between the 7.5 and 15 doses. In contrast, with the LSF inoculum cellobiose supplementation only affected k values, which were quadratically increased (P = 0.043) and had maximal values for the 7.5 dose. A quadratic effect (P ≤ 0.018) of cellobiose supplementation was observed for total VFA production at 24 h when CEL and PEC were fermented, obtaining the maximal VFA production for the 7.5 dose of cellobiose. Total VFA production for CEL was greater with LSF than with HSF inoculum (20.7 vs. 12.9 mmol/L; P = 0.014), but the opposite was found for WS (3.97 vs. 6.21 mmol/L; P = 0.005). The use of LSF-inoculum for CEL fermentation sharply reduced acetate (P = 0.001) and increased butyrate proportions (P ≤ 0.001) compared with the HSF-inoculum. A positive relationship between total VFA caecal concentrations in rabbits receiving the same experimental treatments and in vitro values was only observed when WS was used as substrate (r = 0.90; P = 0.015; n = 6). The results suggest that experimental factors influenced the fermentative activity of caecal digesta, but the observed response differed with the incubated substrate, being the CEL the most affected. From the results found in this PhD Thesis it can be concluded that a doses of 7.0-7.5 g of cellobiose/L decreased the mortality when the animals where fed with diets low in soluble fibre content. This positive effect might be related with the increased in the butyrate proportion observed in vivo and in vitro. In contrast, a concentration higher than 7.0 g of cellobiose/L, increased the mortality independently of the soluble fibre content. Accordingly, it would be interesting to evaluate the synergic potential of the cellobiose with other beneficial oligosaccharides (e.g., xylo-oligosaccharides) in order to reduce mortality below the threshold of 10%.