Materials and methods to avoid major change in the type of cellulose or

142 T . Gidenne, R. Bellier Livestock Production Science 63 2000 141 –152 Table 1 fibre to prevent digestive troubles mainly diarrhoea, Composition of the experimental diets but associated to a negative effect on growth per- Diet Control HC HC 1 P ˆ formance Ma ı tre et al., 1990. Conversely, dietary starch could also play a substantial role in interaction Ingredients g kg diet with fibre, as it is incompletely hydrolysed in the Wheat straw 120 150 140 Wheat bran 270 50 small intestine of the weanling rabbit Blas et al., Sugar Beet pulp 200 1988; Blas et al., 1994 causing alteration of the Sunflower meal 250 50 50 caeco–colic digestion associated with digestive dis- Soya bean meal 35 98 162 turbances. Wheat 560 400 367 a Therefore, the reduction of starch supply should Minerals and vitamins 35 32 31 be explored without excessive reduction in the Chemical composition g kg DM digestible energy and without modifying the supply Organic matter 928 925 924 in low digested fibre fraction. Because the caeco– Starch 361 290 256 colic fermentation is relatively short 7–12 h; Giden- Crude protein N 3 6.25 176 167 179 ne, 1997, the use of rapidly fermented fibre i.e., Gross Energy MJ kg DM 17.96 18.13 17.87 Water insoluble cell-wall 280 341 338 digestible fibre replacing available carbohydrates Neutral-detergent fibre 286 339 327 could be of interest to supply energy to the host via Acid-detergent fibre 149 150 163 caecal microbial activity, without alteration of rate of Acid-detergent lignin 40 30 27 b passage. The level of degradation for hemicellulose Hemicellulose 137 189 164 fractions 30–45 and for pectins 70 in the Uronic acids 27 18 59 c Pectins 30 19 82 rabbit classes these cell wall constituents in the a ‘‘digestible fibre’’ compared to low-digested fibres Contained: g kg, respectively for C, HC; HC 1 P diets calcium carbonate and bicalcium phosphate 21.0, 17.5, 18.0, such as lignocellulose Gidenne et al., 1998a. The DL -methionine 0.5, 1.5, 1.5, L -lysine 1.5, 1.0, 0.0 salt 7.0, 7.0, present study assessed two ways of starch replace- 6.5; and 5.0 g kg diet of vitamin premix retinol 540 mg kg, ment either mainly through a supply of hemicellulose cholecalcipherol 5 mg kg, a-tocopherol 3.3 mg kg, robenidine or with a mix of hemicellulose and pectins. Further- 13.2 mg kg. b more, we aimed to develop a new approach to obtain Hemicellulose fraction estimated by difference between NDF and ADF. a more comprehensive information on caecal diges- c Water insoluble pectins: calculated from tabulated data on tion, that included simultaneous in-vivo measure- ingredients IO7, 1993. ment of rate of passage, nutrient digestion and caecal microbial activity CMA, over a substantial period of the rabbit growth 6–10 wk old. ADF, mainly provided by one source wheat straw

2. Materials and methods to avoid major change in the type of cellulose or

lignin. The ratio digestible protein digestible energy 2.1. Diets and feeding was maintained constant among the diets, by adjust- ing the incorporation of soya bean meal and wheat. Three experimental diets were prepared Table 1 in a pelleted form with raw material classically used 2.2. Animals, housing and experimental protocol in rabbit feed formulation, to obtain a variation in the level of ‘‘digestible’’ fibre: a control diet, and two Three groups of 17 New Zealand white rabbits diets where a portion of starch was substituted either were allocated after controlling the effect of litter by hemicellulose evaluated as NDF-ADF supplied origin at weaning 28 d old to one of the ex- mainly by wheat bran HC diet, or by perimental diets. They were housed in individual ‘‘hemicellulose 1 pectins’’ through a supply in beet metabolism cages 55 3 40 cm and submitted to a pulp HC 1 P diet. In addition, the three diets have 12 h light 7:00 to 19:00 and 12 h dark schedule. a similar level of low-digestible fibre lignocellulose, After a seven-day adaptation period to the diet, nine T . Gidenne, R. Bellier Livestock Production Science 63 2000 141 –152 143 rabbits of each group were surgically fitted with whole tract was calculated according to MRT 5 M t i i T-cannula in the caecum as described by Bellier et M , where t was the time that has elapsed between i i al. 1995. Sampling of caecal digesta and diges- marker administration and the ith defecation and M i tibility measurements were performed after a one was the quantity of marker excreted in faeces. MRT week recovery period. Apparent faecal digestibility includes the minimal transit time TTm, which was coefficients were calculated from individual and the time that has elapsed between dosing and the first daily total collection during six consecutive days 42 marker appearance in the faeces. TTm reflects the to 48 d of age. retention time of digesta without a delay in the Samples of caecal digesta were individually col- mixing compartments, so it represents the rate of lected in vivo via the cannula at 45, 50, 60 and 70 passage in the tubular segment of the tract, i.e., days of age. Rabbits were placed in a special mainly in the small intestine and also in the distal hammock for 10 min, and digesta drained by gravity colon Gidenne, 1994. in sampling tubes Gidenne and Bellier, 1992. Caecal volume was determined in-vivo according Sampling time was fixed at the end of the caecot- to the following marker dilution procedure, on six 51 rophy period 12:00 when inter-individual varia- rabbits per group. The concentration of chromium- tions are lower Bellier et al., 1995. After sampling, EDTA was determined in samples of caecal digesta the caecal material was immediately subjected to the collected 1, 2, 4 and 6 h after the introduction of the determination of pH. Portions of caecal digesta marker dose between 11:30 and 12:00. A linear sample 5 to 10 g fresh matter were placed in tubes adjustment of the decrease in marker concentration containing 2, v v H PO or H SO storage was performed. The caecal volume was then ob- 3 4 2 4 solution 1 and 2 ml tube, respectively for further tained by extrapolating to zero time the curve of analyses of short chain fatty acids SCFAs and tracer concentration against time Leng and ¨ ammoniacal nitrogen NH , and stored at 2 258C. Hornicke, 1976, and expressed in g of fresh matter. 3 51 A solution of chromium-EDTA and cell wall The caecal pool of SCFAs was calculated by multip- 141 particles labelled with cerium Gidenne, 1994 lying the SCFA concentration mM l and the liquid were used as liquid- and solid-phase markers. Cell content of the caecum in ml, obtained from caecal wall particles were prepared from each diet, and volume and dry matter determination. corresponded to the NDF residue after sieving on a 0.05 mm screen. Digesta mean retention time of 2.3. Analytical methods solid and liquid phase was measured on six cannu- lated animals per group, by following the kinetics of Dry matter was determined on feeds and faeces by the faecal excretion of the two markers, using an heating for 24 h at 1038C. Organic matter OM was automatic faecal sampler Automatic Sampler; API, determined by ashing at 5508C for 5 h. Measure- Castanet, France. First, rate of passage between ments of fibre fractions neutral detergent fibre: caecum and rectum was recorded during a five-day NDF, acid detergent fibre: ADF, acid detergent period from 48 to 52 d old, following the adminis- lignin: ADL were made according to Van Soest et al. tration at 11:30 of the two markers in the caecal 1991 using an amylolytic pre-treatment with a cannula. Then, rate of passage in the whole tract was thermostable amylase, and water insoluble cell walls ´ obtained on the same cannulated rabbits, during a were obtained according to Carre and Brillouet six-day period from 53 to 57 d old following the 1989. Nitrogen was determined by DUMAS com- oral administration at 8:30 of markers. A dose of bustion method using Leco apparatus Model FP- labelled particle corresponded to 100 mg DM 428, Leco Corp., St. Joseph, MI, USA, and con- activity 5 2 kBq and was given orally using a verted to crude protein using the factor 6.25. Gross modified syringe 1-ml model. The marker con- energy was measured by an adiabatic calorimeter centrations were determined in dried faeces using a PARR Instrument, Moline, IL, USA. Ammonia gamma spectrometer Packard Instrument, Model concentration was determined spectrophotometrically 5530, Downersgrove, IL, USA. at 660 nm according to Verdow et al. 1977. In The digesta mean retention time MRT in the feeds, uronic acids were determined colorimetrically 144 T . Gidenne, R. Bellier Livestock Production Science 63 2000 141 –152 by the m-phenylphenol method Blumenkrantz and 3. Results Asboe-Hansen, 1973. Starch was hydrolysed en- zymatically and the resultant glucose was measured According to the objectives of the formulation, the by using the hexokinase EC 2.7.1.1-glucose-6- three experimental diets were similar in their levels phosphate dehydrogenase NAD EC1.1.1.49 sys- of lignocellulose meanly 155g ADF kg, Table 1. ¨ tem Boehringer Mannheim. SCFAs were analysed They differed mainly by their level of starch high by gas–liquid chromatography CP9000, Chrom- for control in balance with the hemicellulose or pack, Middelburg, The Netherlands on a semi-capil- pectin level high for HC and HC 1 P, although the lary column Bellier, 1994. total fibre level NDF or WICW differed slightly 10 among diets. 2.4. Statistical analyses 3.1. Effect of the cannulation and of the diet on Rabbits having an abnormally low feed intake 3 intake and growth SD under mean during the measurements were discarded from the statistical analyses. A two ways Among the six groups of rabbits, only one animal analysis of variance was done using the general receiving HC diet died because of an acute diarrhoea linear model ‘‘GLM’’ procedure Statistical Analysis at 48 d of age. Following the implantation of the System, 1988, with the effect of cannulation and cannula the rabbits recovered a normal feed intake diets and interaction between diet and cannulation as within four or five days, so that from 28 to 45 d of main effects for the parameters of intake and growth. age the daily feed intake was only 10 lower for Digestibility and rate of passage data were examined cannulated animals. This resulted in a lower live by one-way analysis of variance ANOVA. weight gain 230 for cannulated animals com- The data on the caecal content composition Table pared to control Table 2. From 45 to 70 days old, 6 and Fig. 1 were examined to evaluate the effect of the daily feed intake and the weight gain did not the age, diet and age 3 diet interactions. The effect differ significantly among cannulated or control of the individual was controlled in this model, to group. In addition, interaction between the effect of take into account that the measurements four sam- the diet and the group of rabbits was not significant ples at four successive ages were repeated on the whatever the parameter. same animals. The significance of the effect of the Dietary treatments affected the intake as well as diet was thus calculated using the mean square of the the weight gain, particularly during the two weeks intra-individual variations as an error term split-plot after weaning. The feed intake was significantly design. Least-square means comparisons were per- higher 1 10 for HC and ‘‘HC 1 P’’ groups from formed using the PDIFF test of GLM. Data from four to six weeks of age, but resulted in a sig- unhealthy animals or animals having an abnormal nificantly higher weight gain only for ‘‘HC 1 P’’ SCFA pattern i.e., with a propionate butyrate ratio group Table 2. Consequently the feed conversion over 2 were not included in the analysis. The in was not significantly different P 5 0.29 among the vivo SCFA pattern from 6 to 10 weeks of age was three groups for the period 4–6 wk mean 2.00, but statistically analysed after pooling the results from it was significantly lower P 5 0.031 for the control the three diets, as no interaction between the diet and group compared to HC 1 P during the 6–10 weeks the age of the rabbit was found. In addition, the old period respectively, 2.73 vs. 3.06. effect of age on CMA was analysed according to the GLM procedure linear model. The partial correla- tion coefficients between the parameters of the caecal 3.2. Nutrient intake and digestibility fermentation and the digestion of the nutrients over the period 6–10 wk old were obtained through a Food intake variations combined with differences multivariate analysis of variance taking into account in the dietary chemical composition produced exagg- the effect of the diet. erated difference in nutrient intake. Thus, although T . Gidenne, R. Bellier Livestock Production Science 63 2000 141 –152 145 Table 2 Effect of caecal cannulation and of dietary treatment on feed intake and growth 1 Effect of the cannulation Effect of diets Statistical difference 2 n 5 23 n 5 24 n 5 16 n 5 15 n 5 16 Cannulation Control Cannulation Control HC HC 1 P RMSE Cannulation Diet 3 Diet Live weight g At 28 days old 509 570 540 534 545 47 , 0.001 0.70 0.94 a ab b At 45 days old 1052 941 955 974 1055 92 , 0.001 0.011 0.68 Intake g d kg LW a b b 28 to 45 days old 85.4 74.6 74.5 82.0 83.3 7.8 , 0.001 0.005 0.29 a ab b 45 to 70 days old 69.8 68.0 65.6 70.1 71.3 5.7 0.25 0.024 0.74 Weight gain g d a a b 28 to 45 days old 39.0 26.5 29.6 31.8 36.4 4.5 , 0.001 , 0.001 0.54 45 to 70 days old 36.6 34.8 35.7 34.3 37.2 4.8 0.21 0.28 0.49 1 Implantation of a caecal cannula at five weeks old. 2 Number of rabbits. RMSE 5 Root mean square error. a, b: Means, between diets, having a common superscript did not differ significantly at the level P 5 0.05. the dietary ADF level was similar, the ADF intake versely the starch intake is reduced by around 20 increased by 20 for HC 1 P group Table 3. for these two groups. Compared to control group, the intake of hemicellul- Organic matter digestibility did not differ sig- ose increased by four units 1 40 for HC group, nificantly from control when starch is replaced while the intake of pectin tripled for HC 1 P. Re- mainly by hemicellulose and pectin HC 1 P group, Table 3 Effect of dietary treatment on the intake of nutrients total and digestible, from 45 to 70 days old Daily intake Control HC HC 1 P Statistical analysis 1 g d kg LW, air dry basis n 5 8 n 5 7 n 5 7 RMSE P . F Raw intake 66.0 68.4 71.4 6.3 0.25 Total nutrients Organic matter 54.5 55.8 58.9 5.2 0.29 Crude protein 10.3 10.1 11.4 1.0 0.053 a b b Starch 21.2 17.6 16.3 1.7 , 0.001 a b b NDF 16.8 20.6 20.8 1.8 , 0.001 a b b ADF 8.7 9.2 10.3 1.0 0.004 2 a c b Hemicellulose 8.0 11.5 10.4 0.9 , 0.001 2 b a c Pectins 1.6 0.9 5.3 0.40 , 0.001 Digestible nutrients Organic matter 38.9 37.2 41.1 3.7 0.20 Crude protein 8.6 8.5 9.4 0.9 0.18 a a b NDF 4.2 4.4 6.4 0.8 , 0.001 a a b ADF 1.1 0.9 1.8 0.4 0.001 2 a a b Hemicellulose 3.1 3.4 4.6 0.4 , 0.001 1 a, b: See Table 2. 2 See Table 1. 146 T . Gidenne, R. Bellier Livestock Production Science 63 2000 141 –152 Table 4 Effect of dietary treatment on the total tract apparent digestibility Control HC HC 1 P Statistical analysis 1 n 5 8 n 5 7 n 5 7 RMSE P . F a b a Organic matter 0.722 0.660 0.696 0.020 , 0.001 Crude protein 0.848 0.840 0.822 0.023 0.083 a b a Energy 0.711 0.654 0.689 0.020 , 0.001 ab b a Neutral-detergent fibre 0.252 0.204 0.303 0.042 , 0.001 ab a a Acid-detergent fibre 0.125 0.090 0.165 0.050 0.019 2 a b a Hemicellulose 0.389 0.294 0.440 0.039 , 0.001 Nutritive value air dry basis Digestible protein, DP g kg 133 124 131 Digestible energy, DE MJ kg 11.35 10.48 10.98 DP DE g MJ 11.7 11.8 11.9 1 a, b: See Table 2. RMSE: Root mean square error. 2 See Table 1. although there was a 2 unit decrease Table 4. In The minimum transit time was identical between contrast, the replacement of starch by hemicellulose solid and liquid phase, thus only mean values for the led to a 6 unit decrease in OM digestibility. From 6 two phases were given in the Table 5. TTm for the to 10 wk old, the intake in digestible OM was similar whole tract was meanly 4.4 h from which 50 among diets Table 3 and logically led to a similar accounted for caeco–rectal transit. TTm remained weight gain Table 2. Crude protein digestibility or unaffected by the dietary treatments. intake in digestible protein were not significantly Contrary to the liquid phase, the MRT of the solid affected by dietary treatments. In accordance with phase was affected by the diets, particularly in the the aims of the dietary formulation the ratio DP DE caeco–colic segment when it decreased P 5 0.008 was similar among the diets. Whatever the diet, the by 50 for ‘‘HC 1 P’’ group compared to control. hemicellulosic fraction was digested at a higher rate Although not statistically significant high interin- compared to lignocellulose, reaching a level of 44 dividual variations MRT for the total tract tended to for HC 1 P group. Compared to control diet, the be lower P 5 0.077 for HC 1 P group. TTm was NDF digestibility was improved with the addition of not significantly different among the three groups, pectin, while it decreased with the addition of indicating therefore that the change in total and hemicellulose mainly from wheat bran. This re- caeco–rectal MRT occurred in the mixing compart- sulted in a high quantity of fibre degraded g d for ments, and mainly in the caecum and proximal HC 1 P group whatever the fibre fraction Table 3, colon. whereas fibre degradation was similar for control and HC groups. 3.4. Caecal fermentation and microbial activity 3.3. Rate of passage Whatever the variable, no significant interactions were found between the effect of age and the effect The rate of passage in the whole tract of the of the diet Table 6, so these two effects will be digesta solid phase was meanly 15.5 h, from which presented separately hereafter. at least the two-thirds was attributable to the caeco– colic segment Table 5. The MRT of the liquid 3.4.1. Effect of age phase was three-fold higher mean 48 h, and more The in-vivo total SCFA concentrations in the than 80 originated from the caeco–colic segment. caecum augmented linearly P 5 0.03 by 20 from T . Gidenne, R. Bellier Livestock Production Science 63 2000 141 –152 147 Table 5 Mean retention time MRT of solid and liquid phase of digesta in the whole tract and caeco–colic segment of the growing rabbit, according to dietary treatment Control HC HC 1 P Statistical analysis 1 n 5 5 n 5 4 n 5 5 RMSE P . F 2 ab a b Live weight g 1349 1234 1480 93 0.014 2 a a b Feed intake g d 95.2 94.5 116.7 10.7 0.013 MRT : whole tract h Solid phase 18.0 15.7 13.1 2.6 0.077 Liquid phase 51.0 49.4 44.8 5.3 0.21 MRT : caecum–rectum h a b b Solid phase 14.7 9.6 7.8 2.0 0.008 Liquid phase 42.3 42.4 38.1 4.6 0.31 TTm : minimal transit time Whole tract h 4.6 3.9 4.6 2.3 0.72 Caecum–rectum h 2.1 2.4 2.4 1.2 0.92 1 a, b: See Table 2. 2 Mean values for the period 48–57 days of age. Table 6 In-vivo measurement from 6 to 10 weeks old, of pH, ammonia and short-chain fatty acids SCFAs concentration mmol l in the caecal content of rabbits according to dietary treatment 1 Effect of the diet Statistical analysis 2 n 5 19 n 5 14 n 5 26 Diet 3 Control HC HC 1 P RMSE Diet Age 3 age Total SCFAs 54.6 58.4 62.2 8.8 0.091 0.022 0.83 4 a ab b Acetate 44.4 49.7 53.0 7.5 0.041 0.129 0.85 Propionate 2.7 2.7 3.2 0.5 0.086 0.78 0.95 Butyrate 6.8 5.4 5.5 1.5 0.35 , 0.001 0.75 5 Minor SCFAs 0.8 0.7 0.7 0.1 0.80 0.41 0.37 Propionate:butyrate 0.46 0.57 0.67 0.18 0.31 0.002 0.80 Ammonia 4.0 3.6 3.8 1.7 0.98 0.54 0.59 pH 6.50 6.57 6.35 0.23 0.090 0.67 0.84 1 Means from samples covering the period 45 to 70 days old. 2 a, b: See Table 2. 3 Comparison of in vivo sampling according to age at 45, 50, 60 and 70 days old. 4 Linear effect of the age P , 0.05 5 Isobutyrate 1 valerate 1 isovalerate. 45 d to 70 d of age Fig. 1, because of a significant 0.001 with age from 0.72 at 45 d to 0.36 at 70 d. linear increase P 5 0.03 in acetate from 48.4 to Ammonia or pH were not significantly affected by 52.5 mmol l and butyrate from 4.3 to 8.9 mmol l the age Table 6. levels. From 45 to 70 d of age, we registered a two-fold increase of the butyrate molar proportion 3.4.2. Effect of the diet Fig. 1, associated with a slight decrease of the Compared to control, the total SCFA levels in acetate molar proportion 25. Consequently the HC 1 P group were 8 units higher P 5 0.09 as the ratio propionate: butyrate declined linearly P , level of acetate increased by 20 P 5 0.04. On the 148 T . Gidenne, R. Bellier Livestock Production Science 63 2000 141 –152 Fig. 1. In vivo caecal SCFA pattern according to the age of the rabbits. Values are means of the three diets. a, b: Means having a common superscript did not differ significantly at the level P 5 0.05. 1 Linear effect of the age P , 0.05. other hand, levels of butyrate propionate or ammonia parameters SCFA levels or molar proportion, ex- remained unaffected by dietary treatments. In return, cept the level of ammonia that was positively related the acetate molar proportion was significantly lower to crude protein digestibility r 5 0.59, P 5 0.02. P 5 0.034 for control compared to HC and HC 1 P A relatively low number of animals was involved group respectively, 81.6 – 85.1 – 85.1, while in the in-vivo determination of the caecal volume the butyrate molar proportion tended to be higher and SCFA pool Table 7, because the marker P 5 0.07 for control 12.1 compared to the two dilution procedure is relatively difficult to manage other groups mean 9.1. and time consuming. Therefore, only large differ- The relationship between caecal fermentation and ences among treatments could appeared as signifi- the digestion of the main nutrients OM, CP, NDF, cant. At 70 d of age, the digesta content of the ADF and NDF-ADF has been assessed by calculat- caecum corresponds meanly to 55g of fresh matter ing the partial correlation coefficients r between that represented 5 of the live weight. The caecal pH, NH or SCFA levels and the digestibility content reached 71g for HC 1 P that was 80 over 3 coefficients or the daily quantity of nutrients di- the value for HC group. Therefore the SCFA pool gested. Digestibility or nutrients intake were not was significantly higher for HC 1 P 1 65 com- significantly correlated with any of the fermentation pared to the two other groups. T . Gidenne, R. Bellier Livestock Production Science 63 2000 141 –152 149 Table 7 In-vivo measurement of the digesta content and of SCFA pool in the caecum of growing rabbits 7 wk old according to dietary treatment Control HC HC 1 P Statistical analysis 1 n 5 4 n 5 3 n 5 5 RMSE P . F Live weight g 1120 1013 1204 99 0.075 ab a b Caecal digesta content g 59.2 39.3 71.4 12.4 0.020 SCFAs mM l 59.8 65.0 68.5 10.0 0.52 2 a a b SCFA pool mM 2.3 2.2 3.8 0.66 0.013 1 a, b: See Table 2. 2 SCFAs mM l 3 liquid caecal content ml.

4. Discussion higher than in hard faeces, Luick et al. 1992 that are