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Dietary protein and fermentable carbohydrates contents influence growth performance and intestinal characteristics in newly weaned pigs
Livestock Science 108 (2007) 194 – 197 www.elsevier.com/locate/livsci
Dietary protein and fermentable carbohydrates contents influence growth performance and intestinal characteristics in newly weaned pigs ☆ P. Bikker a,⁎, A. Dirkzwager a,1 , J. Fledderus a , P. Trevisi b,2 , I. le Huërou-Luron b , J.P. Lallès b , A. Awati c,3 a
Schothorst Feed Research, P.O. Box 533, 8200 NA Lelystad, The Netherlands Institut National de la Recherche Agronomique, UMR SENAH, 35590, Saint-Gilles, France Animal Nutrition Group, Wageningen University and Research Centre, P.O. Box 9101, 6700 HB Wageningen, The Netherlands b
c
Abstract Increasing the dietary amount of fermentable carbohydrates (FC) may counteract the negative effects of protein fermentation in newly weaned piglets. To study this hypothesis, 272 newly weaned piglets were allotted to 4 dietary treatments in a 2 × 2 factorial arrangement with low and high FC (7.5 and 13.5%) and crude protein (CP, 15 and 22%) content as respective factors. Intestinal histology, enzyme activity, microbiota and fermentation products were determined in 8 pigs per treatment 7 days post-weaning. In the 4 wk experimental period, interactions between dietary CP and FC content were found for feed intake (P = 0.022), daily gain (P = 0.001), and gain:feed (P = 0.033). The high-FC content reduced daily gain by 50 g/d in the low-CP diet, whereas the FC content did not affect growth performance in the high-CP diet. Over the 4 wk experimental period, daily gain (350 g/d) and feed intake (519 g/d) were highest for piglets on the low-CP low-FC diet. The high-FC content resulted in an increase in number of lactobacilli (P = 0.047) and a decrease of total coliforms (P = 0.06) in the small intestine. It increased the lactic acid content (P = 0.08) and reduced the ammonia content (P = 0.04) in the small intestine and increased the VFA content in the colon (P = 0.009). The reduction in CP content reduced ammonia concentration in the small intestine (P = 0.003). We concluded that dietary FC influenced microbial population and fermentation products in the gut. However, this was not reflected in an increased growth performance. © 2007 Elsevier B.V. All rights reserved. Keywords: Carbohydrates; Fermentation; Growth performance; Intestinal health; Protein; Weaned piglets
☆ This paper is part of the special issue entitled “Digestive Physiology in Pigs” guest edited by José Adalberto Fernández, Mette Skou Hedemann, Bent Borg Jensen, Henry Jørgensen, Knud Erik Bach Knudsen and Helle Nygaard Lærke. ⁎ Corresponding author. E-mail address: [email protected] (P. Bikker). 1 Present address: Gezondheidsdienst voor Dieren, P.O. Box 9, 7400 AA Deventer, The Netherlands. 2 On doctoral leave from DIPROVAL, University of Bologna, via f.lli Rosselli 107, 42100 Reggio Emilia, Italy. 3 Present address: Riddet Centre, Massey University, Private bag 11222, Palmerston North, New Zealand.
1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.01.057
P. Bikker et al. / Livestock Science 108 (2007) 194–197
1. Introduction Excess of crude protein (CP) in the diet of newly weaned pigs may stimulate protein fermentation and encourage proliferation of pathogenic bacteria in the gastrointestinal tract (Ball and Aherne, 1987). Reduction of protein fermentation can be achieved by lowering the amount of CP in the diet (Nyachoti et al., 2006) or by increasing the amount of dietary fermentable carbohydrates (FC) (Awati, 2005). The aim of this experiment was to assess whether the negative effect of protein fermentation in piglets could be reduced by either lowering the CP level or increasing the amount of FC in the feed in newly weaned pigs and to determine the consequences for growth performance.
195
carbohydrates were defined as sum of the fermentable non-starch polysaccharides and the ileal indigestible fraction of native starch according to CVB (2003). Fermentable carbohydrates were included by increasing the amount of wheat middlings, sugar beet pulp and native potato starch at the expense of wheat and maize. Animal fat and soybean oil were added to equalize the net energy content. Crude protein level was increased by inclusion of wheat gluten meal and maize gluten meal at the expense of tapioca, potato protein and synthetic amino acids. All diets contained 9.9 MJ of NE and 1.0 g ileal digestible lysine per kg. Piglets had free access to feed and water throughout the experiment. 2.3. Measurements and analyses
The experimental protocol used in this study was approved by the ethical committee of Schothorst Feed Research, Lelystad, The Netherlands. The experiment was conducted with 272 Yorkshire × Landrace pigs, initially weighing 8.7 ± 0.17 kg at weaning at an average age of 26 ± 0.8 days. At weaning the pigs were allotted to one of four dietary treatments with low- and high-CP and FC content, according to a 2 × 2 factorial arrangement with 10 replicates per treatment. Four replicates started with 8 piglets per pen and 6 replicates with 6 piglets per pen. The piglets were housed in pens (2.00 × 1.10 m) in environmentally controlled battery units.
Feed intake and weight of piglets were recorded per pen at d 0 (weaning), 7, 14, and 28, and daily gain, feed intake and gain:feed were calculated per pen. In addition, from the pens with 8 piglets, 2 piglets with an average body weight (in total 8 piglets per treatment) were euthanized and dissected on d 7 for determination of intestinal characteristics. An intestinal segment of about 20 cm was obtained from the mid jejunum. The specific activity of maltase, sucrase and aminopeptidase-N were determined in the small intestinal mucosa (Marion et al., 2005) and villus length and crypt depth were determined as described by Goodlad et al. (1991). In digesta samples from ileum and colon the amount of NH3, counts of coliform bacteria (on 3MTM PetrifilmTM) and lactobacilli (on Man Rogosa Sharpe agar), lactic acid and volatile fatty acids (VFA) pattern (by gas chromatography) were determined.
2.2. Diets
2.4. Statistical analysis
Four experimental diets were formulated with low (15%) or high (22%) CP content combined with low (7.5%) or high (13.5%) FC content. Fermentable
Pen was the experimental unit for ADFI, ADG and G:F. Analysis of variance was performed using the statistical software of Genstat (GenStat, 2000). Data
2. Materials and methods 2.1. Animals and treatments
Table 1 Effects of dietary crude protein (CP) and fermentable carbohydrates (FC) content on growth performance of piglets during 4 wks following weaning Dietary treatment
P-values
SEM
CP content
Low
High
FC content
Low
High
Low
High
CP
FC
CP × FC
Initial BW, kg Final BW, kg Feed intake, g/d Body gain, g/d Gain:feed
8.71 18.85a 519a 350a 0.67a
8.70 17.40b 478b 300b 0.63b
8.71 17.79b 466b 313b 0.67a
8.71 17.83b 478b 315b 0.66a
0.881 0.152 0.022 0.149 0.048
1.000 0.003 0.192 0.002 b0.001
0.764 0.002 0.022 0.001 0.033
Values are means of 10 pens per dietary treatment. Within a row, means without a common superscript letter differ (P b 0.05).
a,b,c,
0.00 0.21 11 7 0.01
196
P. Bikker et al. / Livestock Science 108 (2007) 194–197
Table 2 Effects of dietary crude protein (CP) and fermentable carbohydrates (FC) content on bacterial counts (log CFU/g) and fermentation products in jejunum and colon of piglets, determined at d 7 after weaning Dietary treatment CP content
Low
FC content
Low
P-values
SEM
High High
Low
Jejunum Lactobacilli Coliforms Ammonia, mg/L Lactic acid, mmol/L Total VFA, mmol/L
7.91 7.61 32.7 12.3 13.2
8.25 7.10 22.9 18.3 10.2
7.66 8.15 73.0 16.6 15.7
Colon Lactobacilli Coliforms Ammonia, mg/L Lactic acid, mmol/L Total VFA, mmol/L
9.31 8.26 356 0.99 74.3
9.11 8.37 196 0.91 80.9
9.14 8.64 376 1.27 68.8
High
CP
FC
CP × FC
8.55 6.78 44.1 26.8 11.9
0.937 0.823 0.003 0.160 0.344
0.047 0.063 0.049 0.080 0.126
0.354 0.377 0.315 0.635 0.867
0.15 0.23 6.2 2.5 1.2
9.20 7.77 339 0.85 95.1
0.798 0.757 0.138 0.747 0.454
0.682 0.282 0.076 0.456 0.009
0.438 0.175 0.255 0.621 0.102
0.09 0.17 34 0.18 4.0
Values are means of 8 piglets per dietary treatment.
were analyzed as a 2 × 2 factorial arrangement of treatments, with dietary CP and FC content as respective factors. Statistical analysis of microbiota, fermentation products, histology and enzymology was performed using the data from 8 dissections per treatment, with piglet as the experimental unit.
FC tended to increase the concentration of lactic acid in the jejunum and concentration of total VFA in the colon. Activity of digestive enzymes and gut morphology were not significantly influenced by dietary treatments.
3. Results
We hypothesized that a reduction in dietary CP and an increase in FC content would reduce the risk of protein fermentation and improve intestinal health and growth performance. Indeed both decreasing the CP content and increasing the FC content of the diet reduced the ammonia concentration in the small intestine. Furthermore, effects of high FC on bacteria and fermentation products indicate a shift in the composition of microbial population in response to dietary substrate. This was in agreement with Konstantinov et al. (2004) and was suggested to be beneficial to protect piglets from pathogenic E. coli adhesion. The low-CP diets also reduced protein fermentation, but this was not coincided by any effect on coliforms or lactobacilli, nor on VFA production. Nyachoti et al. (2006) also found no effect of lowering the dietary CP content (from 23 to 17%) on total VFA concentration and on bacterial population in the jejunum. Despite these effects of an increase in FC content on intestinal characteristics, increasing the FC level did not improve growth performance during the first 4 wks after weaning. We speculate that nutrient availability (energy and/or amino acids) from fibrous ingredients in high-FC diets may have been suboptimal because of the immature digestive capacity in newly weaned piglets.
3.1. Animal performance In the 4 wk experimental period, increasing the FC content of the low-CP diet reduced feed intake, daily gain and gain:feed, whereas increasing the FC content of the high-CP diet did not affect growth performance (Table 1). Furthermore, the reduction in CP content improved feed intake and daily gain of piglets fed the low-FC diets, but not of piglets fed the high-FC diets where it reduced gain:feed ratio. 3.2. Intestinal characteristics The effects of CP and FC were presented in Table 2. Interactions between CP and FC contents were not significant. A low-CP level reduced the amount of ammonia in the jejunum, without significant effects on other characteristics. An increase in the level of dietary FC increased counts of lactobacilli and tended to lower counts of coliforms in the jejunum, but not in the colon. High dietary FC decreased the concentration of ammonia in the jejunum and tended to decrease the ammonia concentration in the colon. In addition, high
4. Discussion
P. Bikker et al. / Livestock Science 108 (2007) 194–197
5. Conclusion We conclude that increasing dietary fermentable carbohydrates can induce a shift in microbial population, reduce ammonia content and increase lactic acid and VFA contents in the digestive tract. However, this does not necessarily result in an increased growth performance of weaned piglets. Acknowledgement This work has been carried out with financial support of the European Community specific RTD programme “Quality of Life and Management of Living Resources” research project HEALTHYPIGUT (QLK5-CT20000– 00522). References Awati, A. 2005. Prebiotics in piglet nutrition? Fermentation kinetics along the GI tract. PhD Diss., Wageningen University, The Netherlands. Ball, R.O., Aherne, F.X., 1987. Influence of dietary nutrient density, level of feed intake and weaning age on young pigs. II. Apparent
197
nutrient digestibility and incidence and severity of diarrhea. Can. J. Anim. Sci. 67, 1105–1115. CVB, 2003. Veevoedertabel [Feedstuff Table, Nutritional Value of Feed Ingredients]. Centraal Veevoederbureau, Lelystad, The Netherlands. GenStat, 2000. GenStat for Windows, Release 4.2. 5th ed. VSN International Ltd., Oxford. Goodlad, R.A., Levi, S., Lee, C.Y., Mandir, N., Hodgson, H., Wright, N.A., 1991. Morphometry and cell proliferation in endoscopic biopsies: evaluation of a technique. Gastroenterology 101, 1235–1241. Konstantinov, S.R., Awati, A., Smidt, H., Williams, B.A., Akkermans, A.D.L., de Vos, W.M., 2004. Specific response of a novel and abundant Lactobacillus amylovorus-like phylotype to dietary prebiotics in the ileum and colon of weaning piglets. Appl. Environ. Microbiol. 70, 3821–3830. Marion, J., Petersen, Y.M., Romé, V., Thomas, F., le Dividich, J., Sangild, P.T., le Huërou-Luron, I., 2005. Early weaning stimulates intestinal brush border enzyme activities in piglets, mainly at posttranscriptional level. J. Pediatr. Gastroenterol. Nutr. 41, 401–410. Nyachoti, C.M., Omogbenigun, F.O., Rademacher, M., Blank, G., 2006. Performance responses and indicators of gastrointestinal health in early-weaned pigs fed low-protein amino acid-supplemented diets. J. Anim. Sci. 84, 125–134.
Dietary protein and fermentable carbohydrates contents influence growth performance and intestinal characteristics in newly weaned pigs ☆ P. Bikker a,⁎, A. Dirkzwager a,1 , J. Fledderus a , P. Trevisi b,2 , I. le Huërou-Luron b , J.P. Lallès b , A. Awati c,3 a
Schothorst Feed Research, P.O. Box 533, 8200 NA Lelystad, The Netherlands Institut National de la Recherche Agronomique, UMR SENAH, 35590, Saint-Gilles, France Animal Nutrition Group, Wageningen University and Research Centre, P.O. Box 9101, 6700 HB Wageningen, The Netherlands b
c
Abstract Increasing the dietary amount of fermentable carbohydrates (FC) may counteract the negative effects of protein fermentation in newly weaned piglets. To study this hypothesis, 272 newly weaned piglets were allotted to 4 dietary treatments in a 2 × 2 factorial arrangement with low and high FC (7.5 and 13.5%) and crude protein (CP, 15 and 22%) content as respective factors. Intestinal histology, enzyme activity, microbiota and fermentation products were determined in 8 pigs per treatment 7 days post-weaning. In the 4 wk experimental period, interactions between dietary CP and FC content were found for feed intake (P = 0.022), daily gain (P = 0.001), and gain:feed (P = 0.033). The high-FC content reduced daily gain by 50 g/d in the low-CP diet, whereas the FC content did not affect growth performance in the high-CP diet. Over the 4 wk experimental period, daily gain (350 g/d) and feed intake (519 g/d) were highest for piglets on the low-CP low-FC diet. The high-FC content resulted in an increase in number of lactobacilli (P = 0.047) and a decrease of total coliforms (P = 0.06) in the small intestine. It increased the lactic acid content (P = 0.08) and reduced the ammonia content (P = 0.04) in the small intestine and increased the VFA content in the colon (P = 0.009). The reduction in CP content reduced ammonia concentration in the small intestine (P = 0.003). We concluded that dietary FC influenced microbial population and fermentation products in the gut. However, this was not reflected in an increased growth performance. © 2007 Elsevier B.V. All rights reserved. Keywords: Carbohydrates; Fermentation; Growth performance; Intestinal health; Protein; Weaned piglets
☆ This paper is part of the special issue entitled “Digestive Physiology in Pigs” guest edited by José Adalberto Fernández, Mette Skou Hedemann, Bent Borg Jensen, Henry Jørgensen, Knud Erik Bach Knudsen and Helle Nygaard Lærke. ⁎ Corresponding author. E-mail address: [email protected] (P. Bikker). 1 Present address: Gezondheidsdienst voor Dieren, P.O. Box 9, 7400 AA Deventer, The Netherlands. 2 On doctoral leave from DIPROVAL, University of Bologna, via f.lli Rosselli 107, 42100 Reggio Emilia, Italy. 3 Present address: Riddet Centre, Massey University, Private bag 11222, Palmerston North, New Zealand.
1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.01.057
P. Bikker et al. / Livestock Science 108 (2007) 194–197
1. Introduction Excess of crude protein (CP) in the diet of newly weaned pigs may stimulate protein fermentation and encourage proliferation of pathogenic bacteria in the gastrointestinal tract (Ball and Aherne, 1987). Reduction of protein fermentation can be achieved by lowering the amount of CP in the diet (Nyachoti et al., 2006) or by increasing the amount of dietary fermentable carbohydrates (FC) (Awati, 2005). The aim of this experiment was to assess whether the negative effect of protein fermentation in piglets could be reduced by either lowering the CP level or increasing the amount of FC in the feed in newly weaned pigs and to determine the consequences for growth performance.
195
carbohydrates were defined as sum of the fermentable non-starch polysaccharides and the ileal indigestible fraction of native starch according to CVB (2003). Fermentable carbohydrates were included by increasing the amount of wheat middlings, sugar beet pulp and native potato starch at the expense of wheat and maize. Animal fat and soybean oil were added to equalize the net energy content. Crude protein level was increased by inclusion of wheat gluten meal and maize gluten meal at the expense of tapioca, potato protein and synthetic amino acids. All diets contained 9.9 MJ of NE and 1.0 g ileal digestible lysine per kg. Piglets had free access to feed and water throughout the experiment. 2.3. Measurements and analyses
The experimental protocol used in this study was approved by the ethical committee of Schothorst Feed Research, Lelystad, The Netherlands. The experiment was conducted with 272 Yorkshire × Landrace pigs, initially weighing 8.7 ± 0.17 kg at weaning at an average age of 26 ± 0.8 days. At weaning the pigs were allotted to one of four dietary treatments with low- and high-CP and FC content, according to a 2 × 2 factorial arrangement with 10 replicates per treatment. Four replicates started with 8 piglets per pen and 6 replicates with 6 piglets per pen. The piglets were housed in pens (2.00 × 1.10 m) in environmentally controlled battery units.
Feed intake and weight of piglets were recorded per pen at d 0 (weaning), 7, 14, and 28, and daily gain, feed intake and gain:feed were calculated per pen. In addition, from the pens with 8 piglets, 2 piglets with an average body weight (in total 8 piglets per treatment) were euthanized and dissected on d 7 for determination of intestinal characteristics. An intestinal segment of about 20 cm was obtained from the mid jejunum. The specific activity of maltase, sucrase and aminopeptidase-N were determined in the small intestinal mucosa (Marion et al., 2005) and villus length and crypt depth were determined as described by Goodlad et al. (1991). In digesta samples from ileum and colon the amount of NH3, counts of coliform bacteria (on 3MTM PetrifilmTM) and lactobacilli (on Man Rogosa Sharpe agar), lactic acid and volatile fatty acids (VFA) pattern (by gas chromatography) were determined.
2.2. Diets
2.4. Statistical analysis
Four experimental diets were formulated with low (15%) or high (22%) CP content combined with low (7.5%) or high (13.5%) FC content. Fermentable
Pen was the experimental unit for ADFI, ADG and G:F. Analysis of variance was performed using the statistical software of Genstat (GenStat, 2000). Data
2. Materials and methods 2.1. Animals and treatments
Table 1 Effects of dietary crude protein (CP) and fermentable carbohydrates (FC) content on growth performance of piglets during 4 wks following weaning Dietary treatment
P-values
SEM
CP content
Low
High
FC content
Low
High
Low
High
CP
FC
CP × FC
Initial BW, kg Final BW, kg Feed intake, g/d Body gain, g/d Gain:feed
8.71 18.85a 519a 350a 0.67a
8.70 17.40b 478b 300b 0.63b
8.71 17.79b 466b 313b 0.67a
8.71 17.83b 478b 315b 0.66a
0.881 0.152 0.022 0.149 0.048
1.000 0.003 0.192 0.002 b0.001
0.764 0.002 0.022 0.001 0.033
Values are means of 10 pens per dietary treatment. Within a row, means without a common superscript letter differ (P b 0.05).
a,b,c,
0.00 0.21 11 7 0.01
196
P. Bikker et al. / Livestock Science 108 (2007) 194–197
Table 2 Effects of dietary crude protein (CP) and fermentable carbohydrates (FC) content on bacterial counts (log CFU/g) and fermentation products in jejunum and colon of piglets, determined at d 7 after weaning Dietary treatment CP content
Low
FC content
Low
P-values
SEM
High High
Low
Jejunum Lactobacilli Coliforms Ammonia, mg/L Lactic acid, mmol/L Total VFA, mmol/L
7.91 7.61 32.7 12.3 13.2
8.25 7.10 22.9 18.3 10.2
7.66 8.15 73.0 16.6 15.7
Colon Lactobacilli Coliforms Ammonia, mg/L Lactic acid, mmol/L Total VFA, mmol/L
9.31 8.26 356 0.99 74.3
9.11 8.37 196 0.91 80.9
9.14 8.64 376 1.27 68.8
High
CP
FC
CP × FC
8.55 6.78 44.1 26.8 11.9
0.937 0.823 0.003 0.160 0.344
0.047 0.063 0.049 0.080 0.126
0.354 0.377 0.315 0.635 0.867
0.15 0.23 6.2 2.5 1.2
9.20 7.77 339 0.85 95.1
0.798 0.757 0.138 0.747 0.454
0.682 0.282 0.076 0.456 0.009
0.438 0.175 0.255 0.621 0.102
0.09 0.17 34 0.18 4.0
Values are means of 8 piglets per dietary treatment.
were analyzed as a 2 × 2 factorial arrangement of treatments, with dietary CP and FC content as respective factors. Statistical analysis of microbiota, fermentation products, histology and enzymology was performed using the data from 8 dissections per treatment, with piglet as the experimental unit.
FC tended to increase the concentration of lactic acid in the jejunum and concentration of total VFA in the colon. Activity of digestive enzymes and gut morphology were not significantly influenced by dietary treatments.
3. Results
We hypothesized that a reduction in dietary CP and an increase in FC content would reduce the risk of protein fermentation and improve intestinal health and growth performance. Indeed both decreasing the CP content and increasing the FC content of the diet reduced the ammonia concentration in the small intestine. Furthermore, effects of high FC on bacteria and fermentation products indicate a shift in the composition of microbial population in response to dietary substrate. This was in agreement with Konstantinov et al. (2004) and was suggested to be beneficial to protect piglets from pathogenic E. coli adhesion. The low-CP diets also reduced protein fermentation, but this was not coincided by any effect on coliforms or lactobacilli, nor on VFA production. Nyachoti et al. (2006) also found no effect of lowering the dietary CP content (from 23 to 17%) on total VFA concentration and on bacterial population in the jejunum. Despite these effects of an increase in FC content on intestinal characteristics, increasing the FC level did not improve growth performance during the first 4 wks after weaning. We speculate that nutrient availability (energy and/or amino acids) from fibrous ingredients in high-FC diets may have been suboptimal because of the immature digestive capacity in newly weaned piglets.
3.1. Animal performance In the 4 wk experimental period, increasing the FC content of the low-CP diet reduced feed intake, daily gain and gain:feed, whereas increasing the FC content of the high-CP diet did not affect growth performance (Table 1). Furthermore, the reduction in CP content improved feed intake and daily gain of piglets fed the low-FC diets, but not of piglets fed the high-FC diets where it reduced gain:feed ratio. 3.2. Intestinal characteristics The effects of CP and FC were presented in Table 2. Interactions between CP and FC contents were not significant. A low-CP level reduced the amount of ammonia in the jejunum, without significant effects on other characteristics. An increase in the level of dietary FC increased counts of lactobacilli and tended to lower counts of coliforms in the jejunum, but not in the colon. High dietary FC decreased the concentration of ammonia in the jejunum and tended to decrease the ammonia concentration in the colon. In addition, high
4. Discussion
P. Bikker et al. / Livestock Science 108 (2007) 194–197
5. Conclusion We conclude that increasing dietary fermentable carbohydrates can induce a shift in microbial population, reduce ammonia content and increase lactic acid and VFA contents in the digestive tract. However, this does not necessarily result in an increased growth performance of weaned piglets. Acknowledgement This work has been carried out with financial support of the European Community specific RTD programme “Quality of Life and Management of Living Resources” research project HEALTHYPIGUT (QLK5-CT20000– 00522). References Awati, A. 2005. Prebiotics in piglet nutrition? Fermentation kinetics along the GI tract. PhD Diss., Wageningen University, The Netherlands. Ball, R.O., Aherne, F.X., 1987. Influence of dietary nutrient density, level of feed intake and weaning age on young pigs. II. Apparent
197
nutrient digestibility and incidence and severity of diarrhea. Can. J. Anim. Sci. 67, 1105–1115. CVB, 2003. Veevoedertabel [Feedstuff Table, Nutritional Value of Feed Ingredients]. Centraal Veevoederbureau, Lelystad, The Netherlands. GenStat, 2000. GenStat for Windows, Release 4.2. 5th ed. VSN International Ltd., Oxford. Goodlad, R.A., Levi, S., Lee, C.Y., Mandir, N., Hodgson, H., Wright, N.A., 1991. Morphometry and cell proliferation in endoscopic biopsies: evaluation of a technique. Gastroenterology 101, 1235–1241. Konstantinov, S.R., Awati, A., Smidt, H., Williams, B.A., Akkermans, A.D.L., de Vos, W.M., 2004. Specific response of a novel and abundant Lactobacillus amylovorus-like phylotype to dietary prebiotics in the ileum and colon of weaning piglets. Appl. Environ. Microbiol. 70, 3821–3830. Marion, J., Petersen, Y.M., Romé, V., Thomas, F., le Dividich, J., Sangild, P.T., le Huërou-Luron, I., 2005. Early weaning stimulates intestinal brush border enzyme activities in piglets, mainly at posttranscriptional level. J. Pediatr. Gastroenterol. Nutr. 41, 401–410. Nyachoti, C.M., Omogbenigun, F.O., Rademacher, M., Blank, G., 2006. Performance responses and indicators of gastrointestinal health in early-weaned pigs fed low-protein amino acid-supplemented diets. J. Anim. Sci. 84, 125–134.