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Effects of benzoic acid on performance and ecology of gastrointestinal microbiota in weanling piglets
Livestock Science 108 (2007) 210 – 213 www.elsevier.com/locate/livsci
Effects of benzoic acid on performance and ecology of gastrointestinal microbiota in weanling piglets ☆ D. Torrallardona a,⁎, I. Badiola b , J. Broz c a
c
IRTA, Mas de Bover, Ctra. Reus-El Morell Km. 3,8 E-43120 Constantí (Tarragona), Spain b CReSA Foundation, Campus de Bellaterra, Edifici V, 08193 Bellaterra, Spain DSM Nutritional Products, Animal Nutrition and Health R&D, CH-4002 Basel, Switzerland
Abstract The effects of feeding benzoic acid on weanling pig performance and on the ecology of their gastrointestinal microbiota were evaluated. One hundred and forty-four piglets (4 weeks of age) were used. Half of the piglets were fed a control diet and half were fed the same diet with 0.5% benzoic acid, and their performance was measured for 28 days. At the end of the trial, twelve piglets from each treatment were euthanised and samples of ileal and caecal digesta were collected. The digesta samples were cultured to quantify Lactobacillus spp., Escherichia coli, Enterococcus spp. and Clostridium perfringens and they were also studied by restriction fragment length polymorphism to evaluate the biodiversity degree and the similarity between the profiles of the microbiota. Benzoic acid improved piglets’ performance, and this was associated with a greater ileal microbiota biodiversity. © 2007 Elsevier B.V. All rights reserved. Keywords: Benzoic acid; Weaning piglets; Intestinal microbiota; Alternatives to antibiotics
1. Introduction Antimicrobial growth promoters have been used for many decades to modulate the re-adaptation of gastrointestinal microbiota, which occurs in piglets at weaning (Collier et al., 2003). However, the recent ban on their use in the European Union led to the investigation of new alternatives such as organic acids (Partanen, 2001). In vitro studies have shown that, of several organic acids tested, benzoic acid had the strongest antimicrobial ef-
☆ 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. Tel.: +34 977 32 84 24; fax: +34 977 34 40 55. E-mail address: [email protected] (D. Torrallardona).
1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.01.062
fect (Knarreborg et al., 2002), but unfortunately little is known about its efficacy in vivo. The aim of this study was to test benzoic acid as a supplement for weaning pig diets and to study its effects on the microbial ecology of their gastrointestinal tract. 2. Materials and methods IRTA's Ethical Committee on Animal Experimentation (CEEA) approved the experimental procedures with animals described in this study. 2.1. Experimental A total of 144 piglets (Landrace × Landrace; mixed sexes) were used. The pigs were weaned at 4 weeks of age with an average initial live body weight of 8.9 kg (SD 1.55) and were housed in two weaning rooms (24
D. Torrallardona et al. / Livestock Science 108 (2007) 210–213 Table 1 Composition of the experimental diet (g/kg as fed) Ingredients
Basal diet
Maize Barley Extruded soybeans Soybean meal (48% CP) Potato protein concentrate Sweet milk whey Lard DL-methionine L-lysine–HCl L-threonine Calcium carbonate Dicalcium phosphate Salt Vit–min complex a Maize starch or benzoic acid
300 360 62 80 50 100 12 0.7 2.7 0.3 6.9 13.8 1.9 4 5
a Providing per kg of diet: vitamin A: 10,000 IU; vitamin D3: 2000 IU; vitamin E: 15 mg; thiamin: 1.3 mg; riboflavin: 3.5 mg; vitamin B12: 0.025 mg; vitamin B6: 1.5 mg; calcium pantothenate: 10 mg; nicotinic acid: 15 mg; biotin: 0.1 mg; folic acid: 0.6 mg; vitamin K3: 2 mg; Fe: 80 mg as iron sulfate; Cu: 6 mg as copper sulfate; Co: 0.75 mg as cobalt sulfate; Zn: 185 mg as zinc oxide; Mn: 60 mg as manganese sulfate; I: 0.75 mg as potassium iodate; Se: 0.10 mg as sodium selenite; ethoxiquin: 0.15 mg.
and 12 pens respectively) equipped with automatic heating, forced ventilation and completely slatted floors. The temperature at the start of the trial was set at 27 °C and was gradually reduced to 23 °C by day 28. At the start of the experiment, the animals were distributed by their initial body weight into 18 blocks of two pens (4 pigs per pen) according to a randomised complete block design with two experimental treatments: negative control (NC) and benzoic acid (BA). Feed and pigs were weighed on day 0, 14 and 28 of trial. Average daily weight gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (FGR) were calculated for each pen. At the end of the trial (day 28), twelve pairs of piglets were selected, so that within each pair, one animal was assigned to each treatment, the two animals belonged to the same litter and they had a similar weight at the start of the trial. These pigs were euthanised by an intravenous injection of 150 to 200 mg/kg BW of sodium pentobarbitone (Dolethal, Vétoquinol, S.A., Madrid, Spain). Samples of ileal and caecal digesta were obtained for the quantification of Lactobacillus spp., Escherichia coli, Enterococcus spp. and Clostridium perfringens as described previously (Torrallardona et al., 2003). Additionally, a second aliquot of these samples was also used to study the ecology of the microbiota by restriction fragment length polymorphism (RFLP) according to
211
the procedures described by Pérez de Rozas et al. (2004). From the RFLP profiles, the biodiversity degree and the similarity between profiles were evaluated. Additionally, urine samples were obtained directly from the urinary bladder with a syringe for the measurement of pH and hippuric acid concentration according to Tomokuni and Ogata (1972). 2.2. Diets The experimental diets were formulated to meet or exceed the animals’ requirements (NRC, 1998). They were formulated to contain 12 g total lysine and 13.5 MJ ME per kg. Feed was presented in pelleted form and offered ad libitum. The NC diet contained 5 g/kg of maize starch, which was replaced by the same amount of benzoic acid (VevoVitall®, DSM Nutritional Products) in the BA diet. All other ingredients were included at an identical concentration for the two diets (Table 1). 2.3. Calculations and statistical analysis The performance parameters were analysed as a randomised complete block design with 2 treatments and 18 blocks, using the pen as the experimental unit. The blocks took into account the initial weight of the animals and pen location. Urine pH and hippuric acid concentration for each treatment were analysed using the individual values by one-way ANOVA. Microbiota data was analysed using a mixed linear model in order to take into account the correlation between the samples obtained from the same pig. The fixed effects of treatment (NC and BA), gastrointestinal
Table 2 Effect of benzoic acid supplementation on the productive parameters of weanling pigs
Body weight (kg)
Weight gain (g/day)
Feed intake (g/day)
Feed:gain ratio
Days postweaning
Control
Benzoic acid (0.5%)
Pooled SE
0 14 28 0–14 14–28 0–28 0–14 14–28 0–28 0–14 14–28 0–28
8.9 11.9 a 18.9 a 209 a 506 a 358 a 324 764 a 544 a 1.62 a 1.52 1.54 a
8.9 12.6 b 20.6 b 261 b 574 b 417 b 356 838 b 597 b 1.40 b 1.47 1.44 b
0.03 0.21 0.35 14.2 13.2 12.4 14.1 20.2 15.6 0.065 0.031 0.035
ab Values in the same row with different letters are significantly different (P b 0.05).
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D. Torrallardona et al. / Livestock Science 108 (2007) 210–213
Table 3 Effect of benzoic acid supplementation on the intra-group similarity between microbiota RFLP profiles (%), the biodiversity degree and microbial contents (log cfu/g) in ileal and caecal digesta of pigs 28 days post-weaning Treatment
Similarity (%)
Biodiversity degree
Lactobacilli (log cfu/g)
Enterococci (log cfu/g)
E. coli (log cfu/g)
C. perfringens (log cfu/g)
Ileum Control Benzoic acid
47.1 c 48.9 c
637 a 1277 b
7.0 bc 6.9 c
2.8 c 3.7 bc
1.7 b 1.7 b
0.8 1.0
Caecum Control Benzoic acid Pooled SE
63.3 b 66.5 a 1.11
8.1 a 7.8 ab 0.32
3.6 ab 4.7 a 0.41
2.7 ab 3.3 a 0.36
1.0 1.1 0.22
Response a
T⁎ S⁎⁎⁎
S⁎⁎
S⁎
S⁎⁎
–
757 ab 631 a 176.2 T × S⁎
abc Values in the same column with different letters are significantly different (P b 0.05). a Responses were analysed using a mixed linear model using treatment (T), gastrointestinal tract segment (S) and their interaction (T × S) as fixed effects: ⁎P b 0.05, ⁎⁎⁎P b 0.01, ⁎⁎⁎P b 0.001.
tract segment (ileum and caecum) and their interaction were considered. 3. Results and discussion The addition of benzoic acid to the diet resulted in improved animal performance (Table 2). Particularly, it increased (P b 0.05) ADG in each experimental period, it also increased (P b 0.05) ADFI during the second half (14–28 days) and the whole experimental period, and finally, it improved (P b 0.05) FGR during the first half (0–14 days) and throughout the whole trial. Benzoic acid addition increased (P b 0.001) urinary hippuric acid concentration from 455 to 741 mg/100 ml (pooled SE = 0.18) as a result of the conjugation of benzoic acid with glycine and this tended (P b 0.1) to reduce urinary pH from 5.9 to 5.4 (pooled SE = 0.18). The analysis of microbiota's intra-group similarity (comparison of the RFLP profiles within each treatment and gastrointestinal segment) indicates a higher homogeneity (P b 0.05) in the caecal microbiota of BA than NC animals, but no differences in the ileal microbiota (Table 3). The ileal vs. caecal similarity (comparison of the RFLP profiles of the two gastrointestinal segments within each treatment) showed a higher (P b 0.001) similarity in the BA than in the NC group (53 vs. 48%). However, further work is required to establish the relevance of these observations. Pigs fed BA showed a higher (P b 0.05) degree of biodiversity of gastrointestinal microbiota at the ileal level than the pigs from the control group (Table 3). This, together with the better performance could be an indication that a higher degree of biodiversity is associated with healthier microbiota. This is in disagreement with the results of Canibe et al. (2005) who
observed that the addition of formic acid to grower pig diets reduced biodiversity at the same time that performance tended to improve. Traditional culture methods (Table 3) revealed that the addition of BA increased (P b 0.05) the number of Enterococci by 1.02 log cfu/g, and that caecal digesta had higher (P b 0.05) counts of Lactobacilli, E. coli and Enterococci than ileal digesta. No interactions between GIT segment and diet were observed (P N 0.05). 4. Conclusions It is concluded that the addition of benzoic acid at 0.5% to the diet of weanling pigs improves performance. This addition is also accompanied by a higher biodiversity of the ileal microbiota and a closer similarity between the ileal and caecal microbiota of the piglets. Acknowledgements The authors wish to thank the technical support received from A.M. Pérez de Rozas, A. Miquel and Ll. Llauradó. The assistance of IRTA's farm staff in looking after the animals is also acknowledged. IRTA's Animal Nutrition group is supported by CIRIT (Grup de Recerca de Qualitat, Ref. GRQ93-9804). References Canibe, N., Højberg, O., Højsgaard, S., Jensen, B.B., 2005. Feed physical form and formic acid addition to the feed affect the gastrointestinal ecology and growth performance of growing pigs. J. Anim. Sci. 83, 1287–1302. Collier, C.T., Smiricky-Tjardes, M.R., Albin, D.M., Wubben, J.E., Gabert, V.M., Deplancke, B., Bane, D., Anderson, D.B., Gaskins, H.R., 2003. Molecular ecological analysis of porcine ileal
D. Torrallardona et al. / Livestock Science 108 (2007) 210–213 microbiota responses to antimicrobial growth promoters. J. Anim. Sci. 81, 3035–3045. Knarreborg, A., Miquel, N., Granli, T., Jensen, B.B., 2002. Establishment and application of an in vitro methodology to study the effects of organic acids on coliform and lactic acid bacteria in the proximal part of the gastrointestinal tract of piglets. Anim. Feed Sci. Technol. 99, 131–140. NRC, 1998. Nutrient Requirements of Swine, tenth ed. National Academy Press, Washington, DC. Partanen, K., 2001. Organic acids — their efficacy and modes of action in pigs. In: Piva, A., Bach Knudsen, K.E., Lindberg, J.E. (Eds.), Gut Environment of Pigs. Nottingham University Press, Nottingham, UK, pp. 201–217.
213
Pérez de Rozas, A.M., Roca, M., Carabaño, R., de Blas, C., Francesch, M., Brufau, J., Martín-Orúe, S.M., Gasa, J., Campoy, S., Barbé, J., Badiola, I., 2004. A comparative study of intestinal microbial diversity from birds, pigs and rabbits by restriction fragment length polymorphism analysis. Reprod Nutr. Dev. 44 (Suppl. 1), S4. Tomokuni, K., Ogata, M., 1972. Direct colorimetric determination of hippuric acid in urine. Clin. Chem. 18, 349–351. Torrallardona, D., Conde, R., Badiola, I., Polo, J., Brufau, J., 2003. Effect of fishmeal replacement with spray-dried animal plasma and colistin on intestinal structure, intestinal microbiology, and performance of weanling pigs challenged with Escherichia coli K99. J. Anim. Sci. 81, 1220–1226.
Effects of benzoic acid on performance and ecology of gastrointestinal microbiota in weanling piglets ☆ D. Torrallardona a,⁎, I. Badiola b , J. Broz c a
c
IRTA, Mas de Bover, Ctra. Reus-El Morell Km. 3,8 E-43120 Constantí (Tarragona), Spain b CReSA Foundation, Campus de Bellaterra, Edifici V, 08193 Bellaterra, Spain DSM Nutritional Products, Animal Nutrition and Health R&D, CH-4002 Basel, Switzerland
Abstract The effects of feeding benzoic acid on weanling pig performance and on the ecology of their gastrointestinal microbiota were evaluated. One hundred and forty-four piglets (4 weeks of age) were used. Half of the piglets were fed a control diet and half were fed the same diet with 0.5% benzoic acid, and their performance was measured for 28 days. At the end of the trial, twelve piglets from each treatment were euthanised and samples of ileal and caecal digesta were collected. The digesta samples were cultured to quantify Lactobacillus spp., Escherichia coli, Enterococcus spp. and Clostridium perfringens and they were also studied by restriction fragment length polymorphism to evaluate the biodiversity degree and the similarity between the profiles of the microbiota. Benzoic acid improved piglets’ performance, and this was associated with a greater ileal microbiota biodiversity. © 2007 Elsevier B.V. All rights reserved. Keywords: Benzoic acid; Weaning piglets; Intestinal microbiota; Alternatives to antibiotics
1. Introduction Antimicrobial growth promoters have been used for many decades to modulate the re-adaptation of gastrointestinal microbiota, which occurs in piglets at weaning (Collier et al., 2003). However, the recent ban on their use in the European Union led to the investigation of new alternatives such as organic acids (Partanen, 2001). In vitro studies have shown that, of several organic acids tested, benzoic acid had the strongest antimicrobial ef-
☆ 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. Tel.: +34 977 32 84 24; fax: +34 977 34 40 55. E-mail address: [email protected] (D. Torrallardona).
1871-1413/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.livsci.2007.01.062
fect (Knarreborg et al., 2002), but unfortunately little is known about its efficacy in vivo. The aim of this study was to test benzoic acid as a supplement for weaning pig diets and to study its effects on the microbial ecology of their gastrointestinal tract. 2. Materials and methods IRTA's Ethical Committee on Animal Experimentation (CEEA) approved the experimental procedures with animals described in this study. 2.1. Experimental A total of 144 piglets (Landrace × Landrace; mixed sexes) were used. The pigs were weaned at 4 weeks of age with an average initial live body weight of 8.9 kg (SD 1.55) and were housed in two weaning rooms (24
D. Torrallardona et al. / Livestock Science 108 (2007) 210–213 Table 1 Composition of the experimental diet (g/kg as fed) Ingredients
Basal diet
Maize Barley Extruded soybeans Soybean meal (48% CP) Potato protein concentrate Sweet milk whey Lard DL-methionine L-lysine–HCl L-threonine Calcium carbonate Dicalcium phosphate Salt Vit–min complex a Maize starch or benzoic acid
300 360 62 80 50 100 12 0.7 2.7 0.3 6.9 13.8 1.9 4 5
a Providing per kg of diet: vitamin A: 10,000 IU; vitamin D3: 2000 IU; vitamin E: 15 mg; thiamin: 1.3 mg; riboflavin: 3.5 mg; vitamin B12: 0.025 mg; vitamin B6: 1.5 mg; calcium pantothenate: 10 mg; nicotinic acid: 15 mg; biotin: 0.1 mg; folic acid: 0.6 mg; vitamin K3: 2 mg; Fe: 80 mg as iron sulfate; Cu: 6 mg as copper sulfate; Co: 0.75 mg as cobalt sulfate; Zn: 185 mg as zinc oxide; Mn: 60 mg as manganese sulfate; I: 0.75 mg as potassium iodate; Se: 0.10 mg as sodium selenite; ethoxiquin: 0.15 mg.
and 12 pens respectively) equipped with automatic heating, forced ventilation and completely slatted floors. The temperature at the start of the trial was set at 27 °C and was gradually reduced to 23 °C by day 28. At the start of the experiment, the animals were distributed by their initial body weight into 18 blocks of two pens (4 pigs per pen) according to a randomised complete block design with two experimental treatments: negative control (NC) and benzoic acid (BA). Feed and pigs were weighed on day 0, 14 and 28 of trial. Average daily weight gain (ADG), average daily feed intake (ADFI) and feed to gain ratio (FGR) were calculated for each pen. At the end of the trial (day 28), twelve pairs of piglets were selected, so that within each pair, one animal was assigned to each treatment, the two animals belonged to the same litter and they had a similar weight at the start of the trial. These pigs were euthanised by an intravenous injection of 150 to 200 mg/kg BW of sodium pentobarbitone (Dolethal, Vétoquinol, S.A., Madrid, Spain). Samples of ileal and caecal digesta were obtained for the quantification of Lactobacillus spp., Escherichia coli, Enterococcus spp. and Clostridium perfringens as described previously (Torrallardona et al., 2003). Additionally, a second aliquot of these samples was also used to study the ecology of the microbiota by restriction fragment length polymorphism (RFLP) according to
211
the procedures described by Pérez de Rozas et al. (2004). From the RFLP profiles, the biodiversity degree and the similarity between profiles were evaluated. Additionally, urine samples were obtained directly from the urinary bladder with a syringe for the measurement of pH and hippuric acid concentration according to Tomokuni and Ogata (1972). 2.2. Diets The experimental diets were formulated to meet or exceed the animals’ requirements (NRC, 1998). They were formulated to contain 12 g total lysine and 13.5 MJ ME per kg. Feed was presented in pelleted form and offered ad libitum. The NC diet contained 5 g/kg of maize starch, which was replaced by the same amount of benzoic acid (VevoVitall®, DSM Nutritional Products) in the BA diet. All other ingredients were included at an identical concentration for the two diets (Table 1). 2.3. Calculations and statistical analysis The performance parameters were analysed as a randomised complete block design with 2 treatments and 18 blocks, using the pen as the experimental unit. The blocks took into account the initial weight of the animals and pen location. Urine pH and hippuric acid concentration for each treatment were analysed using the individual values by one-way ANOVA. Microbiota data was analysed using a mixed linear model in order to take into account the correlation between the samples obtained from the same pig. The fixed effects of treatment (NC and BA), gastrointestinal
Table 2 Effect of benzoic acid supplementation on the productive parameters of weanling pigs
Body weight (kg)
Weight gain (g/day)
Feed intake (g/day)
Feed:gain ratio
Days postweaning
Control
Benzoic acid (0.5%)
Pooled SE
0 14 28 0–14 14–28 0–28 0–14 14–28 0–28 0–14 14–28 0–28
8.9 11.9 a 18.9 a 209 a 506 a 358 a 324 764 a 544 a 1.62 a 1.52 1.54 a
8.9 12.6 b 20.6 b 261 b 574 b 417 b 356 838 b 597 b 1.40 b 1.47 1.44 b
0.03 0.21 0.35 14.2 13.2 12.4 14.1 20.2 15.6 0.065 0.031 0.035
ab Values in the same row with different letters are significantly different (P b 0.05).
212
D. Torrallardona et al. / Livestock Science 108 (2007) 210–213
Table 3 Effect of benzoic acid supplementation on the intra-group similarity between microbiota RFLP profiles (%), the biodiversity degree and microbial contents (log cfu/g) in ileal and caecal digesta of pigs 28 days post-weaning Treatment
Similarity (%)
Biodiversity degree
Lactobacilli (log cfu/g)
Enterococci (log cfu/g)
E. coli (log cfu/g)
C. perfringens (log cfu/g)
Ileum Control Benzoic acid
47.1 c 48.9 c
637 a 1277 b
7.0 bc 6.9 c
2.8 c 3.7 bc
1.7 b 1.7 b
0.8 1.0
Caecum Control Benzoic acid Pooled SE
63.3 b 66.5 a 1.11
8.1 a 7.8 ab 0.32
3.6 ab 4.7 a 0.41
2.7 ab 3.3 a 0.36
1.0 1.1 0.22
Response a
T⁎ S⁎⁎⁎
S⁎⁎
S⁎
S⁎⁎
–
757 ab 631 a 176.2 T × S⁎
abc Values in the same column with different letters are significantly different (P b 0.05). a Responses were analysed using a mixed linear model using treatment (T), gastrointestinal tract segment (S) and their interaction (T × S) as fixed effects: ⁎P b 0.05, ⁎⁎⁎P b 0.01, ⁎⁎⁎P b 0.001.
tract segment (ileum and caecum) and their interaction were considered. 3. Results and discussion The addition of benzoic acid to the diet resulted in improved animal performance (Table 2). Particularly, it increased (P b 0.05) ADG in each experimental period, it also increased (P b 0.05) ADFI during the second half (14–28 days) and the whole experimental period, and finally, it improved (P b 0.05) FGR during the first half (0–14 days) and throughout the whole trial. Benzoic acid addition increased (P b 0.001) urinary hippuric acid concentration from 455 to 741 mg/100 ml (pooled SE = 0.18) as a result of the conjugation of benzoic acid with glycine and this tended (P b 0.1) to reduce urinary pH from 5.9 to 5.4 (pooled SE = 0.18). The analysis of microbiota's intra-group similarity (comparison of the RFLP profiles within each treatment and gastrointestinal segment) indicates a higher homogeneity (P b 0.05) in the caecal microbiota of BA than NC animals, but no differences in the ileal microbiota (Table 3). The ileal vs. caecal similarity (comparison of the RFLP profiles of the two gastrointestinal segments within each treatment) showed a higher (P b 0.001) similarity in the BA than in the NC group (53 vs. 48%). However, further work is required to establish the relevance of these observations. Pigs fed BA showed a higher (P b 0.05) degree of biodiversity of gastrointestinal microbiota at the ileal level than the pigs from the control group (Table 3). This, together with the better performance could be an indication that a higher degree of biodiversity is associated with healthier microbiota. This is in disagreement with the results of Canibe et al. (2005) who
observed that the addition of formic acid to grower pig diets reduced biodiversity at the same time that performance tended to improve. Traditional culture methods (Table 3) revealed that the addition of BA increased (P b 0.05) the number of Enterococci by 1.02 log cfu/g, and that caecal digesta had higher (P b 0.05) counts of Lactobacilli, E. coli and Enterococci than ileal digesta. No interactions between GIT segment and diet were observed (P N 0.05). 4. Conclusions It is concluded that the addition of benzoic acid at 0.5% to the diet of weanling pigs improves performance. This addition is also accompanied by a higher biodiversity of the ileal microbiota and a closer similarity between the ileal and caecal microbiota of the piglets. Acknowledgements The authors wish to thank the technical support received from A.M. Pérez de Rozas, A. Miquel and Ll. Llauradó. The assistance of IRTA's farm staff in looking after the animals is also acknowledged. IRTA's Animal Nutrition group is supported by CIRIT (Grup de Recerca de Qualitat, Ref. GRQ93-9804). References Canibe, N., Højberg, O., Højsgaard, S., Jensen, B.B., 2005. Feed physical form and formic acid addition to the feed affect the gastrointestinal ecology and growth performance of growing pigs. J. Anim. Sci. 83, 1287–1302. Collier, C.T., Smiricky-Tjardes, M.R., Albin, D.M., Wubben, J.E., Gabert, V.M., Deplancke, B., Bane, D., Anderson, D.B., Gaskins, H.R., 2003. Molecular ecological analysis of porcine ileal
D. Torrallardona et al. / Livestock Science 108 (2007) 210–213 microbiota responses to antimicrobial growth promoters. J. Anim. Sci. 81, 3035–3045. Knarreborg, A., Miquel, N., Granli, T., Jensen, B.B., 2002. Establishment and application of an in vitro methodology to study the effects of organic acids on coliform and lactic acid bacteria in the proximal part of the gastrointestinal tract of piglets. Anim. Feed Sci. Technol. 99, 131–140. NRC, 1998. Nutrient Requirements of Swine, tenth ed. National Academy Press, Washington, DC. Partanen, K., 2001. Organic acids — their efficacy and modes of action in pigs. In: Piva, A., Bach Knudsen, K.E., Lindberg, J.E. (Eds.), Gut Environment of Pigs. Nottingham University Press, Nottingham, UK, pp. 201–217.
213
Pérez de Rozas, A.M., Roca, M., Carabaño, R., de Blas, C., Francesch, M., Brufau, J., Martín-Orúe, S.M., Gasa, J., Campoy, S., Barbé, J., Badiola, I., 2004. A comparative study of intestinal microbial diversity from birds, pigs and rabbits by restriction fragment length polymorphism analysis. Reprod Nutr. Dev. 44 (Suppl. 1), S4. Tomokuni, K., Ogata, M., 1972. Direct colorimetric determination of hippuric acid in urine. Clin. Chem. 18, 349–351. Torrallardona, D., Conde, R., Badiola, I., Polo, J., Brufau, J., 2003. Effect of fishmeal replacement with spray-dried animal plasma and colistin on intestinal structure, intestinal microbiology, and performance of weanling pigs challenged with Escherichia coli K99. J. Anim. Sci. 81, 1220–1226.