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Effects of chito-oligosaccharide supplementation on the growth performance, nutrient digestibility, intestinal morphology, and fecal shedding of Escherichia coli and Lactobacillus in weaning pigs

Effects of chito-oligosaccharide supplementation on the growth performance, nutrient... ABSTRACT A total of 50 weaning pigs (16 d of age; 4.72 ± 0.23 kg of BW) were selected to investigate the effect of dietary chito-oligosaccharide (COS) supplementation on growth performance, fecal shedding of Escherichia coli and Lactobacillus, apparent digestibility, and small intestinal morphology. Pigs housed in individual metabolic cages were assigned randomly to 5 treatments (n = 10), including 1 basal diet (control), 3 diets with COS supplementation (100, 200, and 400 mg/kg), and 1 diet with chlortetracycline (CTC) supplementation (80 mg/kg). Fresh fecal samples were collected to evaluate shedding of E. coli and Lactobacillus on d 0, 7, 14, and 21 postweaning. Fresh fecal samples collected from each cage from d 19 to 21 were stored frozen for determination of apparent total tract digestibility. On d 21, all pigs were killed to collect the middle sections of the duodenum, jejunum, and ileum for determination of mucosa morphology. Supplementation of COS at 100 and 200 mg/kg and supplementation of CTC improved (P < 0.05) overall ADG, ADFI, and G:F in comparison with the control. Supplementation of COS at 200 mg/kg as well as supplementation of CTC increased (P < 0.05) apparent total tract digestibility of DM, GE, CP, crude fat, Ca, and P, whereas COS at 100 mg/kg increased (P < 0.05) the digestibility of DM, Ca, and P in comparison with the control diet. Pigs receiving diets supplemented with COS or CTC had a decreased (P < 0.05) incidence of diarrhea and decreased diarrhea scores compared with control pigs. Fecal samples from pigs receiving diets supplemented with COS had greater (P < 0.05) Lactobacillus counts than those from control pigs and pigs receiving diets supplemented with CTC on d 14 and 21. However, supplementation of COS at 200 mg/kg and supplementation of CTC decreased (P < 0.05) E. coli counts in the feces on d 21 compared with the control diet. Dietary supplementation of COS at 200 mg/kg and of CTC increased (P < 0.05) the villus height and villus:crypt ratio at the ileum and jejunum, and COS at 100 mg/kg also increased (P < 0.05) the villus height in the ileum compared with the control diet. The current results indicated that dietary supplementation of COS at 100 and 200 mg/kg enhanced growth performance by increasing apparent digestibility, decreasing the incidence of diarrhea, and improving small intestinal morphology. INTRODUCTION Weaning is a critical stage for pigs because of alterations in the gastrointestinal tract architecture and function, as well as changes in adapting to enteric microbiota and immune responses (Pluske et al., 1997; Spreeuwenberg et al., 2001; Boudry et al., 2004; Mao et al., 2005). Pigs can be affected by weaning stress, such as nutritional, environmental, and social stresses, which can cause depressed growth performance, nutrient malabsorption, and a high incidence of diarrhea (Barnett et al., 1989; Hedemann and Jensen, 2004; Yuan et al., 2006). Antibiotics, as growth promoters and therapeutic medicines to decrease the susceptibility to infectious diseases, have been widely used in animal production for many years (Barton, 2000). However, issues with bacterial antibiotic resistance may cause problems for human health (Bach Knudsen, 2001; Smith et al., 2002). Many alternatives to the use of antibiotics have been suggested, introduced, and tested (Turner et al., 2001). Consumption of functional oligosaccharides has been shown to improve growth performance and enhance the host health status (Gibson and Roberfroid, 1995). Certain oligosaccharides, including galacto-oligosaccharide, manno-oligosaccharide, and fructo-oligasaccharide, may improve growth performance in young pigs (Davis et al., 2004; Miguel et al., 2004). Chito-oligosaccharide (COS) can be efficiently derived by chemical and enzymatic hydrolysis of poly-chitosan, which is the second most abundant carbohydrate polymer in nature (Knaul et al., 1999). More recently, COS has been shown to have immune-enhancing characteristics (Okamoto et al., 2003), antibacterial activity (Matsuhashi and Kume, 1997; Jeon et al., 2001), and protection against pathogenic infections (Rhoades et al., 2006). A previous study showed that a diet containing COS at 250 mg/kg increased serum GH, IGF-I, and protein synthesis, which in turn might have caused improved growth performance in weaned pigs (Tang et al., 2005). Huang et al. (2005) and Li et al. (2007) showed that dietary supplementation of COS at 100 mg/kg effectively increased nutrient digestibility and weight gain by improving gut health and modulating the balance of microbial flora in broilers. However, the effect of dietary COS supplementation on growth performance, intestinal structure, and microbial flora in weaned pigs is still largely unknown. Therefore, this experiment was conducted to test the effect of dietary COS supplementation on growth performance, intestinal structure, and fecal shedding of total Escherichia coli and Lactobacillus in weaning pigs. MATERIALS AND METHODS The animal care protocol was approved by the China Agricultural University Animal Care and Use Committee. Preparation and Composition of Chito-oligosaccharide Preparation and composition of the chito-oligosaccharide supplement (GlycoBio Company, Dalian, China) have been described previously (Li et al., 2007). Briefly, the different oligomers contained in the COS supplement and a standard sample prepared by mixing 6 chitosan oligomers (Sigma, St. Louis, MO) were separated and quantified by HPLC with an evaporative light-scattering detector (model 301; ESA, Chelmsford, MA) and column (4.6 × 250 mm, Asahipak NH2P-50-4E; Shodex, Tokyo, Japan). The results indicated that the COS supplement contained 40% COS and 60% cyclodextrin as a carrier. This COS is composed of 5 oligomers with an average molecular weight of 1,500 Da, which were identified as a combination of chitobiose, chitotriose, chitotetrose, chitopentose, and chitohexose, with concentrations of 0.58, 2.51, 4.49, 5.80, and 2.21 mg/mL, respectively, when 20 mg of COS sample was diluted with 1 mL of water. Water solubility of the COS supplement was greater than 99% (Li et al., 2007). Animals and Experimental Design Fifty newly weaned barrows (Large White × Landrace; initial BW = 4.72 ± 0.23 kg; 16 d of age) were selected from a commercial pig farm (Beijing, China) and transported to China Agricultural University. All pigs were housed in individual metabolism cages (0.7 × 1.7 m) in a temperature-controlled nursery (27 to 28°C) during the entire 3-wk experimental period. Pigs had access to feed and water ad libitum. Barrows were assigned to 5 treatments (n = 10) according to litter and initial BW, in a randomized complete block design. The 5 treatments were 1) a basal diet (negative control group), 2) 3 diets with COS supplementation (100, 200, and 400 mg/kg), and 3) a diet with chlortetracycline (CTC) supplementation (80 mg/kg; positive control group). The basal diet was formulated to meet the nutrient requirements suggested by NRC (1998) and contained no antibiotics or COS (Table 1). Chromium oxide (0.30%) was added to all diets as an indigestible marker. Table 1. Ingredient and chemical composition of the experimental diet, as-fed basis Item  Amount  Diet ingredient, %      Corn, yellow  67.16      Soybean meal, dehulled, 45% CP  22.65      Fish meal, 65% CP  3.00      Spray-dried porcine plasma  3.00      Dicalcium phosphate  1.58      Limestone  0.67      L-Lys·HCl, 98% Lys  0.29      DL-Met  0.04      Sweetener  0.02      Salt  0.30      Vitamin and mineral premix1  1.00      Chromic oxide  0.30  Analyzed nutritional content,2 %      CP  20.00      Ca  0.80      P  0.73      Lys  1.40      Met  0.40  Item  Amount  Diet ingredient, %      Corn, yellow  67.16      Soybean meal, dehulled, 45% CP  22.65      Fish meal, 65% CP  3.00      Spray-dried porcine plasma  3.00      Dicalcium phosphate  1.58      Limestone  0.67      L-Lys·HCl, 98% Lys  0.29      DL-Met  0.04      Sweetener  0.02      Salt  0.30      Vitamin and mineral premix1  1.00      Chromic oxide  0.30  Analyzed nutritional content,2 %      CP  20.00      Ca  0.80      P  0.73      Lys  1.40      Met  0.40  1 Vitamin and mineral premix provided the following per kilogram of diet: vitamin A, 12,000 IU as vitamin A acetate; vitamin D, 2,500 IU as vitamin D3; 30 IU of vitamin E; 12 μg of vitamin B12; vitamin K, 3 mg as menadione sodium bisulfate; D-pantothenic acid, 15 mg as calcium pantothenate; 40 mg of nicotinic acid; choline chloride, 400 mg as choline chloride; Mn, 40 mg as manganous oxide; Zn, 100 mg as zinc oxide; Fe, 90 mg as iron sulfate; Cu, 8.8 mg as copper oxide; I, 0.35 mg as ethylenediamine dihydroiodide; and Se, 0.3 mg as sodium selenite. 2 Calculated ME content: 3.20 Mcal/kg. View Large Table 1. Ingredient and chemical composition of the experimental diet, as-fed basis Item  Amount  Diet ingredient, %      Corn, yellow  67.16      Soybean meal, dehulled, 45% CP  22.65      Fish meal, 65% CP  3.00      Spray-dried porcine plasma  3.00      Dicalcium phosphate  1.58      Limestone  0.67      L-Lys·HCl, 98% Lys  0.29      DL-Met  0.04      Sweetener  0.02      Salt  0.30      Vitamin and mineral premix1  1.00      Chromic oxide  0.30  Analyzed nutritional content,2 %      CP  20.00      Ca  0.80      P  0.73      Lys  1.40      Met  0.40  Item  Amount  Diet ingredient, %      Corn, yellow  67.16      Soybean meal, dehulled, 45% CP  22.65      Fish meal, 65% CP  3.00      Spray-dried porcine plasma  3.00      Dicalcium phosphate  1.58      Limestone  0.67      L-Lys·HCl, 98% Lys  0.29      DL-Met  0.04      Sweetener  0.02      Salt  0.30      Vitamin and mineral premix1  1.00      Chromic oxide  0.30  Analyzed nutritional content,2 %      CP  20.00      Ca  0.80      P  0.73      Lys  1.40      Met  0.40  1 Vitamin and mineral premix provided the following per kilogram of diet: vitamin A, 12,000 IU as vitamin A acetate; vitamin D, 2,500 IU as vitamin D3; 30 IU of vitamin E; 12 μg of vitamin B12; vitamin K, 3 mg as menadione sodium bisulfate; D-pantothenic acid, 15 mg as calcium pantothenate; 40 mg of nicotinic acid; choline chloride, 400 mg as choline chloride; Mn, 40 mg as manganous oxide; Zn, 100 mg as zinc oxide; Fe, 90 mg as iron sulfate; Cu, 8.8 mg as copper oxide; I, 0.35 mg as ethylenediamine dihydroiodide; and Se, 0.3 mg as sodium selenite. 2 Calculated ME content: 3.20 Mcal/kg. View Large Body weight and feed consumption of individual pigs were determined weekly (on d 0, 7, 14, and 21) to calculate ADG, ADFI, and G:F. From 0600 to 0630 h in the morning of d 0, 7, 14, and 21, fecal samples were collected from the rectum with sterile rubber gloves and placed in sterile plastic tubes with lids. The samples were stored in a freezer at −20°C until analysis for counts of E. coli and Lactobacillus. On d 19, 20, and 21, approximately 50 g of fecal sample from each pig was collected immediately after defecation, placed in a sterile plastic bottle, and stored at −20°C until analysis for nutrient digestibility. On d 21, after an overnight fast, pigs were electronically stunned and killed, and tissue samples from the small intestine were collected immediately. To minimize potential errors associated with the timing of sample collection, the procedure was conducted simultaneously by 5 different groups of people, with each group in charge of 1 of the 5 treatments. The middle sections of the duodenum, jejunum, and ileum were aseptically isolated, flushed with a 0.9% salt solution, fixed with 10% formaldehyde-phosphate buffer, and kept at 4°C for microscopic assessment of mucosal morphology. Chemical Analyses Diet and fecal samples were dried in an oven (60°C; 24 h for diet and 48 h for feces) and then homogenized with a laboratory grinder (0.5-mm screen for diets and 1.0-mm screen for feces) before analysis. Dietary and fecal DM, CP, crude fat (CF), Ca, and P contents were analyzed according to AOAC (1997). Gross energy was measured by an automatic adiabatic oxygen bomb calorimeter (Parr Instrument Co., Moline, IL). Chromium content was measured with an atomic absorption spectrophotometer (Hitachi Z-5000; Hitachi Ltd., Tokyo, Japan) according to the procedure of Williams et al. (1962). Digestibility of DM, CP, CF, Ca, and P were determined by using the indicator method of Sauer and de Lange (1992) as follows: ADF = 1 − [(Cr2O3D × NF)/(Cr2O3F × ND)] × 100%, where ADF is the apparent total tract digestibility (%) of CP, CF, Ca, and P; Cr2O3D is the chromic oxide concentration in the assay diet (g/kg); NF is the concentration of a nutrient in the feces (g/kg); Cr2O3F is the concentration of chromic oxide in the feces (g/kg); and ND is the concentration of a nutrient in the assay diet (g/kg). Diarrhea Incidence and Diarrhea Score The incidence of diarrhea in piglets was observed and recorded 3 times per day during the study. To assess the severity of diarrhea, feces from each pig were scored by determining the moisture content according to the method of Hart and Dobb (1988). Scores were 0, normal, firm feces; 1, possible slight diarrhea; 2, definitely unformed, moderately fluid feces; or 3, very watery and frothy diarrhea. A cumulative diarrhea score per diet and day was then calculated (Montagne et al., 2004). The occurrence of diarrhea was defined as maintaining fecal scores of 2 and 3 for 2 consecutive days. Diarrhea incidence was calculated according to the formula reported by Huang et al. (2004) and Sun et al. (2007): diarrhea incidence (%) = number of pigs with diarrhea/(number of pigs × total experimental days) × 100, where “number of pigs with diarrhea” was the total number of pigs with diarrhea observed each day. Bacteriological Methods In vitro survival of Lactobacillus and E. coli was determined according to the methods of Guo et al. (2006) and Mikkelsen et al. (2003) with certain modifications. In brief, before enumeration, frozen fecal samples were incubated at 4°C for 10 h. Thereafter, 1 g of digesta was taken from each sample and serially diluted 10-fold with sterile physiological saline, resulting in dilutions ranging from 10−1 to 10−8 for enumeration. Escherichia coli was cultivated on MacConkey agar (Beijing Haidian Microbiological Culture Factory, Beijing, China). Lactobacillus was cultured in MRS agar (De Man, Rogosa, Sharpe agar; Oxoid Ltd., Hampshire, UK). Each dilution was determined in triplicate and the result was the average of 3 replicates. Lactobacillus was inoculated in Hungate roll tubes, and E. coli was fostered in plates. All tubes and plates were incubated at 37°C for 36 h. The microbial enumerations of digesta were expressed as log10 colony-forming units per gram. Bacteria were enumerated by a visual count of colonies by using the best replicate set from dilutions that resulted in 30 to 300 colonies per plate or tube. Small Intestinal Morphology Villus height and crypt depth were measured according to the method of Li et al. (1990) with some modifications. Briefly, fixed intestinal samples were prepared by using conventional paraffin embedding techniques. Samples were sectioned at a 6-μm thickness and stained with hematoxylin and eosin. Villus height and crypt depth were measured at 40× magnification with a microscope (Olympus CK40; Olympus Optical Company, Shenzhen, China). A minimum of 15 well-oriented, intact villi were selected to measure in triplicate for each pig. Statistical Analysis Data for growth performance, apparent total track digestibility, small intestinal morphology, and fecal shedding of E. coli and Lactobacillus were subjected to ANOVA as a randomized complete block design by using the GLM procedure (SAS Inst. Inc., Cary, NC). The pen was considered the experimental unit. Differences among treatment means were evaluated by using the PDIFF option of SAS. Results were presented as least squares means with SEM. Data for diarrhea incidence and diarrhea score were analyzed by using a chi-square contingency test (Cody and Smith, 1991; Owusu-Asiedu et al., 2003). Data were reported as a cumulative score for all pigs on each day. All nonparametric analyses were performed by using StatView (SAS Inst. Inc.). In addition, the ADG data were further analyzed by broken-line analysis to determine the optimal supplementation level of COS (Parr et al., 2003; Yuan et al., 2006). Probability values of less than 0.05 were used as the criterion for statistical significance. RESULTS Growth Performance During the first week (d 0 to 7), dietary supplementation of COS at 200 mg/kg and dietary supplementation of CTC improved (P < 0.05) ADG, ADFI, and G:F compared with the negative control diet (Table 2). No difference was detected for ADG, ADFI, and G:F between pigs fed diets with COS at 200 mg/kg and pigs fed diets with CTC. Supplementation of COS at 100 and 400 mg/kg did not increase ADG and ADFI but did increase (P < 0.05) G:F in comparison with the negative control diet. During the second week (d 7 to 14), the inclusion of COS at 100 and 200 mg/kg as well as the inclusion of CTC increased (P < 0.05) ADG and ADFI compared with the negative control diet. No differences in ADG, ADFI, and G:F were detected between the dietary supplementation of COS at 400 mg/kg and the negative control diet. There was no difference in ADG, ADFI, and G:F among pigs fed diets with COS at 100 and 200 mg/kg and pigs fed diets with CTC. Feed consumption of pigs fed diets with COS at 100 and 200 mg/kg COS and with CTC was greater (P < 0.05) than that of pigs fed the diet with COS at 400 mg/kg. During the third week (d 14 to 21), dietary supplementation of COS at 100 and 200 mg/kg and dietary supplementation of CTC increased (P < 0.05) ADG, ADFI, and G:F compared with the negative control diet. Dietary supplementation of COS at 400 mg/kg increased (P < 0.05) G:F compared with the negative control diet but did not affect ADG and ADFI. Pigs fed diets with COS at 100 and 200 mg/kg and the diet with CTC had greater (P < 0.05) ADFI than those fed the diet with COS at 400 mg/kg or the negative control diet. Table 2. Effects of dietary chito-oligosaccharide (COS) on the growth performance of weaning pigs1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  BW, kg      d 0  4.7  4.8  4.7  4.7  4.7  0.23  0.887      d 7  5.8  5.9  6.0  5.9  6.0  0.24  0.528      d 14  7.8b  8.1a  8.2a  8.0ab  8.1a  0.32  0.038      d 21  10.7b  11.2a  11.3a  11.0ab  11.3a  0.48  0.004  ADG, g      d 0 to 7  159c  168bc  175ab  165bc  180a  14  0.007      d 7 to 14  285c  307ab  320a  295bc  313a  21  0.002      d 14 to 21  411c  445ab  450a  428bc  452a  30  0.003      d 0 to 21  285c  307ab  315a  296bc  315a  19  <0.001  ADFI, g      d 0 to 7  236b  241ab  252a  236b  254a  17  0.029      d 7 to 14  442b  460a  466a  443b  467a  21  0.003      d 14 to 21  630b  661a  662a  636b  669a  29  0.002      d 0 to 21  436b  454a  460a  438b  463a  19  <0.001  G:F, g/g      d 0 to 7  0.67b  0.70a  0.69a  0.70a  0.71a  0.02  0.009      d 7 to 14  0.65a  0.67a  0.69a  0.67a  0.67a  0.02  0.002      d 14 to 21  0.65b  0.67a  0.69a  0.67a  0.68a  0.02  0.030      d 0 to 21  0.66b  0.68a  0.69a  0.68a  0.68a  0.02  <0.001      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  BW, kg      d 0  4.7  4.8  4.7  4.7  4.7  0.23  0.887      d 7  5.8  5.9  6.0  5.9  6.0  0.24  0.528      d 14  7.8b  8.1a  8.2a  8.0ab  8.1a  0.32  0.038      d 21  10.7b  11.2a  11.3a  11.0ab  11.3a  0.48  0.004  ADG, g      d 0 to 7  159c  168bc  175ab  165bc  180a  14  0.007      d 7 to 14  285c  307ab  320a  295bc  313a  21  0.002      d 14 to 21  411c  445ab  450a  428bc  452a  30  0.003      d 0 to 21  285c  307ab  315a  296bc  315a  19  <0.001  ADFI, g      d 0 to 7  236b  241ab  252a  236b  254a  17  0.029      d 7 to 14  442b  460a  466a  443b  467a  21  0.003      d 14 to 21  630b  661a  662a  636b  669a  29  0.002      d 0 to 21  436b  454a  460a  438b  463a  19  <0.001  G:F, g/g      d 0 to 7  0.67b  0.70a  0.69a  0.70a  0.71a  0.02  0.009      d 7 to 14  0.65a  0.67a  0.69a  0.67a  0.67a  0.02  0.002      d 14 to 21  0.65b  0.67a  0.69a  0.67a  0.68a  0.02  0.030      d 0 to 21  0.66b  0.68a  0.69a  0.68a  0.68a  0.02  <0.001  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs. NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large Table 2. Effects of dietary chito-oligosaccharide (COS) on the growth performance of weaning pigs1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  BW, kg      d 0  4.7  4.8  4.7  4.7  4.7  0.23  0.887      d 7  5.8  5.9  6.0  5.9  6.0  0.24  0.528      d 14  7.8b  8.1a  8.2a  8.0ab  8.1a  0.32  0.038      d 21  10.7b  11.2a  11.3a  11.0ab  11.3a  0.48  0.004  ADG, g      d 0 to 7  159c  168bc  175ab  165bc  180a  14  0.007      d 7 to 14  285c  307ab  320a  295bc  313a  21  0.002      d 14 to 21  411c  445ab  450a  428bc  452a  30  0.003      d 0 to 21  285c  307ab  315a  296bc  315a  19  <0.001  ADFI, g      d 0 to 7  236b  241ab  252a  236b  254a  17  0.029      d 7 to 14  442b  460a  466a  443b  467a  21  0.003      d 14 to 21  630b  661a  662a  636b  669a  29  0.002      d 0 to 21  436b  454a  460a  438b  463a  19  <0.001  G:F, g/g      d 0 to 7  0.67b  0.70a  0.69a  0.70a  0.71a  0.02  0.009      d 7 to 14  0.65a  0.67a  0.69a  0.67a  0.67a  0.02  0.002      d 14 to 21  0.65b  0.67a  0.69a  0.67a  0.68a  0.02  0.030      d 0 to 21  0.66b  0.68a  0.69a  0.68a  0.68a  0.02  <0.001      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  BW, kg      d 0  4.7  4.8  4.7  4.7  4.7  0.23  0.887      d 7  5.8  5.9  6.0  5.9  6.0  0.24  0.528      d 14  7.8b  8.1a  8.2a  8.0ab  8.1a  0.32  0.038      d 21  10.7b  11.2a  11.3a  11.0ab  11.3a  0.48  0.004  ADG, g      d 0 to 7  159c  168bc  175ab  165bc  180a  14  0.007      d 7 to 14  285c  307ab  320a  295bc  313a  21  0.002      d 14 to 21  411c  445ab  450a  428bc  452a  30  0.003      d 0 to 21  285c  307ab  315a  296bc  315a  19  <0.001  ADFI, g      d 0 to 7  236b  241ab  252a  236b  254a  17  0.029      d 7 to 14  442b  460a  466a  443b  467a  21  0.003      d 14 to 21  630b  661a  662a  636b  669a  29  0.002      d 0 to 21  436b  454a  460a  438b  463a  19  <0.001  G:F, g/g      d 0 to 7  0.67b  0.70a  0.69a  0.70a  0.71a  0.02  0.009      d 7 to 14  0.65a  0.67a  0.69a  0.67a  0.67a  0.02  0.002      d 14 to 21  0.65b  0.67a  0.69a  0.67a  0.68a  0.02  0.030      d 0 to 21  0.66b  0.68a  0.69a  0.68a  0.68a  0.02  <0.001  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs. NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large Over the entire experimental period, dietary supplementation of COS at 100 and 200 mg/kg and dietary supplementation of CTC increased (P < 0.05) ADG by 7.7, 10.5, and 10.5%, respectively, compared with the negative control diet. The ADFI of pigs fed diets with COS at 100 and 200 mg/kg and the diet with CTC were 3.9, 5.2, and 5.8% greater (P < 0.05), respectively, than that of the negative control group. The G:F of pigs fed diets with COS at 100 and 200 mg/kg and pigs fed diets with CTC were 3.3, 4.4, and 3.9% greater (P < 0.05), respectively, than that of pigs fed the negative control diet. The provision of COS at 400 mg/kg increased (P < 0.05) G:F by 3.4% but did not affect the ADG and ADFI compared with the negative control diet. The broken-line analysis indicated that the COS supplementation level for the maximal ADG of pigs during the 21-d feeding period was 158.8 mg/kg (Figure 1). Figure 1. View largeDownload slide Broken-line analysis of ADG of pigs given diets with different supplemental levels of chito-oligosaccharide (COS) during the entire study period of 21 d. The broken-line analysis indicated the breakpoint as 158.8 mg/kg, showing that the maximal ADG can be obtained by supplementation of 158.8 mg of COS/kg (P < 0.05, R2 = 0.37). Figure 1. View largeDownload slide Broken-line analysis of ADG of pigs given diets with different supplemental levels of chito-oligosaccharide (COS) during the entire study period of 21 d. The broken-line analysis indicated the breakpoint as 158.8 mg/kg, showing that the maximal ADG can be obtained by supplementation of 158.8 mg of COS/kg (P < 0.05, R2 = 0.37). Apparent Total Tract Digestibility The apparent digestibility of DM, GE, CP, CF, Ca, and P did not differ between the diet with COS at 200 mg/kg and the diet with CTC, but they were greater (P < 0.05) than those in the negative control group (Table 3). Dietary supplementation of COS at 100 mg/kg increased (P < 0.05) the digestibility of DM, Ca, and P compared with the negative control diet. Compared with the negative control diet, dietary supplementation of COS at 400 mg/kg increased (P < 0.05) the digestibility of Ca and P. There was no difference in the digestibility of Ca and P among groups with dietary supplementation of COS or CTC. Table 3. Effects of dietary chito-oligosaccharide (COS) supplementation on the apparent digestibility (%) of DM, GE, CP, crude fat (CF), Ca, and P for experimental piglets1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  GE  82.5c  83.5bc  84.8ab  83.3bc  85.4a  2.02  0.006  DM  81.4c  83.3ab  83.7ab  82.6bc  84.3a  1.94  0.006  CP  74.7b  76.4ab  77.4a  76.3ab  78.1a  2.64  0.043  Ca  52.0b  56.3a  57.6a  56.0a  56.4a  3.81  0.010  P  44.1b  46.1a  47.5a  46.0a  46.5a  2.77  0.022  CF  72.2c  72.8bc  73.6ab  72.9bc  74.2a  1.23  <0.001      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  GE  82.5c  83.5bc  84.8ab  83.3bc  85.4a  2.02  0.006  DM  81.4c  83.3ab  83.7ab  82.6bc  84.3a  1.94  0.006  CP  74.7b  76.4ab  77.4a  76.3ab  78.1a  2.64  0.043  Ca  52.0b  56.3a  57.6a  56.0a  56.4a  3.81  0.010  P  44.1b  46.1a  47.5a  46.0a  46.5a  2.77  0.022  CF  72.2c  72.8bc  73.6ab  72.9bc  74.2a  1.23  <0.001  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large Table 3. Effects of dietary chito-oligosaccharide (COS) supplementation on the apparent digestibility (%) of DM, GE, CP, crude fat (CF), Ca, and P for experimental piglets1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  GE  82.5c  83.5bc  84.8ab  83.3bc  85.4a  2.02  0.006  DM  81.4c  83.3ab  83.7ab  82.6bc  84.3a  1.94  0.006  CP  74.7b  76.4ab  77.4a  76.3ab  78.1a  2.64  0.043  Ca  52.0b  56.3a  57.6a  56.0a  56.4a  3.81  0.010  P  44.1b  46.1a  47.5a  46.0a  46.5a  2.77  0.022  CF  72.2c  72.8bc  73.6ab  72.9bc  74.2a  1.23  <0.001      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  GE  82.5c  83.5bc  84.8ab  83.3bc  85.4a  2.02  0.006  DM  81.4c  83.3ab  83.7ab  82.6bc  84.3a  1.94  0.006  CP  74.7b  76.4ab  77.4a  76.3ab  78.1a  2.64  0.043  Ca  52.0b  56.3a  57.6a  56.0a  56.4a  3.81  0.010  P  44.1b  46.1a  47.5a  46.0a  46.5a  2.77  0.022  CF  72.2c  72.8bc  73.6ab  72.9bc  74.2a  1.23  <0.001  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large Diarrhea Incidence and Diarrhea Score Diarrhea occurred mainly during the second week of the experiment (d 8 to 14). The occurrence of diarrhea lasted 3 to 5 d, and no pig was observed with diarrhea after d 13. The incidence of diarrhea and diarrhea score were decreased in pigs fed diets with COS (P < 0.05) compared with those fed the control diet (Table 4). There were no differences among the COS treatments and the CTC treatment in diarrhea incidence or diarrhea score. Table 4. Effects of dietary chito-oligosaccharide (COS) supplementation on diarrhea incidence and the fecal shedding of Escherichia coli and Lactobacillus (log cfu/g of wet digesta) in weaning pigs1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Lactobacillus      d 0  7.37  7.34  7.39  7.40  7.42  0.16  0.820      d 7  6.88  7.05  7.10  7.07  6.94  0.24  0.190      d 14  6.72b  7.07a  7.11a  7.06a  6.65b  0.40  0.009      d 21  6.62b  7.15a  7.22a  7.13a  6.66b  0.52  0.010  E. coli      d 0  3.72  3.74  3.77  3.75  3.78  0.14  0.891      d 7  3.87  3.68  3.65  3.69  3.55  0.28  0.162      d 14  3.95  3.56  3.53  3.58  3.38  0.46  0.082      d 21  3.87a  3.57ab  3.53b  3.62ab  3.38b  0.36  0.035  Diarrhea incidence,2 %  13.8a  6.7b  6.2b  9.0b  5.7b    <0.05  Diarrhea score2  19a  9b  8b  10b  8b    <0.05      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Lactobacillus      d 0  7.37  7.34  7.39  7.40  7.42  0.16  0.820      d 7  6.88  7.05  7.10  7.07  6.94  0.24  0.190      d 14  6.72b  7.07a  7.11a  7.06a  6.65b  0.40  0.009      d 21  6.62b  7.15a  7.22a  7.13a  6.66b  0.52  0.010  E. coli      d 0  3.72  3.74  3.77  3.75  3.78  0.14  0.891      d 7  3.87  3.68  3.65  3.69  3.55  0.28  0.162      d 14  3.95  3.56  3.53  3.58  3.38  0.46  0.082      d 21  3.87a  3.57ab  3.53b  3.62ab  3.38b  0.36  0.035  Diarrhea incidence,2 %  13.8a  6.7b  6.2b  9.0b  5.7b    <0.05  Diarrhea score2  19a  9b  8b  10b  8b    <0.05  a,b Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. 2 Means are different by a chi-square contingency test. View Large Table 4. Effects of dietary chito-oligosaccharide (COS) supplementation on diarrhea incidence and the fecal shedding of Escherichia coli and Lactobacillus (log cfu/g of wet digesta) in weaning pigs1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Lactobacillus      d 0  7.37  7.34  7.39  7.40  7.42  0.16  0.820      d 7  6.88  7.05  7.10  7.07  6.94  0.24  0.190      d 14  6.72b  7.07a  7.11a  7.06a  6.65b  0.40  0.009      d 21  6.62b  7.15a  7.22a  7.13a  6.66b  0.52  0.010  E. coli      d 0  3.72  3.74  3.77  3.75  3.78  0.14  0.891      d 7  3.87  3.68  3.65  3.69  3.55  0.28  0.162      d 14  3.95  3.56  3.53  3.58  3.38  0.46  0.082      d 21  3.87a  3.57ab  3.53b  3.62ab  3.38b  0.36  0.035  Diarrhea incidence,2 %  13.8a  6.7b  6.2b  9.0b  5.7b    <0.05  Diarrhea score2  19a  9b  8b  10b  8b    <0.05      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Lactobacillus      d 0  7.37  7.34  7.39  7.40  7.42  0.16  0.820      d 7  6.88  7.05  7.10  7.07  6.94  0.24  0.190      d 14  6.72b  7.07a  7.11a  7.06a  6.65b  0.40  0.009      d 21  6.62b  7.15a  7.22a  7.13a  6.66b  0.52  0.010  E. coli      d 0  3.72  3.74  3.77  3.75  3.78  0.14  0.891      d 7  3.87  3.68  3.65  3.69  3.55  0.28  0.162      d 14  3.95  3.56  3.53  3.58  3.38  0.46  0.082      d 21  3.87a  3.57ab  3.53b  3.62ab  3.38b  0.36  0.035  Diarrhea incidence,2 %  13.8a  6.7b  6.2b  9.0b  5.7b    <0.05  Diarrhea score2  19a  9b  8b  10b  8b    <0.05  a,b Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. 2 Means are different by a chi-square contingency test. View Large Fecal Shedding of E. coli and Lactobacillus Lactobacillus and E. coli in the feces of each treatment were determined at 4 different time points (d 0, 7, 14, and 21) during the study (Table 4). Lactobacillus counts on d 14 and 21 indicated that pigs fed diets supplemented with COS at 100, 200, and 400 mg/kg had greater (P < 0.05) Lactobacillus counts than did pigs in the negative control and CTC treatments. In addition, dietary supplementation of COS at 200 mg/kg and dietary supplementation of CTC decreased (P < 0.05) the counts of E. coli shed in the feces on d 21 compared with the negative control diet. No difference was detected in E. coli counts among groups with supplementation of COS at 100, 200, and 400 mg/kg or with supplementation of CTC at all 4 time points. Small Intestinal Morphology No effect of dietary supplementation of COS or CTC was observed in villus height, crypt depth, or the villus height:crypt ratio at the duodenum (Table 5). By contrast, dietary supplementation of COS at 200 mg/kg and dietary supplementation of CTC increased (P < 0.05) the villus height and villus:crypt ratio at the ileum and jejunum compared with those of the negative control group. Pigs fed the diet with COS at 100 mg/kg had greater (P < 0.05) villus heights at the ileum than did pigs in the negative control group. Table 5. Effects of chito-oligosaccharide (COS) supplementation on small intestinal morphology1     Levels of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Villus height, μm      Duodenum  407.2  412.5  414.1  411.7  416.1  8.07  0.142      Jejunum  406.2c  411.4abc  416.3ab  410.0bc  417.7a  8.70  0.013      μIleum  330.5c  343.9ab  350.9ab  339.8bc  353.4a  16.00  0.006  Crypt depth, μm      Duodenum  220.5  214.7  214.2  215.2  212.0  10.22  0.447      Jejunum  227.7  224.8  224.2  227.9  224.2  6.70  0.543      Ileum  204.0  203.7  201.4  205.1  200.2  7.66  0.614  Villus height:crypt depth      Duodenum  1.85  1.92  1.94  1.92  1.97  0.11  0.178      Jejunum  1.78b  1.83ab  1.86a  1.80b  1.86a  0.06  0.005      Ileum  1.62c  1.69abc  1.75ab  1.66bc  1.77a  0.12  0.023      Levels of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Villus height, μm      Duodenum  407.2  412.5  414.1  411.7  416.1  8.07  0.142      Jejunum  406.2c  411.4abc  416.3ab  410.0bc  417.7a  8.70  0.013      μIleum  330.5c  343.9ab  350.9ab  339.8bc  353.4a  16.00  0.006  Crypt depth, μm      Duodenum  220.5  214.7  214.2  215.2  212.0  10.22  0.447      Jejunum  227.7  224.8  224.2  227.9  224.2  6.70  0.543      Ileum  204.0  203.7  201.4  205.1  200.2  7.66  0.614  Villus height:crypt depth      Duodenum  1.85  1.92  1.94  1.92  1.97  0.11  0.178      Jejunum  1.78b  1.83ab  1.86a  1.80b  1.86a  0.06  0.005      Ileum  1.62c  1.69abc  1.75ab  1.66bc  1.77a  0.12  0.023  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large Table 5. Effects of chito-oligosaccharide (COS) supplementation on small intestinal morphology1     Levels of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Villus height, μm      Duodenum  407.2  412.5  414.1  411.7  416.1  8.07  0.142      Jejunum  406.2c  411.4abc  416.3ab  410.0bc  417.7a  8.70  0.013      μIleum  330.5c  343.9ab  350.9ab  339.8bc  353.4a  16.00  0.006  Crypt depth, μm      Duodenum  220.5  214.7  214.2  215.2  212.0  10.22  0.447      Jejunum  227.7  224.8  224.2  227.9  224.2  6.70  0.543      Ileum  204.0  203.7  201.4  205.1  200.2  7.66  0.614  Villus height:crypt depth      Duodenum  1.85  1.92  1.94  1.92  1.97  0.11  0.178      Jejunum  1.78b  1.83ab  1.86a  1.80b  1.86a  0.06  0.005      Ileum  1.62c  1.69abc  1.75ab  1.66bc  1.77a  0.12  0.023      Levels of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Villus height, μm      Duodenum  407.2  412.5  414.1  411.7  416.1  8.07  0.142      Jejunum  406.2c  411.4abc  416.3ab  410.0bc  417.7a  8.70  0.013      μIleum  330.5c  343.9ab  350.9ab  339.8bc  353.4a  16.00  0.006  Crypt depth, μm      Duodenum  220.5  214.7  214.2  215.2  212.0  10.22  0.447      Jejunum  227.7  224.8  224.2  227.9  224.2  6.70  0.543      Ileum  204.0  203.7  201.4  205.1  200.2  7.66  0.614  Villus height:crypt depth      Duodenum  1.85  1.92  1.94  1.92  1.97  0.11  0.178      Jejunum  1.78b  1.83ab  1.86a  1.80b  1.86a  0.06  0.005      Ileum  1.62c  1.69abc  1.75ab  1.66bc  1.77a  0.12  0.023  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large DISCUSSION There have been several attempts to demonstrate the use of certain types of oligosaccharides as a potential alternative antibiotic in enhancing animal growth and improving animal microbial flora (Patterson and Burkholder, 2003). Collins and Gibson (1999) provided an overall review of the benefits of oligosaccharides, including N-acetylglucosamine, oligofructose, lactulose, and certain glycoproteins, as prebiotics to improve gut health. Furthermore, several supplemental oligosaccharides, such as galacto-oligasaccharide and manno-oligosaccharide, have been shown to improve growth performance in young pigs (Davis et al., 2004; Miguel et al., 2004). However, there is still little knowledge of the possible benefits of COS. In fact, COS is the second most abundant carbohydrate polymer in nature (Knaul et al., 1999). In this study, COS was supplemented at 3 levels. These levels were selected based on results from a previous study, which showed that early-weaned pigs fed a diet containing COS at 250 mg/kg had increased plasma GH and IGF-I concentrations, which may improve growth and feed conversion efficiency (Tang et al., 2005). In this study, dietary supplementation of COS at 100 and 200 mg/kg as well as dietary supplementation of CTC at 80 mg/kg seemed to increase ADG, ADFI, and G:F during the entire 3-wk experimental period. The improved growth performance in this experiment was similar to that in our previous study (Li et al., 2007), in which COS supplementation improved the growth of broilers. Huang et al. (2005) also demonstrated that dietary COS supplementation improved the growth of broilers. However, in the current study pigs fed the diet supplemented with COS at 400 mg/kg increased only G:F without affecting ADG and ADFI, indicating a potential adverse effect of COS supplementation on growth performance at high levels. A previous study with broilers (Huang et al., 2005) also showed that growth performance did not improve with COS supplementation (dose-dependently), especially at high supplementation levels. It has been suggested that one possible reason for the improved growth performance with dietary COS supplementation is increased feed intake (Tang et al., 2005). Increased energy supply from increased feed intake can increase serum GH and IGF-I concentrations (Clemmons and Underwood, 1991; Steele et al., 1995), both of which can improve growth (Florini et al., 1996). In the current study, feed intake and total tract apparent digestibility of major nutrients were increased by dietary supplementation of COS at 200 mg/kg, which may have contributed to the increased ADG of pigs. Previous studies with broilers also showed that ADG could be improved, possibly by the increased feed intake and total tract apparent digestibility, when broilers were fed a diet with COS (Huang et al., 2005; Li et al., 2007). However, it is not known how dietary COS might increase feed consumption. Oligosaccharides are usually defined as prebiotics that can selectively stimulate the growth of health-promoting bacteria, with a beneficial impact on host health (Gibson and Roberfroid, 1995). Thus, Lactobacillus and Bifidobacterium are deemed target organisms because of their potential to inhibit the growth of putrefactive and pathogenic bacteria (Paton et al., 2006). In the current study, dietary COS supplementation successfully increased the population of Lactobacillus and decreased the counts of E. coli in fecal samples from pigs on d 14 and 21. In agreement with our findings, Li et al. (2007) also indicated that dietary COS supplementation improved the gastrointestinal Lactobacillus population, whereas it reduced the E. coli population. We also showed that dietary COS supplementation at 200 mg/kg and dietary CTC supplementation were more effective in reducing the fecal E. coli counts than were other treatments on d 21. Pluske et al. (1997) reported that a healthy gut environment (e.g., low counts of enteropathogenic E. coli) could affect the voluntary feed intake, which may have contributed to the increased growth rate with the COS treatment at 200 mg/kg and with the CTC treatment. All these shifts in the population of Lactobacillus and E. coli indicated that COS may act as a dietary prebiotic ingredient (Crittenden, 1999; Flickinger et al., 2000). It has been demonstrated that the efficacy of dietary prebiotic supplementation in the intestinal ecosystem can be related to the condition of the intestinal microflora and to body immunity (Hidaka et al., 1986; Howard et al., 1995; Orban et al., 1997), which may explain some of the reasons for conflicting results among research publications with dietary prebiotic supplementation (Orban et al., 1997; White et al., 2002; Mikkelsen et al., 2003). The exact mechanism through which COS may alter the type of intestinal bacteria remains uncertain. One possible explanation may be that COS serves as a growth promoter for lactic acid bacteria (Lee et al., 2002). In addition, COS has been shown to be effective in inhibiting the growth and activity of E. coli, even though those results were largely dependent on the molecular weight of COS used in the studies (Matsuhashi and Kume, 1997; Tsai et al., 2000). In the current study, the molecular weight of COS was between 103 and 104 Da, which has been shown to modulate the immune response effectively and reduce the establishment of pathogens in the gut (Rhoades et al., 2006). In the present study, pigs fed a control diet had a dramatic decline in Lactobacillus counts, an intensive augmentation of the E. coli population, and coincidently displayed a greater incidence of diarrhea with greater diarrhea scores than pigs supplemented with COS and CTC. Mathew et al. (1996) indicated that weaning may lead to a dramatic decline in Lactobacillus counts in the gut and induce invasion by other bacteria, such as enteropathogenic E. coli (Hampson et al., 1985; Madec et al., 1998), both of which directly contribute to diarrhea. The COS has been shown to be effective in reducing the adhesion of certain strains of enteropathogenic E. coli to intestinal cells but not to nontoxigenic, commensal flora (Rhoades et al., 2006); thus, the decrease in total E. coli counts may have been resulted from the decreased number of enteropathogenic E. coli. Therefore, one possible reason for the decreased diarrhea incidence and diarrhea score in the groups supplemented with COS may be the high concentration of Lactobacillus (Silva et al., 1987; Blomberg et al., 1993) and decreased population of E. coli in the intestinal tract (Oli et al., 1998; Li et al., 2007). The villus:crypt ratio is a useful criterion to estimate the nutrient digestion and absorption capacity of the small intestine (Montagne et al., 2003). Maximal digestion and absorption occurred as the villus:crypt ratio increased (Pluske et al., 1996) in newly weaned pigs. In this study, dietary supplementation with COS at 200 mg/kg or with CTC increased the villus height and villus:crypt ratio in the jejunum and ileum. These results were consistent with previous studies in broilers (Wang et al., 2003) and rats (He et al., 2006). Other types of oligosaccharides, such as mannan-oligosaccharide and fructooligosaccharide, have also been shown to exert positive effects on gut morphology in turkeys (Savage et al., 1996) and pigs (Spencer et al., 1997). However, the exact mechanism is still unknown. One possible explanation related to the increased villus heights with dietary COS supplementation could be N-acetylglucosamine, which is a common component of receptor-active oligosaccharides (Klemm and Schembri, 2000; Ofek et al., 2003). N-Acetylglucosamine is a basic component of COS (Kim and Rajapakse, 2005). Previous studies have shown that N-acetylglucosamine is a common component of many mammalian glycoconjugates, particularly of mucins (Podolsky, 1985), which can be used as receptors preventing a wide range of bacteria from binding to the gut tissue of host animals. Thus, the N-acetylglucosamine abundance in COS may cause binding of COS to certain types of bacteria (Klemm and Schembri, 2000; Ofek et al., 2003), possibly interfering with their colonization in the gut (Stanley et al., 2000; Rhoades et al., 2006). A decrease in intraluminal bacteria has been shown to improve the proliferation of epithelial cells to increase villi in the gut (Mourao et al., 2006), thereby leading to enhanced intestinal morphology. Thus, the increased villus heights with dietary COS supplementation may lead to enhanced nutrient digestibility in weaned pigs (Pluske et al., 1996). In conclusion, COS can be an effective alternative to the use of antibiotic growth promoters to increase growth through enhancing small intestinal structure, preventing diarrhea, and modifying fecal shedding of E. coli and Lactobacillus. On the basis of the current study, COS would be most effective in improving the growth of nursery pigs when supplemented in the diet at a concentration of 158.8 mg/kg. However, further study is needed to investigate the exact mechanism by which supplemented COS can affect the gut microflora and intestinal morphology of weaning pigs. LITERATURE CITED AOAC 1997. Official Methods of Analysis.  16th ed. 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Effects of chito-oligosaccharide supplementation on the growth performance, nutrient digestibility, intestinal morphology, and fecal shedding of Escherichia coli and Lactobacillus in weaning pigs

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Copyright 2008 Journal of Animal Science
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0021-8812
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1525-3163
DOI
10.2527/jas.2007-0668
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18502883
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Abstract

ABSTRACT A total of 50 weaning pigs (16 d of age; 4.72 ± 0.23 kg of BW) were selected to investigate the effect of dietary chito-oligosaccharide (COS) supplementation on growth performance, fecal shedding of Escherichia coli and Lactobacillus, apparent digestibility, and small intestinal morphology. Pigs housed in individual metabolic cages were assigned randomly to 5 treatments (n = 10), including 1 basal diet (control), 3 diets with COS supplementation (100, 200, and 400 mg/kg), and 1 diet with chlortetracycline (CTC) supplementation (80 mg/kg). Fresh fecal samples were collected to evaluate shedding of E. coli and Lactobacillus on d 0, 7, 14, and 21 postweaning. Fresh fecal samples collected from each cage from d 19 to 21 were stored frozen for determination of apparent total tract digestibility. On d 21, all pigs were killed to collect the middle sections of the duodenum, jejunum, and ileum for determination of mucosa morphology. Supplementation of COS at 100 and 200 mg/kg and supplementation of CTC improved (P < 0.05) overall ADG, ADFI, and G:F in comparison with the control. Supplementation of COS at 200 mg/kg as well as supplementation of CTC increased (P < 0.05) apparent total tract digestibility of DM, GE, CP, crude fat, Ca, and P, whereas COS at 100 mg/kg increased (P < 0.05) the digestibility of DM, Ca, and P in comparison with the control diet. Pigs receiving diets supplemented with COS or CTC had a decreased (P < 0.05) incidence of diarrhea and decreased diarrhea scores compared with control pigs. Fecal samples from pigs receiving diets supplemented with COS had greater (P < 0.05) Lactobacillus counts than those from control pigs and pigs receiving diets supplemented with CTC on d 14 and 21. However, supplementation of COS at 200 mg/kg and supplementation of CTC decreased (P < 0.05) E. coli counts in the feces on d 21 compared with the control diet. Dietary supplementation of COS at 200 mg/kg and of CTC increased (P < 0.05) the villus height and villus:crypt ratio at the ileum and jejunum, and COS at 100 mg/kg also increased (P < 0.05) the villus height in the ileum compared with the control diet. The current results indicated that dietary supplementation of COS at 100 and 200 mg/kg enhanced growth performance by increasing apparent digestibility, decreasing the incidence of diarrhea, and improving small intestinal morphology. INTRODUCTION Weaning is a critical stage for pigs because of alterations in the gastrointestinal tract architecture and function, as well as changes in adapting to enteric microbiota and immune responses (Pluske et al., 1997; Spreeuwenberg et al., 2001; Boudry et al., 2004; Mao et al., 2005). Pigs can be affected by weaning stress, such as nutritional, environmental, and social stresses, which can cause depressed growth performance, nutrient malabsorption, and a high incidence of diarrhea (Barnett et al., 1989; Hedemann and Jensen, 2004; Yuan et al., 2006). Antibiotics, as growth promoters and therapeutic medicines to decrease the susceptibility to infectious diseases, have been widely used in animal production for many years (Barton, 2000). However, issues with bacterial antibiotic resistance may cause problems for human health (Bach Knudsen, 2001; Smith et al., 2002). Many alternatives to the use of antibiotics have been suggested, introduced, and tested (Turner et al., 2001). Consumption of functional oligosaccharides has been shown to improve growth performance and enhance the host health status (Gibson and Roberfroid, 1995). Certain oligosaccharides, including galacto-oligosaccharide, manno-oligosaccharide, and fructo-oligasaccharide, may improve growth performance in young pigs (Davis et al., 2004; Miguel et al., 2004). Chito-oligosaccharide (COS) can be efficiently derived by chemical and enzymatic hydrolysis of poly-chitosan, which is the second most abundant carbohydrate polymer in nature (Knaul et al., 1999). More recently, COS has been shown to have immune-enhancing characteristics (Okamoto et al., 2003), antibacterial activity (Matsuhashi and Kume, 1997; Jeon et al., 2001), and protection against pathogenic infections (Rhoades et al., 2006). A previous study showed that a diet containing COS at 250 mg/kg increased serum GH, IGF-I, and protein synthesis, which in turn might have caused improved growth performance in weaned pigs (Tang et al., 2005). Huang et al. (2005) and Li et al. (2007) showed that dietary supplementation of COS at 100 mg/kg effectively increased nutrient digestibility and weight gain by improving gut health and modulating the balance of microbial flora in broilers. However, the effect of dietary COS supplementation on growth performance, intestinal structure, and microbial flora in weaned pigs is still largely unknown. Therefore, this experiment was conducted to test the effect of dietary COS supplementation on growth performance, intestinal structure, and fecal shedding of total Escherichia coli and Lactobacillus in weaning pigs. MATERIALS AND METHODS The animal care protocol was approved by the China Agricultural University Animal Care and Use Committee. Preparation and Composition of Chito-oligosaccharide Preparation and composition of the chito-oligosaccharide supplement (GlycoBio Company, Dalian, China) have been described previously (Li et al., 2007). Briefly, the different oligomers contained in the COS supplement and a standard sample prepared by mixing 6 chitosan oligomers (Sigma, St. Louis, MO) were separated and quantified by HPLC with an evaporative light-scattering detector (model 301; ESA, Chelmsford, MA) and column (4.6 × 250 mm, Asahipak NH2P-50-4E; Shodex, Tokyo, Japan). The results indicated that the COS supplement contained 40% COS and 60% cyclodextrin as a carrier. This COS is composed of 5 oligomers with an average molecular weight of 1,500 Da, which were identified as a combination of chitobiose, chitotriose, chitotetrose, chitopentose, and chitohexose, with concentrations of 0.58, 2.51, 4.49, 5.80, and 2.21 mg/mL, respectively, when 20 mg of COS sample was diluted with 1 mL of water. Water solubility of the COS supplement was greater than 99% (Li et al., 2007). Animals and Experimental Design Fifty newly weaned barrows (Large White × Landrace; initial BW = 4.72 ± 0.23 kg; 16 d of age) were selected from a commercial pig farm (Beijing, China) and transported to China Agricultural University. All pigs were housed in individual metabolism cages (0.7 × 1.7 m) in a temperature-controlled nursery (27 to 28°C) during the entire 3-wk experimental period. Pigs had access to feed and water ad libitum. Barrows were assigned to 5 treatments (n = 10) according to litter and initial BW, in a randomized complete block design. The 5 treatments were 1) a basal diet (negative control group), 2) 3 diets with COS supplementation (100, 200, and 400 mg/kg), and 3) a diet with chlortetracycline (CTC) supplementation (80 mg/kg; positive control group). The basal diet was formulated to meet the nutrient requirements suggested by NRC (1998) and contained no antibiotics or COS (Table 1). Chromium oxide (0.30%) was added to all diets as an indigestible marker. Table 1. Ingredient and chemical composition of the experimental diet, as-fed basis Item  Amount  Diet ingredient, %      Corn, yellow  67.16      Soybean meal, dehulled, 45% CP  22.65      Fish meal, 65% CP  3.00      Spray-dried porcine plasma  3.00      Dicalcium phosphate  1.58      Limestone  0.67      L-Lys·HCl, 98% Lys  0.29      DL-Met  0.04      Sweetener  0.02      Salt  0.30      Vitamin and mineral premix1  1.00      Chromic oxide  0.30  Analyzed nutritional content,2 %      CP  20.00      Ca  0.80      P  0.73      Lys  1.40      Met  0.40  Item  Amount  Diet ingredient, %      Corn, yellow  67.16      Soybean meal, dehulled, 45% CP  22.65      Fish meal, 65% CP  3.00      Spray-dried porcine plasma  3.00      Dicalcium phosphate  1.58      Limestone  0.67      L-Lys·HCl, 98% Lys  0.29      DL-Met  0.04      Sweetener  0.02      Salt  0.30      Vitamin and mineral premix1  1.00      Chromic oxide  0.30  Analyzed nutritional content,2 %      CP  20.00      Ca  0.80      P  0.73      Lys  1.40      Met  0.40  1 Vitamin and mineral premix provided the following per kilogram of diet: vitamin A, 12,000 IU as vitamin A acetate; vitamin D, 2,500 IU as vitamin D3; 30 IU of vitamin E; 12 μg of vitamin B12; vitamin K, 3 mg as menadione sodium bisulfate; D-pantothenic acid, 15 mg as calcium pantothenate; 40 mg of nicotinic acid; choline chloride, 400 mg as choline chloride; Mn, 40 mg as manganous oxide; Zn, 100 mg as zinc oxide; Fe, 90 mg as iron sulfate; Cu, 8.8 mg as copper oxide; I, 0.35 mg as ethylenediamine dihydroiodide; and Se, 0.3 mg as sodium selenite. 2 Calculated ME content: 3.20 Mcal/kg. View Large Table 1. Ingredient and chemical composition of the experimental diet, as-fed basis Item  Amount  Diet ingredient, %      Corn, yellow  67.16      Soybean meal, dehulled, 45% CP  22.65      Fish meal, 65% CP  3.00      Spray-dried porcine plasma  3.00      Dicalcium phosphate  1.58      Limestone  0.67      L-Lys·HCl, 98% Lys  0.29      DL-Met  0.04      Sweetener  0.02      Salt  0.30      Vitamin and mineral premix1  1.00      Chromic oxide  0.30  Analyzed nutritional content,2 %      CP  20.00      Ca  0.80      P  0.73      Lys  1.40      Met  0.40  Item  Amount  Diet ingredient, %      Corn, yellow  67.16      Soybean meal, dehulled, 45% CP  22.65      Fish meal, 65% CP  3.00      Spray-dried porcine plasma  3.00      Dicalcium phosphate  1.58      Limestone  0.67      L-Lys·HCl, 98% Lys  0.29      DL-Met  0.04      Sweetener  0.02      Salt  0.30      Vitamin and mineral premix1  1.00      Chromic oxide  0.30  Analyzed nutritional content,2 %      CP  20.00      Ca  0.80      P  0.73      Lys  1.40      Met  0.40  1 Vitamin and mineral premix provided the following per kilogram of diet: vitamin A, 12,000 IU as vitamin A acetate; vitamin D, 2,500 IU as vitamin D3; 30 IU of vitamin E; 12 μg of vitamin B12; vitamin K, 3 mg as menadione sodium bisulfate; D-pantothenic acid, 15 mg as calcium pantothenate; 40 mg of nicotinic acid; choline chloride, 400 mg as choline chloride; Mn, 40 mg as manganous oxide; Zn, 100 mg as zinc oxide; Fe, 90 mg as iron sulfate; Cu, 8.8 mg as copper oxide; I, 0.35 mg as ethylenediamine dihydroiodide; and Se, 0.3 mg as sodium selenite. 2 Calculated ME content: 3.20 Mcal/kg. View Large Body weight and feed consumption of individual pigs were determined weekly (on d 0, 7, 14, and 21) to calculate ADG, ADFI, and G:F. From 0600 to 0630 h in the morning of d 0, 7, 14, and 21, fecal samples were collected from the rectum with sterile rubber gloves and placed in sterile plastic tubes with lids. The samples were stored in a freezer at −20°C until analysis for counts of E. coli and Lactobacillus. On d 19, 20, and 21, approximately 50 g of fecal sample from each pig was collected immediately after defecation, placed in a sterile plastic bottle, and stored at −20°C until analysis for nutrient digestibility. On d 21, after an overnight fast, pigs were electronically stunned and killed, and tissue samples from the small intestine were collected immediately. To minimize potential errors associated with the timing of sample collection, the procedure was conducted simultaneously by 5 different groups of people, with each group in charge of 1 of the 5 treatments. The middle sections of the duodenum, jejunum, and ileum were aseptically isolated, flushed with a 0.9% salt solution, fixed with 10% formaldehyde-phosphate buffer, and kept at 4°C for microscopic assessment of mucosal morphology. Chemical Analyses Diet and fecal samples were dried in an oven (60°C; 24 h for diet and 48 h for feces) and then homogenized with a laboratory grinder (0.5-mm screen for diets and 1.0-mm screen for feces) before analysis. Dietary and fecal DM, CP, crude fat (CF), Ca, and P contents were analyzed according to AOAC (1997). Gross energy was measured by an automatic adiabatic oxygen bomb calorimeter (Parr Instrument Co., Moline, IL). Chromium content was measured with an atomic absorption spectrophotometer (Hitachi Z-5000; Hitachi Ltd., Tokyo, Japan) according to the procedure of Williams et al. (1962). Digestibility of DM, CP, CF, Ca, and P were determined by using the indicator method of Sauer and de Lange (1992) as follows: ADF = 1 − [(Cr2O3D × NF)/(Cr2O3F × ND)] × 100%, where ADF is the apparent total tract digestibility (%) of CP, CF, Ca, and P; Cr2O3D is the chromic oxide concentration in the assay diet (g/kg); NF is the concentration of a nutrient in the feces (g/kg); Cr2O3F is the concentration of chromic oxide in the feces (g/kg); and ND is the concentration of a nutrient in the assay diet (g/kg). Diarrhea Incidence and Diarrhea Score The incidence of diarrhea in piglets was observed and recorded 3 times per day during the study. To assess the severity of diarrhea, feces from each pig were scored by determining the moisture content according to the method of Hart and Dobb (1988). Scores were 0, normal, firm feces; 1, possible slight diarrhea; 2, definitely unformed, moderately fluid feces; or 3, very watery and frothy diarrhea. A cumulative diarrhea score per diet and day was then calculated (Montagne et al., 2004). The occurrence of diarrhea was defined as maintaining fecal scores of 2 and 3 for 2 consecutive days. Diarrhea incidence was calculated according to the formula reported by Huang et al. (2004) and Sun et al. (2007): diarrhea incidence (%) = number of pigs with diarrhea/(number of pigs × total experimental days) × 100, where “number of pigs with diarrhea” was the total number of pigs with diarrhea observed each day. Bacteriological Methods In vitro survival of Lactobacillus and E. coli was determined according to the methods of Guo et al. (2006) and Mikkelsen et al. (2003) with certain modifications. In brief, before enumeration, frozen fecal samples were incubated at 4°C for 10 h. Thereafter, 1 g of digesta was taken from each sample and serially diluted 10-fold with sterile physiological saline, resulting in dilutions ranging from 10−1 to 10−8 for enumeration. Escherichia coli was cultivated on MacConkey agar (Beijing Haidian Microbiological Culture Factory, Beijing, China). Lactobacillus was cultured in MRS agar (De Man, Rogosa, Sharpe agar; Oxoid Ltd., Hampshire, UK). Each dilution was determined in triplicate and the result was the average of 3 replicates. Lactobacillus was inoculated in Hungate roll tubes, and E. coli was fostered in plates. All tubes and plates were incubated at 37°C for 36 h. The microbial enumerations of digesta were expressed as log10 colony-forming units per gram. Bacteria were enumerated by a visual count of colonies by using the best replicate set from dilutions that resulted in 30 to 300 colonies per plate or tube. Small Intestinal Morphology Villus height and crypt depth were measured according to the method of Li et al. (1990) with some modifications. Briefly, fixed intestinal samples were prepared by using conventional paraffin embedding techniques. Samples were sectioned at a 6-μm thickness and stained with hematoxylin and eosin. Villus height and crypt depth were measured at 40× magnification with a microscope (Olympus CK40; Olympus Optical Company, Shenzhen, China). A minimum of 15 well-oriented, intact villi were selected to measure in triplicate for each pig. Statistical Analysis Data for growth performance, apparent total track digestibility, small intestinal morphology, and fecal shedding of E. coli and Lactobacillus were subjected to ANOVA as a randomized complete block design by using the GLM procedure (SAS Inst. Inc., Cary, NC). The pen was considered the experimental unit. Differences among treatment means were evaluated by using the PDIFF option of SAS. Results were presented as least squares means with SEM. Data for diarrhea incidence and diarrhea score were analyzed by using a chi-square contingency test (Cody and Smith, 1991; Owusu-Asiedu et al., 2003). Data were reported as a cumulative score for all pigs on each day. All nonparametric analyses were performed by using StatView (SAS Inst. Inc.). In addition, the ADG data were further analyzed by broken-line analysis to determine the optimal supplementation level of COS (Parr et al., 2003; Yuan et al., 2006). Probability values of less than 0.05 were used as the criterion for statistical significance. RESULTS Growth Performance During the first week (d 0 to 7), dietary supplementation of COS at 200 mg/kg and dietary supplementation of CTC improved (P < 0.05) ADG, ADFI, and G:F compared with the negative control diet (Table 2). No difference was detected for ADG, ADFI, and G:F between pigs fed diets with COS at 200 mg/kg and pigs fed diets with CTC. Supplementation of COS at 100 and 400 mg/kg did not increase ADG and ADFI but did increase (P < 0.05) G:F in comparison with the negative control diet. During the second week (d 7 to 14), the inclusion of COS at 100 and 200 mg/kg as well as the inclusion of CTC increased (P < 0.05) ADG and ADFI compared with the negative control diet. No differences in ADG, ADFI, and G:F were detected between the dietary supplementation of COS at 400 mg/kg and the negative control diet. There was no difference in ADG, ADFI, and G:F among pigs fed diets with COS at 100 and 200 mg/kg and pigs fed diets with CTC. Feed consumption of pigs fed diets with COS at 100 and 200 mg/kg COS and with CTC was greater (P < 0.05) than that of pigs fed the diet with COS at 400 mg/kg. During the third week (d 14 to 21), dietary supplementation of COS at 100 and 200 mg/kg and dietary supplementation of CTC increased (P < 0.05) ADG, ADFI, and G:F compared with the negative control diet. Dietary supplementation of COS at 400 mg/kg increased (P < 0.05) G:F compared with the negative control diet but did not affect ADG and ADFI. Pigs fed diets with COS at 100 and 200 mg/kg and the diet with CTC had greater (P < 0.05) ADFI than those fed the diet with COS at 400 mg/kg or the negative control diet. Table 2. Effects of dietary chito-oligosaccharide (COS) on the growth performance of weaning pigs1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  BW, kg      d 0  4.7  4.8  4.7  4.7  4.7  0.23  0.887      d 7  5.8  5.9  6.0  5.9  6.0  0.24  0.528      d 14  7.8b  8.1a  8.2a  8.0ab  8.1a  0.32  0.038      d 21  10.7b  11.2a  11.3a  11.0ab  11.3a  0.48  0.004  ADG, g      d 0 to 7  159c  168bc  175ab  165bc  180a  14  0.007      d 7 to 14  285c  307ab  320a  295bc  313a  21  0.002      d 14 to 21  411c  445ab  450a  428bc  452a  30  0.003      d 0 to 21  285c  307ab  315a  296bc  315a  19  <0.001  ADFI, g      d 0 to 7  236b  241ab  252a  236b  254a  17  0.029      d 7 to 14  442b  460a  466a  443b  467a  21  0.003      d 14 to 21  630b  661a  662a  636b  669a  29  0.002      d 0 to 21  436b  454a  460a  438b  463a  19  <0.001  G:F, g/g      d 0 to 7  0.67b  0.70a  0.69a  0.70a  0.71a  0.02  0.009      d 7 to 14  0.65a  0.67a  0.69a  0.67a  0.67a  0.02  0.002      d 14 to 21  0.65b  0.67a  0.69a  0.67a  0.68a  0.02  0.030      d 0 to 21  0.66b  0.68a  0.69a  0.68a  0.68a  0.02  <0.001      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  BW, kg      d 0  4.7  4.8  4.7  4.7  4.7  0.23  0.887      d 7  5.8  5.9  6.0  5.9  6.0  0.24  0.528      d 14  7.8b  8.1a  8.2a  8.0ab  8.1a  0.32  0.038      d 21  10.7b  11.2a  11.3a  11.0ab  11.3a  0.48  0.004  ADG, g      d 0 to 7  159c  168bc  175ab  165bc  180a  14  0.007      d 7 to 14  285c  307ab  320a  295bc  313a  21  0.002      d 14 to 21  411c  445ab  450a  428bc  452a  30  0.003      d 0 to 21  285c  307ab  315a  296bc  315a  19  <0.001  ADFI, g      d 0 to 7  236b  241ab  252a  236b  254a  17  0.029      d 7 to 14  442b  460a  466a  443b  467a  21  0.003      d 14 to 21  630b  661a  662a  636b  669a  29  0.002      d 0 to 21  436b  454a  460a  438b  463a  19  <0.001  G:F, g/g      d 0 to 7  0.67b  0.70a  0.69a  0.70a  0.71a  0.02  0.009      d 7 to 14  0.65a  0.67a  0.69a  0.67a  0.67a  0.02  0.002      d 14 to 21  0.65b  0.67a  0.69a  0.67a  0.68a  0.02  0.030      d 0 to 21  0.66b  0.68a  0.69a  0.68a  0.68a  0.02  <0.001  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs. NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large Table 2. Effects of dietary chito-oligosaccharide (COS) on the growth performance of weaning pigs1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  BW, kg      d 0  4.7  4.8  4.7  4.7  4.7  0.23  0.887      d 7  5.8  5.9  6.0  5.9  6.0  0.24  0.528      d 14  7.8b  8.1a  8.2a  8.0ab  8.1a  0.32  0.038      d 21  10.7b  11.2a  11.3a  11.0ab  11.3a  0.48  0.004  ADG, g      d 0 to 7  159c  168bc  175ab  165bc  180a  14  0.007      d 7 to 14  285c  307ab  320a  295bc  313a  21  0.002      d 14 to 21  411c  445ab  450a  428bc  452a  30  0.003      d 0 to 21  285c  307ab  315a  296bc  315a  19  <0.001  ADFI, g      d 0 to 7  236b  241ab  252a  236b  254a  17  0.029      d 7 to 14  442b  460a  466a  443b  467a  21  0.003      d 14 to 21  630b  661a  662a  636b  669a  29  0.002      d 0 to 21  436b  454a  460a  438b  463a  19  <0.001  G:F, g/g      d 0 to 7  0.67b  0.70a  0.69a  0.70a  0.71a  0.02  0.009      d 7 to 14  0.65a  0.67a  0.69a  0.67a  0.67a  0.02  0.002      d 14 to 21  0.65b  0.67a  0.69a  0.67a  0.68a  0.02  0.030      d 0 to 21  0.66b  0.68a  0.69a  0.68a  0.68a  0.02  <0.001      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  BW, kg      d 0  4.7  4.8  4.7  4.7  4.7  0.23  0.887      d 7  5.8  5.9  6.0  5.9  6.0  0.24  0.528      d 14  7.8b  8.1a  8.2a  8.0ab  8.1a  0.32  0.038      d 21  10.7b  11.2a  11.3a  11.0ab  11.3a  0.48  0.004  ADG, g      d 0 to 7  159c  168bc  175ab  165bc  180a  14  0.007      d 7 to 14  285c  307ab  320a  295bc  313a  21  0.002      d 14 to 21  411c  445ab  450a  428bc  452a  30  0.003      d 0 to 21  285c  307ab  315a  296bc  315a  19  <0.001  ADFI, g      d 0 to 7  236b  241ab  252a  236b  254a  17  0.029      d 7 to 14  442b  460a  466a  443b  467a  21  0.003      d 14 to 21  630b  661a  662a  636b  669a  29  0.002      d 0 to 21  436b  454a  460a  438b  463a  19  <0.001  G:F, g/g      d 0 to 7  0.67b  0.70a  0.69a  0.70a  0.71a  0.02  0.009      d 7 to 14  0.65a  0.67a  0.69a  0.67a  0.67a  0.02  0.002      d 14 to 21  0.65b  0.67a  0.69a  0.67a  0.68a  0.02  0.030      d 0 to 21  0.66b  0.68a  0.69a  0.68a  0.68a  0.02  <0.001  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs. NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large Over the entire experimental period, dietary supplementation of COS at 100 and 200 mg/kg and dietary supplementation of CTC increased (P < 0.05) ADG by 7.7, 10.5, and 10.5%, respectively, compared with the negative control diet. The ADFI of pigs fed diets with COS at 100 and 200 mg/kg and the diet with CTC were 3.9, 5.2, and 5.8% greater (P < 0.05), respectively, than that of the negative control group. The G:F of pigs fed diets with COS at 100 and 200 mg/kg and pigs fed diets with CTC were 3.3, 4.4, and 3.9% greater (P < 0.05), respectively, than that of pigs fed the negative control diet. The provision of COS at 400 mg/kg increased (P < 0.05) G:F by 3.4% but did not affect the ADG and ADFI compared with the negative control diet. The broken-line analysis indicated that the COS supplementation level for the maximal ADG of pigs during the 21-d feeding period was 158.8 mg/kg (Figure 1). Figure 1. View largeDownload slide Broken-line analysis of ADG of pigs given diets with different supplemental levels of chito-oligosaccharide (COS) during the entire study period of 21 d. The broken-line analysis indicated the breakpoint as 158.8 mg/kg, showing that the maximal ADG can be obtained by supplementation of 158.8 mg of COS/kg (P < 0.05, R2 = 0.37). Figure 1. View largeDownload slide Broken-line analysis of ADG of pigs given diets with different supplemental levels of chito-oligosaccharide (COS) during the entire study period of 21 d. The broken-line analysis indicated the breakpoint as 158.8 mg/kg, showing that the maximal ADG can be obtained by supplementation of 158.8 mg of COS/kg (P < 0.05, R2 = 0.37). Apparent Total Tract Digestibility The apparent digestibility of DM, GE, CP, CF, Ca, and P did not differ between the diet with COS at 200 mg/kg and the diet with CTC, but they were greater (P < 0.05) than those in the negative control group (Table 3). Dietary supplementation of COS at 100 mg/kg increased (P < 0.05) the digestibility of DM, Ca, and P compared with the negative control diet. Compared with the negative control diet, dietary supplementation of COS at 400 mg/kg increased (P < 0.05) the digestibility of Ca and P. There was no difference in the digestibility of Ca and P among groups with dietary supplementation of COS or CTC. Table 3. Effects of dietary chito-oligosaccharide (COS) supplementation on the apparent digestibility (%) of DM, GE, CP, crude fat (CF), Ca, and P for experimental piglets1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  GE  82.5c  83.5bc  84.8ab  83.3bc  85.4a  2.02  0.006  DM  81.4c  83.3ab  83.7ab  82.6bc  84.3a  1.94  0.006  CP  74.7b  76.4ab  77.4a  76.3ab  78.1a  2.64  0.043  Ca  52.0b  56.3a  57.6a  56.0a  56.4a  3.81  0.010  P  44.1b  46.1a  47.5a  46.0a  46.5a  2.77  0.022  CF  72.2c  72.8bc  73.6ab  72.9bc  74.2a  1.23  <0.001      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  GE  82.5c  83.5bc  84.8ab  83.3bc  85.4a  2.02  0.006  DM  81.4c  83.3ab  83.7ab  82.6bc  84.3a  1.94  0.006  CP  74.7b  76.4ab  77.4a  76.3ab  78.1a  2.64  0.043  Ca  52.0b  56.3a  57.6a  56.0a  56.4a  3.81  0.010  P  44.1b  46.1a  47.5a  46.0a  46.5a  2.77  0.022  CF  72.2c  72.8bc  73.6ab  72.9bc  74.2a  1.23  <0.001  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large Table 3. Effects of dietary chito-oligosaccharide (COS) supplementation on the apparent digestibility (%) of DM, GE, CP, crude fat (CF), Ca, and P for experimental piglets1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  GE  82.5c  83.5bc  84.8ab  83.3bc  85.4a  2.02  0.006  DM  81.4c  83.3ab  83.7ab  82.6bc  84.3a  1.94  0.006  CP  74.7b  76.4ab  77.4a  76.3ab  78.1a  2.64  0.043  Ca  52.0b  56.3a  57.6a  56.0a  56.4a  3.81  0.010  P  44.1b  46.1a  47.5a  46.0a  46.5a  2.77  0.022  CF  72.2c  72.8bc  73.6ab  72.9bc  74.2a  1.23  <0.001      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  GE  82.5c  83.5bc  84.8ab  83.3bc  85.4a  2.02  0.006  DM  81.4c  83.3ab  83.7ab  82.6bc  84.3a  1.94  0.006  CP  74.7b  76.4ab  77.4a  76.3ab  78.1a  2.64  0.043  Ca  52.0b  56.3a  57.6a  56.0a  56.4a  3.81  0.010  P  44.1b  46.1a  47.5a  46.0a  46.5a  2.77  0.022  CF  72.2c  72.8bc  73.6ab  72.9bc  74.2a  1.23  <0.001  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large Diarrhea Incidence and Diarrhea Score Diarrhea occurred mainly during the second week of the experiment (d 8 to 14). The occurrence of diarrhea lasted 3 to 5 d, and no pig was observed with diarrhea after d 13. The incidence of diarrhea and diarrhea score were decreased in pigs fed diets with COS (P < 0.05) compared with those fed the control diet (Table 4). There were no differences among the COS treatments and the CTC treatment in diarrhea incidence or diarrhea score. Table 4. Effects of dietary chito-oligosaccharide (COS) supplementation on diarrhea incidence and the fecal shedding of Escherichia coli and Lactobacillus (log cfu/g of wet digesta) in weaning pigs1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Lactobacillus      d 0  7.37  7.34  7.39  7.40  7.42  0.16  0.820      d 7  6.88  7.05  7.10  7.07  6.94  0.24  0.190      d 14  6.72b  7.07a  7.11a  7.06a  6.65b  0.40  0.009      d 21  6.62b  7.15a  7.22a  7.13a  6.66b  0.52  0.010  E. coli      d 0  3.72  3.74  3.77  3.75  3.78  0.14  0.891      d 7  3.87  3.68  3.65  3.69  3.55  0.28  0.162      d 14  3.95  3.56  3.53  3.58  3.38  0.46  0.082      d 21  3.87a  3.57ab  3.53b  3.62ab  3.38b  0.36  0.035  Diarrhea incidence,2 %  13.8a  6.7b  6.2b  9.0b  5.7b    <0.05  Diarrhea score2  19a  9b  8b  10b  8b    <0.05      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Lactobacillus      d 0  7.37  7.34  7.39  7.40  7.42  0.16  0.820      d 7  6.88  7.05  7.10  7.07  6.94  0.24  0.190      d 14  6.72b  7.07a  7.11a  7.06a  6.65b  0.40  0.009      d 21  6.62b  7.15a  7.22a  7.13a  6.66b  0.52  0.010  E. coli      d 0  3.72  3.74  3.77  3.75  3.78  0.14  0.891      d 7  3.87  3.68  3.65  3.69  3.55  0.28  0.162      d 14  3.95  3.56  3.53  3.58  3.38  0.46  0.082      d 21  3.87a  3.57ab  3.53b  3.62ab  3.38b  0.36  0.035  Diarrhea incidence,2 %  13.8a  6.7b  6.2b  9.0b  5.7b    <0.05  Diarrhea score2  19a  9b  8b  10b  8b    <0.05  a,b Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. 2 Means are different by a chi-square contingency test. View Large Table 4. Effects of dietary chito-oligosaccharide (COS) supplementation on diarrhea incidence and the fecal shedding of Escherichia coli and Lactobacillus (log cfu/g of wet digesta) in weaning pigs1     Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Lactobacillus      d 0  7.37  7.34  7.39  7.40  7.42  0.16  0.820      d 7  6.88  7.05  7.10  7.07  6.94  0.24  0.190      d 14  6.72b  7.07a  7.11a  7.06a  6.65b  0.40  0.009      d 21  6.62b  7.15a  7.22a  7.13a  6.66b  0.52  0.010  E. coli      d 0  3.72  3.74  3.77  3.75  3.78  0.14  0.891      d 7  3.87  3.68  3.65  3.69  3.55  0.28  0.162      d 14  3.95  3.56  3.53  3.58  3.38  0.46  0.082      d 21  3.87a  3.57ab  3.53b  3.62ab  3.38b  0.36  0.035  Diarrhea incidence,2 %  13.8a  6.7b  6.2b  9.0b  5.7b    <0.05  Diarrhea score2  19a  9b  8b  10b  8b    <0.05      Level of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Lactobacillus      d 0  7.37  7.34  7.39  7.40  7.42  0.16  0.820      d 7  6.88  7.05  7.10  7.07  6.94  0.24  0.190      d 14  6.72b  7.07a  7.11a  7.06a  6.65b  0.40  0.009      d 21  6.62b  7.15a  7.22a  7.13a  6.66b  0.52  0.010  E. coli      d 0  3.72  3.74  3.77  3.75  3.78  0.14  0.891      d 7  3.87  3.68  3.65  3.69  3.55  0.28  0.162      d 14  3.95  3.56  3.53  3.58  3.38  0.46  0.082      d 21  3.87a  3.57ab  3.53b  3.62ab  3.38b  0.36  0.035  Diarrhea incidence,2 %  13.8a  6.7b  6.2b  9.0b  5.7b    <0.05  Diarrhea score2  19a  9b  8b  10b  8b    <0.05  a,b Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. 2 Means are different by a chi-square contingency test. View Large Fecal Shedding of E. coli and Lactobacillus Lactobacillus and E. coli in the feces of each treatment were determined at 4 different time points (d 0, 7, 14, and 21) during the study (Table 4). Lactobacillus counts on d 14 and 21 indicated that pigs fed diets supplemented with COS at 100, 200, and 400 mg/kg had greater (P < 0.05) Lactobacillus counts than did pigs in the negative control and CTC treatments. In addition, dietary supplementation of COS at 200 mg/kg and dietary supplementation of CTC decreased (P < 0.05) the counts of E. coli shed in the feces on d 21 compared with the negative control diet. No difference was detected in E. coli counts among groups with supplementation of COS at 100, 200, and 400 mg/kg or with supplementation of CTC at all 4 time points. Small Intestinal Morphology No effect of dietary supplementation of COS or CTC was observed in villus height, crypt depth, or the villus height:crypt ratio at the duodenum (Table 5). By contrast, dietary supplementation of COS at 200 mg/kg and dietary supplementation of CTC increased (P < 0.05) the villus height and villus:crypt ratio at the ileum and jejunum compared with those of the negative control group. Pigs fed the diet with COS at 100 mg/kg had greater (P < 0.05) villus heights at the ileum than did pigs in the negative control group. Table 5. Effects of chito-oligosaccharide (COS) supplementation on small intestinal morphology1     Levels of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Villus height, μm      Duodenum  407.2  412.5  414.1  411.7  416.1  8.07  0.142      Jejunum  406.2c  411.4abc  416.3ab  410.0bc  417.7a  8.70  0.013      μIleum  330.5c  343.9ab  350.9ab  339.8bc  353.4a  16.00  0.006  Crypt depth, μm      Duodenum  220.5  214.7  214.2  215.2  212.0  10.22  0.447      Jejunum  227.7  224.8  224.2  227.9  224.2  6.70  0.543      Ileum  204.0  203.7  201.4  205.1  200.2  7.66  0.614  Villus height:crypt depth      Duodenum  1.85  1.92  1.94  1.92  1.97  0.11  0.178      Jejunum  1.78b  1.83ab  1.86a  1.80b  1.86a  0.06  0.005      Ileum  1.62c  1.69abc  1.75ab  1.66bc  1.77a  0.12  0.023      Levels of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Villus height, μm      Duodenum  407.2  412.5  414.1  411.7  416.1  8.07  0.142      Jejunum  406.2c  411.4abc  416.3ab  410.0bc  417.7a  8.70  0.013      μIleum  330.5c  343.9ab  350.9ab  339.8bc  353.4a  16.00  0.006  Crypt depth, μm      Duodenum  220.5  214.7  214.2  215.2  212.0  10.22  0.447      Jejunum  227.7  224.8  224.2  227.9  224.2  6.70  0.543      Ileum  204.0  203.7  201.4  205.1  200.2  7.66  0.614  Villus height:crypt depth      Duodenum  1.85  1.92  1.94  1.92  1.97  0.11  0.178      Jejunum  1.78b  1.83ab  1.86a  1.80b  1.86a  0.06  0.005      Ileum  1.62c  1.69abc  1.75ab  1.66bc  1.77a  0.12  0.023  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large Table 5. Effects of chito-oligosaccharide (COS) supplementation on small intestinal morphology1     Levels of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Villus height, μm      Duodenum  407.2  412.5  414.1  411.7  416.1  8.07  0.142      Jejunum  406.2c  411.4abc  416.3ab  410.0bc  417.7a  8.70  0.013      μIleum  330.5c  343.9ab  350.9ab  339.8bc  353.4a  16.00  0.006  Crypt depth, μm      Duodenum  220.5  214.7  214.2  215.2  212.0  10.22  0.447      Jejunum  227.7  224.8  224.2  227.9  224.2  6.70  0.543      Ileum  204.0  203.7  201.4  205.1  200.2  7.66  0.614  Villus height:crypt depth      Duodenum  1.85  1.92  1.94  1.92  1.97  0.11  0.178      Jejunum  1.78b  1.83ab  1.86a  1.80b  1.86a  0.06  0.005      Ileum  1.62c  1.69abc  1.75ab  1.66bc  1.77a  0.12  0.023      Levels of dietary COS, mg/kg        Item  NC  100  200  400  PC  SEM  P-value  Villus height, μm      Duodenum  407.2  412.5  414.1  411.7  416.1  8.07  0.142      Jejunum  406.2c  411.4abc  416.3ab  410.0bc  417.7a  8.70  0.013      μIleum  330.5c  343.9ab  350.9ab  339.8bc  353.4a  16.00  0.006  Crypt depth, μm      Duodenum  220.5  214.7  214.2  215.2  212.0  10.22  0.447      Jejunum  227.7  224.8  224.2  227.9  224.2  6.70  0.543      Ileum  204.0  203.7  201.4  205.1  200.2  7.66  0.614  Villus height:crypt depth      Duodenum  1.85  1.92  1.94  1.92  1.97  0.11  0.178      Jejunum  1.78b  1.83ab  1.86a  1.80b  1.86a  0.06  0.005      Ileum  1.62c  1.69abc  1.75ab  1.66bc  1.77a  0.12  0.023  a–c Means in the same row with different superscripts differ (P < 0.05). 1 Values are means of 10 individually caged pigs (3 replicates per pig). NC = pigs in the negative control group were fed a basal diet; PC = pigs in the positive control group were fed a basal diet supplemented with 80 mg/kg of chlortetracycline. View Large DISCUSSION There have been several attempts to demonstrate the use of certain types of oligosaccharides as a potential alternative antibiotic in enhancing animal growth and improving animal microbial flora (Patterson and Burkholder, 2003). Collins and Gibson (1999) provided an overall review of the benefits of oligosaccharides, including N-acetylglucosamine, oligofructose, lactulose, and certain glycoproteins, as prebiotics to improve gut health. Furthermore, several supplemental oligosaccharides, such as galacto-oligasaccharide and manno-oligosaccharide, have been shown to improve growth performance in young pigs (Davis et al., 2004; Miguel et al., 2004). However, there is still little knowledge of the possible benefits of COS. In fact, COS is the second most abundant carbohydrate polymer in nature (Knaul et al., 1999). In this study, COS was supplemented at 3 levels. These levels were selected based on results from a previous study, which showed that early-weaned pigs fed a diet containing COS at 250 mg/kg had increased plasma GH and IGF-I concentrations, which may improve growth and feed conversion efficiency (Tang et al., 2005). In this study, dietary supplementation of COS at 100 and 200 mg/kg as well as dietary supplementation of CTC at 80 mg/kg seemed to increase ADG, ADFI, and G:F during the entire 3-wk experimental period. The improved growth performance in this experiment was similar to that in our previous study (Li et al., 2007), in which COS supplementation improved the growth of broilers. Huang et al. (2005) also demonstrated that dietary COS supplementation improved the growth of broilers. However, in the current study pigs fed the diet supplemented with COS at 400 mg/kg increased only G:F without affecting ADG and ADFI, indicating a potential adverse effect of COS supplementation on growth performance at high levels. A previous study with broilers (Huang et al., 2005) also showed that growth performance did not improve with COS supplementation (dose-dependently), especially at high supplementation levels. It has been suggested that one possible reason for the improved growth performance with dietary COS supplementation is increased feed intake (Tang et al., 2005). Increased energy supply from increased feed intake can increase serum GH and IGF-I concentrations (Clemmons and Underwood, 1991; Steele et al., 1995), both of which can improve growth (Florini et al., 1996). In the current study, feed intake and total tract apparent digestibility of major nutrients were increased by dietary supplementation of COS at 200 mg/kg, which may have contributed to the increased ADG of pigs. Previous studies with broilers also showed that ADG could be improved, possibly by the increased feed intake and total tract apparent digestibility, when broilers were fed a diet with COS (Huang et al., 2005; Li et al., 2007). However, it is not known how dietary COS might increase feed consumption. Oligosaccharides are usually defined as prebiotics that can selectively stimulate the growth of health-promoting bacteria, with a beneficial impact on host health (Gibson and Roberfroid, 1995). Thus, Lactobacillus and Bifidobacterium are deemed target organisms because of their potential to inhibit the growth of putrefactive and pathogenic bacteria (Paton et al., 2006). In the current study, dietary COS supplementation successfully increased the population of Lactobacillus and decreased the counts of E. coli in fecal samples from pigs on d 14 and 21. In agreement with our findings, Li et al. (2007) also indicated that dietary COS supplementation improved the gastrointestinal Lactobacillus population, whereas it reduced the E. coli population. We also showed that dietary COS supplementation at 200 mg/kg and dietary CTC supplementation were more effective in reducing the fecal E. coli counts than were other treatments on d 21. Pluske et al. (1997) reported that a healthy gut environment (e.g., low counts of enteropathogenic E. coli) could affect the voluntary feed intake, which may have contributed to the increased growth rate with the COS treatment at 200 mg/kg and with the CTC treatment. All these shifts in the population of Lactobacillus and E. coli indicated that COS may act as a dietary prebiotic ingredient (Crittenden, 1999; Flickinger et al., 2000). It has been demonstrated that the efficacy of dietary prebiotic supplementation in the intestinal ecosystem can be related to the condition of the intestinal microflora and to body immunity (Hidaka et al., 1986; Howard et al., 1995; Orban et al., 1997), which may explain some of the reasons for conflicting results among research publications with dietary prebiotic supplementation (Orban et al., 1997; White et al., 2002; Mikkelsen et al., 2003). The exact mechanism through which COS may alter the type of intestinal bacteria remains uncertain. One possible explanation may be that COS serves as a growth promoter for lactic acid bacteria (Lee et al., 2002). In addition, COS has been shown to be effective in inhibiting the growth and activity of E. coli, even though those results were largely dependent on the molecular weight of COS used in the studies (Matsuhashi and Kume, 1997; Tsai et al., 2000). In the current study, the molecular weight of COS was between 103 and 104 Da, which has been shown to modulate the immune response effectively and reduce the establishment of pathogens in the gut (Rhoades et al., 2006). In the present study, pigs fed a control diet had a dramatic decline in Lactobacillus counts, an intensive augmentation of the E. coli population, and coincidently displayed a greater incidence of diarrhea with greater diarrhea scores than pigs supplemented with COS and CTC. Mathew et al. (1996) indicated that weaning may lead to a dramatic decline in Lactobacillus counts in the gut and induce invasion by other bacteria, such as enteropathogenic E. coli (Hampson et al., 1985; Madec et al., 1998), both of which directly contribute to diarrhea. The COS has been shown to be effective in reducing the adhesion of certain strains of enteropathogenic E. coli to intestinal cells but not to nontoxigenic, commensal flora (Rhoades et al., 2006); thus, the decrease in total E. coli counts may have been resulted from the decreased number of enteropathogenic E. coli. Therefore, one possible reason for the decreased diarrhea incidence and diarrhea score in the groups supplemented with COS may be the high concentration of Lactobacillus (Silva et al., 1987; Blomberg et al., 1993) and decreased population of E. coli in the intestinal tract (Oli et al., 1998; Li et al., 2007). The villus:crypt ratio is a useful criterion to estimate the nutrient digestion and absorption capacity of the small intestine (Montagne et al., 2003). Maximal digestion and absorption occurred as the villus:crypt ratio increased (Pluske et al., 1996) in newly weaned pigs. In this study, dietary supplementation with COS at 200 mg/kg or with CTC increased the villus height and villus:crypt ratio in the jejunum and ileum. These results were consistent with previous studies in broilers (Wang et al., 2003) and rats (He et al., 2006). Other types of oligosaccharides, such as mannan-oligosaccharide and fructooligosaccharide, have also been shown to exert positive effects on gut morphology in turkeys (Savage et al., 1996) and pigs (Spencer et al., 1997). However, the exact mechanism is still unknown. One possible explanation related to the increased villus heights with dietary COS supplementation could be N-acetylglucosamine, which is a common component of receptor-active oligosaccharides (Klemm and Schembri, 2000; Ofek et al., 2003). N-Acetylglucosamine is a basic component of COS (Kim and Rajapakse, 2005). Previous studies have shown that N-acetylglucosamine is a common component of many mammalian glycoconjugates, particularly of mucins (Podolsky, 1985), which can be used as receptors preventing a wide range of bacteria from binding to the gut tissue of host animals. Thus, the N-acetylglucosamine abundance in COS may cause binding of COS to certain types of bacteria (Klemm and Schembri, 2000; Ofek et al., 2003), possibly interfering with their colonization in the gut (Stanley et al., 2000; Rhoades et al., 2006). A decrease in intraluminal bacteria has been shown to improve the proliferation of epithelial cells to increase villi in the gut (Mourao et al., 2006), thereby leading to enhanced intestinal morphology. Thus, the increased villus heights with dietary COS supplementation may lead to enhanced nutrient digestibility in weaned pigs (Pluske et al., 1996). In conclusion, COS can be an effective alternative to the use of antibiotic growth promoters to increase growth through enhancing small intestinal structure, preventing diarrhea, and modifying fecal shedding of E. coli and Lactobacillus. On the basis of the current study, COS would be most effective in improving the growth of nursery pigs when supplemented in the diet at a concentration of 158.8 mg/kg. However, further study is needed to investigate the exact mechanism by which supplemented COS can affect the gut microflora and intestinal morphology of weaning pigs. LITERATURE CITED AOAC 1997. Official Methods of Analysis.  16th ed. 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