Influence of acidified drinking water on growth performance and gastrointestinal function of broilers

Influence of acidified drinking water on growth performance and gastrointestinal function of... ABSTRACT The ban on the use of antibiotic feed additives as growth promoters compelled the researchers for exploring the future utility of other alternatives. This experiment was designed to evaluate the effect of acidified drinking water on growth performance, gastrointestinal pH, digestive enzymes, intestinal histomorphology, and cecum microbial counting of the broiler chicken. A total of 540 one-day-old male broilers (Arbor Acre) were randomly assigned to 5 treatments, with 6 replicates of 18 chicks per replicate. Broilers received diets and water as follows: NC (negative control, basal diet, normal water), PC (positive control, basal diet + 8 ppm colistin sulfate + 8 ppm enduracidin, normal water), A1 (basal diet, continuous supply of acidified water during whole experiment period), A2 (basal diet, intermittent acidification of water during 0 to 14 d, 22 to 28 d, and 36 to 42 d), and A3 [basal diet, intermittent acidification of water (24 h/d from 0 to 14 d and from 10:00 am to 4:00 pm on d 15 to 42)]. During the entire period, the acidified groups (A1, A2, and A3) and PC group showed improve on weight gain, average daily gain and feed conversion ratio compared to NC group (P < 0.05). The pH in crop, proventriculus and ileum at 43 d declined by 0.04, 1.03, 1.23; 0.55, 0.69, 0.70; and 0.63, 0.74, 1.21 in A1, A2, and A3 group, respectively. There was a significant decline of lipase activity in the PC and acidified groups compared to NC group. The A2 group had higher villus height in jejunum than NC group. The PC and acidified groups reduced (P < 0.05) the total aerobic bacteria count of cecum when contrasted to NC group. Therefore, we conclude that acidified drinking water can improve growth performance, compensate for gastric acidity, and control pathogenic bacteria in broilers and may be considered as a potential alternative to improve production parameters. Discontinuous supply of acidified water had the same or even better influence on broilers compared to continuous supply. INTRODUCTION The ban on the use of antibiotic feed additives as growth promoters increased problems in poultry performance, increased feed conversion ratio (FCR), and augmented the incidence of certain infectious diseases (Dibner and Richards, 2005). This scenario has urged researchers to investigate the ability of alternatives in poultry production. The antimicrobial properties of organic acids, for instance, lowering the pH of drinking water and buffering capacity of the feed have a beneficial effect on the physiology of the crop and proventriculus (Van et al., 2006). Dietary supplementation of several organic acids has been found to promote growth performance, mineral absorption, feed efficiency, and phytate-P utilization (Boling-Frankenbach et al., 2001). Other significant benefits related to dietary acidification are as follows: improved exocrine pancreas function, increased cellular turnover (Dibner and Buttin, 2002), enhanced gastric proteolysis, improved protein and amino acids digestibility (Symeon et al., 2010), and decreased Escherichia coli counts in the small intestine of laying hens (Moharrery and Mahzonieh, 2005). Dietary acidifiers can cause corrosion of processing equipment and volatilization during the granulating process (Zhu et al., 2014). Therefore, it is presumed that providing organic acid through drinking water would reduce these problems (Wales et al., 2010). Acidified water is of particular importance in the feed withdrawal period before slaughter, when birds have a greater chance of becoming infected (Ramirez et al., 1997; Byrd et al., 1998; Corrier et al., 1999). Studies showed that feed withdrawal causes changes in the crop, which are characterized by lactic acid reduction, pH increase, and a consequent increase of Salmonella contamination (Corrier et al., 1999). Another benefit of supplying organic acid through water vs through feed is more effective in increasing feed intake during heat stress. Organic acids added to drinking water had been previously reported to improve swine growth performance and feed efficiency, as well as reduce pathogenic bacteria counts (Cole et al., 1968). Recently acidified drinking water has been used to improve broiler performance (Chaveerach et al., 2004; Cornelison et al., 2005; Aclkgoz et al., 2011). Escherichia coli is a common pathogenic bacteria in all types and age categories of poultry, which is often used as indicators of overall food quality and the hygienic conditions (Scheinberg et al., 2017). It is crucial to reduce the microbial population of these pathogenic bacteria to prevent or reduce economic losses. The use of acidified drinking water will not only disinfect the water itself (Ritskes-Hoitinga et al., 1998; Krug et al., 2012), but also lead to improved performance and immunological parameters in the birds (Byrd et al., 2001; Chaveerach et al., 2004; Aclkgoz et al., 2011; Hamed and Hassan, 2013). Thus, the object of the current research was to evaluate the effects of acidified water on growth performance, gastrointestinal pH, digestive enzymes, intestinal histomorphology, and intestinal microflora of broilers. MATERIALS AND METHODS Experimental Design, and Treatments The protocol was reviewed and approved by the Animal Care and Use Committee of China Agricultural University. All procedures were performed strictly in accordance with the guidelines of recommendations in the Guide for Experimental Animals of the Ministry of Science and Technology (Beijing, China), and all efforts were made to minimize suffering. In the experiment, a total of 540 male broiler chicks (0 d of age) with similar body weight (BW) were randomly divided into 5 treatment groups, with 6 replicates of 18 chicks each, in a completely randomized design. The 5 treatments were as follows (Figure 1): NC (negative control, basal diet, normal water), PC (positive control, basal diet + 8 ppm colistin sulfate + 8 ppm enduracidin, normal water), A1 (basal diet, continuous supply of acidified water during whole experiment period), A2 (basal diet, intermittent acidification of water during 0 to 14 d, 22 to 28 d, and 36 to 42 d) and A3 [basal diet, intermittent acidification of water (24 h/d from 0 to 14 d and from 10:00 am to 4:00 pm on d 15 to 42)]. Drinking water was acidified with a liquid acidifier (Lupro-Mix NC, produced by BASF Co., Ltd., consisting of propionic acid, ammonium propionate, formic acid and ammonium formate as active ingredients). The pH was reduced from 7.8 to 4.2 in the acidified water. Figure 1. View largeDownload slide Experimental treatments and timeline of acidified water supply. NC, negative control; PC, positive control; A1, whole supply acidified water; A2, supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3, supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d. Figure 1. View largeDownload slide Experimental treatments and timeline of acidified water supply. NC, negative control; PC, positive control; A1, whole supply acidified water; A2, supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3, supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d. The Arbor Acre male broilers were obtained from a commercial hatchery, and raised in pens (120 × 120 × 60 cm; 18 broilers/pen), which were equipped with raised-wire floor. All birds had access to feed and water ad libitum. The whole experimental period lasted 42 d, composed of a starter period (0 to 21 d) and a finisher period (22 to 42 d). The basal diet (Table 1) was formulated to meet the nutrient requirements of the National Research Council (1994). The water supplying systems of different groups were independent. Birds were vaccinated on the hatchery against Marek's disease vaccine at 0 d, and on the farm against Newcastle disease at 7 d of age and infectious bronchitis disease at 21 d of age, respectively. A 24-h lighting regime was carried out during the first 3 d, and 23 h of lighting with 1 h of darkness was used from 4 d of age onward. Mean air temperature of animal chamber maintained at approximately 35°C during the first week, and then decreased gradually to get a constant temperature of 25°C during the rest of the trial. The relative humidity was maintained between 65 and 70% Table 1. Composition and nutrient levels of basal diet. Composition  Percentage (%)  Nutrients value1  Content    0 to 21 d  22 to 42 d    0 to 21 d  22 to 42 d  Corn  57.67  59.80  Crude protein (%)  20.51  19.28  Soybean meal  28.30  25.65  Apparent metabolizable energy (Mcal/kg)  3000  3100  Extruded soybean  8.00  8.00  Calcium (%)  1.01  0.90  Soybean oil  1.90  3.00  Total phophorus (%)  0.66  0.63  Limestone  1.30  1.10  Non-phytic phosphorus (%)  0.43  0.41  Dicalcium phosphate  1.70  1.60  Methionine (%)  0.56  0.42  Salt  0.30  0.30  Methionine + Cystine (%)  0.91  0.76  Lysine-HCl (98.5%)  0.10  –  Lysine (%)  1.15  1.01  DL-Methionine  0.25  0.12  Tryptophan (%)  0.24  0.22  Theronine  0.05  –  Theronine (%)  0.81  0.72  Vitamins premix2  0.03  0.03        50% Choline chloride  0.10  0.10        Micro-Mineral premix3  0.30  0.30        Total  100.00  100.00        Composition  Percentage (%)  Nutrients value1  Content    0 to 21 d  22 to 42 d    0 to 21 d  22 to 42 d  Corn  57.67  59.80  Crude protein (%)  20.51  19.28  Soybean meal  28.30  25.65  Apparent metabolizable energy (Mcal/kg)  3000  3100  Extruded soybean  8.00  8.00  Calcium (%)  1.01  0.90  Soybean oil  1.90  3.00  Total phophorus (%)  0.66  0.63  Limestone  1.30  1.10  Non-phytic phosphorus (%)  0.43  0.41  Dicalcium phosphate  1.70  1.60  Methionine (%)  0.56  0.42  Salt  0.30  0.30  Methionine + Cystine (%)  0.91  0.76  Lysine-HCl (98.5%)  0.10  –  Lysine (%)  1.15  1.01  DL-Methionine  0.25  0.12  Tryptophan (%)  0.24  0.22  Theronine  0.05  –  Theronine (%)  0.81  0.72  Vitamins premix2  0.03  0.03        50% Choline chloride  0.10  0.10        Micro-Mineral premix3  0.30  0.30        Total  100.00  100.00        1Determined values except for apparent metabolizable energy. 2Provided per kilogram of diet: vitamin A, 15,000 IU; vitamin D3, 3000 IU; vitamin E, 20 IU; vitamin K3, 2.18 mg; thiamine, 2.15 mg; riboflavin, 8.00 mg; pyridoxine, 4.40 mg; vitamin B12 0.02 mg; calcium pantothenate, 25.60 mg; nicotinic acid, 65.80 mg; folic acid, 0.96 mg; biotin, 0.20 mg. 3Provided per kilogram of diet: Fe, 100 mg; Cu, 8 mg; Zn, 78 mg; Mn, 105 mg; I, 0.5 mg; Se, 0.3 mg. View Large Table 1. Composition and nutrient levels of basal diet. Composition  Percentage (%)  Nutrients value1  Content    0 to 21 d  22 to 42 d    0 to 21 d  22 to 42 d  Corn  57.67  59.80  Crude protein (%)  20.51  19.28  Soybean meal  28.30  25.65  Apparent metabolizable energy (Mcal/kg)  3000  3100  Extruded soybean  8.00  8.00  Calcium (%)  1.01  0.90  Soybean oil  1.90  3.00  Total phophorus (%)  0.66  0.63  Limestone  1.30  1.10  Non-phytic phosphorus (%)  0.43  0.41  Dicalcium phosphate  1.70  1.60  Methionine (%)  0.56  0.42  Salt  0.30  0.30  Methionine + Cystine (%)  0.91  0.76  Lysine-HCl (98.5%)  0.10  –  Lysine (%)  1.15  1.01  DL-Methionine  0.25  0.12  Tryptophan (%)  0.24  0.22  Theronine  0.05  –  Theronine (%)  0.81  0.72  Vitamins premix2  0.03  0.03        50% Choline chloride  0.10  0.10        Micro-Mineral premix3  0.30  0.30        Total  100.00  100.00        Composition  Percentage (%)  Nutrients value1  Content    0 to 21 d  22 to 42 d    0 to 21 d  22 to 42 d  Corn  57.67  59.80  Crude protein (%)  20.51  19.28  Soybean meal  28.30  25.65  Apparent metabolizable energy (Mcal/kg)  3000  3100  Extruded soybean  8.00  8.00  Calcium (%)  1.01  0.90  Soybean oil  1.90  3.00  Total phophorus (%)  0.66  0.63  Limestone  1.30  1.10  Non-phytic phosphorus (%)  0.43  0.41  Dicalcium phosphate  1.70  1.60  Methionine (%)  0.56  0.42  Salt  0.30  0.30  Methionine + Cystine (%)  0.91  0.76  Lysine-HCl (98.5%)  0.10  –  Lysine (%)  1.15  1.01  DL-Methionine  0.25  0.12  Tryptophan (%)  0.24  0.22  Theronine  0.05  –  Theronine (%)  0.81  0.72  Vitamins premix2  0.03  0.03        50% Choline chloride  0.10  0.10        Micro-Mineral premix3  0.30  0.30        Total  100.00  100.00        1Determined values except for apparent metabolizable energy. 2Provided per kilogram of diet: vitamin A, 15,000 IU; vitamin D3, 3000 IU; vitamin E, 20 IU; vitamin K3, 2.18 mg; thiamine, 2.15 mg; riboflavin, 8.00 mg; pyridoxine, 4.40 mg; vitamin B12 0.02 mg; calcium pantothenate, 25.60 mg; nicotinic acid, 65.80 mg; folic acid, 0.96 mg; biotin, 0.20 mg. 3Provided per kilogram of diet: Fe, 100 mg; Cu, 8 mg; Zn, 78 mg; Mn, 105 mg; I, 0.5 mg; Se, 0.3 mg. View Large Growth Performance On 21 and 42 d, the BW and feed intake of broilers were recorded. The average daily feed intake (ADFI), average daily gain (ADG), and feed: gain ratio (F:G) were calculated. Sampling Procedure At the morning on 43 d, 12 birds close to the average weight from each treatment group (2 birds per replicate) were randomly selected, slaughtered by cervical dislocation and manually eviscerated, trying not to rupture the digestive tract segments. Digesta from gastrointestinal segments, namely the crop, gizzard, proventriculus, duodenum, jejunum, ileum, and cecum, were separately taken. Immediately upon obtaining the digesta from each section, the pH was determined with a pH meter (SFK Inc., Kolding, Denmark) as described by Chaveerach et al. (2004). The contents of the duodenum were gently collected to determine the digestive enzyme activities (Wu et al., 2013). The 2-cm-long segments from the duodenum (the middle part of the duodenal loop), the jejunum (the middle part between the end point of duodenal loop and Meckel's diverticulum) and the ileum (5 cm after Meckel's diverticulum) were sampled to measure morphology. Then digesta were collected from the cecum and stored immediately at −20°C for microbiological analysis. Digestive Enzyme Activities of Duodenal Contents The duodenal digesta samples were diluted 10 ×, based on the sample weight, with ice-cold PBS (pH 7.0), homogenized for 60 s, and sonicated for 1 min with 3 cycles at 30 s intervals. The sample was then centrifuged at 6000 g for 15 min at 4°C. The supernatants were divided into aliquots and stored at −70°C for the enzyme activity assays. The activities of trypsin, chymotrypsin, lipase, and amylase were measured according to the methods described by Lhoste et al. (1993). Morphological Measurement of the Duodenal, Jejunal, and Ileal Mucosa Three cross-sections for each intestinal segment (duodenum, jejunum, and ileum) were fixed with formalin solution and were prepared using the standard paraffin embedding procedures by sectioning 5 μm thickness and staining with hematoxylin and eosin. A total of 15 intact, well-oriented crypt-villus units were measured in each type of tissue for each sample. Villus height and crypt depth (CD) were determined using an image processing and analyzing system (version 6.0, Image-Pro Plus) and expressed in micrometer (μm). Microbiological Analyses The 1 g of sample was removed from the cecal digesta and then serially diluted (1:10) in 9 mL aliquots for maximum recovery of diluents (MRD, Oxoid, Basingstoke, UK), and spread (0.1 mL aliquots) on to selective agars for the preparation of a 10-fold dilution. The total aerobic bacteria and E. coli as pathogenic and hygiene indicators were isolated using brain heart infusion agar (BHI, Oxoid) with 5% defibrinated sheep blood and MacConkey (MC) agar, respectively, followed by aerobic incubation at 37°C for 24 h. All bacteria were counted and expressed as total colony forming unit (CFU)/g digesta, per gram of sample and results, were presented as log10 transformed data. Statistical Analyses The experiment was carried out as a completely randomized design with 5 treatments. Statistical analyses of all growth performance, histomorphology, pH, and enzyme data were subjected to one-way ANOVA using statistical analysis systems (SAS) statistical software package (Version 8, SAS Institute, Cary, NC, USA), and post hoc comparison of the means using Turkey (equal variances) test at P < 0.05. For log-transformed data of microbial counts were subjected to analyses of variance, and differences between treatments assessed by Dunnett's T3 (unequal variances) tests at P < 0.05. RESULTS Growth Performance Throughout the entire period (0 to 21 d, 22 to 42 d, and 0 to 42 d), the broilers in PC and acidified water groups (A1, A2, and A3) showed higher final BW, ADG, and lower F: G compared to NC group (P < 0.05; Table 2). The ADFI of the birds in PC and acidified water groups was significantly lower during 0 to 21 d (P < 0.05) and significantly higher during 22 to 42 d (P < 0.05) than those in NC group, but no significant difference existed among all groups during 0 to 42 d (P > 0.05). Table 2. Effects of acidified drinking water on growth performance of broilers during 0 to 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  BW (g)                0 d  43.3  43.5  43.5  43.5  43.4  0.02  0.25  21 d  748c  795a,b  780b  787a,b  810a  3.49  <0.001  42 d  2,289b  2,584a  2,590a  2,589a  2,606a  14.9  <0.001  ADG (g)                0 to 21 d  33.5c  35.8a,b  35.1b  35.4a,b  36.5a  0.17  <0.001  22 to 42 d  73.4b  85.2a  86.2a  85.8a  85.5a  0.63  <0.001  0 to 42 d  53.5b  60.5a  60.6a  60.6a  61.0a  0.36  <0.001  ADFI (g)                0 to 21 d  54.2a  50.7b  49.7b  50.4b  50.9b  0.24  <0.001  22 to 42 d  148b  156a  156a  157a  158a  0.88  0.057  0 to 42 d  101  103  103  104  105  0.44  0.34  F:G (g of feed/g of gain)                0 to 21 d  1.62a  1.42b  1.42b  1.42b  1.39b  0.01  <0.001  22 to 42 d  2.01a  1.83b  1.81b  1.83b  1.85b  0.01  <0.001  0 to 42 d  1.89a  1.71b  1.70b  1.71b  1.71b  0.01  <0.001  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  BW (g)                0 d  43.3  43.5  43.5  43.5  43.4  0.02  0.25  21 d  748c  795a,b  780b  787a,b  810a  3.49  <0.001  42 d  2,289b  2,584a  2,590a  2,589a  2,606a  14.9  <0.001  ADG (g)                0 to 21 d  33.5c  35.8a,b  35.1b  35.4a,b  36.5a  0.17  <0.001  22 to 42 d  73.4b  85.2a  86.2a  85.8a  85.5a  0.63  <0.001  0 to 42 d  53.5b  60.5a  60.6a  60.6a  61.0a  0.36  <0.001  ADFI (g)                0 to 21 d  54.2a  50.7b  49.7b  50.4b  50.9b  0.24  <0.001  22 to 42 d  148b  156a  156a  157a  158a  0.88  0.057  0 to 42 d  101  103  103  104  105  0.44  0.34  F:G (g of feed/g of gain)                0 to 21 d  1.62a  1.42b  1.42b  1.42b  1.39b  0.01  <0.001  22 to 42 d  2.01a  1.83b  1.81b  1.83b  1.85b  0.01  <0.001  0 to 42 d  1.89a  1.71b  1.70b  1.71b  1.71b  0.01  <0.001  Each value is a mean of duplicate assays. SEM = standard error of the mean; BW = body weight; ADG = average daily gain; ADFI = average daily feed intake; F: G = feed: gain ratio. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a–cMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance (ANOVA) using Tukey's test. View Large Table 2. Effects of acidified drinking water on growth performance of broilers during 0 to 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  BW (g)                0 d  43.3  43.5  43.5  43.5  43.4  0.02  0.25  21 d  748c  795a,b  780b  787a,b  810a  3.49  <0.001  42 d  2,289b  2,584a  2,590a  2,589a  2,606a  14.9  <0.001  ADG (g)                0 to 21 d  33.5c  35.8a,b  35.1b  35.4a,b  36.5a  0.17  <0.001  22 to 42 d  73.4b  85.2a  86.2a  85.8a  85.5a  0.63  <0.001  0 to 42 d  53.5b  60.5a  60.6a  60.6a  61.0a  0.36  <0.001  ADFI (g)                0 to 21 d  54.2a  50.7b  49.7b  50.4b  50.9b  0.24  <0.001  22 to 42 d  148b  156a  156a  157a  158a  0.88  0.057  0 to 42 d  101  103  103  104  105  0.44  0.34  F:G (g of feed/g of gain)                0 to 21 d  1.62a  1.42b  1.42b  1.42b  1.39b  0.01  <0.001  22 to 42 d  2.01a  1.83b  1.81b  1.83b  1.85b  0.01  <0.001  0 to 42 d  1.89a  1.71b  1.70b  1.71b  1.71b  0.01  <0.001  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  BW (g)                0 d  43.3  43.5  43.5  43.5  43.4  0.02  0.25  21 d  748c  795a,b  780b  787a,b  810a  3.49  <0.001  42 d  2,289b  2,584a  2,590a  2,589a  2,606a  14.9  <0.001  ADG (g)                0 to 21 d  33.5c  35.8a,b  35.1b  35.4a,b  36.5a  0.17  <0.001  22 to 42 d  73.4b  85.2a  86.2a  85.8a  85.5a  0.63  <0.001  0 to 42 d  53.5b  60.5a  60.6a  60.6a  61.0a  0.36  <0.001  ADFI (g)                0 to 21 d  54.2a  50.7b  49.7b  50.4b  50.9b  0.24  <0.001  22 to 42 d  148b  156a  156a  157a  158a  0.88  0.057  0 to 42 d  101  103  103  104  105  0.44  0.34  F:G (g of feed/g of gain)                0 to 21 d  1.62a  1.42b  1.42b  1.42b  1.39b  0.01  <0.001  22 to 42 d  2.01a  1.83b  1.81b  1.83b  1.85b  0.01  <0.001  0 to 42 d  1.89a  1.71b  1.70b  1.71b  1.71b  0.01  <0.001  Each value is a mean of duplicate assays. SEM = standard error of the mean; BW = body weight; ADG = average daily gain; ADFI = average daily feed intake; F: G = feed: gain ratio. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a–cMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance (ANOVA) using Tukey's test. View Large The final BW and ADG of PC group were significantly higher than A2 group (P < 0.05) but didn’t have significant differences when compared to A1 and A3 groups during 0 to 21 d. There was no difference in growth performance between PC and any acidified groups (P > 0.05) during 22 to 42 d and 0 to 42 d. And no difference was founded in growth performance between the acidified groups (P > 0.05) during any period. pH of Gastrointestinal Segments In comparison to NC group, the pH in crop, proventriculus and ileum declined significantly (P < 0.05) by 0.55, 0.69, and 0.70 in A2 group, and 0.63, 0.74, and 1.21 in A3 group, respectively, whereas A1 and PC groups only decreased the pH of the proventriculus (by 1.03 and 0.68, respectively) and ileum (by 1.23 and 0.81; P < 0.05; Table 3). Among 3 acidified groups, A2 and A3 groups had significantly lower digesta pH in crop and gizzard (P < 0.05), and A2 group had significantly higher pH in duodenum (P < 0.05) than A1 group, whereas there was no significant difference in ileum among 3 acidified groups (P > 0.05). The pH value in the jejunum and cecum was not affected by the treatments (P > 0.05). Table 3. Effect of acidified water at pH of gastrointestinal tract of broilers on day 42. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Crop  5.83a  5.79a  5.28b  5.20b  6.11a  0.07  <0.001  Gizzard  3.43a,b  3.56a  2.99b  3.09b  3.27a,b  0.06  0.004  Proventriculus  5.05a  4.02b  4.36b  4.31b  4.37b  0.07  <0.001  Duodenum  6.02a,b  5.88b  6.23a  6.17a,b  6.19a,b  0.04  0.023  Jejunum  6.33  6.27  6.19  6.20  6.33  0.03  0.41  Ileum  7.71a  6.48b  7.01b  6.50b  6.90b  0.09  <0.001  Cecum  6.53  6.37  6.55  6.37  6.50  0.05  0.64  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Crop  5.83a  5.79a  5.28b  5.20b  6.11a  0.07  <0.001  Gizzard  3.43a,b  3.56a  2.99b  3.09b  3.27a,b  0.06  0.004  Proventriculus  5.05a  4.02b  4.36b  4.31b  4.37b  0.07  <0.001  Duodenum  6.02a,b  5.88b  6.23a  6.17a,b  6.19a,b  0.04  0.023  Jejunum  6.33  6.27  6.19  6.20  6.33  0.03  0.41  Ileum  7.71a  6.48b  7.01b  6.50b  6.90b  0.09  <0.001  Cecum  6.53  6.37  6.55  6.37  6.50  0.05  0.64  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a–dMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance (ANOVA) using Tukey's test. View Large Table 3. Effect of acidified water at pH of gastrointestinal tract of broilers on day 42. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Crop  5.83a  5.79a  5.28b  5.20b  6.11a  0.07  <0.001  Gizzard  3.43a,b  3.56a  2.99b  3.09b  3.27a,b  0.06  0.004  Proventriculus  5.05a  4.02b  4.36b  4.31b  4.37b  0.07  <0.001  Duodenum  6.02a,b  5.88b  6.23a  6.17a,b  6.19a,b  0.04  0.023  Jejunum  6.33  6.27  6.19  6.20  6.33  0.03  0.41  Ileum  7.71a  6.48b  7.01b  6.50b  6.90b  0.09  <0.001  Cecum  6.53  6.37  6.55  6.37  6.50  0.05  0.64  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Crop  5.83a  5.79a  5.28b  5.20b  6.11a  0.07  <0.001  Gizzard  3.43a,b  3.56a  2.99b  3.09b  3.27a,b  0.06  0.004  Proventriculus  5.05a  4.02b  4.36b  4.31b  4.37b  0.07  <0.001  Duodenum  6.02a,b  5.88b  6.23a  6.17a,b  6.19a,b  0.04  0.023  Jejunum  6.33  6.27  6.19  6.20  6.33  0.03  0.41  Ileum  7.71a  6.48b  7.01b  6.50b  6.90b  0.09  <0.001  Cecum  6.53  6.37  6.55  6.37  6.50  0.05  0.64  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a–dMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance (ANOVA) using Tukey's test. View Large Digestive Enzyme Activity in Duodenum The activities of trypsin, chymotrypsin and amylase were not influenced by any experimental treatments (P > 0.05) (Table 4). There was a decrease (P < 0.05) in lipase activity in PC and all acidified water groups when compared to NC group (P < 0.05). Table 4. Effects of acidified water at the digestive enzyme activities (U/mgprot) in the duodenal contents of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Trypsin  4000  2809  2692  4926  3657  390  0.34  Chymotrypsin  6.94  5.38  4.47  6.12  4.66  0.44  0.34  Lipase  96.8a  46.6b  31.4b  50.6b  45.3b  5.90  <0.001  Amylase  5.97  5.04  3.72  6.38  4.94  0.57  0.67  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Trypsin  4000  2809  2692  4926  3657  390  0.34  Chymotrypsin  6.94  5.38  4.47  6.12  4.66  0.44  0.34  Lipase  96.8a  46.6b  31.4b  50.6b  45.3b  5.90  <0.001  Amylase  5.97  5.04  3.72  6.38  4.94  0.57  0.67  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tukey's test. View Large Table 4. Effects of acidified water at the digestive enzyme activities (U/mgprot) in the duodenal contents of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Trypsin  4000  2809  2692  4926  3657  390  0.34  Chymotrypsin  6.94  5.38  4.47  6.12  4.66  0.44  0.34  Lipase  96.8a  46.6b  31.4b  50.6b  45.3b  5.90  <0.001  Amylase  5.97  5.04  3.72  6.38  4.94  0.57  0.67  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Trypsin  4000  2809  2692  4926  3657  390  0.34  Chymotrypsin  6.94  5.38  4.47  6.12  4.66  0.44  0.34  Lipase  96.8a  46.6b  31.4b  50.6b  45.3b  5.90  <0.001  Amylase  5.97  5.04  3.72  6.38  4.94  0.57  0.67  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tukey's test. View Large Intestinal Histomorphology As shown in Table 5, in comparison to NC group, PC group significantly increased the CD in duodenum (P < 0.05) and didn’t affect any other indices in intestinal histomorphology (P > 0.05). The A1 group had significantly higher CD in jejunum (P < 0.05) and the lower ratio of villous height (VH) to CD (V/C) in jejunum and ileum (P < 0.05) than NC group, and significantly higher CD in duodenum and jejunum (P < 0.05) and lower V/C in 3 small intestinal segments (P < 0.05) than PC group. The A2 group had significant higher VH and higher V/C in jejunum than NC group (P < 0.05), and higher VH in jejunum and higher CD in duodenum than PC group (P < 0.05). The A3 group had significantly higher CD in ileum and lower V/C in jejunum and ileum than NC group (P < 0.05), and higher CD in duodenum and ileum and lower V/C in the 3 small intestinal segments than PC group (P < 0.05). Among 3 acidified groups, A2 group had higher VH in jejunum and higher V/C in jejunum and ileum than A1 and A3 groups. Table 5. Effects of acidified drinking water at the intestinal histomorphology of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Duodenum  VH (μm)  416  435  455  444  461  5.80  0.11  CD (μm)  122a,b  128a  124a,b  127a  115b  1.52  0.021  V/C (μm/μm)  3.40b  3.39b  3.69a,b  3.49b  4.03a  0.07  0.009  Jejunum  VH (μm)  350b  351b  390a  359b  364b  3.94  0.002  CD (μm)  116a,b  130a  121a,b  129a,b  115b  1.86  0.012  V/C (μm/μm)  3.02b  2.70c  3.23a  2.80c  3.17a,b  0.05  <0.001  Ileum  VH (μm)  318  302  310  309  313  2.51  0.41  CD (μm)  110b  111b  108b  121a  109b  1.34  0.003  V/C (μm/μm)  2.90a  2.74b  2.88a  2.54c  2.86a  0.03  <0.001  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Duodenum  VH (μm)  416  435  455  444  461  5.80  0.11  CD (μm)  122a,b  128a  124a,b  127a  115b  1.52  0.021  V/C (μm/μm)  3.40b  3.39b  3.69a,b  3.49b  4.03a  0.07  0.009  Jejunum  VH (μm)  350b  351b  390a  359b  364b  3.94  0.002  CD (μm)  116a,b  130a  121a,b  129a,b  115b  1.86  0.012  V/C (μm/μm)  3.02b  2.70c  3.23a  2.80c  3.17a,b  0.05  <0.001  Ileum  VH (μm)  318  302  310  309  313  2.51  0.41  CD (μm)  110b  111b  108b  121a  109b  1.34  0.003  V/C (μm/μm)  2.90a  2.74b  2.88a  2.54c  2.86a  0.03  <0.001  Each value is a mean of duplicate assays. SEM = standard error of the mean; VH = villus height; CD = crypt depth. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tukey's test. View Large Table 5. Effects of acidified drinking water at the intestinal histomorphology of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Duodenum  VH (μm)  416  435  455  444  461  5.80  0.11  CD (μm)  122a,b  128a  124a,b  127a  115b  1.52  0.021  V/C (μm/μm)  3.40b  3.39b  3.69a,b  3.49b  4.03a  0.07  0.009  Jejunum  VH (μm)  350b  351b  390a  359b  364b  3.94  0.002  CD (μm)  116a,b  130a  121a,b  129a,b  115b  1.86  0.012  V/C (μm/μm)  3.02b  2.70c  3.23a  2.80c  3.17a,b  0.05  <0.001  Ileum  VH (μm)  318  302  310  309  313  2.51  0.41  CD (μm)  110b  111b  108b  121a  109b  1.34  0.003  V/C (μm/μm)  2.90a  2.74b  2.88a  2.54c  2.86a  0.03  <0.001  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Duodenum  VH (μm)  416  435  455  444  461  5.80  0.11  CD (μm)  122a,b  128a  124a,b  127a  115b  1.52  0.021  V/C (μm/μm)  3.40b  3.39b  3.69a,b  3.49b  4.03a  0.07  0.009  Jejunum  VH (μm)  350b  351b  390a  359b  364b  3.94  0.002  CD (μm)  116a,b  130a  121a,b  129a,b  115b  1.86  0.012  V/C (μm/μm)  3.02b  2.70c  3.23a  2.80c  3.17a,b  0.05  <0.001  Ileum  VH (μm)  318  302  310  309  313  2.51  0.41  CD (μm)  110b  111b  108b  121a  109b  1.34  0.003  V/C (μm/μm)  2.90a  2.74b  2.88a  2.54c  2.86a  0.03  <0.001  Each value is a mean of duplicate assays. SEM = standard error of the mean; VH = villus height; CD = crypt depth. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tukey's test. View Large Cecal Microflora The A3 group reduced (P < 0.05) the total aerobic bacteria count in cecum, when compared to NC group, and had no significant difference to PC group (P > 0.05; Table 6). The acidified groups showed a tendency (P < 0.10) to reduce counts of E. coli when compared to NC group, but this effect was not statistically significant. There was no difference among A1, A2, and PC groups in counts of the total aerobic bacteria and E. coli counts in the cecum. Table 6. Effect of acidified water at cecal microflora (Log CFU/g) of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Total aerobic bacteria  7.87a  7.09a,b  7.00a,b  6.76b  6.95a,b  0.12  0.025  Escherichia coli  7.35  6.71  6.58  6.22  6.57  0.13  0.067  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Total aerobic bacteria  7.87a  7.09a,b  7.00a,b  6.76b  6.95a,b  0.12  0.025  Escherichia coli  7.35  6.71  6.58  6.22  6.57  0.13  0.067  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am–to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tamhane's T2 (unequal variances) test. View Large Table 6. Effect of acidified water at cecal microflora (Log CFU/g) of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Total aerobic bacteria  7.87a  7.09a,b  7.00a,b  6.76b  6.95a,b  0.12  0.025  Escherichia coli  7.35  6.71  6.58  6.22  6.57  0.13  0.067  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Total aerobic bacteria  7.87a  7.09a,b  7.00a,b  6.76b  6.95a,b  0.12  0.025  Escherichia coli  7.35  6.71  6.58  6.22  6.57  0.13  0.067  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am–to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tamhane's T2 (unequal variances) test. View Large DISCUSSION Acidification of drinking water has been previously shown to have a beneficial effect on water quality and growth performance in laying hens (Rehman et al., 2013). The present study showed that water acidification improved BW, ADG, and F:G of broilers as effective as the antibiotics growth promoter compound of 8 ppm colistin sulfate + 8 ppm enduracidin in diet. Our results are similar to those of Pesti et al. (2004) and Chaveerach et al. (2004), who reported that acidified water improved growth performance and BW gain and boosted the digestion of feed in comparison to regular water. Desai et al. (2007) also found that broilers are receiving blends of formic and propionic acids in drinking water had improved weight gain and feed conversion. Pesti et al. (2004) reported that organic acids significantly reduced the pH of all gastrointestinal tract segments in broilers. But in other studies, the pH reduction mainly happened in the upper gut such as crop (Byrd et al., 2001), and the cecal pH kept stable (Waldroup et al., 1995). Similarly, in this study, A2 and A3 groups had significantly decreased pH value for crop, proventriculus, and ileum when compared to NC group, whereas A1 and PC groups only had decreased pH in the proventriculus and ileum. In contrast, Cornelison et al. (2005) and Watkins et al. (2004), by using different acids, found no significant effect on the gizzard pH of broilers and turkeys. That may be a result of the difference in acid type and concentration. It has been demonstrated that organic acids concentrations decreased further down the digestive tract as a result of absorption and metabolism (Bolton and Dewar, 1965). Our experiment showed that the A2 group had higher villi height and V/C in the jejunum, the A1 group had deeper CD in jejunum and lower V/C in jejunum and ileum, and the A3 group had deeper crypts and lower V/C in the jejunum and ileum. These are similar to the results of Garcia et al. (2007), who observed the highest villi and deepest crypts in the jejunum in broilers fed formic acid in their diets. The current study is in agreement with the findings of Loddi et al. (2004) and Pelicano et al. (2005), who observed that the jejunum had the increased villus heights with the majority of the organic acidifiers. The cation of organic acids may play an important role in the modulation of intestinal histomorphology. Paul et al. (2007) discovered that organic acid salt supplementation significantly increased (P < 0.05) the villus height of different segments of small intestine and decreased the intestinal microbial load, which also decreased the presence of toxins that are related to changes in intestinal morphology in broilers. In this study, we observed that the acidified water (A1, A2, and A3 groups) had inhibitory effects on the aerobic bacterial count and the E. coli population in cecum contents. Similarly, Hamed and Hassan (2013) also observed that 7 d post-infection, Japanese quails that consumed an organic acid mixture and acetic acid water had (P < 0.05) reduced total bacterial counts in the ceca. Subsequent studies suggested that drinking water containing organic acids was helpful in reducing pathogens in the broiler (Hassan et al., 2010; Hamed and Hassan, 2013). Bunnik et al. (2012) also observed that the gizzard and stomach of birds drinking acidified water were more acidic and might decrease the bacteria that were present in the gastrointestinal tract, especially in the lower gastrointestinal tract. Organic acids provide a suitable pH in the gut, which increases beneficial bacteria, and decreases harmful bacteria of broilers (Roser, 2006). Meanwhile, accumulation of acid cations released from the anions in the higher pH environment inside cells is also an important mechanism by which organic acids inhibit bacterial growth (Carpenter and Roadbent, 2009). The antibacterial action of organic acids has been attributed to cytoplasmic acidification from proton discharge, causing inhibition of acid sensitive enzymes, for instance those engaged with glycolysis (Davidson, 2001). However, there is sufficient evidence to confirm that the accumulation of acid anions may be more inhibitory for cell development, especially in mild acidic conditions above pH 5.0 (Russell, 1992; Roe et al., 2002). Lipid-permeable, weak acids dissociate into the cytoplasm, resulting in decline of pH and the accumulation of anions (Salmond et al., 1984). The accumulation of anions can cause an osmotic problem for the cell if it leads to an increase in cell turgor pressure. The cell may recompense for this by discharging the anion itself, or by bringing down the concentration of other cellular anions, mainly glutamate (Roe et al., 1998). Such anion altercation would be a factor in decreasing intracellular pH and inhibition of cell function. Thus, lower pathogenic bacteria population in cecum contents under nearly same pH values in acidified groups could be explained by the lower pH in the upper gastrointestinal gut and higher acid anions (such as formate ion and propionate ion) in whole gut contents. The findings of the current study showed that there was a significant (P < 0.05) decline in lipase enzymes in PC and all acidified groups in comparison to NC group, whereas there was no significant difference on other enzymes.There are different factors affecting enzyme activity. It is known that there was modulation of the activity of various digestive enzymes in the same chicken species (Maria et al., 2014). Various reports point out that the concentration of digestive enzymes in poultry change with age (Krogdahl and Sell, 1989; Pubols, 1991; Noy and Sklan, 1995). Duodenal lipase activity in young chicks increases 20 times between 4 and 21 d of age (Noy and Sklan, 1995) in chickens. Gastric lipase enzyme has been appeared to be significantly inhibited by protonated free fatty acids (Borel et al., 1994; Wang et al., 2017). Previous studies on lipoprotein models (Miller and Small, 1982; Miller and Small, 1983; Miller and Small, 1987) and a current study consuming dietary mixtures (Borel et al., 1994) have exposed that 2 to 5 mole % of the droplet surface lipid is triacylglycerol, so allowing lipase action at the surface of the lipid droplet. Thereby, fatty acids inhibition may also a cause of reduced lipid activity. It has been found, when chickens were fed diets with variable protein and carbohydrate contents, that the activities of the digestive enzymes are modulated by the availability of the substrates (Siddons, 1972; Biviano et al., 1993). Additionally, research conducted by Hulan and Bird (1972) exposed that increase in dietary carbohydrates and fat enhanced the activity of amylase and lipase enzymes, whereas the dietary addition of inorganic compounds, including natural zeolite and minerals, also produced substantial morphological and enzymatic variations in the intestine of broiler chickens (Incharoen et al., 2009; Ruttanavut and Yamauchi, 2010). Several researchers (Osman, 1982; Kihara and Sakata, 1997) also reported that EDTA addition causes a decrease in lipase activity and found that the optimum pH for intestinal lipase is 9. It has already been observed that acidified water reduces intestinal pH, so as a result of being at lower than optimal pH lipase enzyme activity might reduce. On the other hand, the intestinal microflora population is thought to change the intestinal environment and produce enzymes and other beneficial substances in the intestines (Marteau and Rambaud, 1993). Acidified water is known to reduce bacterial load so that lesser enzymes being available in the body from bacteria might be responsible for lower enzyme activity. However, the effects of acidified drinking water on lipase activity need more research. This study shows that acidified drinking water can alter pH in the gut and has a positive effect on the microflora balance of the gastrointestinal tract and the growth performance by reducing harmful gut bacteria. The morphological changes of intestinal mucosa could partly explain the improvement of FCR by acidified drinking water. Moreover, compared to continuous supply of acidified water, discontinuous supply of acidified water had the same or even better influence on broilers performance. It may be due to host adaptation to constant water acidification by reducing endogenous acid production or may be due to an adaptive tolerance response. A mildly acidic environmental stress (pH 5.5 to 4.5) produces response in several bacteria and offers protection towards subsequent exposure to deadly stress (pH < 4.0). This acid-tolerance response has been recognized and studied in an extensive variety of gram-negative and gram-positive bacterial species, confirming that exposure to mildly acidic pH induces the expression of several acid-shock proteins that encourage bacterial survival in subsequent extreme acid environments (Booth et al., 2002). The main defense mechanism, which defends the cell from acid, is alterations in membrane composition (Chang and Cronan, 1999; Jordan et al., 1999; Yuk and Marshall, 2004) and changes in internal pH homeostasis systems (Park et al., 1996; Richard and Foster, 2004). In this way, intermittent acidic water supply might change the internal pH and bacterial growth, and the subsequent decline in bacterial growth might produce a positive effect on the health of the animal. ACKNOWLEDGMENTS We greatly appreciate the support of National Key Research and Development Program of China (grant No. 2017YFD0500500), Public Sector (Agriculture) Scientific Research of China (grant No. 201403047) and National Key Technology Research and Development Program of China (grant No. 2011BAD26B04 and 2012BAD39B01). REFERENCES Aclkgoz Z., Bayraktar H., Aclkgoz Z., Bayraktar H., Altan Ö.. 2011. Effects of formic acid administration in the drinking water on performance, intestinal microflora and carcass contamination in male broilers under high ambient temperature. Anim Feed Sci Technol . 24: 87– 94. Biviano A. B., Del Rio C. M., Phillips D. L.. 1993. Ontogenesis of intestine morphology and intestinal disaccharidases in chickens (Gallus gallus) fed contrasting purified diets. J. Camp. Physiol. B . 163: 508– 518. Boling-Frankenbach S., Snow J., Parsons C., Baker D.. 2001. The effect of citric acid on the calcium and phosphorus requirements of chicks fed corn-soybean meal diets. 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Influence of acidified drinking water on growth performance and gastrointestinal function of broilers

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pey212
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Abstract

ABSTRACT The ban on the use of antibiotic feed additives as growth promoters compelled the researchers for exploring the future utility of other alternatives. This experiment was designed to evaluate the effect of acidified drinking water on growth performance, gastrointestinal pH, digestive enzymes, intestinal histomorphology, and cecum microbial counting of the broiler chicken. A total of 540 one-day-old male broilers (Arbor Acre) were randomly assigned to 5 treatments, with 6 replicates of 18 chicks per replicate. Broilers received diets and water as follows: NC (negative control, basal diet, normal water), PC (positive control, basal diet + 8 ppm colistin sulfate + 8 ppm enduracidin, normal water), A1 (basal diet, continuous supply of acidified water during whole experiment period), A2 (basal diet, intermittent acidification of water during 0 to 14 d, 22 to 28 d, and 36 to 42 d), and A3 [basal diet, intermittent acidification of water (24 h/d from 0 to 14 d and from 10:00 am to 4:00 pm on d 15 to 42)]. During the entire period, the acidified groups (A1, A2, and A3) and PC group showed improve on weight gain, average daily gain and feed conversion ratio compared to NC group (P < 0.05). The pH in crop, proventriculus and ileum at 43 d declined by 0.04, 1.03, 1.23; 0.55, 0.69, 0.70; and 0.63, 0.74, 1.21 in A1, A2, and A3 group, respectively. There was a significant decline of lipase activity in the PC and acidified groups compared to NC group. The A2 group had higher villus height in jejunum than NC group. The PC and acidified groups reduced (P < 0.05) the total aerobic bacteria count of cecum when contrasted to NC group. Therefore, we conclude that acidified drinking water can improve growth performance, compensate for gastric acidity, and control pathogenic bacteria in broilers and may be considered as a potential alternative to improve production parameters. Discontinuous supply of acidified water had the same or even better influence on broilers compared to continuous supply. INTRODUCTION The ban on the use of antibiotic feed additives as growth promoters increased problems in poultry performance, increased feed conversion ratio (FCR), and augmented the incidence of certain infectious diseases (Dibner and Richards, 2005). This scenario has urged researchers to investigate the ability of alternatives in poultry production. The antimicrobial properties of organic acids, for instance, lowering the pH of drinking water and buffering capacity of the feed have a beneficial effect on the physiology of the crop and proventriculus (Van et al., 2006). Dietary supplementation of several organic acids has been found to promote growth performance, mineral absorption, feed efficiency, and phytate-P utilization (Boling-Frankenbach et al., 2001). Other significant benefits related to dietary acidification are as follows: improved exocrine pancreas function, increased cellular turnover (Dibner and Buttin, 2002), enhanced gastric proteolysis, improved protein and amino acids digestibility (Symeon et al., 2010), and decreased Escherichia coli counts in the small intestine of laying hens (Moharrery and Mahzonieh, 2005). Dietary acidifiers can cause corrosion of processing equipment and volatilization during the granulating process (Zhu et al., 2014). Therefore, it is presumed that providing organic acid through drinking water would reduce these problems (Wales et al., 2010). Acidified water is of particular importance in the feed withdrawal period before slaughter, when birds have a greater chance of becoming infected (Ramirez et al., 1997; Byrd et al., 1998; Corrier et al., 1999). Studies showed that feed withdrawal causes changes in the crop, which are characterized by lactic acid reduction, pH increase, and a consequent increase of Salmonella contamination (Corrier et al., 1999). Another benefit of supplying organic acid through water vs through feed is more effective in increasing feed intake during heat stress. Organic acids added to drinking water had been previously reported to improve swine growth performance and feed efficiency, as well as reduce pathogenic bacteria counts (Cole et al., 1968). Recently acidified drinking water has been used to improve broiler performance (Chaveerach et al., 2004; Cornelison et al., 2005; Aclkgoz et al., 2011). Escherichia coli is a common pathogenic bacteria in all types and age categories of poultry, which is often used as indicators of overall food quality and the hygienic conditions (Scheinberg et al., 2017). It is crucial to reduce the microbial population of these pathogenic bacteria to prevent or reduce economic losses. The use of acidified drinking water will not only disinfect the water itself (Ritskes-Hoitinga et al., 1998; Krug et al., 2012), but also lead to improved performance and immunological parameters in the birds (Byrd et al., 2001; Chaveerach et al., 2004; Aclkgoz et al., 2011; Hamed and Hassan, 2013). Thus, the object of the current research was to evaluate the effects of acidified water on growth performance, gastrointestinal pH, digestive enzymes, intestinal histomorphology, and intestinal microflora of broilers. MATERIALS AND METHODS Experimental Design, and Treatments The protocol was reviewed and approved by the Animal Care and Use Committee of China Agricultural University. All procedures were performed strictly in accordance with the guidelines of recommendations in the Guide for Experimental Animals of the Ministry of Science and Technology (Beijing, China), and all efforts were made to minimize suffering. In the experiment, a total of 540 male broiler chicks (0 d of age) with similar body weight (BW) were randomly divided into 5 treatment groups, with 6 replicates of 18 chicks each, in a completely randomized design. The 5 treatments were as follows (Figure 1): NC (negative control, basal diet, normal water), PC (positive control, basal diet + 8 ppm colistin sulfate + 8 ppm enduracidin, normal water), A1 (basal diet, continuous supply of acidified water during whole experiment period), A2 (basal diet, intermittent acidification of water during 0 to 14 d, 22 to 28 d, and 36 to 42 d) and A3 [basal diet, intermittent acidification of water (24 h/d from 0 to 14 d and from 10:00 am to 4:00 pm on d 15 to 42)]. Drinking water was acidified with a liquid acidifier (Lupro-Mix NC, produced by BASF Co., Ltd., consisting of propionic acid, ammonium propionate, formic acid and ammonium formate as active ingredients). The pH was reduced from 7.8 to 4.2 in the acidified water. Figure 1. View largeDownload slide Experimental treatments and timeline of acidified water supply. NC, negative control; PC, positive control; A1, whole supply acidified water; A2, supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3, supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d. Figure 1. View largeDownload slide Experimental treatments and timeline of acidified water supply. NC, negative control; PC, positive control; A1, whole supply acidified water; A2, supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3, supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d. The Arbor Acre male broilers were obtained from a commercial hatchery, and raised in pens (120 × 120 × 60 cm; 18 broilers/pen), which were equipped with raised-wire floor. All birds had access to feed and water ad libitum. The whole experimental period lasted 42 d, composed of a starter period (0 to 21 d) and a finisher period (22 to 42 d). The basal diet (Table 1) was formulated to meet the nutrient requirements of the National Research Council (1994). The water supplying systems of different groups were independent. Birds were vaccinated on the hatchery against Marek's disease vaccine at 0 d, and on the farm against Newcastle disease at 7 d of age and infectious bronchitis disease at 21 d of age, respectively. A 24-h lighting regime was carried out during the first 3 d, and 23 h of lighting with 1 h of darkness was used from 4 d of age onward. Mean air temperature of animal chamber maintained at approximately 35°C during the first week, and then decreased gradually to get a constant temperature of 25°C during the rest of the trial. The relative humidity was maintained between 65 and 70% Table 1. Composition and nutrient levels of basal diet. Composition  Percentage (%)  Nutrients value1  Content    0 to 21 d  22 to 42 d    0 to 21 d  22 to 42 d  Corn  57.67  59.80  Crude protein (%)  20.51  19.28  Soybean meal  28.30  25.65  Apparent metabolizable energy (Mcal/kg)  3000  3100  Extruded soybean  8.00  8.00  Calcium (%)  1.01  0.90  Soybean oil  1.90  3.00  Total phophorus (%)  0.66  0.63  Limestone  1.30  1.10  Non-phytic phosphorus (%)  0.43  0.41  Dicalcium phosphate  1.70  1.60  Methionine (%)  0.56  0.42  Salt  0.30  0.30  Methionine + Cystine (%)  0.91  0.76  Lysine-HCl (98.5%)  0.10  –  Lysine (%)  1.15  1.01  DL-Methionine  0.25  0.12  Tryptophan (%)  0.24  0.22  Theronine  0.05  –  Theronine (%)  0.81  0.72  Vitamins premix2  0.03  0.03        50% Choline chloride  0.10  0.10        Micro-Mineral premix3  0.30  0.30        Total  100.00  100.00        Composition  Percentage (%)  Nutrients value1  Content    0 to 21 d  22 to 42 d    0 to 21 d  22 to 42 d  Corn  57.67  59.80  Crude protein (%)  20.51  19.28  Soybean meal  28.30  25.65  Apparent metabolizable energy (Mcal/kg)  3000  3100  Extruded soybean  8.00  8.00  Calcium (%)  1.01  0.90  Soybean oil  1.90  3.00  Total phophorus (%)  0.66  0.63  Limestone  1.30  1.10  Non-phytic phosphorus (%)  0.43  0.41  Dicalcium phosphate  1.70  1.60  Methionine (%)  0.56  0.42  Salt  0.30  0.30  Methionine + Cystine (%)  0.91  0.76  Lysine-HCl (98.5%)  0.10  –  Lysine (%)  1.15  1.01  DL-Methionine  0.25  0.12  Tryptophan (%)  0.24  0.22  Theronine  0.05  –  Theronine (%)  0.81  0.72  Vitamins premix2  0.03  0.03        50% Choline chloride  0.10  0.10        Micro-Mineral premix3  0.30  0.30        Total  100.00  100.00        1Determined values except for apparent metabolizable energy. 2Provided per kilogram of diet: vitamin A, 15,000 IU; vitamin D3, 3000 IU; vitamin E, 20 IU; vitamin K3, 2.18 mg; thiamine, 2.15 mg; riboflavin, 8.00 mg; pyridoxine, 4.40 mg; vitamin B12 0.02 mg; calcium pantothenate, 25.60 mg; nicotinic acid, 65.80 mg; folic acid, 0.96 mg; biotin, 0.20 mg. 3Provided per kilogram of diet: Fe, 100 mg; Cu, 8 mg; Zn, 78 mg; Mn, 105 mg; I, 0.5 mg; Se, 0.3 mg. View Large Table 1. Composition and nutrient levels of basal diet. Composition  Percentage (%)  Nutrients value1  Content    0 to 21 d  22 to 42 d    0 to 21 d  22 to 42 d  Corn  57.67  59.80  Crude protein (%)  20.51  19.28  Soybean meal  28.30  25.65  Apparent metabolizable energy (Mcal/kg)  3000  3100  Extruded soybean  8.00  8.00  Calcium (%)  1.01  0.90  Soybean oil  1.90  3.00  Total phophorus (%)  0.66  0.63  Limestone  1.30  1.10  Non-phytic phosphorus (%)  0.43  0.41  Dicalcium phosphate  1.70  1.60  Methionine (%)  0.56  0.42  Salt  0.30  0.30  Methionine + Cystine (%)  0.91  0.76  Lysine-HCl (98.5%)  0.10  –  Lysine (%)  1.15  1.01  DL-Methionine  0.25  0.12  Tryptophan (%)  0.24  0.22  Theronine  0.05  –  Theronine (%)  0.81  0.72  Vitamins premix2  0.03  0.03        50% Choline chloride  0.10  0.10        Micro-Mineral premix3  0.30  0.30        Total  100.00  100.00        Composition  Percentage (%)  Nutrients value1  Content    0 to 21 d  22 to 42 d    0 to 21 d  22 to 42 d  Corn  57.67  59.80  Crude protein (%)  20.51  19.28  Soybean meal  28.30  25.65  Apparent metabolizable energy (Mcal/kg)  3000  3100  Extruded soybean  8.00  8.00  Calcium (%)  1.01  0.90  Soybean oil  1.90  3.00  Total phophorus (%)  0.66  0.63  Limestone  1.30  1.10  Non-phytic phosphorus (%)  0.43  0.41  Dicalcium phosphate  1.70  1.60  Methionine (%)  0.56  0.42  Salt  0.30  0.30  Methionine + Cystine (%)  0.91  0.76  Lysine-HCl (98.5%)  0.10  –  Lysine (%)  1.15  1.01  DL-Methionine  0.25  0.12  Tryptophan (%)  0.24  0.22  Theronine  0.05  –  Theronine (%)  0.81  0.72  Vitamins premix2  0.03  0.03        50% Choline chloride  0.10  0.10        Micro-Mineral premix3  0.30  0.30        Total  100.00  100.00        1Determined values except for apparent metabolizable energy. 2Provided per kilogram of diet: vitamin A, 15,000 IU; vitamin D3, 3000 IU; vitamin E, 20 IU; vitamin K3, 2.18 mg; thiamine, 2.15 mg; riboflavin, 8.00 mg; pyridoxine, 4.40 mg; vitamin B12 0.02 mg; calcium pantothenate, 25.60 mg; nicotinic acid, 65.80 mg; folic acid, 0.96 mg; biotin, 0.20 mg. 3Provided per kilogram of diet: Fe, 100 mg; Cu, 8 mg; Zn, 78 mg; Mn, 105 mg; I, 0.5 mg; Se, 0.3 mg. View Large Growth Performance On 21 and 42 d, the BW and feed intake of broilers were recorded. The average daily feed intake (ADFI), average daily gain (ADG), and feed: gain ratio (F:G) were calculated. Sampling Procedure At the morning on 43 d, 12 birds close to the average weight from each treatment group (2 birds per replicate) were randomly selected, slaughtered by cervical dislocation and manually eviscerated, trying not to rupture the digestive tract segments. Digesta from gastrointestinal segments, namely the crop, gizzard, proventriculus, duodenum, jejunum, ileum, and cecum, were separately taken. Immediately upon obtaining the digesta from each section, the pH was determined with a pH meter (SFK Inc., Kolding, Denmark) as described by Chaveerach et al. (2004). The contents of the duodenum were gently collected to determine the digestive enzyme activities (Wu et al., 2013). The 2-cm-long segments from the duodenum (the middle part of the duodenal loop), the jejunum (the middle part between the end point of duodenal loop and Meckel's diverticulum) and the ileum (5 cm after Meckel's diverticulum) were sampled to measure morphology. Then digesta were collected from the cecum and stored immediately at −20°C for microbiological analysis. Digestive Enzyme Activities of Duodenal Contents The duodenal digesta samples were diluted 10 ×, based on the sample weight, with ice-cold PBS (pH 7.0), homogenized for 60 s, and sonicated for 1 min with 3 cycles at 30 s intervals. The sample was then centrifuged at 6000 g for 15 min at 4°C. The supernatants were divided into aliquots and stored at −70°C for the enzyme activity assays. The activities of trypsin, chymotrypsin, lipase, and amylase were measured according to the methods described by Lhoste et al. (1993). Morphological Measurement of the Duodenal, Jejunal, and Ileal Mucosa Three cross-sections for each intestinal segment (duodenum, jejunum, and ileum) were fixed with formalin solution and were prepared using the standard paraffin embedding procedures by sectioning 5 μm thickness and staining with hematoxylin and eosin. A total of 15 intact, well-oriented crypt-villus units were measured in each type of tissue for each sample. Villus height and crypt depth (CD) were determined using an image processing and analyzing system (version 6.0, Image-Pro Plus) and expressed in micrometer (μm). Microbiological Analyses The 1 g of sample was removed from the cecal digesta and then serially diluted (1:10) in 9 mL aliquots for maximum recovery of diluents (MRD, Oxoid, Basingstoke, UK), and spread (0.1 mL aliquots) on to selective agars for the preparation of a 10-fold dilution. The total aerobic bacteria and E. coli as pathogenic and hygiene indicators were isolated using brain heart infusion agar (BHI, Oxoid) with 5% defibrinated sheep blood and MacConkey (MC) agar, respectively, followed by aerobic incubation at 37°C for 24 h. All bacteria were counted and expressed as total colony forming unit (CFU)/g digesta, per gram of sample and results, were presented as log10 transformed data. Statistical Analyses The experiment was carried out as a completely randomized design with 5 treatments. Statistical analyses of all growth performance, histomorphology, pH, and enzyme data were subjected to one-way ANOVA using statistical analysis systems (SAS) statistical software package (Version 8, SAS Institute, Cary, NC, USA), and post hoc comparison of the means using Turkey (equal variances) test at P < 0.05. For log-transformed data of microbial counts were subjected to analyses of variance, and differences between treatments assessed by Dunnett's T3 (unequal variances) tests at P < 0.05. RESULTS Growth Performance Throughout the entire period (0 to 21 d, 22 to 42 d, and 0 to 42 d), the broilers in PC and acidified water groups (A1, A2, and A3) showed higher final BW, ADG, and lower F: G compared to NC group (P < 0.05; Table 2). The ADFI of the birds in PC and acidified water groups was significantly lower during 0 to 21 d (P < 0.05) and significantly higher during 22 to 42 d (P < 0.05) than those in NC group, but no significant difference existed among all groups during 0 to 42 d (P > 0.05). Table 2. Effects of acidified drinking water on growth performance of broilers during 0 to 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  BW (g)                0 d  43.3  43.5  43.5  43.5  43.4  0.02  0.25  21 d  748c  795a,b  780b  787a,b  810a  3.49  <0.001  42 d  2,289b  2,584a  2,590a  2,589a  2,606a  14.9  <0.001  ADG (g)                0 to 21 d  33.5c  35.8a,b  35.1b  35.4a,b  36.5a  0.17  <0.001  22 to 42 d  73.4b  85.2a  86.2a  85.8a  85.5a  0.63  <0.001  0 to 42 d  53.5b  60.5a  60.6a  60.6a  61.0a  0.36  <0.001  ADFI (g)                0 to 21 d  54.2a  50.7b  49.7b  50.4b  50.9b  0.24  <0.001  22 to 42 d  148b  156a  156a  157a  158a  0.88  0.057  0 to 42 d  101  103  103  104  105  0.44  0.34  F:G (g of feed/g of gain)                0 to 21 d  1.62a  1.42b  1.42b  1.42b  1.39b  0.01  <0.001  22 to 42 d  2.01a  1.83b  1.81b  1.83b  1.85b  0.01  <0.001  0 to 42 d  1.89a  1.71b  1.70b  1.71b  1.71b  0.01  <0.001  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  BW (g)                0 d  43.3  43.5  43.5  43.5  43.4  0.02  0.25  21 d  748c  795a,b  780b  787a,b  810a  3.49  <0.001  42 d  2,289b  2,584a  2,590a  2,589a  2,606a  14.9  <0.001  ADG (g)                0 to 21 d  33.5c  35.8a,b  35.1b  35.4a,b  36.5a  0.17  <0.001  22 to 42 d  73.4b  85.2a  86.2a  85.8a  85.5a  0.63  <0.001  0 to 42 d  53.5b  60.5a  60.6a  60.6a  61.0a  0.36  <0.001  ADFI (g)                0 to 21 d  54.2a  50.7b  49.7b  50.4b  50.9b  0.24  <0.001  22 to 42 d  148b  156a  156a  157a  158a  0.88  0.057  0 to 42 d  101  103  103  104  105  0.44  0.34  F:G (g of feed/g of gain)                0 to 21 d  1.62a  1.42b  1.42b  1.42b  1.39b  0.01  <0.001  22 to 42 d  2.01a  1.83b  1.81b  1.83b  1.85b  0.01  <0.001  0 to 42 d  1.89a  1.71b  1.70b  1.71b  1.71b  0.01  <0.001  Each value is a mean of duplicate assays. SEM = standard error of the mean; BW = body weight; ADG = average daily gain; ADFI = average daily feed intake; F: G = feed: gain ratio. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a–cMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance (ANOVA) using Tukey's test. View Large Table 2. Effects of acidified drinking water on growth performance of broilers during 0 to 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  BW (g)                0 d  43.3  43.5  43.5  43.5  43.4  0.02  0.25  21 d  748c  795a,b  780b  787a,b  810a  3.49  <0.001  42 d  2,289b  2,584a  2,590a  2,589a  2,606a  14.9  <0.001  ADG (g)                0 to 21 d  33.5c  35.8a,b  35.1b  35.4a,b  36.5a  0.17  <0.001  22 to 42 d  73.4b  85.2a  86.2a  85.8a  85.5a  0.63  <0.001  0 to 42 d  53.5b  60.5a  60.6a  60.6a  61.0a  0.36  <0.001  ADFI (g)                0 to 21 d  54.2a  50.7b  49.7b  50.4b  50.9b  0.24  <0.001  22 to 42 d  148b  156a  156a  157a  158a  0.88  0.057  0 to 42 d  101  103  103  104  105  0.44  0.34  F:G (g of feed/g of gain)                0 to 21 d  1.62a  1.42b  1.42b  1.42b  1.39b  0.01  <0.001  22 to 42 d  2.01a  1.83b  1.81b  1.83b  1.85b  0.01  <0.001  0 to 42 d  1.89a  1.71b  1.70b  1.71b  1.71b  0.01  <0.001  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  BW (g)                0 d  43.3  43.5  43.5  43.5  43.4  0.02  0.25  21 d  748c  795a,b  780b  787a,b  810a  3.49  <0.001  42 d  2,289b  2,584a  2,590a  2,589a  2,606a  14.9  <0.001  ADG (g)                0 to 21 d  33.5c  35.8a,b  35.1b  35.4a,b  36.5a  0.17  <0.001  22 to 42 d  73.4b  85.2a  86.2a  85.8a  85.5a  0.63  <0.001  0 to 42 d  53.5b  60.5a  60.6a  60.6a  61.0a  0.36  <0.001  ADFI (g)                0 to 21 d  54.2a  50.7b  49.7b  50.4b  50.9b  0.24  <0.001  22 to 42 d  148b  156a  156a  157a  158a  0.88  0.057  0 to 42 d  101  103  103  104  105  0.44  0.34  F:G (g of feed/g of gain)                0 to 21 d  1.62a  1.42b  1.42b  1.42b  1.39b  0.01  <0.001  22 to 42 d  2.01a  1.83b  1.81b  1.83b  1.85b  0.01  <0.001  0 to 42 d  1.89a  1.71b  1.70b  1.71b  1.71b  0.01  <0.001  Each value is a mean of duplicate assays. SEM = standard error of the mean; BW = body weight; ADG = average daily gain; ADFI = average daily feed intake; F: G = feed: gain ratio. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a–cMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance (ANOVA) using Tukey's test. View Large The final BW and ADG of PC group were significantly higher than A2 group (P < 0.05) but didn’t have significant differences when compared to A1 and A3 groups during 0 to 21 d. There was no difference in growth performance between PC and any acidified groups (P > 0.05) during 22 to 42 d and 0 to 42 d. And no difference was founded in growth performance between the acidified groups (P > 0.05) during any period. pH of Gastrointestinal Segments In comparison to NC group, the pH in crop, proventriculus and ileum declined significantly (P < 0.05) by 0.55, 0.69, and 0.70 in A2 group, and 0.63, 0.74, and 1.21 in A3 group, respectively, whereas A1 and PC groups only decreased the pH of the proventriculus (by 1.03 and 0.68, respectively) and ileum (by 1.23 and 0.81; P < 0.05; Table 3). Among 3 acidified groups, A2 and A3 groups had significantly lower digesta pH in crop and gizzard (P < 0.05), and A2 group had significantly higher pH in duodenum (P < 0.05) than A1 group, whereas there was no significant difference in ileum among 3 acidified groups (P > 0.05). The pH value in the jejunum and cecum was not affected by the treatments (P > 0.05). Table 3. Effect of acidified water at pH of gastrointestinal tract of broilers on day 42. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Crop  5.83a  5.79a  5.28b  5.20b  6.11a  0.07  <0.001  Gizzard  3.43a,b  3.56a  2.99b  3.09b  3.27a,b  0.06  0.004  Proventriculus  5.05a  4.02b  4.36b  4.31b  4.37b  0.07  <0.001  Duodenum  6.02a,b  5.88b  6.23a  6.17a,b  6.19a,b  0.04  0.023  Jejunum  6.33  6.27  6.19  6.20  6.33  0.03  0.41  Ileum  7.71a  6.48b  7.01b  6.50b  6.90b  0.09  <0.001  Cecum  6.53  6.37  6.55  6.37  6.50  0.05  0.64  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Crop  5.83a  5.79a  5.28b  5.20b  6.11a  0.07  <0.001  Gizzard  3.43a,b  3.56a  2.99b  3.09b  3.27a,b  0.06  0.004  Proventriculus  5.05a  4.02b  4.36b  4.31b  4.37b  0.07  <0.001  Duodenum  6.02a,b  5.88b  6.23a  6.17a,b  6.19a,b  0.04  0.023  Jejunum  6.33  6.27  6.19  6.20  6.33  0.03  0.41  Ileum  7.71a  6.48b  7.01b  6.50b  6.90b  0.09  <0.001  Cecum  6.53  6.37  6.55  6.37  6.50  0.05  0.64  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a–dMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance (ANOVA) using Tukey's test. View Large Table 3. Effect of acidified water at pH of gastrointestinal tract of broilers on day 42. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Crop  5.83a  5.79a  5.28b  5.20b  6.11a  0.07  <0.001  Gizzard  3.43a,b  3.56a  2.99b  3.09b  3.27a,b  0.06  0.004  Proventriculus  5.05a  4.02b  4.36b  4.31b  4.37b  0.07  <0.001  Duodenum  6.02a,b  5.88b  6.23a  6.17a,b  6.19a,b  0.04  0.023  Jejunum  6.33  6.27  6.19  6.20  6.33  0.03  0.41  Ileum  7.71a  6.48b  7.01b  6.50b  6.90b  0.09  <0.001  Cecum  6.53  6.37  6.55  6.37  6.50  0.05  0.64  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Crop  5.83a  5.79a  5.28b  5.20b  6.11a  0.07  <0.001  Gizzard  3.43a,b  3.56a  2.99b  3.09b  3.27a,b  0.06  0.004  Proventriculus  5.05a  4.02b  4.36b  4.31b  4.37b  0.07  <0.001  Duodenum  6.02a,b  5.88b  6.23a  6.17a,b  6.19a,b  0.04  0.023  Jejunum  6.33  6.27  6.19  6.20  6.33  0.03  0.41  Ileum  7.71a  6.48b  7.01b  6.50b  6.90b  0.09  <0.001  Cecum  6.53  6.37  6.55  6.37  6.50  0.05  0.64  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a–dMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance (ANOVA) using Tukey's test. View Large Digestive Enzyme Activity in Duodenum The activities of trypsin, chymotrypsin and amylase were not influenced by any experimental treatments (P > 0.05) (Table 4). There was a decrease (P < 0.05) in lipase activity in PC and all acidified water groups when compared to NC group (P < 0.05). Table 4. Effects of acidified water at the digestive enzyme activities (U/mgprot) in the duodenal contents of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Trypsin  4000  2809  2692  4926  3657  390  0.34  Chymotrypsin  6.94  5.38  4.47  6.12  4.66  0.44  0.34  Lipase  96.8a  46.6b  31.4b  50.6b  45.3b  5.90  <0.001  Amylase  5.97  5.04  3.72  6.38  4.94  0.57  0.67  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Trypsin  4000  2809  2692  4926  3657  390  0.34  Chymotrypsin  6.94  5.38  4.47  6.12  4.66  0.44  0.34  Lipase  96.8a  46.6b  31.4b  50.6b  45.3b  5.90  <0.001  Amylase  5.97  5.04  3.72  6.38  4.94  0.57  0.67  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tukey's test. View Large Table 4. Effects of acidified water at the digestive enzyme activities (U/mgprot) in the duodenal contents of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Trypsin  4000  2809  2692  4926  3657  390  0.34  Chymotrypsin  6.94  5.38  4.47  6.12  4.66  0.44  0.34  Lipase  96.8a  46.6b  31.4b  50.6b  45.3b  5.90  <0.001  Amylase  5.97  5.04  3.72  6.38  4.94  0.57  0.67  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Trypsin  4000  2809  2692  4926  3657  390  0.34  Chymotrypsin  6.94  5.38  4.47  6.12  4.66  0.44  0.34  Lipase  96.8a  46.6b  31.4b  50.6b  45.3b  5.90  <0.001  Amylase  5.97  5.04  3.72  6.38  4.94  0.57  0.67  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tukey's test. View Large Intestinal Histomorphology As shown in Table 5, in comparison to NC group, PC group significantly increased the CD in duodenum (P < 0.05) and didn’t affect any other indices in intestinal histomorphology (P > 0.05). The A1 group had significantly higher CD in jejunum (P < 0.05) and the lower ratio of villous height (VH) to CD (V/C) in jejunum and ileum (P < 0.05) than NC group, and significantly higher CD in duodenum and jejunum (P < 0.05) and lower V/C in 3 small intestinal segments (P < 0.05) than PC group. The A2 group had significant higher VH and higher V/C in jejunum than NC group (P < 0.05), and higher VH in jejunum and higher CD in duodenum than PC group (P < 0.05). The A3 group had significantly higher CD in ileum and lower V/C in jejunum and ileum than NC group (P < 0.05), and higher CD in duodenum and ileum and lower V/C in the 3 small intestinal segments than PC group (P < 0.05). Among 3 acidified groups, A2 group had higher VH in jejunum and higher V/C in jejunum and ileum than A1 and A3 groups. Table 5. Effects of acidified drinking water at the intestinal histomorphology of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Duodenum  VH (μm)  416  435  455  444  461  5.80  0.11  CD (μm)  122a,b  128a  124a,b  127a  115b  1.52  0.021  V/C (μm/μm)  3.40b  3.39b  3.69a,b  3.49b  4.03a  0.07  0.009  Jejunum  VH (μm)  350b  351b  390a  359b  364b  3.94  0.002  CD (μm)  116a,b  130a  121a,b  129a,b  115b  1.86  0.012  V/C (μm/μm)  3.02b  2.70c  3.23a  2.80c  3.17a,b  0.05  <0.001  Ileum  VH (μm)  318  302  310  309  313  2.51  0.41  CD (μm)  110b  111b  108b  121a  109b  1.34  0.003  V/C (μm/μm)  2.90a  2.74b  2.88a  2.54c  2.86a  0.03  <0.001  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Duodenum  VH (μm)  416  435  455  444  461  5.80  0.11  CD (μm)  122a,b  128a  124a,b  127a  115b  1.52  0.021  V/C (μm/μm)  3.40b  3.39b  3.69a,b  3.49b  4.03a  0.07  0.009  Jejunum  VH (μm)  350b  351b  390a  359b  364b  3.94  0.002  CD (μm)  116a,b  130a  121a,b  129a,b  115b  1.86  0.012  V/C (μm/μm)  3.02b  2.70c  3.23a  2.80c  3.17a,b  0.05  <0.001  Ileum  VH (μm)  318  302  310  309  313  2.51  0.41  CD (μm)  110b  111b  108b  121a  109b  1.34  0.003  V/C (μm/μm)  2.90a  2.74b  2.88a  2.54c  2.86a  0.03  <0.001  Each value is a mean of duplicate assays. SEM = standard error of the mean; VH = villus height; CD = crypt depth. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tukey's test. View Large Table 5. Effects of acidified drinking water at the intestinal histomorphology of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Duodenum  VH (μm)  416  435  455  444  461  5.80  0.11  CD (μm)  122a,b  128a  124a,b  127a  115b  1.52  0.021  V/C (μm/μm)  3.40b  3.39b  3.69a,b  3.49b  4.03a  0.07  0.009  Jejunum  VH (μm)  350b  351b  390a  359b  364b  3.94  0.002  CD (μm)  116a,b  130a  121a,b  129a,b  115b  1.86  0.012  V/C (μm/μm)  3.02b  2.70c  3.23a  2.80c  3.17a,b  0.05  <0.001  Ileum  VH (μm)  318  302  310  309  313  2.51  0.41  CD (μm)  110b  111b  108b  121a  109b  1.34  0.003  V/C (μm/μm)  2.90a  2.74b  2.88a  2.54c  2.86a  0.03  <0.001  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Duodenum  VH (μm)  416  435  455  444  461  5.80  0.11  CD (μm)  122a,b  128a  124a,b  127a  115b  1.52  0.021  V/C (μm/μm)  3.40b  3.39b  3.69a,b  3.49b  4.03a  0.07  0.009  Jejunum  VH (μm)  350b  351b  390a  359b  364b  3.94  0.002  CD (μm)  116a,b  130a  121a,b  129a,b  115b  1.86  0.012  V/C (μm/μm)  3.02b  2.70c  3.23a  2.80c  3.17a,b  0.05  <0.001  Ileum  VH (μm)  318  302  310  309  313  2.51  0.41  CD (μm)  110b  111b  108b  121a  109b  1.34  0.003  V/C (μm/μm)  2.90a  2.74b  2.88a  2.54c  2.86a  0.03  <0.001  Each value is a mean of duplicate assays. SEM = standard error of the mean; VH = villus height; CD = crypt depth. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tukey's test. View Large Cecal Microflora The A3 group reduced (P < 0.05) the total aerobic bacteria count in cecum, when compared to NC group, and had no significant difference to PC group (P > 0.05; Table 6). The acidified groups showed a tendency (P < 0.10) to reduce counts of E. coli when compared to NC group, but this effect was not statistically significant. There was no difference among A1, A2, and PC groups in counts of the total aerobic bacteria and E. coli counts in the cecum. Table 6. Effect of acidified water at cecal microflora (Log CFU/g) of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Total aerobic bacteria  7.87a  7.09a,b  7.00a,b  6.76b  6.95a,b  0.12  0.025  Escherichia coli  7.35  6.71  6.58  6.22  6.57  0.13  0.067  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Total aerobic bacteria  7.87a  7.09a,b  7.00a,b  6.76b  6.95a,b  0.12  0.025  Escherichia coli  7.35  6.71  6.58  6.22  6.57  0.13  0.067  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am–to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tamhane's T2 (unequal variances) test. View Large Table 6. Effect of acidified water at cecal microflora (Log CFU/g) of broilers on 42 d. Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Total aerobic bacteria  7.87a  7.09a,b  7.00a,b  6.76b  6.95a,b  0.12  0.025  Escherichia coli  7.35  6.71  6.58  6.22  6.57  0.13  0.067  Item  NC  A1  A2  A3  PC  Pooled SEM  P value  Total aerobic bacteria  7.87a  7.09a,b  7.00a,b  6.76b  6.95a,b  0.12  0.025  Escherichia coli  7.35  6.71  6.58  6.22  6.57  0.13  0.067  Each value is a mean of duplicate assays. SEM = standard error of the mean. NC = negative control; A1 = whole supply acidified water; A2 = supply acidified water during 0 to 14 d, 22 to 28 d, and 36 to 42 d; A3 = supply acidified water 24 h during 0 to 14 d and 10:00 am–to 4:00 pm during 15 to 42 d; PC = positive control. a,bMeans in lines without same superscripts were significantly different (P < 0.05). The differences between means and the effects of treatments were determined by one-way analysis of variance(ANOVA) using Tamhane's T2 (unequal variances) test. View Large DISCUSSION Acidification of drinking water has been previously shown to have a beneficial effect on water quality and growth performance in laying hens (Rehman et al., 2013). The present study showed that water acidification improved BW, ADG, and F:G of broilers as effective as the antibiotics growth promoter compound of 8 ppm colistin sulfate + 8 ppm enduracidin in diet. Our results are similar to those of Pesti et al. (2004) and Chaveerach et al. (2004), who reported that acidified water improved growth performance and BW gain and boosted the digestion of feed in comparison to regular water. Desai et al. (2007) also found that broilers are receiving blends of formic and propionic acids in drinking water had improved weight gain and feed conversion. Pesti et al. (2004) reported that organic acids significantly reduced the pH of all gastrointestinal tract segments in broilers. But in other studies, the pH reduction mainly happened in the upper gut such as crop (Byrd et al., 2001), and the cecal pH kept stable (Waldroup et al., 1995). Similarly, in this study, A2 and A3 groups had significantly decreased pH value for crop, proventriculus, and ileum when compared to NC group, whereas A1 and PC groups only had decreased pH in the proventriculus and ileum. In contrast, Cornelison et al. (2005) and Watkins et al. (2004), by using different acids, found no significant effect on the gizzard pH of broilers and turkeys. That may be a result of the difference in acid type and concentration. It has been demonstrated that organic acids concentrations decreased further down the digestive tract as a result of absorption and metabolism (Bolton and Dewar, 1965). Our experiment showed that the A2 group had higher villi height and V/C in the jejunum, the A1 group had deeper CD in jejunum and lower V/C in jejunum and ileum, and the A3 group had deeper crypts and lower V/C in the jejunum and ileum. These are similar to the results of Garcia et al. (2007), who observed the highest villi and deepest crypts in the jejunum in broilers fed formic acid in their diets. The current study is in agreement with the findings of Loddi et al. (2004) and Pelicano et al. (2005), who observed that the jejunum had the increased villus heights with the majority of the organic acidifiers. The cation of organic acids may play an important role in the modulation of intestinal histomorphology. Paul et al. (2007) discovered that organic acid salt supplementation significantly increased (P < 0.05) the villus height of different segments of small intestine and decreased the intestinal microbial load, which also decreased the presence of toxins that are related to changes in intestinal morphology in broilers. In this study, we observed that the acidified water (A1, A2, and A3 groups) had inhibitory effects on the aerobic bacterial count and the E. coli population in cecum contents. Similarly, Hamed and Hassan (2013) also observed that 7 d post-infection, Japanese quails that consumed an organic acid mixture and acetic acid water had (P < 0.05) reduced total bacterial counts in the ceca. Subsequent studies suggested that drinking water containing organic acids was helpful in reducing pathogens in the broiler (Hassan et al., 2010; Hamed and Hassan, 2013). Bunnik et al. (2012) also observed that the gizzard and stomach of birds drinking acidified water were more acidic and might decrease the bacteria that were present in the gastrointestinal tract, especially in the lower gastrointestinal tract. Organic acids provide a suitable pH in the gut, which increases beneficial bacteria, and decreases harmful bacteria of broilers (Roser, 2006). Meanwhile, accumulation of acid cations released from the anions in the higher pH environment inside cells is also an important mechanism by which organic acids inhibit bacterial growth (Carpenter and Roadbent, 2009). The antibacterial action of organic acids has been attributed to cytoplasmic acidification from proton discharge, causing inhibition of acid sensitive enzymes, for instance those engaged with glycolysis (Davidson, 2001). However, there is sufficient evidence to confirm that the accumulation of acid anions may be more inhibitory for cell development, especially in mild acidic conditions above pH 5.0 (Russell, 1992; Roe et al., 2002). Lipid-permeable, weak acids dissociate into the cytoplasm, resulting in decline of pH and the accumulation of anions (Salmond et al., 1984). The accumulation of anions can cause an osmotic problem for the cell if it leads to an increase in cell turgor pressure. The cell may recompense for this by discharging the anion itself, or by bringing down the concentration of other cellular anions, mainly glutamate (Roe et al., 1998). Such anion altercation would be a factor in decreasing intracellular pH and inhibition of cell function. Thus, lower pathogenic bacteria population in cecum contents under nearly same pH values in acidified groups could be explained by the lower pH in the upper gastrointestinal gut and higher acid anions (such as formate ion and propionate ion) in whole gut contents. The findings of the current study showed that there was a significant (P < 0.05) decline in lipase enzymes in PC and all acidified groups in comparison to NC group, whereas there was no significant difference on other enzymes.There are different factors affecting enzyme activity. It is known that there was modulation of the activity of various digestive enzymes in the same chicken species (Maria et al., 2014). Various reports point out that the concentration of digestive enzymes in poultry change with age (Krogdahl and Sell, 1989; Pubols, 1991; Noy and Sklan, 1995). Duodenal lipase activity in young chicks increases 20 times between 4 and 21 d of age (Noy and Sklan, 1995) in chickens. Gastric lipase enzyme has been appeared to be significantly inhibited by protonated free fatty acids (Borel et al., 1994; Wang et al., 2017). Previous studies on lipoprotein models (Miller and Small, 1982; Miller and Small, 1983; Miller and Small, 1987) and a current study consuming dietary mixtures (Borel et al., 1994) have exposed that 2 to 5 mole % of the droplet surface lipid is triacylglycerol, so allowing lipase action at the surface of the lipid droplet. Thereby, fatty acids inhibition may also a cause of reduced lipid activity. It has been found, when chickens were fed diets with variable protein and carbohydrate contents, that the activities of the digestive enzymes are modulated by the availability of the substrates (Siddons, 1972; Biviano et al., 1993). Additionally, research conducted by Hulan and Bird (1972) exposed that increase in dietary carbohydrates and fat enhanced the activity of amylase and lipase enzymes, whereas the dietary addition of inorganic compounds, including natural zeolite and minerals, also produced substantial morphological and enzymatic variations in the intestine of broiler chickens (Incharoen et al., 2009; Ruttanavut and Yamauchi, 2010). Several researchers (Osman, 1982; Kihara and Sakata, 1997) also reported that EDTA addition causes a decrease in lipase activity and found that the optimum pH for intestinal lipase is 9. It has already been observed that acidified water reduces intestinal pH, so as a result of being at lower than optimal pH lipase enzyme activity might reduce. On the other hand, the intestinal microflora population is thought to change the intestinal environment and produce enzymes and other beneficial substances in the intestines (Marteau and Rambaud, 1993). Acidified water is known to reduce bacterial load so that lesser enzymes being available in the body from bacteria might be responsible for lower enzyme activity. However, the effects of acidified drinking water on lipase activity need more research. This study shows that acidified drinking water can alter pH in the gut and has a positive effect on the microflora balance of the gastrointestinal tract and the growth performance by reducing harmful gut bacteria. The morphological changes of intestinal mucosa could partly explain the improvement of FCR by acidified drinking water. Moreover, compared to continuous supply of acidified water, discontinuous supply of acidified water had the same or even better influence on broilers performance. It may be due to host adaptation to constant water acidification by reducing endogenous acid production or may be due to an adaptive tolerance response. A mildly acidic environmental stress (pH 5.5 to 4.5) produces response in several bacteria and offers protection towards subsequent exposure to deadly stress (pH < 4.0). This acid-tolerance response has been recognized and studied in an extensive variety of gram-negative and gram-positive bacterial species, confirming that exposure to mildly acidic pH induces the expression of several acid-shock proteins that encourage bacterial survival in subsequent extreme acid environments (Booth et al., 2002). The main defense mechanism, which defends the cell from acid, is alterations in membrane composition (Chang and Cronan, 1999; Jordan et al., 1999; Yuk and Marshall, 2004) and changes in internal pH homeostasis systems (Park et al., 1996; Richard and Foster, 2004). In this way, intermittent acidic water supply might change the internal pH and bacterial growth, and the subsequent decline in bacterial growth might produce a positive effect on the health of the animal. ACKNOWLEDGMENTS We greatly appreciate the support of National Key Research and Development Program of China (grant No. 2017YFD0500500), Public Sector (Agriculture) Scientific Research of China (grant No. 201403047) and National Key Technology Research and Development Program of China (grant No. 2011BAD26B04 and 2012BAD39B01). REFERENCES Aclkgoz Z., Bayraktar H., Aclkgoz Z., Bayraktar H., Altan Ö.. 2011. Effects of formic acid administration in the drinking water on performance, intestinal microflora and carcass contamination in male broilers under high ambient temperature. Anim Feed Sci Technol . 24: 87– 94. Biviano A. B., Del Rio C. M., Phillips D. L.. 1993. Ontogenesis of intestine morphology and intestinal disaccharidases in chickens (Gallus gallus) fed contrasting purified diets. J. Camp. Physiol. B . 163: 508– 518. Boling-Frankenbach S., Snow J., Parsons C., Baker D.. 2001. The effect of citric acid on the calcium and phosphorus requirements of chicks fed corn-soybean meal diets. 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Poultry ScienceOxford University Press

Published: Jun 1, 2018

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