Effect of organic acids or probiotics alone or in combination on growth performance, nutrient digestibility, enzyme activities, intestinal morphology and gut microflora in broiler chickens

2.1 Growth performance

A total of 1,440 one-day-old broiler chicks (Ross308) were individually weighed and randomly assigned to four groups. Each group contained five replicates, with 72 chicks per replicate (36 males and 36 females). The chicks were reared in two phases (starter phase, days 0–21 and grower phase, days 22–35). The nutrient composition for both starter and grower phases was formulated to be similar to the requirement of the birds according to the NRC (1994) recommendations as shown in Table 1. This control diet (CD) was supplemented with 2 g/kg organic acids (CDOA, 1 kg contained 320 g fumaric acid, 30 g formic acid, 130 g lactic acid, 30 g propionic acid and 10 g citric acid), 2 g/kg probiotics (CDP, 1 kg contained 30 g bacillus spores, bacillus count ≥5 × 10¹¹ CFU/kg, 620 g dried fermentation products and nutritive media, 347 g sodium sulphate and 3 g anticaking agent) or its combination (CDOAP, 2 g/kg organic acids and 2 g/kg probiotics). The chicks for both starter and grower phases were raised in an open-house system and fed ad libitum with the basal CD or the experimental diets. Drinking water was available ad libitum throughout the experiment. The chicks fed either the CD or the experimental diets were weighed 21 and 35 days after starting the experiment. Feed intake (FI) and body weight gain (BWG) were recorded and used to compute the FCR per chick. For economical efficiency, feed cost (FC), feed cost of production (FCP) and total feed cost (TFC) of the experimental diets were also evaluated. The mortality rate of chicks was recorded throughout the experiment, and all the treatments were similar and had 98.35% survival (on average) at the end of the period studied. All the experimental protocols describing the management and care of animals in this study were reviewed and approved by the Animal Care and Use Committee of the Institute of Animals for Scientific Purposes Development, National Research Council of Thailand (Code U1-01783-2558).

2.2 Nutrient digestibility

At day 35, a total of 20 male chicks with similar body weights were tested to measure the digestibility of their dietary treatments (n = 5 per treatment). All the chicks were randomly assigned into individual metabolic cages (40 cm width × 40 cm length × 45 cm height). The four grower phase dietary treatments were randomly allotted to individual cages for a total period of 8 days, consisting of 5 days of diet acclimation followed by 3 days during which the feed digestibility was measured. During days 40 to 42, all the excreta from each cage was collected and the total FI was measured for each cage individually. The excreta from the metabolic cages, which was weighed separately every day during the sample collection period, was collected in plastic trays and stored in a deep freezer until required for analysis (Kirkpinar, Açikgöz, Bozkurt, & Ayhan, 2004). At the end of the three-day collection period, the excreta from each replicate was mixed, ground and representative samples were then taken for proximate composition determination according to the methods of AOAC (2000). The procedures utilized for the determination of dry matter and nutrients were determined in accordance with the methods described by Banerjee (1978).

2.3 Digestive enzyme extraction

The same sample chicks from the digestibility studies were used for digestive enzyme activity assay (n = 5 per treatment) at the age of 42 days. Each bird was sacrificed by cervical dislocation, and the proventriculi of the chicks were dissected. Intestinal sections were carefully removed, identified and dissected on ice as follows: duodenum (divided from gizzard to pancreo-biliary ducts), jejunum (divided from pancreo-biliary ducts to Meckel’s diverticulum) and ileum (divided from Meckel’s diverticulum to ileo-caecal junction). To extract the broiler digestive enzymes and quantify the protein concentration, their frozen proventriculi and intestines (including the duodenum, jejunum and ileum) were weighed, cut into small pieces and then homogenized in 0.2 M phosphate buffer pH 7 (1:5 w/v), using a microhomogenizer (THP-220; Omni International, Kennesaw, GA, USA). The homogenate was centrifuged at 13,000 × g, at 4°C for 20 min, and aliquots prepared which were kept at −20°C until use. The protein concentration from a crude enzyme extract was compared to a standard curve of bovine serum albumin (BSA), within a linear range, according to the standard method of Lowry, Rosenbrough, Farr, and Randall (1951).

2.4 Digestive enzyme assays

Proventriculus extract only was used for assaying pepsin activity (EC 3.4.23.1) while three intestinal extracts were used to assay the activities of trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21.1), α-amylase (EC 3.2.1.1), cellulase (EC 3.1.1.4) and lipase (EC 3.1.1.3). All analyses were conducted at 37°C with a preferred pH as described in previous reports (Gabriel, Mallet, & Leconte, 2003; Ghalehkandi et al., 2011; Jang, Ko, Kang, & Lee, 2007; Mahmood, Khan, Sarwar, & Nisa, 2008; Shin, Han, Ji, Kim, & Lee, 2008). The pepsin activity was assayed using casein as a substrate, according to the method of Rungruangsak and Utne (1981). The L-tyrosine standard was used against the absorbance of liberated product at 720 nm. The activities of trypsin and chymotrypsin were assayed using N-benzoyl-L-Arg-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrates, respectively, according to the method described by Rungruangsak-Torrissen, Moss, Andresen, Berg, and Waagbo (2006). The colorimetric determination of p-nitroanilide was taken at 410 nm against liberated product. The activities of α-amylase and cellulase were determined using soluble starch and carboxymethylcellulose as substrates, according to the method of Areekijseree et al. (2004) and Mendels and Weber (1969) respectively. The concentration of products was quantified against standard maltose and glucose, respectively, at 540 nm. Lipase activity was determined according to the method of Winkler and Stuckmann (1979) using p-nitrophenyl palmitate as the substrate. The colorimetric detection was measured at 410 nm against the standard linear range of p-nitrophenol.

2.5 Excreta thermal properties

Digestible (low temperature) and indigestible (high temperature) elements after digestion and absorption were monitored by the thermal properties of the excreta (Kanghae et al., 2017; Wattanakul, Thongprajukaew, Songnui, Satjarak, & Kanghae, 2017). Fresh broiler excreta (n = 5 per treatment) was collected from trays during the last 3 days prior to the end of the experiment. The thermal properties of the excreta were determined according to the method described by Wattanakul et al. (2017) with slight modifications. A three-milligram sample was placed in an aluminium pan, sealed, allowed to equilibrate at room temperature and then heated from 20 to 500°C at a rate of 10°C/min, against an empty reference pan. The thermal parameters, in terms of onset (T_o), peak (T_p) and conclusion (T_c) temperatures and enthalpy (ΔH), were determined using a differential scanning calorimeter (DSC7; Perkin Elmer, Waltham, MA, USA).

2.6 Intestinal microanatomy and digestive microflora counts

At day 35, a total of 80 chicks consisting of 20 chicks (10 males and 10 females) per treatment were randomly selected. The chicks were sacrificed by cervical dislocation, and a 2-cm duodenal segment of each small intestine was collected, fixed in Bouin fluid, dehydrated with a standard alcohol–toluene sequence and embedded in paraffin wax. Five-micrometre slices were prepared and stained with haematoxylin and eosin. The intestinal villi with their crypts of Lieberkühn from each segment were individually measured in 70 microscopic fields using an image analysis system. The villus height to crypt depth ratio was also calculated for each segment (Santin et al., 2001).

Digesta samples were collected from the ileum and caecum. Contaminating micro-organisms were removed by immersing the surface sample in 70:100 (v/v) ethanol, then immersing them in sterile distilled water for 30 s (Ghanbari, Rezaei, Jami, & Nazari, 2009). The samples were suspended (1:10 w/v) in normal saline solution (0.85:100 w/v) and were homogenized on a Stomacher laboratory blender for 1 min and then 10-fold serially diluted. One hundred microlitres of the dilution was plated onto eosin methylene blue agar (EMB) as well as Lactobacilli MRS Agar (MRS). The EMB plates were incubated at 37°C for 18 hr. The MRS agar was incubated at 37°C for 48 hr under anaerobic condition. The lactobacilli and coliform bacteria were counted using a colony counter. The results were expressed as the log₁₀ of colony-forming units (CFU) per gram of content.

2.7 Statistical analysis

All the obtained data were analysed in a completely randomized design using the PROC UNIVARIATE procedure (SAS 2015 version 9.4; SAS Institute, Cary, NC, USA). The statistical analyses were conducted using one-way ANOVA for all the results. The effects of dietary supplementation on digestive enzyme activities at different parts of the GI tract were analysed using two-way ANOVA with treatment, intestinal segments and their interactions as the factors. Means were separated by Duncan’s multiple range test (Duncan, 1955). Variability in the data was expressed as the pooled standard error (SE), and results were considered to be statistically significant where p < .05.

3.1 Growth performance and economical efficiency

There were no significant (i.e., p > .05) differences based on the dietary treatments in growth performance during the starter phase or the grower phase (Table 2). Neither the organic acids nor the probiotics alone nor their combination affected FI, BWG and FCR, and the majority of the data were similar for all the treatments. However, during the starter phase (days 0–21), the chicks fed on CDOA, CDP and CDOAP showed slightly higher BWG and FCR than those fed on the CD although the difference was not significant (p > .05). FC, FCP and TFC of the experimental diets were relatively high in the CDOAP and CDP treatments followed by the CDOA, relative to the CD (Table 2).

3.2 Nutrient digestibility

The digestion coefficients of the dry matter, crude protein, ether extract, ash and nitrogen-free extract were not significantly different in the chicks fed on different treatments (p > .05, Table 3). Nonetheless, the crude fibre digestibility in the chicks fed on CDOA and CDOAP showed significantly higher fibre digestion than that in the chicks fed the CD (p < .05) for the period from 35 to 42 days.

3.3 Specific activities of digestive enzymes

Supplementation with probiotics or organic acids alone or in combination had no effect on the specific activities of any of the observed enzymes nor on the specific activity of amylase to trypsin, the A/T ratio (Table 4). The specific activities of trypsin, chymotrypsin and amylase were highest in the duodenum followed by the jejunum and the ileum. The reverse trend was observed for cellulase, for which the highest specific activity was in the ileum. There were no significant (p > .05) differences in the lipase-specific activity nor in the A/T ratio across the three intestinal segments. Based on a two-way ANOVA, there was no effect on the A/T ratio or digestive enzyme activities from dietary supplementation alone nor in combination with the intestinal segment in which the measurement was taken, while intestinal segments alone had significant (p < .05) effects on all the observed enzymes, except for lipase and the A/T ratio.

3.4 Thermal characteristics of excreta

The thermal properties of the available nutrients present in excreta, in terms of T_o, T_p, T_c, T_c–T_o and ΔH, were not affected by dietary supplementation with probiotics or organic acids alone or in combination (Table 5).