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

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


A feeding trial was conducted to determine the effect of organic acids or probiotics alone or in combination on growth performance, nutrient digestibility, enzyme activity, intestinal morphology and gut microflora in broiler chickens (Ross308). A completely randomized design was used, with 1,440 broiler chicks across four treatments and five replications of 72 chicks each. The chicks in the control treatment were fed on a control diet (CD), whereas for the other treatment groups, the CD was supplemented with 0.2 g/kg organic acids (CDOA), probiotics (CDP) or a combination of organic acids and probiotics (CDOAP). All the chicks were fed ad libitum during the feeding trial throughout 35 days. A total of 20 chicks were randomly allotted to individual metabolic cages to measure the nutrient digestibility (35–42 days) and the digestive enzyme activities (42 days). The intestinal morphology and gut microflora of 80 chicks were examined at the end of experiment. There were no significant (p > .05) differences in the feed intake, body weight gain or feed conversion ratio of the chicks across the four dietary treatments. The crude fibre digestibility was significantly increased in chicks fed on CDOA or CDOAP relative to CD (p < .05). Nutrient utilization, in terms of digestive enzyme activities and excreta thermal property, was unchanged by any supplementation. The chicks fed on the CDOAP had significantly higher duodenal villi height and crypt depth than the chicks fed on CDOA (p < .05). This dietary treatment dramatically improved gut microflora by decreasing the population of Escherichia coli and increasing the Lactobacillus spp.:E. coli ratio. Based on our investigations, supplementation of organic acids and probiotics in chick diets can increase the ability to digest crude fibre and villus height and decrease intestinal E. coli without impairing growth performance.


The gastrointestinal (GI) tract system is of high importance in the production of animals, contributing to growth rate and successful feed efficiency. The optimal gut health is directly involved in the balance of gut microflora, the control of the immune system and its inflammation, the protection of enteric pathogens and the digestion and absorption of nutrients. While the global production of poultry meat has been growing fast, the use of antibiotics has significantly improved animal health by lowering the incidence of diseases (Diarra & Malouin, 2014), enhancing growth and feed efficiency as well as reducing mortality in broiler production. However, their use was introduced without rigorous testing (Graham, Boland, & Silbergeld, 2007) and in recent years, the nature of antibiotic use in poultry production has changed considerably because of concerns about potential negative consequences for human health caused by their use (Singer & Hofacre, 2006). For example, the European Union has banned the use of growth-promoting antibiotics (AGPs) and their use is being reassessed in the United States. Therefore, current research is focused on finding alternatives to antibiotics in the production of sustainable animal food products to allay environmental and food safety concerns (Kogut & Arsenault, 2016) as well as finding alternative supplements, such as organic acids and probiotics.

The acidification of diets using various weak organic acids such as formic, fumaric, propionic, lactic and sorbic acid has been reported to decrease colonization by pathogens and the production of toxic metabolites, as well as improving the digestibility of protein and of Ca, P, Mg and Zn which can serve as substrates in intermediary metabolism (Hassan, Mohamed, Youssef, & Hassan, 2010; Kirchgessner & Roth, 1988). In the extensive rearing of broiler chickens, organic acid supplements in their diet can enhance their utilization of nutrients and improve their growth and feed conversion efficiency (Agboola, Omidiwura, Odu, Popoola, & Iyayi, 2015). Organic acids and their salts can penetrate a bacterium’s cell wall and disrupt its normal physiology as well as reducing the pH of the digesta, increasing pancreatic secretion and can also have trophic effects on the mucosa of the GI tract of animals (Adil, Banday, Bhatm, Mir, & Rehman, 2010). Moreover, the creation of an acidic environment (pH 3.5–4.0) in the gut of broiler chickens by organic acids favours the development of lactobacilli and inhibits the replication of Escherichia coli, Salmonella and other gram-negative bacteria (Chowdhury et al., 2009; Kopecký, Hrnčár, & Weis, 2012), thus improving their gut health and feed conversion ratio (FCR).

Probiotics are viable micro-organisms which provide beneficial effects to their host by modifying the intestinal microbiota (Fatufe & Matanmi, 2011). In livestock nutrition, Bacillus, Enterococcus and Saccharomyces are the most common organisms used (Simon, Jadamus, & Vahjen, 2001). The mode of action of probiotics in poultry includes maintaining normal intestinal microflora by competitive exclusion antagonism, lowering the pH through acid fermentation, competing for mucosal attachment and nutrients, producing bacteriocins, stimulating the immune system associated with the gut and increasing the production of short-chain fatty acids (Sarangi et al., 2016). Both Pirgozliev, Murphy, Owens, George, and McCannin (2008) and Agboola et al. (2015) have reported the beneficial effects of dietary additives (organic acids and probiotics) on energy and protein utilization in poultry. Both constituents are able to modify the microflora which can be the origin of GI sickness and favour a healthy intestinal microflora (Çelik, Mutluay, & Uzatici, 2007; Conway & Wang, 1997; Youssef, Mostafa, & Abdel-Wahab, 2017). Nevertheless, there is a paucity of information on the efficacy of combining organic acids and probiotics and the objective of this study was to evaluate their effect alone and in combination on growth performance, nutrient digestibility, enzyme activities, intestinal morphology and gut microflora in broilers. The practical findings from this study might help to improve the production of 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).

Table 1. Ingredient and chemical composition of the basal diets used for feeding broiler chicks
Ingredient and composition Basal diet
Starter phase Grower phase
  1. aTrace mineral premix, 1 kg of diet contained 149.6 mg of manganese, 125.1 mg of zinc, 16.5 mg of iron, 1.7 mg of copper, 1.05 mg of iodine, 0.25 mg of selenium, a minimum of 6.27 mg of calcium and a maximum of 8.69 mg of calcium. The carrier is calcium carbonate, and the premix contains less than 1% mineral oil.
  2. bVitamin premix, 1 kg of diet contained 11,023 IU of vitamin A, 3,858 IU of vitamin D3, 46 IU of vitamin E, 0.0165 mg of B12, 5.845 mg of riboflavin, 45.39 mg of niacin, 20.21 mg of D-pantothenic acid, 477.67 mg of choline, 1.47 mg of menadione, 1.75 mg of folic acid, 7.17 mg of pyridoxine, 2.94 mg of thiamine and 0.55 mg of biotin. The carrier is ground rice hulls.
Ingredient (g/kg)
Corn 510 530
Rice bran 100 100
Soybean meal 264.3 243.5
Fish meal 60 60
Palm oil 36.6 41.6
Dicalcium phosphate 9.5 6.9
Limestone 9.3 8.1
Sodium chloride 3.5 3.5
Mineralsa 2.5 2.5
Vitaminsb 2.5 2.5
DL-Methionine 1.8 1.4
Calculated composition (g/kg)
Crude protein 215 200
Calcium 10 9
Non-phytic phosphorus 4.5 4
Lysine 11.5 10.7
Methionine 5.5 5.0
Metabolizable energy (MJ/kg) 12.4 12.8

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 (= 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 (= 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 while three intestinal extracts were used to assay the activities of trypsin (EC, chymotrypsin (EC, α-amylase (EC, cellulase (EC and lipase (EC 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 (= 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 (To), peak (Tp) and conclusion (Tc) 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).

Table 2. Growth performance of the broilers fed different dietary treatments
Period (day) Parameter Treatment SEM p-Value
  1. CD, control diet; CDOA, control diet supplemented with organic acids; CDP, control diet supplemented with probiotics; CDOAP, control diet supplemented with organic acids and probiotics; FI, feed intake; BWG, body weight gain; FCR, feed conversion ratio; FC, feed cost; FCP, feed cost of production; TFC, total feed cost.
  2. aFCP (USD/kg) = Feed cost × FCR.
  3. bTFC (USD/total gain) = Starter (gain × FCP) + grower (gain × FCP).
0–21 FI (g/b) 692.53 689.95 689.99 694.44 6.22 .619
BWG (g/b) 449.80 471.84 458.35 450.30 25.03 .489
FCR 1.54 1.46 1.52 1.55 0.08 .368
FC (USD/kg) 0.53 0.54 0.55 0.56
FCP (USD/kg)a 0.82 0.79 0.84 0.87
22–35 FI (g/b) 881.09 880.16 885.53 890.86 34.36 .958
BWG (g/b) 823.59 775.02 779.55 773.85 31.45 .068
FCR 1.06 1.13 1.13 1.15 0.05 .078
FC (USD/kg) 0.52 0.53 0.55 0.55
FCP (USD/kg)a 0.56 0.60 0.62 0.63
0–35 FI (g/b) 1,573.63 1,570.11 1,575.53 1,585.30 34.45 .909
BWG (g/b) 1,273.39 1,246.86 1,237.91 1,224.15 39.90 .292
FCR 1.30 1.30 1.32 1.35 0.04 .395
FCP (USD/kg)a 0.65 0.67 0.70 0.72
TFC (USD/total gain)b 0.83 0.84 0.87 0.89

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.

Table 3. Digestion coefficients of broilers fed different dietary treatments
Parameter Treatment SEM p-Value
  1. CD, control diet; CDOA, control diet supplemented with organic acids; CDP, control diet supplemented with probiotics; CDOAP, control diet supplemented with organic acids and probiotics.
  2. Significant differences in each row are indicated by different superscripts (p < .05).
Dry matter 0.88 0.87 0.86 0.87 3.15 .833
Crude protein 0.75 0.75 0.74 0.76 3.74 .875
Ether extract 0.77 0.76 0.76 0.78 5.00 .862
Crude fibre 0.70b 0.75a 0.72ab 0.76a 3.16 .030
Ash 0.74 0.74 0.72 0.75 3.81 .727
Nitrogen-free extract 0.83 0.84 0.82 0.83 4.46 .962

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.

Table 4. Specific activities (mU/mg protein) of protein-, carbohydrate- and lipid-digesting enzymes in various part of broiler GI tract
Factor Proventriculus Intestine
Pepsin Trypsin Chymotrypsin Amylase Cellulase Lipase Amylase:Trypsin ratio
  1. CD, control diet; CDOA, control diet supplemented with organic acids; CDP, control diet supplemented with probiotics; CDOAP, control diet supplemented with organic acids and probiotics.
  2. Significant differences in each column are indicated by different superscripts (p < .05).
CD 12.83 4.24 3.79 2,294.41 450.08 167.70 591.69
CDOA 10.79 3.98 3.27 2,398.38 434.16 132.80 694.61
CDP 10.23 5.65 4.66 2,257.42 406.95 146.18 490.06
CDOAP 11.21 4.37 3.42 2,158.19 487.84 129.81 533.24
SEM 1.45 0.63 0.85 210.68 21.93 25.01 59.42
Duodenum 5.75a 5.85a 2,424.99a 400.25b 114.03 536.50
Jejunum 4.41ab 3.84a 2,558.96a 429.36b 155.32 656.04
Ileum 3.53b 1.67b 1,847.20b 504.66a 163.03 539.66
SEM 0.54 0.74 182.45 18.99 21.65 51.46
Treatment (T) 0.628 0.253 0.666 0.881 0.082 0.700 0.098
Segment (S) 0.021 0.001 0.019 0.001 0.238 0.184
T × S 0.483 0.290 0.227 0.375 0.293 0.679

3.4 Thermal characteristics of excreta

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

Table 5. The thermal transition characteristics of faeces collected from broilers when fed control or experimental diets, supplemented with probiotics or organic acids alone or in combination
Thermal parameter Treatment SEM p-Value
  1. CD, control diet; CDOA, control diet supplemented with organic acids; CDP, control diet supplemented with probiotics; CDOAP, control diet supplemented with organic acids and probiotics; To, onset temperature; Tp, peak temperature; Tc, conclusion temperature; TcTo, temperature range; ΔH, transition enthalpy.
To (°C) 91.90 89.47 98.01 91.02 3.39 .344
Tp (°C) 112.13 111.75 111.67 112.29 3.17 .999
Tc (°C) 131.21 133.56 132.44 135.42 3.65 .867
TcTo (°C) 39.31 44.09 34.44 44.40 4.15 .325
ΔH (J/g) 1,550.11 1,543.21 1,503.18 1,551.12 11.39 .365




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