Journal of Applied Poultry Research

Volume 30, Issue 3, September 2021, 100179

Effect of dietary supplementation of acidifiers and essential

oils on growth performance and intestinal health of broiler
R. Emili Vinolya, U. Balakrishnan , B. Yasir, S. Chandrasekar

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https://doi.org/10.1016/j.japr.2021.100179
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SUMMARY
The present study was conducted to evaluate the efficacy of combinations of organic, inorganic acidifier and essential
oil blends on growth performance and intestinal health of broilers. A total of 1,200 Vencobb 400 male and female
chicks were randomly distributed to 6 treatment groups with 10 replicates of 20 birds each. Treatments groups were
Control (C) without acidifier, combination of inorganic acid/organic acids (T1), combinations of organic acids (T2),
combination of organic acids and essential oil (T3), combination of organic acids and medium chain fatty acids (T4) and
double salted organic acid (T5). Corn-soybean diet supplemented with 5% meat and bone meal was fed to provoke
challenges on intestinal health conditions. Body weight gain, feed intake, feed conversion ratio, and livability were
measured until 42 d on weekly basis. On d 42, intestinal lesion scoring and ileal microbial populations of birds (Total
coliforms, Clostridium perfringens, lactic acid bacteria) were assessed. On d 42, combination of organic acids (T2) and
organic acids with essential oil (T3) had shown significantly improved body weight and average daily gain over control
(C) (P < 0.05). Combination of organic acids (T2) also showed significantly improved feed conversion by 5 points over
control (P < 0.05). In terms of microbiological parameters, all the treatments except T5 had shown significantly lower (P
< 0.05) bacterial enteritis lesions and Clostridial populations, over control. Based on the outcome of trial results,
combination of organic acids (T2) and combination with essential oil (T3) had shown enhanced performance with
better economic return under challenged dietary conditions with animal protein source. The findings of this study shed
light on the supplementation of organic acids acidifiers as an able substitute to improve the performance of broilers
without the use of feed antibiotics.

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Key words
organic acids; acidifiers; broiler performance; intestinal health

DESCRIPTION OF PROBLEM
The acidifiers in animal diets are well proven to reduce the rate of disease occurrence in animal intestinal tract thereby
improving the productive performance. The role of acidifiers in feed helps to maintain the optimal intestinal pH for an
efficient proteolytic digestive enzyme activity and antimicrobial activity on intestinal pathogens to reduce subclinical
infections (Yadav et al., 2016; Khan and Iqbal, 2016; Sugiharto, 2016). Organic acids in the literature have a particular
proclivity to kill Gram negative bacteria. This may be due to higher cell pH which causes the dissociation of the acid,
releasing H+ ions which lowers the pH of the microbial cell, also causing metabolic problems because of remaining
anions. Several types of acidifiers are available for use in animal feed applications as organic acids, short chain fatty
acids (SCFA), medium chain fatty acids (MCFA), inorganic acids and salt of organic acids.

SCFA are organic acids with one to seven carbons with a carboxylic group (formic, acetic, propionic, butyric acid), either
with a hydroxyl group (lactic, malic, tartaric, and citric acid) or with double bonds (fumaric and sorbic acid). The
antimicrobial activity of SCFA in the gut depends on its molecular size, its dissociation constant (pKa), pH of gut and
specifics of the microorganism (Mroz et al., 2006). Common acidifiers used in animal feed have pKa value between 3
and 5 (Dibner and Buttin, 2002). The undissociated organic acid molecules are lipophilic in nature and penetrate into
the semipermeable bacterial membrane. Inside the bacterial cytoplasm, the organic acids dissociate and release the
proton (H+) which inhibits the ATP synthesis and other metabolic process of microbial cells (Mroz et al., 2006). The
accumulation of anions of the organic acids is also toxic to the cell metabolites and disrupt the bacterial membranes
(Freese et al., 1973; Russell, 1992). Some acid-tolerant, Gram positive beneficial bacteria namely Lactobacillus sp. and
Bifidobacterium sp. can survive at lower pH and their intracellular potassium can neutralize the acid anions and provide
resistance to acidifiers (Russell and Diez-Gonzalez, 1998). Thus, organic acids create the eubiotic intestinal environment
by selectively inhibiting the growth of pathogenic microbes like coliforms without affecting beneficial microbes
population. Some organic acids are also play an intermediate role on animal metabolism, for example, fumaric and
citric acids are intermediate compounds of the tricarboxylic acid (Partanen and Mroz, 1999; Pryde et al., 2002). Salt
forms of organic acids (sodium, potassium, and calcium) are used in acidifier products considering less odor, less
corrosion and more water solubility than free acids. One percent of potassium diformate in the diet of weaning piglets
can boost secretion of hydrochloric and lactic acid through increased mRNA expression of H+-K+ -ATPase and gastrin
receptors by 2 to 3 fold in the oxyntic mucosa of stomach (Xia et al., 2016).

Hydrochloric acid, sulphuric acid and phosphoric acid are inorganic acids used as feed acidifiers. These are more
economical compared to organic acids but relatively more corrosive and difficult to handle for feed applications. The
blend of acids is more effective than using individual acids (Namkung et al., 2004; Kil et al., 2011). Namkung et al. (2004)
found differences in intestinal microbiota, immunological blood parameters like lymphocytes, monocytes Interleukins-
1 in weaned pigs when administrated with a combination of organic acids (acetic, propionic, phosphoric, citric blend)
or with lactic acid. Similarly, organic acids showed better results in combination with phosphoric acid than individual
components in formulation (Hashemi et al., 2014). Hashemi et al. (2014) showed that organic acids with phosphoric acid
treatment at 0.15% inclusion rate had increased 6% of body weight of broilers. In recent years, SCFA-based organic
acidifiers are used in combination with other bio actives mostly essential oil (EO) and MCFA.

EO are volatile aromatic compounds extracted from plants, mainly containing terpenes and their derivatives. Essential
oils carry a broad spectrum of antimicrobial actives against both gram positive and negative bacteria. The mode of
action of essential oils is poorly understood. However, different studies suggesting that lipophilicity of EO disrupts the
bacteria cell membrane integrity and eventually leads to microbial death (Helander et al., 1998). Essential oil has been
shown to possess antimicrobial effect against beneficial bacteria like Lactobacilli at high inclusion levels which is an
undesirable character for animal feed applications (Horosova et al., 2006).

MCFA are a group of organic acid bio actives used with SCFA. These are fatty acids with 6 to 12 carbon length. Lauric
acid, capric acid and caprylic acids are well known to have potent antimicrobial activity against a broad spectrum of
intestinal gram-positive microbes like Clostridium, Listeria, streptococcus (Jackman et al., 2020). The antimicrobial
activity of MCFA is extends to gram-positive, fungi and absorption of MCFA by intestinal enterocytes provides energy to
birds. It is considered that SCFA and MCFA together have a synergistic effect on bacteria, whereby MCFA damages
microorganism's cell walls, thus allowing SCFA access into the bacterial cytoplasm to produce an antibacterial effect
(Kim and Rhee, 2013). The concept of gut health includes intestinal morphological integrity, intestinal physiological,
efficient immune response and adequate beneficial microbiota to produce energy for host cells by fermenting the
complex polysaccharides (Carrasco et al., 2019). The birds with gut pathogens need to utilize significant energy for their
immune response to avoid disease conditions which leads to compromised performance (Carrasco et al., 2019).

Protein sources, calcium carbonate, and dicalcium phosphate in poultry feed are the responsible components to
poultry feed pH to alkaline side, these can strongly influence the buffering capacity of feed (Gilani et al., 2013). Not only
plant-based protein sources, animal proteins like meat and bone meal (MBM), fishmeal and blood meal are being used
in poultry feed from the animal rendering industry by products due to their cost advantage and superior amino acid
content (Bozkurt et al., 2004). MBM is the one of the common animal protein sources used by feed industries
(Bozkurt et al., 2004). The buffering capacity of MBM is higher than soybean meal protein. Ten g of soybean meal
requires 21.15 mL of 1M HCl to reduce pH 6.6 to 5, whereas meat meal requires 24.0 mL to reduce pH 5.7 to 5.0
(Lawlor et al., 2005). MBM inclusion rates above 4% may potentially favor the overgrowth of enteric pathogens,
including C. perfringens. The protein molecules, elastin, collage, keratin in MBM can be the substrate for Clostridium
perfingens. High levels of elastin, collagen and keratin from MBM are refractory to gastric digestion and may act as
nutrient substrates for C. perfringens. Such proteins are metabolized by C. perfringens by putrefactive fermentation,
producing trimethylamine and ammonia that affect gut health and increase the pH of digesta (Zanu et al., 2020). High
protein and clostridium growth lead to an unfavorable intestinal microbiota population which impacts animal
performance. The aim of the current study was to identify suitable acidifiers from different feed acidifiers like organic
acids, organic acids with inorganic acid/ EO/MCFA, salt of organic acid to enhance broiler performance and gut health
when bird fed with MBM.

MATERIALS AND METHODS

Experimental Design
All the animal studies were carried out in accordance to guidelines proposed and approved by the committee for
Certificate in Poultry Farming (CPF, Registration number: IGNOU-38011P). The trial was conducted on Vencobb 400
broilers for a period of 42 d using a completely randomized design in a deep litter pen system at the Tropical Institute
of Livestock Management and Health, Gurgaon, India. Rice husk was used as litter material. One-day-old chicks were
procured locally. Vent sexing was carried out in 100% of birds to segregate male and female birds. Male and female
birds were equally distributed in all test groups. The birds were weighed individually, wing banded, and randomly
segregated into groups. The birds were vaccinated for Newcastle Disease Virus (NDV) and Infectious Bursal Disease
(IBD). The NDV vaccine was given through intraocular passage on fifth day (primary dose) and 20th day (booster dose)
of trial. IBD vaccine was given through intraocular passage on 10th day (Primary dosage) and 21st day (booster dose).
The experimental design included 6 treatment groups, containing 10 replicates with 20 birds (10 males and 10 females)
in each replicate. A control treatment was kept without any acidifier treatment. Diets were formulated as per breed
specification recommended by National Research Council (1994). Three feed formulations of maize soya mash diet with
MBM were prepared according to the phases of life of the bird: pre-starter (d 1–d 10), starter (d 11–d 20), and finisher
(d 21– 42) (Table 1). The presence of total coliforms and C. perfingens were confirmed in feed before dietary treatments.
The groups were treated with different test products at their recommended dosages and a control group without
acidifier treatment. The details of 5 test products are mainly combination of organic acids/inorganic acids (SCFA blends
with inorganic acids, T1), combination of organic acids (SCFA blends, T2), combination of organic acids and essential oil
(SCFA blends with EO, T3), combination of organic acids and medium chain fatty acids (SCFA blends with MCFA, T4)
and double salted organic acid (Sodium diformate, T5), which are provided in Table 2. All diets were mash feed and fed
ad libitum to the birds throughout the study period. The nutritional composition of diet fed on different growth phases
was given Table 3.

Table 1. Feed composition for different growth stages.

Ingredients (%) Pre-starter Starter Finisher

Maize 57.0 59.7 62.0

Soybean meal 37.8 33.8 30.0

Rice bran oil 1.25 2.55 3.90

Calcite 1.6 1.6 1.6

Di calcium phosphate 1.15 1.0 1.1

Salt 0.25 0.25 0.25

Soda bicarbonate 0.15 0.15 0.15

Lysine hydrochloride 0.05 0.07 0.07

DL Methionine 0.24 0.24 0.22

Meat and bone meal (MBM) 0.3 0.4 0.5

Phytase 5,000 FTU / g 0.01 0.01 0.01

Vitamin mix 0.05 0.05 0.05

Mycotoxin binder 0.1 0.1 0.1

Total 100 100 100

Table 2. Details of the different acidifier treatments.

Treatment Recommended dosage /

groups Acidifier categories Key ingredients MT of feed

C Control - -

T1 Inorganic and organic acids in Phosphoric acid, fumaric acid, lactic acid, citric acid 1 kg
blends

T2 Combination of organic acids Fumaric, lactic, benzoic, propionic, formic acid 1 kg

T3 Combination of organic acids Fumaric, lactic, benzoic, formic, propionic, citric acid and 1 kg
/ essential oil cinnamaldehyde

T4 Combination of organic acids Lactic acid, propionic acid, citric acid, benzoic acid, 1 kg
/ MCFA formic acid and capric, caprylic acid
Treatment Recommended dosage /
groups Acidifier categories Key ingredients MT of feed

T5 Double salted organic acid Sodium di formate 1.5 kg

Table 3. The nutritional composition of diet on different stage of growth.

Nutrients Pre-starter Starter Finisher

Metabolizable (Kcal/kg) 2850 2950 3050

Fat (%) 3.35 4.67 6.04

Crude fiber (%) 3.15 3.00 2.84

Crude protein (%) 21.75 20.20 18.70

Lysine (g/kg) 1.10 1.02 0.93

Methionine (g/kg) 0.54 0.52 0.48

Cystine (g/kg) 0.29 0.28 0.26

Arginine (g/kg) 1.37 1.26 1.15

Histidine (g/kg) 0.54 0.50 0.47

Leucine (g/kg) 1.68 1.59 1.50

Isoleucine (g/kg) 0.84 0.78 0.71

Threonine (g/kg) 0.75 0.69 0.64

Tryptophan (g/kg) 0.23 0.21 0.19

Valine (g/kg) 0.94 0.87 0.81

Calcium (g/kg) 1.00 0.99 0.99

Phosphorus (g/kg) 1.04 0.97 0.99

Chloride (g/kg) 0.19 0.19 1.94

Sodium (g/kg) 0.16 0.16 0.16

Performance Parameters
Average cumulative body weight (BW) of each replicate (each pen) was measured every week. Feed intake of each
replicate (each pen) was measured and the cumulative feed conversion ratio (FCR) was calculated. The mortality of the
birds was recorded daily.

Intestinal Scoring for Bacterial Enteritis

Intestinal lesion scoring was performed on d 42 of the trial by a trained veterinary pathologist. One male bird from 5
replicates of all the test groups was sampled based on their BW, similar to mean BW of test groups. The birds were
euthanized by cervical dislocation and the gastrointestinal tract was taken out with reference provided by
National Research Council (2010). The intestine was cut open aseptically. Whole part of intestines of birds was scored
for subclinical or clinical bacterial enteritis lesions. Dysbacteriosis scoring was done based by considering only 4 most
associated symptoms: ballooning of gut, redness of serosal/mucosal surface, flaccidity of gut immediate after
dissecting, and presence of undigested feed particle. The score was given on a scale of 0 to 4 based on the description
and observations given in Teirlynck et al. (2011).

Microbial Analysis of Ileal Content

When the birds were sacrificed for intestinal lesion scoring, the digesta (0.5 g) from the proximal end of ileum were
collected aseptically and immediately transferred to Cairy Blair transport solid media (Hiculture Transport swab W/
Cary- Blair Medium in propylene tube (MS202), Himedia, India).These samples were transported to the laboratory in
ice packs within 12h time periods. Microbial analysis of the ileal digesta was carried out for the counts of total
coliforms, C. perfringens and lactic acid bacteria. One bird from 5 replicates of each test group was analyzed. The ileal
digesta was serially diluted and plated on different culture medias that is, Hichrome coliform agar (Hi media
laboratories, India), C. perfringensselective supplement (Merck, India) and MRS agar (Merck, India) in duplicates. The
total coliforms were enumerated from Hichrome coliform agar plates after 24 h by incubation at 37˚C. C. perfringens
colonies were enumerated from perfringens agar plates after 24 h of incubation at 37°C in anaerobic condition. The
lactic acid bacterial colonies were enumerated from MRS agar after 48 h of incubation at 37˚C in anaerobic condition
(Feng et al., 2016).

Histomorphology Studies
Intestinal tissue from the duodenum and jejunum were collected from birds sampled for lesion scoring. The collected
tissues were preserved in 10% formalin and analyzed for villi length, crypt depth of intestinal enterocytes by
histomorphology studies at Madras Veterinary College, Chennai. The tissue samples were trimmed and transverse
sections of 4 to 6 microns were stained with hematoxylin and eosin. All slides were evaluated for necrotic lesions and
histomorphology on an Olympus BX 51 microscope (Olympus Corporation, Tokyo, Japan). The slides were digitalized
using video image software (DP2 BSW for Olympus BX 51). The images were analyzed to measure the crypt depth and
villi height. The villus length was measured from the villus tip to the bottom, excluding the intestinal crypt. Six villi and
crypts were measured per sample, and the mean was calculated.

Statistical Analysis
All the parameters were analyzed across all test groups by ANOVA analysis using STATGRAPHICS plus 5.1. Differences at
P < 0.05 were considered significant.

RESULTS AND DISCUSSION

The MBM is the most common animal protein being used in poultry industries. The studies done by Bozkurt et
al. (2004) and Caires et al. (2010) reported that inclusion of MBM at 5% or lesser did not showed negative performance.
But, two studies from Hendriks et al. (2002) and Davis et al. (2015) reported that the nutritive values like crude protein
and amino acid digestibility of MBM samples vary greatly because of the differences in their origin and processing
conditions. High protein content and indigestible amino acids in MBM could increase intestinal pH to alkaline side and
buffering capacity of dietary acidifier required to maintain intestinal pH for complete digestion. Failing to that affects
negatively on the bird's performance by creating more microbial pathogenic load in the lower part of the intestinal gut.
It is essential to use a suitable acidifier in feed which has 5% MBM to maintain feed buffering capacity and to avoid
establishment of pathogenic microbes in the intestine. The current study was aimed to identify the suitable feed
acidifier from different categories being used in feed with inclusion of 5% MBM. Common types of acidifiers available
in commercial products like SCFA blends with inorganic acids (T1), SCFA blends (T2), SCFA blends with EO (T3), SCFA
blends with MCFA (T4), sodium diformate (T5) were studied in broilers. The pH of all test formulation ranges between
2 and 3. The total coliforms and C.perfingens in feed before acidifier treatments in diet was approximately 3 log cfu/g, 2
log cfu/g respectively.
Body Weight and FCR
Body weight and feed intake was assessed on weekly basis. SCFA blends (T2), SCFA blends with EO (T3) showed
significantly higher body weight (P < 0.05) than control group (Table 4) during the starter and finisher periods. The rest
of the tested groups did not show any significance over control. On the 42nd d of rearing, SCFA blends (T2, 2,386 g)
showed significantly higher body weight and lower FCR by 5 points than control (2,278 g). These results agree with
studies done by Hassan et al. (2010) who tested two commercial SCFA organic acid products and confirmed almost 5.5%
improvement in BW and 12 points lower FCR by 35 days of growth.

Table 4. The effect of acidifier on growth performance of broiler birds.

Prestarter (0–14 d)

Groups BW (g) FI (g) FCR (no unit) Livability (%)

C 482 550 1.14 98.5

T1 483 557 1.15 99.0

T2 487 549 1.13 99.0

T3 490 560 1.14 99.0

T4 480 554 1.16 99.5

T5 484 555 1.15 98.5

SEM 3.776 2.863 0.0092 -

P-value 0.4477 0.094 0.3941 -

Starter (15–28 d)

C 1,355b 1,903 1.40 98.0

T1 1,362b 1,910 1.40 99.0

T2 1,391 1,914 1.38 98.0

T3 1,400ab 1,926 1.38 98.0

T4 1,359b 1,918 1.41 99.5

T5 1,376ab 1,912 1.39 97.5

SEM 24.34 11.050 0.0109 -

P-value 0.0023 0.7632 0.1024 -

Entire trial duration (0-42 d)

C 2,278b 3,786 1.66ab 96.5

T1 2,333ab 3,833 1.64abc 99.0

T2 2,386 3,833 1.61c 98.0

T3 2,395 3,903 1.63bc 98.0

T4 2,345ab 3,817 1.63bc 99.5

T5 2,314b 3,872 1.67a 96.5

SEM 24.35 30.271 0.01532 -

 Prestarter (0–14 d)

Groups BW (g) FI (g) FCR (no unit) Livability (%)

P-value 0.0252 0.117 0.0351 -

Abbreviations: BW, body weight (g); FI, feed intake (g); FCR, feed conversion ratio (g of feed weight/ g of body weight).The superscript
letters within the same column denotes the statistical differences at confidence level of P < 0.05.

Similarly, SCFA blends with EO (T3, 2395g) showed significantly higher body weight (60 g–110 g/bird, P < 0.05) than
control (2,278 g) group (Table 4). This result is also consistent with studies reported by Tiihonen et al. (2010) that the EO
blends contained thymol and cinnamaldehyde could enhance BW by 4.5% in Ross broiler birds.

Other test group, SCFA and inorganic acid blends (T1) did not show improvement in higher BW or lower FCR. These
results are consistent with results from Loh et al. (2007) study with organic acids and phosphoric acid blend. SCFA
blends with MCFA (T4) did not show either increased body weight or lower FCR than control group.

T1 and T4 has neither shown increased BW gain nor FCR reduction in the current study. This is due to the lower total
acid content in the product compared to other treatments. Recently, Abdelli et al. (2020) has compared SCFA+EO (T3),
SCFA+MCFA, MCFA+EO in bird's performance and demonstrated production performance improvement in terms of BW
gain and FCR for all 3 groups. Sodium diformate (T5) treated group negatively impacted the bird's growth in current
study. Canibe et al. (2001) reported that there is no body weight and FCR improvement observed in pigs supplemented
with potassium formate. This could due to low solubility and buffering capacity of salt form compared to the pure form
at intestinal conditions. However, the reduction in intestinal pathogenic bacteria was obtained without affecting
intestinal morphology.

Livability
The mortality rate of birds by 42 days of rearing was shown in Table 4. Treatments T1, T2, T3 showed 1 to 2% mortality.
T4 showed 0.5% mortality rate than control (3.5%).

Intestinal lesion scoring and ileal microbial population

The ileal microbiota was analyzed from all test groups for total coliforms, Clostridium, and lactic acid bacteria (Ahmed et
al., 2014). The results from current study indicates that test groups, SCFA blends with inorganic acid (T1), SCFA blends
(T2), SCFA blends with EO, SCFA blends with MCFA (T4) were efficient in maintaining intestinal health by recording
lower bacterial enteritis infection (Figure 1) and lower C. perfingens populations in the ileum (Figure 2). The treatments
T1, T4 reduced ileal C. perfingens by 2 logs cfu/g and T2, T3 reduced by 4 logs cfu/g when compared to control (Figure
2). These results are consistent with results from Baltic et al (2019), Abdelli et al. (2020) and Pham et al. (2020).
C.perfingens a gram-positive bacterium was reported to be inhibited by essential oil as well as MCFA (Baltic et al., 2019;
Pham et al., 2020). Abdelli et al (2020) reported that SCFA blend with MCFA treatment had lower clostridial population
than treatments with SCFA and EO blends. But the current study result showed that SCFA blends with EO (T3) had
better anti-clostridial effect than the treatment with SCFA blend with MCFA (T4). This could be due to the difference in
total acid content of 2 groups. Treatments T3, T4 had a total acid content of 87% and 50%, respectively. The observations
from current study and Abdelli et al. (2020) provides the insight that the efficacy due to total acid content and presence
of other bioactives (MCFA, EO).
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Figure 1. Intestinal lesion scores for bacterial enteritis in different treatment groups. The results are mean ± SE of 5
replicates from each treatment (n = 5) and different alphabets denote statistical differences at confidence level of P <
0.05.

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Figure 2. Clostridium perfringens count in ileal content of birds on 42nd day of rearing. The counts are average of values
obtained from 5 birds from each treatment group. The results are mean ± SE of 10 replicates from each treatment (n = 5)
and different alphabets denote statistical differences at confidence level of P < 0.05.

In the current study, total coliforms were quantified from ileal contents of birds from different treatments. There is no
significant reduction in coliforms between different treatment groups except the sodium diformate treated group (T5)
when compared to control (Figure 3). These results are not in agreement with studies by Hassan et al. (2010),
Tiihonen et al. (2010), Natsir et al. (2017), and Abdelli et al. (2020) who have shown better antimicrobial efficacy of SCFA,
EO, MCFA effect against E. coli, Salmonella in chicken intestine. The reason for inconclusive result from the current study
is unknown.
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Figure 3. Total coliforms count in ileal content from one bird each from 5 replicates of all test groups on 42nd day of
rearing. The results are mean ± SE of 5 replicates from each treatment (n = 5) and different alphabets denote statistical
differences at confidence level of P < 0.05.

The effect of test groups toward the beneficial intestinal microbiota was analyzed in the current study by quantification
of lactic acid bacteria in ileal contents (Figure 4). The birds from control and sodium formate treated group (T5)
showed 3 log cfu/g of lactic acid bacteria in Ileum. The treatments, SCFA blends with inorganic acids(T1), SCFA blends
(T2), SCFA blends with EO (T3) and SCFA blends with MCFA (T4) did increase LAB population by 1 log cfu/g, 2 log cfu/g,
2.5 log cfu/g, >3log cfu/g respectively than control (Figure 4). Similar results were demonstrated in reports from
Natsir et al. (2017) and Abdelli et al. (2020). The inclusion of combination of SCFA, MCFA, and nature identical aromatic
compounds at the dosage rate of 0.17% to 0.2% showed increased Lactobacillus to 0.26 log in ileum and 2.5 log in cecum
of Ross Broiler (Abdelli et al., 2020). Natsir et al. (2017) study in Lohmann broilers, acidifier treatment with/without
herbal extract and encapsulated form at the dosage rate of 1.2% showed increased lactic acid bacteria by 1log cfu,
decreased E. coli, Salmonella species by 1.29 log cfu and 0.83 log cfu in jejunum. Tiihonen et al. (2010) study in Ross
broilers treated with essential oil (cinnamaldehyde 5ppm and thymol 15ppm) showed a 2 log reduction of C.
perfingens, 1 log reduction in E. coli, and 1log increase in lactic acid bacteria compared the untreated group. But there
was decline of 1 log reduction in lactic acid bacteria from d 21 to 42 in the essential oil treatment. This is evidence that
the dosage of essential oil plays a crucial role in maintaining lactic acid bacteria population in intestinal gut.

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Figure 4. Lactic acid bacterial counts in ileal content of birds from one bird each from 5 replicates of all test groups on
42nd day of rearing. The results are mean ± SE of 5 replicates from each treatment (n = 5) and different alphabets
denote statistical differences.

Histomorphology Studies
The effect of different test groups on intestinal tissues was studied by histomorphology analysis on ileal and jejunal
tissue segments. The villi length (VL) and crypt depth (CD) of intestinal enterocytes were measured and the results are
given in Table 5.

Table 5. Villi height and crypt depth of duodenum and jejunum tissues of one bird from 3 replicates of all test groups on
42nd day of rearing. The results are indicated as mean ± SE, n = 6 and different alphabets denote statistical differences
at confident level of P < 0.05.

Test group Duodenum Jejunum

Villi height (µm) Crypt depth (µm) Crypt:Depth Ratio Villi height (µm) Crypt depth (µm) Crypt:Depth Ratio

Control 822 ± 133c 94 ± 12cd 8.74 858 ± 74b 78 ± 5c 11.00

T1 1175 ± 24ab 177 ± 14ab 6.64 1142 ±172ab 120 ± 25ab 9.52

T2 987 ± 172abc 105 ± 11cd 9.40 1255 ± 119a 94 ± 14bc 13.35

T3 1230 ± 48a 152 ± 33bc 8.09 1043 ± 60ab 127 ± 8ab 8.21

T4 1242 ± 89a 136 ± 5bc 9.13 1029 ± 106ab 140 ± 20a 7.35

T5 923 ± 97bc 81 ± 15d 11.40 826 ± 146b 76 ± 7c 10.87

The superscript letters within the same column denotes the statistical differences at confidence level of P < 0.05.

In duodenal tissues, all treatments except SCFA blends (T2) and sodium formate (T5) showed higher villi length
compared to control. SCFA blends with inorganic acid (T1) SCFA blends with EO (T3), SCFA blends with MCFA (T4)
showed 43%, 50%, and 53% increase in VL compared to control (822 µm) and T1, T4 showed higher VL/CD ratio of 9.40,
9.13, respectively compared control (8.74). These results showed treatments T1 and T4 were efficient in maintaining
intestinal morphology at duodenum. Higher VL and low CD indicate the larger site for nutrient absorption (Pelicano et
al., 2005). In jejunum SCFA blends (T2) showed significantly higher VL by 46% than control. The VL/CD ratio of SCFA
blends (T2) was higher (13.35) compared to control (11.00). No other treatment enhanced VL/CD ratio in jejunum than
control. The obtained results agree with reported results from Baltic et al (2019) and Abdelli et al. (2020). The VL/CD was
enhanced by essential oil treatment (5.01) than untreated group (3.26) at duodenum (Baltic et al., 2019). The same
result was observed in ileum, cecum in that study. Similarly, Abdelli et al. (2020) reports that VL/CD was enhanced by
organic acid/EO combination (6.10), organic acid/EO/MCFA combination (5.82), EO/MCFA (4.89) than control (3.79).

In the current study with 5% MBM, the group supplemented with sodium diformate (T5) had negatively impacted
broiler performance and intestinal micro flora. This result contradict the reports from Canibe et al. (2001) and
Abdelhady et al. (2015). SCFA blends (T2) and SCFA blends with EO (T3) which contains nearly 85% total acid content
showed both performance and intestinal benefits. SCFA blends contains inorganic (T1), SCFA blends with MCFA (T4)
contain nearly 50% total acid content showed only intestinal microbiological benefits but no improvement in animal
performance. This is evidence that total acid content in dietary acidifier products play an important role apart from
their secondary active molecules like EO or MCFA. However, there are two challenges to increase acid content in T1, T4.
The pH of SCFA with inorganic acid (T1) is 1.8. Increasing acid content in T1 to 85%, will lower intestinal pH drastically
and product can impose strong acidic action on bird's intestine. Similarly, increasing the total organic acids in SCFA
blend with MCFA (T4) may be beneficial but may not be economically viable considering price of MCFA molecules.

CONCLUSIONS AND APPLICATIONS

1. A dietary acidifier product in any diet formulation should have potent buffering capacity and intestinal
antimicrobial activity to deliver good gut environment and overall health benefits to birds.

2. The current study shows that SCFA blends as a stand-alone product (T2) or SCFA blends with EO (T3) can be more
suitable solution for efficient animal performance in comparison with other acidifier combinations in poultry feed
with animal protein (MBM) inclusion in the diet.

3. The study results also confirmed that double salted organic acid negatively impacts the performance of birds in
MBM diet.

DISCLOSURES
The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter
discussed in the manuscript.

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