Article

Influence of Dietary Supplementation with Yeast Culture and

Microencapsulated Butyric Acid on Growth Performance,
Carcass Traits, Gut Health, and Immune Status in Broilers
Azhar Nazir 1 , Ehsaan Ullah Khan 1, * , Muhammad Muneeb 1 , Shafqat Nawaz Qaisrani 1 , Saima Naveed 1 ,
Sohail Ahmad 2 , Rao Muhammad Kashif Yameen 2 , Ali R. Al Sulaiman 3 , Rashed A. Alhotan 4
and Ala E. Abudabos 5, *

1 Department of Animal Nutrition, Faculty of Animal Production and Technology, University of Veterinary and
Animal Sciences, Lahore 54000, Pakistan; 2019-mphil-2243@uvas.edu.pk (A.N.);
2022-mphil-1331@uvas.edu.pk (M.M.); shafqat.qaisrani@uvas.edu.pk (S.N.Q.);
saimamahad@uvas.edu.pk (S.N.)
2 Department of Poultry Production, Faculty of Animal Production and Technology, University of Veterinary
and Animal Sciences, Lahore 54000, Pakistan; sohail.ahmad@uvas.edu.pk (S.A.);
2015-phd-1058@uvas.edu.pk (R.M.K.Y.)
3 Environmental Protection Technologies Institute, Sustainability and Environment Sector, King Abdulaziz City
for Science and Technology, P.O. Box 6086, Riyadh 11442, Saudi Arabia; arsuliman@kacst.gov.sa
4 Department of Animal Production, College of Food and Agriculture Sciences, King Saud University,
P.O. Box 2460, Riyadh 11451, Saudi Arabia; ralhotan@ksu.edu.sa
5 Department of Agriculture, School of Agriculture and Applied Sciences, Alcorn State University, 1000 ASU
Drive, Lorman, MS 39096-7500, USA
* Correspondence: ehsaan@uvas.edu.pk (E.U.K.); aabudabos@alcorn.edu (A.E.A.)

Simple Summary: Modern poultry farming encounters difficulties in enhancing growth,

feed efficiency, and disease resistance while minimizing dependence on antibiotics. This
study examined the potential benefits of integrating yeast culture, which boosts immune
and nutrient absorption, and butyric acid, which aids in digestion, on broiler health and
Academic Editor: Patrick Butaye productivity. The study assessed the impact of various diets—having these supplements
Received: 12 March 2025 either alone or in combination—on gut health, growth, immunity, and meat quality. The
Revised: 6 April 2025 findings indicated that the combination of both additives resulted in the most significant
Accepted: 10 April 2025
changes, including increased weight, improved feed conversion, enhanced immunolog-
Published: 12 April 2025
ical responses, and healthier digestive systems. Chickens consuming this diet exhibited
Citation: Nazir, A.; Khan, E.U.;
enhanced nutrient absorption and diminished levels of pathogenic bacteria in their in-
Muneeb, M.; Qaisrani, S.N.; Naveed,
S.; Ahmad, S.; Yameen, R.M.K.; Al
testines, hence lowering the risk of illnesses. These findings demonstrate that employing
Sulaiman, A.R.; Alhotan, R.A.; such dietary interventions can improve poultry farming—benefiting through increased
Abudabos, A.E. Influence of Dietary efficiency and reduced costs—while simultaneously promoting consumer health by de-
Supplementation with Yeast Culture creasing the use of antibiotics in food production. This approach enhances sustainable
and Microencapsulated Butyric Acid poultry production, fostering improved animal welfare and environmental conservation.
on Growth Performance, Carcass
Traits, Gut Health, and Immune Status
Abstract: The study aimed to examine the effects of dietary supplementation with mi-
in Broilers. Vet. Sci. 2025, 12, 359.
https://doi.org/10.3390/
croencapsulated butyric acid (EBA) and yeast culture (YC) in broiler diets. A total of 450
vetsci12040359 Ross-308 broiler chicks were selected and randomly allocated to five dietary treatments
with six replicates (15 birds per replicate) in a complete block design. The experimental
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
diets included the following treatments: (1) Negative control (NC) with basal diet without
This article is an open access article any additives. (2) Positive control (PC) with basal diet + 0.2 g/kg enramycin. (3) EBA, basal
distributed under the terms and diet + 0.3 g/kg EBA. (4) YC, basal diet + 1 g/kg YC. (5) EBA+YC, basal diet + 0.3 g/kg EBA
conditions of the Creative Commons and 1 g/kg YC. The results indicated a non-significant effect on feed intake (FI) during the
Attribution (CC BY) license experiment periods. However, the EBA+YC treatment exhibited significantly increased
(https://creativecommons.org/
body weight gain (BWG), better feed conversion ratio (FCR), and enhanced carcass traits
licenses/by/4.0/).

Vet. Sci. 2025, 12, 359 https://doi.org/10.3390/vetsci12040359

Vet. Sci. 2025, 12, 359 2 of 14

(p < 0.05) compared to other treatments. A significant effect was observed for the immune
organ weights and ND titters. Villus height (VH) and the ratio of villus height-to-crypt
depth (VH: CD) were noted for EBA+YC across all other treatments. Ileal microbial analysis
revealed a significantly lower count of E. coli and Salmonella in the ileal digesta of broiler
chickens in the EBA+YC treatment compared to the NC group (p < 0.05). In conclusion,
dietary supplementation with any supplement positively influences the broiler’s perfor-
mance, carcass characteristics, gut health, and immune status over the NC group. More
pronounced improvements were obtained from the EBA+YC group, indicating that EBA
and YC had a synergistic effect on broilers.

Keywords: yeast culture; microencapsulated butyric acid; AGP alternative; broiler perfor-
mance; gut health

1. Introduction
Gut health has been intensively researched, as the gut is deemed the primary loca-
tion for nutrient digestion and absorption. Deteriorated gut health can decrease nutrient
digestion and absorption, thus impacting poultry health and production performance [1].
Consequently, sustaining a healthy gut is essential for optimal well-being and productivity.
Modern breeding plans and improved rearing systems have enabled broiler chickens to
achieve about 2 kg body weight within 35 days [2]. With the advancement of the poultry
industry and the need to ensure the sustainability of the food chain, there is a growing
interest in achieving a better growth output with promising efficiency of feed utilization
in broilers. The commercial broiler strains have the genetic potential of achieving higher
growth rates at the least feed consumption, representing a better feed conversion ratio
(FCR) [3]. However, this output capacity of broiler strains can only be put across by
providing complete broiler rations that are nutritionally balanced and help the intestinal
environment support maximum digestion and absorption of dietary nutrients [4]. The
digestive system is widely recognized as vulnerable to numerous pathogens [5]. Conse-
quently, a healthy gut environment for optimal production output can be better maintained
with the help of different dietary additives [6]. Antibiotic growth promoters (AGPs) have
been widely used in poultry production to enhance growth performance and maintain
gut ecosystem balance, primarily due to their affordability and widespread availability [7].
However, in 2006, the European Union banned the use of AGPs in animal feeds due to their
residual effects and transfer of drug-resistant genes [8]. Hence, the search for alternatives
to AGPs is receiving significant interest [9]. These alternatives include but are not limited
to prebiotics, probiotics, organic acids, yeasts, and enzymes [10]. Continuous research into
an appropriate feed additive(s) that promotes broiler growth, is non-hazardous, and is
cost-effective for optimizing gut health is vital.
Yeast, in many forms—i.e., fermented yeast, breweries or distillery yeast, and commer-
cial yeast—is among the common feed additives in poultry diets [11]. Yeasts include both
unicellular and multicellular species. Similarly, yeast size varies greatly, from 3–4 µm to
over 40 µm [12]. Yeast is considered antagonistic to detrimental microbes, thereby causing
a barrier effect and possibly helping protect the intestinal mucosa against assaulting germs.
Furthermore, yeast fractions stimulate the host animal’s immune response [13]. The yeast
culture (YC) is an important yeast product in the broiler industry. A YC can be defined as a
distinct micro-ecological product consisting of a mixture of biomass with living yeast and
other fermentation metabolites. The extracellular metabolites, like organic acids, alcohols,
peptides, and esters, are the major constituents of YC [14]. The fermentation products of Sac-
Vet. Sci. 2025, 12, 359 3 of 14

charomyces cerevisiae, also called baker’s yeast, are widely used as YC [15]. YC was reported
to provide protein, amino acids, trace essential minerals, and vitamins for broilers [12]. YC
possesses both in vitro and in vivo antagonistic activity against the population of several
pathogenic microbes [16]. YC supplementation significantly improves growth performance
and cecal microbial community in broiler birds [17]. Other studies have also revealed
valuable effects of dietary YC addition to poultry diets, such as improved performance [18],
blood characteristics [19], humoral immunity response, and carcass characteristics [20].
Organic acids (OAs) are also effective additives in broiler feed and have been proven
to improve broiler performance [21]. Organic acids are an extensive category of essential
compounds used in the body’s basic metabolic processes [22]. The OAs are associated
with several beneficial attributes in broilers: buffering the broiler’s diet, restricting harmful
microorganisms in the intestine by altering the pH, raising the nutrients available from
the diet, and improving immune responses in poultry [8]. Only the short-chain OAs (C1 –
C7 ) are specified for antimicrobial activity and may be produced through carbohydrate
fermentation in the large intestine of broilers [22]. Among the OAs, the most common
are short-chain fatty acids such as monocarboxylic acids, propionic, acetic, formic, and
butyric acids, which may exist in their esterified form with calcium, sodium, and potassium
salts [23]. Salts have advantages over acids because they are odorless and easy to handle in
feed manufacturing [7]. Butyric acid (BA) is the most commonly used OA supplement in
the broiler diet. Attributes for its use include better bioavailability, improved gut health,
higher nutrient absorption, and enterocytes’ ease of absorbing more nutrients [24]. The
intestinal villi can utilize BA as a readily available energy source that accelerates the
differentiation and multiplication of villus cells [25]. This phenomenon improves mucosal
nutrient absorption capacity and broiler feed efficiency [26]. Assembling of the host cell
peptides is also stimulated by the presence of BA in the intestine, which further triggers
cellular proliferation and encourages the development and repair of the gut [27]. It was
proven that BA is un-dissociated at low pH and lipophilic, resulting in diffusion across the
bacterial cell membranes, reducing the harmful microbial population [1]. Uncoated BA
may hence get absorbed by the crop and proventriculus of the broiler, limiting its efficacy
in the small intestine [28]. This issue, however, can be resolved through modern techniques
like microencapsulation. The encapsulation of BA (EBA) through palm fat results in a
slow release during transport through the intestinal tract [29] and ensures its beneficial
utilization on the proper site of interest, that is, the duodenal area of the intestine [30].
As described, dietary supplementation with YC also alters the gut pH through its
metabolites and microbiota modulation, i.e., secreting OAs like acetic acid and lactic
acid. This drop in pH is detrimental to the survival of pathogens in the poultry gut [31].
Furthermore, yeast cells prevent pathogen colonization, modulate the host’s immune
response, and maintain gut microbial homeostasis. The BA, like other OAs, also lowers
the intestinal pH, improves nutrient absorption, and reduces pathogenic microorganisms
in the gut [32]. Thus, their combined use can create synergy to boost broiler performance.
The solitary effects of YC and OAs in broiler gut health optimization have been well
documented in the existing literature. However, to the best of our knowledge, limited
studies are available on their synergistic effects in broilers. Therefore, the current study
aimed to compare the single and combined action of YC and EBA supplementation on
broiler performance, carcass traits, immune response, and gut health. It was hypothesized
that supplementing broiler diets with the combination of YC and EBA could better maintain
the gut microbial balance and serve as a viable substitute for AGPs. This approach may
have significant financial benefits for poultry producers by improving gut health, increasing
feed efficiency, and reducing the medication costs of the flocks.
Vet. Sci. 2025, 12, 359 4 of 14

2. Materials and Methods

2.1. Ethical Approval
The experiment was carried out over 35 days at the Poultry Research and Training
Centre, a floor-rearing broiler facility at the University of Veterinary and Animal Sciences
(UVAS), Ravi Campus (C-block), Pattoki, Pakistan. All procedures conformed with the
rules established by the Ethical Review Committee, UVAS, Lahore, Pakistan (Approval
number: 196; Date: 16 March 2022).

2.2. Experimental Design

A total of 450 straight-run broiler chicks (Ross-308) with similar initial body weight
were selected; then, chicks were divided into five treatments with six replicates of 15 birds
each (90 chicks per treatment) in a completely randomized design. Treatment one was
a basal diet without supplements (negative control (NC)). Treatment two served as the
positive control (PC); the diet was provided with an AGP (EnraLiv® , Enramycin 4%) at
0.2 g/kg. The diet in treatment three was supplemented with EBA (ButiPEARL® , 45%
Calcium Butyrate) at 0.3 g/kg. The diet in treatment four was supplemented with YC
(GroPro® , Baker’s yeast derivative) at 1 g/kg. The diet in treatment five was supplemented
with a combined EBA (0.3 g/kg) and YC (1 g/kg) (Table 1). The EBA and YC preparations
used in this study were in powder form and added to the diet during manufacturing at the
feed processing unit.

Table 1. Experimental layout.

Phase
No. Treatment Total Number of Birds
Starter Phase (1–21 d) and Grower
Phase (22–35 d)
1. NC Basal diet without any additive No of treatments = 5
No of replicates = 6
2. PC Basal diet +Enramycin at 0.2 g/kg
Experimental units = 5 × 6 = 30
Basal diet + Microencapsulated Butyric No of birds/replicate = 15
3. EBA
acid at 0.3 g/kg Total birds = 5 × 6 × 15 = 450
4. YC Basal diet + Yeast culture at 1 g/kg
Ross-308
(Straight-run)
Basal diet + Microencapsulated Butyric
5. EBA+YC
acid (0.3 g/kg) + Yeast Culture (1 g/kg) Completely Randomized
Design (CRD)
NC = Negative Control, without any additive; PC = Positive Control, Enramycin at 0.2 g/kg; EBA = Microencap-
sulated Butyric Acid at 0.3 g/kg. YC = Yeast Culture at 1 g/kg; EBA+YC = Combined Microencapsulated Butyric
Acid and Yeast Culture at 0.3 g/kg and 1 g/kg, respectively. All the supplements were added at the mixer level
and were part of pellets/crumbles.

2.3. Bird Husbandry

The chicks were maintained according to the standard managemental practices of Ross-
308 [33]. Briefly, before the placement of chicks, the house was preheated, and minimum
ventilation was maintained. House temperature was initially maintained at 33 ◦ C with
the help of an electric brooder and decreased gradually by 2.8 ◦ C per week until day 21.
The relative humidity (RH) was stabilized at 65%. Later on, the environmental conditions
(temperature, RH, and ventilation) were adjusted in line with the bird’s behavior and age.
According to the ambient conditions during the experiment, the transitional ventilation
system was mainly used after brooding. The house was kept under a 23 h light period
throughout the experiment. Rice husk was used as litter material and evenly spread to a
depth of approximately 2–5 cm on the floor. The bedding material was racked on alternative
Vet. Sci. 2025, 12, 359 5 of 14

days to maintain its quality. Feed and fresh clean drinking water supply were given ad
libitum throughout the experiment. The diets were prepared following Ross-308 nutrient
specifications [34]. A broiler starter diet was fed from 1 to 21 d and a grower diet from 22
to 35 d (Table 2). All the feed ingredients and compounded diets were analyzed in the lab
according to the standard protocols of AOAC.

Table 2. Basal diet feed formulation (as fed basis %).

Ingredients (%) Starter (1–21 d) Grower (22–35 d)

Corn 44.56 49.52
Soybean Meal 25.0 22.5
Canola Meal 11.0 11.0
Rice Polish 8.00 5.00
Corn Gluten Meal 30% 5.00 3.50
Canola Oil 2.96 5.25
L-Lysine HCL 0.45 0.35
DL-Methionine 0.21 0.18
Common Salt 0.50 0.50
Dicalcium phosphate 0.22 0.20
Limestone 2.00 2.00
Minerals and Vitamin
0.10 0.10
Premix *
Total (%) 100 100
Calculated Nutrients
Metabolizable energy (ME,
3000 3200
Kcal/kg)
Crude protein, % 23.00 21.5
Ether extract, % 5.93 6.92
Crude fiber, % 4.63 4.63
Calcium % 0.96 0.87
Phosphorus % 0.48 0.43
Dig-Lysine % 1.28 1.15
Dig-Methionine % 0.51 1.47
Dig-Threonine % 0.86 0.77
Analyzed Nutrients (%)
Crude Protein 22.96 20.99
Crude fiber 4.61 4.64
Ether extract 5.91 6.93
Crude ash 1.63 1.63
Calcium 0.96 0.87
Phosphorus 0.47 0.44
* Fe (sulfate), 40 mg; I (iodide), 0.15 mg; Cu (sulfate), 16 mg; Se (selenate), 0.3 mg; Mn (oxide and sulfate), 120 mg;
Zn (oxide and sulfate), 100 mg; mineral oil, 3.75 mg; and cereal-based carrier, 128 mg; vit. A, 12,000 IU; vit. D3,
5000 IU; vit. K, 3 mg; vit. E, 75 mg; vit. B2, 8 mg; vit. B3, 55 mg; vit. B1, 3 mg; vit. B6, 5 mg; vit. B12, 16 µg; vit. B9,
2 mg; vit. B5, 13 mg; biotin, 200 µg; mineral oil, 2.5 mg; and cereal-based carrier, 149 mg.

2.4. Parameters Studied

Growth performance was measured as earlier explained [35]. Briefly, feed intake (FI),
body weight gain (BWG), and feed conversion ratio (FCR) were determined. Mortality
was taken daily and used to determine livability. Three birds per pen (18/treatment)
were picked randomly at the end of the trial for further analysis. The sampled birds were
individually weighed and were then slaughtered by exsanguination following electrical
stunning. Subsequently, they were scalded, mechanically wet-plucked, processed, and
eviscerated. The dressed weight was computed by dividing the eviscerated weight by
the pre-slaughter BW and represented as a proportion [36]. On days 21 and 35, blood
Vet. Sci. 2025, 12, 359 6 of 14

samples were collected from the wing veins of 18 birds per treatment for immune status.
The samples were centrifuged at 2000 rpm to harvest serum. For Newcastle disease
virus (NDV) antibody titer testing, the serum was aliquoted and stored at −20 ◦ C in the
experimental laboratory of the Department of Microbiology UVAS-Lahore. The antibody
titers against NDV were determined using hemagglutination-inhibition (HI) [37]. The
immune organ weights were also measured to assess the development of the immune
system in birds. On day 35, 18 birds per treatment were sampled for microbial enumeration
at the end of the experiment. One g digesta was taken from the ileum portion, transferred
to sterile tubes containing PBS (phosphate buffer solution), and carefully taken to the
laboratory for enumeration of the microbial population. Each sample (1 g) was tenfold
serially diluted in a sterilized normal saline solution. Tenfold serially diluted samples were
poured on Petri plates of selective media. Salmonella was grown on Brilliant Green agar
media (Oxoid Basingstoke, UK), and E. coli on McConkey agar media (Oxoid, Basingstoke,
UK). The colonies of these bacteria were quantified using a colony counter. Total colony-
forming units were calculated by multiplying the mean number of colonies formed and
the inverse of the dilution factor as previously outlined [38]. For studying the intestinal
morphological changes, a 2 cm portion from the duodenum region (distal to the duodenal
loop) was removed as the sample. The intestinal segments were washed and fixed in
10% formalin (48 h). The tissue samples were dehydrated in different dilutions of ethyl
alcohol and then embedded in paraffin wax. Tissue sections (about 5 µm) were made using
a microtome and mounted on a glass slide. A routine staining process was carried out
by utilizing hematoxylin and eosin stains. The VH and crypt depth (CD) were observed
through the 10X objective by using a light microscope. Measurements were taken and
standardized with software (PixelPro® 3.2TM , Labo America Inc., Fremont, NY, USA).
A total of 10 well-oriented villi for each region were selected for measurement. Values
obtained were averaged and used for statistical analysis.

2.5. Statistical Analysis

Pen replicates denoted the experimental unit for performance, while other analyses
treated each bird as a separate unit. Data analysis was conducted using SAS 9.4 software
(SAS Inc., Cary, NC, USA). Data underwent analysis through a one-way analysis of vari-
ance, with Tukey’s test used for multiple comparisons. Results were deemed significantly
different at p values ≤ 0.05.

3. Results
3.1. Growth Performance
The results revealed a non-significant impact on the FI of birds in all the groups
(p > 0.05). Feed intake during the starter (1–21 d) and grower phases (22–35 d) and in the
overall period (1–35 d) was not affected by treatment. Improved BWG was reported during
the starter, the grower, and the overall period due to feed supplementation compared to
the un-supplemented diet, NC. The highest BWG was obtained from the group that had
received EBA+YC compared to all other treatments (p < 0.001). The results showed that the
EBA+YC group had 2.74%, 5.97%, 6.44%, and 15.29% higher BWG compared to EBA, PC,
YC, and NC treatments, respectively. Starter, grower, and overall FCR were significantly
improved when diets were supplemented compared to the un-supplemented group (NC).
The best improvements with FCR were obtained from birds that received EBA+YC and
EBA for the starter period; EBA+YC, EBA, and PC for the grower period; and EBA+YC and
EBA for the cumulative period (p < 0.01, p < 0.001, p < 0.001, respectively). A substantial
difference in the livability percentage of broiler chickens was observed. The NC possessed
the lowest (p < 0.05) livability percentages in comparison to all other treatments (Table 3).
Vet. Sci. 2025, 12, 359 7 of 14

Table 3. Effects of different treatments on growth performance of broilers.

Treatments
Parameter p-Value
NC PC EBA YC EBA+YC
Initial BW (g) 45.0 ± 0.02 45.0 ± 0.02 45.0 ± 0.02 45.0 ± 0.02 45.0 ± 0.02 0.790
d 1–21
BW (g) 844.1 d ± 6.82 872.5 c ± 6.98 899.8 b ± 7.09 875.6 c ± 7.28 922.5 a ± 7.33 0.001
BWG (g) 799.1 d ± 6.81 827.5 c ± 6.99 854.8 b ± 7.07 830.6 c ± 7.28 877.5 a ± 7.34 0.001
FI (g) 1132.7 ± 3.62 1149.7 ± 8.90 1150.7 ± 3.55 1139.4 ± 7.55 1153.3 ± 10.96 0.277
FCR 1.417 a ± 0.01 1.389 ab ± 0.02 1.346 bc ± 0.01 1.372 ab ± 0.02 1.314 c ± 0.02 0.007
d 22–35
BW (g) 1873.1 d ± 15.84 2033.8 c ± 16.20 2096.3 b ± 16.88 2025.0 c ± 15.94 2152.5 a ± 17.21 0.001
BWG (g) 1029.0 d ±11.33 1161.3 c ± 9.33 1196.5 b ± 10.46 1149.4 c ± 10.37 1230.0 a ± 10.66 0.001
FI (g) 2089.3 ± 10.35 2097.0 ± 20.02 2100.9 ± 3.63 2098.8 ± 24.62 2117.7 ± 27.38 0.882
FCR 2.030 a ± 0.01 1.806 bc ± 0.02 1.756 bc ± 0.02 1.826 b ± 0.04 1.722 c ± 0.04 0.001
d 1–35
BWG (g) 1828.1 d ± 15.82 1988.8 c ± 16.21 2051.3 b ± 16.86 1980.0 c ± 15.95 2107.5 a ± 17.22 0.001
FI (g) 3222.0 ± 12.61 3246.6 ± 26.62 3251.6 ± 3.83 3238.2 ± 31.29 3271.0 ± 38.21 0.745
FCR 1.763 a ± 0.01 1.632 b ± 0.02 1.585 bc ± 0.01 1.635 b ± 0.03 1.552 c ± 0.03 0.001
Liv (%) 92.5 b ± 0.67 94.3 ab ± 0.92 95.6 ab ± 0.86 93.5 ab ± 1.11 96.1 a ± 1.40 0.113
a,b,c,dMeans in the same row with different superscripts differ (p < 0.05). NC = Negative Control, without any
additive; PC = Positive Control, Enramycin at 0.2 g/kg; EBA = Microencapsulated Butyric Acid at 0.3 g/kg;
YC = Yeast Culture at 1 g/kg; EBA+YC = Combined Microencapsulated Butyric Acid & Yeast Culture at 0.3 g/kg
and at 1 g/kg, respectively; BW = body weight; BWG = body weight gain; FI = feed intake; FCR = feed conversion
ratio; Liv = livability.

3.2. Carcass Traits

Table 4 presents the results of the treatment effects on the broiler’s carcass charac-
teristics at d 35. The results revealed that the highest carcass yield was obtained from
broilers that had received EBA+YC, which was significantly different from the NC and
PC groups (p < 0.001). Insignificant differences were reported for EBA, YC, and EBA+YC
(p > 0.05). Breast muscle yield followed the same trend as carcass yield; higher breast yield
was obtained from EBA, YC, and EBA+YC (p < 0.001). Breast yield from the PC group was
intermediate with no significant difference from the EBA+YC or NC groups. The NC group
had the lowest breast muscle yield. Leg quarter yield showed no significant differences
between treatments (p > 0.05). Similarly, heart, liver, and gizzard absolute weights showed
no significant differences between all treatments (p > 0.05). The broilers supplemented with
EBA+YC had considerably more bursal development when compared to the NC and PC
groups (p < 0.05). The spleen weight was significantly higher for EBA+YC compared to the
NC, PC, and YC (p < 0.01), but it was similar to the EBA group (Table 4).

Table 4. Carcass yield and some internal organ weights as a percentage of pre-slaughter weight in
broiler chickens at 35 d.

Treatments
Parameter p-Value
NC PC EBA YC EBA+YC
Carcass yield (%) 68.7 c ± 0.40 70.0 b ± 0.34 70.6 ab ± 0.33 70.5 ab ± 0.34 71.3 a ± 0.34 0.001
Leg quarter (%) 23.4 ± 0.27 23.5 ± 0.26 23.7 ± 0.22 23.6 ± 0.24 24.0 ± 0.23 0.150
Breast weight (%) 24.7 b ± 0.24 25.5 ab ± 0.25 26.3 a ± 0.25 26.5 a ± 0.23 26.5 a ± 0.22 0.001
Heart weight (g) 8.5 ± 0.14 8.7 ± 0.16 8.8 ± 0.15 8.8 ± 0.07 8.9 ± 0.11 0.338
Liver weight (g) 41.7 ± 0.28 41.8 ± 0.41 42.0 ± 0.41 42.1 ± 0.25 42.2 ± 0.27 0.841
Gizzard weight (g) 33.5 ± 0.55 33.7 ± 0.21 33.9 ± 0.19 33.8 ± 0.23 34.0 ± 0.20 0.796
Spleen (g) 2.31 c ± 0.07 2.56 bc ± 0.11 2.82 ab ± 0.10 2.63 bc ± 0.15 3.00 a ± 0.10 0.003
Bursa (g) 1.79 c ± 0.10 1.91 bc ± 0.11 2.15 ab ± 0.09 2.05 abc ± 0.11 2.29 a ± 0.08 0.043
a,b,c
Means in the same row with different superscripts differ (p < 0.05). NC = Negative Control, without any
additive; PC = Positive Control, Enramycin at 0.2 g/kg; EBA = Microencapsulated Butyric Acid at 0.3 g/kg;
YC = Yeast Culture at 1 g/kg; EBA+YC = Combined Microencapsulated Butyric Acid & Yeast Culture at 0.3 g/kg
and at 1 g/kg, respectively.
 Vet. Sci. 2025, 12, 359 8 of 14

3.3. Intestinal Morphology

A significant increase in VH, CD, and VH: CD was reported due to treatments
(p < 0.001, 0.05, and 0.001, respectively). The highest VH (1776.2 µm) and VH: CD (7.3) were
in broilers fed with the EBA+YC diet, which was higher than all other treatments except for
VH: CD for the EBA group (Table 5). On the other hand, the highest CD (284.0) (p < 0.05)
was observed in broilers with basal diet-fed without any supplementation (Table 5).

Table 5. Effects of different treatments on intestinal morphology of commercial broilers (35 d).

Treatments
Parameter p-Value
NC PC EBA YC EBA+YC
VH (µm) 1126.8 d ± 33.09 1393.8 c ± 32.83 1568.0 b ± 35.40 1495.8 b ± 31.29 1776.2 a ± 33.26 0.001
CD (µm) 284.0 a ± 21.07 283.8 a ± 18.53 235.2 b ± 7.76 277.5 ab ± 10.87 243.7 ab ± 6.33 0.050
VH: CD 4.09 c ± 0.33 5.03 b ± 0.38 6.69 a ± 0.20 5.42 b ± 0.20 7.30 a ± 0.14 0.001
a,b,c,d
Means in the same row with different superscripts differ (p < 0.05). NC = Negative Control, without any
additive; PC = Positive Control, Enramycin at 0.2 g/kg; EBA = Microencapsulated Butyric Acid at 0.3 g/kg;
YC = Yeast Culture at 1 g/kg; EBA+YC = Combined Microencapsulated Butyric Acid & Yeast Culture at 0.3 g/kg
and at 1 g/kg, respectively; VH = villus height; CD = crypt depth; VH: CD = Villus height into crypt depth ratio.

3.4. Immune Status

The results showed a notable impact (p < 0.05) of added YC, EBA, and EBA+YC on
the development and maturity of immune organs and NDV titers on d 21 and 35. The
highest (p < 0.001) ND titers at d 21 and 35 were observed in the EBA+YC and EBA groups
Vet. Sci. 2025, 12, x FOR PEER REVIEW 9 of 15
(Figure 1). Moreover, NDV titers were higher for the PC and YC groups compared to the
NC (p < 0.001).

8 p = 0.001 p = 0.001 a
a ab
7 bc
ab c
6 bc
c d
5
ND Titer

d
4
3
2
1
0
d 21 d 35

NC PC EBA YC EBA+YC
Figure 1. Effects of dietary treatments on the antibody titer against Newcastle disease in broiler
Figure 1. Effects
chickens of dietary
on days 21 and 35. a,b,c,d Means
treatments on the antibody
in the titerwith
same row against Newcastle
different disease
superscripts in broiler
differ (p < 0.05).
chickens on days 21 and 35. a,b,c,d Means in the same row with different superscripts differ (p < 0.05).
3.5. Intestinal Microbial Profile
The results
3.5. Intestinal regarding
Microbial Profile the influence of different treatments on intestinal microbial
counts of broilers
The results are described
regarding in Figure
the influence 2. The results
of different indicated
treatments on aintestinal
significant (p < 0.001)
microbial
decreased number of E. coli and Salmonella in the ileal digesta of broiler chickens
counts of broilers are described in Figure 2. The results indicated a significant (p < 0.001) for all
supplemented
decreased numbergroups compared
of E. coli to the NC.
and Salmonella in The EBA+YC
the ileal group
digesta lowest Salmonella
had thechickens
of broiler for all
count compared to all other groups and the lowest E. Coli count compared
supplemented groups compared to the NC. The EBA+YC group had the lowest Salmonella to all groups
except
count the EBA
compared togroup (p <groups
all other 0.001). and the lowest E. Coli count compared to all groups
except the EBA group (p < 0.001).

10 p = 0.001 p = 0.001
a
8
unt
 decreased number of E. coli and Salmonella in the ileal digesta of broiler chickens for all
supplemented groups compared to the NC. The EBA+YC group had the lowest Salmonella
Vet. Sci. 2025, 12, 359 count compared to all other groups and the lowest E. Coli count compared to all groups
9 of 14
except the EBA group (p < 0.001).

10 p = 0.001 p = 0.001
a
8

Microbial Count
b b
6 c c a
b b
4 c

2
d
0
E. coli Salmonella

NC PC EBA YC EBA+YC
Figure 2. The effects of treatments on the ileal microbial count at 35 days (log10 CFU/g). a,b,c,d Means
Figure 2. The
in the sameeffects of treatments
row with on the ileal microbial
different superscripts differ (p < count
0.05). at 35 days (log10 CFU/g). a,b,c Means
in the same row with different superscripts differ (p < 0.05).
4. Discussion
In poultry production, sub-therapeutic antibiotics are frequently used to encourage
growth and guard against bacterial infections. However, restrictions on their usage in
animal production have prompted the development of alternatives to AGPs. The individual
potentials of yeast-based products and organic acids are well described in the existing
literature. However, their combined application has been limited, and only a few studies
have examined the synergistic effects. Thus, this study aimed to assess the synergistic
effects of YC and microencapsulated BA supplementation alone and in combination on
performance, carcass traits, immune status, and gut health in broilers.
The non-significant difference in the FI among all the treatments obtained in this study
is in alignment with the findings of Panda et al. [39], who concluded that supplementation
of 0.05% butyric acid showed a non-significant difference in FI over the whole experimental
period (1–35 d). Similarly, Zhen et al. [17] concluded that broiler FI was not affected by
YC supplementation at different levels. The findings of this study are also consistent with
those of Chand and Khan [40], who found that FI was unaffected by single-cell protein of
yeast at the different levels during all broiler growth phases, while butyric acid increased
the FI. This improvement in FI could be due to protection against gut pathogens through
competitive exclusion, enhanced nutrient utilization, and improved growth feed efficiency
by supplementing broiler diets with butyric acid [41].
The current study showed that treatments had a significant effect on the BWG of
broilers during all phases. In line with a previous study, [38] found similar results of
EBA on the BWG in broilers; they explained that organic acid’s low pH and antibacterial
qualities prevent harmful gut microorganisms and lessen the production of detrimental
byproducts; moreover, the improvement in the BWG of broilers was attributed to improv-
ing the digestibility of protein and energy. Furthermore, the supplementation of diets
with propionic acid and butyric acid (0.2% and 0.3%) significantly increased the BWG of
broiler chickens [42]. Additionally, the findings of our study regarding improved BWG
concur with [43,44], who credited this increase in BWG to the optimized intestinal environ-
ment when supplementing broiler diets with butyric acid. In agreement with the results
obtained herein, another group of researchers reported improved BWG when yeast was
supplemented [45,46]. The yeast improved digestion, gut health, and nutrient absorption,
resulting in better BWG. In the current study, there was an improvement in FCR due to
EBA, YC, and their combination in all growth phases. Rationally, a higher BWG at a similar
Vet. Sci. 2025, 12, 359 10 of 14

FI brought out the variation of FCR among the treatments. Organic acid supplementation
at 2 g/kg [43,44] and encapsulated calcium butyrate at 0.2, 0.3, or 0.4 g/kg were reported
to improve FCR [46]. The beneficial effects of EBA on intestinal microbiota, gut morphol-
ogy, and digestive processes may be responsible for this increase in FCR [47]. Regarding
the addition of YC in the broiler diet, our findings correlate with Alqahtani [10], who
reported that YC supplementation at 0.25 g/kg can restore the growth performance of
broiler chickens during a C. perfringens challenge, especially FCR. The YC contains various
components, including β-glucans, which have been confirmed to positively influence the
intestinal health of broiler chickens through diverse mechanisms.
High carcass and breast yields were obtained from EBA or YC independently, but
the combination of the two additives (EBA+YC) further improved carcass yield. Butyrate
was reported to improve carcass yield [46–48]. The results may be because of the ability
of organic acids to enhance protein digestion, affect intestinal cell morphology, stimulate
pancreatic secretions, act as a substrate for intermediate metabolism, improve nutrient
retention, and control electrolyte balance in the intestine. These factors will provide the host
animal with more nutrients for protein accretion. Other reports found that YC improved
carcass and breast yield [49]. Contrary to the earlier findings, a non-significant difference
in the carcass yield and the relative weight of giblets across all the treatments was noted
by [19].
The immune system plays a significant role in poultry health regulation and disease
prevention. In this study, the titer against NDV was higher for all treatments compared to
the NC. This could be due to the reduced intestinal pH, increased intestinal integrity, and
improved microbial balance in the intestine with OAs use, which leads to the enhancement
of immune response. The results obtained herein aligned with those of [29,50], who
concluded that sodium butyrate supplementation resulted in significantly higher NDV
antibody titers in broilers and layers. Moreover, the current outcomes are consistent with
those of Gao [51] and Muthusamy [52], who found that adding YC significantly improved
the broilers’ antibody titer against NDV and improved the immune status in broilers. This
may be explained by the beneficial effects of YC on preserving the physiological balance
of immunopotent cells and, consequently, creating a robust immune system environment.
This suggests that YC might increase antibody production by the humoral immune system.
The intestinal mucosa is coated with more antibodies that shield villi from damage. It was
suggested that the oligosaccharides found in YC walls might attach to viruses and function
as vaccine adjuvants in YC-treated birds to raise antibody titers [53].
The findings of this study showed that treatments, especially EBA+YC, have a notable
effect on the gut microbiota of broilers. In this study, the supplementation of EBA reduced
Salmonella and E. coli counts in the small intestine. This might be explained by the lower
gut pH caused by OAs, which is detrimental to the growth of acid-intolerant coliforms.
Butyrate, among other SCFAs, has a higher efficacy against acid-intolerant coliforms like
Salmonella and E. coli. The outcomes are similar to those of [39], who proposed that the
E. coli population was significantly decreased in the gut with butyrate supplemented to
broilers. When butyrate is supplemented to broilers, it rapidly releases sodium ions in
the bird’s stomach; butyrate is swiftly transformed into the undissolved form known
as butyric acid [54]. Since butyric acid is highly lipophilic and may permeate bacterial
membranes, this form is the one that exhibits antimicrobial activity. Butyrate can prevent
harmful bacteria from colonizing the lower portion of the intestinal system by blocking the
production of genes that cause the invasion of the epithelial cells [55]. Findings of another
study by Sun et al. [46] reported that YC supplementation to broilers significantly reduced
the count of E. coli in comparison to the control group.
Vet. Sci. 2025, 12, 359 11 of 14

An essential indicator of intestinal health is the ratio of VL to CD. The results ob-
tained for the intestinal morphometrics in this study agree with those of Ahsan et al. [2],
who found that butyric acid supplementation significantly improved VH and VH: CD.
The improvements in intestinal morphological characteristics are explained by butyrate
absorption by the enterocytes and their function as an energy source, which could be
related to the better gut health of birds. It can be inferred that the EBA and YC used
have improved the intestinal absorptive area by enhancing VH. Increased VH and deeper
crypts happen because of increased growth of the enterocytes and elongation of the villi.
The ingestion of butyrate is known to alter the microstructure of intestines, and butyrate
enhances enterocyte development, differentiation, and proliferation [27]. Similarly, the
findings of another study [47] stated that sodium butyrate linearly improved the VH and
VH: CD with increasing levels in the feed. Similarly, broilers fed EBA (0.3 g/kg) presented
higher VH, whereas the CD was decreased compared to the control birds [46]. A high
VH to CD ratio indicates a long villus in which the epithelium is sufficiently matured and
functionally active, in combination with a shallow crypt with constant cell renewal. The
effect on VH could be due to better pancreatic fluid secretion, as well as better digestion
and absorption of dietary nutrients. Contrary to the findings of our study, Levy et al. [30]
found non-significant variations in VH, CD, and VH: CD in the small intestine of broilers
fed a diet having 0.3 g/kg EB compared to the NC. Considering the addition of YC in
the broiler diet, our results are aligned with the results of different groups [12,15,45] who
reported that broilers fed different levels of YC showed an increase in VH, CD, and VH: CD.
In contrast to the above results, [56] concluded that YC supplementation to the broiler’s
diet under heat stress resulted in a non-significant change in the amount of VH and CD in
the small intestine.

5. Conclusions
The results of the present study demonstrated that EBA and YC supplements to the
broiler’s diet independently enhanced the performance of broilers by improving BWG and
FCR without negatively affecting FI. However, when these two additives were combined
in treatment 5 (EBA+YC), there was a synergistic effect, and the positive response was
more pronounced on performance. Additionally, this combination improved carcass traits,
immune response, and intestinal morphometrics while effectively lowering the burden of
harmful pathogenic bacteria in the ileum. The findings suggest that EBA and YC supple-
mentation can be a good substitute for AGPs, promoting sustainable poultry production.

Author Contributions: Conceptualization and methodology: A.N. and E.U.K.; investigation and
validation, M.M. and A.E.A.; supervision: E.U.K., S.N.Q. and S.A., data curation and formal analysis,
S.A. and R.A.A.; writing—original draft preparation, review, and editing, E.U.K., S.A., M.M., S.N.,
R.M.K.Y. and A.E.A.; funding acquisition, resources, and project administration, A.R.A.S., R.A.A.,
A.E.A. and E.U.K. All authors have read and agreed to the published version of the manuscript.

Funding: This project was funded by Researchers Supporting Project number [RSPD2025R581], King
Saud University, Riyadh, Saudi Arabia.

Institutional Review Board Statement: This research adhered to ethical guidelines provided by the
Ethical Review Committee of UVAS, Lahore, ensuring the well-being of the birds throughout the
study (Approval no: 196; Date: 16 March 2022).

Informed Consent Statement: Not applicable.

Data Availability Statement: The authors declare that all the data and materials used in this study
comply with field standards and are available on demand.

Conflicts of Interest: The authors declare that they have no conflicts of interest.
Vet. Sci. 2025, 12, 359 12 of 14

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