Open Life Sciences 2021; 16: 311–322

Research Article

Lihuan Zhang*, Ruonan Zhang, Hao Jia, Zhiwei Zhu, Huifeng Li, Yueyue Ma
Supplementation of probiotics in water beneficial growth
performance, carcass traits, immune function, and antioxidant
capacity in broiler chickens
https://doi.org/10.1515/biol-2021-0031
received April 29, 2020; accepted January 04, 2021
1 Introduction
Abstract: This study aims to investigate the effects of Chicken meat, which contains unsaturated fatty acids,
commercial probiotic supplementation in water on the oleic acid and linoleic acids, reduces low-density lipopro-
performance parameters, carcass traits, immune function, teins and cholesterol, which are harmful to humans.
and antioxidant capacity of broiler chicks. In the experi- Hence, the large-scale and intensive development of the
ment, 120 Arbor Acres (AA) broilers (60 male and 60 female) broiler chicken industry is blooming along with the rising
were randomly allocated into four groups (G) – G1: basal demand for chicken [1]. However, in order to meet the
diet and G2, G3, and G4: basal diet with 1% Lactobacillus large demand for broilers in the market, antibiotics that
casei, 1% L. acidophilus, and 1% Bifidobacterium in the
promote the growth of broilers are abused [2,3]. Excessive
water, lasting 42 days. The experimental results revealed
use of antibiotics brings about many problems such as
that probiotic additives produced positive impacts on body
drug resistance in animals and drug residues in livestock
weight, average daily feed intake (ADFI), and average daily
products, which threatens the sustainable development
weight gain for female chicks, whereas these probiotics sig-
of human and nature, and it has emerged as a severe food
nificantly reduced ADFI and the feed conversion ratio of
security issue [4]. To address this problem, several coun-
male chicks (P < 0.05). Probiotics efficiently improved evis-
tries have legislated and banned the application of anti-
cerated yield and breast yield while reducing the abdominal
biotics as growth promoters in feeds [5]. Consequently,
fat (P < 0.05) for the male broiler chicks. A marked increase
the selection of growth promoters as a replacement for
was observed in the weight of the spleen, bursa of Fabricius,
antibiotics has become a hot topic in feed research.
and thymus in the treatment group (P < 0.05). Besides,
The United Nations Food and Agriculture Organization
probiotics produced a significant effect on the concentra-
and the World Health Organization have defined probiotics
tions of immune-related proteins (P < 0.05) and markedly
as “living microorganisms which when administered in
increased the concentrations of antioxidase and digestive
adequate amount confer health benefits on the host” [6].
enzymes when compared with the control (P < 0.05).
The probiotics, Lactobacillus, Lactococcus, Bifidobacterium,
The addition of probiotics dramatically reduced the total
and Saccharomyces, are commonly used in laboratory
counts of Escherichia coli and Salmonella and increased
animal experiments [7]. Probiotics are usually produced
the quantity of Lactobacilli (P < 0.05). The results of the
in the feed industry through the processes of isolation, cul-
present study demonstrated an increase in growth perfor-
tivation, and fermentation and can be used as additives in
mance, carcass traits, immune function, gut microbial
feed production [8,9]. Multiple works of literature have
population, and antioxidant capacity by supplementing
reported the diverse benefits of probiotic supplementation
1% probiotics (L. casei, L. acidophilus, and Bifidobacterium)
in breeding commercial animals, including the increase in
in the water for broilers.
feed conversion ratio (FCR) and weight gain, egg/milk yield,
Keywords: broiler chicks, probiotics, growth performance, and the reduction in morbidity and mortality [10–12]. Pro-
carcass traits, gut microbiota biotics advance the growth performance, nutrient digesti-
bility, and FCR; improve the gastrointestinal microecological
 environment; and enhance the internal immunity as well
* Corresponding author: Lihuan Zhang, College of Life Science, as antioxidant capacity, thereby inhibiting the adhesion
Shanxi Agricultural University, Jinzhong 030801, Shanxi Province,
of pathogenic bacteria in broiler chickens [13–16]. In addi-
China, tel: +86-13903446909, e-mail: [email protected]
Ruonan Zhang, Hao Jia, Zhiwei Zhu, Huifeng Li, Yueyue Ma: College
tion, probiotics were used to compact the negative effects
of Life Science, Shanxi Agricultural University, Jinzhong 030801, of heat stress [8], salmonella [17], detoxification of nitrate
Shanxi Province, China [2,16], and aflatoxin [3,18]. The substitute of veterinary

Open Access. © 2021 Lihuan Zhang et al., published by De Gruyter. This work is licensed under the Creative Commons Attribution 4.0
International License.
312  Lihuan Zhang et al.

drug probiotics therefore is of great significance to Table 1: Composition and nutrient levels of basal diets (on dry
enhance the production scale and green development of matter basis) %
broilers.
As the previous study reported, multiple probiotics Ingredient (%) 1–21 days 22–42 days
of age of age
seem to positively affect the production and physiological
traits of broiler chickens. However, of which Lactobacillus Corn 55.75 62.00
casei, L. acidophilus, and Bifidobacterium are the most Soybean meal (44%) 35.00 25.70
Fish meal 3.00 7.00
useful probiotics for farmers; and their effect on male
Soybean oil 3.00 2.50
and female broilers requires further exploration. To assess Salt 0.30 0.20
the effects of the three probiotics on male and female Limestone 1.00 1.00
broilers at different growth stages, the present study aimed CaHPO4 1.50 1.00
to investigate the effects of probiotic additives in the water Choline chloride 0.15 0.10
on growth performance, carcass traits, immune function, Mineral premixa 0.10 0.20
Vitamin premixb 0.20 0.30
gut microbiota, and antioxidant capacity of broilers.
Total 100.00 100.00
Nutrient levelsc
Metabolizable energy 12.47 12.73
(MJ/kg)
2 Materials and methods Crude protein (%) 21.42 20.46
Calcium (%) 1.01 1.04
Available phosphorus (%) 0.46 0.48
2.1 Probiotics Lysine (%) 1.22 1.18
Methionine (%) 0.35 0.38
In this research, as a probiotic feed additive, L. casei, Met + Cys 0.71 0.69
L. acidophilus, and Bifidobacterium were selected as the
experimental strains. The effective live bacteria content
a
Mineral premix provided per kilogram of diet: Cu (CuSO4·5H2O) 8 mg,
of all probiotics was greater than 5 × 109 CFU/g. All bacteria Fe (FeSO4·7H2O) 50 mg, Zn (ZnSO4·7H2O) 45 mg, Mn (MnSO4·H2O)
65 mg, Se (Na2 SeO3) 1 mg, Ca(IO3)2 1 mg, and Ca3(PO4)2 0.2 mg.
were purchased from Shanghai Xianlong Biotechnology b
Vitamin premix provided per kilogram of diet: VA 40000000 IU, VD3
Co., Ltd. (Shanghai, China). 11000000 IU, VE 80000 IU, VK3 12 g, VB1 10 g, VB2 22 g, VB6 15 g, VB12
100 mg, folic acid 4 g, biotin 300 mg, niacinamide 100 g, pantothenic
acid 50 g, and antioxidant 500 mg. c Nutrition levels were calculated
values.
2.2 Basal diet and the calculated nutrient
composition

The corn–soybean basal diet was prepared according to were supplied with distilled water with a basal diet as
the National Research Council recommendations (NRC, the control group (G1). In the treatment of a single pro-
1994) and the Chinese Chicken Feeding Standards (2004). biotic group, 1% (10 mL of probiotics per liter of distilled
Basal diets’ formula and chemical composition are shown water) L. casei (G2), L. acidophilus (G3), and bifidobac-
in Table 1. terium (G4) were added into daily drinking water. To
ensure the activity and the effect of the probiotic prepara-
tion, the individual strain was refrigerated at 4°C, and
2.3 Experimental design every single strain of probiotic was mixed every morning
before use. The whole experimental feeding period was
A total of 120 1-day-old Arbor Acres (AA) broiler chicks for 42 days.
(60 male and 60 female) were selected for the experi-
ment. On the first day, male and female chicks were iden- Ethical approval: The research related to animal use has
tified by the feather sexing method and randomly divided been complied with all the relevant national regulations
into four groups (30 each) with similar mean body weight and institutional policies for the care and use of animals
(BW), three replicates of each group, and 10 chicks and approved by the Shanxi Agricultural University Ethics
(5 males and 5 females) in each replicate. The broilers Committee.
 Effects of probiotic supplementation in broilers  313

2.4 Feeding management Carcass yield was calculated as a percentage of the pre-live
BW of the broilers. The indexes of these immune organs
All the experiments were carried out in a fully enclosed were expressed as immune organ fresh weight (g)/live BW (kg).
three-tiered chicken coop (length × width × height, 140 cm × Five milliliters of arterial blood of the chickens were col-
70 cm × 40 cm). The chicken coop was thoroughly cleaned lected and centrifuged at 3,000× g for 15 min to obtain the
and disinfected prior to the experiment. The environ- serum. The jejunum tissue was carefully rinsed with phos-
mental conditions in the cage were set following the phate buffer, and partial tissue was transferred with the
requirements of the “AA Broiler Breeding Management serum for preservation at −80°C. For the measurement of
Manual”. Feeding and drinking water (free from antibio- the intestinal microbial population, 1 g of the composite
tics) were provided ad libitum. The room temperature was gut sample was accurately weighed in a sterile environment.
kept at 33°C during the first 3 days of age. Thereafter, the
temperature was decreased by 3°C per week to reach 24°C
at 21 days of age. The temperature was subsequently main-
2.7 ELISA and biochemical tests
tained at ∼24°C until the end of the experiment. Twenty-four
hour lighting was provided on day 1, followed by 23 h/day,
ELISA kit (Shanghai Meilan, China) was used to deter-
with 1 h of darkness from 19:00 to 20:00. The chicks were
mine the concentrations of immune-related proteins (IL-2,
fed regularly, the health status was observed, and the feed
IL-10, IgA, and IgG) and digestive enzymes (amylase, lipase,
consumption of each group was accurately recorded. The
and trypsin) [20]. The contents of superoxide dismutase
chicks were inoculated with Marek’s disease vaccine at
(SOD), glutathione peroxidase (GSH-Px), total antioxidant
the hatchery, infectious bronchitis virus, influenza at
capacity (T-AOC), and malondialdehyde (MDA) were deter-
day 7, Gumboro disease vaccine at days 14 and 24, and
mined by biochemical assay on the basis of the instruction
Newcastle disease vaccine at days 7 and 18.
of the chemical kits purchased from Nanjing Jiancheng
Bioengineering Institute (Nanjing, China). All determination
procedures and calculation formulas were carried out in
2.5 Performance traits strict accordance with the manufacturer’s instructions.

The average BW of each group was measured and recorded

at 21 and 42 days of age. The average daily weight gain
2.8 Gut microbial population
(ADG), the average daily feed intake (ADFI), and the FCR
in each growth period were calculated based on the
The samples of the chickens were diluted with 9 mL of
experimental records. The weight of dead broiler chickens
0.9% saline solution and mixed in a vortex for 15 min.
was included to calculate the average weight gain, feed
The viable numbers of bacteria in the samples were sub-
intake, and FCR.
sequently counted by plating serial 10-fold dilutions
(in 1% peptone solution) into eosin methylene blue
agar, Salmonella–Shigella agar, and lactobacilli de Man,
2.6 Sample collection and analysis
Rogosa, and Sharpe agar plates (to isolate Escherichia coli,
Salmonella, and Lactobacillus). E. coli and Salmonella were
At 21 and 42 days of age, the broilers were electrically
aerobically cultured in a 37°C incubator for 24 h before
stunned and exsanguinated to obtain tissues. Selected
colony counting, and Lactobacillus was anaerobically incu-
chickens were not given feed 12 h before slaughter, whereas
bated at the same temperature for 48 h after colony counting.
constant access to water was ensured [19]. Five female and
Bacterial colony-forming units (CFUs) in the petri dishes
five male broilers were randomly selected from each group
were counted using a colony counter. The counts were
for slaughtering carcass, characteristic evaluation, artificial
expressed as log 10 CFUs per gram of digesta (log10 CFU/g).
anatomy, and weighing. All parameters of live BW, dressed
weight, eviscerated weight, half-eviscerated weight (evis-
cerated weight with giblet), breast muscle, leg muscle,
abdominal fat, and immune organ (bursa of Fabricius, 2.9 Statistical analyses
thymus, and spleen) were excised and weighed individu-
ally. The calculation method of carcass traits was conducted The experimental data were compiled by Excel 2016. The
following the description of Ghasemi-Sadabadi et al. [14]. data were expressed as mean ± standard error of the
314  Lihuan Zhang et al.

mean (mean ± SEM, n = 5). The one-way ANOVA using treatment of probiotic efficiently elevated the BW, ADG,
SPSS 20 software (SPSS Inc., IL, USA) was used to ana- and the ADFI in female chickens when compared with the
lyze the different treatment groups, and Tukey’s test with control, whereas no impact was observed on the FCR. In
mean separations was applied to determine the signifi- the probiotic group, the ADFI and FCR of male chickens
cant differences. A two-tailed t test with a p value of <0.05 significantly reduced, while BW significantly increased.
was considered significant. During the 0–42 days, probiotic supplementation mark-
edly increased the BW, ADG, and ADFI in female
chickens and significantly improved BW and FCR in
male chickens.
3 Results

3.1 Production performance 3.2 Carcass traits

As shown in Table 2, the supplement of probiotics to daily The carcass traits of broilers such as dressed yield, evis-
drinking water had no obvious effect on ADG, ADFI, and cerated yield, half-eviscerated yield, breast muscle, leg
FCR of both male and female chickens in their early muscle, and abdominal fat were tested. As illustrated in
growth period (0–21 days). From day 22 to 42, the Figure 1, no notable difference was revealed amid groups

Table 2: Effects of probiotic supplementation on the growth performance parameters in male and female broiler chickens

Items Treatments Statistical parameters

G1 G2 G3 G4 SEM p value

1–21 days (male)

BW, g 507 481 490 517 10.4 0.299
ADFI, g/day 42.7 41.4 41.7 42.0 3.76 0.826
ADG, g/day 21.6 20.1 20.7 22.0 2.35 0.453
FCR, kg/kg 1.98 2.06 2.02 1.91 0.08 0.085
1–21 days (female)
BW, g 569 536 546 578 15.2 0.484
ADFI, g/day 42.65 42.79 42.35 41.59 3.24 0.697
ADG, g/day 24.7 23.2 23.6 25.2 1.56 0.144
FCR, kg/kg 1.73 1.85 1.79 1.65 0.06 0.092
22–42 days (male)
BW, g 1,426b 1,527a 1,454b 1,543a 20.1 0.020
ADFI, g/day 125a 106b 114b 109b 5.08 0.019
ADG, g/day 43.3 49.8 45.9 48.8 3.91 0.086
FCR, kg/kg 2.60a 2.13b 2.48a 2.23b 0.15 0.003
22–42 days (female)
BW, g 1,542c 1,787a 1,769ab 1,824a 25.8 <0.0001
ADFI, g/day 115b 127a 128a 128.5a 5.26 0.028
ADG, g/day 46.4b 59.5a 58.2a 59.33a 4.85a 0.037
FCR, kg/kg 2.48 2.14 2.20 2.16 0.18 0.061
1–42 days (male)
BW, g 1,452b 1,533a 1,462b 1,548a 21.7 0.031
ADFI, g/day 84.0a 73.9b 77.8ab 75.3b 3.12 0.048
ADG, g/day 32.4 34.9 33.2 35.4 2.45 0.069
FCR, kg/kg 2.59 2.12 2.34 2.13 0.11 0.039
1–42 days (female)
BW, g 1,563 1,790 1,764 1,825 23.1 <0.0001
ADFI, g/day 78.9b 85.1a 85.3a 85.0a 2.97 0.042
ADG, g/day 35.5b 41.4a 40.9a 42.3a 2.86 0.029
FCR, kg/kg 2.19 2.04 2.08 2.01 0.09 0.225

BW: average body weight; ADFI: average daily feed intake; ADG: average daily weight gain; FCR: feed conversion ratio = ADFI/ADG. Mean
values (± SEM, n = 5) within each row of different treatments with common superscript do not differ significantly at p < 0.05. G1: control
group, and G2–G4: treatment groups of 1% (probiotic, 10 mL/L water) L. casei, L. acidophilus, and Bifidobacterium, respectively.
 Effects of probiotic supplementation in broilers  315

Figure 1: Carcass traits of male and female broilers fed probiotics on days 21 and 42, respectively. These data (mean ± SEM, n = 5) were
analyzed using one-way ANOVA, followed by post hoc Tukey’s test. The “**” indicated when the treatment was compared to the control and
p < 0.05. G1: control group; G2–G4: treatment groups with 1% (probiotic, 10 mL/L water) L. casei, L. acidophilus, and Bifidobacterium,
respectively.

of day 21. On day 42, the probiotic-supplemented male organ index of male and female broiler chickens in G2,
chickens had a significantly higher eviscerated yield, G3, and G4 groups, respectively. On day 21, compared
while a dramatically lower abdominal fat percentage with the control group, the supplemented probiotics
than the control. Interestingly, the percentage of chest (G2, G3, and G4) markedly increased the thymus index
muscle in female chickens was only significantly increased in female broiler chickens, and a dramatic increase was
in the G4 (Bifidobacterium) group. No significant difference revealed in the spleen index of the male broilers. The G2
was observed in half-eviscerated yield and leg muscle. manifested a significant increase in the thymus index of
No significant difference was observed in half-eviscerated male chickens. An increase in the spleen index of the
yield and leg muscle percentage compared with the control male broilers was observed in the G2 and G3, while that
group. of the female broilers was increased only in the G3. Com-
pared with the control group, the bursa of Fabricius index
suggested no significant difference. On day 42, the
3.3 Immune function thymus indexes of male and female broilers in the G2
and G3 groups and the G4 group were significantly higher
The data presented in Figure 2a show that supplemented than those of the control. The spleen index of female
probiotics produced different effects on the lymphoid broilers in G2 and G4 showed a higher percentage, while
316  Lihuan Zhang et al.

Figure 2: Effects of probiotics on immune function in male and female broiler chickens at 21 and 42 days. (a) The index of lymphoid organs in
male and female broiler chickens. Immune organ index = immune organ fresh weight (g)/live weight (kg). (b) The immune-related protein in
serum was detected by the ELISA kit. All data (mean ± SEM, n = 5) were analyzed using one-way ANOVA followed by post hoc Tukey’s test.
** represents the treatment group compared to the control group and indicates p < 0.05. * indicates p < 0.01. G1: control group and G2–G4:
treatment with 1% (probiotic, 10 mL/L water) L. casei, L. acidophilus, and Bifidobacterium, respectively.

male broilers in G3 showed a higher percentage. L. acid- and G3 was higher than that in the control group. How-
ophilus (G3) had a significant positive impact on the ever, the concentration of IL-10 in female chickens was
bursa index in male broilers, while, in female broilers, decreased in G3 and G4. On day 42, the feeding with
Bifidobacterium (G4) had a significant positive impact on probiotics (G2, G3, and G4) greatly enhanced the concen-
bursa index. tration of IgA and IgG in male and female broilers. The
Furthermore, the effects of probiotics on concentra- concentration of IL-2 showed no significant difference
tions of immune-related proteins (IL-2, IL-10, IgA, and among all treatment groups. As for roosters, feeding
IgG) in broilers’ serum were shown (Figure 2b). On day with L. casei (G2) significantly increased the IL-10 con-
21, G3 and G4 had a higher concentration of IgA in male centration, while the L. acidophilus (G3) significantly
and female broiler chickens; and all three kinds of pro- decreased the concentration of IL-10 (Figure 2b).
biotics significantly enhanced the IgG concentrations in
female chickens. L. acidophilus (G3) and Bifidobacterium
(G4) remarkably increased the IL-2 concentration in male 3.4 Digestive enzymes and gut microbial
chickens, while L. casei remarkably decreased the IL-2 populations
concentration. In the female chickens, a significant
increase in IL-2 concentration was observed in G2 and The lipase contents of the male and female broilers fed
G4. The concentration of IL-10 in male chickens in G2 with probiotics (G2 and G4) on day 21 were higher than
 Effects of probiotic supplementation in broilers  317

days 21 and 42, an increase in Lactobacilli was observed

in the male and female chickens when compared with the
control. Of the three probiotic groups, L. casei (G2) was
the most effective. Additionally, supplementation of pro-
biotics for male and female broiler chickens effectively
reduced the number of harmful E. coli and Salmonella
bacteria in the gut.

3.5 Antioxidative activity

For the analysis of antioxidative activity following broilers

being fed with probiotics, the concentration of T-AOC,
SOD, GSH-Px, and MDA in serum on 21 and 42 days
(Figure 4) was detected as illustrated. The results
revealed that the effects of probiotic supplementation
on the concentration of SOD and T-AOC were greater
than that on GSH-Px. Over half of the treatment groups
showed a conspicuous increase in SOD and T-AOC con-
centration, while changes in GSH-Px concentration were
observed in several groups. Moreover, probiotic treat-
ment alone significantly reduced the concentration of
MDA in the male and female broilers in contrast with
the control at 21 days of age. Feeding with L. casei (G2)
or Bifidobacterium (G4) intensely reduced MDA concen-
tration on day 42. These data demonstrated that probiotic
supplementation positively affected antioxidant activity
in broilers.

4 Discussion
The multistrain probiotic containing 1 × 108 CFU/g of
Figure 3: The concentration of digestive enzymes in digestive tract L. casei, L. acidophilus, B. thermophilum, and Enterococcus
contents of jejunum was detected by the ELISA kit. All data (mean ± fed (0.900 g/kg) to broilers had positive effect on chickens
SEM, n = 5) were analyzed using one-way ANOVA followed by post
[21]. The L. casei P-8 was added to the chickens’ drinking
hoc Tukey’s test. ** represents the treatment group compared to the
control group and indicates p < 0.05. * indicates p < 0.01. G1: control
water at a final concentration of 2 × 106 CFU/mL and
group and G2–G4: treatment groups of 1% (probiotic, 10 mL/L water) showed improved weight gain, feed intake, and feed effi-
L. casei, L. acidophilus, and Bifidobacterium, respectively. ciency [20]. The probiotic mixture (L. acidophilus, L. casei,
Bifidobacterium bifidum, and Enterococcus faecium) supple-
ment at 2 × 108 CFU/kg improved FCR [22]. In summary,
that of the control group (Figure 3). On day 42, the probiotics are beneficial to broiler growth when the density
contents of lipase in female chickens fed with L. casei of the viable bacteria is greater than 105 CFU/g. In this
(G2) and Bifidobacterium (G4) were also significantly study, the density of viable bacteria of L. casei, L. acido-
increased. In addition, supplementation of L. casei (G2) philus, and Bifidobacterium was greater than 5 × 109 CFU/
and Bifidobacterium (G4) had positive effects on trypsin mL. One percent probiotic supplement in water could
concentration during the whole age period. ensure that probiotics have effect on broiler growth. These
The effects of supplementation of probiotics on gut findings confirmed the positive effects of probiotics on
microbial populations are illustrated in Table 3. On both the growth performance of broilers, consistent with the
318  Lihuan Zhang et al.

Figure 4: The concentration of antioxidant enzymes and oxidation products in serum was detected by the ELISA kit. All data (mean ± SEM,
n = 5) were analyzed using one-way ANOVA followed by post hoc Tukey’s test. ** represents the treatment group compared to the control
group and indicates p < 0.05. * indicates p < 0.01. G1: control group and G2–G4: treatment groups of 1% (probiotic, 10 mL/L water) L. casei,
L. acidophilus, and Bifidobacterium, respectively.

Table 3: Effects of probiotic addition on gut microbial population (log10 CFU/g) in male and female broiler chickens at 21 and 42 days

Items Treatments Statistical parameters

G1 G2 G3 G4 SEM p value

1–21 day (male)

E. coli 5.02a 4.26c 4.63b 4.30c 0.078 <0.0001
Salmonella 4.39a 4.01b 3.98b 3.92b 0.059 0.0007
Lactobacillus 6.46b 7.25a 6.81ab 7.08a 0.147 0.0032
1–21 day (female)
E. coli 5.25a 4.47b 5.08ab 4.61b 0.109 0.0029
Salmonella 4.63a 4.13b 4.22b 4.36b 0.084 0.0005
Lactobacillus 6.04c 7.12a 6.65b 7.03a 0.139 <0.0001
22–42 day (male)
E. coli 5.72a 5.04b 4.91b 5.58a 0.136 0.0015
Salmonella 4.69a 4.48ab 4.03b 3.92b 0.092 0.0024
Lactobacillus 6.85b 7.49b 7.55a 6.97b 0.164 0.0071
22–42 day (female)
E. coli 5.36a 4.72b 5.10ab 5.09ab 0.141 0.0266
Salmonella 4.99a 4.41b 4.90a 3.97c 0.121 0.0002
Lactobacillus 6.08c 7.26a 6.54b 6.73b 0.130 0.0008

Mean values (± SEM, n = 5) within each row of different treatments with common superscript do not differ significantly at p < 0.05.
G1: control group and G2–G4: treatment groups of 1% (10 mL of probiotics per liter of distilled water) L. casei, L. acidophilus, and
Bifidobacterium, respectively.
 Effects of probiotic supplementation in broilers  319

previously reported studies [15,23,24]. Furthermore, the probiotic mix (L. acidophilus, L. casei, and B. thermo-
supplement of single probiotic or mixed probiotic feed addi- philum) had a significant effect on the carcass yield, thigh
tives could remarkably improve broilers’ growth perfor- yield, and abdominal fat in male and female Ross 308
mance and reduce their FCR under normal, stress, disease, chickens [14]. On the contrary, Qorbanpour et al. (2018)
and other challenging conditions [2,3,8,16–18]. Broilers fed reported that the weights of carcass, breast, and thighs in
with the L. casei and L. acidophilus exhibited an increase in chickens were not significantly influenced by dietary
ADG [25]. Conversely, others have stated that probiotics had treatments with multistrain probiotics (L. acidophilus,
no positive effect on broilers’ performance [26]. Our data L. casei, E. faecium, and B. thermophilum) [21]. Therefore,
indicated that the addition of probiotics in water signifi- we chose to add a single L. acidophilus, L. casei, and
cantly improved the FCR of male broilers, while no signifi- B. thermophilum to explore the influence of probiotic on
cant effect was observed on FCR in female broilers. The broiler chickens. Our findings revealed that the addition
inconsistent roles of probiotics among these studies of probiotics effectively increased the eviscerated yield
including the present study may be related to the type, and reduced the abdominal fat rate in male broilers.
dosage of probiotics, and the breed of the broilers. The Hence, it seems that the use of probiotic increased diges-
positive effects of L. casei and Bifidobacterium on the growth tion and absorption due to higher intestinal microbial
performance of broilers were higher than that of L. acido- population and gut health, and the balance of absorbed
philus in our study. It is pertinent to mention that FCR is nutrients increased and caused decreasing abdominal
very crucial in the poultry sector as it reflects the efficient fat. However, no significant difference was observed in
utilization of nutrients [27]. The amylase, protease, and dressed yield, breast yield, and thigh yield. The difference
lipase produced by probiotic preparation could degrade in carcass traits between male and female broilers was
plant carbohydrates and complex substances in the feed, probably related to the utilization of nutrients under dif-
so that the carbohydrates were better absorbed and utilized ferent probiotic treatments. Differences in nutrient utili-
by the intestine, thereby improving the feed efficiency [28]. zation between male and female broilers were previously
In the present study, the treatment of probiotics increased reported [31]
the concentration of digestive enzymes in the jejunum of The lymphatic organs of poultry are the spleen, bursa
broilers in varying degrees, indirectly interpreting possible of Fabricius, and thymus, and their weight directly reflects
reasons for FCR improvement. Low FCR treated by L. casei the strength of the internal immune function [32]. It has
and Bifidobacterium seemed to be related to a high concen- been reported that single probiotic treatment or in combi-
tration of digestive enzymes. nation can increase the weight of the spleen, thymus, and
As a probiotic, lactobacillus is suitable for domestic bursa of Fabricius in broilers [33]. In the present study,
animals, because it can inhibit the growth of pathogenic different probiotics exerted the greatest impact on the
bacteria and promote the growth of nonpathogenic bacteria thymus index and spleen index of broilers. Lactic acid
by producing different metabolites, thereby improving the bacteria were widely reported to enhance the immune
intestinal microecological environment [2,3,8,16–18]. The system of animals by positively affecting the innate and
commonly encountered pathogenic or zoonotic bacteria in adaptive immune responses [34]. The contents of IgG
poultry farming are E. coli, Salmonella enterica, Campylo- and IgA in serum are important markers to estimate the
bacter jejuni, and Clostridium perfringens [5]. In the present changes in animal immune function. The administration
study, supplementation of L. casei, L. acidophilus, and Bifi- of Lactobacillus spp. could efficiently activate the immu-
dobacterium in water reduced the abundance of E. coli and nity of mucosa in chickens by increasing the levels of IgA
Salmonella and increased the abundance of Lactobacillus. and IgG [35]. These confirmed our results that the treat-
The results were consistent with the findings of other ment of probiotics increased the levels of IgG and IgA
researchers who had observed an improvement in the intest- in the serum of broilers at all growth stages. IL-2 and IL-
inal microbial population in broilers fed with probiotics 10 were the cytokines that reflected the immune level
[29,30]. In general, supplementation of probiotics in water secreted by Th1 and Th2 cells [36]. Wang et al. (2015)
could ameliorate the structure of the gut microflora. reported that L. plantarum strain P-8 activated the protec-
Eviscerated yield, breast muscle yield, leg muscle tive immune response in broilers, and the upregulated IL-2
yield, and abdominal fat rate were essential indicators and the downregulated IL-10 transcriptions were detected
for evaluating the slaughtering performance of broilers. in an age-dependent manner [20]. Another study reported
The addition of probiotics increased the carcass yield of that feeding broilers with Bacillus subtilis increased the
broiler chickens as previously reported [14,15]. Ghasemi- expression of IL-2 and IL-10 [37]. In this study, IL-2 con-
Sadabadi et al. (2019) reported that the supplement of centration increased while the IL-10 concentration
320  Lihuan Zhang et al.

increased or decreased in different treatment groups. Author contributions: L.Z. designed and performed the
These results suggested that feeding with probiotics experiments and wrote this article. R.Z. and H.J. per-
probably had an effect on broiler chickens’ immune formed the experiments and analyzed the data; Y.M.
function. Besides, the different effects of probiotics on drew the pictures using software; and Z.Z. and H.L.
the immune system of broilers were probably correlated revised and edited the manuscript.
with the differences in fermentation substrates and pro-
ducts of the three probiotics, resulting in different Conflict of interest: The authors state no conflict of
degrees of immune response between male and female interest.
broilers.
To eliminate excessive free radicals, levels of anti- Data availability statement: The data sets generated during
oxidant enzymes scavenging free radicals in vivo will and/or analyzed during the current study are available
increase and indirectly reflect the degree of oxidative from the corresponding author on reasonable request.
stress [38]. SOD, CAT, MDA, and GSH-Px are all anti-
oxidant enzymes. The ability of the human body to sca-
venge free radicals can be determined by measuring its
activity [38]. T-AOC level reflects the total antioxidant
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