Effects of honeycomb extract on the growth performance, carcass traits,

immunity, antioxidant function and intestinal microorganisms of yellow bantam

broilers

W. J. Song ,*,1 Q. L. Song,*,2 X. L. Chen,*,1 G. H. Liu ,y,* Z. H. Zou,* J. Tan,* L. X. Liu,* and
Y. B. Zeng*
*
Institute of Animal Husbandry and Veterinary Science, Jiangxi Academy of Agricultural Sciences, Nanchang
330200, P. R. China; and yFeed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, P. R.
China

ABSTRACT Although many studies have already to the control group, with all 3 HE addition groups hav-
described the physiological effects of bee products, such ing statistically identical values to the antibiotic group.
as honey, propolis, pollen, and royal jelly, on livestock HE implementation dramatically increased spleen index,
farming, the health benefits of the honeycomb are still serum immunoglobulin A (IgA), immunoglobulin M
not fully understood. The problem of drug residues and (IgM,), glutathione peroxide (GSH-Px), superoxide
bacterial resistance caused by the abuse of antibiotics is dismutase (SOD), total antioxidant capacity (T-
becoming increasingly serious. For this reason, a safe, AOC), and total cecum bacteria and Lactobacillus com-
green substitute has to be sought. We conducted a com- pared to the control group, numerically at the same level
parative study of honeycomb extract (HE) and an anti- as, or even better than, the antibiotic group. HE and
biotic on growth performance, carcass traits, immunity, CTE both markly reduced serum malondialdehyde
antioxidant function and intestinal microorganisms of (MDA) concentration compared to the control group,
yellow bantam broilers. A total of four hundred eighty with higher concentrations of HE reducing the effect
21-day-old female yellow bantam broilers were randomly more dramatically than antibiotics. Both HE and CTE
divided into 5 groups of 6 replicates of 16 birds each. The significantly raised dressed yield compared to the control
5 groups were as follows, with birds receiving a basal diet group. In summary, HE, as a potential antibiotic alter-
supplemented with 150 ppm (mg/kg) of chlortetracy- native, improved growth performance, carcass traits,
cline (CTE), a basal diet without HE (control group), immune function, serum antioxidant capacity and intes-
and a basal diet with 0.1%, 0.15%, or 0.2% HE for tinal microorganisms in yellow bantam broilers. Accord-
60 days. The results showed that HE addition signifi- ing to the cubic regression analyses, the recommended
cantly increased average daily feed intake (ADFI), supplemental dose of HE was calculated to be 0.15 to
average daily gain (ADG), decrease feed gain ratio 0.17% for female yellow bantam broilers between 21 and
(F/G) from 21 to 80 and 51 to 80 days of age compared 80 d of age.
Key words: honeycomb extract, growth performance, immune function, intestinal microorganisms, yellow bantam
broilers
2022 Poultry Science 101:101811
https://doi.org/10.1016/j.psj.2022.101811

INTRODUCTION making bacteria more resistant to drugs, and the conse-

quences of this are a threat to people's health
The widespread use of antibiotics has greatly (Dipendra et al., 2017; Seal et al., 2013). Broilers are
improved the growth performance of livestock and one of the fastest growing and very heavily farmed spe-
poultry, while the misuse of antibiotics in animal feed cies in the livestock industry, yet the addition of antibi-
may result in antibiotic residues in animal products, otics in farming has long been a common means used
to address intestinal problems in broilers
Ó 2022 Published by Elsevier Inc. on behalf of Poultry Science Asso- (Jadhav et al., 2015), but with the development of the
ciation Inc. This is an open access article under the CC BY-NC-ND times, the disadvantages of antibiotic use have been
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Received November 30, 2021. realized. Therefore, the search for alternatives to antibi-
Accepted February 22, 2022. otics in broiler feed is an increasing concern
1
These authors contributed equally to this work. (Tayeri et al., 2018; He et al., 2019).
2
Corresponding author: [email protected]

1
2 SONG ET AL.

Honeycomb also called Nidus Vespae or Beehive MATERIALS AND METHODS

(the hive of Polistes olivaceous, P. japonicus Saussure
and Parapolybiavaria fabricius) is a nontoxic and
Animals and Raw Material Preparation
natural resinous bee byproduct. Honeycomb contains Animal care and procedures were carried out in accor-
many kinds of residues of bee products, such as bees- dance with the Chinese Animal Welfare Guidelines and
wax, cocoons, honey, pollen, propolis, and royal jelly, approved by the Animal Care and Use Committee of the
and has been recognized to have several physiological Jiangxi Academy of Agricultural Sciences under
and biochemical properties, including antibacterial, approval number 2010-JXAAS-XM-01. The original
antioxidant, antimutagenic, antitumor, anti-inflam- honeycombs used in this study were provided by the
matory, and bactericidal properties (Gekker et al., Jiangxi Academy of Agricultural Sciences, China. The
2005; Li et al., 2008; Alvarez-Suarez et al., 2010; honeycomb was mixed with alcohol in the ratio of 1:2,
Cheng et al., 2011; Astani et al., 2013; Talas et al., incubated overnight, 100°C for 1 h, then filtered, cooled
2014; Zhao et al., 2015). These properties may be and concentrated to obtain HE. The contents of total
attributed to the active components in honeycombs, flavonoids, polysaccharides and polyphenols in HE was
such as flavonoids, polysaccharides and polyphenols 8.63 mg/g, 0.490 g/100 g, and 98.6 mg/g, respectively.
(Alani et al., 2019). In recent years, with the large-
scale development of apiculture, bee products have
been widely used in livestock and poultry production. Birds, Diets, and Management
Many studies have shown that the use of bee byprod-
ucts as additives in animal feed can improve the A total of four hundred eighty 1-day-old female yellow
growth and productive performance of the animals. A bantam broilers were purchased from a local hatchery
significant increase in body weight, feed consumption, (Nanchang, Jiangxi), and after an acclimatization
and feed conversion ratio and a significant decrease period (0−20 days of age), the birds were randomly allo-
in mortality was observed when chicks were fed prop- cated to 5 treatment groups with 6 replicates, each con-
olis (Shalmany and Shivazad, 2006). Propolis can taining 16 birds. The treatments were as follows: the
relieve the injury caused by heat stress and improve antibiotic group received a basal diet complemented
the quality of animal products under heat stress con- with 150 ppm (mg/kg) chlortetracycline (CTE), and
ditions (Seven, 2008). Inspite of bee byproducts's the other groups received a basal diet supplemented
broad applications, few studies have investigated the with 0.0% (control group), 0.1%, 0.15%, and 0.2% HE,
effect of honeycomb extract (HE) on animals, partic- respectively. The basal diet (in pellet form) was devel-
ularly in poultry. oped according to the Nutrition Research Council
Many countries, such as China, Turkey, Canada, (1994) that met the requirements for native breed
Argentina, and Iran, are large bee producers with large- broilers (Table 1). Throughout the whole experimental
scale bee colonies (Shahbandeh, 2021). In addition, hon-
eycombs have a certain cost advantage compared with
other bee byproducts. The substantial honeycomb yields Table 1. Composition and nutrient levels of the basal diet (air-
provided the material resources for the utilization of dry basis).
honeycomb residue as an antibiotic substitute in Contents
broilers. Items 21−50 d 51−80 d
The yellow bantam broiler is a breed bred from the
Basal ingredients
introduction of the dw gene (Stewart et al., 1984) into Corn, % 64.00 65.00
high-quality yellow bantam broilers, which has charac- Soybean oil, % 3.00 4.00
teristics such as high fertility, feed conversion and dis- Soybean meal (42.0% CP), % 26.00 26.00
Fish meal, % 2.00 -
ease resistance, low space occupation higher. Yellow Premix1, % 5.00 5.00
bantam broilers are popular with Chinese consumers for Total, % 100.00 100.00
their tender meat and unique flavor. The effect of HE on Nutrient level2
Metabolism energy, MJ/kg 12.66 13.43
the growth of yellow bantam broilers has not been Crude protein, % 18.38 17.15
reported yet. Previous results from our laboratory have Calcium, % 0.90 0.70
shown that HE can improve the growth performance of Total phosphorus, % 0.60 0.45
Lysine, % 1.17 1.07
broiler (data unpublished). Therefore, the aim of this Methionine, % 0.45 0.42
study was to evaluate the effects of HE on growth per- Cysteine, % 0.31 0.30
formance, immune and antioxidant capacity and intesti- Tryptophan, % 0.21 0.19
Threonine, % 0.70 0.64
nal microorganisms of yellow bantam broiler, as well as Arginine, % 1.21 1.13
its alternative role to antibiotics, to provide a theoretical 1
The premix provided the following per kilogram of diet: vitamin A:
basis and reference for further application of HE. In 12500 IU; vitamin D3: 500 IU; vitamin E: 25 mg; vitamin K3: 3 mg; vita-
addition, research on the substitution of HE for antibiot- min B1: 3 mg; vitamin B2: 8 mg; vitamin B6: 7 mg; vitamin B12: 0.03 mg;
ics can improve food safety and quality and promote D-pantothenic acid: 20 mg; niacin: 50 mg;biotin:0.1 mg; folic acid:1.5 mg;
Fe:100 mg;Cu:8 mg;Zn:100 mg;Mn:100 mg; I: 0.6 mg; Se: 0.16 mg.
green, healthy and sustainable development of livestock 2
Estimated from the Chinese feed database, which provides tables of
and poultry farming. feed composition and nutritive values in China (2015 26th edition).
 EFFECTS OF HONEYCOMB EXTRACT ON BROILERS 3
period (60 d), the birds were fed in individual cages with weights; eviscerated weight = half-eviscerated weight -
nets and housed in naturally ventilated windowed coops (heart + liver + proventriculus + gizzard + fat around
with temperatures between 23°C and 26°C, relative abdomen and gizzard + head + neck + claws) weights.
humidity between 65 and 75%, and illumination for Dressed yield = dressed weight/body weight £ 100%;
24 h/d (20 lux). Diets and water were provided ad libi- half-eviscerated yield = half-eviscerated weight/body
tum. Immunization and disinfection were carried out as weight £ 100%; eviscerated yield = eviscerated weight/
per routine procedures. body weight £ 100%; breast muscle yield = breast mus-
cle weight/eviscerated weight £ 100%; leg muscle yield
= thigh muscle weight/eviscerated weight £ 100%;
Sample Collection and Carcass Trait abdominal fat yield = abdominal and gizzard/eviscer-
Evaluation ated weight £ 100%. The immune organ index was
recorded and expressed relative to body weight as fol-
All birds were fasted (water available) for 12 h and the
lows: immune organ index = immune organ weight/
body weight and feed intakes of each replicate was
body weight £ 100%.
recorded on 21, 51, and 80 d. The average daily gain
(ADG), average daily feed intake (ADFI) and feed to
gain ratio (F/G) were calculated from the body weight Intestinal Microbial Count
and feed intake data. Daily mortality records (if avail-
able) were maintained for each treatment. At the end of Following the collection of blood samples, the birds
the experimental period, two broilers were randomly were euthanized by cervical dislocation and a postmor-
selected from each replicate based on their average body tem examination was carried out. In the lab, on a sterile
weight after a 12 h fast. Blood samples were collected operating table, a sample of approximately 0.5 g of the
from wing veins. These samples were placed in test tubes chyme in cecum was collected, mixed with 10 mL of
and centrifuged at 3,000g for 15 min to separate the 0.9% NaCl solution and then diluted to different gradi-
serum, which was then stored at 80°C for future deter- ent concentrations. Finally, 100 ml of the bacterial dilu-
mination of serum parameters. tions were inoculated onto the corresponding agar
medium plates. Total bacteria, E. coli, Lactobacillus
and yeast were coated on nutrient agar (NA), eosin
Serum Parameters methylene blue agar (EMB), man rogosa sharpe
The concentration of serum Immunoglobulin A medium (MRS), and yeast peptone dextrose adenine
(IgA), Immunoglobulin G (IgG), Immunoglobulin M (YPDA) (Qingdao Hope Bio-Technology Co., Ltd.
(IgM) were determined using a BS-420 automatic bio- Qingdao, China) solid media, respectively. Total bacte-
chemical analyser (Mindray, Shenzhen, China) with the ria and E. coli were incubated for 24 h at 37°C, yeast
appropriate detection kits (BioSino Bio-Technology & was incubated for 36 h at 30°C in SPX-250BSH-II incu-
Science Inc., Beijing, China). The activities of superox- bator (Xinmiao, Shanghai, China) and Lactobacillus for
ide dismutase (SOD), glutathione peroxidase (GSH- 36 h in a 37°C YQX-II anaerobic incubator (Shanghai
Px), the total antioxidant capacity (T-AOC), and the Haixiang Inareumenr & Equipment Factory, Shanghai,
concentration of malondialdehyde (MDA) were deter- China). At the end of the incubation, 2 replicates of
mined with commercial assay kits (Jiancheng Bioengi- each sample were counted. The number of colonies was
neering, Nanjing, China) using a DR-200BS enzymatic determined by the following formula: colonies per gram
analyser (Hiwell diatek instruments Co., LTD, Wuxi, of intestinal contents = Lg [(average number of
China) according to the protocols of the manufacturers. colonies £ dilution times £ 10 mL/0.1 mL)/weight of
Sample quality control and testing parameter settings the organism].
were strictly in accordance with the product specifica-
tion. The results were normalized to liter of serum and
are shown as U/L, nmol/L, and g/L, respectively. Statistical Analysis
Statistical analyses were performed by SPSS 24.0
Carcass Traits and Immune Organ Index (SPSS Inc., Chicago, IL). Data were subjected to one-
way ANOVA, linear, quadratic, and cubic tests of vari-
To assess carcass traits, at the end of the experiment ance and Levene's test. After ANOVA was significant,
(80 d of age), 2 birds of average weight were randomly significant differences between treatments were deter-
selected from each replicate, weighed individually and mined by Tukey's multiple range test. The polynomial
sacrificed after 12 h of feed deprivation. Birds were dis- comparison method was used to test for linear and qua-
sected manually to determine carcass, breast muscle, leg dratic effects at the supplementary HE level. A cubic
muscle, and abdominal fat weights and yields. All yields regression (Y = b3 £ X3+b2 £ X2 + b £ X + constant)
were calculated as follows. Dressed weight = body was fitted to the response of the dependent variable to
weight - blood weight - feathers weight; half-eviscerated the level of HE supplemented in the diet. The extreme
weight = dressed weight - (trachea + esophagus + crop response of HE was defined according to the derived
+ intestine + spleen + pancreas + gall bladder + repro- equation of the cubic regression. Each broiler was con-
ductive organs + gizzard contents and membranes) sidered as one experimental unit in each replicate. The
4 SONG ET AL.

significance level was set at P < 0.05. All values are eviscerated was observed between the 0.15%, 0.2% HE
expressed as mean and standard error of the mean added, and antibiotic groups (P > 0.05). All treatments
(SEM). did not have any statistically significant effects on evis-
cerated yield, breast muscle yield, leg muscle yield, or
abdominal fat yield (P > 0.05).
RESULTS
Growth Performance Immune Organ Index
Growth performance data are presented in Table 2. As shown in Table 4, broilers receiving dietary supple-
Results showed that dietary HE and CTE supplementa- mentation of 0.1%, 0.15%, 0.2% HE or CTE exhibited a
tion had significant increase in ADFI, ADG but a higher spleen index compared to the control group (P <
decrease in F/G, compared to the control group (P < 0.05) and there was no significant difference between
0.05), within d 21 to 80 and d 51 to 80. There was no dif- broilers fed CTE or different concentrations of HE (P >
ference in ADFI, ADG, and F/G between the 0.15% and 0.05). The varying treatments of the diets did not affect
0.2% HE supplemented groups and the antibiotic group the thymus index and the bursa index (P > 0.05).
(P > 0.05).

Serum Immunoglobulin Content

Carcass Traits
Serum immunoglobulin levels in broilers are summa-
Carcass traits of broilers are shown in Table 3. Com- rized in Table 5. Serum IgA levels were significantly
pared to the control group, broiler dressed yield (P < 0.05) increased in broilers supplemented with
increased significantly (P < 0.05) in the 0.1% and 0.2% 0.1%, 0.15%, 0.2% HE or CTE in the diet compared to
HE groups and half-eviscerated yield increased signifi- the control group. Of which, dietary supplementation of
cantly (P < 0.05) in the all 3 HE groups. The dressed 0.15% HE had the most significant effect on raising
yield and half-eviscerated of broilers increased linearly serum IgA levels (P < 0.05). Serum IgM was signifi-
and quadratically (P < 0.05) with higher HE addition. cantly increased in broilers supplemented with 0.15,
No significant differences in dressed yield or half- 0.2% HE or CTE in the diet compared to the control

Table 2. Effect of honeycomb extract on the growth performance of broilers.

Honeycomb extract, % P-Value

Item AB 0.0(CON) 0.1 0.15 0.2 SEM ANOVA Linear Quadratic
21−50 d
ADFI, g/d 50.90 50.47 50.52 50.60 50.57 0.076 0.415 0.614 0.880
ADG, g/d 20.23 19.09 20.04 19.92 20.32 0.154 0.065 0.007 0.022
F/G 2.52 2.65 2.53 2.54 2.49 0.020 0.116 0.012 0.038
51−80 d
ADFI, g/d 79.12a 72.52b 78.77a 78.25a 77.46a 0.589 0.001 0.005 <0.001
ADG, g/d 20.59a 16.56b 19.88a 20.28a 20.51a 0.389 0.002 <0.001 <0.001
F/G 3.86b 4.43a 3.98b 3.86b 3.79b 0.065 0.013 0.001 0.003
21−80 d
ADFI, g/d 64.91a 61.91b 63.38ab 64.58a 63.40ab 0.302 0.010 0.168 0.034
ADG, g/d 20.36a 18.00c 19.45b 19.80ab 19.98ab 0.175 <0.001 <0.001 <0.001
F/G 3.19b 3.45a 3.26b 3.26b 3.17b 0.023 <0.001 <0.001 <0.001
Values are represented as the mean and SEM (n = 6).
AB: antibiotic group; ADG: average daily gain; ADFI: average daily feed intake; CON: control group; HE: honeycomb extract; F/G: feed to gain ratio;
SEM: standard error of the mean.
a-c
Means within a row lacking a common superscript differ significantly (P < 0.05).

Table 3. Effect of honeycomb extract on slaughter performance of broilers.

Honeycomb extract, % P-Value

Item AB 0.0(CON) 0.1 0.15 0.2 SEM ANOVA Linear Quadratic
ab b a ab a
Dressed yield, % 88.57 88.09 89.33 89.19 89.61 0.181 0.048 0.011 0.030
Half-eviscerated yield, % 81.66ab 80.66b 82.03a 82.50a 82.29a 0.193 0.018 0.004 0.008
Eviscerated yield, % 66.62 65.98 66.73 67.52 66.88 0.202 0.197 0.068 0.115
Breast muscle yield, % 31.35 32.45 30.74 32.00 31.07 0.370 0.599 0.307 0.495
Leg muscle yield, % 36.34 38.04 37.48 36.55 37.34 0.419 0.705 0.444 0.675
Abdominal fat yield, % 9.17 8.63 10.29 9.62 9.95 0.308 0.474 0.218 0.314
Values are the means and SEM of 12 broilers (2 broilers per replicate).
AB: antibiotic group; CON: control group; HE: honeycomb extract; SEM: standard error of the mean.
a-b
Means within a row lacking a common superscript differ significantly (P < 0.05).
 EFFECTS OF HONEYCOMB EXTRACT ON BROILERS 5
Table 4. Effects of honeycomb extract on the organ indices of broilers.

Honeycomb extract, % P-Value

Item AB 0.0(CON) 0.1 0.15 0.2 SEM ANOVA Linear Quadratic
Spleen index, % 0.45 a
0.27 b
0.38 a
0.40 a
0.42 a
0.015 <0.001 <0.001 0.001
Thymus index, % 0.25 0.23 0.28 0.27 0.25 0.009 0.422 0.386 0.132
Bursa index, % 0.14 0.13 0.15 0.15 0.15 0.005 0.588 0.225 0.229
Values are the means and SEM of 12 broilers (2 broilers per replicate).
AB: antibiotic control group; CON: control group; HE: honeycomb extract; SEM: standard error of the mean.
a-b
Means within a row lacking a common superscript differ significantly (P < 0.05).

group (P < 0.05). Serum IgM was increased in broilers Cecal Microorganisms
supplemented with 0.15%, 0.2% HE or CTE diets com-
pared to the control group (P < 0.05). There was no dif- The data in Table 7 show the effect of HE on broiler
ference in serum IgA, IgG, and IgM between the HE and cecal microorganisms. The total number of bacteria in
CTE supplemented groups (P > 0.05). the cecal increased significantly (P < 0.05) with the
addition of HE to the diet compared to the control
group. Lactobacillus was higher in the 0.2% HE added
group than that in the control group (P < 0.05). For the
Serum Antioxidant Activity of Broilers above bacteria count, the antibiotic group had similar
results, but the abundance of total cecal bacteria, E.
Table 6 shows the effect of HE supplementation in the coli, yeast and Lactobacillus did not change between the
diet on the activity of serum antioxidant enzymes and antibiotic and HE added groups in broilers (P > 0.05).
MDA content in broilers. All 3 HE-addition groups and
antibiotic group had significant effects on GSH-Px, T-
AOC, SOD, and MDA (P < 0.05). Serum GSH-Px, T- Optimal HE Level
AOC, and SOD activities increased linearly and
quadratically (P < 0.05) with increasing HE levels, As shown in Table 8, the data of the ADG from 21 to
MDA concentration decreased linearly and quadrati- 50 d; ADFI, ADG, and F/G from 51 to 80 d; ADFI,
cally with increasing HE levels. Dietary supplementa- ADG, and F/G from 21 to 80 d, dressed yield, half-evis-
tion with 0.15% HE resulted in the greatest effect on cerated yield; spleen index; serum IgA, IgM, GSH-Px,
reducing serum MDA compared to the antibiotic group MDA, T-AOC, and SOD levels, cecum total bacteria,
(P < 0.05), and the 0.15% and 0.2% HE supplemented Lactobacillus count were selected for further analysis by
groups showed higher GSH-Px, T-AOC, and SOD activ- cubic regressions related to the HE level. The optimal
ities (P < 0.05). HE levels that minimized F/G from 51 to 80 d, F/G

Table 5. Effects of dietary honeycomb extract on the serum immunoglobulin contents of broilers.

Honeycomb extract, % P-Value

Item AB 0.0(CON) 0.1 0.15 0.2 SEM ANOVA Linear Quadratic
IgA, g/L 2.26 b
2.10c
2.20b
2.35a
2.23b
0.016 <0.001 <0.001 <0.001
IgG, g/L 4.21 4.09 4.17 4.31 4.28 0.027 0.078 0.007 0.027
IgM, g/L 1.63abc 1.57c 1.61bc 1.70a 1.65ab 0.011 0.004 0.001 0.003
Values are the means and SEM of 12 broilers (2 broilers per replicate).
AB: antibiotic group; CON: control group; HE: honeycomb extract; IgA: immunoglobulin A; IgG: immunoglobulin G; IgM: immunoglobulin M; SEM:
standard error of the mean.
a-c
Means within a row lacking a common superscript differ significantly (P < 0.05).

Table 6. Effects of honeycomb extract on serum antioxidant capacity in broilers.

Honeycomb extract, % P-Value

Item AB 0.0(CON) 0.1 0.15 0.2 SEM ANOVA Linear Quadratic
GSH-Px, U/mL 522.70 c
372.46 e
443.08 d
668.19 a
603.89 b
15.413 <0.001 <0.001 <0.001
MDA, nmol/mL 4.69bc 5.37a 4.94ab 3.99d 4.23cd 0.113 <0.001 <0.001 <0.001
T-AOC, U/mL 10.07b 8.03c 9.40b 12.94a 12.53a 0.303 <0.001 <0.001 <0.001
SOD, U/mL 86.40b 74.52b 82.09b 113.87a 102.40a 2.641 <0.001 <0.001 <0.001
Values are the means and SEM of 12 broilers (2 broilers per replicate).
AB: antibiotic group; CON: control group; GSH-Px: glutathione peroxide; HE: honeycomb extract; MDA: malondialdehyde; SEM: standard error of
the mean; SOD: superoxide dismutase; T-AOC: total antioxidant capacity.
a-e
Means within a row lacking a common superscript differ significantly (P < 0.05).
6 SONG ET AL.

Table 7. Effects of honeycomb extract on cecum microorganisms of broilers.

Honeycomb extract, % P-Value

Item AB 0.0(CON) 0.1 0.15 0.2 SEM ANOVA Linear Quadratic
ab b a a a
Total bacteria, lg (CFU/g) 8.12 7.69 8.28 8.31 8.49 0.086 0.032 0.003 0.011
Escherichia coli, lg (CFU/g) 7.00 7.30 6.84 7.08 6.97 0.072 0.371 0.192 0.258
Yeast, lg (CFU/g) 7.46 7.10 7.28 7.59 7.80 0.081 0.057 0.003 0.010
Lactobacillus, lg (CFU/g) 8.12b 7.69b 8.51ab 8.48ab 9.09a 0.142 0.030 0.001 0.007
Values are the means and SEM of 12 broilers (2 broilers per replicate).
AB: antibiotic group; CON: control group; HE: honeycomb extract; lg: base-10 logarithm. SEM, standard error of the mean.
a-b
Means within a row lacking a common superscript differ significantly (P < 0.05).

Table 8. Optimal HE level based on the equation model parameter.

Stages Parameter estimate

2
HE level /% Items Constant b1 b2 b3 R P Optimal
51−80 d ADFI 72.520 143.439 1025.833 2161.111 0.564 0.001 0.104
ADG 16.562 59.654 330.617 655.444 0.564 0.001 0.200
F/G 4.426 6.591 24.567 -37.556 0.440 0.010 0.200
21−80 d ADFI 61.906 104.339 1067.517 2915.222 0.365 0.031 0.177
ADG 18.002 60.437 596.450 1718.778 0.699 <0.001 0.156
F/G 3.446 5.255 48.817 147.000 0.580 0.001 0.129
Dressed yield 88.094 38.192 364.083 1055.000 0.154 0.059 0.149
Half-eviscerated yield 80.655 8.851 102.125 528.056 0.196 0.021 0.163
Spleen index 0.266 2.146 12.560 28.651 0.437 0.003 0.200
IgA 2.096 5.483 99.750 345.000 0.499 <0.001 0.160
IgM 1.566 2.860 50.458 170.278 0.302 0.001 0.163
GSH-Px 372.464 8063.872 129295.333 415951.111 0.869 <0.001 0.169
MDA 5.367 30.571 515.792 1672.500 0.542 <0.001 0.170
T-AOC 8.027 111.853 1839.917 5840.556 0.700 <0.001 0.173
SOD 74.515 1226.401 19212.875 61918.611 0.498 <0.001 0.167
Total bacteria 7.692 14.436 118.417 330.556 0.357 0.029 0.200
Lactobacillus 7.685 28.403 296.250 947.222 0.452 0.011 0.134
The data were fitted by a cubic equation, the dependent variables were the Items, and the independent variable was the HE level. b1, b2, and b3 indi-
cate the coefficients of the cubic equation. R2, fitting degrees of regression model.
ADFI: average daily feed intake; ADG: average daily gain; F/G: feed to gain ratio; GSH-Px: glutathione peroxide; HE: honeycomb extract; IgA: immu-
noglobulin A; IgG: immunoglobulin G; IgM: immunoglobulin M; MDA: malondialdehyde; SOD: superoxide dismutase; T-AOC: total antioxidant
capacity.

from 21 to 80 d and serum MDA content in broilers were Broilers have an underdeveloped sense of taste but a
0.200%, 0.129%, and 0.170%, respectively. The maxi- relatively acute sense of smell. HE has an aromatic odor
mum responses of ADG, ADFI from 51 to 80 d, ADG, and a stimulating effect on salivary and gastric glands,
ADFI from 21 to 80 d, dressed yield, half-eviscerated and the addition of HE to the diet increased the ADFI of
yield, spleen index, serum IgA, IgM, GSH-Px, T-AOC, broilers, suggesting that HE may have some food-induc-
SOD levels, and cecum total bacteria, Lactobacillus ing effects. Many herbal extracts can slow down the
count were observed at 0.10%, 0.20%, 0.18%, 0.16%, emptying of the gastrointestinal tract (Manza-
0.15% 0.16%, 0.20%, 0.16%, 0.16%, 0.17%, 0.17%, nilla, 2004), stimulate the secretion of digestive juices
0.17%, 0.20%, and 0.134%, respectively. Based on the (saliva, bile, mucus) (Platel, 2004) and increase the
above indices, the optimal HE level was 0.15 to 0.17%. activity of digestive enzymes (Platel, 1996), G of the ani-
mals, which i/G of the animals, which is one of the possi-
ble reasons why HE leads to an increase in ADG and
FCR in broilers. Many studies have confirmed that the
DISCUSSION addition of substances such as palygorskite-based anti-
The honeycomb and its extracts have a variety of bacterial agent (Zha et al. 2021) and cetylpyridinium-
pharmacological activities, including antibacterial, anti- montmorillonite (Ke et al. 2014) to feed can improve
inflammatory, antiviral, antitumor, and anesthetic feed utilization in broilers in the later and whole stages
effects (Jin, 2007), and have been widely used in tradi- of growth. We also found in our study that the number
tional Chinese medicine (TCM). HE contains flavo- of intestinal probiotics became more abundant with the
noids, polysaccharides, enzymes, peptides and other addition of HE, which may also promote the absorption
unique active ingredients that have growth-promoting of nutrients in the feed.
effects on animals (Yan et al., 2006). In this study, Flavonoids (Kamboh, 2013), polyphenols (Denli, 2004)
improved feed intake and growth performance may have and polysaccharides (Yang, 2019) contained in HE have
been associated with good health, digestion and absorp- been studied for growth promotion. In addition,
tion (Tayeb et al., 2014). nutrients are fully and completely digested (Seven, 2008;
 EFFECTS OF HONEYCOMB EXTRACT ON BROILERS 7
Abass et al., 2017) with the help of components such as encountered during growth. The thymus, bursa and
4-hydroxybenzoic acid and benzoic acid in HE, so spleen are the most important immune organs in poul-
broilers fed HE supplements show higher ADG and lower try and it is generally accepted that immunosuppres-
F/G. Bee products have beneficial effects on broiler sion is associated with a decrease in the relative
intestinal morphophysiology by increasing the contact weight of immune organs, while an increase in the rel-
area between the small intestine and chime ative weight of immune organs reflects an increase in
(Prakatur et al., 2019), thus promoting feed absorption. immunity (Leticia et al., 2006). In this study, the
Most importantly, HE maintains broiler health by alter- addition of HE to the broiler diet had no effect on the
ing the microbial community structure and creating a thymus and bursa, which was somewhat of a depar-
microbial balance in the gastrointestinal tract. Previous ture from the previous findings (Emmanuel et al.,
studies have reported that diets supplemented with prop- 2016). May be due to differences in age. The bursa
olis, royal jelly, honey, and bee pollen can improve the begins to develop from hatching and reaches its maxi-
growth performance of quails, and that adding honey to mum size at 8 to 10 wk of age. It then enters the
the drinking water of broilers can increase weight gain regression process, which is completed at 6 to 7 mo of
and feed intake and reduce feed conversion ratio age (Cazaban et al., 2015). In contrast, a linear and
(Babaei et al., 2016; Emmanuel et al., 2016). The find- quadratic increase in spleen index was observed with
ings of the above study corroborate the results we the addition of HE to the diet. This is similar to the
obtained. relevant results of previous authors who found that
In this study, HE showed a cumulative effect on HE can enhance immune function by directly stimu-
broiler growth performance as time progressed. During lating the proliferation of thymus and spleen cells
d 21 to 50, HE had no effect on ADFI and F/G, with a (Wei et al., 1996). HE promotes spleen growth and
modest increase in ADG. From d 51 to 80, HE increased development, increases spleen index, raises serum IgG,
ADFI, ADG, and decreased F/G. These results suggest IgA, and IgM levels and enhances immune function in
that the increase in early growth performance may be broilers. Honey bee by-products can fight viral infec-
due to nutrient deposition and that the increase in later tions through immunomodulation and can signifi-
growth performance may be mainly due to feeding stim- cantly improve humoral and cellular immunity by
ulation. The growth-promoting effect of HE in yellow inhibiting the formation of prostaglandins, which are
bantam broiler was linearly and quadratically correlated considered to be a highly immunoreactive substance
with the level of supplementation, and it may be related (Taheri et al., 2005). Therefore, our findings suggest
to the concentration of different levels of active ingre- that HE significantly increases serum IgA and IgM
dients such as flavonoids. Studies have shown that high levels, thus enhancing the immune function of broiler
concentrations of flavonoids can be directly absorbed in chickens, which is also similar to the findings of some
the intestine and then exert relevant effects in gastroin- previous studies on honeycomb (Zhu et al., 1999).
testinal epithelial cells, endocrine cells, and immune The antioxidant activity of HE in vitro (Hou et al.,
cells, but low concentrations of flavonoids cannot be 2011) has been confirmed by previous studies. Honey-
directly absorbed and must be metabolized by microbial comb has favorable antioxidant and free oxygen radical
breakdown into substances such as valerolactone and scavenging abilities, including the ability to scavenge
phenolic acids before they can be absorbed by intestinal superoxide anions and hydroxyl radicals in vitro
cells (Appeldoorn, 2009; Zhang, 2016). (Cheng et al., 2012). Both the aqueous and ethanolic
Carcass traits are a major indicator to evaluate poul- extracts of honeycomb have beneficial free radical scav-
try productivity and meat production performance. The enging abilities. The fat-soluble and water-soluble com-
honeycomb contains propolis, bee pollen, flavonoids, ponents of honeycomb possess antioxidant activity. This
and polysaccharides, therefore, the suggested enhance- study showed that HE significantly increased the
ment of slaughter performance in broilers may be related enzyme activities in broiler serum, which is consistent
to the influence of these active ingredients. In the pres- with the findings of Wan (2013), that concluded that
ent study, HE showed linear and quadratic improve- honeycomb significantly increased the activity of SOD
ments in dressed yield and half-eviscerated yield in and GSH-Px in serum, reduced MDA content and
broilers, and these effects may be closely related to improved the antioxidant capacity of broilers. Propolis,
improved growth performance. Previous studies have royal jelly, and honey have good antioxidant properties
pointed out that polysaccharides can significantly (Kumazawa et al., 2004; Hang et al., 2008). The antioxi-
dressed yield and half-eviscerated yield of broilers dant activity of HE may be due to propolis and other
(Sun et al., 2017), flavonoids can modify carcass traits in antioxidant components. This is because of the presence
broilers (Yang et al., 2014), propolis can significantly of antioxidant peptides (Mckibben and Engeseth, 2002;
boost carcass yield and breast weight in broilers Guo et al., 2009), as well as phenolic compounds includ-
(Seven et al., 2008), and the addition of bee pollen can ing flavonoids, phenolic acids, caffeic acid, phenethyl
significantly lift dressed yields with or without edible esters, and terpenoids. They contribute to the metabo-
bowels (Abood and Ezzat, 2018). lism and enhance the organic acids influenced by total
With the ban on antibiotics in poultry diets, there polyphenols, thus playing an important role in the neu-
will be a greater reliance on the poultry's own immune tralization and absorption of free radicals
system to combat the various threats that may be (Balkanska et al., 2017).
8 SONG ET AL.

HE has a good antibacterial effect, which has been and research direction for our subsequent in-depth
studied and confirmed by many authors. HE has an study.
inhibitory effect on the growth of Staphylococcus
aureus, and this inhibition increases with increasing
HE concentration (Gong et al., 2008). The aqueous CONCLUSION
extract of honeycomb showed some inhibitory effect In summary, this study shows that dietary supple-
on Bacillus brevis, Staphylococcus aureus, E. coli, mentation of HE can improve growth performance, car-
Bacillus cereus, Bacillus subtilis, and Salmonella cass traits and serum antioxidant capacity, modulate
(Zhu et al., 1999). The alcoholic extract of the honey- immune function and increase intestinal probiotic popu-
comb showed good concentration-dependent inhibition lations in female yellow bantam broilers. The effect of
of S. aureus, Staphylococcus epidermidis, Pseudomo- HE in broiler diets was at the same level as the addition
nas aeruginosa, Streptococcus haemolyticus B, and of antibiotics to broiler diets, and even performed better
Streptococcus pneumoniae (Cheng, 2012). The cecum in some indicators. It is a potential and valuable alterna-
is the part of the intestine with the highest probability tive to antibiotics. According to cubic regressions, most
of microbial colonization (Shi, 2019), so this experi- indicators were in the best zone when HE was added in
ment investigated the effect of HE on the microbiology the range of 0.15 to 0.17%.
of the cecum of yellow bantam broilers. The balance of
the intestinal flora of livestock plays an essential role
in optimizing production performance. The intestinal AUTHOR CONTRIBUTIONS
microorganisms consist mainly of pathogenic and pro-
Wenjing SONG, Qiongli SONG and Xiaolian CHEN
biotic bacteria. Most pathogenic bacteria are aerobic,
designed the experiment and the analyzed data. Wenj-
while probiotic bacteria are anaerobic. In this study,
ing SONG, Qiongli SONG and Linxiu LIU conducted
HE promote the proliferation of Lactobacillus in the
the experiments. Wenjing SONG and Jia TAN drafted
cecum of broiler, which is in general agreement with
the article. Guohua LIU and Zhiheng ZOU supervised
Zhang, 2011, who found that honeycomb could pro-
the experiments and revised the manuscript. All the
mote the proliferation anaerobic bacteria in the gut of
coauthors have seen a draft of the manuscript and agree
broiler. Higher doses of honeycomb in the diet signifi-
with its publication.
cantly reduced the number of Enterobacteria isolated
from the chicken crop (Krocko et al., 2012). Our
results showed that HE had a very slight inhibitory ACKNOWLEDGMENTS
effect on E. coli in the cecum of broiler, which may be
due to the level of HE in the diet. In addition, E. coli This work was supported by the Technical System of
is a Gram-negative bacterium and the honeycomb the National Broiler Industry (CARS-42-Z10), the Sci-
showed better inhibitory effect on Gram-positive bac- ence and Technology Plan of Jiangxi Province
teria than Gram-negative bacteria (Sforcin et al., (20202BBFL63027) and the Key Research and develop-
2002; Chu and Hu, 2012; Bílikova et al., 2015). There- ment program of Jiangxi Province (20212BBF63025).
fore, the mechanisms by which HE improves growth
performance may be regulation of the gut microbiota, DISCLOSURES
nutrient savings due to reduced numbers of competing
microorganisms, reduction or elimination of microor- We declare that we have no financial and personal
ganisms that cause subclinical infections, and reduc- relationships with other people or organizations that
tion of growth-inhibiting toxins or metabolites can inappropriately influence our work, there is no pro-
produced by the gut microbiota. fessional or other personal interest of any nature or kind
It is worth noting that the above indicators do not in any product, service and/or company that could be
exist independently of each other, meaning that there is construed as influencing the position presented in, or the
an interaction between them. The increase in the num- review of, the manuscript entitled, “Effects of honey-
ber of intestinal probiotic bacteria breaks down the comb extract on the growth performance, carcass traits,
nutrients in the feed more completely, and the products immunity, antioxidant function and intestinal microor-
obtained from the complete breakdown provide the ganisms of yellow bantam broilers”.
intestinal probiotic bacteria with a greater and more
adapted source of energy. The increased immunity also
SUPPLEMENTARY MATERIALS
provides for the growth and intestinal microorganisms
of broilers. The end result is that the addition of HE Supplementary material associated with this article
improves all evaluation indicators in broilers and is no can be found in the online version at doi:10.1016/j.
less effective than the commonly used antibiotics. psj.2022.101811.
Finally, as this experiment did not clarify the chemical
components of HE by techniques such as mass spectrom-
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