Italian Journal of Animal Science

ISSN: (Print) 1828-051X (Online) Journal homepage: https://www.tandfonline.com/loi/tjas20

Effects of dietary probiotic (Bacillus subtilis)

supplementation on productive performance,
immune response and egg quality characteristics
in laying hens under high ambient temperature

Moataz Fathi, Ibrahim Al-Homidan, Abdelaziz Al-Dokhail, Tarek Ebeid,

Osama Abou-Emera & Ahmed Alsagan

To cite this article: Moataz Fathi, Ibrahim Al-Homidan, Abdelaziz Al-Dokhail, Tarek
Ebeid, Osama Abou-Emera & Ahmed Alsagan (2018) Effects of dietary probiotic (Bacillus
subtilis) supplementation on productive performance, immune response and egg quality
characteristics in laying hens under high ambient temperature, Italian Journal of Animal
Science, 17:3, 804-814, DOI: 10.1080/1828051X.2018.1425104

To link to this article: https://doi.org/10.1080/1828051X.2018.1425104

© 2018 The Author(s). Published by Informa Published online: 16 Jan 2018.

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ITALIAN JOURNAL OF ANIMAL SCIENCE, 2018
VOL. 17, NO. 3, 804–814
https://doi.org/10.1080/1828051X.2018.1425104

PAPER

Effects of dietary probiotic (Bacillus subtilis) supplementation on productive

performance, immune response and egg quality characteristics in laying
hens under high ambient temperature
Moataz Fathia,b , Ibrahim Al-Homidana, Abdelaziz Al-Dokhaila, Tarek Ebeida,c, Osama Abou-Emeraa,b and
Ahmed Alsagand
a
Department of Animal Production and Breeding, College of Agriculture and Veterinary Medicine, Qassim University, Al-Qassim, Saudi
Arabia; bDepartment of Poultry Production, Faculty of Agriculture, Ain Shams University, Cairo, Egypt; cDepartment of Poultry
Production, Faculty of Agriculture, Kafrelsheikh University, Kafr El-Sheikh, Egypt; dKing Abdulaziz City for Science and Technology,
Riyadh, Saudi Arabia

ABSTRACT ARTICLE HISTORY

A two level factorial experiment (3
3) was designed to evaluate the effect of dietary inclusion Received 12 October 2017
level of probiotic and breed on productive performance, immune status and egg quality charac- Revised 2 January 2018
teristics in laying hens reared under high ambient temperature. A total of 216, 32-week-old lay- Accepted 4 January 2018
ing hens representing three different breeds (White Leghorn, Saudi black and Saudi brown) were
KEYWORDS
randomly assigned to three dietary treatments (0, 200 and 400 g/t feed) in a 90-d experiment. Probiotic; egg quality;
The current results indicated that the inclusion of probiotics in layer diets did not appear to laying hens; immunity;
cause any adverse effects on egg production traits compared with non-treated hens. Dietary Saudi chickens
supplementation with probiotics at 400 ppm level significantly (p < .05) increased shell thickness
compared to non-treated laying hens. Moreover, an improvement in eggshell quality and break-
ing strength in hens fed a diet containing probiotics was observed under high ambient tempera-
ture. Also, plasma cholesterol and triglyceride were reduced in laying hens fed a diet
supplemented with probiotics. Furthermore, probiotic inclusion significantly increased IgM
immunoglobulin concentration in laying hens fed a diet containing either 200 or 400 ppm com-
pared with untreated hens. Concerning breed effect, Saudi black laying hens recorded signifi-
cantly (p < .02) higher egg mass compared with the other breeds. Also, both Saudi chicken
breeds exhibited significantly higher cell-mediated response and IgM concentration compared
with Leghorn hens.

Introduction birds (M€anner and Wang 1991; Zulkifli et al. 2000). It

has been shown that probiotic supplementation is
Probiotics as feed additives are increasingly used in
more beneficial during stressful conditions (Jin et al.
animal and poultry feeding in commercial manner. 1997).
Improved growth performance, reduced mortality and The inclusion of probiotic in the diet has been
enhanced immunocompetence broiler chickens are found to improve egg production and food conversion
well defined (Attia et al. 2011). The effectiveness of ratio in several studies (Panda et al. 2008; Youssef
probiotic administration in laying hens may depend et al. 2013; Chung et al. 2015). Furthermore, giving
on several factors, including microbial species compos- probiotics to laying hens has been found to improve
ition, supplemental dose, method and frequency of eggshell quality and reduced the number of damaged
administration, diet composition, bird’s age, genotype eggs (Mikulski et al. 2012; Zhang et al. 2012). Likewise,
(breed) and environmental stress factors (Mikulski Zhang et al. (2013) reported that the dietary supple-
et al. 2012). It is well known that heat stress in laying mentation of 0.01% probiotic improved egg produc-
hens has a severe negative impact on eggshell quality. tion and egg quality. Conversely, some contradictorily
Many investigators have reported that dietary pro- results have been reported on the effects of probiotic
biotic supplementation could reduce heat stress in supplementation on egg yield and feed efficiency

CONTACT Prof. Moataz Fathi [email protected] Department of Animal Production and Breeding, College of Agriculture and Veterinary
Medicine, Qassim University, Al-Qassim 51452, Saudi Arabia
ß 2018 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which
permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
 ITALIAN JOURNAL OF ANIMAL SCIENCE 805

(Zarei et al. 2011; Afsari et al. 2014; Sobczak and Table 1. Ingredients and composition of the basal diet.
Kozłowski 2015). Ingredient (%)
The supplementation of probiotics to diet of laying Yellow corn 60.2
Soybean meal (44% CP) 26.0
hens may play an important role in altering the lipid Limestone 8.7
metabolism of chickens. Many investigators have Di-calcium phosphatea 1.7
Vitamin–mineral Premixb 0.3
pointed out that probiotics could reduce the choles- Sodium bicarbonate 0.23
terol content of egg yolk (Li et al. 2006; Mikulski et al. Salt (NaCl) 0.24
2012) and serum (Kalavathy et al. 2003). DL-Methionine 0.13
Vegetable oil 2.5
Probiotics have been used to stimulate the immune Calculated analysis (%, unless otherwise noted)
system in poultry. Numerous studies have concluded Metabolisable energy (kcal/kg diet) 2875
Crude protein 16.6
that using probiotics as feed additives exerts a benefi- Ca 3.77
cial influence on immune response and health status Available phosphorous 0.45
a
in both layer- and meat-type strain of chickens (Willis Dicalcium phosphate contained: 16% phosphorous and 23% calcium.
b
Premix provided per kilogram of diet: vitamin A (transretinyl acetate),
and Reid 2008; Vila et al. 2009; Shivaramaiah et al. 10,000 IU; vitamin D3 (cholecalciferol), 3000 IU; vitamin E (all-rac-a-toc-
2011). Fong et al. (2015) observed an improved opherol), 30 IU; menadione, 1.3 mg; thiamine, 2.2 mg; riboflavin, 8 mg;
nicotinamide, 40 mg; choline chloride, 400 mg; calcium pantothenate,
immune response in the cells of healthy subjects 10 mg; pyridoxine, 4 mg; biotin, 0.04 mg; folic acid, 1 mg; vitamin B12
treated with Lactobacillus ramnosus in vitro. An (cobalamin), 0.013 mg; Fe (from ferrous sulphate), 80 mg; Cu (from cop-
per sulphate), 8.0 mg; Mn (from manganese sulphate), 110 mg; Zn (from
increase in serum bactericidal activity was observed in zinc oxide), 60 mg; I (from calcium iodate), 1.1 mg; Se (from sodium sel-
laying hens given a diet supplemented with probiotics enite), 0.3 mg.
(Forte et al. 2016). Therefore, the present study aimed
at evaluating productive performance, immune status Laying performance
and egg quality of laying hens from different chicken
Feed intake was recorded on a replicate basis for the
breeds fed a diet supplemented with varied concentra-
whole experimental period. Feed conversion ratio (FCR)
tions of probiotic under high ambient temperature.
was calculated at the end of the experiment on the basis
of the amount of feed consumed in gram divided by
egg mass in gram. Damaged eggs, including broken,
Materials and methods
cracked and shell-less eggs, were recorded as they
Experimental design, birds and dietary treatments occurred. All eggs were collected and recorded on a
A total of 216, 36-week-old laying hens were allocated daily basis. Eggs were individually weighed and egg
in a two-level factorial experimental design (3
3), mass was calculated for the whole experimental period.
consisting of three concentrations of dietary probiotic
(0, 200 and 400 g/t feed) containing 4
109 cfu/g of Egg quality
Bacillus subtilis and three different breeds (White
Leghorn, Saudi black and Saudi brown). Each sub- At 48 weeks of age, 48 eggs from each dietary treat-
group had eight replicates (experimental cages) of ment per breed were collected to assess internal and
three hens each. The birds were placed in wire cages external egg quality characteristics. Egg width and egg
(60 cm
45 cm
43 cm, L
W
H) under lighting length were measured in mm using electronic digital
schedule of 17 h/d light cycle. The average of high Vernier calliper (±0.01 mm). Egg shape was calculated
ambient temperature and relative humidity during the according to the following formula:
whole experimental period was 34 ± 1.5  C and 55%, Egg shape ¼ ½egg width=egg length
100
respectively. Feed and water were provided ad libitum
throughout the 90-d experimental period. The basal Following collection, the breaking strength for
diet was formulated to contain approximately 16.6% intact eggs was determined in kg/cm2 using Egg Force
crude protein and 2875 ME kcal/kg in a typical layer ReaderTM, Orka Food Technology Ltd, St. Paul, MN.
diet (Table 1). Three probiotic levels (0, 200 and Also, egg weight, Haugh unit and yolk colour were
400 ppm) were mixed with diet every week. measured automatically using Egg AnalyzerTM manu-
Conventional breeding and management procedures factured by Orka Food Technology Limited, St. Paul,
were applied throughout the experimental period. All MN. The liquid contents were put aside and the shell
birds were provided with similar environmental and plus membranes were washed under running water to
hygienic conditions. The care and handling of the lay- remove adhering albumen. The wet eggshell was left
ing hens were in accordance with regulations of ani- for 24 h at room temperature for drying and then
mal care committee of Qassim University. weighed to the nearest 0.01 g. The relative weight of
806 M. FATHI ET AL.

dry eggshell was calculated on the basis of egg Statistical analysis

weight. To measure shell thickness, pieces from three
Data were subjected to a two-way ANOVA with pro-
different regions (two poles and equator) of each egg-
biotic level and breed as fixed effects using JMP Ver.
shell with intact membranes were measured with a
11 (SAS Institute 2013). The model applied is as fol-
dial gauge micrometer to the nearest 0.01 mm.
lows:
Yijk ¼ l þ Pi þ Bj þ ðPBÞij þ eijk
Concentrations of plasma immunoglobulins
where Yijk is the observation taken on the kth individ-
The effects of probiotic administration and breed type
ual, m is the overall mean, Pi is a fixed effect of the ith
on the humoural status of the bird by quantifying IgA,
probiotic level, Bj is a fixed effect of the jth breed,
IgM, and IgY levels in blood plasma were measured.
(PB)ij is the interaction between probiotic level and
At the end of the experiment, 10 hens per sub-group
breed, eijk is the random error assumed to be inde-
were randomly chosen and blood samples were col-
pendent normally distributed with mean ¼ 0 and
lected from the wing vein in heparinised tubes. Blood
variance ¼ r2.
samples were subsequently stored in ice, centrifuged
Data given in percentage were subjected to arcsine
at 2500
g for 10 min at 4  C, and the harvested
transformation before statistical analysis, however, the
plasma was stored at 20  C until antibody analyses.
actual data are listed. All results are presented as
Plasma IgA, IgM and IgY concentrations were deter-
mean and the pooled SEM. The significance of differ-
mined in appropriately diluted samples by a sandwich
ELISA using microtitre plates and chicken-specific IgA, ence among the groups was assessed using Tukey’s
IgM, and IgY ELISA quantitation kits (GenWay Biotech test. Significance was set as p < .05.
Inc., San Diego, CA).
Results
Cell-mediated immunity Egg production traits and damaged egg ratio as
Cell-mediated immunity in vivo was evaluated by affected by dietary probiotic level and breed are
injection of the mitogen phytohemagglutinin-P (PHA- shown in Table 2. At the beginning of the experiment,
P) into the wattle. At 48 weeks of age, 15 laying there were no statistically differences in the average
hens from each treatment per breed were randomly body weight-laying hens between dietary treatments
assigned. Each hen was intradermally injected in within each breed (data not shown). During the
the right wattle with 100 mg phytohemagglutinin-P experimental period, no hen died in each treatment
(Sigma Chemical Co., St. Louis, MO) in 0.1 mL of ster- within each breed. As shown in Table 2, there was no
ile saline. The initial wattle thickness was measured statistically significant difference in egg production
with a constant tension calliper before injection and performance among dietary treatments. Insignificant
at 24, 48 and 72 h after PHA-P injection. The resultant decreased feed intake (p < .08) was observed in both
swelling response in the wattle was calculated as the groups fed a diet supplemented with probiotics com-
difference between its thickness before and after pared to group receiving the control diet. The lowest
injection. feed intake (9789 g) was found in group that con-
sumed diet containing 200 ppm probiotics, while the
highest (10,406 g) was recorded in the control group.
Blood biochemical analysis There was no significant difference in FCR between
At the end of the experimental period, 10 blood hens fed probiotic-supplemented diets and hens fed
samples from each treatment group per breed were the control diet. Probiotic supplementation did not
withdrawn for blood biochemical analysis. Plasma affect damaged egg ratio. However, non-significant
was harvested as mentioned above. Plasma total reduction in damaged eggs was detected (22% and
protein, albumen, total cholesterol and triglyceride 39% in groups supplemented with 200 and 400 ppm
were spectrophotometerically determined using com- probiotics, respectively). With respect to breed effect,
mercial kits (Stanbio Laboratory, Boerne, TX). The the Saudi black laying hens recorded significantly
globulin was calculated as the difference between (p < .02) higher egg mass compared with the other
the total protein and albumen. Serum triiodothyron- breeds. The worst performance was found in Saudi
ine (T3) and thyroxine (T4) concentrations were brown breed.
measured using commercial ELISA kits (BioCheckV R, The effects of dietary probiotic supplementation
Foster City, CA). and breed type on internal and external egg quality
 ITALIAN JOURNAL OF ANIMAL SCIENCE 807

Table 2. Effect of probiotic dietary inclusion and breed on egg production traits.
Trait
Factor Egg No Egg weight, g Egg mass, g Feed intake, g FCR Damaged egg ratio
Probiotic level (P)
0 ppm 62.8 51.9 3252.8 10405.9 3.2 2.71
200 ppm 58.2 51.5 2966.6 9789.4 3.3 2.11
400 ppm 65.4 51.8 3380.7 10283.0 3.0 1.66
Breed (B)
Leghorn 56.9b 55.6a 3173.0b 9605.9b 3.0b 1.21b
Black 69.1a 50.7b 3499.6a 10,637.0a 3.0b 1.94b
Brown 60.4b 48.9b 2927.5b 10,235.3a 3.5a 3.34a
SEM 1.38 0.53 67.7 158.6 0.05 0.34
p Value
P .46 .96 .28 .08 .59 .42
B .04 .01 .02 <.01 .04 .03
P
B .14 .65 .03 .20 .07 .33
Means in the same column with no common letters differ significantly (p < .05).
a,b

FCR: feed conversion ratio (g feed: g egg mass).

Table 3. Effect of probiotic level and breed on internal and external egg quality characteristics of laying hens.
Trait
Factor Yolk % Yolk colour Haugh unit Egg shape Shell weight, g Eggshell % Shell thickness (mm
102) Shell strength (kg/cm2)
Probiotic level (P)
0 ppm 30.9 9.1 58.5 76.5 5.0 9.5b 39.7b 3.8b
200 ppm 30.7 9.3 59.0 76.0 5.2 10.2a 40.1ab 4.3a
400 ppm 30.9 9.1 56.1 75.3 5.2 10.2a 40.8a 4.2a
Breed (B)
Leghorn 30.9ab 8.7b 53.8b 74.8b 5.5a 10.2 40.3 4.0
Black 31.9a 9.4a 57.9ab 76.4a 5.1ab 9.8 39.8 4.1
Brown 29.7b 9.4a 61.9a 76.6a 4.8b 9.9 40.5 4.2
SEM 0.27 0.14 0.84 0.250 0.05 0.09 0.200 0.05
p Value
P .9 .7 .3 .1 .1 <.01 .05 <.01
B <.01 .05 <.01 <.01 <.01 .3 .3 .7
P
B .5 .9 .6 <.01 <.01 .03 .4 .9
a,b
Means in the same column with no common letters differ significantly (p < .05).

measurements of laying hens are presented in Table 3. significant difference due to breed effect. As shown in
No significant differences were found between the diet- Figure 1, a significant interaction between probiotic
ary groups with respect to yolk%, yolk colour and albu- level and breed was observed for shell weight, shell
men quality expressed as Haugh unit score. Both thickness and egg-shape index. The highest shell weight
probiotic levels significantly (p < .01) increased relative and shell thickness were detected in Leghorn given
weight of eggshell compared with the control group. 200 ppm probiotics and in black given 400 ppm
Likewise, shell thickness significantly (p < .05) improved probiotics.
in laying hens fed a diet containing 400 ppm probiotic Cellular and humoural immune responses of the
when compared with those fed a basal diet. While the laying hens as affected by probiotic administration
shell thickness of hens fed on 200 ppm probiotic was level and breed are shown in Table 4. The laying hens
intermediate. Therefore, as a consequence, the inclusion fed a diet with the level of 400 ppm inclusion resulted
of probiotic in both levels significantly (p < .05) in a numerical improvement in cellular immune
increased eggshell strength compared with the control response (PHA-P) at all tested times compared with
group (4.27, 4.17 and 3.82 kg/cm2, respectively). the other groups. Moreover, probiotic inclusion signifi-
Regarding breed effect, it could be noticed that the cantly (p < .01) increased IgM immunoglobulin concen-
native breeds (black and brown) laid eggs with signifi- tration at both probiotic levels (200 and 400 ppm). On
cantly (p < .01) darker yolk colour than those of Leghorn the contrary, this trend did not exist in both IgA and
hens. Also, they produced eggs with rounded shape IgY immunoglobulins. In terms of breeds, the results
compared with those of Leghorn. The brown breed obviously revealed that the Saudi native breeds (black
recorded lower relative yolk and higher HU compared and brown) had a significant increase in swelling
to the other two breeds. In terms of eggshell quality response, particularly at earlier stage of PHA-P test.
characteristics, the results revealed that there was no Concerning the interaction between breed and
808 M. FATHI ET AL.

Figure 1. Interaction between probiotic level and breed of eggshell traits.

Table 4. Effect of probiotic level and breed on cell mediated response and plasma immunoglobulins concentration of laying
hens.
Swelling response Immunoglobulin
Factor Difference 24 h Difference 48 h Difference 72 h IgA IgM IgY
Probiotic level (P)
0 ppm 0. 88 0.55 0.45 0.43 0.87c 4.61
200 ppm 0.74 0.52 0.42 0.43 0.96b 4.65
400 ppm 0.96 0.65 0.50 0.45 1.06a 4.69
Breed (B)
Leghorn 0.57b 0.44b 0.37 0.44 0.68c 4.62
Black 1.00a 0.61a 0.47 0.41 1.29a 4.70
Brown 1.01a 0.67a 0.53 0.46 0.92b 4.63
SEM 0.05 0.04 0.03 0.0098 0.0301 0.0266
p Value
P .14 .26 .53 .62 <.01 .41
B <.01 .02 .07 .19 <.01 .40
P
B .04 .04 .03 .90 .74 <.01
a,b,c
Means in the same column with no common letters differ significantly (p < .05).

probiotic level, a significant effect on cell-mediated globulin, while no significant difference was observed
immunity was found. The best swelling responses at in total protein. After 90-d of probiotic supplementa-
24, 48 and 72 h were detected in black
400 and tion, a significant difference was found in blood chol-
brown
0 (Figure 2). The highest IgY concentration esterol. As shown in Table 5, cholesterol level was
was detected in Leghorn
200, black
0 and black significantly (p < .05) reduced by probiotic inclusion.

400 (Figure 3). This decrease was associated with increased dietary
Biochemical blood plasma parameters as affected probiotic concentration. On the one hand, blood chol-
by probiotic concentration and type of breed in laying esterol level was significantly affected by the inter-
hens are presented in Table 5. Plasma total protein, action between probiotic level and breed (Figure 4).
albumen and globulin concentrations were not signifi- The lowest cholesterol level was found in brown breed
cantly affected due to dietary probiotic supplementa- fed a diet supplemented with 400 ppm probiotics. On
tion. On the basis of breed factor, there were the other hand, probiotic supplementation had no
significant differences among breeds for albumen and effect on serum triglyceride level. There were no
 ITALIAN JOURNAL OF ANIMAL SCIENCE 809

Figure 2. Interaction between probiotic level and breed of cell mediated response.

Figure 3. Interaction between probiotic level and breed of immunoglobulin IgY.

significant differences in cholesterol and triglyceride Discussion

due to breed effect. The statistical analysis revealed
Laying performance
that there was no interaction between dietary treat-
ment and breed for all blood parameters except for In congruent with our findings, it was speculated that
cholesterol level (Table 5). In terms of thyroid hor- FCRs were not affected by probiotics in some reports
mones, no significant differences in blood plasma T3 (Nahashon et al. 1994; Mohan et al. 1995; Tortuero
and T4 concentrations were observed among treat- and Fernandez 1995). Similarly, numerous studies have
ments or breeds. concluded that probiotics used as feed additives did
810 M. FATHI ET AL.

Table 5. Effect of probiotic level and breed on biochemical blood plasma parameters of laying hens.
Parameter
Factor T. Protein, g/dL Albumen, g/dL Globulin, g/dL Cholesterol, mg/dL Triglyceride, mg/dL T3 T4
Probiotic level (P)
0 ppm 6.7 4.7 2.0 166.2a 1660.8 1.9 5.5
200 ppm 6.5 4.4 2.4 152.8b 1643.5 1.9 5.6
400 ppm 7.0 4.6 2.0 145.5c 1626.8 1.9 5.6
Breed (B)
Leghorn 6.9 4.1b 2.8a 154.8 1541.3 1.9 5.7
Black 6.7 4.7a 2.0b 157.9 1722.9 1.9 5.5
Brown 6.5 4.9a 1.7b 151.6 1658.8 1.9 5.4
SEM 0.11 0.10 0.12 4.31 47.8 0.01 0.10
p Value
P .1 .5 .2 .05 .30 .72 .95
B .4 <.01 <.01 .57 .96 .71 .43
P
B .2 .07 .8 .02 .26 .21 .25
a,b,c
Means in the same column with no common letters differ significantly (p < .05).

Figure 4. Interaction between probiotic level and breed of plasma cholesterol.

not affect egg production traits (Arpasova et al. 2012; previous reports may be related to differences in pro-
Afsari et al. 2014; Sobczak and Kozłowski 2015). biotic level, breed type and the hen’s age. Significant
Beneficial effects on the damaged egg ratio resulting interaction between probiotic level and breed type in
from probiotic supplementation are in agreement with egg mass confirms that performance of laying hens
the report of Balevi et al. (2001), who indicated a receiving probiotics may be differ according to geno-
similar probiotic supplementation. On the one hand, type or breed used in the current experiment.
we suggest that the increase in shell thickness per-
centage and in turn, the reduction in number of dam-
Egg quality assessment
aged eggs in hens receiving probiotics may have been
caused by increase calcium retention. This hypothesis Our findings have been confirmed by many reports
was suggested by Balevi et al. (2001). On the other (Mikulski et al. 2012; Zhang et al. 2012; Sobczak and
hand, several studies confirmed that the inclusion of Kozłowski 2015). Conversely, Mahdavi et al. (2005) and
probiotic to the diet has been found to improve egg Mohebbifar et al. (2013) did not find significant effects
production and feed conversion ratio (Panda et al. for dietary probiotic inclusion on egg quality. Probiotic
2008; Youssef et al. 2013; Chung et al. 2015). However, supplementation seems to have a positive effect on
the difference between the present study and the the eggshell percentage. A positive effect of probiotic
 ITALIAN JOURNAL OF ANIMAL SCIENCE 811

supplementation on eggshell quality characteristics mucosal immune system, which responds to antigenic
has been reported (Li et al. 2006; Abdelqader et al. stimuli by secreting immunoglobulin (IgA). Diet sup-
2013). Swia˛tkiewicz et al. (2010) attributed the positive plemented with 0.1% Lactobacillus acidophilus induced
effects of probiotics on eggshell quality parameters to the best immune response compared with 0.05%
the increased intestinal availability of Ca. The benefi- Bacillus subtilis in laying hens (Forte et al. 2016). The
cial effects on eggshell thickness and strength improvement in immune response found in Saudi
observed in the current study were directly associated chicken breeds compared with Leghorn chickens is in
with the reduction in the number of damaged eggs. agreement with the results of Fathi et al. (2017).
Under heat stress, it is well known that the eggshell Likewise, Saudi laying hens significantly (p < .01)
strength is impaired and in turn, the incidence of recorded higher IgM concentration compared to
cracked eggs exceeded. Therefore, many researchers Leghorn counterparts. In consistent with our results,
have focussed their investigations toward improving Osei-Amponsah et al. (2013) confirmed that the local
eggshell quality using genetic, nutritional and bio- Ghanaian ecotypes of chickens were superior to exotic
logical approaches. Probiotic supplementation is the breeds in terms of their ability to respond to SRBC
effective applied procedure in birds suffering from sev- antigens. In a comparison of humoural immunity due
eral stressors. Insignificant results of eggshell quality to breed, Fathi et al. (2017) reported that there was no
due to breed effect are in consistence with the results significant difference among breeds (Saudi, Leghorn
of Najib and Al-Yousef (2014) and Al-Homod (2016). and Lohmann) for antibody levels against Newcastle
disease virus vaccine. Regarding the other types of
immunoglobulins (IgA and IgY), the current results did
Immune response
not show significant differences among breeds.
Inflammatory response to PHA-P injected in wattle, as Contrary to our results, Mountzouris et al. (2010)
an indication for cell-mediated immunity, did not differ reported that the use of probiotics may modulate the
significantly as affected by probiotic supplementation systemic immune system by increasing the total levels
level. Immunoglobulin (chicken IgY, IgA and IgM) lev- of serum IgG in broilers and be indicative of the over-
els in serum are indicative of the humoural immune all humoural immune status of the bird. Koenen et al.
status of chickens (Mountzouris et al. 2010). As well (2002) also explored the effects of probiotics in the
established, IgM is a potent complement activating systemic humoural immune response, and found that
antibody and since complement is needed for the different Lactobacillus spp. increase the levels of IgG in
generation of a normal antibody response (Parmentier laying hens.
et al. 2004). Generally, enhancing immunity resulting
from probiotic administration has been reported by
Blood plasma biochemical concentrations
many investigators (Fong et al. 2015; Attia et al. 2017).
Laying hens received a diet containing multi strains of The present findings are in agreement with those of
probiotics exhibited a higher antibody production Dimcho et al. (2005), who found that probiotic inclu-
against SRBC compared with non-treated hens during sion did not affect the serum total protein concentra-
high environmental temperature (Asli et al. 2007). tion of chickens. Also, Alkhalf et al. (2010) postulated
Higher antibody production against Newcastle disease that the serum concentrations of total protein and
virus (NDV) was observed in the group fed a diet sup- albumin were not significantly affected by dietary pro-
plemented with Lactobacillus acidophilus compared to biotic supplementation. The levels of total protein,
the control group (Forte et al. 2016). Under heat stress, albumin and globulin obtained in the present study
dietary supplementation of probiotics improved are within the normal physiological values reported by
humoural immunity against Newcastle disease virus Meluzzi et al. (1992) and Attia et al. (2011, 2016). It is
and infectious bursal disease virus (Sohail et al. 2010). generally known that blood plasma total protein plays
Contrarily, probiotic supplementation did not affect key roles in the maintenance of colloid osmotic pres-
specific antibody synthesis to ND vaccine antigen sure, as a rapid substitute for indispensable amino
administered via drinking water (Balevi et al. 2001). acids, assuring glucose through gluconeogenesis, in
This non-stimulation of humoural immunity by pro- transport of minerals and hormones, in forming
biotic may be attributable to the non-host specific enzymes and the immune system in the organism.
strains, species or even genera of the microorganisms. Therefore, blood plasma proteins have an exceptional
Perdigon et al. (1990) showed that treating lagers with significance in homeostasis maintenance. Moreover,
Lactobacillus supplementation increased cellularity of albumen serves as the most favourable source of
Peyer’s patches and this indicated a stimulation of the amino acids for synthesis of tissue proteins in the
812 M. FATHI ET AL.

period of quick somatic growth of birds, especially Conclusions

under feed restricted conditions (Yaman et al. 2000;
In conclusion, diet supplementation with probiotics
Filipoviae et al. 2007).
did not improve laying performance. A beneficial
Reducing blood cholesterol level due to probiotic
effect resulting from probiotic supplementation on
inclusion has also been reported in broilers fed a diet
eggshell quality was observed without affecting
supplemented with prebiotic and probiotic (Sohail
internal egg quality traits under high ambient tem-
et al. 2010) and layers supplemented with probiotic
perature. Also, plasma cholesterol and triglyceride
(Kurtoglu et al. 2004; Sobczak and Kozłowski 2015). On
reduced in laying hens fed a diet supplemented with
the one hand, manipulation of enteric microflora with
probiotics. Furthermore, probiotic inclusion signifi-
probiotics may also play an important role in altering
cantly increased IgM immunoglobulin concentration in
the lipid metabolism of chickens as various studies
laying hens fed a diet containing either 200 or
have shown that probiotics could reduce cholesterol
400 ppm compared with unsupplemented hens.
content in egg yolk and serum (Jin et al. 1998;
Concerning breed effect, Saudi black laying hens
Kalavathy et al. 2003; Li et al. 2006). On the other
recorded significantly higher egg mass compared with
hand, Kurtoglu et al. (2004), Zarei et al. (2011) and
the other breeds. Also, both Saudi chicken breeds
Mohebbifar et al. (2013) reported that the dietary pro-
exhibited higher cell-mediated response and IgM con-
biotic supplementation did not affect serum choles-
centration compared with Leghorn hens.
terol or triglyceride. Insignificantly decreased serum
triglyceride level is in consistency with Zhang et al.
(2012) who reported that using Bacillus subtilis at level Disclosure statement
of 400 g/t of diet significantly (p < .05) reduced serum
The authors alone are responsible for the content and writ-
triglyceride level (1760.6 mgdL1) compared with ing of this article.
untreated group (2158.8 mgdL1). Our results con-
cerning blood plasma T3 and T4 concentrations in lay-
ing hens fed a diet supplemented with probiotics are ORCID
in agreement with Chotinsky and Mihaylov (2013), Moataz Fathi http://orcid.org/0000-0001-9207-3861
who noted that the quantity of T4 did not change sig-
nificantly, while blood serum T3 concentration
increased with the supplementation of Lactobacillus
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