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Citation: Eltahan, H.M.; Kang, C.W.;
Chowdhury, V.S.; Eltahan, H.M.;
Abdel-Maksoud, M.A.; Mubarak, A.;
Lim, C.I. Cold Drinking Water Boosts
the Cellular and Humoral Immunity
in Heat-Exposed Laying Hens.
Animals 2023,13, 580. https://
doi.org/10.3390/ani13040580
Academic Editor: Radiah C. Minor
Received: 8 December 2022
Revised: 19 January 2023
Accepted: 31 January 2023
Published: 7 February 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
animals
Article
Cold Drinking Water Boosts the Cellular and Humoral
Immunity in Heat-Exposed Laying Hens
Hatem M. Eltahan 1, 2, * , Chang W. Kang 3, Vishwajit S. Chowdhury 4, Hossam M. Eltahan 1,
Mostafa A. Abdel-Maksoud 5, Ayman Mubarak 5and Chun Ik Lim 6,*
1Animal Production Research Institute, Agriculture Research Center, Agriculture Ministry,
Sakha, Kafr El-Sheikh 33717, Egypt
2Postdoc at the Department of Animal Science, Jeonbuk National University, Jeonju 54896, Republic of Korea
3College of Veterinary Medicine, Jeonbuk National University, Jeonju 54596, Republic of Korea
4Division for Experimental Natural Science, Faculty of Arts and Science, Graduate School of Bioresource and
Bioenvironmental Science, Kyushu University, Fukuoka 819-0395, Japan
5
Botany and Microbiology Department, College of Science, King Saud University, Riyadh 11451, Saudi Arabia
6
Poultry Research Institute, National Institute of Animal Science, RDA, Pyeongchang 25342, Republic of Korea
*Correspondence: hatem_eltahan2002@yahoo.com (H.M.E.); dlacjsdlr@naver.com (C.I.L.);
Tel.: +20-1005122758 (H.M.E.); +82-63-270-2638 (C.I.L.); Fax: +82-63-270-2612 (C.I.L.)
Simple Summary:
The current study shows that using cold water under high ambient temperature
(CW: 15
±
1
◦
C; HT: HT: 35
±
1
◦
C) in heat-exposed laying hens is capable of maintaining productive
efficiency and immune-suppressing under heat stress. The feed intake and egg production were
enhanced after using the cold water under heat stress. Moreover, the cold water restored the decline
in the level of B-cell, helper T cells, and the ratio of helper/cytotoxic T cells in peripheral blood
mononuclear cells, as well as the concentration of IL-2, IFN-
γ
, and immunoglobulin G in plasma.
Therefore, cold water is one of the mechanisms that can be considered under heat stress.
Abstract:
This study aimed to investigate the effects of cold drinking water on cellular and humoral
immunity in heat-exposed laying hens. One hundred and eight laying hens at 19 weeks old were
placed into three treatments with six replicates of six hens in each group as follows: (1) hens were
provided with normal drinking water (NW) under the control of thermoneutral temperature (CT:
25
±
1
◦
C; CT + NW), (2) hens were provided with NW under high ambient temperature (HT:
35
±
1
◦
C; HT + NW) for 8 h/d for a month, and (3) hens were treated under HT with cold drinking
water (CW: 15
±
1
◦
C; HT + CW) for 8 h/d for a 4-weeks. Then, the feed consumption, egg production,
egg weight, feed conversion ratio, and blood immune parameters were investigated. The results
showed that cold drinking water (CW) caused a significant (p< 0.05) recovery in the reduction of
food intake and egg production due to heat stress; however, there was no significant effect (p> 0.05)
on egg weight and feed conversion ratio. Moreover, CW significantly (p< 0.05) restored the immune-
suppressing effects of heat stress on the contents of peripheral blood mononuclear cells, including
B-cell (BU-Ia), helper T cell (CD4), and the ratio of helper/cytotoxic T cell (CD4/CD8). In addition,
CW significantly (p< 0.05) recovered the reduction on the level of mRNA expression of interleukin-2
(IL-2) and interferon-gamma (IFN-
γ
), as well as significantly (p< 0.05) restored the reduction of
plasma concentration of IL-2, IFN-
γ
and immunoglobulin G in heat-stressed laying hens. These
results prove that CW increased heat dissipation and enhanced feed intake, egg production, and
cellular and humoral immunity in heat-exposed laying hens.
Keywords: cold drinking water; laying hens; heat stress; immunity; cytokines
1. Introduction
High ambient temperature (HT) in the summer season is one of the major environmen-
tal challenges facing the poultry industry worldwide. Commercial chickens for intensive
Animals 2023,13, 580. https://doi.org/10.3390/ani13040580 https://www.mdpi.com/journal/animals
Animals 2023,13, 580 2 of 10
meat and egg production are more sensitive to diseases due to a reduction in immune
response throughout genetic selection improvement [
1
,
2
]. Heat stress harms all age groups
of chickens, including young chicks [
3
], broilers of market age [
4
], and adult layers [
5
].
Several studies have demonstrated that heat stress negatively impacts feed intake, body
weight, behaviours, egg production, eggshell quality, gut integrity, immunity, and mortal-
ity [6–9]. It has been reported that high temperature triggers the hypothalamus’s appetite
centre to send nerve impulses that inhibit feed intake [
10
]. Moreover, hens decrease their
daily feed intake by roughly 1% to 1.5% for every degree over the thermoneutral zone
(20 to 25 ◦C) [11].
Furthermore, numerous studies have indicated that heat stress has immunosuppres-
sive effects in laying hens. The immune system of chickens consists of innate immunity,
nonspecific lymphocytes (such as a macrophage, neutrophil, natural killer cell, or dendritic
cell), and adaptive immunity, including cellular (T cells) and humoral (B cells) immunity.
B and T cells are the white blood cells made in the bone marrow. B cells mature in the
bone marrow, while the T cells travel to the thymus and mature there. The humoral B cells
can transform into plasmocytes helping signal plasma cells to create three different types
of antibodies called immunoglobulin G, M, and A (IgG, M, and A, respectively), which
effectively coat or kill particular microorganisms. Furthermore, the cellular (T cells) and
humoral (B cells) interact together via chemical messengers known as cytokines, which
include interleukin-2 (IL-2) and interferon-gamma (IFN-
γ
), as well as many other com-
ponents [
12
]. In addition, IL-2 has been shown to boost T cell proliferation and IFN-
γ
to
improve adaptive cellular immunity [13].
It has been demonstrated that heat stress reduces innate and adaptive immunity in
hens, as measured through phagocytic response and serum antibody titers [
14
–
16
]. Heat
stress, for example, lowered bursal weight and the number of lymphocytes inside the
bursal cortex and medulla sections, as well as the number of circulating lymphocytes,
and increased the number of heterophils [
8
]. Moreover, heat stress reduced the relative
weights of the spleen and thymus [
17
,
18
], declined the liver weights [
19
,
20
], decreased
the response of systemic humoral immunity [
9
], and reduced the lymphocytes and IgA-
secreting cells in the intestinal tract [
6
]. Therefore, it is critical to discover beneficial
methods and techniques to alleviate heat stress-induced adverse effects on immunological
functioning in laying hens.
Water is one of the nutrients with several positive benefits, making it the most crucial
and essential nutrient for overall health. Water aids in the regulation of body temperature,
feed digestion and absorption, nutrient transfer, and waste disposal from the body [
21
].
Usually, under average normal ambient temperature, the birds consume water approx-
imately 1.6 to 2.0 times more than feed based on their weight [
22
]. During heat stress,
water consumption quadrupled to increase heat dissipation via convection, conduction,
and radiation [
23
]. According to NRC, water consumption rises roughly 7% for every 1
◦
C
increase in ambient temperature over 21 ◦C in broilers [24].
Cold drinking water (CW) has been shown to maintain high feed intake and egg
production in hens under heat stress (30
◦
C) [
25
,
26
]. On the other hand, CW immersions in
the human body three times a week for six weeks increased the total T lymphocytes (CD3),
helper T cells (CD4), and B lymphocytes while suppressing cytotoxic T cells (CD8) [
27
].
Furthermore, Tipton et al. [
28
] demonstrated the advantages of using cold water for heat
dissipation and immunological enhancement in humans. However, it is yet unknown
whether CW has any protective effect on laying hen immunity when exposed to heat stress.
In this study, we examined how cold water affects the activity of immune cells, such
as cellular immunity (CD3, CD4, CD8, IL-2, and IFN-
γ
) and humoral immunity (B-cell and
IgG), in laying hens.
Animals 2023,13, 580 3 of 10
2. Materials and Methods
2.1. Animal Housing and Management
The layers were housed in metal wire cages randomly. The dimensions of each
cage were 60
×
60
×
40 cm (2 layers/cage). The cages were divided into two-block
chambers (
4×4.2 ×2.6 m
) for a control thermoneutral temperature (CT: 25
±
1
◦
C) and HT
(35
±
1
◦
C) for eight h/day (h/d). Each birdcage was equipped with a feeding and drinking
station. For the cold drinking water group, water at 15.0
±
1
◦
C was passed through the
chiller directly to the cup and nipple waterer as described elsewhere [
29
]. The present
study was conducted following the guidelines for animal experiments at the Faculty of
Agriculture of Jeonbuk National University and followed the ethical approvement number
2021-0168.
2.2. Experimental Design
A total of one hundred and eight 19-week-old Hy-line brown layers weighing 1.4
to 1.8 kg were divided into three treatment groups with six replicates of six hens in each
group. The first group was treated with normal drinking water (NW) under CT (25
±
1
◦
C;
CT + NW
). Then, the second group was given NW under HT (35
±
1
◦
C) for 8 h/d for a
month (HT + NW). Finally, the third group was treated with cold drinking water (15
±
1
◦
C,
CW) under HT for 8 h/d for a month (HT + CW). For four weeks, the CW treatment was
paired with HT treatment for 8 h daily from 11 a.m. to 7 p.m., then kept on the NW until
the next day. The hens were placed for adaptation in the experimental chambers two weeks
before the experiment started. The hens were exposed to 17 h of light per day, and the
humidity was 60% during the investigation, while water and commercial feed, according
to [25] (Table 1), were available ad libitum.
Table 1. The ingredient and nutrient composition of experimental diets.
Ingredient (%)
Corn 55.700
Hard Red Winter Wheat 3.820
Wheat bran 10.220
Soybean meal (48%) 19.370
Monocalcium phosphate (Ca 18%, p21%) 0.830
Limestone (Ca 38.5%) 9.460
Iodized salt 0.300
DL-methionine (99%) 0.100
Vitamin premix 10.100
Mineral premix 20.100
Total 100
Calculated nutrient composition
Metabolizable energy (kcal/kg) 2750
Crude protein (%) 16.00
Calcium (%) 4.00
Total phosphorus (%) 0.69
Available phosphorus (%) 0.40
Lysine (%) 0.80
Methionine (%) 0.36
Cysteine (%) 0.29
Arginine (%) 0.99
1
Vitamin supplement provided per kilogram of diet: vitamin A, 10,000 IU; vitamin D3, 2500 IU; vitamin E, 20 IU;
vitamin B1 1.5 mg; vitamin B2 5.0 mg; vitamin B6, 0.15 mg; vitamin B12 15.0 mg; choline, 300 mg; pantothenate,
12 mg; nicotinic acid, 50 mg; biotin, 0.15 mg; folic acid, 1.5 mg.
2
Mineral supplemented provided per kilogram of
diet: Fe, 60 mg, Cu,10 mg; Zn 80 mg; Mn, 110 mg; Iodine, 0.48 mg; Se, 0.40 mg.
2.3. Data and Sample Collection
The feed was provided twice daily. The feed intake was measured by collecting and
weighing the leftovers in the feeder and calculating the average feed intake for each weekly
Animals 2023,13, 580 4 of 10
interval in replications, while daily water intake was determined by measuring water on
the morning of the first day and weighing it back the next morning. Moreover, eggs were
collected and recorded daily at 4 p.m. Weekly eggs were pooled and analysed related
to the number of hens for each cage in each replication. The feed conversion ratio was
calculated using weekly feed consumption divided by the weekly egg mass for the four
weeks, while egg mass per hen per day was calculated as the average egg production
percentage multiplied by the average daily egg weight. Furthermore, blood samples were
collected before the experiment and after the 15th and 30th days of the trial to examine the
immune cells in peripheral blood mononuclear cells (PBMC).
2.4. Isolation of Plasma and PBMC
Fifteen ml of chicken blood was obtained from ten hens of each treatment in a sterile
glass tube containing 150 IU of preservative-free heparin (Sigma, St Louis, MO, USA).
Plasma and PBMCs consisting of lymphocytes, monocytes, and polymorphs were isolated
with the standard Ficoll-Hypaque (Histopaque, Sigma) density gradient centrifugation
method to the manufacturer’s instructions. One aliquot of 5
×
10
6
cells was used imme-
diately for RNA extraction, and the remaining (usually
≥
10
×
10
6
) cells were used to
determine the concentration of cytokines content.
2.5. Isolation of PBMC Total RNA and Quantitative Real-Time PCR
Total RNA was extracted from the chicken PBMCs using the modified guanidinium
thiocyanate-phenol-chloroform method, according to Gauthier et al. [
30
]. According to
the manufacturer’s instructions, cDNA was synthesized using 1
µ
g of total RNA and the
PrimeScript
®
RT reagent Kit with gDNA Eraser (Takara, Shiga, Japan). All primers were
evaluated using routine PCR and gel electrophoresis before real-time PCR (TaKaRa PCR
Thermal Cycler Dices, Takara, Shiga, Japan). The expression of chicken IL-2 and IFN-
γ
in the PBMC were quantified with real-time PCR following the steps that are written
elsewhere in Eltahan et al. [
3
]. The primer sequences are presented in Table 2. Relative
mRNA expressions have been calculated by comparing the thermal cycles needed to
generate threshold amounts of product (PCR-ct). PCR-ct was calculated for the chicken
IL-2 and IFN-
γ
, as well as the chicken RNA polymerase-II (RP-II) as a housekeeping gene,
since it was confirmed that the RP-II expression level was not altered under the current
experimental conditions. The IL-2 and IFN-
γ
mRNA expression were calculated as 2-
∆∆
PCR-ct, as described elsewhere by Schmittgen and Livak [31].
Table 2. Primers used for real-time PCR.
Gene Accession No. Sequences 50-30(Forward/Reverse) Annealing
Temperature (◦C)
Product
Size (bp)
INF-γNM_205149.2 50-TGTAGCTGACGGTGGACCT-30/
50-ATGTGTTTGATGTGCGGCTT-3060 147
IL-2 NM_204153.2 50-ACTCTGCAGTGTTACCTGGG-30/
50-CCGGTGTGATTTAGACCCGT-3060 140
RP-II NM_001006448.2 50-CGACGGTTTGATTGCACCTG-30/
50-CAATGCCAGTCTCGCTAGTTC-3064 161
Primers were designed with Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast) accessed on
23 November 2021
for interleukins-2 (IL-2), while accessed on 28 October 2021 for interferon-gamma (INF-
γ
) and
accessed on 23 September 2021 for RNA polymerase II (RP-II). According to Eltahan et al. [
3
], RP-II has been
selected as an internal control gene constantly expressed during heat stress.
2.6. Statistical Analysis
The study data were subjected to compare the effects of providing cold drinking
water and normal drinking water on the productivity of laying hens under average or
HT using one-way analysis of variance (ANOVA) software (v 9.4; SAS Institute, 2016,
Gary, CA, USA) [
32
]. We chose p< 0.05 as the minimum acceptable significance level
except regarding the blood analysis. Results are shown as mean, standard deviation, and
coefficient of variance.
Animals 2023,13, 580 5 of 10
3. Results
3.1. Production Performance Following CW Administration under Heat Stress Exposure in Hens
The egg production and feed intake were significantly (p< 0.05) reduced under
HT+NW compared with CT+NW in laying hens (Table 2). However, the CW of 15 ±1◦C
significantly (p< 0.05) improved the adverse effects of HT on egg production and feed
intake. In contrast, water intake increased from HT under the NW and CW compared
with CT. In addition, there was no significant difference between the HT or CW on the egg
weight and feed conversion ratio (Table 3).
Table 3.
The effect of cold drinking water on the performance of laying hens after heat stress exposure.
Treatments Egg Production (%) Egg Weight (g) Daily Feed Intake (g) Daily Water Intake (mL) Feed Conversion (%)
CT + NW 91.17 ±1.04 a62.75 ±0.39 108.93 ±0.62 a217 ±24.0 b1.90 ±0.02
HT + NW 85.07 ±1.50 b62.21 ±0.61 102.95 ±1.03 b355 ±73.8 a1.95 ±0.04
HT + CW 89.11 ±0.66 a61.84 ±0.71 107.81 ±0.39 a304 ±63.8 a1.96 ±0.02
p-value 0.04 0.583 0.045 0.047 0.876
Values are means
±
SEM in the number of 24 laying chickens used in each treatment. Different superscripts in the
same column indicate significant differences at p< 0.05 between the treatments. CT + NW, control thermoneutral
temperature (25
±
1
◦
C) with regular tap water treatment (25
±
1
◦
C); HT + NW, high temperature (35
±
1
◦
C)
with regular tap water treatment (25
±
1
◦
C); HT + CW, high temperature (35
±
1
◦
C) with cold water treatment
(15 ±1◦C).
3.2. The Contents of PBMCs Following CW Administration under Heat Stress Exposure in Hens
The percentage of B cells significantly (p< 0.05) declined after exposure to the HT for
15 and 30 days in laying hens (Table 4). However, we found that CW treatment (HT + CW)
significantly eliminated the adverse effect of HT on the number of B cell lymphocytes after
15 and 30 days in heat-stressed laying hens. In addition, the number of CD4 and the ratio
between CD4/CD8 have shown the same significant (p< 0.05) pattern of the reduction
under heat stress treatment (HT + NW). This reduction due to HT exposure was impeded
significantly (p< 0.05) by the CW treatment (HT + CW) in heat-stressed laying hens after
15 and 30 days (Table 4). In contrast, there were no significant differences in the percentage
of CD3 and CD8 in PBMCs in connection to the HT exposure or CW treatments (Table 4).
Table 4.
Effects of cold drinking water on the contents of PBMC lymphocyte subsets in laying hens
under heat stress.
Period and Treatments BU-Ia (%) CD3 (%) CD4 (%) CD8 (%) CD4/CD8
0 day
CT + NW 2.76 ±0.43 11.61 ±2.23 48.80 ±7.64 23.08 ±3.43 2.12 ±0.36
HT + NW 2.80 ±0.35 13.60 ±2.28 48.65 ±6.57 23.60 ±4.97 2.17 ±0.28
HT + CW 2.67 ±0.45 14.95 ±2.49 47.35 ±9.12 23.30 ±5.53 2.07 ±0.23
p-value 0.583 0.656 0.523 0.324 0.243
15 days
CT + NW 3.32 ±0.46 a13.38 ±1.39 63.52 ±7.87 a21.55 ±4.66 3.03 ±0.35 a
HT + NW 2.56 ±0.41 b15.73 ±3.94 52.96 ±5.25 b21.21 ±5.59 2.51 ±0.25 b
HT + CW 4.23 ±0.52 a15.67 ±2.40 62.00 ±7.77 a21.77 ±4.00 2.95 ±0.29 a
p-value 0.038 0.234 0.037 0.891 0.048
30 days
CT + NW 3.98 ±0.48 a15.66 ±4.88 63.21 ±7.58 a16.76 ±5.03 3.95 ±0.32 a
HT + NW 2.04 ±0.34 b13.68 ±2.83 47.27 ±7.29 b16.12 ±3.54 2.93 ±0.21 b
HT + CW 4.31 ±0.52 a14.03 ±2.54 63.80 ±6.78 a16.76 ±3.34 4.05 ±0.34 a
p-value 0.026 0.675 0.027 0.853 0.046
Values are means
±
SEM in the number of 10 laying chickens used in each group. Different superscripts in the
same column indicate significant differences at p< 0.05 between the treatments in the same period. CT + NW,
thermoneutral control temperature (25
±
1
◦
C) with regular tap water treatment (25
±
1
◦
C); HT + NW, high
temperature (35
±
1
◦
C) with standard tap water treatment (25
±
1
◦
C); HT + CW, high temperature (35
±
1
◦
C)
with cold water treatment (15
±
1
◦
C); PBMC, peripheral blood mononuclear cells; BU-Ia, B-cells; CD3, number of
lymphocytes in PBMC; CD4, helper T cells; CD8, cytotoxic T cells; PBMC, peripheral blood mononuclear cells.
Animals 2023,13, 580 6 of 10
3.3. The mRNA Expression of IL-2 and IFN-γin PBMCs and Concentrations of Plasma Immune
Parameters Following CW Administration under Heat Stress Exposure in Hens
The mRNA expressions of IL-2 and IFN-
γ
in PBMC significantly (p< 0.05) declined
after 15 and 30 days of heat exposure in hens under the HT + NW group. However, the CW
treatment under HT (HT + CW) significantly (p< 0.05) recovered the heat stress-induced
decline of the mRNA expressions of IL-2 and IFN-
γ
in PBMC after 15 and 30 days of heat
exposure in hens (Table 5). The levels of IL-2, IFN-
γ
, and IgG concentrations in the plasma
significantly (p< 0.05) declined after 30 days of heat exposure (HT+ NW) in hens (Table 5).
The analysis has shown that CW treatment (HT + CW) showed a tendency (p= 0.09) to
restore the adverse high-temperature effects on the concentration of IL-2, IFN-
γ
, and IgG
in plasma after 15 days of administration. It was interesting to note that CW treatment
significantly (p< 0.05) recovered the adverse effect of HT on the concentration level of
plasma IL-2, IFN-γ, and IgG after 30 days of exposure to heat stress in hens (Table 5).
Table 5.
Effects of cold drinking water on the mRNA expression of cytokines in PBMC and immune
parameter concentration in plasma of laying hens after heat stress exposure.
Period and
Treatments
The Expression of Cytokines mRNA in PBMC The Concentration of Cytokines and IgG in Plasma
IL-2 IFN-γIL-2 (ng/mL) IFN-γ(ng/mL) IgG (mg/mL)
0 day
CT + NW 1.01 ±0.23 0.99 ±0.31 2.45 ±0.62 1.15 ±0.31 3.84 ±0.56
HT + NW 1.08 ±0.18 0.98 ±0.22 2.54 ±0.59 1.01 ±0.22 3.72 ±0.63
HT + CW 1.14 ±0.32 1.12 ±0.36 2.45 ±0.69 1.12 ±0.27 3.79 ±0.73
p-value 0.983 0.354 0.845 0.987 0.998
15 days
CT + NW 4.46 ±0.82 a7.31 ±1.45 a2.84 ±0.55 1.31 ±0.49 4.06 ±0.77
HT + NW 2.84 ±0.55 b3.59 ±0.99 b1.95 ±0.48 0.83 ±0.29 2.76 ±0.69
HT + CW 4.43 ±0.71 a6.33 ±1.32 a2.36 ±0.54 1.05 ±0.46 3.69 ±0.92
p-value 0.038 0.024 0.278 0.144 0.068
30 days
CT + NW 5.44 ±0.97 a5.19 ±1.02 a2.64 ±0.67 a1.79 ±0.43 a4.18 ±0.79 a
HT + NW 1.34 ±0.63 b1.44 ±0.77 b0.78 ±0.63 b0.37 ±0.19 b1.71 ±0.61 b
HT + CW 4.12 ±0.86 a5.03 ±1.22 a2.31 ±0.76 a1.87 ±0.40 a3.52 ±0.73 a
p-value 0.024 0.029 0.047 0.049 0.038
Values are means
±
SEM in the number of 10 laying chickens used in each group. Different superscripts in the
same column indicate significant differences at p< 0.05 between the treatments in the same period. CT + NW,
thermoneutral control temperature (25
±
1
◦
C) with regular tap water treatment (25
±
1
◦
C). HT + NW, high
temperature (35
±
1
◦
C) with standard tap water treatment (25
±
1
◦
C). HT + CW, high temperature (35
±
1
◦
C)
with cold water treatment (15
±
1
◦
C); IL-2, interleukins-2; IFN-
γ
, interferon-gamma; IgG, immunoglobulin G;
PBMC, peripheral blood mononuclear cells.
4. Discussion
In this study, we demonstrated that the CW could overcome the detrimental effects of
summer heat stress on the hen’s productivity and immune system, including CD3, CD4,
CD8, IL-2, and IFN-γin PBMCs in the plasma.
It has been elucidated that heat stress reduced the feed intake as a starting point for
the detrimental effects on productivity. It caused a decline in body weight, feed conversion
ratio, egg production, egg quality, and immune response in laying hens [
33
]. These findings
are matching with our current study results (Table 3), indicating that exposure to the HT
significantly (p< 0.05) decreased the feed intake, which might be the result of suppressed
metabolic heat production under heat stress. Furthermore, we found a reduction in egg
production due to exposure to HT that showed similarity with several studies on laying
hens [
6
,
34
]. According to Guterrez et al. [
25
], chilled water (16
±
0.5
◦
C) increased feed in-
take, which reflects increased calcium intake and, consequently, egg production, compared
to un-chilled water (23
±
2.5
◦
C) under constant temperature at 30
◦
C in the laying hens.
We observed that CW (15
±
1
◦
C) significantly (p< 0.05) recovered the detrimental effects of
heat stress on feed intake and egg production (Table 3). Moreover, Lim et al. [
35
] reinforced
Animals 2023,13, 580 7 of 10
the hypothesis that CW significantly (p< 0.05) raised the egg production in Hy-line layer
hens under heat stress.
It has been observed that broilers exposed to acute heat stress reduce their feed
intake [
36
] and increase water intake to efficiently control their body temperature with
evaporative cooling (panting) [
37
]. We found that HT increases the water intake and reduces
feed intake while using CW slightly declines water intake and consequently increases feed
intake. Therefore, cold water under heat stress treatment might reduce the core body
temperature through conduction and convection, which could increase feed intake and,
consequently, egg production. It will be interesting to investigate the body temperature
and the appetite hormone signalling in future research to explain the improvement of egg
production after using cold water under heat stress. In contrast, previous research [
5
,
38
]
indicated a decline in egg weight and feed conversion ratio in heat-stressed laying hens.
We found no significant change in egg weight and feed conversion ratio under HT or CW
treatment in heat-stressed laying hens. This could be due to our research’s short duration
of one month.
Previously, several studies have demonstrated that heat stress has an immunosup-
pressing impact on laying hens. In agreement with Aengwanich [
8
], who reported that heat
stress decreased bursal weight (the organ for B lymphocyte development, proliferation,
and differentiation in chicken), we discovered that heat stress dramatically decreased the
percentage of B cells in PBMC after 15 and 30 days of exposure to HT (Table 4). Moreover,
Oznurlu et al. [
39
] have reported that embryonic or post-hatch heat stress leads to bursal
follicular atrophy, which could affect B cell production. Another study found that B cells
were reduced in both bursal and blood under heat stress [
40
]. The immunosuppressive
status of layers after exposure to heat stress in the current investigation was highlighted
by the same pattern of reduction in helper T cells (CD4) and the ratio between helper to
cytotoxic T cells (CD4/CD8) in PBMC after 15 and 30 days of exposure to heat stress. Our
results have similarities with the previous studies [
41
,
42
], which have reported that there
had been a reduction in the number of circulating lymphocytes in laying hens under heat
stress. Our results showed that CW recovered the harmful effect of HT on declining the
percentage of BU-Ia, CD4, and CD4/CD8 in PBMC after 15 and 30 days (Table 4). These
health advantages are believed to be a consequence of the physiological and biochemical
responses that occur from drinking CW, such as the body’s heat loss through conduction
which decreases tissue temperature and, hence, reduces the metabolic rate and oxygen
requirement of this cooled tissue [43].
Moreover, we found that gene expression of IL-2 and IFN-
γ
in PBMC significantly
decreased after 15 and 30 days of exposure to HT (Table 5), which provides additional
evidence on the immune-suppressing effects of heat stress in laying hens. In addition, our
results have shown that CW can restore the reduction of gene expression IL-2 and IFN-
γ
in PBMC (Table 5) patterns under heat exposure and enhance the laying hen’s immunity.
The current results regarding the concentration of cytokines in plasma have strengthened
our hypothesis about CW improving the recovery of the detrimental effects of heat stress
(Table 5) on the concentration of cytokines IL-2, IFN-
γ
, and IgG after 30 days but not on
15 days in laying hens’ plasma.
5. Conclusions
Our observations suggest that using CW effectively contributed to overcoming the
adverse heat stress effects in the decline of the immune cell contents, including cellular
immunity CD4, CD8, IL-2, and IFN-
γ
and humoral immunity of BU-Ia and IgG in PBMC
and plasma in heat-stressed laying hens.
Author Contributions:
H.M.E. (Hatem M. Eltahan), C.I.L. and V.S.C. designed the study. H.M.E.
(Hatem M. Eltahan), C.I.L. and C.W.K. conducted the research; H.M.E. (Hatem M. Eltahan), H.M.E.
(Hossam M. Eltahan), M.A.A.-M., A.M. and V.S.C. wrote and revised the paper. All authors have
read and agreed to the published version of the manuscript.
Animals 2023,13, 580 8 of 10
Funding:
This research was funded by the Korean government (MSIT) grant number [2019K2A9A1A-
09081549]. As well this work was funded by the Korea Institute of Planning and Evaluation for
Technology (IPET) in Food, Agriculture, and Forestry through the Agriculture, Food and Rural Affairs
Convergence Technologies Program for Educating Global Creative Leader, funded by the Ministry
of Agriculture, Food and Rural Affairs grant number [MAFRA; 716002-7]. In addition, the research
was supported by King Saud University Researchers Supporting Project number (RSPD2023R725),
Riyadh, Saud Arabia.
Institutional Review Board Statement:
The study was conducted following the Declaration of
Helsinki and approved by the Faculty of Agriculture Ethics Committee, Department of Animal
Science, Jeonbuk National University. The ethical approvement number was (JBNU 2021-0168), and
the date on December 2021.
Informed Consent Statement: Not applicable.
Data Availability Statement:
On fair request, the corresponding author will provide data that
support the study’s conclusions.
Acknowledgments:
The authors wish to acknowledge the helpful suggestions of members of the
Department of Animal Science, faculty of agriculture, Jeonbuk National University, Republic of
Korea. Furthermore, the Animal Production Research Institute members for their helpful suggestions.
In addition, the authors extend their appreciation to the Researchers Supporting Project number
(RSPD2023R725) King Saud University, Riyadh, Saud Arabia.
Conflicts of Interest: The authors declare no conflict of interest.
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