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. 2021 Apr 24;7(3):621–630. doi: 10.1016/j.aninu.2021.01.003

Zinc alleviates the heat stress of primary cultured hepatocytes of broiler embryos via enhancing the antioxidant ability and attenuating the heat shock responses

Tingting Li a,1, Wengang He b,c,1, Xiudong Liao b, Xi Lin d, Liyang Zhang b, Lin Lu b, Yanli Guo c, Zongping Liu e, Xugang Luo a,∗
PMCID: PMC8334375  PMID: 34401540

Abstract

Zinc (Zn) has been shown to attenuate the adverse effects of heat stress on broilers, but the mechanisms involving this process remain unclear. We aimed to investigate possible protective mechanisms of Zn on primary cultured hepatocytes of broiler embryos subjected to heat stress. Three experiments were conducted. In Exp. 1, hepatocytes were treated with 0, 50, 100, 200, or 400 μmol/L added Zn as inorganic Zn sulfate (iZn) for 12, 24 or 48 h. In Exp. 2, cells were exposed to 40 °C (a normal temperature [NT]) and 44 °C (a high temperature [HT]) for 1, 2, 4, 6, or 8 h. In Exp. 3, cells were preincubated with 0 or 50 μmol/L Zn as iZn or organic Zn lysine chelate (oZn) for 8 h under NT, and then incubated with the same Zn treatments under NT or HT for 4 or 6 h. The biomarkers of antioxidative status and heat stress in cells were measured. The results in Exp. 1 indicated that 50 μmol/L Zn and 12 h incubation were the optimal conditions for increasing antioxidant ability of hepatocytes. In Exp. 2, the 4 or 6 h incubation under HT was effective in inducing heat shock responses of hepatocytes. In Exp. 3, HT elevated (P < 0.01) malondialdehyde content and expressions of heat shock protein 70 (HSP70) mRNA and protein, as well as HSP90 mRNA. However, Zn supplementation increased (P < 0.05) copper zinc superoxide dismutase (CuZnSOD) activity and metallothionein mRNA expression, and effectively decreased (P < 0.05) the expressions of HSP70 mRNA and protein, as well as HSP90 mRNA. Furthermore, oZn was more effective (P < 0.05) than iZn in enhancing CuZnSOD activity of hepatocytes under HT. It was concluded that Zn (especially oZn) could alleviate heat stress of broiler hepatocytes via enhancing their antioxidant ability and attenuating heat shock responses.

Keywords: Broiler hepatocyte, Zinc, Heat stress, Antioxidant ability, Heat shock response

1. Introduction

Heat stress in environmental stresses is a major concern for poultry industry because of the resulting poor egg production, depressed feed intake and feed efficiency along with high mortality (Liao et al., 2018; Zhu et al., 2015b, 2017a). In addition to decreased productive performance, heat stress was also reported to disturb the redox balance and induce oxidative stress with the excessive generation of reactive oxygen species (ROS) in broilers (Lin et al., 2006; Tan et al., 2010; Xie et al., 2015) and laying hens (Lin et al., 2008; Naziroglu et al., 2000). In response to oxidative stress, organisms have evolved many defense mechanisms including endogenous antioxidants and stress proteins. Copper zinc superoxide dismutase (CuZnSOD) and metallothionein (MT) are considered endogenous ROS scavengers that confer protection against oxidative stress in organisms (Alscher et al., 2005; Volkova et al., 2012). The heat shock proteins (HSP) are a set of stress proteins synthesized in response to various stresses, including heat stress (Evdonin et al., 2009; Xie et al., 2014). Heat stress induces synthesis of HSP in broilers to aid in thermotolerance and antioxidant stress (Gu et al., 2012; Soleimani et al., 2012). However, this abrupt rise in HSP expression also reflects cellular potential heat stress damage (Tedeschi et al., 2015). Therefore, CuZnSOD, MT and HSP have been broadly used as molecular biomarkers to assess the potential cellular and physiological effects of heat stress or antioxidant nutrients (Cao et al., 2015; Jang et al., 2014; Sahin et al., 2012; Wang et al., 2016; Wu et al., 2011; Yin et al., 2018).

Zinc (Zn) is an essential trace element for living organisms that acts as a cofactor for over 300 enzymes (Vallee et al., 1990). Zinc can be not stored in the animal's body; hence, there is a need for daily supplementation of Zn through the diet. Dietary supplementation with Zn is beneficial, especially in heat stress condition that induces production of ROS (Sahin et al., 2005, 2009). In broiler breeder hens, we have demonstrated that supplementation with Zn in maternal diets effectively eliminated the adverse effects of maternal heat stress on embryonic development and growth performance of offspring (Zhu et al., 2017a, 2017b). The requirement of Zn increases during heat stress. Dietary supplementation of Zn from 30 to 60 mg/kg, equating to the total level of Zn in diets from 70 to120 mg/kg, could improve feed intake, body weight, feed conversation radio, and carcass traits in broilers reared under heat stress condition (Salamat et al., 2019; Chand et al., 2014; Kucuk et al., 2003; Rama Rao et al., 2016; Ramiah et al., 2019). However, the exact mechanisms of the protective effect of Zn on broilers against heat stress are little known. Recent studies in chickens have demonstrated that dietary supplementation with either inorganic or organic Zn could elevate the mRNA abundances of MT and CuZnSOD, as well as the CuZnSOD activity and MT concentration in the liver (Azad et al., 2017; Zhang et al., 2017, 2018). Furthermore, the organic Zn was more effective than the inorganic Zn in augmenting CuZnSOD activity and MT concentration in the liver (Zhang et al., 2017, 2018). Inorganic ZnSO4 is the common supplemental form of Zn in diets for poultry. Nevertheless, organic Zn sources, especially Zn amino acid complexes or chelates, have been receiving more and more attention because of their better absorption and higher bioavailabilities (Kwiecień et al., 2017; Suo et al., 2015). Series of studies from our laboratory have further indicated that absorption and bioavailability of organic Zn sources were closely related to their chelation strengths, and the organic Zn with a moderate chelation strength was most available in broilers (Huang et al., 2009, Huang et al., 2013; Yu et al., 2010, 2017) and heat-stressed laying broiler breeders (Liao et al., 2018). The liver is not only a key organ in chickens' response to heat stress (Lin et al., 2006; Mackei et al., 2020), but also a sensitive tissue to reflect the Zn status of broilers (Azad et al., 2017). Accordingly, primary cultured broiler hepatocytes may be an ideal in vitro model for exploring the anti-heat stress mechanisms of Zn. Therefore, we hypothesized that Zn, especially the organic Zn with a moderate chelation strength, might protect primary cultured hepatocytes of broiler embryos against heat stress via enhancing the antioxidant ability and attenuating the heat shock responses. The objective of the present study was to investigate the effect of Zn, especially the organic Zn with a moderate chelation strength, on antioxidant status and expressions of HSP in primary cultured hepatocytes of broiler embryos exposed to heat stress.

2. Materials and methods

All experimental procedures were approved by the Animal Management Committee (in charge of animal welfare issue) of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (IAS-CAAS, Beijing, China), and experiments were performed in accordance with the guidelines. Ethical approval on animal survival was given by the animal ethics committee of IAS-CAAS.

2.1. Isolation and cultivation of primary hepatocytes of broiler embryos

One hundred and fourteen 30-week-old female Arbor Acres broiler breeders with similar weights were purchased from a commercial hatching factory (Huadu Broiler Breeding Corp., Hebei, China) and kept in stainless-steel cages (length 50 cm × width 50 cm × height 50 cm) with 2 birds per cage. All broiler breeder hens were fed a Zn-deficient semi-purified diet containing Zn at 7.13 mg/kg (analysed value) from 30 to 38 weeks in order to obtain the low-Zn eggs so that the primary hepatocytes isolated from the embryos could be Zn-deficient, and thus more sensitive to Zn supplementation (Sun et al., 2018). During this period, lighting and feeding management followed the Arbor Acres breeder management guidelines, and birds had ad libitum access to tap water with no detectable Zn. Healthy fertilized eggs with similar weights were collected from 36- to 38-week-old broiler breeder hens. They were incubated at 38 °C and (50 ± 2)% relative humidity for 14 d. Primary cultured hepatocytes of broiler embryos were prepared according to the previous method (Zhou et al., 2005) with some modifications. Briefly, after anesthesia in cold phosphate-buffered saline (PBS), the livers of 14-d-old chick embryos were collected, washed 3 to 4 times with PBS formulated without magnesium and calcium, and then minced and digested with 0.12% collagenase type II in a shaking water bath (90 cycles/min) at 37 °C for 20 min with frequent pipetting to facilitate cell dissociation. The digested cells were then dispersed by pipettes and filtered to remove large debris using 150-μm and 70-μm meshes. Cell suspensions were centrifuged 3 times at 200 × g at 4 °C, each time for 5 min. The combined pellets were resuspended in the complete culture medium, consisting of L-15 medium (Invitrogen, Carlsbad, CA, USA), supplemented with 10% fetal bovine serum (FBS), 10 μg/mL transferrin, 10 μg/mL vitamin C, 10−6 mol/L dexamethasone, 10−6 mol/L insulin, and 1% antibiotics (100 IU/mL penicillin and 100 mg/mL streptomycin). Cell viability was assessed by the Trypan Blue exclusion test and was not less than 90% for each preparation. Evaluation of the cells by using light microscopy showed that about 95% of the collected cells were hepatocytes (Appendix Fig. 1). Hepatocytes were plated in 6-well cell culture plates (Corning Life Sciences, Lowell, MA, USA) at 1 × 106 cells per well in 2 mL of the complete culture medium. Cells were cultured at physiological temperature (40 °C) of chickens (Han et al., 2010) in a humidified incubator (Model 3110 series; Thermo Electron Corporation, Marietta, OH, USA) with 100% air. The medium was replaced with L-15 medium containing 5% FBS at 24 h after plating and then daily before further treatment. Seventy-two hours (100% confluence) after plating was taken as time 0, at which the different treatments were performed. In each experiment, there were 6 replicates of 6-well cells per replicate for each treatment.

2.2. Treatments of hepatocytes, sample collections and preparation

Experiment 1 was conducted to estimate the optimal dose and effective incubation time of Zn necessary to strengthen antioxidant system of hepatocytes. In this trial, the hepatocytes were incubated in the Zn-unsupplemented fresh serum-free basal L-15 medium containing no detectable Zn (control) and the basal medium supplemented with 50, 100, 200, or 400 μmol/L Zn as inorganic Zn sulfate (iZn; reagent-grade ZnSO4·7H2O, containing 22.5% Zn by analysis) for 12, 24 or 48 h, respectively. Zinc concentrations and incubation time points used in this study were based on previous studies (Muthuraman et al., 2014; Wang et al., 2013). At the end of each incubation time, the medium was removed and the cell monolayer was washed with ice-cold PBS 3 times. The cells in the first 3 wells from each replicate were scraped in ice-cold saline, and then pooled and sonicated at 4 °C for 2 min. Lysates were centrifuged at 1,000 × g at 4 °C for 10 min to harvest the supernatants for CuZnSOD activity analysis. The cells in the second 3 wells from each replicate were scraped in TRIzol reagent, and then pooled and frozen (−80 °C) for analyses of CuZnSOD and MT mRNA expressions.

Experiment 2 was conducted to measure lactate dehydrogenase (LDH) activity and HSP expressions of hepatocytes subjected to heat stress to estimate the optimal heat-stressed time range. In this experiment, hepatocytes were incubated in the fresh complete L-15 medium at 40 °C (a normal temperature [NT]) and 44 °C (a high temperature [HT]) for 1, 2, 4, 6, or 8 h, respectively. Culture temperatures and heat-stressed time points used in this study were based on previous studies (Han et al., 2010; Liu et al., 2012). At the end of each culture time, the medium from each replicate was collected, pooled and stored at −20 °C for LDH activity analysis. Meanwhile, the cells were harvested by the above mentioned method in Exp. 1 for analyses of HSP70 and HSP90 mRNA expressions.

Experiment 3 was performed to investigate the effect of Zn source on the antioxidant status and HSP expressions of hepatocytes exposed to heat stress. The results in Exp. 1 showed that the cells exposed to the serum-free L-15 medium containing 50 μmol/L added Zn as Zn sulfate for 12 h had the best antioxidant ability. Additionally, the results in Exp. 2 indicated that HT affected (P < 0.0001) HSP70 and HSP90 mRNA expressions, and the incubation time of 4 or 6 h under HT was effective in increasing the mRNA expressions of these 2 genes. Therefore, in Exp. 3, the cells were firstly preincubated with the Zn-unsupplemented fresh serum-free basal L-15 medium containing no detectable Zn (control) and the basal medium supplemented with 50 μmol/L Zn as either iZn or organic Zn lysine chelate (oZn) for 8 h at NT (40 °C). The oZn, synthesized at a 2:1 molar ratio of lysine to Zn, contained 8.28% Zn by analysis, which was the same as that used in a previous study of Sun et al. (2018). The quotient of formation (Qf) of the oZn was 62.1, which is categorized as a moderate chelation strength (Holwerda et al., 1995). Lysine concentration in each treatment was balanced by supplementation of additional l-lysine monohydrochoride. And then, culture mediums in the all of the above preincubated cell wells were not changed, and the half of them continued to be incubated under NT, but the other half were transferred to the other incubator and incubated at HT (44 °C) for another 4 or 6 h. At the end of each incubation time, the cells in the first 2 wells from each replicate were collected and prepared by the above-mentioned method in Exp. 1 for determining CuZnSOD activity, MT and malondialdehyde (MDA) contents. The cells in the second 2 wells from each replicate were harvested and prepared by the above-mentioned method in Exp. 1 for analyses of CuZnSOD, MT, HSP70 and HSP90 mRNA expressions. The cells in the third 2 wells from each replicate were lysed in radio-immunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China), and then pooled and sonicated at 4 °C for 2 min. Lysates were centrifuged at 12,000 × g at 4 °C for 5 min to obtain the supernatants, and then supernatants were frozen (−80 °C) for analyses of HSP70 and HSP90 protein expressions.

2.3. Measurement of Zn contents

The Zn contents in Zn sources, the Zn-deficient semi-purified diet and tap water were determined by inductively coupled plasma emission spectroscope (model IRIS Intrepid II; Thermal Jarrell Ash, Waltham, MA, USA) after wet digestions with HNO3 and HClO4, as described by Huang et al. (2009).

2.4. Determinations of LDH activity, MDA and MT levels and CuZnSOD activity

The LDH activity was analyzed according to the instructions in the commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The MDA levels were determined using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The MT concentration was determined by the method of ELISA with commercial kits (Mlbio, Shanghai, China). The CuZnSOD activity was determined using a commercial assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

2.5. RNA extraction, reverse transcription and real-time quantitative PCR

Total RNA was isolated from each sample using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instruction. The purity, concentration, and integrity of the RNA were checked using a NanoDrop 2000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), respectively. Single-strand cDNA was synthesized using a PrimeScriptTM RT Master Mix kit (Takara, Dalian, China). Real-time quantitative PCR was performed using an Applied Biosystems 7500 Real-Time PCR System (Life Technologies, Foster, CA, USA) using SYBR-Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA). The amplification started with initial denaturation step at 95 °C for 30 s followed by 40 cycles at 95 °C for 5 s and an annealing step at 60 °C for 34 s, at which point fluorescence was acquired. Finally, a dissociation curve to test PCR specificity was generated by one cycle at 95 °C for 15 s followed by 60 °C for 1 min and ramped to 95 °C with acquired fluorescence. Specific primers (Table 1) for CuZnSOD, MT, HSP70, HSP90, β-actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed based on sequences retrieved from the National Center for Biotechnology Information (NCBI) database. Gene expression levels were normalized to those of β-actin and GAPDH to attain relative expression levels using the 2−ΔΔCt method.

Table 1.

Primers used for the target and reference genes.

Genes Forward Reverse
CuZnSOD 5′-GGAGGAGTGGCAGAAGT-3′ 5′-TAAACGAGGTCCAGCAT-3′
MT 5′-AAGGGCTGTGTCTGCAAGGA-3′ 5′-CTTCATCGGTATGGAAGGTACAAA-3′
HSP70 5′-CGTCAGTGCTGTGGACAAGAGTA-3′ 5′-CCTATCTCTGTTGGCTTCATCCT-3′
HSP90 5′-GAGTTTGACTGACCCGAGCA-3′ 5′-TCCCTATGCCGGTATCCACA-3′
β-actin 5′-ACCTGAGCGCAAGTACTCTGTCT-3′ 5′-CATCGTACTCCTGCTTGCTGAT-3′
GAPDH 5′-CTTTGGCATTGTGGAGGGTC-3′ 5′-ACGCTGGGATGATGTTCTGG-3′
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CuZnSOD = copper zinc superoxide dismutase; MT = metallothionein; HSP70 = heat-shock protein 70; HSP90 = heat-shock protein 90; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

2.6. Western blotting

Proteins of hepatocytes were extracted using lysis buffer and the concentration determination was performed by using a bicinchninic acid (BCA) Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). Total proteins (30 μg) were separated on a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to a polyvinylidene difluoride membrane (Merck-Millipore, Bedford, MA, USA). After blocked in 5% bovine serum albumin (BSA) blocking solution for 1 h at room temperature, the membrane was incubated overnight at 4 °C with primary antibodies against HSP70 (Abcam, diluted 1:5,000), HSP90 (Abcam, diluted 1:5,000) and GAPDH (Abcam, diluted 1:5,000). Subsequently, the membrane was developed with a horseradish peroxides conjugated goat anti-rabbit secondary antibody (Abcam; diluted 1:10,000). Protein bands were visualized using enhanced chemiluminescence substrate (Pierce Biotechnology, Rockford, IL, USA) and quantified with an ImageQuant LAS4000 scanner (GE Healthcare Life Sciences, Buckinghamshire, UK), and analyzed with TotalLab Quant Software (TotalLab, Newcastle, UK). The GAPDH protein was used to normalize the expression levels of target proteins.

2.7. Statistical analyses

Data from the Exp. 1 or 2 were subjected to two-way ANOVA using the general linear model (GLM) procedure of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). Data from the Exp. 3 were subjected to three-way ANOVA using GLM procedure of SAS 9.2 (SAS Institute Inc., Cary, NC, USA). The replicate served as the experimental unit. Differences among means were tested by the least significant difference (LSD) method, and the statistical significance was set at P ≤ 0.05.

3. Results

3.1. The optimal dose and effective incubation time for strengthening the antioxidant ability of primary cultured hepatocytes of broiler embryos (Exp. 1)

There was an interaction (P = 0.0002) between incubation time and added Zn level in CuZnSOD activity (Table 2). At 12 h incubation, 50 μmol/L added Zn increased (P < 0.03) CuZnSOD activity compared with the control and all other added Zn levels, whereas 100 and 200 μmol/L added Zn decreased (P < 0.018) it compared with the control, and no differences (P > 0.14) were observed among 100, 200 and 400 μmol/L added Zn and between 0 and 400 μmol/L added Zn. At 24 h incubation, the CuZnSOD activity was higher (P < 0.015) at 50 μmol/L added Zn than at 200 and 400 μmol/L added Zn, and there were no differences (P > 0.17) among 0, 50 and 100 μmol/L added Zn and among 0, 100, 200 and 400 μmol/L added Zn. At 48 h incubation, there were no differences (P > 0.15) in CuZnSOD activity among all Zn levels. Incubation time affected (P < 0.02) CuZnSOD mRNA expression in hepatocytes (Table 2). Cells at either 12 or 24 h incubation had higher (P < 0.017) CuZnSOD mRNA expression than those at 48 h with no difference (P > 0.57) between those at 12 and 24 h. Added Zn level affected (P < 0.0001) MT mRNA expression (Table 2). Compared with the control, the addition of Zn elevated (P < 0.0001) MT mRNA expressions, but no differences (P > 0.18) were observed among 50, 100, 200 and 400 μmol/L of added Zn. Based on the above combined results, 50 μmol/L of added Zn induced the optimal responses of CuZnSOD and MT expressions at 12 h incubation.

Table 2.

Effects of incubation time and added Zn level on the CuZnSOD activity, and mRNA expressions of CuZnSOD and MT in primary cultured hepatocytes of broiler embryos (Exp. 1).

Item Added Zn level, μmol/L CuZnSOD activity, U/mg prot. CuZnSOD mRNA1, RQ MT mRNA1, RQ
Incubation time 2
 12 h 0 53.8 ± 1.50b 1.30 ± 0.11 0.280 ± 0.048
50 59.3 ± 2.16a 0.958 ± 0.097 1.85 ± 0.38
100 48.0 ± 1.65c 0.662 ± 0.045 1.72 ± 0.22
200 45.5 ± 1.13c 1.37 ± 0.39 2.00 ± 0.30
400 51.5 ± 1.40 bc 1.65 ± 0.15 2.88 ± 0.56
 24 h 0 52.3 ± 2.26 bc 1.29 ± 0.63 0.126 ± 0.028
50 53.7 ± 1.10b 1.80 ± 0.56 1.96 ± 0.77
100 50.5 ± 2.17 bc 1.33 ± 0.46 1.55 ± 0.49
200 43.5 ± 2.06c 1.49 ± 0.47 1.42 ± 0.18
400 45.9 ± 1.95c 0.622 ± 0.215 2.84 ± 1.22
 48 h 0 46.0 ± 1.40c 0.682 ± 0.094 0.077 ± 0.021
50 45.0 ± 1.84c 0.773 ± 0.151 0.700 ± 0.154
100 43.4 ± 0.92c 0.643 ± 0.064 1.06 ± 0.12
200 47.2 ± 1.27c 0.770 ± 0.161 2.17 ± 0.25
400 47.2 ± 1.58c 0.757 ± 0.197 1.41 ± 0.38
Incubation time 3, h
 12 51.6 ± 1.11 1.19 ± 0.11a 1.75 ± 0.21
 24 49.2 ± 1.12 1.31 ± 0.21a 1.63 ± 0.36
 48 45.8 ± 0.65 0.725 ± 0.060b 1.12 ± 0.16
Added Zn level 4, μmol/L
 0 50.7 ± 1.26 1.10 ± 0.22 0.167 ± 0.031b
 50 52.7 ± 1.72 1.18 ± 0.21 1.48 ± 0.32a
 100 47.4 ± 1.12 0.878 ± 0.166 1.44 ± 0.19a
 200 45.4 ± 0.91 1.24 ± 0.21 1.84 ± 0.18a
 400 48.2 ± 1.14 1.01 ± 0.15 2.34 ± 0.48a
P-value
 Incubation time <0.0001 0.016 0.121
 Added Zn level <0.0001 0.712 <0.0001
 Interaction 0.0002 0.216 0.511
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CuZnSOD = copper zinc superoxide dismutase; MT = metallothionein.

a, b, c Mean values within a column with unlike superscript letters are significantly different (P < 0.05).

1

The CuZnSOD or MT mRNA abundances were calculated as the relative quantity (RQ) of the CuZnSOD or MT mRNA to the geometric mean of β-actin and GAPDH mRNA; RQ = 2-ΔΔCt (Ct = threshold cycle).

2

Each value represents the mean ± SE of 6 replicates (n = 6).

3

Each value represents the mean ± SE of 30 replicates (n = 30).

4

Each value represents the mean ± SE of 18 replicates (n = 18).

3.2. The optimal heat exposure time for establishing heat stress model with primary cultured hepatocytes of broiler embryos (Exp. 2)

An interaction between culture temperature and incubation time affected (P < 0.002) LDH activity, HSP70 and HSP90 mRNA expressions (Table 3). Compared with NT, HT had no effect (P > 0.05) on LDH activity when cells were treated for 1 and 2 h, but increased (P < 0.0001) LDH activity at 4, 6 or 8 h of incubation. Under NT, incubation time had no effect (P > 0.95) on HSP70 and HSP90 mRNA expressions. However, under HT, heat exposure time increased (P < 0.034) cellular HSP70 mRNA expression within 4 h with a peak at 4 h followed by a gradual decline, and it also increased (P < 0.023) cellular HSP90 mRNA expression within 6 h with a peak at 6 h followed by a significant (P < 0.0008) decline. Overall, on the base of the consideration of continued viability of cells, the optimal heat exposure time for responses of HSP70 and HSP90 mRNA to HT was 4 or 6 h.

Table 3.

Effects of culture temperature (CT) and incubation time (IT) on LDH activity in the culture medium and mRNA expressions of HSP70 and HSP90 in primary cultured hepatocytes of broiler embryos (Exp. 2).

Item IT, h LDH activity, U/L HSP70 mRNA1, RQ HSP90 mRNA1, RQ
CT 2
 NT 1 471 ± 13.3 cd 0.118 ± 0.016c 0.208 ± 0.040c
2 429 ± 12.8d 0.115 ± 0.015c 0.208 ± 0.070c
4 455 ± 33.2 cd 0.138 ± 0.012c 0.325 ± 0.033c
6 460 ± 18.5 cd 0.155 ± 0.018c 0.193 ± 0.060c
8 503 ± 24.6c 0.150 ± 0.019c 0.277 ± 0.056c
 HT 1 424 ± 4.49d 3.12 ± 0.19c 2.04 ± 0.39c
2 426 ± 16.8d 11.8 ± 3.00b 7.65 ± 2.64b
4 635 ± 15.6b 28.3 ± 6.76a 7.12 ± 1.05b
6 663 ± 14.4b 27.2 ± 4.00a 14.2 ± 1.88a
8 755 ± 15.7a 22.6 ± 4.08a 7.29 ± 2.55b
CT 3
 NT 464 ± 9.71 0.135 ± 0.007 0.242 ± 0.024
 HT 581 ± 25.4 18.6 ± 2.46 7.66 ± 1.10
IT 4, h
 1 448 ± 9.25 1.62 ± 0.46 1.13 ± 0.33
 2 427 ± 10.1 5.97 ± 2.27 3.93 ± 1.69
 4 545 ± 31.3 14.2 ± 5.21 3.72 ± 1.10
 6 561 ± 32.6 13.7 ± 4.50 7.19 ± 2.29
 8 629 ± 40.5 11.3 ± 3.90 3.78 ± 1.61
P-value
 CT <0.0001 <0.0001 <0.0001
 IT <0.0001 0.0002 0.0019
 CT × IT <0.0001 0.0001 0.0018
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LDH = lactate dehydrogenase; HSP70 = heat-shock protein 70; HSP90 = heat-shock protein 90; NT = normal temperature, 40 °C; HT = high temperature, 44 °C.

a, b, c, d Mean values within a column with unlike superscript letters are significantly different (P < 0.05).

1

The HSP70 or HSP90 mRNA abundances were calculated as the relative quantity (RQ) of the HSP70 or HSP90 mRNA to the geometric mean of β-actin and GAPDH mRNA; RQ = 2-ΔΔCt. (Ct = threshold cycle).

2

Each value represents the mean ± SE of 6 replicates (n = 6).

3

Each value represents the mean ± SE of 30 replicates (n = 30).

4

Each value represents the mean ± SE of 12 replicates (n = 12).

3.3. Effects of Zn source, incubation time and culture temperature on the antioxidant status and HSP expressions of primary cultured hepatocytes of broiler embryos (Exp. 3)

The MDA content and CuZnSOD mRNA expression were affected (P < 0.01) by culture temperature. Compared with NT, HT increased (P < 0.01) MDA content and CuZnSOD mRNA expression. For CuZnSOD activity, an interaction (P < 0.04) between culture temperature and Zn source was observed (Table 4). At NT, iZn increased (P < 0.005) CuZnSOD activity compared with the CON, with no differences (P > 0.09) between the 2 Zn sources and between the CON and oZn; however, at HT, oZn increased (P < 0.05) CuZnSOD activity compared with the CON and iZn, with no difference (P > 0.47) between the CON and iZn (Table 5). As for the MT mRNA expression, a three-way interaction (P < 0.01) among incubation time, culture temperature and Zn source were observed (Table 4). At NT and for either 4 or 6 h of incubation time, iZn increased (P < 0.0004) MT mRNA expression compared with the CON and oZn, with no difference (P > 0.42) between the CON and oZn. The similar results were observed in hepatocytes subjected to HT for 6 h. However, when hepatocytes were exposed to HT for 4 h, either iZn or oZn increased (P < 0.004) MT mRNA expression compared with the CON, with no difference (P > 0.93) between the 2 Zn sources. Overall, HT caused oxidative damage, whereas the addition of Zn as either source might enhance hepatic antioxidant ability of chick hepatocytes.

Table 4.

Effects of zinc source, incubation time (IT) and culture temperature (CT) on the antioxidant status of primary cultured hepatocytes of broiler embryos (Exp. 3).

Item CT Zn source MDA content, nmol/mg prot. MT content, pg/mg prot. CuZnSOD activity, U/mg prot. CuZnSOD mRNA1, RQ MT mRNA1, RQ
IT 2
 4 h NT CON 0.580 ± 0.040 98.2 ± 6.96 59.7 ± 1.63 0.857 ± 0.164 0.651 ± 0.161d
iZn 0.627 ± 0.051 109 ± 5.78 69.5 ± 1.96 0.840 ± 0.055 2.01 ± 0.35b
oZn 0.567 ± 0.055 102 ± 4.74 63.0 ± 3.11 1.06 ± 0.15 0.681 ± 0.046d
HT CON 0.713 ± 0.057 96.4 ± 6.18 57.9 ± 2.92 1.26 ± 0.17 0.521 ± 0.083d
iZn 0.675 ± 0.045 89.4 ± 4.94 62.5 ± 1.12 0.956 ± 0.071 1.65 ± 0.26 bc
oZn 0.818 ± 0.106 102 ± 4.58 70.2 ± 4.25 1.27 ± 0.15 1.68 ± 0.35 bc
 6 h NT CON 0.827 ± 0.049 92.5 ± 7.85 57.8 ± 2.87 0.756 ± 0.083 0.497 ± 0.068d
iZn 0.636 ± 0.029 96.7 ± 7.64 63.3 ± 1.60 0.850 ± 0.122 1.75 ± 0.21b
oZn 0.570 ± 0.040 104 ± 10.7 60.9 ± 2.60 0.850 ± 0.103 0.785 ± 0.060d
HT CON 0.850 ± 0.099 93.3 ± 5.23 60.8 ± 2.83 1.23 ± 0.21 0.624 ± 0.075d
iZn 0.813 ± 0.080 96.4 ± 3.80 60.0 ± 2.42 1.36 ± 0.09 3.06 ± 0.60a
oZn 0.855 ± 0.012 100 ± 5.33 62.8 ± 2.52 1.48 ± 0.27 1.00 ± 0.08 cd
IT 3
 4 h 0.663 ± 0.028 99.5 ± 2.35 63.8 ± 1.28 1.04 ± 0.06 1.20 ± 0.14
 6 h 0.759 ± 0.031 97.2 ± 2.72 60.9 ± 1.00 1.09 ± 0.08 1.29 ± 0.18
CT 3
 NT 0.634 ± 0.017b 100 ± 2.97 62.4 ± 1.09 0.868 ± 0.047b 1.06 ± 0.12
 HT 0.788 ± 0.030a 96.3 ± 2.04 62.4 ± 1.25 1.26 ± 0.07a 1.42 ± 0.19
Zn source 4
 CON 0.743 ± 0.038 95.1 ± 3.07 59.1 ± 1.25 1.02 ± 0.09 0.575 ± 0.051
 iZn 0.688 ± 0.030 97.9 ± 3.05 63.8 ± 1.13 1.00 ± 0.06 2.12 ± 0.21
 oZn 0.703 ± 0.041 102 ± 3.19 64.2 ± 1.66 1.16 ± 0.10 1.04 ± 0.12
P-value
 IT 0.069 0.571 0.061 0.573 0.565
 CT 0.006 0.250 0.998 <0.0001 0.018
 Zn source 0.695 0.320 0.012 0.256 <0.0001
 IT × CT 0.879 0.434 0.733 0.089 0.207
 CT × Zn source 0.299 0.510 0.037 0.813 0.221
 IT × CT × Zn source 0.619 0.695 0.323 0.611 0.004
Open in a new tab

MDA = malondialdehyde; MT = metallothionein; CuZnSOD = copper zinc superoxide dismutase; NT = normal temperature, 40 °C; HT = high temperature, 44 °C; CON = no Zn addition; iZn = inorganic Zn sulphate; oZn = organic Zn lysine chelate.

a, b, c, d Mean values within a column with unlike superscript letters are significantly different (P < 0.05).

1

The CuZnSOD or MT mRNA abundances were calculated as the relative quantity (RQ) of the CuZnSOD or MT mRNA to the geometric mean of β-actin and GAPDH mRNA; RQ = 2-ΔΔCt (Ct = threshold cycle).

2

Each value represents the mean ± SE of 6 replicates (n = 6).

3

Each value represents the mean ± SE of 36 replicates (n = 36).

4

Each value represents the mean ± SE of 24 replicates (n = 24).

Table 5.

Effects of culture temperature (CT) and zinc source on CuZnSOD activity of primary cultured hepatocytes of broiler embryos (Exp. 3).1

Item NT
 HT
 P-value
CON iZn oZn CON iZn oZn
CuZnSOD, U/mg prot. 58.8 ± 1.59c 66.4 ± 1.52 ab 62.0 ± 1.96 abc 59.4 ± 1.98c 61.2 ± 1.33 bc 66.5 ± 2.61a 0.037
Open in a new tab

NT = normal temperature, 40 °C; HT = high temperature, 44 °C; CON = no Zn addition; iZn = inorganic Zn sulphate; oZn = organic Zn lysine chelate; CuZnSOD = copper zinc superoxide dismutase.

a, b, c, Mean values within a row with unlike superscript letters are significantly different (P < 0.05).

1

Each value represents the mean ± SE of 12 replicates (n = 12).

The incubation time × culture temperature interaction (P = 0.006) and the culture temperature × Zn source interaction (P = 0.029) were detected for HSP70 mRNA expression (Table 6). Compared with NT, HT greatly increased (P < 0.0001) HSP70 mRNA expression (Table 7, Table 8). Under NT, there were no differences (P > 0.07) between 4 and 6 h of incubation time and among the 3 Zn source treatments, however, at HT, HSP70 mRNA expression decreased (P < 0.03) over time (Table 7), and iZn reduced (P < 0.05) HSP70 mRNA expression compared with the CON and oZn, with no difference (P > 0.85) between the CON and oZn (Table 8). A three-way interaction among incubation time, culture temperature and Zn source affected (P < 0.04) the expressions of HSP70 protein and HSP90 mRNA (Table 6). Compared with NT, for either 4 or 6 h of incubation time, HT largely increased (P < 0.0001) the expression levels of HSP70 protein (also as shown in Fig. 1) and HSP90 mRNA. At NT, for either 4 or 6 h of incubation time, no differences (P > 0.70) in HSP70 protein expression were observed among the 3 Zn source treatments; however, at HT, either iZn or oZn decreased (P < 0.0001) HSP70 protein expression at 6 h of heat exposure, with no differences (P > 0.33) between the 2 Zn sources at 6 h and among the 3 Zn source treatments at 4 h. At NT, for either 4 or 6 h of incubation time, oZn decreased (P < 0.005) HSP90 mRNA expression compared with the CON and iZn, with no difference (P > 0.15) between the CON and iZn; however, at HT, iZn decreased (P < 0.0003) HSP90 mRNA expression compared with the CON and oZn at 4 h of heat exposure, with no differences (P > 0.58) between the CON and oZn at 4 h and among the 3 Zn source treatments at 6 h. Overall, HT largely induced heat shock responses, whereas the addition of Zn as either source might attenuate the heat shock responses of chick hepatocytes.

Table 6.

Effects of zinc source, incubation time (IT) and culture temperature (CT) on the expressions of heat stress proteins in primary cultured hepatocytes of broiler embryos (Exp. 3).

Item CT Zn source HSP70 mRNA1, RQ HSP90 mRNA1, RQ HSP70 protein2, AU HSP90 protein2, AU
IT3
 4 h NT CON 0.044 ± 0.012 0.255 ± 0.051c 0.188 ± 0.028c 0.977 ± 0.174
iZn 0.069 ± 0.021 0.169 ± 0.020c 0.192 ± 0.015c 1.06 ± 0.19
oZn 0.049 ± 0.008 0.117 ± 0.014d 0.155 ± 0.020c 1.11 ± 0.21
HT CON 27.3 ± 4.91 10.5 ± 1.78a 1.22 ± 0.18b 1.03 ± 0.18
iZn 14.9 ± 1.84 4.22 ± 0.93b 1.21 ± 0.15b 1.20 ± 0.12
oZn 26.8 ± 4.77 9.77 ± 1.44a 1.19 ± 0.21b 1.23 ± 0.14
 6 h NT CON 0.083 ± 0.005 0.266 ± 0.043c 0.168 ± 0.019c 0.992 ± 0.094
iZn 0.069 ± 0.010 0.203 ± 0.039c 0.245 ± 0.063c 1.23 ± 0.15
oZn 0.045 ± 0.005 0.076 ± 0.006d 0.162 ± 0.029c 0.855 ± 0.114
HT CON 16.7 ± 2.95 6.96 ± 1.14a 2.23 ± 0.40a 1.31 ± 0.22
iZn 14.1 ± 3.31 5.86 ± 0.86a 1.24 ± 0.19b 1.13 ± 0.08
oZn 16.8 ± 2.32 5.89 ± 0.62a 1.03 ± 0.05b 1.02 ± 0.15
IT 4
 4 h 11.5 ± 2.33 4.17 ± 0.84 0.691 ± 0.099 1.10 ± 0.07
 6 h 8.04 ± 1.56 3.21 ± 0.57 0.847 ± 0.144 1.09 ± 0.05
CT4
 NT 0.123 ± 0.005 0.181 ± 0.017 0.185 ± 0.014 1.04 ± 0.06
 HT 19.4 ± 1.62 7.20 ± 0.59 1.35 ± 0.11 1.15 ± 0.06
Zn source 5
 CON 11.1 ± 2.78 4.49 ± 1.04 0.951 ± 0.204 1.08 ± 0.08
 iZn 7.29 ± 1.75 2.61 ± 0.60 0.722 ± 0.120 1.15 ± 0.07
 oZn 10.9 ± 2.68 3.96 ± 0.93 0.634 ± 0.110 1.05 ± 0.08
P-value
 IT 0.743 0.248 0.064 0.846
 CT <0.0001 <0.0001 <0.0001 0.194
 Zn source 0.512 0.0001 0.016 0.671
 IT × CT 0.006 0.733 0.111 0.848
 CT × Zn source 0.029 <0.0001 0.013 0.771
 IT × CT × Zn source 0.241 0.033 0.006 0.372
Open in a new tab

HSP70 = heat-shock protein 70; HSP90 = heat-shock protein 90; NT = normal temperature, 40 °C; HT = high temperature, 44 °C; CON = no Zn addition; iZn = inorganic Zn sulphate; oZn = organic Zn lysine chelate.

a, b, c, d Mean values within a column with unlike superscript letters are significantly different (P < 0.05).

1

The HSP70 or HSP90 mRNA abundances were calculated as the relative quantity (RQ) of the HSP70 or HSP90 mRNA to the geometric mean of β-actin and GAPDH mRNA; RQ = 2-ΔΔCt (Ct = threshold cycle).

2

The GAPDH was used to normalize the expression level of the HSP70 or HSP90 as an arbitrary unit (AU).

3

Each value represents the mean ± SE of 6 replicates (n = 6).

4

Each value represents the mean ± SE of 36 replicates (n = 36).

5

Each value represents the mean ± SE of 24 replicates (n = 24).

Table 7.

Effects of culture temperature (CT) and incubation time (IT) on HSP70 mRNA expression of primary cultured hepatocytes of broiler embryos (Exp. 3).1

Item NT
 HT
 P-value
4 h 6 h 4 h 6 h
HSP70 mRNA2, RQ 0.054 ± 0.008c 0.066 ± 0.005c 23.0 ± 2.47a 15.9 ± 1.47b 0.006
Open in a new tab

NT = normal temperature, 40 °C; HT = high temperature, 44 °C; HSP70 = heat-shock protein 70.

a, b, c, Mean values within a row with unlike superscript letters are significantly different (P < 0.05).

1

Each value represents the mean ± SE of 18 replicates (n = 18).

2

The HSP70 mRNA abundances were calculated as the relative quantity (RQ) of the HSP70 mRNA to the geometric mean of β-actin and GAPDH mRNA; RQ = 2-ΔΔCt (Ct = threshold cycle).

Table 8.

Effects of culture temperature (CT) and zinc source on HSP70 mRNA expression of primary cultured hepatocytes of broiler embryos (Exp. 3).1

Item NT
 HT
 P-value
CON iZn oZn CON iZn oZn
HSP70 mRNA2, RQ 0.062 ± 0.009c 0.069 ± 0.011c 0.047 ± 0.004c 22.0 ± 3.16a 14.5 ± 1.81b 21.8 ± 2.94a 0.029
Open in a new tab

NT = normal temperature, 40 °C; HT = high temperature, 44 °C; CON = no Zn addition; iZn = inorganic Zn sulphate; oZn = organic Zn lysine chelate; HSP70 = heat-shock protein 70.

a, b, c, Mean values within a row with unlike superscript letters are significantly different (P < 0.05).

1

Each value represents the mean ± SE of 12 replicates (n = 12).

2

The HSP70 mRNA abundances were calculated as the relative quantity (RQ) of the HSP70 mRNA to the geometric mean of β-actin and GAPDH mRNA; RQ = 2-ΔΔCt (Ct = threshold cycle).

Fig. 1.

Fig. 1

Open in a new tab

Representative immunoblots demonstrating heat-shock protein 70 (HSP70) and heat-shock protein 90 (HSP90) of primary cultured hepatocytes of broiler embryos subjected to varying incubation time (IT), culture temperature (CT) and Zn source (Exp. 3). NT = normal temperature, 40 °C; HT = high temperature, 44 °C; CON = no Zn addition; iZn = inorganic Zn sulphate; oZn = organic Zn lysine chelate; GAPDH = glyceraldehyde-3-phosphate dehydrogenase.

4. Discussion

Chickens are highly susceptible to heat stress and have a narrow thermal comfort zone. Under the hot environment, the birds are prone to heat stress which would reduce growth performance and meat quality. In broilers, we have demonstrated that dietary supplementation of Zn could alleviate the adverse effects of heat stress (Zhu et al., 2017a, 2017b). However, the exact mechanisms of the protective effects of Zn on broilers are unclear. Our hypothesis that Zn, especially the organic Zn with a moderate chelation strength, might protect primary cultured hepatocytes of broiler embryos against heat stress via enhancing the antioxidant ability and attenuating the heat shock responses has been supported by the results in the present study. The present study has demonstrated that either iZn or oZn supplementation could alleviate the adverse effects of heat stress on primary cultured hepatocytes of broiler embryos, and further indicated that Zn might exert its anti-heat stress effects via enhancing the antioxidant defense system and attenuating the heat shock responses of the above primary cultured hepatocytes. Furthermore, oZn appeared to be more effective than iZn in improving the antioxidant status of hepatocytes under high temperature. The aforementioned findings might provide a more effective nutritional strategy for protecting broilers from heat stress.

The liver is not only a key organ in chickens’ response to heat stress (Lin et al., 2006; Mackei et al., 2020), but also a sensitive tissue to reflect the Zn status of broilers (Azad et al., 2017). And also, evidence suggests that liver would be the most sensitive organ synthesizing HSP in response to hyperthermia (Xie et al., 2014) and also expressing high antioxidant enzyme activities (Azad et al., 2017) in broilers. Therefore, the primary cultured broiler hepatocytes may be a good stress model, which could be used to evaluate the protective effects and elucidate the mechanisms of Zn against heat stress on the liver tissue. The CuZnSOD and MT are important endogenous antioxidants that scavenge ROS, and their expression levels reflect the antioxidant ability of cells (Alscher et al., 2005; Volkova et al., 2012). In Exp. 1, we found that the optimal responses of CuZnSOD and MT occurred when hepatocytes were exposed to Zn (ZnSO4; 50 μmol/L) for 12 h, suggesting that a Zn concentration of 50 μmol/L and an incubation time of 12 h could be the optimal condition necessary to strengthen the antioxidant defense system of hepatocytes. Both HSP70 and HSP90 are useful biomarkers for assessing cellular potential heat damages (Tedeschi et al., 2015). The results from Exp. 2 showed that HT affected HSP70 and HSP90 mRNA expressions of hepatocytes, and the heat exposure time of either 4 or 6 h with HT treatment was effective in increasing the mRNA expressions of the 2 genes. Taken together, primary broiler hepatocytes could be an effective model for further exploring the protective mechanisms of Zn against heat stress.

Heat stress is believed to promote the generation of ROS thereby causing oxidative stress in broilers (Lin et al., 2006; Tan et al., 2010; Xie et al., 2015) and laying hens (Lin et al., 2008; Naziroglu et al., 2000). The increased level of lipid peroxide (marked by MDA) in heat-stressed hepatocytes observed in the present study indicated the involvement of free radicals and confirmed a state of oxidative stress (Habashy et al., 2019). However, at the same time, heat stress led to up-regulation of genes that encode for CuZnSOD and MT in heat-stressed hepatocytes. According to previous studies, the up-regulation of CuZnSOD and MT is the mechanism utilized by cells to manage potential cytotoxicity induced by heat stress (Habashy et al., 2018; Wang et al., 2016). Therefore, the damage to the hepatocellular membrane resulting from free radicals may have induced the corresponding increase in its antioxidant response owing to the cellular self-protective function during heat stress. In addition to antioxidant genes, rapidly synthesized HSP are another endogenous mechanism by which living cells adapt to heat stress (Garbuz et al., 2017). Evidence suggests that HSP, especially HSP70, might be involved in the development of thermotolerance in broiler chickens (Vinoth et al., 2018; Yahav et al., 1997). Overexpression of HSP70 obviously improved the antioxidant defense system of broilers during heat stress (Gu et al., 2012). In the present study, abrupt rises in HSP70 mRNA and protein expression levels, as well as HSP90 mRNA expression in hepatocytes exposed to heat stress, might favour an anti-heat stress response. However, this up-regulated response might depend on the presence of ROS and/or oxidant stress induced by heat stress (Slimen et al., 2014). Overall, the above results suggest that heat stress might interrupt the redox balance and HSP homoeostasis, thereby causing adverse effects on broilers and primary cultured hepatocytes of broiler embryos.

Zinc is an important nutritional antioxidant trace element that can help to eliminate free radicals via Zn-containing enzymes and proteins. Numerous investigators have noted that Zn is a crucial component of CuZnSOD, and the primary enzyme in cells that plays a fundamental role in the detoxification of superoxide free radicals and in the protection of cells against oxidative stress (Yuan et al., 2011). Further evidence shows that the activity of CuZnSOD can be regulated by the availability of its component trace elements, including Zn and Cu ions (Kopeć et al., 2013). The present study showed that the addition of Zn as either iZn or oZn significantly increased CuZnSOD activity of hepatocytes. Our results are consistent with Azad et al. (2017) and Liu et al. (2015), who reported that organic or inorganic Zn addition in the feed elevated CuZnSOD activity in the liver of broilers. Moreover, our results indicated that oZn was more effective than iZn in improving antioxidant status of heat-stressed hepatocytes with elevated CuZnSOD activity, which might be attributed to the fact that the bioavailability of moderately chelated organic Zn is higher than that of inorganic ZnSO4 for broilers (Huang et al., 2008; Huang et al., 2013; Yu et al., 2010, 2017). Consistent with this explanation, Zhang et al. (2017) reported that organic Zn glycinate had a better effect on hepatic antioxidant status than inorganic ZnSO4 in broiler breeders, due to the increased Zn retention in the liver resulting from a higher bioavailability of Zn glycinate. Additionally, Zn is an effective inducer of MT, a cysteine-rich protein acting as a free radical scavenger (Alscher et al., 2005). In vivo, previous researches have indicated that dietary supplementation with Zn could increase MT mRNA expression in the liver of broilers (Azad et al., 2017; Zhang et al., 2017; Zhu et al., 2017b). In vitro, it has been reported that the induction of MT conferred protection against ROS in liver (Garla et al., 2017), kidney (Sharma et al., 2002) and islet (Li et al., 2004) cells. In the present study, either iZn or oZn increased MT mRNA expression in heat-stressed hepatocytes, suggesting that Zn-treated hepatocytes might be better prepared to deal with oxidative stress induced by heat stress.

Heat shock, as a promoter of oxidative stress, creates a redox imbalance by increasing the generation of ROS (Yang et al., 2010). Subsequent cellular damage caused by the accumulation of ROS has been considered as a key factor for the activation of HSP genes (Mahmoud et al., 2003; Sahin et al., 2003). When cells are subjected to heat shock with an increase in lipid peroxidation, HSP accumulate and might serve as a type of tissue biomarkers for potential stress damage (Tedeschi et al., 2015). Thus, HSP expression levels might be regarded as a kind of responses to damage resulting from a strong stress to the organism (Burdon et al., 1987). In addition, the increased activities of SOD and catalase likely scavenge free radicals that inhibit the expression of HSP proteins and improve cell survival (Omar et al., 1993). In the present study, the addition of Zn as either iZn or oZn could attenuate heat shock responses of hepatocytes exposed to heat stress, which was evidenced by the decrease in the expressions of HSP70 mRNA and protein, as well as HSP90 mRNA. The possible reasons may be that Zn enhanced CuZnSOD activity and MT expression, thereby lowering ROS levels and weakening stress-induced heat shock responses. Similarly, it has been reported that the reduced induction of HSP70 protein occurred in selenium -fed chickens because of the higher glutathione peroxidase activity in the liver (Mahmoud et al., 2003). Increased activity of manganese superoxide dismutase in manganese-fed broilers was also accompanied by a decline of HSP70 expression under high temperatures (Zhu et al., 2015a).

5. Conclusions

This study revealed that heat stress-induced oxidative damage evidenced by the elevated MDA content and the up-regulated expressions of HSP70 mRNA and protein, as well as HSP90 mRNA. Zinc supplementation enhanced antioxidant ability with increased CuZnSOD activity and up-regulated MT mRNA expression against oxidative damage, and effectively attenuated the heat shock responses with down-regulated expressions of HSP70 mRNA and protein, as well as HSP90 mRNA. Furthermore, oZn appeared to be more effective than iZn in improving the antioxidant status of hepatocytes at high temperatures. It is suggested that Zn, especially oZn, could alleviate the heat stress of primary cultured hepatocytes of broiler embryos via enhancing the antioxidant ability and attenuating the heat shock responses of above primary hepatocytes. Considering that the in vitro cellular model cannot completely mimic the in vivo environment, additional in vivo studies are necessary to verify the protective effect of supplemental Zn on the liver of broilers at high temperatures.

Author contributions

T. T. Li: data curation, writing - original draft preparation. W. G. He: investigation. X. D. Liao: formal analysis. X. Lin: conceptualization. L. Y. Zhang: investigation. L. Lu: methodology. Y. L. Guo: methodology. Z. P. Liu: resources. X. G. Luo: supervision, writing - review & editing.

Conflict of interest

We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, and there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the content of this paper.

Acknowledgements

The present study was supported by the Key International Cooperation Program of the National Natural Science Foundation of China (project no. 31110103916; Beijing, China), the National Natural Science Foundation of China (project no. 31972583; Beijing, China), the China Agricultural Research System (project no. CARS-41; Beijing, China), and the Agricultural Science and Technology Innovation Program (ASTIP-IAS09; Beijing, China).

Footnotes

Peer review under responsibility of Chinese Association of Animal Science and Veterinary Medicine.

Supplementary data to this article can be found online at https://doi.org/10.1016/j.aninu.2021.01.003.

Appendix

The following is the Supplementary data to this article:

Multimedia component 1
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References

  1. Alscher D.M., Braun N., Biegger D., Stuelten C., Gawronski K., Mürdter T.E. Induction of metallothionein in proximal tubular cells by zinc and its potential as an endogenous antioxidant. Kidney Blood Press Res. 2005;28(3):127–133. doi: 10.1159/000084921. [DOI] [PubMed] [Google Scholar]
  2. Azad S.K., Shariatmadari F., Karimi Torshizi M.A., Ahmadi H. Effect of zinc concentration and source on performance, tissue mineral status, activity of superoxide dismutase enzyme and lipid peroxidation of meat in broiler chickens. Anim Prod Sci. 2017 doi: 10.1071/an15758. [DOI] [Google Scholar]
  3. Burdon R.H., Gill V.M., Rice-Evans C. Oxidative stress and heat shock protein induction in human cells. Free Radic Res Commun. 1987;3(1–5):129–139. doi: 10.3109/10715768709069778. [DOI] [PubMed] [Google Scholar]
  4. Cao Y., Li Y.S., Li Z.J., Wang F., Li C.M. Dietary zinc may attenuate heat-induced testicular oxidative stress in mice via up-regulation of CuZnSOD. Genet Mol Res. 2015;14(4):16616–16626. doi: 10.4238/2015.December.11.9. [DOI] [PubMed] [Google Scholar]
  5. Chand N., Naz S., Khan A., Khan S., Khan R.U. Performance traits and immune response of broiler chicks treated with zinc and ascorbic acid supplementation during cyclic heat stress. Int J Biometeorol. 2014;58(10):2153–2157. doi: 10.1007/s00484-014-0815-7. [DOI] [PubMed] [Google Scholar]
  6. Evdonin A.L., Medvedeva N.D. The extracellular heat shock protein 70 and its functions. Tsitologiia. 2009;51(2):130–137. [PubMed] [Google Scholar]
  7. Garbuz D.G. Regulation of heat shock gene expression in response to stress. Mol Biol (Mosk) 2017;51(3):400–417. doi: 10.7868/S0026898417020100. [DOI] [PubMed] [Google Scholar]
  8. Garla R., Kango P., Gill N.K., Garg M.L. Induction of metallothionein in rat liver by zinc exposure: a dose and time dependent study. Protein J. 2017;36(5):433–442. doi: 10.1007/s10930-017-9737-7. [DOI] [PubMed] [Google Scholar]
  9. Gu X.H., Hao Y., Wang X.L. Overexpression of heat shock protein 70 and its relationship to intestine under acute heat stress in broilers: 2. Intestinal oxidative stress. Poultry Sci. 2012;91(4):790–799. doi: 10.3382/ps.2011-01628. [DOI] [PubMed] [Google Scholar]
  10. Habashy W.S., Milfort M.C., Rekaya R., Aggrey A.E. Cellular antioxidant enzyme activity and biomarkers for oxidative stress are affected by heat stress. Int J Biometeorol. 2019;63(12):1569–1584. doi: 10.1007/s00484-019-01769-z. [DOI] [PubMed] [Google Scholar]
  11. Habashy W.S., Milfort M.C., Rekaya R., Aggrey S.E. Expression of genes that encode cellular oxidant/antioxidant systems are affected by heat stress. Mol Biol Rep. 2018;45(3):389–394. doi: 10.1007/s11033-018-4173-0. [DOI] [PubMed] [Google Scholar]
  12. Han A.Y. Chinese Academy of Agricultural Sciences; 2010. Effects of heat stress on calcium signal transduction of lymphocytes from broilers and the regulation by chromium picolinate. [Doctor Degree Thesis Dissertation] [Google Scholar]
  13. Holwerda R.A., Albin R.C., Madsen F.C. Chelation effectiveness of zinc proteinates demonstrated. Feedstuffs. 1995;67(25):12–13. [Google Scholar]
  14. Huang Y.L., Lu L., Li S.F., Luo X.G., Liu B. Relative bioavailabilities of organic zinc sources with different chelation strengths for broilers fed a conventional corn-soybean meal diet. J Anim Sci. 2009;87(6):2038–2046. doi: 10.2527/jas.2008-1212. [DOI] [PubMed] [Google Scholar]
  15. Huang Y.L., Lu L., Xie J.J., Li S.F., Li X.L., Liu S.B. Relative bioavailabilities of organic zinc sources with different chelation strengths for broilers fed diets with low or high phytate content. Anim Feed Sci Technol. 2013;179:144–148. [Google Scholar]
  16. Jang I.S., Ko Y.H., Moon Y.S., Sohn S.H. Effects of vitamin C or E on the pro-inflammatory cytokines, heat shock protein 70 and antioxidant status in broiler chicks under summer conditions. Asian-Australas J Anim Sci. 2014;27(5):749–756. doi: 10.5713/ajas.2013.13852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kopeć W., Jamroz D., Wiliczkiewicz A., Biazik E., Pudlo A., Hikawczuk T. Influence of different histidine sources and zinc supplementation of broiler diets on dipeptide content and antioxidant status of blood and meat. Br Poultry Sci. 2013;54(4):454–465. doi: 10.1080/00071668.2013.793295. [DOI] [PubMed] [Google Scholar]
  18. Kucuk O., Sahin N., Sahin K. Supplemental zinc and vitamin A can alleviate negative effects of heat stress in broiler chickens. Biol Trace Elem Res. 2003;94(3):225–235. doi: 10.1385/BTER:94:3:225. [DOI] [PubMed] [Google Scholar]
  19. Kwiecień M., Winiarska-Mieczan A., Milczarek A., Milczarek A., Klebaniuk R. Biological response of broiler chickens to decreasing dietary inclusion levels of zinc glycine chelate. Biol Trace Elem Res. 2017;175(1):204–213. doi: 10.1007/s12011-016-0743-y. [DOI] [PubMed] [Google Scholar]
  20. Li X.Y., Chen H.N., Epstein P.N. Metallothionein protects islets from hypoxia and extends islet graft survival by scavenging most kinds of reactive oxygen species. J Biol Chem. 2004;279(1):765–771. doi: 10.1074/jbc.M307907200. [DOI] [PubMed] [Google Scholar]
  21. Liao X.D., Li W.X., Zhu Y.W., Zhang L.Y., Lu L., Lin X. Effects of environmental temperature and dietary zinc on egg production performance, egg quality and antioxidant status and expression of heat-shock proteins in tissues of broiler breeders. Br J Nutr. 2018;120(1):3–12. doi: 10.1017/S0007114518001368. [DOI] [PubMed] [Google Scholar]
  22. Lin H., De Vos D., Decuypere E., Buyse J. Dynamic changes in parameters of redox balance after mild heat stress in aged laying hens (Gallus gallus domesticus) Comp Biochem Physiol C Toxicol Pharmacol. 2008;147(1):30–35. doi: 10.1016/j.cbpc.2007.07.005. [DOI] [PubMed] [Google Scholar]
  23. Lin H., Decuypere E., Buyse J. Acute heat stress induces oxidative stress in broiler chickens. Comp Biochem Physiol A Mol Integr Physiol. 2006;144(1):11–17. doi: 10.1016/j.cbpa.2006.01.032. [DOI] [PubMed] [Google Scholar]
  24. Liu Z.H., Lu L., Wang R.L., Lei H.L., Li S.F., Zhang L.Y. Effects of supplemental zinc source and level on antioxidant ability and fat metabolism-related enzymes of broilers. Poultry Sci. 2015;94(11):2686–2694. doi: 10.3382/ps/pev251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Liu Zj, Lv Y.J., Zhang M., Yue Z.H., Tang S., Isiam A. Hsp110 expression changes in rat primary myocardial cells exposed to heat stress in vitro. Genet Mol Res. 2012;11(4):4728–4738. doi: 10.4238/2012.November.29.1. [DOI] [PubMed] [Google Scholar]
  26. Mackei M., Molnár A., Nagy S., Pál L., Kővágó C., Gálfi P. Effects of acute heat stress on a newly established chicken hepatocyte-nonparenchymal cell co-culture model. Animals (Basel) 2020;10(9):409. doi: 10.3390/ani10030409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mahmoud K.Z., Edens F.W. Influence of selenium sources on age-related and mild heat stress-related changes of blood and liver glutathione redox cycle in broiler chickens (Gallus domesticus) Comp Biochem Physiol B Biochem Mol Biol. 2003;136(4):921–934. doi: 10.1016/s1096-4959(03)00288-4. [DOI] [PubMed] [Google Scholar]
  28. Muthuraman P., Ramkumar K., Kim D.H. Analysis of dose-dependent effect of zinc oxide nanoparticles on the oxidative stress and antioxidant enzyme activity in adipocytes. Appl Biochem Biotechnol. 2014;174(8):2851–2863. doi: 10.1007/s12010-014-1231-5. [DOI] [PubMed] [Google Scholar]
  29. Naziroglu M., Sahin K., Simsek H., Aydilek N., Ertas O.N. The effects of food withdrawal and darkening on lipid peroxidation of laying hens in high ambient temperatures. Dtsch Tierarztl Wochenschr. 2000;107(5):199–202. [PubMed] [Google Scholar]
  30. Omar R., Pappolla M. Oxygen free radicals as inducers of heat shock protein synthesis in cultured human neuroblastoma cells: relevance to neurodegenerative disease. Eur Arch Psychiatr Clin Neurosci. 1993;242(5):262–267. doi: 10.1007/BF02190384. [DOI] [PubMed] [Google Scholar]
  31. Rama Rao S.V., Prakash B., Raju M., Panda A.K., Kumari R.K., Reddy E.P.K. Effect of supplementing organic forms of zinc, selenium and chromium on performance, anti-oxidant and immune responses in broiler chicken reared in tropical summer. Biol Trace Elem Res. 2016;172(2):511–520. doi: 10.1007/s12011-015-0587-x. [DOI] [PubMed] [Google Scholar]
  32. Ramiah S.K., Awad E.A., Mookiah S., Idrus Z. Effects of zinc oxide nanoparticles on growth performance and concentrations of malondialdehyde, zinc in tissues, and corticosterone in broiler chickens under heat stress conditions. Poultry Sci. 2019;98(9):3828–3838. doi: 10.3382/ps/pez093. [DOI] [PubMed] [Google Scholar]
  33. Sahin K., Kucuk O. Zinc supplementation alleviates heat stress in laying Japanese quail. J Nutr. 2003;133(9):2808–2811. doi: 10.1093/jn/133.9.2808. [DOI] [PubMed] [Google Scholar]
  34. Sahin K., Orhan C., Akdemir F., Tuzcu M., Iben C., Sahin N. Resveratrol protects quail hepatocytes against heat stress: modulation of the Nrf2 transcription factor and heat shock proteins. J Anim Physiol Anim Nutr. 2012;96(1):66–74. doi: 10.1111/j.1439-0396.2010.01123.x. [DOI] [PubMed] [Google Scholar]
  35. Sahin K., Smith M.O., Onderci M., Sahin N., Gursu M.F., Kucuk O. Supplementation of zinc from organic or inorganic source improves performance and antioxidant status of heat-distressed quail. Poultry Sci. 2005;84(6):882–887. doi: 10.1093/ps/84.6.882. [DOI] [PubMed] [Google Scholar]
  36. Sahin N., Tuzcu M., Ozercan I., Sahin K., Prasad A.S., Kucuk O. Zinc picolinate in the prevention of leiomyoma in Japanese quail. J Med Food. 2009;12(6):1368–1374. doi: 10.1089/jmf.2008.0287. [DOI] [PubMed] [Google Scholar]
  37. Salamat A.H., Ghasemi H.A. Effect of different sources and contents of zinc on growth performance, carcass characteristics, humoral immunity and antioxidant status of broiler chickens exposed to high environmental temperatures. Livest Sci. 2019;223:76–83. [Google Scholar]
  38. Sharma R., Sharma M., Datta P.K., Savin V.J. Induction of metallothionein-I protects glomeruli from superoxide-mediated increase in albumin permeability. Exp Biol Med (Maywood) 2002;227(1):26–31. doi: 10.1177/153537020222700105. [DOI] [PubMed] [Google Scholar]
  39. Slimen I.B., Najar T., Ghram A., Dabbebi H., Mrad M.B., Abdrabbah M. Reactive oxygen species, heat stress and oxidative-induced mitochondrial damage. A review. Int J Hyperther. 2014;30(7):513–523. doi: 10.3109/02656736.2014.971446. [DOI] [PubMed] [Google Scholar]
  40. Soleimani A.F., Zulkifli I., Hair-Bejo M., Omar A.R., Raha A.R. The role of heat shock protein 70 in resistance to Salmonella enteritidis in broiler chickens subjected to neonatal feed restriction and thermal stress. Poultry Sci. 2012;91(2):340–345. doi: 10.3382/ps.2011-01703. [DOI] [PubMed] [Google Scholar]
  41. Sun X.M., Lu L., Liao X.D., Zhang L.Y., Lin X., Luo X.G. Effect of in ovo zinc injection on the embryonic development and epigenetics-related indices of zinc-deprived broiler breeder eggs. Biol Trace Elem Res. 2018;185(2):456–464. doi: 10.1007/s12011-018-1260-y. [DOI] [PubMed] [Google Scholar]
  42. Suo H.Q., Lu L., Zhang L.Y., Zhang X.Y., Li H., Lu Y.F. Relative bioavailability of zinc-methionine chelate for broilers fed a conventional corn-soybean meal diet. Biol Trace Elem Res. 2015;165(2):206–213. doi: 10.1007/s12011-015-0252-4. [DOI] [PubMed] [Google Scholar]
  43. Tan G.Y., Yang L., Fu Y.Q., Feng J.H., Zhang M.H. Effects of different acute high ambient temperatures on function of hepatic mitochondrial respiration, antioxidative enzymes, and oxidative injury in broiler chickens. Poultry Sci. 2010;89(1):115–122. doi: 10.3382/ps.2009-00318. [DOI] [PubMed] [Google Scholar]
  44. Tedeschi J.N., Kennington W.J., Berry O., Whiting S., Meekan M., Mitchell N.J. Increased expression of Hsp70 and Hsp90 mRNA as biomarkers of thermal stress in loggerhead turtle embryos (Caretta Caretta) J Therm Biol. 2015;47:42–50. doi: 10.1016/j.jtherbio.2014.11.006. [DOI] [PubMed] [Google Scholar]
  45. Vallee B.L., Auld D.S. Zinc coordination, function, and structure of zinc enzymes and other proteins. Biochemistry. 1990;29(24):5647–5659. doi: 10.1021/bi00476a001. [DOI] [PubMed] [Google Scholar]
  46. Vinoth A., Thirunalasundari T., Shanmugam M., Uthrakumar A., Suji S., Rajkumar U. Evaluation of DNA methylation and mRNA expression of heat shock proteins in thermal manipulated chicken. Cell Stress Chaperones. 2018;23(2):235–252. doi: 10.1007/s12192-017-0837-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Volkova I.V., Sukhova L.L., Davydov V.V., Goloborod’ko A.V. The activity of the first line enzymes of the antioxidant defence in the liver of pubertal rats during stress. Biomed Khim. 2012;58(5):573–578. doi: 10.18097/pbmc20125805573. [DOI] [PubMed] [Google Scholar]
  48. Wang X.W., Dong W.P., Yuan B.B., Yang Y.C., Yang D.P., Lin X. Vitamin E confers cytoprotective effects on cardiomyocytes under conditions of heat stress by increasing the expression of metallothionein. Int J Mol Med. 2016;37(5):1429–1436. doi: 10.3892/ijmm.2016.2543. [DOI] [PubMed] [Google Scholar]
  49. Wang Y.H., Li K.J., Mao L., Hu X., Zhao W.J., Hu A. Effects of exogenous zinc on cell cycle, apoptosis and viability of MDAMB231, HepG2 and 293 T cells. Biol Trace Elem Res. 2013;154(3):418–426. doi: 10.1007/s12011-013-9737-1. [DOI] [PubMed] [Google Scholar]
  50. Wu C., Zhang W., Mai K., Xu W., Zhong X.L. Effects of dietary zinc on gene expression of antioxidant enzymes and heat shock proteins in hepatopancreas of abalone Haliotis discus hannai. Comp Biochem Physiol C Toxicol Pharmacol. 2011;154(1):1–6. doi: 10.1016/j.cbpc.2011.03.003. [DOI] [PubMed] [Google Scholar]
  51. Xie J.J., Tang L., Lu L., Zhang L.Y., Lin X., Liu H.C. Differential expression of heat shock transcription factors and heat shock proteins after acute and chronic heat stress in laying chickens (Gallus gallus) PloS One. 2014;9(7) doi: 10.1371/journal.pone.0102204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Xie J.J., Tang L., Lu L., Zhang L.Y., Lin X., Liu H.C. Effects of acute and chronic heat stress on plasma metabolites, hormones and oxidant status in restrictedly fed broiler breeders. Poultry Sci. 2015;94(7):1635–1644. doi: 10.3382/ps/pev105. [DOI] [PubMed] [Google Scholar]
  53. Yahav S., Shamai A., Haberfeld A., Horev G., Hurwitz S., Einat M. Induction of thermotolerance in chickens by temperature conditioning: heat shock protein expression. Ann N Y Acad Sci. 1997;813:628–636. doi: 10.1111/j.1749-6632.1997.tb51757.x. [DOI] [PubMed] [Google Scholar]
  54. Yang L., Tan G.Y., Fu Y.Q., Feng J.H., Zhang M.H. Effects of acute heat stress and subsequent stress removal on function of hepatic mitochondrial respiration, ROS production and lipid peroxidation in broiler chickens. Comp Biochem and Physiology Toxicol C Pharmacol. 2010;151(2):204–208. doi: 10.1016/j.cbpc.2009.10.010. [DOI] [PubMed] [Google Scholar]
  55. Yin B., Tang S., Sun J.R., Zhang X.H., Xu J., Di L.J. Vitamin C and sodium bicarbonate enhance the antioxidant ability of H9C2 cells and induce HSPs to relieve heat stress. Cell Stress Chaperones. 2018;23(4):735–748. doi: 10.1007/s12192-018-0885-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yu Y., Lu L., Li S.F., Zhang L.Y., Luo X.G. Organic zinc absorption by the intestine of broilers in vivo. Br J Nutr. 2017;117(8):1086–1094. doi: 10.1017/S0007114517001040. [DOI] [PubMed] [Google Scholar]
  57. Yu Y., Lu L., Wang R.L., Lin X., Luo X.G., Liu B. Effects of zinc source and phytate on zinc absorption by in situ ligated intestinal loops of broilers. Poultry Sci. 2010;89(10):2157–2165. doi: 10.3382/ps.2009-00486. [DOI] [PubMed] [Google Scholar]
  58. Yuan J., Xu Z., Huang C., Zhou S., Guo Y. Effect of dietary Mintrex-Zn/Mn on performance, gene expression of Zn transfer proteins, activities of Zn/Mn related enzymes and fecal mineral excretion in broiler chickens. Anim Feed Sci Technol. 2011;168(1–2):72–79. [Google Scholar]
  59. Zhang L., Wang J.S., Wang Q., Li K.X., Guo T.Y., Xiao X. Effects of maternal zinc glycine on mortality, zinc concentration, and antioxidant status in a developing embryo and 1-day-old chick. Biol Trace Elem Res. 2018;181(2):323–330. doi: 10.1007/s12011-017-1028-9. [DOI] [PubMed] [Google Scholar]
  60. Zhang L., Wang Y.X., Xiao X., Wang J.S., Wang Q., Li K.X. Effects of zinc glycinate on productive and reproductive performance, zinc concentration and antioxidant status in broiler breeders. Biol Trace Elem Res. 2017;178(2):320–326. doi: 10.1007/s12011-016-0928-4. [DOI] [PubMed] [Google Scholar]
  61. Zhou C., Zhang C. Protective effects of antioxidant vitamins on Aroclor 1254-induced toxicity in cultured chicken embryo hepatocytes. Toxicol Vitro. 2005;19(5):665–673. doi: 10.1016/j.tiv.2005.03.010. [DOI] [PubMed] [Google Scholar]
  62. Zhu Y.W., Li W.X., Lu L., Zhang L.Y., Ji C., Lin X. Impact of maternal heat stress in conjunction with dietary zinc supplementation on hatchability, embryonic development, and growth performance in offspring broilers. Poultry Sci. 2017;96(7):2351–2359. doi: 10.3382/ps/pew481. [DOI] [PubMed] [Google Scholar]
  63. Zhu Y.W., Liao X.D., Lu L., Li W.X., Zhang L.Y., Ji C., Lin X., Liu H.C., Odle J., Luo X.G. Maternal dietary zinc supplementation enhances the epigenetic-activated antioxidant ability of chick embryos from maternal normal and high temperatures. Oncotarget. 2017;8(12):19814–19824. doi: 10.18632/oncotarget.15057. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zhu Y.W., Lu L., Li W.X., Zhang L.Y., Ji C., Lin X. Effect of dietary manganese on antioxidant status and expression levels of heat-shock proteins and factors in tissues of laying broiler breeders under normal and high environmental temperatures. Br J Nutr. 2015;114(12):1965–1974. doi: 10.1017/S0007114515003803. [DOI] [PubMed] [Google Scholar]
  65. Zhu Y.W., Xie J.J., Li W.X., Lu L., Zhang L.Y., Ji C. Effects of environmental temperature and dietary manganese on egg production performance, egg quality, and some plasma biochemical traits of broiler breeders. J Anim Sci. 2015;93(7):3431–3440. doi: 10.2527/jas.2015-8956. [DOI] [PubMed] [Google Scholar]

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