A systematic review on the modulation of heat shock protein 70 in broilers at different stages of growth under thermal stress

141 Czech Journal of Animal Science, 71, 2026 (4): 141–158

Review https://doi.org/10.17221/1/2026-CJAS © The authors. This work is licensed under a Creative Commons Attribution 4.0 International (CC-BY 4.0) license.

Supported by the Malaysian Research University Network (MRUN) [International Research Grant Scheme (MIRGS) under the Grant No. 5539320].

A systematic review on the modulation of heat shock protein 70 in broilers at different stages of growth under thermal stress

Hudu Ramalan Abdullahi1,4 , Abubakar Abubakar Ahmed1,2 ,

Suriya Kumari Ramiah1,3 , Hasliza Abu Hassim1,2 , Yong Meng Goh1,2*

1Laboratory of Sustainable Animal Production and Biodiversity, Institute of Tropical

Agriculture and Food Security, University of Putra Malaysia, UPM Serdang,

Selangor, Malaysia 2Department of Preclinical Sciences, Faculty of Veterinary Medicine, University of Putra

Malaysia, UPM Serdang, Selangor, Malaysia 3School of Veterinary Medicine, International Medical University, MRANTI Technology Park,

Bukit Jalil, WP Kuala Lumpur, Malaysia 4Department of Animal Science, Federal University of Lafia, Lafia, Nasarawa State, Nigeria

*Corresponding author: ymgoh@upm.edu.my Citation: Abdullahi H.R., Ahmed A.A., Ramiah S.K., Hassim H.A., Goh Y.M. (2026): A systematic review on the modu- lation of heat shock protein 70 in broilers at different stages of growth under thermal stress. Czech J. Anim. Sci., 71:

141–158.

Abstract: This systematic review synthesises evidence from published articles investigating nutritional strategies to modulate heat shock protein 70 (HSP70). A total of 1 616 records were identified from four databases and snowballing. After screening and eligibility assessment, 25 studies comprising 29 reports published between 2004 and 2025 were included. The analysis shows that targeted interventions particularly antioxidants (vitamins C,

E, and selenium), amino acids and their derivatives (methionine, betaine), and phytogenic compounds effec- tively downregulate HSP70 expression in a dose or tissue-dependent manner. This modulation is associated with improved growth performance, enhanced redox balance, and normalised stress hormone profiles. Key findings indicate that synergistic combinations outperform single additives in suppressing HSP70 and improving carcass yield under chronic heat stress (32–38 °C); the early-life nutrient delivery modulates HSP70 expression and influ- ences post-hatch thermotolerance; and organic mineral or methionine hydroxy analogue are more effective than inorganic forms. However, efficacy varies by life stage, with most studies focused on Ross and Cobb genotypes, and is influenced by gut microbiota interactions. Major research gaps remain, including defining stage-specific optimal HSP70 thresholds, clarifying links between HSP70 modulation, immunity or gut health, understanding long-term effects of early-life nutritional programming, and determining the influence of administration routes on nutrient efficacy.

Keywords: antioxidant; gene expression; intervention; nutrient; thermotolerance

142 Review Czech Journal of Animal Science, 71, 2026 (4): 141–158 https://doi.org/10.17221/1/2026-CJAS

INTRODUCTION Poultry farming is a cornerstone of global ag- riculture, critically underpinning food security, economic development, and rural livelihoods (Tallentire et al. 2016). Driven by rising demand for animal protein, technological advancements, and the pursuit of production efficiency, the in- dustry has evolved significantly from traditional backyard systems to intensive commercial opera- tions. This transformation is particularly evident in broiler production, where genetic selection and optimised nutrition have yielded remarkable im- provements in growth rates, feed conversion ra- tios (FCR), and disease resistance. However, these gains come with vulnerabilities: the high metabolic rates of fast-growing genotypes increase metabol- ic heat output, rendering them highly susceptible to heat stress (HS), a challenge amplified by global warming (Nawaz et al. 2021; Gouda et al. 2024).

The poultry sector faces persistent challenges, including disease outbreaks, environmental sus- tainability, and the need for cost-effective alterna- tive feed sources (Wongtangtintharn et al. 2025).

Among these, heat stress has emerged as a criti- cal constraint, especially in the tropical regions where poultry farming is  economically vital (Wongtangtintharn et al. 2025). Heat stress disrupts physiological homeostasis, leading to reduced feed intake, impaired growth performance, increased mortality, and heightened disease susceptibility (Zulkifli et al. 2018). It also induces significant oxidative stress, damaging cellular structures (e.g. through lipid peroxidation) and compromising the immune function (Shehata et al. 2020; Abdel- Moneim et al. 2021; Qin et al. 2023).

At the molecular level, a key adaptive response to HS is the induction of heat shock protein 70 (HSP70). This cytoprotective chaperone prevents protein denaturation and aggregation, maintains cellular integrity, mitigates HS-induced inflamma- tion (e.g. by reducing IL-6 and TNF-α), and reduces oxidative damage (Goel et al. 2021; Balakrishnan et  al. 2023). Chronic HS (typically 32–35  °C) upregulates HSP70 in critical tissues like the liver, jejunum, and pectoralis major muscle. However, this protective response is energetically costly and can divert resources away from growth (Teyssier et al. 2022; Balakrishnan et al. 2023). Broilers ex- hibiting higher basal HSP70 levels often demon- strate better survivability, highlighting its dual role as both a biomarker of stress severity and a poten- tial therapeutic target (Zulkifli et al. 2018). Despite its importance, the regulation of HSP70 and its interactions with other physiological pathways, particularly under nutritional interventions, re- main incompletely understood (Abdel-Moneim et al. 2021; Nawaz et al. 2021).

Nutritional strategies offer a promising avenue for mitigating HS impacts. Supplementation of antiox- idants (e.g. ascorbic acid, selenium) and probiotics has shown a potential to enhance antioxidant de- fences, modulate immune responses, and improve thermotolerance, partly by modulating HSP70 ex- pression (Kumbhar et al. 2018; Shakeri et al. 2020;

Wasti et al. 2020). However, the efficacy of these interventions is highly variable, influenced by fac- tors such as dosage, broiler life stage, supplement form (organic vs inorganic), and interactions with the gut microbiota (Deng et al. 2022; Apalowo et al.

2024). Critically, broilers exhibit distinct metabolic and physiological responses to HS across differ- ent growth stages, necessitating the development of stage-specific dietary approaches.

Despite growing research, a significant gap exists in comprehensive reviews synthesising recent find- ings on the application of these nutritional strategies in the modulation of heat stress in broilers of differ- ent stages. This systematic review, therefore, aims to critically analyse current knowledge on HS im- pacts in broilers, focusing on the roles of HSPs, oxidative stress pathways, and nutritional interven- tions. Specific objectives are to (i) assess the efficacy of amino acids and other nutrients in mitigating HS; (ii) analyse the relationship between HSP70 regu- lation and key performance, health, and stress pa- rameters; and (iii) identify research gaps and future directions for developing sustainable HS manage- ment strategies. By integrating findings from recent studies, this review will provide actionable insights for researchers, nutritionists, and poultry producers striving to enhance broiler welfare and productivity in heat-challenged environments.

MATERIAL AND METHODS Inclusion and exclusion criteria

This review followed the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta- Analyses) guidelines. It included only published

143 Czech Journal of Animal Science, 71, 2026 (4): 141–158

Review https://doi.org/10.17221/1/2026-CJAS peer-reviewed journal articles in English, without applying any restrictions on publication dates.

Studies that focused on other non-nutritional in- terventions, were not on broiler birds and did not measure HSP70 were excluded. This systematic review focuses on broiler chickens (population) subjected to heat stress conditions, evaluating the impact of nutritional supplementation (interven- tion), including vitamins, minerals, amino acids, phytogenics, and probiotics, on the modulation of heat shock protein 70 (HSP70). Interventions have been compared against non-supplemented control groups or baseline pre-treatment mea- surements (Comparison), with all groups exposed to standardised heat stress protocols. Primary outcomes include changes in HSP70 expression (measured via mRNA levels or protein concen- tration), while secondary outcomes encompass performance metrics such as growth rate, feed efficiency, and mortality (outcome). Finally, only articles indexed in  the Web of  Science (WOS) database were included to ensure a high level of credibility.

Literature search, screening and selection The search process was performed twice using a consistent strategy across all selected databases.

The initial round of searches was carried out between

June 15th and June 22nd, 2025, and a follow-up search took place from June 27th to July 5th, 2025, to validate and update the results. All records retrieved during both phases underwent a rigorous screening process, including full-text and abstract reviews, to deter- mine their eligibility based on predefined criteria.

Literature searches were conducted using Google Scholar (https://scholar.google.com/),

Scopus (https://www.scopus.com), ScienceDirect (https://www.sciencedirect.com/), and Pub- Med (https://pubmed.ncbi.nlm.nih.gov). The com- prehensiveness of searched articles was enhanced via a snowballing approach by examining the refer- ence lists of relevant articles identified through the databases (Saba et al. 2024; Ishaq et al. 2025; Saba et al. 2025). Details of keywords used in carrying out a comprehensive search based on PICO headings in the various databases can be found in Table 1.

Table 1. Search keywords from different databases based on PICO headings

Component Description Google Scholar ("heat shock protein 70" OR HSP70) AND ("broilers" OR "broiler chicken") AND ("nutrients" OR "dietary supplements" OR "feed additives" OR "vitamins" OR "minerals" OR "amino acids" OR "ascorbic acid" OR

"betaine" OR "selenium") AND ("modulate" OR "regulate" OR "expression" OR "suppressions") AND ("developmental stage" OR "embryonic" OR "in ovo" OR "starter" OR "grower" OR "finisher" OR

"age-dependent") AND ("heat stress" OR "thermal stress") –layers –breeders –ruminants –swine

PubMed ("heat-shock proteins" OR "HSP70 heat-shock proteins" OR "heat shock protein 70" OR "HSP70") AND ("Chickens" OR "broilers" OR "Gallus gallus") AND ("dietary supplements" OR "nutrient" OR "feed addi- tives" OR "amino acids" OR "vitamins" OR "minerals" OR "ascorbic acid" OR "betaine" OR "polyphenols")

AND ("modulate" OR "regulate" OR "expression") AND ("growth and development" OR "embryonic structures" OR "in ovo" OR "starter" OR "grower" OR "finisher") AND ("heat stress sisorders" OR

"heat stress" OR "thermal stress") NOT ("layer" OR "turkey" OR "duck")

Scopus ("heat shock protein 70" OR "HSP70") AND ("broilers" OR "broiler chicken") AND ("nutrients" OR "dietary supplements" OR "functional feed" OR "antioxidants" OR "ascorbic acid" OR "betaine" OR "chromium" OR

"selenium" OR "zinc") AND ("modulate" OR "regulate" OR "gene expression" OR "protein synthesis”) AND ("developmental stage" OR "life stage" OR "embryonic" OR "early posthatch" OR "starter" OR "grower" OR

"finisher") AND ("heat stress" OR "thermal stress" OR "high temperature stress") AND NOT ("layer" OR

"breeder" OR "swine" OR "fish") ScienceDirect ("HSP70" OR "heat shock protein 70") AND ("broilers" OR "meat chickens") AND ("nutritional interven- tion" OR "dietary strategy" OR "nutraceutical" OR "phytogenic" OR "vitamin" OR "trace minerals" OR "me- thionine" OR "lysine") AND ("modulate" OR "downregulate" OR "suppression") AND ("growth phase" OR

"embryonic development" OR "hatching stage" OR "starter phase" OR "grower phase" OR "finisher phase")

AND ("heat stress" OR "heat challenge" OR "hyperthermia") AND NOT ("layer" OR "turkey")

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Figure 1 At the end of  the literature search, a  total of 1 616 records were identified. After initial title screening, 747 duplicates were removed, and 689 re- cords were excluded for not meeting the inclusion criteria, leaving 180 records for abstract screen- ing (Figure 1). Of these, 120 records were excluded based on abstract relevance, and 60 records were selected for full-text retrieval. All 60 full texts were successfully retrieved and assessed for eligibility.

During full-text screening, 33 records were exclud- ed for the following reasons: not focusing on broil- ers (n = 6), not addressing HSP70 (n = 11), not being related to heat stress (n = 10), being duplicates (n = 3), or not being published in English (n = 1),

Figure 1. PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flow chart of literature search, screening and selection process

Identification of studies via databases and snowballing

Records identified from:

Google Scholar (n = 732) Scopus (n = 244) Science Direct (n = 632)

Snowballing (n = 8) Total (n = 1 616) Records removed from title screening:

Duplicates removed (n = 747) Records removed due to ineligibility (n = 689)

Records excluded (n = 120) Records not retrieved (n = 0)

Records excluded:

Not broiler (n = 6) Not HSP70 (n = 11) Not heat stress (n = 10)

Are duplicates (n = 3) Not in English (n = 1) Not indexed in WOS (n = 4)

Studies included in review (n = 25) Reports from included studies (n = 29)

Records assessed for eligibility (n = 60) Records sought for retrieval (n = 60)

Records screened by abstracts (n = 180) Identification

Screening Included 145 Czech Journal of Animal Science, 71, 2026 (4): 141–158

Review https://doi.org/10.17221/1/2026-CJAS or not indexed in the Web of Science database (4).

Ultimately, 25 studies were included in the review, comprising a total of 29 relevant reports.

Identification, screening, and selection of article

Most of the search results from each database were exported as CSV files into the Rayyan QCRI online platform (https://www.rayyan.ai/), apart from re- sults obtained through snowballing. To remove du- plicates, two key steps were followed: verifying the presence of relevant studies and discarding those not aligning with the inclusion criteria. Thereafter, titles and abstracts were screened using Rayyan. After completing the initial screening on Rayyan, the full texts of selected studies were retrieved for in-depth evaluation (Ouzzani et al. 2016; Saba et al. 2023).

These articles were carefully reviewed, and relevant data were systematically extracted using Microsoft

Excel 365 (Redmond, WA, USA). Articles not previ- ously screened in Rayyan had their abstracts inde- pendently reviewed prior to the full-text analysis.

As the comprehensive review of the full texts pro- gressed, several studies were excluded due to failure to meet the predefined eligibility criteria.

Data extraction and synthesis The full-text versions of all identified articles were downloaded as PDFs for further review. Relevant information was tabulated and analysed descrip- tively using Microsoft Office Excel 365. General in- formation about the included studies was extracted, including treatments used, broiler stage, dependent variables, study design, sample size, broiler geno- type, age of birds (days), heat-stress conditions (tem- perature/duration), nutrient type, nutrient dose, duration of supplementation, control group details,

HSP70 measurement method and tissue used.

RESULTS AND DISCUSSION Experimental design and intervention details

For most of the studies, randomised control trials (RCT, 72%) were used, with the randomised facto- rial design (RFD, 16%) (Figure 2). Other designs include randomised block design (RBD), in vitro, and in vivo testing. Antioxidants constituted 48% of interventions, primarily ascorbic acid (200–

600 mg/kg diet or 100–300 mg/bird/day), vitamins (100–250 mg/kg), and selenium (0.2–0.3 mg/kg).

Amino acids/derivatives (31% of studies) included methionine, betaine, and leucine. Minerals featured chromium (0.1–1.2 mg/kg CrMet or 1 200 µg/kg picolinate) and zinc/manganese. Other agents included melatonin (0.5 mg/kg) and probiotics (Table 2). Furthermore, about 80% of studies used long-term supplementation (≥35 days). In ovo inter- ventions involved single embryonic injections, e.g. leucine (Han et al. 2019). All controls received basal diets without additives under identical heat stress (32–38 °C). Three studies included thermoneutral controls (24–25 °C) (Mahmoud et al. 2004b; Toplu et al. 2014; Abo-Samaha et al. 2021).

For HSP70 measurement, most studies (65%) quantified mRNA via qRT-PCR (SYBR® Green/

TAQMan), often normalised to GAPDH or β-actin (Table 2). The tissue-specific analysis dominated (liver: 40%; muscle: 30%; intestine: 15%). For protein detection, studies mostly (35%) used ELISA (com- mercial chicken HSP70 kits) or Western blot (e.g. recombinant HSP70 standards: Mahmoud et al.

2004a). Only two studies combined mRNA/pro- tein assessment (Zhu et al. 2015; Belal et al. 2018).

Figure 2. Experimental design of included studies relat- ing to the modulation of heat stress in broilers at differ- ent stages

RBD = randomised block design; RCT = randomised control trials; RFD = randomised factorial design

1, 4% 4, 16% 1, 4% 1, 4% 18, 72% RCT RBD In vivo

RFD In vitro 146 Review Czech Journal of Animal Science, 71, 2026 (4): 141–158 https://doi.org/10.17221/1/2026-CJAS

Table 2. Nutrient used, duration, control and HSP70 measurement method in the selected studies

Nutrient type Nutrient dose Supplementation duration

Control group details HSP70 measurement method/ tissue used

Reference Ascorbic acid (AA) 200 mg/kg diet days 1–33 basal diet qRT-PCR (liver)

Abo-Samaha et al. (2021) Ascorbic acid (AA) 500 mg/kg diet days 1–28 basal diet

Western blot (heart tissue) Mahmoud et al. (2004a)

Ascorbic acid (AA) 100–300 mg/bird 42-day trial basal diet

ELISA kit (plasma) Mishra et al. (2015) Chromium-methionine

0–1.20 mg/kg diet days 22–43 basal diet RT-qPCR (Pectoralis major)

Dalolio et al. (2024) Inorganic & organic Mn 120 mg Mn/kg diet

32–45 weeks basal diet RT-qPCR & Western blot: liver, heart, and breast

Zhu et al. (2015) Vitamin E and selenium 100–150 mg/kg Se

0.2–0.3 mg/kg Vit E days 42/ trial period basal diet chicken HSP70 ELISA kit from serum

Bora et al. (2024) l-Ascorbic acid (vitamin C) 500 mg/kg of diet days 22–42 basal diet immunohistochemistry assay: liver, kidneys and brain

Toplu et al. (2014) l-Methionine and l-cysteine dissolved in saline

5.90 mg/l-methionine 3.40 mg/l-cysteine days 17.5 of incubation negative and positive controls

ELISA & qPCR: mRNA expression in tissues and serum

Elnesr et al. (2019) l-Leucine (l-leu) 34.5 µmol l-leu per egg in 500 µl sterile water injection at day 7 of incubation sterile water injection qPCR: mRNA expression in diencephalon, liver, muscle

Han et al. (2019) Probiotic (Saccharomyces cerevisiae); ascorbic acid

1 g/kg of feed; ascorbic acid: 200 mg/kg of feed days 1–35 basal diet

RT-qPCR from serum Sumanu et al. (2023) Selenium-enriched probiotics (SP)

0.30 mg Se/kg feed (total Se: 0.41 mg/kg) days 1–42 basal diet qRT-PCR from heart tissue

Khan et al. (2016) Serotonin 20 µg/egg single injection before incubation normal saline (0.9% NaCl) injection qPCR from brain tissues

KHasti et al. (2025) Vitamin C (l-ascorbic acid) 1 g/kg basal diet days 1–42 basal diet qPCR from liver, heart and kidney

Albokhadaim et al. (2019) Sodium selenite, vitamin E

Se: 0.2 mg/kg; VE: 250 mg/kg days 1–42 basal diet real-time PCR of the breast muscles

Kumbhar et al. (2018) Selenium yeast (Sel-Plex®) 0.2 mg/kg feed days 1–42 basal diet

ELISA (liver tissue) Mahmoud and Edens (2005) 147 Czech Journal of Animal Science, 71, 2026 (4): 141–158

Review https://doi.org/10.17221/1/2026-CJAS Nutrient type

Nutrient dose Supplementation duration Control group details

HSP70 measurement method/ tissue used Reference dl-Methionine + l-carnitine

200 mg/kg diet days 1–42 basal diet qRT-PCR (mRNA level) in liver and hypothalamus

Ghasemi and Nari (2023) MHA + l-carnitine 200 mg/kg diet

+ MHA days 1–42 basal diet qRT-PCR (mRNA level) in liver and hypothalamus

Ghasemi and Nari (2023) Recombinant chicken leptin

8 µg/kg per hour 6 h saline solution RT-PCR/Southern blot analysis

Figueiredo et al. (2007) Recombinant chicken leptin

10–1 000 ng/ml 24 h untreated cells RT-PCR/Southern blot analysis

Figueiredo et al. (2007) Ascorbic acid 200, 400, 600 mg/kg diet days 1–42 basal diet

RT-qPCR in liver tissue Biswas et al. (2024) Taurine

0.1% in water 5 weeks no taurine qRT-PCR (mRNA) and

Western blot (protein) from liver and breast Belal et al. (2018)

Inorganic phosphorus (P) 0.50% available P 3 weeks no P

Northern blot (mRNA) Mahmoud et al. (2004b) Vitamin C

300 mg/kg diet days 1–42 basal diet qRT-PCR (mRNA) from liver, brain, heart and spleen

Hajati et al. (2015) Vitamin C 0.2% in all periods days 1–42 basal diet real-time PCR (qPCR) with β-actin as a control from the liver

Erfani et al. (2021) Methionine hydroxy analogue (MHA)

0.46% (starter), 0.36% (grower), 0.32% (finisher) days 1–42 basal diet real-time PCR (qPCR) with β-actin as a control from the liver

Erfani et al. (2021) MHA + Vitamin C MHA: 0.36–0.46%

Vit C: 0.2–0.32% days 1–42 basal diet real-time PCR (qPCR) with β-actin as a control form liver

Erfani et al. (2021) Amino acid-chelated trace minerals not specified

42 days basal diet qPCR (RNA isolation + SYBR Green) from blood

Baxter et al. (2020) Inorganic Zn sulphate (iZn), organic Zn lysine chelates (oZn)

50 µmol/l 8 h preincubation + 4 or 6 h incubation basal diet real-time quantitative PCR,

Western blot Li et al. (2021) Amino acids (lysine, methionine, etc.)

80–110% of Aviagen AA specifications 36 days basal diet qRT-PCR (Power SYBR Green) on pectoralis muscles

Alhotan et al. (2021) Table 2 to be continued 148 Review

Czech Journal of Animal Science, 71, 2026 (4): 141–158 https://doi.org/10.17221/1/2026-CJAS

Sample characteristics Commercial Ross strains (predominantly

Ross 308) were used in 44% of studies (11/25), fol- lowed by Cobb variants (Cobb 500: 16%; Cobb 400:

8%) and Arbor Acres (16%). Only two studies used unspecified commercial broilers. This reflects the industry alignment with high-yield genetic lines.

About 84% of studies (21/25) tested aged birds, while 16% (4/25) included embryonic stages, for example, in ovo injections at ED7-18 (Elnesr et al.

2019; Han et al. 2019) or starter phases, 0–10 days (Alhotan et al. 2021). Zhu et al. (2015) studied lay- ing broiler breeders (32–45 weeks), while Li et al. (2021) used embryonic hepatocytes (in vitro). Bird numbers ranged widely, with small-scale testing involving 40–96 birds (Figueiredo et al. 2007;

Han et al. 2019) and large-scale testing involv- ing 240–720 birds (e.g. Ghasemi and Nari 2023;

Biswas et al. 2024). As per replication, most stud- ies used pen replicates (4–8 birds/pen; 3–6 rep- licates/treatment), while some studies reported unclear replication. Moreover, about 70% of the studies specified male-only cohorts to eliminate sex-based variability. All birds were reared under standard commercial pre-trial conditions, with heat stress applied at defined ages (32–38 °C from days 21–42) (Table 3). Most studies re- ported that water was provided ad libitum to all experimental birds throughout the heat stress ex- posure period, consistent with standard poultry research protocols and essential for supporting evaporative cooling, thermoregulatory balance, and animal welfare while avoiding confounding dehydration stress.

Other parameters Research assessing HSP70 (heat shock pro- tein 70) in broilers commonly evaluates it along- side several key categories of dependent variables, reflecting the multifaceted impact of heat stress.

These include physiological and hormonal stress markers, growth and production performance, an- tioxidant status, oxidative stress, haematological and biochemical blood parameters, immune and inflammatory markers, molecular markers (beyond

HSP70), tissue integrity, and morphology. A prima- ry focus is on indicators of the stress response itself.

This consistently includes measuring the plasma or serum levels of corticosterone (CORT or CS), the major avian stress hormone (Mahmoud et al.

2004a,b; Mishra et al. 2015; Toplu et al. 2014; Elnesr et al. 2019). Body temperature is a direct physi- ological indicator that is also monitored (Toplu et al. 2014; Dalolio et al. 2024).

Hormonal assessments sometimes extend to thy- roid hormones (Erfani et al. 2021), linking stress to metabolic regulation. Organ weights, particular- ly lymphoid organs (thymus, spleen, bursa) (Hassan and Assim 2020; Bora et al. 2024; Dalolio et al.

2024) and sometimes the heart or liver, serve as in- dicators of immune status and metabolic strain.

Fear-related behaviour (tonic immobility) is also assessed as a stress indicator (Toplu et al. 2014).

Direct measures of economic impact are frequently correlated with HSP70. These include body weight (BW), body weight gain (BWG or weight gain), feed intake (FI), feed efficiency ratio (FCR), per- formance index (PI), body performance efficiency index (BPEI), and mortality (Mahmoud and Edens

2005; Mishra et al. 2015; Baxter et al. 2020; Abo- Samaha et al. 2021; Alhotan et al. 2021; Erfani et al.

2021; Ghasemi and Nari 2023; Biswas et al. 2024; Bora et al. 2024; Dalolio et al. 2024).

Carcass characteristics such as dressing percent- age, prime cuts yield, and giblet weights are also evaluated as end-point production traits (Baxter et al. 2020; Bora et al. 2024). Given the strong link between heat stress and oxidative damage, numer- ous studies measure markers of antioxidant defence and lipid peroxidation. Key variables include malo- ndialdehyde (MDA) levels (a marker of lipid per- oxidation), activities of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), glu- tathione peroxidase (GSH-Px), and levels of gluta- thione (GSH, TGSH, GSH/GSSG ratio) (Mahmoud and Edens 2005; Zhu et al. 2015; Kumbhar et al.

2018; Albokhadaim et al. 2019; Elnesr et al. 2019;

Li et al. 2021; Ghasemi and Nari 2023; Sumanu et al. 2023). The oxidative DNA damage biomarker

8-OHdG is also measured (Sumanu et al. 2023).

In studies where they were reported, corticoste- rone levels were typically measured using validated radioimmunoassay (RIA) or enzyme-linked immu- nosorbent assay (ELISA) kits on plasma or serum samples collected via wing vein, jugular, or car- diac puncture, centrifuged at 1 500–3 000 × g for

10–15 min at 4 °C, stored at –20 °C or –80 °C, and assayed in duplicate with coefficients of variation below 5–6%.

149 Czech Journal of Animal Science, 71, 2026 (4): 141–158

Review https://doi.org/10.17221/1/2026-CJAS Table 3. Broiler production stages, sample size used and genotype in the selected studies under heat stress conditions

Stage of production Sample size Genotype Heat stress condition

Reference Grower (33 days) 96 (4 × 3 reps of 8 birds)

Cobb 500 34 °C from day 21 through day 24 Abo-Samaha et al. (2021)

Grower phase (28 days) 160 (2 × 2 factorial) Arbor Acres

41 °C (over 3.5 h/3 days) Mahmoud et al. (2004a) Grower phase (42 days)

96 (4 × 2 reps, 12 birds) Cobb 400 40 ± 5.0 °C heat stress

Mishra et al. (2015) Grower to finisher (22–43 days) experiment 1: 336 broilers

Cobb 500 33 °C for 12 h/day for 21 days Dalolio et al. (2024)

Broiler breeders (18–45 weeks) 144 (6 × 6 reps, 4 birds)

Arbor Acres 32 ± 1 °C from 32 to 45 weeks Zhu et al. (2015)

Grower phase (0 to 42 days) 240 (4 × 3 reps, 20 birds)

Cobb 400 36.5 °C with 92.5% RH Bora et al. (2024) Grower phase (0 to 42 days)

320 (4 × 4 reps of 20 birds) Ross 308 35 °C for 6 h/day

Toplu et al. (2014) Embryonic stage (1–18) 150 eggs (50 per group)

Ross broiler 39.6 °C for 6 h daily Elnesr et al. (2019)

Grower (0 to 35 days) 56 (14 groups of 4 birds) Ross 308 not explicitly stated

Sumanu et al. (2023) Grower phase (0 to 42 days) 200 (4 × 5 reps of 10 birds)

Ross 308 34 ± 1 °C with 60% to 80% RH Khan et al. (2016)

Embryogenesis (ED 13–18) 120 eggs Ross 308 39.5 °C for 2 h, 55–65% RH

KHasti et al. (2025) Grower phase (0 to 42 days) 200 (50/group of 4 birds)

Ross 308 34 °C for 8 hours/day Albokhadaim et al. (2019)

Grower phase (0 to 42 days) 200 (50/group in 4 reps)

Ross 308 34 °C to 36 °C for the first 14 days Kumbhar et al. (2018)

Grower phase (0 to 42 days) 128 (64/group in 2 reps)

Arbor Acres 40 °C for 1 h Mahmoud and Edens (2005)

Grower phase (0 to 42 days) 720 (5 × 6 reps, 24 birds)

Ross 308 34 °C for the first 3 days Ghasemi and Nari (2023)

Grower phase (0 to 42 days) 192 (4 × 6 reps × 8 birds) not specified

35.4 °C, 79% RH Biswas et al. (2024) Grower (0 to 35 days)

40 (10 × 4) Ross broilers 34 °C for 3 weeks at 60% RH

Belal et al. (2018) Grower phase (0 to 21 days) 60 (10 × 6) not stated

41 °C for 60 min Mahmoud et al. (2004b) Grower phase (0 to 42 days)

60 (12 × 5) Cobb-500 34 ± 1 °C with 65% to 70%, 5 h

Hajati et al. (2015) Grower phase (0 to 42 days) 400 (5 reps of 20 birds)

Ross 308 32–34 °C with 60 and 70% RH Erfani et al. (2021)

Grower phase (0 to 42 days) 480 (12 × 24 reps, 20 birds)

Cobb 500 35 °C and 20% to 30% RH Baxter et al. (2020)

Embryo hepatocytes in vitro 6 reps per treatment Arbor Acres

44 °C for 1, 2, 4, 6, or 8 h Li et al. (2021) Starter phase (0 to 10 days)

576 (24 reps, 6 chicks) Ross 308 32 °C for 8 h daily

Alhotan et al. (2021) Grower phase (0 to 21 days) 5 per group

Ross strain 37 °C at 5% humidity Figueiredo et al. (2007)

Grower phase (0 to 42 days) 3 per group Cobb strain

37 °C at 5% humidity Figueiredo et al. (2007) Embryonic stage

120 eggs (60/group) Ross strain 37.6 °C with 58–68% RH

Han et al. (2019) ED = embryonic days; reps = replicates; RH = relative humidity

150 Review Czech Journal of Animal Science, 71, 2026 (4): 141–158 https://doi.org/10.17221/1/2026-CJAS

The blood analysis provides a snapshot of the overall physiological state. Commonly assessed parameters include haemoglobin (Hb), red blood cell count (RBCs), packed cell volume (PCV), white blood cell count (WBC), heterophil/lym- phocyte (H/L) ratio (a stress leukogram), glucose, total protein, liver enzymes (SGOT/AST, SGPT/

ALT), lipid profile (triglycerides, cholesterol, HDL,

LDL, VLDL), and electrolytes (Toplu et al. 2014; Hajati et al. 2015; Hassan and Assim 2020; Erfani et al. 2021). Immune function assessment includes measuring cytokine gene expression (Abo-Samaha et al. 2021), antibody titres (e.g. against Newcastle

Disease Virus – NDV) (Toplu et al. 2014), inter- leukin (IL-10) levels (Sumanu et al. 2023), general assessments of immunity (Ghasemi and Nari 2023;

Biswas et al. 2024), and inflammation markers (Baxter et al. 2020). Lymphoid organ weights also indirectly reflect the immune status.

Molecular markers (beyond HSP70) Research often explores the broader molecu- lar response by measuring the gene or protein expression of other heat shock proteins (HSP60,

HSP90) and heat shock factors (HSF1, HSF3) (Figueiredo et al. 2007; Zhu et al. 2015; Khan et al.

2016; Albokhadaim et al. 2019; KHasti et al. 2025).

Expression of genes related to antioxidant defence (MnSOD, GPx1, GPx4, selenoproteins, GSH-Px), metabolism (mTOR pathway genes: mTOR, S6K1,

4EBP1; IGF-1; amino acid transporters; aqua- porins – AQPs), and selenoprotein P (SelP) are also evaluated (Zhu et al. 2015; Khan et al. 2016;

Kumbhar et al. 2018; Elnesr et al. 2019; Han et al.

2019; Alhotan et al. 2021; Dalolio et al. 2024). Heart histopathology provides a tissue-level insight (Khan et al. 2016).

Tissue integrity and morphology Some studies delve into structural changes, examining intestine morphology (villus height, crypt depth) (Erfani et al. 2021) and intestinal in- tegrity markers (Baxter et al. 2020). In summary,

HSP70 serves as a central biomarker for cellular stress response in poultry research, consistently evaluated alongside core indicators of physiological stress (corticosterone, body temperature), growth performance (BW, BWG, FI, FCR), and oxida- tive stress/antioxidant status (MDA, SOD, GSH).

It is further contextualised by measures of immune function, haematology, blood biochemistry, car- cass traits, and the expression of related molecular pathways (other HSPs/HSFs, antioxidant genes, and metabolic genes).

Heat shock protein 70 modulation This systematic review comprehensively assessed the impact of nutrient supplements on heat stress modulation in broilers of different growth stages, specifically with respect to HSP70, and the relation- ship with other parameters such as physiological and hormonal stress markers, growth and produc- tion performance, antioxidant status and oxidative stress, haematological and biochemical blood pa- rameters, immune and inflammatory markers, mo- lecular markers (beyond HSP70), tissue integrity and morphology. Various research gaps were iden- tified with recommendations for future research.

The induction of heat shock protein 70 (HSP70) is a hallmark response to thermal stress in broil- ers, with most studies in this review identifying it as a critical biomarker for evaluating the heat stress resilience. Numerous interventions, from di- etary antioxidants to amino acid supplements, have demonstrated the potential to attenuate HSP 70 ex- pression. For instance, supplementation of ascorbic acid (AA) significantly downregulated HSP 70 and pro-inflammatory cytokines, though the weight gain was not significantly affected in the short post- stress recovery period (Abo-Samaha et al. 2021).

Similarly, organic selenium (Se) and vita- min E (VE) combinations consistently reduced

HSP 70 mRNA or protein levels across multiple studies (Kumbhar et al. 2018; Bora et al. 2024), underscoring their synergistic antioxidant roles.

Importantly, tissue specificity emerged as a recur- ring theme. HSP 70 expression was often assessed in breast muscle (Dalolio et al. 2024), or hypothala- mus (Figueiredo et al. 2007), reflecting differential stress responses. However, the lack of protein-level validation in many mRNA-based studies (e.g. Han et al. 2019; KHasti et al. 2025) limits interpretabil- ity, highlighting the need for multi-level molecular assessments at different stages of broiler chicken production (starter and finisher phases), which needs to be investigated.

151 Czech Journal of Animal Science, 71, 2026 (4): 141–158

Review https://doi.org/10.17221/1/2026-CJAS Antioxidant and micronutrient interventions

Antioxidants such as vitamins C and E, selenium, chromium (Cr), and zinc (Zn) have shown promise in enhancing thermotolerance by reducing oxida- tive damage and HSP70 expression. Dalolio et al. (2024) demonstrated that chromium methionine (CrMet) enhanced metabolic efficiency and down- regulated HSP70, while Zhu et al. (2015) showed that organic manganese improved antioxidant enzyme activity and reduced HSP70 expression in muscle tissues. Organic mineral sources, particu- larly selenium and zinc, appear superior to their inorganic counterparts in terms of bioavailability and stress mitigation (Mahmoud and Edens 2005;

Li et al. 2021). Nevertheless, Bora et al. (2024) cau- tioned that the form of supplementation (organic vs inorganic) was often unspecified, and controlled comparisons were scarce.

Role of amino acids and in ovo interventions Specific amino acids, notably methionine, leu- cine, and tryptophan, have been investigated for their regulatory effects on HSP70. Han et al. (2019) found that the in ovo l-leucine injection modu- lated arginine and lysine metabolism, attenuating

HSP70/90 expression via mTOR signalling, albeit in a tissue-specific manner. Methionine analogues such as MHA [2-hydroxy-4-(methylthio)butanoic acid] showed superior effects under heat stress compared to dl-methionine, with improved anti- oxidant profiles and lowered HSP70 (Erfani et al.

2021). Despite these promising results, many in ovo studies lacked the post-hatch performance data (e.g. Elnesr et al. 2019; Han et al. 2019), which limits practical translation.

Phytogenic and hormonal modulation of HSP 70 Phytogenic-like compounds including taurine (Belal et al. 2018) and citrulline (amino acid deriva- tives; Ghasemi and Nari 2023) have demonstrated efficacy in modulating HSP70 expression, often accompanied by improved growth or antioxidant status. Likewise, serotonin and leptin have been investigated for their central roles in stress adapta- tion. KHasti et al. (2025) reported that serotonin increased HSP70/90 mRNA expression, indicating a potential neuromodulatory effect, while leptin was shown to act indirectly via the central nervous system to regulate HSP70 (Figueiredo et al. 2007).

Although these findings open novel avenues, most hormonal studies were constrained by small sample sizes and limited mechanistic validation at the pro- tein level, necessitating further work.

Synergistic supplementation strategies A growing body of evidence supports the use of combined supplementation strategies to achieve maximal mitigation of heat-induced stress. For example, vitamin E + selenium (Bora et al. 2024) showed superior HSP70 suppression versus single agents, while the in ovo injection of sulphur amino acids (methionine and cysteine) in heat-stressed embryonated eggs enhanced antioxidant markers and GSH-Px expression while reducing HSP70 ex- pression, corticosterone levels, and plasma lipids in newly hatched broilers (Elnesr et al. 2019). This suggests it may be an effective strategy to miti- gate heat and oxidative stress effects; moreover, combining MHA and citrulline restored the per- formance and minimised the oxidative stress mark- ers better than either supplement alone (Ghasemi and Nari 2023). Similarly, Khan et al. (2016) found that combining selenium and probiotics suppressed

HSP70 expression and improved cardiac resilience, suggesting synergistic effects at both molecular and physiological levels. Despite this, few studies explored interaction effects formally, and dose-re- sponse data were often missing or limited (Baxter et al. 2020; Kadim and Alhamadani 2023).

Early life conditioning and developmental plasticity

Early thermal conditioning has emerged as a non- nutritional strategy to build the heat resilience.

Toplu et al. (2014) demonstrated that early expo- sure to 36 °C for 24 h at 5 days of age was more effective than vitamin C in reducing HSP70 and improving the tissue protection. This finding sup- ports the theory of developmental plasticity, where- in controlled early-life stress induces adaptive responses that persist later in life. Nevertheless,

152 Review Czech Journal of Animal Science, 71, 2026 (4): 141–158 https://doi.org/10.17221/1/2026-CJAS long-term implications and performance metrics were not reported in most such studies, leaving critical gaps regarding practical utility.

Inverse association with performance and growth Elevated HSP70 (mRNA or protein) is often as- sociated with impaired growth performance under heat stress (HS). Studies report significant increas- es in HSP70 alongside reduced body weight (BW), body weight gain (BWG), and feed efficiency (FCR) in HS groups compared to thermoneutral controls (Abo-Samaha et al. 2021). Conversely, interven- tions that successfully reduce HSP 70 expression or concentration [e.g. ascorbic acid (AA), chro- mium methionine (CrMet), selenium, betaine, vi- tamins, and melatonin] consistently demonstrate concomitant improvements in BW, BWG, FCR, car- cass yield, and reduced mortality (Mahmoud and

Edens 2005; Mishra et al. 2015; Elnesr et al. 2019;

Erfani et al. 2021; Ghasemi and Nari 2023; Biswas et al. 2024; Bora et al. 2024; Dalolio et al. 2024). This inverse relationship positions HSP 70 as a reliable biomarker of HS-induced performance loss.

Strong link to physiological stress markers The HSP70 elevation is frequently associated with key stress hormones, particularly corticoste- rone (CORT). Higher HSP70 consistently coincides with higher plasma CORT levels (Mahmoud et al.

2004a,b; Toplu et al. 2014; Mishra et al. 2015), and interventions reducing HSP70 also lower CORT (Elnesr et al. 2019; Baxter et al. 2020; Ghasemi and

Nari 2023; Dalolio et al. 2024). HSP70 also correlates positively with the stress leukogram marker het- erophil/lymphocyte (H/L) ratio (Toplu et al. 2014) and often with hyperglycaemia and dyslipidaemia (Toplu et al. 2014; Elnesr et al. 2019; Erfani et al.

2021). This underscores the role of HSP70 as part of the integrated physiological stress response.

Association with oxidative stress and antioxidant response

Elevated HSP70 is frequently associated with increased oxidative damage (higher malondial- dehyde – MDA) and often with reduced antioxi- dant enzyme activity (SOD, CAT, GPx, GSH) (Zhu et al. 2015; Albokhadaim et al. 2019; Sumanu et al.

2023). Interventions that mitigate oxidative stress (e.g. Mn, Zn, vitamins, selenium, melatonin, and betaine) typically reduce both MDA and HSP70 while enhancing the antioxidant enzyme activ- ity (Mahmoud and Edens 2005; Zhu et al. 2015;

Kumbhar et al. 2018; Elnesr et al. 2019; Li et al.

2021; Ghasemi and Nari 2023). More importantly, injecting sulphur amino acids (methionine and cys- teine) into embryonated eggs exposed to heat stress improved the antioxidant status and upregulated

GSH-Px gene expression, while decreasing HSP70 expression, corticosterone levels, and lipid parame- ters in newly hatched broiler chicks. This approach shows a potential for reducing the negative impacts of heat and oxidative stress. This highlights the in- terconnectedness of heat shock response and other oxidative stress pathways.

Complex relationship with immunity The link between HSP70 and immune outcomes is less consistent. While elevated HSP70 often asso- ciates with immune suppression, e.g. reduced anti- body titres (Toplu et al. 2014), HSP70 reduction via supplements sometimes correlates with improved immune organ weights like spleen (Bora et al. 2024) or humoral/cell-mediated immunity (Ghasemi and

Nari 2023; Biswas et al. 2024) and reduced inflam- mation (Baxter et al. 2020). However, some stud- ies found no significant effect on lymphoid organs despite HSP70 reduction (Zhu et al. 2015; Dalolio et al. 2024). The pro-inflammatory cytokine ele- vation alongside HSP70 (Abo-Samaha et al. 2021) suggests context-dependent roles, potentially re- lated to the severity/duration of stress or tissue lo- cation. Anti-inflammatory IL-10 showed a variable response (Sumanu et al. 2023).

The magnitude and direction of HSP70 change, and its relationship with outcomes, can be tissue specific. For example, leucine supplementation re- duced HSP70 in the diencephalon and muscle but increased it in the liver under HS (Han et al. 2019).

Similarly, the efficacy of interventions in modu- lating HSP70 and associated outcomes varies.

Optimal doses are often identified, for example, CrMet at 0.80 mg/kg (Dalolio et al. 2024), betaine at 600 mg/kg (Biswas et al. 2024), and some supple- 153

Czech Journal of Animal Science, 71, 2026 (4): 141–158

Review https://doi.org/10.17221/1/2026-CJAS ments show gender-dependent effects (Figueiredo et al. 2007). Serotonin increased HSP70 mRNA ex- pression while reducing neuronal damage (KHasti et al. 2025), contrasting with the general inverse performance relationship.

Critical considerations While there exist strong associations, most data demonstrate correlation. The HSP70 induction is a protective mechanism; its reduction often indi- cates successful stress mitigation rather than being the direct cause of improved outcomes. Outcomes depend on whether HSP70 is measured at the mRNA or protein level, in specific tissues (liver, muscle, brain, blood) or systemically (serum), and the timing relative to stress exposure. HSP70 is one component of a complex network, e.g. interacting with other HSPs, HSFs, antioxidant systems, and hormones like IGF-1 and melatonin (Dalolio et al.

2024). Its relationship with outcomes reflects this integration. Genotype, age, diet composition, and specific HS conditions significantly influence re- sults, making direct comparisons between studies challenging. Some studies lack direct performance measures alongside molecular data (Zhu et al. 2015;

Han et al. 2019).

LIMITATIONS AND RESEARCH GAPS A major limitation across studies is the inconsis- tent reporting of performance metrics (e.g. weight gain, feed conversion ratio), despite their centrality to poultry production. Furthermore, many stud- ies relied solely on the mRNA analysis of HSP70 without corresponding protein validation, weaken- ing the physiological inference. Additionally, small sample sizes, single genotypes, and short trial du- rations undermine the generalisability of several findings (e.g. Sumanu et al. 2023; Bora et al. 2024;

KHasti et al. 2025). Environmental variability, such as natural vs controlled heat stress, also compli- cates cross-study comparison. Moreover, the form of nutrient supplements (e.g. organic vs inorganic

Se) was inconsistently described, calling for stan- dardisation.

Significant knowledge gaps persist regard- ing HSP70 dynamics and heat stress responses across broiler development stages. While most research focuses on acute heat stress during fin- isher phases (21–42 days), critical deficiencies exist in understanding early life and chronic exposure scenarios. The role of HSP70 in immune devel- opment, thermoregulation, and growth plasticity during the vulnerable post-hatch and chick stages remains poorly defined, despite hints of early im- pacts (Erfani et al. 2021). Similarly, the potential for the in ovo modulation of HSP70 (via nutrients or temperature) to program lasting stress resilience and performance outcomes requires investigation, as embryonic studies like Han et al. (2019) often lack performance linkages. Furthermore, the effects of prolonged, subclinical heat exposure on HSP70 adaptation and tolerance mechanisms throughout the growth cycle are inadequately characterised.

Beyond stage-specific gaps, the causal relation- ships between HSP70 modulation and physiologi- cal outcomes remain elusive. While there exist correlations, it is unclear whether reduced HSP70 expression directly enhances growth or merely serves as a biomarker of successful stress mitiga- tion through other pathways, such as enhanced antioxidant capacity or hormone regulation. The tissue-specific roles of HSP70 (e.g. in liver metab- olism versus muscle growth versus neural stress integration) and their varying importance across development stages are also underexplored, despite evidence of tissue divergence (Han et al. 2019).

The mechanistic dissection of HSP70 interactions with other heat shock proteins (HSPs), heat shock factors (HSFs), and key signalling pathways (e.g. mTOR, IGF-1, selenoproteins) is needed through- out growth.

The relationship between HSP70 and immune function presents unresolved complexities.

Conflicting findings exist on whether HSP70 el- evation suppresses or activates specific immune functions (e.g. innate vs adaptive) at different ages (Abo-Samaha et al. 2021 vs Toplu et al. 2014), and its impact on gut integrity and microbiota com- position, critically linked to immunity, across de- velopment stages is largely unknown, with Baxter et al. (2020) being a notable exception. Translating mechanistic understanding into practical strategies faces hurdles. Standardised, stage-optimised pro- tocols (e.g. for starter vs grower phases) for dosing key nutrients and compounds (e.g. betaine, CrMet, vitamins) are lacking, despite the clear evidence of dose-dependency (Biswas et al. 2024; Dalolio et al. 2024). Synergistic approaches combining anti- 154

Review Czech Journal of Animal Science, 71, 2026 (4): 141–158 https://doi.org/10.17221/1/2026-CJAS oxidants, amino acids, probiotics, and management practices require rigorous, stage-specific efficacy testing. Furthermore, the long-term consequences of early-life HSP70 modulation on productivity and welfare remain unknown.

Addressing these gaps necessitates specific meth- odological improvements: standardising HSP70 reporting (units, tissues), conducting dose-opti- misation studies for emerging agents (e.g. mela- tonin), and directly comparing embryonic versus grower-phase intervention efficacy. The current research focus on Ross 308/Cobb strains in finish- er stages underscores the limited understanding of HSP70 modulation in early development and non-industrial genotypes. Future studies must pri- oritise expanding embryonic and starter-phase co- horts, including both sexes and diverse genotypes, and ensuring adequate sample sizes and replication.

FUTURE DIRECTIONS Figure 3 gives a summary of the future directions in understanding the modulation of heat stress via nutritional strategies based on HSP 70. To address the critical knowledge gaps, future research must adopt a comprehensive developmental perspective, moving beyond isolated studies in finisher broil- ers. Longitudinal investigations profiling HSP70 expression (mRNA/protein), stress hormones (e.g. corticosterone – CORT), redox status, and immune markers from embryo to market age under con- trolled thermal challenges are essential. These stud- ies should establish stage-specific HSP70 thresholds that differentiate adaptive responses from detri- mental stress levels, providing crucial benchmarks for intervention timing and efficacy evaluation.

Understanding the mechanistic approaches re- quires leveraging multi-omics studies (transcrip- tomics, proteomics, and metabolomics) to map

HSP70-interacting networks and identify key re- silience drivers across tissues and developmental stages. Concurrently, exploring the epigenetic regulation (e.g. DNA methylation) of HSP70 and associated genes will illuminate mechanisms un- derlying the lifelong stress tolerance programming.

This mechanistic foundation is vital for designing targeted nutritional interventions optimised for specific developmental windows: in ovo delivery for embryonic programming, neonatal inoculation

Figure 3. Future directions in research for the modulation of heat shock protein 70 at different stages of broilers

Targeted nutritional strategies • Stage-specific supplementation

• Synergistic combinations to target HSP70, redox balance and immune function

Optimising sulphur amino acid (SAA) interventions • Improving on dosage and administration routes

• Using different combinations of SAA Genetics and sustainability

• HSP70-linked markers for resilience • Cost effective strategies for real world applications

Precision management • Cyclic thermal conditioning

• Age-tailored ventilation systems HSP70 modulation

Gut health and immunity • HSP70 roles on intestinal barriers function and lymphoid organs

Multi-omics mechanistic insights • HSP70 roles on intestinal barrier function and lymphoid organs

Developmental framework • Longitudinal HSP70 thresholds from embryo to market age

• Benchmark for timing and efficacy of interventions

155 Czech Journal of Animal Science, 71, 2026 (4): 141–158

Review https://doi.org/10.17221/1/2026-CJAS or starter diets enhancing early thermotolerance, and finisher strategies preserving carcass yield un- der heat stress.

Advanced mitigation strategies should focus on developing and testing synergistic combina- tions of compounds (e.g. methionine + cysteine or betaine + vitamin C + selenium) at optimised, stage-specific doses to holistically address per- formance, HSP70 dynamics, redox balance, and immunity. The potential of candidate modulators – such as serotonin precursors (KHasti et al. 2025), and phytogenics to influence HSP70 across ages warrants thorough exploration. In addition, the in ovo inoculation of SAA requires further investi- gation due to its potential in heat stress modulation (Elnesr et al. 2019). Furthermore, integrating these nutritional approaches with precision management techniques (e.g. cyclic heat conditioning, age-tai- lored ventilation control) offers a promising avenue for enhanced resilience.

A dedicated focus on immunity and gut health is  paramount. Research must define how the

HSP70 modulation influences gut barrier integrity, microbiome composition, and mucosal immunity at different stages, building upon initial findings like Baxter et al. (2020). Clarifying the dual role of HSP70 in pro- and anti-inflammatory signalling within key immune organs (e.g. thymus, spleen) during development is also critical to resolve ex- isting inconsistencies. Finally, breeding and sus- tainability efforts should aim to identify genetic markers linked to favourable HSP70 expression patterns and associated stress resilience traits for selective breeding programmes. Concurrently, rig- orous assessment of the long-term sustainability and cost-efficacy of HSP70-modulating strategies across the entire production cycle is necessary for practical implementation.

CONCLUSION This systematic review establishes heat shock protein 70 (HSP70) as a critical biomarker and therapeutic target for mitigating heat stress (HS) in broilers. Collectively, the evidence demonstrates that targeted nutritional interventions, particular- ly those rich in antioxidants (e.g. vitamins C, E, and selenium) and balanced in key amino acids (sulphur-containing, branched-chain ones) effec- tively modulate HSP70 expression and enhance thermotolerance, thereby preserving productivity under thermal challenge. However, the interactions between nutrients and HSP70 are complex, exhib- iting significant tissue specificity and dependence on stress severity and duration.

While these strategies offer substantial promise, a deeper understanding of long-term physiologi- cal effects, tissue-specific molecular interactions, inoculation route and timing, and the metabolic trade-offs associated with HSP70 induction is es- sential. Moving forward, translating this knowledge into practical, scalable solutions requires future research focused on (i) refining stage-specific nu- tritional protocols to optimise the protective ben- efits of HSP70 while minimising its energetic costs; (ii) establishing standardised methodologies for

HSP70 assessment; and (iii) validating approaches across diverse genotypes and commercial settings.

Implementing these evidence-based, nutritionally modulated HSP70 strategies is paramount for the poultry industry to enhance broiler resilience, wel- fare, and economic sustainability in the face of es- calating global temperatures.

Acknowledgement The authors would like to express gratitude to the

Ministry of Higher Education Malaysia through the Higher Institution Centre of Excellence (HICoE) scheme (UPM.ITAFos.800-3/8/HICoE (Phase II), all staff from Laboratory of Sustainable Animal

Production and Biodiversity, Institute of Tropical

Agriculture and Food Security, Department of  Preclinical Sciences, Faculty of  Veterinary

Medicine, University of  Putra Malaysia and Nigerian Tertiary Education Trust Fund (TETfund) for their support.

Conflict of interest The authors declare no conflict of interest.

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Received: January 4, 2026 Accepted: March 26, 2026

Published online: April 30, 2026