As global temperatures continue to rise, heat stress (HS) has become a significant challenge in animal production, particularly in tropical or subtropical regions. A study published over 2 decades ago estimated $128 - $165 million annual economic losses from HS in the poultry industry in the United States, with projections indicating these losses will continue to increase (St-Pierre et al., 2003). The commercial poultry strains selected for rapid growth rates or high egg production are negatively impacted by HS because of their increased metabolic rates, higher heat production, and reduced tolerance to heat (Gogoi et al., 2021; Nawaz et al., 2023). The adverse effects of HS on chickens housed in cage system include slower growth, lower egg production rate and quality, greater disease susceptibility, and higher mortality rates (Tang et al., 2021; Kim et al., 2024). Under the HS condition, chickens exhibit various behavioral and physiological responses, such as panting and wing lifting as means of evaporative cooling (Richards, 1970; Kim et al., 2021), and reduced feed intake as an adaptive strategy to decrease the internal heat generation (Renaudeau et al., 2012; de Souza et al., 2016). However, panting causes hyperventilation and significant carbon dioxide loss, which eventually results in respiratory alkalosis characterized by decreased H⁺, increased HCO₃ ⁻, and elevated blood pH (Teeter et al., 1985; Franco-Jimenez et al., 2007). Evidence suggests that acid-base imbalance resulted from HS affects the eggshell quality by decreasing calcium and carbonate secretion in shell gland (Mongin, 1978; Oluwagbenga and Fraley, 2023). It has been demonstrated that respiratory alkalosis developed because of hyperventilation adversely affects the activity of an enzyme carbonic anhydrase, important for eggshell formation (Ahmad and Sarwar, 2006). However, the severity of acid-base disturbance and the importance of maintaining acid-base homeostasis during HS condition in chickens is less appreciated. Moreover, HS in chickens is often accompanied by increased mitochondrial reactive oxygen species (ROS) production, ultimately developing into oxidative stress (Mujahid et al., 2005; Akbarian et al., 2016). Although low levels of ROS are important in various signaling pathways, over production of ROS results in oxidative damage to carbohydrate, proteins, fatty acids and DNA (Habashy et al., 2019; Katerji et al., 2019). In chicken ovary, oxidative stress was shown to promote follicular cell apoptosis by downregulating genes involved in cell survival and hormone synthesis while upregulating the pro-apoptotic factors (Wang et al., 2021).

To improve layer chicken welfare, the U.S. egg industry targets to converting 66% of its national hen inventory from conventional cage (CC) housing to cage-free (CF) housing by 2026 (UEP, 2024). However, studies have reported that the current CF system is 36% more expensive (Matthews and Sumner, 2015) and 7–8 times dustier (Zhao et al., 2015), emits higher levels of ammonia, and enriches more pathogens (Jones et al., 2015) than the CC system. While the negative impacts of HS on the productivity and wellbeing of laying chickens are well documented, the mechanistic basis underlying such outcomes is still poorly understood. Specifically, very few studies have directly corelated the physiological changes and transcriptomic responses with the observed reduction in egg production and quality during HS conditions (Kim et al., 2024; Habashy et al., 2025). Additionally, most of the studies to date have been conducted using CC systems, however, as the industry is shifting toward the CF system, it is essential to characterize how HS impacts laying chickens under these newer housing conditions. In this study, we investigated the effects of chronic HS on commercial layers housed in a CF system, with particular emphasis on egg production, egg quality, feed intake, blood physiological parameters and selected gene expressions. Importantly, we have corelated the physiological changes in blood parameters and transcriptomic alterations of key genes in uterus (shell gland) directly involved in egg formation to determine how HS-mediated molecular alterations contribute to the observed production losses. The findings of this study will help identify nutritional and managemental interventions aimed at correcting the observed biochemical and molecular alterations, thereby mitigating the adverse effects of chronic HS in commercial laying chickens.

This study was conducted to evaluate the effects of chronic HS on growth performance, egg production, egg quality, blood biochemical indices, and the expression of selected genes in shell glands in Hy-Line W-36 birds housed in a CF system. Our findings revealed that HS negatively influenced body weight, feed intake, early egg production, egg weight, and shell quality. In addition, HS altered blood acid–base balance and iCa levels as well as downregulated key shell gland genes involved in mineralization. These findings provide further insight into how prolonged HS compromises the productivity and physiology of commercial layers, particularly under CF system.

In the present study, chronic HS significantly reduced BW in laying hens compared to TN controls. This reduction can be attributed to the combined effects of decreased feed intake and the additional energy expenditure required for thermoregulation. Under thermal stress, birds redirect available energy toward dissipating excess heat rather than growth (Papanikolaou et al., 2025), resulting in a negative energy balance. Previous studies have reported that HS leads to the mobilization of body reserves and breakdown of muscle protein to meet production demands (Furukawa et al., 2016; Ma et al., 2021), which aligns with the reduction in BW observed in our trial. Although BW tended to be lower in HS birds at weeks 1 and 3, no statistically significant differences were observed compared to TN birds. In contrast, FI in HS birds remained significantly lower throughout the study period. Despite BW and FI are generally corelated, reductions in FI might not always perfectly translate into proportional BW loss as it can also be affected by residual feed efficiency traits and metabolic needs (Willems et al., 2013; Lewis and Emmans, 2020).

The significant reduction of FI in HS birds compared to TN birds might be related with the anorexic effects of heat exposure. This suppression of appetite has been linked to elevated secretion of leptin and adiponectin, as well as the expression of their receptors. Leptin, in turn, activates the hypothalamic axis and suppresses feed intake (Bernabucci et al., 2009; Morera et al., 2012). Despite this reduction of FI observed in the HS group, the FCR remained comparable with the TN group in our study, which aligns with reports from previous findings (Bozkurt et al., 2012; Kim et al., 2020). However, there are lots of inconsistent findings regarding the effect of HS on feed efficiency. Some studies have demonstrated a worsened FCR under HS conditions (He et al., 2018), whereas other studies have observed an improved FCR in HS birds (Barrett et al., 2019; Kim et al., 2024) which has been linked with the mobilization of body reserves to sustain egg production (Song et al., 2012). These discrepancies in FCR are likely the results of different experimental conditions, including the duration and severity of heat exposure, as well as differences in bird age and housing system.

The decrease in egg weights observed in HS chickens compared to controls in our study is consistent with previous findings reporting a similar trend of egg weight reduction under HS conditions (Mashaly et al., 2004; Allahverdi et al., 2013). This reduction in total egg weight has been linked with the reduction in yolk weight (Chen et al., 2023) and albumen weight (Barrett et al., 2019). In this study, HS birds exhibited an initial decline in HDEP% during the first 3 weeks, which then recovered by fourth week. Previous studies have also reported that continuous exposure to HS severely decreases HDEP% of layer chickens (Rozenboim et al., 2007; Kim et al., 2024). In another study, only continuous HS significantly reduced HDEP%, while cyclic HS (8 h a day) did not significantly affect the HDEP% compared to controls (Mashaly et al., 2004). Together, these studies suggest that the duration of heat exposure plays a critical role in determining its effects on laying performance.

The detrimental effects of HS on eggshell quality are well documented, and have been associated with acid-base imbalance, impaired nutrient absorption, and altered calcium metabolism (Mahmoud et al., 1996; Bahadoran et al., 2018). In this study, eggs from HS birds exhibited significantly lower shell breaking strength and shell weight, which potentially could increase the egg loss during handling and transportation. Together, these results confirm that prolonged HS compromises eggshell integrity, in line with earlier reports of decreased shell strength and thickness (Kim et al., 2020; Kim et al., 2022).

Blood biochemical parameters may provide valuable insights into the physiological condition associated with the egg mass and eggshell formation. Previous research has demonstrated that HS disrupts calcium metabolism and decreases calcium concentration in blood plasma, leading to thinner eggshells (El-Tarabany, 2016; Cruvinel et al., 2021). Herein, we observed reduced blood pH in HS birds that could be the result of multiple physiological disturbances, including hyperventilation-induced respiratory alkalosis followed by metabolic compensation, impaired renal function reducing acid-base regulation, and increased lactic acid accumulation due to tissue hypoxia (Aryal et al., 2025). However, the PCO₂ level was increased in HS birds despite the expectation of excessive CO₂ loss due to panting. This paradox may indicate a shift from respiratory to metabolic disturbances during prolonged HS. Initially, panting reduces PCO₂, leading to respiratory alkalosis, but over time, fatigue of respiratory muscles, reduced ventilation efficiency, or impaired gas exchange due to pulmonary damage may hinder CO₂ elimination (Brugaletta et al., 2022; Wu et al., 2023). Furthermore, increased metabolic rate and potential tissue hypoxia under HS may contribute to elevated endogenous CO₂ production, outweighing respiratory losses and ultimately resulting in higher circulating PCO₂ concentrations (de Souza et al., 2016; Lian et al., 2021). In addition, the increased level of iCa observed in HS birds may be attributed to the reduction in blood pH in those birds. There is an established inverse relationship between Ca²⁺ levels and blood pH (Axelrod, 1961; McCudden, 2013). As blood pH decreases (acidosis), proton ion (H⁺) competes with Ca²⁺ for binding sites on plasma proteins, particularly albumin, resulting in a greater proportion of calcium remaining in its free, ionized form (Figure 7).

HS condition induces excessive ROS production, resulting in oxidative damage to proteins, lipids, carbohydrates, and DNA (Emami et al., 2020). One possible mechanism by which HS results in oxidative stress in internal organs, including the intestinal and reproductive tract, is the redistribution of blood flow towards the external surface (skin) of the body to promote heat dissipation. This compensatory response reduces blood flow to internal organs, resulting in hypoxic condition and subsequent development of oxidative and nitrosative stress (Rostagno, 2020). Moreover, study from Li et al. (2020) suggested that HS-induced elevation in corticosterone likely increases oxidative stress, that in turn activated the FasL/Fas and TNF-α apoptotic pathways in follicular cells, ultimately impairing follicular development and egg production. Similarly, separate study has demonstrated that oxidative stress reduced the expression of genes associated with cell survival and steroidogenic function, including SIRT1, while increasing the expression of pro-apoptotic regulators P53 and FoxO1 in chicken ovary, thereby reducing ovarian function and laying performance (Wang et al., 2021). In the present study, the observed downregulation of genes associated with uterine ion transport and biomineralization (CALB1, SLC4A9, and OPN) may similarly reflect oxidative injury and subsequent apoptosis occurring within uterine tissues under HS condition. Consistent with this, a previous study reported that HS downregulated key uterine ion transporter genes (ITPR3, SLC4A4, and SLC4A9), impairing uptake of Ca²⁺ and HCO₃ ⁻ into uterine epithelial cells (Bahadoran et al., 2018). This disruption may explain the elevated circulating iCa observed in our study, alongside reduced egg weight and weakened shell strength. Consistently, reductions in eggshell quality have also been linked to decreased CALB1 localization in eggshell gland and intestine (Ebeid et al., 2012) and to altered serum electrolytes levels during HS (Kim et al., 2024).

In conclusion, chronic HS exhibited detrimental effects in layer chickens housed in CF system. These adverse effects include reduced BW, feed consumption, initial egg production rate, egg weight, and shell breaking strength. These changes were accompanied by acid–base imbalance and altered calcium metabolism, reflected as lower blood pH and elevated PCO₂ and iCa levels. Future studies are warranted to delineate the mechanism of HS-induced oxidative injury in chicken reproductive tract and underlying alterations in production performance, immune responses, disease susceptibility and gut microbiota diversity in HS chickens raised in CF system. Moreover, development and field-scale evaluation of effective nutritional supplements and physical cooling methods in commercial settings will be critical for improving resilience to HS in commercial laying operations.