Rapidly growing broilers are susceptible to various environmental stressors, including cold stress, which is one of the limiting factors for broiler production in northern China. Previous studies have indicated that the increase in cardiovascular disease mortality is positively correlated with a decrease in environmental temperature [1]. Cold stress can also lead to heart hypertrophy, interstitial fibrosis and reduced heart ejection fraction [2]. It is worth noting that exposure to cold stress cause neuroendocrine disruption and increased heat production, accompanied by the generation of heart oxidative stress [3]. Additionally, cold stress has been reported to impair heart energy status by reducing heart energy reserves and fatty acid oxidation capacity in broilers [4].

The ability of broilers to respond to cold stress depends on the appropriate involvement of the central and peripheral nervous systems, coordinating hormonal and immune regulation among multiple organ systems to establish homeostasis [5]. The hypothalamic-pituitary-adrenal (HPA) axis and hypothalamic-pituitary-thyroid (HPT) axis are integral components of the neuroendocrine system, secreting hormones such as corticosterone (CORT) and liothyronine (T3) to regulate responses to cold stress, including energy balance, thermoregulation and cold-induced thermogenesis [6]. CORT serves as the primary effector hormone of the HPA axis, reflecting the level of HPA axis activation under cold stress and influencing nearly all physiological systems, thereby redistributing energy or nutrient resources within biological systems. For instance, previous research has shown that cold stress significantly increases CORT levels in the blood of piglets and mice, accompanied by elevated levels of glucose and lipid substances, aiding in their adaptation to cold stress [7]. Cold stress also activates the HPT axis, subsequently leading to elevated levels of T3 and thyroxine (T4) in the blood [8]. Xie's study [9] found that cold stress accelerates the conversion of T4 to T3 in laying hens, thereby increasing blood T3 level to enhance catabolic metabolism, increase heat production, and promote the synthesis of intracellular proteins, RNA and DNA, thereby resisting cold stress damage. Therefore, investigating the changes in neuroendocrine hormones in broilers under cold stress is of significant importance for studying their physiological responses to cold stress and metabolic changes.

The adenosine monophosphate (AMP)-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) pathway is essential for restoring internal balance following stress stimulation. It is generally believed that when there is a relative increase in intracellular AMP or adenosine diphosphate (ADP) levels, the AMPK pathway stimulates downstream genes, such as peroxisome proliferators-activated receptor α (PPARα), carnitine palmitoyltransferase1 (CPT1) and acyl-CoA oxidase (ACO), thereby inhibiting synthetic metabolic pathways and promoting catabolic pathways to replenish adenosine triphosphate (ATP) supply in broilers [10]. Zhou et al found that cold stress induces enhanced expression of the AMPK gene in the intestines of broilers and upregulates the expression of intestinal tight junction genes, demonstrating a potential link between cold stress, AMPK and intestinal barrier function [11]. Study also has found that the expression levels of AMPKα, PPARα and CPT1 in broiler muscle were increased under nutrient deficiency conditions, which was thought to be related to the increased level of fatty acid oxidation [12]. As important downstream genes and regulatory nodes of the AMPK pathway, mTOR and its target genes exert effects that are precisely opposite to those of the AMPK pathway. Li et al [4] reported that in mice fed high glucose, the liver AMPK pathway was inhibited, while sterol-regulatory element binding protein (SREBP) and its downstream stearoyl-coA desaturase (SCD) and acetyl-coA carboxylase (ACC) were activated, resulting in non-alcoholic fatty liver in the mice. RNA sequencing results on chicken DF-1 cells revealed downregulation of genes including oxidative phosphorylation, ribosomes and mTOR after low-temperature stimulation. These genes primarily impact ribosomal translation and mitochondrial electron transport in DF-1 cells [13]. Although the role of the AMPK/mTOR pathway in skeletal muscle and liver has been well studied, its role in other metabolically active organs (such as the heart) remains largely unknown.

The detrimental effects of cold stress on heart function have been widely acknowledged, but research on how to alleviate cold-induced heart injury is relatively scarce. However, our previous studies have demonstrated that appropriate intermittent cold stimulation can significantly improve broiler performance and immune function, effectively mitigating acute cold stress (ACS) injury [14–16]. Therefore, we hypothesized that appropriate intermittent cold stimulation could regulate the AMPK/mTOR pathway by affecting neuroendocrine hormone secretion, thereby regulating heart lipid metabolism and enhancing cold adaptability of broilers.

The heart requires a significant amount of energy to maintain its contractile activity. Fatty acids are the preferred substrate for energy metabolism in the heart, with over 70% of energy derived from them [17]. As an organ with high lipid metabolic rate, the heart needs to maintain a high energy conversion efficiency for effective contraction in a low temperature environment [18]. Therefore, heart lipid metabolism is one of the important factors in adapting to cold environments and meeting specific energy demands. However, it remains unclear whether intermittent cold stimulation directly impacts broilers heart lipid metabolism and subsequently their adaptability to ACS.

Evidence suggests that the activity of hormones and molecules related to energy metabolism undergoes seasonal or temperature-induced changes. These processes are crucial for maintaining body temperature and generating heat in warm-blooded animals in cold temperature [19]. T3 is the active metabolite of thyroid hormone. Research indicates that in cold environments, T3 can significantly upregulate the expression of Uncoupling protein 1 (UCP1), leading to the uncoupling of ATP synthesis from oxidative phosphorylation, resulting in the release of energy in the form of heat. Therefore, the increase in T3 concentration in cold environments is believed to contribute to improved cold tolerance in animals [20]. CORT, the primary glucocorticoid in birds, increases under stress conditions to mobilize energy resources for survival, but prolonged exposure to high levels can negatively affect cellular immunity and production performance in broilers [11,21]. In present study, at 36d, both T3 and CORT concentrations in the CS3 group were significantly lower than in the CS0 group, while there were no significant differences in these indicators between the CS5 group and the CS0 group. This suggests that the intermittent cold stimulation protocol is mild and does not activate the HPT axis and HPA axis in broilers. Additionally, research by Wen et al. demonstrates that cold acclimation at 5°C significantly increases serum T3 level in striped hamsters and significantly enhances mitochondrial state-4 respiration and lipid breakdown capacity to maintain body temperature, providing further evidence for the involvement of T3 hormone in the cold acclimation process [22]. At Y12, the T3 levels in the CS3 group and CS5 group were significantly higher than that in the CS0 group. Therefore, we believe that broilers subjected to intermittent cold stimulation in the early stage can quickly activate the HPT axis under ACS conditions to regulate internal energy allocation and adapt to ACS. Ge et al. found that mice exposed to 4°C for 5 hours exhibited significantly higher plasma CORT levels compared to mice kept at room temperature (25°C), accompanied by an increase in the organism's anti-inflammatory levels. This to some extent enhanced the cold adaptability of the mice [23]. However, in a study by Hu, a high level of CORT can induce stress responses in broilers [24]. In present study, at Y12, the CORT levels in the CS3 group and CS5 group were significantly lower than that in the CS0 group. This may be because broilers in the CS3 and CS5 groups may have already adapted to the cold, resulting in a lower degree of activation of the HPA axis and thus not eliciting intense stress responses.

AMPK, a phylogenetically conserved serine/threonine protein kinase, is gaining attention as a crucial pathway for intracellular energy regulation. Its activation relies on the upstream phosphorylation of the α subunit at Thr-172 by LKB1. AMPK activation in the heart leads to downstream changes in gene expression, regulating fatty acid oxidation, glucose transport and protein metabolism, thereby increasing ATP production [25]. PPARα, a downstream gene of AMPKα, maintains cellular energy homeostasis by enhancing the expression of key genes involved in β-oxidation [26]. Experimental evidence demonstrates that mice with chronic AMPK activation exhibit high fatty acid oxidation capacity, leading to decreased triglyceride (TG) levels in the liver [27]. In the study by Wu, crayfish subjected to 9°C cold stress showed significant upregulation of AMPKα gene expression in the hepatopancreas, accompanied by downregulation of lipogenesis gene expression [28]. Furthermore, increased phosphorylation levels of AMPKα can mediate mitochondrial homeostasis, promote the function of CPT1 in fatty acid transport, and alleviate energy stress induced by low temperatures in pigs [29]. In the present study, contrary to previous research results, at 36d, mRNA levels of AMPKα and ACO, as well as the protein level of PPARα in the hearts of broilers in the CS3 and CS5 groups, were significantly reduced compared to those in the CS0 group. This indicates that the AMPK pathway in the hearts of broilers in the CS3 and CS5 groups was not activated, suggesting that intermittent mild cold stimulation does not affect the lipid decomposition capacity of broilers hearts. However, at Y12, compared to the CS0 and CS3 groups, mRNA levels of CPT1 and ACO, as well as the p-AMPKα/AMPKα protein level, were significantly upregulated in the CS5 group. In summary, compared to the CS0 and CS3 groups, the CS5 group is capable of decomposing more lipids for energy supply during ACS, thus preventing lipid metabolism disorders in the heart and maintaining normal function.

It is generally believed that the activation of AMPKα can stimulate TSC, thereby inhibiting the expression of mTOR and suppressing the synthesis of intracellular fatty acids [30]. As a downstream molecule of mTOR, SREBP maintains lipid metabolic homeostasis by positively regulating FAS and ACC [31]. ACC catalyzes the first step in FAS, converting acetyl-CoA to malonyl-CoA, while FAS synthesizes long-chain fatty acids from acetyl-CoA and malonyl-CoA. Therefore, the levels and activity of ACC and FAS determine the capacity for FAS [32,33]. Wang's research found that sustained activation of AMPKα in cold environments attenuates hepatic cell autophagy and apoptosis mediated by the PI3K/mTOR pathway, thereby enhancing the ability of fish to cope with cold stress [34]. Moreover, studies have shown that mTOR knockout in mice leads to disruptions in heart lipid metabolism and dysfunction [35]. Additionally, cold acclimation inhibits the activity of ACC and FAS in the liver of animals, enabling the liver to maintain its capacity to regulate lipid metabolism under high energy demand [32]. At 36d, except for TSC, all other mTOR pathway genes in the CS3 and CS5 groups were significantly lower than those in the CS0 group. This indicates that the intermittent cold stimulation can effectively induce adaptive changes in lipid synthesis metabolism in the hearts of broilers, thereby maintaining the energy homeostasis of broilers to adapt to cold stress. However, at Y12, compared to the 50d, the mRNA levels of mTOR and ACC were significantly downregulated in the CS0 group, while the mRNA and protein levels of FAS were significantly downregulated in the CS3 group. This indicates that the synthesis metabolism of heart fat in both CS0 and CS3 groups of broilers was inhibited by ACS.

Research has shown that the SREBP-SCD axis regulates the biosynthesis of monounsaturated fatty acids, thereby modulating the fluidity of cell membranes to maintain critical biophysical properties [36]. Additionally, the expression of the SCD gene is closely associated with low temperatures and plays a significant role in enhancing the adaptability of organisms [37]. In present study, at Y12, the mRNA expression level of SCD in the CS5 group was significantly higher than CS0 and CS3 groups, and significantly increased compared to 50d. This suggests that broilers subjected to intermittent cold stimulation can effectively mobilize the SREBP-SCD axis in the heart during ACS to enhance cell membrane fluidity and resist cold stress damage.

It is worth noting that studies have shown that hormones from both the HPA and HPT axes can activate the AMPK/mTOR pathway [20,38,39]. Research has revealed that CORT upregulates the gene expression of the AMPKα and mTOR in broilers [24]. Additionally, T3 enhances β-oxidation in mice primary brown adipocytes while diminishing mTOR activity [8]. However, the potential link between cold stress, neuroendocrine responses and heart lipid metabolism requires further investigation. In this study, at Y12, both the CS3 and CS5 groups exhibited significantly higher levels of T3 hormone and p-AMPKα/AMPKα compared to the CS0 group, while the CORT levels in CS3 and CS5 groups were significantly lower than in the CS0 group. Therefore, we speculate that it may be T3 rather than CORT that mediates the changes in gene expression of the AMPK pathway in the hearts of broilers under cold stress, thereby affecting heart fatty acid oxidation capacity. However, the specific regulatory mechanisms involved require further investigation.

The findings of present study showed that intermittent cold stimulation at 3°C lower than the normal rearing temperature for 5 h can regulate hormone levels of the HPT and HPA axis, activate AMPK pathway and inhibit the mTOR pathway to maintain their heart lipid metabolism homeostasis. However, the above regulating effects were not significant with intermittent cold stimulation for 3h. Thus, intermittent cold stimulation at 3°C lower than the normal rearing temperature for 5h could help broilers adapt to the cold temperature environment by promoting heart lipid metabolism.