1. Introduction
Heat stress is the most significant environmental challenge for the modern poultry industry worldwide. Among monogastric animals, birds are particularly susceptible to high temperatures compared to other monogastric animals, which is primarily due to their feather coverage and absence of sweat glands [
1]. Kocaman and Balnave et al. reported that the optimal temperature range for laying hens is typically between 16 and 25 °C [
2,
3]. Heat stress could occur when birds adapted to lower ambient temperatures encounter rising temperatures above 25 °C. This phenomenon leads to detrimental effects on the physiological and performance traits of poultry [
4]. The total annual economic loss caused by heat stress in the United States poultry industry was estimated to be between USD 128 and 165 million [
5]. Additionally, in many countries, poultry routinely experience temperatures reaching 32 °C and higher. Especially in the southern regions of China, where summers are long, the high temperatures disrupt the thermoregulation process in poultry [
6]. Xinyi Huaixiang chicken, a special breed found in the western part of Guangdong Province, southern China, possesses commercial advantages such as huge market demand, extensive feeding, and a huge development prospect. It serves as the favorite chicken breed of local farmers in Guangdong and contributes significantly to egg production [
7].
Overall egg quality is important for both poultry breeders and consumers in the global egg industry. When assessing egg quality, two main aspects are taken into consideration: external and internal traits [
8,
9]. External characteristics, such as egg weight and the condition of the shell, play a critical role in determining consumers’ acceptability [
10]. Simultaneously, the interior parameters of eggs, specifically yolk index, including yolk weight, yolk color, yolk amino acid, and so on, hold significant significance as good nutrition criteria in the egg production industry.
In recent decades, climate change has notably contributed to an increase in the occurrence of hot days. It has been generally thought that high temperatures have detrimental effects on poultry egg production. Many studies have mainly focused on examining the effects of heat stress on the egg’s external qualities, while studies investigating the effects of heat stress on internal egg quality and the underlying mechanisms of local chickens under heat exposure are limited in the present literature. Therefore, this research aimed to investigate the impact of high temperature on the internal egg yolk quality traits, mainly including yolk weight, yolk color, yolk lipids, and yolk amino acid, and the potential mechanisms of Huaixiang chickens.
4. Discussion
This experiment was primarily performed to study the impact of high ambient temperature on the internal egg yolk quality and the potential mechanisms in Huaixiang chicken. The intensity and duration of heat exposure play a crucial role in determining the detrimental effects on these growth parameters [
18]. Previous studies have indicated that heat stress reduced feed intake and affected the digestibility of different diets [
19]. In the current study, our results showed that eggs from the NT group were heavier than those of the high-ambient-temperature group. These results were similar to Star, who reported that heat stress significantly decreased egg weight compared with the hens in the control chamber [
20]. It is also consistent with other reports that have demonstrated that heat stress decreased egg weight in commercial laying hens [
21,
22]. However, these findings are in contrast to the previous results by Barrett et al., which found that the heat-stressed (HS) group had increased egg weight compared to the pre-heat-stressed (pre-HS) group in laying hens [
23]. These differences in results may be attributed to heat stress causing feed intake reduction in chickens, limiting the accumulation of egg nutrients and subsequently reducing egg weight.
Egg yolk color is a crucial visual feature that significantly influences consumer perception and has an essential role in the marketing and quality evaluation of the eggs. Apart from enhancing aesthetic appeal, egg yolk color serves as an indicator of an egg’s nutritional composition. Previous studies showed that yolks with deeper hues generally contain higher levels of essential nutrients, such as vitamins A, D, E, and omega-3 fatty acids [
12,
24]. Additionally, the uniform yellow color of the yolk signifies the health of the chicken flock on the farm. The yolk color primarily depends on the diet of the chickens, specifically the type and amount of consumed xanthophyll pigments [
25]. In our study, we found a significant reduction in yolk color in the high-temperature group (HT) compared with the normal-temperature group (NT) of Huaixiang chickens in the experiment. This finding differs from the study conducted by Gholizadeh, who reported that heat stress had no significant impact on egg color with a basal diet [
23]. One possible explanation for our research findings is that heat stress reduced feed intake and digestive capacity, resulting in decreased pigment intake and ultimately leading to a reduction in yolk color. Furthermore, heat stress may have damaged liver function, consequently decreasing the synthesis of β-carotene and egg yolk pigmentation. Our study also revealed that high-temperature treatment remarkably elevated the yolk triglyceride (TG) level of Huaixiang chicken. Meanwhile, limited studies showed the mechanisms of heat exposure on the yolk lipid profile of laying hens in the literature. Our results indicated that heat stress significantly enhanced lipid deposits in the yolk of Huaixiang chicken. The phenomenon might be due to adipose tissue lacking fat mobilization, while its ability to respond to lipolysis stimuli was reduced [
26].
In order to further explore the underlying mechanisms of heat stress on internal egg yolk quality, we investigated the effect of heat stress on the liver function and hepatic lipid metabolism of Huaixiang chickens. The liver is regarded as an organ that plays a significant role in avian growth and thermoregulation, making it a compelling subject of study due to its high susceptibility to heat stress. Organ weight can serve as an indicator of organ development, thereby reflecting its functionality. In our study, we observed that heat stress remarkably reduced the absolute and relative liver weight in Huaixiang chickens. The results indicated that high temperature detrimentally affects liver growth and development in the local chickens. These findings are consistent with those of Chen et al. who reported that heat stress reduced liver weight in broilers when compared to a control group [
27]. Furthermore, Liu et al. demonstrated that heat exposure for two weeks reduced the liver weight and liver index of the broilers [
28]. The essential cause behind the harmful influence of high temperature is the reduction in feed intake and nutrients available for the organ growth. In contrast, Ma et al. demonstrated a significant increase in the relative liver weight following heat exposure in broiler chickens when compared to the normal-temperature group [
29]. This abnormal increase in liver weight could be attributed to compensatory hypertrophy in response to heat stress to make up for the worse liver function caused by heat exposure.
In poultry, the liver plays a pivotal role as the primary organ for fat synthesis. To gain insights into the impact of heat stress, we conducted histological analysis of the liver using hematoxylin and eosin (HE) staining. Our results unveiled significant vacuolar degeneration within hepatocytes in the HT group of Huaixiang chickens. Similar to our findings, Liu et al. observed that heat stress induced liver tissue damage, including the infiltration of lymphocytes and neutrophils in broilers [
28]. Given the liver’s crucial role in digestion and nutrient absorption, such hepatic damage has the potential to impede these vital processes in laying hens. Furthermore, our study revealed that heat stress led to a remarkable increase in the TG levels in the liver, signaling abnormal hepatic fat deposition in chickens. Intriguingly, after six weeks of heat exposure, we noted a substantial reduction in total cholesterol (TC) levels in the HT group’s livers compared to the non-temperature-treated (NT) group. These parallel changes in lipid profiles, characterized by elevated TG levels and decreased TC levels in both the yolk and liver, suggest a potential interconnected and coordinated regulation of lipid metabolism between these two vital organs. In contrast, Yin et al. reported an opposing trend, as they observed a significant increase in TC levels in heat-stressed birds, which is accompanied by a simultaneous decrease in TG levels when compared to birds maintained under normal conditions [
30]. The potential reasons for the elevation of hepatic TG levels and the decrease in TC levels induced by heat stress can be analyzed as follows. Firstly, during heat stress, the body requires more energy to cope with the stress response and maintain normal functions. To meet this demand, the liver increases fat breakdown, leading to an elevation in triglyceride levels. Additionally, heat stress leads to the depletion of glycogen, the storage form of glucose, as it is consumed to meet energy needs. In response, the liver utilizes fatty acids for energy production, leading to enhanced triglyceride synthesis and accumulation. Furthermore, heat stress can induce alterations in the levels of hormones and cytokines, which may in turn suppress cholesterol synthesis in the liver. These observations provide a plausible explanation for how heat stress can lead to metabolic disorders, potentially stemming from excessive fat deposition during the experiment.
Some previous studies have reported that heat stress may promote fat synthesis and deposition in broilers [
31,
32]. Further investigating the underlying mechanisms requires establishing a precise understanding of the interplay between lipid metabolism in the yolk and liver, which contributes to a comprehensive understanding of the physiological responses in avian species during heat stress. Our study analyzed the levels of lipid-related genes in the liver. Our results demonstrated that heat stress significantly increased the mRNA levels of sterol regulatory element-binding protein 1c (SREBP-1c), Ace-CoA carboxylase (ACACA), and fatty acid synthase (FASN) when compared to the NT group in the livers of the Huaixiang chickens. Notably, the mRNA expression levels of live X receptor alpha (LXRα) showed an increasing trend under heat stress conditions. The increased expression of SREBP-1c correlates with increased TG levels in the liver [
28]. Both LXRα and SREBP-1c can further activate the transcription of the key genes associated with fat synthesis, including FASN and ACACA [
33,
34]. ACACA plays a pivotal role in catalyzing the synthesis of malonyl-CoA, while FASN determines the speed of fatty acid synthesis [
35]. Similarly, Lu et al. have demonstrated that heat exposure markedly enhanced the mRNA levels of SREBP-1c, ACACA, and FASN in the liver [
36]. Consequently, it is plausible that heat stress activates the lipid signaling pathway in the liver, ultimately leading to yolk fat synthesis in Huaixiang chickens. These findings collectively shed light on the potential mechanisms underlying heat stress-induced lipid changes in egg yolks.
The effect of heat stress on amino acids in egg yolk has not been reported until now. To further explore the underlying mechanisms of heat stress on internal egg yolk quality, we investigated the effect of heat stress on yolk amino acids (AAs) of Huaixiang chickens. The concentration of free amino acids primarily hinges on the balance between the formation and degradation of amino acids in the tissues. In our study, we observed a significant reduction in the total amino acid levels in the yolk of the HT group when compared to the NT group. The pathophysiological changes induced by heat stress may increase the amino acid energy supplement requirement and heat shock protein synthesis of laying hens, which leads to the decrease in AA concentration in egg yolk under heat stress conditions [
37]. Additionally, we found that the cysteine (Cys) and tyrosine (Tyr) levels were obviously higher in the HT group than in the NT group during our experiment. Cys is recognized as a potential marker and a critical intracellular antioxidant for heat stress. However, research on the specific role of cysteine under heat stress conditions remains limited. Consequently, further studies are imperative to elucidate its functions in heat-stressed animals. Cervantes et al. reported that the serum Cys concentration decreased in heat-stressed growing pigs [
38]. The decline in cysteine concentration may be associated with its role as a component of glutathione, a crucial antioxidant compound, along with glutamate (Glu) and glycine (Gly). Cysteine plays a pivotal role in scavenging and neutralizing reactive oxygen species (ROS). ROS production escalates within cells under heat stress conditions, resulting in a higher demand for antioxidants [
39]. Cys is a pivotal amino acid that can be catabolized through various pathways, including gluconeogenesis, to produce energy. Moreover, Cervantes et al. also demonstrated that heat stress significantly reduced the essential AA Tyr level in the serum of growing pigs. These results suggest that cells may remove these essential AAs to generate body proteins to relieve the effects of heat stress [
38]. We speculate that Cyr and Tyr level changes in egg yolk may be associated with reduced feed intake and nutrient absorption, which are common phenomena in HS birds [
40,
41]. The increased demand for energy during heat stress can trigger enhanced amino acid catabolism, including Cys and Tyr, consequently resulting in a reduced concentration of these amino acids in the egg yolk. Further research is needed to explore strategies for enhancing the availability of essential amino acid in heat-stressed birds. Such efforts could ultimately improve egg yolk quality and the overall health of chickens.