Hepatic lipid metabolomics in response to heat stress in local broiler chickens breed (Huaixiang chickens)

Abstract High‐temperature environment‐induced heat stress (HS) is a hazard environmental element for animals, leading to dramatic changes in physiological and metabolic function. However, the metabolomic‐level mechanisms underlying lipid metabolism in liver of slow‐growing broilers are still obscure. The present study investigated the effects of HS on hepatic lipidomics in Chinese indigenous slow‐growing broilers (Huaixiang chickens). The study includes two treatments, each treatment had 5 replicates with 4 broilers per cage, where a total of 40 eight‐week‐old female Huaixiang chickens (average initial body weight of 840.75 ± 20.79 g) were randomly divided into normal temperature (NT) and HS groups for 4 weeks, and the broilers of NT and HS groups were exposed to 21.3 ± 1.2℃ and 32.5 ± 1.4℃ respectively. The relative humidity of the two groups was maintained at 55%–70%. The liquid chromatography‐mass spectrometry (LC‐MS)‐based metabolomics were conducted to evaluate the changes in hepatic lipidomics of broilers. The results showed that there were 12 differential metabolites between the two treatments. Compared with the NT group, HS group reduced the levers of hepatic phosphatidylcholine (PC) (16:0/16:0), PC (16:0/18:2), triglyceride (TG) (16:0/16:1/18:1), TG (18:0/18:1/20:4) (VIP > 1 and p < 0.05), while increased PC (18:1/20:3), PC (18:0/18:1), PC (18:1/18:1), PC (18:0/22:5), dimethyl‐phosphatidyl ethanolamine (dMePE) (14:0/18:3), dMePE (18:0/18:1) and dMePE (16:0/20:3) levels (Variable Importance in the Projection; VIP > 1 and p < 0.05). In addition, according to the analysis of metabolic pathway, the pathways of linoleic acid, alpha‐linolenic acid, glycerolipid and glycerophospholipid metabolism were involved in the effects of HS on hepatic lipid metabolism of broilers (p < 0.05). In conclusion, HS altered the hepatic lipid metabolism mainly through linoleic acid, alpha‐linolenic acid, glycerolipid and glycerophospholipid metabolism pathway in indigenous broilers. These findings provided novel insights into the role of HS on hepatic lipidomics in Chinese indigenous broiler chickens.


| INTRODUC TI ON
Under economic stimulus, the high stocking density causes HS, which can adversely affect the health and production of animals, especially in summer (Lolli et al., 2010;Quinteiro-Filho et al., 2010). Broilers are particularly sensitive to HS (Mascarenhas et al., 2018;Sakamoto et al., 2020). Therefore, HS has been considered to be one of the main environmental factors affecting broilers production. HS can reduce feed intake, slow down the growth rate and lead to systemic immune disorders in broilers (Goo et al., 2019;Liu et al., 2019). In addition, HS decreased the basal metabolic rate and significantly changed the level of fat metabolism in broilers, resulting in excessive fat deposition (Sato et al., 2006;Shim et al., 2016). The liver is one of the important lipid metabolic organs in broilers, which has crucial functions such as secretion of bile, storage of hepatic glycogen and detoxification (Yarru et al., 2009). Lipid metabolism is closely related to the maintenance of dynamic energy balance and the physiological function of broilers (He et al., 2018). When broilers under HS, the energy balance in the body is destroyed, and too much energy is used for fat storage, thus damaging hepatic tissue and function (Lu et al., 2019). Previous studies in the liver of broilers have shown that HS altered the expression and the activity of various enzymes and played vital role in the regulation of lipid metabolism (Flees et al., 2017;Tang et al., 2015). Meanwhile, HS induced changes in certain hepatic parameters and lipid metabolites in broilers, indicating that hepatic lipid metabolites are sensitive and valuable parameters for lipid metabolism research in broilers exposed to HS (Shim et al., 2006). In this regard, Lu et al. (2019) demonstrated that HS increases the lever of hepatic triglycerides (TG), total cholesterol (TCHO) and fatty acid synthase (FAS) in Arbor Acres (AA) broilers.
In recent years, metabolomics has become the leading means of systems biology research, and has been widely used in biomedical, pharmaceutical and toxicological research. Gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) have been extensively used in metabolic profiling analysis of various samples due to its high resolution and detection sensitivity (Bujak et al., 2015;Li et al., 2018). According to earlier reports, it has been suggested that the mechanism of lipid metabolism regulation and the specific lipid metabolites can be researched by lipidomics analysis in various life phenomena (Cai et al., 2011). Lipids have many important biological functions, and abnormal lipid metabolism can cause many animal diseases (Hertzel et al., 2008). The identification and comprehensive analysis of lipid metabolites in organisms at the lipidomic and systematic level can reveal the changes in lipid metabolism and related regulatory mechanisms under internal or external stimulation (Shevchenko & Simons, 2010). However, there are limited reports on the effects of HS on hepatic lipid metabolism in broilers based at lipidomic level, especially for the indigenous slow-growing broilers.
Indigenous yellow-feathered broilers are gradually favoured by consumers because of the excellent meat quality. Huaixiang chicken is a famous Chinese indigenous yellow-feathered slow-growing broiler breed. It has the advantages of rough feeding tolerance, good foraging ability and strong disease resistance, which is widely raised in South China Liu et al., 2019;Shi et al., 2016). In addition, this breed of chicken has the advantages of tender meat and low content of fat. The high environmental temperature in South China often causes HS in indigenous slow-growing broilers, which may affect their lipid metabolism and leads to excessive fat deposition, thus negatively influencing the carcass traits, meat quality and health status. However, few studies have focused on the effects of HS on hepatic lipid metabolomics in Huaixiang chicken. Hence, this study attempted to analyse the characteristics of lipid metabolism in the liver of Huaixiang chickens under HS through lipid metabolomics study, and to lay a foundation for the screening of lipid biomarkers in indigenous broilers under HS. It also expanded understandings of the effects of HS on hepatic lipidomics in Chinese indigenous yellow-feathered broiler chickens.

| Animals, diet and experimental procedures
A total of 40 eight-week-old female Huaixiang chickens (average initial body weight of 840.75 ± 20.79 g) were randomly divided into two treatments that included normal temperature (NT) and heat stress (HS) groups for 4 weeks. Each treatment had 5 replicates with 4 broilers per cage, and there was no significant difference in initial body weight between repetitions and treatments. The broilers of NT and HS groups were exposed to 21.3 ± 1.2℃ and 32.5 ± 1.4℃, respectively, from 9 a.m. to 5 p.m., which lasted for 8 hr every day.
The relative humidity of the two groups was maintained at 55%-70% during the whole experimental period. All the birds were raised in the chicken house with environmental control equipment, the temperature and humidity are controlled by equipment. The temperature and humidity data were recorded at 6.30 a.m., 9 a.m., 12 a.m., 2 p.m., 5 p.m., 8 p.m. and 11 p.m. every day from six different locations in the chicken house. The cage size was 90 (length) × 70 (width) × 40 (height) cm. All birds were given ad libitum access to feed and water. During the experimental period, all broilers were fed with the diets (Table 1)

| Sampling and preparation
At the end of the trial, five birds (one bird from each repeat) were randomly selected from each treatment and killed by cervical dislocation. Subsequently, the liver samples were collected immediately, then the liver samples were immersed in liquid nitrogen and stored at −80℃ for further determination and analysis Liu et al., 2020).
For lipid extraction, chloroform/methanol solution was added to each sample. The solution was mixed by a vortex mixer for 5 min.
Then, the mixture was centrifuged at 8000 g for 10 min at 4℃. The supernatant was put into the clean test tube, and the precipitates were extracted twice with 2 ml chloroform/methanol solution. All the supernatants were dried by N2 and then dissolved with chloroform/methanol (2:1, v:v). The supernatant (200 μl) was transferred to vials for detection. Dionex UltiMate 3000 (UHPLC)-Thermo Orbitrap Elite was used for liquid LC-MS analysis.
Mass spectrometry was operated in both positive and negative ion modes. The parameters optimized were as follows. Positive mode, Heater Temp 300℃; Sheath Gas Flow rate 45 arb; Aux Gas

| Metabolomic data analysis
Raw data were converted the common (mz.data) format by Agilent Masshunter Qualitative Analysis B.08.00 software (Agilent Technologies, USA). In the R software platform, the XCMS program was used in peak identification, retention time correction and automatic integration pre-treatment. Then, the data were subjected to internal standard normalization . After editing, the data matrices were import into SIMCA-P 13.0 (Umetrics, Umea, Sweden), mean centred and scaled to Pareto variance. Then, multivariate analysis was conducted. Data were analysed by partial leastsquare discriminant analysis (PLS-DA), and orthogonal projections to latent structures discriminant analysis (OPLS-DA). Differential metabolites were screened out by Variable Importance in the Projection (VIP) value of OPLS-DA model (VIP ≥ 1) and independent sample t test (p < 0.05).
The differential metabolites of HS and NT groups were mapped to the Kyoto Encyclopedia of Genes and Genomes Identifier (KEGG ID) by online software MetaboAnalyst. A pathway analysis was implemented. House mouse (Mus musculus) was selected as a model organism. The significant pathways (p < 0.05) were selected using KEGG.

| PLS-DA and OPLS-DA analysis
The PLS-DA scores plots are depicted in Figure

| Analysis of related metabolic pathways
As indicated in Table 4, pathway analysis showed that HS changed  to the HS showed excessive fat deposition, which indicated that the process of lipid synthesis and metabolism has been changed in broilers (Ryder et al., 2004). The metabolic process of lipids in the body mainly includes the metabolism of triglycerides, phospholipids, cholesterol and plasma lipoproteins (Zeng et al., 2017). Previous studies have shown that HS inhibited hepatocyte proliferation, promoted hepatocyte apoptosis and induced hepatocyte necrosis in mice (Li et al., 2012;Thompson et al., 2014). It has also suggested that the hepatocytes of broilers exposed to HS showed "steatosis" (Lu et al., 2019). Lipid metabolism is closely related to hepatic function, and abnormal metabolic pathway may be related to the effects of HS on hepatic function (Lv et al., 2018). Specifically, the result of this study showed that the hepatic TG content was decreased by chronic HS (4 weeks). As we all know, TG is one of the components of lipids, and the main function of TG is to supply and store energy, which can fix and protect internal organs of animals (Das et al., 2014). In vivo, TG synthesized are mainly in the liver, followed by adipose tissue (Almeida et al., 2013). It has been reported that HS broilers have reduced feed intake, resulting in energy deficiency, which in turn leads to lipid metabolism disorders (Cornejo et al., 2007). Therefore, the decrease in TG content may be due to the decrease in feed intake and the damage of hepatic function caused by HS. The low level of TG may also be due to species of broilers, diet  (Zhou et al., 2018). It is suggested that the TG level in liver is related to the duration of HS, however, the specific mechanism needs to be further studied.

| D ISCUSS I ON
In addition, lipids are the main substances in cells and have a variety of biological functions, including the construction of biofilms, the regulation of energy conversion and the transmission of cell signals (Ottaviani et al., 2011). Therefore, they are involved in a variety of biological processes, such as cell growth and differentiation. The phospholipid is an important part of biomembrane and the main lipid carrier in blood, which plays a pivotal role in activating cells and maintaining metabolism (Wang et al., 2003).
Moreover, phospholipids can promote fat metabolism, prevent fatty liver and reduce serum cholesterol (Li et al., 2010). As the main sources of phospholipids, PC and PE have crucial functions on the integrity of cell and organelle membranes (Luo et al., 2018).
Also, it has been reported that dMePE was formed by methylation of phosphatidylethanolamine (PE) by S-adenosylmethionine, and the upregulation of dMePE level may be related to the increase in methylase activity. Li et al. (2012) found that HS destroys the function of hepatic cell membrane, which affects the value-added ability of cells. In addition, previous studies have shown that changes in the levels of PC and PE are related to steatohepatitis (Li et al., 2006). Therefore, it can be inferred that the damage of HS  . VIP, variable importance in the projection; FC, ratio of mean peak area of the HS group to the mean peak area of the NT group; ↑, metabolites with higher concentrations in the HS group than in the NT group with FC > 1; ↓, metabolites with lower concentrations in the HS group than in the NT group with FC < 1.
to cell membrane and hepatic function may be due to the changes in hepatic PC and dMePE levels. Regarding the mechanism of the effects of HS on the levels of hepatic phospholipids, it has been suggested that HS can increase the activity of phospholipase A2 (PLA2) and promote the decomposition of phospholipids, thereby causing damage to cell membranes (Sahebkar, 2013). Therefore, the development of PLA2 inhibitors in future researches may help to ameliorate HS-induced impairment of phospholipids metabolism and cell membrane function in broilers. On the other hand, previous studies have shown that the chemical activators (phenformin and AICAR) of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) inhibit both PC biosynthetic pathways in isolated hepatocytes (Kasturi et al., 2006). Also, it has been indicated that HS activates the activity of AMPK in piglet hepatocytes and reduces the synthesis of phospholipids, TG and cholesterol in hepatocytes  F I G U R E 3 Schematic overview of alpha-Linolenic acid metabolic pathway and some related metabolites in heat-stressed broilers. Red, metabolites in HS group versus NT group upregulation F I G U R E 4 Schematic overview of glycerolipid metabolic pathway and some related metabolites in heat-stressed broilers. Blue, metabolites in HS group versus NT group downregulation F I G U R E 5 Schematic overview of glycerophospholipid metabolic pathway and some related metabolites in heat-stressed broilers. Red, metabolites in HS group versus NT group upregulation

| CON CLUS IONS
In conclusion, HS altered the level of 12 hepatic lipid metabolites and affected hepatic linoleic acid, alpha-linolenic acid and glycerolipid, and glycerophospholipid metabolic pathways in indigenous broilers.
This may be the mechanism of lipid metabolism that HS-induced hepatic dysfunction. The current findings provided new insights into the effects of HS on lipid metabolism at metabolomic level in indigenous slow-growing broilers.

E TH I C S A PPROVA L
The present study was carried out at the College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang, China. The experimental protocol used in the study was approved by the Animal Care Committee of Guangdong Ocean University, Zhanjiang, China.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest. Wen-Chao Liu: Funding acquisition.

DATA AVA I L A B I L I T Y S TAT E M E N T
All public data generated or analysed during this study are included in the study.