Impact of Chromium Picolinate on Breast Muscle Metabolomics and Glucose and Lipid Metabolism-Related Genes in Broilers Under Heat Stress

1. Introduction

Heat stress is an increasingly harmful consequence caused by global warming, which has a wide range of detrimental effects on poultry production which includes reducing feed intake, growth rate, and feed conversion ratio, suboptimal carcass quality; excessive abdominal adiposity[1,2,3], while also exerting a detrimental effect on the immunity and organ function in broilers[4,5,6].

PPAR is a member of the nuclear receptor family involved in regulating lipid metabolism, lipoprotein metabolism, and glucose homeostasis[7]. One of the subtypes PPARα plays a crucial role in modulating gene expression associated with both peroxisomal and mitochondrial fatty acid oxidation. It also influences genes involved in fatty acid synthesis, transport, oxidation, and storage. By doing so, it contributes to the reduction of lipid accumulation and is intricately linked to the regulation of glucose and lipid metabolism as well as overall energy homeostasis[8]. The activation of PPARα can increase hepatic insulin activity, and reduce body fat deposition[9].

Chromium is an essential trace mineral commonly used as a dietary supplement for both humans and animals. Studies indicate that heat stress triggers the release of chromium from tissues, increases its excretion through urine, and decreases its retention, leading to chromium deficiency in the body[10]. As a result, the requirement for chromium becomes more critical. As is well known, heat stress can increase circulating concentrations of corticosterone in broilers. There is evidence showing that corticosterone can reduce insulin sensitivity in broilers, resulting in insulin resistance, which is associated with many diseases such as type II diabetes, obesity, and fatty liver disease[11]. In this case, chromium is known to enhance insulin sensitivity in broilers[12]. Supplementing the diet of broilers with chromium picolinate under heat stress has been shown to enhance growth performance [13], increase carcass yield[14], decrease abdominal fat deposition[15], improve the weights of immune organs[16], increase insulin, and decrease serum glucose[14].

As regards the skeletal muscle, the most relevant muscle in fast-growing broilers, accounting for 20–25% of their live body weight, which is considered a highly metabolically active tissue deeply involved in glucose and lipid metabolism[17]. Analyzing metabolomics in breast muscle could provide detailed information on glucose and lipid metabolism.

Our previous study showed that PPARα and ABCA1 were reduced in expression in heat-stressed broilers, which suggested that the PPARα gene may play a critical role in the mechanism of lipid metabolism under heat stress[18]. However, in light of previous studies, it can be found that researchers mainly focus on the lipid metabolism of the chromium effect. However, it is largely unknown how Cr supplementation impacts the glucose metabolism and lipid metabolism in the skeletal muscle and these related genes. Addtionally, it remains unclear whether the chromium supplementation can impact the subtype of PPAR, which in turn regulate glucose metabolism and lipid metabolism.

Therefore, the current study aimed to explore the effect of chromium on growth performance, blood parameters, breast muscle metabolism, and related gene expression under heat stress in broilers. We hypothesize that chromium dietary supplementation could affect the glucose and lipid metabolism, and PPARα may play a role in mitigating the impact of chromium under heat stress.

2. Materials and Methods

2.1. Experimental Design, Animals and Housing

A total of 220 one-day-old Arbor Acres male chickens were reared in 22 one-tier elevated cages (0.8 m × 0.8 m × 0.4 m) to 28 days old. The corn-soybean meal starter diet that meets NRC (1994) requirements for Arbor Acres chickens for this period was obtained from Huadu Commercial Company (Beijing, China) and served ad libitum with through feeders (0.82 m long × 0.07 m wide × 0.06 m deep). The water was available with nipple drinkers. The set of all environmental parameters followed the Arbor Acres management handbook. At the age of 29 d, 180 healthy male birds with similar body weight (1150 ± 70 g) were selected and randomly assigned to three treatments: 0 (heat stress group), 400, and 800 µg Cr/kg of supplemental diet (chromium picolinate) and transferred to three climate chambers. The environmental chambers were of identical size, each containing 6 cages with 10 chickens each. A cage was seen as a replicate. All treatments were conducted under a constant ambient temperature of 31 ± 1°C. The parameters of the chambers were set as follows: the humidity remained at 60 ± 7%, the airflow rate was under 0.5 m/s, and the ammonia concentration was controlled under 5 ppm. The lighting program was 24L:0D. The experiment lasted from 14 days to 42 days of age.

2.2. Diet

The basal diet was primarily composed of corn and soybean meal, and the composition is presented in Table 1. The heat stress group was subjected to the basal diet, while the diets of the other 2 groups were mixed with 400 and 800 µg Cr/kg chromium picolinate, respectively. Chickens were fed ad libitum and provided with free access to fresh drinking water throughout the experimental period. All diets were mixed thoroughly before feeding, and representative samples were analyzed for crude protein, metabolizable energy, and amino acid content to ensure diet consistency.

2.3. Measurements

Growth Performance: The body weight was determined at the start and the end of the experiment. The feed intake was recorded on a daily basis during the experiment. The average daily gain (ADG), average daily feed intake (ADFI), and feed conversion ratio (FCR) were calculated.

Body core temperature: 2 birds per replicate were randomly selected for the body core temperature determination by a thermometer once a week during the experiment.

Sample collection: At the end of the experiment, all chickens fasted for 12 h prior to the sampling. Thereafter, two chickens per replicate were selected based on their body weight closest to the cage average. The blood collection was performed using a blood collection needle at the wing vein. Subsequently, chickens were euthanized by CO₂ asphyxiation. The left breast muscle and abdominal fat were weighed, and their respective proportions were calculated as a percentage of body weight. The liver was collected and placed in a cryotube, immediately transferred to liquid nitrogen, and subsequently stored in a -80°C freezer.

LC-MS analysis: Breast muscle samples were analyzed using hydrophilic interaction liquid chromatography (HILIC) with an ACQUITY UPLC BEH Amide column (1.7 μm, Waters). Electrospray ionization (ESI) was applied in both positive and negative modes. The mobile phase system consisted of 25 mM ammonium acetate and 25 mM ammonium hydroxide in water (A), and acetonitrile (B). The gradient elution began with 85% B for 1 min, gradually decreased to 65% over 11 min, quickly dropped to 40% within 0.1 min and held for 4 min, followed by an increase to 85% within 0.1 min and a re-equilibration period of 5 min. For reversed-phase liquid chromatography (RPLC), an ACQUITY UPLC HSS T3 column (1.8 μm) was used. In positive mode, the mobile phase consisted of water and acetonitrile, both containing 0.1% formic acid, whereas in negative mode, 0.5 mM ammonium fluoride was added instead. The gradient profile was: 1% B for 1.5 min, increased to 99% over 11.5 min, maintained for 3.5 min, dropped to 1% within 0.1 min, and then equilibrated for 3.4 min. The column was operated at 25 °C with a flow rate of 0.3 mL/min, and 2 μL of each sample was injected for analysis.

Blood analysis: Serum samples were obtained by centrifugation at 3000×g for 20 min at 4°C. The fasting blood glucose was measured by using the Roche glucometer (Accu-Chek Performa, Roche Diagnostics, USA). The fasting insulin was determined by a kit that was obtained from Nanjing Jiancheng Institute of Bioengineering (Nanjing Jiancheng, Nanjing, China). The Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated by a formulation as follows: HOMA-IR = FBG (mmol/L) × FINS (mU/L) / 22.5. The levels of free fatty acids, cholesterol, and corticosterone (CORT) were measured using an automatic biochemical analyzer (Hitachi 7600, Hitachi Corporation, Japan). All the procedures were carried out according to the protocol of the manufacturer.

qPCR: Total RNA from breast muscle samples was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. cDNA synthesis and real-time quantitative PCR were performed on a LightCycler 96 instrument (Roche, Basel, Switzerland) using standard qPCR procedures. Transcript levels of ABCA1, ABCG1, PPARα, and PGC-1α were quantified in both liver and breast muscle tissues. Gene-specific primers were designed based on GenBank sequences (see Table 2), and β-actin was employed as the reference gene for normalization. Relative gene expression was calculated using the 2^-ΔΔCt method.

2.4. Statistical Analysis

All data were analyzed using a one-way ANOVA based on a single-factor experimental design, followed by Duncan’s multiple range test for post-hoc comparisons, implemented in SAS 9.2 software (SAS Institute Inc., Cary, NC, USA). Results were expressed as mean ± standard deviation (SD), with statistical significance defined at P < 0.05. For metabolomics, orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to differentiate between groups, and metabolites with a variable importance in projection (VIP) score ≥ 1 and P < 0.05 (from independent sample t-tests) were considered significantly different. Identified differential metabolites between the HS and NT groups were annotated using KEGG IDs and analyzed through MetaboAnalyst. The pathways with P < 0.05 were deemed significantly affected.

3. Results

3.1. Growth Performance

All chickens remained free from diseases, and no mortality occurred during the experiment. As shown in Table 3, the ADFI and ADG in the 400 and 800 µg Cr/kg groups were significantly higher than those in the HS group (P < 0.05); the FCR was lower than the HS group. Additionally, the 400 and 800 µg Cr/kg groups both exhibited a significant increase in breast muscle rate and a significant decrease in abdominal fat rate compared to the HS group (P < 0.05). However, no significant differences in these parameters were observed between the 400 and 800 µg Cr/kg groups (P > 0.05).

3.2. LC-MS Analysis

The scores (OPLS-DA) plot in Figure 1 shows a clear separation between two groups, indicating distinct metabolic profiles. In the OPLS-DA model, the positive mode scores plot yielded R²X=0.470, R²Y=0.999, and Q²=0.779, while the negative mode scores plot resulted in R²X=0.359, R²Y=0.994, and Q²=0.854.

A total of 91 metabolites were identified as significantly different between the HS group and the group receiving 400 µg Cr/kg, as determined by OPLS-DA (VIP ≥ 1) combined with independent sample t-test (P < 0.05). Detailed information on these differential metabolites is provided in Table 5. Relative to the HS group, 35 differential metabolites were upregulated and 56 differential metabolites were downregulated in the 400 µg Cr/kg group. The key differential metabolites involved in metabolic pathways include citric acid, L-aspartic acid, L-glutamine, L-proline, and (R)-(+)-2-Pyrrolidone-5-carboxylic acid, phosphatidylcholine (14:0/20:4[8Z,11Z,14Z,17Z]), 9,10,13-trihydroxy-octadecadienoic acid, phosphatidylcholine (22:6[4Z,7Z,10Z,13Z,16Z,19Z]), phosphatidylserine (18:0/20:0), and a series of lysophosphatidylcholine (LysoPC) species, including LysoPC (16:0), LysoPC (20:4[5Z,8Z,11Z,14Z]), LysoPC (18:1[9Z]), LysoPC (17:0), LysoPC (18:0), LysoPC (P-18:1[9Z]), LysoPC (20:1[11Z]), and LysoPC (P-18:0). Compared with the HS group, the concentrations of citric acid, L-aspartic acid, L-aspartic acid, L-glutamine, L-proline, (R)-(+)-2-pyrrolidone-5-carboxylic acid, PC(14:0/20:4[8Z,11Z,14Z,17Z]), 9,10,13-Trihydroxy-octadecadienoic acid were higher, and others were lower in the 400 µg Cr/kg group (P < 0.05).

As shown in Figure 2, pathway analysis revealed that supplementation with 400 µg Cr/kg had a significant impact on 9 metabolic pathways compared with the HS group (P < 0.05). These pathways include alanine, aspartate, and glutamate metabolism; D-glutamine and D-glutamate metabolism; arginine biosynthesis; linoleic acid metabolism; glycine, serine, and threonine metabolism; glyoxylate and dicarboxylate metabolism; aminoacyl-tRNA biosynthesis; ABC transporters; and glycerophospholipid metabolism.

Table 4. Differential metabolites involved in metabolic pathways in metabolomics in breast muscle of HS and EG broilers.

Metabolites	VIP(HS&EG)	P-value	Change
Citric acid	5.92	0.04	↑
L-aspartic acid	6.94	0.02	↑
L-glutamine	3.91	0.02	↑
L-proline	5.82	0.03	↑
(R)-(+)-2-Pyrrolidone-5-carboxylic acid	5.23	0.02	↑
Phosphatidylcholine (14:0/20:4[8Z,11Z,14Z,17Z])	3.94	0.05	↑
9,10,13-trihydroxy-octadecadienoic acid	4.69	0.002	↑
Phosphatidylcholine (22:6[4Z,7Z,10Z,13Z,16Z,19Z])	5.35	0.05	↓
Phosphatidylserine (18:0/20:0)	3.59	0.02	↓
LysoPC (16:0)	10.43	0.005	↓
LysoPC (20:4[5Z,8Z,11Z,14Z])	3.56	<0.001	↓
LysoPC (18:1[9Z])	5.97	0.03	↓
LysoPC (17:0)	8.95	0.002	↓
LysoPC (18:0)	6.93	0.02	↓
LysoPC (P-18:1[9Z])	8.38	<0.001	↓
LysoPC (20:1[11Z])	3.56	0.02	↓
LysoPC (P-18:0)	5.80	0.03	↓

Table 4. Differential metabolites involved in metabolic pathways in metabolomics in breast muscle of HS and EG broilers.

Metabolites	VIP(HS&EG)	P-value	Change
Citric acid	5.92	0.04	↑
L-aspartic acid	6.94	0.02	↑
L-glutamine	3.91	0.02	↑
L-proline	5.82	0.03	↑
(R)-(+)-2-Pyrrolidone-5-carboxylic acid	5.23	0.02	↑
Phosphatidylcholine (14:0/20:4[8Z,11Z,14Z,17Z])	3.94	0.05	↑
9,10,13-trihydroxy-octadecadienoic acid	4.69	0.002	↑
Phosphatidylcholine (22:6[4Z,7Z,10Z,13Z,16Z,19Z])	5.35	0.05	↓
Phosphatidylserine (18:0/20:0)	3.59	0.02	↓
LysoPC (16:0)	10.43	0.005	↓
LysoPC (20:4[5Z,8Z,11Z,14Z])	3.56	<0.001	↓
LysoPC (18:1[9Z])	5.97	0.03	↓
LysoPC (17:0)	8.95	0.002	↓
LysoPC (18:0)	6.93	0.02	↓
LysoPC (P-18:1[9Z])	8.38	<0.001	↓
LysoPC (20:1[11Z])	3.56	0.02	↓
LysoPC (P-18:0)	5.80	0.03	↓

3.3. Blood Paraments

Table 5 shows the levels of CORT, free fatty acids, and cholesterol in serum in the 400 and 800 µg Cr/kg groups were significantly lower (P < 0.05), compared with the HS group (P < 0.05), whereas there was no significant difference in the 400 and 800 µg Cr/kg group (P > 0.05). As shown in Figure 3, the fasting blood glucose and HOMA-IR in the 400 and 800 µg Cr/kg groups were significantly lower than those in the HS group (P < 0.05), and the fasting insulin in the 400 and 800 µg Cr/kg groups was significantly higher than that in the HS group (P < 0.05), whereas there was no significant difference in the 400 and 800 µg Cr/kg groups (P > 0.05).

Table 5. Effects of 400 and 800 µg Cr/kg of chromium picolinate supplementation on blood parameters in broilers under heat stress from 29 d to 42 d of age.

Items2	0	400	800	P-value
Cholesterol (mmol/L)	4.80a ± 0.11	3.57b ± 0.52	3.21b ± 0.26	<0.01
Free fatty acids (μmol/L)	106.68a ± 3.24	89.06b ± 1.46	106.68b ± 3.24	<0.01
Corticosterone (ng/mL)	15.78a ± 0.51	12.14b ± 0.72	12.14 b ± 1.22	0.02

Table 5. Effects of 400 and 800 µg Cr/kg of chromium picolinate supplementation on blood parameters in broilers under heat stress from 29 d to 42 d of age.

Items2	0	400	800	P-value
Cholesterol (mmol/L)	4.80a ± 0.11	3.57b ± 0.52	3.21b ± 0.26	<0.01
Free fatty acids (μmol/L)	106.68a ± 3.24	89.06b ± 1.46	106.68b ± 3.24	<0.01
Corticosterone (ng/mL)	15.78a ± 0.51	12.14b ± 0.72	12.14 b ± 1.22	0.02

3.4. Genes Associated with Glycolipid Homeostasis

As shown in Figure 4, the expression of PGC-1α, PPARα, ABCA1 and ABCG1 in the liver in the 400 µg Cr/kg group was significantly higher than in the HS group (P < 0.05). As shown in Figure 5, the expression of PGC-1α, PPARα, ABCA1 and ABCG1 mRNA in breast muscle in the 400 µg Cr/kg group was significantly higher than in the HS group (P < 0.05), whereas there was no significant difference of ABCG1 in breast muscle between the two groups (P > 0.05).

4. Discussion

The current study was performed under heat stress at 31°C for 4 weeks. Our findings demonstrated that dietary Cr provision, regardless of the dosage, both increased the growth performance, mitigated the glucose and lipid metabolism disorders.

Provision of Cr in broiler has been proven to mitigate the adverse impact on performance caused by heat stress[19]. Evidences has indicated that the use of Cr supplementation as a nutritional strategy to alleviate growth performance[20] via improved nutrient metabolism [14,21], immune responses[22], antioxidant function and stress response[20,23] in broiler chickens exposed to heat stress. Our results demonstrated that dietary supplementation with 400 and 800 µg Cr/kg significantly improved the average daily feed intake (ADFI) and average daily gain (ADG) of broilers while reducing the feed conversion ratio (FCR). Moreover, Cr significantly increased the breast muscle yield and decreased the abdominal fat rate, suggesting a beneficial effect on carcass composition and meat quality. These findings were similar to studies conducted bySahin, et al. [24] andSahin, Hayirli, Orhan, Tuzcu, Akdemir, Komorowski and Sahin [19] , they found feed intake and body mass gain were enhanced after provision of 200, 400, 800, or 1200 μg Cr as Cr pic/kg diet.Sahin, Sahin, Onderci, Gursu and Cikim [24] found that abdominal fat was decreased in chicks supplemented with 4 × 105 μg Cr as Cr pic/kg diet.Zha, et al. [25] found FCR to be decreased in chicks supplemented with 500 μg Cr as Cr pic/kg diet or 500 μg Cr as Cr nano/kg diet and found that chicks supplemented with 500 μg Cr as Cr nano/kg diet had increased breast muscle mass. However, the results of the current study are not consistent with those ofAmatya, et al. [26] andMoeini, et al. [27]. The conflicting results among these studies could be attributed, in part, to the differing chromium sources and administration dosages.

In our study, Cr supplementation significantly lowered fasting blood glucose (FBG) and the HOMA-IR while markedly increasing fasting insulin levels. These results indicate that Cr enhances insulin sensitivity and glucose homeostasis under HS conditions. The reduction in HOMA-IR suggests that Cr mitigates insulin resistance, which is commonly observed during heat stress due to glucocorticoid overproduction and metabolic stress[28]. Chromium is generally accepted to be the active component in glucose tolerance factor, which increases the sensitivity of tissue receptors to insulin, resulting in increased glucose uptake by cells[29,30]. The observed improvements in growth performance and carcass traits may be attributed to the ability of Cr to enhance insulin sensitivity and promote efficient nutrient utilization, particularly glucose and lipid metabolism. Previous studies have reported that Cr, particularly in the form of chromium picolinate, chromium propionate, and chromium chloride, enhances insulin receptor activity and facilitates glucose transport, thereby improving feed efficiency and lean tissue deposition in poultry under thermal stress conditions[14,31]. Furthermore, Cr supplementation has been shown to reduce lipid peroxidation and oxidative damage caused by HS, thereby supporting better muscle development and reducing fat accumulation[32].

Heat stress can stimulate the hypothalamus-pituitary-adrenal axis, causing an increase in the blood corticosterone concentration[33]. Studies have shown that excessive secretion of glucocorticoids such as corticosterone can trigger oxidative damage leading to muscle protein hydrolysis and significantly affect glucose metabolism, lipid metabolism, and muscle development[34]. In the present study, broilers supplemented with 400 and 800 µg Cr/kg exhibited significantly lower serum levels of corticosterone (CORT), free fatty acids (FFA), and cholesterol compared to the control group. It is reported that increasing chromium supplementation in broilers linearly reduces serum corticosterone concentrations[10]. The decreased FFA and cholesterol levels might be attributed to decreased glucocorticoid secretion, as demonstrated in a study by Sahin, Sahin, Onderci, Gursu and Cikim [24].

In this study, supplementation with 400 µg Cr/kg under heat stress altered metabolic profiles in breast muscle metabolomics. Citric acid plays a critical role in the TCA cycle[35]. In addition, the L-aspartic acid and L-glutamine are involved in the malate-aspartate shuttle system, which promotes energy production[36]. On the other hand, L-glutamine and L-proline are vital components of the amino acid metabolism pathway and are involved in the antioxidant system[37], suggesting that Cr might reduce amino acid degradation by maintaining oxidative stability. PC is the important component of the cell membrane. One of the possible explanations is that the Cr might promote choletone and PC external to the cell by activating the ABC transport protein pathway. 9,10,13-Trihydroxy-octadecadienoic acid is the product of linoleic acid metabolism and has anti-inflammatory and antioxidant effects[38]. Therefore, the elevated levels of citric acid, L-aspartic acid, and L-glutamine suggest the TCA cycle, amino acid metabolism, and linoleic acid metabolism might be improved by Cr supplementation. In the KEGG results, the enrichment of pathways confirmed our supposition. Dietary supplementation with 400 µg Cr/kg significantly influenced nine metabolic pathways, including alanine, aspartate, and glutamate metabolism; D-glutamine and D-glutamate metabolism; arginine biosynthesis; linoleic acid metabolism; glycine, serine, and threonine metabolism; glyoxylate and dicarboxylate metabolism; aminoacyl-tRNA biosynthesis; ABC transporter pathways; and glycerophospholipid metabolism. We hypothesize that Cr might enhance amino acid metabolism by promoting the biosynthesis and utilization of key amino acids such as glutamine, arginine, and glycine, which are essential for protein synthesis, nitrogen balance, and antioxidant defense under HS conditions. Improved glycine, serine, and threonine metabolism may enhance the production of glutathione, a critical antioxidant, thereby reducing oxidative stress and cellular damage caused by HS. Similarly, the upregulation of linoleic acid metabolism indicates enhanced lipid mobilization and utilization, which may contribute to the reduction in abdominal fat rate observed in Cr-supplemented broilers.

Our previous results indicated that heat stress can reduce PPARα level in liver and suggested that PPAR might play an intermediate role in regulating lipid metabolism in broilers[18]. In the present study, the results indicated that the 400 µg Cr as pic/kg supplementation increased PPARα level to a large extent, which indicated that it helped to alleviate the adverse effects of heat stress on lipid metabolism. The beneficial effects of Cr on metabolic pathways are likely mediated through the activation of energy-regulating signaling cascades, such as the AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α) pathways. Previous studies have demonstrated that Cr can activate AMPK, a key cellular energy sensor, which in turn promotes glucose uptake, fatty acid oxidation, and mitochondrial biogenesis[39]. The increased expression of PGC-1α, PPARα, ABCA1, and ABCG1 observed in the current study further supports this mechanism, indicating that Cr enhances hepatic and muscular energy metabolism and lipid transport. Moreover, the significant upregulation of ABCA1 and ABCG1 in the liver suggests that Cr enhances cholesterol efflux and reverse cholesterol transport (RCT). ABCA1 and ABCG1 are membrane transporters responsible for exporting excess cholesterol to high-density lipoproteins (HDL), facilitating its transport to the liver for clearance[40]. The increased expression of these transporters indicates that Cr promotes cholesterol homeostasis and reduces the risk of hepatic lipid overload under HS conditions. Notably, the elevated expression of ABCA1 and ABCG1 in the liver suggests that Cr promotes cholesterol efflux and lipid clearance[41], contributing to improved lipid homeostasis under heat stress, which is consistent with our earlier statement. Interestingly, the expression of ABCG1 in the breast muscle was not significantly altered by Cr supplementation. This could be attributed to the low basal expression levels of ABCG1 in skeletal muscle tissue or its relatively weaker role in lipid transport compared to the liver.

5. Conclusions

In conclusion, chromium supplementation might improve energy metabolism and lipid transport in the liver and muscles by upregulating the expression of genes related to glucose and lipid metabolism, thereby alleviating the damage to the glucose and lipid metabolic function and growth performance under heat stress conditions.