Biogenic Selenium Nanoparticles Synthesized Using Alginate Oligosaccharides Attenuate Heat Stress-Induced Impairment of Breast Meat Quality via Regulating Oxidative Stress, Metabolome and Ferroptosis in Broilers

Selenium (Se) is an indispensable trace element with versatile functions in antioxidant defense in poultry. In our previous study, we synthesized a novel type of biogenic selenium nanoparticle based on alginate oligosaccharides (SeNPs-AOS), and found that the particles are sized around 80 nm with an 8% Se content, and the dietary addition of 5 mg/kg of SeNPs-AOS could effectively alleviate the deleterious effects of heat stress (HS) in broilers, but it is still unclear whether SeNPs-AOS can improve the meat quality. Therefore, the aim of this study was to evaluate the protective effects of SeNPs-AOS on breast meat quality in heat-stressed broilers, and explore the relevant mechanisms. Birds at the age of 21 days were randomly divided into four groups with six replicates per group (eight broilers per replicate) according to a 2 × 2 experimental design, using HS (33 ± 2 °C, 10 h/day vs. thermoneutral, TN, under 23 ± 1.5 °C) and SeNPs-AOS (5 mg/kg feed vs. no inclusion) as variables. The results showed that dietary SeNPs-AOS decreased the cooking loss (p < 0.05), freezing loss (p < 0.001), and shear force (p < 0.01) of breast muscle in heat-stressed broilers. The non-targeted metabolomics analysis of the breast muscle identified 78 differential metabolites between the HS and HS + SeNPs-AOS groups, mainly enriched in the arginine and proline metabolism, β-alanine metabolism, D-arginine and D-ornithine metabolism, pantothenate, and CoA biosynthesis pathways (p < 0.05). Meanwhile, supplementation with SeNPs-AOS increased the levels of the total antioxidant capacity (T-AOC), the activities of catalase (CAT) and glutathione peroxidase (GSH-Px), and decreased the content of malondialdehyde (MDA) in the breast muscle (p < 0.05) in broilers under HS exposure. Additionally, SeNPs-AOS upregulated the mRNA expression of CAT, GPX1, GPX3, heme oxygenase-1 (HO-1), masculoaponeurotic fibrosarcoma G (MafG), MafK, selenoprotein W (SELENOW), SELENOK, ferritin heavy polypeptide-1 (FTH1), Ferroportin 1 (Fpn1), and nuclear factor erythroid 2-related factor 2 (Nrf2) (p < 0.05), while it downregulated Kelch-like ECH-associated pro-36 tein 1 (Keap1) and prostaglandin-endoperoxide Synthase 2 (PTGS2) expression (p < 0.05) in broilers under HS. These findings demonstrated that the dietary addition of SeNPs-AOS mitigated HS-induced oxidative damage and metabolite changes in the breast muscle of broilers, which may be related to the regulation of the Nrf2 signaling pathway and selenoprotein synthesis. In addition, SeNPs-AOS upregulated the breast muscle gene expression of anti-ferroptosis-related molecules in broilers under HS, suggesting that SeNPs-AOS can be used as novel Se supplements against HS in broilers.


Introduction
Currently, in the context of global warming, heat stress (HS) has become a prime environmental stressor in broiler production [1,2].HS not only adversely affects the growth and feed conversion rate of broilers [3] but also has a profound influence on muscle physiology and metabolism, ultimately impairing the chicken quality [4].For instance, a lower pH, higher shear force, and freezing loss of the muscle were observed in broilers under HS [5,6].Reactive oxygen species (ROS) accumulation is one of the main reasons for poorer meat quality in broilers subjected to HS.It has been confirmed that HS caused oxidative damage to muscle by generating excessive ROS, thus leading to the oxidation of muscle fatty acids and proteins, reduction of flavor, and the shortened shelf life of chicken [7].Therefore, oxidative damage is considered to be the major factor of meat spoilage in broilers under HS, which is manifested by meat discoloration, a decrease in the water retention capacity, and the loss of nutritional value [8].In addition, ferroptosis is usually accompanied by oxidative stress due to the phospholipid peroxidation of cell membranes [9].Hence, the muscle cells are prone to ferroptosis under the oxidative conditions caused by HS, thereby exacerbating the impairment of muscle edible quality, which is a new framework of chicken quality reduction caused by thermal feeding conditions [10].Notably, nutritional manipulation is an available option and has been used to minimize the HS-induced negative effects on the meat quality of broilers [11].
Selenium (Se) is an essential trace element for poultry and a component of selenomethionine, selenocysteine, and their oxidized forms, which are also substrates of various enzymes, including the antioxidant glutathione peroxidase (GPx) and other enzymes with antioxidant functions [12].Meanwhile, Se acts as a co-factor for antioxidants and is involved in free radical scavenging, protects enzymes and nucleic acids from the deleterious impacts of ROS, and prevents cell membranes and organelles from lipid peroxidation, thereby greatly alleviating oxidative damage in heat-stressed broilers [13].On the other hand, it has been demonstrated that dietary Se deficiency induces oxidative stress, inflammatory damage, and apoptosis in a variety of tissues [14], and adversely affects broiler performance [15].As a new form of Se supplement, selenium nanoparticles (SeNPs) can maximize the beneficial effects of Se because of their high surface activity and nanoscale effect [16].Compared with organic and inorganic selenium, SeNPs have lower toxicity and higher biological activity [17].Studies have shown that the dietary inclusion of SeNPs could increase the content of polyunsaturated fatty acids (PUFAs) and protect lipids from ROS damage, and that SeNPs have better resistance to oxidative stress than inorganic and organic selenium in broilers [18].However, SeNPs are unstable in storage and easily form precipitation, which influence their application in practice [19,20].Conversely, biogenic SeNPs has been proven to be more stable in storage and more conducive to practical application compared to ordinary SeNPs [21], and dietary biogenic SeNPs have been reported to improve antioxidant capacity and meat quality in broilers [22,23].
Alginate oligosaccharides (AOSs) are naturally occurring anionic polymers isolated from brown seaweed, which have antioxidant and other biological activities [24].We previously synthesized a new type of biogenic SeNP using AOS, and we speculated that the preparation of biogenic SeNPs using AOSs possesses both Se and AOS bioactivity simultaneously, and the efficacy of Se can be enhanced through nano-size effects [25].However, the protective effects of SeNPs-AOS on breast meat quality in heat-stressed broilers and the mode of action are still unclear.Therefore, the present study aimed to evaluate whether SeNPs-AOS could improve the antioxidant capacity, metabolite composition, and ferroptosis to ameliorate the HS-induced impairment of breast meat quality in broilers.

Biosynthesis of SeNPs-AOS
The biosynthesis of SeNPs-AOS was achieved by reacting Na 2 SeO 3 with Vc, while AOS served as a polymer template for the reaction.The reaction conditions, including reaction temperature, concentration of AOS, and reaction time, were selected through orthogonal experiments.It was found that the stable SeNPs-AOS products could be prepared under the conditions of AOS concentration of 400 mg/mL, reaction temperature of 60 • C, and reaction time of 30 min.Then, the SeNPs-AOS were characterized using scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS), and demonstrated that the Se particle size was 80 nm and the Se content was 8%.In addition, supplementation with 5 mg/kg SeNPs-AOS had a significant anti-HS effect through an in vivo experiment.The specific preparation process, chemical components of AOS, and other details were described in our previous study [25].

Birds, Experimental Design, and Management
A total of 210 1-day-old unsexed Arbor Acres broilers (CP group, Zhanjiang, China) were raised to 21 days of age, from which 192 broilers with similar body weight were selected and divided into four groups according to a 2 × 2 trial design: thermoneutral group (TN), TN + SeNPs-AOS group, heat stress group (HS), and HS + SeNPs-AOS group.Each group had six replicates with eight chickens per replicate.The TN group and HS group were fed a basal diet (without antibiotics), and the additive group was supplemented with 5 mg/kg SeNPs-AOS in basal diet.The basal diet was formulated with reference to the NRC (1994), and the basal diet composition and nutritional levels are shown in Table S1.The Se content of the basal diet was 0.282 mg/kg, and the Se content of SeNPs-AOS added group was 0.696 mg/kg.
The feeding experimental period was 21 days, and the chickens were managed by free feeding and watering.The ambient temperature of the TN groups was maintained at 23 ± 1.5 • C; the ambient temperature of the HS group (8:00-18:00, 10 h/day) was maintained at 33 ± 2 • C, and ambient temperature was the same as that of the TN group during 18:00-8:00.The relative humidity was maintained at 60-75% for all groups.

Sample Collection
Samples of the breast muscle were collected on the 21st day of the experiment (42-days-old).From the replicate cages of each treatment group, one chicken weighing close to the average weight of the cage was randomly selected for slaughter, and the left breast muscle was collected for meat quality testing, while the right breast muscle was collected and stored in the refrigerator at −80 • C for the determination of metabolome, antioxidant capacity, and gene expression.

Measurement of Meat Quality
The breast muscle rate was measured according to the following formula: total breast muscle weight/live body weight.The pH of the breast muscle was determined by a pH meter after broiler slaughter; the brightness (L*), yellowness (b*), and redness (a*) values of the breast muscle were determined by a meat colorimeter within 2 h of slaughter.Determination of drip loss, freezing loss, and cooking loss was based on the report of Khajeh Bami et al. [26].A regularly shaped 3 cm (length) × 2 cm (width) × 1 cm (thickness) meat sample was taken, weighed (W1), and then hung along the direction of the muscle fibers in a refrigerator at 4 • C.After 24 h, it was taken out to absorb the surface water with filter paper and weighed (W2), and the drip loss = (W1 − W2)/W1.Within 2 h of slaughter sampling, 5 g of muscle sample (W1) was placed in a −20 • C refrigerator for 24 h, and thawed under room temperature conditions and then the surface water was absorbed with filter paper and weighed (W2); freezing loss = (W1 − W2)/W1.After slaughter, about 15 g of regular shaped meat samples were cut and weighed after removing the fascia (W1), heated in a water bath at 80 • C for exactly 30 min, then the meat samples were taken out and cooled statically to room temperature, and weighed after drying the surface water with filter paper (W2); cooking loss = (W1 − W2)/W1.After determination of cooking loss, regularly shaped 3 cm (length) × 1 cm (width) × 1 cm (thickness) meat samples were cut from each sample, and the shear force was determined using the method of Chen et al. [6].

Non-Targeted Metabolomics Analysis
A 2 g breast muscle sample was collected from each slaughtered broiler, which were sent to Suzhou PANOMIX Biomedical Technology Co., Ltd.(Suzhou, China) for the determination of non-targeted metabolomics.The samples were processed and subjected to liquid chromatography (LC)-mass spectrum (MS) detection.The assay methods and data analysis are referenced to Yue et al. [27].Briefly, the breast samples were first used for metabolite extraction, and then LC-MS determination was performed.The LC analysis was conducted using Vanquish UHPLC System (Thermo Fisher Scientific, Waltham, MA, USA).MS detection of metabolites was carried out using Orbitrap Exploris 120 (Thermo Fisher Scientific, Waltham, MA, USA).The raw data were firstly converted to mzXML format using MSConvert in ProteoWizard software package (La Jolla, CA, USA, v3.0.8789).The Ropls software (Auckland, New Zealand, v2.1) was used for multivariate data analyses and modeling.Differential metabolites were subjected to pathway enrichment analysis using MetaboAnalyst software (Edmonton, AB, Canada, v3.0).

Determination of Antioxidant, Selenoprotein, and Ferroptosis-Related Gene Expression
Total RNA from breast muscle was extracted according to the TRIzol total RNA extraction reagents and instructions.RNA was reverse transcribed into cDNA using the reverse transcription kit HiScript II Q RT SuperMix for qPCR from Nanjing Vazyme Bioscience and Technology Company Limited, Nanjing, China (Item No. R223-01), ChamQ Universal SYBR qPCR Master Mix (Item No. Q711-02) was used for qPCR experiments, qPCR reaction system was Mix (10 µL), H 2 O (8.2 µL), cDNA (1 µL), and F/R (0.4 µL/0.4 µL), and the information on the primers is presented in Table S2.Specific sequences of the related genes were quantified by qPCR, and the relative mRNA expression of the related genes was calculated by the 2 −∆∆CT method, the β-actin was used as the internal reference gene.

Statistical Analysis
All data except metabolomics were analyzed using two-way analysis of variance (ANOVA) of general linear modelling (GLM) procedure in SAS software (v9.4), and Tukey's test was used to compare the significance of differences between groups.Metabolomic data analysis was described in Section 2.5.A value of 0.05 ≤ p < 0.10 indicates that the difference tends to be significant, p < 0.05 indicates that the difference is significant, and p < 0.01 indicates that the difference is highly significant.

Meat Quality
As shown in Table 1, the shear force (p < 0.01), cooking loss (p < 0.01), and freezing loss (p < 0.05) were elevated, and the pH (0 min) (p < 0.05) was reduced in the HS group, the dietary SeNPs-AOS decreased the freezing loss (p < 0.01) and shear force (p < 0.01) of the breast muscle in the broilers under HS.There was an interactive effect of temperature and SeNPs-AOS on the cooking loss, freezing loss, and shear force of the breast muscle (p < 0.01).; SEM, standard error; temp., temperature; a,b,c different superscript letters suggest a significant difference between groups; 0.05 ≤ p < 0.10 indicates that the difference tends to be significant, p < 0.05 indicates that the difference is significant, p < 0.01 indicates that the difference is highly significant.

Multivariate Statistical Analysis
Based on the positive and negative ion patterns, all the QC samples were densely distributed on the PCA analysis plot, indicating good reproducibility (Figure 1A,B), while all the blue Q2 points in the PLS-DA (R2Y = 0.91, Q2Y = 0.33) plot were lower than the original blue Q2 points on the far right, suggesting that the results were reliable and valid (Figure 1C,D).
dietary SeNPs-AOS decreased the freezing loss (p < 0.01) and shear force (p < 0.01) of the breast muscle in the broilers under HS.There was an interactive effect of temperature and SeNPs-AOS on the cooking loss, freezing loss, and shear force of the breast muscle (p < 0.01).  b,c different superscript letters suggest a significant difference between groups; 0.05 ≤ p < 0.10 indicates that the difference tends to be significant, p < 0.05 indicates that the difference is significant, p < 0.01 indicates that the difference is highly significant.

Multivariate Statistical Analysis
Based on the positive and negative ion patterns, all the QC samples were densely distributed on the PCA analysis plot, indicating good reproducibility (Figure 1A,B), while all the blue Q2 points in the PLS-DA (R2Y = 0.91, Q2Y = 0.33) plot were lower than the original blue Q2 points on the far right, suggesting that the results were reliable and valid (Figure 1C,D).

Differential Metabolites Identification
The metabolite difference between the groups was analyzed by heatmaps and a volcano diagram, and a total of 312 metabolites were identified in both the positive and negative ion modes (Figure 2A,B).A total of 84 differential metabolites were detected between the TN and HS group; 78 differential metabolites were found between the HS and HS + SeNPs-AOS group (Figure 2C,D).These differential metabolites mainly include fatty acids, organic acids, and amino acids.Compared with the TN group, 39 metabolites were upregulated and 45 metabolites were downregulated in the HS group, including elevated cholesterol and decreased L-histidine, L-glutamine, and L-malic acid (Table S3).Compared to the HS group, there were 35 metabolites elevated and 43 metabolites reduced in the HS + SeNPs-AOS group, including the elevated L-malate, L-glutamate, and inosine (Table S4).

Metabolic Pathways of Differential Metabolites
In order to explore the metabolic pathways of the differential metabolites between groups, an enrichment analysis of 89 differential metabolites were performed (Figure 3A).Among the 20 KEGG pathways, six metabolic pathways were significantly enriched (p < 0.05), including β-alanine metabolism, pantothenic acid and CoA biosynthesis, lysine degradation, arginine biosynthesis, linoleic acid metabolism, as well as alanine, aspartate, and glutamate metabolism (Figure 3B,C).

Differential Metabolites Identification
The metabolite difference between the groups was analyzed by heatmaps and a volcano diagram, and a total of 312 metabolites were identified in both the positive and negative ion modes (Figure 2A,B).A total of 84 differential metabolites were detected between the TN and HS group; 78 differential metabolites were found between the HS and HS + SeNPs-AOS group (Figure 2C,D).These differential metabolites mainly include fatty acids, organic acids, and amino acids.Compared with the TN group, 39 metabolites were upregulated and 45 metabolites were downregulated in the HS group, including elevated cholesterol and decreased L-histidine, L-glutamine, and Lmalic acid (Table S3).Compared to the HS group, there were 35 metabolites elevated and 43 metabolites reduced in the HS + SeNPs-AOS group, including the elevated L-malate, L-glutamate, and inosine (Table S4).

Metabolic Pathways of Differential Metabolites
In order to explore the metabolic pathways of the differential metabolites between groups, an enrichment analysis of 89 differential metabolites were performed (Figure 3A).Among the 20 KEGG pathways, six metabolic pathways were significantly enriched (p < 0.05), including β-alanine metabolism, pantothenic acid and CoA biosynthesis, lysine degradation, arginine biosynthesis, linoleic acid metabolism, as well as alanine, aspartate, and glutamate metabolism (Figure 3B,C).

Antioxidant Capacity
As shown in Table 2, HS decreased the level of T-AOC, reduced the activities of the T-SOD, CAT, and GSH-Px enzymes, and increased the content of MDA (p < 0.01).Dietary SeNPs-AOS elevated the activities of the CAT, GST, and GSH-Px enzymes, and decreased the MDA content in the breast muscle of the broilers under HS (p < 0.05).There was an interaction between temperature and SeNPs-AOS on the T-AOC, CAT, MDA, and GSH-Px (p < 0.05).

Antioxidant Capacity
As shown in Table 2, HS decreased the level of T-AOC, reduced the activities of the T-SOD, CAT, and GSH-Px enzymes, and increased the content of MDA (p < 0.01).Dietary SeNPs-AOS elevated the activities of the CAT, GST, and GSH-Px enzymes, and decreased the MDA content in the breast muscle of the broilers under HS (p < 0.05).There was an interaction between temperature and SeNPs-AOS on the T-AOC, CAT, MDA, and GSH-Px (p < 0.05).
SeNPs-AOS, biogenic selenium nanoparticles synthesized from alginate oligosaccharides; TN group, thermoneutral zone; HS group, heat stress; CON group, control group, basal diet without addition of SeNPs-AOS; SeNPs-AOS group, basal diet with addition of 5 mg/kg SeNPs-AOS; T-AOC, total antioxidant capacity; T-SOD, total superoxide dismutase; CAT, catalase; MDA, malondialdehyde; GSH-Px, glutathione peroxidase; GST, glutathione-S-transferase; temp., temperature; data are expressed as mean (6 replicates per treatment group, n = 6); SEM, standard error; a,b,c different superscript letters suggest a significant difference between groups ; 0.05 ≤ p < 0.10 indicates that the difference tends to be significant, p < 0.05 indicates that the difference is significant, p < 0.01 indicates that the difference is highly significant.a,b,c different superscript letters suggest a significant difference between groups; 0.05 ≤ p < 0.10 indicates that the difference tends to be significant, p < 0.05 indicates that the difference is significant, p < 0.01 indicates that the difference is highly significant.

Figure 4.
Effect of SeNPs-AOS on mRNA expression of breast muscle-related genes in heat-stressed broilers.TN, thermoneutral; TN + SeNPs-AOS, basal diet + SeNPs-AOS (5 mg/kg); HS, heat stress control; HS+ SeNPs-AOS, basal diet + SeNPs-AOS; P T , ambient temperature main effect p value; P Se , SeNPs-AOS main effect p value; P T×Se , p value for the interaction effect between ambient temperature and SeNPs-AOS.a,b,c Different superscript letters suggest a significant difference between groups; 0.05 ≤ p < 0.10 indicates that the difference tends to be significant, p < 0.05 indicates that the difference is significant, p < 0.01 indicates that the difference is highly significant.

Discussion
Chicken has become the main animal source food for humans worldwide.Nevertheless, global warming makes broilers susceptible to HS and results in a decrease in chicken quality [28].HS conditions adversely affect glycolytic metabolism, protein synthesis, and fat deposition in broilers, and simultaneously over-produces reactive oxygen species (ROS), thereby impairing the meat quality [29].Extensive studies have confirmed the deleterious influences of HS on the chicken quality in terms of increased drip loss, cooking loss, and muscle brightness, and reduced pH and redness [30][31][32].Additionally, HS reduces feed intake, resulting in a shortage of myoglobin and other proteins that regulate meat color, which in turn induces an increase in meat brightness and a lower degree of redness [33].After slaughter, a large amount of lactic acid from glycolysis accumulates in the muscle, which reduces muscle pH, leading to protein denaturation and ultimately reducing the muscle tethering capacity [34].Furthermore, free radicals and lipid peroxides produced by HS cause a decrease in antioxidant activity in the muscle, thereby destroying the structure and function of the biofilm and leading to the water loss and atrophy of muscle cells [35].In this study, HS boosted the breast shear force, cooking loss, and freezing loss, while it reduced the breast redness and pH (0 min) values, which are consistent with previous studies [36][37][38][39].It is interesting that dietary SeNPs-AOS reduced the shear force and freezing loss of breast muscle in the heat-stressed broilers.Similarly, Mohamedde et al. [40] indicated that supplementation with inorganic Se and bacterial selenoproteins reduced the drip loss, cooking loss, and shear force of breast muscle.This may be associated with the respective antioxidant functions of Se and AOS in SeNPs-AOS.Also, the biogenic SeNPs could enhance Se bioactivity through nanoscale effects to promote the antioxidant properties [41].Se is a key component of Se-containing proteins and enzymes, such as GSH-Px [42,43], which play a crucial role in the antioxidant defense system [44].It has been reported that dietary Se enhanced the GPx activity of muscle, which contributed to the improvement of meat quality [45].Zhou et al. [46] suggested that supplementation with SeNPs was effective in upgrading the Se content of the tissues as well as the meat quality.In addition, in AOS as the degradation products of alginate, the antioxidant activity has been demonstrated [47].Hence, it is reasonable to speculate that SeNPs-AOS reduced the adverse effects of HS on meat quality and may be partial via improving the antioxidant capacity of breast muscle.
Metabolites are another important indicator of meat quality as they can characterize muscle composition and metabolic status [48].Metabolomics analysis is a new technique for detecting changes in endogenous metabolites affected by external stimulated or internal disturbances, which enables the diagnosis and prediction of metabolite change [49].LC-MS, as one of the assays to detect metabolites, is able to provide a comprehensive coverage of substances and is considered to be a suitable technique for studying metabolism [50].Numerous studies have documented that HS reduced meat quality by altering energy metabolism, amino acid transport, glycolysis, intramuscular fat deposition, and protein synthesis [36,[51][52][53].In our study, a total of 312 metabolites were identified based on metabolomics analysis, mainly including organic acids, sugars, and amino acids, which were enriched in amino acid and organic acid metabolism using the KEGG analysis.The content of free amino acids, unsaturated fatty acids, and nucleotides (inosine-5 -monophosphate [IMP], adenosine-5 -monophosphate [AMP], and inosine) has been reported as detection indicators for meat flavor [54].Amino acids (glutamic acid, aspartic acid, glycine, arginine, etc.) are not only essential for proteins, but also influence the synthesis of other muscle components, which are important for the specific flavor of meat [55].Meanwhile, amino acids and their derivatives can increase the concentration of free amino acids, enhance the antioxidant function and immunity, and indirectly affect meat quality [56].Broilers under HS conditions have altered amino acid metabolism and accelerated protein breakdown, thus decreasing protein deposition and ultimately impairing the meat quality [57].On the other hand, HS adversely affects lipid metabolism and reduces the lipolytic capacity of animals, leading to metabolic disorders as well as fat deposition [58].In this study, the glutamine, histidine, and L-malate were decreased and the cholesterol were increased by HS exposure.Glutamate and inosine are the taste-active components of chicken and there is a synergistic effect between them [59].L-glutamic acid is closely related to the meat quality (e.g., shear force, cooking loss, drip loss) and when L-glutamic acid is elevated, the shear force, cooking loss, and drip loss are decreased.It is worth mentioning that the present findings demonstrated that dietary SeNPs-AOS elevated the L-glutamate and inosine levels of breast muscle in heat-stressed broilers.This is in agreement with Tian et al. [60], who showed that supplementation with yeast Se increased the concentration of glutamate and flavor amino acids in the meat, which could contribute to the enhancement of meat quality.Therefore, the regulation of amino acid metabolism is one of the reasons why dietary SeNPs-AOS alleviate HS-induced breast muscle quality damage.
HS generates large amounts of ROS, and when the balance between the production and elimination of ROS is disrupted, the body's antioxidant level decreases, which in turn induces cellular oxidative stress [61].The present study indicated that HS decreased the level of T-AOC, reduced the activity of the T-SOD, CAT, and GSH-Px enzymes, and increased the content of MDA.Antioxidant enzymes are important factors for maintaining redox balance, and the scavenging of free radicals in the muscle depends on a variety of antioxidant enzymes, including SOD, CAT, and GSH-Px [62].MDA, as a lipid peroxide, is widely recognized as a biomarker of oxidative stress [63].Supplementation with Se has been reported to enhance the antioxidant capacity in the muscle and other organs [64].Consistently, we found that dietary SeNPs-AOS increased the levels of T-AOC, elevated CAT and GSH-Px activities, and decreased the MDA content, thus easing the oxidative damage of breast muscle caused by HS.Hence, the findings of antioxidant performance confirmed that the beneficial effect of SeNPs-AOS on meat quality could be attributed to the improvement of the peroxidation state in the breast muscle.
Selenium acts as an essential trace element in animals and plays a key role in various biological functions, mainly in the form of selenoproteins, which exhibit wide-ranging physiological functions in the body [65].Twenty-four selenoproteins have been identified in broilers (SELENOK, SELENOS, SELENOT, etc.).These proteins are involved in the maintenance of redox homeostasis [66].In the present study, HS reduced the mRNA expression of selenoproteins such as GPx1, GPx3, SELENOS, SELENOW, etc.This is similar to the results of Cao et al. [67], who demonstrated that HS downregulated the expression of GPx2, GPx6, Txnrd, Selh, Selm, and Selx in IPEG-J2 cells.However, our research found that dietary supplementation with SeNPs-AOS upregulated the mRNA expression of selenoprotein genes including GPx3, SELENOW, and SELENOK.In agreement, a previous study suggested that dietary selenite promoted the mRNA expression of GPx1, SELENOW, and SELENOP in an animal model [68], indicating that supplementation with SeNPs-AOS ameliorate oxidative damage also through promoting selenoprotein biosynthesis.The Nrf2/Keap1 is a key pathway of the oxidative stress response.In the physiological condition, Nrf2 binds to Keap1 in the cytoplasm, but when the cell undergoes oxidative stress, Nrf2 disassembles from Keap1 and translocates to the nucleus, then activates the expression of a series of downstream antioxidant genes (including selenoprotein coding genes) by binding to antioxidant elements (AREs) to enhance the antioxidant capacity of the cell [69].In this experiment, HS downregulated the mRNA relative expression of CAT, SOD1, GSTA3, MafF, Nrf2 and other genes, and upregulated the mRNA expression of Keap1.Notably, dietary SeNPs-AOS upregulated the mRNA expression of Nrf2 and the downstream antioxidant related genes.Similar to our study, a previous report demonstrated that dietary biogenic SeNPs were able to protect the mouse intestinal barrier function from oxidative stress by activating Nrf2 signaling and its downstream genes [70].Additionally, it should be noted that the AOS could also reduce the oxidative damage through activating the Nrf2 pathway [71].Therefore, Se and AOS may both contribute to SeNPs-AOS in alleviating breast oxidative stress by targeting Nrf2 signaling, and, unlike the traditional forms of selenium, its unique nanoscale effect may enhance such beneficial regulatory roles.
Ferroptosis is a novel form of cell death, mainly caused by the accumulation of excessive iron-dependent lipid peroxidation, which is also an important adverse consequence of oxidative stress and can exacerbate the impairment of meat quality [72].Nrf2 is not only a key regulator of cellular antioxidant, but also can inhibit ferroptosis by modulating the expression of genes involved in iron metabolism (e.g., FTH1 and Fpn1) and glutathione synthesis and metabolism (e.g., GPX4 and SLC7A11) [73].The results of this experiment showed that HS reduced the expression of GPX4 and SLC7A11, and increased the expres-sion of the PTGS2 gene.Conversely, supplementation with SeNPs-AOS raised the levels of FTH1, Fpn1, and lowered the levels of PTGS2.Iron transport mechanisms are capable of maintaining intracellular iron homeostasis [74].Under normal physiological conditions, almost all iron in the body is tightly bound to transferrin and ferritin [75].FTH1 is a negative regulator of ferroptosis [76] and protects cells from ferroptosis by rapidly converting Fe 2+ to Fe 3+ and storing iron in ferritin [77].As an iron transporter, Fpn1 can alleviate ferroptosis by reducing the overload of Fe 2+ in the cell [78].PTGS2 is a biomarker of ferroptosis [79] and plays a role as a pivotal gene in the biology of ferroptosis [80], and the downregulation of the PTGS2 expression level suggests the alleviation of ferroptosis.Previously, Zhao et al. [81] have shown that supplementation with Se reduced ferroptosis in heart cells.It has been found that Se supplementation could activate the Nrf2/transferrin signaling pathway to suppress ferroptosis [82,83], suggesting that SeNPs-AOS could inhibit oxidative stress by regulating the Nrf2 pathway, which might also suppress ferroptosis through targeting Nrf2/transferrin pathway in the breast muscle of heat-stressed broilers.However, a Prussian blue staining and intracellular iron accumulation assay for the breast muscle were not performed in this study, so the anti-ferroptosis effects of SeNPs-AOS and the mechanism of action still need further confirmation.

Conclusions
Collectively, we prepared biogenic SeNPs-AOS that could ameliorate HS-induced meat quality loss through reducing oxidative stress in the breast muscle of broilers.The beneficial effect may be associated with the activation of the Nrf2 signaling pathway and the promotion of selenoprotein synthesis.Additionally, SeNPs-AOS improved the breast meat quality in relation to the regulation of metabolites (especially the amino acid metabolism), and SeNPs-AOS could also upregulate the expression of the Nrf2/transferrin pathwayassociated anti-ferroptosis genes of the breast muscle in broilers under HS (Figure 5).The current findings have provided novel insights into SeNPs-AOS, which serve as a new type of antioxidant to improve the meat quality in broilers raised in summer and/or tropical areas.

Supplementary Materials:
The following supporting information can be downloaded at www.mdpi.com/xxx/s1:Table S1: Basal diet composition and ingredients (22-42 d); Table S2: Primers used in this study for quantitative real-time PCR; Table S3: Effect of heat stress on various differential metabolites in the breast muscle of broilers; Table S4: Effect of biogenic selenium nanoparticles synthesized from alginate oligosaccharides (SeNPs-AOS) on various differential metabolites in the breast muscle of broilers under heat stress.

Figure 1 .
Figure 1.Multivariate statistical analysis between groups.BMT, thermoneutral group; BMTSE, thermoneutral SeNPs-AOS-added group; BMH, heat stress control group; BMHSE, heat stress SeNPs-AOS-added group; (A) principal component analysis (PCA) score plot-based positive ion mode results; (B) PCA score plot-based negative ion mode results; (C) PLS-DA substitution test plot based on the positive ion model; (D) PLS-DA substitution test plot based on the negative ion model.In Figure 1C,D, green dots represent R2 (the proportion of data variance or variation that the current model can explain), blue dots represent Q2 (the proportion of data variance that the current model can predict), dashed lines represent regression analysis lines, and solid lines represent horizontal and vertical coordinates starting from 0.

Figure 1 .
Figure 1.Multivariate statistical analysis between groups.BMT, thermoneutral group; BMTSE, thermoneutral SeNPs-AOS-added group; BMH, heat stress control group; BMHSE, heat stress SeNPs-AOS-added group; (A) principal component analysis (PCA) score plot-based positive ion mode results; (B) PCA score plot-based negative ion mode results; (C) PLS-DA substitution test plot based on the positive ion model; (D) PLS-DA substitution test plot based on the negative ion model.In Figure 1C,D, green dots represent R2 (the proportion of data variance or variation that the current model can explain), blue dots represent Q2 (the proportion of data variance that the current model can predict), dashed lines represent regression analysis lines, and solid lines represent horizontal and vertical coordinates starting from 0.

Figure 2 .
Figure 2. Analysis and identification of differential metabolites in the breast muscle between groups.BMT, thermoneutral group; BMTSE, thermoneutral SeNPs-AOS-added group; BMH, heat stress control group; BMHSE, heat stress SeNPs-AOS-added group; (A) heatmap clustering of different groups in positive ion mode; (B) heatmap clustering of different groups in negative ion mode; (C) volcano map of differential metabolites in normothermic thermoneutral and heat-stressed groups; (D) volcano map of differential metabolites in heat stress and heat stress control groups.

Figure 2 .
Figure 2. Analysis and identification of differential metabolites in the breast muscle between groups.BMT, thermoneutral group; BMTSE, thermoneutral SeNPs-AOS-added group; BMH, heat stress control group; BMHSE, heat stress SeNPs-AOS-added group; (A) heatmap clustering of different groups in positive ion mode; (B) heatmap clustering of different groups in negative ion mode; (C) volcano map of differential metabolites in normothermic thermoneutral and heat-stressed groups; (D) volcano map of differential metabolites in heat stress and heat stress control groups.

Figure 3 .
Figure 3. Intergroup enrichment analysis of differential metabolites and metabolic pathways.BMT, thermoneutral group; BMTSE, thermoneutral SeNPs-AOS-added group; BMH, heat stress control group; BMHSE, heat stress SeNPs-AOS-added group; (A) heatmap of differential metabolites; (B) pathway prediction of differential metabolites based on KEGG analysis; (C) pathway enrichment of differential metabolites, the color of the dots is related to the p-value, with darker colors indicating smaller p-values and lighter colors indicating larger p-values; the size of the dots represents the impact value, and a larger impact value indicates a larger dot.

Figure 3 .
Figure 3. Intergroup enrichment analysis of differential metabolites and metabolic pathways.BMT, thermoneutral group; BMTSE, thermoneutral SeNPs-AOS-added group; BMH, heat stress control group; BMHSE, heat stress SeNPs-AOS-added group; (A) heatmap of differential metabolites; (B) pathway prediction of differential metabolites based on KEGG analysis; (C) pathway enrichment of differential metabolites, the color of the dots is related to the p-value, with darker colors indicating smaller p-values and lighter colors indicating larger p-values; the size of the dots represents the impact value, and a larger impact value indicates a larger dot.

Figure 4 .
Figure 4.Effect of SeNPs-AOS on mRNA expression of breast muscle-related genes in heat-stressed broilers.TN, thermoneutral; TN + SeNPs-AOS, basal diet + SeNPs-AOS (5 mg/kg); HS, heat stress control; HS+ SeNPs-AOS, basal diet + SeNPs-AOS; PT, ambient temperature main effect p value; PSe, SeNPs-AOS main effect p value; PT×Se, p value for the interaction effect between ambient temperature and SeNPs-AOS.a,b,c Different superscript letters suggest a significant difference between groups; 0.05 ≤ p < 0.10 indicates that the difference tends to be significant, p < 0.05 indicates that the difference is significant, p < 0.01 indicates that the difference is highly significant.

Antioxidants 2023 , 17 Figure 5 .
Figure 5. Proposed mechanism for SeNPs-AOS (biogenic selenium nanoparticles synthesized using alginate oligosaccharides) to attenuate heat stress-induced impairment of breast meat quality in broilers.The upward red arrow indicates the promoting effect of SeNPs-AOS, while the downward blue arrow indicates the reducing effect of SeNPs-AOS.

Figure 5 .
Figure 5. Proposed mechanism for SeNPs-AOS (biogenic selenium nanoparticles synthesized using alginate oligosaccharides) to attenuate heat stress-induced impairment of breast meat quality in broilers.The upward red arrow indicates the promoting effect of SeNPs-AOS, while the downward blue arrow indicates the reducing effect of SeNPs-AOS.

Table 1 .
Effect of SeNPs-AOS on breast meat quality of heat-stressed broilers.

Table 1 .
Effect of SeNPs-AOS on breast meat quality of heat-stressed broilers.AOS, biogenic selenium nanoparticles synthesized from alginate oligosaccharides; TN group, thermoneutral zone; HS group, heat stress; CON group, control group, basal diet without addition of SeNPs-AOS; SeNPs-AOS group, basal diet with addition of 5 mg/kg SeNPs-AOS; data are expressed as mean (6 replicates per treatment group, n = 6); SEM, standard error; temp., temperature;

Table 2 .
Effect of SeNPs-AOS on antioxidant capacity of heat-stressed broiler breast muscle.