Dietary supplementation of alpha-lipoic acid mitigates the negative effects of heat stress in broilers

Sanjeev Wasti; Nirvay Sah; Chin N Lee; Rajesh Jha; Birendra Mishra

doi:10.1371/journal.pone.0254936

. 2021 Jul 26;16(7):e0254936. doi: 10.1371/journal.pone.0254936

Dietary supplementation of alpha-lipoic acid mitigates the negative effects of heat stress in broilers

Sanjeev Wasti ¹, Nirvay Sah ¹, Chin N Lee ¹, Rajesh Jha ¹, Birendra Mishra ^1,^*

Editor: Arda Yildirim²

PMCID: PMC8312949 PMID: 34310622

Abstract

Heat stress accounts for substantial economic loss in the poultry industry by altering the health and performance of chickens. Alpha-lipoic acid (ALA) is a water and fat-soluble antioxidant which is readily absorbed from the intestine resulting in maximum bioavailability. Moreover, ALA acts as a coenzyme in glucose metabolism and helps generate other antioxidants. Considering these benefits, we hypothesized that dietary supplementation of ALA would help mitigate heat stress in poultry. A total of 72 Day-old broiler chicks were randomly assigned into three treatment groups: no heat stress (NHS), heat stress with basal diet (HS), and heat stress with alpha-lipoic acid (HS+ALA); each treatment group had 6 replicate pens with 4 birds in each pen (n = 24/group). The allocated birds were raised under standard husbandry practices for 3 weeks. After 21 d, birds in the HS and HS+ALA groups were exposed to heat stress (33°C for 8 hours during the day) for 3 weeks, while the NHS group was reared under normal conditions (22–24°C). The HS+ALA group received a basal finisher diet fortified with ALA (500 mg/kg) during the treatment period (22 to 42 d), while other birds were provided with the basal finisher diet. Weekly body weight and feed intake were recorded. The cecum digesta for volatile fatty acids (VFAs) analysis and 16S rRNA sequencing for the gut microbiota analysis; and the ileum tissue samples for histological and gene expression analyses were collected on d 42. Exposure to heat stress decreased (P<0.05) average daily gain (ADG) and final body weight (FBW) in the HS group compared to the NHS group, the supplementation of ALA improved (P<0.05) ADG and FBW in heat-stressed birds. Furthermore, birds in the HS+ALA group had increased (P<0.05) expression of HSP90, PRDX1, GPX3, SOD2, OCLN, and MUC2 genes and higher (P<0.05) concentrations of major VFAs (acetate, propionate, and butyrate). The dietary ALA supplementation also improved the villus height and villus height to crypt depth ratio in the HS+ALA group. The microbial diversity analysis revealed significant abundance (P<0.05) of beneficial bacteria Lactobacillus and Peptostreptococcaceae in the cecum of the ALA group. These results indicate that dietary ALA supplementation effectively mitigates the negative effects of heat stress in broilers by improving the expression of heat-shock, tight-junction, antioxidants, and immune-related genes in the intestine, improving villus structures, increasing concentration of major VFAs, and enriching the beneficial microbiota.

Introduction

Poultry meat is a good source of white meat containing a low amount of fat and high proteins. Considering this benefit of poultry meat, the consumers’ preference for poultry meat has rapidly increased in the past decade. This demand has been largely fulfilled by intensive selection and breeding of the chickens for rapid growth and heavier breast muscle [1]. However, these improved breeding are accompanied by one major problem, i.e., highly susceptible to higher environmental temperature due to higher metabolic rate, lack of sweat glands, and the presence of feathers [2]. Thus, with the rising global temperature, heat stress is a significant problem in the poultry industry resulting in massive economic loss in the tropical regions [3]. Heat stress exhibits negative effects on poultry health and production performance by altering different physiological, neuroendocrine, immunological, and behavior responses [2]. As a result, there has been a huge surge in the research regarding mitigating the heat stress in poultry. Researchers have recently tried and tested different compounds such as polyphenols and antioxidants to ameliorate the heat stress in poultry [2, 4–7].

Alpha-lipoic acid (ALA), also known as Universal antioxidant, is produced in small amounts inside the cell [8]. ALA is both water and fat-soluble antioxidants, are readily absorbed from the intestine, and can easily cross the blood-brain barrier resulting in optimum bioavailability. Besides this, both ALA and dihydrolipoic acid (DHLA)- the reduced form of ALA, can quench free radicals both in the liquid and aqueous domains [9]. Moreover, ALA and DHLA also possess metal-chelating activity, acts as a coenzyme in glucose metabolism, and generates other antioxidants such as ascorbate, vitamin E, and glutathione (GST) [8, 10]. Considering these benefits, ALA has recently gained huge attention in normal and heat stress conditions as a potential feed supplement in poultry.

Studies have shown that dietary fortification of ALA was able to improve the growth performance indices, immunological and biochemical characteristics, lipid metabolism, and oxidative stress in the poultry [9]. Likewise, the storability of poultry meat and meat product was also found to increase with the fortification of ALA in the feed [9]. However, there has not been any study focusing on ALA supplementation on gastrointestinal physiology and the growth performance in heat-stressed broiler birds. Thus, this study was carried out to unveil ALA supplementation’s mitigatory effects on the growth performance, immune parameters, gut microbiota, and gut health of heat-stressed broiler birds.

Material and methods

Birds and husbandry

The animal experimentation was carried out at the small animal facility of the University of Hawaii at Manoa, and the experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of Hawaii (Approval no. 17–2605). A total of 72 day-old Cobb-500 unsexed chicks were sourced from a local hatchery (Asagi Hatchery Inc., Honolulu, HI). On d 1, birds were weighed individually, winged tagged, and placed equally and randomly to one of 3 treatments with 6 replicates of each treatment (n = 24 birds/treatment; 4 birds/pen). The treatment groups were: 1) No heat stress (NHS), 2) Heat stress with basal diet (HS), and 3) heat stress with alpha-lipoic acid (HS+ALA). For the first 21 d, all the birds were raised following the standard broiler rearing guidelines on the floor pen system with access to ad libitum feed and water. After 21 d, birds in the HS, and HS+ALA were exposed to the heat of 33–35°C (8 am to 6 pm) and 21–22°C (6 pm to 8 am) with 50% relative humidity for 3 weeks. Birds in the NHS group were reared at the normal room temperature (22°C-24°C) with 50% relative humidity. The lighting regime was 23 h light and 1 h dark periods. The size of the pen was 1 m x 0.71 m, and the stocking density was 1500 cm²/bird.

Diets

Birds were fed the corn-soybean meal-based mash diets in two phases, starter (1–21 d) and finisher (22–42 d), to meet the nutrients requirements of broilers [11]. The energy and protein requirements of the diet was met following the NRC (1994); however, Ca and P level was lower than the commercial requirements. All the birds were provided with the normal starter diet for the first 14 d; afterward, 500 mg/kg ALA was supplemented on the starter diet of the HS+ALA group from 14 to 21 d, while the other two groups (HS and NHS) were provided with the normal starter diet. From 22 to 42 d, NHS and HS birds were fed with the basal finisher diets, and 500 mg/kg ALA was supplemented in the finisher diet of the HS+ALA group. The dose rate of ALA was considered as 500 mg/kg feed based on a previous study [12] in the broiler birds. The diet formulation and their nutrient profiles are presented in Table 1.

Table 1. Ingredients and nutrient composition of the experimental diets.

	Starter		Finisher
Ingredients %	Basal	Basal+ALA	Basal	Basal+ ALA
Corn	54.86	54.86	63.14	63.14
SBM	39.5	39.5	29.6	29.6
Soybean oil	2	2	4.5	4.5
Limestone	1.27	1.27	0.85	0.85
Monocalcium phosphate	0.75	0.75	0.5	0.5
Lysine	0.23	0.23	0.18	0.18
Methionine	0.14	0.14	0.12	0.12
Threonine	0.2	0.2	0.16	0.16
NaCl	0.43	0.43	0.35	0.35
Sodium bicarbonate	0.12	0.12	0.1	0.1
Vitamin + Mineral mix^*	0.5	0.5	0.5	0.5
ALA, mg/kg (top dressing)	0	500	0	500
Calculated nutrient content, %
MEn, kcal/kg	2909	2909	3203	3203
CP	22.09	22.09	18.07	18.07
Ca	0.75	0.75	0.52	0.52
Total P	0.57	0.57	0.47	0.47
Available P	0.3	0.3	0.23	0.23
Lysine	1.39	1.39	1.10	1.10
Methionine	0.48	0.48	0.41	0.41
Cystine	0.43	0.43	0.38	0.38
Threonine	1.03	1.03	0.85	0.85
Tryptophan	0.33	0.33	0.26	0.26
Methionine + Cysteine	0.91	0.91	0.8	0.8
Arginine	1.61	1.61	1.31	1.31
Valine	1.22	1.22	1.03	1.03
Isoleucine	0.93	0.93	0.76	0.76
Leucine	1.89	1.89	1.63	1.63
dig Lys	1.25	1.25	0.99	0.99
dig Met	0.45	0.45	0.39	0.39
dig Thr	0.85	0.85	0.69	0.69
NDF	9.13	9.13	8.78	8.78
CF	3.97	3.97	3.46	3.46
Na	0.22	0.22	0.18	0.18
Cl	0.30	0.30	0.25	0.25
Choline (mg/kg)	1419	1419	1200	1200

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*Providing the following (per kg of diet): vitamin A (trans-retinyl acetate), 10,000 IU; vitamin D₃ (cholecalciferol), 3,000 IU; vitamin E (all-rac-tocopherol-acetate), 30 mg; vitamin B₁, 2 mg; vitamin B₂, 8 mg; vitamin B₆, 4 mg; vitamin B₁₂ (cyanocobalamin), 0.025 mg; vitamin K₃ (bisulphatemenadione complex), 3mg; choline (choline chloride), 250 mg; nicotinic acid, 60 mg; pantothenic acid (D-calcium pantothenate), 15 mg; folic acid, 1.5 mg; betaíne anhydrous, 80 mg; D-biotin, 0.15 mg; zinc (ZnO), 80 mg; manganese (MnO), 70 mg; iron (FeCO₃), 60 mg; copper (CuSO₄·5H₂O), 8 mg; iodine (KI), 2 mg; selenium (Na₂SeO₃), 0.2 mg.

Growth performance

The birds were individually weighed on d 1, 7, 14, 21, 28, 35, and 42. Feed provided to the individual pen was noted, and leftover feed in the pen was also weighed weekly. The feed intake per replicate pen was determined by subtracting the leftover feed over to the total feed consumed during the week. The mortality of the birds was monitored daily. The ADG, ADFI, and FCR were calculated after adjusting for mortality, if any.

Sample collection

At 42 d, two birds/pen (n = 12) from each treatment group were selected randomly and were euthanized by carbon dioxide asphyxiation. A small piece of the ileum (5 cm posterior to the ileocecal transition) was collected (n = 6/treatment; one from each pen), snap-frozen in liquid nitrogen, and stored at -80°C for subsequent gene expression study. The cecum was excised, snap-frozen, and placed at -80°C for microbiota (n = 6/treatment; one from each pen) and VFA (n = 12/treatment; two from each pen) analysis while, approximately 1 cm of the ileum sample (6 cm posterior to the ileocecal junction) was excised, washed with normal saline and preserved in 10% neutral buffered formalin (NBF, pH 7) for the ileum histomorphology analysis (n = 4/treatment).

Total RNA extraction

The 50–100 mg of frozen ileum tissues were used for the total RNA extraction. The total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. The RNA concentration was determined using NanoDrop one (Thermo Fisher Scientific, Madison, WI), while quality was accessed by running RNA samples on 2% agarose gel. Extracted RNA was stored at -80°C until further analysis.

Quantitative real-time PCR (qPCR) assay

The specific oligonucleotide primers used for the qPCR assay were designed using the NCBI Primer-Blast tool and are presented in Table 2. The gene expression analysis was carried out as previously described [13]. In short, for cDNA synthesis, 1 μg of total RNA (20 μL reaction of reverse transcriptase mixture) was reverse transcribed using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The newly synthesized cDNA (20 μL) was diluted (25X) with 480 μL of nuclease-free water and was stored at -20°C until further analysis. The qPCR was carried out by using StepOne Plus real-time PCR system (Applied Biosystems, Foster City, CA) where10 μL reaction mixture containing 3 μL of cDNA, 5 μL of PowerUp SYBR Green Master Mix (Applied Biosystems, Foster City, CA), and 1 μL each of forward and reverse primers specific to the gene target was used. The amplification conditions were 50°C for 2 minutes (hold), 95°C for 2 minutes (hold), followed by 40 repeat cycles of 95°C for 15 seconds (denaturation), 60°C for 15 seconds (annealing), and 72°C for 1 minute (extension). Based on the consistency of expression in the ileum tissue, β-actin was chosen for normalization. The target genes were analyzed in duplicates, and an average value was taken from the experimental replicate. The expression level of target genes was determined using the cycle threshold (Ct) values and changes in the gene expression were calculated by the 2^-ΔΔCt method compared to the control group. The relative mRNA expression was normalized to the endogenous reference gene B-actin.

Table 2. Primers used to quantify the expression of the genes by qPCR.

Gene	Accession no.	Primer Sequence	Amplicon (bp)
SOD1	NM_205064.1	F: `CAACACAAATGGGTGTACCA`	119
		R: `CTCCCTTTGCAGTCACATTG`
SOD2	NM_204211.1	F: `CCTTCGCAAACTTCAAGGAG`	160
		R: `AGCAATGGAATGAGACCTGT`
GPX1	NM_001277853.2	F: `AATTCGGGCACCAGGAGAA`	101
		R: `CTCGAACATGGTGAAGTTGG`
GPX3	NM_001163232.2	F: `GAGGGAGAAGGTGAAATGCT`	192
		R: `CCCAGCTCATTTTGTAGTGC`
TXN	NM_205453.1	F: `GGCAATCTGGCTGATTTTGA`	79
		R: `ACCATGTGGCAGAGAAATCA`
PRDX1	NM_001271932.1	F: `GGTATTGCATACAGGGGTCT`	101
		R: `AGGGTCTCATCAACAGAACG`
NRF2	NM_205117.1	F: `CCCTGCCCTTAGAGATTAGAC`	248
		R: `CAAGTTCATGTCCTTTTCTCTGC`
HSF1	NM_001305256.1	F: `AAGGAGGTGCTCCCAAAGTA`	221
		R: `TTCTTTATGCTGGACACGCTG`
HSF3	NM_001305041.1	F: `TTCAGCGATGTGTTTAACCCT`	244
		R: `GGAGGTCTTTTGGATCCTCT`
HSP90	NM_001109785.1	F: `GATAACGGTGAACCTTTGGG`	120
		R: `GGGTAGCCAATGAACTGAGA`
HSP70	NM_001006685.1	F: `TCTCATCAAGCGTAACACCAC`	104
		R: `TCTCACCTTCATACACCTGGAC`
OCLN	NM_205128	F: `CCGAGGACAGCCCTCAATAC`	82
		R: `CTTTGGTAGTCTGGGCTCCG`
CLDN1	NM_001013611	F: `TACCCCAAAAATGCCCCCTC`	109
		R: `GCGGCATTGTAGTGTCCTCT`
MUC2	NM_001318434	F: `GTGGTCTGTGTGGCAACTT`	71
		R: `GTCTCTTGCAGCCCATTCCT`
IL4	NM_001030693	F: `TGTGCCCACGCTGTGCTTACA`	155
		R: `CTTGTGGCAGTGCTGGCTCTCC`

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*F = Forward; R = Reverse.

Ileum histomorphology

The 10% NBF fixed ileal tissues were shipped to the Histology core facility, John A. Burns School of Medicine, UH Manoa, where samples were dehydrated with the series of ethanol solutions (70%, 80%, 95%, and 100%) and embedded in paraffin blocks. The embedded ileum tissue was sectioned at 6 μm thickness and stained with hematoxylin and eosin staining. A total of 18 well oriented villus-crypts unit per sample was selected and observed under an 8× objective lens by using an Olympus microscope (U-TV0.63XC, Tokyo, Japan). Different intestinal morphological parameters such as villus height (VH)- distance from the tip of villus to the crypt, crypt depth (CD)- distance from villus base to the muscularis mucosa, and villus height to crypt depth ratio (VH/CD) [14] were measured along with the apparent villus surface area by using Infinite Analyze software (Lumenera Corporation, Ottawa, ON, Canada).

Volatile fatty acid

The VFA analysis was carried out as previously described [15]. The VFA profile was analyzed by using gas chromatography (Trace 1300, Thermo Scientific, Waltham, MA) equipped with a flame ionization detector (FID), AS 1310 series automatic liquid sampler, and a 30 m × 0.53 mm internal diameter column (Stabilwax-DA, Restek, Restek Corporation, Bellefonte, PA). Helium was supplied as a carrier gas at a flow rate of 14.5 mL/min, and run time was set for 15 min. The temperature of the injector port and detector port was set at 200°C and 240°C, respectively. Trimethyl acetate (TMA) was used as an internal standard. Data handling and processing were performed on ChromeleonTM 7.2 software (Thermo Scientific, Waltham, MA).

DNA extraction and 16S rRNA gene sequencing

Total genomic DNA was extracted from the cecal content using the QIAamp® DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instruction. The extracted bacterial DNA concentration was determined using NanoDrop one (Thermo Fisher Scientific, Madison, WI) and stored at -20°C until further analysis. The V3 and V4 hypervariable regions of the 16S rRNA gene were amplified as outlined in Illumina 16S Metagenomic Sequencing Library guideline (Illumina) with the following modifications: Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Life Technologies Corporation, Grand Island, NY) was used to set up the PCR reaction, and Mag-Bind Total Pure NGS beads (Omega Bio-Tek) were used for PCR Clean-Ups, and 35 cycles were used in the PCR. Briefly, 16S rRNA sequencing involved the following steps: 1) 1^st stage PCR: Amplicon PCR, 2) PCR Clean-Up, 3) 2^nd Stage PCR: Index PCR, 4) 2^nd PCR Clean-Up 5) Library quantification, normalization, and pooling, and 6) Library denaturing and MiSeq sample loading. The specific sequence of 16S rRNA gene used for amplified were forward primer (5′-CCTACGGGNGGCWGCAG-3′) and reverse primer (5′GACTACHVGGGTATCTAATCC- 3′) [16]. Finally, amplicons were normalized and pooled, and subsequently sequenced on the Illumina MiSeq sequencer at the University of Hawaii at Manoa in Genomics, Proteomics, and Bioinformatics core facility.

Sequencing processing

The Qiagen CLC Genomics Workbench 12.0.1 and the CLC Microbial Genomics module was used for microbial bioinformatics. The sequencing analysis procedures were followed as described in the OTU clustering step-by-step tutorial (Qiagen, Hilden, Germany). The sequencing files (ending with ‘fastq’) were imported in the CLC workbench; files were then paired, setting the minimum distance to 200 and maximum distance to 500. The reads were then trimmed, and the read with the low coverage was removed from the analysis. Thus, obtained reads were clustered as operational taxonomical units (OTUs), based on 97% sequence similarity against the Greengenes v13_8 97% database using the CLC Microbial Genomics module. Chimeras and OTUs with lower abundance (less than 10 reads) were removed. For alpha and beta diversity analysis, the phylogenetic tree was constructed using a maximum likelihood approach based on multiple sequence alignment (MSA) of the OTU sequences generated by MUSCLE in the workbench. The Simpson’s index and Shanon entropy were calculated to determine alpha diversity, while the unweighted UniFrac and weighted UniFrac distances were calculated to determine beta diversity. Permutational multivariate analysis of variance (PERMANOVA) analysis was carried out to measure the significance of beta diversity. Differences in microbiome among treatment groups were analyzed by one-way ANOVA to determine significant difference at different taxa level.

Statistical analysis

Growth performance, gene expression, ileum histomorphology, and significantly abundant microbiome were analyzed using the R-studio and presented as mean ± SEM. These data were analyzed by using one-way analysis of variance (ANOVA), and mean separation between treatment groups was done by the Tukey-Kramer test. Kruskal-Wallis pairwise test and pairwise PERMANOVA test were carried out for alpha and beta diversity to determine the potential difference between treatment groups using the CLC Microbial Genomics module. The spearman correlation analysis was conducted to analyze associations between differential abundance taxonomic groups and measured change parameters using statistical software JMP v14 (SAS Institute Inc., Cary, NC). Values with P<0.05 were regarded as statistically significant.

Results

Effects of alpha-lipoic acid in growth performance

The growth performance of birds in different treatment groups is shown in Fig 1. Supplementation of the ALA significantly improved (P<0.05) the final bodyweight in the heat-stressed broiler birds as compared to the HS group. There was no significant difference (P>0.05) in body weight between the treatment groups until 28 days. However, on 35 d, body weight was significantly decreased (P<0.05) in the HS group compared to the NHS group; dietary ALA supplementation in the heat-stressed broiler birds could not significantly improve the body weight. Nevertheless, on d 42, dietary ALA supplementation significantly improved (P<0.05) the bodyweight in the heat-stressed broiler birds. During the heat stress period (21–42 d), the ADFI was significantly lowered (P<0.05) in both HS and HS+ALA groups as compared to the NHS group. The ADG, on the other hand, was significantly lowered (P<0.05) in the HS group as compared to the NHS group, and dietary supplementation of ALA significantly improved (P<0.05) ADG in heat-stressed broiler birds during the heat stress period (21–42 d). FCR was significantly high (P<0.05) in the HS group and the ALA group compared to the NHS group. Dietary ALA supplementation was not able to significantly improve (P>0.05) the FCR in the heat-stressed broiler birds. There was no mortality in the NHS group, while one bird died in the HS group and the ALA group.

Effects of ALA on the intestinal gene expression

Expression of heat shock protein-related genes

The mRNA expression of heat shock protein-related genes (HSF1, HSF3, HSP70, and HSP90) in the ileum of birds in different treatment groups is shown in Fig 2(A). The mRNA expression of HSF1, HSF3, and HSP70 remains unchanged across the treatment groups. The mRNA expression of HSP90 was significantly decreased (P<0.05) in the HS group as compared to the NHS group, while dietary ALA supplementation significantly increased (P<0.05) the mRNA expression of HSP90 in heat-stressed broiler birds.

Expression of tight-junction related genes

The mRNA expression of the tight-junction related genes (OCLN and CLDN1) in the ileum of birds in different treatment groups is shown in Fig 2(B). The heat stress significantly decreased (P<0.05) the mRNA expression of OCLN in the HS group as compared to the NHS group, while dietary ALA supplementation significantly increased (P<0.05) the expression of OCLN in heat-stressed broiler birds. The mRNA expression of CLDN1 remains unchanged between treatment groups.

Expression of antioxidant related genes

The mRNA expression of the antioxidant-related genes (SOD1, SOD2, PRDX1, TXN, GPX1, GPX3, and NRF2) in the ileum of birds in different treatment groups are shown in Fig 2(C). The mRNA expression of the SOD2 and PRDX1 was significantly decreased (P<0.05) in the HS group as compared to the NHS group, while ALA supplementation significantly increased (P<0.05) their expressions in heat-stressed birds. Dietary ALA supplementation in the heat-stressed birds significantly increased (P<0.05) the mRNA expression of GPX3 as compared to the NHS group. The mRNA expression of the SOD1, TXN, GPX1, and NRF2 remain unchanged between the treatment groups.

Expression of immune-related genes

The mRNA expression of the immune-related genes (IL4 and MUC2) between different treatment groups is shown in Fig 2(D). Dietary ALA supplementation in the heat-stressed broiler birds significantly increased (P<0.05) the expression of MUC2 in heat-stressed broiler birds, as compared to the heat-stressed birds provided with just basal diet (HS group). The mRNA expression of the IL4 remained unchanged between the treatment groups.

Ileum histomorphology

The ileum histomorphology of birds in different treatment groups is shown in Fig 3. Villus height and Villus height to crypt depth ratio were significantly lowered (P<0.05) in the HS group as compared to the NHS group, while ALA supplementation significantly improved (P>0.05) these parameters in heat-stressed broiler birds. The villus surface area was significantly decreased (P<0.05) in the heat-stressed birds compared to the NHS group. Dietary ALA supplementation did not improve (P>0.05) the surface area in heat-stressed broiler birds. The crypt depth remained unchanged across the treatment groups.

Volatile fatty acids

The major VFA in the cecal digesta of birds in different treatment groups is shown in Fig 4. The amount of propionate was significantly decreased (P<0.05) in the HS groups as compared to the NHS groups. Simultaneously, dietary ALA supplementation significantly increased (P<0.05) propionate concentration in the cecal digesta compared to the HS group. The amount of acetate was significantly decreased (P<0.05) in the HS group as compared to the NHS group, while dietary ALA supplementation in heat-stressed broiler birds significantly increased its amount as compared to the HS group. Dietary ALA supplementation in the heat-stressed broiler birds also significantly increased (P<0.05) the concentration of butyrate as compared to the other two groups. Overall, total VFA was significantly lower (P<0.05) in the HS group as compared to the NHS group, and dietary ALA supplementation significantly increased (P<0.05) the concentration of the total VFA.

Alpha and beta diversity of cecal microbiota

Shannon entropy was significantly higher (P<0.05) in the HS+ALA group as compared to the NHS group, while no differences (P>0.05) were observed in the Simpson index between these two groups (Fig 5). The unweighted UniFrac based PCoA revealed that bacterial composition in cecal content was significantly different (PERMANOVA analysis, P value = 0.01687) between the treatments, while no difference was observed in weighted UniFrac (Fig 6).

Fig 5 — (A) Shannon entropy, and (B) Simpson’s index.

Fig 6 — (A) weighted UniFrac, and (B) unweighted UniFrac.

Cecal microbial composition

At the phylum level, HS and HS+ALA groups were dominated by Firmicutes (63%, 63%), followed by Bacteroidetes (36%, 35%), while the NHS group has an almost equal abundance of Firmicutes (49%) and Bacteroidetes (50%) (Fig 7(A)). At the class level, the taxon-based analysis revealed that Clostridia and Bacteroidia were dominant classes across different treatment groups. However, these dominant taxa were not significantly different (P>0.05) across treatment groups (Fig 7(B)). At the order level, Clostridales and Bacteroidales were dominant in different treatment groups (Fig 7(C)). These dominant orders were not statistically different between treatments. Dietary ALA supplementation, however, significantly increased (P<0.05) Lactobacillales in the heat-stressed birds (Fig 7(D)) as compared to the HS group.

Fig 7 — (A) Relative abundance at Phylum level, (B) Relative abundance at Class level, (C) Relative abundance at the Order level, and (D) Significantly abundance of *Lactobacillales*.

The Porphyromonadaceae, Ruminococcaceae, Lachnospiraceae, and unknown Family of Clostridiales were the dominant family found across the treatment groups. The families Lactobacillaceae and Peptostreptococcaceae were significantly enriched (P<0.05) in the HS+ALA group compared to other groups (Fig 8).

At the genus level, Parabacteroides was the most dominant genus across all groups- HS (29%), HS+LA (24%), and NHS (44%), followed by unknown family_Clostridales. The relative abundance of these dominant genera was not significantly different among the groups. Dietary ALA supplementation, however, significantly enriched (P<0.05) the Lactobacillus and unknown genus of Peptostreptococcaceae in the heat-stressed birds (Fig 9).

Correlation between the differential microbial species and measured parameters

The significant correlations observed after performing Spearman correlation analysis are shown in Table 3. The results showed that the expression of SOD2 and OCLN were positively (P<0.05) associated with order Lactobacillales. Acetate, butyrate, and total VFAs were positively correlated (P<0.05) with order Lactobacillales, family Lactobacillaceae, family Peptostreptococcaceae, genus Lactobacillus, and unknown_genus_Peptostreptococcaceae; While propionate was positively correlated (P<0.05) only with order Lactobacillales.

Table 3. Spearman correlation between the differential microbial species and changed measure parameters.

Variables	Differential enriched microbes	Spearman ρ	Prob>\|ρ
SOD2	O_Lactobacillales	0.5119	0.0299*
OCLN	O_Lactobacillales	0.4891	0.0394*
Acetate	O_Lactobacillales	0.5595	0.0158*
Acetate	F_Lactobacillaceae	0.7004	0.0012*
Acetate	F_Peptostreptococcaceae	0.5199	0.0270*
Acetate	G_Lactobacillus	0.7004	0.0012*
Acetate	G_Unknown_Genus_Peptostreptococcaceae	0.5199	0.0270*
Propionate	O_Lactobacillales	0.4871	0.0404*
Butyrate	O_Lactobacillales	0.5429	0.0199*
Butyrate	F_Lactobacillaceae	0.6715	0.0023*
Butyrate	F_Peptostreptococcaceae	0.4795	0.0441*
Butyrate	G_Lactobacillus	0.6715	0.0023*
Butyrate	G_Unknown_Genus_Peptostreptococcaceae	0.4795	0.0441*
Total VFA	O_Lactobacillales	0.5905	0.0099*
Total VFA	F_Lactobacillaceae	0.7128	0.0009*
Total VFA	F_Peptostreptococcaceae	0.471	0.0485*
Total VFA	G_Lactobacillus	0.7128	0.0009*
Total VFA	G_Unknown_Genus_Peptostreptococcaceae	0.471	0.0485*

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Discussion

This study was carried out to evaluate the effectiveness of ALA (500 mg/kg of feed) to mitigate the negative effects of heat stress in poultry by observing growth performance along with different GI physiological parameters. Dietary supplementation of the ALA (500 mg/kg) significantly improved the final body weight, ADG, expressions of antioxidant, heat-shock, tight-junction, and immune-related genes, increased concentration of VFAs, and enriched beneficial gut microbiota in the heat-stressed broiler birds.

Heat stress negatively affects the production performance of broilers by decreased feed intake and growth [2, 17]. We also observed a decreased growth performance of broilers under heat stress. Several nutritional strategies are employed to mitigate the negative effects of heat stress in poultry. Among them, dietary supplementation of ALA as potential feed additives during heat stress has gained popularity in recent years and is found effective in mitigating the negative effects of heat stress in poultry [9]. In accord with those, we also observed significant improvement in the final body weight and ADG in heat-stressed broiler birds supplemented with ALA.

Further, we were interested in delineating the underlying GI physiological changes, leading to improved growth performance in heat-stressed birds supplemented with ALA. The intestine is an important organ that helps in nutrient digestion and absorption. High environmental temperature is found to have detrimental effects on the intestine [18]. Amongst different intestine parts, the ileum, a terminal part of the small intestine, is mainly susceptible to heat stress [19]. Different groups of genes (heat shock protein, antioxidant, tight junction, and immune-related genes) were then analyzed to detail the effects of ALA on heat-stressed broiler birds.

Heat shock proteins (HSPs)–chaperons–are the proteins that help properly fold the protein during the stress condition and possess cytoprotective action [20]. Different HSPs and their transcriptional factors were analyzed to determine the protective action of ALA during stress. Unlike previous studies [19, 21], in acute stress, where expression of HSP90 was higher in heat-stressed birds, expression HSP90 was lowered in this study. There have been varied expressions of HSFs and HSPs regarding tissue sample and the stress duration (i.e., acute vs. chronic stress) [22]. The spatiotemporal expression study of the HSPs and HSFs is therefore warranted. The lower expression of these genes in our study may be due to the exhaustion of the protective mechanism within the cells during chronic stress. Interestingly, dietary supplementation of ALA significantly increased the expression of HSP90.

Antioxidants are the molecules that scavenge the free radicals produced inside the cell. Heat stress elicits oxidative stress resulting in excess production of free radicals within the cell [23]. Therefore, different antioxidant-related genes were analyzed in this study to determine the effectiveness of ALA at the cellular level. The expressions of SOD2, PRDX1, and GPX3 were significantly improved in the heat-stressed broiler birds supplemented with ALA. SOD2 is mitochondrial manganese (Mn) containing enzymes that dismutase the superoxide radicals into hydrogen peroxide [24]. GPX3 reduces hydroperoxides and H₂O₂ by glutathione [25], while PRDX1 reduces H₂O₂ and hydroperoxides by using thioredoxin [26]. Like this study, previous studies have also demonstrated beneficial effects of ALA in improving total antioxidant capacity and antioxidant enzyme activity in oxidative stress conditions in broilers [27, 28].

The expression of tight junction protein and immune-related genes was then analyzed. The OCLN, a transmembrane protein, is one of the major tight junction protein that regulates paracellular permeability and also plays a role in barrier functions [29]. Heat stress impairs the tight-junction protein in the intestine by reducing the blood flow and ultimately generating hypoxia in the intestine [30]. In light of that, the OCLN was lowered in the heat-stressed broiler birds in this study, while ALA supplementation improved the expression of OCLN in heat-stressed broiler birds. Besides, MUC2 expression was also improved in ALA supplemented heat-stressed broiler birds. Mucin is essential in protecting the gut from pathogens, acidic environment, digestive enzymes, and nutrient digestion and absorption [31]. These results indicate that ALA supplementation improved the intestinal membrane integrity and protected the gut from pathogen and acidic environment, which might be one reason for improving the body weight and ADG in heat-stressed birds.

Heat stress impairs the intestinal morphology, decreases villus height, and villus height to crypt depth ratio [32–34]. In this study, heat stress altered the villus’ morphology in the ileum, which may be due to intestinal ischemia, leading to epithelial shedding [35]. Although not statistically significant, the dietary ALA supplementation improved villus height and villus height to crypt depth ratio in heat-stressed birds. The improvement observed in the villus structure was possibly due to the VFAs produced in the gut as VFAs are exerting trophic effects on the intestinal morphology [36]. This observation drove us to analyze the major VFAs produced in the gut of heat-stressed birds. All major VFAs (acetate, propionate, and butyrate) concentrations were significantly increased in heat-stressed broiler birds supplemented with ALA. The improvement in the VFAs in heat-stressed birds was possibly due to the change in the microbiota as VFA is the microbial fermentation product [37]. Thus, we directed our study towards analyzing the microbiota changes in heat-stressed birds supplemented with ALA.

Dietary ALA supplementation significantly improved the relative abundance of Lactobacillales (order), Lactobacillaceace (family), Peptostreptococcaceae (family), Lactobacillus, and unknown genus of Peptostreptococcaceae in the heat-stressed broiler birds.

Peptostreptococcaceae belong to the phylum Firmicutes and play a role in gut homeostasis [38]. They are also found to produce VFAs in the intestine [39]. Spearman’s correlation analysis in this study too revealed a positive association between Peptostreptococcaceae and acetate, butyrate, and total VFAs. Lactobacillus is the gram-positive bacteria that produce lactic acid, which reduces the pH and prevents the growth of pathogenic bacteria [40]. Thus, significant dominance of the Lactobacillus probably reduced the risk of pathogen amplification and invasion in heat-stressed birds supplemented with ALA. Besides, lactobacillus is found to have antioxidant capacity and remove the ROS to mitigate the damage induced by oxidative stress [41]. Moreover, Lactobacillus is also considered as a potent probiotic to improve gut health [42, 43]. Additionally, a positive association was observed between Lactobacillus and different gut health parameters such as expressions of SOD2 and OCLN; and concentration of acetate, propionate, butyrate, and total VFAs. Considering these results, improved body weight and ADG in heat-stressed broilers supplemented with the ALA can be mainly attributed to improved gut microbiota and VFAs. More specifically, Lactobacillus and Peptostreptococcaceae might have played a vital role in improving growth performance in heat-stressed birds.

The alteration of the microbiota on supplementation of ALA can be attributed to the difference in acquisition and use of the lipoic acid among the different microbial species. There is diversity in the lipoate metabolism among different microbial species. One such example is a difference in lipoate metabolism between Helicobacter pylori and Pseudomonas aeruginosa. Protein encoding the lipoate metabolism is absent in the H. pylori, while P. aeruginosa contains the genome that encodes both the lipoate synthesis and lipoate scavenging enzymes as well as the components of the five lipoate complexes [44].

Conclusion

The results of this study revealed that the dietary supplementation of ALA (500 mg/kg) significantly improved the final body weight and ADG; improved expressions of antioxidant, heat shock, immune-related, and tight-junction related genes in the ileum; increased concentration of VFAs and enriched the beneficial bacteria in the gut of heat-stressed broilers. Thus, ALA supplementation can be considered as one of the potential strategies to mitigate heat stress in broilers.

Supporting information

S1 Data

(XLSX)

Click here for additional data file.^{(11.4KB, xlsx)}

Acknowledgments

We sincerely thank Socorro Tauyan for helping in animal experimentation, Dr. Amit Singh in sampling, and Dr. Mohammad Arif for providing the facilities for Microbiome Analysis.

Data Availability

Additional supplemental files having the minimal data set is submitted along with this manuscript.

Funding Statement

This work was supported by a Start-up grant from CTAHR University of Hawaii at Manoa, and USDA Multistate (2052R) to B.M.

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PLoS One. doi: 10.1371/journal.pone.0254936.r001

Decision Letter 0

Arda Yildirim

9 Apr 2021

PONE-D-21-05713

Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Poultry

PLOS ONE

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Firstly, thank you for your suggestion as an academic editor. This MS deals with an interesting and important topic in poultry production. Nevertheless there are still some points of concern from the reviewers, before the manuscript can be accepted for publication. Please make MS title specific because of focusing on animal material that is broiler. As there are some points unclear to me regarding the trial execution, sampling and statements in the MS, I recommend major revision.

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Reviewer #1: The design and objectives of this study is interesting, and it is expected to contribute further to the knowledge base of improving broiler health by providing nutritional strategies to tackle heat-stress problem. The authors have done good work to make a comprehensive story of microbiota, immunity, heat stress parameters and performance. However, authors need to revise their work and correct some inherent concerns in the feed formulation. The other general comments and minor corrections are listed pointwise.

Major comments

1. What standard requirement was followed for this diet formulation? The calcium and phosphorus are extremely low.

The starter diet has high protein but comparatively low energy which is not as the right recommended ratio.

2. Were the birds weighed individually to show the standard deviation in each pen?

3. Confirm whether the gene expression calculation was based on relative standard curve or the comparative CT

method. Describe more specifically what represents the gene expression value.

4. Line 153. The citation for embedding is not properly attributed. The reviewer was not able to locate the information in

the cited article or in the reference attributed in the cited article!

5. Since the authors have stated that ALA would have improved performance via microbiota-VFA modulation, it is

expected that they provide some insight into why ALA potentially modify microbiota.

Minor comments

Line 17: add ‘is’ between ‘which’ and ‘readily’

Line 20: State clearly the adaptation and allocation period along with the replication of the treatments.

Line 48: Better say heavier breast meat. Also, it should be improved breeding and not improved chicken.

Line 50: Insert ‘the’ between ‘and’ and ‘presence’.

Line 51: High temperature is the problem of tropical condition in the context of chicken.

Line 59: Check whether it will be is or are with ALA.

Line 82: Use n for experimental unit. Either state total number of birds per treatment or just mention birds per experimental unit.

Line 91: Remove ‘with’ after ‘fed’.

Line 110: Change ‘reducing’ to ‘subtracting’.

Line 158: Is it muscularis mucosae or submucosa?

Line 200-2001: Were the reads trimmed before pairing or after pairing?

Line 227: Add significance statistics.

Line 238: Remove numerical improvement. This means there was high variation among the replicates.

Line 244-245: Check grammar.

Line 262: Change ‘remains’ to ‘remain’.

Line 273: Use correct preposition after ‘compared’. Add significance statistics.

Line 288: Remove ‘total’ before ‘concentration’.

Line 298: Change ‘have’ to ‘had’.

Line 305: Add ‘the’ before ‘heat-stressed’.

Line 378: Is decreasing crypt depth an impairment?

Figure 5: Define the box plot. State whether the whiskers are error or data range. If the whiskers are range, then why are no data points on NHS box plot whisker?

Figure 7: What is the unit of relative abundance of Lactobacillales? Is it any fractions?

Reviewer #2: Dear Authors Regarding the manuscript title Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Poultry

The scientific background of the topic was well mentioned in the introduction part. The experiment design, as well as the replicates and methods used, were very good. The results obtained were presented in tables well discussed with other author’s results. However, some observation in the present paper should be corrected and add to improve the quality of the paper.

• The title (Dietary Supplementation of Alpha-lipoic A 1 cid Mitigates the Negative Effects of Heat Stress in Poultry) it will be better if you replacing Poultry to broilers

• Table 1 Ingredients and nutrient composition of the experimental diets, need to carful checking for the following :

1- SBM , add the crude protein level 44 or 46%?

2- MEn, kcal/kg, Ca, digP , not according to the Cobb-500 requirement.

• Introduction and Discussion

Need some other references about the impact of heat stress and Growth and Lipid Metabolism Marker Genes in Broiler Chickens in poultry I recommend you read the following references:

Saki Shimamoto 1,2, Kiriko Nakamura 1, Shozo Tomonaga 3, Satoru Furukawa 4, Akira Ohtsuka and Daichi Ijiri. Effects of Cyclic High Ambient Temperature and Dietary Supplementation of Orotic Acid, a Pyrimidine Precursor, on Plasma and Muscle Metabolites in Broiler Chickens. Metabolites 2020, 10, 189; doi:10.3390/metabo10050189

Saleh, Ahmed A.; Shukry, Mustafa; Farrag, Foad; Soliman, Mohamed M.; Abdel-Moneim, Abdel-Moneim E. (2021) "Effect of Feeding Wet Feed or Wet Feed Fermented by Bacillus licheniformis on Growth Performance, Histopathology and Growth and Lipid Metabolism Marker Genes in Broiler Chickens" Animals 11, no. 1: 83.

Ahmed A. Saleh , Mohammed S. Eltantawy , Esraa M. Gawish , Hassan H. Younis , Khairy A. Amber , Abd El-Moneim E. Abd El-Moneim & Tarek A. Ebeid (2020) Impact of Dietary Organic Mineral Supplementation on Reproductive Performance, Egg Quality Characteristics, Lipid Oxidation, Ovarian Follicular Development, and Immune Response in Laying Hens Under High Ambient Temperature. Biological Trace Element Research. 195:506–514.

Inoue H, Shimamoto S, Takahashi H, et al. Effects of astaxanthin-rich dried cell powder from Paracoccus carotinifaciens on carotenoid composition and lipid peroxidation in skeletal muscle of broiler chickens under thermo-neutral or realistic high temperature conditions. Anim Sci J. 2018;00:1–8.

Ahmed A. Saleh , Abeer A. Kirrella, Mahmoud A. O. Dawood, Tarek A. Ebeid (2019) Effect of dietary inclusion of cumin seed oil on the performance, egg quality, immune response and ovarian development in laying hens under high ambient temperature. Animal Physiology and Animal Nutrition. 103(6):1810-1817.

• Results: it will be better if you present the data for performance in table.

Reviewer #3: Heat stress is a practical problem to broiler chicks which results in over $128 million in losses for the poultry industry in the United States. The manuscript by Wasti et al examines whether using alpha lipoic acid (ALA) mitigated negative effects of heat stress in poultry chickens. The results document the beneficial effects of ALA in broiler chickens by improving gut health. The manuscript has been written in a well-orchestrated manner. However, some points have to be discussed and manuscript could be accepted only after major revisions.

General Comments:

Line 91: Remove the space error after “phases”, and also throughout the manuscript.

Line 97: Add space between “study[9]”

Line 111: No need to explain abbreviation ADG again once it already mentioned in the abstract section.

Line 238: Dietary ALA supplementation o was able to improve the FCR. What is supplementation o? Please remove it from text.

Specific Comments:

Comment 1: Figure 1,2,3,4 No word about sample size based on which the means and SEM were computed. Please add.

Comment 2: Most of the figures do not have the superscripts. Authors have to correct those figures.

Comment 3: The exact computed P-value at least for significant ones should be shown not only (P<0.05) which is a theoretical value.

Comment 4: Can authors please describe more for antioxidant defence system and its significance in poultry?

Comment 5: Please elaborate the ALA mechanism in improving growth performance of heat stress chickens in the discussion section.

Comment 6: Authors have not mentioned any dehydration-related parameters which is the most important factor to study the heat stress-related study as this causes dehydration in animals.

Comment 7: During dehydration, mainly epithelial cells hampered. Tight junction plays a major role during heat stress. The authors focused on the claudin and occludin molecules. Results showing that insignificant changes in claudin molecule but occludin is significantly higher in the treatment group. Tight junction maintains by both of these molecules. It is assuming from this result that due to heat stress, epithelial cells in the HS group individuals may experience dehydration condition which may lead to the loss of experimental animals. Is there so? If no, then how it will be justified? However, how it will be justified about the findings of villus height (higher in NHS), crypt depth (higher in NHS), and villus surface area (same in HS and ALA+HS) in relation to claudin and occludin level? Meantime, IL-4 is reduced which is an anti-inflammatory marker in the HS group.

Comment 8: Insert superscripts in Heat shock protein-related graph (Figure 2A). How it is possible the same level of HSP70 in the NHS and HS groups? It seems that the protein folding process will be the same in the control and HS group of animals. Then how HSP90 levels get reduced in the HS group?

Comment 9: Peroxiredoxin level showing different levels in the graph but it is showing the same superscripts? Why?

Comment 10: Most of the antioxidant-related parameter levels are higher in the treatment group. Meantime SOD1 and SOD2 are higher in the NHS group. How it will be justified?

Reviewer #4: It is a well-written manuscript. The study design and data collection are sufficient. The only concern is the number of animals used in the current study is low, and the growth performance data may not be representative. There are some minor comments:

Please indicate the sample number/statistical unit in each result. For example, if you collect two samples from each replicate. The data need to be averaged within each replicate upon analysis.

Is there any reason why ALA was not fed to the birds from the beginning of the trial?

It would be great if the author could present the morality from each treatment since it may impact the performance calculation due to low birds no.

Please also describe the raising environment, such as pen size and type (batter, cage or floor pen, litter, etc. )

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Sahil Kalia

Reviewer #4: Yes: Chongxiao Chen

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PLoS One. 2021 Jul 26;16(7):e0254936. doi: 10.1371/journal.pone.0254936.r002

Author response to Decision Letter 0

19 May 2021

Reviewer #1:

The design and objectives of this study is interesting, and it is expected to contribute further to the knowledge base of improving broiler health by providing nutritional strategies to tackle heat-stress problem. The authors have done good work to make a comprehensive story of microbiota, immunity, heat stress parameters and performance. However, authors need to revise their work and correct some inherent concerns in the feed formulation. The other general comments and minor corrections are listed pointwise.

Response: We highly appreciate the reviewer's thorough reading and insightful comments and suggestions on our manuscript. Based on the reviewer's suggestions, we have modified our manuscript, and highlighted it in “yellow” in the text of the manuscript.

Major comments

1. What standard requirement was followed for this diet formulation? The calcium and phosphorus are extremely low. The starter diet has high protein but comparatively low energy which is not as the right recommended ratio.

Response: Although official guidelines are still NRC 1994, we believe that is outdated. So, we formulate a ration based on the commercial birds' recommendation. Yet, mention as “meet or exceed the recommended requirements of NRC” (which is common among poultry researchers). In such a case, for starters, we target to have 21-22% CP and ~2900 Kcal/kg MEn diets. Also, there is debate on the Ca and P requirements with no any concrete recommendation. So, we are using a relatively lower dose of Ca and P in our formulation for broiler ration and go at the upper level in layer ration (to be safe). It seems that there was a little low in the finisher, though. However, we believe that it will not affect the result of this study, as it was similar in both the heat-stressed and control birds.

2. Were the birds weighed individually to show the standard deviation in each pen?

Response: Yes, birds were weighed individually in each pen.

3. Confirm whether the gene expression calculation was based on relative standard curve or the comparative CT method. Describe more specifically what represents the gene expression value.

Response: Gene expression calculation in our study was based on the comparative CT method. We confirmed the specificity of primers by running the melt curve, and gel electrophoresis for the specific product size.

4. Line 153. The citation for embedding is not properly attributed. The reviewer was not able to locate the information in the cited article or in the reference attributed in the cited article.

Response: Additional information has now been added, and the cited reference has been deleted from the text.

5. Since the authors have stated that ALA would have improved performance via microbiota-VFA modulation, it is expected that they provide some insight into why ALA potentially modify microbiota.

Response: Thank you for your suggestion. We have added explanation for the change in microbiota on supplementation of the VFA.

“The alteration of the microbiota on supplementation of ALA can be attributed to the difference in acquisition and use of the lipoic acid among the different microbial species. There is diversity in the lipoate metabolism among different microbial species. One such example is in between Helicobacter pylori and Pseudomonas aeruginosa. Protein encoding the lipoate metabolism is absent in the H. pylori, while P. aeruginosa contains the genome that encodes both the lipoate synthesis and lipoate scavenging enzymes as well as the components of the five lipoate complexes”.

Minor comments

Line 17: add ‘is’ between ‘which’ and ‘readily’

Response: Corrected

Line 20: State clearly the adaptation and allocation period along with the replication of the treatments.

Response: Additional information about the replication and the adaptation is now provided in the text (Line 21-22).

Line 48: Better say heavier breast meat. Also, it should be improved breeding and not improved chicken.

Response: We have made changes as per the reviewer’s suggestion.

Line 50: Insert ‘the’ between ‘and’ and ‘presence’.

Response: ‘the’ is now added in the text between ‘and’ and ‘presence’.

Line 51: High temperature is the problem of tropical condition in the context of chicken.

Response: We have now mentioned tropical regions in the text.

Line 59: Check whether it will be is or are with ALA.

Response: It is ‘is’.

Line 82: Use n for experimental unit. Either state total number of birds per treatment or just mention birds per experimental unit.

Response: It is now used in the text.

Line 91: Remove ‘with’ after ‘fed’.

Response: It is now removed from the text.

Line 110: Change ‘reducing’ to ‘subtracting’.

Response: It is now changed.

Line 158: Is it muscularis mucosae or submucosa?

Response: Crypt depth was measured from the villus base to the muscularis mucosa.

Line 200-2001: Were the reads trimmed before pairing or after pairing?

Response: It was paired and then trimmed.

Line 227: Add significance statistics.

Response: It is now added in the text.

Line 238: Remove numerical improvement. This means there was high variation among the replicates.

Response: The numerical improvement is now removed from the text.

Line 244-245: Check grammar.

Response: We have corrected the sentence.

Line 262: Change ‘remains’ to ‘remain’.

Response: The change is made in the text.

Line 273: Use correct preposition after ‘compared’. Add significance statistics.

Response: Correct preposition and statistical statistics is now added in the text.

Line 288: Remove ‘total’ before ‘concentration’.

Response: It is now removed from the text.

Line 298: Change ‘have’ to ‘had’.

Response: It is now changed in the text.

Line 305: Add ‘the’ before ‘heat-stressed’.

Response: It is now added in the text.

Line 378: Is decreasing crypt depth an impairment?

Response: It depends on the context. We have removed the word from the text.

Figure 5: Define the box plot. State whether the whiskers are error or data range. If the whiskers are range, then why are no data points on NHS box plot whisker?

Response: Thank you for the feedback. We have generated the new boxplot images using the R. In this boxplot; the whiskers represent the range of the data.

Figure 7: What is the unit of relative abundance of Lactobacillales? Is it any fractions?

Response: Yes, it is a fraction of Lactobacillales present in the particular treatment group.

Reviewer #2:

Dear Authors Regarding the manuscript title Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Poultry. The scientific background of the topic was well mentioned in the introduction part. The experiment design, as well as the replicates and methods used, were very good. The results obtained were presented in tables well discussed with other author’s results. However, some observation in the present paper should be corrected and add to improve the quality of the paper.

• The title (Dietary Supplementation of Alpha-lipoic A 1 cid Mitigates the Negative Effects of Heat Stress in Poultry) it will be better if you replacing Poultry to broilers

Response: We have made changes in our title according to reviewer’s suggestion.

• Table 1 Ingredients and nutrient composition of the experimental diets, need to carful checking for the following :

1- SBM , add the crude protein level 44 or 46%?

2- MEn, kcal/kg, Ca, digP , not according to the Cobb-500 requirement.

Response: 1. We agreed with the reviewer that SBM is typically labeled as 44 to 48, depending on supplies. However, we used the analyzed value, which was 45% (although claimed 46%). So, we suggest not adding the protein value in the ingredient, like others, rather provide the nutrient content in the formulated diets.

2. “Although official guidelines is still NRC 1994, we believe that is outdated. So, we formulate ration based on the commercial birds' recommendation. Yet, mention as “meet or exceed the recommended requirements of NRC” (which is common among poultry researchers). For starters, we target to have 21-22% CP and ~2900 Kcal/kg MEn diets. Also, there is debate on the Ca and P requirements with no any concrete recommendation. So, we are using a relatively lower dose of Ca and P in our formulation for broiler ration and go at the upper level in layer ration (to be safe). It seems that there was a little low in the finisher, though. However, we believe that it will not affect the result of this study, as it was similar in both heat-stressed and normal burds group.”

• Introduction and Discussion

Need some other references about the impact of heat stress and Growth and Lipid Metabolism Marker Genes in Broiler Chickens in poultry I recommend you read the following references:

Response: We have now included the following references in our manuscript.

• Saki Shimamoto 1,2, Kiriko Nakamura 1, Shozo Tomonaga 3, Satoru Furukawa 4, Akira Ohtsuka and Daichi Ijiri. Effects of Cyclic High Ambient Temperature and Dietary Supplementation of Orotic Acid, a Pyrimidine Precursor, on Plasma and Muscle Metabolites in Broiler Chickens. Metabolites 2020, 10, 189; doi:10.3390/metabo10050189

• Saleh, Ahmed A.; Shukry, Mustafa; Farrag, Foad; Soliman, Mohamed M.; Abdel-Moneim, Abdel-Moneim E. (2021) "Effect of Feeding Wet Feed or Wet Feed Fermented by Bacillus licheniformis on Growth Performance, Histopathology and Growth and Lipid Metabolism Marker Genes in Broiler Chickens" Animals 11, no. 1: 83.

• Ahmed A. Saleh , Mohammed S. Eltantawy , Esraa M. Gawish , Hassan H. Younis , Khairy A. Amber , Abd El-Moneim E. Abd El-Moneim & Tarek A. Ebeid (2020) Impact of Dietary Organic Mineral Supplementation on Reproductive Performance, Egg Quality Characteristics, Lipid Oxidation, Ovarian Follicular Development, and Immune Response in Laying Hens Under High Ambient Temperature. Biological Trace Element Research. 195:506–514.

• Inoue H, Shimamoto S, Takahashi H, et al. Effects of astaxanthin-rich dried cell powder from Paracoccus carotinifaciens on carotenoid composition and lipid peroxidation in skeletal muscle of broiler chickens under thermo-neutral or realistic high temperature conditions. Anim Sci J. 2018;00:1–8.

• Ahmed A. Saleh , Abeer A. Kirrella, Mahmoud A. O. Dawood, Tarek A. Ebeid (2019) Effect of dietary inclusion of cumin seed oil on the performance, egg quality, immune response and ovarian development in laying hens under high ambient temperature. Animal Physiology and Animal Nutrition. 103(6):1810-1817.

• Results: it will be better if you present the data for performance in table.

Response: For better visibility of growth performance data, we presented the findings in bar graphs. A supplemental file is now submitted along with the manuscript, which includes performance data in a tabular format.

Reviewer #3:

Heat stress is a practical problem to broiler chicks which results in over $128 million in losses for the poultry industry in the United States. The manuscript by Wasti et al examines whether using alpha lipoic acid (ALA) mitigated negative effects of heat stress in poultry chickens. The results document the beneficial effects of ALA in broiler chickens by improving gut health. The manuscript has been written in a well-orchestrated manner. However, some points have to be discussed and manuscript could be accepted only after major revisions.

General Comments:

Line 91: Remove the space error after “phases”, and also throughout the manuscript.

Response: We have now corrected it throughout the manuscript.

Line 97: Add space between “study[9]”

Response: Added

Line 111: No need to explain abbreviation ADG again once it already mentioned in the abstract section.

Response: Corrected

Line 238: Dietary ALA supplementation o was able to improve the FCR. What is supplementation o? Please remove it from text.

Response: Corrected

Specific Comments:

Comment 1: Figure 1,2,3,4 No word about sample size based on which the means and SEM were computed. Please add.

Response: We appreciate the reviewer for pointing out this issue. Information about the sample size is now provided in the figure.

Comment 2: Most of the figures do not have the superscripts. Authors have to correct those figures.

Response: Thank for your suggestion. However, we have used the different letters/ superscripts only in the figures where there is statistically significance. We believe that will make the reader easily visualize the main findings of the figure, and there is no point in using the superscript in the figures where there is not statistically significant.

Comment 3: The exact computed P-value at least for significant ones should be shown not only (P<0.05) which is a theoretical value.

Response: Thank you for your suggestion. We have now included that information in the supplemental data provide along with this manuscript.

Comment 4: Can authors please describe more for antioxidant defense system and its significance in poultry?

Response: Thanks for your suggestion. Information about the antioxidant defense system is provided in the discussion section.

Comment 5: Please elaborate the ALA mechanism in improving growth performance of heat stress chickens in the discussion section.

Response: Besides observing the growth performance, the objective of this paper was to delineate the mechanism the beneficial aspects of the ALA in heat-stressed birds. We have tried to incorporate the mechanism by measuring different parameters – VFAs, gene expression, histomorphology and microbiota. All these aspects have been discussed in the discussion section. Moreover, the mechanism by which ALA helped in the improvement of microbiota is also now included in the manuscript.

Comment 6: Authors have not mentioned any dehydration-related parameters which is the most important factor to study the heat stress-related study as this causes dehydration in animals.

Response: Yes, we agreed with the reviewer’s point that we should have included dehydration-related parameters such as – water intake etc. We were not able to precisely collect those data because of our animal experimentation facility. All the birds in HS group were provided with ad libitum of water. We are, however, planning to include those parameters in our future experiments.

Response: Yes, there was mortality. One of the birds died in the HS and the ALA group. Besides Occludin and Claudin, there are a lot of other molecules that affect the morphology of the villi in the intestine. Occludin and claudin are only a few tight-junction proteins chosen to observe the beneficial effect of ALA in heat-stressed birds. A comprehensive study needs to be undertaken if we want to delineate the exact mechanism of how HS is affecting the villus height and crypt depth at the molecular level. We won’t be able to come up with a concrete interpretation just by observing these two molecules.

IL-4 has many biological roles inside the cell. It is involved in the T-cell proliferation and differentiation of B cells into the plasma cells. Besides, IL-4 also induces B-cell class switching to IgE. Taken together, decreased IL-4 level in the HS group shows the lower immunity of the birds during the high temperature.

Response: Studies have revealed that the expression of these molecules varies with the intensity of the stress, i.e., either acute or chronic. In our study, we did not find a significant difference in the expression of HSP70 between the treatment groups. In our opinion, to justify the same level of expression of the HSP70 gene in between NHS and HS, spatiotemporal expression of this gene needs to be carried out, which can come up with any conclusive statements about its expression pattern. Moreover, in our follow-up study, we plan to study the spatiotemporal expression of these genes in heat-stressed birds.

Comment 9: Peroxiredoxin level showing different levels in the graph but it is showing the same superscripts? Why?

Response: It is so because of the error bars (variation). If you look at the error bars of NHS and HS+ALA groups, they coincide, which indicates that they were not statistically different. That is the reason the same superscript was given in between two groups – NHS and HS+ALA.

Comment 10: Most of the antioxidant-related parameter levels are higher in the treatment group. Meantime SOD1 and SOD2 are higher in the NHS group. How it will be justified?

Response: The higher level of the SOD1 and SOD2 in the NHS group observed in this study was in agreement with our expectation. The higher of SOD1 and SOD2 indicates the high amount of this antioxidant inside the cell; when we exposed the animal to the heat stress condition, the relative expression of these genes went down, which we expect in the heat stress. Again, on supplementing ALA, the level of these genes went up. These findings reflect the perfect antioxidant mechanism inside the stressed cell.

Reviewer #4:

It is a well-written manuscript. The study design and data collection are sufficient. The only concern is the number of animals used in the current study is low, and the growth performance data may not be representative. There are some minor comments:

Response: We highly appreciate the reviewer's thorough reading and insightful comments and suggestions on our manuscript. Based on the reviewer's suggestions, we have modified our manuscript and highlighted it in “yellow” in the text of the manuscript.

Please indicate the sample number/statistical unit in each result. For example, if you collect two samples from each replicate. The data need to be averaged within each replicate upon analysis.

Response: The sample number is now mentioned below the figure in the result section.

Is there any reason why ALA was not fed to the birds from the beginning of the trial?

Response: The objective of this study was to observe the mitigatory effects of ALA in heat-stressed broilers. We exposed the birds to the heat stress condition only after 21 days, so we choose to provide them only during the heat stress period. Also, in a real-life practical scenario, farmers will prefer to supplement the products only at the HS time.

It would be great if the author could present the morality from each treatment since it may impact the performance calculation due to low birds no.

Response: We really appreciate your comment regarding mortality. One bird died from the HS group and the HS+ALA group. This information is now provided in the result section.

Please also describe the raising environment, such as pen size and type (batter, cage or floor pen, litter, etc. )

Response: The birds were raised on the floor pen system, the size of the individual pen was 1 m x 0.71 m, and the stocking density was 1500 cm2/bird. These informations are now added to the text.

Attachment

Submitted filename: Reviewers response_PONE-D-21-05713R1.pdf

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PLoS One. doi: 10.1371/journal.pone.0254936.r003

Decision Letter 1

Arda Yildirim

25 Jun 2021

PONE-D-21-05713R1

Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Broilers

PLOS ONE

Dear Dr. Mishra,

Please provide clear information on whether the non-standard requirement of a commercial hybrid broiler for calcium and phosphorus (available) values is met.

Please submit your revised manuscript by Aug 09 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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We look forward to receiving your revised manuscript.

Kind regards,

Arda Yildirim, Ph.D.

Academic Editor

PLOS ONE

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Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: 1. More specifically, the calcium and phosphorus values are not formulated according to the commercial birds recommendation (in this case Cobb 500). So, the authors answer is not correct. It is true that there is variation in the inclusion level of calcium and phosphorus. The authors must mention in the manuscript that they formulated a low calcium and phosphorus diet and if possible, should provide the reason for such formulation. Low Ca and P do not necessarily keep the birds safe. This is not going to affect the outcome of this study but would provide a wrong reference for other researchers that low Ca and P diet would be required to yield a standard growth performance. The authors should provide correct information. They cannot cite one reference for meeting requirement and provide reason that it is outdated, and they considered commercial recommendation, surprisingly which was even not followed. Cobb guidelines state Ca requirement to be 0.9% in the starter and 0.8% in the finisher, available phosphorus is around 0.45%. The authors have formulated the diet containing 0.75% Ca in the starter and 0.52% in the finisher, while the available phosphorus is 0.3% in the starter and 0.23% in the finisher. There is a typo: it should be available phosphorus and not dig. If the authors provide correct information in the draft and state the reason for this inclusion level then it would be ok otherwise it would be understood as a mistake in their feed formulation.

2. Also, authors should have removed 'standard curve method' from line 146 if they calculated gene expression by the comparative CT method. Figure 2 does not show any fold change (as stated in line 147) as none of the treatment has a normalized value of 1. So, it would be better to remove the fold change from the materials and methods. Besides, the authors should mention the treatment (may be the control group) that was used to calculate the ΔΔ CT.

Reviewer #2: Dear Authors Regarding the manuscript title Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Broilers

The scientific background of the topic was well mentioned in the introduction part. The experiment design, as well as the replicates and methods used, were very good. The results obtained were presented in tables well discussed with other author’s results. In addition the authors answered all inquiries and the manuscript may accept in this form.

Reviewer #3: (No Response)

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Sahil Kalia

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PLoS One. 2021 Jul 26;16(7):e0254936. doi: 10.1371/journal.pone.0254936.r004

Author response to Decision Letter 1

2 Jul 2021

Reviewer #1

More specifically, the calcium and phosphorus values are not formulated according to the commercial birds’ recommendation (in this case Cobb 500). So, the authors answer is not correct. It is true that there is variation in the inclusion level of calcium and phosphorus. The authors must mention in the manuscript that they formulated a low calcium and phosphorus diet and if possible, should provide the reason for such formulation. Low Ca and P do not necessarily keep the birds safe. This is not going to affect the outcome of this study but would provide a wrong reference for other researchers that low Ca and P diet would be required to yield a standard growth performance. The authors should provide correct information. They cannot cite one reference for meeting requirement and provide reason that it is outdated, and they considered commercial recommendation, surprisingly which was even not followed. Cobb guidelines state Ca requirement to be 0.9% in the starter and 0.8% in the finisher, available phosphorus is around 0.45%. The authors have formulated the diet containing 0.75% Ca in the starter and 0.52% in the finisher, while the available phosphorus is 0.3% in the starter and 0.23% in the finisher. There is a typo: it should be available phosphorus and not dig. If the authors provide correct information in the draft and state the reason for this inclusion level then it would be ok otherwise it would be understood as a mistake in their feed formulation.

Response: We highly appreciate the reviewer’s meticulous review and pointing out about the feed formulation (following the recent commercial standard). Yes, we agree with the reviewer’s point that we have used lower amount of the calcium and phosphorus than that is recommended for the commercial requirements. We have added this information in our revised manuscript. (Line 94-95). The objective of this study is focused on mitigating heat stress using nutritional supplementation (Alpha lipoic acid). So, our focus was on meeting the energy and protein requirement following the NRC (1994) guidelines and examining at the variables related to heat stress. We agree with the reviewer that we should have formulate diets based on commercial requirement, which we believe is a valuable input for executing our future projects. In this study, however, we believe that lower Ca and P level will not affect the tested variable. So, the finding of the study still holds scientific merits as it provides valuable information on mitigation of heat stress.

Corrections:

• Birds were fed the corn-soybean meal-based diets in two phase, starter (1-21d) and finisher (22-42d). The energy and protein requirements of the diet was met following the NRC (1994); however, Ca and P level was used at lower level than the commercial requirements (Line 94-95).

• We have corrected “dig-P” to “available phosphorous” (in the Table-1, Line 102)

Again, thank you so much for your critical review for the refinement of this manuscript, and we will follow the recent NRC guidelines in our future study as per reviewer’s recommendation.

Response: Standard curve has been removed from the method section, and was modified

In line 147-150: The expression level of target genes was determined using the cycle threshold (Ct) values and changes in the gene expression were calculated by the 2-ΔΔCt method compared to the control group. The relative mRNA expression was normalized to the endogenous reference gene B-actin.

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PLoS One. doi: 10.1371/journal.pone.0254936.r005

Decision Letter 2

Arda Yildirim

7 Jul 2021

Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Broilers

PONE-D-21-05713R2

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PLoS One. doi: 10.1371/journal.pone.0254936.r006

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16 Jul 2021

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Dietary Supplementation of Alpha-lipoic Acid Mitigates the Negative Effects of Heat Stress in Broilers

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PERMALINK

Dietary supplementation of alpha-lipoic acid mitigates the negative effects of heat stress in broilers

Sanjeev Wasti

Nirvay Sah

Chin N Lee

Rajesh Jha

Birendra Mishra

Roles

Abstract

Introduction

Material and methods

Birds and husbandry

Diets

Table 1. Ingredients and nutrient composition of the experimental diets.

Growth performance

Sample collection

Total RNA extraction

Quantitative real-time PCR (qPCR) assay

Table 2. Primers used to quantify the expression of the genes by qPCR.

Ileum histomorphology

Volatile fatty acid

DNA extraction and 16S rRNA gene sequencing

Sequencing processing

Statistical analysis

Results

Effects of alpha-lipoic acid in growth performance

Fig 1. Effects of ALA on the growth performance of heat-stressed broilers.

Effects of ALA on the intestinal gene expression

Expression of heat shock protein-related genes

Fig 2. Effects of ALA supplementation on the ileal gene expression of heat-stressed broilers.

Expression of tight-junction related genes

Expression of antioxidant related genes

Expression of immune-related genes

Ileum histomorphology

Fig 3. Effects of ALA supplementation on the ileum histomorphology of the heat-stressed broilers.

Volatile fatty acids

Fig 4. Effects of ALA supplementation on the major volatile fatty acids in the cecal digesta of the heat-stressed broilers.

Alpha and beta diversity of cecal microbiota

Fig 5. Box plot showing the effects of ALA supplementation on microbial alpha diversity in heat-stressed broilers.

Fig 6. Effects of ALA supplementation on microbial beta diversity in heat-stressed broilers.

Cecal microbial composition

Fig 7. Cecal microbial compositions.

Fig 8. Cecal microbial composition at the family level. (A) Relative abundance of microbiota, and (B) Significantly abundance microbiota within the family level.

Fig 9. Cecal microbial composition at the genus level. (A) Relative average abundance of microbiota, and (B) significantly abundance microbiota at the genus level.

Correlation between the differential microbial species and measured parameters

Table 3. Spearman correlation between the differential microbial species and changed measure parameters.

Discussion

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Acceptance letter

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Associated Data

Supplementary Materials

Data Availability Statement

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