Limitation and Potential Effects of Different Levels of Aging Corn on Performance, Antioxidative Capacity, Intestinal Health, and Microbiota in Broiler Chickens

Simple Summary Corn is an important ingredient and staple food in China; thus, corn storage has a certain importance to ensure domestic food resources. Normally, corn has been stored for 3 or more years under the proper storage conditions in national barns before it is used as a feed ingredient. This study aimed to investigate the effect of different levels of aging corn (AC) on performance, antioxidative capacity, intestinal health, and microbiota in broilers. In the present study, AC grains were stored for 4 years under the proper storage conditions at the national storage facility. The results indicated that a lower level of AC diet showed improved performance and overall bird health than a higher level of AC, and comparable with a normal corn diet. However, antioxidative capacity is reduced by AC diets. Abstract Three-hundred and sixty-day-old male broilers underwent three treatments with six replicates of 20 birds per treatment. The experimental diets included NC: normal corn diet; ACL: lower level (39.6–41.24%) of AC; and ACH: a higher level (56.99–59.12%) of AC. During phase 1 (0–21 d), broilers fed on AC showed lower (p < 0.05) body weight (BW), body weight gain (BWG), and feed conversion ratio (FCR) as compared with the NC group. During phase 2 (22–42 d), the NC group and ACL group showed better (p < 0.05) BW, BWG, and FCR than the ACH group. The footpad lesion score (p = 0.05) and litter moisture percentage (p < 0.05) were found to be higher in the ACH group. During phase 1, the ACL group showed a lower level of malondialdehyde (MDA) contents (p < 0.05) in serum; moreover, catalase (CAT) (p < 0.05) and glutathione peroxidase (GSH-Px) activities (p < 0.05) were found lower in both AC-containing groups. During phase 2, CAT activity in serum was found higher (p < 0.05) in the ACH group. During phase 1, the NC group showed higher CAT (p = 0.05), GSH-Px (p < 0.05), and superoxide dismutase (SOD) activity (p = 0.03); however, it showed lower MDA (p < 0.05) and total-antioxidative capability (T-AOC) (p < 0.05) in the liver. During phase 1, in breast muscle, CAT, SOD, and T-AOC were higher (p < 0.05) in the NC group. During phase 1, total cholesterol and high-density lipoprotein were found to be lower (p < 0.05) in the ACL group. Similarly, triglyceride and low-density lipoprotein were found to be lower (p < 0.05) in the ACL group than the ACH group. During phase 1, villus height was found to be higher (p < 0.05) in the ACH group. Moreover, the goblet cell (GC) was found to be higher (p < 0.05) in the NC group than the ACL group. During phase 2, GC was found to be higher (p < 0.05) in the ACL group. In ileal digesta, during phase 1, acetic acid, propionic acid, and butyric acid (BA) levels were found to be higher (p < 0.05) in the ACL group. In cecal digesta, BA was significantly lower (p < 0.05) in the NC group.


Aging Corn
Normal corn and AC originated from the national barns in Yinchuan, China, and Changchun, China, which had been stored for 6 months and 4 years, respectively. All corn samples were stored in brick structures, and some phytochemical properties were determined ( Table 2). According to the methods described by AOAC International [28], the Animals 2021, 11, 2832 4 of 18 dry matter (DM), crude protein (CP), and crude fiber (CF) were analyzed by oven drying (Method No. 934.01), Kjeldahl (Method No. 990. 3), and Soxhlet fat analysis (Method No. 920.39), respectively. Gross energy was analyzed by adiabatic bomb calorimetry (Parr Instrument Company, Moline, IL, USA). The quantity of potassium hydroxide (KOH) required for acid neutralization in a 100 g sample was used for the analysis of titratable acidity (GB/T 20570-2015). The POD and CAT activity, as well as malondialdehyde (MDA) contents, were measured by using precise detection kits from Nanjing Jiancheng Bioengineering Institute (Nanjing, China) with a Multiskan Spectrum Reader (Model 1500; Thermo Scientific, Nyon, Switzerland). Deoxynivalenol, zearalenone, and aflatoxin were measured by using the national standard methods, i.e., SN/T 1571-2005, GB/T 28716-2012, and GB/T 30955-2014, respectively. Similarly, the standard method for fatty acids (FAs) was used for its quantification (GB 5009.168-2016).

Growth Performance
Individual broilers, after 12 h fasting, body weight (BW), and the amount of feed intake (FI; offered-refused) by pen were measured at the end of each phase (1 and 2) at 21 and 42 d. Then, average body weight (ABW), average body weight gain (ABWG), average feed intake (AFI), and feed conversion ratio (FCR) by pen were calculated. Birds were monitored to account for their morbidity (health status) and mortality on a daily basis. During the experiment, the BW of dead birds was included in growth performance calculations.

Footpad Dermatitis Score
Footpad dermatitis scoring was conducted at the end of phase 1 and phase 2 by visual inspection of the footpad of both feet of all live birds in each pen. A seven-point scoring system was followed, according to the procedure of Mayne et al. [29]. The better quality of the footpad is associated with a lower footpad dermatitis score. The total footpad score (TFPS) for each pen was calculated as follows.

Litter Moisture Percentage
Litter samples were taken from each pen at the end of phase 1 and phase 2. Four subsamples were taken from each pen from the surface to the full depth of the accumulation at locations shown in Figure 1. Sub-samples were taken from the middle of each side except the side with the door, avoiding the drinker and feeders. Side sub-samples were taken approximately 30 cm from the wall at the side of the pen. The four sub-samples were mixed per pen for analysis for moisture. Litter moisture was determined by placing litter samples in a forced-air drying oven at 60 • C for more than 24 h. inspection of the footpad of both feet of all live birds in each pen. A seven-point scoring system was followed, according to the procedure of Mayne et al. [29]. The better quality of the footpad is associated with a lower footpad dermatitis score. The total footpad score (TFPS) for each pen was calculated as follows.

Litter Moisture Percentage
Litter samples were taken from each pen at the end of phase 1 and phase 2. Four subsamples were taken from each pen from the surface to the full depth of the accumulation at locations shown in Figure 1. Sub-samples were taken from the middle of each side except the side with the door, avoiding the drinker and feeders. Side sub-samples were taken approximately 30 cm from the wall at the side of the pen. The four sub-samples were mixed per pen for analysis for moisture. Litter moisture was determined by placing litter samples in a forced-air drying oven at 60 °C for more than 24 h.

Sample Collections
After phases 1 and 2, two birds from each replicate (12 broilers from each treatment), with BW near to the average BW of the pen, were selected and slaughtered by severing their jugular vein. Before slaughtering, the samples of blood were collected through the jugular vein, and afterward, for serum collection, these blood samples were centrifuged (2000× g, 10 min, 4 °C). The collected serum was then stored at −20 °C for further biochemical parameter analysis. After serum was collected from 12 birds, from them, 6 birds were slaughtered by severing their jugular vein. Liver tissue, breast muscle, and gastrointestinal tract (GIT) samples were collected. Serum, liver, and breast muscle samples were stored at −20 °C for antioxidant activity analysis. The GIT was immediately dissected after euthanization, and a half portion of jejunum was stored in 4% paraformaldehyde solution of histological analysis. However, another half portion of jejunum was used for jejunum mucosa flash-frozen in liquid nitrogen −80 °C until gene expression analysis. Moreover, from the same birds, ileal and cecal digesta samples were collected by squeezing them

Sample Collections
After phases 1 and 2, two birds from each replicate (12 broilers from each treatment), with BW near to the average BW of the pen, were selected and slaughtered by severing their jugular vein. Before slaughtering, the samples of blood were collected through the jugular vein, and afterward, for serum collection, these blood samples were centrifuged (2000× g, 10 min, 4 • C). The collected serum was then stored at −20 • C for further biochemical parameter analysis. After serum was collected from 12 birds, from them, 6 birds were slaughtered by severing their jugular vein. Liver tissue, breast muscle, and gastrointestinal tract (GIT) samples were collected. Serum, liver, and breast muscle samples were stored at −20 • C for antioxidant activity analysis. The GIT was immediately dissected after euthanization, and a half portion of jejunum was stored in 4% paraformaldehyde solution of histological analysis. However, another half portion of jejunum was used for jejunum mucosa flash-frozen in liquid nitrogen −80 • C until gene expression analysis. Moreover, from the same birds, ileal and cecal digesta samples were collected by squeezing them gently and stored at −20 • C for short-chain fatty acid (SCFA) analysis. On days 21 and 42, the remaining six birds were slaughtered to collect cecal digesta and this was stored at −80 • C for microbial community analysis.

Antioxidant Activity
Six serum, liver, and breast muscle samples from each treatment were used to analyze MDA content, the enzymatic activity of CAT, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and the total antioxidative capability (T-AOC) using commercial assay kits (Nanjing Jiancheng Institute, Nanjing, China) and procedure described previously by Che et al. [30].

Serum Profile
Six serum samples from each treatment were used to analyze triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) concentrations, as well as alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, which were important indices to evaluate lipid metabolism and liver function, using an automatic biochemistry analyzer Hitachi 7020 (Hitachi High Technologies Inc., Tokyo, Japan) with kits being purchased from the Nanjing Jiancheng Bioengineering Research Institute, Nanjing, China. Fixed segments in 4% neutral buffered paraformaldehyde solution were rinsed in ethyl alcohol and embedded in paraffin wax. The samples were cut (5 µm) using a propeller slicer (Leica-2016, Leica Inc., Bensheim, Germany), with 3 slices per treatment, and stained using the hematoxylin and eosin method. The micrographs were taken using a microscope (BA400Digital, Motic China Group Co. Ltd., Xiamen, China) was used to take micrograph, and measurements were performed for villus height (VH), crypt depth (CD), and calculated ratio of villus height to crypt depth (VH:CD), as well as count the goblet cells (GC) and calculate the number of goblet cells per unit area by using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD, USA) for each structure per slice. The tip of the villus to the villus-crypt junction was defined as VH, whereas the CD was measured from the depth of the invagination to adjacent villi [31].

Jejunum Mucosal mRNA Gene Expression
After phase I and II, from jejunal mucosa, total RNA was extracted by using a TRIzol reagent kit (Takara, Dalian, China), and synthesis of cDNA was completed by using the reagent kit (PrimeScript RT, Takara, Kusatsu, Japan). Primers for Tight Junction Protein (TJP), 3 genes in association with the intestinal barrier including Claudin1 (CLDN1), Zonula Occludens-1 (ZO-1), Occludin (OCLN), and β-actin (housekeeping gene) were designed using Primer Express 3.0 (Applied Biosystems, Waltham, MA, USA; Table 3). Real-time quantitative PCR was performed according to Livak and Schmittgen, [32]. At 21 and 42 d, proportions of the volatile fatty acid (VFA), i.e., acetic acid (AA), propionic acid (PA), and butyric acid (BA), were determined from ileal and cecal digesta by using the HPLC system following the method Qin et al. [35] with some modifications. Approximately 0.5 g ileal and cecal contents were gently transferred into a micro-centrifuge tube containing 2 ml of ultrapure water. The solution was thoroughly mixed using a vortex mixer and centrifuged at 5000 rpm for 10 min at 4 • C. Taking 1 mL supernatant added 0.2 mL ice-cold 25% (w/v) meta-phosphoric acid solution, incubated at 4 • C for 30 min, and again centrifuged @12,000 rpm for 10 min. The supernatant was filtered through 0.22 µm Animals 2021, 11, 2832 7 of 18 syringe filters. The VFA contents of this filtrate were measured using a gas chromatograph (CP-3800, Varian, Palo Alto, CA, USA).

16S rDNA Gene Amplicons Analysis
At d 21 and 42, cecal digesta samples were subjected for extraction of DNA by using QIAamp PowerFecal DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA concentration and quality were checked using a NanoDrop Spectrophotometer. The Novo gene platform (Illumina Hiseq, Novogene Bioinformation Technology, Beijing, China) was used to performed 16S rDNA gene amplicons analysis. All methods including extraction of DNA, 16S rRNA sequencing, processing of sequences, and analysis of data were performed according to Qin et al. [36]. Concisely, sterile water was used to dilute DNA up to 10 ng/µL. The 16S rRNA genes of distinct regions (16S V4) were amplified using a specific primer (515F GTGCCAGCMGCCGCGGTAA; 806R GGACTACHVGGGTWTCTAAT) with the unique barcodes. The Phusion High-Fidelity PCR Master Mix (New England Biolabs) was used for the accomplishment of all PCR reactions. The end products of PCR were mixed in equal density ratios. Then, Qiagen Gel Extraction Kit (Qiagen) was used for purification of these PCR mixture products. Sequencing libraries were generated using TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) following the manufacturer's recommendations, and index codes were added. The Agilent Bioanalyser 2100 system and the Qubit 2.0 Fluorometer (Thermo Scientific) were used to assess the quality of these libraries. Lastly, the sequencing of eligible libraries was completed on an Illumina HiSeq 2500 platform and generated the 250 bp paired-end reads. QIIME quality filters were used for the filtration of selected reads. Sequences with ≥97% similarity were assigned to the same optimal taxonomic units (OTUs). The relative abundance of each OTU was examined at different taxonomic levels. Diversity within communities (Alpha diversity) calculations and taxonomic community assessments were performed by Qiime 1.7.0, and Beta diversity included both unweighted and weighted Unifrac distances calculated with 10 times subsampling; distances were visualized by principal component analysis (PCA; Lozupone and Knight) [37], and the separation was tested using R in Anosim.

Statistical Analysis
The experiment was a completely randomized design with a pen as the experimental unit. For serum parameter, antioxidative parameters, intestinal characteristics, short-chain fatty acids, and diversity and structure of the cecal microbiota analysis, the randomly selected birds were the experimental unit, and results were analyzed by a one-way analysis of variance (ANOVA) using the GLM procedure of SAS (SAS Institute Inc., Cary, NC, USA). Differences among means were tested with Duncan's multiple range tests. p ≤ 0.05 is considered significant.

Growth Performance
The growth performance responses of the broiler chickens under different experimental groups are shown in Table 4. During phase 1, the ACL group has lowered (p < 0.05) ABW and AWBG from NC and ACH groups, whereas ACH groups have lowered (p < 0.05) ABW and AWBG from the NC group, and similarly FCR was poor (p < 0.05) in ACL group from NC diet, and numerically poor from the ACH group. On 42 d, ABW was significantly (p < 0.05) and numerically lower in the ACH group from the NC group and ACL group, respectively. Similarly, during phase 2 and the whole experiment (1-42 d), AWBG and FCR were found to be poor (p < 0.05) in the ACH group among all dietary treatments. The FI and mortality were not significantly different among treatments during phase 1, phase 2, and during the whole experiment (1-42 d). Overall, during the whole experimental period, a lower level (39.6-41.24%) of AC has shown comparable performance with normal corn, whereas a high level (56.99-59.12%) of AC has shown lower performance than other dietary treatments.

Footpad Dermatitis Score and Litter Quality
Both footpads were gross examined on the d 21 and d 42 for allotting the proper score according to their pathological condition (Table 5). A similar trend was observed in TFPS between experimental groups after phase 1 and phase 2. After 21 d, TFPS was significantly lower (p = 0.05) in the ACL group from the ACH group and numerically lower from the NC group. Similarly, after 42 d, TFPS in the ACL group was numerically lower and higher from ACH and NC groups, respectively. Litter moisture percentage (%) was also measured on 21 d and 42 d (Table 5) and found similar results as TFPS. On d 21, litter moisture was numerically lower in the ACL from NC and ACH groups. After 42, the litter moisture percentage was significantly higher (p < 0.05) in the ACH group than in the NC and ACL groups. Convincingly, a higher level (56.99-59.12%) of AC in the diet caused adverse effects on footpad dermatitis score and litter moisture as compared with normal corn and lower levels (39.6-41.24%) of AC.

Antioxidant Activity
The enzymatic activity of CAT, SOD, GSH-Px, T-AOC, and MDA contents were measured in serum, breast muscle, and liver on d 21 and 42 (Table 6). On d 21, CAT (p < 0.05) and GSH-Px (p < 0.05) were found higher in the NC group than ACL and ACH groups, and MDA was found lower (p < 0.05) in the ACL group than NC and ACH groups in serum; however, SOD and T-AOC were not significantly different among all treatment groups. For the liver, the NC group shows higher CAT (p = 0.05) than the ACH group, and higher GSH-Px (p < 0.05) than ACL and ACH groups, similarly SOD was found higher (p < 0.05) in the NC group than ACL group; however, MDA found lower (p < 0.05) in the NC group from all other treatment, and TAOC also found lower (p = 0.05) in the NC group than the ACL group. For breast muscles, CAT was higher (p < 0.05) in the NC group than the ACH group, and SOD and TAOC were found higher (p < 0.05) in the NC group than all other treatment groups. However, the lowest TAOC was found in the ACH group. Moreover, GSH-Px and MDA were found to be similar among all the treatment groups.  On d 42, CAT was found to be higher (p < 0.05) in the ACH group from other treatment groups; however, T-AOC was found higher (p < 0.05) in the NC group than ACL group in serum, whereas GSH-Px, SOD, and MDA were found similar among all treatment groups in serum. However, for the liver, all antioxidant indices were found to be similar among all dietary treatments. For breast muscle, CAT was found to be higher (p < 0.05) in the NC group than other treatment groups, and SOD was found to be higher (p < 0.05) in the ACH group than other treatment groups. Other indices including GSH-Px, MDA, and TAOC were found to be similar among all treatment groups.

Serum Profile
Results of important indices to evaluate liver function (AST and ALT) and lipid metabolism (TC, TG, HDL-C, and LDL-C) through serum analysis during both phases 1 and 2 are shown in Table 7. During phase 1, both AST and ALT were found to be similar among all dietary treatments. However, TC and HDL-C were found to be lower (p < 0.05) in the ACL group than NC and ACH groups. Similarly, TG and LDL-C were found to be lower (p < 0.05) in the ACL group from the ACH group. Although during phase 2, serum levels of AST, ALT, TC, TG, HDL-C, and LDL-C were found to be non-significantly different from each other, the ACL group has a lower value for all serum indices.  1 Normal corn diet (NC); low aging corn (ACL); high aging corn (ACH). 2 Level of serum alanine aminotransferase (ALT); level of serum aspartate aminotransferase (AST); Level of serum total cholesterol (TC); level of serum total triglyceride (TG); level of serum high-density lipoprotein (HDL-C); and level of serum low-density lipoprotein (LDL-C). The data are presented as the mean of n = 6 for each group. a,b Mean within each row with different superscripts differ significantly (p < 0.05).

Intestinal Morphology and mRNA Gene Expression
On day 21, jejunal morphological results were observed to be higher VH in the ACH group from NC and ACL group (p < 0.05) shown in Table 8. Similarly, VH:CD was also found to be higher (p < 0.05) in the ACH group from the NC group, whereas CD was found to be similar among the dietary treatments. Moreover, the GC was found to be higher in the NC group (p < 0.05; Table 8; Figure 2) than in the ACL group. On day 42, VH:CD was found higher in the NC group than ACL and ACH groups (p < 0.05), and GC was found higher in the ACL group than NC and ACH groups (p < 0.05), whereas VH and CD were found to be similar among all the treatments. Jejunal mRNA gene expression for TJP results (Table 8) indicated that CLDN1, ZO-1, and OCLN were not significant among all dietary treatments during both phases 1 and 2, whereas the ACL group has shown more improved results than the ACH group, and these results are comparable with the NC group.

Ileal and Cecal VFA Contents
In ileal digesta, after phase I, AA level was higher (p < 0.05) in the ACL group than NC group and similar with the ACH group; moreover, PA and BA levels were found significantly (p < 0.05) higher in the ACL group from all other treatment groups (Table 9).

Ileal and Cecal VFA Contents
In ileal digesta, after phase I, AA level was higher (p < 0.05) in the ACL group than NC group and similar with the ACH group; moreover, PA and BA levels were found significantly (p < 0.05) higher in the ACL group from all other treatment groups (Table 9). After phase II, AA and PA were found similar among all the experimental groups; however, BA was significantly lower in the NC group than all other treatment groups (p < 0.05). In cecal digesta, AA and PA were found to be similar among all the experimental groups; however, BA was significantly lower in the NC group than the ACH treatment group (p < 0.05) after phase I, whereas after phase II, PA and BA were found to be lower in the NC group than the ACL group (p < 0.05) and similar to the ACH group. The level of AA was found to be similar among all experimental groups.

Cecal Microbial Community
The microbial communities were compared in the cecum among five dietary groups, using Illumina Hiseq high-throughput sequencing. On day 21, a total of 1,083,368 sequencing reads were obtained from the cecal digesta samples, and through cutting and filtering of reads, an average of 83,336 reads was measured per sample, and an average of 78,125 valid data was obtained after quality control. The effective rate of quality control was 93.8%. The sequences were clustered into OTUs (operational taxonomic units) with 97% identity. At the phylum level, the most dominant species were Firmicutes, Proteobacteria, and Bacteroidetes, and their abundance was not significantly different among all experimental groups ( Figure 3A). At the genus level, dominant but significantly similar, the species present were Enterococcus, Faecalibacterium, and Parabacteroides ( Figure 3B). Similarly, at the species level, the dominant species were Parabacteroides distasonis, Bacteroides uniformis, and Lactobacillus salivarius, and were found to be similar among the all-treatment groups. On day 42, a total of 1,546,555 sequencing reads were obtained from the cecal digesta samples, and through cutting and filtering of reads, an average of 85,919 reads was measured per sample, and an average of 81,499 valid data was obtained after quality control. The effective rate of quality control was 94.8%. The sequences were clustered into OTUs (operational taxonomic units) with 97% identity, and a total of 3110 OTUs were obtained. At the phylum level, the most dominant species were Firmicutes, Bacteroidetes, and Proteobacteria, and their abundance was not significantly different among all experimental groups ( Figure 3D), whereas Fusobacteria (p < 0.05) was significantly higher in the ACL group as compared with all other treatments. At the genus level, the dominant species were Bacteroides, Faecalibacterium, and Phyllobacterium, and Bacteroides and Phyllobacterium were found to be statistically similar, whereas Faecalibacterium was significantly higher (p < 0.05) in the NC group than other dietary treatments ( Figure 3E). At the species level, the dominant species were Bacteroides uniformis, Bacteroides plebeius, and E. Coli, and found to be similar among all treatment groups. Moreover, all other species were found to be non-significantly different from each other ( Figure 3F). After phase 1, the result of alpha (α) diversity including observed species (OS), Shannon (SH), Simpson (SI), Chao1 (CH), abundance-based coverage estimator metric (ace), good coverage (gc), and phylogenetic distance (PD) were found to be similar among all dietary treatments (Table 10). However, after phase 2, the result of all parameters of αdiversity except PD was found similar among all experimental groups (Table 10). PD was observed to be higher (p < 0.05) in the ACL group (Table 10). Additionally, the relationships between communities of various bacteria belonging to different treatments were characterized by PCA, and the results exhibited that there was no significant difference among microbial communities of cecal digesta from different experimental groups during both phase 1 and phase 2.

Discussion
Various studies have designated storage conditions and time had adverse effects on cereal grains, specifically their chemical composition [6,38,39], as storage time is directly proportional to the acidity value of stored corn [40]. The results of the present study corroborated earlier findings that AC fat acidity was found to be higher when compared with corn that had been stored for a few months. These findings indicated that prolonged storage is not suitable for AC. Moreover, others scientists reported that lipid present in AC can be oxidized and formed hydroperoxides (H 2 O 2 ) [41], and CAT and POD were decreased during grain storage [3]. These findings can be used as an indicator of cereal grain deterioration during storage, i.e., rice [42]. The AC quality evaluation mainly depends on the POD activity, and FAs' value includes the important parameters of the quality evaluation of corn storage. In the present study, after chemical evaluation of AC, it was observed that the FAs' value and POD activity of AC were found to be higher and lower, respectively, than normal corn. The AC came from the grain national depot, therefore the storage conditions were suitable, and the content of mycotoxins was not considerable, thus the adverse effects of the AC-containing diet on broilers were caused by changes in the corn nutritional components, such as FA oxidation.
In the present study, the performance was affected during both the starter phase and growing phase in broilers fed on the lower and higher level of AC, respectively. The BW and BWG were found to be highest in the normal corn diet, followed by a higher AC level diet, and the lowest was found in a lower AC level diet. The higher AC level diet showed higher BW and BWG than the lower AC level diet during the starter period because it contains a higher level of soybean, as this diet is based on corn-soybean, whereas other diets based on corn-wheat-soybean diets, and soybean meal is a major source of protein due to its higher digestibility and amino acid availability [43]. However, during the growing phase, a higher AC diet attained the lowest BW and BWG, as well as having poorer FCR than the other groups. Interestingly, a lower level of AC diet shows improved FCR even from the normal corn diet. That indicated the positive effect of the lower level of AC as compared with a higher level of AC. A similar result was reported in ducks as a higher level of AC produced a more adverse effect on duck health as compared with a lower level of AC [23]. Another report in layers [25] as AC produced no negative effect on production performance. The performance results are interlinked with intestinal morphology as it was found to be balanced during the starter phase in broilers fed on a higher level of AC, whereas during the growing phase, it was found improved in broilers fed on the lower level of AC. As improved intestinal morphology can enhance digestibility, this ultimately improves performance in birds [44]. Therefore, it can be assumed that a lower level of AC could enhance digestibility and reduce the water content in excreta, as high moisture in excreta is a major cause of footpad lesions in broilers [45]. Improved intestinal health and digestibility could be a reason for lower litter moisture and footpad score in broilers fed on lower level of AC as compared with a higher level of AC.
The antioxidant capacity was found to be lower in serum, liver, and breast muscle by the inclusion of both higher and lower quantities of AC during the starter and growing period. These results indicating that the storage of corn can decrease the activity of antioxidant enzymes due to the presence of FFA, because FFA oxidized easily to produce H 2 O 2 , and these can affect the activities of enzymes such as POD and CAT in maize [13]. Similar results were reported as old corn lowers the serum antioxidative capacity in broilers [46]. Under the condition of oxidative stress, the activities of T-AOC, GSH-Px, CAT, and SOD in the serum were decreased; however, the MDA contents were increased [43]. Long-term corn storage could cause a negative impact on birds' health. Ducks fed AC diets were more likely to obtain oxidative damage, which resulted in reduced growth performance [23,47]. Moreover, MDA in serum and liver was found higher with a higher level of AC in the diet. MDA is a lipid peroxidation (LPO) and the main product of degradation, which reflects the lipid peroxidation (damage) in the body, and cells are attacked by free radicals. The MDA content was also found to be higher in stored corn, which can cause lipid peroxidation in broilers [21]. Liu et al. [46] also reported higher serum MDA content in broilers fed on the old corn.
In the growing animals, nutritional status is mainly reflected by their serum biochemical parameters [48]. Our results showed that a lower level of AC influences lipid metabolism during the starter phase. The lower TG in AC groups may be attained by lowering the FAs synthesis inside the liver and the activation of peroxisome proliferator activating receptor α, which enhances β-oxidation of FAs [49,50]. In this study, a lower level of AC significantly reduced various forms or types of cholesterol including TC, HDL-C, and LDL-C in serum. In another study, rats were fed on oxidized oil containing products of lipid peroxidation, and resulted in reducing cholesterol and triacylglycerols concentrations in the plasma and liver [51]. Likewise, Koch et al. [52] reported that oxidized oil significantly decreased the contents of VLDL-C, HDL-C, and TC in the liver and plasma. A similar effect of AC on serum lipid profile was reported in layer [22].
The villus inside the small intestine, i.e., the jejunum, is a major site for digestion of food and absorption of nutrients in monogastric animals. Consequently, the proper functioning of gut microbiota, immune system, and nutrition, could be achieved by a healthy mucosa of the small intestine. During the growing phase, a higher level of AC had adverse effects on intestinal morphology as compared with a lower level of AC and NC groups. Similar results have been reported by various scientists [47,53]. It can be assumed that lipid peroxidation in a higher level of AC might stimulate the jejunal mucosa and causes oxidative stress that ultimately increases the energy demand of birds as the free radicals damage the mucosa of the small intestine. The VH:CD is considered to play a key role during digestion and absorption. In the present study, a higher level of AC shows lower VH:CD in the jejunum indicated that AC in diets may damage the intestinal mucous to some extent and causes the reduction in the absorptive capacity. Moreover, intestinal morphology, tight junction proteins have also played key roles in maintaining the integrity of the intestinal barrier and regulating intestinal permeability [54]. In the present study, although no significant effect was observed between all dietary treatments, the ACL group has shown improve TJP than the ACH group.
In broilers, the major concentration of analyzed SCFAs of ileal and cecal chyme can be varied mainly due to the ingredients of chicken feed [55]. The present study provides novel information regarding ileal and cecal SCFAs in broilers fed on the higher and lower level of AC as it has never been tested previously. During both dietary phases, all SCFAs in ileum and cecum were found to be higher in broilers fed on a diet containing AC aging corn as compared with boilers fed on the diet containing normal corn. Mainly SCFAs are produced in the GIT due to the fermentation of complex carbohydrates [56]. Therefore, it could be assumed that AC might contain the complex carbohydrates that could be contributed to the production of SCFAs in the GIT of broilers. No study has been reported regarding the effect of AC on the intestinal microbial community in the broiler. Although microbiota including taxonomy, alpha diversity, and beta diversity were found to be similar among all the treatment and control groups, the SCFAs improved the intestinal health in broilers fed AC, especially the lower level of AC. Various researchers reported the positive effect of SCFAs on intestinal health in broilers [57] and pigs [58].

Conclusions
After phase 1, the higher level (56.9%) of AC had improved the performance compared with the lower level (39.6%); however, after phase 2, the higher level (59.12%) had more negative effects on performance and broilers' health than lower level (41.24%). As the higher level (59.12%) showed lower performance, antioxidant capacity, and lipid profile, which interlinked with decreased intestinal health that causes more water in dropping and resulting in higher footpad necrosis. However, a lower level (41.24%) of AC diet showed a more improved performance than a higher level (59.12%) of AC and was comparable with a normal corn diet. However, antioxidative capacity was found to be lower in both AC groups. Better performance and overall bird health could be obtained from lower levels (39.6-41.24%) of AC in the broiler diet by using some antioxidants as a supplement in the broiler diet.