All research procedures were proved by the animal care committee of Henan Agricultural University (No. HNND 20190306).

A total of 480 1-d-old male Arbor Acres (AA) broiler chickens were purchased from a local hatchery and housed in a temperature- and humidity-controlled room. The initial room temperature was set at approximately 34°C and subsequently reduced to 24°C by 20 d. The light program was 23 L:1 D during d 10 to 39. Birds were vaccinated again Newcastle disease-infectious and bronchitis on d 4. Of note, a completed random of single factor was introduced to this experiment, where birds were fed the same starter diets until 10 d and then were allocated into one of three groups based on their BW including i) the low-level wheat addition group, in which the wheat addition levels were 15% and 25% on grower and finisher periods, respectively, ii) the medium-level wheat group with the 30% and 40% of wheat in the grower and finisher periods, respectively, and iii) the high-level wheat group, where the grower and finisher diets contained 55.8% and 62.4% of wheat, respectively. Each group included 8 pens with 20 broiler chickens per pen. The diets were formulated to meet the nutrient requirements of AA broiler chickens [26] and were supplied as pellets (Table 1). The broiler chickens were weighed at d 10 and 39, and the feed intake and the number of dead or culled birds from 10 to 39 d were recorded on a replicate basis. The feed conversion rate (FCR) was expressed as the ratio of feed consumption to weight gain and the mortality was calculated in the whole trial.

Sampling was performed at 39 d. To ensure the consistency and minimize the variability due to sampling time, the selection of broilers, and feeding time before slaughter, the birds were fasted for 12 h and all samples were taken between 8:00 AM and 10:00 AM in the pen of bird order. In detail, one bird was selected based on the average BW of each pen, and blood was collected from the jugular vein for separating serum. After euthanasia, the liver and abdominal fat were taken and then put into a formalin solution for histology analysis. The duodenum, jejunum, ileum, and cecum were dissected, and the length and weight of intestinal segments were obtained after removing the thyme. Ileal mucosa was collected from the middle of the ileum, and stored in a sterile, RNase-/DNase-free EP tube at −80°C for gene expression determination. Enzyme-treated EP tubes were used to transfer fresh cecal contents for 16S rRNA microbiome sequencing. The tibias were immediately dissected from soft tissues and utilized to examine the bone properties of the tibia.

According to the description of Lebednikaitė and colleagues [27], the serum concentrations of calcium (Ca), phosphorus (P), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), CHO, TG, HDL-c, and LDL-c were analyzed using an automatic biochemical analyzer (Beckman Coulter AU5800; Beckman Coulter Inc., Brea, CA, USA), with corresponding commercial kits purchased from Bioengineering Institute (Nanjing, China), respectively. The concentrations of interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) were determined using commercial kits following the manufacturer’s instructions. Bone turnover markers in serum including alkaline phosphatase (ALP), procollagen type I N-terminal propeptide (PINP), and C-terminal cross-linked telopeptide of type I collagen (CTx) were quantified with available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

The fixed liver and abdominal fat samples were dehydrated with an alcohol gradient of 500, 700, 800, 950, and 1,000 mL/L and then embedded in paraffin using the HistoCore Arcadia H system (Leica, Shanghai, China). The 5-μm sections were prepared and stained with hematoxylin and eosin (H&E staining). The histopathological characteristics and changes of broiler livers and abdominal were observed with an optical microscope (Bio-Rad Laboratories, Hercules, CA, USA). The data on abdominal fat underwent quantitative processing using Image-Pro Plus (IPP 6.0; Media Cybernetics, Silver Spring, MD, USA), and the adipocyte size per treatment group was measured as the adipocyte cross-sectional surface area following the methodology described by Corino et al [28], in which fifteen adipocytes were randomly selected from the same slice field to measure their area and mean diameter.

Referring to the methods of previous studies [29], the length and circumference of the middle part of the left tibia were measured by vernier caliper and flexible ruler, respectively. These tibias were then weighed after being dried with filter paper, and the relative tibia fresh weights were calculated as the ratio of fresh weight to BW. In addition, the Seedor index, an instructor of bone mineralization, was determined by the ratio of bone weight to length. Subsequently, the tibia was immersed in ethyl ether, air-dried at room temperature, and oven-dried at 105°C for 24 h for quantifying tibial fat-free weight.

Biomechanical testing was performed by the 3-point bending method with a TA-XT Plus Texture Analyzer (TA. XT. Plus; Stable Micro Systems, Surrey, UK) at a constant 50 kg load cell according to the previous description [29] Loading proceeded at the mid-point of the right tibia with a constant rate (5 mm/min) up to the breaking of the bone. Force-displacement data were obtained to calculate bone stiffness, yield load, ultimate strength, and area-under-the-curve (AUC), in which the yield point and stiffness were defined as the load at which the load-deformation relationship ceased to be linear and the slope of the linear portion of the load-displacement curve.

The total genomic DNA was extracted from cecum digesta, followed by quality evaluation with electrophoresis using 0.8% agarose gels. The DNA concentration was quantified with a Tecan F200 microplate reader (Infinite F200; Tecan, Männedorf, Switzerland). The V4 region of the 16S rRNA gene was then amplified using specific primers (F: 5′-CCTA CGGGRSGCAGCAG-3′; R: 5′-GGACTACVVGGGTATC TAATC -3′). The polymerase chain reaction (PCR) products were purified and sequenced on the Illumina MiSeq platform. The two-ended sequences were spliced using FLASH [30]. Each sample sequence was then isolated from raw reads based on the barcode using QIIME (version 1.9.0), with the Barcode sequence truncated. The UPARSE algorithm was used to cluster the operational taxonomic units at a 97% threshold value. The alpha diversity was estimated with the Chao 1, Simpson, and Shannon indexes and Observed species, and beta diversity at the genus level was estimated by calculating Bray-Curtis dissimilarity and visualized with principal coordinates analysis (PCoA). The microbial taxa that differed between the different groups were identified using linear discriminant analysis (LDA) with a threshold of 2.0.

Statistical analysis was carried out using IBM SPSS statistic 26 (Statistical Packages for Social Science 26; IBM, Corporation, Armonk, NY, USA). Data was checked for normality via the Shapiro-Wilk test. One-way analysis of variance with Tukey’s test or Kruskal-Wallis with Dunn’s test for normally or non-normally distributed data, respectively, were used to evaluate the statistical differences of biological parameters. Pearson’s correlation analysis was performed to determine the correlation between tibia ultimate strength and lipid metabolism. The alterations in microbial composition following 16S rRNA amplicon sequencing were determined in R using the Microbiota Process package for community analysis (version 3.17). A statistically significant and tendency was considered when a p<0.05 and p≤0.10, respectively.

The effects of different levels of addition with wheat on the growth performance of broiler chickens are presented in Table 2. In comparison to the low-level wheat group, the increased wheat addition tended to reduce the BW (p = 0.059) at 39 d and weight gain (p = 0.050) from 10 to 39 d, whereas the high-level wheat group showed a significant increase in feed consumption and decrease in FCR as compared to the low- and medium-level wheat diets (both p<0.001). Of note, the incorporation of wheat in diets did not significantly change the mortality during 1 to 39 d.

The effect of dietary treatments on digestive organ development is presented in Figure 1. Different levels of wheat supplementation in the diets of broiler chickens have little effect on the weight of glandular stomach, gizzard, small intestine, and cecum. Meanwhile, the length of the intestine was examined, and found the dietary wheat supplementation had not apparently changed the absolute and relative length of the duodenum, ileum, and cecum, but the birds fed the high-level wheat diet exhibited a significantly longer jejunum when compared to those fed the low- and medium-levels wheat diets (p = 0.031; Figure 1C).

Considering the significantly increased feed intake and jejunal length, as well as the compromised FCR due to the addition of high-level wheat in the diet, the alterations in cecal microbiota between the low- and high-level wheat groups were determined and showed that the flora richness of each sample increased with the increase of sequencing depth, and the end of the curve tended to be flat, indicating that the amount of sequencing data was reasonable (Figure 2A). There were no significant differences between the low- and high-level wheat groups in the alpha diversity, evidenced by comparable Observed species, Chao1 (p = 0.08), Shannon, and Simpson indexes (Figure 2B). PCoA showed a clear separation between the two groups (Figure 2C), where PCoA1 and PCoA2 accounted for 28% and 14.8% of the variation in microbial diversity, respectively. At the genus level, the high-level wheat diet decreased the abundance of Ruminococcin, Bacteroidetes, and Lactobacillus, whereas it increased the proportion of Helicobacter as compared to the low wheat diets group (Figure 3D, 3E). The LDA showed significant enrichment of Bacteroidetes in the low-level wheat group and Verrucomicroblale in the high-level wheat group in the cecal microbiota (Figure 2F).

When compared with the low- and medium-level wheat diets, the number of fat cells in the high-level wheat diet was significantly higher in the same field of view (Figure 3A). However, there was no significant effect on the adipocyte area, and mean diameter at different levels of wheat treatment (Figure 3B, 3C). With the increased wheat levels in the diets, the contents of CHO, TG, and HDL-c in serum were significantly increased (p<0.05). Meanwhile, the serum concentrations of GLU and LDL-c were similar among the experimental groups (Table 3).

The effects of different levels of addition with wheat on the bone properties of broiler chickens are presented in Table 4. The experimental treatments failed to affect the length and circumference of the tibia. The supplementation of wheat neither changed fresh weight, relative weight, nor fat-free weight in this study. There were no significant differences between the three groups regarding the Seedor index (Table 4). Moreover, the bone mechanical parameters including yield load, ultimate strength, stiffness, and AUC were assessed according to Figure 4A, and the results showed that the stiffness was not changed by dietary wheat supplementation (Figure 4B). The yield load (p<0.05) and ultimate strength (p = 0.059) of the tibia were elevated by the increased wheat levels (Figures 4C, 4D). However, no apparent differences were found regarding AUC among the three groups (Figure 4E).

To evaluate the relationship between bone ultimate strength, yield load, and serum glycolipid levels, a Pearson’s correlation matrix was performed and showed that the ultimate strength of bone tended to positively correlate with serum CHO content (p = 0.095). Similarly, a trend between yield load and serum TG (p = 0.043) and CHO (p = 0.078) was also observed. No other significant changes were noted in the determined parameters (Figure 5).

There was no significant effect on serum Ca and P at different levels of wheat treatment (Figures 6A, 6B). The outcomes of serum bone formation markers revealed that the concentration of P1NP and ALP were similar among the three wheat diets (Figures 6C, 6D). Nevertheless, Serum CTx level, representing bone resorption, was decreased by dietary high-level wheat diet when compared with the low-level wheat group (p = 0.051; Figure 6B).

Effects of dietary treatments on liver microstructure of broiler chickens Figure 7A, the histological analysis showed normal hepatocytes without necrosis. The cells were arranged in order, and the nuclei were large and round with even cytoplasm across all treatment groups. With the increase in dietary wheat content, the inflammatory cell infiltrating area in liver tissue decreased, the hepatocyte boundary increased, and the number of neutrophils decreased in the high-level wheat group. Reflection on the serum biochemical indexes, the high-level wheat diet decreased serum AST activities when compared with the medium-level wheat group. There were no significant differences in terms of the levels of ALT and TP, as well as the relative of the liver among the three groups (Figures 7B–7D). In addition, the outcomes of serum inflammatory factor revealed that the dietary wheat inclusion did not change the anti-inflammatory cytokine IL-10 and pro-inflammatory factors IL-1β, IL-6, and TNF-α concentration in the present study (Figures 7E–7H).

With the rapid development of poultry farming, the shortage of global supply driving the rising price of corn demands seeking its replacement in the animal husbandry. Wheat has been widely used as energy feed in animal husbandry, especially in Europe, implying wheat has become the primary choice to replace corn. However, the influences of wheat-based diets on BW or weight gain are inconclusive. The improved weight gain in the broiler chickens fed wheat diets was observed as compared to those receiving corn-based diets [14,15]. The positive role might be attributed to the corporation of insoluble NSP in ingredients or the higher weight mainly derived from the visceral organs [15]. No apparent effects on the BW were also found in broiler chickens fed an inclusion of 40% wheat in a corn-based diet [11] and growing-finishing pigs that consumed a corn-soybean meal-based diet with 10% to 60% of wheat [12]. In this study, the increased wheat levels in the diet led to a decline in BW and weight gain, which was confirmed in a previous study indicating that the wheat-based diets compromised the BW of broiler chickens when compared to those offered the corn-based diets [13]. It was reported that a diet enriched in NSPs, such as wheat and barley, could increase the thyme viscosity, reducing feed consumption and growth performance [31]. In comparison to the corn-based diet, a wheat-based one caused a 3 times increase in viscosity of ileal digesta in broiler chickens [6]. Therefore, the delayed transit time and gut motility due to higher viscousity hindering feed intake is warranted in higher NSP-contained diets [5], including the wheat-based diet. A study on broiler chickens found that the supplementation of a wheat-based diet impaired the feed intake and reduced the ratio of feed consumption to weight gain from 25 to 42 d in broiler chickens [11]. In accordance with previous research on birds [14], the feed consumption of birds in high-wheat diets was higher than those fed other diets in the current study, thereby bringing about an inferior FCR. The possible explanation includes the palatability of wheat to animals [32] and the supplementation of enzymes [33]. Of note, incorporating NSP-degrading enzymes, particularly xylanase, into wheat-based diets could damage fiber structures, resulting in a decrease in intestinal viscosity and subsequently increasing nutrient digestibility of birds and promoting foraging [34,35]. Collectively, under the condition of supplementing exogenous enzymes, the medium-level wheat inclusion did not obviously decrease growth performance, when the supplemented level of wheat extended to 57.77% and 62.38% in grower and finisher diets, respectively, the wheat treatment increased feed intake and compromised FCR of broilers chickens.

To uncover the mechanism underlying the supplemented levels of wheat on the growth performance of broiler chickens, the development of gastrointestinal tract and cecal microbiota were detected. It was pointed out that wheat bran contains a lot of insoluble dietary fiber, which has been shown to stimulate gizzard development [36]. The wheat diet increased the weight of the cecum, and the length of the duodenum and ileum as compared to the corn diet [37]. Analogously, an absolute longer jejunum was noticed in the high proportion of wheat group than that in other groups in the present study. Moreover, the composition of gut microbiota is closely associated with the digestion and absorption of nutrients, immune, and intestinal barrier functions of animals, and it is also influenced by the diet type [15,38]. The crude fiber contained in wheat could be directly fermented by microbiota as the energy material, reflecting that the abundance of species of gut microbiota depends on the dietary fiber types and levels [39]. Incorporation of corn-based diets, and the supplementation of wheat-based diets increased the account of Escherichia coli, a well-known harmful bacterium [11], and decreased the proportion of Ruminococcus in the caeca microbiota profile of broiler chickens [6]. A study on growing pigs found that the feeding of wheat-based diets decreased the levels of Escherichia-Shigella and increased the proportion of beneficial bacteria such as Bifidobacterium and Lactobacillus at the genus level [38]. The discrepancy among these researchers may imply that the alterations of microbial composition rely on the dietary components, trial objects, environment, etc. In this study, the high wheat addition decreased the abundance of Ruminococcin, Bacteroidetes, and Lactobacillus, whereas it increased the proportion of Helicobacter as compared to the low wheat groups. Bacteroides play important roles in the degradation of polysaccharides and oligosaccharides [40,41]. Lactobacillus is widely distributed in domestic birds, and oral supplementation with Lactobacillus improved gut microbiota and stimulated intestinal improvement of broiler chickens, thereby promoting growth performance [42,43]. On the contrary, Helicobacter, a gram-negative bacterium, is often found to cause inflammation and ulceration [44]. Therefore, the decreased abundance of Bacteroidetes and Lactobacillus, along with increased the proportion of Helicobacter in the high-level wheat diet might result in compromised growth performance.

Wheat serves as an energy source for broiler chickens and has a higher level of starch [3]. Previous studies have found that the supplementation of wheat fermentation products caused the reduction of serum TG and LDL-c and resulted in increased HDL-c [18]. Nevertheless, other outcomes suggested that serum TG, TC, and LDL-c levels were increased significantly after feeding refined wheat flour for 16 weeks, which was accompanied by decreased HDL-c levels in model rats [19]. The results are similar to the data in this experiment, i.e., the increase of wheat substitution levels increased the contents of CHO, TG, and HDL-c in serum, indicating this increased fat deposition. H&E staining also showed an increase in the size of adipocytes in the high group. These results suggest that higher levels of wheat in broiler feed can lead to an increase in fat accumulation.

Of note, a link between lipid metabolism and bone turnover was observed. Guo et al [22] showed that leg disorders are associated with compromised growth, bone quality, bone structure, and lipid metabolism. The probable mechanism might be partly due to the common origin of adipocytes and osteoblasts, both originating from MSCs [20]. MSCs present in bone marrow possess the ability to differentiate into osteoblasts and adipocytes, which play a crucial role in bone formation and the adipose tissue component within the marrow, respectively. In this study, the correlation analysis of serum glycolipid profile and bone properties in broiler chickens showed a linear correlation between serum CHO and both ultimate strength and yield load of tibia, the content of TG levels also positively related to tibial yield load. In this context, an inverse relationship between bone marrow adiposity and bone mass has been noticed in bone diseases such as osteoporosis [21]. These data suggested that it is necessary to further explore the dynamic relationship between MSCs, osteoblasts, and adipocytes for revealing bone metabolism and adipose tissue development.

In this study, feeding the medium- and high-level wheat increased the yield load and ultimate strength of the tibia, suggesting that the diets contained appropriate levels of wheat combining with complex enzymes could enhance the mechanical properties of the tibia of broiler chickens. The positive effects of wheat addition on tibia are mainly because of the enzyme addition. Research by Olgun et al [23] found that wheat-based feeding negatively affects the biomechanical properties of mineralization and tibia of hens but adding enzymes decreases this negative effect of wheat. The replacement of corn with wheat in laying diets without affecting the quality of the eggshell or bone mineral content, can improve the strength and quality of the bones after supplementation with xylanase in these diets [24,25]. Similarly, Kiarie et al [14] reported decreased phosphorus retention in hens fed on wheat-based diets, but increased phosphorus retention in broiler chickens with enzymes (xylanase) added to feed. In this study, different levels of wheat had no significant effect on the levels of serum Ca and P in broiler chickens. Considering the bone turnover, serum PINP and ALP levels, both secreted by osteoblasts, can be used as an indicator of new bone formation [45]. In this experiment, using different levels of wheat in the diet had no significant effect on the serum content of P1NP and ALP in broiler chickens. CTx is a marker of bone resorption, and its level reflects the bone resorption activity of osteoclasts [46]. The high-level wheat substitution group resulting in decreased CTx content implied that the positive roles of wheat administration on tibial strength might partially be attributed to reduced bone absorption.

It is well-established that the goal of animal husbandry is to optimize animal health and food security rather than simple production, therefore, the liver function and inflammatory status were seriously reconsidered. By evaluating the healthy status of hepatocytes and systemic inflammation, the present data suggests that the supplementation of high-level wheat did not impair liver function, evidenced by decreased AST activity, a biomarker of early liver injury, and similar serum levels of ALT, TP, and inflammatory cytokines in this study. Moreover, the high levels of wheat inclusion could reduce liver inflammatory cell infiltration in broiler chickens, which was consistent with previous findings that wheat malt extracts (100 or 200 mg/kg) reduce the degree of hepatological histological injury and serum ALT and AST in a dose-dependent manner [47]. Therefore, in this experiment, the dose of wheat in the diets of broiler chickens had no deleterious effects on liver health and inflammatory status.