Effects of Dietary Supplementation of Chinese Yam Polysaccharide on Carcass Composition, Meat Quality, and Antioxidant Capacity in Broilers

Simple Summary The purpose of this experiment was to investigate the effects of yam polysaccharide as a feed additive on the meat quality and antioxidant capacity of broilers. Our results showed that dietary yam polysaccharide could improve carcass performance, color difference, and shear force and enhance the antioxidant capacity of broilers. Abstract The study aimed to evaluate the influences of the dietary supplementation of Chinese yam polysaccharide (CYP) on the carcass performance, antioxidant capacity, and meat quality of broilers. Three hundred and sixty healthy 1-day-old broilers with similar body weight (39 ± 1 g, gender balanced) were randomly divided into four groups (control, CYP1, CYP2, and CYP3 groups). In the control group, broilers were fed a basal diet with CYP, and the CYP1, CYP2, and CYP3 groups were fed diets supplemented with 250, 500, and 1000 mg/kg CYP, respectively. There were three replicates in each group, 30 birds in each replicate, and the feeding trial lasted for 48 days. Statistical analysis was performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) by one-way analysis of variance. The results showed that compared with the control group, dietary supplementation with 500 mg/kg CYP can improve live weight, half-eviscerated carcass percentage, eviscerated carcass percentage, and thigh muscle percentage. Moreover, dietary supplementation with 500 mg/kg CYP can improve the contents of total antioxidant capacity (T-AOC), total superoxide dismutase (T-SOD), glutathione peroxidase (GPX), and glutathione s-transferase (GST) in serum (p < 0.05). Meanwhile, the mRNA expression levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase 1 (HO-1), quinone oxidoreductase (NQO1), superoxide dismutase 1 (SOD1), glutathione peroxidase 1 (GPX1), and catalase (CAT) in the liver; the mRNA expression levels of HO-1, NQO1, GPX1, and CAT in the breast muscle; and the mRNA expression levels of NQO1, SOD1, and CAT in the thigh muscle of broilers in the CYP2 group were significantly increased (p < 0.05). In addition, the yellowness and shear force of the thigh and breast muscles and the content of malondialdehyde (MDA) in the serum of broilers in the control group were higher than that in the CYP2 groups (p < 0.05). The results demonstrated that the CYP2 group had the best effect on improving meat quality. In conclusion, dietary supplementation with 500 mg/kg CYP can improve the meat quality of broilers by improving carcass quality, meat color, shear force, and antioxidant capacity.


Introduction
In recent years, because of the characteristics of fast growth, short feeding cycle, and high feed conversion rate of broilers, they have become the second largest breeding industry in China after pig breeding. In actual production, farmers usually adopt the mode

Animal Care
In this study, all bird feeding programs were approved by the Animal Care and Use Committee of the Henan Institute of Science and Technology (No. 2020HIST018, Xinxiang, China).

Experimental Diets and Animal Management
Three hundred and sixty healthy 1-day broilers with similar body weight (39 ± 1 g, sex balance) were purchased from Henan Fengyuan Poultry Co., Ltd., Xinxiang, China, and the broilers were randomly divided into four groups: basal diet (CON group) and basal diet supplemented with 250, 500, 1000 mg/kg CYP (CYP1, CYP2, and CYP3 groups). Each group consisted of three replicates with 30 broilers per replicate. Additionally, data related to feed intake were prepared for publication as part of another article, and data related to average daily feed intake (g/d) are as follows: 1-28 d (control groups, 23.50 ± 0.75; CYP1 groups, 27.36 ± 0.23; CYP2 groups, 28.88 ± 0.03; and CYP3 groups, 26.06 ± 0.01), 29-48 d (control groups, 89.16 ± 1.38; CYP1 groups, 97.72 ± 0.02; CYP2 groups, 98.78 ± 0.09; and CYP3 groups, 97.19 ± 0.03), 1-48 d (control groups, 56.33 ± 0.50; CYP1 groups, 62.54 ± 0.14; CYP2 groups, 63.83 ± 0.02; and CYP3 groups, 61.62 ± 0.04). The nutritional requirements Animals 2023, 13, 503 3 of 11 of broilers from the National Research Council were used to formulate the basal diet, and it was fed for 48 days [17]. Table 1 shows the composition and the levels of nutrients in the basal diet. Experimental broilers were reared in cages, and feed was available ad libitum. During the rearing period, all broilers were kept under constant light for 24 h, and room temperature was at 32 • C for the first 3 days and then gradually cooled to 26 • C on the 21st day. The crude CYP used in the experiment was purchased from Shaanxi Hana Biological Technology Co., Ltd., Xi'an, China, with a particle size pass 80 mesh, drying loss weight less than 5%, and polysaccharide content greater than or equal to 30%, and its monosaccharide types included glucose, 99.48%, and galactose, 0.52%).

Slaughter Performance Measurement
At 48 days of age, 24 broilers of similar live weights (6 broilers in each group, sex balanced) were randomly selected. After fasting for 12 h, they were weighed, and their venous blood from the wing was placed in centrifuge tubes without anticoagulant and allowed to condense overnight at 4 • C. Serum was collected after centrifugation (3000 g, 10 min, at 4 • C) and stored at −70 • C until analysis. Then, birds were slaughtered according to the procedures presented by Geldenhuys et al. [18]. After bloodletting, feather extraction and beak shell and foot skin removal, the carcass weight was taken. Viscera were removed; only the heart, liver, glandular stomach, and muscle stomach (excluding contents and keratin membrane) were retained, and abdominal fat was called half-eviscerated weight. The heart, liver, glandular stomach, muscular stomach, abdominal fat, head, and feet (the foot is divided from the hock joint, and the head is cut off from the link of the first cervical spine) were removed and then weighted to determine the dressing percentage. The thigh and breast muscles were taken out and weighed, and the percentages of the breast and thigh muscles were calculated. The meat samples were refrigerated at 4 • C for 24 h, and then the breast muscle and thigh muscle were divided into two parts. One was used to measure the meat color and shear force, while the other was frozen with liquid nitrogen and stored at −70 • C until analysis. The carcass-performance-related calculation formula is as follows: Dressing percentage (%) = (carcass weight/live weight) × 100%; Half-eviscerated carcass percentage (%) = (half-eviscerated weight/live weight) ×100%; Eviscerated carcass percentage (%) = (eviscerated weight/live weight) × 100%; Breast muscle percentage (%) = (weight of breast muscle on both sides/live weight) × 100%; Thigh muscle percentage (%) = (weight of thigh muscle on both sides/live weight) × 100%.

Color Measurement
After being stored at 4 • C for 24 h, the meat colors (including lightness, redness, and yellowness, which are L*, a*, and b* values) were determined by a colorimeter (Konica Minolta CR-410, Sensing Inc., Osaka, Japan). White tiles (L* 93.65, a* −0.88, and b* 3.24) were used as standard.

Shear Force Measurement
Shear force was measured with a Warner-Bratzler shear device (Zwick Roell Group, Ulm, Baden Wuerttemberg, Germany) in accordance with the method described by Liu et al. [19]. The thawed thigh and breast muscles were cut into three cubes of approximately 1 × 1 × 3 cm 3 along the direction of the myofibrils. A shear instrument was used for longitudinal shear along the direction of the vertical myofibril, the shear force value was recorded, and the average value was calculated several times.

Serum Antioxidant Measurement
Serum was collected after centrifugation of blood samples clotted overnight and loaded into 1.5 mL of sterile tubes. Then the concentrations of glutathione peroxidase (GPX), total superoxide dismutase (T-SOD), glutathione s-transferase (GST), and malondialdehyde (MDA) in the serum of broilers were measured by the ELISA kit purchased from the Nanjing Jing Bioengineering Research Institute (Nanjing, China). The microassay was used to measure the total antioxidant capacity (T-AOC). The specific steps are as follows: preheat (30 min, adjust wavelength to 593 nm) → calibration → add sample to test tube, stand for 10 min → add sample (96-well plate) → measure OD value → calculate. Table 2 shows primer sequences designed according to the known sequences stored in GenBank. Gene expressions of nuclear factor-erythroid 2-related factor 2 (Nrf2), quinone oxidoreductase (NQO1), heme oxygenase 1 (HO-1), catalase (CAT), superoxide dismutase 1 (SOD1), and glutathione peroxidase 1 (GPX1) in liver and breast and thigh muscles were measured by quantitative real-time polymerase chain reaction (qRT-PCR). TRIzol reagent was used to extract total RNA from breast and thigh muscles and liver (Takara Bio Inc., Tokyo, Japan). The concentration and purity of the extracted RNA were measured using a spectrophotometer (Implen, Westlake Village, CA, USA) at an optical density of 1.8 ≤ 260/280 ≤ 2.2. The synthesis of complementary DNA (cDNA) obtained using the PrimeScript™ RT Reagent Kit (Takara Bio Inc., Tokyo, Japan). qRT-PCR was performed with the ViiA™ 7 Real-Time PCR System using a SYBR Green RT-PCR kit from Bio-Rad (Hercules, CA, USA). The mRNA expression levels were calculated by the 2 −∆∆CT method.

Statistical Analysis
Statistical analysis was performed by one-way ANOVA using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Duncan's multiple range tests were used for multiple comparison among groups. Data were expressed as mean ± SEM (standard error of means), and there was a significant difference at p < 0.05. Table 2. Primer sequences for quantitative RT-PCR analysis.

Genes (Accession) 1 Primer Sequence Length (bp)
Nrf2 ( Table 3 shows the effects of the dietary addition of CYP on broilers' slaughter performance. The liver weight, half-eviscerated carcass percentage, eviscerated carcass percentage, and thigh muscle percentage of broilers in the CYP2 group were higher than those in the control group (p < 0.05). Meanwhile, the CYP2 group had a higher eviscerated carcass percentage and thigh muscle percentage compared with the CYP3 group. No differences in live weight and half-eviscerated percentage were determined between the CYP1, CYP2, and CYP3 groups (p > 0.05). Furthermore, dressing percentage and breast muscle percentage were not different among all groups (p > 0.05).  Table 4 shows the effects of the dietary addition of CYP on the meat quality of broilers. In our study, the values of L* and a* were not differences in the thigh and breast muscles of broilers (p > 0.05). The b* value of the CYP2 group was lower than that of the control group (p < 0.05), and the shear force of the thigh and breast muscles of the broilers in all experimental groups (CYP1, CYP2, and CYP3 groups) was lower than those in the control group (p < 0.05). Furthermore, the CYP1 and CYP2 groups had lower shear force of thigh muscle compared with the CYP3 group, and the CYP1 group had a lower b* value of the thigh muscle compared with the CYP2 and CYP3 groups, respectively (p < 0.05).

Antioxidant Enzyme Activities
As shown in Table 5, the activities of GST, GPX, and T-AOC in the serum of all experimental groups were higher than those of the control group (p < 0.01), while the activity of MDA was on the contrary (p < 0.01). Meanwhile, the activity of GST in the CYP3 group was higher than that in the CYP1 group and lower than that in the CYP2 group (p < 0.05). The activity of T-SOD in the control and CYP1 groups was significantly lower than that in the CYP2 and CYP3 groups (p < 0.05). Among all experimental groups, no differences in serum T-SOD, T-AOC, GPX, and MDA were determined (p > 0.05).

Gene Expression
As shown in Figures 1-3, compared with the control group, in the liver, all experimental groups upregulated the Nrf2, HO-1, NQO1, and SOD1 mRNA expression levels (p < 0.05), and the CYP2 group upregulated the GPX1 and CAT mRNA expression levels (p < 0.05). In the breast muscle, all experimental groups upregulated the HO-1 mRNA expression levels (p < 0.05), the CYP1 and CYP2 groups upregulated the NQO1 mRNA expression levels (p < 0.05), and the CYP2 group upregulated the CAT and GPX1 mRNA expression levels compared with the control group (p < 0.05). Meanwhile, there was no significant difference in Nrf2 and SOD1 mRNA expression (p > 0.05) among all groups (control, CYP1, CYP2, and CYP3 groups). In the thigh muscle, the CYP2 group upregulated the HO-1, NQO1, SOD1, and CAT mRNA expression levels compared with the control group (p < 0.05), while Nrf2 and GPX1 mRNA expressions were not significantly different among all groups (p > 0.05). pression levels (p < 0.05), the CYP1 and CYP2 groups upregulated the NQO1 mRNA expression levels (p < 0.05), and the CYP2 group upregulated the CAT and GPX1 mRNA expression levels compared with the control group (p < 0.05). Meanwhile, there was no significant difference in Nrf2 and SOD1 mRNA expression (p > 0.05) among all groups (control, CYP1, CYP2, and CYP3 groups). In the thigh muscle, the CYP2 group upregulated the HO-1, NQO1, SOD1, and CAT mRNA expression levels compared with the control group (p < 0.05), while Nrf2 and GPX1 mRNA expressions were not significantly different among all groups (p > 0.05).

Discussion
A measure of differences in nutrient deposition in tissues is carcass traits [20]. Furthermore, the performance of meat production can be measured by slaughter and total carcass yield [9,21]. In our study, dietary CYP supplementation could significantly im-

Discussion
A measure of differences in nutrient deposition in tissues is carcass traits [20]. Furthermore, the performance of meat production can be measured by slaughter and total

Discussion
A measure of differences in nutrient deposition in tissues is carcass traits [20]. Furthermore, the performance of meat production can be measured by slaughter and total carcass yield [9,21]. In our study, dietary CYP supplementation could significantly improve the live weight, half-eviscerated percentage, eviscerated carcass percentage, and thigh muscle percentage of broilers, and the group of 500 mg/kg CYP had the best effect. There is evidence that blueberry extract as a feed additive of natural plant extracts can improve the growth performance and carcass characteristics of broiler breast and thigh meat in the study of Ölmez et al. [22]. A previous study also found that the dressing, carcass, and breast and thigh weight of quail can be improved by dietary supplementation with clove oil (Syzygium aromaticum) [23]. Furthermore, the results of Miao et al. indicated that expanded chitosan significantly increased the slaughter rate and lean meat rate of growing-finishing pigs [24]. Our results are similar to previous studies, suggesting that the dietary supplementation of CYP may be used as a potential feed additive to improve the broilers' carcass traits.
The appearance, texture, and other physiological characteristics of meat products directly affect consumers' willingness to buy meat products. In a study of the New Zealand meat market, Moore et al. found that color value is considered an indicator of meat safety and quality [25], and it has been reported that a higher a* value and lower L* and b* values are more popular [26]. Shear force, an important indicator to evaluate meat tenderness, is one of the major attributes of sensation that determine whole acceptability, and the related study has shown that lower shear force and better meat quality generally reflect thinner muscle fibers and higher muscle moisture content [27,28]. We found that the dietary addition of 250 mg/kg CYP and 500 mg/kg CYP can reduce the b* value in the breast and thigh muscles of broilers at 48 days, but there is no difference in L* and a* values, which was opposite to the result of Wang et al. [29] and similar to that of Wang et al. [30]. Furthermore, our study showed that dietary supplementation with CYP resulted in lower shear force in broilers after slaughter, and the 500 mg/kg CYP group has the best result. A similar result was found in the effect of Camellia oleifera cake polysaccharides on meat quality in yellow broilers [29]. Therefore, it may be a way to improve the meat quality of broilers that add CYP to regulate the color and shear force of meat.
The antioxidant status is an important factor that affects host health, and the previous study showed that MDA is the primary degradation product of lipid peroxidation; its content is correlated with oxidative damage [31]. The opposite is that GPX and superoxide SOD are usually regarded as enzyme-free radical scavengers in cells and the important antioxidant enzyme that converts superoxide anion to hydrogen peroxide in cellular antioxidant reactions [32,33]. In addition, previous studies have suggested that CYP can improve oxidation stability. According to Zhu et al., CYP can effectively remove DPPH, ABTS+, and ·OH free radicals, and its scavenging ability on DPPH free radicals is equivalent to ascorbic acid [34]. Chen et al. successfully prepared yam oligosaccharides by hydrolyzing them with H 2 O 2 , and the results showed that yam oligosaccharides were a suitable hydroxyl radical scavenger [35]. Our study found that dietary addition with 500 mg/kg CYP can improve serum SOD and GPX activities and decrease the activity of serum MAD of broilers, thus improving the activity of serum T-AOC of broilers, which was similar to the study of Tan in 2014 [36], which found that the dietary supplementation of CYP could significantly improve the activity of GPX and decrease the content of serum MDA in yellow chickens. In addition, in poultry muscle, due to the lower fat content and high content of polyunsaturated fatty acids (PUFA), the unsaturated degree of the muscle membrane is increased. Therefore, poultry meat is easily oxidized, leading to a decrease in meat quality and nutritional value. In addition, it can reduce meat tenderness by reducing the activity of calcium-activating enzymes [37]. Lipid oxidation increases the permeability of cell membranes and decreases the water retention of muscles [38]. Thus, an antioxidant balance Animals 2023, 13, 503 9 of 11 is essential for normal physiological and metabolic function in animals. Nrf2, a member of the basic leucine zipper transcription factor family, is a crucial transcription factor that maintains the balance of reductant-oxidant and signal transduction, which has a cellular protective effect on oxidative stress [39,40]. In addition, Nrf2 can regulate NQO1, CAT, GPX, SOD, and a series of downstream antioxidant-related genes. HO-1 is a target gene regulated by the Nrf2 signaling pathway and is an antioxidant enzyme. HO-1 was found to have antioxidant, anti-inflammatory, immunomodulatory, and antiapoptotic effects, and can decompose heme to produce biliverdin, free iron, and carbon monoxide (CO). Furthermore, reduced bilirubin and byproduct biliverdin can effectively remove activated oxygen to resist peroxide free radicals, hydroxyl groups, oxides, and peroxynitrite [41]. Ho-1 promoter activity can be directly regulated by Nrf2. In this study, in the 500 mg/Kg CYP group, Nrf2 in the liver had a higher level of mRNA expression, and the expression levels of the NQO1, HO-1, SOD1, GAX1, and CAT genes downstream of Nrf2 were also upregulated. Although the expression level of Nrf2 in the breast muscle and the thigh muscle showed an upward trend, the difference was not significant; the expression levels of the downstream genes HO-1, NQO1, and CAT were significantly increased; and the expression levels of GPX1 in the breast muscle and SOD1 in the thigh muscle were also significantly increased. The results were similar to previous studies of natural polysaccharides in broilers [42,43]. Therefore, our results suggested that dietary supplementation with CYP can improve the antioxidant capacity of broilers by regulating the activity of serum antioxidant enzymes and the mRNA expression of related genes in the Nrf2 signaling pathway.

Conclusions
The dietary addition of yam polysaccharide had positive effects on the slaughter performance, serum antioxidant capacity, antioxidant-related genes in the liver and muscle, and meat quality of broilers. In addition, compared with 250 mg/kg and 100 mg/kg in the diet, the effect of 500 mg/kg is more prominent. These results indicate that dietary 500 mg/kg CYP can significantly improve the meat quality of broilers by changing slaughter performance, b* value, shear force, and antioxidant capacity.