*2.7. Statistical Analysis*

The experimental values were analyzed by GLM procedure of SAS® 9.3 software (SAS Inst. Inc., Cary, NC, USA). The pen was used as the experimental unit for the analysis of growth performance, and an individual chicken was considered as the experimental unit for measuring the blood, meat quality, and carcass trait samplings. The difference of means was tested by Tukey test. The effect of dietary SK supplementation and stocking densities and their interactions were determined. A significant difference was expressed either *p* < 0.01 or *p* < 0.05.

#### **3. Results**

#### *3.1. Antioxidant Factors*

There was a significantly lower (*p* < 0.01) concentration of MDA in the serum with the supplementation of SK (Table 2). The serum concentrations of catalase (*p* < 0.05) and SOD (*p* < 0.05) were increased by supplementation of SK in the diet, while there was no difference between the SD treatments. The addition of dietary SK resulted in a greater (*p* < 0.01) TAC in the serum; however, the SD treatments did not change the TAC capacity. The low SD (*p* < 0.05) and supplementation of SK (*p* < 0.01) decreased the content of MDA in the leg muscle. The concentrations of catalase (*p* < 0.01) and SOD (*p* < 0.05) were increased by supplementation of SK in the diet, while there was a decrease in the concentration of SOD in the high SD treatment. There was an increased (*p* < 0.01) TAC in the leg muscle of

chickens with increased supplementation of SK; however, there were no significant effects of SD on TAC in the leg muscle.

**Table 2.** Effect of dietary SK and SD on antioxidant activity of serum and leg muscle in Korean native chicken.


SEM, standard error of means; SD, stocking density effect; SK, *Sophora koreensis* supplementation effect; SD × SK, stocking density × *Sophora koreensis* supplementation effect interaction; MDA, malondialdehyde; SOD, Superoxide dismutase; TAC, total antioxidant capacity.

#### *3.2. ABTS-Reducing Activity*

The antioxidant capacity result of serum indicated that ABTS-reducing activity was enhanced in the SK-supplemented treatments (Figure 1). No difference in ABTS-reducing activity was detected between the SD treatments. The SK-supplemented treatments showed an increased (*p* < 0.05) ABTS-reducing activity of leg muscles compared with the non-SKsupplemented treatments; however, there was a greater ABTS-reducing activity of leg muscle in the SK-supplemented treatments.

**Figure 1.** *Cont*.

**Figure 1.** *Cont*.

**Figure 1.** ABTS radical scavenging capacity (%) of different stocking density (SD) on serum (**a**) and leg muscle (**b**), and *Sophora koreensis* (SK) supplementation on serum (**c**) and leg muscle (**d**) of Korean native chicken. Non-SK, basal diet; SK, basal diet + 20 ppm SK; Asterisks (\*) indicate statistical significance (*p* < 0.05).

#### *3.3. Meat Color and Meat Quality*

The effect of diets and SD on breast meat color and quality of chickens was shown in Table 3. There were no breast meat redness, lightness, and yellowness responses to supplementation of SK in the diet and rearing in different stocking densities. The higher water holding capacity of breast muscle was shown for the SK-supplemented treatments (*p* < 0.01); however, there was no change among the SD treatments. A lower cooking loss and breast muscle pH was reported in the SK-supplemented treatments (*p* < 0.01), although there were no changes in cooking loss and pH of breast muscle between the SD treatments. The shear force was not affected by treatments.

#### *3.4. Growth Response, Carcass Traits, Immune Organ Ratio*

Table 4 shows the influences of diets and SD on growth performance, carcass traits, and relative organ weight. The effect of SK supplementation on improving final BW, BW gain, and feed intake of chickens was significant (*p* < 0.01). There was no difference in feed conversion ratio between the SK treatments. There was no difference in final BW and BW gain between the SD treatments; however, the feed intake and feed conversion ratio of chickens were adversely affected by the high SD. The carcass yield, breast meat, and drumsticks showed no dietary SK effects; however, a lower (*p* < 0.01) abdominal fat was shown in the SK-supplemented treatments. The carcass yield, breast meat, drumstick percentage, and abdominal fat were decreased in the high SD treatments. There was no change in the relative weight of the liver. There were significant interactions between the SD and SK in increasing the relative weight of the spleen and bursa Fabricius. The main effects shown were that the relative weight of the spleen and bursa Fabricius were

increased by supplementation of SK, but decreased by increasing the SD. There were no differences in the percentage of the spleen to bursa Fabricius and relative weight of thyroid between the treatments.

**Table 3.** Effect of dietary SK and SD on breast meat color and quality in Korean native chicken.


SEM, standard error of means; SD, stocking density effect; SK, *Sophora koreensis* supplementation effect; SD × SK, stocking density × *Sophora koreensis* supplementation effect interaction.

**Table 4.** Effect of dietary SK and SD on growth performance, carcass traits, and relative weights of organs in Korean native chicken.


SEM, standard error of means; SD, stocking density effect; SK, *Sophora koreensis* supplementation effect; SD × SK, stocking density × *Sophora koreensis* supplementation effect interaction; BW, bodyweight; FI, feed intake; FCR, feed conversion ratio.

#### *3.5. Blood Profile*

The effects of dietary SK on blood profile are shown in Table 5. Results indicated that total cholesterol and glucose levels were decreased in the high SD treatment but that the concentration of blood total cholesterol was increased in the SK-supplemented treatments. There was no change in concentration of total protein, triglyceride, GPT, GOT, albumin, phosphate, and calcium among the treatments. The blood corticosterone level was significantly higher (*p* < 0.05) in the high SD treatment; however, corticosterone level was decreased in the SK-supplemented treatments.


**Table 5.** Effect of dietary SK and SD on blood profile in Korean native chicken.

SEM, standard error of means; SD, stocking density effect; SK, *Sophora koreensis* supplementation effect; SD × SK, stocking density × *Sophora koreensis* supplementation effect interaction; GPT, glutamic pyruvic transaminase; GOT, glutamate oxaloacetate transaminase.

### **4. Discussion**

*Sophora koreensis* has been known as a traditional herb to treat rheumatoid issues in Korea because of its antioxidant capacity [13,24]. The antioxidant enzymes including SOD, GPx, and catalase are the first factors against antioxidant reactions [11,12]. Antioxidant effects of scopoletin were shown earlier [16,17]. An increase in the aforementioned enzyme's production capacity can improve the antioxidant system by controlling the production of ROS. Superoxide dismutase is an important enzyme in the protection of cells from adverse effects of ROS [25]. In the current study, decreased concentration of MDA and increased TAC in the serum of chickens treated with SK may be related to the increased concentrations of catalase and SOD, which reduce the formation of peroxides and hydroperoxides in fat tissues [7]. The increased activities of catalase and SOD in the serum of broiler chickens fed SK diets show its potential to scavenge free radicals during the stressful condition of high SD. It has been reported that the stressful condition decreases the SOD and catalase production, which in turn increases MDA production [26]. There is a positive correlation between high MDA concentrations and lipid peroxidation [7,8]. Meanwhile, the reduction of lipid peroxidation in the thigh muscle may be reflected in the decrease of MDA concentration in the SK treatments. Scopoletin also showed high inhibitory activities against anti-inflammatory cytokines by decreasing TNF-α, IL-1β, and IL-6 secretions [17,19,27]. It has been reported that the anti-inflammatory influences of scopoletin are associated with a decrease in free radicals production. The production of MDA, as an important indicator of lipid peroxidation, is due to the exposure of free radicals to the plasma membrane [7,8,28]. Lee et al. [29] reported that the MDA levels were significantly increased in mice under alcoholic food stress, but that the supplementation of scopoletin prevented the increase in MDA concentration compared with the control treatment. Several challenge experiments with high inflammatory condition confirmed that the decrease in inflammation mediates cell damage, lipid peroxidation, and increases the inactivation of antioxidant enzymes [9,10,28]. Several researchers have reported that high SD could increase the stress level and decrease the antioxidant status by increasing MDA and decreasing SOD concentrations in serum [1,2,30]. Although increased catalase and TAC seem to be consistent in the plasma and leg muscle in the SK-supplemented treatments, this trend is missing for chickens in the SD treatments. However, chickens in the SD treatments showed a higher MDA and lower SOD content in the leg muscle. Although the decrease in the SOD concentration was in line with the increased MDA in the leg muscle, the concentration of the aforementioned parameters in the serum was not in agreement with the leg muscle results.

Although scopoletin increases the antioxidant status [17], literature did not study the effects of supplemental scopoletin on meat quality related to SD stress in chickens focusing on the antioxidant capacity. Therefore, this study aimed to test the influences

of scopoletin on the antioxidant capacity of meat, plasma, and the possible interactions with the quality and color of meat. Although the scavenging capacity of scopoletin against ABTS in chicken meat has not been studied, an in vitro study on the antioxidant role of phenolic compounds reported that the α-diphenyl-β-picrylhydrazyl radical-scavenging activity of scopoletin was around 11,800 times higher than vitamin C, making it a potent antioxidant compound [31]. The increased scavenging capacity of ABTS of meat due to scopoletin was in line with an enhanced TAC in the leg muscle, indicating the reduction of oxidative damage in muscle tissues with the presence of scopoletin. Furthermore, an increased SOD, as well as decreased MDA, content in the serum and meat confirm the capability of scopoletin in decreasing oxidative stress. Therefore, the result of the current study showed that the supplementation of 20 ppm scopoletin adequately increased the ABTS-reducing activity in chickens.

In the present study, the abdominal fat percentage of chicken was shown to be lower in the SK-supplemented group than in the non-supplemented group, which was in line with the results reported by Rajaei et al. [32], who reported that the addition of 5 mL/L of noni juice, as a rich source of scopoletin, in drinking water had a significant effect in decreasing abdominal fat content in broiler chickens. Scopoletin is known as a stimulator of fatty acid oxidative genes including PPARa, Acsl1, CPT, Acox, and Acaa1a, and an inhibitor of lipogenic genes such as sterol regulatory element-binding protein-1c and fatty acid synthase in the white adipose tissue and liver in rat [29,33]. The reduced abdominal fat may be due to the supplementation of scopoletin as a bioactive component that reduces oxidative stress and improves carbohydrate and fat metabolism. Serum corticosterone can reflect the welfare status of chickens with the environment [34], and several production and behavioral parameters can be under the influence of corticosterone hormone. Hosseindoust et al. [5] reported that stress hormone is a decisive index for deposition of protein and carcass percentage in chickens. Supplementation with *Morinda citrifolia* L. as a rich source of scopoletin increased the absorption of amino acids, which subsequently increased the carcass rate of broiler chickens [32]. In the current study, the decrease of abdominal fat in the SK-supplemented treatments may not be due to the serum corticosterone because of the insignificant difference in breast meat and drumstick. However, chickens in the SD treatments showed a lower breast meat and drumstick percentage as well. The lower protein deposition may decrease the percentage of muscle to fat and be responsible for the higher relative abdominal fat. There is a positive correlation between corticosterone concentration and abdominal, thigh, or cervical adipose tissue's fat contents [35]; however, the degradation of skeletal muscle can be increased when the serum corticosterone concentration increases [36]. Therefore, the lower protein deposition in muscular organs including breast muscle and drumstick may be responsible for the higher relative abdominal fat.

The health status and BW of animals are under the adverse effect of SD stress, which may cause economic loss [1,2]. Corticosterone is one of the most common end-products of stress and will be secreted to the blood during stressful environments [5,34,37]. Long-time exposure to restraints disrupts the hypothalamic-pituitary-adrenal axis and increases the concentrations of plasma corticosterone [38]. Consistently, the current study showed that the supplementation of SK in the diet during SD stress led to the decrease of corticosterone in the serum. The excessive production of ROS compromises cell growth by degrading cytoskeletal proteins, as well as causing lipids peroxidation during stressful conditions [9]. Our result is in line with those from other researchers who have employed SD stress in poultry [34].

Our study showed that dietary SK supplementation improved the meat quality of chickens by increasing water holding capacity and decreasing cooking loss. The water holding capacity is known as a determinantal factor of meat quality [3]. The significantly lower cooking loss in SK-supplemented treatments may be due to the lower serum corticosterone level. It was reported that excessive levels of serum corticosterone not only reduced the BW gain through reducing anabolism and increasing catabolism processes [39], but also

induced lipid peroxidation [40,41], which may influence meat quality. The protective roles of antioxidant enzymes and controlling the effects of free radical damage seem essential to reduce the adverse influences of stocking density stress. Antioxidant properties of scopoletin may be reflected in the MDA concentration of meat. The lower concentration of serum corticosterone may indicate that the chickens fed SK diets had relatively lower stress regardless of SD because the SD did not decrease cooking loss of breast meat. There is a positive relationship between corticosterone production and the secretion of inflammatory cytokines including IL-1b, IL-6, IL-10, IL-12-a, and IL-18 [19,37], which can adversely affect meat quality. Corticosterone secretion has been shown to be a factor to decrease meat quality by degrading protein in muscle and decreasing the fatty acid transport protein expression [35]. Song et al. [42] stated that the concentration of uric acid, as a factor to show protein catabolism in tissues, increased in blood when the concentration of corticosterone increased in the blood. In addition, the production of ROS can be increased by corticosteroid hormones [43]. The high antioxidant status leads to lower exposure to hydroxyl and peroxyl radicals and possibly the protection of lipid tissues from oxidation through chelating free radicals [4,6]. The result of radical scavenging capacity in this study shows that scopoletin is a potent antioxidant factor to protect fatty acids oxidation and increase meat quality.

The BW gain of chickens in the high SD treatments was 11.9% lower than the low SD treatments. In addition, feed intake was 13.9% lower in the high SD treatments. The reduction of weight gain and feed intake may show that the SD stress adversely affected the performance of broiler chickens. There are important biomarkers such as corticosterone that decrease growth performance during a stressful period [34,38]. The greater BWG in SK-supplemented groups may be because of the antioxidant effects of scopoletin, which may stimulate protein synthesis.

#### **5. Conclusions**

In conclusion, to improve radical scavenging capacity and controlling lipids peroxidation, the antioxidant capacity of broiler chickens can be improved by SK supplementation during high SD stress. Dietary SK improved meat quality through increasing ABTS radical scavenging capacity in the serum and leg muscle. In addition, lower abdominal fat and higher immune organs weight were shown in chickens fed SK. Therefore, our study suggests that SK is a practically useful feed additive to improve the meat quality and weight gain of chickens regardless of SD stress.

**Author Contributions:** Conceptualization and writing—original draft preparation, A.H., S.H.H., H.K.K. and J.S.K.; methodology, A.H. and S.H.H.; software, H.K.K.; validation, S.H.H.; formal analysis, S.H.H.; investigation, S.H.H., H.K.K. and H.T.; resources, A.H.; data/table curation, H.K.K., H.T. and H.L.; writing—review and editing, J.Y.M., E.J.C. and J.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ012821012021)" Rural Development Administration, Republic of Korea.

**Institutional Review Board Statement:** The experiment was approved by the Institutional Animal Care and Use Committee, Kangwon National University (KW-170519-1).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

