Effect of dietary supplementation with flavonoid from Scutellaria baicalensis Georgi on growth performance, meat quality and antioxidative ability of broilers

1. lntroduction

Flavonoids (FNs) from plants have several biological properties, such as antioxidant, anti-in flammatory and antibacterial protective effects on cells and organs of animals(Liang and Han 2003; Zhang et al. 2011). Numerous studies have shown that FNs have beneficial effects on performance of animals (Jenkins and Atwal 1995; Payne et al. 2001;Jiang et al. 2007).

It has been reported that dietary supplementation with the FN from Scutellaria baicalensis Georgi (SBGFN) could improve growth performance, immune function and intestinal health of broilers (Mou et al. 2010; Liang et al. 2012). The S. baicalensis Georgi (SBG) is a kind of traditional Chinese medicine, whose main effective component is FN. The FN has a special structure which can chelate with metal ions like zinc (Zn). As an essential trace element for animals,Zn can promote growth and development, raise the power of immunity system and maintain the cell membrane’s integrity of animals (Liang 2006). Wang et al. (2004)found that Zn chelate with baicalin was more effective than baicalin in inhibiting the activity of HIV-1 entry into host cells.When chelated with Zn, baicalin has a lower cytotoxicity compared with baicalin itself in vitro, and behaves more effective on scavenging free radicals and protecting the red cell membranes from oxidative damage. It is also reported that Zn chelate with baicalin had anti-in flammatory and anti-allergic efficiencies in mice (Jia et al. 1994). We hypothesized that SBGFN might be more effective for animals when it is chelated with Zn. The SBGFN-Zn as one new product might have a good application prospect in improving performances of animals. However, to our knowledge, the information is lacking on the effect of SBGFN-Zn on performances of broilers and other animals.Therefore, the present study was conducted to investigate the effect of dietary supplementation with SBGFN as SBGFN-Zn on growth performance, carcass characteristics,meat quality, immune function and antioxidation of broilers.

2. Materials and methods

2.1. Experimental design and treatments

A completely randomized design was used in this study.There were a total of 5 dietary treatments, including a SBGFN-unsupplemented corn-soybean meal basal diet(control) or the basal diet supplemented with 60, 120, 180 or 240 mg SBGFN kg–1 in the form of SBGFN-Zn, respectively.

老父亲没有继续跟儿子争辩下去，而是让小儿子拿来一条锁链去把狗拴住。可是狗没犟几下就把铁链挣断了。父亲拾过铁链递到儿子手中，“你好好看看！”父亲语重心长地说道：“一条链子有几十个扣子，其中只锈坏了一个，而其他的完好，却仍然拴不住一条狗啊！”

2.2. Animals

All experimental procedures were approved by the Animal Management Committee (in charge of animal welfare issue) of the Institute of Animal Science, Chinese Academy of Agricultural Sciences (IAS-CAAS, Beijing, China) and performed in accordance with the guidelines. Ethical approval on animal survival was given by the Animal Ethics Committee of IAS-CAAS. A total of 450 one-d-old Arbor Acres commercial male broiler chicks (Huadu Broiler Company, Beijing, China) were randomly assigned into 5 treatments with 6 replicate cages each with 15 birds per cage based upon body weight for 42 d. Broilers were maintained on a 24-h constant light schedule. Feeding management throughout the experiment followed the Arbor Acres broilers management guidelines. Feed and tap water were available ad libitum. Broilers were vaccinated with the Newcastle vaccine VA/GA at 7 and 24 d of age, and the H5N1 vaccine at 11 and 28 d of age, respectively. These vaccines were purchased from Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences. The numbers of dead broilers were recorded daily, and feed intake and weight gain of broilers per cage were measured at 21 and 42 d of age to calculate the average daily gain (ADG),average daily feed intake (ADFI), feed:gain ratio (F/G) and mortality.

2.3. Diets

The contents of Zn and Ca in feed ingredients and diet samples were measured by inductively coupled plasma emission spectroscope (model IRIS Intrepid II, Thermal Jarrell Ash, Waltham, MA, USA) after wet digestions with HNO3 and HCIO4 as described by Luo et al. (2007).Concentrations of CP in feed ingredient or diet samples were determined using Official Analytical Chemists methods(AOAC 1990). The pH values of breast and thigh muscles were tested immediately after the birds were slaughtered using a Model pH-211 meter (Hanna Instruments Inc.,Padova, Italy) equipped with a spear electrode. The meat color (L＊, a＊ and b＊) was determined using an automatic colorimeter (model SC-80C; Beijing Kangguang Instrument Co., Beijing, China). The meat color was measured at 3 points of every meat after slaughter. The breast muscle and thigh muscle were excised (1 cm×1 cm×5 cm) and weighed and then placed in a plastic bag and stored at 4°C.After 24 h, the muscle was removed from the plastic bag wiped with filter paper, and weighed to evaluate drip loss and expressed as a percentage of initial muscle weight.At 24 h after slaughter, the breast and thigh muscles were heated in plastic bags in a water bath at 80 to 85°C until the temperature of the meat center reached 75°C. They were then taken out and cooled to room temperature and excised(1 cm×1 cm×5 cm) to measure the shear force at 3 points of every meat with a C-LM Muscle Tenderness Instrument(Northeast Agricultural University, Heilongjiang, China). The concentration of MDA, activity of T-SOD and GSH-Px in serum, liver and muscle samples of broilers were determined using the detection kits of MDA, T-SOD and GSH-Px (Nanjing Jiancheng BioEngineering Institute, Nanjing, China). The antibody titers against Newcastle and H5N1 vaccines were measured using the haemagglut-ination inhibition test as described by Meulemans et al. (1987). The analysis of whole blood T lymphocyte proliferation was carried out by MTT assay (Mosmann 1983). The mean optical density was determined for bipartite samples, and the proliferation responses were expressed as a mean stimulation index obtained by dividing the mean optical density (OD) of stimulated cells by OD of unstimulated cells (SI).

Table 1 Composition and nutrient levels of the basal diets for broilers (as-fed basis)

1) Reagent grade. 2) Provided per kilogram of diet for days 1–21: vitamin A (all-trans retinol acetate) 12 500 IU, cholecalciferol 3 750 IU, vitamin E (allrac-α-tocopherol acetate) 20 IU, vitamin K3 2.5 mg, vitamin B1 2.5 mg, vitamin B2 8 mg, vitamin B6 2.5 mg, vitamin B12 0.015 mg,pantothenic acid calcium 12.5 mg, niacin 32.5 mg, folic acid 1.25 mg, biotin 0.125 mg, choline 700 mg, Cu (CuSO4·5H2O)8 mg, Mn (MnSO4·H2O) 110 mg, Fe (FeSO4·7H2O) 60 mg, I (KI)0.35 mg, Se (Na2SeO3) 0.15 mg. Provided per kilogram of diet for days 22–42: vitamin A (all-trans retinol acetate) 10 000 IU,cholecalciferol 3 400 IU, vitamin E (all-rac-α-tocopherol acetate)12.8 IU, vitamin K3 1.6 mg, vitamin B1 0.8 mg, vitamin B2 6.8 mg, vitamin B6 1.6 mg, vitamin B12 0.008 mg, pantothenic acid calcium 8 mg, niacin 26 mg, folic acid 0.8 mg, biotin 0.126 mg,choline 500 mg, Cu (CuSO4·5H2O) 8 mg, Mn (MnSO4·H2O)80 mg, Fe (FeSO4·7H2O) 60 mg, I (KI) 0.35 mg, Se (Na2SeO3)0.15 mg. 3) Zn sulfate or flavonoid from Scutellaria baicalensis Georgi-Zn added in place of equivalent weights of corn starch. 4) Measured values based on triplicate determination.

Item Day 1–21 Day 22–42 Ingredients (%)Corn 56.39 59.78 Soybean meal 35.61 32.95 Soybean oil 3.50 3.50 CaCO3 2.10 1.50 NaCl1) 0.30 0.30 DL-Met1) 0.23 0.13 Micronutrients2) 0.38 0.24 Cornstarch+Zn3) 0.20 0.20 Total 100 100 Nutrient composition (%)ME (MJ kg–1) 12.37 12.57 CP4) 21.40 20.16 Lys 1.10 1.03 Met 0.54 0.43 Met+Cys 0.90 0.76 Ca4) 1.00 0.90 Zn (mg kg–1)4) 88.01 61.85 Nonphytate P4) 0.45 0.35 1.29 1.40 CaHPO4 1)1)

2.4. Sample collections and preparations

Supplemental SBGFN did not affect (P＞0.20) ADG (33.4, 79.9 and 58.8 g d–1), ADfi(46.1, 156 and 101 g d–1), F/G (1.38,1.95 and 1.72 g g–1) and mortality (1.56, 0.92 and 2.44%)of broilers during 1 to 21, 22 to 42 and 1 to 42 d of age,respectively (values represent the means of all treatments).

Many studies have shown that FNs could promote the growth performance of broilers (Liang et al. 2012; Ma et al.2015). Liang et al. (2012) found that diets supplemented with skullcap flavone enhanced ADG and ADfiof broilers.Ma et al. (2015) also reported that flavones of sea buckthorn fruits improved ADFI, ADG and final body weight of broilers.However, in the present study, we found that the diets supplemented with SBGFN as SBGFN-Zn did not influence the growth performance of broilers, which was similar to the reports of Kuhn et al. (2004) and Solcan et al. (2013).These differences might be due to different diets, type of chicks, growth phases, or different supplementation (Wang and Han 1994; Li et al. 1999; Ren et al. 2001; Whitten and Patisaul 2001).

2.5. Sample analyses

The corn-soybean meal basal diets (Table 1) were formulated to meet or exceed the National Research Council(NRC 1994) requirements for broilers for all nutrients.The SBGFN-Zn (containing 85% SBGFN and 6.25% Zn,respectively; Beijing Huamuweiye Technology Co., Ltd.,China) was added to the basal diet according to the above experimental treatments. The SBGFN was isolated from SBG. Each treatment diet was supplemented with 60 or 40 mg of Zn kg–1 of diet for broilers from 1 to 21 or 22 to 42 d of age by adjusting the usage of corn starch and Znsulfate according to the Zn content from added SBGFN-Zn,respectively. Each supplemental SBGFN level was mixed with corn-starch to the same weight and then mixed with each aliquot of the basal diet. The diets were fed in mash form.

2.6. Statistical analyses

Data were analyzed using the general linear model procedure of the SAS Institute (release 9.2; SAS Inst.Inc., Cary, NC, USA). The replicate cage served as the experimental unit. Percentage data were transformed to arcsine for analysis. Differences among means were tested by the least significant difference method and the P＜0.05 was considered to be statistically significant.

Supplemental SBGFN affected (P＜0.03) T-SOD and GSHPx activity in liver of broilers at 42 d of age, but did not affect(P＞0.11) MDA concentration either in liver or breast muscle,T-SOD and GSH-Px activity in breast muscle of broilers at 42 d of age as well as serum antioxidant parameters of broilers at 21 and 42 d of age (Tables 3 and 4). Broilers fed the diet supplemented with 180 mg SBGFN kg–1 had higher (P＜0.03) liver T-SOD activity than those fed the diets supplemented with 0, 60 and 120 mg SBGFN kg–1 with no differences (P＞0.27) between broilers fed the diets supplemented with 180 and 240 mg SBGFN kg–1. Broilers fed the diet supplemented with 180 mg SBGFN kg–1 had higher (P＜0.02) liver GSH-Px activity than those fed the diets supplemented with all other levels of SBGFN, and no differences were observed between the control broilers and those fed the diets supplemented with 60, 120 or 240 mg SBGFN kg–1.

3. Results

3.1. Growth performance

The samples of feed ingredients and diets from all the treatments were taken and submitted for analyses of crude protein (CP), calcium (Ca) and Zn before the initiation of the trial to con firm CP, Ca and Zn contents in diets. At 21 and 42 d of age, birds were individually weighed following an 12-h fast and 2 birds from each replicate cage were chosen based on average body weight (BW). Whole blood samples were taken into heparinized tubes from each of two birds via a wing vein for lymphocyte proliferation analysis. Another blood sample was also collected into tubes without anticoagulant, and then was centrifuged at 3 000×g for 10 min at 4°C to harvest serum samples for analyses of antibody titers against Newcastle and H5N1 vaccines, malondialdehyde (MDA) concentration as well as of total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px) activity. At 42 d of age, the birds were slaughtered by cervical dislocation. The breast and thigh muscles were used to determine the meat color and the pH value after slaughter, and then they were placed in a plastic bag and stored at 4°C for 24 h to determine the drip loss of the muscle, and then cooked to determine the shear force of the muscle, respectively. The percentages of spleen and thymus were calculated by using their weights to divide the live BW. The liver and right breast muscle were subsequently taken out and then frozen at –20°C for analyses of liver and breast muscle MDA concentration as well as T-SOD and GSH-Px activity. All samples from 2 birds in each replicate were pooled into one sample in equal ratios before analysis.

Supplemental SBGFN affected (P＜0.01) drip loss in thigh muscle, but did not affect (P＞0.05) L＊, a＊ and b＊ values, shear force and pH value in both breast and thigh muscles as well as drip loss in breast muscle of broilers (Table 2). Chicks fed the diets supplemented with 120, 180 and 240 mg SBGFN kg–1 had lower (P＜0.03) drip loss of thigh muscle than those fed the control diet, whereas no difference (P＞0.06) in drip loss was found between the control chicks and those fed the diet supplemented with 60 mg SBGFN kg–1.

3.2. Meat quality

受精卵孵化所用海水经过两级沉淀过滤并消毒后，通过砂滤罐，再经200目过滤网袋过滤进入培育池，并投放8mg/L的EDTA用以络合重金属离子。水温控制在18℃，盐度28.6‰，除了适宜的温度、盐度等基本水质条件外，充足的溶氧是提高孵化率最重要的因素，对虾受精卵为沉性卵，密度稍大于海水，静水时沉入水底，动荡时才悬浮水中。静水中卵粒沉于池底，极易造成局部缺氧出现死卵。由于激烈充气会损伤受精卵，充气强度需弱一些，但溶解氧不低于5mg/L。为防止孵化过程局部缺氧，需定时翻卵，每2～3h翻卵一次。受精卵经过30h左右孵化出无节幼体，孵化率在90%以上。

3.3. lmmune function

Supplemental SBGFN had no effect (P＞0.05) on the spleen(0.119) and thymus (0.452) percentages and serum antibody titers against H5N1 (7.73log2) at 42 d of age, against Newcastle (3.85 and 2.61log2) and blood T-lymphocyte proliferation (0.890 and 0.939 SI) of broilers at 21 and 42 d of age, respectively (values represent the means of all treatments).

3.4. Antioxidation

燕麦作为长日照作物，日照时长对其生殖生长有重要影响。杨才等[2]于1978—1979年在海南冬季进行了皮、裸燕麦的加光试验，研究发现，同一品种不加光的燕麦不能抽穗结实，而加光处理的品种与北方春播燕麦的生育性状相接近，能够正常完成生育期。随着燕麦育种工作的展开，杨才等采用四倍体大燕麦（A.magna）、大粒裸燕麦（A.nuda）、普通栽培燕麦（A.sativa）三种间聚合杂交法，结合北育南繁、温室加代、自然选择等措施，育成了300余个对短日照不敏感的燕麦新种质及高代品系，在海南冬季短日照不加光条件下连续5年都取得了种植的成功[3]。

4. Discussion

纸艺，即以纸质材料为主要表现的艺术作品，它的最大特点是能够充分展现纸的特有材质美，不像传统的绘画艺术，纸只不过是艺术家笔下色彩的陪衬。“纸艺”的表现形式有很多，如：剪纸、拼贴纸艺、折纸、纸雕、纸塑等。其中，折纸和剪纸在培养幼儿的动手能力、发展幼儿智力、训练幼儿做事的认真精神等方面所起到的作用，也大有异曲同工之妙。

Table 2 Effect of dietary flavonoid from Scutellaria baicalensis Georgi (SBGFN) on meat quality of broilers at 42 d of age1)

1) L＊, lightness; a＊, redness; b＊, yellowness. Values represent means of 6 replicates (n=6). Means with different letters within the same column differ (P＜0.04).

Added SBGFN level(mg kg–1)Thigh muscle 0 57.5 8.01 18.2 56.1 9.39 15.5 2.70 2.26 4.85 7.18 a 6.53 6.63 60 59.2 7.44 17.9 56.5 9.06 14.3 2.80 1.97 4.43 5.65 ab 6.53 6.59 120 58.6 7.53 18.2 56.7 9.08 15.4 2.79 1.80 4.02 5.33 bc 6.53 6.60 180 58.9 6.80 19.3 58.4 8.99 16.3 2.50 2.02 4.39 4.19 bcd 6.51 6.59 240 58.7 7.32 18.2 57.4 8.56 15.8 3.60 1.92 4.21 3.52 d 6.51 6.61 Pooled SEM 1.1 0.38 0.6 0.6 0.25 0.4 0.38 0.14 0.49 0.56 0.03 0.02 P-value 0.83 0.31 0.63 0.11 0.50 0.06 0.31 0.24 0.81 ＜0.01 0.97 0.63 Breast muscle Thigh muscle Shear force (kg) Drip loss (%) pH value L＊ a ＊ b＊ L ＊ a ＊ b＊ Breast muscle Thigh muscle Breast muscle Thigh muscle Breast muscle

Table 3 Effect of dietary flavonoid from Scutellaria baicalensis Georgi (SBGFN) on antioxidant parameters in serum of broilers1)

1) MDA, malondialdehyde; T-SOD, total superoxide dismutase; GSH-Px, glutathione peroxidase.Values represent means of 6 replicates (n=6).

Added SBGFN level(mg kg–1)MDA (nmol mg–1 protein) T-SOD (NU mL–1) GSH-Px (U mL–1)Day 21 Day 42 Day 21 Day 42 Day 21 Day 42 0 3.18 5.54 176 230 183 771 60 4.30 4.02 199 233 181 566 120 3.08 4.56 209 249 176 640 180 3.00 5.39 212 257 207 756 240 3.44 3.81 196 240 210 659 Pooled SEM 0.39 0.58 11 13 23 64 P-value 0.15 0.12 0.21 0.56 0.74 0.16

Table 4 Effect of dietary flavonoid from Scutellaria baicalensis Georgi (SBGFN) on antioxidant parameters in liver and breast muscle of broilers at 42 d of age1)

1) MDA, malondialdehyde; T-SOD, total superoxide dismutase; GSH-Px, glutathione peroxidase. Values represent means of 6 replicates (n=6). Means with different letters within the same column differ (P＜0.03).

Added SBGFN level(mg kg–1)MDA (nmol mg–1 protein) T-SOD (NU mg–1 protein) GSH-Px (U mg–1 protein)Liver Breast muscle Liver Breast muscle Liver Breast muscle 0 0.719 0.220 196 b 27.2 29.4 b 3.58 60 0.697 0.252 224 b 28.7 31.7 b 4.32 120 0.523 0.228 216 b 24.2 31.8 b 3.99 180 0.640 0.203 279 a 29.4 43.3 a 3.82 240 0.576 0.215 248 ab 27.4 30.4 b 3.48 Pooled SEM 0.096 0.015 20 1.6 2.9 0.68 P-value 0.62 0.26 0.02 0.25 0.02 0.91

Drip loss was also an important index to evaluate meat quality (Otto et al. 2006). Moisture loss could take away the heme and cause the loss of soluble flavor substance in meat (Savage et al. 1990; Luciano et al. 2009). Therefore,the moisture closely linked with the physical form, flavor and color of meat. The results from the present study showed that supplemental SBGFN-Zn decreased drip loss of thigh muscle, which was consistent with the study of Li et al.(2008), who found that flavones of sea buckthorn could decrease the drip loss in thigh muscle of broilers. The drip loss has been shown to be negatively related to antioxidant capability and immune function. The decreased drip loss may be due to increased antioxidative ability in the chickens or immunoregulatory effects of FNs through involvement of humoral immune components (Middleton 1996; Young et al.2003; Shen et al. 2007). The exact mechanism remains unclear and needs to be further studied in the future.However, the results from the present study indicated that dietary supplementation with SBGFN as SBGFN-Zn had no effect on immune responses of broilers.

It has long been recognized that FNs have antioxidant activity, which have the ability to scavenge free radicals,superoxide and hydroxyl radicals by single-electron transfer(RiceEvans et al. 1996; Choi et al. 2002). The FNs could block apoptosis in human umbilical vein endothelial cells through decreasing reactive oxygen species, increasing glutathione and protecting DNA damage (Gong et al. 2010).The results from our current study demonstrated that dietary supplementation of SBGFN in the form of SBGFN-Zn increased liver T-SOD and GSH-Px activity of broilers. The T-SOD and GSH-Px are important and widely existing freeradical scavenging enzymes in the body, and they can clear the superoxide and lipid hydroperoxide, reduce the damage of organic hydrogen peroxide to the body (Ma 2008). Fang et al. (1991) reported that FN-Zn could scavenge superoxide and inhibit membrane peroxidation of red blood cell in vitro.The above-mentioned previous results and those from the present study demonstrated that the FN-Zn chelate has strong antioxidant ability.

5. Conclusion

The results from the current study indicate that dietary supplementation with 120, 180 and 240 mg SBGFN kg–1 as the SBGFN-Zn chelate improved meat quality, while the addition of 180 mg SBGFN kg–1 promoted antioxidative ability of broilers. Therefore, the SBGFN-Zn as one new product might have a good application prospect in improving both meat quality and antioxidative ability of broilers.

我在寻访团中年龄最小，同学们都是一些95后。突出的困难就是没有改革开放40年的完整经历，没有在社会工作的真实体验。

Acknowledgements

This work was supported by the earmarked fund for China Agriculture Research System (CARS-41), and the Agricultural Science and Technology Innovation Program,China (ASTIP-IAS08).

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