Effects of Ginkgo biloba leaves (Ginkgo biloba) and Ginkgo biloba extract on nutrient and energy utilization of broilers

Effects of Ginkgo biloba leaves (Ginkgo biloba) and Ginkgo biloba extract on nutrient and energy... Abstract An experiment using 112 21-day-old male Arbor Acres broilers that were randomly allocated to 7 treatments with 8 replicates in a completely randomized design was conducted to assess the effects of Ginkgo biloba leaves (Ginkgo biloba, GL) and Ginkgo biloba extract (EGB) on utilization of nutrients of broiler chickens. The dietary treatments were corn-soybean meal based diets: 1) T1, control diet; 2) T2, T1 + 20 g/kg GL; 3) T3, T1 + 40 g/kg GL; 4) T4, T1 + 60 g/kg GL; 5) T5, T1 + 0.4 g/kg EGB; 6) T6, T1 + 0.8 g/kg EGB; and 7) T7, T1 + 1.2 g/kg EGB. Endogenous losses were obtained from another 16 broilers. Excreta samples were collected to analyze the dry matter (DM), organic matter (OM), ether extract (EE), crude protein (CP), gross energy (GE), and amino acids (AA), and GE was analyzed for computation of AME and TME. As compared with those of the control treatment, the apparent digestibility (AD) and true digestibility (TD) of EE, CP, Thr, Val, Ile, Leu, Phe, Lys, His, and Arg were quadratically (P < 0.05) increased; moreover, the AD and TD of Met was linearly (P < 0.05) increased as the concentration of the EGB in the diet increased. Increasing GL from 0 to 60 g/kg of diet linearly (P < 0.05) increased the AD and TD of EE, Thr, Val, Leu, His, and Met and tended (0.05 < P < 0.1) to increase the TME, TMEn, and Arg. Supplementation of EGB increased (P < 0.05) AD and TD of EE, Thr, Val, Ile, Leu, Phe, His, and Arg and tended (0.05 < P < 0.1) to increase Lys as compared with those in the GL-supplemented groups. Dietary supplementation of GL and EGB improved the utilization of nutrients of broiler chickens in a dose-dependent manner, and the optimum supplementation levels of GL or EGB in the diet of broilers was 60 or 0.8 g/kg of diet, respectively. INTRODUCTION After banning of antibiotic growth promoters, alternative sources of feed additives are the continuous research interest of the animal nutritionist (Salim et al., 2013; Kim et al., 2016). Efficacious, environmentally friendly, and consumer favorable feed additives are necessary to animal productions (Wan et al., 2017). Recently, herbs or plant extracts have been incorporated into the diet to improve animal productivity by enhancing the production performance and the quality of food derived from those animals (Yan et al., 2011). Plant-derived feed additives, which contain natural active compounds, can enhance growth performance and improve intestinal health and function common to poultry (Aziza et al., 2010). Moreover, several studies pertaining to dietary plant origin products used in chicken diets have been published previously because of natural, non-toxic substances with multiple biological activities (Fei et al., 2008). Ginkgo biloba leaves (Ginkgoaceae, Ginkgo biloba, GL) are from an ancient Chinese tree that has been cultivated and held sacred for its health-promoting properties. There is substantial experimental evidence to support the view that Ginkgo biloba extract (EGB), which is the leaf extract of Ginkgo biloba, has many pharmacological effects (Kazumasa et al., 2002; Chen et al., 2011; Zhang et al., 2013). In recent years, GL have attracted increasing attention as a functional food ingredient because they contain numerous bioactive constituents, such as flavonoids, bilobalides, and polyphenols (Chen et al., 2003; Yao et al., 2004; Niu et al., 2016). Most of the studies indicated that supplementation of GL products had favorable influences on feed efficiency, growth performance, intestinal morphology and absorption functions, and immune responses without adverse effects in broiler chickens (Zhang et al., 2012; Yu et al., 2015; Zhang et al., 2015). And in laying hens, diets supplemented with Ginkgo leaves fermented with Aspergillus niger increased egg production, and improved the lipid metabolism (Zhao et al., 2013). Moreover, EGB, which contains 24% flavonoid glycosides, 6% terpene lactones, and less than 5 ppm ginkgolic acid (Mahadevan and Park, 2008, Mohammad and Anvari, 2015), has various biological activities and different pharmacological effects, including antioxidation (Liu et al., 2006; Zhao et al., 2011), anti-inflammation (Zeybek et al., 2003; Zhou et al., 2006), and modulation of immune response (Zhao et al., 2011). Despite these findings, there has been little research on the use of GL and EGB as feed additives for nutrient and energy utilization of broilers fed a corn-soybean diet. Information is also lacking on isolating the active ingredients in GL and then using them as growth promoters in broilers. The objective of this research was to assess the optimum dose of GL and EGB feed in broiler diets and to evaluate the effects of supplementation of GL and EGB on utilization of nutrients of broiler chickens. MATERIALS AND METHODS Preparation of Ginkgo Biloba Leaves and Ginkgo Biloba Extract GL were provided by Rui Kangsheng Biology Co., Ltd. (Linyi, China) and dried at 65°C in a mechanical drier (20226A, Shanghai Yangguang Laboratory Instruments Co. Ltd., Shanghai, China), The dried GL were subsequently milled with a disc mill (FFC-15, Shandong Changlin Machinery Group Co. Ltd, Linshu, Shandong, China) to pass a screen size of 0.25 mm and stored in the dark in airtight plastic bags at ambient temperature (21 to 27°C). The GL used in this study contained 90, 15, 5.48, and 4.9% of dry matter (DM), crude protein (CP), ether extract (EE), and crude ash (CA), respectively, and 1.83 Mcal/kg gross energy (GE), which were analyzed in our laboratory according to AOAC (1999). EGB also was obtained from Rui Kangsheng Biology Co., Ltd. (Linyi, China); the extract was obtained from the GL by ethanol as an extracting solvent, and EGB was stored in the dark in airtight plastic bags at ambient temperature (21 to 27°C). The components of flavonoids and terpene lactones were analyzed using HPLC and have been identified as the main bioactive constituents in GL and EGB (Ahlemeyer and Krieglstein, 2003). High-performance liquid chromatography The chemical compositions of the GL and EGB were carried out by HPLC (Waters alliance 2695, Milford, MA, USA) analysis. The content of flavonol glycosides was determined by using the HPLC-UV method, and the mixture of 50% methanol and 50% phosphoric acids solution of 0.5% concentration (V/V) as the mobile phase. The flow rate was 1.0 mL/min. Detection was performed at a wavelength of 360 nm with the column maintained at a constant temperature (30°C). The content of terpene lactones was developed using HPLC-ELSD means. The mobile phase consisted of methanol and water (30:70, V/V), and the flow rate was 1.0 mL/minute. The drift tube temperature was 105°C, the flow rate of the carrier gas was 2.9 mL/min, and the column (250 mm × 4.6 mm, 5.0 μm) temperature was 25°C. Quercetin, Kaempferol, Isorhamnetin, Ginkgolide A, Ginkgolide B, Ginkgolide C, and Bilobalide were used as external standards, and the retention times and concentrations of each ingredient in the EGB were determined relative to the standards. The content of GL and EGB contained 0.54 and 30.96% total flavonoid glycosides, and 0.18 and 8.91% terpene lactones, respectively. Animals, experiment design, and management A total of 112 21-day-old male Arbor Acres broiler chicks with similar body weight were purchased from a commercial hatchery (Xiling Family Farm, Tai’an, China), individually weighed, and then randomly distributed into 7 dietary treatments with 8 replicates (2 chicks/each, according to the requirement of the experiment animals in Animal Nutrition, Mcdonald et al., 2010) in a completely randomized design. Each broiler was housed in individual wire cages equipped with water troughs and feeders. The dietary treatments were corn-soybean meal based diets, and the diets were made as followings: 1) T1, control diet; 2) T2, T1 + 20 g/kg GL; 3) T3, T1 + 40 g/kg GL; 4) T4, T1 + 60 g/kg GL; 5) T5, T1 + 0.4 g/kg EGB; 6) T6, T1 + 0.8 g/kg EGB; and 7) T7, T1 + 1.2 g/kg EGB. The experiment was designed to match the total flavonoid glycosides and terpene lactones in GL- and EGB-supplemented treatments (Table 1). The diets were made as wheat bran was replaced with GL and were formulated to meet or exceed nutrient requirements of Arbor Acres growing broilers (Feeding Standard of Chicken of the People's Republic of China; NY/T 33–2004). Diet compositions are shown in Table 1. GL and EGB were first mixed with the premix and then mixed with other ingredients and stored in covered containers before feeding. Moreover, the endogenous contribution was calculated from excreta collected from 16 broilers that underwent the same procedure without being fed. All broilers were continuously provided with uniform light for 24 h, and room temperature was maintained at 22°C. Body weight was recorded by cage at the end of the adaptation period (28 d) and the test period (31 d). The animal care and use protocol was reviewed and approved by Shandong Agricultural University Animal Nutrition Research Institute (Tai’an, Shandong, China). Table 1. Ingredients and nutrient composition of experimental diets.1     Dietary GL concentration (%)  Dietary EGB concentration (%)2  Item  control  2  4  6  0.04  0.08  0.12  Ingredients, %  Corn  57.3  57.3  57.3  57.3  57.3  57.3  57.3  Soybean meal (CP, 46%)  28.4  28.4  28.4  28.4  28.4  28.4  28.4  Soy oil  4.60  4.60  4.60  4.60  4.60  4.60  4.60  Wheat bran  6.00  4.00  2.00  0.00  6.00  6.00  6.00  GL  0.00  2.00  4.00  6.00  0.00  0.00  0.00  EGB  0.00  0.00  0.00  0.00  0.04  0.08  0.12  Dicalcium phosphate  1.20  1.20  1.20  1.20  1.20  1.20  1.20  Limestone powder  0.95  0.95  0.95  0.95  0.95  0.95  0.95  Sodium Chloride  0.21  0.21  0.21  0.21  0.21  0.21  0.21  L- lysine -HCl  0.15  0.15  0.15  0.15  0.15  0.15  0.15  DL-methionine  0.19  0.19  0.19  0.19  0.19  0.19  0.19  Premix3  1.00  1.00  1.00  1.00  1.00  1.00  1.00  Total  100  100  100  100  100  100  100  Analysis nutrient content  ME, kcal/kg  3140  3144  3149  3153  3140  3140  3140  Crude protein  19.33  19.35  19.36  19.37  19.33  19.33  19.33  Calcium  0.86  0.86  0.86  0.86  0.86  0.86  0.86  Available phosphorus  0.35  0.35  0.36  0.37  0.35  0.35  0.35  Flavonoid glycosides  0.00  1.18  2.39  3.60  1.20  2.40  3.60  Terpene lactones  0.00  0.36  0.72  1.08  0.36  0.71  1.07  Sodium Chloride  0.30  0.30  0.30  0.30  0.30  0.30  0.30  Lysine  1.07  1.08  1.08  1.09  1.07  1.07  1.07  Methionine  0.48  0.48  0.49  0.50  0.48  0.48  0.48      Dietary GL concentration (%)  Dietary EGB concentration (%)2  Item  control  2  4  6  0.04  0.08  0.12  Ingredients, %  Corn  57.3  57.3  57.3  57.3  57.3  57.3  57.3  Soybean meal (CP, 46%)  28.4  28.4  28.4  28.4  28.4  28.4  28.4  Soy oil  4.60  4.60  4.60  4.60  4.60  4.60  4.60  Wheat bran  6.00  4.00  2.00  0.00  6.00  6.00  6.00  GL  0.00  2.00  4.00  6.00  0.00  0.00  0.00  EGB  0.00  0.00  0.00  0.00  0.04  0.08  0.12  Dicalcium phosphate  1.20  1.20  1.20  1.20  1.20  1.20  1.20  Limestone powder  0.95  0.95  0.95  0.95  0.95  0.95  0.95  Sodium Chloride  0.21  0.21  0.21  0.21  0.21  0.21  0.21  L- lysine -HCl  0.15  0.15  0.15  0.15  0.15  0.15  0.15  DL-methionine  0.19  0.19  0.19  0.19  0.19  0.19  0.19  Premix3  1.00  1.00  1.00  1.00  1.00  1.00  1.00  Total  100  100  100  100  100  100  100  Analysis nutrient content  ME, kcal/kg  3140  3144  3149  3153  3140  3140  3140  Crude protein  19.33  19.35  19.36  19.37  19.33  19.33  19.33  Calcium  0.86  0.86  0.86  0.86  0.86  0.86  0.86  Available phosphorus  0.35  0.35  0.36  0.37  0.35  0.35  0.35  Flavonoid glycosides  0.00  1.18  2.39  3.60  1.20  2.40  3.60  Terpene lactones  0.00  0.36  0.72  1.08  0.36  0.71  1.07  Sodium Chloride  0.30  0.30  0.30  0.30  0.30  0.30  0.30  Lysine  1.07  1.08  1.08  1.09  1.07  1.07  1.07  Methionine  0.48  0.48  0.49  0.50  0.48  0.48  0.48  1The control group was fed the basal diet. The other treatment diets were the same basal diet supplemented with 0.4 g/kg, 0.8 g/kg, 1.2 g/kg EGB, respectively, and for GL treatments, supplemented with 20 g/kg, 40 g/kg, 60 g/kg GL of diet by replacing the equivalent amount of wheat bran in the basal diet formulation. 2GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. 3Supplied per kilogram of diet: vitamin A, 8,050 IU; cholecalciferol, 3,000 IU; vitamin E, 30 mg; vitamin K3, 5 mg; thiamin, 2.58 mg; riboflavin, 12.5 mg; pyridoxine, 4.5 mg; vitamin B12, 0.018 mg; choline chloride, 800 mg; calcium pantothenate, 13 mg; niacin, 45 mg; biotin, 0.20 mg; folic acid, 1.20 mg; Mn, 100 mg; Fe, 80 mg; Zn, 58 mg; Cu, 8.8 mg; I, 0.374 mg; Se, 0.28 mg. View Large Sampling The broilers were distributed randomly and enclosed in individual metabolic cages labeled with the respective dietary treatments, which allowed total collection of excreta from each individual separately. Each dietary treatment was provided with the same corresponding experimental diets (T1, T2, T3, T4, T5, T6, or T7). The endogenous treatment was fed the control diet. The entire experiment contained the adaptation (7 d: 21 d to 27 d of age) period and the test period (3 d: 28 d to 30 d of age). During the adaptation period, animals were gradually adapted to the experimental diet. After the adaptation period, the test diets of T1 to T7 were given for 3 d and the excreta were collected at the same time. In addition, the broilers in the endogenous treatment were fed only water for 48 h after the initial 24-hour fasting period to determine the endogenous nutrient production. Feathers and shredded dry skin were removed carefully before excreta were analyzed. Broilers were fed ad libitum (expect the endogenous treatment) and had free access to water throughout the whole experiment. Collection and analyses of samples A sample of feed was prepared for determination of DM intake and subsequent chemical analysis during the collection period. Excreta were collected daily for 72 h, with the endogenous treatment for 48 h, then pooled, weighed, and mixed for each replication of 2 broilers, and stored at −20°C until all samples were analyzed. All chemical analyses were performed in duplicate. The diets and the fresh excreta were analyzed for N according to the Kjeldahl N determination method to determine CP content (total N × 6.25 = CP). The feed and excreta samples were dried at 65°C and ground to pass through a 0.5 mm screen and then stored in wild-mouth bottles to analyze for DM, CA, EE, and GE based on the Association of Official Analytical Chemists procedures (AOAC, 1999). After freeze-drying, feed and excreta samples were analyzed for amino acid (AA, except tryptophan). The AME and TME values of the feedstuffs and excreta were calculated by the method described by Sibbald (1976). Organic matter (OM) was calculated by content of crude ash. All samples were analyzed for DM dried at 105°C for 48 h, and then burned in a muffle furnace (SX2 box-type resistance furnace, Yantai, China) for 3 h at 550°C, and the ashes were used to determine the content of crude ash. The GE was determined by adiabatic bomb calorimetry (WHR-15 Oxygen bomb calorimeter, Changsha, China). The AA concentrations were assayed with Hitachi 835 type high-speed automatic amino acid analyzer. Shandong Agricultural University (Shandong, China) provides the place to analyze the samples. Calculation and statistical analysis Apparent digestibility (AD) and true digestibility (TD) of DM, OM, EE, CP, GE, and AA were calculated using the following equations:   \begin{equation*}{\rm{AD}}\,\left( \% \right) = \left[ {\left( {{\rm{E}}1 - {\rm{E}}2} \right)/{\rm{E}}1} \right]{\rm{ }} \times 100 \end{equation*}   \begin{equation*} {\rm{TD}}\,\left( \% \right) = \left[ {\left( {{\rm{E}}1 - {\rm{E}}2 + {\rm{E}}3} \right)/{\rm{E}}1} \right]{\rm{ }} \times 100\end{equation*} Where: E1 is the total amount (mg) of the nutrient (DM, OM, EE, or CP) and GE (MJ) in the diet that was fed to each bird; E2 is the total amount (mg) of the corresponding nutrient (DM, OM, EE, or CP) and GE (MJ) in excreta collected during the 3-day period; E3 is the total amount (mg) of the corresponding endogenous nutrient (DM, OM, EE, or CP) and GE (MJ) in excreta collected for a 48-hour period without being fed. AME and AME with that of endogenous nitrogen (N) losses (AMEn), and TME and TME with that of endogenous N losses (TMEn) (DM basis) were calculated using the following equations:   \begin{equation*} {\rm{AME}}\,\left( {{\rm{MJ}}/{\rm{kg}}} \right) = \left( {{\rm{E}}4 - {\rm{E}}5} \right)/{\rm{E}}7 \end{equation*}   \begin{equation*} {\rm{TME}}\,\left( {{\rm{MJ}}/{\rm{kg}}} \right) = \left( {{\rm{E}}4-{\rm{E}}5 + {\rm{E}}6} \right)/{\rm{E}}7 \end{equation*}   \begin{equation*} {\rm{AMEn}} = {\rm{AME}} - {\rm{RN}} \times 34.39 \end{equation*}   \begin{equation*} {\rm{TMEn}} = {\rm{TME}} - {\rm{RN}} \times 34.39 \end{equation*} Where: E4 is the total amount (MJ) of energy in the diet that was fed to each bird; E5 is the total amount (MJ) of energy in excreta collected during the 3-day period; E6 is the total amount (MJ) of energy in excreta collected for a 48-hour period without being fed; E7 is the feed intake of the diet that was fed to each broiler (DM basis). RN is the apparent N retention calculated as the difference between N intake and N output. All statistical analyses were performed using the GLM procedure of SAS (SAS Institute, 2000). The data were analyzed as a completely randomized design with individual unit as the random factor to examine the overall effect of treatments. Orthogonal polynomial contrasts were used to determine linear and quadratic responses to the increasing levels of GL and EGB in diets. Statements of significance were P < 0.05, and statistical tendencies were indicated if the P-value was between 0.05 and 0.10. RESULTS Body Weight All broilers appeared healthy, and no mortality occurred throughout the entire experimental period (data not shown); body weight was not significantly affected by the addition of GL and EGB in diets (Table 2). Table 2. Effects of GL and EGB on body weight (g/bird) of broilers fed diets.1 Item  GL  EGB  21 d  28 d  31 d  T1  0  0  845.8  1392.5  1652.9  T2  20 g/kg    846.3  1393.8  1653.2  T3  40 g/kg    846.7  1396.4  1654.9  T4  60 g/kg    848.9  1397.2  1655.6  T5    0.4 g/kg  846.3  1394.5  1652.6  T6    0.8 g/kg  848.3  1396.4  1656.7  T7    1.2 g/kg  847.8  1392.5  1656.8  SEM      2.510  1.990  2.496  P      0.99  0.89  0.97  Item  GL  EGB  21 d  28 d  31 d  T1  0  0  845.8  1392.5  1652.9  T2  20 g/kg    846.3  1393.8  1653.2  T3  40 g/kg    846.7  1396.4  1654.9  T4  60 g/kg    848.9  1397.2  1655.6  T5    0.4 g/kg  846.3  1394.5  1652.6  T6    0.8 g/kg  848.3  1396.4  1656.7  T7    1.2 g/kg  847.8  1392.5  1656.8  SEM      2.510  1.990  2.496  P      0.99  0.89  0.97  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Apparent Digestibility and True Digestibility of Dry Matter, Organic Matter, Ether Extract, Crude Protein, and Gross Energy of Diets The effects of supplementation of GL and EGB on AD of DM, OM, EE, CP, and GE in broilers fed a corn-soybean diet are shown in Table 3. The AD of DM, OM, and GE in all treatments had common digestibility. However, as the levels of EGB increased from 0 to 1.2 g/kg of diet, the AD of EE and CP was quadratically (P < 0.05) increased compared with that of control treatments. As compared with the control treatments, the AD of EE was linearly (P < 0.05) and quadratically (P < 0.05) increased as the levels of GL increased from 0 to 60 g/kg of diet. Addition of EGB in the broiler diets increased (P < 0.05) AD of EE compared with that in the GL-supplemented groups. Results for TD follow the same pattern as the results for AD (Table 4). Table 3. Effects of GL and EGB on AD of DM, OM, EE, CP, and GE of broilers fed diets.1 Item  GL  EGB  DM, %  OM, %  EE, %  CP, %  GE, %  T1  0  0  74.2  77.2  62.4  74.0  75.4  T2  20 g/kg    74.9  77.8  62.8  74.2  75.6  T3  40 g/kg    74.9  77.8  70.1  74.4  75.7  T4  60 g/kg    74.9  77.9  70.9  74.5  75.5  T5    0.4 g/kg  74.8  77.4  70.9  76.2  75.3  T6    0.8 g/kg  74.8  77.7  71.4  76.4  75.5  T7    1.2 g/kg  74.0  75.9  71.0  70.7  75.5    SEM    0.680  0.826  1.05  0.966  0.793  Control    0  74.2  77.2  62.4  74.0  75.4  With EGB    Pooled  74.5  77.0  71.1  74.4  75.5  With GL    Pooled  74.9  77.8  67.9  74.4  75.6  P-value      Control vs. EGB  0.51  0.18  <0.001  0.005  0.98  EGB  Linear  0.91  0.30  <0.001  0.039  0.66      Quadratic  0.31  0.11  <0.001  0.002  0.90      Control vs. GL  0.84  0.96  <0.001  0.99  0.99  GL  Linear  0.46  0.67  <0.001  0.71  0.94      Quadratic  0.66  0.86  <0.001  0.93  0.95  EGB vs. GL    0.48  0.25  0.007  0.96  0.84  Item  GL  EGB  DM, %  OM, %  EE, %  CP, %  GE, %  T1  0  0  74.2  77.2  62.4  74.0  75.4  T2  20 g/kg    74.9  77.8  62.8  74.2  75.6  T3  40 g/kg    74.9  77.8  70.1  74.4  75.7  T4  60 g/kg    74.9  77.9  70.9  74.5  75.5  T5    0.4 g/kg  74.8  77.4  70.9  76.2  75.3  T6    0.8 g/kg  74.8  77.7  71.4  76.4  75.5  T7    1.2 g/kg  74.0  75.9  71.0  70.7  75.5    SEM    0.680  0.826  1.05  0.966  0.793  Control    0  74.2  77.2  62.4  74.0  75.4  With EGB    Pooled  74.5  77.0  71.1  74.4  75.5  With GL    Pooled  74.9  77.8  67.9  74.4  75.6  P-value      Control vs. EGB  0.51  0.18  <0.001  0.005  0.98  EGB  Linear  0.91  0.30  <0.001  0.039  0.66      Quadratic  0.31  0.11  <0.001  0.002  0.90      Control vs. GL  0.84  0.96  <0.001  0.99  0.99  GL  Linear  0.46  0.67  <0.001  0.71  0.94      Quadratic  0.66  0.86  <0.001  0.93  0.95  EGB vs. GL    0.48  0.25  0.007  0.96  0.84  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Table 4. Effects of GL and EGB on TD of DM, OM, EE, CP, and GE of broilers fed diets.1 Item  GL  EGB  DM, %  OM, %  EE, %  CP, %  GE, %  T1  0  0  78.0  77.9  63.0  76.8  78.6  T2  20 g/kg    78.6  77.9  63.4  76.9  78.7  T3  40 g/kg    78.6  78.0  70.7  77.1  78.5  T4  60 g/kg    78.7  78.2  71.5  77.0  78.9  T5    0.4 g/kg  78.7  78.9  71.5  79.3  78.5  T6    0.8 g/kg  78.8  78.5  72.0  79.6  78.7  T7    1.2 g/kg  77.9  79.1  71.6  76.0  78.5    SEM    0.499  0.864  0.793  0.681  0.681  Control    0  78.0  77.9  63.0  76.8  78.6  With EGB    Pooled  78.5  78.8  71.7  78.3  78.6  With GL    Pooled  78.6  78.0  65.5  77.0  78.7  P-value      Control vs. EGB  0.53  0.70  <0.001  0.003  0.99  EGB  Linear  0.96  0.35  <0.001  0.63  0.99      Quadratic  0.33  0.61  <0.001  0.001  0.98      Control vs. GL  0.69  0.99  <0.001  0.99  0.99  GL  Linear  0.31  0.82  <0.001  0.77  0.83      Quadratic  0.49  0.97  <0.001  0.94  0.96  EGB vs. GL    0.78  0.20  0.002  0.044  0.88  Item  GL  EGB  DM, %  OM, %  EE, %  CP, %  GE, %  T1  0  0  78.0  77.9  63.0  76.8  78.6  T2  20 g/kg    78.6  77.9  63.4  76.9  78.7  T3  40 g/kg    78.6  78.0  70.7  77.1  78.5  T4  60 g/kg    78.7  78.2  71.5  77.0  78.9  T5    0.4 g/kg  78.7  78.9  71.5  79.3  78.5  T6    0.8 g/kg  78.8  78.5  72.0  79.6  78.7  T7    1.2 g/kg  77.9  79.1  71.6  76.0  78.5    SEM    0.499  0.864  0.793  0.681  0.681  Control    0  78.0  77.9  63.0  76.8  78.6  With EGB    Pooled  78.5  78.8  71.7  78.3  78.6  With GL    Pooled  78.6  78.0  65.5  77.0  78.7  P-value      Control vs. EGB  0.53  0.70  <0.001  0.003  0.99  EGB  Linear  0.96  0.35  <0.001  0.63  0.99      Quadratic  0.33  0.61  <0.001  0.001  0.98      Control vs. GL  0.69  0.99  <0.001  0.99  0.99  GL  Linear  0.31  0.82  <0.001  0.77  0.83      Quadratic  0.49  0.97  <0.001  0.94  0.96  EGB vs. GL    0.78  0.20  0.002  0.044  0.88  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large AME, AMEn, TME, and TMEn of Diets As shown in Table 5, there were no significant effects of GL or EGB supplementation in the diets on the AME, AMEn, TME, and TMEn, but compared with those of the control treatments, TME and TMEn in GL-supplemented treatments had a quadratically increasing (0.05 < P < 0.1) tendency. Table 5. Effects of GL and EGB on AME, AMEn, TME, and TMEn of broilers fed diets.1 Item  GL  EGB  AME MJ/kg.DM  AMEn MJ/kg.DM  TME MJ/kg.DM  TMEn MJ/kg.DM  T1  0  0  13.2  10.9  13.9  11.0  T2  20 g/kg    13.3  11.1  13.9  11.2  T3  40 g/kg    13.4  11.1  13.8  11.1  T4  60 g/kg    13.4  11.2  13.8  11.3  T5    0.4 g/kg  13.2  11.0  13.7  11.2  T6    0.8 g/kg  13.3  11.1  13.7  11.3  T7    1.2 g/kg  13.4  11.2  13.8  11.2    SEM    0.175  0.190  0.149  0.191  Pooled  Control  0  13.2  10.9  13.9  11.0  EGB  Pooled  13.3  11.1  13.7  11.2  GL  Pooled  13.3  11.1  13.8  11.2  P-value      Control vs. EGB  0.86  0.83  0.92  0.88  EGB  Linear  0.38  0.35  0.82  0.46      Quadratic  0.68  0.64  0.83  0.72      Control vs. GL  0.82  0.83  0.99  0.82  GL  Linear  0.34  0.36  0.80  0.38      Quadratic  0.63  0.64  0.061  0.058  EGB vs. GL    0.82  0.96  0.34  0.48  Item  GL  EGB  AME MJ/kg.DM  AMEn MJ/kg.DM  TME MJ/kg.DM  TMEn MJ/kg.DM  T1  0  0  13.2  10.9  13.9  11.0  T2  20 g/kg    13.3  11.1  13.9  11.2  T3  40 g/kg    13.4  11.1  13.8  11.1  T4  60 g/kg    13.4  11.2  13.8  11.3  T5    0.4 g/kg  13.2  11.0  13.7  11.2  T6    0.8 g/kg  13.3  11.1  13.7  11.3  T7    1.2 g/kg  13.4  11.2  13.8  11.2    SEM    0.175  0.190  0.149  0.191  Pooled  Control  0  13.2  10.9  13.9  11.0  EGB  Pooled  13.3  11.1  13.7  11.2  GL  Pooled  13.3  11.1  13.8  11.2  P-value      Control vs. EGB  0.86  0.83  0.92  0.88  EGB  Linear  0.38  0.35  0.82  0.46      Quadratic  0.68  0.64  0.83  0.72      Control vs. GL  0.82  0.83  0.99  0.82  GL  Linear  0.34  0.36  0.80  0.38      Quadratic  0.63  0.64  0.061  0.058  EGB vs. GL    0.82  0.96  0.34  0.48  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Apparent Digestibility and True Digestibility of Essential Amino Acid The effects of supplementation of GL and EGB on AD and TD of essential AA in broilers fed diets are shown in Tables 6 and 7. The AD and TD of Thr, Val, Ile, Leu, Phe, Lys, His, and Arg were quadratically (P < 0.05) increased, the utilization of was linearly (P < 0.05) increased as the concentration of EGB in the diet increased compared with those of the control treatment. Addition of GL quadratically (P < 0.05) increased the AD and TD of Thr, Val, Leu, His, and Met and tended (0.05 < P < 0.1) to increase Arg as the concentration of GL in the diet increased compared with those of the control treatment. Supplementation of EGB in the broiler diets increased (P < 0.05) the AD and TD of Thr, Val, Ile, Leu, Phe, His, and Arg and tended (0.05 < P < 0.1) to increase Lys compared with those in the GL-supplemented groups. Table 6. Effects of GL and EGB on apparent digestibility of essential amino acids of broilers fed diets.1 Item  GL  EGB  Thr, %  Val, %  Ile, %  Leu, %  Phe, %  Lys, %  His, %  Arg, %  Met, %  T1  0  0  82.2  80.2  82.5  87.4  86.3  85.6  88.9  92.6  84.5  T2  20 g/kg    80.1  77.3  81.2  85.8  86.0  85.9  86.0  91.1  85.5  T3  40 g/kg    81.5  78.5  81.6  85.9  85.7  86.4  87.0  91.6  93.6  T4  60 g/kg    82.7  80.3  82.7  87.6  86.1  87.4  88.6  91.9  87.8  T5    0.4 g/kg  83.7  81.1  84.2  88.9  88.4  87.5  91.2  93.5  89.2  T6    0.8 g/kg  87.7  84.8  87.5  90.7  89.5  90.0  92.2  94.9  84.8  T7    1.2 g/kg  85.0  82.8  84.2  89.2  86.8  86.0  89.9  93.6  89.8    SEM    0.788  0.955  0.817  0.563  0.638  0.690  0.532  0.378  1.010  Pooled    Control    80.6  77.7  81.4  86.1  86.1  86.1  86.3  91.3  87.2    EGB    85.5  82.9  85.3  89.6  88.2  87.8  91.1  94.0  88.0    GL    81.4  78.7  81.8  86.4  85.9  86.6  87.2  91.5  89.0  P-value      Control vs. EGB  <0.001  0.014  0.002  0.004  0.004  <0.001  <0.001  0.001  0.001  EGB  Linear  0.006  0.020  0.054  0.017  0.46  0.36  0.18  0.023  0.053      Quadratic  0.002  0.027  0.005  0.003  0.002  0.001  <0.001  0.002  0.16      Control vs. GL  0.071  0.076  0.43  0.021  0.92  0.21  0.002  0.061  <0.001  GL  Linear  0.41  0.75  0.73  0.85  0.69  0.037  0.97  0.37  0.015      Quadratic  0.052  0.045  0.25  0.007  0.80  0.102  0.001  0.051  0.005  EGB vs. GL    <0.001  <0.001  <0.001  <0.001  <0.001  0.065  <0.001  <0.001  0.35  Item  GL  EGB  Thr, %  Val, %  Ile, %  Leu, %  Phe, %  Lys, %  His, %  Arg, %  Met, %  T1  0  0  82.2  80.2  82.5  87.4  86.3  85.6  88.9  92.6  84.5  T2  20 g/kg    80.1  77.3  81.2  85.8  86.0  85.9  86.0  91.1  85.5  T3  40 g/kg    81.5  78.5  81.6  85.9  85.7  86.4  87.0  91.6  93.6  T4  60 g/kg    82.7  80.3  82.7  87.6  86.1  87.4  88.6  91.9  87.8  T5    0.4 g/kg  83.7  81.1  84.2  88.9  88.4  87.5  91.2  93.5  89.2  T6    0.8 g/kg  87.7  84.8  87.5  90.7  89.5  90.0  92.2  94.9  84.8  T7    1.2 g/kg  85.0  82.8  84.2  89.2  86.8  86.0  89.9  93.6  89.8    SEM    0.788  0.955  0.817  0.563  0.638  0.690  0.532  0.378  1.010  Pooled    Control    80.6  77.7  81.4  86.1  86.1  86.1  86.3  91.3  87.2    EGB    85.5  82.9  85.3  89.6  88.2  87.8  91.1  94.0  88.0    GL    81.4  78.7  81.8  86.4  85.9  86.6  87.2  91.5  89.0  P-value      Control vs. EGB  <0.001  0.014  0.002  0.004  0.004  <0.001  <0.001  0.001  0.001  EGB  Linear  0.006  0.020  0.054  0.017  0.46  0.36  0.18  0.023  0.053      Quadratic  0.002  0.027  0.005  0.003  0.002  0.001  <0.001  0.002  0.16      Control vs. GL  0.071  0.076  0.43  0.021  0.92  0.21  0.002  0.061  <0.001  GL  Linear  0.41  0.75  0.73  0.85  0.69  0.037  0.97  0.37  0.015      Quadratic  0.052  0.045  0.25  0.007  0.80  0.102  0.001  0.051  0.005  EGB vs. GL    <0.001  <0.001  <0.001  <0.001  <0.001  0.065  <0.001  <0.001  0.35  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Table 7. Effects of GL and EGB on true digestibility of essential amino acids of broilers fed diets.1 Item  GL  EGB  Thr, %  Val, %  Ile, %  Leu, %  Phe, %  Lys, %  His, %  Arg, %  Met, %  T1  0  0  82.7  80.5  82.7  87.7  86.4  86.0  89.0  92.6  84.6  T2  20 g/kg    80.5  77.6  81.4  86.1  86.3  86.2  86.3  91.3  85.6  T3  40 g/kg    81.9  78.9  81.8  86.1  86.0  86.8  87.3  91.8  93.7  T4  60 g/kg    83.2  80.6  82.8  87.8  86.6  87.8  89.1  92.7  88.0  T5    0.4 g/kg  84.2  81.4  84.4  89.1  88.7  87.8  91.4  93.7  89.3  T6    0.8 g/kg  88.1  85.1  87.7  90.9  89.8  90.3  92.4  95.1  85.6  T7    1.2 g/kg  85.5  83.1  84.5  89.5  87.1  86.5  90.2  93.8  89.1    SEM    0.787  0.954  0.819  0.563  0.629  0.689  0.525  0.377  0.976  Pooled    Control    82.7  80.5  82.7  87.7  86.4  86.0  89.0  92.6  84.6    EGB    85.9  83.2  85.5  89.8  88.5  88.2  91.3  94.2  88.0    GL    81.9  79.0  82.0  86.7  86.3  86.9  87.6  91.9  89.1  P-value      Control vs. EGB  <0.001  0.015  0.002  0.004  0.003  <0.001  <0.001  <0.001  0.004  EGB  Linear  0.006  0.020  0.051  0.016  0.34  0.32  0.13  0.009  0.076      Quadratic  0.002  0.027  0.005  0.004  0.001  0.002  <0.001  <0.001  0.19      Control vs. GL  0.065  0.073  0.50  0.020  0.91  0.21  <0.001  0.039  <0.001  GL  Linear  0.39  0.74  0.84  0.85  0.87  0.037  0.65  0.66  0.014      Quadratic  0.048  0.044  0.32  0.007  0.82  0.10  <0.001  0.020  0.005  EGB vs. GL    <0.001  <0.001  <0.001  <0.001  0.002  0.053  <0.001  <0.001  0.10  Item  GL  EGB  Thr, %  Val, %  Ile, %  Leu, %  Phe, %  Lys, %  His, %  Arg, %  Met, %  T1  0  0  82.7  80.5  82.7  87.7  86.4  86.0  89.0  92.6  84.6  T2  20 g/kg    80.5  77.6  81.4  86.1  86.3  86.2  86.3  91.3  85.6  T3  40 g/kg    81.9  78.9  81.8  86.1  86.0  86.8  87.3  91.8  93.7  T4  60 g/kg    83.2  80.6  82.8  87.8  86.6  87.8  89.1  92.7  88.0  T5    0.4 g/kg  84.2  81.4  84.4  89.1  88.7  87.8  91.4  93.7  89.3  T6    0.8 g/kg  88.1  85.1  87.7  90.9  89.8  90.3  92.4  95.1  85.6  T7    1.2 g/kg  85.5  83.1  84.5  89.5  87.1  86.5  90.2  93.8  89.1    SEM    0.787  0.954  0.819  0.563  0.629  0.689  0.525  0.377  0.976  Pooled    Control    82.7  80.5  82.7  87.7  86.4  86.0  89.0  92.6  84.6    EGB    85.9  83.2  85.5  89.8  88.5  88.2  91.3  94.2  88.0    GL    81.9  79.0  82.0  86.7  86.3  86.9  87.6  91.9  89.1  P-value      Control vs. EGB  <0.001  0.015  0.002  0.004  0.003  <0.001  <0.001  <0.001  0.004  EGB  Linear  0.006  0.020  0.051  0.016  0.34  0.32  0.13  0.009  0.076      Quadratic  0.002  0.027  0.005  0.004  0.001  0.002  <0.001  <0.001  0.19      Control vs. GL  0.065  0.073  0.50  0.020  0.91  0.21  <0.001  0.039  <0.001  GL  Linear  0.39  0.74  0.84  0.85  0.87  0.037  0.65  0.66  0.014      Quadratic  0.048  0.044  0.32  0.007  0.82  0.10  <0.001  0.020  0.005  EGB vs. GL    <0.001  <0.001  <0.001  <0.001  0.002  0.053  <0.001  <0.001  0.10  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Apparent Digestibility and True Digestibility of Non-Essential Amino Acid The effects of supplementation of GL and EGB on AD and TD of non-essential AA in broilers fed the diets are shown in Tables 8 and 9. The AD and TD of Asp, Ser, Glu, Gly, Ala, and Pro were quadratically (P < 0.05) increased as the concentration of EGB in the diet increased, compared with those of the control treatment. The AD and TD of Ser, Glu, Tyr, and Cys were quadratically (P < 0.05) increased as the concentration of GL in the diet increased compared with those in the control treatment. Supplementation of EGB in the broiler diets increased (P < 0.05) the AD and TD of Asp, Ser, Glu, Gly, Ala, Tyr, Pro, and Cys compared with those in the GL-supplemented groups. Table 8. Effects of GL and EGB on apparent digestibility of non-essential amino acids of broilers fed diets.1 Item  GL  EGB  Asp, %  Ser, %  Glu, %  Gly, %  Ala, %  Tyr, %  Pro, %  Cys, %  T1  0  0  84.5  85.7  90.0  74.0  75.6  86.0  86.8  73.7  T2  20 g/kg    83.1  84.4  87.9  73.7  75.0  84.4  85.2  76.6  T3  40 g/kg    83.9  86.0  88.8  75.7  74.4  83.8  85.6  83.3  T4  60 g/kg    84.9  87.6  90.1  75.7  77.5  86.3  86.9  83.7  T5    0.4 g/kg  85.5  87.9  90.8  76.2  75.9  87.1  88.9  76.4  T6    0.8 g/kg  89.2  90.6  92.8  79.7  81.8  88.3  89.4  76.6  T7    1.2 g/kg  87.2  88.3  91.4  79.2  79.1  86.4  87.6  77.6    SEM    0.700  0.609  0.438  1.363  1.309  0.747  0.623  2.009  Pooled    Control    83.4  84.9  88.2  74.1  75.3  84.6  85.4  77.6    EGB    87.3  88.9  91.7  78.4  79.0  87.3  88.6  76.8    GL    84.0  86.0  88.9  75.0  75.6  84.8  85.9  81.2  P-value      Control vs. EGB  <0.001  <0.001  0.001  0.040  0.015  0.22  0.021  0.71  EGB  Linear  0.003  0.005  0.010  0.008  0.023  0.51  0.33  0.27      Quadratic  0.003  <0.001  0.003  0.002  0.048  0.15  0.007  0.52      Control vs. GL  0.23  <0.001  0.001  0.39  0.22  0.013  0.11  <0.001  GL  Linear  0.51  0.008  0.57  0.13  0.33  0.95  0.79  <0.001      Quadratic  0.14  <0.001  <0.001  0.32  0.14  0.006  0.053  <0.001  EGB vs. GL    <0.001  <0.001  <0.001  0.007  0.008  <0.001  <0.001  0.020  Item  GL  EGB  Asp, %  Ser, %  Glu, %  Gly, %  Ala, %  Tyr, %  Pro, %  Cys, %  T1  0  0  84.5  85.7  90.0  74.0  75.6  86.0  86.8  73.7  T2  20 g/kg    83.1  84.4  87.9  73.7  75.0  84.4  85.2  76.6  T3  40 g/kg    83.9  86.0  88.8  75.7  74.4  83.8  85.6  83.3  T4  60 g/kg    84.9  87.6  90.1  75.7  77.5  86.3  86.9  83.7  T5    0.4 g/kg  85.5  87.9  90.8  76.2  75.9  87.1  88.9  76.4  T6    0.8 g/kg  89.2  90.6  92.8  79.7  81.8  88.3  89.4  76.6  T7    1.2 g/kg  87.2  88.3  91.4  79.2  79.1  86.4  87.6  77.6    SEM    0.700  0.609  0.438  1.363  1.309  0.747  0.623  2.009  Pooled    Control    83.4  84.9  88.2  74.1  75.3  84.6  85.4  77.6    EGB    87.3  88.9  91.7  78.4  79.0  87.3  88.6  76.8    GL    84.0  86.0  88.9  75.0  75.6  84.8  85.9  81.2  P-value      Control vs. EGB  <0.001  <0.001  0.001  0.040  0.015  0.22  0.021  0.71  EGB  Linear  0.003  0.005  0.010  0.008  0.023  0.51  0.33  0.27      Quadratic  0.003  <0.001  0.003  0.002  0.048  0.15  0.007  0.52      Control vs. GL  0.23  <0.001  0.001  0.39  0.22  0.013  0.11  <0.001  GL  Linear  0.51  0.008  0.57  0.13  0.33  0.95  0.79  <0.001      Quadratic  0.14  <0.001  <0.001  0.32  0.14  0.006  0.053  <0.001  EGB vs. GL    <0.001  <0.001  <0.001  0.007  0.008  <0.001  <0.001  0.020  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Table 9. Effects of GL and EGB on true digestibility of non-essential amino acids of broilers fed diets.1 Item  GL  EGB  Asp, %  Ser, %  Glu, %  Gly, %  Ala, %  Tyr, %  Pro, %  Cys, %  T1  0  0  84.9  86.1  90.2  75.0  76.1  86.5  87.5  73.7  T2  20 g/kg    83.5  84.7  88.1  74.1  75.4  84.6  85.4  76.6  T3  40 g/kg    84.2  86.4  89.0  76.1  74.9  84.1  85.9  83.3  T4  60 g/kg    85.3  88.0  90.3  76.1  78.0  87.2  87.2  83.7  T5    0.4 g/kg  85.9  88.3  91.1  76.7  76.4  87.4  89.1  76.4  T6    0.8 g/kg  89.5  90.9  93.0  80.1  82.2  88.6  89.6  79.0  T7    1.2 g/kg  87.7  88.7  91.7  79.6  79.6  86.8  87.9  77.6    SEM    0.699  0.609  0.438  1.377  1.309  0.729  0.622  1.882  Pooled    Control    84.9  86.1  90.2  75.0  76.1  86.5  87.5  73.7    EGB    87.7  89.3  91.9  78.8  79.4  87.6  88.9  77.6    GL    84.3  86.4  89.1  75.5  76.1  85.3  86.2  81.2  P-value      Control vs. EGB  <0.001  <0.001  0.001  0.078  0.016  0.29  0.057  0.42  EGB  Linear  0.003  0.005  0.009  0.015  0.021  0.59  0.56  0.16      Quadratic  0.002  <0.001  0.003  0.044  0.048  0.22  0.024  0.26      Control vs. GL  0.21  0.001  <0.001  0.50  0.22  <0.001  0.15  <0.001  GL  Linear  0.49  0.007  0.54  0.26  0.31  0.64  0.86  <0.001      Quadratic  0.13  <0.001  <0.001  0.49  0.14  <0.001  0.023  <0.001  EGB vs. GL    <0.001  <0.001  <0.001  0.045  0.003  0.001  <0.001  0.032  Item  GL  EGB  Asp, %  Ser, %  Glu, %  Gly, %  Ala, %  Tyr, %  Pro, %  Cys, %  T1  0  0  84.9  86.1  90.2  75.0  76.1  86.5  87.5  73.7  T2  20 g/kg    83.5  84.7  88.1  74.1  75.4  84.6  85.4  76.6  T3  40 g/kg    84.2  86.4  89.0  76.1  74.9  84.1  85.9  83.3  T4  60 g/kg    85.3  88.0  90.3  76.1  78.0  87.2  87.2  83.7  T5    0.4 g/kg  85.9  88.3  91.1  76.7  76.4  87.4  89.1  76.4  T6    0.8 g/kg  89.5  90.9  93.0  80.1  82.2  88.6  89.6  79.0  T7    1.2 g/kg  87.7  88.7  91.7  79.6  79.6  86.8  87.9  77.6    SEM    0.699  0.609  0.438  1.377  1.309  0.729  0.622  1.882  Pooled    Control    84.9  86.1  90.2  75.0  76.1  86.5  87.5  73.7    EGB    87.7  89.3  91.9  78.8  79.4  87.6  88.9  77.6    GL    84.3  86.4  89.1  75.5  76.1  85.3  86.2  81.2  P-value      Control vs. EGB  <0.001  <0.001  0.001  0.078  0.016  0.29  0.057  0.42  EGB  Linear  0.003  0.005  0.009  0.015  0.021  0.59  0.56  0.16      Quadratic  0.002  <0.001  0.003  0.044  0.048  0.22  0.024  0.26      Control vs. GL  0.21  0.001  <0.001  0.50  0.22  <0.001  0.15  <0.001  GL  Linear  0.49  0.007  0.54  0.26  0.31  0.64  0.86  <0.001      Quadratic  0.13  <0.001  <0.001  0.49  0.14  <0.001  0.023  <0.001  EGB vs. GL    <0.001  <0.001  <0.001  0.045  0.003  0.001  <0.001  0.032  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large DISCUSSION According to our results, supplementation of GL and EGB in broiler diets improved the nutrient and energy utilization of broilers. The results in this study indicate that addition of GL and EGB has a positive effect on broilers. GL and EGB contain approximately 30 kinds of flavonoids and their derivatives and terpenoids, such as ginkgolide A, ginkgolide B, ginkgolide C, and bilobalide (Kleijnen and Knipschild, 1992, Liu et al., 2006; Zhang et al., 2012). The enhanced nutrient and energy utilization of broilers in GL- or EGB-supplemented groups observed in this study may be partially attributed to the effects of flavonoid contents. It has been reported that flavonoids are more easily and rapidly absorbed in the intestines (Izumi et al., 2000). Further detailed study is required on the flavonoid mode of action. Diets supplemented with 100 g/kg of fermented GL residues had the greatest beneficial effects on growth performance, nutrient digestibility, and immune function in weaned piglets (Zhou et al., 2015). The growth performance, feed efficiency, and intestinal morphology were significantly improved in groups supplemented with Aspergillus or Bac licheniformis-fermented GL (Zhang et al., 2012; Yu et al., 2015). The total flavonoids and terpenoids of A. niger-fermented-G. biloba leaves are most likely the key compounds responsible for the influences on the small intestinal morphology and immune responses without adverse effects in broiler chickens (Zhang et al., 2013). These improve growth performance and intestinal morphology of broilers in GL- or EGB-supplemented treatments and might result from the change of bacterial community structure in the intestines or the growth of intestinal villus (Zhou et al., 2006; Petrolli et al., 2012). The height of intestinal villi and deeper crypts reflect the surface area for nutrient absorption (Xu et al., 2003; Hu et al., 2011). All of these factors may have favorable effects on broiler health, which may partially contribute to the affected nutrient and energy utilization of broilers in this study. GL or EGB fed to broilers improved intestinal health and the utilization rate of nutrients. Furthermore, the findings were that dietary supplementation of GL and EGB increased the utilization of nutrient and energy in broilers in a dose-dependent manner. Zhang et al. (2012) reported that supplementation of 3.5 to 5 g/kg and 7 to 10 g/kg Aspergillus niger-fermented GL in the starter and grower diets, respectively, improved feed efficiency, intestinal morphology, digestion, and absorption function of broilers. In the current study, the use of 5 g/kg (in the starter phase) and 10 g/kg (in the grower phase) of fermented GL products had a positive influence on growth performance, lipid metabolism, and antioxidant capacity (Cao et al., 2012). Supplementation of 0.25 to 1.0 g/kg EGB evidently elevated the activities of SOD and GSH-Px, and at the same time, reduced the content of MDA, which indicated that the effects were exerted in a dose-dependent manner (Liu et al., 2006). The results of this study showed that supplementation of 20 to 60 g/kg GL or 0.4 to 0.8 g/kg EGB in diets increased nutrient and energy utilization in a dose-dependent manner. However, inclusion levels of EGB up to 1.2 g/kg reduced the nutrients and energy utilization and that of some essential and non-essential AA (Chamorro et al., 2012). This is likely due to different concentrations of the active substances and the toxic effect of the high level of dietary EGB. Ginkgolic acid has been recognized as a hazardous compound with suspected cytotoxic and allergenic properties, which may have adverse effects on the nutrients and energy utilization of the high level of dietary EGB in poultry (Yu et al., 2015). The efficacy of GL and its extracts also could be affected by other factors, such as the diet type, animal age, hygiene, environmental factors, and so on (Amad et al., 2011). In the present study, the addition of 60 g/kg GL in broiler diets is the optimum concentration compared to those of GL-supplemented treatments, and this may be mostly due to the smaller concentration gradient; further study is required to continue to increase the concentration of GL in diets and evaluate effects on nutrient and energy utilization. Supplementation of GL and EGB in broiler diets increased the utilization of AA compared with that of control treatments. Ward et al. (2002) reported that supplementation of fermented GL increased the growth performance, intestinal morphology, digestion, and absorption function, which have been ascribed to the increased contents of flavones, CP, and AA, decreases in ginkgolic acid, and the synergistic reactions among various pharmacologically active constituents found in the leaf. In the present study, adding EGB in broiler diets improved the utilization of AA, except Thr and Cys, whereas the utilization of Leu, His, Met, Ser, Glu, and Cys were increased in the GL-supplemented treatments, but other AA were not. This may imply that EGB potentiates the function of digestion and absorption by purifying the active constituent concentrations. However, relatively high dietary concentrations of flavones obtained with supplementation of these ingredients reduced the performance of chickens (Chamorro et al., 2012). An interesting finding of this study was that the isolation of the effective compounds affected efficacy of GL in increasing the utilization of nutrients in broilers. It appeared that under the same concentrations of the active substances in GL and EGB, supplementation of EGB had a better effect than that in GL-supplemented treatments. The constituent of GL contains simple ginkgolic acids, which are harmful to animals. After an ethanol extraction process, some compounds are enriched (flavonoids, terpene lactones), while others (ginkgolic acids) are removed (Teris et al., 2009). This may imply that the components in the EGB were more digestible than those present in GL and better able to exert their biological effects. Therefore, the isolation of GL is responsible for the effect of EGB, but the leaves have inferior effect, which means the isolation of effective compounds is necessary. In fact, EGB has a high price but little addition in diets and had a better effect on utilization of nutrient and energy compared with GL-supplemented diets. The results of this study indicate that supplementation with GL or EGB increased the utilization of nutrient and energy of broiler chickens. CONCLUSIONS Supplementation of 20 to 60 g/kg GL and 0.4 to 0.8 g/kg EGB of diet, respectively, increased nutrient and energy utilization of broilers. This study showed that optimum concentrations of GL and EGB in the diets of broiler chickens were 60 g/kg or 0.8 g/kg of diet, respectively, for enhancing the nutrient and energy utilization, and further research is needed on the effects of other livestock industries. The results of this study indicate that GL and EGB could be used as feed additives for improving the nutrient and energy utilization of broilers. 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Effects of Ginkgo biloba leaves (Ginkgo biloba) and Ginkgo biloba extract on nutrient and energy utilization of broilers

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© 2018 Poultry Science Association Inc.
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0032-5791
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Abstract

Abstract An experiment using 112 21-day-old male Arbor Acres broilers that were randomly allocated to 7 treatments with 8 replicates in a completely randomized design was conducted to assess the effects of Ginkgo biloba leaves (Ginkgo biloba, GL) and Ginkgo biloba extract (EGB) on utilization of nutrients of broiler chickens. The dietary treatments were corn-soybean meal based diets: 1) T1, control diet; 2) T2, T1 + 20 g/kg GL; 3) T3, T1 + 40 g/kg GL; 4) T4, T1 + 60 g/kg GL; 5) T5, T1 + 0.4 g/kg EGB; 6) T6, T1 + 0.8 g/kg EGB; and 7) T7, T1 + 1.2 g/kg EGB. Endogenous losses were obtained from another 16 broilers. Excreta samples were collected to analyze the dry matter (DM), organic matter (OM), ether extract (EE), crude protein (CP), gross energy (GE), and amino acids (AA), and GE was analyzed for computation of AME and TME. As compared with those of the control treatment, the apparent digestibility (AD) and true digestibility (TD) of EE, CP, Thr, Val, Ile, Leu, Phe, Lys, His, and Arg were quadratically (P < 0.05) increased; moreover, the AD and TD of Met was linearly (P < 0.05) increased as the concentration of the EGB in the diet increased. Increasing GL from 0 to 60 g/kg of diet linearly (P < 0.05) increased the AD and TD of EE, Thr, Val, Leu, His, and Met and tended (0.05 < P < 0.1) to increase the TME, TMEn, and Arg. Supplementation of EGB increased (P < 0.05) AD and TD of EE, Thr, Val, Ile, Leu, Phe, His, and Arg and tended (0.05 < P < 0.1) to increase Lys as compared with those in the GL-supplemented groups. Dietary supplementation of GL and EGB improved the utilization of nutrients of broiler chickens in a dose-dependent manner, and the optimum supplementation levels of GL or EGB in the diet of broilers was 60 or 0.8 g/kg of diet, respectively. INTRODUCTION After banning of antibiotic growth promoters, alternative sources of feed additives are the continuous research interest of the animal nutritionist (Salim et al., 2013; Kim et al., 2016). Efficacious, environmentally friendly, and consumer favorable feed additives are necessary to animal productions (Wan et al., 2017). Recently, herbs or plant extracts have been incorporated into the diet to improve animal productivity by enhancing the production performance and the quality of food derived from those animals (Yan et al., 2011). Plant-derived feed additives, which contain natural active compounds, can enhance growth performance and improve intestinal health and function common to poultry (Aziza et al., 2010). Moreover, several studies pertaining to dietary plant origin products used in chicken diets have been published previously because of natural, non-toxic substances with multiple biological activities (Fei et al., 2008). Ginkgo biloba leaves (Ginkgoaceae, Ginkgo biloba, GL) are from an ancient Chinese tree that has been cultivated and held sacred for its health-promoting properties. There is substantial experimental evidence to support the view that Ginkgo biloba extract (EGB), which is the leaf extract of Ginkgo biloba, has many pharmacological effects (Kazumasa et al., 2002; Chen et al., 2011; Zhang et al., 2013). In recent years, GL have attracted increasing attention as a functional food ingredient because they contain numerous bioactive constituents, such as flavonoids, bilobalides, and polyphenols (Chen et al., 2003; Yao et al., 2004; Niu et al., 2016). Most of the studies indicated that supplementation of GL products had favorable influences on feed efficiency, growth performance, intestinal morphology and absorption functions, and immune responses without adverse effects in broiler chickens (Zhang et al., 2012; Yu et al., 2015; Zhang et al., 2015). And in laying hens, diets supplemented with Ginkgo leaves fermented with Aspergillus niger increased egg production, and improved the lipid metabolism (Zhao et al., 2013). Moreover, EGB, which contains 24% flavonoid glycosides, 6% terpene lactones, and less than 5 ppm ginkgolic acid (Mahadevan and Park, 2008, Mohammad and Anvari, 2015), has various biological activities and different pharmacological effects, including antioxidation (Liu et al., 2006; Zhao et al., 2011), anti-inflammation (Zeybek et al., 2003; Zhou et al., 2006), and modulation of immune response (Zhao et al., 2011). Despite these findings, there has been little research on the use of GL and EGB as feed additives for nutrient and energy utilization of broilers fed a corn-soybean diet. Information is also lacking on isolating the active ingredients in GL and then using them as growth promoters in broilers. The objective of this research was to assess the optimum dose of GL and EGB feed in broiler diets and to evaluate the effects of supplementation of GL and EGB on utilization of nutrients of broiler chickens. MATERIALS AND METHODS Preparation of Ginkgo Biloba Leaves and Ginkgo Biloba Extract GL were provided by Rui Kangsheng Biology Co., Ltd. (Linyi, China) and dried at 65°C in a mechanical drier (20226A, Shanghai Yangguang Laboratory Instruments Co. Ltd., Shanghai, China), The dried GL were subsequently milled with a disc mill (FFC-15, Shandong Changlin Machinery Group Co. Ltd, Linshu, Shandong, China) to pass a screen size of 0.25 mm and stored in the dark in airtight plastic bags at ambient temperature (21 to 27°C). The GL used in this study contained 90, 15, 5.48, and 4.9% of dry matter (DM), crude protein (CP), ether extract (EE), and crude ash (CA), respectively, and 1.83 Mcal/kg gross energy (GE), which were analyzed in our laboratory according to AOAC (1999). EGB also was obtained from Rui Kangsheng Biology Co., Ltd. (Linyi, China); the extract was obtained from the GL by ethanol as an extracting solvent, and EGB was stored in the dark in airtight plastic bags at ambient temperature (21 to 27°C). The components of flavonoids and terpene lactones were analyzed using HPLC and have been identified as the main bioactive constituents in GL and EGB (Ahlemeyer and Krieglstein, 2003). High-performance liquid chromatography The chemical compositions of the GL and EGB were carried out by HPLC (Waters alliance 2695, Milford, MA, USA) analysis. The content of flavonol glycosides was determined by using the HPLC-UV method, and the mixture of 50% methanol and 50% phosphoric acids solution of 0.5% concentration (V/V) as the mobile phase. The flow rate was 1.0 mL/min. Detection was performed at a wavelength of 360 nm with the column maintained at a constant temperature (30°C). The content of terpene lactones was developed using HPLC-ELSD means. The mobile phase consisted of methanol and water (30:70, V/V), and the flow rate was 1.0 mL/minute. The drift tube temperature was 105°C, the flow rate of the carrier gas was 2.9 mL/min, and the column (250 mm × 4.6 mm, 5.0 μm) temperature was 25°C. Quercetin, Kaempferol, Isorhamnetin, Ginkgolide A, Ginkgolide B, Ginkgolide C, and Bilobalide were used as external standards, and the retention times and concentrations of each ingredient in the EGB were determined relative to the standards. The content of GL and EGB contained 0.54 and 30.96% total flavonoid glycosides, and 0.18 and 8.91% terpene lactones, respectively. Animals, experiment design, and management A total of 112 21-day-old male Arbor Acres broiler chicks with similar body weight were purchased from a commercial hatchery (Xiling Family Farm, Tai’an, China), individually weighed, and then randomly distributed into 7 dietary treatments with 8 replicates (2 chicks/each, according to the requirement of the experiment animals in Animal Nutrition, Mcdonald et al., 2010) in a completely randomized design. Each broiler was housed in individual wire cages equipped with water troughs and feeders. The dietary treatments were corn-soybean meal based diets, and the diets were made as followings: 1) T1, control diet; 2) T2, T1 + 20 g/kg GL; 3) T3, T1 + 40 g/kg GL; 4) T4, T1 + 60 g/kg GL; 5) T5, T1 + 0.4 g/kg EGB; 6) T6, T1 + 0.8 g/kg EGB; and 7) T7, T1 + 1.2 g/kg EGB. The experiment was designed to match the total flavonoid glycosides and terpene lactones in GL- and EGB-supplemented treatments (Table 1). The diets were made as wheat bran was replaced with GL and were formulated to meet or exceed nutrient requirements of Arbor Acres growing broilers (Feeding Standard of Chicken of the People's Republic of China; NY/T 33–2004). Diet compositions are shown in Table 1. GL and EGB were first mixed with the premix and then mixed with other ingredients and stored in covered containers before feeding. Moreover, the endogenous contribution was calculated from excreta collected from 16 broilers that underwent the same procedure without being fed. All broilers were continuously provided with uniform light for 24 h, and room temperature was maintained at 22°C. Body weight was recorded by cage at the end of the adaptation period (28 d) and the test period (31 d). The animal care and use protocol was reviewed and approved by Shandong Agricultural University Animal Nutrition Research Institute (Tai’an, Shandong, China). Table 1. Ingredients and nutrient composition of experimental diets.1     Dietary GL concentration (%)  Dietary EGB concentration (%)2  Item  control  2  4  6  0.04  0.08  0.12  Ingredients, %  Corn  57.3  57.3  57.3  57.3  57.3  57.3  57.3  Soybean meal (CP, 46%)  28.4  28.4  28.4  28.4  28.4  28.4  28.4  Soy oil  4.60  4.60  4.60  4.60  4.60  4.60  4.60  Wheat bran  6.00  4.00  2.00  0.00  6.00  6.00  6.00  GL  0.00  2.00  4.00  6.00  0.00  0.00  0.00  EGB  0.00  0.00  0.00  0.00  0.04  0.08  0.12  Dicalcium phosphate  1.20  1.20  1.20  1.20  1.20  1.20  1.20  Limestone powder  0.95  0.95  0.95  0.95  0.95  0.95  0.95  Sodium Chloride  0.21  0.21  0.21  0.21  0.21  0.21  0.21  L- lysine -HCl  0.15  0.15  0.15  0.15  0.15  0.15  0.15  DL-methionine  0.19  0.19  0.19  0.19  0.19  0.19  0.19  Premix3  1.00  1.00  1.00  1.00  1.00  1.00  1.00  Total  100  100  100  100  100  100  100  Analysis nutrient content  ME, kcal/kg  3140  3144  3149  3153  3140  3140  3140  Crude protein  19.33  19.35  19.36  19.37  19.33  19.33  19.33  Calcium  0.86  0.86  0.86  0.86  0.86  0.86  0.86  Available phosphorus  0.35  0.35  0.36  0.37  0.35  0.35  0.35  Flavonoid glycosides  0.00  1.18  2.39  3.60  1.20  2.40  3.60  Terpene lactones  0.00  0.36  0.72  1.08  0.36  0.71  1.07  Sodium Chloride  0.30  0.30  0.30  0.30  0.30  0.30  0.30  Lysine  1.07  1.08  1.08  1.09  1.07  1.07  1.07  Methionine  0.48  0.48  0.49  0.50  0.48  0.48  0.48      Dietary GL concentration (%)  Dietary EGB concentration (%)2  Item  control  2  4  6  0.04  0.08  0.12  Ingredients, %  Corn  57.3  57.3  57.3  57.3  57.3  57.3  57.3  Soybean meal (CP, 46%)  28.4  28.4  28.4  28.4  28.4  28.4  28.4  Soy oil  4.60  4.60  4.60  4.60  4.60  4.60  4.60  Wheat bran  6.00  4.00  2.00  0.00  6.00  6.00  6.00  GL  0.00  2.00  4.00  6.00  0.00  0.00  0.00  EGB  0.00  0.00  0.00  0.00  0.04  0.08  0.12  Dicalcium phosphate  1.20  1.20  1.20  1.20  1.20  1.20  1.20  Limestone powder  0.95  0.95  0.95  0.95  0.95  0.95  0.95  Sodium Chloride  0.21  0.21  0.21  0.21  0.21  0.21  0.21  L- lysine -HCl  0.15  0.15  0.15  0.15  0.15  0.15  0.15  DL-methionine  0.19  0.19  0.19  0.19  0.19  0.19  0.19  Premix3  1.00  1.00  1.00  1.00  1.00  1.00  1.00  Total  100  100  100  100  100  100  100  Analysis nutrient content  ME, kcal/kg  3140  3144  3149  3153  3140  3140  3140  Crude protein  19.33  19.35  19.36  19.37  19.33  19.33  19.33  Calcium  0.86  0.86  0.86  0.86  0.86  0.86  0.86  Available phosphorus  0.35  0.35  0.36  0.37  0.35  0.35  0.35  Flavonoid glycosides  0.00  1.18  2.39  3.60  1.20  2.40  3.60  Terpene lactones  0.00  0.36  0.72  1.08  0.36  0.71  1.07  Sodium Chloride  0.30  0.30  0.30  0.30  0.30  0.30  0.30  Lysine  1.07  1.08  1.08  1.09  1.07  1.07  1.07  Methionine  0.48  0.48  0.49  0.50  0.48  0.48  0.48  1The control group was fed the basal diet. The other treatment diets were the same basal diet supplemented with 0.4 g/kg, 0.8 g/kg, 1.2 g/kg EGB, respectively, and for GL treatments, supplemented with 20 g/kg, 40 g/kg, 60 g/kg GL of diet by replacing the equivalent amount of wheat bran in the basal diet formulation. 2GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. 3Supplied per kilogram of diet: vitamin A, 8,050 IU; cholecalciferol, 3,000 IU; vitamin E, 30 mg; vitamin K3, 5 mg; thiamin, 2.58 mg; riboflavin, 12.5 mg; pyridoxine, 4.5 mg; vitamin B12, 0.018 mg; choline chloride, 800 mg; calcium pantothenate, 13 mg; niacin, 45 mg; biotin, 0.20 mg; folic acid, 1.20 mg; Mn, 100 mg; Fe, 80 mg; Zn, 58 mg; Cu, 8.8 mg; I, 0.374 mg; Se, 0.28 mg. View Large Sampling The broilers were distributed randomly and enclosed in individual metabolic cages labeled with the respective dietary treatments, which allowed total collection of excreta from each individual separately. Each dietary treatment was provided with the same corresponding experimental diets (T1, T2, T3, T4, T5, T6, or T7). The endogenous treatment was fed the control diet. The entire experiment contained the adaptation (7 d: 21 d to 27 d of age) period and the test period (3 d: 28 d to 30 d of age). During the adaptation period, animals were gradually adapted to the experimental diet. After the adaptation period, the test diets of T1 to T7 were given for 3 d and the excreta were collected at the same time. In addition, the broilers in the endogenous treatment were fed only water for 48 h after the initial 24-hour fasting period to determine the endogenous nutrient production. Feathers and shredded dry skin were removed carefully before excreta were analyzed. Broilers were fed ad libitum (expect the endogenous treatment) and had free access to water throughout the whole experiment. Collection and analyses of samples A sample of feed was prepared for determination of DM intake and subsequent chemical analysis during the collection period. Excreta were collected daily for 72 h, with the endogenous treatment for 48 h, then pooled, weighed, and mixed for each replication of 2 broilers, and stored at −20°C until all samples were analyzed. All chemical analyses were performed in duplicate. The diets and the fresh excreta were analyzed for N according to the Kjeldahl N determination method to determine CP content (total N × 6.25 = CP). The feed and excreta samples were dried at 65°C and ground to pass through a 0.5 mm screen and then stored in wild-mouth bottles to analyze for DM, CA, EE, and GE based on the Association of Official Analytical Chemists procedures (AOAC, 1999). After freeze-drying, feed and excreta samples were analyzed for amino acid (AA, except tryptophan). The AME and TME values of the feedstuffs and excreta were calculated by the method described by Sibbald (1976). Organic matter (OM) was calculated by content of crude ash. All samples were analyzed for DM dried at 105°C for 48 h, and then burned in a muffle furnace (SX2 box-type resistance furnace, Yantai, China) for 3 h at 550°C, and the ashes were used to determine the content of crude ash. The GE was determined by adiabatic bomb calorimetry (WHR-15 Oxygen bomb calorimeter, Changsha, China). The AA concentrations were assayed with Hitachi 835 type high-speed automatic amino acid analyzer. Shandong Agricultural University (Shandong, China) provides the place to analyze the samples. Calculation and statistical analysis Apparent digestibility (AD) and true digestibility (TD) of DM, OM, EE, CP, GE, and AA were calculated using the following equations:   \begin{equation*}{\rm{AD}}\,\left( \% \right) = \left[ {\left( {{\rm{E}}1 - {\rm{E}}2} \right)/{\rm{E}}1} \right]{\rm{ }} \times 100 \end{equation*}   \begin{equation*} {\rm{TD}}\,\left( \% \right) = \left[ {\left( {{\rm{E}}1 - {\rm{E}}2 + {\rm{E}}3} \right)/{\rm{E}}1} \right]{\rm{ }} \times 100\end{equation*} Where: E1 is the total amount (mg) of the nutrient (DM, OM, EE, or CP) and GE (MJ) in the diet that was fed to each bird; E2 is the total amount (mg) of the corresponding nutrient (DM, OM, EE, or CP) and GE (MJ) in excreta collected during the 3-day period; E3 is the total amount (mg) of the corresponding endogenous nutrient (DM, OM, EE, or CP) and GE (MJ) in excreta collected for a 48-hour period without being fed. AME and AME with that of endogenous nitrogen (N) losses (AMEn), and TME and TME with that of endogenous N losses (TMEn) (DM basis) were calculated using the following equations:   \begin{equation*} {\rm{AME}}\,\left( {{\rm{MJ}}/{\rm{kg}}} \right) = \left( {{\rm{E}}4 - {\rm{E}}5} \right)/{\rm{E}}7 \end{equation*}   \begin{equation*} {\rm{TME}}\,\left( {{\rm{MJ}}/{\rm{kg}}} \right) = \left( {{\rm{E}}4-{\rm{E}}5 + {\rm{E}}6} \right)/{\rm{E}}7 \end{equation*}   \begin{equation*} {\rm{AMEn}} = {\rm{AME}} - {\rm{RN}} \times 34.39 \end{equation*}   \begin{equation*} {\rm{TMEn}} = {\rm{TME}} - {\rm{RN}} \times 34.39 \end{equation*} Where: E4 is the total amount (MJ) of energy in the diet that was fed to each bird; E5 is the total amount (MJ) of energy in excreta collected during the 3-day period; E6 is the total amount (MJ) of energy in excreta collected for a 48-hour period without being fed; E7 is the feed intake of the diet that was fed to each broiler (DM basis). RN is the apparent N retention calculated as the difference between N intake and N output. All statistical analyses were performed using the GLM procedure of SAS (SAS Institute, 2000). The data were analyzed as a completely randomized design with individual unit as the random factor to examine the overall effect of treatments. Orthogonal polynomial contrasts were used to determine linear and quadratic responses to the increasing levels of GL and EGB in diets. Statements of significance were P < 0.05, and statistical tendencies were indicated if the P-value was between 0.05 and 0.10. RESULTS Body Weight All broilers appeared healthy, and no mortality occurred throughout the entire experimental period (data not shown); body weight was not significantly affected by the addition of GL and EGB in diets (Table 2). Table 2. Effects of GL and EGB on body weight (g/bird) of broilers fed diets.1 Item  GL  EGB  21 d  28 d  31 d  T1  0  0  845.8  1392.5  1652.9  T2  20 g/kg    846.3  1393.8  1653.2  T3  40 g/kg    846.7  1396.4  1654.9  T4  60 g/kg    848.9  1397.2  1655.6  T5    0.4 g/kg  846.3  1394.5  1652.6  T6    0.8 g/kg  848.3  1396.4  1656.7  T7    1.2 g/kg  847.8  1392.5  1656.8  SEM      2.510  1.990  2.496  P      0.99  0.89  0.97  Item  GL  EGB  21 d  28 d  31 d  T1  0  0  845.8  1392.5  1652.9  T2  20 g/kg    846.3  1393.8  1653.2  T3  40 g/kg    846.7  1396.4  1654.9  T4  60 g/kg    848.9  1397.2  1655.6  T5    0.4 g/kg  846.3  1394.5  1652.6  T6    0.8 g/kg  848.3  1396.4  1656.7  T7    1.2 g/kg  847.8  1392.5  1656.8  SEM      2.510  1.990  2.496  P      0.99  0.89  0.97  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Apparent Digestibility and True Digestibility of Dry Matter, Organic Matter, Ether Extract, Crude Protein, and Gross Energy of Diets The effects of supplementation of GL and EGB on AD of DM, OM, EE, CP, and GE in broilers fed a corn-soybean diet are shown in Table 3. The AD of DM, OM, and GE in all treatments had common digestibility. However, as the levels of EGB increased from 0 to 1.2 g/kg of diet, the AD of EE and CP was quadratically (P < 0.05) increased compared with that of control treatments. As compared with the control treatments, the AD of EE was linearly (P < 0.05) and quadratically (P < 0.05) increased as the levels of GL increased from 0 to 60 g/kg of diet. Addition of EGB in the broiler diets increased (P < 0.05) AD of EE compared with that in the GL-supplemented groups. Results for TD follow the same pattern as the results for AD (Table 4). Table 3. Effects of GL and EGB on AD of DM, OM, EE, CP, and GE of broilers fed diets.1 Item  GL  EGB  DM, %  OM, %  EE, %  CP, %  GE, %  T1  0  0  74.2  77.2  62.4  74.0  75.4  T2  20 g/kg    74.9  77.8  62.8  74.2  75.6  T3  40 g/kg    74.9  77.8  70.1  74.4  75.7  T4  60 g/kg    74.9  77.9  70.9  74.5  75.5  T5    0.4 g/kg  74.8  77.4  70.9  76.2  75.3  T6    0.8 g/kg  74.8  77.7  71.4  76.4  75.5  T7    1.2 g/kg  74.0  75.9  71.0  70.7  75.5    SEM    0.680  0.826  1.05  0.966  0.793  Control    0  74.2  77.2  62.4  74.0  75.4  With EGB    Pooled  74.5  77.0  71.1  74.4  75.5  With GL    Pooled  74.9  77.8  67.9  74.4  75.6  P-value      Control vs. EGB  0.51  0.18  <0.001  0.005  0.98  EGB  Linear  0.91  0.30  <0.001  0.039  0.66      Quadratic  0.31  0.11  <0.001  0.002  0.90      Control vs. GL  0.84  0.96  <0.001  0.99  0.99  GL  Linear  0.46  0.67  <0.001  0.71  0.94      Quadratic  0.66  0.86  <0.001  0.93  0.95  EGB vs. GL    0.48  0.25  0.007  0.96  0.84  Item  GL  EGB  DM, %  OM, %  EE, %  CP, %  GE, %  T1  0  0  74.2  77.2  62.4  74.0  75.4  T2  20 g/kg    74.9  77.8  62.8  74.2  75.6  T3  40 g/kg    74.9  77.8  70.1  74.4  75.7  T4  60 g/kg    74.9  77.9  70.9  74.5  75.5  T5    0.4 g/kg  74.8  77.4  70.9  76.2  75.3  T6    0.8 g/kg  74.8  77.7  71.4  76.4  75.5  T7    1.2 g/kg  74.0  75.9  71.0  70.7  75.5    SEM    0.680  0.826  1.05  0.966  0.793  Control    0  74.2  77.2  62.4  74.0  75.4  With EGB    Pooled  74.5  77.0  71.1  74.4  75.5  With GL    Pooled  74.9  77.8  67.9  74.4  75.6  P-value      Control vs. EGB  0.51  0.18  <0.001  0.005  0.98  EGB  Linear  0.91  0.30  <0.001  0.039  0.66      Quadratic  0.31  0.11  <0.001  0.002  0.90      Control vs. GL  0.84  0.96  <0.001  0.99  0.99  GL  Linear  0.46  0.67  <0.001  0.71  0.94      Quadratic  0.66  0.86  <0.001  0.93  0.95  EGB vs. GL    0.48  0.25  0.007  0.96  0.84  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Table 4. Effects of GL and EGB on TD of DM, OM, EE, CP, and GE of broilers fed diets.1 Item  GL  EGB  DM, %  OM, %  EE, %  CP, %  GE, %  T1  0  0  78.0  77.9  63.0  76.8  78.6  T2  20 g/kg    78.6  77.9  63.4  76.9  78.7  T3  40 g/kg    78.6  78.0  70.7  77.1  78.5  T4  60 g/kg    78.7  78.2  71.5  77.0  78.9  T5    0.4 g/kg  78.7  78.9  71.5  79.3  78.5  T6    0.8 g/kg  78.8  78.5  72.0  79.6  78.7  T7    1.2 g/kg  77.9  79.1  71.6  76.0  78.5    SEM    0.499  0.864  0.793  0.681  0.681  Control    0  78.0  77.9  63.0  76.8  78.6  With EGB    Pooled  78.5  78.8  71.7  78.3  78.6  With GL    Pooled  78.6  78.0  65.5  77.0  78.7  P-value      Control vs. EGB  0.53  0.70  <0.001  0.003  0.99  EGB  Linear  0.96  0.35  <0.001  0.63  0.99      Quadratic  0.33  0.61  <0.001  0.001  0.98      Control vs. GL  0.69  0.99  <0.001  0.99  0.99  GL  Linear  0.31  0.82  <0.001  0.77  0.83      Quadratic  0.49  0.97  <0.001  0.94  0.96  EGB vs. GL    0.78  0.20  0.002  0.044  0.88  Item  GL  EGB  DM, %  OM, %  EE, %  CP, %  GE, %  T1  0  0  78.0  77.9  63.0  76.8  78.6  T2  20 g/kg    78.6  77.9  63.4  76.9  78.7  T3  40 g/kg    78.6  78.0  70.7  77.1  78.5  T4  60 g/kg    78.7  78.2  71.5  77.0  78.9  T5    0.4 g/kg  78.7  78.9  71.5  79.3  78.5  T6    0.8 g/kg  78.8  78.5  72.0  79.6  78.7  T7    1.2 g/kg  77.9  79.1  71.6  76.0  78.5    SEM    0.499  0.864  0.793  0.681  0.681  Control    0  78.0  77.9  63.0  76.8  78.6  With EGB    Pooled  78.5  78.8  71.7  78.3  78.6  With GL    Pooled  78.6  78.0  65.5  77.0  78.7  P-value      Control vs. EGB  0.53  0.70  <0.001  0.003  0.99  EGB  Linear  0.96  0.35  <0.001  0.63  0.99      Quadratic  0.33  0.61  <0.001  0.001  0.98      Control vs. GL  0.69  0.99  <0.001  0.99  0.99  GL  Linear  0.31  0.82  <0.001  0.77  0.83      Quadratic  0.49  0.97  <0.001  0.94  0.96  EGB vs. GL    0.78  0.20  0.002  0.044  0.88  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large AME, AMEn, TME, and TMEn of Diets As shown in Table 5, there were no significant effects of GL or EGB supplementation in the diets on the AME, AMEn, TME, and TMEn, but compared with those of the control treatments, TME and TMEn in GL-supplemented treatments had a quadratically increasing (0.05 < P < 0.1) tendency. Table 5. Effects of GL and EGB on AME, AMEn, TME, and TMEn of broilers fed diets.1 Item  GL  EGB  AME MJ/kg.DM  AMEn MJ/kg.DM  TME MJ/kg.DM  TMEn MJ/kg.DM  T1  0  0  13.2  10.9  13.9  11.0  T2  20 g/kg    13.3  11.1  13.9  11.2  T3  40 g/kg    13.4  11.1  13.8  11.1  T4  60 g/kg    13.4  11.2  13.8  11.3  T5    0.4 g/kg  13.2  11.0  13.7  11.2  T6    0.8 g/kg  13.3  11.1  13.7  11.3  T7    1.2 g/kg  13.4  11.2  13.8  11.2    SEM    0.175  0.190  0.149  0.191  Pooled  Control  0  13.2  10.9  13.9  11.0  EGB  Pooled  13.3  11.1  13.7  11.2  GL  Pooled  13.3  11.1  13.8  11.2  P-value      Control vs. EGB  0.86  0.83  0.92  0.88  EGB  Linear  0.38  0.35  0.82  0.46      Quadratic  0.68  0.64  0.83  0.72      Control vs. GL  0.82  0.83  0.99  0.82  GL  Linear  0.34  0.36  0.80  0.38      Quadratic  0.63  0.64  0.061  0.058  EGB vs. GL    0.82  0.96  0.34  0.48  Item  GL  EGB  AME MJ/kg.DM  AMEn MJ/kg.DM  TME MJ/kg.DM  TMEn MJ/kg.DM  T1  0  0  13.2  10.9  13.9  11.0  T2  20 g/kg    13.3  11.1  13.9  11.2  T3  40 g/kg    13.4  11.1  13.8  11.1  T4  60 g/kg    13.4  11.2  13.8  11.3  T5    0.4 g/kg  13.2  11.0  13.7  11.2  T6    0.8 g/kg  13.3  11.1  13.7  11.3  T7    1.2 g/kg  13.4  11.2  13.8  11.2    SEM    0.175  0.190  0.149  0.191  Pooled  Control  0  13.2  10.9  13.9  11.0  EGB  Pooled  13.3  11.1  13.7  11.2  GL  Pooled  13.3  11.1  13.8  11.2  P-value      Control vs. EGB  0.86  0.83  0.92  0.88  EGB  Linear  0.38  0.35  0.82  0.46      Quadratic  0.68  0.64  0.83  0.72      Control vs. GL  0.82  0.83  0.99  0.82  GL  Linear  0.34  0.36  0.80  0.38      Quadratic  0.63  0.64  0.061  0.058  EGB vs. GL    0.82  0.96  0.34  0.48  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Apparent Digestibility and True Digestibility of Essential Amino Acid The effects of supplementation of GL and EGB on AD and TD of essential AA in broilers fed diets are shown in Tables 6 and 7. The AD and TD of Thr, Val, Ile, Leu, Phe, Lys, His, and Arg were quadratically (P < 0.05) increased, the utilization of was linearly (P < 0.05) increased as the concentration of EGB in the diet increased compared with those of the control treatment. Addition of GL quadratically (P < 0.05) increased the AD and TD of Thr, Val, Leu, His, and Met and tended (0.05 < P < 0.1) to increase Arg as the concentration of GL in the diet increased compared with those of the control treatment. Supplementation of EGB in the broiler diets increased (P < 0.05) the AD and TD of Thr, Val, Ile, Leu, Phe, His, and Arg and tended (0.05 < P < 0.1) to increase Lys compared with those in the GL-supplemented groups. Table 6. Effects of GL and EGB on apparent digestibility of essential amino acids of broilers fed diets.1 Item  GL  EGB  Thr, %  Val, %  Ile, %  Leu, %  Phe, %  Lys, %  His, %  Arg, %  Met, %  T1  0  0  82.2  80.2  82.5  87.4  86.3  85.6  88.9  92.6  84.5  T2  20 g/kg    80.1  77.3  81.2  85.8  86.0  85.9  86.0  91.1  85.5  T3  40 g/kg    81.5  78.5  81.6  85.9  85.7  86.4  87.0  91.6  93.6  T4  60 g/kg    82.7  80.3  82.7  87.6  86.1  87.4  88.6  91.9  87.8  T5    0.4 g/kg  83.7  81.1  84.2  88.9  88.4  87.5  91.2  93.5  89.2  T6    0.8 g/kg  87.7  84.8  87.5  90.7  89.5  90.0  92.2  94.9  84.8  T7    1.2 g/kg  85.0  82.8  84.2  89.2  86.8  86.0  89.9  93.6  89.8    SEM    0.788  0.955  0.817  0.563  0.638  0.690  0.532  0.378  1.010  Pooled    Control    80.6  77.7  81.4  86.1  86.1  86.1  86.3  91.3  87.2    EGB    85.5  82.9  85.3  89.6  88.2  87.8  91.1  94.0  88.0    GL    81.4  78.7  81.8  86.4  85.9  86.6  87.2  91.5  89.0  P-value      Control vs. EGB  <0.001  0.014  0.002  0.004  0.004  <0.001  <0.001  0.001  0.001  EGB  Linear  0.006  0.020  0.054  0.017  0.46  0.36  0.18  0.023  0.053      Quadratic  0.002  0.027  0.005  0.003  0.002  0.001  <0.001  0.002  0.16      Control vs. GL  0.071  0.076  0.43  0.021  0.92  0.21  0.002  0.061  <0.001  GL  Linear  0.41  0.75  0.73  0.85  0.69  0.037  0.97  0.37  0.015      Quadratic  0.052  0.045  0.25  0.007  0.80  0.102  0.001  0.051  0.005  EGB vs. GL    <0.001  <0.001  <0.001  <0.001  <0.001  0.065  <0.001  <0.001  0.35  Item  GL  EGB  Thr, %  Val, %  Ile, %  Leu, %  Phe, %  Lys, %  His, %  Arg, %  Met, %  T1  0  0  82.2  80.2  82.5  87.4  86.3  85.6  88.9  92.6  84.5  T2  20 g/kg    80.1  77.3  81.2  85.8  86.0  85.9  86.0  91.1  85.5  T3  40 g/kg    81.5  78.5  81.6  85.9  85.7  86.4  87.0  91.6  93.6  T4  60 g/kg    82.7  80.3  82.7  87.6  86.1  87.4  88.6  91.9  87.8  T5    0.4 g/kg  83.7  81.1  84.2  88.9  88.4  87.5  91.2  93.5  89.2  T6    0.8 g/kg  87.7  84.8  87.5  90.7  89.5  90.0  92.2  94.9  84.8  T7    1.2 g/kg  85.0  82.8  84.2  89.2  86.8  86.0  89.9  93.6  89.8    SEM    0.788  0.955  0.817  0.563  0.638  0.690  0.532  0.378  1.010  Pooled    Control    80.6  77.7  81.4  86.1  86.1  86.1  86.3  91.3  87.2    EGB    85.5  82.9  85.3  89.6  88.2  87.8  91.1  94.0  88.0    GL    81.4  78.7  81.8  86.4  85.9  86.6  87.2  91.5  89.0  P-value      Control vs. EGB  <0.001  0.014  0.002  0.004  0.004  <0.001  <0.001  0.001  0.001  EGB  Linear  0.006  0.020  0.054  0.017  0.46  0.36  0.18  0.023  0.053      Quadratic  0.002  0.027  0.005  0.003  0.002  0.001  <0.001  0.002  0.16      Control vs. GL  0.071  0.076  0.43  0.021  0.92  0.21  0.002  0.061  <0.001  GL  Linear  0.41  0.75  0.73  0.85  0.69  0.037  0.97  0.37  0.015      Quadratic  0.052  0.045  0.25  0.007  0.80  0.102  0.001  0.051  0.005  EGB vs. GL    <0.001  <0.001  <0.001  <0.001  <0.001  0.065  <0.001  <0.001  0.35  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Table 7. Effects of GL and EGB on true digestibility of essential amino acids of broilers fed diets.1 Item  GL  EGB  Thr, %  Val, %  Ile, %  Leu, %  Phe, %  Lys, %  His, %  Arg, %  Met, %  T1  0  0  82.7  80.5  82.7  87.7  86.4  86.0  89.0  92.6  84.6  T2  20 g/kg    80.5  77.6  81.4  86.1  86.3  86.2  86.3  91.3  85.6  T3  40 g/kg    81.9  78.9  81.8  86.1  86.0  86.8  87.3  91.8  93.7  T4  60 g/kg    83.2  80.6  82.8  87.8  86.6  87.8  89.1  92.7  88.0  T5    0.4 g/kg  84.2  81.4  84.4  89.1  88.7  87.8  91.4  93.7  89.3  T6    0.8 g/kg  88.1  85.1  87.7  90.9  89.8  90.3  92.4  95.1  85.6  T7    1.2 g/kg  85.5  83.1  84.5  89.5  87.1  86.5  90.2  93.8  89.1    SEM    0.787  0.954  0.819  0.563  0.629  0.689  0.525  0.377  0.976  Pooled    Control    82.7  80.5  82.7  87.7  86.4  86.0  89.0  92.6  84.6    EGB    85.9  83.2  85.5  89.8  88.5  88.2  91.3  94.2  88.0    GL    81.9  79.0  82.0  86.7  86.3  86.9  87.6  91.9  89.1  P-value      Control vs. EGB  <0.001  0.015  0.002  0.004  0.003  <0.001  <0.001  <0.001  0.004  EGB  Linear  0.006  0.020  0.051  0.016  0.34  0.32  0.13  0.009  0.076      Quadratic  0.002  0.027  0.005  0.004  0.001  0.002  <0.001  <0.001  0.19      Control vs. GL  0.065  0.073  0.50  0.020  0.91  0.21  <0.001  0.039  <0.001  GL  Linear  0.39  0.74  0.84  0.85  0.87  0.037  0.65  0.66  0.014      Quadratic  0.048  0.044  0.32  0.007  0.82  0.10  <0.001  0.020  0.005  EGB vs. GL    <0.001  <0.001  <0.001  <0.001  0.002  0.053  <0.001  <0.001  0.10  Item  GL  EGB  Thr, %  Val, %  Ile, %  Leu, %  Phe, %  Lys, %  His, %  Arg, %  Met, %  T1  0  0  82.7  80.5  82.7  87.7  86.4  86.0  89.0  92.6  84.6  T2  20 g/kg    80.5  77.6  81.4  86.1  86.3  86.2  86.3  91.3  85.6  T3  40 g/kg    81.9  78.9  81.8  86.1  86.0  86.8  87.3  91.8  93.7  T4  60 g/kg    83.2  80.6  82.8  87.8  86.6  87.8  89.1  92.7  88.0  T5    0.4 g/kg  84.2  81.4  84.4  89.1  88.7  87.8  91.4  93.7  89.3  T6    0.8 g/kg  88.1  85.1  87.7  90.9  89.8  90.3  92.4  95.1  85.6  T7    1.2 g/kg  85.5  83.1  84.5  89.5  87.1  86.5  90.2  93.8  89.1    SEM    0.787  0.954  0.819  0.563  0.629  0.689  0.525  0.377  0.976  Pooled    Control    82.7  80.5  82.7  87.7  86.4  86.0  89.0  92.6  84.6    EGB    85.9  83.2  85.5  89.8  88.5  88.2  91.3  94.2  88.0    GL    81.9  79.0  82.0  86.7  86.3  86.9  87.6  91.9  89.1  P-value      Control vs. EGB  <0.001  0.015  0.002  0.004  0.003  <0.001  <0.001  <0.001  0.004  EGB  Linear  0.006  0.020  0.051  0.016  0.34  0.32  0.13  0.009  0.076      Quadratic  0.002  0.027  0.005  0.004  0.001  0.002  <0.001  <0.001  0.19      Control vs. GL  0.065  0.073  0.50  0.020  0.91  0.21  <0.001  0.039  <0.001  GL  Linear  0.39  0.74  0.84  0.85  0.87  0.037  0.65  0.66  0.014      Quadratic  0.048  0.044  0.32  0.007  0.82  0.10  <0.001  0.020  0.005  EGB vs. GL    <0.001  <0.001  <0.001  <0.001  0.002  0.053  <0.001  <0.001  0.10  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Apparent Digestibility and True Digestibility of Non-Essential Amino Acid The effects of supplementation of GL and EGB on AD and TD of non-essential AA in broilers fed the diets are shown in Tables 8 and 9. The AD and TD of Asp, Ser, Glu, Gly, Ala, and Pro were quadratically (P < 0.05) increased as the concentration of EGB in the diet increased, compared with those of the control treatment. The AD and TD of Ser, Glu, Tyr, and Cys were quadratically (P < 0.05) increased as the concentration of GL in the diet increased compared with those in the control treatment. Supplementation of EGB in the broiler diets increased (P < 0.05) the AD and TD of Asp, Ser, Glu, Gly, Ala, Tyr, Pro, and Cys compared with those in the GL-supplemented groups. Table 8. Effects of GL and EGB on apparent digestibility of non-essential amino acids of broilers fed diets.1 Item  GL  EGB  Asp, %  Ser, %  Glu, %  Gly, %  Ala, %  Tyr, %  Pro, %  Cys, %  T1  0  0  84.5  85.7  90.0  74.0  75.6  86.0  86.8  73.7  T2  20 g/kg    83.1  84.4  87.9  73.7  75.0  84.4  85.2  76.6  T3  40 g/kg    83.9  86.0  88.8  75.7  74.4  83.8  85.6  83.3  T4  60 g/kg    84.9  87.6  90.1  75.7  77.5  86.3  86.9  83.7  T5    0.4 g/kg  85.5  87.9  90.8  76.2  75.9  87.1  88.9  76.4  T6    0.8 g/kg  89.2  90.6  92.8  79.7  81.8  88.3  89.4  76.6  T7    1.2 g/kg  87.2  88.3  91.4  79.2  79.1  86.4  87.6  77.6    SEM    0.700  0.609  0.438  1.363  1.309  0.747  0.623  2.009  Pooled    Control    83.4  84.9  88.2  74.1  75.3  84.6  85.4  77.6    EGB    87.3  88.9  91.7  78.4  79.0  87.3  88.6  76.8    GL    84.0  86.0  88.9  75.0  75.6  84.8  85.9  81.2  P-value      Control vs. EGB  <0.001  <0.001  0.001  0.040  0.015  0.22  0.021  0.71  EGB  Linear  0.003  0.005  0.010  0.008  0.023  0.51  0.33  0.27      Quadratic  0.003  <0.001  0.003  0.002  0.048  0.15  0.007  0.52      Control vs. GL  0.23  <0.001  0.001  0.39  0.22  0.013  0.11  <0.001  GL  Linear  0.51  0.008  0.57  0.13  0.33  0.95  0.79  <0.001      Quadratic  0.14  <0.001  <0.001  0.32  0.14  0.006  0.053  <0.001  EGB vs. GL    <0.001  <0.001  <0.001  0.007  0.008  <0.001  <0.001  0.020  Item  GL  EGB  Asp, %  Ser, %  Glu, %  Gly, %  Ala, %  Tyr, %  Pro, %  Cys, %  T1  0  0  84.5  85.7  90.0  74.0  75.6  86.0  86.8  73.7  T2  20 g/kg    83.1  84.4  87.9  73.7  75.0  84.4  85.2  76.6  T3  40 g/kg    83.9  86.0  88.8  75.7  74.4  83.8  85.6  83.3  T4  60 g/kg    84.9  87.6  90.1  75.7  77.5  86.3  86.9  83.7  T5    0.4 g/kg  85.5  87.9  90.8  76.2  75.9  87.1  88.9  76.4  T6    0.8 g/kg  89.2  90.6  92.8  79.7  81.8  88.3  89.4  76.6  T7    1.2 g/kg  87.2  88.3  91.4  79.2  79.1  86.4  87.6  77.6    SEM    0.700  0.609  0.438  1.363  1.309  0.747  0.623  2.009  Pooled    Control    83.4  84.9  88.2  74.1  75.3  84.6  85.4  77.6    EGB    87.3  88.9  91.7  78.4  79.0  87.3  88.6  76.8    GL    84.0  86.0  88.9  75.0  75.6  84.8  85.9  81.2  P-value      Control vs. EGB  <0.001  <0.001  0.001  0.040  0.015  0.22  0.021  0.71  EGB  Linear  0.003  0.005  0.010  0.008  0.023  0.51  0.33  0.27      Quadratic  0.003  <0.001  0.003  0.002  0.048  0.15  0.007  0.52      Control vs. GL  0.23  <0.001  0.001  0.39  0.22  0.013  0.11  <0.001  GL  Linear  0.51  0.008  0.57  0.13  0.33  0.95  0.79  <0.001      Quadratic  0.14  <0.001  <0.001  0.32  0.14  0.006  0.053  <0.001  EGB vs. GL    <0.001  <0.001  <0.001  0.007  0.008  <0.001  <0.001  0.020  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large Table 9. Effects of GL and EGB on true digestibility of non-essential amino acids of broilers fed diets.1 Item  GL  EGB  Asp, %  Ser, %  Glu, %  Gly, %  Ala, %  Tyr, %  Pro, %  Cys, %  T1  0  0  84.9  86.1  90.2  75.0  76.1  86.5  87.5  73.7  T2  20 g/kg    83.5  84.7  88.1  74.1  75.4  84.6  85.4  76.6  T3  40 g/kg    84.2  86.4  89.0  76.1  74.9  84.1  85.9  83.3  T4  60 g/kg    85.3  88.0  90.3  76.1  78.0  87.2  87.2  83.7  T5    0.4 g/kg  85.9  88.3  91.1  76.7  76.4  87.4  89.1  76.4  T6    0.8 g/kg  89.5  90.9  93.0  80.1  82.2  88.6  89.6  79.0  T7    1.2 g/kg  87.7  88.7  91.7  79.6  79.6  86.8  87.9  77.6    SEM    0.699  0.609  0.438  1.377  1.309  0.729  0.622  1.882  Pooled    Control    84.9  86.1  90.2  75.0  76.1  86.5  87.5  73.7    EGB    87.7  89.3  91.9  78.8  79.4  87.6  88.9  77.6    GL    84.3  86.4  89.1  75.5  76.1  85.3  86.2  81.2  P-value      Control vs. EGB  <0.001  <0.001  0.001  0.078  0.016  0.29  0.057  0.42  EGB  Linear  0.003  0.005  0.009  0.015  0.021  0.59  0.56  0.16      Quadratic  0.002  <0.001  0.003  0.044  0.048  0.22  0.024  0.26      Control vs. GL  0.21  0.001  <0.001  0.50  0.22  <0.001  0.15  <0.001  GL  Linear  0.49  0.007  0.54  0.26  0.31  0.64  0.86  <0.001      Quadratic  0.13  <0.001  <0.001  0.49  0.14  <0.001  0.023  <0.001  EGB vs. GL    <0.001  <0.001  <0.001  0.045  0.003  0.001  <0.001  0.032  Item  GL  EGB  Asp, %  Ser, %  Glu, %  Gly, %  Ala, %  Tyr, %  Pro, %  Cys, %  T1  0  0  84.9  86.1  90.2  75.0  76.1  86.5  87.5  73.7  T2  20 g/kg    83.5  84.7  88.1  74.1  75.4  84.6  85.4  76.6  T3  40 g/kg    84.2  86.4  89.0  76.1  74.9  84.1  85.9  83.3  T4  60 g/kg    85.3  88.0  90.3  76.1  78.0  87.2  87.2  83.7  T5    0.4 g/kg  85.9  88.3  91.1  76.7  76.4  87.4  89.1  76.4  T6    0.8 g/kg  89.5  90.9  93.0  80.1  82.2  88.6  89.6  79.0  T7    1.2 g/kg  87.7  88.7  91.7  79.6  79.6  86.8  87.9  77.6    SEM    0.699  0.609  0.438  1.377  1.309  0.729  0.622  1.882  Pooled    Control    84.9  86.1  90.2  75.0  76.1  86.5  87.5  73.7    EGB    87.7  89.3  91.9  78.8  79.4  87.6  88.9  77.6    GL    84.3  86.4  89.1  75.5  76.1  85.3  86.2  81.2  P-value      Control vs. EGB  <0.001  <0.001  0.001  0.078  0.016  0.29  0.057  0.42  EGB  Linear  0.003  0.005  0.009  0.015  0.021  0.59  0.56  0.16      Quadratic  0.002  <0.001  0.003  0.044  0.048  0.22  0.024  0.26      Control vs. GL  0.21  0.001  <0.001  0.50  0.22  <0.001  0.15  <0.001  GL  Linear  0.49  0.007  0.54  0.26  0.31  0.64  0.86  <0.001      Quadratic  0.13  <0.001  <0.001  0.49  0.14  <0.001  0.023  <0.001  EGB vs. GL    <0.001  <0.001  <0.001  0.045  0.003  0.001  <0.001  0.032  Notes: Data are means for 8 replicates of 2 chicks per pen. 1GL = Ginkgo biloba leaves; EGB = Ginkgo biloba extract. View Large DISCUSSION According to our results, supplementation of GL and EGB in broiler diets improved the nutrient and energy utilization of broilers. The results in this study indicate that addition of GL and EGB has a positive effect on broilers. GL and EGB contain approximately 30 kinds of flavonoids and their derivatives and terpenoids, such as ginkgolide A, ginkgolide B, ginkgolide C, and bilobalide (Kleijnen and Knipschild, 1992, Liu et al., 2006; Zhang et al., 2012). The enhanced nutrient and energy utilization of broilers in GL- or EGB-supplemented groups observed in this study may be partially attributed to the effects of flavonoid contents. It has been reported that flavonoids are more easily and rapidly absorbed in the intestines (Izumi et al., 2000). Further detailed study is required on the flavonoid mode of action. Diets supplemented with 100 g/kg of fermented GL residues had the greatest beneficial effects on growth performance, nutrient digestibility, and immune function in weaned piglets (Zhou et al., 2015). The growth performance, feed efficiency, and intestinal morphology were significantly improved in groups supplemented with Aspergillus or Bac licheniformis-fermented GL (Zhang et al., 2012; Yu et al., 2015). The total flavonoids and terpenoids of A. niger-fermented-G. biloba leaves are most likely the key compounds responsible for the influences on the small intestinal morphology and immune responses without adverse effects in broiler chickens (Zhang et al., 2013). These improve growth performance and intestinal morphology of broilers in GL- or EGB-supplemented treatments and might result from the change of bacterial community structure in the intestines or the growth of intestinal villus (Zhou et al., 2006; Petrolli et al., 2012). The height of intestinal villi and deeper crypts reflect the surface area for nutrient absorption (Xu et al., 2003; Hu et al., 2011). All of these factors may have favorable effects on broiler health, which may partially contribute to the affected nutrient and energy utilization of broilers in this study. GL or EGB fed to broilers improved intestinal health and the utilization rate of nutrients. Furthermore, the findings were that dietary supplementation of GL and EGB increased the utilization of nutrient and energy in broilers in a dose-dependent manner. Zhang et al. (2012) reported that supplementation of 3.5 to 5 g/kg and 7 to 10 g/kg Aspergillus niger-fermented GL in the starter and grower diets, respectively, improved feed efficiency, intestinal morphology, digestion, and absorption function of broilers. In the current study, the use of 5 g/kg (in the starter phase) and 10 g/kg (in the grower phase) of fermented GL products had a positive influence on growth performance, lipid metabolism, and antioxidant capacity (Cao et al., 2012). Supplementation of 0.25 to 1.0 g/kg EGB evidently elevated the activities of SOD and GSH-Px, and at the same time, reduced the content of MDA, which indicated that the effects were exerted in a dose-dependent manner (Liu et al., 2006). The results of this study showed that supplementation of 20 to 60 g/kg GL or 0.4 to 0.8 g/kg EGB in diets increased nutrient and energy utilization in a dose-dependent manner. However, inclusion levels of EGB up to 1.2 g/kg reduced the nutrients and energy utilization and that of some essential and non-essential AA (Chamorro et al., 2012). This is likely due to different concentrations of the active substances and the toxic effect of the high level of dietary EGB. Ginkgolic acid has been recognized as a hazardous compound with suspected cytotoxic and allergenic properties, which may have adverse effects on the nutrients and energy utilization of the high level of dietary EGB in poultry (Yu et al., 2015). The efficacy of GL and its extracts also could be affected by other factors, such as the diet type, animal age, hygiene, environmental factors, and so on (Amad et al., 2011). In the present study, the addition of 60 g/kg GL in broiler diets is the optimum concentration compared to those of GL-supplemented treatments, and this may be mostly due to the smaller concentration gradient; further study is required to continue to increase the concentration of GL in diets and evaluate effects on nutrient and energy utilization. Supplementation of GL and EGB in broiler diets increased the utilization of AA compared with that of control treatments. Ward et al. (2002) reported that supplementation of fermented GL increased the growth performance, intestinal morphology, digestion, and absorption function, which have been ascribed to the increased contents of flavones, CP, and AA, decreases in ginkgolic acid, and the synergistic reactions among various pharmacologically active constituents found in the leaf. In the present study, adding EGB in broiler diets improved the utilization of AA, except Thr and Cys, whereas the utilization of Leu, His, Met, Ser, Glu, and Cys were increased in the GL-supplemented treatments, but other AA were not. This may imply that EGB potentiates the function of digestion and absorption by purifying the active constituent concentrations. However, relatively high dietary concentrations of flavones obtained with supplementation of these ingredients reduced the performance of chickens (Chamorro et al., 2012). An interesting finding of this study was that the isolation of the effective compounds affected efficacy of GL in increasing the utilization of nutrients in broilers. It appeared that under the same concentrations of the active substances in GL and EGB, supplementation of EGB had a better effect than that in GL-supplemented treatments. The constituent of GL contains simple ginkgolic acids, which are harmful to animals. After an ethanol extraction process, some compounds are enriched (flavonoids, terpene lactones), while others (ginkgolic acids) are removed (Teris et al., 2009). This may imply that the components in the EGB were more digestible than those present in GL and better able to exert their biological effects. Therefore, the isolation of GL is responsible for the effect of EGB, but the leaves have inferior effect, which means the isolation of effective compounds is necessary. In fact, EGB has a high price but little addition in diets and had a better effect on utilization of nutrient and energy compared with GL-supplemented diets. The results of this study indicate that supplementation with GL or EGB increased the utilization of nutrient and energy of broiler chickens. CONCLUSIONS Supplementation of 20 to 60 g/kg GL and 0.4 to 0.8 g/kg EGB of diet, respectively, increased nutrient and energy utilization of broilers. This study showed that optimum concentrations of GL and EGB in the diets of broiler chickens were 60 g/kg or 0.8 g/kg of diet, respectively, for enhancing the nutrient and energy utilization, and further research is needed on the effects of other livestock industries. The results of this study indicate that GL and EGB could be used as feed additives for improving the nutrient and energy utilization of broilers. 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Poultry ScienceOxford University Press

Published: Apr 1, 2018

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