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Animal Science Journal (2015) 86, 790–799

doi: 10.1111/asj.12361

ORIGINAL ARTICLE Effects of dietary supplementation of fermented Ginkgo biloba L. residues on growth performance, nutrient digestibility, serum biochemical parameters and immune function in weaned piglets Hao ZHOU, Chengzhang WANG, Jianzhong YE, Hongxia CHEN and Ran TAO Institute of Chemical Industry of Forest Products, CAF, Nanjing, Jiangsu, China

ABSTRACT This study evaluated the effects of fermented Ginkgo biloba L. residues (FGBLR) on growth performance, nutrient digestibility, serum biochemical parameters and immune function in weaned piglets. Pigs were allotted to five dietary treatments, including negative control (NC: antibiotic free basal diet), positive control (PC) (NC + 30 mg apramycin/kg) and FGBLR-50, 100, 150 (NC + 50, 100, 150 g FGBLR/kg). Pigs in FGBLR-100 and PC treatments showed increased final body weight, average daily gain, gain:feed and apparent total tract digestibility of dry matter, N and gross energy (P < 0.05) compared with NC, FGBLR-50 and FGBLR-150 treatments, In addition, pigs fed with FGBLR-100 diet showed higher serum total protein, albumin, alkaline phosphatase, glucose, hemoglobin, total iron, total iron binding capacity, superoxide dismutase and glutathione superoxide dismutase levels, and lower serum blood urea nitrogen, malondialdehyde, glutamicpyruvic transaminase, glutamic-oxalacetic transaminase, triglyceride and total cholesterol levels than those fed with PC and NC diets (P < 0.05). Moreover, feeding FGBLR-100 could increase levels of immunoglobulin G (IgG), IgA and IgM, as well as lymphocyte transformation rates, ratio of CD4+ to CD8+ cells and proportions of CD2+, CD4+, B, major histocompatibility complex (MHC)-I and MHC-II cells, and can decrease proportion of CD8+ cells in blood of piglets compared with PC and NC groups (P < 0.05). These results indicate that dietary supplementation with 10% of FGBLR showed greatest beneficial effects on growth performance, nutrient digestibility, serum biochemical parameters and immune function in weaned piglets, which were superior to antibiotic supplemental diets.

Key words: fermented Ginkgo biloba L. residue, growth performance, nutrient digestibility, serum biochemical parameters and immune function, weaned piglet.

INTRODUCTION Recently, phytogenic feed additives have been wildly suggested to livestock producers as alternatives to antibiotic growth promoters, as the misuse of antibiotic growth promoters could promote bacterial resistance and result in less efficient antibiotic treatments for human and animal diseases (Windisch et al. 2008; Ao et al. 2011a). Several European countries have banned the use of antibiotics in the animal diet (Simon et al. 2003); also, antibiotics supplementation will be banned in China in the near future. Previous studies have documented that herbal feed additives could increase growth performance, feed efficiency and immune-related blood characteristics, and could also exert anti-bacterial, anti-viral and anti-oxidative effects in livestock (Cho et al. 2006; Huang et al. 2010). © 2015 Japanese Society of Animal Science

Therefore, considerable effort has been devoted to find effective and safe herbal alternatives to promote growth and prevent disease in livestock. Ginkgo biloba L. (GBL), which is a traditional natural medicinal herb in China, is well known for its cholesterol lowering, antioxidant, antiviral, antiinflammatory, immunostimulant and cardiovascular improving properties (Cao et al. 2002; Ding et al. 2009). Ginkgo biloba L. residues (GBLR) are the by-products of leaves generated from flavonoids

Correspondence: Chengzhang Wang, Institute of Chemical Industry of Forest Products, CAF, Nanjing, Jiangsu 210042, China. (Email: [email protected]) Received 6 September 2014; accepted for publication 16 October 2014.

EFFECTS OF GBLR IN WEANED PIGLETS

extraction of GBL. Traditionally, they are directly discarded, which represents a major cause for environmental pollution and also an important loss of biomass. GBLR contain many active components such as flavonoids, polysaccharides, proteins, terpenoids and mineral substances (Li et al. 2012). The utilization of GBLR as animal feed is undoubtedly a good way of recycling this by-product. Probiotics strains are commonly used to ferment feeds for many years with beneficial effects (Boguhn et al. 2006). Presently Saccharomyces and Aspergillus are the major probiotic strains applied in fermentation of feed (Chen et al. 2009). They can produce high amounts of enzymes such as cellulase, protease, hydrolases, amylase and lipases, which can increase the digestibility of protein, fibre and starch, improve gastrointestinal health, and decrease anti-nutrients in the feedstuffs (Canibe & Jensen 2003; Mathivanan et al. 2006). In previous studies, fermented feeds such as fermented wheat, fermented soybean meals, fermented mulberry leaf and fermented grains have been reported to improve growth performance, feed efficiency, nutrient digestibility, immunity and blood parameters in livestock (Scholten et al. 2002; Feng et al. 2007a; Huyen et al. 2012; Cho et al. 2013). Till now, it has only been reported Aspergillus nigerfermented GBL can improve growth performance, egg quality, lipid metabolism and immunity in laying hens (Zhao et al. 2013), but the effects of femented Ginkgo biloba L. residues (FGBLR) in livestock have not yet been investigated. Therefore, this study was conducted to determine the effects of the GBLR fermented with Candida tropicalis and Aspergillus oryzae on growth performance, nutrient digestibility, immune response and blood characteristics in weaned piglets.

MATERIALS AND METHODS Preparation of FGBLR The FGBLR were provided by Pizhou Xinyuan Biological Products Co., Ltd. (Xuzhou, China). Dried GBLR were soaked in distilled water (40% moisture content) for 1 h. Hydrated GBLR were then cooked in a steam tank at 60–70°C for 1 h. Thereafter, GBLR were cooled to room temperature, supplemented with 2% maltose and peptone, and then inoculated with Candida tropicalis and Aspergillus oryzae at an initial count of 105 colony-forming units (cfu)/g, respectively. After fermentation for 96 h at 28–30°C, the fermented samples were ground in a jet mill and refrigerated (4°C) until they were mixed in the experimental diets. The nutritional analysis of the GBLR before and after fermentation are shown in Table 1.

Experimental design, animals, housing and diets A total of 120 castrated piglets (Duroc × Landrace × Yorkshire) with an average initial body weight (BW) of Animal Science Journal (2015) 86, 790–799

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Table 1 Main nutritional ingredients in Ginkgo biloba L. residues before and after fermentation

Items

Before fermentation

Total flavonoids (mg/g) Polysaccharides (g/kg) Protein (g/kg) fibre (g/kg) Total ginkgolic acid (g/kg) Total amino acid (g/kg) Indispensable amino acid (g/kg) Ferment cells number (cfu/g) Cellulase (IU/g) Glucosidase (IU/g) Protease (IU/g)

28.35 8.62 123.5 162.4 3.86 81.72 31.25 0 0 0 0

After fermentation 34.12 13.25 348.6 82.6 0.11 230.54 87.61 2.5 × 109 1768.26 4520.32 3345.68

7.6 ± 0.35 kg (weaned at day 21) were used in a 42-day experiment. Piglets were randomly allocated into one of five treatments with six replications (pens) each consisting of four pigs (two barrows and two gilts) in a randomized complex block design according to their BW and sex. The dietary treatments included: negative control (NC: antibiotic free basal diet); positive control (PC; NC + 30 mg apramycin /kg); FGBLR-50 (NC + 50 g FGBLR /kg); FGBLR-100 (NC + 100 g FGBLR /kg); and FGBLR-150 (NC + 150 g FGBLR /kg). The dosage of FGBLR was designed for the control of fiber content and to meet the protein content in the experimental diets, and the dosage was also designed by our preliminary experiment. The experimental diets used in this experiment were formulated to meet or exceed NRC (1998) recommendations for all nutrients (Table 2). The pigs were housed in an environmentally controlled, slatted-floor facility in 30 adjacent pens (1.80 × 1.80 m) and were allowed ad libitum access to feed and water through a self-feeder and nipple drinker throughout the experimental period.

Sampling and measurements Individual pig BW and the amount of feed consumed were recorded weekly for calculating average daily gain (ADG), average daily feed intake (ADFI) and gain/feed (G/F) ratio. The apparent total tract digestibility (ATTD) of dry matter (DM), nitrogen (N) and gross energy(GE) was determined using chromic oxide (0.2%) as an inert indicator. Pigs were fed diets mixed with chromic oxide from day 36 to day 42. Fresh fecal grab samples were collected via massaging the rectum from two pigs (one barrow and one gilt) randomly selected from each pen from days 39 to 42. All fecal and feed samples from one pen were pooled and mixed, after which a representative sample was stored in a freezer at−20°C until analysis. Prior to chemical analysis, the fecal samples were dried at 70°C for 72 h and finely ground to pass through a 1-mm screen. The procedures utilized for the determination of DM, N and GE digestibility were conducted in accordance with the methods established by the AOAC (2000). Chromium levels were analyzed using UV absorption spectrophotometry (UV-1201; Shimadzu, Kyoto, Japan). Nitrogen was measured using a Kjeltec 2300 analyzer (Foss Tecator AB, Hoeganaes, Sweden). The gross energy was determined by measuring the heat of combustion in the samples using a Parr 6100 oxygen bomb calorimeter (Parr Instrument Co., Moline, IL, USA). The ATTD of DM, N and GE were © 2015 Japanese Society of Animal Science

792 H. ZHOU et al.

Table 2 Ingredient composition and analyzed nutrient content of experimental diets†

Item

NC/g

PC/g

FGBLR-50/g

FGBLR-100/g

FGBLR-150/g

Wheat Fermented Ginkgo biloba L. residues (FGBLR) Soybean meal, 460 g CP/kg Lactose Canola oil Soy protein concentrate, 560g CP/kg Herring fish meal, 700 g CP/kg Limestone Celite‡ Mono/dicalcium phosphate Vitamin premix§ Mineral premix¶ Salt L-Lysine HCl, 780 g/kg DL-methionine, 980 g/kg L-threonine, 980 g/kg L-tryptophan, 980 g/kg Choline chloride, 600 g/kg Analyzed composition(g/kg) Dry matter Ash Crude protein Crude fibre Crude fat Calcium Phosphorus Metabolizable energy (MJ/kg)

570.0 – 200.0 50.0 30.0 50.0 50.0 9.1 8.0 8.2 5.0 5.0 5.0 – 0.4 0.4 – 0.3

570.0 – 200.0 50.0 30.0 50.0 50.0 9.1 8.0 8.2 5.0 5.0 5.0 – 0.4 0.4 – 0.3

520.0 50.0 200.0 50.0 30.0 50.0 50.0 9.1 8.0 8.0 5.0 5.0 5.0 1.0 0.3 0.6 0.1 0.3

470.0 100.0 200.0 50.0 30.0 50.0 50.0 9.1 8.0 8.0 5.0 5.0 5.0 1.5 0.3 0.8 0.2 0.3

420.0 150.0 200.0 50.0 30.0 50.0 50.0 9.1 8.0 8.0 5.0 5.0 5.0 2.0 0.3 1.0 0.3 0.3

901.3 63 246 16 46 6.8 5.7 17.4

903.8 65 242 16 44 6.8 5.7 17.2

902.5 68 236 17 42 6.9 5.8 17.5

905.4 66 220 22 41 6.8 5.7 17.4

904.7 68 205 27 42 6.7 5.8 17.5

†Dietary treatment: (1) NC: antibiotic free basal diet; (2) PC: NC + 30 mg apramycin/kg; (3) FGBLR-50: NC + 50g FGBLR/kg; (4) FGBLR-100: NC + 100g FGBLR/kg; (5) FGBLR-150: NC + 150 g FGBLR /kg. CP, crude protein. ‡Celite 281 (World Minerals Inc., Santa Barbara, CA, USA) used as acid insoluble ash. §Supplied per kilogram of diet: 7500 IU of vitamin A, 750 IU of vitamin D, 50 IU of vitamin E, 37.5 mg of niacin, 15 mg of pantothenic acid, 2.5 mg of folacin, 5 mg of riboflavin, 1.5 mg of pyridoxine, 2.5 mg of thiamine, 4 mg of vitamin K, 0.25 mg of biotin and 0.02 mg of vitamin B12. ¶Supplied per kilogram of diet: 125 mg of Zn, 50 mg of Cu, 75 mg of Fe, 25 mg of Mn, 0.5 mg of I and 0.3 mg of Se.

calculated using indirect methods described by Fenton and Fenton (1979). On the last day of the experiment, two pigs were randomly selected from each pen (one gilt and one barrow) and bled via jugular venipuncture. Blood samples were collected into vacuum tubes containing no additive, and tubes containing vitamin K3 ethylenediaminetetraacetic acid (K3EDTA) to obtain serum and whole blood samples, respectively. The serum was separated by centrifugation for 30 min at 2000 × g at 4°C, and the aliquot was stored at −4°C(within 24 h) prior to assay. The whole blood samples for immune function analysis were packaged on ice on the day of sampling and then transported to the laboratory in 30 min at 4°C to be analyzed immediately. For the serum biochemical parameters, the levels of serum total protein (TP), albumin (ALB), urea nitrogen (BUN), alkaline phosphatase (ALP), glucose (GLU), hemoglobin (Hb), total iron (TI), total iron binding capacity (TIBC), triglyceride (TG), total cholesterol (TCHO), superoxide dismutase (SOD), glutathione superoxide dismutase (GSHPx), malondialdehyde (MDA), glutamic-pyruvic transaminase (ALT) and glutamic-oxalacetic transaminase (AST) were analyzed by an automatic biochemistry blood analyzer (HITACHI 7020, Hitachi, Tokyo, Japan). For immune function analysis, the levels of serum immunoglobulins (IgG, IgA and IgM) were determined using an © 2015 Japanese Society of Animal Science

ELISA test kit (Diagnostic Automation/Cortez Diagnostics, Inc., Los Angeles, CA, USA); and with the whole blood samples, lymphocyte transformation rates and lymphocyte subpopulations, including CD2+, CD4+ and CD8+ cells, B-cells, and major histocompatibility complex (MHC) classes I and II were measured. The lymphocyte transformation rates were determined using the method reported by Mosmann (1983). The porcine lymphocyte subpopulations were measured using flow cytometry in conjunction with a Calibur fluorescence-activated cell sorter and the CellQuest program (Becton Dickinson, Franklin Lakes, NJ, USA) using monoclonal antibodies (Southern Biotech, Birmingham, AL, USA) that were specifically reactive to porcine MHC classes I and II, cluster of differentiation antigens positive cells 2, 4 and 8 (CD2+, CD4+ and CD8+, respectively) and B-cells.

Statistical analysis All the data were analyzed using one-way analysis of variance (ANOVA) (SPSS 19.0: SPSS Inc, Chicago, IL, USA). Least Significant Difference and Duncan’s multiple comparison methods were used to compare statistical differences (P < 0.05) between treatments. The results were presented as means and pooled standard error of the mean (SEM). Animal Science Journal (2015) 86, 790–799

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RESULTS Growth performance and nutrient digestibility

Serum biochemical parameters

The ingredient composition and analyzed nutrient content of the five dietary treatments are shown in Table 1. The FGBLR were used to replace 5%, 10% and 15% of wheat; their effects in piglets were compared with normal basal diet and antibiotic supplemental diet. The nutritional analyses of the GBLR before and after fermentation are shown in Table 2. After fermentation, the content of fiber was reduced by 49.1%, the contents of flavonoids, protein, polysaccharides and amino acid were increased by 20.6%, 182.9%, 53.7% and 182.1%, respectively, and the content of ginkgolic acid was decreased sharply from 0.386% to 0.011% in GBLR. The effects of FGBLR on growth performance of weaned piglets are presented in Table 3. The final BW, ADG and G:F ratio of pigs in PC, FGBLR-50, FGBLR100 and FGBLR-150 groups were higher (P < 0.01) than those in the NC group. Pigs fed with FGBLR-100 and PC diets showed higher (P < 0.05) final BW, ADG and G:F ratio than those fed with FGBLR-50 and FGBLR-150 diets. Furthermore, pigs in the FGBLR100 group showed higher final BW, ADG and G:F ratio compared with PC treatment, but the difference between them was not significant. In addition, no differences in ADFI among treatments were observed. The effects of FGBLR on nutrient digestibility of weaned piglets are presented in Table 4. Pigs in PC, FGBLR-50, FGBLR-100 and FGBLR-150 groups showed greater (P < 0.05) DM, N and GE digestibility compared with NC treatment. Moreover, the DM, N and GE digestibility of pigs in PC and FGBLR-100 treatments were higher than those in FGBLR-50 and FGBLR-150 treatments. In addition, pigs in FGBLR100 treatment showed higher DM, N and GE digest-

The results of serum biochemical parameters are presented in Table 5. The levels of serum TP, ALB, ALP and GLU were increased in the PC, FGBLR-50, FGBLR-100 and FGBLR-150 groups and differed significantly from the control group (P < 0.01); levels of serum TP, ALB, ALP and GLU in PC and FGBLR-100 groups were higher (P < 0.05) than that in FGBLR-50 and FGBLR-150 groups; there was no difference between the FGBLR-50 and FGBLR-150 groups; and the levels of serum TP, ALB, ALP and GLU were highest in the FGBLR-100 group and were significantly different from the PC group (P < 0.05). In addition, the BUN level was decreased in the PC, FGBLR-50, FGBLR-100 and FGBLR-150 groups and differed significantly from the control group (P < 0.01); BUN level in PC and FGBLR-100 groups was lower (P < 0.05) than those in FGBLR-50 and FGBLR-150 groups; there was no significant difference between the FGBLR-50 and FGBLR-150 groups; and the BUN level was lowest in the FGBLR-100 group and was significantly different from the PC group (P < 0.05). The concentrations of serum Hb, TI and TIBC in the PC, FGBLR-50, FGBLR-100 and FGBLR-150 groups were significantly higher (P < 0.01) than that in the control group; and the FGBLR-100 and FGBLR-150 groups showed greater (P < 0.05) Hb, TI and TIBC concentrations than the PC and FGBLR-50 groups; the Hb, TI and TIBC concentrations were highest in the FGBLR-100 group, but there was no significant difference between the FGBLR-100 and FGBLR-150 groups. The levels of serum SOD and GSH-Px were increased in the PC, FGBLR-50, FGBLR-100 and FGBLR-150 groups and differed significantly from the control group (P < 0.01); the FGBLR-100 and

ibility compared with PC treatment, but there was no significant difference between them.

Table 3 Effect of fermented Ginkgo biloba L. residues on growth performance in weaned piglets

Items

NC

PC

FGBLR-50

FGBLR-100

FGBLR-150

SE†

P-value

Initial BW, kg Final BW, kg ADG, g ADFI, g G:F

7.61 28.24c 491c 774 0.635b

7.63 29.56a 521a 765 0.683a

7.62 28.96b 507b 768 0.662b

7.61 29.72a 525a 765 0.687a

7.59 28.96b 506b 766 0.660b

0.35 0.29 6.89 7.99 0.009

0.001 0.001 0.804 0.003

†Pooled standard error. a,b,cMeans in the same row with different superscripts differ (P < 0.05). ADFI, average daily feed intake; ADG, average daily gain; BW, body weight; NC, negative control; PC, positive control; FGBLR, femented Ginkgo biloba L. residues; G:F, gain:feed.

Table 4 Effect of fermented Ginkgo biloba L. residues on nutrient digestibility in weaned piglets

Items, % DM N GE

NC

PC c

78.90 79.10c 79.30c

FGBLR-50 a

82.08 82.34a 82.34a

b

80.56 80.84b 81.16b

FGBLR-100

FGBLR-150

SE†

P-value

a

b

0.63 0.78 0.65

0.006 0.009 0.005

82.26 82.66a 82.66a

80.72 80.92b 81.06b

†Pooled standard error. a,b,cMeans in the same row with different superscripts differ (P < 0.05). NC, negative control; PC, positive control; DM, dry matter; FGBLR, femented Ginkgo biloba L. residues; GE, gross energy; N, nitrogen.

Animal Science Journal (2015) 86, 790–799

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794 H. ZHOU et al.

Table 5 Effects of fermented Ginkgo biloba L. residues on the serum biochemical parameters in weaned pigs

Items TP (g/L) ALB (g/L) BUN (mmol/L) ALP (U/L) GLU (mmol /L) Hb (g/L) TI (mg/L) TIBC (mg/L) TG (g/L) TCHO (g/L) SOD (U/mL) GSH-Px (U/mL) MDA (nmol/mL) ALT (U/L) AST (U/L)

NC

PC d

50.27 46.63d 4.12a 117.7d 3.88d 105.0c 2.04c 5.91c 0.66a 2.27a 304.0c 112.3c 7.65a 18.18a 17.47a

FGBLR-50 b

c

58.59 50.74b 3.35c 148.9b 4.87b 112.9b 2.58b 6.82b 0.64a 2.25a 339.0b 124.9b 6.72b 17.70a 16.61a

55.07 49.50c 3.74b 140.0c 4.56c 111.2b 2.50b 6.76b 0.52b 2.14b 343.3b 122.5b 6.81b 15.86b 13.84b

FGBLR-100

FGBLR-150

a

c

61.10 51.83a 3.11b 154.2a 5.01a 116.0a 2.85a 7.20a 0.46c 2.03c 369.9a 131.1a 6.49c 13.88c 10.66c

54.09 48.60c 3.85b 135.5c 4.46c 115.1a 2.81a 7.13a 0.40d 1.91d 365.4a 129.2a 6.58c 13.84c 10.58c

SE†

P-value

0.54 0.52 0.06 2.18 0.05 0.81 0.05 0.06 0.01 0.05 2.56 1.17 0.05 0.38 0.46

0.006 0.008 0.007 0.008 0.006 0.005 0.008 0.005 0.008 0.006 0.007 0.009 0.009 0.005 0.006

†Pooled standard error. a,b,c,dMeans in the same row with different superscripts differ (P < 0.05). ALB, albumin; ALP, alkaline phosphatase; ALT, glutamic-pyruvic transaminase; AST, glutamic-oxalacetic transaminase; BUN, blood urea nitrogen; FGBLR, femented Ginkgo biloba L. residues;TP, total protein; GLU, glucose; GSH-Px, glutathione superoxide dismutase; Hb, hemoglobin; MDA, malondialdehyde; NC, negative control; PC, positive control; SOD, superoxide dismutase; TCHO, total cholesterol; TG, triglyceride; TI, total iron; TIBC, total iron binding capacity.

Table 6 Effects of fermented Ginkgo biloba L. residues on the immune function in weaned pigs

Items IgG (μg/mL) IgA (μg/mL) IgM (μg/mL) Lymphocyte transformation rate (%) Lymphocyte subpopulation‡ (%) CD2+ CD4+ CD8+ CD4+:CD8+ B-cells MHC-I MHC-II

NC

PC c

FGBLR-50 b

b

FGBLR-100

FGBLR-150

SE†

P-value

a

b

136.6 35.32c 12.69c 53.09c

170.2 40.79b 15.09b 55.91b

167.7 38.76b 14.75b 54.84b

181.9 45.49a 16.86a 58.71a

168.7 39.38b 14.92b 55.61b

1.41 0.98 0.68 0.58

0.005 0.008 0.006 0.007

18.59d 23.72d 35.20a 0.67d 26.75c 25.47c 26.49c

23.54b 27.55b 30.80c 0.89b 29.29b 27.22b 28.38b

20.64c 25.27c 32.44b 0.78c 28.78b 27.06b 28.41b

25.44a 29.17a 29.38d 0.99a 31.65a 29.76a 30.73a

21.64c 25.85c 32.82b 0.79c 30.82a 28.86a 29.92a

0.82 0.50 0.66 0.01 0.68 0.64 0.59

0.005 0.008 0.006 0.006 0.008 0.007 0.009

†Pooled standard error. ‡Lymphocyte subpopulation: Cluster of differentiation of antigen 2, 4, 8 (CD2+, CD4+, and CD8+, respectively), B-cells, CD4+: CD8+, major histocompatability complex (MHC) classes I, and II. a,b,c,dMeans in the same row with different superscripts differ (P < 0.05). FGBLR, femented Ginkgo biloba L. residues; NC, negative control; PC, positive control.

FGBLR-150 groups showed greater (P < 0.05) SOD and GSH-Px levels than the PC and FGBLR-50 groups, and the FGBLR-100 group showed the highest SOD and GSH-Px levels, but there was no significant difference between the FGBLR-100 and FGBLR-150 groups. Moreover, the MDA level was decreased in the PC, FGBLR-50, FGBLR-100 and FGBLR-150 groups and differed significantly from the control group (P < 0.01); the FGBLR-100 and FGBLR-150 groups showed lower (P < 0.05) MDA level than the PC and FGBLR-50 groups; the FGBLR-100 group showed the lowest MDA level, and there were no significant differences between the FGBLR-100 and FGBLR-150 groups. The concentrations of serum TG and TCHO in the FGBLR-50, FGBLR-100 and FGBLR-150 groups were significantly decreased (P < 0.01) than those in the PC and NC groups; there was no significant difference © 2015 Japanese Society of Animal Science

between the PC and NC groups; and the FGBLR-50, FGBLR-100 and FGBLR-150 groups showed a linear reduction in the TG and TCHO concentrations (P < 0.05). The levels of serum ALT and AST in the FGBLR-50, FGBLR-100 and FGBLR-150 groups were significantly decreased (P < 0.01) than that in the PC and NC groups; and the FGBLR-100 group showed the lowest ALT and AST levels.

Immune function The results of immune function are presented in Table 6. The levels of serum IgG, IgA and IgM in the PC, FGBLR-50, FGBLR-100 and FGBLR-150 groups were significantly higher (P < 0.01) than that in the control group; and the FGBLR-100 group showed greater (P < 0.05) IgG, IgA and IgM levels than the PC, FGBLR-50 and FGBLR-150 groups. Meanwhile, the lymphocyte transformation rate in the PC, FGBLR-50, Animal Science Journal (2015) 86, 790–799

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FGBLR-100 and FGBLR-150 groups were increased and differed significantly from the NC group (P < 0.01); and the FGBLR-100 group showed higher (P < 0.05) lymphocyte transformation rate than the PC, FGBLR-50 and FGBLR-150 groups. The proportions of CD2+ and CD4+ cells were significantly increased in the PC, FGBLR-50, FGBLR-100 and FGBLR-150 groups compared with NC group (P < 0.01), and the FGBLR-100 group showed the highest proportions of CD2+ and CD4+ cells and differed significantly from the PC, FGBLR-50 and FGBLR-150 groups (P < 0.05). Moreover, the proportions of CD8+ cells were significantly decreased in the PC, FGBLR-50, FGBLR-100 and FGBLR-150 groups compared with the NC group (P < 0.01), and the FGBLR-100 group showed the lowest proportions of CD8+ cells and differed significantly from the PC, FGBLR-50 and FGBLR-150 groups (P < 0.05). Furthermore, the ratio of CD4+ to CD8+ cells in the PC, FGBLR-50, FGBLR-100 and FGBLR-150 groups were significantly higher (P < 0.01) than that in the NC group; and the FGBLR-100 group showed the highest ratio of CD4+ to CD8+ cells and differed significantly from the PC, FGBLR-50 and FGBLR-150 groups (P < 0.05). In addition, the proportions of B-cells, MHC-I and MHC-II in the PC, FGBLR-50, FGBLR-100 and FGBLR-150 groups were significantly increased compared with the NC group (P < 0.01), and the FGBLR100 and FGBLR-150 groups showed higher (P < 0.05) proportions of B-cells, MHC-I and MHC-II than the PC and FGBLR-50 groups.

DISCUSSION GBLR is the by-product generated from extraction of GBL. Every year about 40 000 tons of GBLR are discarded in China, which represents an important loss of biomass. GBLR is known to be rich in flavonoids, polysaccharides, terpenoids, vitamins, amino acids and mineral substances. However, GBLR contain high content of fibers with low digestibility and contain the hazardous compounds of ginkgolic acid with suspected cytotoxic and allergenic properties, which restricts its application in animal feeding. Fermentation is a useful tool for bioconversion of cellulosic biomass to decrease their fiber content, reduce toxic constituents, and increase their protein content and nutritional value by production of useful enzymes (Ng et al. 2011). In the present study, GBLR were fermented with Candida tropicalis and Aspergillus oryzae; after fermentation, the content of nutrients were significantly increased, and the content of fiber and toxic ingredients were decreased sharply in GBLR. Previous studies have reported fermented GBL can improve growth performance and immunity in chicks (Zhang et al. 2012). However, no reference is available for the use of Animal Science Journal (2015) 86, 790–799

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FGBLR in livestock feeding. So, the present study was designed to evaluate the effects of FGBLR on growth performance, nutrient digestibility, serum biochemical parameters and immune function in weaned piglets, with the view of its potential inclusion in pig diets as an alternative feed resource and antibiotic alternative for feeding livestock.

Growth performance and nutrient digestibility In the present study, diets supplemented with FGBLR improved significantly the growth performance of piglets. Our results showed that pigs fed with FGBLR (replacing 5%, 10% and 15% of wheat, respectively) or antibiotic supplemental diets, had greater final BW, ADG and G:F than those fed with NC diet. It was generally considered that addition of antibiotics to the diet was a good way to improve the growth performance of swine and poultry because of its antimicrobial effect and health-promoting effect (Mateo et al. 2006). However, pigs fed with diets supplemented with 10% FGBLR showed higher final BW, ADG and G:F than those fed with PC diet. Previous studies have documented that fermented herb additives, such as fermented water plantain, red ginseng and green tea, could improve growth performance, feed efficiency and nutrient digestibility in livestock (Ao et al. 2011b; Hossain et al. 2012; Hossain & Yang 2014), and the reason for the enhanced performance may be that fermented herbs could improve the flavor and palatability of feed, increase the activity of digestive enzymes of the gastrointestinal tract and the nutrient utilization in livestock (Czech et al. 2009). In our study, it showed that increasing dietary inclusion of FGBLR from 10% to 15% induced a decreased growth performance of weaned pigs, which is due to the increased fiber content in pig diets, which can decrease protein and energy digestibility and thereby reduce growth performance of pigs. In addition, our results showed there is no difference in ADFI among all the dietary treatments, this could be due to the taste and palatability of the feed. The results of the study showed that diets supplemented with FGBLR can significantly increase the ATTD in the piglets, which may partly explain the improvement in growth performance observed. The ATTD of DM, N and GE in piglets fed with FGBLR (replacing 5%, 10% and 15% of wheat, respectively) or antibiotic supplemental diets were significantly higher than those fed with NC diet, and pigs fed with diets supplemented with 10% FGBLR showed highest ATTD of DM, N and GE. Previous studies have demonstrated that fermented herbs with Candida tropicalis or Aspergillus oryzae can produce a number of hydrolytic enzymes (such as α-amylase, cellulase, maltase, protease and so on), and improve the gastrointestinal ecosystem, digestive enzyme activities and health © 2015 Japanese Society of Animal Science

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status of digestive system in weaned pigs, which can contribute to the higher nutrient digestibility (Lyberg et al. 2006; Feng et al. 2007b).

Serum biochemical parameters In the current study, it is suggested that diets supplemented with FGBLR could induce positive effects on serum biochemical parameters in piglets. The serum concentrations of TP, ALB and BUN are commonly regarded as indicators of protein synthesis and metabolism which are related to the growth performance in piglets, increased TP and ALB levels indicated more proteins were synthesized and absorbed, and BUN is the product of protein degradation in vivo; decreased BUN indicated more proteins were synthesized with amino acids (Kim et al. 2013). ALP is the important enzyme which contributes to the absorption of Ca and P and protein synthesis, and serum GLU level is the frequently used indicator of energy supply ability (Wang et al. 2011). Piglets fed with FGBLR (replacing 5%, 10% and 15% of wheat, respectively) or antibiotic supplemental diets showed significantly higher serum TP, ALB, ALP and GLU levels than those fed with NC diet, and they showed significantly lower serum BUN level compared with the NC group. These results are in accordance with previous studies which have demonstrated that fermented herbs can improve serum TP, ALP and GLU levels in pigs by improving intestinal morphology and digestive enzyme activities and nutrient metabolism (Lee et al. 2009). Meanwhile, these positive results can partly explain the observed improvement of growth performance and nutrient digestibility in pigs fed with FGBLR supplemental diets. In addition, pigs fed with FGBLR-100 diets showed higher serum TP, ALB, ALP and GLU levels and lower serum BUN level than those fed with FGBLR-150 diets; this could be due to the increased fiber content in pig diets, which may decrease protein synthesis and absorption. The serum Hb, TI and TIBC levels are commonly regarded as the indicators of body Fe status (Rincker et al. 2004). We found that the levels of Hb, TI and TIBC in piglets fed with FGBLR (replacing 10% and 15% of wheat, respectively) supplemental diets were significantly higher than those fed with antibiotic supplemental diets and NC diet. It has been reported the GBL extracts can increase serum Hb and TI levels in rats or human bodies, which could be ascribed to elevated iron incorporation via the enzyme ferrochelatase to form heme (Abdel-Baieth 2009). Moreover, pigs fed with FGBLR-100 diets showed higher serum Hb, TI and TIBC levels than those fed with FGBLR-150 diets; this may be due to the increased fiber content which decreased the absorption of Fe. A high plasma concentration of TCHO and TG is considered as a risk factor in humans for coronary © 2015 Japanese Society of Animal Science

heart disease and atherosclerosis, which are strongly related to the dietary intake of pork (Hu et al. 2011). It has been reported that GBL have lowering effects on TCHO and TG in a variety of studies (Ting et al. 2011); also, some researchers found that Saccharomyces and Aspergillus strains are able to decrease blood TCHO and TG by directly binding TCHO or TG into the small intestine during fat digestion before they can be absorbed into the blood (Krasowska et al. 2007). The results of our study showed that piglets fed with diets containing 5%, 10% and 15% of FGBLR showed a linear reduction in the TCHO and TG concentrations, and they showed significantly lower serum TCHO and TG levels than those fed with antibiotic supplemental diet and NC diet. This reduction could be explained by the reduced absorption or synthesis of TCHO in the gastrointestinal tract, and the reduced excretion of TG from liver cells (Velasco et al. 2010). The enzymes ALT and AST provide information regarding liver function; increased AST and ALT activity suggests liver disease, infection, parasitism or trauma (Lander et al. 2003). Our results indicate that piglets fed with diets containing 5%, 10% and 15% of FGBLR showed significantly lower serum ALT and AST levels than those fed with antibiotic supplemental diet and NC diet, which demonstrated the hepatoprotective effects of FGBLR. This result is consistent with previous a study which has suggested the active compounds of polyprenols in GBL have obvious hepatoprotective effects (Parimoo et al. 2014). SOD and GSH-Px are the main antioxidant enzymes in the body, which contribute to the antioxidant activity; they can protect cells from free radicals which can cause the oxidation of bio-molecules and lead to cell injury and death (Fridovich 1995). MDA is one of the most frequently used indicators of lipid peroxidation; increased MDA can be interpreted as a result of cellular membrane damage initially caused by increased formation of radicals (Niedernhofer et al. 2003). In the present study, the piglets fed with FGBLR (replacing 10% and 15% of wheat, respectively) supplemental diets showed significantly higher serum SOD and GSH-Px levels than those fed with PC and NC diets, and they showed significantly lower serum MDA level compared with PC and NC group. These results indicate FGBLR have obvious antioxidant and anti-stress abilities, which are consistent with a previous study which has reported that GBL could elevate SOD and GSH-Px activity and reduce lipid peroxide and MDA levels in blood, heart, brain and liver of rats (Kobus et al. 2009). In addition, pigs fed with FGBLR-100 diets showed higher serum SOD and GSH-Px levels and lower serum MDA level than those fed with FGBLR150 diets, this could be due to the increased fiber content in pig diets, which may decrease the absorption and digestibility of nutrient substance and thereby reduce antioxidant and anti-stress abilities of pigs. Animal Science Journal (2015) 86, 790–799

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Immune function It is well documented that the active compounds of flavones and terpenes in GBL have immune enhancing activities that include promotion of lymphocyte synthesis, cytokine release, immunoglobulin levels and phagocytosis activity (Zhao et al. 2011). In the present study, diets supplemented with FGBLR enhanced both humoral and cellular immunity of piglets. The serum antibody level is a useful indicator of humoral immunity. Actually, IgG, IgA and IgM are key components of the humoral immunity in all mammals, which are the major serum immunoglobulins that protect the extravascular compartment against pathogenic virus and microorganisms (Kong et al. 2007). A notable finding of the present study was that the serum concentrations of IgG, IgA and IgM in pigs fed with FGBLR (replacing 10% of wheat) supplemental diets were significantly higher than those fed with PC and NC diets. Previous research has indicated that FGBL could increase serum IgG, IgA and IgM levels in chicks, and this beneficial effect might be due to the influence on pathogenic microorganisms’ growth in the gastrointestinal ecosystem (Zhang et al. 2013). Lymphocyte transformation ratio and lymphocyte subpopulation numbers are the important indices for evaluation of the cell immune function in mammals. Our study showed that the lymphocyte transformation rate in pigs fed with FGBLR (replacing 10% of wheat) supplemental diets were significantly higher than those fed with PC and NC diets, which suggested that FGBLR could ameliorate the immune function of pigs through the improvement of lymphocyte proliferation and transformation, and such a result was in accordance with the elevation of serum concentration of IgG, IgA and IgM. Lymphocyte subpopulations, including CD2+, CD4+, CD8+ cells, B-cell, MHC-I and II cells, belong to the adaptive immune system and perform a wide array of functions in immune regulation, inflammation and protective immune responses (Gerner et al. 2009). In the present study, it indicated that pigs fed with FGBLR (replacing 10% of wheat) supplemental diets showed significantly higher numbers of CD2+ and CD4+ cells, higher ratio of CD4+ to CD8+ cells, and lower number of CD8+ cells than those fed with PC and NC diets; moreover, pigs fed with FGBLR (replacing 10% and 15% of wheat, respectively) supplemental diets showed significantly higher number of B-cell, MHC-I and II cells than those fed with PC and NC diets. These results demonstrated that feeding diets supplemented with FGBLR could enhance immunity by changing the lymphocyte populations in piglets. Moreover, pigs fed with FGBLR-100 diets showed higher levels of humoral and cellular immunity index than those fed with FGBLR-150 diets; this may be also Animal Science Journal (2015) 86, 790–799

due to the increased fiber content in pig diets, which may decrease the absorption and digestibility of nutrient substance and thereby reduce immune function of pigs.

Conclusions In summary, the results obtained in the present study indicate that FGBLR supplemental diets can improve the growth performance and nutrient digestibility (DM, N and GE), induce positive effects on serum biochemical parameters, and enhance both humoral and cellular immunity in piglets. Meanwhile, diets containing 10% of FGBLR showed greatest beneficial effects on growth performance, nutrient digestibility, serum biochemical parameters and immune function in weaned piglets, which were superior to antibiotic supplemental diets. Therefore, the FGBLR could be considered as partial substitution of basic diets, growth promoter and antibiotic alternative in weaned piglets.

ACKNOWLEDGMENTS The authors gratefully acknowledge the Special Fund of Scientific Research from Chinese Academy of Forestry (CAFYBB2012015) and the financial support by the Production and Research Project of Jiangsu Province (BY2013014).

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