Dietary supplementation with Clostridium butyricum modulates serum lipid metabolism, meat quality, and the amino acid and fatty acid composition of Peking ducks

Dietary supplementation with Clostridium butyricum modulates serum lipid metabolism, meat... ABSTRACT The aim of this study was to investigate the effects of Clostridium butyricum (C. butyricum) on the performance, serum lipid metabolism, muscle morphology, meat quality, and fatty acid profiles of Peking ducks. A total of 1,500 Peking ducks were randomly divided into five groups with five replicates and were fed a non-antibiotic basal diet (Control) or a basal diet supplemented with either 200, 400, or 600 mg/kg of C. butyricum (2.0 × 109 CFU/g) or 150 mg of aureomycin/kg for 42 d. Compared with the control group, supplementation with C. butyricum increased the average daily weight gain but reduced the feed/gain ratio from 1 to 42 d of age. Similarly, dietary C. butyricum increased the activities of antioxidant enzymes but decreased the malondialdehyde (MDA) and lipid metabolites concentration. C. butyricum supplementation increased the muscle pH value at 45 min postmortem, the redness of the meat, and the contents of inosine acid (IMP) and intramuscular fat (IMF) in Peking ducks. By contrast, C. butyricum supplementation lowered the lightness, drip loss, and the shear force of breast meat. Supplementation with C. butyricum increased the concentrations of essential amino acids and flavor amino acids, as well as arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and total polyunsaturated fatty acids (PUFA) in breast muscle. Dietary C. butyricum could positively improve performance, lipid metabolism, meat quality, and the amino acid and fatty acid composition in a dose-dependent manner. Therefore, C. butyricum is proposed as a feasible alternative feed additive for the production of healthier Peking duck meat with favorable properties. INTRDUCTION Increased attention is being paid to meat quality and flavor as well as its nutritional value by consumers (Cheng et al., 2017b). Duck meat, especially prepared as roast duck, is traditional food in China that has remained popular over time. Its relatively low fat content and high concentration of polyunsaturated fatty acids (PUFA), make it an important food source throughout the world (Kamboh and Zhu, 2013). In addition, the fatty acid profile and fat content of this meat can easily be improved using dietary strategies (Tavarez et al., 2011). In recent decades, to maintain health and increase production efficiency, antibiotics have been used widely in poultry production. However, the sub-therapeutic and prophylactic usage of antibiotics has led to health concerns, not least the emergence of drug resistance (Meng et al., 2010; Sen et al., 2012). In recent years, considering the severe consequences of antibiotics use on animal and human health, the search for alternatives has become a real challenge (Zhang and Kim, 2014). Clostridium butyricum (C. butyricum) is a Gram-positive anaerobe that can produce butyric acid and form spores, which exists in the intestines of healthy animals and humans (Juan et al., 2017). C. butyricum had been widely used in animal production, as it was indicated to improve growth performance, feed efficiency, and antioxidant capability in animals (Awad et al., 2009; Zhang et al., 2011; Duan et al., 2017). Similarly, experiments in ruminants have evidenced that C. butyricum addition can improve feed efficiency and enhance some digestive enzyme activities (Chilliard and Ferlay, 2004). Research has also shown that dietary supplementation with C. butyricum promotes the immune status and is of benefit to the intestinal flora of broilers (Awad et al., 2009; Zhao et al., 2013; Cheng et al., 2017c). Dietary supplementation with C. butyrium achieved similar or better results in terms of improving performance, promoting immune function, and benefiting cecal microflora in Escherichia. coli K88-challenged birds (Gao et al., 2012; Zhang et al., 2016). Furthermore, supplementation with synbiotics in the broiler diet that comprised probiotics (B. subtilis, B. licheniformis and C. butyricum) could increase meat quality, such as increasing the pH24h value (muscle pH value at 24 h postmortem), and decrease cooking loss in breast muscle (Hossain et al., 2016). In addition to regulatory functions on animal health, it has been reported that dietary supplementation with C. butyricum only could also improve meat quality in broilers, which may result from its modulation of nutrient digestibility and adsorption, as well as muscular fatty acid composition and antioxidant status (Liao et al., 2015; Zhang et al., 2017). C. butyricum therefore been proposed as a suitable alternative to antibiotics to improve the growth performance and immune status of cherry valley ducks (Zhuang et al., 2015). Nevertheless, it remains to be determined whether dietary C. butyricum supplementation can improve meat quality and flavor, and the amino acid and fatty acid composition of Peking Ducks. Therefore, our study was conducted to evaluate the effects of C. butyricum supplementation on the performance, serum lipid metabolism, muscle morphology, muscular antioxidant capacity, meat quality, and amino acid and fatty acid composition of breast meat in Peking ducks. We aimed to explore the possible beneficial effects of C. butyricum addition on Peking duck meat and determine whether C. butyricum could replace commonly used antibiotics as an alternative feed additive for widespread use in the duck industry for the production of healthier Peking duck meat with favorable for consumers. MATERIALS AND METHODS Ethics Statement The present study was approved by the ethics committee and conducted according to the Guidelines for Experimental Animals of China Agricultural University (Beijing, China). Dietary Treatments and Feeding A total of 1500 1-d-old male Peking ducks were purchased from a commercial hatchery (Beijing, China) and reared in 2-tier cages at the Experimental Center of China Agricultural University. The ducks were ad libitum divided into five groups (five replicates with sixty ducks each), and housed in an environmentally controlled house. The initial temperature was maintained at 35°C during the first week, and then gradually reduced as the birds aged until it reached 25°C. All ducks were allowed free access to feed and water, and exposed to 24 h of constant light throughout the whole experimental period. The control group was given a corn-soybean basal diet for 42 d. The nutrient levels of the diets were formulated to meet or exceed the standards (NRC, 1994) (Table 1). The other four groups were fed a basal diet supplemented with 200 mg/kg (low dose group, LG), 400 mg/kg (middle dose group, MG), or 600 mg/kg (high dose group, HG) of C. butyricum (2.0 × 109 CFU/g) or 150 mg of aureomycin/kg (antibiotic group, AG), respectively. The bacterial strain C. butyricum (batch No. 20,170,325,003) was provided by Beijing Shine Biology Technology Co., Ltd., China. Table 1. Composition and nutrient levels of the basal diets (air-dry basis). Items  1–21 d  22–42 d  Ingredients (%)  Corn  56.00  60.24  Soybean meal  32.69  24.67  Wheat middling  5.00  9.00  Soybean oil  2.10  1.80  Phytases  0.02  0.02  Dicalcium phosphate  1.00  1.60  Limestone  1.50  1.20  DL-Methionine  0.15  0.12  L-Lysine  0.20  0.10  Vitamin premix1  0.02  0.02  Trace mineral premix2  0.20  0.20  NaCl  0.35  0.30  Choline chloride (50%)  0.24  0.20  Ethoxyquin (33%)  0.03  0.03  Maifanite  0.50  0.50  Total  100  100  Nutrient levels3 (%)  ME (MJ/kg)  12.31  12.53  Crude protein (%)  19.52  16.83  Lysine (%)  1.12  0.87  Methionine (%)  0.46  0.39  Calcium (%)  0.88  0.89  Available phosphorus (%)  0.29  0.39  Total phosphorus (%)  0.54  0.62  Methionine+Cysteine (%)  0.79  0.69  Items  1–21 d  22–42 d  Ingredients (%)  Corn  56.00  60.24  Soybean meal  32.69  24.67  Wheat middling  5.00  9.00  Soybean oil  2.10  1.80  Phytases  0.02  0.02  Dicalcium phosphate  1.00  1.60  Limestone  1.50  1.20  DL-Methionine  0.15  0.12  L-Lysine  0.20  0.10  Vitamin premix1  0.02  0.02  Trace mineral premix2  0.20  0.20  NaCl  0.35  0.30  Choline chloride (50%)  0.24  0.20  Ethoxyquin (33%)  0.03  0.03  Maifanite  0.50  0.50  Total  100  100  Nutrient levels3 (%)  ME (MJ/kg)  12.31  12.53  Crude protein (%)  19.52  16.83  Lysine (%)  1.12  0.87  Methionine (%)  0.46  0.39  Calcium (%)  0.88  0.89  Available phosphorus (%)  0.29  0.39  Total phosphorus (%)  0.54  0.62  Methionine+Cysteine (%)  0.79  0.69  1The vitamin premix provided the following per kilogram of diet: vitamin A, 12,500 IU; vitamin D3, 3500 IU; vitamin E, 20 IU; vitamin K3, 2.65 mg; thiamin, 2.00 mg; riboflavin, 6.00 mg; pyridoxin, 3.00 mg; VB12, 0.025 mg; biotin, 0.0325 mg; folic acid, 12.00 mg; pantothenic acid, 50 mg; nicotinic acid, 50.00 mg. 2The mineral premix provided the following per kg of diet: Cu, 6 mg; Fe, 80 mg; Zn, 40 mg; Mn, 100 mg; Se, 0.15 mg; I, 0.35 mg. 3Calculated values. View Large Table 1. Composition and nutrient levels of the basal diets (air-dry basis). Items  1–21 d  22–42 d  Ingredients (%)  Corn  56.00  60.24  Soybean meal  32.69  24.67  Wheat middling  5.00  9.00  Soybean oil  2.10  1.80  Phytases  0.02  0.02  Dicalcium phosphate  1.00  1.60  Limestone  1.50  1.20  DL-Methionine  0.15  0.12  L-Lysine  0.20  0.10  Vitamin premix1  0.02  0.02  Trace mineral premix2  0.20  0.20  NaCl  0.35  0.30  Choline chloride (50%)  0.24  0.20  Ethoxyquin (33%)  0.03  0.03  Maifanite  0.50  0.50  Total  100  100  Nutrient levels3 (%)  ME (MJ/kg)  12.31  12.53  Crude protein (%)  19.52  16.83  Lysine (%)  1.12  0.87  Methionine (%)  0.46  0.39  Calcium (%)  0.88  0.89  Available phosphorus (%)  0.29  0.39  Total phosphorus (%)  0.54  0.62  Methionine+Cysteine (%)  0.79  0.69  Items  1–21 d  22–42 d  Ingredients (%)  Corn  56.00  60.24  Soybean meal  32.69  24.67  Wheat middling  5.00  9.00  Soybean oil  2.10  1.80  Phytases  0.02  0.02  Dicalcium phosphate  1.00  1.60  Limestone  1.50  1.20  DL-Methionine  0.15  0.12  L-Lysine  0.20  0.10  Vitamin premix1  0.02  0.02  Trace mineral premix2  0.20  0.20  NaCl  0.35  0.30  Choline chloride (50%)  0.24  0.20  Ethoxyquin (33%)  0.03  0.03  Maifanite  0.50  0.50  Total  100  100  Nutrient levels3 (%)  ME (MJ/kg)  12.31  12.53  Crude protein (%)  19.52  16.83  Lysine (%)  1.12  0.87  Methionine (%)  0.46  0.39  Calcium (%)  0.88  0.89  Available phosphorus (%)  0.29  0.39  Total phosphorus (%)  0.54  0.62  Methionine+Cysteine (%)  0.79  0.69  1The vitamin premix provided the following per kilogram of diet: vitamin A, 12,500 IU; vitamin D3, 3500 IU; vitamin E, 20 IU; vitamin K3, 2.65 mg; thiamin, 2.00 mg; riboflavin, 6.00 mg; pyridoxin, 3.00 mg; VB12, 0.025 mg; biotin, 0.0325 mg; folic acid, 12.00 mg; pantothenic acid, 50 mg; nicotinic acid, 50.00 mg. 2The mineral premix provided the following per kg of diet: Cu, 6 mg; Fe, 80 mg; Zn, 40 mg; Mn, 100 mg; Se, 0.15 mg; I, 0.35 mg. 3Calculated values. View Large Measurements Performance Parameters. Body weight (BW) and feed intake were recorded for each replicate at 42 d of age. Average body weight (ABW), along with average daily gain (ADG), average daily feed intake (ADFI), and the feed conversion ratio (FCR) were calculated. Sample Collection. Ducks (12 birds per group) with a BW similar to the mean BW of each replicates were selected at 42 d. Blood samples were taken aseptically from the jugular vein and were centrifuged at 4000 × g for 10 min. Serum was separated and stored at –20°C for further analysis. All ducks were euthanized after sodium pentobarbitone (50 mg/kg BW) anesthesia. Mid-segments of breast muscle were collected and cut into section fixed in 4% paraformaldehyde solution for morphology measurements. The breast muscle from the same side carcass was removed according to the standard method of dissection. They were divided into two parts respectively: one was quickly frozen at –20°C for later determination of fatty acid composition, and another stored at 4°C was used to evaluate meat quality. The breast muscles (100 mg) were homogenized using a glass Teflon homogenizer with 900 ml of cold 0.9% NaCl solution (1:9). The homogenates were centrifuged at 3500–4000 g for 10 min at 4°C. The supernatants were collected for antioxidant assays. Lipid Metabolites and Antioxidant Parameters. The serum lipid metabolites of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, very-low density lipoprotein (v-LDL), and non-esterified fatty acid (NEFA), and the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX), as well as concentrations of glutathione (GSH) and malondialdehyde (MDA) in breast muscle, were measured using the corresponding commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) following manufacturer's instructions. Meat Quality. Initial pH45 min, middle pH24 h, and ultimate pH48 h post-mortem were measured using a Testo 206 pH meter (Testo Limited, Alton, Hampshire, UK) according to the manufacturer's recommendations. The meat color (L* = lightness; a* = redness; and b* = yellowness) was measured at 24 h post-mortem by a spectrocolorimeter (model WSCS, Shanghai Shenguang Ltd., China) using the CIELAB system according to the manufacturer's recommendations. The shear force was determined using the Warner-Bratzler shear method as described (Jiang et al., 2011; Kiarie et al., 2014). An additional 20 g of meat sample was placed in a polyethylene container and the drip loss was evaluated after 24 h of storage at 4°C (Taylor and Dant, 1971; Young et al., 2004). The drip loss at 24 h post-mortem was calculated as a percentage of the initial weight. Muscle Morphology. Tissue samples were freshly prepared and fixed in 4% paraformaldehyde solution for 24 h. The fixed muscles were dehydrated using a graded series of ethanol, cleared using xylene, and then embedded in paraffin. Sections (4 μm thick) from transverse specimens of the Control, MG, and AG were stained with the standard hematoxylin-eosin (HE) method to observe muscle morphology (Zhu et al., 2009). The HE stained sections were analyzed under an optical microscopy (XSP-BM16C, Optical Instrument Factory, Shanghai, China). Oil Red O Staining and Intramuscular Fat Content. Frozen section of breast muscle was made using a Leica kryostat (Leica CM3050S, Leica instrument GmbH, Germany), fixed with 10% paraformaldehyde for 30 min, incubated with Oil Red O solution for 15 min, then the distribution of fat between muscle tissue was observed using a light microscopy. The staining was assessed by Beijing Epsilon Bio-technology Co., Ltd, Beijing, China. The intramuscular fat (IMF) and inosine acid (IMP) contents were determined as described elsewhere (Zerehdaran et al., 2004; Cui et al., 2012). The results are expressed as a percentage of the wet weight of the breast muscle. Amino Acid and Fatty Acid Analysis. The contents of 17 types of amino acids were measured in the freeze-dried, fat-free meat samples by ion-exchange chromatography of the acidhydrolyzed protein. Each sample was placed in a sealed tube and treated with 6 M HCl in an oil bath at 110°C for 32 h. Sodium citrate buffers were used to separate the amino acids before assessment on a Beckman amino acid analyzer (Model 6300) (Elgasim and Alkanhal, 1992). The fatty acid composition of breast muscles was measured by gas chromatography (Trace GC Ultra, GC-2010 Shimadzu, Tokyo, Japan) (Folch et al., 1957). Total lipid extracts were transmethylated into fatty acid methyl esters and separated using a HP 6890 gas chromatograph equipped with a flame-ionized detector and a DB-23 capillary column (0.25 mm × 60 m × 0.25 μm; Supelco, Bellefonte, PA, USA). The column, injector, and detector temperatures were set as 195°C, 225°C and 250°C, respectively. Helium was used at a flow rate of 1.5 cm3/min. Nonadecanoic acid (C19:0, Buchs, Switzerland) was used as the internal standard. Fatty acids were evaluated by comparing their retention times with authentic standards. The concentrations were performed as milligrams per 100 g. Statistical Analysis The data were analyzed using one-way ANOVA and Duncan's multiple test for multiple comparisons by the SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). Results were presented as means with their SEM. A value of P < 0.05 was indicated to be statistically significant. RESULTS Performance All ducks appeared healthy throughout the entire experimental period. In MG and AG the ducks showed greater (P < 0.05) AMB at 42 d compared with those in the control group. Ducks fed with C. butyricum or antibiotics showed improved ADG (P < 0.01) compared with the control group. ADFI in the LG, MG, and AG (P < 0.05), and FCR in the LG, MG, HG, and AG were lower (P < 0.01) than those in the control group from 1–42 d (Table 2). Table 2. Effects of Clostridium butyricum on growth performance of Pekin ducks at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  AMB (kg)  2.96a  3.07a,c  3.24b,d  3.08a,c  3.12c,d  0.028  0.019  ADG (g/d)  77.4a  79.6b  81.8c  80.1d  78.9e  0.390  <0.001  ADFI (g/d)  152.0a  146.0b  144.0b  151.0a  144.0b  1.018  0.001  FCR (g/g)  1.96a  1.83b  1.76c  1.88d  1.82b  0.019  <0.001  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  AMB (kg)  2.96a  3.07a,c  3.24b,d  3.08a,c  3.12c,d  0.028  0.019  ADG (g/d)  77.4a  79.6b  81.8c  80.1d  78.9e  0.390  <0.001  ADFI (g/d)  152.0a  146.0b  144.0b  151.0a  144.0b  1.018  0.001  FCR (g/g)  1.96a  1.83b  1.76c  1.88d  1.82b  0.019  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3ABW: average body weight, ADG: average daily gain, ADFI: average daily feed intake, FCR: feed conversion ratio. View Large Table 2. Effects of Clostridium butyricum on growth performance of Pekin ducks at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  AMB (kg)  2.96a  3.07a,c  3.24b,d  3.08a,c  3.12c,d  0.028  0.019  ADG (g/d)  77.4a  79.6b  81.8c  80.1d  78.9e  0.390  <0.001  ADFI (g/d)  152.0a  146.0b  144.0b  151.0a  144.0b  1.018  0.001  FCR (g/g)  1.96a  1.83b  1.76c  1.88d  1.82b  0.019  <0.001  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  AMB (kg)  2.96a  3.07a,c  3.24b,d  3.08a,c  3.12c,d  0.028  0.019  ADG (g/d)  77.4a  79.6b  81.8c  80.1d  78.9e  0.390  <0.001  ADFI (g/d)  152.0a  146.0b  144.0b  151.0a  144.0b  1.018  0.001  FCR (g/g)  1.96a  1.83b  1.76c  1.88d  1.82b  0.019  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3ABW: average body weight, ADG: average daily gain, ADFI: average daily feed intake, FCR: feed conversion ratio. View Large Lipid Metabolic Status The contents of LDL, v-LDL, and NEFA in all the C. butyricum-supplemented groups were higher than those in the control groups; but there were no significant differences between C. butyricum-supplemented groups and the control group in terms of LDL, v-LDL, and NEFA concentrations in the serum at 42 d. However, the contents of TC in the LG, HG, and AG were higher than in the control group (P < 0.05). The concentration of serum HDL decreased significantly (P < 0.05) only in the MG at 42 d, whereas the concentration of TG in the serum in both the HG and AG tended to decrease at 42 d (Figure 1). Figure 1. View largeDownload slide Effects of Clostridium butyricum on serum lipid metabolism in Pekin duck at 42 d of age. Values are mean ± SD of 12 independent determinations; the superscript different letters indicate that there are significant differences (P < 0.05) between any two groups. TC: total cholesterol; TG: triglyceride; HDL: high-density lipoprotein cholesterol; LDL: low-density lipoprotein cholesterol; v-LDL: very low-density lipoprotein cholesterol; NEFA: non-esterified fatty acid. Figure 1. View largeDownload slide Effects of Clostridium butyricum on serum lipid metabolism in Pekin duck at 42 d of age. Values are mean ± SD of 12 independent determinations; the superscript different letters indicate that there are significant differences (P < 0.05) between any two groups. TC: total cholesterol; TG: triglyceride; HDL: high-density lipoprotein cholesterol; LDL: low-density lipoprotein cholesterol; v-LDL: very low-density lipoprotein cholesterol; NEFA: non-esterified fatty acid. Muscular Oxidative Status Overall, the T-AOC was significantly higher (P < 0.05) with the MG diet whereas significantly lower (P < 0.05) in the MG and AG diets as compared to the control group; the same trends were observed for muscular T-SOD. The activities of muscular GSH-PX and CAT were significantly higher (P < 0.05) with the MG diet than with the other diets and the control group. The contents of muscular GSH increased significantly (P < 0.05) in the LG, MG, and AG diets compared with the control group. Supplementation of C. butyricum didn’t influence the accumulation of MDA in the duck breast muscle at 42 d significantly (P = 0.104) (Table 3). Table 3. Muscular antioxidant status in Pekin duck at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  T-AOC (U/mg prot)  1.08a  1.08a  1.27b  0.85c  0.59d  0.049  <0.001  T-SOD (U/mg prot)  8.05a  7.86a  9.40b  6.45c  5.48c  0.303  <0.001  GSH-PX (U/mg prot)  85.02a  115.53b,c  122.93b,d  104.81a,c,d  115.99b,d  4.175  0.026  CAT (U/mg prot)  7.04a  7.36a,c  7.79b,c  7.62a,c  10.16d  1.261  <0.001  GSH (mg/g prot)  0.77a  1.04b,c  1.07b  0.88a,c  1.11b  0.035  0.015  MDA (nmol/mg prot)  0.312  0.197  0.225  0.254  0.273  0.014  0.104  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  T-AOC (U/mg prot)  1.08a  1.08a  1.27b  0.85c  0.59d  0.049  <0.001  T-SOD (U/mg prot)  8.05a  7.86a  9.40b  6.45c  5.48c  0.303  <0.001  GSH-PX (U/mg prot)  85.02a  115.53b,c  122.93b,d  104.81a,c,d  115.99b,d  4.175  0.026  CAT (U/mg prot)  7.04a  7.36a,c  7.79b,c  7.62a,c  10.16d  1.261  <0.001  GSH (mg/g prot)  0.77a  1.04b,c  1.07b  0.88a,c  1.11b  0.035  0.015  MDA (nmol/mg prot)  0.312  0.197  0.225  0.254  0.273  0.014  0.104  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3T-AOC: total antioxidant capacity; T-SOD: total superoxide dismutase; GSH-PX: glutathione peroxidase; CAT: catalase; GSH: glutathione; MDA: malonaldehyde. View Large Table 3. Muscular antioxidant status in Pekin duck at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  T-AOC (U/mg prot)  1.08a  1.08a  1.27b  0.85c  0.59d  0.049  <0.001  T-SOD (U/mg prot)  8.05a  7.86a  9.40b  6.45c  5.48c  0.303  <0.001  GSH-PX (U/mg prot)  85.02a  115.53b,c  122.93b,d  104.81a,c,d  115.99b,d  4.175  0.026  CAT (U/mg prot)  7.04a  7.36a,c  7.79b,c  7.62a,c  10.16d  1.261  <0.001  GSH (mg/g prot)  0.77a  1.04b,c  1.07b  0.88a,c  1.11b  0.035  0.015  MDA (nmol/mg prot)  0.312  0.197  0.225  0.254  0.273  0.014  0.104  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  T-AOC (U/mg prot)  1.08a  1.08a  1.27b  0.85c  0.59d  0.049  <0.001  T-SOD (U/mg prot)  8.05a  7.86a  9.40b  6.45c  5.48c  0.303  <0.001  GSH-PX (U/mg prot)  85.02a  115.53b,c  122.93b,d  104.81a,c,d  115.99b,d  4.175  0.026  CAT (U/mg prot)  7.04a  7.36a,c  7.79b,c  7.62a,c  10.16d  1.261  <0.001  GSH (mg/g prot)  0.77a  1.04b,c  1.07b  0.88a,c  1.11b  0.035  0.015  MDA (nmol/mg prot)  0.312  0.197  0.225  0.254  0.273  0.014  0.104  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3T-AOC: total antioxidant capacity; T-SOD: total superoxide dismutase; GSH-PX: glutathione peroxidase; CAT: catalase; GSH: glutathione; MDA: malonaldehyde. View Large Quality of Breast Muscle No significant differences were found among the treatment groups for pH24 h and pH48 h, whereas the pH45 min of the breast muscle was significantly higher in the ducks fed with the LG, MG, HG, and AG diets compared with the control (P < 0.01). The lightness of meat color of breast muscle was significantly lower (P < 0.05) with the HG diet in comparison with the control group. A higher (P < 0.05) redness for meat color and a lower (P < 0.01) drip loss and shear force of breast muscle (P = 0.002) were measured for the C. butyricum-supplemented diet and the antibiotic-supplemented diet as compared to the control group. Compared with the no C. butyricum-supplemented diet, the yellowness of meat color presented no differences (P = 0.280). (Table 4) Table 4. Effects of Clostridium butyricum on breast meat quality in Pekin duck at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  pH45 min  6.22a  6.33b,d  6.37c  6.32b  6.36c,d  0.011  <0.001  pH24 h  5.89a,c  5.92a,d  5.90a,e  5.86c,e  5.97b,d  0.012  0.018  pH48 h  5.83  5.86  5.83  5.84  5.89  0.010  0.292  Lightness (L*)  47.40a,c  48.56a  45.99b,c  45.04b  46.61a,b,c  0.366  0.017  Redness (a*)  18.36a  18.46a,c  19.86b  19.42b,c  19.62b  0.181  0.010  Yellowness (b*)  6.51  5.45  5.22  5.32  5.73  0.204  0.280  Shear force (N/cm2)  45.80a  33.97b  31.14b  32.66b  36.78b  1.380  0.002  Drip loss (%)  10.76a  7.91b  8.09b  9.98a  7.51b  0.302  <0.001  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  pH45 min  6.22a  6.33b,d  6.37c  6.32b  6.36c,d  0.011  <0.001  pH24 h  5.89a,c  5.92a,d  5.90a,e  5.86c,e  5.97b,d  0.012  0.018  pH48 h  5.83  5.86  5.83  5.84  5.89  0.010  0.292  Lightness (L*)  47.40a,c  48.56a  45.99b,c  45.04b  46.61a,b,c  0.366  0.017  Redness (a*)  18.36a  18.46a,c  19.86b  19.42b,c  19.62b  0.181  0.010  Yellowness (b*)  6.51  5.45  5.22  5.32  5.73  0.204  0.280  Shear force (N/cm2)  45.80a  33.97b  31.14b  32.66b  36.78b  1.380  0.002  Drip loss (%)  10.76a  7.91b  8.09b  9.98a  7.51b  0.302  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3pH45 min: muscle pH value at 45 min postmortem; pH24 h: muscle pH value at 24 h postmortem; pH48 h: muscle pH value at 48 h postmortem. View Large Table 4. Effects of Clostridium butyricum on breast meat quality in Pekin duck at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  pH45 min  6.22a  6.33b,d  6.37c  6.32b  6.36c,d  0.011  <0.001  pH24 h  5.89a,c  5.92a,d  5.90a,e  5.86c,e  5.97b,d  0.012  0.018  pH48 h  5.83  5.86  5.83  5.84  5.89  0.010  0.292  Lightness (L*)  47.40a,c  48.56a  45.99b,c  45.04b  46.61a,b,c  0.366  0.017  Redness (a*)  18.36a  18.46a,c  19.86b  19.42b,c  19.62b  0.181  0.010  Yellowness (b*)  6.51  5.45  5.22  5.32  5.73  0.204  0.280  Shear force (N/cm2)  45.80a  33.97b  31.14b  32.66b  36.78b  1.380  0.002  Drip loss (%)  10.76a  7.91b  8.09b  9.98a  7.51b  0.302  <0.001  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  pH45 min  6.22a  6.33b,d  6.37c  6.32b  6.36c,d  0.011  <0.001  pH24 h  5.89a,c  5.92a,d  5.90a,e  5.86c,e  5.97b,d  0.012  0.018  pH48 h  5.83  5.86  5.83  5.84  5.89  0.010  0.292  Lightness (L*)  47.40a,c  48.56a  45.99b,c  45.04b  46.61a,b,c  0.366  0.017  Redness (a*)  18.36a  18.46a,c  19.86b  19.42b,c  19.62b  0.181  0.010  Yellowness (b*)  6.51  5.45  5.22  5.32  5.73  0.204  0.280  Shear force (N/cm2)  45.80a  33.97b  31.14b  32.66b  36.78b  1.380  0.002  Drip loss (%)  10.76a  7.91b  8.09b  9.98a  7.51b  0.302  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3pH45 min: muscle pH value at 45 min postmortem; pH24 h: muscle pH value at 24 h postmortem; pH48 h: muscle pH value at 48 h postmortem. View Large Muscle Morphology Muscle morphology is observed in Figure 2. In the Control group, the muscle fibers were disordered with uneven thickness. The fibers were degeneration and the spaces between muscle fibers were widened. In MG group, the muscle fibers are arranged in a wavy pattern, with uniform thickness and tightness. In the AG group, the muscle fibers were slightly disordered with the uneven thickness, and the fibers became thin. Figure 2. View largeDownload slide The microstructure of the breast muscle of Pekin ducks at 42 d of age (N = 6). Control 1: magnification × 100; Control 2: magnification × 200; Control 3: magnification × 400. MG1: magnification × 100; MG2: magnification × 200; MG3: magnification × 400. AG1: magnification × 100; AG2: magnification × 200; AG3: magnification × 400. Figure 2. View largeDownload slide The microstructure of the breast muscle of Pekin ducks at 42 d of age (N = 6). Control 1: magnification × 100; Control 2: magnification × 200; Control 3: magnification × 400. MG1: magnification × 100; MG2: magnification × 200; MG3: magnification × 400. AG1: magnification × 100; AG2: magnification × 200; AG3: magnification × 400. IMF and IMP Contents In the Oil Red O stained tissue, an increased number of red lipid droplets were observed in the MG compared with the control group or the AG. This indicated that addition of C. butyricum can improve the quantity of IMF between the muscle fibers or muscle cells (Figure 3). Figure 3. View largeDownload slide The Oil Red O stain of breast muscle fibril of Pekin ducks at 42 d of age (N = 6). Control 1: magnification × 100; Control 2: magnification × 200; Control 3: magnification × 400. MG1: magnification × 100; MG2: magnification × 200; MG3: magnification × 400. AG1: magnification × 100; AG2: magnification × 200; AG3: magnification × 400. Figure 3. View largeDownload slide The Oil Red O stain of breast muscle fibril of Pekin ducks at 42 d of age (N = 6). Control 1: magnification × 100; Control 2: magnification × 200; Control 3: magnification × 400. MG1: magnification × 100; MG2: magnification × 200; MG3: magnification × 400. AG1: magnification × 100; AG2: magnification × 200; AG3: magnification × 400. The contents of IMP were significantly increased (P < 0.01) in response to the MG and HG diets and the AG diet compared with those fed with the control diet. Similarly, the contents of IMF were significantly higher (P < 0.01) in response to the MG and HG diets compared with the control group. No significant differences (P > 0.05) were found between the AG diet and the control group diet (Figure 4). Figure 4. View largeDownload slide Contents of IMP and IMF in breast muscle of Pekin duck at 42 d of age (N = 6). Values are mean ± SD of 12 independent determinations; the superscript different letters indicate that there are significant differences (P < 0.05) between any two groups. IMP: inosine acid; IMF: intramuscular fat. Figure 4. View largeDownload slide Contents of IMP and IMF in breast muscle of Pekin duck at 42 d of age (N = 6). Values are mean ± SD of 12 independent determinations; the superscript different letters indicate that there are significant differences (P < 0.05) between any two groups. IMP: inosine acid; IMF: intramuscular fat. Amino Acid and Fatty Acid Composition The effect of C. butyricum dietary supplementation on the animo acid profile of duck breast muscle is presented in Table 5. Significant differences (P < 0.05) were observed in some animo acid concentrations in the supplemented groups compared with control groups. For total amino acids (TAA), the TAA concentrations significantly increased (P < 0.05) when fed with the MG, HG, and AG diets compared with the control diet, whereas no significant differences (P > 0.05) were found in the contents of other essential amino acids (EAA) among all of the treatments. Interestingly, with MG and HG diets, the contents of glycine, tyrosine, and the total flavor amino acids (FAA, including glycine, alanine, aspartic acid, glutamic acid, phenylalanine, and tyrosine) significantly increased (P < 0.05) compared with the control diet but no significant differences (P > 0.05) were found in the other four FAA contents among all of the treatments. Table 5. Effects of Clostridium butyricum on amino acid composition in breast muscle of Pekin duck at 42 d of age.2 Parameters3  Groups      (ng/mg)  Control  LG  MG  HG  AG  SEM1  P value  Glycine  134.79a  110.67b  169.21c  156.66c  102.26b  7.292  <0.001  Alanine  419.05  334.67  420.03  372.13  377.65  11.741  0.082  Serine  151.02a  132.19b  196.82c  168.21d  186.94c  6.584  <0.001  Proline  157.90a,c  175.59b  157.14c  180.30b  91.64d  8.536  <0.001  Valine  76.00a  71.11a  77.49a  76.81a  102.28a  3.145  0.002  Cysteine  82.37a  79.71a  110.84b  103.52c  110.93b  3.712  <0.001  Isoleucine  2.40a  2.52a  7.23b  3.26a  3.19a  0.507  <0.001  Asparagine  42.24a,c  36.83a  44.12c  44.29c  67.02b  2.917  0.001  Aspartic acid  40.57a  33.72b  44.09a  43.23a  45.74a  1.108  0.017  Glutamine  88.48a,d  73.53a,c  60.34b,c  98.56d  89.72a,d  4.378  0.014  Glutamic acid  377.30  351.00  444.43  373.86  427.94  13.891  0.151  Methionine  93.74a  65.80a,c  136.54b  60.07c  88.71a,c  8.218  0.003  Histidine  36.87a  36.79a  42.72a  39.60a  51.45b  1.731  0.010  Phenylalanine  42.65a  31.67b  46.46a  44.03a  44.69a  1.645  0.007  Arginine  58.06a,c  48.72a  60.69c  70.79b  77.39b  2.934  0.033  Tyrosine  184.67a  202.77a  336.94b  337.29b  199.34a  20.314  0.037  Tryptophan  14.45a,c  13.96a  16.18b,c  18.37d  20.97e  0.731  <0.001  Lysine  200.98  179.64  236.86  143.01  192.84  12.659  0.213  Leucine  51.15a  52.54a  79.19b  67.91c  90.76d  4.189  <0.001  Threonine  91.02a,c  80.18a,d  97.04b,c  96.31b,c  75.43d  2.823  0.015  FAA  1199.04a  1064.50b  1461.15c  1327.20d  1197.62a  38.939  <0.001  EAA  683.05  592.01  840.18  557.60  688.46  44.892  0.898  TAA  2345.79a  2113.62b  2784.37c  2498.37a  2446.90a  63.857  0.023  Parameters3  Groups      (ng/mg)  Control  LG  MG  HG  AG  SEM1  P value  Glycine  134.79a  110.67b  169.21c  156.66c  102.26b  7.292  <0.001  Alanine  419.05  334.67  420.03  372.13  377.65  11.741  0.082  Serine  151.02a  132.19b  196.82c  168.21d  186.94c  6.584  <0.001  Proline  157.90a,c  175.59b  157.14c  180.30b  91.64d  8.536  <0.001  Valine  76.00a  71.11a  77.49a  76.81a  102.28a  3.145  0.002  Cysteine  82.37a  79.71a  110.84b  103.52c  110.93b  3.712  <0.001  Isoleucine  2.40a  2.52a  7.23b  3.26a  3.19a  0.507  <0.001  Asparagine  42.24a,c  36.83a  44.12c  44.29c  67.02b  2.917  0.001  Aspartic acid  40.57a  33.72b  44.09a  43.23a  45.74a  1.108  0.017  Glutamine  88.48a,d  73.53a,c  60.34b,c  98.56d  89.72a,d  4.378  0.014  Glutamic acid  377.30  351.00  444.43  373.86  427.94  13.891  0.151  Methionine  93.74a  65.80a,c  136.54b  60.07c  88.71a,c  8.218  0.003  Histidine  36.87a  36.79a  42.72a  39.60a  51.45b  1.731  0.010  Phenylalanine  42.65a  31.67b  46.46a  44.03a  44.69a  1.645  0.007  Arginine  58.06a,c  48.72a  60.69c  70.79b  77.39b  2.934  0.033  Tyrosine  184.67a  202.77a  336.94b  337.29b  199.34a  20.314  0.037  Tryptophan  14.45a,c  13.96a  16.18b,c  18.37d  20.97e  0.731  <0.001  Lysine  200.98  179.64  236.86  143.01  192.84  12.659  0.213  Leucine  51.15a  52.54a  79.19b  67.91c  90.76d  4.189  <0.001  Threonine  91.02a,c  80.18a,d  97.04b,c  96.31b,c  75.43d  2.823  0.015  FAA  1199.04a  1064.50b  1461.15c  1327.20d  1197.62a  38.939  <0.001  EAA  683.05  592.01  840.18  557.60  688.46  44.892  0.898  TAA  2345.79a  2113.62b  2784.37c  2498.37a  2446.90a  63.857  0.023  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3FAA: flavor amino acid; EAA: essential amino acid; TAA: total amino acids. View Large Table 5. Effects of Clostridium butyricum on amino acid composition in breast muscle of Pekin duck at 42 d of age.2 Parameters3  Groups      (ng/mg)  Control  LG  MG  HG  AG  SEM1  P value  Glycine  134.79a  110.67b  169.21c  156.66c  102.26b  7.292  <0.001  Alanine  419.05  334.67  420.03  372.13  377.65  11.741  0.082  Serine  151.02a  132.19b  196.82c  168.21d  186.94c  6.584  <0.001  Proline  157.90a,c  175.59b  157.14c  180.30b  91.64d  8.536  <0.001  Valine  76.00a  71.11a  77.49a  76.81a  102.28a  3.145  0.002  Cysteine  82.37a  79.71a  110.84b  103.52c  110.93b  3.712  <0.001  Isoleucine  2.40a  2.52a  7.23b  3.26a  3.19a  0.507  <0.001  Asparagine  42.24a,c  36.83a  44.12c  44.29c  67.02b  2.917  0.001  Aspartic acid  40.57a  33.72b  44.09a  43.23a  45.74a  1.108  0.017  Glutamine  88.48a,d  73.53a,c  60.34b,c  98.56d  89.72a,d  4.378  0.014  Glutamic acid  377.30  351.00  444.43  373.86  427.94  13.891  0.151  Methionine  93.74a  65.80a,c  136.54b  60.07c  88.71a,c  8.218  0.003  Histidine  36.87a  36.79a  42.72a  39.60a  51.45b  1.731  0.010  Phenylalanine  42.65a  31.67b  46.46a  44.03a  44.69a  1.645  0.007  Arginine  58.06a,c  48.72a  60.69c  70.79b  77.39b  2.934  0.033  Tyrosine  184.67a  202.77a  336.94b  337.29b  199.34a  20.314  0.037  Tryptophan  14.45a,c  13.96a  16.18b,c  18.37d  20.97e  0.731  <0.001  Lysine  200.98  179.64  236.86  143.01  192.84  12.659  0.213  Leucine  51.15a  52.54a  79.19b  67.91c  90.76d  4.189  <0.001  Threonine  91.02a,c  80.18a,d  97.04b,c  96.31b,c  75.43d  2.823  0.015  FAA  1199.04a  1064.50b  1461.15c  1327.20d  1197.62a  38.939  <0.001  EAA  683.05  592.01  840.18  557.60  688.46  44.892  0.898  TAA  2345.79a  2113.62b  2784.37c  2498.37a  2446.90a  63.857  0.023  Parameters3  Groups      (ng/mg)  Control  LG  MG  HG  AG  SEM1  P value  Glycine  134.79a  110.67b  169.21c  156.66c  102.26b  7.292  <0.001  Alanine  419.05  334.67  420.03  372.13  377.65  11.741  0.082  Serine  151.02a  132.19b  196.82c  168.21d  186.94c  6.584  <0.001  Proline  157.90a,c  175.59b  157.14c  180.30b  91.64d  8.536  <0.001  Valine  76.00a  71.11a  77.49a  76.81a  102.28a  3.145  0.002  Cysteine  82.37a  79.71a  110.84b  103.52c  110.93b  3.712  <0.001  Isoleucine  2.40a  2.52a  7.23b  3.26a  3.19a  0.507  <0.001  Asparagine  42.24a,c  36.83a  44.12c  44.29c  67.02b  2.917  0.001  Aspartic acid  40.57a  33.72b  44.09a  43.23a  45.74a  1.108  0.017  Glutamine  88.48a,d  73.53a,c  60.34b,c  98.56d  89.72a,d  4.378  0.014  Glutamic acid  377.30  351.00  444.43  373.86  427.94  13.891  0.151  Methionine  93.74a  65.80a,c  136.54b  60.07c  88.71a,c  8.218  0.003  Histidine  36.87a  36.79a  42.72a  39.60a  51.45b  1.731  0.010  Phenylalanine  42.65a  31.67b  46.46a  44.03a  44.69a  1.645  0.007  Arginine  58.06a,c  48.72a  60.69c  70.79b  77.39b  2.934  0.033  Tyrosine  184.67a  202.77a  336.94b  337.29b  199.34a  20.314  0.037  Tryptophan  14.45a,c  13.96a  16.18b,c  18.37d  20.97e  0.731  <0.001  Lysine  200.98  179.64  236.86  143.01  192.84  12.659  0.213  Leucine  51.15a  52.54a  79.19b  67.91c  90.76d  4.189  <0.001  Threonine  91.02a,c  80.18a,d  97.04b,c  96.31b,c  75.43d  2.823  0.015  FAA  1199.04a  1064.50b  1461.15c  1327.20d  1197.62a  38.939  <0.001  EAA  683.05  592.01  840.18  557.60  688.46  44.892  0.898  TAA  2345.79a  2113.62b  2784.37c  2498.37a  2446.90a  63.857  0.023  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3FAA: flavor amino acid; EAA: essential amino acid; TAA: total amino acids. View Large Significant differences (P < 0.05) were observed in the concentrations of several fatty acids among the different treatment groups compared with control groups. For saturated fatty acid (SFA), the total contents were significantly lower (P < 0.05) in the ducks fed with LG and MG diets but were higher (P < 0.05) with the AG diet in comparison with the control diet. Palmitic acid (C16:0), stearic acid (C18:0), and docosanoic acid (C22:0) were significantly decreased with the MG diet compared with control diet. However, the other SFAs showed no significant differences (P > 0.05) among all of the treatment groups. For monounsaturated fatty acids (MUFA), the contents of myristic dilute acid (C14:1), oleic acid (C18:1n-9c), gondoic acid (C20:1n-9), and total MUFA were significantly higher (P < 0.05) in the MG compared to control group. For PUFA, the contents of eicosadienoic acid (C20:2n-6), dihomo-gammalinolenic acid (DGLA: C20:3n-6), arachidonic acid (AA: C20:4n-6), eicosapentaenoic acid (EPA: C20:5n-3), docosahexaenoic acid (DHA: C22:6n-3), and the total PUFA content significantly increased (P < 0.05) in the ducks fed with MG diet compared with the control diet. By contrast, the contents of other unsaturated fatty acids showed no significant differences (P > 0.05) in breast muscle among all of the five groups (Table 6). Table 6. Effects of Clostridium butyricum on fatty acid composition in breast muscle of Pekin ducks at 42 d of age.2 Parameters3  Groups      (μg/g)  Control  LG  MG  HG  AG  SEM1  P value  C10:0  17.57a,c  18.25a,d  17.74a,e  17.27c,e  18.92b,d  0.179  0.005  C11:0  16.23a  16.59a  15.02b  16.12a  21.28c  0.592  <0.001  C12:0  19.76a,c  20.56a,d  19.95a,e  19.45c,e  21.30b,d  0.204  0.018  C13:0  21.11a,c  20.79a,d  20.31a,e  19.77c,e  21.68b,d  0.208  0.011  C14:0  22.32  23.25  22.17  22.69  24.16  0.271  0.098  C15:0  20.45a,c  21.21a,d  20.53a,e  20.20c,e  21.81b,d  0.196  0.024  C16:0  134.82a  114.43b  114.39b  156.52c  180.30d  7.185  <0.001  C17:0  20.40  21.48  20.37  20.17  21.31  0.212  0.150  C18:0  238.16  217.63  218.89  252.39  290.63  10.792  0.174  C20:0  17.27  17.85  16.79  16.92  18.16  0.228  0.259  C22:0  17.02  17.76  16.62  16.63  17.79  0.199  0.120  C24:0  20.88a,c  21.59a,d  20.66a,e  20.49c,e  22.38b,d  0.227  0.017  ⊙SFA  564.98a  531.39a  523.78a  598.61a,c  679.67b,c  18.366  0.015  C14:1  9.00a  9.51b  9.63b  8.85a  9.01a  0.091  0.001  C16:1  23.02  23.38  33.96  26.92  19.51  1.876  0.123  C18:1n-9  231.99a,d  371.60b,c  349.29a,c  207.71d  206.29d  23.900  0.023  C20:1  19.76  20.24  21.59  20.39  19.40  0.329  0.288  C22:1n-9  18.91a,d  19.57a,c  20.15b,c  18.63d  19.01a,d  0.182  0.026  ⊙MUFA  302.67a  444.31b  434.62b  282.50a  273.40a  25.427  0.027  C18:2n-6  206.05  145.42  250.05  231.43  164.51  18.287  0.354  C18:3n-6  17.56a,d  18.22a,c  18.77b,c  17.48d  17.71a,d  0.153  0.008  C18:3n-3  21.64  22.13  27.00  24.57  21.67  0.872  0.212  C20:2n-6  28.07a  26.09a  33.77b  27.39a  25.02a  1.035  0.035  C20:3n-6  30.15a,b  28.60a  35.52b  27.93a  26.38a  1.087  0.042  C20:4n-6  124.39  98.48  154.75  128.56  104.00  7.004  0.050  C20:3n-3  19.56  20.75  20.63  19.37  19.50  0.219  0.079  C20:5n-3  19.09a,d  20.26a,c  20.67b,c  18.58d  18.89d  0.267  0.018  C22:6n-3  21.78a,c  23.25a,d  24.20b,d  21.71c  21.16c  0.352  0.007  ⊙PUFA  488.32  403.21  618.61  517.02  418.84  28.602  0.053  PUFA/SFA  0.86a  0.76a  1.18b  0.85a  0.63a  0.054  0.001  n-3PUFA  82.10a  86.39a,c  92.43b,c  84.23a,d  81.22a,d  1.201  0.002  n-6PUFA  406.22a  316.82b  526.18c  432.79d  319.47b  21.080  <0.001  Parameters3  Groups      (μg/g)  Control  LG  MG  HG  AG  SEM1  P value  C10:0  17.57a,c  18.25a,d  17.74a,e  17.27c,e  18.92b,d  0.179  0.005  C11:0  16.23a  16.59a  15.02b  16.12a  21.28c  0.592  <0.001  C12:0  19.76a,c  20.56a,d  19.95a,e  19.45c,e  21.30b,d  0.204  0.018  C13:0  21.11a,c  20.79a,d  20.31a,e  19.77c,e  21.68b,d  0.208  0.011  C14:0  22.32  23.25  22.17  22.69  24.16  0.271  0.098  C15:0  20.45a,c  21.21a,d  20.53a,e  20.20c,e  21.81b,d  0.196  0.024  C16:0  134.82a  114.43b  114.39b  156.52c  180.30d  7.185  <0.001  C17:0  20.40  21.48  20.37  20.17  21.31  0.212  0.150  C18:0  238.16  217.63  218.89  252.39  290.63  10.792  0.174  C20:0  17.27  17.85  16.79  16.92  18.16  0.228  0.259  C22:0  17.02  17.76  16.62  16.63  17.79  0.199  0.120  C24:0  20.88a,c  21.59a,d  20.66a,e  20.49c,e  22.38b,d  0.227  0.017  ⊙SFA  564.98a  531.39a  523.78a  598.61a,c  679.67b,c  18.366  0.015  C14:1  9.00a  9.51b  9.63b  8.85a  9.01a  0.091  0.001  C16:1  23.02  23.38  33.96  26.92  19.51  1.876  0.123  C18:1n-9  231.99a,d  371.60b,c  349.29a,c  207.71d  206.29d  23.900  0.023  C20:1  19.76  20.24  21.59  20.39  19.40  0.329  0.288  C22:1n-9  18.91a,d  19.57a,c  20.15b,c  18.63d  19.01a,d  0.182  0.026  ⊙MUFA  302.67a  444.31b  434.62b  282.50a  273.40a  25.427  0.027  C18:2n-6  206.05  145.42  250.05  231.43  164.51  18.287  0.354  C18:3n-6  17.56a,d  18.22a,c  18.77b,c  17.48d  17.71a,d  0.153  0.008  C18:3n-3  21.64  22.13  27.00  24.57  21.67  0.872  0.212  C20:2n-6  28.07a  26.09a  33.77b  27.39a  25.02a  1.035  0.035  C20:3n-6  30.15a,b  28.60a  35.52b  27.93a  26.38a  1.087  0.042  C20:4n-6  124.39  98.48  154.75  128.56  104.00  7.004  0.050  C20:3n-3  19.56  20.75  20.63  19.37  19.50  0.219  0.079  C20:5n-3  19.09a,d  20.26a,c  20.67b,c  18.58d  18.89d  0.267  0.018  C22:6n-3  21.78a,c  23.25a,d  24.20b,d  21.71c  21.16c  0.352  0.007  ⊙PUFA  488.32  403.21  618.61  517.02  418.84  28.602  0.053  PUFA/SFA  0.86a  0.76a  1.18b  0.85a  0.63a  0.054  0.001  n-3PUFA  82.10a  86.39a,c  92.43b,c  84.23a,d  81.22a,d  1.201  0.002  n-6PUFA  406.22a  316.82b  526.18c  432.79d  319.47b  21.080  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid. View Large Table 6. Effects of Clostridium butyricum on fatty acid composition in breast muscle of Pekin ducks at 42 d of age.2 Parameters3  Groups      (μg/g)  Control  LG  MG  HG  AG  SEM1  P value  C10:0  17.57a,c  18.25a,d  17.74a,e  17.27c,e  18.92b,d  0.179  0.005  C11:0  16.23a  16.59a  15.02b  16.12a  21.28c  0.592  <0.001  C12:0  19.76a,c  20.56a,d  19.95a,e  19.45c,e  21.30b,d  0.204  0.018  C13:0  21.11a,c  20.79a,d  20.31a,e  19.77c,e  21.68b,d  0.208  0.011  C14:0  22.32  23.25  22.17  22.69  24.16  0.271  0.098  C15:0  20.45a,c  21.21a,d  20.53a,e  20.20c,e  21.81b,d  0.196  0.024  C16:0  134.82a  114.43b  114.39b  156.52c  180.30d  7.185  <0.001  C17:0  20.40  21.48  20.37  20.17  21.31  0.212  0.150  C18:0  238.16  217.63  218.89  252.39  290.63  10.792  0.174  C20:0  17.27  17.85  16.79  16.92  18.16  0.228  0.259  C22:0  17.02  17.76  16.62  16.63  17.79  0.199  0.120  C24:0  20.88a,c  21.59a,d  20.66a,e  20.49c,e  22.38b,d  0.227  0.017  ⊙SFA  564.98a  531.39a  523.78a  598.61a,c  679.67b,c  18.366  0.015  C14:1  9.00a  9.51b  9.63b  8.85a  9.01a  0.091  0.001  C16:1  23.02  23.38  33.96  26.92  19.51  1.876  0.123  C18:1n-9  231.99a,d  371.60b,c  349.29a,c  207.71d  206.29d  23.900  0.023  C20:1  19.76  20.24  21.59  20.39  19.40  0.329  0.288  C22:1n-9  18.91a,d  19.57a,c  20.15b,c  18.63d  19.01a,d  0.182  0.026  ⊙MUFA  302.67a  444.31b  434.62b  282.50a  273.40a  25.427  0.027  C18:2n-6  206.05  145.42  250.05  231.43  164.51  18.287  0.354  C18:3n-6  17.56a,d  18.22a,c  18.77b,c  17.48d  17.71a,d  0.153  0.008  C18:3n-3  21.64  22.13  27.00  24.57  21.67  0.872  0.212  C20:2n-6  28.07a  26.09a  33.77b  27.39a  25.02a  1.035  0.035  C20:3n-6  30.15a,b  28.60a  35.52b  27.93a  26.38a  1.087  0.042  C20:4n-6  124.39  98.48  154.75  128.56  104.00  7.004  0.050  C20:3n-3  19.56  20.75  20.63  19.37  19.50  0.219  0.079  C20:5n-3  19.09a,d  20.26a,c  20.67b,c  18.58d  18.89d  0.267  0.018  C22:6n-3  21.78a,c  23.25a,d  24.20b,d  21.71c  21.16c  0.352  0.007  ⊙PUFA  488.32  403.21  618.61  517.02  418.84  28.602  0.053  PUFA/SFA  0.86a  0.76a  1.18b  0.85a  0.63a  0.054  0.001  n-3PUFA  82.10a  86.39a,c  92.43b,c  84.23a,d  81.22a,d  1.201  0.002  n-6PUFA  406.22a  316.82b  526.18c  432.79d  319.47b  21.080  <0.001  Parameters3  Groups      (μg/g)  Control  LG  MG  HG  AG  SEM1  P value  C10:0  17.57a,c  18.25a,d  17.74a,e  17.27c,e  18.92b,d  0.179  0.005  C11:0  16.23a  16.59a  15.02b  16.12a  21.28c  0.592  <0.001  C12:0  19.76a,c  20.56a,d  19.95a,e  19.45c,e  21.30b,d  0.204  0.018  C13:0  21.11a,c  20.79a,d  20.31a,e  19.77c,e  21.68b,d  0.208  0.011  C14:0  22.32  23.25  22.17  22.69  24.16  0.271  0.098  C15:0  20.45a,c  21.21a,d  20.53a,e  20.20c,e  21.81b,d  0.196  0.024  C16:0  134.82a  114.43b  114.39b  156.52c  180.30d  7.185  <0.001  C17:0  20.40  21.48  20.37  20.17  21.31  0.212  0.150  C18:0  238.16  217.63  218.89  252.39  290.63  10.792  0.174  C20:0  17.27  17.85  16.79  16.92  18.16  0.228  0.259  C22:0  17.02  17.76  16.62  16.63  17.79  0.199  0.120  C24:0  20.88a,c  21.59a,d  20.66a,e  20.49c,e  22.38b,d  0.227  0.017  ⊙SFA  564.98a  531.39a  523.78a  598.61a,c  679.67b,c  18.366  0.015  C14:1  9.00a  9.51b  9.63b  8.85a  9.01a  0.091  0.001  C16:1  23.02  23.38  33.96  26.92  19.51  1.876  0.123  C18:1n-9  231.99a,d  371.60b,c  349.29a,c  207.71d  206.29d  23.900  0.023  C20:1  19.76  20.24  21.59  20.39  19.40  0.329  0.288  C22:1n-9  18.91a,d  19.57a,c  20.15b,c  18.63d  19.01a,d  0.182  0.026  ⊙MUFA  302.67a  444.31b  434.62b  282.50a  273.40a  25.427  0.027  C18:2n-6  206.05  145.42  250.05  231.43  164.51  18.287  0.354  C18:3n-6  17.56a,d  18.22a,c  18.77b,c  17.48d  17.71a,d  0.153  0.008  C18:3n-3  21.64  22.13  27.00  24.57  21.67  0.872  0.212  C20:2n-6  28.07a  26.09a  33.77b  27.39a  25.02a  1.035  0.035  C20:3n-6  30.15a,b  28.60a  35.52b  27.93a  26.38a  1.087  0.042  C20:4n-6  124.39  98.48  154.75  128.56  104.00  7.004  0.050  C20:3n-3  19.56  20.75  20.63  19.37  19.50  0.219  0.079  C20:5n-3  19.09a,d  20.26a,c  20.67b,c  18.58d  18.89d  0.267  0.018  C22:6n-3  21.78a,c  23.25a,d  24.20b,d  21.71c  21.16c  0.352  0.007  ⊙PUFA  488.32  403.21  618.61  517.02  418.84  28.602  0.053  PUFA/SFA  0.86a  0.76a  1.18b  0.85a  0.63a  0.054  0.001  n-3PUFA  82.10a  86.39a,c  92.43b,c  84.23a,d  81.22a,d  1.201  0.002  n-6PUFA  406.22a  316.82b  526.18c  432.79d  319.47b  21.080  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid. View Large DISCUSSION At present, consumers are paying increasingly attention to the nutritional value and health benefits of the food they eat and this also applies to duck meat (Liao et al., 2015). In the experiments described here, ducks fed a C. butyricum inclusion diet showed an increased average body weight, along with an increased average daily gain and feed conversion ratio compared with the control group and the antibiotic group, indicating that it is feasible for C. butyricum to replace antibiotics as a growth promoter. Previous reports have also shown that a C. butyricum supplementation diet could enhance immune function, promote nutrient digestion, and help to balance intestinal microflora of animals (Murayama et al., 1995; Yang et al., 2012; Chen et al., 2013). These findings may therefore explain why ducks fed with a C. butyricum diet present higher growth performance compared with the control group and the antibiotic supplemented group. The color value of duck meat is a most important attribute relating to meat freshness and quality. Some authors have previously reported that dietary supplementation with probiotics can improve meat quality in animals. Suo et al. (Suo et al., 2012) reported that meat quality, as measured by the pH45 min, meat color and the shore force, were all significantly enhanced after dietary supplementation of pigs with L. plantarum ZJ316. Endo and Nakano confirmed that the characteristics of meat quality were all improved with dietary supplementation of probiotics in broilers (Endo and Nakano, 1999). Their findings may help explain the results of our study in which the addition of C. butyricum to the diet significantly increased the pH45 min, pH24 h, and a* value and the difference in a* value for meat color may have been due to the dietary supplementation of probiotics of Peking ducks. Similarly, the present study revealed that the addition of C. butyricum could decrease the L* value and b* value of the breast muscle in Peking ducks and subsequently increase their market price. It is well known that pH is one of the most important post-slaughter factors affecting the drip loss (water-holding capacity) of duck meat (Yang et al., 2017). Swatland demonstrated that drip loss of meat was associated with the ultimate pH and the speed of pH decline (Swatland, 1995). A higher ultimate pH is thought to lead to lower drip losses (Di Luca et al., 2016). Drip loss can be used as an indicator to evaluate the quality deviations associated with the tempo and extent of postmortem glycolysis in muscle. Therefore, a dietary C. butyricum-induced improvement in water-holding capacity and better meat quality of Peking ducks could be beneficial for poultry production. Recent studies have revealed the potential reduction of oxidative stress of dietary additive probiotics (Kai et al., 2004; Martarelli et al., 2011), and therefore dietary supplementation with C. butyricum could be provided to reduce intramuscular oxidative damages in Peking ducks, thereby improving the meat quality. In our study, the observed increase in antioxidant enzymatic activity (T-SOD, CAT, and GSH-PX) in muscle might be related to the dietary supplementation with C. butyricum. Relatively, T-AOC levels increased in all groups supplemented with C. butyricum even at the lowest level (200 mg/kg) of C. butyricum. With increasing levels of C. butyricum the MDA content showed a decreasing trend in muscle. In addition, several reports have revealed that some probiotic strains could influence the activity of antioxidants and might be useful in alleviating systemic oxidative stress either through stimulating the immune system and reducing inflammation or preventing infection by intestinal pathogens thereby reducing inflammation and related oxidative damages (Cheng et al., 2017c). In our study, dietary C. butyricum did not significantly influence the concentrations in serum of total cholesterol, v-LDL, and NEFA in Peking ducks. However, the dietary levels of C. butyricum significantly reduced the concentrations of TG, HDL-C, and LDL-C in serum compared with control groups. High serum concentrations of TG and LDL-C are recognized as an independent risk factor for atherosclerosis, which may be the main risk factor of coronary heart disease (Virani et al., 2011). However, Fukushima and Nakano found that dietary supplementation with a mixture of probiotics resulted in a higher HDL concentration and a lower LDL cholesterol concentration in the serum of rats (Fukushima and Nakano, 1996). Therefore, our results suggest that dietary C. butyricum is an effective health promoting agent to improve lipid metabolism in live ducks. With the strengthening economy, living standards have been improved in China, and along with this the demands on the food industry in terms of flavor and nutrition have increased. IMP is one of the main components responsible the umami flavor of meat and the content of IMP can determine the freshness of the meat after cooking (Jung et al., 2013). In our study, the addition of dietary C. butyricum increased the content of IMP in the breast muscle of Peking ducks in a dose-dependent manner. The flavor was more pronounced when the lipid levels were high in the breast meat. IMF is used to improve the tenderness of the meat in two ways. On the one hand, it cuts off the crosslinking structure between the fiber bundles, and on the other hand, it is beneficial to the fracture of muscle fibers during the chewing process (Chartrin et al., 2006; Qiu et al., 2017). The increasing content of IMF in the breast muscle supplemented with dietary C. butyricum also improved the meat flavor, making it more popular among consumers. Muscle tenderness is negatively related to the diameter of the muscle fibers but positively related to muscle density (Calkins et al., 1981; Zhu et al., 2012). The biological characteristics of muscle fibers are therefore an important quantitative index of meat quality, which is used to assess properties of duck meat and is closely related to the color of meat, the water content and pH value (Felício et al., 2013; Wu et al., 2015). Glycine, alanine, aspartic acid, glutamic acid, phenylalanine, and tyrosine are referred to collectively as the flavor amino acid (FAA) (Sabagh et al., 2016). Their composition and content directly affects the freshness of food. The increased levels of FAA following C. butyricum supplementation in our study improved the flavor of the duck meat, making it more favorable to consumers. Fatty acid alterations via dietary means may provide an effective way to obtain healthy duck products for human consumption. The current study revealed that dietary supplementation with C. butyricum could change the fatty acid composition and decreased the contents of SFA in duck meat. Over recent years, researchers have paid increasing attention to the modulation of fatty acid profiles in poultry meat as cardiovascular heart disease are considered closely associated with the contents of SFA intake in daily diet (Ramasamy et al., 2006), and hence from a healthy perspective, meat that contains relatively lower SFA contents could help in reducing the risk of cardiovascular heart diseases (Cheng et al., 2017a). Therefore, much attention should be paid toward the alteration of Peking duck products to improve the meat quality and flavor via dietary means, such as adding antioxidants and probiotics, to meet consumers’ needs and improve their health (Ivanovic et al., 2012; Yuan et al., 2012). The levels of docosahexaenoic acid (DHA, C22:6, n-3) in the breast meat of ducks fed with C. butyricum were significantly higher than the control. This can be explained by the modulation effects of dietary C. butyricum treatment. DHA is responsible for hypolipidemic and neuroprotective effects (Ramasamy et al., 2009). Overall, it was not only the meat quality (color and flavor) that was improved but also the contents of EPA (C20:5, n-3) and PUFA that were increased in breast muscle via the stimulative effects of C. butyricum supplemented in the duck diet. The increased contents of PUFA may be due to the enhancement of the antioxidant defenses system in ducks fed a diet supplemented with C. butyricum, although this requires further study. The enrichment of duck meat with long chain PUFA by supplementation with C. butyricum may be beneficial to human health and could replace the need for marine sources of EPA and DHA in the diet of poultry. Supplementation with dietary C. butyricum enhanced the ratio of PUFA: SFA by changing the fatty acid composition in breast muscle. Moreover, no adverse effects on the growth performance of Peking ducks were found after dietary supplementation with C. butyricum. Thus, the present results led to the conclusion that the widespread dietary supplementation of ducks with C. butyricum might be a feasible strategy to reduce the SFA contents and enhance the MUFA and PUFA contents of Peking duck meat. The resulting alterations in the fatty acid composition of meat may satisfy the nutritional demands of human consumers. To the best of our knowledge, this is the first comprehensive report on the influence of a C. butyricum-supplemented diet on the quality and flavor of Peking duck meat. In summary, it is well established that dietary C. butyricum can have a positive effect on animal health and meat quality by affecting PUFA deposition and metabolic processes in the breast muscle; however, further studies are required to investigate the effects of C. butyricum supplementation on cooked duck meat. CONCLUSIONS Overall, supplementation of C. butyricum for 42 d enhanced the growth performance, promoted meat quality and antioxidant activities, and improved the IMP and IMF contents of ducks, but decreased the triglyceride and HDL-C and LDL-C contents of Peking ducks. Such a diet is therefore able to enrich meat with FAA, EPA and DHA and increase the PUFA to SFA ratio. Consumption of such duck meat products may be beneficial to human health while also offering an enhanced flavor. Furthermore, dietary supplementation with probiotics may contribute to reduced drug resistance in poultry. Further studies are needed to investigate the effects of C. butyricum supplementation on fatty acid profile of cooked duck meat. Acknowledgements This research was supported by Beijing Agriculture Innovation Consortium (BAIC04-2018). The authors declare that they have no competing interests. REFERENCES Awad W. A., Ghareeb K., Abdel-Raheem S., Bohm J.. 2009. 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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Poultry Science Oxford University Press

Dietary supplementation with Clostridium butyricum modulates serum lipid metabolism, meat quality, and the amino acid and fatty acid composition of Peking ducks

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Oxford University Press
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© 2018 Poultry Science Association Inc.
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0032-5791
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1525-3171
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10.3382/ps/pey162
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Abstract

ABSTRACT The aim of this study was to investigate the effects of Clostridium butyricum (C. butyricum) on the performance, serum lipid metabolism, muscle morphology, meat quality, and fatty acid profiles of Peking ducks. A total of 1,500 Peking ducks were randomly divided into five groups with five replicates and were fed a non-antibiotic basal diet (Control) or a basal diet supplemented with either 200, 400, or 600 mg/kg of C. butyricum (2.0 × 109 CFU/g) or 150 mg of aureomycin/kg for 42 d. Compared with the control group, supplementation with C. butyricum increased the average daily weight gain but reduced the feed/gain ratio from 1 to 42 d of age. Similarly, dietary C. butyricum increased the activities of antioxidant enzymes but decreased the malondialdehyde (MDA) and lipid metabolites concentration. C. butyricum supplementation increased the muscle pH value at 45 min postmortem, the redness of the meat, and the contents of inosine acid (IMP) and intramuscular fat (IMF) in Peking ducks. By contrast, C. butyricum supplementation lowered the lightness, drip loss, and the shear force of breast meat. Supplementation with C. butyricum increased the concentrations of essential amino acids and flavor amino acids, as well as arachidonic acid (AA), docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and total polyunsaturated fatty acids (PUFA) in breast muscle. Dietary C. butyricum could positively improve performance, lipid metabolism, meat quality, and the amino acid and fatty acid composition in a dose-dependent manner. Therefore, C. butyricum is proposed as a feasible alternative feed additive for the production of healthier Peking duck meat with favorable properties. INTRDUCTION Increased attention is being paid to meat quality and flavor as well as its nutritional value by consumers (Cheng et al., 2017b). Duck meat, especially prepared as roast duck, is traditional food in China that has remained popular over time. Its relatively low fat content and high concentration of polyunsaturated fatty acids (PUFA), make it an important food source throughout the world (Kamboh and Zhu, 2013). In addition, the fatty acid profile and fat content of this meat can easily be improved using dietary strategies (Tavarez et al., 2011). In recent decades, to maintain health and increase production efficiency, antibiotics have been used widely in poultry production. However, the sub-therapeutic and prophylactic usage of antibiotics has led to health concerns, not least the emergence of drug resistance (Meng et al., 2010; Sen et al., 2012). In recent years, considering the severe consequences of antibiotics use on animal and human health, the search for alternatives has become a real challenge (Zhang and Kim, 2014). Clostridium butyricum (C. butyricum) is a Gram-positive anaerobe that can produce butyric acid and form spores, which exists in the intestines of healthy animals and humans (Juan et al., 2017). C. butyricum had been widely used in animal production, as it was indicated to improve growth performance, feed efficiency, and antioxidant capability in animals (Awad et al., 2009; Zhang et al., 2011; Duan et al., 2017). Similarly, experiments in ruminants have evidenced that C. butyricum addition can improve feed efficiency and enhance some digestive enzyme activities (Chilliard and Ferlay, 2004). Research has also shown that dietary supplementation with C. butyricum promotes the immune status and is of benefit to the intestinal flora of broilers (Awad et al., 2009; Zhao et al., 2013; Cheng et al., 2017c). Dietary supplementation with C. butyrium achieved similar or better results in terms of improving performance, promoting immune function, and benefiting cecal microflora in Escherichia. coli K88-challenged birds (Gao et al., 2012; Zhang et al., 2016). Furthermore, supplementation with synbiotics in the broiler diet that comprised probiotics (B. subtilis, B. licheniformis and C. butyricum) could increase meat quality, such as increasing the pH24h value (muscle pH value at 24 h postmortem), and decrease cooking loss in breast muscle (Hossain et al., 2016). In addition to regulatory functions on animal health, it has been reported that dietary supplementation with C. butyricum only could also improve meat quality in broilers, which may result from its modulation of nutrient digestibility and adsorption, as well as muscular fatty acid composition and antioxidant status (Liao et al., 2015; Zhang et al., 2017). C. butyricum therefore been proposed as a suitable alternative to antibiotics to improve the growth performance and immune status of cherry valley ducks (Zhuang et al., 2015). Nevertheless, it remains to be determined whether dietary C. butyricum supplementation can improve meat quality and flavor, and the amino acid and fatty acid composition of Peking Ducks. Therefore, our study was conducted to evaluate the effects of C. butyricum supplementation on the performance, serum lipid metabolism, muscle morphology, muscular antioxidant capacity, meat quality, and amino acid and fatty acid composition of breast meat in Peking ducks. We aimed to explore the possible beneficial effects of C. butyricum addition on Peking duck meat and determine whether C. butyricum could replace commonly used antibiotics as an alternative feed additive for widespread use in the duck industry for the production of healthier Peking duck meat with favorable for consumers. MATERIALS AND METHODS Ethics Statement The present study was approved by the ethics committee and conducted according to the Guidelines for Experimental Animals of China Agricultural University (Beijing, China). Dietary Treatments and Feeding A total of 1500 1-d-old male Peking ducks were purchased from a commercial hatchery (Beijing, China) and reared in 2-tier cages at the Experimental Center of China Agricultural University. The ducks were ad libitum divided into five groups (five replicates with sixty ducks each), and housed in an environmentally controlled house. The initial temperature was maintained at 35°C during the first week, and then gradually reduced as the birds aged until it reached 25°C. All ducks were allowed free access to feed and water, and exposed to 24 h of constant light throughout the whole experimental period. The control group was given a corn-soybean basal diet for 42 d. The nutrient levels of the diets were formulated to meet or exceed the standards (NRC, 1994) (Table 1). The other four groups were fed a basal diet supplemented with 200 mg/kg (low dose group, LG), 400 mg/kg (middle dose group, MG), or 600 mg/kg (high dose group, HG) of C. butyricum (2.0 × 109 CFU/g) or 150 mg of aureomycin/kg (antibiotic group, AG), respectively. The bacterial strain C. butyricum (batch No. 20,170,325,003) was provided by Beijing Shine Biology Technology Co., Ltd., China. Table 1. Composition and nutrient levels of the basal diets (air-dry basis). Items  1–21 d  22–42 d  Ingredients (%)  Corn  56.00  60.24  Soybean meal  32.69  24.67  Wheat middling  5.00  9.00  Soybean oil  2.10  1.80  Phytases  0.02  0.02  Dicalcium phosphate  1.00  1.60  Limestone  1.50  1.20  DL-Methionine  0.15  0.12  L-Lysine  0.20  0.10  Vitamin premix1  0.02  0.02  Trace mineral premix2  0.20  0.20  NaCl  0.35  0.30  Choline chloride (50%)  0.24  0.20  Ethoxyquin (33%)  0.03  0.03  Maifanite  0.50  0.50  Total  100  100  Nutrient levels3 (%)  ME (MJ/kg)  12.31  12.53  Crude protein (%)  19.52  16.83  Lysine (%)  1.12  0.87  Methionine (%)  0.46  0.39  Calcium (%)  0.88  0.89  Available phosphorus (%)  0.29  0.39  Total phosphorus (%)  0.54  0.62  Methionine+Cysteine (%)  0.79  0.69  Items  1–21 d  22–42 d  Ingredients (%)  Corn  56.00  60.24  Soybean meal  32.69  24.67  Wheat middling  5.00  9.00  Soybean oil  2.10  1.80  Phytases  0.02  0.02  Dicalcium phosphate  1.00  1.60  Limestone  1.50  1.20  DL-Methionine  0.15  0.12  L-Lysine  0.20  0.10  Vitamin premix1  0.02  0.02  Trace mineral premix2  0.20  0.20  NaCl  0.35  0.30  Choline chloride (50%)  0.24  0.20  Ethoxyquin (33%)  0.03  0.03  Maifanite  0.50  0.50  Total  100  100  Nutrient levels3 (%)  ME (MJ/kg)  12.31  12.53  Crude protein (%)  19.52  16.83  Lysine (%)  1.12  0.87  Methionine (%)  0.46  0.39  Calcium (%)  0.88  0.89  Available phosphorus (%)  0.29  0.39  Total phosphorus (%)  0.54  0.62  Methionine+Cysteine (%)  0.79  0.69  1The vitamin premix provided the following per kilogram of diet: vitamin A, 12,500 IU; vitamin D3, 3500 IU; vitamin E, 20 IU; vitamin K3, 2.65 mg; thiamin, 2.00 mg; riboflavin, 6.00 mg; pyridoxin, 3.00 mg; VB12, 0.025 mg; biotin, 0.0325 mg; folic acid, 12.00 mg; pantothenic acid, 50 mg; nicotinic acid, 50.00 mg. 2The mineral premix provided the following per kg of diet: Cu, 6 mg; Fe, 80 mg; Zn, 40 mg; Mn, 100 mg; Se, 0.15 mg; I, 0.35 mg. 3Calculated values. View Large Table 1. Composition and nutrient levels of the basal diets (air-dry basis). Items  1–21 d  22–42 d  Ingredients (%)  Corn  56.00  60.24  Soybean meal  32.69  24.67  Wheat middling  5.00  9.00  Soybean oil  2.10  1.80  Phytases  0.02  0.02  Dicalcium phosphate  1.00  1.60  Limestone  1.50  1.20  DL-Methionine  0.15  0.12  L-Lysine  0.20  0.10  Vitamin premix1  0.02  0.02  Trace mineral premix2  0.20  0.20  NaCl  0.35  0.30  Choline chloride (50%)  0.24  0.20  Ethoxyquin (33%)  0.03  0.03  Maifanite  0.50  0.50  Total  100  100  Nutrient levels3 (%)  ME (MJ/kg)  12.31  12.53  Crude protein (%)  19.52  16.83  Lysine (%)  1.12  0.87  Methionine (%)  0.46  0.39  Calcium (%)  0.88  0.89  Available phosphorus (%)  0.29  0.39  Total phosphorus (%)  0.54  0.62  Methionine+Cysteine (%)  0.79  0.69  Items  1–21 d  22–42 d  Ingredients (%)  Corn  56.00  60.24  Soybean meal  32.69  24.67  Wheat middling  5.00  9.00  Soybean oil  2.10  1.80  Phytases  0.02  0.02  Dicalcium phosphate  1.00  1.60  Limestone  1.50  1.20  DL-Methionine  0.15  0.12  L-Lysine  0.20  0.10  Vitamin premix1  0.02  0.02  Trace mineral premix2  0.20  0.20  NaCl  0.35  0.30  Choline chloride (50%)  0.24  0.20  Ethoxyquin (33%)  0.03  0.03  Maifanite  0.50  0.50  Total  100  100  Nutrient levels3 (%)  ME (MJ/kg)  12.31  12.53  Crude protein (%)  19.52  16.83  Lysine (%)  1.12  0.87  Methionine (%)  0.46  0.39  Calcium (%)  0.88  0.89  Available phosphorus (%)  0.29  0.39  Total phosphorus (%)  0.54  0.62  Methionine+Cysteine (%)  0.79  0.69  1The vitamin premix provided the following per kilogram of diet: vitamin A, 12,500 IU; vitamin D3, 3500 IU; vitamin E, 20 IU; vitamin K3, 2.65 mg; thiamin, 2.00 mg; riboflavin, 6.00 mg; pyridoxin, 3.00 mg; VB12, 0.025 mg; biotin, 0.0325 mg; folic acid, 12.00 mg; pantothenic acid, 50 mg; nicotinic acid, 50.00 mg. 2The mineral premix provided the following per kg of diet: Cu, 6 mg; Fe, 80 mg; Zn, 40 mg; Mn, 100 mg; Se, 0.15 mg; I, 0.35 mg. 3Calculated values. View Large Measurements Performance Parameters. Body weight (BW) and feed intake were recorded for each replicate at 42 d of age. Average body weight (ABW), along with average daily gain (ADG), average daily feed intake (ADFI), and the feed conversion ratio (FCR) were calculated. Sample Collection. Ducks (12 birds per group) with a BW similar to the mean BW of each replicates were selected at 42 d. Blood samples were taken aseptically from the jugular vein and were centrifuged at 4000 × g for 10 min. Serum was separated and stored at –20°C for further analysis. All ducks were euthanized after sodium pentobarbitone (50 mg/kg BW) anesthesia. Mid-segments of breast muscle were collected and cut into section fixed in 4% paraformaldehyde solution for morphology measurements. The breast muscle from the same side carcass was removed according to the standard method of dissection. They were divided into two parts respectively: one was quickly frozen at –20°C for later determination of fatty acid composition, and another stored at 4°C was used to evaluate meat quality. The breast muscles (100 mg) were homogenized using a glass Teflon homogenizer with 900 ml of cold 0.9% NaCl solution (1:9). The homogenates were centrifuged at 3500–4000 g for 10 min at 4°C. The supernatants were collected for antioxidant assays. Lipid Metabolites and Antioxidant Parameters. The serum lipid metabolites of triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, very-low density lipoprotein (v-LDL), and non-esterified fatty acid (NEFA), and the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX), as well as concentrations of glutathione (GSH) and malondialdehyde (MDA) in breast muscle, were measured using the corresponding commercial assay kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China) following manufacturer's instructions. Meat Quality. Initial pH45 min, middle pH24 h, and ultimate pH48 h post-mortem were measured using a Testo 206 pH meter (Testo Limited, Alton, Hampshire, UK) according to the manufacturer's recommendations. The meat color (L* = lightness; a* = redness; and b* = yellowness) was measured at 24 h post-mortem by a spectrocolorimeter (model WSCS, Shanghai Shenguang Ltd., China) using the CIELAB system according to the manufacturer's recommendations. The shear force was determined using the Warner-Bratzler shear method as described (Jiang et al., 2011; Kiarie et al., 2014). An additional 20 g of meat sample was placed in a polyethylene container and the drip loss was evaluated after 24 h of storage at 4°C (Taylor and Dant, 1971; Young et al., 2004). The drip loss at 24 h post-mortem was calculated as a percentage of the initial weight. Muscle Morphology. Tissue samples were freshly prepared and fixed in 4% paraformaldehyde solution for 24 h. The fixed muscles were dehydrated using a graded series of ethanol, cleared using xylene, and then embedded in paraffin. Sections (4 μm thick) from transverse specimens of the Control, MG, and AG were stained with the standard hematoxylin-eosin (HE) method to observe muscle morphology (Zhu et al., 2009). The HE stained sections were analyzed under an optical microscopy (XSP-BM16C, Optical Instrument Factory, Shanghai, China). Oil Red O Staining and Intramuscular Fat Content. Frozen section of breast muscle was made using a Leica kryostat (Leica CM3050S, Leica instrument GmbH, Germany), fixed with 10% paraformaldehyde for 30 min, incubated with Oil Red O solution for 15 min, then the distribution of fat between muscle tissue was observed using a light microscopy. The staining was assessed by Beijing Epsilon Bio-technology Co., Ltd, Beijing, China. The intramuscular fat (IMF) and inosine acid (IMP) contents were determined as described elsewhere (Zerehdaran et al., 2004; Cui et al., 2012). The results are expressed as a percentage of the wet weight of the breast muscle. Amino Acid and Fatty Acid Analysis. The contents of 17 types of amino acids were measured in the freeze-dried, fat-free meat samples by ion-exchange chromatography of the acidhydrolyzed protein. Each sample was placed in a sealed tube and treated with 6 M HCl in an oil bath at 110°C for 32 h. Sodium citrate buffers were used to separate the amino acids before assessment on a Beckman amino acid analyzer (Model 6300) (Elgasim and Alkanhal, 1992). The fatty acid composition of breast muscles was measured by gas chromatography (Trace GC Ultra, GC-2010 Shimadzu, Tokyo, Japan) (Folch et al., 1957). Total lipid extracts were transmethylated into fatty acid methyl esters and separated using a HP 6890 gas chromatograph equipped with a flame-ionized detector and a DB-23 capillary column (0.25 mm × 60 m × 0.25 μm; Supelco, Bellefonte, PA, USA). The column, injector, and detector temperatures were set as 195°C, 225°C and 250°C, respectively. Helium was used at a flow rate of 1.5 cm3/min. Nonadecanoic acid (C19:0, Buchs, Switzerland) was used as the internal standard. Fatty acids were evaluated by comparing their retention times with authentic standards. The concentrations were performed as milligrams per 100 g. Statistical Analysis The data were analyzed using one-way ANOVA and Duncan's multiple test for multiple comparisons by the SPSS version 20.0 (SPSS Inc., Chicago, IL, USA). Results were presented as means with their SEM. A value of P < 0.05 was indicated to be statistically significant. RESULTS Performance All ducks appeared healthy throughout the entire experimental period. In MG and AG the ducks showed greater (P < 0.05) AMB at 42 d compared with those in the control group. Ducks fed with C. butyricum or antibiotics showed improved ADG (P < 0.01) compared with the control group. ADFI in the LG, MG, and AG (P < 0.05), and FCR in the LG, MG, HG, and AG were lower (P < 0.01) than those in the control group from 1–42 d (Table 2). Table 2. Effects of Clostridium butyricum on growth performance of Pekin ducks at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  AMB (kg)  2.96a  3.07a,c  3.24b,d  3.08a,c  3.12c,d  0.028  0.019  ADG (g/d)  77.4a  79.6b  81.8c  80.1d  78.9e  0.390  <0.001  ADFI (g/d)  152.0a  146.0b  144.0b  151.0a  144.0b  1.018  0.001  FCR (g/g)  1.96a  1.83b  1.76c  1.88d  1.82b  0.019  <0.001  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  AMB (kg)  2.96a  3.07a,c  3.24b,d  3.08a,c  3.12c,d  0.028  0.019  ADG (g/d)  77.4a  79.6b  81.8c  80.1d  78.9e  0.390  <0.001  ADFI (g/d)  152.0a  146.0b  144.0b  151.0a  144.0b  1.018  0.001  FCR (g/g)  1.96a  1.83b  1.76c  1.88d  1.82b  0.019  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3ABW: average body weight, ADG: average daily gain, ADFI: average daily feed intake, FCR: feed conversion ratio. View Large Table 2. Effects of Clostridium butyricum on growth performance of Pekin ducks at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  AMB (kg)  2.96a  3.07a,c  3.24b,d  3.08a,c  3.12c,d  0.028  0.019  ADG (g/d)  77.4a  79.6b  81.8c  80.1d  78.9e  0.390  <0.001  ADFI (g/d)  152.0a  146.0b  144.0b  151.0a  144.0b  1.018  0.001  FCR (g/g)  1.96a  1.83b  1.76c  1.88d  1.82b  0.019  <0.001  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  AMB (kg)  2.96a  3.07a,c  3.24b,d  3.08a,c  3.12c,d  0.028  0.019  ADG (g/d)  77.4a  79.6b  81.8c  80.1d  78.9e  0.390  <0.001  ADFI (g/d)  152.0a  146.0b  144.0b  151.0a  144.0b  1.018  0.001  FCR (g/g)  1.96a  1.83b  1.76c  1.88d  1.82b  0.019  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3ABW: average body weight, ADG: average daily gain, ADFI: average daily feed intake, FCR: feed conversion ratio. View Large Lipid Metabolic Status The contents of LDL, v-LDL, and NEFA in all the C. butyricum-supplemented groups were higher than those in the control groups; but there were no significant differences between C. butyricum-supplemented groups and the control group in terms of LDL, v-LDL, and NEFA concentrations in the serum at 42 d. However, the contents of TC in the LG, HG, and AG were higher than in the control group (P < 0.05). The concentration of serum HDL decreased significantly (P < 0.05) only in the MG at 42 d, whereas the concentration of TG in the serum in both the HG and AG tended to decrease at 42 d (Figure 1). Figure 1. View largeDownload slide Effects of Clostridium butyricum on serum lipid metabolism in Pekin duck at 42 d of age. Values are mean ± SD of 12 independent determinations; the superscript different letters indicate that there are significant differences (P < 0.05) between any two groups. TC: total cholesterol; TG: triglyceride; HDL: high-density lipoprotein cholesterol; LDL: low-density lipoprotein cholesterol; v-LDL: very low-density lipoprotein cholesterol; NEFA: non-esterified fatty acid. Figure 1. View largeDownload slide Effects of Clostridium butyricum on serum lipid metabolism in Pekin duck at 42 d of age. Values are mean ± SD of 12 independent determinations; the superscript different letters indicate that there are significant differences (P < 0.05) between any two groups. TC: total cholesterol; TG: triglyceride; HDL: high-density lipoprotein cholesterol; LDL: low-density lipoprotein cholesterol; v-LDL: very low-density lipoprotein cholesterol; NEFA: non-esterified fatty acid. Muscular Oxidative Status Overall, the T-AOC was significantly higher (P < 0.05) with the MG diet whereas significantly lower (P < 0.05) in the MG and AG diets as compared to the control group; the same trends were observed for muscular T-SOD. The activities of muscular GSH-PX and CAT were significantly higher (P < 0.05) with the MG diet than with the other diets and the control group. The contents of muscular GSH increased significantly (P < 0.05) in the LG, MG, and AG diets compared with the control group. Supplementation of C. butyricum didn’t influence the accumulation of MDA in the duck breast muscle at 42 d significantly (P = 0.104) (Table 3). Table 3. Muscular antioxidant status in Pekin duck at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  T-AOC (U/mg prot)  1.08a  1.08a  1.27b  0.85c  0.59d  0.049  <0.001  T-SOD (U/mg prot)  8.05a  7.86a  9.40b  6.45c  5.48c  0.303  <0.001  GSH-PX (U/mg prot)  85.02a  115.53b,c  122.93b,d  104.81a,c,d  115.99b,d  4.175  0.026  CAT (U/mg prot)  7.04a  7.36a,c  7.79b,c  7.62a,c  10.16d  1.261  <0.001  GSH (mg/g prot)  0.77a  1.04b,c  1.07b  0.88a,c  1.11b  0.035  0.015  MDA (nmol/mg prot)  0.312  0.197  0.225  0.254  0.273  0.014  0.104  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  T-AOC (U/mg prot)  1.08a  1.08a  1.27b  0.85c  0.59d  0.049  <0.001  T-SOD (U/mg prot)  8.05a  7.86a  9.40b  6.45c  5.48c  0.303  <0.001  GSH-PX (U/mg prot)  85.02a  115.53b,c  122.93b,d  104.81a,c,d  115.99b,d  4.175  0.026  CAT (U/mg prot)  7.04a  7.36a,c  7.79b,c  7.62a,c  10.16d  1.261  <0.001  GSH (mg/g prot)  0.77a  1.04b,c  1.07b  0.88a,c  1.11b  0.035  0.015  MDA (nmol/mg prot)  0.312  0.197  0.225  0.254  0.273  0.014  0.104  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3T-AOC: total antioxidant capacity; T-SOD: total superoxide dismutase; GSH-PX: glutathione peroxidase; CAT: catalase; GSH: glutathione; MDA: malonaldehyde. View Large Table 3. Muscular antioxidant status in Pekin duck at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  T-AOC (U/mg prot)  1.08a  1.08a  1.27b  0.85c  0.59d  0.049  <0.001  T-SOD (U/mg prot)  8.05a  7.86a  9.40b  6.45c  5.48c  0.303  <0.001  GSH-PX (U/mg prot)  85.02a  115.53b,c  122.93b,d  104.81a,c,d  115.99b,d  4.175  0.026  CAT (U/mg prot)  7.04a  7.36a,c  7.79b,c  7.62a,c  10.16d  1.261  <0.001  GSH (mg/g prot)  0.77a  1.04b,c  1.07b  0.88a,c  1.11b  0.035  0.015  MDA (nmol/mg prot)  0.312  0.197  0.225  0.254  0.273  0.014  0.104  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  T-AOC (U/mg prot)  1.08a  1.08a  1.27b  0.85c  0.59d  0.049  <0.001  T-SOD (U/mg prot)  8.05a  7.86a  9.40b  6.45c  5.48c  0.303  <0.001  GSH-PX (U/mg prot)  85.02a  115.53b,c  122.93b,d  104.81a,c,d  115.99b,d  4.175  0.026  CAT (U/mg prot)  7.04a  7.36a,c  7.79b,c  7.62a,c  10.16d  1.261  <0.001  GSH (mg/g prot)  0.77a  1.04b,c  1.07b  0.88a,c  1.11b  0.035  0.015  MDA (nmol/mg prot)  0.312  0.197  0.225  0.254  0.273  0.014  0.104  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3T-AOC: total antioxidant capacity; T-SOD: total superoxide dismutase; GSH-PX: glutathione peroxidase; CAT: catalase; GSH: glutathione; MDA: malonaldehyde. View Large Quality of Breast Muscle No significant differences were found among the treatment groups for pH24 h and pH48 h, whereas the pH45 min of the breast muscle was significantly higher in the ducks fed with the LG, MG, HG, and AG diets compared with the control (P < 0.01). The lightness of meat color of breast muscle was significantly lower (P < 0.05) with the HG diet in comparison with the control group. A higher (P < 0.05) redness for meat color and a lower (P < 0.01) drip loss and shear force of breast muscle (P = 0.002) were measured for the C. butyricum-supplemented diet and the antibiotic-supplemented diet as compared to the control group. Compared with the no C. butyricum-supplemented diet, the yellowness of meat color presented no differences (P = 0.280). (Table 4) Table 4. Effects of Clostridium butyricum on breast meat quality in Pekin duck at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  pH45 min  6.22a  6.33b,d  6.37c  6.32b  6.36c,d  0.011  <0.001  pH24 h  5.89a,c  5.92a,d  5.90a,e  5.86c,e  5.97b,d  0.012  0.018  pH48 h  5.83  5.86  5.83  5.84  5.89  0.010  0.292  Lightness (L*)  47.40a,c  48.56a  45.99b,c  45.04b  46.61a,b,c  0.366  0.017  Redness (a*)  18.36a  18.46a,c  19.86b  19.42b,c  19.62b  0.181  0.010  Yellowness (b*)  6.51  5.45  5.22  5.32  5.73  0.204  0.280  Shear force (N/cm2)  45.80a  33.97b  31.14b  32.66b  36.78b  1.380  0.002  Drip loss (%)  10.76a  7.91b  8.09b  9.98a  7.51b  0.302  <0.001  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  pH45 min  6.22a  6.33b,d  6.37c  6.32b  6.36c,d  0.011  <0.001  pH24 h  5.89a,c  5.92a,d  5.90a,e  5.86c,e  5.97b,d  0.012  0.018  pH48 h  5.83  5.86  5.83  5.84  5.89  0.010  0.292  Lightness (L*)  47.40a,c  48.56a  45.99b,c  45.04b  46.61a,b,c  0.366  0.017  Redness (a*)  18.36a  18.46a,c  19.86b  19.42b,c  19.62b  0.181  0.010  Yellowness (b*)  6.51  5.45  5.22  5.32  5.73  0.204  0.280  Shear force (N/cm2)  45.80a  33.97b  31.14b  32.66b  36.78b  1.380  0.002  Drip loss (%)  10.76a  7.91b  8.09b  9.98a  7.51b  0.302  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3pH45 min: muscle pH value at 45 min postmortem; pH24 h: muscle pH value at 24 h postmortem; pH48 h: muscle pH value at 48 h postmortem. View Large Table 4. Effects of Clostridium butyricum on breast meat quality in Pekin duck at 42 d of age.2 Parameters3  Control  LG  MG  HG  AG  SEM1  P value  pH45 min  6.22a  6.33b,d  6.37c  6.32b  6.36c,d  0.011  <0.001  pH24 h  5.89a,c  5.92a,d  5.90a,e  5.86c,e  5.97b,d  0.012  0.018  pH48 h  5.83  5.86  5.83  5.84  5.89  0.010  0.292  Lightness (L*)  47.40a,c  48.56a  45.99b,c  45.04b  46.61a,b,c  0.366  0.017  Redness (a*)  18.36a  18.46a,c  19.86b  19.42b,c  19.62b  0.181  0.010  Yellowness (b*)  6.51  5.45  5.22  5.32  5.73  0.204  0.280  Shear force (N/cm2)  45.80a  33.97b  31.14b  32.66b  36.78b  1.380  0.002  Drip loss (%)  10.76a  7.91b  8.09b  9.98a  7.51b  0.302  <0.001  Parameters3  Control  LG  MG  HG  AG  SEM1  P value  pH45 min  6.22a  6.33b,d  6.37c  6.32b  6.36c,d  0.011  <0.001  pH24 h  5.89a,c  5.92a,d  5.90a,e  5.86c,e  5.97b,d  0.012  0.018  pH48 h  5.83  5.86  5.83  5.84  5.89  0.010  0.292  Lightness (L*)  47.40a,c  48.56a  45.99b,c  45.04b  46.61a,b,c  0.366  0.017  Redness (a*)  18.36a  18.46a,c  19.86b  19.42b,c  19.62b  0.181  0.010  Yellowness (b*)  6.51  5.45  5.22  5.32  5.73  0.204  0.280  Shear force (N/cm2)  45.80a  33.97b  31.14b  32.66b  36.78b  1.380  0.002  Drip loss (%)  10.76a  7.91b  8.09b  9.98a  7.51b  0.302  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3pH45 min: muscle pH value at 45 min postmortem; pH24 h: muscle pH value at 24 h postmortem; pH48 h: muscle pH value at 48 h postmortem. View Large Muscle Morphology Muscle morphology is observed in Figure 2. In the Control group, the muscle fibers were disordered with uneven thickness. The fibers were degeneration and the spaces between muscle fibers were widened. In MG group, the muscle fibers are arranged in a wavy pattern, with uniform thickness and tightness. In the AG group, the muscle fibers were slightly disordered with the uneven thickness, and the fibers became thin. Figure 2. View largeDownload slide The microstructure of the breast muscle of Pekin ducks at 42 d of age (N = 6). Control 1: magnification × 100; Control 2: magnification × 200; Control 3: magnification × 400. MG1: magnification × 100; MG2: magnification × 200; MG3: magnification × 400. AG1: magnification × 100; AG2: magnification × 200; AG3: magnification × 400. Figure 2. View largeDownload slide The microstructure of the breast muscle of Pekin ducks at 42 d of age (N = 6). Control 1: magnification × 100; Control 2: magnification × 200; Control 3: magnification × 400. MG1: magnification × 100; MG2: magnification × 200; MG3: magnification × 400. AG1: magnification × 100; AG2: magnification × 200; AG3: magnification × 400. IMF and IMP Contents In the Oil Red O stained tissue, an increased number of red lipid droplets were observed in the MG compared with the control group or the AG. This indicated that addition of C. butyricum can improve the quantity of IMF between the muscle fibers or muscle cells (Figure 3). Figure 3. View largeDownload slide The Oil Red O stain of breast muscle fibril of Pekin ducks at 42 d of age (N = 6). Control 1: magnification × 100; Control 2: magnification × 200; Control 3: magnification × 400. MG1: magnification × 100; MG2: magnification × 200; MG3: magnification × 400. AG1: magnification × 100; AG2: magnification × 200; AG3: magnification × 400. Figure 3. View largeDownload slide The Oil Red O stain of breast muscle fibril of Pekin ducks at 42 d of age (N = 6). Control 1: magnification × 100; Control 2: magnification × 200; Control 3: magnification × 400. MG1: magnification × 100; MG2: magnification × 200; MG3: magnification × 400. AG1: magnification × 100; AG2: magnification × 200; AG3: magnification × 400. The contents of IMP were significantly increased (P < 0.01) in response to the MG and HG diets and the AG diet compared with those fed with the control diet. Similarly, the contents of IMF were significantly higher (P < 0.01) in response to the MG and HG diets compared with the control group. No significant differences (P > 0.05) were found between the AG diet and the control group diet (Figure 4). Figure 4. View largeDownload slide Contents of IMP and IMF in breast muscle of Pekin duck at 42 d of age (N = 6). Values are mean ± SD of 12 independent determinations; the superscript different letters indicate that there are significant differences (P < 0.05) between any two groups. IMP: inosine acid; IMF: intramuscular fat. Figure 4. View largeDownload slide Contents of IMP and IMF in breast muscle of Pekin duck at 42 d of age (N = 6). Values are mean ± SD of 12 independent determinations; the superscript different letters indicate that there are significant differences (P < 0.05) between any two groups. IMP: inosine acid; IMF: intramuscular fat. Amino Acid and Fatty Acid Composition The effect of C. butyricum dietary supplementation on the animo acid profile of duck breast muscle is presented in Table 5. Significant differences (P < 0.05) were observed in some animo acid concentrations in the supplemented groups compared with control groups. For total amino acids (TAA), the TAA concentrations significantly increased (P < 0.05) when fed with the MG, HG, and AG diets compared with the control diet, whereas no significant differences (P > 0.05) were found in the contents of other essential amino acids (EAA) among all of the treatments. Interestingly, with MG and HG diets, the contents of glycine, tyrosine, and the total flavor amino acids (FAA, including glycine, alanine, aspartic acid, glutamic acid, phenylalanine, and tyrosine) significantly increased (P < 0.05) compared with the control diet but no significant differences (P > 0.05) were found in the other four FAA contents among all of the treatments. Table 5. Effects of Clostridium butyricum on amino acid composition in breast muscle of Pekin duck at 42 d of age.2 Parameters3  Groups      (ng/mg)  Control  LG  MG  HG  AG  SEM1  P value  Glycine  134.79a  110.67b  169.21c  156.66c  102.26b  7.292  <0.001  Alanine  419.05  334.67  420.03  372.13  377.65  11.741  0.082  Serine  151.02a  132.19b  196.82c  168.21d  186.94c  6.584  <0.001  Proline  157.90a,c  175.59b  157.14c  180.30b  91.64d  8.536  <0.001  Valine  76.00a  71.11a  77.49a  76.81a  102.28a  3.145  0.002  Cysteine  82.37a  79.71a  110.84b  103.52c  110.93b  3.712  <0.001  Isoleucine  2.40a  2.52a  7.23b  3.26a  3.19a  0.507  <0.001  Asparagine  42.24a,c  36.83a  44.12c  44.29c  67.02b  2.917  0.001  Aspartic acid  40.57a  33.72b  44.09a  43.23a  45.74a  1.108  0.017  Glutamine  88.48a,d  73.53a,c  60.34b,c  98.56d  89.72a,d  4.378  0.014  Glutamic acid  377.30  351.00  444.43  373.86  427.94  13.891  0.151  Methionine  93.74a  65.80a,c  136.54b  60.07c  88.71a,c  8.218  0.003  Histidine  36.87a  36.79a  42.72a  39.60a  51.45b  1.731  0.010  Phenylalanine  42.65a  31.67b  46.46a  44.03a  44.69a  1.645  0.007  Arginine  58.06a,c  48.72a  60.69c  70.79b  77.39b  2.934  0.033  Tyrosine  184.67a  202.77a  336.94b  337.29b  199.34a  20.314  0.037  Tryptophan  14.45a,c  13.96a  16.18b,c  18.37d  20.97e  0.731  <0.001  Lysine  200.98  179.64  236.86  143.01  192.84  12.659  0.213  Leucine  51.15a  52.54a  79.19b  67.91c  90.76d  4.189  <0.001  Threonine  91.02a,c  80.18a,d  97.04b,c  96.31b,c  75.43d  2.823  0.015  FAA  1199.04a  1064.50b  1461.15c  1327.20d  1197.62a  38.939  <0.001  EAA  683.05  592.01  840.18  557.60  688.46  44.892  0.898  TAA  2345.79a  2113.62b  2784.37c  2498.37a  2446.90a  63.857  0.023  Parameters3  Groups      (ng/mg)  Control  LG  MG  HG  AG  SEM1  P value  Glycine  134.79a  110.67b  169.21c  156.66c  102.26b  7.292  <0.001  Alanine  419.05  334.67  420.03  372.13  377.65  11.741  0.082  Serine  151.02a  132.19b  196.82c  168.21d  186.94c  6.584  <0.001  Proline  157.90a,c  175.59b  157.14c  180.30b  91.64d  8.536  <0.001  Valine  76.00a  71.11a  77.49a  76.81a  102.28a  3.145  0.002  Cysteine  82.37a  79.71a  110.84b  103.52c  110.93b  3.712  <0.001  Isoleucine  2.40a  2.52a  7.23b  3.26a  3.19a  0.507  <0.001  Asparagine  42.24a,c  36.83a  44.12c  44.29c  67.02b  2.917  0.001  Aspartic acid  40.57a  33.72b  44.09a  43.23a  45.74a  1.108  0.017  Glutamine  88.48a,d  73.53a,c  60.34b,c  98.56d  89.72a,d  4.378  0.014  Glutamic acid  377.30  351.00  444.43  373.86  427.94  13.891  0.151  Methionine  93.74a  65.80a,c  136.54b  60.07c  88.71a,c  8.218  0.003  Histidine  36.87a  36.79a  42.72a  39.60a  51.45b  1.731  0.010  Phenylalanine  42.65a  31.67b  46.46a  44.03a  44.69a  1.645  0.007  Arginine  58.06a,c  48.72a  60.69c  70.79b  77.39b  2.934  0.033  Tyrosine  184.67a  202.77a  336.94b  337.29b  199.34a  20.314  0.037  Tryptophan  14.45a,c  13.96a  16.18b,c  18.37d  20.97e  0.731  <0.001  Lysine  200.98  179.64  236.86  143.01  192.84  12.659  0.213  Leucine  51.15a  52.54a  79.19b  67.91c  90.76d  4.189  <0.001  Threonine  91.02a,c  80.18a,d  97.04b,c  96.31b,c  75.43d  2.823  0.015  FAA  1199.04a  1064.50b  1461.15c  1327.20d  1197.62a  38.939  <0.001  EAA  683.05  592.01  840.18  557.60  688.46  44.892  0.898  TAA  2345.79a  2113.62b  2784.37c  2498.37a  2446.90a  63.857  0.023  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3FAA: flavor amino acid; EAA: essential amino acid; TAA: total amino acids. View Large Table 5. Effects of Clostridium butyricum on amino acid composition in breast muscle of Pekin duck at 42 d of age.2 Parameters3  Groups      (ng/mg)  Control  LG  MG  HG  AG  SEM1  P value  Glycine  134.79a  110.67b  169.21c  156.66c  102.26b  7.292  <0.001  Alanine  419.05  334.67  420.03  372.13  377.65  11.741  0.082  Serine  151.02a  132.19b  196.82c  168.21d  186.94c  6.584  <0.001  Proline  157.90a,c  175.59b  157.14c  180.30b  91.64d  8.536  <0.001  Valine  76.00a  71.11a  77.49a  76.81a  102.28a  3.145  0.002  Cysteine  82.37a  79.71a  110.84b  103.52c  110.93b  3.712  <0.001  Isoleucine  2.40a  2.52a  7.23b  3.26a  3.19a  0.507  <0.001  Asparagine  42.24a,c  36.83a  44.12c  44.29c  67.02b  2.917  0.001  Aspartic acid  40.57a  33.72b  44.09a  43.23a  45.74a  1.108  0.017  Glutamine  88.48a,d  73.53a,c  60.34b,c  98.56d  89.72a,d  4.378  0.014  Glutamic acid  377.30  351.00  444.43  373.86  427.94  13.891  0.151  Methionine  93.74a  65.80a,c  136.54b  60.07c  88.71a,c  8.218  0.003  Histidine  36.87a  36.79a  42.72a  39.60a  51.45b  1.731  0.010  Phenylalanine  42.65a  31.67b  46.46a  44.03a  44.69a  1.645  0.007  Arginine  58.06a,c  48.72a  60.69c  70.79b  77.39b  2.934  0.033  Tyrosine  184.67a  202.77a  336.94b  337.29b  199.34a  20.314  0.037  Tryptophan  14.45a,c  13.96a  16.18b,c  18.37d  20.97e  0.731  <0.001  Lysine  200.98  179.64  236.86  143.01  192.84  12.659  0.213  Leucine  51.15a  52.54a  79.19b  67.91c  90.76d  4.189  <0.001  Threonine  91.02a,c  80.18a,d  97.04b,c  96.31b,c  75.43d  2.823  0.015  FAA  1199.04a  1064.50b  1461.15c  1327.20d  1197.62a  38.939  <0.001  EAA  683.05  592.01  840.18  557.60  688.46  44.892  0.898  TAA  2345.79a  2113.62b  2784.37c  2498.37a  2446.90a  63.857  0.023  Parameters3  Groups      (ng/mg)  Control  LG  MG  HG  AG  SEM1  P value  Glycine  134.79a  110.67b  169.21c  156.66c  102.26b  7.292  <0.001  Alanine  419.05  334.67  420.03  372.13  377.65  11.741  0.082  Serine  151.02a  132.19b  196.82c  168.21d  186.94c  6.584  <0.001  Proline  157.90a,c  175.59b  157.14c  180.30b  91.64d  8.536  <0.001  Valine  76.00a  71.11a  77.49a  76.81a  102.28a  3.145  0.002  Cysteine  82.37a  79.71a  110.84b  103.52c  110.93b  3.712  <0.001  Isoleucine  2.40a  2.52a  7.23b  3.26a  3.19a  0.507  <0.001  Asparagine  42.24a,c  36.83a  44.12c  44.29c  67.02b  2.917  0.001  Aspartic acid  40.57a  33.72b  44.09a  43.23a  45.74a  1.108  0.017  Glutamine  88.48a,d  73.53a,c  60.34b,c  98.56d  89.72a,d  4.378  0.014  Glutamic acid  377.30  351.00  444.43  373.86  427.94  13.891  0.151  Methionine  93.74a  65.80a,c  136.54b  60.07c  88.71a,c  8.218  0.003  Histidine  36.87a  36.79a  42.72a  39.60a  51.45b  1.731  0.010  Phenylalanine  42.65a  31.67b  46.46a  44.03a  44.69a  1.645  0.007  Arginine  58.06a,c  48.72a  60.69c  70.79b  77.39b  2.934  0.033  Tyrosine  184.67a  202.77a  336.94b  337.29b  199.34a  20.314  0.037  Tryptophan  14.45a,c  13.96a  16.18b,c  18.37d  20.97e  0.731  <0.001  Lysine  200.98  179.64  236.86  143.01  192.84  12.659  0.213  Leucine  51.15a  52.54a  79.19b  67.91c  90.76d  4.189  <0.001  Threonine  91.02a,c  80.18a,d  97.04b,c  96.31b,c  75.43d  2.823  0.015  FAA  1199.04a  1064.50b  1461.15c  1327.20d  1197.62a  38.939  <0.001  EAA  683.05  592.01  840.18  557.60  688.46  44.892  0.898  TAA  2345.79a  2113.62b  2784.37c  2498.37a  2446.90a  63.857  0.023  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3FAA: flavor amino acid; EAA: essential amino acid; TAA: total amino acids. View Large Significant differences (P < 0.05) were observed in the concentrations of several fatty acids among the different treatment groups compared with control groups. For saturated fatty acid (SFA), the total contents were significantly lower (P < 0.05) in the ducks fed with LG and MG diets but were higher (P < 0.05) with the AG diet in comparison with the control diet. Palmitic acid (C16:0), stearic acid (C18:0), and docosanoic acid (C22:0) were significantly decreased with the MG diet compared with control diet. However, the other SFAs showed no significant differences (P > 0.05) among all of the treatment groups. For monounsaturated fatty acids (MUFA), the contents of myristic dilute acid (C14:1), oleic acid (C18:1n-9c), gondoic acid (C20:1n-9), and total MUFA were significantly higher (P < 0.05) in the MG compared to control group. For PUFA, the contents of eicosadienoic acid (C20:2n-6), dihomo-gammalinolenic acid (DGLA: C20:3n-6), arachidonic acid (AA: C20:4n-6), eicosapentaenoic acid (EPA: C20:5n-3), docosahexaenoic acid (DHA: C22:6n-3), and the total PUFA content significantly increased (P < 0.05) in the ducks fed with MG diet compared with the control diet. By contrast, the contents of other unsaturated fatty acids showed no significant differences (P > 0.05) in breast muscle among all of the five groups (Table 6). Table 6. Effects of Clostridium butyricum on fatty acid composition in breast muscle of Pekin ducks at 42 d of age.2 Parameters3  Groups      (μg/g)  Control  LG  MG  HG  AG  SEM1  P value  C10:0  17.57a,c  18.25a,d  17.74a,e  17.27c,e  18.92b,d  0.179  0.005  C11:0  16.23a  16.59a  15.02b  16.12a  21.28c  0.592  <0.001  C12:0  19.76a,c  20.56a,d  19.95a,e  19.45c,e  21.30b,d  0.204  0.018  C13:0  21.11a,c  20.79a,d  20.31a,e  19.77c,e  21.68b,d  0.208  0.011  C14:0  22.32  23.25  22.17  22.69  24.16  0.271  0.098  C15:0  20.45a,c  21.21a,d  20.53a,e  20.20c,e  21.81b,d  0.196  0.024  C16:0  134.82a  114.43b  114.39b  156.52c  180.30d  7.185  <0.001  C17:0  20.40  21.48  20.37  20.17  21.31  0.212  0.150  C18:0  238.16  217.63  218.89  252.39  290.63  10.792  0.174  C20:0  17.27  17.85  16.79  16.92  18.16  0.228  0.259  C22:0  17.02  17.76  16.62  16.63  17.79  0.199  0.120  C24:0  20.88a,c  21.59a,d  20.66a,e  20.49c,e  22.38b,d  0.227  0.017  ⊙SFA  564.98a  531.39a  523.78a  598.61a,c  679.67b,c  18.366  0.015  C14:1  9.00a  9.51b  9.63b  8.85a  9.01a  0.091  0.001  C16:1  23.02  23.38  33.96  26.92  19.51  1.876  0.123  C18:1n-9  231.99a,d  371.60b,c  349.29a,c  207.71d  206.29d  23.900  0.023  C20:1  19.76  20.24  21.59  20.39  19.40  0.329  0.288  C22:1n-9  18.91a,d  19.57a,c  20.15b,c  18.63d  19.01a,d  0.182  0.026  ⊙MUFA  302.67a  444.31b  434.62b  282.50a  273.40a  25.427  0.027  C18:2n-6  206.05  145.42  250.05  231.43  164.51  18.287  0.354  C18:3n-6  17.56a,d  18.22a,c  18.77b,c  17.48d  17.71a,d  0.153  0.008  C18:3n-3  21.64  22.13  27.00  24.57  21.67  0.872  0.212  C20:2n-6  28.07a  26.09a  33.77b  27.39a  25.02a  1.035  0.035  C20:3n-6  30.15a,b  28.60a  35.52b  27.93a  26.38a  1.087  0.042  C20:4n-6  124.39  98.48  154.75  128.56  104.00  7.004  0.050  C20:3n-3  19.56  20.75  20.63  19.37  19.50  0.219  0.079  C20:5n-3  19.09a,d  20.26a,c  20.67b,c  18.58d  18.89d  0.267  0.018  C22:6n-3  21.78a,c  23.25a,d  24.20b,d  21.71c  21.16c  0.352  0.007  ⊙PUFA  488.32  403.21  618.61  517.02  418.84  28.602  0.053  PUFA/SFA  0.86a  0.76a  1.18b  0.85a  0.63a  0.054  0.001  n-3PUFA  82.10a  86.39a,c  92.43b,c  84.23a,d  81.22a,d  1.201  0.002  n-6PUFA  406.22a  316.82b  526.18c  432.79d  319.47b  21.080  <0.001  Parameters3  Groups      (μg/g)  Control  LG  MG  HG  AG  SEM1  P value  C10:0  17.57a,c  18.25a,d  17.74a,e  17.27c,e  18.92b,d  0.179  0.005  C11:0  16.23a  16.59a  15.02b  16.12a  21.28c  0.592  <0.001  C12:0  19.76a,c  20.56a,d  19.95a,e  19.45c,e  21.30b,d  0.204  0.018  C13:0  21.11a,c  20.79a,d  20.31a,e  19.77c,e  21.68b,d  0.208  0.011  C14:0  22.32  23.25  22.17  22.69  24.16  0.271  0.098  C15:0  20.45a,c  21.21a,d  20.53a,e  20.20c,e  21.81b,d  0.196  0.024  C16:0  134.82a  114.43b  114.39b  156.52c  180.30d  7.185  <0.001  C17:0  20.40  21.48  20.37  20.17  21.31  0.212  0.150  C18:0  238.16  217.63  218.89  252.39  290.63  10.792  0.174  C20:0  17.27  17.85  16.79  16.92  18.16  0.228  0.259  C22:0  17.02  17.76  16.62  16.63  17.79  0.199  0.120  C24:0  20.88a,c  21.59a,d  20.66a,e  20.49c,e  22.38b,d  0.227  0.017  ⊙SFA  564.98a  531.39a  523.78a  598.61a,c  679.67b,c  18.366  0.015  C14:1  9.00a  9.51b  9.63b  8.85a  9.01a  0.091  0.001  C16:1  23.02  23.38  33.96  26.92  19.51  1.876  0.123  C18:1n-9  231.99a,d  371.60b,c  349.29a,c  207.71d  206.29d  23.900  0.023  C20:1  19.76  20.24  21.59  20.39  19.40  0.329  0.288  C22:1n-9  18.91a,d  19.57a,c  20.15b,c  18.63d  19.01a,d  0.182  0.026  ⊙MUFA  302.67a  444.31b  434.62b  282.50a  273.40a  25.427  0.027  C18:2n-6  206.05  145.42  250.05  231.43  164.51  18.287  0.354  C18:3n-6  17.56a,d  18.22a,c  18.77b,c  17.48d  17.71a,d  0.153  0.008  C18:3n-3  21.64  22.13  27.00  24.57  21.67  0.872  0.212  C20:2n-6  28.07a  26.09a  33.77b  27.39a  25.02a  1.035  0.035  C20:3n-6  30.15a,b  28.60a  35.52b  27.93a  26.38a  1.087  0.042  C20:4n-6  124.39  98.48  154.75  128.56  104.00  7.004  0.050  C20:3n-3  19.56  20.75  20.63  19.37  19.50  0.219  0.079  C20:5n-3  19.09a,d  20.26a,c  20.67b,c  18.58d  18.89d  0.267  0.018  C22:6n-3  21.78a,c  23.25a,d  24.20b,d  21.71c  21.16c  0.352  0.007  ⊙PUFA  488.32  403.21  618.61  517.02  418.84  28.602  0.053  PUFA/SFA  0.86a  0.76a  1.18b  0.85a  0.63a  0.054  0.001  n-3PUFA  82.10a  86.39a,c  92.43b,c  84.23a,d  81.22a,d  1.201  0.002  n-6PUFA  406.22a  316.82b  526.18c  432.79d  319.47b  21.080  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid. View Large Table 6. Effects of Clostridium butyricum on fatty acid composition in breast muscle of Pekin ducks at 42 d of age.2 Parameters3  Groups      (μg/g)  Control  LG  MG  HG  AG  SEM1  P value  C10:0  17.57a,c  18.25a,d  17.74a,e  17.27c,e  18.92b,d  0.179  0.005  C11:0  16.23a  16.59a  15.02b  16.12a  21.28c  0.592  <0.001  C12:0  19.76a,c  20.56a,d  19.95a,e  19.45c,e  21.30b,d  0.204  0.018  C13:0  21.11a,c  20.79a,d  20.31a,e  19.77c,e  21.68b,d  0.208  0.011  C14:0  22.32  23.25  22.17  22.69  24.16  0.271  0.098  C15:0  20.45a,c  21.21a,d  20.53a,e  20.20c,e  21.81b,d  0.196  0.024  C16:0  134.82a  114.43b  114.39b  156.52c  180.30d  7.185  <0.001  C17:0  20.40  21.48  20.37  20.17  21.31  0.212  0.150  C18:0  238.16  217.63  218.89  252.39  290.63  10.792  0.174  C20:0  17.27  17.85  16.79  16.92  18.16  0.228  0.259  C22:0  17.02  17.76  16.62  16.63  17.79  0.199  0.120  C24:0  20.88a,c  21.59a,d  20.66a,e  20.49c,e  22.38b,d  0.227  0.017  ⊙SFA  564.98a  531.39a  523.78a  598.61a,c  679.67b,c  18.366  0.015  C14:1  9.00a  9.51b  9.63b  8.85a  9.01a  0.091  0.001  C16:1  23.02  23.38  33.96  26.92  19.51  1.876  0.123  C18:1n-9  231.99a,d  371.60b,c  349.29a,c  207.71d  206.29d  23.900  0.023  C20:1  19.76  20.24  21.59  20.39  19.40  0.329  0.288  C22:1n-9  18.91a,d  19.57a,c  20.15b,c  18.63d  19.01a,d  0.182  0.026  ⊙MUFA  302.67a  444.31b  434.62b  282.50a  273.40a  25.427  0.027  C18:2n-6  206.05  145.42  250.05  231.43  164.51  18.287  0.354  C18:3n-6  17.56a,d  18.22a,c  18.77b,c  17.48d  17.71a,d  0.153  0.008  C18:3n-3  21.64  22.13  27.00  24.57  21.67  0.872  0.212  C20:2n-6  28.07a  26.09a  33.77b  27.39a  25.02a  1.035  0.035  C20:3n-6  30.15a,b  28.60a  35.52b  27.93a  26.38a  1.087  0.042  C20:4n-6  124.39  98.48  154.75  128.56  104.00  7.004  0.050  C20:3n-3  19.56  20.75  20.63  19.37  19.50  0.219  0.079  C20:5n-3  19.09a,d  20.26a,c  20.67b,c  18.58d  18.89d  0.267  0.018  C22:6n-3  21.78a,c  23.25a,d  24.20b,d  21.71c  21.16c  0.352  0.007  ⊙PUFA  488.32  403.21  618.61  517.02  418.84  28.602  0.053  PUFA/SFA  0.86a  0.76a  1.18b  0.85a  0.63a  0.054  0.001  n-3PUFA  82.10a  86.39a,c  92.43b,c  84.23a,d  81.22a,d  1.201  0.002  n-6PUFA  406.22a  316.82b  526.18c  432.79d  319.47b  21.080  <0.001  Parameters3  Groups      (μg/g)  Control  LG  MG  HG  AG  SEM1  P value  C10:0  17.57a,c  18.25a,d  17.74a,e  17.27c,e  18.92b,d  0.179  0.005  C11:0  16.23a  16.59a  15.02b  16.12a  21.28c  0.592  <0.001  C12:0  19.76a,c  20.56a,d  19.95a,e  19.45c,e  21.30b,d  0.204  0.018  C13:0  21.11a,c  20.79a,d  20.31a,e  19.77c,e  21.68b,d  0.208  0.011  C14:0  22.32  23.25  22.17  22.69  24.16  0.271  0.098  C15:0  20.45a,c  21.21a,d  20.53a,e  20.20c,e  21.81b,d  0.196  0.024  C16:0  134.82a  114.43b  114.39b  156.52c  180.30d  7.185  <0.001  C17:0  20.40  21.48  20.37  20.17  21.31  0.212  0.150  C18:0  238.16  217.63  218.89  252.39  290.63  10.792  0.174  C20:0  17.27  17.85  16.79  16.92  18.16  0.228  0.259  C22:0  17.02  17.76  16.62  16.63  17.79  0.199  0.120  C24:0  20.88a,c  21.59a,d  20.66a,e  20.49c,e  22.38b,d  0.227  0.017  ⊙SFA  564.98a  531.39a  523.78a  598.61a,c  679.67b,c  18.366  0.015  C14:1  9.00a  9.51b  9.63b  8.85a  9.01a  0.091  0.001  C16:1  23.02  23.38  33.96  26.92  19.51  1.876  0.123  C18:1n-9  231.99a,d  371.60b,c  349.29a,c  207.71d  206.29d  23.900  0.023  C20:1  19.76  20.24  21.59  20.39  19.40  0.329  0.288  C22:1n-9  18.91a,d  19.57a,c  20.15b,c  18.63d  19.01a,d  0.182  0.026  ⊙MUFA  302.67a  444.31b  434.62b  282.50a  273.40a  25.427  0.027  C18:2n-6  206.05  145.42  250.05  231.43  164.51  18.287  0.354  C18:3n-6  17.56a,d  18.22a,c  18.77b,c  17.48d  17.71a,d  0.153  0.008  C18:3n-3  21.64  22.13  27.00  24.57  21.67  0.872  0.212  C20:2n-6  28.07a  26.09a  33.77b  27.39a  25.02a  1.035  0.035  C20:3n-6  30.15a,b  28.60a  35.52b  27.93a  26.38a  1.087  0.042  C20:4n-6  124.39  98.48  154.75  128.56  104.00  7.004  0.050  C20:3n-3  19.56  20.75  20.63  19.37  19.50  0.219  0.079  C20:5n-3  19.09a,d  20.26a,c  20.67b,c  18.58d  18.89d  0.267  0.018  C22:6n-3  21.78a,c  23.25a,d  24.20b,d  21.71c  21.16c  0.352  0.007  ⊙PUFA  488.32  403.21  618.61  517.02  418.84  28.602  0.053  PUFA/SFA  0.86a  0.76a  1.18b  0.85a  0.63a  0.054  0.001  n-3PUFA  82.10a  86.39a,c  92.43b,c  84.23a,d  81.22a,d  1.201  0.002  n-6PUFA  406.22a  316.82b  526.18c  432.79d  319.47b  21.080  <0.001  1Total SEM (N = 12). 2Means with different superscript letters indicate that there are significant differences (P < 0.05) between any two groups in the same row. 3SFA: saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid. View Large DISCUSSION At present, consumers are paying increasingly attention to the nutritional value and health benefits of the food they eat and this also applies to duck meat (Liao et al., 2015). In the experiments described here, ducks fed a C. butyricum inclusion diet showed an increased average body weight, along with an increased average daily gain and feed conversion ratio compared with the control group and the antibiotic group, indicating that it is feasible for C. butyricum to replace antibiotics as a growth promoter. Previous reports have also shown that a C. butyricum supplementation diet could enhance immune function, promote nutrient digestion, and help to balance intestinal microflora of animals (Murayama et al., 1995; Yang et al., 2012; Chen et al., 2013). These findings may therefore explain why ducks fed with a C. butyricum diet present higher growth performance compared with the control group and the antibiotic supplemented group. The color value of duck meat is a most important attribute relating to meat freshness and quality. Some authors have previously reported that dietary supplementation with probiotics can improve meat quality in animals. Suo et al. (Suo et al., 2012) reported that meat quality, as measured by the pH45 min, meat color and the shore force, were all significantly enhanced after dietary supplementation of pigs with L. plantarum ZJ316. Endo and Nakano confirmed that the characteristics of meat quality were all improved with dietary supplementation of probiotics in broilers (Endo and Nakano, 1999). Their findings may help explain the results of our study in which the addition of C. butyricum to the diet significantly increased the pH45 min, pH24 h, and a* value and the difference in a* value for meat color may have been due to the dietary supplementation of probiotics of Peking ducks. Similarly, the present study revealed that the addition of C. butyricum could decrease the L* value and b* value of the breast muscle in Peking ducks and subsequently increase their market price. It is well known that pH is one of the most important post-slaughter factors affecting the drip loss (water-holding capacity) of duck meat (Yang et al., 2017). Swatland demonstrated that drip loss of meat was associated with the ultimate pH and the speed of pH decline (Swatland, 1995). A higher ultimate pH is thought to lead to lower drip losses (Di Luca et al., 2016). Drip loss can be used as an indicator to evaluate the quality deviations associated with the tempo and extent of postmortem glycolysis in muscle. Therefore, a dietary C. butyricum-induced improvement in water-holding capacity and better meat quality of Peking ducks could be beneficial for poultry production. Recent studies have revealed the potential reduction of oxidative stress of dietary additive probiotics (Kai et al., 2004; Martarelli et al., 2011), and therefore dietary supplementation with C. butyricum could be provided to reduce intramuscular oxidative damages in Peking ducks, thereby improving the meat quality. In our study, the observed increase in antioxidant enzymatic activity (T-SOD, CAT, and GSH-PX) in muscle might be related to the dietary supplementation with C. butyricum. Relatively, T-AOC levels increased in all groups supplemented with C. butyricum even at the lowest level (200 mg/kg) of C. butyricum. With increasing levels of C. butyricum the MDA content showed a decreasing trend in muscle. In addition, several reports have revealed that some probiotic strains could influence the activity of antioxidants and might be useful in alleviating systemic oxidative stress either through stimulating the immune system and reducing inflammation or preventing infection by intestinal pathogens thereby reducing inflammation and related oxidative damages (Cheng et al., 2017c). In our study, dietary C. butyricum did not significantly influence the concentrations in serum of total cholesterol, v-LDL, and NEFA in Peking ducks. However, the dietary levels of C. butyricum significantly reduced the concentrations of TG, HDL-C, and LDL-C in serum compared with control groups. High serum concentrations of TG and LDL-C are recognized as an independent risk factor for atherosclerosis, which may be the main risk factor of coronary heart disease (Virani et al., 2011). However, Fukushima and Nakano found that dietary supplementation with a mixture of probiotics resulted in a higher HDL concentration and a lower LDL cholesterol concentration in the serum of rats (Fukushima and Nakano, 1996). Therefore, our results suggest that dietary C. butyricum is an effective health promoting agent to improve lipid metabolism in live ducks. With the strengthening economy, living standards have been improved in China, and along with this the demands on the food industry in terms of flavor and nutrition have increased. IMP is one of the main components responsible the umami flavor of meat and the content of IMP can determine the freshness of the meat after cooking (Jung et al., 2013). In our study, the addition of dietary C. butyricum increased the content of IMP in the breast muscle of Peking ducks in a dose-dependent manner. The flavor was more pronounced when the lipid levels were high in the breast meat. IMF is used to improve the tenderness of the meat in two ways. On the one hand, it cuts off the crosslinking structure between the fiber bundles, and on the other hand, it is beneficial to the fracture of muscle fibers during the chewing process (Chartrin et al., 2006; Qiu et al., 2017). The increasing content of IMF in the breast muscle supplemented with dietary C. butyricum also improved the meat flavor, making it more popular among consumers. Muscle tenderness is negatively related to the diameter of the muscle fibers but positively related to muscle density (Calkins et al., 1981; Zhu et al., 2012). The biological characteristics of muscle fibers are therefore an important quantitative index of meat quality, which is used to assess properties of duck meat and is closely related to the color of meat, the water content and pH value (Felício et al., 2013; Wu et al., 2015). Glycine, alanine, aspartic acid, glutamic acid, phenylalanine, and tyrosine are referred to collectively as the flavor amino acid (FAA) (Sabagh et al., 2016). Their composition and content directly affects the freshness of food. The increased levels of FAA following C. butyricum supplementation in our study improved the flavor of the duck meat, making it more favorable to consumers. Fatty acid alterations via dietary means may provide an effective way to obtain healthy duck products for human consumption. The current study revealed that dietary supplementation with C. butyricum could change the fatty acid composition and decreased the contents of SFA in duck meat. Over recent years, researchers have paid increasing attention to the modulation of fatty acid profiles in poultry meat as cardiovascular heart disease are considered closely associated with the contents of SFA intake in daily diet (Ramasamy et al., 2006), and hence from a healthy perspective, meat that contains relatively lower SFA contents could help in reducing the risk of cardiovascular heart diseases (Cheng et al., 2017a). Therefore, much attention should be paid toward the alteration of Peking duck products to improve the meat quality and flavor via dietary means, such as adding antioxidants and probiotics, to meet consumers’ needs and improve their health (Ivanovic et al., 2012; Yuan et al., 2012). The levels of docosahexaenoic acid (DHA, C22:6, n-3) in the breast meat of ducks fed with C. butyricum were significantly higher than the control. This can be explained by the modulation effects of dietary C. butyricum treatment. DHA is responsible for hypolipidemic and neuroprotective effects (Ramasamy et al., 2009). Overall, it was not only the meat quality (color and flavor) that was improved but also the contents of EPA (C20:5, n-3) and PUFA that were increased in breast muscle via the stimulative effects of C. butyricum supplemented in the duck diet. The increased contents of PUFA may be due to the enhancement of the antioxidant defenses system in ducks fed a diet supplemented with C. butyricum, although this requires further study. The enrichment of duck meat with long chain PUFA by supplementation with C. butyricum may be beneficial to human health and could replace the need for marine sources of EPA and DHA in the diet of poultry. Supplementation with dietary C. butyricum enhanced the ratio of PUFA: SFA by changing the fatty acid composition in breast muscle. Moreover, no adverse effects on the growth performance of Peking ducks were found after dietary supplementation with C. butyricum. Thus, the present results led to the conclusion that the widespread dietary supplementation of ducks with C. butyricum might be a feasible strategy to reduce the SFA contents and enhance the MUFA and PUFA contents of Peking duck meat. The resulting alterations in the fatty acid composition of meat may satisfy the nutritional demands of human consumers. To the best of our knowledge, this is the first comprehensive report on the influence of a C. butyricum-supplemented diet on the quality and flavor of Peking duck meat. In summary, it is well established that dietary C. butyricum can have a positive effect on animal health and meat quality by affecting PUFA deposition and metabolic processes in the breast muscle; however, further studies are required to investigate the effects of C. butyricum supplementation on cooked duck meat. CONCLUSIONS Overall, supplementation of C. butyricum for 42 d enhanced the growth performance, promoted meat quality and antioxidant activities, and improved the IMP and IMF contents of ducks, but decreased the triglyceride and HDL-C and LDL-C contents of Peking ducks. Such a diet is therefore able to enrich meat with FAA, EPA and DHA and increase the PUFA to SFA ratio. Consumption of such duck meat products may be beneficial to human health while also offering an enhanced flavor. Furthermore, dietary supplementation with probiotics may contribute to reduced drug resistance in poultry. Further studies are needed to investigate the effects of C. butyricum supplementation on fatty acid profile of cooked duck meat. Acknowledgements This research was supported by Beijing Agriculture Innovation Consortium (BAIC04-2018). The authors declare that they have no competing interests. REFERENCES Awad W. A., Ghareeb K., Abdel-Raheem S., Bohm J.. 2009. 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Poultry ScienceOxford University Press

Published: May 14, 2018

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