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Effects of riboflavin supplementation on growth performance, carcass traits, and riboflavin status of growing male white Pekin ducks
Animal Feed Science and Technology 209 (2015) 274–279
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Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
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Effects of riboflavin supplementation on growth performance, carcass traits, and riboflavin status of growing male white Pekin ducks J. Tang a,b,1 , J. Hu a,1 , Z.G. Wen a,b , Y. Jiang a,b , H. AL-Kateb c , W. Huang a , Y.M. Guo b , M. Xie a,∗ , S.S. Hou a a b c
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China College of Animal Science and Technology, China Agricultural University, Beijing 100193, China University of the West of England, Centre for Research in Biosciences, Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY, UK
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Article history: Received 24 December 2014 Received in revised form 24 July 2015 Accepted 25 July 2015 Keywords: Ducks Riboflavin Requirement Growth performance
a b s t r a c t An experiment involving six dietary riboflavin concentration (1.38, 2.38, 3.38, 4.38, 5.38, and 6.38 mg/kg) was conducted to determine the effects of riboflavin on performance and riboflavin status of growing male white Pekin ducks in terms of growth performance, carcass traits, and free riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) concentration in the liver and plasma. A basal corn-soybean meal containing 1.38 mg/kg of naturally occurring riboflavin was used and this basal diet was fed with 0, 1, 2, 3, 4, and 5 mg/kg diet of supplemental riboflavin. One-day-old male white Pekin ducks were fed common starter diets from hatch to 14 days of age and then fed the experimental diets from 15 to 35 days of age. A total of 288 15-day-old ducks were allotted to 6 dietary treatments with 6 replicate pens of 8 birds per pen. At 35 days of age, the average daily weight gain (ADG), average daily feed intake (ADFI), gain to feed ratio (G:F), and percentage yield of breast and leg meat, abdominal fat were examined and free riboflavin, FMN, and FAD in plasma or liver were all analyzed. The growth depression, low plasma free riboflavin, liver free riboflavin, liver FMN, and liver FAD concentration were observed in the ducks fed the riboflavin-deficient basal diet (P < 0.05), and these adverse effects were alleviated by riboflavin supplementation. The growing ducks fed the basal diet with no supplementation of riboflavin had the lowest ADG, ADFI, G:F and breast meat yield among all ducks (P < 0.05) and these criteria showed linear or quadratic response to increasing dietary riboflavin (P < 0.05). On the other hand, dietary riboflavin influenced the status of this vitamin in growing ducks. The poorest growth performance of the riboflavin-deficient growing ducks was accompanied with the lowest content of riboflavin, FAD, and FMN in plasma or liver (P < 0.05) but these bad statuses were reversed by increasing dietary riboflavin in which the riboflavin and its derivative increased linearly or quadratically as dietary riboflavin increased (P < 0.05). According to the broken-line model, the riboflavin requirements (based on dietary total riboflavin) of male white Pekin ducks from 15 to 35 days of age for ADG, ADFI, G:F, plasma free riboflavin, liver free riboflavin, and liver FMN were 2.43, 2.48, 2.31, 3.57, 2.78, and 2.83 mg/kg, respectively. © 2015 Elsevier B.V. All rights reserved.
Abbreviations: ADG, average daily weight gain; ADFI, average daily feed intake; G:F, gain to feed ratio; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; HPLC, high performance liquid chromatography; SEM, the standard error of the mean. ∗ Corresponding author. Tel.: +86 10 62816227; fax: +86 10 62815832. E-mail address: [email protected] (M. Xie). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.anifeedsci.2015.07.023 0377-8401/© 2015 Elsevier B.V. All rights reserved.
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1. Introduction Riboflavin is a water-solube vitamin and this vitamin is involved in a range of redox reactions in primary metabolic pathways and electron transfer processes (Powers, 2003; Lienhart et al., 2013), mainly as its biologically active coenzymes forms flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Like other avian species, riboflavin is needed by ducks and the deficiency of this vitamin could lead to high mortality, poor growth, and low retention of tissue riboflavin in starter Pekin ducks (Tang et al., 2013, 2014). Recently, the riboflavin requirements of modern starter Pekin ducks were estimated by Tang et al. (2013, 2014) and they confirmed that the riboflavin recommendation of NRC (1994) during the starter period (4 mg/kg) is still sufficient for modern Pekin duck strains. However, although the riboflavin recommendation of growing Pekin ducks (4 mg/kg) was provided by NRC (1994), no documentation was provided to support this value and the riboflavin requirements of growing ducks are virtually lacking until now. In chicks, the riboflavin requirements of birds decreased as age increased (Norris et al., 1936; Heuser et al., 1938) but this conclusion was not confirmed in ducks, which indicates that the riboflavin requirement of ducks during the growing period may be different from those during starter period. In practice, raising ducks is directed to meat production and carcass yield is usually considered to promote the benefit of duck production, which makes it necessary to evaluate the effects of riboflavin on growth performance and carcass traits of Pekin ducks when the riboflavin requirements of growing ducks were examined. On the other hand, in ducks, poor growth is usually accompanied with low plasma and liver riboflavin or FMN in riboflavin-deficient birds and thus the status of this vitamin could be indicated by tissue riboflavin and its derivative (Tang et al., 2013, 2014), which may reinforce the evaluation of the duck response to increasing dietary riboflavin. Therefore, the objective of the present study was to determine the effects of riboflavin on growth performance, carcass traits, and riboflavin status of growing Pekin ducks and to evaluate the requirements of this vitamin of these birds during growing period. 2. Materials and methods 2.1. Animals and housing All procedures of the present study were approved by the animal care and use committee of the Institute of Animal Sciences of Chinese Academy of Agricultural Sciences. A total of 320 one-day-old male white Pekin ducks obtained from Pekin duck breeding center in Chinese Academy of Agricultural Sciences were randomly allotted to 16 raised plastic-floor pens with 20 birds per pen and then they were raised until 14 days of age. At 14 days of age, after fasting for 12 h, all the ducks were weighed individually and 288 ducks selected from these ducks were divided into six dietary treatments, each containing 6 replicate pens with 8 birds per pen. All pens had similar pen weight and these birds were free access to water and feed from 15 to 35 days of age. During this period, the lighting was continuous, and the temperature was kept at 33 ◦ C from 1 to 3 days of age, and then it was reduced gradually to approximately 25 ◦ C until 14 days of age and was kept at approximately 16–22 ◦ C thereafter. 2.2. Diet All the ducks were raised with common corn-soybean meal starter diet (Table 1) and this diet was formulated to meet the nutrient recommendations for starter ducks provided by Ministry of Agriculture of China (2012). The starter diet included 4 mg/kg of supplemental riboflavin. The basal diet during the growing period was formulated to be riboflavin-deficient (Table 1) and all nutrients except riboflavin met the recommendations for growing ducks provided by Ministry of Agriculture of China (2012). In order to produce six experimental diets, the basal diet were prepared as mash and then supplemented with 0, 1, 2, 3, 4, and 5 mg crystalline riboflavin/kg diet, respectively. All diets were cold-pelleted at room temperature. The crystalline riboflavin (purity, 99%) was obtained from (Sigma–Aldrich, St. Louis, MO, USA). The riboflavin content of this basal diet was determined by high performance liquid chromatography (HPLC) with a fluorescence detector and the value detected was 1.38 mg/kg. The riboflavin concentrations for the six experimental diets were calculated. These values are 1.38, 2.38, 3.38, 4.38, 5.38, and 6.38 mg riboflavin/kg of feed. 2.3. Sample preparation and data collection At 35 days of age, the average daily weight gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F) of each pen were measured. Two ducks were selected randomly from each pen and bled by cardiac puncture. The blood were collected into heparin sodium-containing tubes and centrifuged at 1520 × g for 10 min to obtain plasma. These plasma samples were then stored at −20 ◦ C until analyzed for riboflavin. Afterwards, these ducks were killed by CO2 inhalation and immersed into hot water at 60 ◦ C for 2 min. Then they were scalded, picked, and eviscerated manually. The breast meat (including both the pectoralis major and pectoralis minor), leg meat (including the thighs and the drum sticks), and abdominal fat were all removed from carcasses and weighed. The percentage yield for the breast meat, leg meat, and abdominal fat were expressed as relative weight to live BW at processing. The livers were removed from these birds and immediately stored at −20 ◦ C until analyzed for riboflavin, FMN, and FAD.
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Table 1 Composition of common starter feed from hatch to 14 days of age and riboflavin-deficient basal diet from 15 to 35 days of age (g/kg as-fed). Item
Common starter feed (from hatch to 14 days)
Basal feed (from 15 to 35 days)
Ingredient, g/kg Corn Soybean Corn gluten meal Limestone Dicalcium phosphate Vitamin and trace mineral premix Sodium chloride dl-Methionine l-Lysine·HCl l-Tryptophan
629.5 333.0 – 8.0 15.0 10.0a 3.0 1.5 – –
706.9 150.0 100.0 10.0 16.0 10.0b 3.0 0.8 2.7 0.6
Calculated composition Metabolizable energyc , MJ/kg Crude protein Calcium Nonphytate phosphorus Lysine Methionine Methionine + cysteine Threonine Tryptophan Arginine Riboflavind , mg/kg
12.22 200.2 9.3 3.6 11.1 4.5 7.9 8.3 2.2 13.8 1.69
12.64 175.9 9.9 4.2 9.1 4.0 7.1 6.4 2.0 8.9 1.38
a Supplied per kilogram of total diet: Cu (CuSO4 ·5H2 O), 10 mg; Fe (FeSO4 ·7H2 O), 60 mg; Zn (ZnO), 60 mg; Mn (MnSO4 ·H2 O), 80 mg; Se (NaSeO3 ), 0.3 mg; I (KI), 0.2 mg; choline chloride, 1000 mg; vitamin A (retinyl acetate), 10,000 IU; vitamin D3 (Cholcalciferol), 3000 IU; vitamin E (dl-␣-tocopheryl acetate), 20 IU; vitamin K3 (menadione sodium bisulfate), 2 mg; thiamin (thiamin mononitrate), 2 mg; riboflavin, 4 mg; pyridoxine hydrochloride, 4 mg; cobalamin, 0.02 mg; calcium-d-pantothenate, 20 mg; nicotinic acid, 50 mg; folic acid, 1 mg; biotin, 0.2 mg. b Supplied per kilogram of total diet: Cu (CuSO4 ·5H2 O), 10 mg; Fe (FeSO4 ·7H2 O), 60 mg; Zn (ZnO), 60 mg; Mn (MnSO4 ·H2 O), 80 mg; Se (NaSeO3 ), 0.3 mg; I (KI), 0.2 mg; choline chloride, 750 mg; vitamin A (retinyl acetate), 8000 IU; vitamin D3 (Cholcalciferol), 3000 IU; vitamin E (dl-␣-tocopheryl acetate), 20 IU; vitamin K3 (menadione sodium bisulfate), 2 mg; thiamin (thiamin mononitrate), 1.5 mg; pyridoxine hydrochloride, 3 mg; cobalamin, 0.02 mg; calcium-d-pantothenate, 10 mg; nicotinic acid, 50 mg; folic acid, 1 mg; biotin, 0.2 mg. c The values are calculated according to the AME of chickens (Ministry of Agriculture of China, 2004). d The numbers were analyzed by high performance liquid chromatography.
2.4. Chemical analysis The riboflavin concentration in feed, plasma, and liver, the FMN and FAD content in liver were all determined by reversedphase HPLC according to the methods described previously by Tang et al. (2013, 2014). Prior to the HPLC analysis, feed, liver and plasma samples were prepared according to the method described by Britton et al. (2003), Batey and Eckhert (1990), and Petteys and Frank (2011), respectively. Peaks were identified and quantified by using the pure authentic standards purchased from (Sigma–Aldrich, St. Louis, MO, USA). 2.5. Statistical analyses The one-way ANOVA were performed using SAS software (SAS Institute, 2003), with pen used as the experimental unit for analysis. The linear and quadratic polynomial contrasts were performed to determine the effect of supplemental riboflavin in the duck diet. The variability in the data was expressed as the standard error of the means (SEM) and a probability level of P < 0.05 was considered to be statistically significant. Associations were established between growth performance (ADG, ADFI or G:F) and tissue riboflavin by multivariate correlation analyses. The broken-line regression analysis (Robbins et al., 2006) was used to estimate the riboflavin requirements for ducks. The broken-line model was provided as follows: y = l + u(r − x) where y = growth performance (ADG, ADFI or G:F), plasma riboflavin, liver riboflavin, and liver FMN, x = dietary total riboflavin level (mg/kg), r = riboflavin requirement, l = the response at x = r, u = the slope of the curve. In this model, y = l when x > r. 3. Results and discussion 3.1. Growth performance and carcass traits In the present study, growth depression was still the typical symptom for riboflavin-deficient growing Pekin ducks and it was similar to the phenomena observed in riboflavin-deficient starter ducks (Tang et al., 2013, 2014). Among all ducks, the birds fed the basal diet with no supplementation of riboflavin showed the lowest ADG, ADFI, and G:F (P < 0.05) and all these
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Table 2 Effects of dietary riboflavin on growth performance of male white Pekin ducks from 15 to 35 days of agea . Dietary riboflavin (mg/kg)
ADG (g/d)
ADFI (g/d)
1.38 2.38 3.38 4.38 5.38 6.38 Pooled SEM
54.4 80.3 81.0 81.4 82.1 81.5 1.80
130.4 168.3 169.9 172.5 173.9 171.3 2.92
P-value Riboflavin Riboflavin linear Riboflavin quadratic
<0.001 <0.001 <0.001
<0.001 <0.001 <0.001
G:F (g:g) 0.42 0.48 0.48 0.47 0.47 0.48 0.005 <0.001 <0.001 <0.001
ADG, average daily gain; ADFI, average daily feed intake; G:F, gain to feed ratio; SEM, standard error of the mean.
Table 3 Effects of dietary riboflavin on carcass yield of the 35-day-old male white Pekin ducksa . Dietary riboflavin (mg/kg)
Relative weight (% of processing live BW)b Breast meat
1.38 2.38 3.38 4.38 5.38 6.38 Pooled SEM P-value Riboflavin Riboflavin linear Riboflavin quadratic a b
2.68 4.21 4.10 4.26 4.58 4.38 0.10 <0.001 <0.001 <0.001
Leg meat 9.23 9.44 8.86 8.55 8.94 8.50 0.12 0.135 0.023 0.810
Abdominal fat 0.82 1.14 1.28 1.36 1.31 1.36 0.04 <0.001 <0.001 0.018
SEM, standard error of the mean. The percentage yield is calculated using the following equation: Yield = (breast meat, leg meat, or abdominal fat weight) × 100%/processing live BW.
criteria increased linearly or quadratically as dietary riboflavin increased from 1.38 to 6.38 mg riboflavin/kg (P < 0.05, Table 2). It is showed that the basal diet was riboflavin-deficient and the deficiency of this vitamin was alleviated by supplementation of crystalline riboflavin. However, riboflavin deficiency in growing diets did not cause high mortality and leg paralysis which took place in starter ducks (Tang et al., 2013, 2014), which indicated that riboflavin deficiency caused less damage to growing ducks and these birds needed less riboflavin than starter ducks. In order to estimate riboflavin requirements of growing ducks for growth performance, the broken-line regression was utilized because this regression was used successfully to predict the riboflavin requirements for ducks (Hegsted and Perry, 1948; Tang et al., 2013, 2014), broilers (Ruiz and Harms, 1988; Chung and Baker, 1990), pigs (Frank et al., 1988), and fish (Deng and Wilson, 2003). According to this regression, the riboflavin requirements of growing male white Pekin ducks from 15 to 35 days of age for ADG, ADFI, and G:F were 2.43, 2.48, and 2.31 mg riboflavin/kg diet, respectively (Table 5). These estimated requirements of growing ducks were all lower than those of starter Pekin ducks predicted by Tang et al. (2013) based on corn-corn gluten meal basal diets (3.31 and 5.21 mg/kg for ADG and feed conversion ratio) or Tang et al. (2014) based on corn-soybean meal basal diets (3.01, 2.98, and 2.79 mg/kg for ADG, ADFI, and G:F). This data is consistent with Heuser et al. (1938) who found that the riboflavin requirements of White Leghorn chicks decrease weekly and gradually from 2 to 8 weeks of age. Furthermore, the riboflavin requirements estimated by this study were much lower than the recommendation of NRC (1994) for growing white Pekin ducks from 2 to 7 weeks of age (4 mg/kg). Therefore, our results partly suggested that the riboflavin recommendation of NRC (1994) for growing ducks should be reduced appropriately, although this data is still adequate for duck growth. On the other hand, the growth depression caused by riboflavin deficiency may lead to reduction of carcass yield. For the ducks fed riboflavin-deficient basal diet, the worst growth performance was accompanied with the lowest breast meat and abdominal fat compared with other ducks fed riboflavin-supplemented diets (P < 0.05, Table 3). Fortunately, these ill effects could be reversed because the breast meat increased linearly or quadratically as dietary riboflavin increased (P < 0.05, Table 3). Therefore, considering that breast meat is the most valuable part of carcass when birds are further processed, dietary riboflavin should be taken into account during diet formulation for growing ducks, although it failed to estimate the riboflavin requirement for breast meat due to non-significant broken-line regression (P > 0.05).
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Table 4 Effects of dietary riboflavin on plasma and liver free riboflavin, liver flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) in 35-day-old Pekin ducksa . Dietary riboflavin (mg/kg)
1.38 2.38 3.38 4.38 5.38 6.38 Pooled SEM P-value Riboflavin Riboflavin linear Riboflavin quadratic a
Plasma (nmol/L)
Liver (g/g wet tissue)
Free riboflavin
Free riboflavin
17.4 63.8 96.4 103.0 110.7 104.2 4.8
7.82 10.04 10.70 11.26 10.32 11.46 0.21
<0.001 <0.001 <0.001
FAD
<0.001 <0.001 0.001
FMN
1.91 2.45 2.67 2.40 2.37 2.64 0.06
4.12 5.94 6.76 6.14 7.31 6.89 0.19
<0.001 0.006 0.048
<0.001 <0.001 0.023
SEM, standard error of the mean.
Table 5 Riboflavin requirements of male white Pekin ducks from 15 to 35 days of age based on broken-line regression analysisa . Response criterion
Regression
Requirement (mg/kg)
95% confidence interval (mg/kg)
P-value
R2
ADG ADFI G:F Plasma riboflavin Liver riboflavin Liver FMN
Y = 81.50 − 25.92 × (2.43 − x) Y = 171.9 − 37.9 × (2.48 − x) Y = 0.48 − 0.06 × (2.31 − x) Y = 106.0 − 39.46 × (3.57 − x) Y = 10.94 − 2.22 × (2.78 − x) Y = 6.78 − 1.82 × (2.83 − x)
2.43 2.48 2.31 3.57 2.78 2.83
2.37–2.49 2.30–2.65 2.04–2.59 3.12–4.01 1.63–3.93 1.48–4.20
<0.001 <0.001 <0.001 0.001 0.029 0.036
0.999 0.994 0.956 0.990 0.916 0.891
a
ADG, average daily gain; ADFI, average daily feed intake; G:F, gain to feed ratio; FMN, flavin mononucleotide.
Table 6 Correlation coefficients between growth performance and tissue riboflavina .
ADFI G:F Plasma riboflavin Liver riboflavin Liver FAD Liver FMN a
r P-value r P-value r P-value r P-value r P-value r P-value
ADG
ADFI
G:F
Plasma riboflavin
Liver riboflavin
Liver FAD
0.998 <0.001 0.969 0.002 0.910 0.012 0.919 0.010 0.883 0.020 0.914 0.011
0.951 0.004 0.929 0.007 0.921 0.009 0.860 0.028 0.923 0.009
0.815 0.048 0.887 0.018 0.946 0.004 0.859 0.029
0.92 0.009 0.79 0.059 0.95 0.004
0.88 0.019 0.83 0.039
0.83 0.040
Liver FMN
ADG, average daily gain; ADFI, average daily feed intake; G:F, gain to feed ratio; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide.
3.2. Riboflavin status Riboflavin exists in tissues with the three forms: free riboflavin, FMN, and FAD. Dietary riboflavin could influence the riboflavin status of growing ducks and it was indicated by riboflavin and its derivatives in tissue. The ducks fed the basal diet with no supplementation of riboflavin had the lowest concentration of free riboflavin, FAD, and FMN in plasma or liver (P < 0.05) and these ducks also showed the poorest growth among all ducks (P < 0.05, Table 4). However, these bad statuses could be reversed by increasing dietary riboflavin. The free riboflavin, FAD, and FMN in plasma or liver all increased linearly or quadratically as dietary riboflavin increased (P < 0.05, Table 4), which was also accompanied with the simultaneous improvement of growth performance (Table 3). Therefore, the correlation analysis was used to explain the relationship between growth performance and tissue riboflavin and confirm the validity of plasma riboflavin as indicator of riboflavin status. Our results showed that the ADG, ADFI, and G:F were all positively correlated to riboflavin, FAD, and FMN in liver or plasma (P < 0.05, Table 6), respectively, which indicated that riboflavin deficiency led to growth retardation of ducks. Furthermore, there existed positive correlations between plasma riboflavin and liver riboflavin and FMN (P < 0.05, Table 6) and there was a trend for the correlation between plasma riboflavin and liver FAD (0.05 < P < 0.10, Table 6), which confirmed that the change of plasma riboflavin was a good reflection of riboflavin status in tissues. Similar to previous study in young rainbow trouts and ducks (Woodward, 1985; Tang et al., 2013, 2014), riboflavin and its derivatives in tissue were used to predict riboflavin requirements of growing ducks. According to the broken-line analysis, the riboflavin requirements
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of growing white Pekin ducks for plasma free riboflavin, liver free riboflavin, and liver FMN were 3.57, 2.78, and 2.83 mg riboflavin/kg diet, respectively, and these estimated requirements were greater than those for growth performance in our study (Table 5), which was in agreement with the results in starter ducks (Tang et al., 2013, 2014). Furthermore, these estimated requirements for tissue saturation were much lower than those of starter Pekin ducks estimated by Tang et al. (2013) (3.85 and 3.91 mg/kg for plasma riboflavin and liver riboflavin) or Tang et al. (2014) (4.18, 3.63, and 3.59 mg/kg for plasma riboflavin, liver riboflavin and liver FMN), which also suggested that the riboflavin requirements of growing ducks should be reduced as age increased (Table 6). 4. Conclusion Riboflavin deficiency depressed growth performance and breast meat yield of growing ducks and riboflavin status was worsened at this instance, but these negative effects could be avoided by increasing dietary riboflavin. According to the broken-line analysis, the riboflavin requirements for growing male white Pekin ducks from 15 to 35 days of age for ADG, ADFI, G:F, plasma free riboflavin, liver free riboflavin, and liver FMN were 2.43, 2.48, 2.31, 3.57, 2.78, and 2.83 mg/kg, respectively. Conflict of interest None declared. Acknowledgments This work was sponsored by the earmarked fund for China Agriculture Research System (CARS-43) and the science and technology innovation project of Chinese Academy of Agricultural Sciences (CXGC-IAS-09). References Batey, D.W., Eckhert, C.D., 1990. Identification of FAD, FMN, and riboflavin in the retina by microextraction and high-performance liquid chromatography. Anal. Biochem. 188, 164–167. Britton, N.L., Riter, K.L., Smallidge, R.L., Joseph, H., 2003. Reversed-phase liquid chromatographic determination of riboflavin in feeds. J. AOAC Int. 86, 197–201. Chung, T.K., Baker, D.H., 1990. Riboflavin requirement of chicks fed amino acid and conventional corn-soybean diet. Poult. Sci. 69, 1357–1363. Deng, D.F., Wilson, R.P., 2003. Dietary riboflavin requirement of juvenile sunshine bass (Morone chrysops ♀ × Morone saxatilis ♂). Aquaculture 218, 695–701. Frank, G.R., Bahr, J.M., Easter, R.A., 1988. Riboflavin requirement of lactating swine. J. Anim. Sci. 66, 47–52. Hegsted, D.M., Perry, R.L., 1948. Nutritional studies with the duck V. Riboflavin and pantothenic acid requirements. J. Nutr. 35, 411–417. Heuser, G.F., Wilgus, H.S., Norris, L.C., 1938. The quantitative vitamin-G requirement of chicks. Poult. Sci. 17, 105–108. Lienhart, W., Gudipati, V., Macheroux, P., 2013. The human flavoproteome. Arch. Biochem. Biophys. 535, 150–162. Ministry of Agriculture of China, 2004. Feeding Standard of Chicken. Standards Press of China, Beijing, China. Ministry of Agriculture of China, 2012. Nutrient Requirements of Meat-type Ducks of China. China Agriculture Press, Beijing, China. Norris, L.C., Wilgus, Jr. H.S., Ringrose A.T., Heiman, V., Heuser, G.F., 1936. The vitamin-G requirement of poultry. Cornell Agr. Exp. Sta. Bull. 660. Ithaca, NY. NRC, 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Petteys, B.J., Frank, E.L., 2011. Rapid determination of vitamin B2 (riboflavin) in plasma by HPLC. Clin. Chim. Acta 412, 38–43. Powers, H.J., 2003. Riboflavin (vitamin B-2) and health. Am. J. Clin. Nutr. 77, 1352–1360. Robbins, K.R., Saxton, A.M., Southern, L.L., 2006. Estimation of nutrient requirements using broken-line regression analysis. J. Anim. Sci. 84, E155–E165. Ruiz, N., Harms, R.H., 1988. Riboflavin requirement of broiler fed a corn-soybean diet. Poult. Sci. 67, 794–799. SAS Institute, 2003. SAS User’s Guide: Statistics. Version 9.0. SAS Inst. Inc., Cary, NC. Tang, J., Xie, M., Yang, J., Wen, Z.G., Zhu, Y.W., Huang, W., Hou, S.S., 2013. Riboflavin requirements of white Pekin ducks from hatch to 21 d of age. Br. Poult. Sci. 54, 407–411. Tang, J., Wen, Z.G., Guo, Z.B., Huang, W., Guo, Y.M., Xie, M., Hou, S.S., 2014. Dietary riboflavin supplementation improve the growth performance and antioxidant status of starter white Pekin ducks fed a corn-soybean meal diets. Livest. Sci. 170, 131–136. Woodward, B., 1985. Riboflavin requirement for growth, tissue saturation and maximal flain-dependent enzyme activity in young rainbow trout (Salmo gairdneri) at two temperatures. J. Nutr. 115, 78–84.
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Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci
Short communication
Effects of riboflavin supplementation on growth performance, carcass traits, and riboflavin status of growing male white Pekin ducks J. Tang a,b,1 , J. Hu a,1 , Z.G. Wen a,b , Y. Jiang a,b , H. AL-Kateb c , W. Huang a , Y.M. Guo b , M. Xie a,∗ , S.S. Hou a a b c
Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China College of Animal Science and Technology, China Agricultural University, Beijing 100193, China University of the West of England, Centre for Research in Biosciences, Frenchay Campus, Coldharbour Lane, Bristol BS16 1QY, UK
a r t i c l e
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Article history: Received 24 December 2014 Received in revised form 24 July 2015 Accepted 25 July 2015 Keywords: Ducks Riboflavin Requirement Growth performance
a b s t r a c t An experiment involving six dietary riboflavin concentration (1.38, 2.38, 3.38, 4.38, 5.38, and 6.38 mg/kg) was conducted to determine the effects of riboflavin on performance and riboflavin status of growing male white Pekin ducks in terms of growth performance, carcass traits, and free riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) concentration in the liver and plasma. A basal corn-soybean meal containing 1.38 mg/kg of naturally occurring riboflavin was used and this basal diet was fed with 0, 1, 2, 3, 4, and 5 mg/kg diet of supplemental riboflavin. One-day-old male white Pekin ducks were fed common starter diets from hatch to 14 days of age and then fed the experimental diets from 15 to 35 days of age. A total of 288 15-day-old ducks were allotted to 6 dietary treatments with 6 replicate pens of 8 birds per pen. At 35 days of age, the average daily weight gain (ADG), average daily feed intake (ADFI), gain to feed ratio (G:F), and percentage yield of breast and leg meat, abdominal fat were examined and free riboflavin, FMN, and FAD in plasma or liver were all analyzed. The growth depression, low plasma free riboflavin, liver free riboflavin, liver FMN, and liver FAD concentration were observed in the ducks fed the riboflavin-deficient basal diet (P < 0.05), and these adverse effects were alleviated by riboflavin supplementation. The growing ducks fed the basal diet with no supplementation of riboflavin had the lowest ADG, ADFI, G:F and breast meat yield among all ducks (P < 0.05) and these criteria showed linear or quadratic response to increasing dietary riboflavin (P < 0.05). On the other hand, dietary riboflavin influenced the status of this vitamin in growing ducks. The poorest growth performance of the riboflavin-deficient growing ducks was accompanied with the lowest content of riboflavin, FAD, and FMN in plasma or liver (P < 0.05) but these bad statuses were reversed by increasing dietary riboflavin in which the riboflavin and its derivative increased linearly or quadratically as dietary riboflavin increased (P < 0.05). According to the broken-line model, the riboflavin requirements (based on dietary total riboflavin) of male white Pekin ducks from 15 to 35 days of age for ADG, ADFI, G:F, plasma free riboflavin, liver free riboflavin, and liver FMN were 2.43, 2.48, 2.31, 3.57, 2.78, and 2.83 mg/kg, respectively. © 2015 Elsevier B.V. All rights reserved.
Abbreviations: ADG, average daily weight gain; ADFI, average daily feed intake; G:F, gain to feed ratio; FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; HPLC, high performance liquid chromatography; SEM, the standard error of the mean. ∗ Corresponding author. Tel.: +86 10 62816227; fax: +86 10 62815832. E-mail address: [email protected] (M. Xie). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.anifeedsci.2015.07.023 0377-8401/© 2015 Elsevier B.V. All rights reserved.
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1. Introduction Riboflavin is a water-solube vitamin and this vitamin is involved in a range of redox reactions in primary metabolic pathways and electron transfer processes (Powers, 2003; Lienhart et al., 2013), mainly as its biologically active coenzymes forms flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Like other avian species, riboflavin is needed by ducks and the deficiency of this vitamin could lead to high mortality, poor growth, and low retention of tissue riboflavin in starter Pekin ducks (Tang et al., 2013, 2014). Recently, the riboflavin requirements of modern starter Pekin ducks were estimated by Tang et al. (2013, 2014) and they confirmed that the riboflavin recommendation of NRC (1994) during the starter period (4 mg/kg) is still sufficient for modern Pekin duck strains. However, although the riboflavin recommendation of growing Pekin ducks (4 mg/kg) was provided by NRC (1994), no documentation was provided to support this value and the riboflavin requirements of growing ducks are virtually lacking until now. In chicks, the riboflavin requirements of birds decreased as age increased (Norris et al., 1936; Heuser et al., 1938) but this conclusion was not confirmed in ducks, which indicates that the riboflavin requirement of ducks during the growing period may be different from those during starter period. In practice, raising ducks is directed to meat production and carcass yield is usually considered to promote the benefit of duck production, which makes it necessary to evaluate the effects of riboflavin on growth performance and carcass traits of Pekin ducks when the riboflavin requirements of growing ducks were examined. On the other hand, in ducks, poor growth is usually accompanied with low plasma and liver riboflavin or FMN in riboflavin-deficient birds and thus the status of this vitamin could be indicated by tissue riboflavin and its derivative (Tang et al., 2013, 2014), which may reinforce the evaluation of the duck response to increasing dietary riboflavin. Therefore, the objective of the present study was to determine the effects of riboflavin on growth performance, carcass traits, and riboflavin status of growing Pekin ducks and to evaluate the requirements of this vitamin of these birds during growing period. 2. Materials and methods 2.1. Animals and housing All procedures of the present study were approved by the animal care and use committee of the Institute of Animal Sciences of Chinese Academy of Agricultural Sciences. A total of 320 one-day-old male white Pekin ducks obtained from Pekin duck breeding center in Chinese Academy of Agricultural Sciences were randomly allotted to 16 raised plastic-floor pens with 20 birds per pen and then they were raised until 14 days of age. At 14 days of age, after fasting for 12 h, all the ducks were weighed individually and 288 ducks selected from these ducks were divided into six dietary treatments, each containing 6 replicate pens with 8 birds per pen. All pens had similar pen weight and these birds were free access to water and feed from 15 to 35 days of age. During this period, the lighting was continuous, and the temperature was kept at 33 ◦ C from 1 to 3 days of age, and then it was reduced gradually to approximately 25 ◦ C until 14 days of age and was kept at approximately 16–22 ◦ C thereafter. 2.2. Diet All the ducks were raised with common corn-soybean meal starter diet (Table 1) and this diet was formulated to meet the nutrient recommendations for starter ducks provided by Ministry of Agriculture of China (2012). The starter diet included 4 mg/kg of supplemental riboflavin. The basal diet during the growing period was formulated to be riboflavin-deficient (Table 1) and all nutrients except riboflavin met the recommendations for growing ducks provided by Ministry of Agriculture of China (2012). In order to produce six experimental diets, the basal diet were prepared as mash and then supplemented with 0, 1, 2, 3, 4, and 5 mg crystalline riboflavin/kg diet, respectively. All diets were cold-pelleted at room temperature. The crystalline riboflavin (purity, 99%) was obtained from (Sigma–Aldrich, St. Louis, MO, USA). The riboflavin content of this basal diet was determined by high performance liquid chromatography (HPLC) with a fluorescence detector and the value detected was 1.38 mg/kg. The riboflavin concentrations for the six experimental diets were calculated. These values are 1.38, 2.38, 3.38, 4.38, 5.38, and 6.38 mg riboflavin/kg of feed. 2.3. Sample preparation and data collection At 35 days of age, the average daily weight gain (ADG), average daily feed intake (ADFI), and gain to feed ratio (G:F) of each pen were measured. Two ducks were selected randomly from each pen and bled by cardiac puncture. The blood were collected into heparin sodium-containing tubes and centrifuged at 1520 × g for 10 min to obtain plasma. These plasma samples were then stored at −20 ◦ C until analyzed for riboflavin. Afterwards, these ducks were killed by CO2 inhalation and immersed into hot water at 60 ◦ C for 2 min. Then they were scalded, picked, and eviscerated manually. The breast meat (including both the pectoralis major and pectoralis minor), leg meat (including the thighs and the drum sticks), and abdominal fat were all removed from carcasses and weighed. The percentage yield for the breast meat, leg meat, and abdominal fat were expressed as relative weight to live BW at processing. The livers were removed from these birds and immediately stored at −20 ◦ C until analyzed for riboflavin, FMN, and FAD.
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Table 1 Composition of common starter feed from hatch to 14 days of age and riboflavin-deficient basal diet from 15 to 35 days of age (g/kg as-fed). Item
Common starter feed (from hatch to 14 days)
Basal feed (from 15 to 35 days)
Ingredient, g/kg Corn Soybean Corn gluten meal Limestone Dicalcium phosphate Vitamin and trace mineral premix Sodium chloride dl-Methionine l-Lysine·HCl l-Tryptophan
629.5 333.0 – 8.0 15.0 10.0a 3.0 1.5 – –
706.9 150.0 100.0 10.0 16.0 10.0b 3.0 0.8 2.7 0.6
Calculated composition Metabolizable energyc , MJ/kg Crude protein Calcium Nonphytate phosphorus Lysine Methionine Methionine + cysteine Threonine Tryptophan Arginine Riboflavind , mg/kg
12.22 200.2 9.3 3.6 11.1 4.5 7.9 8.3 2.2 13.8 1.69
12.64 175.9 9.9 4.2 9.1 4.0 7.1 6.4 2.0 8.9 1.38
a Supplied per kilogram of total diet: Cu (CuSO4 ·5H2 O), 10 mg; Fe (FeSO4 ·7H2 O), 60 mg; Zn (ZnO), 60 mg; Mn (MnSO4 ·H2 O), 80 mg; Se (NaSeO3 ), 0.3 mg; I (KI), 0.2 mg; choline chloride, 1000 mg; vitamin A (retinyl acetate), 10,000 IU; vitamin D3 (Cholcalciferol), 3000 IU; vitamin E (dl-␣-tocopheryl acetate), 20 IU; vitamin K3 (menadione sodium bisulfate), 2 mg; thiamin (thiamin mononitrate), 2 mg; riboflavin, 4 mg; pyridoxine hydrochloride, 4 mg; cobalamin, 0.02 mg; calcium-d-pantothenate, 20 mg; nicotinic acid, 50 mg; folic acid, 1 mg; biotin, 0.2 mg. b Supplied per kilogram of total diet: Cu (CuSO4 ·5H2 O), 10 mg; Fe (FeSO4 ·7H2 O), 60 mg; Zn (ZnO), 60 mg; Mn (MnSO4 ·H2 O), 80 mg; Se (NaSeO3 ), 0.3 mg; I (KI), 0.2 mg; choline chloride, 750 mg; vitamin A (retinyl acetate), 8000 IU; vitamin D3 (Cholcalciferol), 3000 IU; vitamin E (dl-␣-tocopheryl acetate), 20 IU; vitamin K3 (menadione sodium bisulfate), 2 mg; thiamin (thiamin mononitrate), 1.5 mg; pyridoxine hydrochloride, 3 mg; cobalamin, 0.02 mg; calcium-d-pantothenate, 10 mg; nicotinic acid, 50 mg; folic acid, 1 mg; biotin, 0.2 mg. c The values are calculated according to the AME of chickens (Ministry of Agriculture of China, 2004). d The numbers were analyzed by high performance liquid chromatography.
2.4. Chemical analysis The riboflavin concentration in feed, plasma, and liver, the FMN and FAD content in liver were all determined by reversedphase HPLC according to the methods described previously by Tang et al. (2013, 2014). Prior to the HPLC analysis, feed, liver and plasma samples were prepared according to the method described by Britton et al. (2003), Batey and Eckhert (1990), and Petteys and Frank (2011), respectively. Peaks were identified and quantified by using the pure authentic standards purchased from (Sigma–Aldrich, St. Louis, MO, USA). 2.5. Statistical analyses The one-way ANOVA were performed using SAS software (SAS Institute, 2003), with pen used as the experimental unit for analysis. The linear and quadratic polynomial contrasts were performed to determine the effect of supplemental riboflavin in the duck diet. The variability in the data was expressed as the standard error of the means (SEM) and a probability level of P < 0.05 was considered to be statistically significant. Associations were established between growth performance (ADG, ADFI or G:F) and tissue riboflavin by multivariate correlation analyses. The broken-line regression analysis (Robbins et al., 2006) was used to estimate the riboflavin requirements for ducks. The broken-line model was provided as follows: y = l + u(r − x) where y = growth performance (ADG, ADFI or G:F), plasma riboflavin, liver riboflavin, and liver FMN, x = dietary total riboflavin level (mg/kg), r = riboflavin requirement, l = the response at x = r, u = the slope of the curve. In this model, y = l when x > r. 3. Results and discussion 3.1. Growth performance and carcass traits In the present study, growth depression was still the typical symptom for riboflavin-deficient growing Pekin ducks and it was similar to the phenomena observed in riboflavin-deficient starter ducks (Tang et al., 2013, 2014). Among all ducks, the birds fed the basal diet with no supplementation of riboflavin showed the lowest ADG, ADFI, and G:F (P < 0.05) and all these
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Table 2 Effects of dietary riboflavin on growth performance of male white Pekin ducks from 15 to 35 days of agea . Dietary riboflavin (mg/kg)
ADG (g/d)
ADFI (g/d)
1.38 2.38 3.38 4.38 5.38 6.38 Pooled SEM
54.4 80.3 81.0 81.4 82.1 81.5 1.80
130.4 168.3 169.9 172.5 173.9 171.3 2.92
P-value Riboflavin Riboflavin linear Riboflavin quadratic
<0.001 <0.001 <0.001
<0.001 <0.001 <0.001
G:F (g:g) 0.42 0.48 0.48 0.47 0.47 0.48 0.005 <0.001 <0.001 <0.001
ADG, average daily gain; ADFI, average daily feed intake; G:F, gain to feed ratio; SEM, standard error of the mean.
Table 3 Effects of dietary riboflavin on carcass yield of the 35-day-old male white Pekin ducksa . Dietary riboflavin (mg/kg)
Relative weight (% of processing live BW)b Breast meat
1.38 2.38 3.38 4.38 5.38 6.38 Pooled SEM P-value Riboflavin Riboflavin linear Riboflavin quadratic a b
2.68 4.21 4.10 4.26 4.58 4.38 0.10 <0.001 <0.001 <0.001
Leg meat 9.23 9.44 8.86 8.55 8.94 8.50 0.12 0.135 0.023 0.810
Abdominal fat 0.82 1.14 1.28 1.36 1.31 1.36 0.04 <0.001 <0.001 0.018
SEM, standard error of the mean. The percentage yield is calculated using the following equation: Yield = (breast meat, leg meat, or abdominal fat weight) × 100%/processing live BW.
criteria increased linearly or quadratically as dietary riboflavin increased from 1.38 to 6.38 mg riboflavin/kg (P < 0.05, Table 2). It is showed that the basal diet was riboflavin-deficient and the deficiency of this vitamin was alleviated by supplementation of crystalline riboflavin. However, riboflavin deficiency in growing diets did not cause high mortality and leg paralysis which took place in starter ducks (Tang et al., 2013, 2014), which indicated that riboflavin deficiency caused less damage to growing ducks and these birds needed less riboflavin than starter ducks. In order to estimate riboflavin requirements of growing ducks for growth performance, the broken-line regression was utilized because this regression was used successfully to predict the riboflavin requirements for ducks (Hegsted and Perry, 1948; Tang et al., 2013, 2014), broilers (Ruiz and Harms, 1988; Chung and Baker, 1990), pigs (Frank et al., 1988), and fish (Deng and Wilson, 2003). According to this regression, the riboflavin requirements of growing male white Pekin ducks from 15 to 35 days of age for ADG, ADFI, and G:F were 2.43, 2.48, and 2.31 mg riboflavin/kg diet, respectively (Table 5). These estimated requirements of growing ducks were all lower than those of starter Pekin ducks predicted by Tang et al. (2013) based on corn-corn gluten meal basal diets (3.31 and 5.21 mg/kg for ADG and feed conversion ratio) or Tang et al. (2014) based on corn-soybean meal basal diets (3.01, 2.98, and 2.79 mg/kg for ADG, ADFI, and G:F). This data is consistent with Heuser et al. (1938) who found that the riboflavin requirements of White Leghorn chicks decrease weekly and gradually from 2 to 8 weeks of age. Furthermore, the riboflavin requirements estimated by this study were much lower than the recommendation of NRC (1994) for growing white Pekin ducks from 2 to 7 weeks of age (4 mg/kg). Therefore, our results partly suggested that the riboflavin recommendation of NRC (1994) for growing ducks should be reduced appropriately, although this data is still adequate for duck growth. On the other hand, the growth depression caused by riboflavin deficiency may lead to reduction of carcass yield. For the ducks fed riboflavin-deficient basal diet, the worst growth performance was accompanied with the lowest breast meat and abdominal fat compared with other ducks fed riboflavin-supplemented diets (P < 0.05, Table 3). Fortunately, these ill effects could be reversed because the breast meat increased linearly or quadratically as dietary riboflavin increased (P < 0.05, Table 3). Therefore, considering that breast meat is the most valuable part of carcass when birds are further processed, dietary riboflavin should be taken into account during diet formulation for growing ducks, although it failed to estimate the riboflavin requirement for breast meat due to non-significant broken-line regression (P > 0.05).
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Table 4 Effects of dietary riboflavin on plasma and liver free riboflavin, liver flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) in 35-day-old Pekin ducksa . Dietary riboflavin (mg/kg)
1.38 2.38 3.38 4.38 5.38 6.38 Pooled SEM P-value Riboflavin Riboflavin linear Riboflavin quadratic a
Plasma (nmol/L)
Liver (g/g wet tissue)
Free riboflavin
Free riboflavin
17.4 63.8 96.4 103.0 110.7 104.2 4.8
7.82 10.04 10.70 11.26 10.32 11.46 0.21
<0.001 <0.001 <0.001
FAD
<0.001 <0.001 0.001
FMN
1.91 2.45 2.67 2.40 2.37 2.64 0.06
4.12 5.94 6.76 6.14 7.31 6.89 0.19
<0.001 0.006 0.048
<0.001 <0.001 0.023
SEM, standard error of the mean.
Table 5 Riboflavin requirements of male white Pekin ducks from 15 to 35 days of age based on broken-line regression analysisa . Response criterion
Regression
Requirement (mg/kg)
95% confidence interval (mg/kg)
P-value
R2
ADG ADFI G:F Plasma riboflavin Liver riboflavin Liver FMN
Y = 81.50 − 25.92 × (2.43 − x) Y = 171.9 − 37.9 × (2.48 − x) Y = 0.48 − 0.06 × (2.31 − x) Y = 106.0 − 39.46 × (3.57 − x) Y = 10.94 − 2.22 × (2.78 − x) Y = 6.78 − 1.82 × (2.83 − x)
2.43 2.48 2.31 3.57 2.78 2.83
2.37–2.49 2.30–2.65 2.04–2.59 3.12–4.01 1.63–3.93 1.48–4.20
<0.001 <0.001 <0.001 0.001 0.029 0.036
0.999 0.994 0.956 0.990 0.916 0.891
a
ADG, average daily gain; ADFI, average daily feed intake; G:F, gain to feed ratio; FMN, flavin mononucleotide.
Table 6 Correlation coefficients between growth performance and tissue riboflavina .
ADFI G:F Plasma riboflavin Liver riboflavin Liver FAD Liver FMN a
r P-value r P-value r P-value r P-value r P-value r P-value
ADG
ADFI
G:F
Plasma riboflavin
Liver riboflavin
Liver FAD
0.998 <0.001 0.969 0.002 0.910 0.012 0.919 0.010 0.883 0.020 0.914 0.011
0.951 0.004 0.929 0.007 0.921 0.009 0.860 0.028 0.923 0.009
0.815 0.048 0.887 0.018 0.946 0.004 0.859 0.029
0.92 0.009 0.79 0.059 0.95 0.004
0.88 0.019 0.83 0.039
0.83 0.040
Liver FMN
ADG, average daily gain; ADFI, average daily feed intake; G:F, gain to feed ratio; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide.
3.2. Riboflavin status Riboflavin exists in tissues with the three forms: free riboflavin, FMN, and FAD. Dietary riboflavin could influence the riboflavin status of growing ducks and it was indicated by riboflavin and its derivatives in tissue. The ducks fed the basal diet with no supplementation of riboflavin had the lowest concentration of free riboflavin, FAD, and FMN in plasma or liver (P < 0.05) and these ducks also showed the poorest growth among all ducks (P < 0.05, Table 4). However, these bad statuses could be reversed by increasing dietary riboflavin. The free riboflavin, FAD, and FMN in plasma or liver all increased linearly or quadratically as dietary riboflavin increased (P < 0.05, Table 4), which was also accompanied with the simultaneous improvement of growth performance (Table 3). Therefore, the correlation analysis was used to explain the relationship between growth performance and tissue riboflavin and confirm the validity of plasma riboflavin as indicator of riboflavin status. Our results showed that the ADG, ADFI, and G:F were all positively correlated to riboflavin, FAD, and FMN in liver or plasma (P < 0.05, Table 6), respectively, which indicated that riboflavin deficiency led to growth retardation of ducks. Furthermore, there existed positive correlations between plasma riboflavin and liver riboflavin and FMN (P < 0.05, Table 6) and there was a trend for the correlation between plasma riboflavin and liver FAD (0.05 < P < 0.10, Table 6), which confirmed that the change of plasma riboflavin was a good reflection of riboflavin status in tissues. Similar to previous study in young rainbow trouts and ducks (Woodward, 1985; Tang et al., 2013, 2014), riboflavin and its derivatives in tissue were used to predict riboflavin requirements of growing ducks. According to the broken-line analysis, the riboflavin requirements
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of growing white Pekin ducks for plasma free riboflavin, liver free riboflavin, and liver FMN were 3.57, 2.78, and 2.83 mg riboflavin/kg diet, respectively, and these estimated requirements were greater than those for growth performance in our study (Table 5), which was in agreement with the results in starter ducks (Tang et al., 2013, 2014). Furthermore, these estimated requirements for tissue saturation were much lower than those of starter Pekin ducks estimated by Tang et al. (2013) (3.85 and 3.91 mg/kg for plasma riboflavin and liver riboflavin) or Tang et al. (2014) (4.18, 3.63, and 3.59 mg/kg for plasma riboflavin, liver riboflavin and liver FMN), which also suggested that the riboflavin requirements of growing ducks should be reduced as age increased (Table 6). 4. Conclusion Riboflavin deficiency depressed growth performance and breast meat yield of growing ducks and riboflavin status was worsened at this instance, but these negative effects could be avoided by increasing dietary riboflavin. According to the broken-line analysis, the riboflavin requirements for growing male white Pekin ducks from 15 to 35 days of age for ADG, ADFI, G:F, plasma free riboflavin, liver free riboflavin, and liver FMN were 2.43, 2.48, 2.31, 3.57, 2.78, and 2.83 mg/kg, respectively. Conflict of interest None declared. Acknowledgments This work was sponsored by the earmarked fund for China Agriculture Research System (CARS-43) and the science and technology innovation project of Chinese Academy of Agricultural Sciences (CXGC-IAS-09). References Batey, D.W., Eckhert, C.D., 1990. Identification of FAD, FMN, and riboflavin in the retina by microextraction and high-performance liquid chromatography. Anal. Biochem. 188, 164–167. Britton, N.L., Riter, K.L., Smallidge, R.L., Joseph, H., 2003. Reversed-phase liquid chromatographic determination of riboflavin in feeds. J. AOAC Int. 86, 197–201. Chung, T.K., Baker, D.H., 1990. Riboflavin requirement of chicks fed amino acid and conventional corn-soybean diet. Poult. Sci. 69, 1357–1363. Deng, D.F., Wilson, R.P., 2003. Dietary riboflavin requirement of juvenile sunshine bass (Morone chrysops ♀ × Morone saxatilis ♂). Aquaculture 218, 695–701. Frank, G.R., Bahr, J.M., Easter, R.A., 1988. Riboflavin requirement of lactating swine. J. Anim. Sci. 66, 47–52. Hegsted, D.M., Perry, R.L., 1948. Nutritional studies with the duck V. Riboflavin and pantothenic acid requirements. J. Nutr. 35, 411–417. Heuser, G.F., Wilgus, H.S., Norris, L.C., 1938. The quantitative vitamin-G requirement of chicks. Poult. Sci. 17, 105–108. Lienhart, W., Gudipati, V., Macheroux, P., 2013. The human flavoproteome. Arch. Biochem. Biophys. 535, 150–162. Ministry of Agriculture of China, 2004. Feeding Standard of Chicken. Standards Press of China, Beijing, China. Ministry of Agriculture of China, 2012. Nutrient Requirements of Meat-type Ducks of China. China Agriculture Press, Beijing, China. Norris, L.C., Wilgus, Jr. H.S., Ringrose A.T., Heiman, V., Heuser, G.F., 1936. The vitamin-G requirement of poultry. Cornell Agr. Exp. Sta. Bull. 660. Ithaca, NY. NRC, 1994. Nutrient Requirements of Poultry. 9th rev. ed. Natl. Acad. Press, Washington, DC. Petteys, B.J., Frank, E.L., 2011. Rapid determination of vitamin B2 (riboflavin) in plasma by HPLC. Clin. Chim. Acta 412, 38–43. Powers, H.J., 2003. Riboflavin (vitamin B-2) and health. Am. J. Clin. Nutr. 77, 1352–1360. Robbins, K.R., Saxton, A.M., Southern, L.L., 2006. Estimation of nutrient requirements using broken-line regression analysis. J. Anim. Sci. 84, E155–E165. Ruiz, N., Harms, R.H., 1988. Riboflavin requirement of broiler fed a corn-soybean diet. Poult. Sci. 67, 794–799. SAS Institute, 2003. SAS User’s Guide: Statistics. Version 9.0. SAS Inst. Inc., Cary, NC. Tang, J., Xie, M., Yang, J., Wen, Z.G., Zhu, Y.W., Huang, W., Hou, S.S., 2013. Riboflavin requirements of white Pekin ducks from hatch to 21 d of age. Br. Poult. Sci. 54, 407–411. Tang, J., Wen, Z.G., Guo, Z.B., Huang, W., Guo, Y.M., Xie, M., Hou, S.S., 2014. Dietary riboflavin supplementation improve the growth performance and antioxidant status of starter white Pekin ducks fed a corn-soybean meal diets. Livest. Sci. 170, 131–136. Woodward, B., 1985. Riboflavin requirement for growth, tissue saturation and maximal flain-dependent enzyme activity in young rainbow trout (Salmo gairdneri) at two temperatures. J. Nutr. 115, 78–84.