Feeding dihydroquercetin to broiler chickens

Feeding dihydroquercetin to broiler chickens by Pirgozliev, V., Westbrook, C., Karagecili, M.R., Karadas, F., Rose, S.P. and Mansbridge, S.C. Copyright, Publisher and Additional Information: This is the authors’ accepted manuscript. The final published version (version of record) is available online via Taylor & Francis Please refer to any applicable terms of use of the publisher. DOI: https://doi.org/10.1080/00071668.2018.1556387 Pirgozliev, V., Westbrook, C., Karagecili, M.R., Karadas, F., Rose, S.P. and Mansbridge, S.C. 2018. Feeding dihydroquercetin to broiler chickens. British Poultry Science. 5 December 2018 1 Feeding dihydroquercetin to broiler chickens 2 V. Pirgozliev1a, C. Westbrook1, S. Woods1, M. R. Karagecili2, F. Karadas2, S.P. Rose1, S. C. 3 Mansbridge1 4 1 The National Institute of Poultry Husbandry, Harper Adams University, Shropshire, UK; 5 2 Department of Animal Science, Yuzuncu Yil University, Van, Turkey 6 a 7 Abstract 1. A total of 80 male Ross 308 broilers were used in a study to investigate the effect 8 of dietary dihydroquercetin (DHQ) on growth performance variables, gastrointestinal tract 9 (GIT) and immune organ development, glutathione peroxidase (GPx) and haemoglobin in 10 blood, hepatic vitamin E content, dietary N-corrected metabolisable energy (AMEn), and 11 nutrient retention coefficients when fed to broiler chickens from 7 to 35 days of age. 12 2. Two treatments were used in this study: control (C) and C + 0.5 g/kg extract of Siberian 13 Larch (Larix sibirica) per kg feed, containing 85 % DHQ. The diets were fed over two feeding 14 phases, a grower phase from 7 to 28 d age, and a finisher phase from 28 to 35 d age. The birds 15 were reared under breeder’s recommended conditions. 16 3. In general, there were no effects of DHQ on growth performance of broiler chickens. 17 However, the results of this experiment have shown that there can be changes in redness colour 18 of the breast meat when DQH is fed. No negative effects of feeding DHQ at 0.5 g/kg diet were 19 observed in this study. 20 4. Supplementation of poultry diets with DHQ under standard industry rearing conditions, did 21 not improve production performance or any of the studied health variables, except an increase 22 of redness index of the breast fillets. Feeding DHQ at different doses and/or under more 23 challenging conditions, e.g. heat stress, may however, bring positive responses. 24 Key words: broilers, dihydroquercetin (DHQ), phenols, growth performance, antioxidants Corresponding author: [email protected] 25 INTRODUCTION 26 27 The popularity of natural antioxidants to protect human and animal health and to increase the 28 shelf life of products from animal origin has increased during the past decade (Weidmann 2012; 29 Iskender et al. 2017). Flavonoids being a major sub-group representing plant polyphenols, are 30 considered antioxidants from natural sources and as such, have been attracting attention for use 31 in animal nutrition (Surai 2014). Dihydroquercetin (DHQ), also known as taxifolin, a flavonoid 32 extracted from various conifers including Siberian Larch (Larix sibirica), longleaf Indian Pine 33 (Pinus roxburghii), Himalayan Cedar (Cedrus deodara) and Chinese Yew (Taxus chinensis 34 var. mairei), has been widely applied as an antioxidant for the surface treatment of fresh meat 35 and fish (Semenova et al. 2008; Ivanov et al. 2009; Balev et al. 2011; Dragoev et al. 2014). 36 Dihydroquercetin has also been incorporated in animal diets in order to enhance production 37 performance. Fomichev et al. (2016) extensively reviewed the effect of DHQ as dietary 38 supplement in animal production and reported enhancement in growth performance and blood 39 variables of poultry and pigs. Research by Nikanova (2017) with piglets further supported the 40 observations of Fomichev et al. (2016). However, Balev et al. (2015) did not find significant 41 difference in growth performance of broilers fed DHQ from day old to 49 days when compared 42 to the control fed birds. Torshkov (2011) reported that the breast meat from 42 d age broilers 43 fed DHQ supplemented diet had higher dry matter, lower fat, lower tryptophan and the same 44 protein content when compared to birds fed control diet only. In addition, Torshkov et al. 45 (2014) found that feeding DHQ to broilers increases the number of red blood cells and 46 haemoglobin concentration compared to control. However, there was no information on growth 47 performance variables in both reports. Omarov et al. (2016) reported increased protein 48 concentration in the organs and tissues of broiler chickens when fed DHQ, however, the 49 experiment was not designed to study the effect of DHQ on growth performance variables. The 50 majority of experiments involving DHQ emphasise more on its impact on the composition of 51 various tissues (muscle, blood), while the information on performance is rarely presented. It is 52 likely that improvements seen in productive performance in some papers may be due to heat 53 stress (Fomichev et al. 2016). Rearing animals at temperatures exceeding their thermal comfort 54 zone (e.g. during summer) may be a reason for depleting levels of tissue antioxidants, thus the 55 antioxidant status of animal may be associated with the mode of action of DHQ. Since DHQ is 56 a natural flavonoid with recognised antioxidant properties, understanding its mode of action 57 may be important for enhancing health and productivity of intensely reared animals. In 58 addition, there is no information on the impact of DHQ on the development of the 59 gastrointestinal tract (GIT), immune organs, dietary available energy and nutrient retention 60 coefficients. 61 The aim of the study was to assess the impact of DHQ on growth performance variables, GIT 62 and immune system organ development, glutathione peroxidase (GPx) and haemoglobin 63 concentration in blood, dietary N-corrected metabolisable energy (AMEn), dry matter (DMR), 64 nitrogen (NR) and fat retention (FR) coefficients when fed to broiler chickens from 7 to 35 65 days of age. 66 MATERIALS AND METHODS 67 Experimental diets 68 Two wheat-soy-based diets were offered to the birds during the experiment. The diets were fed 69 over two feeding phases, a grower phase from 7 to 28 d age, and a finisher phase from 28 to 70 35 d age. The grower and finisher basal diets were formulated to meet breeder’s 71 recommendations (Aviagen Ltd., Edinburgh, UK) (Table 1). All diets included 5 g/kg of TiO2 72 as a marker. The basal diets were then split into two batches that had 1.) no additive (control 73 diet; C) and 2.) 0.5 g/kg extract of Siberian Larch (Larix sibirica) (JSC NPF Flavit, IBI RAS, 74 Pushchino city, Moscow region, Russian Federation 142290). According to the company 75 producer, this extract contains over 85 % pure DHQ. 76 Husbandry and sample collection 77 The experiment was conducted at the National Institute of Poultry Husbandry and approved by 78 the Research Ethics Committee of Harper Adams University. A total of 85 male Ross 308 79 broilers were obtained from a commercial hatchery (Cyril Bason Ltd, Craven Arms, UK), 80 allocated to a single floor pen and offered a standard wheat-based broiler starter feed 81 formulated to meet Ross 308 nutrient requirements (Aviagen Ltd., Edinburgh, UK). At 7 d age, 82 80 of the birds were allocated to 16 raised floor pens (60 x 60 cm) each holding 5 birds. Each 83 of the pens had a solid floor and were equipped with an individual feeder and drinker. Feed 84 and water were fed ad libitum to birds throughout the experiment. Each diet was offered to 85 birds in 8 pens following complete randomisation. The birds were fed the experimental diets 86 from 7 to 35 d age, when the experiment ended. Room temperature and lighting regime met 87 commercial recommendations (Aviagen Ltd, Edinburgh, UK). The well-being of the birds was 88 checked regularly every day. 89 Birds and feed were weighed on days 7, 28 and 35 in order to determine average daily feed 90 intake (FI), average daily weight gain (WG) and to calculate the gain:feed ratio (G:F) on a pen 91 basis. For the last 3 days of the study, the solid floor of each pen was replaced with a wire 92 mesh. Excreta were collected each day for the last three days of the experiment, stored at 4 °C, 93 and a representative subsample was dried at 60 °C and then milled through 0.75 mm screen. 94 At the end of the study, one bird per pen, selected at random, was electrically stunned and 95 blood was obtained in heparin coated tubes from the jugular vein. The organs from the GIT, 96 including proventriculus and gizzard (PG), duodenum, pancreas, jejunum, ileum, caeca and 97 liver, the heart, the spleen and the bursa of Fabricius were weighed. The colour on the surface 98 of the left breast fillet was determined, and the left breast muscles were used to determine the 99 chilling yield. 100 Laboratory Analysis 101 Dry matter (DM) in feed and excreta samples was determined by drying of samples in a forced 102 draft oven at 105 °C to a constant weight (AOAC 2000; method 934.01). Crude protein (6.25 103 × N) in samples was determined by the combustion method (AOAC 2000; method 990.03) 104 using a LECO FP-528 N (Leco Corp., St. Joseph, MI). Oil (as ether extract) was extracted with 105 diethyl ether by the ether extraction method (AOAC 2000; method 945.16) using a Soxtec 106 system (Foss Ltd., Warrington, UK). The gross energy (GE) value of feed and excreta samples 107 was determined in a bomb calorimeter (model 6200; Parr Instrument Co., Moline, IL) with 108 benzoic acid used as the standard. Titanium in feed and excreta was determined as explained 109 by Short et al. (1996). 110 The colour score on the surface of the left breast meat within 5 minutes after slaughter was 111 carried out using a Chroma Meter CR-400 from Konica Minolta (Sunderland, UK) to determine 112 luminance and chromaticity scores using CIELAB scoring (where L* refers to lightness, a* 113 refers to redness, and b* refers to yellowness). Areas were selected that were free of any 114 obvious blood-related defects, such as bruises, haemorrhages, or full blood vessels (Fletcher et 115 al. 2000). Two readings of CIE L*, a*, and b* were obtained for the breast fillet for each bird 116 (2 readings/left side). The chilling yield determined on the left breast of each slaughtered 117 chicken was also determined (Jeong et al. 2011). 118 The glutathione peroxidase in blood was determined using a Ransel GPx kit (Randox 119 Laboratories Ltd., UK) that employs the method based on that of Paglia and Valentine (1967). 120 The concentration of hepatic vitamin E was determined using an HPLC system as previously 121 described (Karadas et al. 2010). 122 Calculations 123 Dietary nutrient retention coefficients were calculated using the following equation: 1 124 / / 125 where exnut is the concentration of the respective nutrient in the excreta, exti is the 126 concentration of titanium dioxide in the excreta, dietnut is the concentration of the respective 127 nutrient in the diet and dietti is the concentration of titanium in the diet. 128 The AMEn value of the experimental diets was determined following the method of Hill and 129 Anderson (1958). 34.39 130 131 where AMEn (MJ/kg) = N-corrected apparent metabolizable energy content of the diet; GE 132 diet and GE ex (MJ/kg) = GE of the diet and excreta, respectively; dietti and exti (%) = titanium 133 in the diet and excreta, respectively; 34.39 (MJ/kg) = energy value of uric acid; and N retained 134 (g/kg) is the N retained by the birds per kilogram of diet consumed. The retained N was 135 calculated as 136 137 where N Diet and N ex (%) = N contents of the diet and excreta, respectively. 138 The relative development of organs was determined as percent by dividing the organ weight to 139 body weight by the respective bird and multiplying by 100 (data not included in tables). 140 Chilling yield of breast meat was determined from 8 carcasses per diet as follows: 141 % 100 % 142 where Post chill breast weight is the weight of the left breast after 24h in a fridge at 4° C and 143 Pre chill breast weight is the weight of the left breast immediately after dissection, respectively. 144 Statistical Analysis 145 Statistical analysis was performed using GenStat 19th edition statistical software (IACR 146 Rothamstead, Hertfordshire, England). A completely randomised one-way analysis of variance 147 was performed to investigate the effect of dietary DHQ on the studied variables. Differences 148 were reported as significant at P < 0.05. 149 150 RESULTS AND DISCUSSION 151 152 All birds were healthy throughout the study period and there was no mortality. There was no 153 effect of treatment on any of the studied growth performance variables (Table 2). 154 The results on AMEn and nutrient retention coefficients are in accordance with previous 155 research (Pirgozliev et al. 2006, 2015; Whiting et al. 2016) and there were no differences 156 (P>0.05) between treatments (Table 3). 157 There were no differences (P>0.05) in the relative weights of the studied organs measured as 158 percentage of body weight (data not in tables) and the results were in agreement with previous 159 research (Dror et al.1977; Abdulla et al. 2016; 2017). 160 The results on chilling yield and the colour score were similar to these reported by Jeong et al. 161 (2011) (Table 4). The breast fillets of the birds fed DHQ had a higher red colour index (a*) 162 compared to the control fed birds (P<0.05). 163 The values for haemoglobin concentration and glutathione peroxidase were in agreement with 164 published reports (Tanaka and Rosenberg 1954; Elagib and Ahmed 2011; Popović et al. 2016) 165 and there were no differences (P>0.05) between diets (Table 5). However, the results did not 166 support the finding of Torshkov et al. (2014) for increased haemoglobin concentration in birds 167 fed DHQ. 168 The values of hepatic vitamin E were in the expected range (Karadas et al. 2010, 2014; Whiting 169 et al. 2018). However, no differences between dietary treatments were observed (P>0.05). 170 In the literature, dietary DHQ concentrations varied between studies and species. In poultry 171 diets the concentration of supplemented DHQ varied from 1 mg per kilogram live weight 172 (Torshkov et al. 2014) to 40 mg per kg live weight (Balev et al. 2015); in calves and cows, 173 from 20 to 200 mg per head daily (Fomichev et al. 2016); in weaning piglets from 10 mg per 174 kg feed (Fomichev et al. 2016), to 50 mg per animal per day (Nikanova 2017). Research by 175 Dunnick and Halley (1992) did not find any toxic effect of quercetin when fed to rats for 6 176 months at concentrations of up to 40000 ppm, and the estimated dose delivered was 177 approximately 40–1900 mg/kg/day. Similarly, DHQ, which is closely related to quercetin in 178 chemical structure, has been shown to be nontoxic when fed to albino rats and humans at much 179 higher levels than in the reported study (Booth and DeEds 1958). There were no treatment- 180 related effects on survival and no treatment-related clinical signs of toxicity for this period. 181 In the reported study, DHQ was added at 0.5 g per kg feed or 500 ppm. On average, birds were 182 consuming approximately, 100 g feed per day, and their average daily weight gain was 183 approximately 60 g. Thus, the average daily consumption of DHQ was 0.05 g per bird, or 0.83 184 g per kilogram daily growth. The lack of adverse effects on birds health further confirms that 185 DHQ is generally safe to use in broiler production. Further exploration of graded levels of 186 dietary DHQ should be considered to optimise the dose required for enhanced production 187 performance. 188 In the reports by Fomichev et al. (2016) and Nikanova (2017), feeding DHQ generally 189 improved the growth performance variables of animals reared in challenging conditions, i.e. 190 high temperature. Fomichev et al. (2016) also reported that the response at later stage of 191 growing, i.e. 42 d old, was more pronounced compared to the early stage of growth (28 d age). 192 However, Balev et al. (2015) reared birds under industry-recognised conditions and did not 193 observe difference in growth performance of broilers fed DHQ for the entire period of 49 days. 194 Heat stress stimulates the release of corticosterone and catecholamines, increase the level of 195 free radicals and initiates lipid peroxidation in cell membranes (Freeman and Crapo 1982). 196 Prochazkova et al. (2011) suggested that flavonoids could prevent injury caused by free 197 radicals by the following mechanisms: direct scavenging of reactive oxygen species (ROS), 198 activation of antioxidant enzymes, metal chelating activity, reduction of a-tocopheryl radicals, 199 inhibition of oxidases, mitigation of oxidative stress caused by nitric oxide, increase in uric 200 acid levels, and increase in antioxidant properties of low molecular antioxidants. The 201 mechanism of flavonoids health-promoting abilities is usually associated with their antioxidant 202 properties (Andriantsitohaina et al. 2012) although recent findings suggest that flavonoids do 203 not behave the same way in vitro and in vivo (Veskoukis et al. 2012). However, in the present 204 study, birds were reared under standard industry recommended conditions, and no challenges 205 were applied, possibly limiting the detection of the benefits of DHQ as an antioxidant. 206 In conclusion, supplementation of poultry diets with 0.5 g DHQ per kg feed, under standard 207 industry rearing conditions, did not improve production performance or any of the studied 208 health variables. However, the redness index of breast fillet was increased. Feeding DHQ at 209 different doses and/or under more challenging conditions, e.g. heat stress, may bring positive 210 responses. 211 212 ACKNOWLEDGEMENT 213 214 Special thanks to Richard James and Rose Crocker for their technical support. 215 216 DISCLOSURE STATEMENT 217 218 The authors reported no potential conflict of interest. 219 220 REFERENCES 221 222 Abdulla, J., Rose, S.P., Mackenzie, A.M., Mirza, M.W. and Pirgozliev, V. 2016. 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The 361 temperature of storage of a batch of wheat distillers dried grains with solubles samples on their 362 nutritive value for broilers. British Poultry Science 59:76-80 363 364 365 366 367 Table 1. Ingredient composition of the control experimental diets (as fed). Ingredients (g/kg) Barley Wheat Soybean meal Full-fat soybeans L Lysine HCL DL Methionine L Threonine Soya oil Limestone Monocalcium phosphate Salt Sodium bicarbonate Premix1 Titanium Dioxide Calculated values (as fed) Crude protein (N x 6.25, g/kg) Crude oil (g/kg) ME (MJ/kg) Calcium (g/kg) Av Phosphorus (g/kg) Av Lysine (g/kg) Lysine (g/kg) Methionine + Cysteine (g/kg) Tryptophan (g/kg) Determined values Dry matter (g/kg) Gross energy (MJ/kg) Crude protein (N x 6.25, g/kg) Crude oil (g/kg) Grower 79 550 230 50 3 3.5 1.5 45 12.5 12.5 2.5 1.5 4 5 Finisher 67 600 190 50 3 3 1.5 47.5 12.5 12.5 2.5 1.5 4 5 201 68 12.99 9.3 4.2 11.8 12.7 9.4 8.5 187 71 13.17 9.2 4.2 10.8 11.6 8.4 7.8 894 17.43 194 69 893 17.39 181 66 The Vitamin and mineral premix contained vitamins and trace elements to meet the requirements specified by 1 368 NRC (1994). All the experimental diets were designed to be low in P. The premix provided (units/kg diet): retinol 369 3600 μg, cholecalciferol 125 μg, α-tocopherol 34 mg, menadione 3 mg, thiamine 2 mg, riboflavin 7 mg, pyridoxine 370 5 mg, cobalamin 15 μg, nicotinic acid 50 mg, pantotenic acid 15 mg, folic acid 1 mg, biotin 200 μg, iron 80 mg, 371 copper 10 mg, manganese 100 mg, cobalt 0.5 mg, zinc 80 mg, iodine 1 mg, selenium 0.2 mg and molybdenium 372 0.5 mg. 373 374 375 376 Table 2. Production performance of broiler chickens fed control or dihydroquercetin (DHQ) supplemented diets. Item Control DHQ SEM (DF=14) P-value Feed Intake 7-35 d (g/b) 2737 2788 29.5 0.268 Weight Gain 7-35 d (g/b) 1609 1666 36.0 0.300 Feed Conversion Efficiency 7-35 d (g/g) 0.588 0.599 0.0104 0.497 Body Weight 35 d age (g) 1735 1790 29.9 0.232 377 378 379 380 381 Table 3. Dietary apparent N-corrected metabolisable energy (AMEn) and nutrient retention coefficients Item Control DHQ SEM (DF=14) P-value AMEn (MJ/kg DM) 13.60 13.52 0.0710 0.470 Dry Matter Retention 0.811 0.808 0.0062 0.574 Nitrogen Retention 0.738 0.737 0.0131 0.963 Fat Retention 0.844 0.835 0.0065 0.244 Dietary AMEn and nutrient retention coefficients were determined between 32 and 35 d of age. 382 383 384 385 Table 4. Chilling yield and surface colour of broiler breast fillets (within 5 minutes after slaughter) fed control or dihydroquercetin (DHQ) supplemented diets. Item Control DHQ SEM (DF=14) P-value Chilling yield (%) 96.26 95.78 0.219 0.138 L* 49.1 52.1 2.83 0.484 a* 2.24 3.42 0.170 0.002 b* 1.02 0.73 0.191 0.322 386 387 388 Table 5. Haemoglobin and glutathione peroxidase (GPx) in blood, and hepatic vitamin E of broiler chickens fed control or dihydroquercetin (DHQ) supplemented diets. Item Control DHQ SEM (DF=14) P-value Haemoglobin (g/l) 132.1 133.6 3.07 0.735 GPx (u/ml RBC) 67.6 64.2 2.85 0.410 Hepatic Vitamin E (µg/g) 19.03 17.03 0.780 0.120