Dietary flaxseed meal and chromium: A nutritional intervention in broiler chicken to 2 study the prospective growth performance, tissue lipid composition and metabolism, 3 health indices, and serum lipid chemistry

12 Background: Flaxseed in a richest terrestrial source of  -3 fatty acid – alpha-linolenic acid 13 (ALA) which can be incorporated in chicken meat when it is included in chicken ration. ALA 14 can further be acted up on by desaturating enzymes to generate PUFA such as EPA and DHA 15 which increase the health value of chicken meat. However, dietary flaxseed results in concurrent 16 increase in lipid oxidation due higher unsaturation and negative impact on chicken growth 17 performance. These negative effects of flaxseed feeding can be reversed by chromium 18 supplementation in broiler chicken. Thus, this study investigated growth performance and 19 efficiency, lipid composition, lipid metabolism, health indices, and serum lipid chemistry of 20 broiler chicken fed flaxseed meal (FSM) and chromium (Cr). 21 Results: Feeding of 100 g FSM exerted negative effects on the growth performance during 22 starter phase only (0-3 weeks) and overall growth efficiency parameters in broiler chicken, 23 whereas, Cr supplementation reversed these negative effects. 100 g FSM reduced abdominal fat 24 in chicken and Cr supplementation linearly decreased it with minimum at 1.5 mg Cr/kg diet. 25 Feeding of 100 g FSM favourably improved the activities of lipid metabolism enzymes which 26 resulted in improved fatty acid profile and health indices of chicken meat. No significant effect 27 of Cr supplementation was observed on lipid metabolism, fatty acid profile, and health indices 28 of chicken meat. 100 g FSM decreased serum total cholesterol, triglyceride, cardiac risk ratio, 29 atherogenic coefficient, and atherogenic index of plasma, whereas, Cr supplementation 30 decreased these parameters linearly with increasing levels. Antioxidant enzyme activities and 31 lipid peroxidation were increased by FSM, whereas, Cr supplementation linearly decreased them 32 with increasing levels; and inverse trend was observed in serum HDL cholesterol levels. 33 Conclusions: Feeding of 100 g FSM exert negative effects on growth performance of young 34 chicken (0-3 weeks), favourably alter lipid metabolism which results in improved fatty profile and health indices of chicken meat. It improves the serum lipid profile and atherogenic 36 indices in broiler chicken, but negatively affects the oxidative stability of lipids. However, Cr 37 supplementation at the rate of 1.5 mg/kg diet successfully overcomes these negative effects of 38 FSM feeding on growth performance and lipid oxidative stability.

[3] which may reflect badly on their growth performance. The Cr deficiency disrupts the normal 48 carbohydrate, protein, and lipid metabolism and reduces the insulin sensitivity in peripheral 49 tissues which results in impairment of growth rate in broiler chicken [4]. Cr supplementation is 50 supposed to improve the growth performance of broiler chicken by apparent involvement in gene 51 expression, whereby it binds nuclear chromatin which results in increase in number of initiation 52 sites and in turn enhances RNA synthesis [5]. Improved growth performance [6], carcass yield, 53 and reduced abdominal fat [3, 4, 6] was reported in broiler chicken supplemented with dietary 54 Cr. Cr supplementation in broiler chicken is an interesting aspect of animal nutrition from 55 physiology and muscle growth point of view because Cr potentiates the effects of insulin which 56 is an anabolic hormone involved in regulation of growth [7]. Compared to ruminants very scanty 57 literature pertaining to Cr supplementation in broiler chicken is available [5]. Thus, because of 58 this insufficient and inconclusive literature on Cr supplementation and its bioavailability there 59 are no dietary Cr recommendations for poultry. 60 8 °C, which was held for 5 min. Then, subsequently the temperature was increased to 240 °C at a 159 rate of 2 °C/min, and held for 60 min. Nitrogen was used as the carrier gas at a flow rate of 1 160 ml/min. The injector and the detector of GC were set at 260 °C. The split ratio was 30:1. Fatty 161 acid standard purchased from Supelcon, Bellefonte-PA contained 37 different FAMEs and 0.5 162 µl was injected into GC to get the standard peaks. The fatty acids were identified by comparing 163 retention time of their peaks with the respective fatty acid methyl ester standards and were 164 expressed as percentage of total fatty acids in feed, breast, and thigh samples. For the analysis of data pertaining to feed intake, FCR, PEF, PER, and EER, each replicate was 217 taken as an experimental unit, whereas, for all other parameters the sampled bird was taken as 218 an experimental unit. All data were tested for normality and homogeneity of variances with the 219 Shapiro-Wilk test and Levene's test respectively prior to analyses. The data were analysed by 220 one way ANOVA using the General Linear Model procedure (IBM SPSS software-20). The 221 significant mean differences were separated by Tukey post-hoc analysis with significance level 222 defined at P < 0.05. Further, to validate the effects of Cr supplementation the data were also 223 subjected to polynomial orthogonal contrast. 224

Growth performance 226
The BWG of birds during 0-3 weeks of age was lower (P = 0.009) in T2 group followed by 227 statistically different T3 and T4 groups, whereas higher BWG was observed in T5 and T1 which 228 did not differ significantly from each other (Table 2). Similarly, the FCR during 0-3 weeks of 229 age was higher (P = 0.006) in T2 group compared to other treatments groups which did not differ 230 significantly from each other. The feed intake and 4-6 week and 0-6 week BWG and FCR of 231 birds were not affected by dietary treatments. The mortality pattern of birds also showed no 232 significant dietary effects (data not shown, only 2 birds died from T3 and 1 bird each from T1,  233   T2, & T4 treatment groups during whole experimental period). 234

Growth efficiency parameters 235
In line with the results of growth performance of birds given above, during 0-3 weeks of age 236 lower PEF (P = 0.008), PER (P = 0.010), and EER (P = 0.007) of birds were observed in group 237 T2 compared to other treatment groups which did not differ significantly from each other (Table  238 3). However, PEF, PER, and EER during 4-6 weeks and 0-6 weeks of age were not affected by 239 dietary treatments. 240

Carcass characteristics and cost economics 241
The carcass characteristics revealed no significant dietary treatment effects except the abdominal 242 fat (Table 4). Lower (P = 0.019) abdominal fat of birds was observed in T5 group followed by 243 statistically similar T4 group. Higher abdominal fat was observed in T1 group followed by 244 statistically different T2 and T3 groups which were not significantly different from T4 group. 245 With respect to control diet (T1) all other treatment groups revealed an increase in cost per kg 246 live weight (P = 0.021) and per kg meat yield (P = 0.018) with higher increase in group T2 and 247 less increase in group T5. However, group T3, and T4 were statistically similar to both T2 and 248 T5. 249

Sensory quality of chicken meat 250
None of the sensory quality attributes of either cooked or raw chicken meat revealed significant 251 dietary effects (Table 5). 252

Fatty acid metabolism of chicken meat 259
The fatty acid metabolism indices of thigh and breast meat revealed that DI (18), DI (16), total 260 DI, and Δ 5 + Δ 6 -desaturase index were lower (P < 0.05) and elongase and thioesterase indices 261 were higher (P < 0.05) in control group T1 compared to other treatments groups which did not 262 differ significantly from each other (Table 8). 263

Health indices of broiler chicken meat 264
The health indices of chicken meat under the influence of FSM and Cr feeding revealed lower 265 (P < 0.01) PUFA:SFA ratio, MUFA:SFA ratio, UFA:SFA ratio, the DFA content, and h/H ratio 266 in control group T1 compared to other treatment groups which did not differ significantly from 267 each other (Table 9). However, higher -6:-3 fatty acid ratio, saturation index, atherogenic 268 index (AI), thrombogenic index (TI), and hypercholesterolemic fatty acids (HFA) were observed 269 in control group T1 compared to other treatment groups which were statistically similar to each 270 other. 271

Serum lipid chemistry and health indices 272
The serum lipid chemistry, antioxidant enzyme activities, and associated health indices are given 273 in Table 10. No significant dietary effects were observed on the serum glucose concentration. on BWG and FCR of birds were observed only up to 3 weeks of age, indicating the age 294 dependence of negative effects of FSM feeding in broiler chicken. Similar age dependence of 295 negative effects of FSM feeding was observed on the PEF, PER, and EEF of birds only up to 3 296 weeks of age. Poor growth performance and efficiency of birds fed FSM indicates their poor 297 efficiency of feed, protein, and energy utilization [36]. Even some old studies maintain that 298 feeding ground flaxseed beyond 7.5% level in broiler chicken reduced the growth and conversion 299 efficiency, explaining the reduction in PER and net protein ratio, which could be due to lower 300 nitrogen and amino acid retention because of the presence of mucilage [16,17]. However, in 301 present study the negative effects of FSM feeding during starter phase seem to be overcome by 302 Cr supplementation. But Cr levels did not differ significantly from each other except that the 303 BWG of birds supplemented with only 1.5 mg Cr/kg diet could match the BWG of control diet 304 fed birds. In earlier studies Cr supplementation in broiler chicken improved BWG and efficiency 305 14 of feed utilization [1,37] The decline of abdominal fat in broiler chicken fed FSM can be attributed to enhanced 311 UFA content in meat at the cost of SFA and former undergo rapid oxidation compared to their 312 saturated counter parts. The FSM also exerts its effect by increasing gut viscosity which hinders 313 the micelle formation and thus diminish lipid uptake, thereby reducing its deposition in the body 314   T1  T2  T3  T4  T5  T1  T2  T3  T4  T5   Maize  537  535  535  535  535  625  622  622  622  622  Flaxseed meal  0.0  100  100  100  100  0.0  100  100  100  100  Soybean  390  290  290  290  290  300  200  200  200  200  Fish meal  30  30  30  30  30  30  30  30  30  30  vegetable oil  14  16  16  16  16  14  16  16  16  16  Limestone  9  9  9  9  9  11  11  11  11