Chromium picolinate can ameliorate the negative effects of heat stress and enhance performance, carcass and meat

traits in broiler chickens by reducing the circulatory cortisol level

f'Journal of the Science of Food and Agriculture

where science meets business

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Saikat Samanta, Sudipto Haldar,* Vijay Bahadur and Tapan K Ghosh

Department of Animal Nutrition, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery SCiences, 37 Kshudiram Bose Sarani, Kolkata 700037, India

Abstract

BACKGROUND: Dietary chromium (Cr3+) supplementation reduces stress in livestock by lowering circulatory cortisol and potentiating the action of insulin. This study aimed to assess the stress-alleviating effects of supplemental cr3+ as chromium picolinate in broilers.

RESULTS: The birds, which were heat stressed and had restricted feed intake between 31 and 40 days of age, were supplemented with 0 (control), 0.5 or 1 mg Cr3+ kg-1 diet from 10 to 40 days of age. Live weight gain, feed efficiency, utilisation efficiency of energy and protein and conversion efficiency of feed protein to muscle protein improved owing to Cr3+ supplementation (P < 0.05), especially during the period of feed restriction. cr3+ supplementation increased hot and eviscerated carcass weights and breast weight (P < 0.05) and reduced ether extract content (P = 0.02, linear effect). Meat protein accretion improved owing to Cr3+ supplementation (P = 0.006, quadratic effect). Relative to its pre-supplementation value, serum cortisol increased by 2.3% in the control group and decreased by 7.6 and 14.3% in the 0.5 and 1 mg Cr3+-supplemented birds respectively (age x diet interaction P = 0.0001).

CONCLUSION: It was concluded that supplemental cr3+ may ameliorate heat stress and augment growth in broilers during periods of physical feed restriction. However, increasing the inclusion of Cr3+ beyond the dose level of 0.5 mg kg-1; diet may not be substantially beneficial.

© 2007 Society of Chemical Industry

Keywords: broiler chickens; carcass fraits; chromium; dose response; cortisol; feed restriction; heat stress; performance

INTRODUCTION

The dietary essentiality of trivalent chromium (C2+) was first demonstrated in 19591 in rats and s~bsequently in humans in 1977.2 In the years to follow, papers on chromium (Cr) in human nutrition in ali kinds of clinical and stress situations were publishedy'-" the main focus being on the association between Cr3+ and diabetes mellitus.P It was only during the late 1990s that c2+ also started to be studied intensively as an essential mineral element in livestock.?

Cr3+ is a component of an oligopeptide called low-molecular-weight Cr-binding substance or chromodulin, which functions as part of the insulinsignalling auto-amplification mechanism." This stimulation of insulin action, which is directly proportional to the c2+ content of chromodulin.f occurs without

changing the concentration of insulin required for halfmaximal activity." Dietary supplementation of c2+ reportedly improved animal metabolism and enhanced production performance and the composition of animal products.l? Dietary c2+ supplementation positively affected growth rate and food conversion in poultry at an inclusion level ranging from 0.1 to 1 mg kg"" diet,u-14 In most of the studies mentioned above, beneficial effects of supplemental c2+ were obtained. However, recommendations regarding the exact dietary inclusion level of Cr3+ in diets of livestock, including poultry, are yet to be established+" and thus need further research.

Cr3+ itself does not have the capacity to cross the cell membranel" and hence has no biological or nutritional value.'? Inorganic. forms of Cr3+ such

• Correspondence to: Sudipto Haldar, Department of Animal Nutrition, Faculty of Veterinary and Animal Sciences, West Bengal University of Animal and Fishery' Sciences, 37 Kshudiram Bose Sarani, Kolkata 700037, India

E-mail: rajhaldar2002@yahoo.com

(Received 12 June 2007; revised version received 17 September 2007; accepted :3 October 2007) Published online 11 December 2007; DOl: 10.1002/jsfa.3146

© 2007 Society of Chemical Industry. J Sci Food Agric 0022-5142/2007/$30.00

S Samanta et al.

as chromic chloride are poorly absorbed because of formation of insoluble chromic oxide, formation of chelates with naturally occurring chelate-forming compounds in feed and interference from other mineral elements.l" The inconsistency in metabolic responses notwithstanding, organic Cr3+ compounds such as Cr picolinate are absorbed and metabolised in animals to a greater extent than their inorganic counterparts.f and hence the former are assumed to be biologically more active than the latter compounds. 19

The present investigation was designed to assess the growth and metabolic responses of broiler chickens fed diets supplemented with Cr3+ as Cr picolinate in graded levels. The birds were exposed to moderate heat stress and their feed intake was restricted. Heat stress is one of the factors that affect broiler performance to a great extent, and supplemental C~+ may effectively alleviate the stress-induced depression of performance.l" The objectives of the present study were (i) to investigate the dose response of broiler chickens receiving supplemental C~+ as Cr picolinate with regards to performance, carcass and meat characteristics and serum metabolites and (ii) to correlate the performance variables and the serum metabolite profile with the circulatory cortisol level of the chickens receiving graded levels of Cr supplementation.

MATERIALS AND METHODS Bird husbandry

Day-old unsexed Cobb 400 broiler chickens (n = 225) were purchased 12h post-hatch from a local hatchery. Immediately after arrival, water containing glucose and electrolytes was offered to the birds, followed by weighing and placing of the birds on litter in pens measuring 1.5 m x 1.5 m. A single pen constituted a replicate, and hence the pens were the experimental units in the present investigation. An initial slaughter was performed on day 1, taking 15 birds at random from the entire flock, and this constituted the initial slaughter group. Subsequently the birds were divided according to their body weight into three designated treatment groups, with each group consisting of seven replicates (n = 10 per replicate). The litter was composed of wood shavings, rice husk and paddy straw. The pens were separated with nylon wire netting, and each pen housed ten birds. The temperature inside the experimental room was kept fixed at 35-36°C during the experiment through heating elements fitted in the room. Light was provided round the clock through incandescent lamps for the first 15 days. A dark period of 1 h was introduced between days 16 and 25 during nights, and after day 25 the lights were turned off altogether for a period of 3 h during nights to reduce the activity of the birds. The birds received vaccination against Marek's disease and Newcastle disease (live B1 strain) immediately after hatching. Subsequent vaccination against Newcastle disease and infectious

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bursal diseases was done on days 7, 14 and 21. Ground com was offered to the birds within 18 h of hatching, and from day 1 a basal starter diet devoid of supplemental Cr3+ was offered replicate-wise to all birds until day 10. From day 11 the birds were fed with diets containing graded levels of Cr3+ as Cr picolinate until day 40. The feed was offered pen-wise along with ad libitum water in bell-type plastic feeders and drinkers. The starter and finisher diets were formulated to meet the requirements of the Cobb 400 broilers-? (Table 1). The birds were fed ad libitum during the starter phase (1-30 days). During the finisher phase (31-40 days), physical feed restriction was induced by adjusting the amount of feed offered in such a way that no orts were left in the feeder after each feeding. To accomplish the feed restriction, only a fixed amount of feed was kept in each pen from day 31 to day 40, with the greater amount of feed being offered in the morning. The amount of feed offered during this period was kept fixed at 110% of the amount offered on day 30. The total amount of feed provided for each pen every day was divided into two equal portions which were offered at 07:00 and 20:00. No mortality was recorded during the experiment.

Dietary treatments

As pointed out earlier, the birds received dietary Cr3+ supplementation for 30 days (days 11-40), and the dietary treatments consisted of feeding the birds a basal diet (control), the basal diet supplemented with 0.5 mg C~+ kg-1 diet or the basal diet supplemented with 1 mg C~+ kg-1 diet (as fed). Cr picolinate (manufactured by Jubilant Organosys, Noida, Uttar Pradesh, India) containing 5 g active trivalent Cr kg-1 was the source of the supplemental Cr3+. To arrive at the desired dose levels, 5 and 109 quantities of Cr picolinate were mixed separately with ground com ('" 1 00 g). The resultant mixtures were further mixed in a mechanical blender with separate 500 g lots of ground com and finally mixed with separate 50 kg lots of the starter and finisher diets to obtain the desired dose levels of 0.5 and 1 mg C~+ kg-1 diet. The unsupplemented basal starter and finisher diets contained 0.18 and 0.2mg kg-1 dry matter (DM) respectively.

Performance traits, carcass analyses and calculation of nutrient utilisation efficiency Replicate-wise feed intake was measured every day. Live weight was recorded replicate-wise at 10 day intervals in the morning before offering food. Replicate-wise live weight gain and feed conversion ratio (FCR, feed intake (g)/live weight gain (g)) were calculated at 10 day intervals, and cumulative live weight gain and FCR between days 1 and 40 were determined.

During the initial slaughter on day 1 the chicks were killed by decapitation and the feather-picked eviscerated carcass was frozen at -20°C for analysis.

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Table 1. Ingredients and chemical composition of basal diet

Starter diet Finisher diet
Component (0-30 days) (31 -40 days)
Ingredients (g kg-1)
Maize 580 620
Soybean meal 350 322
Sunflower cake 24
Fat 30 40
'I Salt 1.47 1.47
Dicalcium phosphate 8.8 9.5
Lysine sulfate 0.3 1.6
DL -Methionine 0.8 1.3
Trace elements" 1.5
Maduramycin 0.5 0.5
Phytase 2500 + 0.26 0.26
NSPase enzymes
Ascorbic acid 0.1 0.1
Toxin binder
Organic acids 1
Vitamin B complex 0.12 0.12
Vitamin AB2D3 0.15 0.15
Nutritive values (g kg-1 dry matter)
Crude proteln'' 210 194
Crude flbre? 37 34
Ether extract? 53 64
Metabolisable energy 12.9 13.4
(MJ kg-1 DM)C
l.yslne? 13 12
Methionine? 5 5
Cystelne? 2.4 2.3
Methionine + cystelne? 7.3 6.8
1- ArginineC 14.8 14
Calciumb 3.5 4.2
Phosphorus? 5.6 5.9
Available phosphorus? 3.1 3.7
Chromium (mg kg-1 0.18 0.2
DM)b • a Contained 40 mg manganese, 30 mg iron, 25 mg zinc, 3.5 mg copper, 0.3 mg iodine, 0.15 mg selenium and 200 mg choline chloride kg-1 .

b Estimated in laboratory.

C Calculated values.

The birds were finally harvested on day 40 by randomly picking two birds from each of the replicates from all treatment groups. After an overnight fast the birds were killed by exsanguination and processed for carcass characteristics, including hot carcass weight (with the head, blood, neck and hocks removed), eviscerated carcass weight, carcass yield (weight of defeathered eviscerated carcass relative to live weight) and yields of breast, frame, legs and wings. The yields of breast, frame and legs were expressed relative to the dressed carcass weight as well. Breast weight included the breast fillet and the tenders (pectoralis major and pectoralis minor), and the frame was defined as the eviscerated body frame with the head, neck, wings, legs and breast removed. The giblets included the liver, heart, lungs and gizzard. The carcass components were stored at -20°C for analyses.

For analysis of the moisture, ash, nitrogen (N), crude protein (CP) and fat (ether extract) contents in the meat the eviscerated carcass of day 1 and

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Chromium supplementation in heat-stressed broiler chickens

the frozen carcass cuts of day 40 were thawed and the muscles were manually separated from the bones, minced mechanically and homogenised in a tissue homogeniser (Remi Motors, Mumbai, India). The homogenised subsamples were finally mixed in a mechanical mixer and analysed for the abovementioned parameters.i" The Nand CP (N x 6.25) content of the carcass was determined using the Kjeldahl distillation procedure. For estimation of the ether extract, moisture-free dry meat samples were ground to pass through a 0.5 mm sieve and extracted with petroleum ether for 24 h, with the ether being changed at intervals of 8 h.21 All values concerned with the chemical composition of the meat were expressed on a fresh weight (FW) basis. Accretion of protein, fat and ash in the meat between days 1 and 40 was calculated.

Serum samples were obtained from the whole blood collected from all birds under each dietary treatment on days 1 and 40. On day 1, samples of whole blood were collected directly in test tubes from the severed jugular vein after the chicks had been decapitated. On day 40, blood samples were collected in sterilised syringes by a silicon catheter inserted into the wing vein through a needle. Immediately after collection the blood was transferred to test tubes, which were placed in ice for 60 min to clot the blood. The serum was separated from the cells by centrifuging the clotted blood at 2350 x g for 10 min (Remi Research R-8C centrifuge, Remi Research Laboratories, Mumbai, India) and harvested in two equal aliquots into 12 mm x 75 mm polystyrene tubes, which were capped and stored at -20°C. One of the aliquots was used for analysis of the metabolites, while the other was used in the cortisol assay. The concentrations of total protein, glucose, cholesterol, triglyceride and uric acid in the serum were determined in a UV -visible spectrophotometer (Systronics 108, Thailand) using commercial kits (Med Source Ozone Biomedicals, Faridabad, Haryana, India). Serum globulin concentration was determined by subtracting the concentration of albumin from that of total protein. All assays were performed in duplicate. An intraassay variation of more than 5% was considered to be invalid and a fresh assay was performed in such cases. Circulatory cortisol was determined by an enzyme immunoassay (Ranskan Sprint ELISA plate reader, Ranbaxy Diagnostics, New Delhi, India) using commercial kits (Equipar Diagnostica, Saronno, Italy). The sample size for cortisol estimation was 200 f1.L and the intra-assay and inter-assay variations were 4 and 9% respectively.

Nutrient utilisation efficiency was calculated based on actual feed intake data during the starter and finisher phases and for the total trial period (1-40 days). Average daily live weight gain (ADG) and daily intake of CP and metabolisable energy (ME) during the different growth periods were calculated. Utilisation efficiency for protein and ME was calculated as the ratio between ADG and intake of

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the corresponding nutrient. Protein conversion ratio was calculated as the ratio between protein intake and protein accretion in 40 days.

Estimation of chromium

For the estimation of dietary Cr3+ concentration, oven-dried samples were ignited in quartz crucibles in a muffle furnace at 400°C for 4h. The ash was treated with concentrated nitric acid under mild heat. The acid-extracted sample was cooled to room temperature and filtered through Whatman No. 1 ashless filter paper. The crucibles were washed with deionised water (MiIlipore Water Purification Systems, Molsheim, France) and the final volume was made up to 10 mL. Cr was analysed in an atomic absorption spectrophotometer (Perkin Elmer A Analyst 100, Massachusetts, Wellesley, USA). A solution of ammonium chloride (20 mL L -1) was added to the standard and the samples to reduce interference due to iron.

Statistical analysis

All data were analysed using the general linear model (GLM) of SPSS,22 and results were expressed in terms of the mean and the pooled standard error of the mean. Diet (concentration of C~+) was the main effect. Orthogonal polynomial contrasts were applied to ascertain the linear and the quadratic dose response. Data related to the nutrient utilisation efficiency and the serum parameters were considered as the response of birds at different ages." Hence the model involved diet and age as the main effects and an age x diet interaction effect. A probability of P < 0.05 was considered to be statistically signiPcant, while a probability of P < 0.1 was described as a trend.

RESULTS

Growth response, feed conversion ratio and efficiency of nutrient utilisation

Live weight of the birds, which was 41.4 ± 0.23 g at 12 h post-hatch, was similar across the dietary treatments until day 30 (P> 0.1), indicating a subtle effect of supplemental C~+ on live weight and live weight gain during the starter phase. Supplemental C~+ elicited a quadratic dose response in terms oflive weight (P = 0.008) and live weight gain (P = 0.01) on day 40, with the effects being heavier in the birds receiving 0.5 mg Cr3+ kg-1 diet. Feed efficiency calculated on day 40 improved quadratically owing to C~+ supplementation (P = 0.01). Significant improvement in total live weight gain (P = 0.008) and cumulative feed efficiency (P = 0.009) also occurred owing to Cr3+ supplementation. However, in all these cases the effects were not linear, rather quadratic, and were heavier in the birds receiving 0.5 mg Cr3+ kg " diet (Table 2).

ADG of the experimental birds was similar (P > 0.1) across the dietary treatments during the starter phase and improved significantly owing to Cr3+ supplementation during the finisher phase (diet effect P = 0.01, diet x age interaction P = 0.02). However, the response was quadratic and heavier in the birds supplemented with 0.5 mg C~+ daily, and this effect was carried over to the pooled data of 1-40 days as well (diet effect P = 0.01, quadratic effect). Compared with the starter phase, the birds consumed more CP and ME during the finisher phase (age effect P = 0.0001) irrespective of the level of Cr3+ supplementation in the diet. However, the level of supplemental C~+ had a subtle effect on the intake of these nutrients (diet effect P> 0.1, diet x age interaction P > 0.1).

Table 2. Growth responses and feed efficiency of broiler chickens fed diet supplemented with C~+ as Cr plcolinate"
Diet (mg c(3+ kg-1 diet) Contrast P value
Measurement 0 0.5 Pooled SE Linear Quadratic
Live weight (g)
Day 10 126 130 125 1.2 0.1 0.62
Day 20 388 384 393 3.9 0.9 0.84
Day 30 890 886 898 11.2 0.79 0.73
Day 40 1514 1755 1602 31.6 0.27 0.008
Live weight gain (g)
Day 10 84 89 83 1.1 0.4 0.73
Day 20 262 258 258 3.7 0.7 0.87
Day 30 503 497 505 9.6 0.91 0.75
Day 40 623 869 704 35.2 0.37 0.01
Total gain 1472 1714 1571 31.6 0.27 0.008
Feed conversion (feed intake (g)/live weight gain (g))
Day 10 1.42 1.42 1.39 0.02 0.1 0.34
Day 20 1.81 1.83 1.84 0.03 0.63 0.92
Day 30 1.96 1.96 1.93 0.04 0.78 0.86
Day 40 2.21 1.65 2.07 0.08 0.51 0.01
Trial mean 2.01 1.73 1.91 0.04 0.34 0.009 a Measurements are pooled means of seven replicates of ten Cobb 400 unsexed broiler chickens. The mean live weight at 12 h post-hatch was 41.4±0.23g.

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Keeping parity with the pattern of live weight gain, the gain/protein intake ratio and gain/ME intake ratio were not affected by supplemental Cr3+ feeding during the starter phase but improved significantly during the finisher phase (diet effect P = 0.02, diet x age interaction P = 0.04). Overall, the effect was quadratic and heavier in the dietary group receiving 0.5 mg supplemental Cr3+ daily for both measurements.

The protein accretion/protein intake ratio, which indicated the conversion efficiency of feed protein to meat protein, improved (P = 0.004) in the birds receiving 0.5 mg supplemental C~+ daily, revealing a quadratic dose response (Table 3).

Carcass and meat traits

Hot carcass weight and eviscerated carcass weight improved quadratically (P = 0.01) owing to C~+ supplementation, with the effects being heavier in the birds receiving 0.5 mg supplemental C~+ daily. The weight of the whole breast (P = 0.002) and that expressed as a % of the eviscerated carcass weight (P = 0.02) increased quadratically owing to C~+ supplementation. The weight of the frame and that of the legs tended to increase with Cr3+ supplementation in the diet (P = 0.07, linear and quadratic effect respectively). However, when expressed as a % of the eviscerated carcass weight, frame weight was found to be higher in the C~+ -supplemented birds (P = 0.04, quadratic effect). The weight of the liver (P = 0.06) and that of the giblets (P = 0.07) tended to increase owing to C~+ supplementation. However, carcass yield as a whole was not affected by dietary C~+ supplementation (Table 4).

Chemical analysis of the meat revealed that its moisture content in the C~+ -supplemegted birds was higher (P = 0.001, linear effect) relative to the control

Chromium supplementation in heat-stressed broiler chickens

group of birds. However, ash and protein contents were similar across the dietary treatments (P> 0.1). Ether extract content of the meat, however, decreased linearly owing to C~+ supplementation (P = 0.02). Meat protein accretion improved significantly owing to C~+ supplementation (P = 0.006, quadratic effect), though accretion of fat and ash in the carcass was not affected by supplemental C~+ feeding (Table 5).

Serum metabolite and cortisol concentration Table 6 depicts the serum metabolite and cortisol profiles in the experimental chickens. The initial serum protein concentration measured on day 1 was similar across the dietary treatments (P> 0.1). On day 40, serum protein tended to be lower in the 0.5 mg C~+ - supplemented birds (P = 0.09, quadratic effect). Serum protein was not affected by the age of the birds (P> 0.1). Overall, serum protein decreased (P = 0.04, quadratic effect) owing to Cr3+ supplementation, and there was a significant age x diet interaction effect (P = 0.006) as well. Serum albumin concentration followed exactly the same pattern observed with serum protein and was lower quadratically on day 40 in the birds supplemented with 0.5 mg Cr3+ than in the other dietary groups (P = 0.03). Overall, serum albumin increased with the age of the birds (P = 0.0001), though a conspicuous age x diet interaction effect was lacking (P = 0.08). Compared with its initial value, serum globulin decreased on day 40 (age effect P = 0.02). However, supplemental C~+ subtly affected the final serum globulin concentration on day 40.

Serum glucose decreased quadratically owing to C~+ supplementation(P = 0.003). Relative to its initial value, serum glucose declined by 22.5% in the 0.5 mg Cr3+ -supplemented group of birds compared with 7.3 and 6.1 % in the control and 1 mg C~+-

Table 3. Efficiency of nutrient utilisation in broiler chickens fed diet supplemented with C~+ as Cr picolinate"
Diet (mg Cr3+ kg-1 diet) Diet effect (contrast P value)
Measurement Age (days) 0 0.5 Pooled SE Age effect Linear Quadratic Diet x age
Live weight gain (g day-1) 1-30 28.3 28.2 28.5 0.37 0.78 0.77
31-40 62.3 86.9 70.4 3.5 0.37 0.01
1-40 34.7 40.6 36.7 0.79 0.0001 0.35 0.01 0.02
Protein intake (g day-1)b 1-30 10.67 10.66 10.68 0.006 0.65 0.35
31-40 26.58 26.79 26.74 0.1 0.55 0.57
1-40 14.65 14.69 14.71 0.03 0.0001 0.53 0.59 0.69
ME intake (MJ day-1)b 1-30 0.657 0.654 0.66 0.001 0.18 0.06
31-40 0.84 0.85 0.85 0.007 0.53 0.57
1-40 0.951 0.954 0.954 0.002 0.0001 0.41 0.78 0.64
Gain/protein intake 1-30 2.65 2.64 2.67 0.04 0.81 0.79
31-40 2.35 3.25 2.64 0.14 0.4 0.02
1-40 2.37 2.77 2.49 0.06 0.53 0.38 0.02 0.04
Gain/ME intake 1-30 43.1 43.0 43.5 0.58 0.79 0.79
31-40 33.9 47.1 38.2 2.1 0.39 0.02
1-40 36.5 42.6 38.5 0.87 0.11 0.37 0.02 0.04
Protein intake (g)/protein . 1-40 1.85 1.52 1.74 0.04 0.26 0.004
accretion (g) 8 Data are pooled by seven replicates of ten Cobb 400 unsexed broiler chickens.

b Means are based on actual feed intake data pooled by replicates (n = 7 per treatment group).

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Table 4. Gross carcass traits of broiler chickens fed diet supplemented with C,.s+ as Cr plcolinate"
Diet (mg C~+ kg-1 diet) Contrast P value
Measurement 0 0.5 Pooled SE Linear Quadratic
Hot carcass (g) 1086 1277 1151 27.5 0.35 0.01
Eviscerated carcass (g) 833 985 8891 21.4 0.29 0.01
Yield of carcass components (g)
Breast 259 324 266 8.1 0.73 0.002
Frame 195 223 221 5.7 0.07 0.23
Legs 295 335 314 7.6 0.32 0.07
Wings 89 97 86 2.9 0.68 0.13
Liver 34 42 38 1.6 0.41 0.06
Giblets 79 95 87 3.1 0.35 0.07
Yield of carcass components (%)b
Breast 32 33 30 0.51 0.21 0.04
Frame 24 23 25 0.36 0.15 0.04
Legs 36 34 35 0.34 0.68 0.12 y
Carcass yield? 60 62 61 0.58 0.67 0.37 a Each treatment group consisted of seven replicates of ten Cobb 400 unsexed broiler chickens with a mean live weight of 41.4 ± 0.23 g at 12 h post-hatch. Slaughter was performed after 40 days feeding trial by taking two birds at random from all seven replicates under each treatment group. The data were pooled replicate-wise for calculation and statistical analyses.

b Weight of the breast, frame and legs relative to the eviscerated carcass weight.

C Weight of the feather-picked eviscerated carcass (with the blood, head, neck and hocks removed) relative to the live weight.

Table 5. Chemical composition (g kg-1 fresh weight) of meat and nutrient accretion (1-40 days) in broiler chickens fed diet supplemented with C,.s+

as Cr picollnate"
Diet (mg C~+ kg-1 diet) Contrast P value
Parameter 0 0.5 Pooled SE Linear Quadratic
... Moisture 712 724 724 1.3 0.001 0.04
Ash 120.2 119.9 120.2 3.17 0.99 0.97
Crude protein 21.6 22.7 21.9 0.37 0.73 0.22
Ether extract 7.4 6.3 6.5 0.25 0.02 0.21
Protein accretion 317.5 389.1 344.5 8.79 0.23 0.006
Fat accretion 109.9 107.1 100.9 3.95 0.37 0.83
Ash accretion 175.9 °204.8 188.6 6.69 0.45 0.13 a Each treatment group consisted of seven replicates of ten Cobb 400 unsexed broiler chickens with a mean live weight of 41.4 ± 0.23 g at 12 h post-hatch. Initial slaughter was performed on day 1, with 15 chicks constituting the initial slaughter group. The birds were finally harvested after 40 days feeding trial by taking two birds at random from all seven replicates under each treatment group. The data were pooled replicate-wise for

calculation and statistical analyses. .

supplemented groups respectively (age effect P = 0.0001, age x diet interaction P = 0.002).

Serum cholesterol was lower in the dietary groups receiving supplemental C~+ compared with the control group of birds when measured on day 40 (P = 0.04, linear effect; P = 0.01, quadratic effect). Relative to its initial value, serum cholesterol changed little in the control group of birds (-1. 9 % ), whereas it declined by 35.8 and 29.4% in the groups supplemented with 0.5 and 1 mg C~+ kg-1 diet respectively (age effect P = 0.0001, age x diet interaction P = 0.0001). The net result was Ii significantly lower mean serum cholesterol concentration in the C~+supplemented groups of birds (P = 0.0001, linear effect).

Conversely, serum triacylglycerol increased with the age of the birds irrespective of dietary treatment (P = 0.0001). However, while triacylglycerol concentration increased by 118% relative to the initial value in the

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control group of birds, it was only 55 and 85% in the 0.5 and 1 mg C~+ -supplemented groups respectively (age x diet interaction P = 0.03). On day 40, serum triacylglycerol was lower in the C~+ -supplemented groups of birds than in the control group (diet effect P = 0.02, linear effect; P = 0.009, quadratic effect).

Serum uric acid concentration, which was similar across the dietary treatments for both periods of measurement, increased with the age of the birds (age effect P = 0.0001).

Cortisol was the other serum parameter affected most by dietary Cr3+ supplementation. The initial (day 1) serum cortisol concentration was found to be higher (P < 0.05) in the birds which later received 1 mg C~+ kg-1 diet, and hence this initial variation was in no way attributable to the dietary treatments. A linear decrease in serum cortisol concentration due to C~+ supplementation was observed on day 40 (diet effect P = 0.0001). Relative to its initial concentration,

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Chromium supplementation in heat-stressed broiler chickens
Table 6. Serum biochemical profile and circulatory cortisol concentration in broiler chickens fed diet supplemented with C~+ as Cr plcollnate=
Diet (mg C~+ kg-1 diet) Diet effect (contrast P value)
Serum analyte Age (days) 0 0.5 Pooled SE Age effect Linear Quadratic Diet x age
Protein (g L -1) 1 34.5 33.1 36.9 1.94 0.1 0.61
40 41.1 35.8 38.2 1.02 0.26 0.09
Trial mean 37.8 34.5 37.6 1.09 0.38 0.0001 0.04 0.006
Albumin (g L -1) 1 17.9 18.9 18.9 0.74 0.6 0.46
40 25.9 21.5 24.1 0.69 0.32 0.03
Trial mean 21.9 20.2 21.4 0.51 0.0001 0.71 0.29 0.08
Globulin (g L -1) 1 17.5 14.1 18.1 2.04 0.07 0.83
40 15.3 14.3 14.1 1.43 0.74 0.9
Trial mean 16.4 14.3 16.1 1.24 0.02 0.91 0.008 0.06
Glucose (mmol L -1) 1 9.6 10.2 9.8 0.12 0.58 0.07
40 8.9 7.9 8.2 0.16 0.57 0.003
Trial mean 9.3 8.9 9.1 0.1 0.0001 0.42 0.14 0.002
Cholesterol (mmol L -1) 1 5.2 5.3 5.1 0.11 0.33 0.37
40 5.1 3.4 3.6 0.08 0.04 0.01
Trial mean 5.1 4.3 4.4 0.07 0.0001 0.0001 0.01 0.0001
Triacylglycerol (mmol L -1) 1 0.61 0.56 0.55 0.03 0.4 0.49
40 1.33 0.87 1.02 0.05 0.02 0.009
Trial mean 0.97 0.71 0.79 0.03 0.0001 0.43 0.007 0.03
Uric acid (prnol L -1) 1 509.1 443.2 406.5 14.3 0.55 0.15
40 297.4 287.1 344.5 23.6 0.43 0.51
Trial mean 652.9 669.8 607.7 31.9 0.0001 0.02 0.26 0.07
Cortisol (mmol L -1) 1 1204.6 1208.9 1287.7 9.8 0.03 0.09
40 1232.1 1116.9 110.3 5.33 0.0001 0.0001
Trial mean 1218.3 1162.9 1195.6 5.56 0.0001 0.11 0.001 0.0001
a Each treatment group consisted of seven replicates of ten Cobb 400 unsexed broiler chickens. Blood was collected on day 1 from the severed
jugular vein of 15 chicks segregated as an initial slaughter group. On day 40, blood was collected from the wing vein of two birds picked at random
" from all seven replicates under each treatment group. The data were pooled replicate-wise for calculation and statistical analyses. serum cortisol.increased by 2.3% in the control group and decreased by 7.6 and 14.3% in the birds receiving 0.5 and 1 mg c2+ kg-1 diet respectively (age x diet

interaction P = 0.0001). G

DISCUSSION

The objective of the present investigation was to evaluate the effects of supplemental Cr3+ on the performance of experimental broiler chickens and to find possible explanations of the observed performance variables through the metabolic changes in the birds. Generally an ambient temperature above 30 DC is considered sufficient to induce heat stress in poultry,23 which negatively influences live weight, feed intake and feed efficiency. 24 The ambient temperature and relative humidity (which averaged 90 ± 1.26% over the duration of the experiment) inside the experimental room were sufficiently high to induce heat stress, and the birds showed visible signs of heat exposure, such as panting and a dispersed distribution within pens. Depression in performance due to heat stress is postulated to be in part due to a reduction in feed intake.25 However, this postulation seemed to have worked little during the present investigation, since feed intake was similar across the dietary treatments until day 30 when the birds were fed ad libitum, and that during days 31-40 could not vary between the groups owing to the

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physical feed restriction imposed during this period. Hence the variation in the performance observed between the dietary groups ought to be due to the Cr3+ -mediated metabolic consequences. Stressors such as high ambient temperature induce a cascade of neural and hormonal events in which cortisol plays a key role.24 Irreversible loss of tissue c2+ occurs owing to stress, which exacerbates a marginal dietary c2+ deficiency." Supplemental c2+ has a role to play in such situations and ameliorates the heat stress,13,14 perhaps by shifting the metabolic balance more towards anabolism through its known involvement in the auto-amplification mechanism of insulin action."

The improvement in the performance variables due to c2+ supplementation was in corroboration With our earlier findings. 14,26 The most intriguing aspect of the present observations was perhaps the enhancement in the efficiency of protein and energy utilisation, which was affected by dietary Cr3+ supplementation during the period of physical feed restriction. It may be postulated that supplemental c2+ might have effectively partitioned the nutrients, especially the energy, between the adipose and lean tissues to sustain an optimum growth rate when the feed intake was restricted. This was in partial contradiction to one of our earlier observations in which supplemental c2+ (as chromic chloride hexahydrate) failed to sustain an optimum growth in energy-starved broiler chickens.F"

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However, unlike in the present investigation, the birds in that experiment were not exposed to any stress per se, which perhaps precluded the supplemental Cr3+ from eliciting its stress-alleviating effects to the fullest extent. Thus the present experiment revealed that dietary supplementation of Cr3+ may be of immense importance in ameliorating physical stress factors such as high ambient temperature and feed restriction in broiler chickens. The latter is particularly important in the poultry industry, because feed restriction is used to yield greater beneficial effects in terms of economising the feeding regimen and production of lean meat. The feed restriction practised in the present investigation was the reverse of the classical restriction approaches. Nevertheless, requirement of nutrients, especially that of amino acids, must have been increased during the feed restriction, and C~+ as Cr' picolinate sustained and facilitated the growth process by increasing the uptake of amino acids by the skeletal muscles.F which in turn enhanced total protein deposition in the muscles of the C~+supplemented birds.

The improvement in hot and eviscerated carcass weights due to C~+ supplementation was inevitable, since these carcass parameters were direct functions of the body weight. The non-significant difference in carcass yield notwithstanding, the enhancement in breast meat yield in the Cr3+ -supplemented birds is noteworthy. A higher breast meat yield supported the higher protein conversion efficiency and protein accretion in the Cr3+ -supplemented birds. Our earlier investigations failed to reveal any substantial effects of supplemental C~+ on breast meat yield. 14,26 However, the aforementioned investigations did not involve Cr picolinate as the source of supplemental C~+. Hence

.,

Crpicolinateis seemingly more effective in enhancing

breast meat yield in comparison with chromic chloride or Cr-yeast complex.

The present investigation revealed that a sort of value addition to broiler meat is possible by dietary supplementation with Cr3+ as Cr picolinate. The linear increment in the moisture content of the C~+ -supplemented meat was indicative of a similar increase in the water-holding capacity of the meat as reported earlier.!" The significantly lower fat content and the improvement in protein accretion indicated that Cr picolinate may be efficiently used for manipulating the carcass composition to yield more lean meat.14,26.28,29 Increased uptake of amino acids at tissue level and increased muscle protein synthesis due to internalisation of insulin in the myocytes through C~+ -mediated potentiation of insulin action have been reported.27,28 It is interesting to note that, despite a linear decrease in carcass fat content, fat accretion was similar across the dietary treatments. There may be a couple of mechanisms involved in this phenomenon. First, the C~+ -supplemented birds had a faster growth rate and a heavier body weight at the end of the trial. As a function of the body weight and growth rate a higher fat accretion was inevitable

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in the' C~+ -supplemented groups despite a lower fat content per unit body weight.26,30 The second possible mechanism involves a rather ambiguous function of the Cr-insulin axis on lipogenesis. A shift in the metabolic balance more towards anabolism due to the Cr3+ -mediated potentiation of insulin action should plausibly increase lipogenesis and decrease lipolysis.!? This may lead to a net increase in fat synthesis. Supplemental Cr3+ reportedly facilitated the interaction between chromodulin and the insulin cell surface receptors and enhanced glucose flux into the adipocytes, causing anet increase in lipogenesis. 19 However, our experimental design was not sensitive enough to detect to what extent the second mechanism operated during the present investigation.

The concentrations of the serum metabolites during the present investigation indicated that the birds were under stress. Apart from an elevation in serum cortisol level during heat stress,31 a reduced plasma protein level and a markedly increased blood glucose level are the other metabolic indices indicating heat stress.32 An elevated serum protein level in the control group of birds compared with that in the Cr3+ -supplemented groups was perhaps suggestive of the heat-stressed condition of the former and its alleviation in the latter as a result of C~+ supplementation. In contrast, in non-stressed broiler chickens an elevation in plasma protein concentration due to C~+ supplementation was reported and the observation was explained by an enhancement in crude protein metabolisability due to C~+ supplernentation.e" However, despite enhanced protein utilisation efficiency, the serum protein level was significantly lower in the Cr3+ -supplemented birds in the present investigation. It was perhaps the heat stress which elevated the serum protein level in the control group. In turn, the findings revealed that dietary supplementation with Cr picolinate might have substantially ameliorated the stress of thermal origin in the experimental chickens.

Increased glucose utilisation due to C~+ supplementation, especially by the adipocytes and the muscle cells, and a resultant lowering of the blood glucose level in broilers have been reported.Uv" Turkeys fed a diet supplemented with C~+ had greater liver glycogen relative to the control group of birds as a result of increased activity of the enzyme glycogen synthatase. Cr3+ facilitated this process by augmenting glutose transport through a potentiated insulin action.P The same mechanism might have operated in this case also to lower the serum glucose level in the C~+supplemented birds. Moreover, the heat stress was yet another contributory factor to the elevation of the serum glucose level in the control group of birds, as reported earlier.P

In non-ruminants, reduced blood cholesterol due to dietary Cr3+ supplementation is one of the most frequently reported responses.!" This may be caused by to an augmented insulin action that reduces lipolysis and increases fatty acid incorporation in the adipocytes.l? An increased lecithin-cholesterol

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metabolism and excretion was perhaps the possible explanation for the above findings.i"

Lowering of circulatory cortisol is one of the typical metabolic consequences of Cr3+ supplementation in livestock.6,10,19 Cortisol secretion increases under stress and, by acting as. an insulin antagonist, cortisol increases blood glucose concentration and reduces glucose utilisation by peripheral tissues.l? Increased blood glucose level stimulates mobilisation of tissue c2+ and its irreversible loss through urine. 34 Supplemental c2+ reverts the process by reducing tissue sensitivity to stress through a potentiated insulin action.I" In the present investigation the decline in serum glucose level almost paralleled that of serum cortisol, which revealed that the effect of heat stress in the c2+ -supplemented birds was ameliorated to yield less glucose in the blood under the influence of the catabolic effects of cortisol.

One of the most intriguing aspects of the present experiment was the lack of linearity and a quadratic dose response with regards to most of the variables studied. However, a similar lack of linearity in dose response has been reported previously in ruminants.Pv'" In the present investigation also the dose response reached a plateau at the dose level of 0.5 mg c2+ kg-1 diet, revealing that a further increment in inclusion level would result in a diminishing return. A diminishing return response can be defined as the decreasing increment in gain as equal increments of nutrients are added to the diet near maximum gain.P? The rectilinear pattern of the dose response curve (not shown) obtained in . the present investigation suggests that the birds approached the maximum gain when 0.5 mg c2+ kg-1 diet was supplemented as Cr picolinateg Nevertheless, further research involving a closer scrutiny of the dose response is needed to define the breakpoint indicating dietary deficiency and sufficiency of supplemental c2+ to achieve an optimum growth response and a greater economic return from broiler chickens.

j

CONCLUSION

It was concluded from the present study that supplementation of diets with c2+ as Cr picolinate may offer a potential management tool in combating heat stress in broiler chickens. Supplemental c2+ may sustain and augment broiler growth rate even when a physical feed restriction is imposed. Dietary Cr3+ supplementation has been found to elicit a significant cortisol-lowering effect in the broilers, and the . performance-enhancing effect is postulated to be mediated through potentiation of insulin action by shifting the metabolic balance more towards anabolism. However, increasing the inclusion of c2+ beyond the dose level of 0.5mgkg-1 diet may not yield any substantial benefit but rather result in a diminishing return.

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Chromium supplementation in heat-stressed broiler chickens

ACKNOWLEDGEMENT

The authors gratefully acknowledge ·],ubilaf).t Organosys Ltd, Vadodara, Gujarat, India for providing the chromium picolinate free of charge.

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