J. Appl. Poult. Res.
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J APPL POULT RES 2009. 18:514-520. doi:10.3382/japr.2008-00141
© 2009 Poultry Science Association
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Research Reports

Impact of stocking density and feeding regimen on broilers: Chicken meat composition, fatty acids, and serum cholesterol levels

U. G. Simsek*,1, I. H. Cerci{dagger}, B. Dalkilic{dagger}, O. Yilmaz{ddagger} and M. Ciftci{dagger}

* Department of Animal Science, {dagger} Department of Animal Nutrition, Faculty of Veterinary Medicine, and {ddagger} Department of Biology, University of Firat, Elazig–Turkey 23119

1 Corresponding author: gsimsek{at}firat.edu.tr


   SUMMARY
TOP
 SUMMARY
DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
The purpose of this study was to evaluate the effects of different stocking densities (22.5, 18.75, 15, 11.25, 7.5 broilers/m2) in ad libitum (Al) and limited- or pair-feeding (Pf) regimens on meat composition; fatty acid profile of the total carcass, breast meat, and thigh meat; and serum cholesterol level in broiler chickens. (All the Pf groups were provided the same amount of feed per broiler, as determined for the Al-fed group with 22.5 broilers/m2.) Limited feeding increased the fat ratio of the chicken meat. Lowering the stocking density reduced the fat ratio and increased the protein ratio of the meat in both feeding regimens. The total saturated fatty acid (SFA) ratio was found to be quite high, whereas the total polyunsaturated fatty acid (PUFA) and n-3 fatty acid ratios were found to be low in the limit-fed broilers. Lowering the stocking density had a variable effect on fatty acid composition of the meat; total SFA and monounsaturated fatty acid ratios decreased, whereas total PUFA, n-3, and n-6 ratios increased in the Al groups. Total SFA and monounsaturated fatty acid ratios increased and total PUFA, n-3, and n-6 ratios decreased in the Pf groups. Serum total and high-density lipoprotein cholesterol levels were reduced with lower stocking densities in the Al groups, but only high-density lipoprotein cholesterol was reduced in the Pf groups. Consequently, stocking density and feeding regimen significantly changed the composition, fatty acid profile, and serum cholesterol level of chicken meat.

Key Words: broiler • stocking density • feeding • meat composition • fatty acid • serum cholesterol level


   DESCRIPTION OF PROBLEM
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Several studies have examined the effects of different stocking densities and feeding programs on performance and welfare parameters in broilers. An increase in the number of birds per unit and limited feeding programs depress growth rate, feed intake, and slaughter yield, and detrimentally affect broiler welfare [1, 2]. Despite the increasing consumer demand for healthy foods, studies about the effect of these factors on meat quality have lessened [3, 4]. However, exercise is an important factor affecting the lipolysis and fatty acid profiles of tissues [5, 6]. In addition, limited feeding programs cause fat synthesis, and hunger changes the metabolism in a way that creates obese individuals [4].

The present study was performed to determine the effects of stocking density and feeding regimen on the composition, fatty acid profile, and serum cholesterol level of chicken meat in ad libitum (Al) and pair-feeding (Pf) regimens.


   MATERIALS AND METHODS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
Experimental Design
This study was performed using 5 different stocking densities for birds fed Al [22.5 (90 birds/pen), 18.75 (75 birds/pen), 15 (60 birds/pen), 11.25 (45 birds/pen), and 7.5 (30 birds/pen) broilers/m2] and 4 different stocking densities for bird fed Pf (18.75, 15, 11.25, and 7.5 broilers/m2), each with 5 replicate pens. Each pen had an area of 4 m2 (2 x 2 m), and provided equal feeder space (264 cm/pen) from pan feeders and equal watering space (90 cm/pen) from bell drinkers. Water was given ad libitum and the photoperiod was 24 h/d. The Pf groups were fed according to the most crowded group (22.5 broilers/m2). For this aim, feed was given ad libitum to 9 treatment groups by measuring the BW on d 4 at the beginning of the study, and daily feed intake was recorded. Beginning from this date, the amount of daily feed intake per broiler in the 22.5 broilers/m2 group was fed for all Pf treatments at different stocking densities from 5 to 42 d. The 22.5 broilers/m2 group was a shared control for both the Al and Pf regimens.

At 42 d, 6 males and 6 females, whose BW were close to the pen average for males and females combined, were slaughtered. Their blood was collected and serum was separated. For chemical analysis of the meat, whole carcasses of 3 of the males and 3 of the females, and the left thigh and half the breast from the remaining 3 males and 3 females were collected. In the carcass group, the whole carcass with bones was minced, whereas in the other groups, the breast and thigh meat were separated from the bones and minced in a meat grinder, homogenized with an electronic blender, and then flash frozen (–40°C, 8 to 10 h) and stored (–20°C, 3 to 4 wk) until analyzed. Fatty acid compositions of diets are given in Table 1Go.


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  		Table 1. Fatty acid1 composition of the diets
 
Chemical Analyses
Protein levels of the total carcass, thigh meat, and breast meat were determined by the macro-Kjeldahl method following the AOAC procedure [7]. Fat levels of these tissues were analyzed by ether extraction [8]. Extraction of lipids from the tissue specimens was performed according to the method of Hara and Radin [9]. For this purpose, a 1-g tissue specimen was homogenized in 10 mL of 3:2 (vol/vol) hexane:isoproropanol mixture for 30 s. Tissue homogenate was centrifuged at 2,260 x g for 10 min; the supernatant was taken for analysis.

Preparation of Fatty Acid Methyl Esters
For the preparation of methyl esters, lipid extract in a hexane:isopropanol phase was placed in 30-mL experiment tubes. Five milliliters of 2% methanolic sulfuric acid was added and the mixture was vortexed. This mixture was left to methylate in a 50°C incubation for 15 h. It was then cooled at room temperature, and 5 mL of 5% sodium chloride was added and mixed. The fatty acid methyl esters that were produced were extracted with 5 mL of hexane. The hexane phase was then removed with a pipette and treated with 5 mL of 2% KHCO3. The solvent in the methyl ester-containing mixture was evaporated at 45°C with a nitrogen flow and dissolved with 1 mL of hexane. All the mixture containing the solvent and hexane was then placed in 2-mL closed autosampler vials and analyzed [10].

Gas Chromatographic Analysis of Fatty Acid Methyl Esters
The fatty acid methyl esters were analyzed in a gas chromatograph [11] with a Machery-Nagel capillary column [12]. During the analysis, column heat was maintained at 120 to 220°C, injection heat was maintained at 240°C, and detector heat was maintained at 280°C. The column heat program was regulated to 220°C from 120°C; the heat increase was set to 5°C/min until reaching 200°C, to 4°C/min from 200 to 220°C, and held at 220°C for 8 min. Nitrogen was the carrier gas and the detector was a flame-ionization detector [11]. Before analysis of the fatty acid methyl esters in the samples, standard fatty acid methyl esters and residence times of each fatty acid were determined. After this determination, the necessary program analysis was made and fatty acid methyl esters mixtures were analyzed.

Analysis of Cholesterol Concentration by HPLC
Cholesterol analysis was carried out according to Katsanidis and Addis [13]. One portion of lipid extract, which was divided into 2 sections and put into tubes with caps, and a 5% KOH solution (prepared in absolute ethanol) was added. After mixing thoroughly, the mixture was held at 85°C for 15 min. The tubes were cooled at room temperature, and 5 mL of pure water was added and vortexed. After phase separation, the upper hexane phase was taken and its solvent was evaporated. It was then dissolved with a nitrogen flow in an acetonitryl:methanol mixture (50:50%, vol/vol) and was placed in autosampler vials and prepared for analysis. For the mobile phase, an acetonitryl:methanol (60:40%, vol/vol) mixture was used. The flow speed of the mobile phase was 1 mL/min. A UV detector was used for the analysis and the wavelength was 202 nm. A Supelcosil LC-18 column was used [14]. The high-density lipoprotein (HDL) cholesterol level was detected with an HDL cholesterol kit [15].

Statistical Analysis
Data were analyzed by 2-way ANOVA using the GLM procedure of SPSS [16], with feeding regimen and stocking density being the main effects. For this purpose, to see effects of stocking density, feeding regimen, and their interaction, the group with 22.5 birds/m2 was excluded from the data set (4 x 2 factorial design). Then, to determine the differences between the group with 22.5 birds/m2 and other stocking densities for the broilers on Al feeding, all Pf groups were first excluded from the data set. The same model was then used for the Pf groups by excluding all Al groups. Data were analyzed by 1-way ANOVA. Significant differences (P ≤ 0.05) were further subjected to Duncan’s multiple range test for separation of means.


   RESULTS AND DISCUSSION
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 
When the influence of feeding regimen on the fat (ether extract) value of chicken meat was examined (Table 2Go), limited feeding was found to increase (P ≤ 0.01) the fat ratios of the total carcass and thigh meat. This result appears to illustrate that limited feeding programs cause fat synthesis in broiler chickens. Limited feeding puts animals under physical stress [17], and in broilers, glucocorticoid corticosterone is secreted under stress [18]. The typical response of the adrenal gland is to balance the blood glucose level by mobilizing glycogen storage [19]. In addition, feeding after hunger causes glucose and insulin secretion and fat synthesis [20]; the blood glucose level is thus stabilized during periods of limited food intake. Immediately after a meal, blood glucose levels increase, which stimulates insulin secretion. Fat is a means of storing energy for periods of limited food supply. Similarly, Zhan et al. [4] confirmed that in broilers that were left hungry for 4 h during a 21-d period, restricted feed at the end of the period resulted in an increased fat ratio in breast meat. The Pf groups at different stocking densities (18.75, 15, 11.25, 7.5 broilers/m2) in this study consumed the same amount of feed per broiler as the Al-fed group with 22.5 broilers/m2, and they finished their feed at different times linked with stocking density; therefore, broilers in these treatments became hungry sooner than others. Fattening in the Pf groups might have resulted from refeeding after hunger in these groups. The protein ratio was also affected (P ≤ 0.05) in the fattened Pf groups.


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  		Table 2. The influence of different stocking densities and feeding regimens on protein (CP) and fat (ether extract) values of raw meat and serum cholesterol levels in broiler chickens
 
Lowering the stocking density reduced the fat ratio of total carcass and thigh meat in both the Al (P ≤ 0.01) and Pf groups (P ≤ 0.05), and increased (P ≤ 0.05) the protein ratio of thigh meat in both the feeding regimens. There were significant density and feeding interactions for fat (P ≤ 0.05) and protein (P ≤ 0.05) ratios of the total carcass and thigh meat. However, no statistical difference in fat and protein ratios of the breast meat were noted (P ≥ 0.05). Lowering the fat deposition could be related to broilers in the lower density group having more movement space. In addition, the effect of physical activity on fat deposition was more significant in the unlimited-fed Al groups than in the limit-fed Pf groups, and limited feeding partially depressed the effect of lowering the stocking density. Similarly, Castellini et al. [5] reported a significant positive correlation between reduced physical activity and fat deposition. Simsek et al. [21] confirmed that increasing the physical activity reduced fat deposition and increased the protein ratio in broiler chickens. Long-term physical activity that increased lipolysis and the muscle load had an important role in lipolysis. Lipolysis was more significant in active muscles than in inactive muscles, as reported by Schrauwen-Hinderling et al. [22]. The increased protein:fat ratios of the total carcass and thigh meat in this study were similar to those of previous researchers [5, 21, 22].

In agreement with reports [23, 24] that physical activity has a significant role in cholesterol levels, especially HDL cholesterol levels, lowering the stocking density reduced (P ≤ 0.05) serum total cholesterol and HDL cholesterol levels in the Al groups (Table 2Go). Although this finding indicated that HDL cholesterol decreased with a lower stocking density, the real reason could be related to the decrease in total cholesterol level. The lower stocking density reduced (P ≤ 0.05) the HDL cholesterol level only in the limit-fed Pf groups.

When fatty acid composition of chicken meat was examined (Table 3Go), it was found that SFA synthesis of the Pf groups caused the increase in the total SFA ratio in the meat (P ≤ 0.01). Previous researchers also reported that fatness had an effect on fatty acid composition of the meat. The SFA and monounsaturated fatty acids (MUFA) increased with increasing fatness [25, 26]. The increase in total SFA ratio of the Pf groups could be related to fatness of these groups. Apart from this, total polyunsaturated fatty acids (PUFA) and n-3 fatty acid ratios were significantly reduced (P ≤ 0.01) in the fattened Pf groups, except for the carcass PUFA ratio. As shown in Table 3Go, the lower stocking density had a variable effect in both feeding regimens. It decreased (P ≤ 0.01) the total SFA and MUFA ratios of chicken meat and increased (P ≤ 0.01) the total PUFA, n-3, and n-6 fatty acid ratios in the Al groups. Similarly, Castellini et al. [5] found that total PUFA and n-3 ratios were significantly increased by an organic production system compared with a conventional system in broilers. However, parallel to lowering the stocking density, total SFA and MUFA ratios were found to increase and total PUFA, n-3, and n-6 fatty acid ratios were found to decrease in the Pf group, except for the SFA ratio of breast meat and the MUFA ratio of thigh meat. These findings might be related to the adaptation of muscle homeostasis to the physical load. From this aspect, membrane composition, and its adaptation mechanism, is of very high importance and is basically influenced by its fatty acid profile, especially by the long-chain PUFA [27]. The lower stocking density was thought to increase the muscle load under the stress of feed restriction and to decrease total PUFA in the limit-fed Pf groups. Significant interactions between density and feeding were obtained in the fatty acids of breast and thigh meat (P ≤ 0.01). In addition to these findings, significant results were focused on thigh meat, breast meat, and the total carcass, respectively (Table 3Go). This finding was in accordance with the report of Szabo et al. [6], who suggested that the physical load of muscles had a significant role in fatty acid composition in rabbits.


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  		Table 3. Influence of different stocking densities and feeding regimens on fatty acid compositions of total carcass, breast, and thigh meat in broiler chickens
 

   CONCLUSIONS AND APPLICATIONS
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
REFERENCES AND NOTES
 
  1. Feeding after a certain period of hunger caused fat deposition in broilers. It increased the SFA ratio in chicken meat and decreased the total PUFA and n-3 fatty acid ratios.
  2. A lower stocking density provided more movement space for chickens; this decreased the fat ratio of meat and increased the protein ratio.
  3. The total PUFA and n-3 and n-6 fatty acid ratios increased with a lower stocking density in the Al feeding regimen. In the Pf groups, the reverse was the case, in which there was feed restriction stress.
  4. For high-quality broiler meat production with a high ratio of unsaturated fatty acids, the following are recommended: no stimulating hormonal mechanism, an Al feeding or limited feeding program with an increased number of meals in a way that does not leave broilers hungry for a long time, and reduced animal numbers per unit area.


   ACKNOWLEDGMENTS
 
This study was supported financially by a grant (106 O 431) from the Scientific and Technical Research Council of Turkey (TUBITAK).


   REFERENCES AND NOTES
TOP
 SUMMARY
 DESCRIPTION OF PROBLEM
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS AND APPLICATIONS
 REFERENCES AND NOTES
 

  1. Saleh, E. A., S. E. Watkins, A. L. Waldroup, and P. W. Waldroup. 2005. Effects of early quantitative feed restriction on live performance and carcass composition of male broilers grown for further processing. J. Appl. Poult. Res. 14:87–93.[Abstract/Free Full Text]
  2. Dozier, W. A., J. P. Thaxton, J. L. Purswell, H. A. Olanrewaju, S. L. Branton, and W. B. Roush. 2006. Stocking density effects on male broilers grown to 1.8 kilograms of body weight. Poult. Sci. 85:344–351.[Abstract/Free Full Text]
  3. Farmer, L. J., G. C. Perry, P. D. Lewis, G. R. Nute, J. R. Piggott, and R. L. S. Patterson. 1997. Responses of two genotypes of chicken to the diets and stocking densities of conventional UK and label rouge production systems. II. Sensory attributes. Meat Sci. 47:77–93.[CrossRef]
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  5. Castellini, C., C. Mugnai, and A. Dal Bosco. 2002. Effect of organic production system on broiler carcass and meat quality. Meat Sci. 60:219–225.[CrossRef]
  6. Szabo, E., R. Romvari, and F. Hedving. 2005. Trainning-induced alteration of the fatty acid profile of rabbit muscle. Acta Vet. Hung. 50:357–364.[CrossRef]
  7. AOAC. 1990. Official Methods of Analysis. 15th ed. Assoc. Off. Anal. Chem., Arlington, VA.
  8. Turkish Standards Institution. 1974. TS.1744: Detection of total fat amount in meat and meat products. Turkish Stand. Inst., Ankara, Turkey.
  9. Hara, A., and N. S. Radin. 1978. Lipid extraction of tissues with a low-toxicity solvent. Anal. Biochem. 90:420–426.[CrossRef][Web of Science][Medline]
  10. Christie, W. W. 1992. Gas Chromatography and Lipids. The Oily Press, Glasgow, Scotland.
  11. 17A Version 3 gas chromatograph with a flame-ionization detector and AOC-20s autoinjector and autosampler, Shimadzu, Kyota, Japan.
  12. 25 m long, 0.25 µm i.d., with a 25-µm Permabond film thickness, Machery-Nagel, Düren, Germany.
  13. Katsanidis, E., and P. B. Addis. 1999. Novel HPLC analysis tocopherols and cholesterol in tissue. Free Radic. Biol. Med. 27:1137–1140.[CrossRef][Web of Science][Medline]
  14. 15 x 4.6 cm, 5 µm i.d., Sigma, St. Louis, MO.
  15. Lot no. 4577, Olympus Diagnostica GmbH, O’Callaghan’s Mills, Co. Clare, Ireland.
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  19. Donaldson, W. E., V. L. Christensen, and K. K. Krueger. 1991. Effects of stressors on blood glucose and hepatic glycogen concentrations in turkey poults. Comp. Biochem. Physiol. 100:945–947.[Medline]
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  21. Simsek, U. G., B. Dalkilic, M. Ciftci, I. H. Cerci, and M. Bahsi. 2009. Effects of enriched housing design on broiler performance, welfare, chicken meat composition and serum cholesterol. Acta Vet. Brno 78:67–74.[CrossRef][Web of Science]
  22. Schrauwen-Hinderling, V. B., L. J. C. Van Loon, R. Kopman, K. Nicolay, W. H. M. Saris, and M. E. Kooi. 2003. Intramyocellular lipid content is increased after exercise in nonexercising human skeletal muscle. J. Appl. Phys. 95:2328–2332.[Abstract/Free Full Text]
  23. Stucchi, A. F., A. H. Terpstra, T. L. Foxall, R. J. Nicolosi, and S. C. Smith. 1991. The effect of exercise on plasma lipids and LDL subclass metabolism in miniature swine. Med. Sci. Sports Exerc. 23:552–561.[Web of Science][Medline]
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