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South African Journal of Animal Science

On-line version ISSN 2221-4062
Print version ISSN 0375-1589

S. Afr. j. anim. sci. vol.45 n.5 Pretoria  2015




Received 25 July 2014
Accepted 2 April 2015
First published online 16 May 2015



# Corresponding author:

^rND^sAlvarez^nI.^rND^sDe La Fuente^nJ.^rND^sCaneque^nV.^rND^sLauzurica^nS.^rND^sPérez^nC.^rND^sDiaz^nM.T.^rND^sCooper^nR.G.^rND^sHorbańczuk^nJ.O.^rND^sCosgrove^nJ.P.^rND^sChurch^nD.F.^rND^sPryor^nW.A.^rND^sDalle Zotte^nA.^rND^sBrand^nT.S.^rND^sHoffman^nL.C.^rND^sSchoon^nK.^rND^sCullere^nM.^rND^sSwart^nR.^rND^sDíaz^nM.T.^rND^sCañeque^nV.^rND^sSánchez^nC.I.^rND^sLauzurica^nS.^rND^sPérez^nC.^rND^sFernandez^nC.^rND^sÁlvarez^nI.^rND^sDe la Fuente^nJ.^rND^sFarouk^nM.M.^rND^sSwan^nJ.E.^rND^sFilgueras^nR.S.^rND^sGatellier^nP.^rND^sZambiazi^nR.C.^rND^sSanté-Lhoutellier^nV.^rND^sFolch^nJ.^rND^sLee^nM.^rND^sSloane Stanley^nG.H.^rND^sGirolami^nA.^rND^sMarsico^nI.^rND^sD'Andrea^nG.^rND^sBraghieri^nA.^rND^sNapolitano^nF.^rND^sCifuni^nG.F.^rND^sHoffman^nL.C.^rND^sHoffman^nL.C.^rND^sJoubert^nM.^rND^sBrand^nT.S.^rND^sManley^nM.^rND^sHoffman^nL.C.^rND^sJones^nM.^rND^sMuller^nN.M.^rND^sJoubert^nE.^rND^sSadie^nA.^rND^sHorbańczuk^nJ.^rND^sSales^nJ.^rND^sCeleda^nT.^rND^sKonecka^nA.^rND^sZiba^nG.^rND^sKawka^nP.^rND^sHorbańczuk^nJ.O^rND^sTomasik^nC.^rND^sCooper^nR.G.^rND^sLeygonie^nC.^rND^sBritz^nT.J.^rND^sHoffman^nL.C.^rND^sLeygonie^nC.^rND^sBritz^nT.J.^rND^sHoffman^nL.C.^rND^sMajewska^nD.^rND^sJakubowska^nM.^rND^sLigocki^nM.^rND^sTarasewicz^nZ.^rND^sSzczerbinska^nD.^rND^sKaramucki^nT.^rND^sSales^nJ.^rND^sPotawska^nE.^rND^sLisiak^nD.^rND^sJózwik^nA.^rND^sPierzchata^nM.^rND^sStrzatkowska^nN.^rND^sPomianowski^nJ.^rND^sWójcik^nA.^rND^sPoawska^nE.^rND^sHorbaczuk^nJ.^rND^sPierzchaa^nM^rND^sStrzakowska^nN.^rND^sJózwik^nA.^rND^sWójcik^nA.^rND^sPomianowski^nJ.^rND^sGutkowska^nK.^rND^sWierzbicka^nA.^rND^sHoffman^nL.C.^rND^sSales^nJ.^rND^sHorbaczuk^nJ.^rND^sSantos-Filho^nJ.M.^rND^sMorais^nS.^rND^sRondina^nD.^rND^sBeserra^nF.^rND^sNeiva^nJ.N.^rND^sMagalhaes^nE.F.^rND^sZymon^nM.^rND^sStrzetelski^nJ.^rND^sPustkowiak^nH.^rND^sSosin^nE.^rND^1A01^nC.W.^sCruywagen^rND^1A01^nT.^sCalitz^rND^1A01^nC.W.^sCruywagen^rND^1A01^nT.^sCalitz^rND^1A01^nC. W^sCruywagen^rND^1A01^nT^sCalitz

In vitro degradation of melamine by ruminal microorganisms



C.W. Cruywagen#; T. Calitz1

Department of Animal Sciences, Stellenbosch University, Private Bag X1, Matieland 7602, South Africa




An in vitro study was conducted to determine the extent of melamine degradation in rumen liquor. Rumen liquor was collected from two ruminally cannulated Holstein cows on four separate dates, one week apart. Erlenmeyer flasks (250 mL) were prepared for incubation by adding 1000 mg of a dairy feed substrate, 100 mg melamine and 100 mL incubation medium, purged with CO2 and fitted with rubber stoppers equipped with one-way gas release valves. The initial melamine concentration was thus 1000 mg/L. The substrates consisted of 600 mg of a commercial dairy concentrate, 200 mg lucerne hay and 200 mg oat hay. The incubation medium consisted of 19 mL rumen liquor, 77 mL of Van Soest buffer and 4 mL of a reducing solution. The flasks were incubated at 39 °C for 0, 6, 24 or 48 hours (two flasks per time in each of four replicates). The 0 h incubation served as a control treatment to enable the calculation of melamine recovery values. For the control treatment (0 h), fermentation was terminated at the onset of the trial by aerating the rumen liquor and submerging the flasks in 50 mm ice. On termination of the incubation, 100 mL 0.2 M perchloric acid was added to each flask in order to dissolve any undegraded melamine. Melamine concentrations were determined by liquid chromatography-tandem mass spectrometry. Melamine degradation was low after 6 hours and 24 hours of incubation (3.2% and 5.5%, respectively) and increased to 13.6% after 48 h of incubation. It was concluded that melamine has low degradability in rumen liquor.

Keywords: Non-protein nitrogen, rumen fermentation, rumen incubation




The industrial chemical melamine (1,3,5-triazine-2,4,6-triamine) is used in manufacturing plastic ware, laminates and paints. Pure melamine has a high nitrogen content (667 g/kg; Merck, 2001), which theoretically equates to a crude protein content (N x 6.25) of 4167 g/kg. This high nitrogen content makes it an attractive adulterant for protein feedstuffs, as demonstrated during the 2008 worldwide pet food recalls and the 2009 tainted infant formula incidents in China.

Following reports by Loosli et al. (1949) that ruminants are able to utilize non-protein nitrogen (NPN) sources to produce essential amino acids, Colbey & Mesler (1958) patented the use of melamine as an NPN source for ruminants. By recognizing the high nitrogen content of melamine and its structural similarities to cyanuric acid, which, according to Clark et al. (1965), proved to be a safe NPN source for sheep, MacKenzie (1966) investigated the potential use of melamine as an NPN source for ruminants. He concluded that melamine was an inefficient NPN source owing to a reduction in apparent nitrogen digestibility, reduced feed intake and the inexplicable deaths of five sheep that were fed 10 g melamine per day. Newton & Utley (1978) found that melamine increased in vitro rumen ammonia concentration, but reported that the rate of melamine hydrolysis in the rumen was insufficient to promote maximum ruminal protein synthesis and concluded that melamine was not an acceptable NPN source for ruminants.

According to Cruywagen et al. (2009), approximately 2% of melamine ingested by dairy cows was excreted via the milk. In a study with sheep to quantify the partitioning of absorbed dietary melamine to various tissues and excretion via faeces and urine, Cruywagen et al. (2011) found that 53.2% of ingested melamine was excreted via urine and 23.3% via faeces. They reported that the melamine residue in muscle tissue accounted for approximately 3.6% of ingested melamine. Of the balance of 18.6%, which was not accounted for, it was found that a small amount was partitioned to other organs and blood, while the authors speculated that the rest could have been degraded in the rumen.

Diversity in the bacterial population is extensive and complex (Russell & Hespell, 1981) with a wide range of substrate affinities. Newton & Utley (1978) reported increased rumen ammonia (NH3) concentrations with the addition of melamine via an in vitro trial. In addition, the in vitro metabolism of melamine to melamine analogues (e.g. ammeline, ammelide, cyanuric acid) by aerobic microorganisms such as Arthrobacter spp., Klebsiella terragena and Pseudomonas spp. was reported by Strong et al. (2002), Shelton et al. (1997) and Jutzi et al. (1982), respectively. Although neither the Arthrobacter spp. nor Klebsiella spp. was isolated from the rumen, Duncan et al. (1999) reported the presence of Pseudomonas aeruginosa in the rumen of sheep. The only documented study that was found on the kinetics of ruminal melamine degradation was that of Sun et al. (2012), who reported that 44.5% of ingested melamine disappeared from the rumen within 12 hours. They explained the disappearance by postulating that it was absorbed via the rumen wall or degraded by the rumen microbes. Their study did not take feed and water intake into account or melamine losses via passage of digesta from the rumen, which could have had a significant effect on ruminal melamine concentration.

The current in vitro study was therefore planned to quantify melamine disappearance in a closed system to exclude the confounding effects of a dynamic in vivo rumen environment.


Materials and Methods

Rumen liquor was collected from two ruminally cannulated lactating Holstein cows in four consecutive runs, one week apart. Cows weighed 719 ± 9.2 (SE) kg and were kept at Welgevallen Experimental Farm of Stellenbosch University. They had free access to oat hay and received 25 kg/d of a commercial semi-complete lactating cow diet (nutrient composition in Table 1). The trial was approved by Stellenbosch University's Research Ethics Committee: Animal Care and Use (ref 10LVCRU02).

Rumen liquor was collected just before the morning feeding at 07:00 and transferred to pre-heated 1L thermos flasks. Approximately 200 g solid material from the rumen was added to each flask. Flasks were filled to the brim before the lid was replaced to prevent aeration. In the laboratory, the rumen liquor of each thermos flask was blended separately in a commercial blender for 20 seconds under a continuous stream of CO2 to maintain anaerobic conditions. For each cow, 500 mL of the blended rumen liquor was filtered through four layers of cheesecloth into a 1L preheated Erlenmeyer flask to obtain a pooled sample. The flask was purged with CO2, fitted with a rubber stopper, and placed in a water bath at 39 °C. The complete procedure was followed in four separate runs to yield four replicates. The mean initial pH of the rumen liquor was 6.35 ± 0.02 (SE).

A series of eight 250 mL Erlenmeyer flasks was prepared for each set of in vitro incubations. Each flask contained 100 mg melamine (melamine 99%, Sigma-Aldrich, St. Louis, Mo), 1000 mg substrate and 100 mL incubation medium. The initial melamine concentration was thus 1000 mg/L. This significant concentration was decided on to ensure that any degradation of melamine would be readily detected. The substrate consisted of 600 mg of a commercial dairy concentrate (provided by Meadow Feeds, Paarl, South Africa), 200 mg lucerne hay and 200 mg oat hay in order to simulate a typical dairy ration of 60 : 40 concentrate to roughage ratio. All substrate ingredients (dairy concentrate, lucerne hay and oat hay) were ground with a laboratory hammer mill (Scientec, South Africa) through a 1 mm screen. Chemical analyses of the substrate ingredients were performed in duplicate according to AOAC (2002) methods for dry matter (DM) (method 934.041), ash (method 942.05), crude protein (CP) (method 990.03) and ether extract (method 920.39). The ANKOM Fiber Analyzer (ANKOM® Technology Corp., Macedon, NY, USA) was used to determine ADF and NDF. Heat-stable alpha-amylase and sodium sulphite were used in the assay. The nutrient composition of the substrate is presented in Table 1.

The incubation medium consisted of 19 mL prepared rumen liquor, 77 mL buffer solution and 4 mL reducing solution. The buffer and reducing solutions were prepared according to Van Soest & Robertson (1985). The flasks were purged with CO2, fitted with rubber stoppers that had one-way gas release valves, and incubated at 39 °C for 0, 6, 24 or 48 hours. In each of the four replicate runs, two flasks were incubated per time to allow for duplicate analysis per replicate. The 0 h served as control treatment to calculate melamine recovery. In this treatment, fermentation was terminated before the onset of the trial by aerating and submerging the Erlenmeyer flasks in 50 mm ice water. Fermentation of the incubated samples was terminated in the same way after the predetermined incubation times. On termination of fermentation, 100 mL 0.2 M perchloric acid was added to each Erlenmeyer flask to dissolve undegraded melamine. Representative rumen liquor samples were taken and stored in airtight containers at -20 °C pending melamine analysis.

An adapted method from Shai et al. (2008) was used for melamine analyses. Thawed rumen liquor samples were centrifuged at 4 500 x g for 5 min. The conditioning of the cation-exchange solid-phase extraction cartridges (Phenomenex Strata SCX; 55 μm, 70 A, 500 mg/3 mL, supplied by Separations, Randburg, South Africa) was done with 6 mL methanol, followed by 6 mL distilled water. The supernatants of the rumen liquor samples (3 mL) were loaded onto the cartridges, together with 100 μL of a 0.5 mg/L stable isotope-labelled melamine (13C3H615N3) internal standard solution (Cambridge Isotope Laboratories Inc., Andover, Mass). Therefore, 0.05 μg of the labelled melamine was loaded onto each cartridge. The cartridges were then washed with 6 mL 0.1 N HCl, followed by 6 mL methanol, and allowed to aspirate under vacuum for 1 min. The melamine was eluted with 6 mL ammonium hydroxide : methanol : dichloromethane (1 : 5 : 5) and collected into clean glass tubes. The resulting extracts were then dried under a stream of nitrogen, re-suspended with 1 mL 50% acetonitrile and transferred to individual vials for analysis. Samples were analysed for melamine by liquid chromatography-tandem mass spectrometry (LC/MSMS) using a Waters API Quattro micro triple quadruple mass spectrometer, coupled to a Waters 2690 HPLC (Waters Corp., Milford, Mass). For this method, the limit of detection for rumen liquor samples was 0.001 mg/kg.

Melamine recovery values were determined from the control (0 h) treatment (with an initial melamine concentration of 1000 mg/L) and found to be 99.4%. The 0 h value was adjusted to 100% (1000 mg/mL) and the concentrations of the incubated samples were adjusted accordingly by multiplying the values by 1.006.

A repeated measures ANOVA with the compound symmetry assumption on the correlation structure over time was applied to analyse responses, using Statistica 64 (2013). The following model was used:

Yij = μ + αi + B(i)j + εij,

where μ is the overall mean, ai the effect of the ith time of measurement, βΜ the effect of the jth cow (or run) at time i and ε0, the random error. The Bonferroni post-hoc procedure was used to discern between significant least square means, and significance was declared at P <0.05.


Results and Discussion

The change in ruminal melamine concentration over time is indicated in Figure 1. As incubation time increased, the melamine concentration decreased gradually. The melamine concentration at 48 h (864 mg/kg) was significantly (P = 0.004) lower compared with previous hours (1000, 963 and 928 mg/kg at 0, 6 and 24 h, respectively). Sun et al. (2012) investigated the kinetics of melamine disappearance from the rumen of dairy cows over 12 hours. They found that melamine concentration decreased exponentially, and that the value at 12 h post ingestion was approximately 50% of the 1 h value. They concluded that 44.5% of ingested melamine was absorbed via the rumen wall or degraded by rumen microbes after 12 hours. However, they did not take into account the passage of digesta from the rumen and feed and water intake, which may have had an effect on ruminal melamine concentration. To relate melamine concentration in a dynamic environment, such as the rumen, directly to degradation may result in an overestimation of degradation rates. In the current study, a closed in vitro system was used and the decrease in melamine concentration over time was probably because of microbial degradation alone.

The extent of melamine degradation (melamine disappearance expressed as % of initial melamine) in the current study is indicated in Figure 2. Initial degradation rate appeared to have been higher than later rates. By 48 h, the amount (%) of melamine that was degraded in rumen liquor (13.6%) was significantly (P = 0.004) higher than at 6 h (3.7%). However, to put these values in context under practical conditions, rumen retention time should be considered. When the accepted rumen passage rate of kp =.08 for lactating dairy cows is applied, the rumen retention time of a high concentrate feed would be 12.5 h (100/8), which would be the corresponding time point for the calculation of effective degradability. In the current study, samples were not incubated specifically for 12.5 h, but the effective degradability of melamine would be a value between 3.7% (6 h incubation) and 7.2% (24 h incubation). Results from the current study would therefore suggest that the effective degradability of melamine is very low at this dosage. The question may arise whether the significant amount of melamine added to the incubation medium (1 g/L) might have over-saturated the N supply and thus decreased degradability values. Taken into account that the melamine would have been mostly insoluble in the buffered medium (solubility is discussed below), that authors postulate that the availability of melamine N would have been low and probably not overwhelming.

Some degradation of melamine in rumen liquor was expected, as Jutzi et al. (1982) reported the hydrolytic cleavage of melamine into ammonia by Pseudomonas sp. strain A in vitro. The presence of Pseudomonas sp. strain A in rumen liquor is unclear. The production of ammonia following melamine cleavage (Jutzi et al., 1982) may also explain the increased ammonia concentration (although not significant) observed in the in vivo and in vitro trials of Newton & Utley (1978). Newton & Utley's (1978) in vitro study notes that higher ammonia concentrations were observed after 24 h of incubation when the melamine substrate was incubated in rumen liquor obtained from a steer-fed melamine compared with rumen liquor from a steer-fed cottonseed. This observation may infer that a period of adaptation may be required by rumen microbes in order to hydrolyse melamine more efficiently.

The estimated effective ruminal melamine degradability of between 3.7% and 7.2% may explain part of the 18% unaccounted for melamine observed in the trial of Cruywagen et al. (2011) who investigated excretion routes of melamine in sheep receiving a melamine-tainted concentrate supplement. McDonald (1981) reported that protein degradation is affected by retention time in the rumen and inversely related to passage rate. Furthermore, small feed particles pass through the rumen with the liquid phase via rumen contractions at a higher rate compared to larger particles (Poppi et al., 2000). This would also apply to melamine and, owing to the small particle size of melamine, it could be expected that the retention time in the rumen would be short and effective degradability very low.

Finally-, ruminal pH may affect melamine solubility and consequently its degradability. In the current study, pH levels stayed fairly constant over the incubation period. Initial pH of the rumen liquid was 6.35 ± 0.02 (SE), whereas the values in the incubation medium were 6.72 ± 0.04 (SE) at 6 h, 6.74 ± 0.05 (SE) at 24 h and 6.75 ± 0.05 (SE) at 48 h. According to Wiwanitkit & Wiwanitkit (2013) melamine is insoluble at a physiological pH (7.35 to 7.45) in the human body. Their determination of solubility was based on the canonical value (CV-CV), which is a variable that determines whether a molecule is soluble. When CV-CV > 0, the molecule would be soluble and when CV-CV < 0 it would be insoluble. These authors reported a series of CV-CV values for melamine that ranged between -0.22 and -0.26 when pH was increased from 6.4 to 8.4. In the current study, it can thus be accepted that melamine would have been mostly insoluble, which could explain the low degradability. Although information could not be found for the exact pH at which melamine becomes soluble, it is possible that when ruminal pH decreases (e.g. when feeding high concentrate diets), the chances of melamine becoming soluble increase. Even so, the relative short ruminal retention time of melamine that would be expected in dairy cows, together with the low solubility of melamine at pH > 6, would result in most of ingested melamine reaching the small intestine intact.



The results of the current trial showed that melamine has a low in vitro degradability in rumen liquor. Because the rumen liquor donor cows received melamine-free diets, the rumen microbes were not adapted to melamine in the substrate. It is speculated that degradability might have been somewhat higher, although still low, if the cows had been exposed to melamine-tainted feed over time. Melamine solubility in relation to pH levels and temperature of the liquid, as well as the availability of certain minerals such as potassium and carbonates, needs to be investigated. In addition, the ability of microbes that have adapted over time to deal with compounds such as melamine and the effect of melamine degradation on fermentation end products warrant further research.



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Received 11 November 2014
Accepted 18 April 2015
First published online 17 May 2015



# Corresponding author:
1 Present address: Tanqua Feeds, Riviersonderend

^rND^sClark^nR.^rND^sBarratt^nE.L.^rND^sKellerman^nJ.H.^rND^sCruywagen^nC.W.^rND^sStander^nM.A.^rND^sAdonis^nM.^rND^sCalitz^nT.^rND^sCruywagen^nC.W.^rND^sVan de Vyver^nW.F.J.^rND^sStander^nM.A.^rND^sDuncan^nS.H.^rND^sDoherty^nC.J.^rND^sGovan^nJ.R.W.^rND^sNeogrady^nS.^rND^sGalfi^nP.^rND^sStewart^nC.S.^rND^sJutzi^nK.^rND^sCook^nM.^rND^sHutter^nR.^rND^sLoosli^nJ.K.^rND^sWilliams^nH.H.^rND^sThomas^nW.E.^rND^sFerris^nF.H.^rND^sMaynard^nL.A.^rND^sMacKenzie^nH.I.^rND^sMcDonald^nI^rND^sNewton^nG.L.^rND^sUtley^nP.R.^rND^sPoppi^nD.P.^rND^sFrance^nJ.^rND^sMcLennan^nS.R.^rND^sRussell^nJ.B.^rND^sHespell^nR.B.^rND^sShelton^nD.R.^rND^sKarns^nJ.S.^rND^sMcCarty^nG.W.^rND^sDurham^nD.R.^rND^sStrong^nL.C.^rND^sRosendahl^nC.^rND^sJohnson^nG.^rND^sSadowsky^nM.J.^rND^sWackett^nL.P.^rND^sSun^nP.^rND^sWang^nJ.Q.^rND^sShen^nJ.S.^rND^sWei^nH.Y.^rND^sWiwanitkit^nS.^rND^sWiwanitkit^nV.^rND^1A01 A02^nT.^sRaphulu^rND^1A01^nC.^sJansen van Rensburg^rND^1A01^nJ.B.J.^svan Ryssen^rND^1A01 A02^nT.^sRaphulu^rND^1A01^nC.^sJansen van Rensburg^rND^1A01^nJ.B.J.^svan Ryssen^rND^1A01 A02^nT^sRaphulu^rND^1A01^nC^sJansen van Rensburg^rND^1A01^nJ. B. J^svan Ryssen

Assessing nutrient adequacy from the crop contents of free-ranging indigenous chickens in rural villages of the Venda region of South Africa



T. RaphuluI, II; C. Jansen van RensburgI, #; J.B.J. van RyssenI

IDepartment of Animal and Wildlife Sciences, University of Pretoria, Pretoria 0002, South Africa
IIDepartment of Agriculture Limpopo, Mara Research Station, P/Bag X2467, Makhado 0920, South Africa




The aim of the study was to evaluate the nutritional status of scavenging chickens by assessing the composition of their crop contents. The study was conducted on 288 free-ranging indigenous chickens from six adjacent rural villages in Venda region of South Africa over three seasons (autumn, winter and spring). The chickens consumed grains, kitchen waste, seeds from the environment, plant materials, worms and insects, and some undistinguishable materials. Household waste accounted for 78.6%, 91.1% and 75.8% and materials of animal origin, including insects and worms, accounted for 7.4%, 10.4% and 16% of the crop content in autumn, winter and spring, respectively. Grains and kitchen waste consumption and macro- and micro-nutrient concentrations varied with season. The crude protein (CP) level of the crop contents of adult chickens in all seasons and the calcium and phosphorus levels in winter corresponded with the requirements of poultry for maintenance and growth, but not egg production. Supplementation of CP to young birds in all seasons and calcium and phosphorus in autumn and spring might be necessary to improve their growth. Concentrations of copper, manganese, zinc and cobalt were above the requirements of poultry, but below their maximum tolerance levels (MTL). Iron concentrations ranged from 2907 mg/kg DM to 6424 mg/kg DM, which are well above MTL, suggesting potential detrimental effects on the birds if the iron in the crop contents is bioavailable. Aluminium concentrations ranged from 2256 mg/kg DM to 4192 mg/kg DM, though aluminium is considered non-toxic. It was concluded that the birds would not suffer from micro-mineral deficiencies, and that a risk of toxicity would depend on the bioavailability of the consumed element.

Keywords: Chemical composition, heavy metals, household waste, nutritional status




In rural communities of Vhembe district in Venda region of South Africa (hereafter Vhembe), poultry production is based on traditional scavenging systems at household level. A local eco-type, the Venda chicken, is the predominant type of chicken. It is adapted to the production system and is an efficient converter of scavengeable feed resources into eggs and meat (Fourie & Grobbelaar, 2003). An improvement in indigenous chicken production in the region could increase the access of rural communities to quality protein in the form of meat and eggs. This should contribute to their health and socio-economic wellbeing. However, any advances in the productivity of free-ranging indigenous chickens would require close attention to nutritional, breeding and health aspects.

In recent studies, the growth potential of the Venda chicken (Norris et al., 2007), and its protein (Mbajiorgu et al., 2011) and metabolizable energy (Alabi et al., 2013) requirements have been investigated under confined optimal management conditions. However, traditional scavenging systems are low input methods in which the production of the animal is usually adjusted to feed availability, implying that the producer has to rely on what is locally and seasonally obtainable (Schiere & De Wit, 1993). Rashid et al. (2004) pointed out that if the capacity of the scavenging feed resource base and seasonal variations are known, more efficient strategies for improved production of scavenging chickens could be developed, though it is impossible to formulate diets without reliable values for nutrient requirements, nutrient composition and daily intake of available feed (Roberts & Gunarantne, 1992).

In many studies worldwide, the physical and nutrient compositions of crop contents have been investigated to obtain information about the feed ingredients that scavenging chickens take in and the nutrient composition of what they consume (Sonaiya, 2004; Goromela et al., 2008; Mekonnen et al., 2010). From this information, the nutritional status of the birds can be estimated, based on the assumption that if the concentration of a nutrient in crop contents indicates a deficiency, the chickens are likely to consume insufficient quantities of that nutrient. It would therefore be possible to obtain reliable information about possible nutrient deficiencies and excesses in the diets. Based on this, recommendations could be made for supplementing nutrients to overcome apparent nutritional problems, which could be implemented if this is feasible under that subsistence husbandry condition.

From investigations of the crop contents of chickens under free-ranging management conditions in rural villages, the general conclusion is that the nutrient composition of crop contents varies widely with season, climatic conditions and locality (Rashid et al., 2004; Sonaiya, 2004; Goromela et al., 2008; Mekonnen et al., 2010). Results from one study cannot be extrapolated to another situation.

Few, if any, studies have been conducted in terms of evaluating the composition of the nutrient intake of free-ranging Venda-type chickens in the rural communities of Vhembe. Such information would assist in identifying possible constraints that inhibit the productivity of the chickens, and could lead to the addition of dietary supplements to meet the nutritional requirements of these chickens and improve their output. The objective of the present study was to establish the nutrient composition of the feed consumed by free-ranging indigenous chickens in Vhembe in various seasons to assess the nutrient adequacy of their diets. Special attention was given to the concentration of mineral elements in their feed, an aspect that has not been covered by similar studies in which the assessment of nutrient adequacy was based on the composition of crop contents.


Materials and Methods

The study was approved by the Animal Use and Care Committee of the University of Pretoria (EC008-08). Two hundred and eighty eight free-ranging indigenous chickens, predominantly of the Venda type (Grobbelaar et al. 2010), were randomly purchased from six adjacent rural villages, namely Tshifudi, Tshidzini, Tshamutshedzi, Tshivhilwi, Tshitereke and Makhuvha, which are situated at latitude S22°48 to S22°53 and longitude E30°28 to E30°42 in Thulamela Municipality, Vhembe, Limpopo, South Africa. Vhembe is regarded as a tropical (humid) region with an average annual rainfall of 820 mm. The peak rainfall occurs in January - February (an average of more than 184 mm per month), while less than 20 mm occurs monthly in the winter period (Mpandeli, 2014). The birds were collected over three seasons (autumn: April; winter: July; and spring: October), 96 chickens per season. Eight young chickens (four males and four females), 10 -16 weeks old, four mature cockerels and four mature hens that have had at least one laying cycle were purchased from each of the six villages. Summer was not included in the study as birds are kept indoors during this period to prevent them from scavenging newly planted crops. The main crops in the area are maize, groundnuts and vegetables.

The birds were caught while scavenging between 14:00 and 17:00, weighed and immediately killed humanely by cervical dislocation. They were eviscerated and the crop contents were collected. The crop contents were categorised visually into eight main components, as presented in Table 1, weighed and oven-dried for 48 h at 60 °C. To measure the chemical composition, the dried crop contents were pooled according to class of bird (sex and age group) and season. Villages constituted the six replications. Dried crop contents were milled to pass through a 0.5 mm screen.

Dry matter (DM), ash, moisture and ether extract (EE) levels were obtained according to AOAC (2000) procedures. Crude protein (CP) was determined by the Dumas combustion procedure (Leco CNS 2000; Leco, St. Joseph, Mich, USA) and crude fibre (CF) with Fibre-Tech apparatus (Robertson & Van Soest, 1981). Calcium (Ca), copper (Cu), iron (Fe), manganese (Mn), zinc (Zn), cobalt (Co), vanadium (V), cadmium (Cd), lead (Pb), chromium (Cr) and aluminium (Al) analyses were done with a Perkin Elmer Atomic Spectrophotometer (Giron, 1973). Phosphorus (P) analysis was carried out with the Spekol 1300 apparatus using the spectrophotometric method (AOAC, 2000). Nitrogen-free extracts (NFE) was calculated by difference as: 1000 - (CP + CF + Ash + EE + moisture). An indirect method of Wiseman (1987) was used to calculate true metabolizable energy (TME) as TME (kcal/kg DM) = 3951 + 54.4 EE% - 88.7 CF% - 40.8 ash%. Apparent metabolizable energy (AME) was calculated by assuming that TME is 8% (Wiseman, 1987) higher than AME. The conversion factor of 238.85 kilocalorie (kcal) equivalents to 1 megajoule (MJ) was used to convert kcal to MJ.

Statistical significance between carcass weight and physical and chemical composition of dry crop contents between treatments (bird age group, sex and season) was determined by an analysis of variance with the GLM model of SAS Institute (SAS, 2010). The following model:

Yijki = μ + Si+ Aj + Xk + (SA)ij + (SX)ik + (AX)jk + Eijkl

was employed and a 5% significant level was used, where Yijk is an observation for a given variable; μ is the general mean common to all observations; Si is the effect due to ith season; Aj is the effect due to the jth age class of chicken; Xk is the effect due to the kth effect of the sex group of chickens; (SA)ij is the interaction effects between the ith season and jth age; (SX)ik is the effects between the jth age and kth sex class; (AX)jk is the effects between the jth age and kth sex group and EUkl is the random error. Differences among means were determined by the least significant difference (LSD) procedure of SAS (2010).



The physical components of the crop contents of the chickens are presented in Table 1.

The household (HH) waste of plant origin could be attributed to kitchen leftovers (porridge and its waste products), maize by-products (bran and maize meal) and brewers' waste from local traditional beer. Dry maize grains are processed at the nearest mill (threshing) or manually to produce maize meal for human consumption, while winnowing and drying are done at the homesteads. The major part of kitchen waste of plant origin was porridge, which is the staple meal of the community.

Percentages of the components of dry crop contents for each season are indicated in Table 2. The main components of the crop contents varied (P <0.05) with season, except for seeds from the environment, grit and undistinguishable materials (P >0.05). There were good supplies of grains in autumn and HH waste of plant origin in spring and winter. The proportion of grains and HH waste of both animal and plant origin to total crop contents varied with season (P <0.05). The presence of grains differed (P <0.05) between seasons, constituting 54.5%, 22.5% and 10.7% of the total in autumn, winter and spring, respectively. The decrease in grains was accompanied by an increase in HH waste of plant origin. The HH waste of plant origin differed (P <0.05) between seasons. Kitchen waste was highest in spring (46.9% HH waste of plant origin), while maize by-products and brewers' waste were highest in winter. The proportion of HH waste of animal origin was highest in winter. Season by age interaction influenced HH waste of animal origin (P <0.05). Some sunflower seeds were observed in the crop contents of birds in autumn when sunflowers were harvested. There was no effect of age, sex and age x sex interaction (P >0.05) on the proportion of the main components of the crop contents. Season x age interaction (P <0.05) was observed for HH waste of animal origin. Adult chickens consumed more HH waste of animal origin (12.28% dry crop content) in winter than growers in autumn (1.42%) and spring (3.98%). Adult chickens tended to consume more HH waste of plant and animal origin and less grains, plant materials, worms and insects than growers. However, in general, differences between age and sex groups were negligible for type of crop component.

Bodyweight of the chickens at slaughter varied with season, age and sex (P <0.05). Adult males had a slaughter weight of 2067 g and adult females a slaughter weight of 1713 g. The heaviest chickens were recorded in winter and the lightest in spring. Weight at slaughter and chemical composition of the crop contents are summarised in Tables 3 and 4.

The NFE and ash levels of the crop contents varied with season (P <0.05) (Table 3). A CP level of 130 g/kg DM (P >0.05) of the crop contents was observed in winter, followed by 118 g CP/kg DM and 113 g CP/kg DM in spring and autumn, respectively. Ash content differed with season (P <0.05), with the lowest ash content being observed in autumn and the highest in spring. Season did not influence (P >0.05) CF and EE levels, but differences (P <0.05) between seasons were recorded for AME and NDF. No significant differences were observed on the effect of age and sex on the chemical composition of the crop contents, except that age (Table 4) had a significant effect on AME content (P <0.05). There was a tendency for the growing chickens to consume feed with a higher CP content and lower CF content than adults. Crop contents from adult birds had a higher (P <0.05) ash content than growers.

The Ca and P levels of the crop contents varied with season. In winter the Ca and P levels were higher (P <0.05) than in the other seasons (Table 5). Age by sex interaction had an effect on Ca in the crop contents (P <0.05). The crop contents of adult females, grower males, adult males and grower females had a Ca content of 11.7 g/kg DM, 7.47 g/kg DM, 6.52 g/kg DM and 3.90 g/kg DM, respectively.

The trace element concentrations of the crop content of the chickens as influenced by season are summarized in Table 5. Aluminium, Pb, Cd, V and Cr concentrations varied with season (P <0.05). The crop contents of Al and V in spring and winter were significantly (P <0.01) higher than those in autumn. Season did not influence Cu, Zn and Co concentrations, but between seasons Fe and Mn concentrations were different (P <0.05). Mean concentrations of the elements over all seasons were (mg/kg DM): Al, 3491; Cd, 0.29; Cr, 30.5; Co, 9.0; Cu, 12.1; Fe, 4049; Pb, 5.4; Mn, 85.6; V, 14.3 and Zn 60.8. Season x age and season x sex interactions on Pb were observed (P <0.05). In autumn, crop contents of growers contained more Pb (10.47 mg/kg DM) than adults (3.65 mg/kg DM), while those obtained from females contained more Pb (10.46 mg/kg DM) than from males (3.65 mg/kg DM).



The bodyweight of the adult birds of 2788 g ± 61.4 g compares well with the mature weight of 2819 g for male Venda chickens reported by Norris et al. (2007) in a growth study in which they were fed optimally to measure their growth curves. The birds in the present study might not all have been pure Venda chickens, but the comparison between adult weights suggests that they received sufficient nutrients to ensure that, at least, they did not suffer permanent stunting during the growth phase. In winter the birds were heavier than in the other seasons (Table 3). This could simply be because of variations in sampling, considering the limited numbers of birds per sex and age groups in each season.

Since the feed components in the crop at a specific time of day represent only a fraction of total DM intake per day, the concentrations of nutrients in the crop content can be used only as an indication of diet composition. In general, differences in chemical composition of crop contents between localities are the result of differences in climate, which are determined by type of vegetation and availability of feed in the environment (Ologbobo, 1990). However, in a review of studies in which the composition of crop contents was evaluated, Goromela et al. (2006) concluded that, on average, diets consumed by scavenging chickens were deficient in energy, protein, Ca and P. In the present study the amount of HH waste accounted for the major proportion of the total crop contents, with 78.6%, 91.1% and 75.8% in autumn, winter and spring, respectively. These results are in agreement with the findings of Goromela et al. (2008) and Rashid et al. (2005). An advantage of this high proportion of household waste is that the birds' owners can add supplements through this portion of the diet if deficiencies are identified.

Alabi et al. (2013) reported that diets containing 12.34 MJ ME/kg DM to 12.91 MJ ME/kg DM supported optimal feed intake, growth rate and feed conversion ratios during the starter and grower phase of Venda chicks under well-controlled management conditions. In the present study the high AME of 12.7 MJ/kg DM to 13.5 MJ/kg DM of crop contents suggests that the fowls had access to diets that were high in energy. These high energy levels are supported by the relatively low CF levels of <40 g CF/kg DM in the crop contents compared with 54 g CF/kg recommended by Gunaratne et al. (1993) for commercial hens. High NFE levels (590 to 649 g/kg), indicating soluble carbohydrates, were recorded (Table 3) and support the high ME levels in the crop content. However, since these high ME levels reflect the quality of the diet and not how much energy the birds consumed per day, it is not possible to conclude that these free-ranging chickens consumed sufficient energy to meet their requirements, especially since a large "activity increment" would probably have to be added to their energy requirements.

According to NRC (1994), the recommended levels of CP in diets for growing chickens (not broilers) range from 150 g/kg DM to 200 g/kg DM and for mature chickens from 100 g/kg DM to 160 g/kg DM. Mbajiorgu et al. (2011) raised Venda chickens in closed confinement from day old to 13 weeks old on diets containing 12.2 MJ ME/kg DM and varying levels of CP. The authors concluded that an energy : protein ratio of between 60 MJ ME/kg and 63 MJ ME/kg protein optimized feed intake, growth rate and feed conversion ratio for this type of chicken. In the present study, the CP levels of the crop contents ranged between 113 g/kg DM and 130 g/kg DM, and did not differ significantly between age groups, while the energy : protein ratio for both adults and growers was 109 MJ ME/kg DM crop contents. It appears therefore as if the birds did not receive adequate levels of dietary protein to support efficient production.

Although amino acid composition is a determining factor in assessing protein sufficiency of birds (Boisen et al., 2000), 7.4%, 10.4% and 16% of the HH waste were from animal origin, plus insects and worms (Table 2), assumedly with a well-balanced amino acid content (Mlcek et al., 2014). This could indicate that the mature birds received sufficient protein. Mwalusanya et al. (2002) recorded that the crop content of chickens in Tanzania contained 104 g CP/kg DM, and Mekonnen et al. (2010) reported a level of 129 - 150 g CP/kg in chickens in Ethiopia. In the present study the highest CP level was observed in winter, the driest season, when there is no abundance of insects and worms. This trend could be because of a higher consumption of household materials of animal origin in winter.

A problem with interpreting nutrient concentrations in crop contents is that the quantity of feed consumed is unknown. Total intake of a nutrient may be insufficient to meet the requirements of the birds, even if the concentration in the crop contents indicates adequacy. In addition, if insufficient quantities are ingested of a nutrient with the highest priority in the body, such as energy and protein, responses to the supplementation of other nutrients might be ineffective, except for those with antioxidant properties (Cronjeet al., 2006).

The ash content of the crop contents of 84, 120 and 139 for autumn, winter and spring, respectively, is well above recommended concentrations given in the NRC (1994). Since the percentage of grit in the crop contents was low, ranging from 0.15% to 1.8%, it is likely that dust and soil that could not be detected in the crop contents, contributed to this high ash content.

The crop contents of adult females, grower males, adult males and grower females had Ca levels of 11.7 g/kg DM, 7.57 g/kg DM, 6.5 g/kg DM and 3.9 g/kg DM, respectively. Total P levels of the crop contents were more constant (3.1 - 4.8 g/kg) and not affected by age, sex or season. Both Ca and total P contents in the crop were relatively low compared with NRC (1994) recommendations for layer- and meat-type poultry. A Ca concentration of approximately 8.8 g/kg DM for broilers and 35 g/kg DM for leghorn layers was recommended by Rama Rao et al. (2003) at low dietary phosphorus levels (3.5 g and 2.8 g of non-phytin P/kg feed, respectively). It is well documented that adult laying hens tend to select feedstuffs with higher levels of Ca than other chicken classes because of their higher Ca requirements to synthesize the eggshell (Payne, 1990; Mwalusanya et al., 2002). Since about 0.50 - 0.80 total P in plant feedstuffs is bound as phytate P (Steiner et al., 2007), which is poorly available to monogastric animals (Pointillart et al., 1984) it can be concluded that the chickens in this study had a low intake of P. Supplementation of Ca and P seems to be necessary to improve the nutritional status of the local chickens, which in turn should improve their productivity.

Identifying deficiencies in the diets of free-ranging chickens in rural communities is valuable in planning a strategy to overcome nutritional problems. However, in most cases the owners of these birds do not have the financial means to purchase supplements for them. Van Ryssen et al. (2014) demonstrated that wood ash is a good source of Ca, and can replace feed lime in the diets of broilers. Calcium supplementation is therefore a possibility in Vhembe, because wood ash is available from homestead fires. However, it is unlikely that pure ash would be consumed by the chickens, and would have to be mixed with other HH waste. In addition, caution when feeding homestead ash was expressed by Van Ryssen & Ndlovu (2003) because of impurities in it. A further problem is that the ideal Ca to non-phytin P ratio in the diet could be disrupted with wood ash supplementation.

The concentration of the transition elements Cu, Fe, Mn, Zn and Co did not vary significantly between seasons. The concentrations of these elements were well above the requirements of meat and leghorn chickens as reported by NRC (1994), and, except for Fe, were within the maximum tolerance level (MTL) as suggested by NRC (2005). The only concern is that high concentrations of Cu, Mn, Zn or Co might be antagonistic to elements that are low in the crop content. It is well documented that soil ingestion by animals can have a significant effect on trace mineral ingestion, implying that the elements in soil are bioavailable. Judson & McFarlane (1998) pointed out that in ruminants under normal grazing conditions ingested soil can be a source of mineral elements such as Fe, iodine (I) and Mn. Neser et al. (1997) found that young calves with a pre-ruminant, that is, a monogastric, digestive system consuming soil high in Mn, developed Mn toxicity owing to the accumulation of Mn in their livers. Suttle et al. (1984) demonstrated that when sheep consumed soils that are high in Fe, Cu absorption was suppressed. It can be assumed that these elements in soil were available to the birds to a certain extent, supplying their requirements.

Iron concentration varied from 2907 mg/kg DM to 6424 mg/kg DM in the crop contents. Suttle (2010) pointed out that all species have high tolerance towards dietary Fe. However, high dietary levels of Fe are antagonistic to the absorption of Cu (Suttle et al., 1984) and Mn (Suttle, 2010). The NRC (2005) concluded that Fe toxicity ranges from 500 mg/kg to 4500 mg/kg, depending on the bioavailability of the Fe source. According to Suttle (2010), tolerable Fe concentrations have been set for poultry at 1000 mg/kg. However, the author indicated that these levels depend on total available Fe intake. Where exogenous Fe sources have low relative biological values, such as in soil, tolerable levels would be much higher. Van Ryssen et al. (1993) recorded excessively high concentrations of Fe (16762 mg/kg DM) in the pure excreta of "backyard" chickens. These samples were collected in the Pietermaritzburg area of South Africa, but not from rural households. They concluded that the Fe must have originated mainly through the consumption of soil.

Prinsen Geerlings et al. (2003) suggested that food prepared in iron cooking pots could be used to overcome Fe deficiency in developing countries. However, in southern Africa there tends to be Fe overload in human beings in rural communities (Walker & Segal, 1999). In Vhembe three-legged cast iron pots are used extensively for cooking. Residues from food prepared in these pots could well be a major source of the high Fe concentration in the crop contents in the present study.

Aluminium concentrations in the crop contents varied from 2256 mg/kg DM to 4192 mg/kg DM. Aluminium is classified as non-toxic (Reilly, 1991), though in the summary of mineral tolerances in animals (NRC, 2005) studies are quoted in which the consumption of certain Al salts elicited toxic symptoms in chickens. A cautious maximum tolerance level of 1000 mg/kg DM was suggested by the NRC (2005). Van Ryssen et al. (1993) recorded a concentration of 9885 mg Al/kg in pure backyard excreta, and concluded that the Al originated from geophagia. Rao & Rao (1995) pointed out that Al contaminates food prepared in aluminium cooking pots, though in Vhembe the use of Al cooking pots is rare. The most likely source of Al in the crop contents is ingested soil, and it is probably not a health concern in free-ranging chickens because of an assumed low Al bioavailability.

The highest concentration of Pb (7.06 mg/kg) was obtained in autumn, but it was lower than the MTL of 10 mg/kg DM suggested by the NRC (2005). Vanadium concentration ranged between 8.36 mg/kg DM. and 18.37 mg/kg DM. Vanadium concentrations in winter and spring were higher than the MTL of 10 mg/kg DM in diets for poultry (NRC, 2005), which might affect egg quality. However, bioavailability of the V source often determines whether the V in the crop contents in winter and spring poses a risk to or a negative effect on the chickens. The mean concentration of Cd ranged between 0.22 mg/kg DM and 0.35 mg/kg DM, and the highest concentration was obtained in winter. Cadmium levels were found to be less than the permissible limit of 0.5 mg/kg DM (NRC, 2005), which indicates that Cd toxicity is not a risk to chickens in Vhembe.



Crop contents of chickens reflect not only the availability of nutrients in the environment, but also the selective feeding habits of birds, which are related to their nutritional requirements. However, crop nutrient contents at any time do not indicate total daily feed intake or utilization of nutrients. Therefore, any recommendation based on crop content of nutrients should take into consideration that crop content is only a guideline, because there are many other important factors such as scavenging area, foraging habit and density of the chickens.

From the concentration of nutrients in the crop contents of free-ranging chickens in Vhembe it could be concluded that the birds probably consume insufficient quantities of protein, Ca and P. It seems feasible to supplement Ca through adding wood ash to their diets, though caution should be taken not to upset the dietary Ca : available P ratio within a class of birds. Although the results suggest that supplementation of high-quality protein and available P would be beneficial to the birds, for practical and economic reasons it might be more difficult to achieve under these subsistence husbandry conditions.

The concentration of Al, Cu, Fe, Mn, Zn and Co in the crop contents were above the requirements of poultry, though below the MTL of the element, except for very high Fe and Al concentrations. It is suggested that most of these elements were obtained through ingesting soil and dust, and consequently would probably have a relatively low bioavailability in the bird, including Al, which is considered non-toxic. However, the high Fe concentrations might have also originated from cast iron pots used for cooking food in the region, and Fe from these pots could have accumulated in the feed consumed by the birds. There seems to be no need for the supplementation of trace minerals in the diets of the birds in the region, though further studies might be necessary to establish the bioavailability of the elements in the feed consumed by the birds.



The authors wish to acknowledge the support of National Research Foundation (NRF) for funding the research work. We are grateful to chicken farmers and Limpopo Department of Agriculture for their support.



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Received 11 September 2014
Accepted 15 April 2015
First published online 21 May 2015



# Corresponding author:

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Fatty acid profile, cholesterol and oxidative status in broiler chicken breast muscle fed different dietary oil sources and calcium levels



N.R. AbdullaI, II; T.C. LohI, III, #; H. AkitI; A.Q. SaziliI, IV; H.L. FooV, VI; R. MohamadV; R. Abdul RahimV; M. EbrahimiVII; A.B. SabowI, II

IDepartment of Animal Science, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
IIDepartment of Animal Resource, University of Salah al- Din, Erbil, Iraq
IIIInstitute of Tropical Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
IVHalal Products Research Institute, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
VDepartment of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Science, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
VIInstitute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
VIIDepartment of Veterinary Preclinical Sciences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia




The aim of the present study was to evaluate the effects of three feeds containing 6% oils: palm oil (PO), soybean oil (SO) and linseed oil (LO); and three calcium levels (NRC recommendation, 1.25% and 1.50%) on the fatty acid profile, lipid oxidation and cholesterol concentrations of broiler breast meat in a 3 χ 3 factorial experiment. A total of 378 one-day-old chicks were randomly assigned to the diets and fed for six weeks. Birds fed diet supplemented with LO, SO and PO had higher proportions of α-linolenic, linoleic and oleic acids, respectively. The LO diet increased the total n-3 fatty acids and decreased the n-6 : n-3 compared with the PO and SO diets. Birds fed the PO diet had higher oxidative stability and cholesterol compared with those fed the SO and LO diets. However, the level of cholesterol in all treatments was within the normal range. The level of calcium and interaction between source of oil and calcium level did not influence lipid oxidation, fatty acid profile and cholesterol level of broiler breast muscle. It can be concluded that dietary LO and SO enhanced n-3 and n-6 polyunsaturated fatty acids, respectively, while dietary PO enhanced the oleic acid and oxidative stability of broiler breast muscle. Thus, this study showed that PO can be used as an alternative oil source in broiler diets with a positive effect on the oxidative stability of chicken meat refrigerated at seven days when compared with vegetable oils that are rich in linoleic and α-linolenic acid.

Keywords: Chicken meat quality, dietary calcium levels, linseed oil, palm oil, soybean oil




Fat and fatty acids in muscle and adipose tissues are among the major factors that influence meat quality, particularly nutritional value and palatability (Coetzee & Hoffman, 2002). Changes in the dietary fatty acid (FA) composition could be reflected in the blood, which in turn would be transported to target organs such as muscle (Aghwan et al., 2014). Poultry meat is considered healthier owing to its relatively lower fat content compared with other animal meat (Brenes & Roura, 2010). Generally, the lipids of the muscle fibres contain a proportion of phospholipids, triacylglycerol and cholesterol (Pikul et al., 1984). The fatty acids of triacylglycerol are made up mainly of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA). Conversely, the fraction of phospholipid has a higher proportion of polyunsaturated fatty acids (PUFA), which is strictly controlled as a component of cellular membranes (De Smet et al., 2004). Red muscles reportedly contain a higher proportion of phospholipids than white muscles and thus a relatively higher amount of PUFA (Wood et al., 2004). However, a higher level of PUFA in muscle membranes is related to increasing susceptibility of meat and meat products to lipid oxidation (Adeyemi & Olorunsanya, 2012b). PUFA undergo rapid oxidative changes, which impair organoleptic characteristics, shorten meat shelf-life and produce off-flavours (Adeyemi & Olorunsanya, 2012a).

Manipulation of PUFA composition without affecting product quality has been a challenge for poultry scientists. Although poultry meat contains high levels of UFA, levels of natural antioxidants such as tocopherols are low (Melton, 1983). On the other hand, the current interest in increasing the n-3 PUFA content of meat and eggs increases the need for additional antioxidant protection. Methods that are effective, safe and of low cost for controlling storage stability of the poultry meat are extremely important to the industry. Recently there has been increased interest in the role of antioxidant nutrients owing to their health benefits in disease conditions such as cancer, coronary heart disease and immune functions (Bendich, 1990; Diplock, 1991). Additionally, the incorporation of vegetable oils that are rich in natural antioxidants has been suggested as an effective and economical means of controlling post-mortem lipid peroxidation and an alternative way of increasing these health-enhancing nutrients in human diets.

Several reports have shown that the level of dietary calcium affects the fatty acid profile of broiler chickens. Earlier studies (Atteh & Leeson, 1983; Ajakaiye et al., 2003) on chicken showed that dietary calcium can form calcium soaps with fatty acids, resulting in lower digestion and absorption of fat and calcium. Hence, supplementation of dietary oil in poultry diets calls for adequate dietary calcium to promote absorption and utilization of dietary fat and calcium and to forestall problems that may jeopardize the well-being of the birds and product quality. Therefore, the objective of this study was to determine the effect of palm oil (PO), soybean oil (SO) and linseed oil (LO) with three levels of calcium : phosphorus on fatty acid profile, lipid oxidation and cholesterol concentration in broiler breast meat.


Materials and Methods

This study was conducted according to the animal ethics guidelines of the Research Policy of University of Putra Malaysia.

A total of 378 one-day-old chicks (Cobb 500) were used for this study. On arrival, the chicks were individually wing banded, weighed and randomly assigned to nine treatment groups. Each group consisted of six replicates, and each replicate was made up of seven chicks. After seven days of the rearing period, all birds were vaccinated with IB-ND live vaccine against infectious bronchitis (IB) and Newcastle Disease (ND) through the intraocular route. The IBD vaccine against infectious bursal disease (IBD) was applied on day 14 of the rearing period through this route. The birds had free access to water and feed. The climatic conditions and lighting programme followed commercial recommendations. The environmental temperature in the first week of life was 35 °C and was then decreased to 28 °C until the end of the experiment. During the first week, 22 h of light were provided with a reduction to 20 h afterwards.

Two basal diets were formulated, namely starter and finisher. The starter diet was fed for the first three weeks, and the finisher was fed for the last three weeks of the experimental period. The compositions of the basal starter and finisher diets are shown in Tables 1 and 2, respectively. The diets contained three types of oil, namely LO, SO and PO as control group, and three levels of calcium : phosphorus, namely 1 : 0.5; 1.25 : 0.63; and 1.5 : 0.75 (NRC, 1994). Palm oil was used as control in this experiment because it is commonly used in poultry production in Malaysia. Diets were formulated to meet the requirements of all nutrients for broiler chickens using FeedLIVE software (FeedLIVE 1.52, Thailand).

At the end of the experiment, 12 birds per treatment (two birds from each cage) were randomly selected and weighed. The birds were slaughtered by neck decapitation, bled and processed. The right pectoralis major (breast) muscles were removed within 45 min of slaughter from all carcasses and separated into four sections. The first section was snap frozen in liquid nitrogen before being stored at -80 °C to determine the fatty acid profile and cholesterol. The other three sections were vacuum packed and kept in a chiller at 4 °C for thiobarbituric acid reactive substance (TBARS) evaluation on the 1st, 3rd and 7th day post mortem. After the ageing period, the muscle samples were snap frozen in liquid nitrogen and stored at -80 °C until further analyses.

The total fatty acids were extracted from breast muscles based on the method Folch et al. (1957), described by Loh et al. (2009), and modified by Ebrahimi et al. (2014), using chloroform : methanol (2 : 1, v/v) containing butylated hydrotoluene to prevent oxidation during sample preparation. The extracted fatty acids were transmethylated to their fatty acid methyl esters (FAME) using 0.66 N KOH in methanol and 14% methanolic boron trifluoride (BF3) (Sigma Chemical Co., St. Louis, Mo, USA) according to the methods by AOAC (1990). The FAME were separated by gas liquid chromatography on an Agilent 7890A GC system (Agilent, Palo Alto, Calif, USA) using a 100 m χ 0.25 mm ID (0.20 μm film thickness) Supelco SP-2560 capillary column (Supelco, Inc., Bellefonte, Phil, USA). One microlitre FAME was injected by an autosampler into the chromatograph, which was equipped with a flame ionization detector (FID). The carrier gas was He, and the split ratio was 10 : 1 after the FAME was injected. The injector temperature was programmed at 250 °C, and the detector temperature was 300 °C. The column temperature programme initially ran at 120 °C, held for 5 min, increased by 2 °C/min to 170 °C, held at 170 °C for 15 min, increased again by 5 °C/min to 200 °C, held at 200 °C for 5 min, then increased again by 2 °C/min to a final temperature 235 °C, and held for 10 min. The FA concentrations were expressed as percentage total identified FA. A reference standard (mix C4 - C24 methyl esters; Sigma-Aldrich, Inc., St. Louis, Mo, USA) and CLA standard mix (CLA cis-9 trans-11 and CLA trans-10, cis-12, Sigma-Aldrich, Inc., St. Louis, Mo, USA) were used to establish recoveries and correction factors to determine individual FA composition.

The determination of muscle cholesterol was carried out using the method described by Rudel & Morris (1973). A crushed meat sample (1 g) was homogenized with 3 mL 95% ethanol and 2 mL 50% potassium hydroxide. The homogenates were incubated in a water bath at 60 °C for 10 min following by cooling to room temperature. A 5 mL hexane was added to homogenates then mixed for 20 s. The final homogenate was raised with additional 3 mL deionised water and vortexed then allowed to settle at room temperature for 15 min to complete phase separation. About 2.5 mL of the upper phase (hexane layer) was transferred into a clean glass tube followed by evaporating the hexane to dryness under nitrogen gas flow at 60 °C. The residue was re-suspended with 4 mL o-phthalaldehyde reagent and kept at room temperature for 10 min followed by adding 2 mL concentrated sulphuric acid slowly, mixing and standing for an additional 10 min at room temperature. Cholesterol standards (Sigma L-4646) were prepared according to the method of Rudel & Morris (1973), to make final concentrations of 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg cholesterol/mL. The concentration of cholesterol and standard were tested at absorbance 550 nm using a spectrophotometer (Secomam, Domont, France).

Lipid peroxidation in muscle was determined using a malondialdehyde assay. Malondialdehyde is a secondary product of lipid peroxidation and is the major substrate in the TBARS test (Pryor, 1991). Measurements were taken on the 1st, 3rd and 7th day of ageing refrigerated at 4 °C. Lipid oxidation was measured using thiobarbituric acid-reactive substances (TBARS) according to the method of Lynch & Frei (1993), modified by Mercier et al. (1998). One gram meat sample was homogenized in 4 mL 0.15 M KCl + 0.1 mM BHT with Ultraturrax (1 min, 6000 rpm). After homogenization, 200 μL of the sample were mixed with TBARS solution and then heated in a water bath at 95 °C for 60 min until a pink colour developed. After cooling, 1 mL distilled water and 3 mL n-butyl alcohol were added to the extracts and homogenized. The mixture was centrifuged at 5000 rpm for 10 min. Absorbance of supernatant was read against an appropriate blank at 532 nm using a spectrophotometer (Secomam, Domont, France). The TBARS value was calculated from a standard curve of 1,1,3,3-tetraethoxypropane and expressed as mg malondialdehyde (MDA)/kg meat.

The experiment followed a three (sources of oil) by three (levels of calcium and phosphorus) factorial arrangement in a completely randomized design. The data obtained for fatty acids and cholesterol were analysed using the generalized linear model of SAS (SAS, 2007 ) while the data obtained for lipid oxidation was subjected to a generalized linear model with sampling time as a repeated measure. Significant differences between treatment means were compared using Duncan's multiple range test.


Results and Discussion

The fatty acid profile of experimental diets is shown in Tables 3 and 4. The supplementation of the diets with PO, SO and LO increased the C18:1n-9, C18:2n-6 and C18:3n-3, respectively. The ratio of n-6 : n-3 was in the order of PO > SO > LO.

The effects of dietary fat sources and calcium levels on the fatty acid composition of the chicken breast muscle are shown in Table 5. It was observed that the fatty acid composition of breast muscle reflected the fatty acid profile of the experimental diet. The major FA detected in the breast muscle and affected by the dietary oil were C18:1n-9, C18:2n-6 and C18:3n-3. The concentration of oleic acid (C18:1 n-9) in the breast meat of broilers fed PO (T1, T2, T3) diet was significantly higher than those groups fed SO (T4, T5, T6) and LO (T7, T8, T9) diets. This observation is similar to the findings of Htin (2006), which indicated that meat from broilers fed a diet containing PO had higher levels of C18:1n-9 compared with birds fed SO and fish oil. The proportion of palmitic acid (C16:0) increased in meat from broiler-fed PO in comparison with those fed diets supplemented with SO and LO. These results are in agreement with the findings of Htin (2006) and Smink et al. (2010), who reported that the levels of palmitic acid increased significantly in broiler breast muscle supplemented with palm oil compared with groups supplemented with SO, coconut oil and sunflower oil. The significant increase in the proportion of oleic and palmitic acids in birds fed the PO diet could be owing to the high oleic and palmitic acid content of PO. Birds fed LO and SO diets had significantly higher α-linolenic and linoleic acids, respectively, compared with those fed PO. The proportion of total saturated and total MUFA of meat samples increased, while total UFA content decreased when PO was incorporated in the diet, resulting in a significantly lower UFA : SFA ratio of the broiler breast muscle compared with those fed SO and LO diets. The highest proportion of total UFA and UFA : SFA ratio was found in birds fed the LO diet. This could be attributed to the high α-linolenic acid content in the fatty acid profile of LO. Regardless of the source of dietary oil, no significant differences were observed in most of the FA in the breast muscle among levels of calcium as well as interaction between source of oil and calcium level.

The n-6 : n-3 ratio differs significantly among sources of oil. Birds fed LO (0.87) had significantly lower n-6 : n-3 compared with PO (9.86) and SO (8.00). These results are in line with those of Crespo & Esteve-Garcia (2002), who found that muscle from broiler diet supplemented with LO had lower n6 : n3 ratio compared to the groups supplemented with tallow, olive and sunflower oil. Similar to present findings, Fébel et al. (2008) reported that birds fed LO had lower n6 : n3 ratio than the birds fed SO .

There are no significant differences between the level of calcium and interaction between fat source and calcium level of the ratio of omega 6 to omega 3 fatty acids of broiler breast (Table 5).

Cholesterol is an important molecule that plays a vital role in membrane structure, as well as being a precursor for the synthesis of molecules such as steroid hormones, vitamin D and bile acids (Dessi & Batetta, 2003; Loh et al., 2013). On the other hand, coronary heart disease and arteriosclerosis are strongly related to the dietary intake of cholesterol (Sacks, 2002). In addition, a strong relationship has been demonstrated between cellular cholesterol concentration and Alzheimer's disease (Michikawa, 2003). The cholesterol content of breast muscle of chickens fed diets containing different oil sources and calcium levels is shown in Table 6. Birds fed PO had higher (P <0.05) cholesterol concentration than those fed LO and SO. However, the cholesterol concentration was within the normal range (40 - 90 mg/100 g) for poultry (Piironen et al., 2002; Valsta et al., 2005; Honikel, 2010). This may be attributed to the decrease in the concentrations of UFAs in birds fed PO compared to those fed LO and SO (Table 5). Furthermore, the PUFA/SFA ratio was much higher in meat of broiler chickens fed SO and LO, which could explain the decrease in cholesterol. This observation agrees the reports of Duraisamy et al. (2013) and Azman et al. (2004), which showed that the level of cholesterol in broiler breast meat decreased when fed a diet containing more UFA rather than SFA. Regardless of the oil source, the concentration of meat cholesterol in the breast muscles of broilers was not significantly different among the calcium levels. Interaction between source of fat and calcium level was not significant.

Lipid oxidation is a major cause of food deterioration and affects the colour, flavour, texture and nutritional value of poultry. Incorporation of natural antioxidants by dietary means may be more effective and practical in controlling lipid-oxidation-related products and providing wholesome nutritious products to health-conscious consumers (Kang et al., 2001). The TBARS values expressed as mg MDA/ kg meat for breast muscles during post-mortem ageing periods of broiler chickens fed different dietary treatments are shown in Table 6. At d 1, 3 and 7 post mortem, a significant difference in lipid oxidation was observed among the dietary oils. Breast muscles from broilers fed a diet supplemented with PO had a lower TBARS value (P <0.05) compared with SO and LO throughout the post-mortem storage. This may be attributed to the decrease in the concentrations of PUFA in meat of birds fed PO, as shown in Table 3. The increase in lipid oxidation in muscle of birds fed SO and LO diet could be due to the increase in PUFA, which promotes formation of free radicals. This observation is in tandem with the report of Rey et al. (2001), Hoz (2004), Wood et al. (2004) and Hugo et al. (2009) which showed that oxidative stability of meat and meat products decreased with increasing concentrations of PUFA. On the other hand, the present result disagrees with the findings of Teye et al. (2006) and Isabel et al. (2003), which showed no significant effect of dietary fat source on TBARS values during storage. Irrespective of the oil source, there was no significant difference among the calcium : phosphorus levels for MDA concentration in the breast muscles of chickens at d 1, 3 and 7 post mortem. There was no significant interaction between oil source and calcium levels for MDA concentration in the breast muscles of chickens.

Generally, lipid oxidation increased with increase in post mortem storage at 4 °C. At d 3 and d 7 post mortem, the TBARS value increased significantly (P <0.05) in all treatment groups, but the increment was lower in birds fed PO compared with other oils (Table 6). These findings are in agreement with those of Coetzee & Hoffman (2001) and Adeyemi & Olorunsanya (2012b) for chicken, who observed that TBARS values of uncooked chicken meat increased gradually during post-mortem storage. However, the threshold value of TBARS (5 mg MDA/kg) for detecting off-odours and off-taste (Insausti et al., 2001) was not reached in the current study.



The results of the current study demonstrated that supplementation of PO, SO and LO increased the proportion of oleic, linoleic and α-linolenic acids, respectively, in broiler breast muscle. Birds fed LO had higher total n-3 and lower n-6: n-3 compared with birds fed PO and SO. Birds fed the PO diet had higher oxidative stability and cholesterol compared with those fed LO and SO. However, the values of cholesterol were within an acceptable range. Calcium level and interaction between calcium level and dietary oil were not significant for fatty acid profile, cholesterol and lipid oxidation. Thus, this study confirms that PO can be used as a vegetable oil in broiler chicken nutrition with positive effects on firmness of meat quality compared with vegetable oils that are rich in linoleic or α-linolenic acid. However, the use of PO in animal feeding may be restricted by its availability in other countries. Further studies on the effects of dietary oils on antioxidant enzyme activities in broiler chicken are suggested.



This project was supported by the Long-Term Research Grant Scheme (LRGS) from the Ministry of Education Malaysia.



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Received 7 January 2015
Accepted 9 March 2015
First published online 25 May 2015


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