Effects of Dietary Inclusion of Oil Sources With or Without Vitamin E on Body Composition and Meat Oxidation Level in Broilers*

The objective of this study was to investigate the effects of rapeseed oil substitution and vitamin E supplemantation on performance and meat quality. 4 replacement levels of rapeseed oil with sunflower oil ( 0, 33.3 , 66.7 and 100%) and 2 levels of vitamin E (0 (50 I.U. from vitamin premix) 300 mg/kg) in a 2 x 3 factorial arrangement for a total of 8 treatments with 4 replicates each, containing 10 day-old male Ross 308 broilers, were examined in the experiment. Dietary treatments had no significant effect on carcass parameters, feed conversion (FCR) and mortality (p>0.05). However, a significant interaction was observed between oil replacement and vitamin E: the negative effect of 100% sunflower oil on BWG (p<0.01) was alleviated by the addition of vitamin E. On the other hand, inclusion of rapeseed oil improved BWG (p<0.01). Rapeseed oil substitution significantly increased amount of total n-3 PUFA and decreased n-6:n-3 ratio in thigh and breast meat (p<0.01). Vitamin E supplemantation contributed to deposition of n-3 PUFA (p<0.01). Replacement of rapeseed oil without vitamin E tended to increase (p<0.01) malonaldehyde production. However, the dietary supplementation of vitamin E markedly (p<0.01) decreased the susceptibility of meat to peroxidation. Inclusion of rapeseed oil did not cause any negative perception on olfactory, texture, and taste of broiler meat. So, it can be concluded that rapeseed oil substitution significantly increased n-3 PUFA deposition without altering performance and sensory properties of broiler meat and, vitamin E had strong potential to prevent the meat lipids from oxidation.

Broiler chickens generally have a high capacity for lipid biosynthesis which has the propensity to become excessively fat. This fat accumulation has significant health implications, as carcass fat of birds is an important source of dietary fat for man (Newman et al., 2002). Therefore, the FA composition of broiler's thigh and breast muscle is an important quality parameter especially with respect to potentially affecting human health from poultry meat consumption. In this regard, n-3 PUFAs such as EPA, docosapentaenoic acid (DPA,, and DHA are some of the most important FA groups (Rahimi et al., 2011).
Fish (especially oily fish such as mackerel, herring, sardine, salmon, trout, and tuna), in spite of containing natural sources of EPA and DHA, has been rarely or never consumed by some people because of its olfactory problems, while demand for other meat productions such as chicken has been increasing (Molendi-Coste et al., 2011;Nyquist et al., 2013). It has been shown that FA composition of broiler meat can be altered by inclusion of different vegetable and fish oil sources in chicken's diet (Lopez-Ferrer et al., 2001a). Therefore, in order to supply human consumer´s diet with n-3 PUFAs and antioxidants such as vitamin E, methods must be utilized to enrich chicken´s diet with EPA and DHA or their precursor linoleic acid (LA; 18:2n-6). During the last four decades many studies have been conducted to manipulate FA profile of broiler muscle tissues in order to increase n-3 PUFA content and decrease n-6 : n-3 ratio (Miller & Robisch, 1969;Zduńczyk et al., 2011;Haug et al., 2011;Rahimi et al., 2011;Nyquist et al., 2013).
The following order shows the oxidative capacity of different meat types: fish > turkey > chicken > pork > beef > lamb. This ranking depends on the content of the PUFA in muscle tissues (Janssens et al., 1999). Negative effect of enriching meat with "good for health" PUFA results from these compond´s sensitivity to oxidaiton that produces "bad for health" pro-oxidant free radicals which may enhance the development of sensory problems. Oxidative damage increases during storage and cooking process based on the time and temperature (Cortinas et al., 2004;Barroeta, 2007;Roux et al., 2011). Inclusion of the vitamin E in broiler diet leads to increased vitamin E in the meat which improves membrane stability. This may boost meat and meat production´s resistance to peroxidation (Coetzee & Hoffman, 2001). That is why the addition of vitamin E has been suggested as a convenient method in improving oxidative stability and increasing the shelf life of meat (Coetzee & Hoffman, 2001;Barroeta, 2007). Diet manipulation or changes in feed processing stages, utilizing methods to decrease feed cost, and quality control of final products during development of new food products often demand sensory evaluation techniques to determine the products acceptability (Srinivassane, 2011). The sensory properties of n-3 FA enriched meat products should be assessed for consumer´s acceptance before introduction into the market. Sensory analysis provides a better understanding of consumer perception of food products (Srinivassane, 2011).
The objective of the current study was to determine the effects of dietary replacing levels of sunflower and rapeseed oils and supplemented or unsupplemented with α-tocopheryl acetate (as a commercial source of vitamin E) on the FA profile and thiobarbituric acid reactive substances (TBARS) level of thigh and breast chicken meat. The other aim of the present study was the determination of the acceptability of n-3 PUFA enriched chicken meat by sensory evaluation.

MATERIALS AND METHODS
All protocols for the experiment were reviewed and approved by the Local Animal Care and Use Committee of Ankara University.

Diet Preparation
Before preparation of the experimental diets, crude protein, crude ash, crude fat and dry matter of corn and soybean were determined (AOAC, 2000). The corn and soybean meal were ground in a mill with a three millimeter sieve for starter (0-3 weeks) periods. Vitamin and mineral premix, vitamin E, lysine, methionine and salt were mixed by a 3 kg capacity laboratory Lödige ® mixer at the department of Feed and Animal Nutrition of University of Ankara. During ration preparation at the Ankara University feed factory, the mixture of micro ingredients were mixed with a fine grined soybean meal. Then, this mixture was added to the corn and soybean meal blending in the mixer. Finally, the experimental oil was slowly poured into the mixer. After a 3-minute mixing period, the diet was packed in 40 kg sacks and stored at a dry place at room temperature (20-22°C) during the trial. The same procedure was followed in preparing the diet for grower-finisher (4-6 weeks) periods with only one difference in which the corn and soybean meal were ground in a mill with a fivemillimeter sieve. Experimental diet and the composition of starter and grower-finisher diets are shown in Table  1 and 2 respectively. The composition of the diets were adjusted according to the NRC (National Research Council, 1994) and met or exceeded the nutritional recommendations of Ross 308 breeders company catalog for broilers.
The dietary oil level was 6 and 7% for starter and grower-finisher periods respectively. At each feeding period rapeseed oil was replaced by 0, 33.3 , 66.7 and 100% levels of sunflower oil. At the same time two different levels of dl-alpha-tocopherol acetate (0 (only

Effects of Dietary Inclusion of Oil Sources With or Without Vitamin E on Body Composition and Meat Oxidation Level in Broilers
50 I.U. comes from vitamin premix) and 300 mg/kg) was also added to each diets. Thus the experiment was conducted as a randomized block design with a factorial arrangement of 4×2. The diets were also analysed for dry matter, protein and crude fat, crude fiber, starch and sugar according to AOAC (2000). The ME content of the diets was then calculated according to the following WPSA Formula (1984); ME [kcal/kg]=[3.69x% crude protein{CP}+8.18×% ether extractable fat{EE}+3.99×% starch+3.11x% sugar]).

Birds and housing
Three hundred and twenty commercial Ross × Ross 308 newly hatched broiler chickens were randomly allocated to eight isocaloric and isonitrogenous dietary treatments with four replicates. Ten chicks were randomly assigned to each of 32 battery wire cages with eight groups of four pens each.

Effects of Dietary Inclusion of Oil Sources With or Without Vitamin E on Body Composition and Meat Oxidation Level in Broilers
fan. Relative humidity was maintained at 50±5%. Initial mean and range of body weight were similar (Table 5) for all pens. Experimental place was artificially ventilated and incandescent light was provided for 24 hours for the first three days and followed by thirty nine days with 23 hours light. Each cage was equipped with two inside nipple drinkers and an outside galvanized feed trough. The birds had ad libitum access to feed (mash form) and water during the experiment. Health status and mortality of birds was controlled daily during the entire experimental period.

Data collection
Performance data, average weekly feed intake (FI) and body weight gain (BWG) were recorded. FCR was calculated from feed consumed per unit of BWG. These data are presented in Table 5 on a periodic basis (1-21, 22-42 and 1-42 day). Mortalities were recorded daily. On day 42 after a 10-hour feed withdrawal, 128 birds (4 birds per replicate) were slaughtered manually and allowed to bleed for 3 minutes. Having soaked in +60°C water for approximatly 50 seconds the carcasses were defeathered mechanically. Carcasses were manually eviscerated and refrigerated to reach +4°C. Then, carcass and carcass parts yield data were collected from 128 broilers (4 birds per replicate) which included carcass yield, thigh, breast, and abdominal fat pad weight.
Carcasses were evaluated as follows: the complete deboned skinless right thigh and breast (pectoralis major + pectoralis minor) meat were removed from the chilled carcasses (4 °C) and frozen at -26°C for 7 days before sensory analyses (olfactory, tenderness and juiciness, taste). Organoleptic characteristics of  Table 1 2 ΣSFA=total saturated fatty acids; ΣMUFA=total monounsaturated fatty acids; ΣPUFA=total polyunsaturated fatty acids.   Table 1 2 BWG -body weight gain, FI -feed intake and FCR -feed conversion ratio a-c means followed by different letters within columns are significantly different (p<0.05).

Effects of Dietary Inclusion of Oil Sources With or Without Vitamin E on Body Composition and Meat Oxidation Level in Broilers
thigh and breast meat were performed with untrained panelists, consisting of 16 bachelor students. Meat samples were prepared according to the method based on Hugo et al. (2009). Before cooking, samples were thawed overnight at 4°C. Meat samples were lightly-salted and steamed (200°C) in an oven, until a constant internal temperature of nearly 90°C was reached. The cooked thighs and breasts were cut into smaller cubes of 2.5 × 2.5 × 2.5 cm and then served to the panelists. Each sample was served in a plate, covered with a square of aluminium foil (which are available at local markets for cooking). Tasting was performed at room temperature (20 -22°C) under white fluorescent lighting. Hedonic scale (1 to 9) was used for acceptability analysis. Numbers 1 to 9 were respectively assigned for dislike extremely, dislike very much, dislike moderately, dislike slightly, neither like nor dislike, like slightly, like moderately, like very much, and like extremely. Three series of questionnaires were prepared based on hedonic scale for the odour, tenderness and juiciness, and taste. Tepid water was used by panellists for palate cleaning between samples. The complete deboned skinless left thighs and breasts (pectoralis major + pectoralis minor) were also collected and two tissue samples were taken from each. The first set of samples were frozen at -26°C for crude protein, crude fat, and FA profile analysis. The second set of samples of left thigh and breast muscle were stored at +4°C for 1, 3, 6 and/or 9 days until TBARS value determination.

Chemical analysis
All 128 left thigh and breast samples from all treatments were used for FA analysis. The FA profile of the experimental oils and diets were also determined. The total fat of oils, diets, thigh and breast muscles were extracted according to the method described by Blight et al. (1959).
The FA profile was determined by means of gas chromatography (Hewlett-Packard Company, Wilmington, DE, A.B.D.) equipped with a BPX70 capillary column (SGE capillary column; length, 25 m; I.D., 0.25 mm, 0.25mm film) and a flame ionization detector. The operating conditions of the gas chromatograph were as follows: the initial temperature was 190 °C for 5 min, increasing by 3 °C/min to 220 °C remained stable at final temperature for 25 min, the injection temperature was 250 °C and the detector temperature was 260 °C. The FA percentage was integrated and then calculated by means of direct normalization of the peak areas. Each FA was identified in the form of a methyl ester by comparing the retention times with the Supelco 37 Component FAME Mix standard acquired at Sigma Interlab A.S. (Interlab S.A. Istanbul, Turkey).
The extent of lipid peroxides in thigh and breast meat samples (1, 3, 6 and/or 9 days of storage at +4°C temperature) was assessed by measuring TBARS according to the method described by Tarladgis et al. (1960).

Statistical Analysis
Statistical analyses of data was conducted as a randomized block design, with a factorial arrangement of 4×2, taking into consideration main effects of inclusion of replacement levels of sunflower oil by rapeseed oil (% 0, % 33.3 , % 66.7 ve % 100) and vitamin E levels (0 and 300 mg/kg) with an equal number of 4 replicates for each treatment. The SAS programme (2001) was used for data analysing. When necessary mean separation was accomplished by using Duncan's multiple-range test a probability value of less than 0.05 was considered significant, unless otherwise noted. All percentage data were subjected to arcsine square root transformation (Steel & Torrie, 1960).

RESULTS AND DISCUSSION
The FA compositions of experimental oils are shown in table (Table 3). The ratio between n-6 and n-3 FA were 671.11 and 2.30 in the sunflower and rapeseed oil, respectively. Rapeseed oil in comparison with sunflower oil contains higher level of MUFA of oleic acid type (61.22 vs 28.81 respectively) and lower level of n-6 PUFA (LA; 18:2n-6) (20.82 vs 60.40 repectively). FA profile of diets (Table 4) clearly reflected the origin of the added oils. As expected, the sunflower oil diet contained a greater proportion of LA (18:2n-6) which was the major FA (60.40%) compared to rapeseed oil diet (20.82%) the only one of the n-6 series.
Body weight gain (BWG), feed intake (FI), and feed conversion ratio (FCR) of chicks fed the different experimental diets are shown in Table 5. Dietary oil level had significant effect on BWG during the starter period of trail so that, birds fed on diet containing 33.3 % replacing level had the highest BWG (p=0.01); the effect of vitamin E and its interaction with dietary oil was not significant for starter period. The interaction of vitamin E and oil was significant in grower-finisher phase (p=0.002) and over the whole period (p=0.003) ( Table 5).
FI was increased in all oil replacing levels (33.3, 66.7 and 100 %) during the starter (p=0.05), grower-finisher (p=0.01) and whole period of the trail (p=0.01). Oil  Table 1 and vitamin E interaction was significant for growerfinisher (p=0.02) and whole period (p=0.03). FCR in the different treatment groups showed no significant differences (Table 5). The vitamin E effect on performance (BWG, FI, and FCR) was also not significant.

Effects of Dietary Inclusion of Oil Sources With or Without Vitamin E on Body Composition and Meat Oxidation Level in Broilers
Advantages of dietary oil in broiler are: a) increasing the absorption and digestion of lipoproteins, b) lower heat increment of fat toward other energy sources such as carbohydrates or proteins, and c) also they assist vitamin A, vitamin E and Ca absorption (Leeson & Atteh, 1995). Rahimi et al. (2011) fed the day old Coob 500 broilers with different levels of full fat canola seed and / or flaxseed for 42 days. They showed that the use of full fat seed in the broiler diet has the lowering (p<0.01) effect on FI. This might be due to, in one hand, the higher fat content diet which reduces the digestion, absorption, and FA synthesis. On the other hand it increases rate of lipid catabolism (Sanz et al., 2000). The results related to FCR in the current study are similar to the findings of Viveros et al. (2009) and Burlikowska et al. (2010) indicating that dietary fat sources had no significant effect on performance parameters (BWG, FI, and FCR). However, Atteh & Leeson (1983) reported that saturation degree of a dietary fat can influence FI, BWG and FCR which results from better availability of energy from PUFA. Oils with PUFA produce higher energy and upgrade the performance, because compared to oils that contain saturated fatty acids they are absorbed easier consequently, as explained by Dvorin et al. (1998), birds fed on diet containing high levels of PUFA have better BWG. Moreover, digestion and absorption of dietary fat depend also on the age of the birds (Burlikowska et al., 2010). The lack of the vitamin E effect on performance in the current study is similar to the findings of Coetzee & Hoffman (2001) and Hsieh et al. ( 2002). As in the current study all dietary treatments were isocaloric and isonitrogenous it seems that dietary vitamin E had no increasing effect on protein or energy utilization in the different treatments. It has been suggested that under commercial conditions, the inclusion of high level of dietary vitamin E is the most beneficial and significantly improved performance will occur more frequently where there is a challenge to the host's defence system (McIlroy et al., 1993). The currrent study was performed in an experimental facility whith high level of hygienic principles. Total mortality during the whole experiment was below 2.5% for all treatments (p>0.05).
No statistical differences were observed for carcass yield or abdominal fat between the control and the test diet groups. Additionally, thigh and breast fat and crude protein percentages were unaffected by treatments ( Table 6).

Effects of Dietary Inclusion of Oil Sources With or Without Vitamin E on Body Composition and Meat Oxidation Level in Broilers
Some studies have shown that total fat content in tissues are reduced by increasing the dietary PUFA level (Cortinas et al., 2004;Shen et al., 2005). High rate of lipid metabolism and low FA synthesis are the reasons of low fat deposition (Sanz et al., 2000). These observations are inconsistent with the current results. There is also evidence that dietary PUFA level had no effect on the intramuscular lipid content of breast meat (Scaife et al., 1994;Crespo & Esteve-Garcia, 2002).
Fat deposition in the tissues is the result of absorption, de novo synthesis, and oxidation of fatty acids. Studies showed that dietary PUFA decreases hepatic lipogenesis and increases the metabolic rhythm, specifically causing a higher β-oxidation rate and inhibits de novo fatty acid synthesis (Shimomura et al., 1990;Sanz et al., 2000;Barroeta, 2007).
In the addition of vitamin E to the diet and the reduction of lipid oxidation, the following must be taken into consideration: vitamin E reduces linolenic acid (LNA; 18:3n-3) oxidation which leads to the reduction of lipogenesis and β-oxidation of FA in liver, which approximately reduces the total lipid deposition in tissues such as thigh and breast. But, in the present study there was no significant difference in the thigh and breast lipid level among experimental groups. Increases in the level of LNA in the body stimulates n-6 PUFA metabolism which affects n-6: n-3 ratio (Zanini et al., 2003). Our findings showed that increases in dietary rapeseed oil level decreased the n-6: n-3 ratio.
FA composition of chicken tissues is a combination of in vivo synthesis of FAs, from carbohydrate and protein precursors, and direct deposition from the diet. SFA and MUFA have this double origin, whereas PUFA deposition depends almost exclusively on dietary supplementation when no essential FA deficiency exists. The main FAs resulting from hepatic lipogenesis are 16:0, 18:0, 18:1n-9 and 16:1n-7 (Villaverde et al., 2006). In the current study the FAs found in thigh and breast meat (Table 7 and 8 respectively) reflected the dietary FA profile so that, increasing dietary PUFA inclusion led to higher deposition of PUFA, while it had the opposite effect on the deposition of SFA and MUFA which as described in literature, is due to an inverse relation between PUFA, SFA and MUFA deposition. Liver is where MUFA is synthesized. The presence of PUFAs in the diet leads to lower conversion of SFA into MUFA due to inhibitary effects of PUFAs on the activity of the enzyme Δ9-desaturase in the liver (Ajuyah et al., 1991;Lopez-Ferrer et al., 2001a,b;Villaverde et al., 2006;Smink et al., 2010). The high concentration of LA (18:2n-6) in the diet with only sunflower oil was reflected in its significantly greater incorporation into thigh and breast muscle compared with the 100% rapeseed oil dietary group (p=0.001). Thigh meat in birds fed on diet containing the 100 % of replacement had the highest level of the LNA (p=0.001) and EPA (p=0.001). The breast meat level of the LNA (p=0.001), EPA (p=0.008), and DHA (p=0.001) were the most highest for the 100 % replacement in comparison to other groups. As it´s shown in Table 4, the relative proportions of thigh and breast incorporation of the mentioned FAs were almost similar to the dietary proportion of LNA, EPA, and DHA. The amount of the ΣSFA was the highest both in thigh and breast meat of birds fed on diets containing only sunflower oil (0% replacing) and as it´s shown in Table 7 and 8 the ΣSFA amount was decreased by increasing the replacement rate of sunflower oil but the effect of vitamin E and it´s interaction with dietary oil were not significant for ΣSFA. In contrast, thigh and breast muscle from birds that recieved only rapeseed oil, had a significantly higher (p=0.001) proportion of ΣPUFA n-3 compared to the group that recieved only sunflower oil. The n-6:n-3 ratio for thigh and breast muscle of 66.7 and 100 % replacing group was the lowest (p= 0.001) compared to other groups. Our findings are in agreement with other studies (Rahimi et al., 2011;Gallardo et al., 2012). More recently Gallardo et al. (2012) fed the broiler chickens with four diets containing 15% oil with different percentages of canola oil (0, 5, 10, 15%), for 31 days. They concluded that canola oil increased the content of n-9 and n-3 FAs and decreased the content of n-6 FAs in meat, fat and plasma.
Our findings also showed that the effect of dietary oil level, vitamin E and their interaction were significant for LNA level in both thigh (p= 0.001, 0.002, 0.002) and breast (p= 0.001, 0.001, 0.02). The effect of vitamin E and the vitamin E × oil interaction were also significant for n-6:n-3 ratio (Table 7 and 8). The effect of vitamin E on tissue FA profile is rather contradictory. Some authors reported a higher level of some n-3 PUFA in thighs (Ahn et al., 1995;Surai & Sparks, 2000) and breasts (Ajuyah et al., 1993;Zanini et al., 2003). Some researchers believe that vitamin E not only protects unsaturated FAs from oxidation, but also stimulates FA synthesis since α-tocopherol quinone, as a product of tocopherol metabolism, is an essential cofactor of the FA desaturases (Infante, 1999). More recently it has been shown that supplementation of vitamin E protected PUFA well, increasing DHA deposition in abdominal tissue (Lu et al., 2014). On the other hand some studies reported no variation in  Table 1 2 ΣSFA=total saturated fatty acids; ΣMUFA=total monounsaturated fatty acids;

Effects of Dietary Inclusion of Oil Sources With or Without Vitamin E on Body Composition and Meat Oxidation Level in Broilers
ΣPUFA=total polyunsaturated fatty acids.
a-e means followed by different letters within columns are significantly different (p<0.05).   Table 1 2 ΣSFA=total saturated fatty acids; ΣMUFA=total monounsaturated fatty acids;

Effects of Dietary Inclusion of Oil Sources With or Without Vitamin E on Body Composition and Meat Oxidation Level in Broilers
ΣPUFA=total polyunsaturated fatty acids.
a-e means followed by different letters within columns are significantly different (p<0.05).

Effects of Dietary Inclusion of Oil Sources With or Without Vitamin E on Body Composition and Meat Oxidation Level in Broilers
FA composition of chicken thighs when broiler diets were supplemented with vitamin E (Hsieh et al., 2002;Cortinas et al., 2004). For exmaple Hsieh et al. (2002) fed the chicks with MUFA / SFA ratio of 1.2 or 4.8 and supplemented with 0, 10 or 20 mg vitamin E kg −1 for 3 weeks. The ∑SFA, ∑MUFA, and ∑PUFA contents in heart, liver, and breast muscle were not affected by dietary vitamin E level. In most studies, results are expressed as percentages (area normalization), which makes comparison of the results difficult. It is necessary to mention that in the current study FAs were quantified based on internal standard to avoid the inaccuracy associated with area normalization (Cortinas et al., 2004). Dietary polyunsaturation level can affect the fat deposit of the body, there is a difference in lipid digestion between non-ruminant and ruminant animals which has an important effect on transference of fats from the diet into the tissue. Broilers fed by diet containing polyunsaturated fats have lower fat deposition in contrast to the diet containing saturated fats. The reason has been explained to be due to higher rate of lipid catabolism and lower FA synthesis (Sanz et al., 2000;Woods & Fearon, 2009). Similar results have been obtained in a study on rats as they showed significantly lower metabolic oxidation of lipids which resulted in lower thermogenesis in tissues of rats fed on saturated fats (Shimomura et al., 1990). In intramuscular fat FA is one of the main components of cellular membrane. As for cells, to ensure the fluidity and permeability of different compounds, they have to maintain their physical chacterictics of the cell membrain. As a result, modification of FA composition of intramuscular fat has been reported to be limited (Lopez-Bote et al., 1997). The TBARS level (mg malonaldehyde/100g tissue) was detected in the thigh and breast meat throughout the 1, 3, 6, and 9 days of storage (Table 9). Thigh and breast muscle from birds fed without vitamin E displayed an increased susceptibility to lipid peroxidation in vitro, as expected, 100 % rapeseed oil supplementation without vitamin E has the greatest impact (p<0.01) on elevating malonaldehyde production. However, the dietary combination of oil and vitamin E at 300 mg/kg markedly (p=0.001) decreased the susceptibility. These findings indicate that dietary inclusion of vitamin E can be an effective way to increase stability of n-3 PUFA enriched broilers meat across the oxidative damage. Surai & Sparks (2000; in different tissues of cockerels Table 9 -Effects of dietary sunflower and rapeseed oil with or without vitamin E on TBARS level of the thigh and breast meat (mg malonaldehyde/100g tissue) 1