Effects of Grape Seed Oil Supplementation to Broilers Diets on Growth Performance, Meat Fatty Acids, Health Lipid Indices and Lipid Oxidation Parameters

: The purpose of this study was to evaluate the effects of grape seed oil (GSO) supplementation to broilers fed polyunsaturated fatty acids (PUFA)-enriched diets on growth performance, color, texture, fatty acid content and lipid peroxidation of meat. The 4-week feeding trial was conducted on 120 Cobb 500 broilers, assigned to three groups and housed in an experimental hall on permanent wood shaves litter. GSO was tested as source of natural antioxidants at different levels, 0% (GSO0, control), 1.5% (GSO1.5) and 3% (GSO3) in the presence of 4% ﬂaxseed meal (FSM) in a completely randomized design. The results show that at the end of the experiment (42 days) the GSO supplementation had no effect ( p < 0.05) on productivity parameters, except the ﬁnal weight which was improved in GSO3 compared to GSO. The thigh meat color indicated a higher degree of lightness ( p < 0.05), but the meat texture was not inﬂuenced ( p < 0.05) by the new tested diets. The GSO diets increased ( p < 0.05) the saturated fatty acid (SFA) content and decreased the PUFA content in the thigh meat. Thigh meat samples from GSO treatments had signiﬁcantly ( p < 0.05) improved oxidative stability. In the breast meat only the concentration of thiobarbituric acid-reactive substances (TBARS) decreased ( p < 0.05). It is concluded that the GSO signiﬁcantly improved the thigh meat oxidative stability.


Introduction
Meat quality is a key evaluation criterion associated with the requirements that must be met to comply with consumers' specifications, standards and expectations. Consumers' ongoing demand for high quality standards in meat production, including safety, health promotion and personal beliefs, requires the development of new strategies to improve meat nutritional value [1,2]. There are several factors that underlie consumer preferences when purchasing a product, some of which are medical, the genetic predisposition to certain diseases, sensitivities or allergies, and others are related to cultural traditions and social evolution [3]. According to the Food and Agriculture Organization (OECD/FAO) [4], broiler meat is in the top of consumer preferences, being the most consumed type of meat due to its price and sustainable production [5]. Current nutritional strategies are based on the use of natural ingredients in the formulation of poultry diets, to improve the nutritional value of the resulting animal products. Therefore, the tendency19 is to enrich the animal products in certain nutrients beneficial for human health, and thus available in an easier way.
Romanian Law 43/2014 for the handling and protection of animals used for experimental purposes, the EU Council Directive 98/58/EC concerning the protection of farmed animals and Directive 2010/63/EU on the protection of animals used for scientific purposes.

Experimental Design and Diets
This study was carried out on 120 14-day-old Cobb 500 unsexed broiler chickens, obtained from a local commercial hatchery. Different levels of grape seed oil (GSO) were tested as a source of natural antioxidants at 0% (GSO0, control), 1.5% (GSO1.5) and 3% (GSO3) levels by substituting the vegetable oil from the basal diet, in the presence of 4% flaxseed meal (FSM) ( Table 1). Flaxseed meal was included in all three diets to enrich them in PUFA. The plant materials (FSM and GSO) used in the new diet formulations were purchased from a local supplier in Romania. The broilers were weighed individually and randomly assigned to three treatments of 40 chicks each in a completely randomized design. According to the Cobb 500 Hybrid growth guide, the feeding program was divided into 2 feeding phases: grower (14-28 days) and finisher (29-42 days) using the HYBRIMIN Futter 5 Nutrition program. Feed and water were available ad libitum. The broilers were housed in semi-intensive system conditions, in a hall divided into 3 experimental spaces as growth boxes of 3 m 2 (each group was housed in a single box) and reared on the floor on permanent wood shaves litter (10-12 cm thick). The temperature and lighting programs were consistent with the recommendations of Hybrid. No mortality was recorded during the overall trial period.

Growth Performance and Broiler Slaughter
Throughout the experimental period (14-42 days) the following parameters were monitored: body weight (BW, g), average daily weight gain (ADWG, g/broiler/day), average daily feed intake (ADFI, g feed/broiler/day) and feed conversion ratio (FCR, g feed/g gain). BW was recorded weekly by weighing every broiler individually; ADWG was calculated by dividing total broiler weight by the number of experimental days; ADFI was calculated on a daily basis by subtracting the amount of rejected feed from the offered feed at the beginning and end of every 24 h (1 day); FCR values were calculated by dividing total feed consumed at total live weight.
At the end of the feeding-trial (42 days), 6 broilers per group were randomly selected and slaughtered by cervical dislocation according to the working protocol approved by the Ethics Commission of INCDBNA, Balotes , ti. Six breast and six thigh meat samples per group were collected and were divided into several sections for each type of determination targeted. They were collected in plastic bags, labeled and distributed for the following determinations: instrumental color measurements; pH assessment; texture profile; fatty acid content; and lipid oxidation parameters.

Antioxidant Capacity
A method adapted from Ninfali et al. [25] was used in order to determine the antioxidant capacity of grape seed oil. The method employed used hexane as solvent to solubilize both oil samples and 2,2-diphenyl-1-picrylhydrazyl (DPPH). The equipment used was a Jasco V 530 UV-VIS spectrometer, controlled with Spectra Manager software. The results were expressed as DPPH equivalents/g of sample, taking into consideration the density of the grape seed oil which was also determined.

Volatile Compounds
The analysis of volatile compounds from grape seed oil was performed by gas chromatography coupled with mass spectrometry (GC-MS), using Thermo Scientific equipment with an automatic Triplus autosampler. The GC-MS system consisted of a Focus GC gas chromatograph coupled with a Polaris Q ion mass spectrometer. The separation was performed on a DB-5MS type capillary column (non-polar stationary phase with 5% phenyl) with a length of 25 m, a diameter of 0.25 mm and 0.25 µm thickness film. The ion source and interface temperatures were 200 and 250 • C, and the detector operated in electron impact mode (70 eV). The detection was performed in the range m/z 35-300, the mass spectrometer operating in full-scan mode. A standard solution of alkanes for GC (even fraction C8-C20 in hexane) was analyzed according to the same temperature program and further used for Kovats retention indices calculation and compounds identification in oil samples. The results were expressed as a percentage.

Instrumental Color Measurements
Determination of meat color parameters in the CIELAB space by measuring the parameters L* (lightness), a* (saturation index in green/red), b* (saturation index in blue/yellow) and c* (metric chroma) and the total color difference (∆E*) was performed according to the method described by Panaite et al. [26] and Mancini et al. [27], using a Konica Minolta CR-400 (Tokyo, Japan) colorimeter. The results were expressed as an average for three measurements/sample.

PH Assessment
The pH of the samples was measured using a Hach HQ30d pH-meter (Hach, Loveland, CO, USA), according to the SR ISO 2917: 2007 standard at 24 h post-mortem. To measure the pH, a meat-water mixture was prepared from 5 g of meat which was cut into pieces with a knife and mixed with 5 mL of distilled water, at neutral pH [28]. At least three replicate measurements for each sample were carried out and the electrode was cleaned after each measurement. Buffer solutions of pH 7 and pH 4 were used for calibration of the pH meter.

Texture Profile Analysis
The chicken meat texture parameters were determined by a double cycle compression performed using a Perten TVT 6700 texturometer (Perten Instruments, Sweden), equipped with a Compression Platen cell. The analysis of the texture profile (TPA) resulting from the application of the compression test highlights as main texture parameters: hardness or firmness, springiness, resilience, cohesiveness and, as secondary parameter, gumminess. The thigh and breast meat samples were cut into cylindrical pieces of 10 mm thickness and 15 mm diameter, after which they were subjected to compression of 50% of the initial height. The principle of the method consisted of applying tension with a stainless-steel cylinder, with a 20 mm diameter on the meat samples, and the automatic recording of the resistance force that they opposed to deformation [28]. The measurements on a sample were repeated at least three times, and the results were expressed as their average.

Lipid Oxidation Parameters Evaluation
Samples of breast and thigh meat were stored for 7 days in the refrigerator at a constant temperature of 4 • C before evaluating the lipid oxidation parameters. In order to carry out the assessment, the samples were minced and cryogenized with liquid nitrogen at a temperature of −180 • C, and therefore were grounded. The oxidative stability of the meat was determined by means of primary lipid degradation parameters, peroxide index, dienes and conjugated trienes, respectively, by secondary parameters, represented by the values of p-anisidine and those of thiobarbituric acid-reactive substances (TBARS), according to the method described by Untea et al. [24]. The lipid peroxidation parameters were spectrophotometrically determined by using a V-530 Jasco (Japan Servo Co. Ltd., Tokyo, Japan) spectrophotometer, as follows: The peroxide value was measured by the ferric thiocyanate method. To the lipid extract sample, a chloroform/methanol solution was added, and the mixture was vortexed. In the next step, xylenol orange solution and FeCl 2 solution were added, the mixture was left for 5 min at room temperature and then measured.
The values of conjugated dienes and trienes were determined using a lipid extract sample dissolved in 2,2,4-trimethylpentane (iso-octane).
The p-anisidine value was determined by a method based on the reaction between p-anisidine and aldehydic compounds present in lipid extract samples in acidic conditions. The lipid extract sample was dissolved in iso-octane, the p-anisidine reagent was added to the cuvette, the sample was placed kept in the dark for 10 min and then the spectra was recorded.
The TBARS values were measured using third derivative spectrophotometry with some modifications [29]. The sample was mixed with trichloroacetic acid and butyrate hydroxytoluene in ethanol, and centrifuged. The aliquots were mixed with aqueous thiobarbituric acid solution in a test tube and further incubated at 80 • C for 50 min. Following incubation, the sample was cooled under running water and measured. TBARS values were calculated against a standard curve obtained with 1,1,3,3-tetramethoxypropane.

Principal Component Analysis
Principal Component Analysis (PCA) was performed to assess the relationships between the evaluated characteristics. The PCA technique converts the original variables into factors, namely principal components, in the conditions of minimal information loss. By applying this technique to the results, the differences and similarities between evaluated charactristics can be observed in the load graph.

Statistical Analysis
All results were statistically processed for each quality parameter of the chicken samples. The analytical data were compared by analysis of variance (ANOVA and t test), using the StatView program for Windows. The results of this experiment are presented as mean values, the differences being considered statistically significant at p < 0.05.

Chemical Composition of Plant Materials
The chemical composition of studied plants materials is shown in Table 2. The volatile compounds identified in grape seed oil (Table 3) showed high contents of ethyl ester of linoleic acid and methyl ester of 10,13-eicosadienoic acid, but also for benzyl benzoate. Based on the structural characteristics of the 24 major components identified in the analyzed grape seed oil, it was possible to classify them by classes of dominant compounds. Thus, it was observed that the esters of acids predominated in the composition of grape seed oil, whether or not they were fatty, in a concentration of approximately 65%. To these were added terpenes and phenols, all these classes of compounds having protective properties against oxidative stress.

Effect of Grape Seed Oil on Growth Performance of Broilers
Growth performance data are shown in Table 4. In the present study, the final body weight of the GSO3 group increased compared to the GSO group, but no significant difference (p > 0.05) was noted for the others' growth parameters, slaughter yield, eviscerated carcass, breast, thigh, liver, heart or gizzard yields compared to GSO group. However, the carcass and organs were higher in GSO1.5 and GSO3 than in GSO. GSO0 = control diet without grape seed oil; GSO1.5 = control diet supplemented with 1.5% grape seed oil; GSO3 = control diet supplemented with 3% grape seed oil; * n = 40; ** n = 6; slaughter yield 1 was calculated as percent of live BW; eviscerated carcass yield 2 was calculated as percent of slaughter yield; muscle and organ yields were calculated as percent of eviscerated carcass. a,b Means within a column with no common superscript differ (p < 0.05); SEM = standard error of the mean.

Effect of Grape Seed Oil on Color Parameters and the pH of Broiler Meat
Color parameters and the pH of broiler meat are shown in Table 5. The grape seed oil inclusion in the new diets influenced the lightness of thigh meat samples, with significantly (p < 0.05) higher values being recorded in the GSO3 group, compared to GSO0. At the same time, the thigh meat had a lower (p < 0.05) pH in groups GSO1.5 and GSO3, in contrast with the GSO0 group.

Effect of Grape Seed Oil on Texture Parameters
The supplementation of new diets with grape seed oil had no effect (p > 0.05) on thigh and breast meat texture parameters (Table 6).  Table 7 shows the meat fatty acid content results. Grape seed oil supplementation to broiler diets led to an increase (p < 0.0001) in the thigh meat SFA content and a decrease in the PUFA level, respectively, PUFA n − 6 and n − 3, in the GSO1.5 group, compared to GSO0 group. A significant decrease (p < 0.05) was also observed for PUFA n − 3 content and an increase (p < 0.05) for the n − 6/n − 3 fatty acid ratio in the group that included the highest level of grape seed oil (GSO3). Regarding the breast meat, there was determined also a significant increase in SFA content in the GSO3 group, but there was an improvement for the PUFA n − 6/n − 3 fatty acids ratio in the same group, compared to the GSO0 group. For health lipid indices of thigh meat, the AI decreased (p = 0.0018) in the GSO3 group, and TI increased (p < 0.0001) in GSO1.5 compared to the GSO0 group. The ratio of h/H was significantly lower (p < 0.0001) in the thigh meat of the GSO1.5 group, compared to GSO0.

Effect of Grape Seed Oil on Lipid Oxidation Parameters
The results presented in Table 8 show that, after 7 days of refrigeration at +4 • C, the thigh meat samples from the GSO1.5 and GSO3 groups had significantly (p < 0.05) improved levels of conjugated dienes and trienes compared to the GSO0 group. Regarding the secondary oxidation parameters, the GSO1.5 group had a significantly (p < 0.05) lower value of p-Anisidine, while GSO3 had the lowest concentration of TBARS, compared to GSO0. The lipid oxidation parameters determined for breast meat samples indicated a significant improvement (p < 0.05) in TBARS concentration in the GSO3 group, compared to the GSO0 group.

The Relationship between Meat Characteristics
In Figure 1 are shown the loadings of the original variables on the first two principal components (PCs), which described 53.95% of the total variance. The variables strongly associated with the first principal component (PC1) were texture characteristics (hardness, gumminess, cohesiveness and resilience), pH and fatty acids in terms of polyunsaturated (PUFA), monounsaturated (MUFA) and saturated fatty acids (SFA), these variables repre-senting 33.95% of the total variance. The second principal component (PC2) accounted for 20.00% of the total variance of color parameters (L*, b* and Chroma), the textural parameter springiness and lipid degradation parameters (dienes, trienes and TBARS) as dominant variables.

Chemical Composition of Plant Materials
Flaxseed meal is recognized worldwide due to its rich composition of PUFA n − 3 fatty acid and alpha-linolenic acid, and also its high-quality protein. Available data in the literature show for flaxseed meal a content higher than 33% for ALA [30]; 42.93% PUFA n − 3; 70.23% total PUFA; and a value of 0.64 for n − 6/n − 3 ratio [31].
Grape seed oil composition is closely related to the grape variety from which it is obtained, environmental and climatic conditions, soil type, harvesting time and seed maturation degree [32,33]. The oil composition includes bioactive compounds, such as free fatty acids, vitamin E (tocopherols and tocotrienols), polyphenols and steroids (campesterol, beta-sitosterol and stigmasterol) [34]. In some countries, grape seed oil is used as a natural food preservative [35]. However, there are poor references in the literature regarding the antioxidant capacity of grape seed oil; available data indicated a concentration of 42.18 mmol of Trolox equivalent/g measured by oxygen radical absorbance capacity assay [20]. GC-MS analysis of grape seed oil allowed in the present study to establish its composition by identifying the main classes of compounds, and based on them the evaluation of the antioxidant potential, necessary to complete the diets with a rich lipid profile. The principal components of the profile established were linoleic acid ethyl ester, benzyl benzoate and 10,13-eicosadienoic acid methyl ester.

Effect of Grape Seed Oil on Growth Performance of Broiler
In our study, although there were no significant effects of grape seed oil inclusion in broiler diets, there was an improvement of final weight in the GSO3 group, compared to the GSO group. The results reveal that between the two experimental groups that included grape seed oil (GSO1.5 and GSO3), a higher level of inclusion (GSO3) led to significantly higher average daily weight gain and body weight gain (p < 0.05). Beneficial effects of grape seed oil use were reported by Tekeli et al. [21] on feed conversion rate, which was improved (p < 0.05) when it included a level of 1.5% GSO in broiler diets. Similar results regarding the feed conversion rate improvement and a significant increase (p < 0.05) in the

Chemical Composition of Plant Materials
Flaxseed meal is recognized worldwide due to its rich composition of PUFA n − 3 fatty acid and alpha-linolenic acid, and also its high-quality protein. Available data in the literature show for flaxseed meal a content higher than 33% for ALA [30]; 42.93% PUFA n − 3; 70.23% total PUFA; and a value of 0.64 for n − 6/n − 3 ratio [31].
Grape seed oil composition is closely related to the grape variety from which it is obtained, environmental and climatic conditions, soil type, harvesting time and seed maturation degree [32,33]. The oil composition includes bioactive compounds, such as free fatty acids, vitamin E (tocopherols and tocotrienols), polyphenols and steroids (campesterol, beta-sitosterol and stigmasterol) [34]. In some countries, grape seed oil is used as a natural food preservative [35]. However, there are poor references in the literature regarding the antioxidant capacity of grape seed oil; available data indicated a concentration of 42.18 mmol of Trolox equivalent/g measured by oxygen radical absorbance capacity assay [20]. GC-MS analysis of grape seed oil allowed in the present study to establish its composition by identifying the main classes of compounds, and based on them the evaluation of the antioxidant potential, necessary to complete the diets with a rich lipid profile. The principal components of the profile established were linoleic acid ethyl ester, benzyl benzoate and 10,13-eicosadienoic acid methyl ester.

Effect of Grape Seed Oil on Growth Performance of Broiler
In our study, although there were no significant effects of grape seed oil inclusion in broiler diets, there was an improvement of final weight in the GSO3 group, compared to the GSO group. The results reveal that between the two experimental groups that included grape seed oil (GSO1.5 and GSO3), a higher level of inclusion (GSO3) led to significantly higher average daily weight gain and body weight gain (p < 0.05). Beneficial effects of grape seed oil use were reported by Tekeli et al. [21] on feed conversion rate, which was improved (p < 0.05) when it included a level of 1.5% GSO in broiler diets. Similar results regarding the feed conversion rate improvement and a significant increase (p < 0.05) in the average of broiler body weight were obtained using 1% grape seed oil [38]. At a supplementation level of 2.5% GSO in diets, no significant effects (p < 0.05) were recorded during the entire experimental period on the broiler production performance [39].

Effect of Grape Seed Oil on Color Parameters and the pH of the Chicken Meat
Meat color is one of the most important buying factors, because depending on it, consumers make a first impression of the product. According to Marcinkowska-Lesiak et al. [40], meat color is considered a freshness and quality indicator of the product. There are several factors that influence the chicken meat color, namely genetic factors, applied diet, muscle fiber characteristics or heme pigments [41]. A high level of PUFA also influences the color and aroma of the meat [42]. In the present study, the new tested diets influenced the chicken meat. Supplementing the broiler diet with a double amount of grape seed oil led to the L* parameter being increased in the GSO3 group compared to the GSO1.5 group, suggesting an increase in thigh meat lightness with an increasing level of oil addition, from 1.5 to 3%. Corroborated with the recorded results for the L* parameter, the values of parameter a* indicated a decrease (p < 0.05) in the red color of thigh meat, compared to the GSO0 and GSO1.5 groups. According to Mirshekar et al. [43], an increased degree of meat lightness is due to a lower pH value, resulting also in higher water losses. It was reported [44] that meat lightness is influenced by collagen deposits on muscles, which are white and accentuate as broilers become older. Another factor that influences the meat color is the myoglobin content increase in the meat depending also on the broiler age. According to Savadkoohi et al. [45], instrumentally measured color changes can be considered visible if ∆E > 2. In this study, the supplementation of broiler diets with GSO led to a visible change in color in the case of thigh meat at the maximum level of inclusion evaluated of 3% (GSO3). The trend was different in the case of breast meat, which had a visibly different color at a lower inclusion level of GSO of 1.5% (GSO1.5), compared to the GSO0 group.
Determining the meat pH is important because there is a correlation between its values and physico-chemical characteristics, such as color and hardness [46]. In this study, the pH measurement indicated lower values in the groups that included grape seed oil (GSO1.5 and GSO3) compared to the GSO0 group, the differences being significant (p < 0.05) for thigh meat. The pH values indicated a high degree of broiler meat freshness and the beneficial effect of GSO inclusion in broiler diets. According to the limits established by different authors regarding the quality of chicken meat reflected by the measured pH value, the results obtained in this study are within the normal limits, between 5.9 and 6.2 [47].

Effect of Grape Seed Oil on Texture Parameters
Along with color, texture is considered an important decision factor for meat quality [48]. The proteolysis of key myofibrillar proteins is responsible for structural integrity in muscle fiber preservation. The weakening of muscle fibers is caused postmortem by proteolytic degradation, and thus, the meat becomes harder [40]. The texture profile analysis of broiler meat, thigh and breast revealed no significant treatment effects, which indicates that no negative effect of grape seed oil feeding was observed in the present study.

Effect of Grape Seed Oil on Meat Fatty Acid Content
This study assessed the quality attributes of chicken meat for consumers, changes in its fatty acid content and the consequent effects on meat oxidative stability under the influence of grape seed oil in the presence of flaxseed meal. The observed variation in the fatty acid composition of broiler meat was due to the differentiated inclusion of GSO in diets, because the level of flaxseed meal was the same in all three treatments. The present study clearly shows that the inclusion of grape seed oil in diets had significant effects, especially on the fatty acids of thigh meat. These effects were visible in the content of increased SFA, lower PUFA and PUFA n − 3, and the increase in n − 6/n − 3 ratio value. Regarding the breast meat, the SFA content increased, but there was an improvement in the ratio of PUFA n − 6/n − 3 between experimental groups (GSO1.5 vs. GSO3).
According to Barroeta [49], the inclusion of PUFA-rich oils in broiler diets results in a lower deposition of body fat, even if PUFA have a higher content of metabolizable energy than SFA. There are several factors that influence the effectiveness of fat utilization in broiler diets, including their chemical composition, inclusion rate or chick age. They are joined by anti-nutritional factors (e.g., tannins, phytic acid and trypsin inhibitor) contained by grape seeds or the raw residues from the GSO extraction industries [50,51]. These compounds, present in grape-derived products, interact with fats by decreasing lipase secretion and consequently lipolysis process, leading to lipid malabsorption [52], and finally affecting the fatty acids profile.
Indices of atherogenicity (AI) and thrombogenicity (TI) and the ratio of hypo-and hyper-cholesterolemia (h/H) are health and nutritional lipid indices [53]. The results obtained in this study provide the overview for a nutritional assessment of fatty acids and their health-related lipid indices. Because the AI index is a significant indicator of cardiovascular disease risk [54], its decrease in thigh meat suggests a reduced risk for broilers to develop this type of disease, corroborated with the level of GSO inclusion in the diet. According to Garaffo et al. [55], the TI index indicates the possibility of clots forming in the blood vessels. The results obtained in the present study show an increased TI value in the GSO1.5 group, but as the level of inclusion in the diet (GSO3) was increased, its value was no longer influenced, being comparable to that obtained in the GSO0 group. In addition to AI and IT indices, the hypo-and hyper-cholesterolemia ratio is an additional index that contributes to the evaluation of the effect that fatty acids have on cholesterol metabolism [53]. The decrease in h/H ratio value in the GSO1.5 group reflects the lower PUFA content in the thigh meat, compared to the GSO0 group.

Effect of Grape Seed Oil on Lipid Oxidation Parameters
It is well known that lipid oxidation is one of the main processes of quality deterioration in meat and meat products [42]. Oxidative stress occurs as a result of an imbalance between the production of various oxidizing chemical species and the natural antioxidant capacity of cells to annihilate these species [56]. The inclusion of grape seed oil at different levels in PUFA-enriched broiler diets has been carried out to manipulate the oxidative stability of meat and to prevent its quality deterioration. In the current study, the antioxidant effect of GSO on meat lipid oxidation could be observed most on thigh meat, compared to the breast meat samples. Apart from the peroxide index, all other lipid degradation parameters of thigh meat were significantly (p < 0.05) improved by the new feed diets. Overall, the inclusion of a 1.5% GSO level indicated for thigh meat the best results regarding the main degradation parameters, conjugated dienes and trienes, but also on one of the secondary ones, p-anisidine. Although there could be observed an improvement for breast meat in terms of lipid oxidation, only the TBARS parameter from the GSO3 group was significantly (p < 0.05) influenced. Unlike thigh meat, for breast the best results were obtained at a rate of 3% GSO inclusion in broiler diets. In a similar study [22] it was shown that the inclusion of 1% GSO led to an improvement in the oxidative status of broiler meat, by significantly (p < 0.05) decreasing the malonaldehyde and peroxide index values. A decrease in the malondialdehyde concentration in broiler meat has also been reported by Bander [38], when using a level of 1% GSO.

The Relationship between Meat Characteristics
PCA results show a bi-plot of the evaluated characteristics of the two PCs and the positive or negative relationship between them. The main contributor characteristics on the first principal component (PC1) were the textural parameters, hardness, gumminess, cohesiveness and resilience, along with the pH and polyunsaturated fatty acids. These characteristics were negatively correlated with the textural parameter springiness and monounsaturated (MUFA) and saturated fatty acids (SFA). Along the PC1, an inverse relationship could be observed between cohesiveness and springiness, which was expected. The second principal component, PC2, showed a close relationship between luminosity (L*), b* parameter and chroma and between dienes, trienes and TBARS lipid degradation parameters. There was a direct significant (p < 0.05) relationship between the b* parameter and chroma (r = 0.965), between b* and L* (r = 0.674), between L* and chroma (r = 0.544) and between dienes and trienes (r = 0.825). Additionally, the secondary parameter of lipid degradation, TBARS, was directly correlated with chroma (r = 0.511), a relationship which can be explained by the fact that lipid oxidation changes meat chroma, affecting the meat quality. Meat hardness was negatively correlated with saturated fatty acids (r = −0.542), whereas a high positive correlation was found between hardness and gumminess (r = 0.954), resilience (r = 0.751) and cohesiveness (r = 0.758). A high negative relationship was found between saturated fatty acids and polyunsaturated fatty acids (r = −0.923).

Conclusions
Under the experimental conditions used in the present study, the results demonstrate that grape seed oil supplementation as a source of natural antioxidants to broilers fed PUFA-enriched diets did not affect the quality attributes of the chicken meat, color, pH and texture, but the content of fatty acids was influenced in the sense of decreasing the PUFA level in thigh meat. However, GSO significantly improved the oxidative stability of thigh meat.