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Introduction
There are many factors influencing broiler meat quality, among which the genetic makeup, animal welfare, pre-slaughter factors and the post-mortem changes of the muscles1. Nowadays, the meat from slow-growing broiler hybrids became more known for its pleasant texture and flavour, less juicy, fitting the current consumer preferences2,3. In Europa these hybrids are adapting faster to the alternative production systems, several lines being available, even if the growth performance of the slow-growing hybrids are less efficient than that of the fast-growing hybrids2,3. Fatty acids contents were found to both hazards and beneficial effects on human health based on type of fats and meat consumption4. Furthermore, consumer concern for high omega - 3 (ω-3 PUFA) foods in their daily diet increased due to their beneficial effect on human health5,6,7.
Animal foods can be enriched in PUFA by feeding the animals diets whose ingredients are high in PUFA. Some of the PUFA-high feed ingredients are the flaxseed8,9 and flaxseed meal10-13, camelina meal14,15, and the rapeseed meal16-19. Flax is an oleaginous plant in which ω-6 / ω-3 ratio is lower than the unit (0.436%), hence, the flax, in all its forms (seeds, oil and meal) is a viable feed ingredient that can increase the PUFA level of the diets8. However, the increased levels of PUFA make the feeds prone to oxidation, which is why the diets have to be supplemented with antioxidants20.
The winery by-products, grape pomace, grape seeds and grape peels, grapeseed cakes or grapeseed meals, have high levels of polyphenols, sources of natural antioxidants. The abundance of active polyphenols in these by-products is of real interest for animal nutrition, because they can be used as natural antioxidants, replacing the synthetic ones21,22.
The grapeseed cakes or meals result from the cold extraction or chemical extraction of the oil. The concentration of polyphenols ranges between 0.642 mg gallic acid equivalents, in the cakes, 3.186 mg gallic acid equivalents, in the grape pomace and 90.41 mg gallic acid equivalents / g sample in the grape seed meal. The antioxidant capacity ranges between 8.554 mM Trolox equivalents, in the grape pomace, 6.241 mM Trolox equivalents, in cakes and 493.07 mM Trolox equivalents /g sample, in the grape seed meal11,23,24. The literature has several studies on the use of winery by-products, such as grape pomace25,26,27 or grapeseed meal28,29 in broiler feeding and on their beneficial effects on broiler performance, protein and amino acids digestibility, blood parameters and meat quality, due to the higher concentration of PUFA and lower lipid peroxidation.
The studies conducted so far have shown that the inclusion of winery by-products in animal feeding, as natural antioxidants, is a good strategy for enhancing the oxidative stability of the animal products, meeting thus consumer requirement for high quality animal foods30,31.
Within this context, the purpose of the present study was to assess the effect of 3 % grapeseed meal, winery by-product, added to Hubbard broilers diet high in polyunsaturated fatty acids due to the dietary 2 % flaxseed meal, on broiler performance, energy profile of the blood plasma, on fatty acids and cholesterol levels and on broiler meat degradation indices.
Material and methods
The feeding trial was conducted in the experimental facilities of the National Research Development Institute for Animal Biology and Nutrition (IBNA-Balotesti, Romania) on a protocol (no. 5122/03.08.2017) approved by the Ethics Commission of the institute in accordance with the EU Directive 2010/63/ EU and Romanian Law regarding Animal Protection.
Animals and experimental design
The 7-wk feeding trial was conducted on 80 Hubbard broiler chicks (14 d), reared on the floor, on wood shavings (10 cm thick). The chicks were weighed individually and assigned to two groups (40 chicks / group), having the average initial weight of 233.6 ± 5.53 g. Each group had four replicates of 10 chicks each. The chicks had free access to the feed and water. There was a 23 h light regimen, the temperature was 22 to 23 oC, and the relative humidity was 60 to 70 % throughout the entire experimental period, according to the Management guide for Hubbard CLASSIC broilers32.
The experimental design was monofactorial, completely randomized. It had two treatments, C (0%) and E (3%) with grapeseed meal applied for the two phases, growing (14-28 d) and finishing (28-63 d), according the feeding requirements of the slow-growing line Hubbard hybrid (Table 1). Both diets (C and E) were enriched in polyunsaturated fatty acids using 2% flaxseed meal. The flaxseed meal and the grapeseed meal were purchased from 2E Prod SRL, Romania. There was a single batch of diet / group for each period: grower and finisher. The growth performance of the broilers: average daily feed intake (g /broiler/d), initial weight (g), final weight (g) was monitored throughout the experimental period (14-63 d). It was calculated the average daily weight gain (g/broiler/d) and feed conversion ratio (g feed/g broiler), on basis of replicate of birds.
Table 1 Compound feeds formulation and chemical analysis
| Grower (14- 28 days) | Finisher (29- 63 days) | ||||
|---|---|---|---|---|---|
| C | E | C | E | ||
| Corn | 49.92 | 44.14 | 44.26 | 47.00 | |
| Wheat | 15.00 | 15.00 | 16.00 | 10.00 | |
| Corn gluten | 4.00 | 4.00 | 4.00 | 3.80 | |
| Soybean meal | 19.85 | 22.00 | 24.00 | 24.00 | |
| Flax meal | 2.00 | 2.00 | 2.00 | 2.00 | |
| Grapeseed meal | - | 3.00 | - | 3.00 | |
| Sunflower oil | 4.40 | 5.00 | 5.00 | 5.46 | |
| Monocalcium phosphate | 1.32 | 1.50 | 1.30 | 1.40 | |
| Calcium carbonate | 1.73 | 1.62 | 1.70 | 1.60 | |
| Salt | 0.34 | 0.34 | 0.34 | 0.34 | |
| Methionine | 0.15 | 0.15 | 0.15 | 0.15 | |
| Lysine | 0.24 | 0.20 | 0.20 | 0.20 | |
| Choline | 0.05 | 0.05 | 0.05 | 0.05 | |
| Premix* | 1.00 | 1.00 | 1.00 | 1.00 | |
| Total | 100 | 100 | 100 | 100 | |
| Chemical analysis results (% DM) | |||||
| Dry matter | 90.18 | 90.16 | 90.00 | 90.11 | |
| Crude protein | 21.80 | 21.87 | 19.12 | 19.20 | |
| Ether extractives | 5.44 | 6.06 | 6.97 | 7.40 | |
| Crude fibre | 4.59 | 4.93 | 4.64 | 4.61 | |
| Lysine | 1.33 | 1.34 | 0.99 | 1.13 | |
| Methionine | 0.43 | 0.41 | 0.38 | 0.39 | |
| Calcium | 0.91 | 0.91 | 0.91 | 0.92 | |
| Total phosphorus | 0.81 | 0.81 | 0.81 | 0.82 | |
| Concentration of total polyphenols (mg GAE / g) | 1.47 | 1.68 | 1.36 | 1.56 | |
| Antioxidant capacity (mM TE / g) | 2.15 | 3.09 | 2.01 | 2.82 | |
| Fatty acids (% of total FAME) | |||||
| Saturated fatty acids (SFA) | 11.04 | 10.43 | 12.89 | 12.30 | |
| Monounsaturated fatty acids (MUFA) | 23.62 | 21.06 | 23.87 | 22.16 | |
| Polyunsaturated fatty acids (PUFA) | 65.33 | 68.51 | 63.10 | 65.47 | |
| α-linolenic acid (ALA) | 4.35 | 4.90 | 5.76 | 6.34 | |
| Omega-3 polyunsaturated fatty acids (ω-3) Omega-6 polyunsaturated fatty acids (ω-6) | 4.46 | 5.01 | 5.89 | 6.47 | |
| 60.87 | 63.50 | 57.21 | 59.00 | ||
| Calculated metabolizable energy (Mj / kg DM)** | 13.18 | 13.20 | 13.39 | 13.40 | |
*Content per kg diet: vitamin A: 11000 IU; vitamin D3: 2000 IU; vitamin E: 27 IU mg; vitamin K3: 3 mg; vitamin B1: 2 mg; vitamin B2: 4 mg; pantothenic acid: 14.85 mg; nicotinic acid: 27 mg; vitamin B6: 3 mg; vitamin B7: 0.04 mg; vitamin B9: 1 mg; vitamin B12: 0.018 mg; vitamin C: 20 mg; manganese: 80 mg; iron: 80 mg; copper: 5 mg; zinc: 60 mg; cobalt: 0.37 mg; iodine: 1.52 mg; selenium: 0.18 mg.
**Metabolizable energy was calculated from the chemical composition
Sampling and chemical analysis
Samples of flaxseed meal and grapeseed meal were collected before feed manufacturing and their chemical composition determined. Grower and finisher compound feed samples were collected and assayed for the basic chemical composition, calcium, phosphorus, lysine, methionine, fatty acids profile, concentration of polyphenols and antioxidant capacity. At the end of the experimental period (63 d of broiler age), six broilers per group were selected randomly.
The blood samples (n= 6) were collected after which the broilers were slaughtered in 6 mL Vacutainer tubes, on anticoagulant heparin-lithium. The blood samples were centrifuged at 3,000 rpm, for 20 min, at +22 oC. The plasma was stored at - 80 oC until analysed. Six individual samples of breast meat and six individual samples of leg meat were formed. Each sample was divided in three. One part was assayed for fatty acids and cholesterol, one sample was refrigerated (+4 °C) for 7 d and thereafter assayed for the fat degradation indices (peroxide value and fat acidity), and the third part was frozen (-18 °C) for one month and thereafter assayed for the fat degradation indices (peroxide value and fat acidity).
The basic chemical composition of the flaxseed meal, of the grapeseed meal and of the compound feeds was determined using methods standardized according to Regulation (EC) no. 152/2009. Dry matter (DM) was determined by the gravimetric method using a Sartorius (Gottingen, Germany) scale and BMT drying closet, ECOCELL Blueline Comfort (Nuremberg, Germany). Crude protein (CP) was determined by the Kjeldahl method using a semiautomatic KJELTEC auto 2300 system - Tecator (Sweden). Ether extractives (EE) were determined by the extraction in organic solvents using a SOXTEC-2055 FOSS system - Tecator (Sweden). Crude fibre (CF) was determined by the method with intermediary filtration using a FIBERTEC 2010 system - Tecator (Sweden). Ash (Ash) was determined by the gravimetric method using a Caloris CL 1206 furnace.
The calcium (Ca) was determined by the titrimetric method according to SR ISO 6491-1:2006. The phosphorus (P) was determined photometrically, according to Regulation (EC) no. 152/2009, using the Jasco V-530 spectrophotometer.
Lysine and methionine of the compound feeds were determined by the liquid chromatographic method, according to Regulation (EC) no. 152/2009, using a Finningan Surveyor Plus HPLC (Thermo-Electron Corporation, Waltham, USA), fitted with PDA detector (Photo Diode Array Detector). The amino acids are separated on a Hypersil BDS C18 column with silica gel (250 ×4.6 mm), particle size 5 μm, with reverse phase and a +45 oC temperature. The results were expressed in g amino acids / 100 g DM.
Concentration of polyphenols and antioxidant capacity of the grapeseed meal and compound feeds. The grapeseed meal samples and the compound feeds samples were first extracted in acidified methanol (methanol: hydrochloric acid = 80:20). The concentration of polyphenols in the methanol extracts was determined by spectrophotometer method33, using a UV-VIS Thermo Scientific spectrophotometer. The results were expressed as mg gallic acid equivalents / g sample (mg GAE / g sample). The antioxidant capacity in the methanol extracts was determined by the DPPH method34, using a UV-VIS Analytik Jena Specord 250 Plus spectrophotometer with thermostatic carousel. The results were expressed in mM Trolox equivalents /g sample (mM TE / g sample).
Metabolic profiles of blood plasma. The blood biochemical parameters were determined from the plasma samples, using a biochemical analyser (Analyzer Chemistry Mindray BS - 130) with ACCENT - 200 kits, according the manufacturer’s instructions.
Fatty acids profile of the feed ingredients, of the compound feeds and of the meat samples. The fat from the meat samples (breast and leg) were extracted, then saponified by reflux boiling in a solution of acidified methanol (2% H2SO4 in methanol), to obtain the fatty acids methyl esters (FAME). The fatty acids methyl esters were added to hexane and concentrated in rotavapor. The resulting samples were analysed by gas chromatographic method, according to the SR CEN ISO/TS 17764-2:2008, using a Perkin Elmer-Clarus 500 chromatograph, fitted with a system for injection into the capillary column, with high polarity stationary phase (BPX70: 60m x 0.25mm inner diameters and 0.25µm thick film); or high polarity cyanopril phases, which have similar resolution for different geometric isomers (THERMO TR-Fame: 120m x 0.25mm ID x 0.25µm film).
Cholesterol level in the meat samples. For the meat samples saponification, it was started with a method described by Dihn et al35, adapted in our laboratory. The meat samples (breast and leg) were saponified by reflux boiling in a solution of methanol and potassium hydroxide (5% KOH in methanol), followed by extraction in petrol ether, concentration in rotavapor, and addition of chloroform. The resulting samples were analysed by gas chromatographic method, according to AOAC International36, using a Perkin Elmer-Clarus 500 chromatograph, with on-column injector (splitting ratio, about 1:100), with programmable column heater; flame ionization detector (FID) and capillary separation column HP-5 (30m, 0.32mm ID, 0.1 µm film) AGILENT.
The peroxide value for the meat samples was determined with the iodometric method according to SR EN ISO 3960:2017. The results were expressed in milliequivalents / kg fat (meq/kg fat). The fat acidity for the meat samples was determined with the volumetric method according to ISO 660:2009. The results were expressed in mg potassium hydroxide / g fat (mg KOH / g fat).
Statistical analysis
The effects of treatments were tested by one-way analysis of variance using the GLM procedure of Minitab software37 with treatment as a fixed effect, according to the model Yi = Ti + ei, where Yi was the dependent variable, Ti is the treatment and ei is the error. When overall F-test was significant, differences between means were declared significant at P < 0.05 using the Tukey comparison test.
Results
Characterisation of flaxseed meal and grapeseed meal
The flaxseed meal, used as feed ingredient high in polyunsaturated fatty acids (PUFA), had: 344.4 g/kg protein, 109.7 g/kg fat, and 20.20 MJ/kg gross energy. The fatty acids profile was: 11.21 g saturated fatty acids (SFA), 20.00 g monounsaturated fatty acids (MUFA), 68.73 g polyunsaturated fatty acids (PUFA), of which 53.35 g ω-3 PUFA, and 15.37 g ω-6 PUFA / 100 g FAME, with ω - 6 / ω - 3 ratio of 0.29. Table 2 shows the basic chemical composition, the fatty acids profile, the concentration of polyphenols and the antioxidant capacity of the grapeseed meal used as natural antioxidant in PUFA-high diets.
Table 2 Content of the main nutrients of grapeseed meal
| Specification | Grapeseed meal |
|---|---|
| Dry matter, % | 918.5 |
| Crude protein, % DM | 129.0 |
| Ether extractives, % DM | 72.2 |
| Gross energy, MJ/kg | 18.55 |
| Concentration of total polyphenols, mg GAE/g | 26.65 |
| Antioxidant capacity, mM TE/g | 148.35 |
| Fatty acids (% of total FAME): SFA | 12.30 |
| MUFA | 20.39 |
| PUFA | 67.14 |
| ω -3 | 0.68 |
| ω -6 | 66.45 |
SFA= saturated fatty acids; MUFA= monounsaturated fatty acids; PUFA= polyunsaturated fatty acids; ω -3- omega 3 polyunsaturated fatty acids; ω -6- omega 6 polyunsaturated fatty acids.
Characterisation of compound feeds
The compound feeds were developed on the basis of the chemical composition of the feed ingredients, being isonitrogenous and isocaloric for each feeding phase, in agreement with the feeding requirements of the Hubbard hybrid (Table 1). Due to the inclusion of 3% grapeseed meal, the compound feed of group E had a higher concentration of polyphenols of 1.68 mg GAE (growing phase), respectively 1.56 mg GAE (finishing phase), compared to the compound feed of group C 1.47 mg GAE (growing phase), respectively 1.36 mg GAE/g diet (finishing phase). Higher results were also determined regarding the antioxidant capacity of the E group compound feed of 3.09 mM TE (growing phase) and 2.82 mM TE (finishing phase), compared to C group which had 2.15 Mm TE (growing phase) and 2.01 mM TE/g diet (finishing phase).
Bird performance
Table 3 shows the effect of using a compound feed with 3% grapeseed meal on the growth performance of the broilers. The values for the entire experimental period (14- 63 d) were higher for group E, than for group C, for the final weight and feed conversion ratio. No mortalities were recorded.
Table 3 Effect of the compound feed with grapeseed meal on the broiler performance: 14- 63 d (mean values)
| Items | C | E | SEM | P-value |
|---|---|---|---|---|
| Average daily feed intake, g/broiler/d | 114.2a | 119.5a | 3.89 | 0.497 |
| Initial weight, g | 233.3a | 233.7a | 3.88 | 0.962 |
| Final weight, g | 2545a | 2728.5a | 48.03 | 0.056 |
| Average daily weight gain, g/broiler/d | 47.18a | 50.70a | 1.04 | 0.090 |
| Feed conversion ratio, g feed/g gain | 2.42a | 2.35a | 0.29 | 0.913 |
| Death rate, % | 0.00 | 0.00 | - | - |
C= Control; E= Control + 3% grapeseed meal; SEM= Standard error of the mean.
a-b Mean values within a row having different superscripts are different (P<0.05).
Metabolic profile of blood plasma
The metabolic profile of the blood plasma, collected at broiler age of 63 d (Table 4), shows significantly (P<0.05) lower energy profile in group E, than in group C, thus, glycaemia decreased by 16.88 %, cholesterol by 24.50 %, and the triglycerides by 34.90 %, in group E, compared to group C, the differences being statistically significant (P<0.05).
Table 4 Effect of compound feed with grapeseed meal on the metabolic profile of blood plasma in broilers of 63 d of age (mean values)
| Items | C | E | SEM | P-value |
|---|---|---|---|---|
| Glycaemia, mg/dL | 271.03a | 225.28b | 7.30 | <0.0001 |
| Cholesterol, mg/dL | 146.82a | 110.85b | 5.99 | <0.0001 |
| Triglycerides, mg/dL | 49.93a | 32.50b | 2.95 | <0.0001 |
C= Control; E= Control + 3% grapeseed meal: SEM= Standard error of the mean.
a-b Mean values within a row having different superscripts are different (P<0.05).
Fatty acid concentration in the broiler meat
The PUFA concentration in the breast meat samples (Table 5) was significantly (P<0.05) higher in group E, than in group C, by 13.02 %. The concentration of ω - 3 PUFA, and of ω - 6 PUFA increased by 15.00 %, and by 12.84 %, in group E, compared to group C (P<0.05). The alfa linolenic acid (ALA) concentration in the breast meat was 1.89 g, in group E, and 1.82 g / 100 g total FAME, in group C (P>0.05). The PUFA concentration in the leg meat samples (Table 5) was significantly (P<0.05) higher in group E, than in group C, by 7.91 %. The concentration of ω -3 PUFA was also higher in group E, than in group C, by 9.66 %, but the difference was not statistically significant (P>0.05). The ALA concentration in the leg meat was 2.56 g in group E, significantly (P<0.05) higher than 1.97 g/ 100g total FAME, in group C.
Table 5 Effect of compound feed with grapeseed meal on the fatty acids concentration in the broiler meat samples (mean values)
| Fatty acids (% of total FAME) | Breast meat | Leg meat | |||||||
|---|---|---|---|---|---|---|---|---|---|
| C | E | SEM | P-value | C | E | SEM | P-value | ||
| SFA | 32.48a | 32.33a | 0.21 | 0.736 | 30.97a | 29.29b | 0.33 | 0.027 | |
| MUFA | 39.09a | 35.83b | 0.54 | < 0.001 | 38.40a | 38.01a | 0.29 | 0.517 | |
| PUFA, of which: | 27.73a | 31.34b | 0.62 | 0.0002 | 30.06a | 32.44b | 0.62 | 0.049 | |
| ALA | 1.82a | 1.89a | 0.03 | 0.276 | 1.97a | 2.56b | 0.11 | 0.002 | |
| ω -3 | 2.80a | 3.22b | 0.08 | 0.003 | 3.00a | 3.29a | 0.11 | 0.205 | |
| ω -6 | 24.84a | 28.03b | 0.55 | 0.0002 | 27.06a | 29.14b | 0.52 | 0.044 | |
| Ratio ω-6 / ω-3 | 8.92a | 8.73a | 0.13 | 0.475 | 9.02a | 8.85a | 0.19 | 0.656 | |
FAME=fatty acid methyl esters; C=Control; E= Control + 3% grapeseed meal; SEM= Standard error of the mean.
SFA-saturated fatty acids; MUFA-monounsaturated fatty acids; PUFA- polyunsaturated fatty acids; ALA -α-linolenic acid; ω-3 -omega-3 polyunsaturated fatty acids; ω-6 - omega-6 polyunsaturated fatty acids;
a-b Mean values within a row having different superscripts are different (P<0.05).
Cholesterol level in the broiler meat
The cholesterol concentration in the breast meat samples (Table 6) were lower in group E, treated with 3 % grapeseed meal, than in group C, but the difference was not statistically significant (P>0.05). For the leg meat, the cholesterol concentration was 30.2 % lower in group E, compared to C (P<0.05).
Table 6 Effect of compound feed with grapeseed meal on the cholesterol level in the broiler meat samples (mean values), (mg / 100 g fresh meat)
| Breast meat | Leg meat | |||||||
|---|---|---|---|---|---|---|---|---|
| C | E | SEM | P-value | C | E | SEM | P-value | |
| 44.28a | 36.49a | 2.05 | 0.052 | 60.91a | 42.52b | 3.76 | 0.006 | |
C= Control; E= Control + 3% grapeseed meal; SEM= Standard error of the mean.
a-b Mean values within a row having different superscripts are different (P<0.05).
Fat degradation indices in the broiler meat
Regarding the fat degradation indices (Table 7), the peroxide value of the breast meat samples, determined after 7 d of refrigeration at +4 oC, was 8.11 meq/kg fat, in groups E, significantly (P<0.05) lower than 8.79 meq, in group C. However, after one month of freezing (-18 oC), their values were similar (P>0.05). Fat acidity was lower in group E, than in group C, both after 7 d of refrigeration (+4 oC), and after one month of freezing (-18 oC), but the difference was not significant (P>0.05).
Table 7 Effect of compound feed with grapeseed meal on the fat degradation indices in the broiler meat samples (mean values)
| Items | Breast meat | ||||
|---|---|---|---|---|---|
| Period | C | E | SEM | P-value | |
| Peroxide value, meq/kg fat | day 7-refrigeration | 8.79a | 8.11b | 0.12 | 0.0002 |
| Fat acidity, mg KOH/g fat | 36.42a | 36.44a | 0.01 | 0.955 | |
| Peroxide value, meq kg fat | 1 month- freezing | 5.08a | 5.15a | 0.02 | 0.161 |
| Fat acidity, mg KOH/g fat | 20.89a | 20.30a | 0.37 | 0.442 | |
| Leg meat | |||||
| Peroxide value, meq/kg fat | day 7-refrigeration | 11.25a | 11.10a | 0.13 | 0.589 |
| Fat acidity, mg KOH/g fat | 43.99a | 40.82b | 0.52 | <0.0001 | |
| Peroxide value, meq/kg fat | 1 month- freezing | 6.39a | 6.23a | 0.04 | 0.043 |
| Fat acidity, mg KOH/g fat | 24.23a | 23.72a | 0.26 | 0.410 | |
C= Control; E= Control + 3% grapeseed meal; SEM= Standard error of the mean.
a-b Mean values within a row having different superscripts are different (P< 0.05).
The peroxide value of the leg meat samples (Table 7) determined after 7 d of refrigeration (+4 oC), and after one month of freezing (-18 oC), was similar in both groups (P>0.05). However, fat acidity after 7 d of refrigeration (+4 oC), 40.82 mg KOH / g fat, in group E, was significantly (P<0.05) lower than 43.99 mg KOH/g fat, in group C. After one month of freezing (-18 oC), the decrease of fat acidity in group E was not statistically significant (P>0.05), compared to group C.
Discussion
The results on the flaxseed meal used as PUFA-high feed ingredient are in agreement with those of Panaite et al11, who reported values of: 32.99 % protein, 9.42 % fat, 19.31 MJ / kg gross energy, 11.07 g SFA, 18.71 g MUFA, 70.23 g PUFA, of which 42.93 g ω-3 PUFA and 27.30 g ω-6 PUFA / 100 g FAME, and ω -6 / ω -3 ratio of 0.64.
The concentration of polyphenols and the antioxidant capacity of the grapeseed meal, used as natural antioxidant, are in agreement with those of Turcu et al29, who studied the effect of 2 % grapeseed meal given to Ross 308 broilers and reported 28.08 mg GAE / g samples, and 145.83 mM Trolox equivalents / g sample, antioxidant capacity. The efficiency of the natural antioxidants depends on their chemical composition which, in turn, depends on the variety of grapes, soil type, agro-climatic factors and wine-making techniques38.
The use of 2 % flaxseed meal, high in PUFA, in the compound feeds for Hubbard broilers, produced close values of the fatty acids profile both in the growing and in the finishing phases. As expected, the use of 3 % grapeseed meal as natural antioxidant, in the formulation for group E, increased the concentration of polyphenols and the antioxidant capacity compared to the feed formulation for group C, in both stages. Thus, the concentration of polyphenols was 14.28 % higher and the antioxidant capacity was 43.72% higher for the growth stage, and 14.70 % and 40.29 % higher, respectively, for the finishing stage. These results are in agreement with those reported by Vlaicu et al28, who studied the effect of 2 % grapeseed meal given to Ross 308 broilers (growth stage) and reported 54.31% higher concentration of polyphenols. Broiler performance for the entire experimental period, show that the use of grapeseed meal in the compound feed formulation for group E did not have adverse effects on the broilers, the differences from the control group not being statistically significant (P>0.05).
The values of the blood plasma energy profile (glycaemia, cholesterol, triglycerides) (Table 4) were significantly (P<0.05) lower in group E, than in the control group. The lower blood cholesterol might be due to the flavonoid content of the grapeseed meal, which inhibits the formation of mycelia within the small intestine, thus reducing the absorption of the intestinal cholesterol39. The results in this study on the blood profile are in agreement with those reported by Abu Hafsa and Ibrahim40 who evaluated the effect of the dietary grapeseed powder (0; 10; 20 and 40 g/kg) given to Cobb 500 broilers. They reported decreases in the following blood parameters: glucose, from 188.42 mg/dL in group C, to 151.39 mg/dL, in the group with 40 g grapeseed powder; cholesterol, from 122.46 mg/dL, in group C, to 95.88 mg/dL, in the group with 40 g grapeseed powder; triglycerides from 67.46 mg/dL, in group C, to 60.75 mg/dL, in the group with 40 g grapeseed powder. Khodayari and Habib26 studied the effect of various levels of dietary grape pomace (0, 2, 4 and 6 %) given to Ross 308 broilers, on broiler performance, lipid peroxidation and blood biochemical parameters, and reported the decrease of blood triglycerides from 52.00 mg/dL, in group C, to 35.33 mg/dL, in the group with 6 % dietary grape pomace. The blood cholesterol decreased from 163.33 mg/dL, in group C, to 129.33 mg/dL, in the group with 6 % dietary grape pomace.
The fact that this study revealed lower values for blood glycaemia, cholesterol and triglycerides in group E, shows a better health state of those broilers, compared to the control group, due to the grapeseed meal added, as antioxidant, to the diet high in polyunsaturated fatty acids.
Broiler meat enhanced in PUFA, particularly in ω -3 PUFA, has established beneficial effects for consumer health4. Kamboh and Zhu41 shows changes in the proportion of fatty acids (decrease in SFA and increase in PUFA), when the broiler diets were treated with bioflavonoids.
The significantly (P<0.05) difference of the breast meat PUFA concentration in group E, can be explained by the fact that the dietary grapeseed meal, due to its antioxidant properties given by the content of polyphenols and flavonoids it slowed down the lipid degradation reactions42,43. Other work44 showed the positive effects of the winery by-products in preventing the oxidation of broiler meat PUFA. They studied the use of an extract of grape seeds and onion, alone or in combination with vitamin E, in the diet formulations for Ross broilers exposed to heat, and reported higher PUFA values in the broiler breast meat, compared to the control group. The significantly (P<0.05) higher concentration of ω -3 PUFA in the breast meat from group E, reported in this study, was also reported29. By the addition of 2 % flaxseed meal and 2 % grapeseed meal to Ross 308 broiler diet, the concentration of ω -3 PUFA was 11.14 % higher in the experimental group than in the control group.
The concentration of PUFA in the leg meat was significantly (P<0.05) different in group E than in group C. Just like for the breast meat samples, for the leg meat sample too, this can be explained by the fact that the dietary grapeseed meal, due to its antioxidant properties given by the content of polyphenols and flavonoids it slowed down the lipid degradation reactions42,43. Significantly (P<0.05) higher PUFA concentrations were also reported by 19.38 %, and 20.02 %, compared to the control group, in the groups of Ross 308 broilers treated with 2 %, and 4 %, respectively, grapeseed meal28. Other teams of researchers27, studied the effect of another winery by-product, the grape pomace, given to broilers in amounts of: 0 %, 5 % and 10 %, reporting increases of PUFA concentration in the broiler leg meat, from 37.0 g, in group C, to 47.4 g, in the group treated with 5 % grape pomace, and to 53.1 g / 100 g total fatty acids, in the group treated with 10 % grape pomace.
The alfa linolenic acid (ALA) is an essential fatty acid because it cannot be synthesized by the organism, the main source being the food. In this study, the ALA concentration in the broiler leg meat samples were significantly (P<0.05) higher in group E than in group C, as also reported by Chamorro et al27, who used grape pomace in the diet formulations for Cobb broilers.
As it is known, cholesterol is a component of fats, playing an important role in the formation of hormones and in vitamin metabolism. Cholesterol synthesis in the liver can be modified by the addition of derivatives of animal fats. Recently, the education and research in food safety increased consumer awareness regarding the health effects of the food cholesterol4 Nutrition can be used to modify the profile of the fatty acids, to decrease ω - 6 / ω - 3 ratio, and the cholesterol level10.
The cholesterol level in the broiler breast and leg meat samples was lower in group E, treated with grapeseed meal. However, only for the leg meat, the decrease was statistically significant: 30.19 %, may be due to high lipid content in leg meat. These results are in contrast with those reported by Yong et al45 who studied the effect of adding 0; 0.25 and 0.5 % grape meal to broiler diets on the cholesterol level in broiler meat. They observed that the cholesterol content of thigh and breast meat was not significantly affected by grape meal supplementation. The antioxidant effect of the grapeseed extract was investigated by Farahat et al46, who used it in concentrations of 125 ppm to 2,000 ppm, in broiler diets, and reported lower values of the total cholesterol than in the broilers treated with synthetic antioxidant, BHT.
The lipid degradation indices showed lower values in the breast and leg meat from group E, both after 7 d of refrigeration and after one month of freezing. However, the decrease was significant only for the 7 d of refrigeration: the peroxide value of the breast meat sample was 7.73 % lower, compared to group C, and fat acidity of the leg meat was 7.21 % lower, compared to group C. Olteanu et al47 reported similar effects, i.e., lower values of fat acidity after 7 d of refrigeration of the meat samples from Cobb 500, treated with 2 % grapeseed added to PUFA-high diet formulations, but the differences from the control group were not statistically significant. The available data from the literature include various information on the ability of grape meal to slow down the lipid peroxidation processes of broiler meat. Chamorro et al27 studied the effect of the dietary grape pomace, and reported increasing oxidative stability of the broiler leg meat after 1 and 4 d of refrigeration, with the increasing dietary grape pomace by MDA decreasing levels. The obtained results showed significant lower differences (P<0.05) from 0.217 µg (C) to 0.112 µg (5 % GP) and 0.114 µg/g meat (10% GP) after 4 d of refrigeration. Kasapidou et al48 concluded that the dietary grape pomace (2.5 % and 10 %) did not influence lipid oxidation of the broiler meat (breast and thigh) samples during 2 - 5 d of refrigeration. Also, it was found that antioxidants improved meat quality of Japanese quail and delayed oxidative rancidity49.
The results obtained in this study show that the grapeseed meal, a winery by-product with a lower price, can be used as natural and non-toxic antioxidant in broiler diets, replacing the synthetic antioxidants like butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA)45
Conclusions and implications
The inclusion of 3% grapeseed meal, as natural antioxidant, in Hubbard broiler diets high in polyunsaturated fatty acids did not affect broiler performance. The feeding quality of the broiler meat (breast and leg) was improved by the higher levels of total PUFA and omega - 3 PUFA, and by the lower cholesterol level. The health state of the broilers was also enhanced by the lower concentrations of cholesterol and triglyceride in the blood. These results highlight the antioxidant properties of the plant additive from the winery industry. The grapeseed meal added to broiler diets high in polyunsaturated fatty acids allowed the production of safe food of beneficial impact on human health.
