Effect of Botanical Extracts on Amino Acid and Fatty Acid Profile of Broiler Meat

The objectives of current study were to evaluate the effect of botanical extracts as antioxidants on amino – and fatty acids profile of broiler meat. In total, 200 one-day-old broiler chicks were divided according to a completely randomized design into eight dietary treatments with five replicates of five chicks each. Dietary treatments were: A positive control (PC) with antibiotics and synthetic antioxidants, B negative control (NC), C NC+Trigonellafoenum-graecum, D -NC+Nigella sativa seed, E -NC+Violaodorata, F -NC+Trachyspermumammi, G -PC+Trachy spermumammi+Trigonellafoenum-graecum+Violaodorata, and H -NC+ Trachyspermumammi, Trigonellafoenum-graecum and Viola odorata. Meat amino – and fatty acid contents were analyzed after slaughter at 35 days of age. Data were submitted to one-way analysis of variance and means were compared by Duncan’s Multiple Range test. The levels of histidine, isoleucine, leucine, lysine and threonine were significantly higher (p≤0.05) in the H, F, E, H and F groups respectively. Arginine, tryptophan, valine, methionine, and phenylalanine were significantly higher (p≤0.05) in the C, F, E, G and H groups. Total essential amino acid levels were highest (40.36%) in C group (p≤0.05). The meat of H broilers presented significantly higher (p≤0.05) unsaturated fatty acids (54.8%) and omega-3 contents (2.77%) and the lowest saturated fatty acid content (44.8%). Group F present the highest linoleic acid and omega-6 levels and group H the highest (p≤0.05) arachidonic and eicosapentaenoic acid (EPA) contents. It was concluded that botanical extracts aid health-beneficial meat production and the simultaneous inclusion of Trachyspermumammi, Trigonellafoenum-graecum, and Viola odorata in broiler diets is a more suitable option than the other dietary regimes.


INTRODUCTION
The poultry industry plays a vital role in supply of healthy meat products to the public.Broiler meat production from broiler is one of the major food-producing enterprises globally.Although broiler exhibits relatively fast growth rate and heavy body size, the nutritional profile of its meat has been criticized under conventional human dietary regimes (Fanatico et al., 2007).
The breast and thigh are the preferred parts of consumers of various regions.However, the chemical composition of meat fat, including its amino acid and fatty acids profile, have been currently intensively researched due to their direct association with cardiovascular diseases in humans.The accretion of amino acids like lysine in skeletal satellite cells in the muscle is largely influenced by the activation of insulinlike growth factor (IGF-1) (Tesseraud et al., 2003).Plant extracts, like that of Aloe vera, can enhance muscle integrity by elevating IGF-1 (Muhammad et al., 2013).It was demonstrated that curcumin extracts enhance the growth performance of pigs by regulating the gene expression of IGF-1 and up regulating the activity of antioxidant enzymes (Upadhaya et al., 2016).Muscular health and growth are well regulated by curcumin feeding (Alamdari et al., 2009).Botanical extracts can aid muscle regeneration and differentiation, reduce muscle atrophy and enhance anti-inflammatory responses (Rondanelli et al., 2016).
Fatty acid composition of dietary fats can affect saturated fatty acids (SFA) as well as unsaturated fatty acids (UFA) levels in broiler breast and thigh muscles (Zhang et al., 2013).Fat sources rich in UFA can be included in broiler diets to produce leaner meat with much better ω-3 profiles.The desired ω-3 fatty acids, such as linolenic acid (LNA), eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA), in the breast and thighs of chicks can be achieved by using omega 3 rich oils (Sadeghi et al., 2012).Unfortunately, UFA-rich oils are subjected to faster oxidative stress and the inclusion of rancid fats with high peroxide value can impair broiler performance and severely affect immunity (McGill et al., 2011).
The dietary addition of antioxidants can improve broiler performance and immunity by reducing thiobarbituric acid levels in liver and increasing serum and liver vitamin A and E concentrations (Taverez et al., 2011).The supplementation of broiler feeds with plant-derived rich in antioxidant compounds enhance oxygen scavenging responses to safeguard fat sources, both outside and inside body.This effect of natural antioxidants reflects results in a decline in cholesterol and triglyceride levels (Ahmed et al., 2015).Phenolic compounds like quercetin and vitamin E decrease fatty acid generation, particularly of SFA (Sohaib et al., 2015).Antioxidant-rich diets potentially inhibit saturated fatty acid levels is by modulating the activity of 9-desaturase enzyme complex, which converts SFA into UFA (Gnoni et al., 2009).A researcher group documented a declining trend in SFA production in the breast muscle of broilers fed gallic acid and linoleic acid (Jung et al., 2010).Similarly, the inclusion genistein and hesperidin in broiler diets reduced SFA level in the breast meat (Kamboh & Zhu, 2013).Black cumin seeds can be used up to 1.5% in layer feed to reduce egg cholesterol and enhance UFA content in egg yolk (Yalc et al., 2009).Chia seed in diet also effective in controlling fat content in meat and significantly lowering SFA content eventually having better SFA: UFA and ω-6:ω-3 ratio in muscles (Ayerza et al., 2002).Feeding layers with black cumin seeds decreased yolk triglyceride content and cholesterol level in up to 40% (Nabiela et al., 2006).
Recent studies have shown that nutrition has a major influence on tissue gene expression.Dietary antioxidant supplements from plant sources can enhance the quality of meat by minimizing oxidative stress on lipids and protein molecules and enhancing the activities of glutathione peroxidase, catalase, and superoxide dismutase, ultimately improving the amino acid and fatty acid profile of meat (Upton et al., 2009).Due to their potential health benefits, consumers are willing to buy meat from broilers fed botanical extracts (Rizzo et al., 2008).After reviewing the potentials of natural extracts, this study aimed at evaluating the effects of the dietary inclusion of botanical extracts on the amino acid and fatty acid profiles of broiler meat.

Preparation of
Trachyspermumammi, Trigonellafoenum-graecum, Nigella sativa and Viola odorata extracts Dried flowers of Viola odorata (sweet violet) and seeds of Trigonellafoenum-graecum (fenugreek), Nigella sativa (black cumin) and Trachyspermumammi (Ajwain) were purchased from local sources.Seeds were ground to 30-mm particle size.The processes of sonication and extraction were performed according to the methodology proposed by Kim & Lee (2002).Briefly, 200 g of each sample were soaked in 2-L flask with 1 L of 80% methanol.After the sonication for 24 hours, samples were filtered, and again submitted to sonication for 24 hours.Filtrates were transferred to round bottom flasks on a rotary evaporator at 40°C and allowed to evaporate until the 300 mL of the contents remained, which then were standardized to 400 mL by adding distilled water.A total of 200 one-d-oldRoss-308 broilers were divided into eight treatments with five replicates of five birds each according to a completely randomized design.Bird were reared on litter at a stocking density Tarar OM, Yaqeen Z, Ali TM

Effect of Botanical Extracts on Amino Acid and Fatty
Acid Profile of Broiler Meat 0.6ft 2 /bird for a period of 35 days.A photoperiod of 22 hours of light and 2 hours of darkness was applied.
Isocaloric (23, 21.12 and 20% crude protein) and isonitrogenous (3000, 3300 and 3200 kcal/kg) diets (starter, grower and finisher respectively) were formulated according to recommendations of the Ross-308 manual using ingredients available in the market.The software FeedLive was used for diet formulation (Table 1).H: NC + supplemented with ajwain, fenugreek and sweet violet extracts, each at 0.5 mL/kg feed.

Parameters evaluated
At 35 days of age, birds were slaughtered, and eviscerated.Carcass yield (%) was calculated by the following equation.

= × Carcass
Carcass weight Live weight % 100 Breast and thighs were cut up from the carcass using sharp knives and their yields (%)were calculated on whole carcass basis using following equations.

Amino Acid Analysis
Breast and thigh meat samples were collected to determine their amino acid profile.Briefly, 50-mg samples were hydrolyzed for 18 h in 6N HCl.The sample container was sealed to prevent oxidation and stored at 110°C for hydrolysis.The amino acid composition of Tarar OM, Yaqeen Z, Ali TM

Effect of Botanical Extracts on Amino Acid and Fatty Acid Profile of Broiler Meat
the meat samples all birds fed the A, B, C, D, E, F, G and H diets was determined according to the method of Schuster (1988) using an amino acid analyzer.Samples were extracted in citrate buffer (2.2 pH) solution followed by filtration to obtain a clear solution of the free amino acids.An aliquot of this clear filtrate was injected into the column (Shim-pack ISC-07/S1504 Na) of the HPLC-based amino acid analyzer (RF-10AXL, Shimadzu Corporation, Tokyo, Japan) equipped with fluorescence detector (FLD-6A).Sodium hypochlorite and o-phthalaldehyde solutions were used as reaction solutions for post-column derivation of amino acids.

Fatty Acid Analysis
For fatty acid analysis, fats were hydrolyzed and fatty acids were derived from total fat content according to the procedure of O'Fallon et al. (2007).Briefly, 50 µL of fat/oil was placed in a Pyrex screw cap tube, and 1 mL of internal standard, 0.7 mL KOH solution and almost 5.3 mL of methanol were added and mixed.Tubes were capped and placed on a preheated water bath at 55°C for 90 minutes.The tubes were then cooled in water followed by H 2 SO 4 addition in the samples.The tubes were then again kept in water bath at 55°C for 1.5 hours, followed by cooling and addition of 3 mL of hexane.Tubes were then placed into a vortex mixer for 5 minutes.The fatty acid methyl ester (FAME) hexane layer (upper layer) was separated, filtered and then analyzed.Gas chromatography was performed in GC-2010 apparatus (Shimadzu Corp., Japan) 07947 equipped with FID detector, split injector and SP-2560 silica fused capillary column (100m x 0.25mm x 0.2µm, Supelco) under the following operating program: injection volume 1 µL at 250°C, detector temperature of 260°C, column temperature of 140°C for 5 minutes and then ramped to 240°C at 4°C per minute, remained stable for 15 minutes.Helium was used as carrier gas at flow rate of 1.12mL/ min and linear velocity of 20 cm/s; split ratio 1:100.
Results were expressed as percentages relative to FID response area.

Statistical Analysis
The collected data were subjected to one-way analysis of variance (Steel et al., 1997)using the SPSS Statistics 20.0 software (IBM Deutschland GmbH, Ehningen, Germany) and means were compared using Duncan's Multiple Range (DMR) test (Duncan, 1955) following the statistical model: Where, Y ij = Observation of dependent variable recorded on i th treatment µ = Population mean Ʈ i = Effect of i th treatment (i = 8 i.eA, B, C, D, E, F, G and H) Ԑ ij = Residual effect of j th observation on i th treatment NID ~ 0, σ 2

Effect of Botanical Extracts on Amino Acid and Fatty Acid Profile of Broiler Meat
have been used to promote muscle health, strength and growth (Nkukwana et al., 2014).Similarly, scientific reviews on the effects of botanical extracts on muscle health (Rondanelli et al., 2016) describe their positive role in muscle growth and integrity.

Essential amino acid profile of broiler meat
The meat contents of essential amino acids, including histidine, isoleucine, leucine, lysine and threonine were significantly (p≤0.05)lower in the negative control group (diet B) compared to all other treatments (Table 3).The breast meat of group H birds presented the highest (p≤0.05)histidine (4%) levels (Table 3).Isoleucine level (4.5%) was significantly (p≤0.05) in the meat of birds fed ajwain extracts (diet F) and leucine (7%; p≤0.05) in the meat of those fed sweet violet extract (diet E).The highest lysine level (9.1%) was achieved with diet F and the lowest with the negative-control diet (B) (Table 3).Threonine (4.2%) levels were higher better (p≤0.05) in the meat of broilers fed ajwain extracts.The breast meat of birds fed fenugreek extract (diet C) presented higher arginine (5.9%; p≤0.05) compared with the other treatments (Table 3).Meat tryptophan values (Table 3) were significantly higher (p≤0.05) in the F-group broilers (1.2%) and valine (4.1%; p≤0.05) in E-group broilers compared with all other treatments.Table 3 shows higher meat methionine content in group G and phenylalanine in group H, both significantly higher (p≤0.05)than in the other groups.The meat of broilers fed sweet violet extract (diets contained the low esttryptophan, methionine and phenylalanine levels (Table 3).The breast meat of group C broilers presented the highest sum of all essential amino acids (40.36%) followed by group H (40.19), both of which were significantly higher (p≤0.05)than other groups.The essential amino acid analysis of the broilers fed negative control diet showed that it contained lower values of many essential amino acids compared with those fed the other tested diets.The feeding of botanical extracts to broilers in general increased the individual levels of essential amino acids, and consequently the sum of all essential amino acid.
The overall improvement of essential amino acid contents in the meat of broilers fed the plant extracts suggest the potential benefits of their inclusion in broiler diets.These results may be due to the presence of bioactive compounds in the evaluated herbs.The findings of the current study are supported by reports of the presence of sulfur and non-sulfur compounds in Allium sativum (Amagase et al., 2001).Some of

Non-essential amino acid profile of broiler meat
There was a significant (p≤0.05)increase in nonessential amino acid levels (46%) in the breast meat of birds fed sweet violet extract (diet E), as shown in Table 4.The meat of broilers fed the E diet meat presented higher proline (4.5%), glycine (6.8%) and alanine (8.2%) contents compared with the other treatments (p≤0.05).The meat from birds fed ajwain extract had significantly (p≤0.05)higher tyrosine (3.6%), cysteine (1.4%) and total amino acid values (80.81%) than that of the other treatments (Table 4).Diet D promoted significantly higher (p≤0.05)aspartic acid values (14.5%) as compared with the other diets (Table 4).The lowest values (p≤0.05) of tyrosine, aspartic acid and, consequently, of total non-essential amino acids were obtained in the meat of broiler fed the G diet (Table 5).
Higher non-essential amino acids levels were obtained in the meat of broilers feds weet violet extract, and the combination of fenugreek, ajwain and sweet violet extracts resulted in a decline in meat non-essential amino acid content.Total amino acids contents were better in all groups supplemented with antioxidants from plant-derived sources.The process of muscle growth and protein accretion in broilers was best described by Tesseraud et al. (2003), who explained that the accretion of amino acids like lysine in skeletal muscle satellite cells is greatly influenced by the activation of insulin-like growth factor (IGF-1).Antioxidant-rich botanical extracts from plants like Aloe vera boost the biological role of IGF-1 to modulate energy kinetics, myoglobin accretion and muscular integrity (Muhammad et al., 2013).Muscle health and growth may be regulated by feeding curcumin, which was shown to improve broiler growth performance by regulating IGF-1 gene expression and enhancing the activity of antioxidant enzymes (Upadhaya et al., 2016;Alamdari et al., 2009).Recently, Rondanelli et al.,(2016) described that botanical extracts may aid muscle regeneration and differentiation, reduce muscle atrophy, and demonstrated it has antiinflammatory and anti-fatigue properties, preventing muscle damage.

Meat Fatty Acid Composition
The results of the meat fatty acid analysis are shown in Table 5. Pentadecanoic acid (C15-1) levels of 18.68% and 20.94% were determined in the B and H groups, respectively, and both were significantly (p≤0.05)higher than all other groups.Palmitic acid (C16) level was highest (18.21%) in the broilers fed the negativecontrol diet (group B) and lowest in those group H (9.09%), both at p≤0.05.The highest (p≤0.05)stearic acid values were obtained in group H (33.8%) and the lowest (p≤0.05) in Groups A (30.61%) and F (30.06%).Oleic acid levels were significantly (p≤0.05)higher in group G (22%) and lower in group B (13.93%), respectively, than in the other groups.Linoleic acid (C18-2n6) values obtained were significantly (p≤0.05)higher in group F and lower in group B (0.21%) than in the other groups.Arachidonic acid (C20) was significantly higher (p≤0.05) in group H chicken fats (0.44%) compared with other groups.Eicosenoic acid (C20-1n9) values in groups C (11.78%) and D (11.93%) were higher (p≤0.05)compared with the other groups.The meat of broilers in group G presented higher eicosatrienoic acid (C20-3n6) levels compared with the other treatments (p≤0.05).The highest eicosapentaenoic acid (EPA: C20-5n) was determined in group H (2.63%; p≤0.05).The highest (p≤0.05)behenic (C22) and docosadienoic (C22-2) acids levels were obtained in broilers fed ajwain extracts.The meat of the broilers fed the negative control diet presented the highest saturated fatty acid (SFA; 55.76%) levels (p≤0.05), and the lowest unsaturated fatty acid (UFA; 41.47%) and polyunsaturated fatty acid (PUFA; 2.24%) levels compared with other groups.Group H presented had significantly higher (p≤0.05)UFA (54.8%) and mono-unsaturated fatty acid (MUFA; 50.71%) levels compared with other all groups (Table 5).Maximum PUFA was observed in group F samples (4.59%) followed by group H (4.5%) and both were significantly better than other groups.Higher omega-3 fatty acid levels were obtained in groups F, G and H. Group H presented the highest omega-3 fatty acid level (2.77%) compared with the other groups (p≤0.05), which was almost three-fold higher than that obtained with the positive control diet (0.91%).The meat of broilers in groups C, D, E, F and G presented 1.04%, 1.0%, 1.22%,1.38%,and 1.43% omega-3 levels, respectively, which were significantly higher than obtained with the positive control diet (p≤0.05).The highest and the lowest omega-6 levels were obtained in groups F (0.68%) and B (0.21%), respectively, both were significantly different from the other groups (p≤0.05).
The inclusion of botanical extracts in broiler diets in the present study resulted in a remarkable increase in the meat levels of unsaturated fatty acids and a clear decline in saturated fats.The meat of the broilers Birds were reared at Research and Development Farm of Sindh Feed, Pvt.Limited, Pakistan.The laboratory analysis of the meat and eggs were performed at the Department of Food Science & Technology, University of Karachi and Pakistan Council for Scientific & Industrial Research (PCSIR) Laboratories Complex, Karachi, Pakistan.
within a column with different lowercase letters are significantly different at (p≤0.05).Tarar OM, Yaqeen Z, Ali TM

Table 2 -
Carcass yield of broilers fed botanical extracts

Table 3 -
Essential Amino acid profile of meat from broilers fed botanical extracts

Table 4 -
Non-Essential Amino acid profile of meat from broilers fed botanical extracts