The Impact of Dietary Linum usitatissimum and Moringa oleifera as Feed Additives on the Fatty Acid Profile, Oxidative Stability, and Composition of Broiler Meat

ABSTRACT

An experiment was conducted to evaluate the effects of supplementing Linum usitatissimum (flaxseed) with Moringa oleifera leaf (moringa) on the carcass quality, fatty acids profile, and oxidative stability of broiler chicken. A total of 240 one-day-old mixed-sex Cobb 500 broilers were assigned to the following four dietary treatments: T₁ - Control diet; T₂ - Flaxseed 0.5% + Moringa 1%; T₃-Flaxseed 1%+ Moringa 1%; and T₄-Flaxseed 1.5% + Moringa 1%. The final body weight gain was similar in all groups after 35 days, with significantly (p<0.05) reduced feed intake in T₄ compared to the control. The thiobarbituric acid reactive substances (TBARS) values of the meat were reduced in the flaxseed and moringa groups (p<0.05) compared to the control. Cholesterol content in thigh meat decreased significantly (p<0.05) in groups T₃ and T₄ compared to the control. In breast and thigh meat, the omega 3 fatty acids were higher, alongside significantly increased polyunsaturated fatty acids, and a lower omega 6 to omega 3 ratio (except in the T₂ group) was observed in the thigh meat of the flaxseed and moringa groups (p<0.05). Overall, the results indicate that diets with up to 1.5% flaxseed and 1% moringa improved meat composition and omega 3 fatty acids and reduced the lipid oxidation of broiler meat.

Keywords:
Broiler chicken; flaxseed; moringa; fatty acid; cholesterol

INTRODUCTION

The poultry industry is one of the fastest-growing animal industries globally. The most desirable aspect of poultry is its sought-after nutritional characteristics, such as high protein, low fat, and relatively high concentrations of polyunsaturated fatty acids (PUFAs) compared to beef or pork (Brenes & Roura, 2010). Red meats such as beef, mutton, and lamb contain larger amounts of saturated fat and cholesterol than white meat (broiler meat). It has been reported that regular red meat consumption is linked to problems such as cardiovascular conditions, stroke, obesity, and type 2 diabetes (Pan et al., 2011; Pan et al., 2012). In recent years, consumption patterns have globally shifted from red to white meat due to the latter’s unsaturated fatty acids (Daniel et al., 2011). PUFAs and omega 3 fatty acids can decrease the risk of heart disease and promote neural development in humans.

Several studies have been conducted to increase the content of PUFAs in chicken meat and eggs by using dietary fat sources such as natural oils containing PUFAs (Kim et al., 2007). Foods containing higher amounts of PUFAs are considered functional and beneficial for the prevention of multiple chronic diseases (Russo, 2009). The beneficial effects of consuming omega 3 fatty acids include reducing heart disease and circulating cholesterol levels, and suppressing inflammation in humans (Puvača et al., 2014). Enriching omega 3 fatty acids in broiler chicken meat is one of the easiest approaches used to incorporate omega 3 PUFAs into standard diets for human nutrition. Many herbs, spices, plant extracts and their essential oils have bioactive compounds, which are divided into primary and secondary metabolites. These metabolites include flavonoids, polyphenols, polypeptides, alkaloids, and saponins, which help inhibit oxidative rancidity, delaying off-flavor development in food and food products (Puvača et al., 2014). The use of safe, non-toxic, and bioactive compound feed additives free of antibiotic residues is currently gaining popularity as an approach to improving broiler performance, since their nutrient contents enhance feed consumption and promotes the intestinal health of broiler chickens (Hashemi & Davoodi, 2010).

Linum usitatissimum L. (flaxseed), known as a functional food, is one of the oldest cultivated crops, widely grown for food, fiber, and oil. Its oil is an excellent source of the omega 3 fatty acid alpha linolenic acid (ALA). It is used in poultry production in the United States to increase the ALA content of commercial table eggs and meat products (Samman et al., 2009; Petrovic et al., 2012; Lopes et al., 2013). Fraeye et al., (2012) have reported that flaxseed effectively converts ALA in poultry meat and eggs due to its high concentration and bioavailability. In its turn, Moringa oleifera (moringa) is the most widely cultivated species of the Moringaceae family in certain Asian countries such as India, Pakistan, Bangladesh, and Afghanistan (Sreelatha & Padma, 2009). Moringa is reported to contain various amino acids, fatty acids, vitamins, and other nutrients (Anwar et al., 2007). The most commonly used part of the plant are its leaves, which are used in many countries as traditional medicine for common diseases. The leaves are rich in vitamins, carotenoids, alkaloids, glucosinolates, isothiocyanates, tannins, and saponins (Leone et al., 2015). ). In addition, moringa leaves are a rich source of natural antioxidant compounds, such as ascorbic acid, flavonoids, phenolics, and carotenoids (Anwar et al., 2007). Moringa leaves have been used to enhance growth performance, feed efficiency, and meat quality in broiler chickens (Sarker et al., 2010; Sharmin et al., 2020).

Adding flaxseed and moringa to poultry diets as feed additives is a notable strategies to increase PUFAs and reduce lipid oxidation in meat. It has been hypothesized that flaxseed and moringa leaf powder ingredients would impart an improved rate of omega 3 fatty acid deposition and oxidative stability to meat with no adverse effects on growth performance. Our previous research with natural feed additives conducted at the Bangladesh Livestock Research Institute suggests that these kinds of additives promotes excellent physiological activity in broiler meat. Therefore, the objectives of the present study were to evaluate the feeding effect of different combinations of flaxseed and moringa on the performance, fatty acid profiles, and oxidative stability of broiler meat.

MATERIALS AND METHODS

Ethical Approval

The experimental protocols, bird management, and sample collection were reviewed and approved by the Animal Care Committee of the Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh (BLRI/SPRDP Project/2019).

Preparation of L. usitatissimum Meal with M. oleifera

Flaxseed (L. usitatissimum) was collected from a local market in Savar, Dhaka, Bangladesh, and subsequently ground into powder using a milling machine. It was then tightly packed in polythene plastic bags, sealed, and kept at room temperature until required. Fresh moringa (M. oleifera) leaves were collected and air dried during the daytime. After four to five days of drying, the leaves were ground to a fine powder. The leaf meal was tightly packaged in polythene plastic bags and kept at room temperature until required. Flaxseed and moringa were analyzed in triplicate for their crude protein, ether extract, moisture, and ash contents as described by the Association of Official Analytical Chemists (2000). The proximate compositions of these additives are presented as supplementary information (S1).

Bird Management and Dietary Treatments

A total of 240 one-day-old mixed-sex Cobb 500 broiler chicks were purchased from Nourish, a commercial hatchery. The chicks were weighed and randomly allocated to sixteen floor pens with four replications, all of which contained rice husk to a depth of 5 cm and were located within an environmentally controlled shed. The experiment was divided into four dietary treatments with four replicates of sixty chicks each. Pens (15 birds) served as the experimental units. The internal temperature of the broiler house was set and maintained at 34 °C for the first week, after which it was gradually reduced to 23 °C at a rate of 3 °C per week, and then maintained at this temperature until the end of the experimental period. The standard lighting program was maintained following Cobb 500 broiler management guidelines (COBB, 2018). The chicks under the trial were vaccinated against the Newcastle disease virus using a commercial vaccine, which was administered through eye drops and drinking water respectively on days four and eighteen of the experimental period. The chicks were inspected daily, and dead birds were removed following mortality recording (pen, date, and body weight [BW]). During the 35-day rearing period, feed and fresh water were provided on an as-needed basis. Feeders and drinkers were cleaned daily to ensure a hygienic supply of water and feed. The birds were managed as per the Cobb manual, and the basal diet was formulated according to the suggested nutrient requirements for commercial Cobb 500 broiler feed. The experimental diet (Table 1) was formulated in mash form and maintained throughout the feeding trial (COBB, 2018). Four dietary treatment groups were produced from the basal feed, as follows: T₁-control diet; T₂-flaxseed 0.5% + moringa 1%; T₃-flaxseed 1% + moringa 1%; and T₄-flaxseed 1.5%+ moringa 1%. The level of moringa inclusion in this experiment was fixed at 1% because, in our previous research, moringa at 1% showed a better performance than other inclusion levels, and we hypothesized that its addition with flaxseed could improve the oxidative stability of broiler meat (Sharmin et al., 2020). Proximate analyses for moisture, crude protein, ash, and ether extract were performed on the experimental diets according to the methods of the Association of Official Analytical Chemists (2000). The metabolizable energy content of the diets was calculated based on the nutrients determined in the proximate analyses. Feed allocation and BW per pen were recorded at placement, as well as at weekly intervals, i.e., seven, fourteen, twenty-one, twenty-eight, and thirty-five days. BW (g) was calculated as final BW minus initial BW; and average daily gains (g) were calculated accordingly. Feed intake (g) was calculated as feed allocated minus feed refused, and the feed conversion ratio (FCR) was calculated as the feed intake (g) per BW (g) on a pen weight basis. Experimental birds were reared in the research shed of the Bangladesh Livestock Research Institute at Savar, and biosecurity measures were strictly followed during the experimental period. The experiment was also subjected to an assessment of broiler chicken health status for its ethical acceptability and was approved by the Ethics Committees of the Bangladesh Livestock Research Institute, Savar, Dhaka.

Table 1
Feed ingredients used for the basal broiler starter (0 to 3 wk) and finisher (4 to 5 wk) diets, with the nutrient composition of the treatments.

Ingredients (%)	Starter diets (0 to 3 wk)				Finisher diets (4 to 5 wk)
	T1	T2	T3	T4	T1	T2	T3	T4
Maize	53.5	53.5	53.5	53.5	56	56	56	56
Rice polish	14	14	14	14	14	14	14	14
Soybean meal	17	17	17	17	15	15	15	15
Soybean oil	2.2	2.2	2.2	2.2	2	2	2	2
Fish meal	5.3	5.3	5.3	5.3	5	5	5	5
Dicalcium phosphate	1.25	1.25	1.25	1.25	1.25	1.25	1.25	1.25
Salt	0.3	0.3	0.3	0.3	0.30	0.30	0.30	0.30
1Vit-min premix	0.25	0.25	0.25	0.25	0.25	0.25	0.25	0.25
Lysin	0.1	0.1	0.1	0.1	0.10	0.10	0.10	0.10
Methionine	0.1	0.1	0.1	0.1	0.10	0.10	0.10	0.10
Protein concentrate	6	4.5	4	3.5	6	4.5	4	3.5
Flaxseed	-	0.5	1	1.5	-	0.5	1	1.5
Moringa		1	1	1	-	1	1	1
Total	100	100	100	100	100	100	100	100
ME (Kcal/kg)	2945	2950	2944	2952	3055	3050	3034	3045
Moisture	10.22	10.8	11.11	10.61	10.22	10.8	11.11	10.61
Crude protein	21.49	20.65	21.68	20.28	19.49	19.65	19.68	19.28
Crude fat	8.09	9.53	9.31	7.67	8.09	9.53	9.31	7.67
Crude fiber	3.45	3.61	3.7	3.75	3.45	3.61	3.7	3.75
Crude ash	8.73	7.77	7.85	8.03	8.73	7.77	7.85	8.03

• ¹
  Vitamin-mineral mixture provided the following nutrients per kilogram of diet: vitamin A, 15,000 IU; vitamin D₃, 1,500 IU; vitamin E, 20.0 mg; vitamin K₃, 0.70 mg; vitamin B₁₂, 0.02 mg; niacin, 22.5 mg; thiamine, 5.0 mg; folic acid, 0.70 mg; pyridoxine, 1.3 mg; riboflavin, 5 mg; pantothenic acid, 25 mg; choline chloride, 175 mg; Mn, 60 mg; Zn, 45 mg; I, 1.25 mg; Se, 0.4 mg; Cu, 10.0 mg; Fe, 72 mg; Co, 2.5 mg. ME (Kcal/kg)- Metabolizable Energy. T₁-Control diet; T₂-Flaxseed 0.5% + Moringa 1%; T₃-Flaxseed 1% + Moringa 1% ; T₄-Flaxseed 1.5% + Moringa 1%.

Slaughter Procedure

At the age of thirty-five days, forty-eight birds per treatment (three per replicate) were randomly selected and individually weighed. Birds were then fasted for eight hours with water offered ad libitum, reweighed and subsequently slaughtered. After bleeding, scalding, plucking, and washing, the feet, heads, necks, and skin were removed. Then, the carcasses were manually eviscerated and cut into wings, breasts, drumsticks (legs), and thighs. The visceral organs and cuts were then weighed individually, and the yields and carcass weight (CW) dressing percentage (CW/BW) were calculated. The breast and thigh muscles were separated from the bones and skin, and were trimmed of adjacent fat and connective tissue. The breast and thigh meat samples from each bird were ground separately using a meat grinder. The samples were subsequently divided into three parts: one for the oxidative stability analysis, another for the proximate analysis, and the third for fatty acid composition and cholesterol analysis. Finally, the samples were kept in zipper bags, after which those selected for oxidative rancidity analysis were refrigerated at 4 °C, while the other samples were stored at -20 °C.

Determination of Fatty Acid Profiles

A direct approach for fatty acid methyl ester (FAME) determination was used to assess the fatty acid contents of breast and thigh meat. The method was a slightly modified version of the one described by O’Fallon et al. (2007). In short, 1 g of minced meat was kept into a 15-mL Falcon tube, followed by the addition of 0.7 mL of 10 N KOH in water and 6.3 mL of methanol. The tube was then incubated in a 55°C water bath for 1.5h, with vigorous hand shaking for 10 s every 30 min to complete spreading, dissolve, and hydrolyze the sample. 0.58 mL of 24 N H₂SO₄ in water was added after cooling in a cold tap water bath. Following inversion mixing of the tube, K₂SO₄ precipitated. The precipitate-containing sample was once more incubated for 1.5 hours at 55°C in a water bath while being vigorously shaken by hand for 10 s every 30 minutes. The tube was chilled in a cold-water bath following FAME synthesis. The tube was then vortex-mixed for 5 min using a multitube vortexer after 3mL of hexane were added. Following a 5 min centrifugation at 3000 g, the top layer (hexane) containing the FAME was dehydrated using anhydrous Na₂SO₄. The extracted and dehydrated hexane was concentrated to 1.5 mL for analysis and put into a GC vial.

The fatty acid composition of the FAME was determined using a Gas Chromatograph (Agilent, 8890A series, USA) equipped with a flame ionization detector and a Hewlett Packard HP-88 capillary column (J&W Scientific, USA), with 60 m of length, 0.25 mm of internal diameter, and 0.20 µm of polyethylene glycol-film thickness. Samples were injected by an auto-sampler (Agilent Technologies 8890, USA). The initial oven temperature was 100 °C, which was held for 1 min, then increased to 175 °C at a rate of 3 °C/min, where it was held for 5 min, and then further increased to 220 C at a rate of 3 °C/min, where it was held for 3 min. Purified air and H₂ were applied at flow rates of 400 mL/min and 40 mL/min, respectively, as the carrier gas, while N₂ was applied at 30 mL/min as the makeup gas. Both the injector and detector temperature were set at 260 °C and the split ratio was 30:1. Fatty acids were identified by comparison of their retention times with those of a standard FAME mixture (Supelco™ 37 Component FAME Mix, 10 mg/mL in CH2Cl2. Catalog Number 47885-U. Supelco, North Harriso Road, Bellefonte, PA 16823-0048, USA).

Determination of Cholesterol

Cholesterol was extracted according to the method of Sharmin et al. (2017), with minor modifications. A five-gram sample was weighed in an extracting tube, to which 10 mL of pyrogallol (6% in ethanol, w/v) and 1.0 mL of 5α-cholestane solution (Sigma Aldrich, MO, USA) were added as an internal standard (1 mg/mL). After sonication for 10 min, 6.0 mL of 60% potassium hydroxide in deionized water was added. The extracting tube was heated at 80°C for one hour in a shaking water bath at 90 rpm (ST30, Nuve, Turkey). After cooling, 15 mL of 2% sodium chloride in deionized water and 15 mL of extracting solvent (hexane: ethylacetate = 85:15, v/v) containing 0.01% butylated hydroxytoluene were added and mixed vigorously for approximately 2 min. The tubes were left undisturbed for 10 min, after which the upper layer was collected into a 50-mL volumetric flask. This extraction step was repeated two more times; extracts were combined and passed through anhydrous sodium sulfate, which acted as a filter to remove water. The volume of the extracts was adjusted to 50 mL with extracting solvent. A 10-mL aliquot of the extract was dried under N₂ gas and re-dissolved in 1.0 mL n-hexane (purity 95%). Extracts were filtered through a 0.25-µm membrane filter (Advantec, PTFE, Duran, Germany) and analyzed using gas chromatography. The cholesterol standard was purchased from TCI (Tokyo, Japan) and used for cholesterol quantification. Cholesterol stock solution (1 mg/mL) was prepared with n-hexane containing butylated hydroxytoluene (0.01%, w/v). Standard curves were obtained by regression analyses using six concentrations of cholesterol standards (0.05, 0.10, 0.20, 0.40, 0.80, and 1.00 mg/mL) for cholesterol quantification.

Cholesterol separation was conducted using an Agilent 8890 Series (Agilent Technology, USA) gas chromatography system equipped with an HP-5 capillary column (30 mm x 0.32 mm x 0.25 µm, Agilent Technologies, Santa Clara, CA, USA) and a flame ionization detector (H₂: 30 mL/min, air: 300 mL/min). The flow rate of the carrier gas (N₂) was 3 mL/min. The injection and detection temperatures were 300 °C and 320 °C, respectively. Cholesterol was separated on a column set at 260 °C. A 1-µL aliquot was injected into the gas chromatography column and was expressed as mg/100 g cholesterol in the meat. Cholesterol data processing was carried out with Open Lab CDS software, and peaks were identified by comparison with the retention times of the standards.

Determination of Oxidative Stability and Proximate Composition

The broiler breast and thigh meat samples used for oxidative stability determination were preserved in a refrigerator. The thiobarbituric acid reactive substance (TBARS) values of the meat samples were assayed at zero, four, seven, fourteen, twenty-one, and twenty-eight days according to the method described by Sarker et al. (2010), with slight modifications. In short, 4 g of meat were homogenized with a vertical homogenizer (OV5, VELP, Italy) at full speed for 1.5 min with 10 mL of a solution containing 20% trichloroacetic acid in 2 M phosphoric acid and 10 mL of distilled water. The mixture was then filtered through Whatman No.1 filter paper (125 mm, Whatman International Ltd., Maidstone, England). Equal amounts of the filtrate (2 mL) and 2-thiobarbituric acid (98% 4, 6 Dihydroxy-2-mercaptopyrimidine, 0.005M in DW) were added. The solution was then heated in a water bath at 80°C (HB-205 SW Hanbaek Scientific Co., Korea) for 30 min. After cooling, the color development was measured at 530 nm with a Lambda 365 UV/Vis spectrophotometer (Perkin Elmer Ltd., USA). The TBARS values are expressed as micromoles of malondialdehyde (MDA) per 100 g of meat sample. Following the procedure outlined by the Association of Official Analytical Chemists (2000), the moisture (930.15), total ash (942.05), crude protein (990.03), and ether extract (991.36) contents of the samples of meat from the breast and thighs were examined in triplicate.

Statistical Analysis

The analytical measurements were made in triplicate, and the results are presented as the average of three analyses and the standard error of mean. The statistical analysis was performed using SPSS (IBM Corp., IBM SPSS Statistics for Windows, Version 27, Armonk, NY, USA) with a one-way ANOVA using the general linear model function. A value of p<0.05 was taken as statistically significant based on Duncan’s multiple range test.

RESULTS

Broilers’ Growth Performance and Meat Composition

The effects of dietary flaxseed and moringa as natural feed additives on broiler growth performance are shown in Table 2. The dietary addition of flaxseed in combination with moringa did not affect the broilers’ growth performance throughout the experimental period. The values of final BW (body weight) and WG (weight gain) among the treatment groups did not show any statistical differences (p>0.05). The group receiving dietary supplementation of flaxseed 1.5% and moringa 1% (group T₄) consumed the lowest (p<0.05) amount of feed compared to the control (see Table 2).

Table 2
Effect of dietary flaxseed and moringa as natural feed additives on broiler growth performance.

Parameter	T1	T2	T3	T4	SEM	p-value
BW(g)
0d	45.62	46.08	45.92	45.71	0.06	0.22
21d	762.04a	773.92a	678.54ab	658.85b	12.61	0.04
35d	1950.50	1977.04	1952.09	1932.11	9.03	0.89
WG (g)
21d	716.42	727.84	632.63	613.15	12.60	0.20
35d	1904.20	1931.10	1906.41	1887.12	9.00	0.25
FI (g/bird)
21d	1256.22	1200.31	1151.13	1150.44	57.88	0.14
35d	3136.16a	3047.42ab	3000.31ab	2972.54b	17.28	0.03
FCR (g:g)
21d	1.75	1.66	1.83	1.89	0.12	0.63
35d	1.65	1.58	1.58	1.58	0.03	0.80

• T₁-Control diet; T₂-Flaxseed 0.5% + Moringa 1%; T₃-Flaxseed 1% + Moringa 1% ; T₄-Flaxseed 1.5% + Moringa 1%. Values are expressed as mean and standard error of means. Means represent four replicates, four birds per replicate. SEM= Standard Error of Mean. BW - Body weight (g); WG - Weight gain; FI - Feed intake (g/bird); FCR - Feed Conversion Ratio.

Table 3 shows the effect of dietary flaxseed and moringa as feed additives on the meat yield traits of broiler chickens. Both the breast and thigh meat in group T₄ showed the highest (p<0.05) relative weights compared to the control group. The addition of dietary feed additives also considerably (p<0.05) improved the meat of the drumsticks: the weight of the T₃ and T₄ birds increased by 3.30% and 18.40%, respectively, in comparison to the control chickens. The abdominal fat content was significantly reduced after the addition of moringa with increased levels of flaxseed (1% and 1.5%, respectively). This trait is indirectly important for increasing meat production.

Table 3
Effect of dietary flaxseed and moringa as feed additives on broiler dressing parameters (percentage in relation to body weight).

Parameters (%)	T1	T2	T3	T4	SEM	p-value
Dressing yield	71.24	70.00	68.65	71.10	0.44	0.70
Breast meat	12.00b	11.44b	12.67ab	13.86a	0.25	0.01
Thigh meat	10.14b	10.64b	11.00ab	11.50a	0.14	0.01
Wing	9.31	9.97	9.11	10.09	0.12	0.72
Drumstick meat	2.12b	2.12b	2.19ab	2.51a	0.04	0.03
Head	2.22	2.43	2.21	2.25	0.07	0.06
Liver	0.49	0.53	0.48	0.47	0.02	0.97
Heart	1.91	2.42	2.02	2.10	0.09	0.39
Gizzard	0.55	0.66	0.67	0.65	0.03	0.41
Abdominal fat	4.99a	4.88ab	4.57b	4.70b	0.14	0.04
Intestine	0.82	0.80	0.78	0.74	0.01	0.80

• T₁-Control diet; T₂-Flaxseed 0.5% + Moringa 1%; T₃-Flaxseed 1% + Moringa 1% ; T₄-Flaxseed 1.5% + Moringa 1%. The values are means (n=4 for each treatment). ^{a, b} Mean with different superscripts within same rows are significantly different (p<0.05) by Duncan’s test, SEM= Standard Error of Mean.

The addition of flaxseed with moringa had no effect on the proximate composition of the breast meat samples (Table 4). The moisture content of the breast meat did not differ between the treatment groups and the control, but a significantly lower moisture content was found in group T₄ compared to the thigh meat in other groups. The crude protein content significantly (p<0.05) increased after the addition of increased levels of flaxseed. The values of crude protein in the thigh meat were found (p<0.05) to be highest in T₄ (22.62%), T₃ (21.92%), and T₂ (20.71%) compared to the control T₁ (20.42). The crude ash content of both breast and thigh meat did not differ from the control group.

Table 4
Effect of dietary flaxseed and moringa as natural feed additives on broiler meat proximate composition (%).

Parameter	T1	T2	T3	T4	SEM	p-value
Breast meat (%)
Moisture	75.30	76.40	76.37	76.01	0.20	0.42
Crude protein	21.65	21.92	22.01	22.98	0.22	0.23
Crude fat	2.55	2.27	3.37	2.21	0.16	0.16
Crude ash	1.63	1.21	1.20	1.26	0.05	0.25
Thigh meat (%)
Moisture	76.55a	76.98a	76.54a	74.81b	0.28	0.002
Crude protein	20.42b	20.71b	21.92ab	22.62a	0.20	0.01
Crude fat	2.23b	2.67b	2.92b	4.07a	0.14	0.00
Crude ash	1.47	1.75	1.40	1.35	0.03	0.31

• T₁-Control diet; T₂-Flaxseed 0.5% + Moringa 1%; T₃-Flaxseed 1% + Moringa 1% ; T₄-Flaxseed 1.5% + Moringa 1%. Means represent four replicates. ^{a, b, c} Mean with different superscripts within same rows are significantly different (p<0.05) by Duncan’s range test. SEM= Standard Error of Mean.

Fatty Acid Profile and Cholesterol in Broiler Meat

The dietary treatments’ effect on the levels of saturated fatty acids, monounsaturated fatty acids, and PUFAs in broiler breast and thigh meat are shown in Tables 5 and 6. The saturated fatty acids percent in the breast meat was significantly (p<0.05) decreased after the addition of feed additives, as compared to the control. The PUFA percentage increased significantly (p<0.05) in the thigh meat in comparison to the control. The sum of omega 3 fatty acid content in the breast meat was found to be highest (p<0.05) in group T₄ when compared to the other groups. The significantly highest value (p<0.05) of stearic acid (6.46 mg/100g) in thigh meat was found in group T₃ compared to the other groups (Table 5). The proportion of linolenic acid and omega 3 fatty acids in the breast meat of broilers that were fed supplemented diets were significantly (p<0.05) higher than those of the broilers fed the control diet. On the other hand, omega 6 fatty acids also decreased for an individual in one of the additive groups. Table 6 shows that the sum of saturated fatty acids and monounsaturated fatty acids in the thigh meat did not differ statistically in the supplemented groups in comparison to group T₁. The arachidic acid content in group T₃ increased significantly (p<0.05) compared to the control (T₁), whereas there was no significant difference in oleic acid content across groups. In the thigh meat of broilers fed treatment diets, linolenic acid contents were significantly increased as compared with group T₁. Although PUFAs and omega 6 fatty acid contents in the thigh meat increased significantly (p<0.05), the omega 6 to omega 3 ratios (p<0.05) in thigh meat also decreased. Significantly decreased cholesterol levels were found in all supplemented compared to the control (34.92 mg/100 g) in broiler thigh meat (Table 6), although no statistical differences were observed in breast meat.

Table 5
Effects of dietary flaxseed and moringa on the fatty acid (g/100g fatty acids) composition and cholesterol contents (mg/100 g) of broiler breast meat.

Parameters (g/100g)	T1	T2	T3	T4	SEM	p-value
Myristic acid (C14:0)	0.51ab	0.27c	0.38c	0.63a	0.02	0.00
Palmitic acid (C16:0)	26.08	24.90	24.25	22.74	0.63	0.13
Stearic acid (C18:0)	3.71b	3.33b	6.46a	3.93b	0.16	0.00
Arachidic acid (C20:0)	0.22b	0.39a	0.37a	0.32ab	0.02	0.01
Behenic acid (C22:0)	0.25c	0.43bc	0.69a	0.53ab	0.07	0.00
Palmitoleic acid (C16:1)	2.43c	4.12a	3.42b	4.34a	0.22	0.00
Oleic Acid (C18:1)	39.42	37.85	34.18	35.62	0.52	0.16
Eicosenoic acid (C20:1)	0.45bc	0.54a	0.51ab	0.20c	0.07	0.00
Linolenic acid (C18:3), ɷ-3	1.20b	0.96b	1.35b	2.51a	0.10	0.00
Eicosapentaenoic acid (C20:5), ɷ-3	0.03b	0.04b	0.08a	0.04b	0.03	0.00
Docosahexaenoic acid (C22:6), ɷ-3	0.05	0.07	0.06	0.06	0.05	0.15
Arachidonic acid (C20:4), ɷ-6	0.35c	1.37a	1.10ab	0.91b	0.09	0.03
Linoleic acid (C18:2), ɷ-6	26.08a	21.56b	23.43ab	22.38b	0.45	0.03
ΣSFA%	31.99a	26.01b	25.01b	25.95b	0.72	0.02
ΣUSFA%	71.09a	70.90a	64.53b	70.55a	1.09	0.01
ΣMUFA %	40.37	42.50	37.49	41.66	0.51	0.16
ΣPUFA %	27.71a	24.75b	26.02ab	25.09ab	0.65	0.00
Σɷ-3	1.29c	1.35bc	1.49b	2.61a	0.01	0.00
Σɷ-6	26.44a	22.93c	24.53b	23.29b	0.31	0.00
Σɷ-6: Σɷ-3	20.50a	16.99b	16.46b	17.01b	0.41	0.00
Cholesterol (mg/100g)	29.84	27.00	29.44	29.86	0.53	0.67

• T₁-Control diet; T₂-Flaxseed 0.5% + Moringa 1%; T₃-Flaxseed 1% + Moringa 1% ; T₄-Flaxseed 1.5% + Moringa 1%. ¹Each value represents the mean of 4 replications with 2 birds/replication. Values are expressed as mean and standard error of means. ΣSFA = saturated fatty acid; ΣMUFA = mono-unsaturated fatty acid; ΣPUFA = Poly-unsaturated fatty acid; Σɷ - 3 = total omega 3 fatty acid; Σɷ - 6 = total omega 6 fatty acid. ^{a, b, c} Mean with different superscripts within same rows are significantly different (p<0.05) by Duncan’s test.

Table 6
Effects of dietary flaxseed and moringa on fatty acid (g/100g fatty acids) composition and cholesterol contents (mg/100 g) of broiler thigh meat.

Parameters (g/100g)	T1	T2	T3	T4	SEM	p-value
Myristic acid (C14:0)	0.02c	0.46b	0.43b	0.71a	0.06	0.00
Palmitic acid (C16:0)	26.81	25.76	26.47	25.21	0.34	0.13
Stearic acid (C18:0)	2.84b	4.54a	4.83a	4.91a	0.30	0.00
Arachidic acid (C20:0)	0.20b	0.20b	0.39a	0.36ab	0.02	0.01
Behenic acid (C22:0)	0.26c	0.66a	0.65ab	0.63bc	0.07	0.00
Palmitoleic acid (C16:1)	3.83a	3.32ab	3.62a	2.41c	0.25	0.01
Oleic Acid (C18:1)	36.56ab	35.57ab	38.83a	38.26ab	0.64	0.17
Eicosenoic acid (C20:1)	0.42b	1.14a	0.25b	0.28b	0.10	0.00
Linolenic acid (C18:3), ɷ-3	0.86c	0.98bc	1.21ab	1.44a	0.07	0.00
Eicosapentaenoic acid (C20:5), ɷ-3	0.07	0.04	0.07	0.03	0.05	0.20
Docosahexaenoic acid (C22:6), ɷ-3	0.07ab	0.04b	0.08a	0.04b	0.01	0.00
Arachidonic acid (C20:4), ɷ-6	1.49a	1.13a	0.08b	0.05b	0.40	0.00
Linoleic acid (C18:2), ɷ-6	19.86b	24.98ab	25.87a	23.12ab	0.55	0.00
ΣSFA%	31.05a	28.97ab	27.84ab	28.74ab	0.42	0.01
ΣUSFA%	67.06ab	69.18ab	70.52a	68.63ab	0.63	0.00
ΣMUFA %	42.26a	41.12ab	40.69ab	41.54ab	0.34	0.03
ΣPUFA %	22.59c	27.14a	27.31a	24.86b	0.59	0.03
Σɷ-3	1.00d	1.03c	1.36b	1.51a	0.02	0.00
Σɷ-6	21.35c	26.11a	25.95ab	23.17bc	0.51	0.00
Σɷ-6: Σɷ-3	21.35b	25.34a	16.65c	16.20c	0.73	0.00
Cholesterol (mg/100g)	34.92a	28.61ab	26.07b	26.31b	0.86	0.02

• T₁-Control diet; T₂-Flaxseed 0.5% + Moringa 1%; T₃-Flaxseed 1% + Moringa 1% ; T₄-Flaxseed 1.5% + Moringa 1%. ¹Each value represents the mean of 4 replications with 2 birds/replication. Values are expressed as mean and standard error of means. ΣSFA = saturated fatty acid; ΣMUFA = mono-unsaturated fatty acid; ΣPUFA = Poly-unsaturated fatty acid; Σɷ - 3 = total omega 3 fatty acid; Σɷ - 6 = total omega 6 fatty acid. ^{a, b, c} Mean with different superscripts within same rows are significantly different (p<0.05) by Duncan’s test.

Oxidative Stability of Broiler Meat

The effects of dietary flaxseed combined with moringa on the oxidative stability of broiler breast and thigh meat are shown in Figures 1 and 2. The treatment groups showed no significant effects on the TBARS values of breast and thigh meat up to day zero (p<0.05). However, after seven days, significantly lower TBARS values were observed in the treated groups’ breast and thigh meat (p<0.05) compared to the control. It was shown that the TBARS values of broiler thigh meat in group T₂ (9.95 μmol MDA/100 g) was significantly lower at fourteen days compared to the control (25.95 μmol MDA/100 g). However, after twenty-one days, all the treated groups’ breast and thigh meat values were significantly lower than that of the control group.

Figure 1
Effects of dietary flaxseed and moringa on thiobarbituric acid value (TBARS) in broiler breast meat. TBARS values are expressed as micromoles of malondialdehyde (MDA) per 100 g of meat (µmol MDA/100 g). The results are expressed as mean±SEM (n=4) using one-way ANOVA followed by Duncan’s test. Significant differences (p<0.05) are represented by different letters over the column.

Figure 2
Effects of dietary flaxseed and moringa on thiobarbituric acid value (TBARS) in the broiler thigh meat. TBARS values are expressed as micromoles of malondialdehyde (MDA) per 100 g of meat (µmol MDA/100 g). The results are expressed as mean±SEM (n=4) using one-way ANOVA followed by Duncan’s test. Significant differences (p<0.05) are represented by different letters over the column.

DISCUSSION

Medicinal plants contain a wide range of bioactive components and can play an important role in biological reactions through their specific modes of action. Flaxseed and moringa are versatile plants that have been used as medicines, spices, oils, food, and feed additives in the livestock and poultry industries (Cui et al., 2018; Mahfuz & Piao, 2019; Nova et al., 2020). The poultry industry has the fastest growth rate in the global meat sector, and is a significant source of lipids and proteins for the dietary needs of the growing human population.

Poultry meat composition can be modified by the diet (Duran-Montgé et al. 2007). Dietary manipulation with the combinations of medicinal plants or spices in poultry is of great interest because it is expected to influence growth performance and enhance nutritional quality and antioxidant status (Sharmin et al. 2020).

In this study, the inclusion levels of feed additives were based on our previous research, and no significant differences were observed in carcass characteristics, weight gain, feed intake, and FCR between groups because of the low level of combined additives (0.5% to 1.5%). In our previous study, birds fed diets with M. oleifera at 1% and Spirulina platensis at 1.5% were observed to have significantly higher BW and decreased feed consumption ratios compared to the control group of broilers for a total period of thirty-five days (Sharmin et al. 2020). According to Zanu et al. (2012), the addition of moringa meals reduced FCR efficiency considerably. In contrast, the addition of 2.5%, 5.0%, and 7.5% of moringa leaf meal in broiler chicken diets had no significant influence on feed intake (Atuahene et al. 2010). It has also been found that using 5% moringa meal in broiler diets notably enhances feed intake and FCR. Paul et al. (2018) concluded that the aqueous extract of the moringa leaves reduced feed intake and enhanced the FCR compared to the use of antibiotics in broiler chickens. The decrease in feed intake might be attributable to the phenolic chemicals found in moringa leaves, such as zeatin, quercetin, kaempferol, and apigenin. The level of feed consumption is a basic and important factor that determines the rate of growth and body composition achieved by animals throughout their life cycles. Faqir et al. (2013) reported that FCR increased with increasing extruded flaxseed levels, and the highest FCR value, found in the high extruded flaxseed treatment, was due to lower weight gain in the broiler birds. These components increase feed utilization and reduce the amount of feed required to satisfy maintenance and production requirements (Teixeira et al. 2014).

As shown in Table 3, no adverse effects were observed in any of the internal organs in response to the combination of flaxseed and moringa. We found higher breast and thigh meat yields in treatment group T₄, the flaxseed 1.5% + moringa 1% group. Vikari (2008) reported a 7.9% breast meat yield when broiler diets were supplemented with plant extracts. Decreasing abdominal fat was observed in our study with the addition of natural feed additives, indicating a variation in lipid metabolism that might be responsible for the depression in growth (Shi et al., 2001). Sangkitikomol et al. (2014) observed that moringa supplementation reduced lipid synthesis in meat. El-Tazi (2014) reported that 5% moringa supplementation in the diet boosted the carcass yield percentage when compared to the control group. In our study, it was found that there was no significant difference in dressing yield. However, Melesse et al. (2011) found that increasing moringa supplementation enhanced carcass yield. Tesfaye et al. (2013) showed that feeding moringa leaf meal increased dressing percentage and thigh and drumstick weight significantly. Rao et al. (2019) reported that the different moringa supplementation levels had no influence on carcass yield. According to Zanu et al. (2012), moringa supplementation did not enhance the dressing percentage.

Crude protein measurement is an important trait in the animal production industry. The crude protein content (Table 4) in the feed additive groups was significantly (p<0.05) increased in the thigh meat from 20.42% (group T₁) to 22.62% (group T₄). It is reported that M. oleifera leaves contain a high protein content with all essential amino acids (Foidl & Paull, 2008). The increased protein content and reduced amounts of tannins, alkaloids, and glycosides in moringa are efficiently digested and may result in improved weight gain in birds (Onu et al., 2011). Crude fat and other proximate components were analyzed for the dietary treatment groups, including T₄ (flaxseed 1.5% + moringa 1%). Breast meat proximate composition, including crude protein, ash, fiber, and crude fat content were recorded and shown in Table 4 in the section above. The reason for the results for breast and thigh meat has not yet been studied, to the best of our knowledge. At present, more attention is being given to new, natural products that have lipid-reducing activity so that cholesterol levels can be controlled (Bostami et al., 2017; Wu Hy et al., 2009).

Von Schacky & Harris (2007) reported that consumers are becoming more conscious of their health and are particularly interested in reducing the risk of cardiovascular and other diseases by consuming more PUFAs, especially omega 3 fatty acids. The composition of poultry meat can be modified by dietary manipulation through fat and oil sources, especially fatty acid profiles and cholesterol content. Duran-Montgé et al. (2007) also reported these changes in meat composition through dietary manipulation. The fatty acid composition of meat products is an important parameter of meat quality. The addition of different levels of flaxseed with a combination of moringa may have the potential to improve fatty acids profiles due to presence in these ingredients of phenolic compounds and organic acids, which have an important role in lipid regulation. The antihyperlipidemic activity of phenolic acids in flaxseed, moringa, and medicinal plants may be attributable to the improvement of the fatty acid content of broiler meat through the lipid homeostasis and fatty acid synthase enzyme (Park et al., 2011). In previous research, these had antioxidant potential and consequently prevented PUFA oxidation in all treated groups (Von Schacky & Harris, 2007). The fatty acid profile of the thigh meat was significantly (p<0.05) enriched in PUFAs in all the treated groups in this research. Both the breast and thigh meat of the treated groups had higher levels of polyunsaturated fatty acids (PUFAs), primarily up-graded linolenic acid. Linolenic acid is an essential fatty acid that acts as the primary precursor of omega 6 PUFAs (Russo, 2009). It can prevent cardiovascular disease (Sposito et al., 2007), which could be beneficial for human health through the consumption of broiler meat. Stanaćev et al. (2013) also reported that the concentration of ALA from flaxseed grain in the edible tissues of poultry, such as skin and meat, was readily increased by increasing the concentration of ALA in the chickens’ diet. The high concentration of these fatty acids in flaxseed may be the reason for the rise in ALA (18:3 omega-3) in the breast and thigh meat in group T₄ (flaxseed 1.5% + moringa 1%) in the current study. Faqir et al. (2013) reported that thigh meat fatty acid profiles were highly enriched by varying concentrations of extruded flaxseed in the diet of birds. The higher addition of linolenic acid in the fatty acid profile of breast meat in group T₄ was due to its high capacity for modification in lipid fraction. Our results are in alignment with the findings of Zuidhof et al. (2009). Incorporation of omega-3 fatty acids from flaxseed into eggs and meat is an efficient way to increase the PUFA content of meat and eggs (Faqir et al. 2013).

Puvača et al. (2014) reported that the introduction of relatively high concentrations of docosahexaenoic acid and eicosapentaenoic acid in chicken diets results in an increased concentration of docosahexaenoic acid and eicosapentaenoic acid in the birds’ tissues. The calculated value of the omega 6 to omega 3 ratio in chicken meat in our experiment was significantly decreased in some groups. In the breast meat, the ratio was significantly different in groups T₁ (control), T₂, T₃, and T₄, with values of 20.50, 16.99, 16.46, and 17.01, respectively. The balance of omega 6 and omega 3 fatty acids is an important factor in decreasing the risk for coronary heart disease in both the primary and secondary prevention of coronary heart disease. A high omega 6 to omega 3 ratio may enhance the risk for both depression and inflammatory diseases (Simopoulos, 2008). According to Simopoulos’s (2008) mini-review, the ratio of omega 6 to omega 3 in different populations is 38-50 in urban India, 16.74 in the US, and 15 in Europe. Dietary supplementation of flaxseed and moringa reduced the ratio of omega 6 to omega 3 fatty acids in thigh meat, except in group T₂, indicating a beneficial effect of flaxseed and moringa on broiler meat quality.

The birds receiving feed additives had significantly lower cholesterol levels in thigh meat than the control group (34.92 mg/100 g). Similar results were found by Olugbemi et al. (2010), who argued that this was probably attributable to the hypocholesterolemic property of the moringa leaf meal. There was a significant reduction (p<0.05) in the cholesterol concentration of broiler thigh meat in birds fed diets with added flaxseed and moringa. Plant-derived secondary metabolites such as saponins can play an important role in reducing cholesterol levels through their action in biological pathways. (Liu et al., 2016). Since plant or starfish-derived saponins bind with cholesterol and make a complex formation, it can be expected that saponin reduces the level of cholesterol in the body (Bidura et al., 2020; Sharmin et al., 2020a). Eskandar et al. (2015) also reported that some saponins can prevent hypercholesterolemia, a phenomenon that results from the complex formation of cholesterol. Our previous results showed that the addition of moringa to feeds at levels of 0.5% to 1.5% tended to reduce the cholesterol levels in egg yolks (Sharmin et al., 2021).

The loss of nutritional and sensory values, as well as the formation of potentially toxic compounds, are caused by lipid oxidation, which affects meat quality and reduces shelf life. Bou et al. (2004) observed that enhancing the antioxidant capability in muscle improves meat quality and extends shelf life. In our experiment, the addition of feed additives in broiler diets with different combinations of flaxseed and moringa significantly reduced lipid oxidation, as indicated by the lower TBARS values of the broiler breast and thigh meat (Figures 1 and 2). This result is consistent with the findings of various researchers (Ahmed et al., 2014; Kanatt et al., 2010; Rajan et al., 2011;Vaithiyanathan et al., 2011). Traber & Atkinson (2007) reported that these types of feed additives are found to be beneficial to combat the adverse effects of oxidative stress. They added that these additions’ principal purpose is to preserve membrane integrity by stopping PUFAs in membrane phospholipids from oxidizing. Since moringa contains highly antioxidant flavonoid and polyphenolic components, it was combined with feed to reduce meat lipid oxidation. The lower plasma MDA levels are another potential reason that moringa has such a high content of antioxidants such as tocopherol, ascorbic acids, flavonoids, and saponins. A number of researchers have demonstrated that moringa reduces lipid peroxidation and leads to a considerably lower MDA levels than control groups that do not consume it (Rao et al., 2019; Karthivashan et al., 2015; Cui et al., 2018). Sangkitikomol et al. (2014) also observed that moringa supplementation reduced lipid synthesis. The interest in polyphenolic antioxidants has increased remarkably in the last decade because of their elevated capacity to scavenge free radicals (Silva et al., 2007). Betti et al. (2009) observed that TBARS values were higher at a higher level of extruded flaxseed (15%) fed to broiler birds. The results of our study are in alignment with the findings of Zanini et al. (2006) and Faqir et al. (2013). Luna et al. (2013) argued that high oxidative deterioration in broiler meat is due to the high concentration of PUFAs in flaxseed. It is reported that increased PUFA content is always associated with oxidation. Interestingly, dietary moringa supplementation also increases PUFAs but decreases MDA in breast muscle during storage. Bou et al. (2004) reported that diets containing natural feed additives improve oxidative stability, which is aligned with our findings. The results from the TBARS test provide evidence that the additive groups could improve oxidative stability in broiler meat. Flaxseed and moringa may intercept and neutralize free radicals, preventing oxidation.

CONCLUSION

The goal of the current study was to determine whether feeding different amounts of flaxseed and moringa affected the growth performance, carcass features, meat composition, fatty acid profiles, cholesterol content, and oxidative stability of broiler chickens. The ratio of omega 6 to omega 3 fatty acids in breast meat was recorded to be significantly (p<0.05) lower in all groups other than the control (T₁), while this ratio in thigh meat was lower in groups T₃ and T₄ compared to the control. Meat yield trait data revealed that breast and thigh meat yields were higher in group T₄. Fatty acid profile data revealed that omega 3 fatty acids were higher in group T₄ compared to the other groups for both breast and thigh meat. Cholesterol contents in thigh meat were lower in group T₄ compared with group T₁. The TBARS values were lower in all other dietary groups compared to group T₁ in both breast and thigh meat. The addition of flaxseed 1.5% + moringa 1% produced the lowest cholesterol levels, even though the thigh meat in group T₄ had a lower moisture content than the other groups.

ACKNOWLEDGEMENTS

This research was funded by the “Strengthening of Poultry Research and Development Project”, of the Bangladesh Livestock Research Institute, Savar, Dhaka, Bangladesh. The authors would like to thank Dr. Nathu Ram Sarker, Executive Director, Krishi Gobeshona Foundation, Dhaka 1207 for the helpful discussions.

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FUNDING

This research was supported by the funding of the Strengthening of Poultry Research and Development Project of the Bangladesh Livestock Research Institute in Savar, Dhaka, under the Ministry of Fisheries and Livestock, Government of the Peoples Republic of Bangladesh.

DATA AVAILABILITY STATEMENT

Data will be made available upon request.

DISCLAIMER/PUBLISHER’S NOTE

The published papers’ statements, opinions, and data are those of the individual author(s) and contributor(s). The editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions, or products referred to in the content.

Corresponding author e-mail address

Md. Sazedul Karim Sarker SPRD Project and Poultry Research Center, Bangladesh Livestock Research Institute, Savar, Dhaka, 1341, Bangladesh. Phone: [phone] Email:[e-mail]

CONFLICT OF INTEREST

No potential conflict of interest is reported by the author(s).

Section Editor:

Rodrigo Garófallo Garcia

Publication Dates

• Publication in this collection
  10 Aug 2026
• Date of issue
  2026

History

• Received
  12 Aug 2025
• Accepted
  09 Apr 2026