Meat quality in broiler chickens fed on cowpea (Vigna unguiculata [L.] Walp) seeds | Scientific Reports

The Animal Ethics Committee approved the study protocol of the National Research and Development Institute for Animal Biology and Nutrition (INCDBNA-IBNA) Baloteşti, Romania, under the EU Directive 2010/63/EU and Romanian Law on Animal Protection. The slaughter of birds was carried out following the applicable rules on handling animals at the time of slaughter, including humane treatment. Also, the methods used in the meat quality tests were carried out by the current and commonly used methodology described in the “Material and methods” section. The study was conducted in compliance with the ARRIVE guidelines. We confirm that all methods on chickens’ were carried out in accordance with relevant guidelines and regulations.

The cowpea seed Aura 26 variety used in this study is part of the collection of genotypes from the Research-Development Station for Plant Culture on Sands, Dăbuleni, Romania, and the National Commission approved registration for horticultural plants, Decision No. 92841/2012.

Birds and diets

The study was conducted on 240 one-day-old healthy broiler chickens (Ross 308) divided into two groups [a control group (SBM) feed a diet with soybean meal as a source of protein, and CWP group (experimental) feed a diet with untreated cowpea seeds], with similar initial weights (46.5 ± 0.23 g) into 6 replications with 20 birds each. The nutritional composition of CWP of the Aura 26 variety is described in Table 5. The feeding program was divided into two feeding phases: starter (days 1–24) and finisher (days 25–42). Diets for each feeding phase were formulated to be isocaloric, isonitrogenous, with similar total lysine, total sulphur amino acids (TSAA; Table 6), calcium and available phosphorous, and to meet or exceed breeder guidelines (Ross 308, Aviagen Ltd., Midlothian, UK). Diets were manufactured in mash form, without the inclusion of growth promoters or antibiotics. However, narasin as a coccidiostat (Monteban G100, Elanco GmbH) and phytase (Axtra PHY 5000 L, Danisco Animal Nutrition, Marlborough, UK) as exogenous enzymes were included in premixes of all two experimental diets. Feed and water were provided ad libitum.

Table 5 Nutritional composition of cowpea (Vigna unguiculata [L.] Walp, Aura 26 variety) seeds added to broiler diets.

Full size table

Table 6 Ingredient and chemical composition of diets (as-fed basis).

Full size table

Chickens were kept in pens on shavings litter in a temperature-controlled room with pens of identical size (1.75 × 1.55 m). Room temperature was maintained at 34 °C for the first 5 days and then gradually reduced according to standard management practices until a temperature of 22 °C by using thermostatically controlled heaters, fans, and adjustable sidewall inlets. Lighting was provided for 23 h/day from 1 to 7D, and from 8D, the light decreased by 1 h a day until 20 h, according to EU legislation (EU Council Directive 2007/43/EC). Broilers were vaccinated at the hatch for Marek’s, Newcastle, and Infectious Bronchitis Disease.

Slaughtering procedures and muscle sampling

At 42 days of age, twelve birds (two birds/pen) from each feeding group (chosen based on pen average final live weight) were individually identified and weighed. The birds were electrically stunned before slaughter, exsanguinated by neck cut, scalded, and eviscerated. Chickens live weight was registered before slaughtering. A total of twelve breasts and twelve thighs were collected on their right and left sides, individually vacuum-sealed and refrigerated (4 ± 1 °C). Meat quality parameters (pH24, and color) were assessed on the Pectoralis major muscle (PM) on the right breast and the Biceps femoris muscle (BF) on the right thigh, while the left breast and thigh meat were frozen at − 20 °C until further analysis (instrumental TPA, proximate chemical composition and FA composition).

Meat quality traits

pH value and instrumental colour measurements

After 24 h cold storage at 4 ± 1 °C, the pH value was measured in triplicate using a Hanna portable pH-meter (model HI 99163, Hanna Instruments, Romania), fitted with a spear-type electrode (FC 099 stainless steel blade tip) and an automatic temperature compensation probe. The color of the muscles was determined using a portable colorimeter (model CR 410, Konica Minolta Inc., Osaka, Japan) calibrated with a white ceramic tile on D65 illuminate. The results were expressed in the CIE Lab color space27. The lightness component is represented by L*, which ranges between 0 to 100, redness by a* and yellowness by b*, both of which have a range of −120 to + 120. For instrumental color determination, three measurements were performed on a fat-free surface area in different locations of each muscle (PM, BF).

Texture profile analysis

The texture measurements of raw chicken muscle samples were analyzed individually by a double cycle compression using a texture analyzer (Model CT3 BROOKFIELD Engineering Laboratories, Inc. MA, USA). To texture analysis, each muscle (PM and BF) was cut into 3-cylinder shapes with a diameter of 20 mm and a height of 15 mm. For an increased accuracy of the parameters reading, there were avoided any large areas of fat. The texture analyzer was equipped with a 50 kg load cell, a cylinder probe of 76.2 × 10 mm to compress the samples and a fixture base table. The probe moved towards the sample at a constant speed of 2.0 mm s−1 (pre-test), 1.0 mm s−1 (test), and 2.0 mm s−1 (post-test). The data was collected using Texture Pro CT Software.

Proximate chemical composition

PM and BF samples were minced, homogenized, and divided into two parts. A portion was used to perform NIR (near-infrared reflectance) spectroscopy analysis. The remaining part was frozen and afterwards analyzed for total lipid extracts and FA composition. NIR data were acquired using a DA6200 meat analyzer (PerkinElmer, Inc. MA, USA), with transmission spectroscopy that uses diode array detectors in the wavelength range of 850 to 1050 nm. Raw minced and homogenized samples from the PM and BF muscles were loaded into a magnetically coupled plastic sample dish of 14 mm height and a volume of 170 mL and analyzed for moisture, protein, fat and collagen contents. To minimize sampling error, we set two duplicates, and each replication was measured twice. Before the sample measurements, a polystyrene check sample was used to verify the optical performance. The averaged spectrum was then used in subsequent analysis.

Fatty acid composition

The FA content was assessed via fatty acid methyl ester (FAME) gas chromatography. The method used for FA composition were done in the same way as previously described by Ciurescu et al.28. In brief, FA from the total lipid extracts was converted to methyl esters by transesterification (in methanol containing 3% concentrated H2SO4, for 4 h at 80 °C). Methyl esters of FA were evaluated in a Perkin Elmer-Clarus 500 chromatograph equipped with a flame ionization detector (FID) fitted with a BPX70 capillary column (60 m × 0.25 mm × 0.25 µm film thickness). The column temperature was programmed at 5 °C/min from 180 to 220 °C. The carrier gas was hydrogen (35 cm/s linear velocities at 180 °C), and the splitting ratio was 1:100. The injector and detector temperatures used were 250 °C and 260 °C, respectively. Peaks were identified by injecting pure FAME standards; quantification was assessed using tridecanoic acid (C13:0) as an internal standard. The results were expressed as the percentage of the total detected FA. The ratio of n-6 PUFA to n-3 PUFA (n-6/n-3 PUFA ratio) was calculated.

Statistical analysis

The data were processed with SPSS Statistics software, v.20.0 for Windows (IBM SPSS Statistics, Armonk, NY, USA). A one-way analysis of variance (ANOVA) was used. Results are reported as means and standard error of the mean (SEM). The effect of the diet on the meat quality parameters was analyzed using Student’s t-tests for independent samples. Significance was declared at P < 0.05. A statistical trend was considered for 0.05 < P ≤ 0.10.

Ethics

The Animal Ethics Committee approved the bird’s care and used protocol at the National Research-Development Institute for Biology and Animal Nutrition (INCDBNA-IBNA), Balotești, Romania, following the principles of EU Directive 2010/63/EU as transposed to Romanian legislation on Animal Protection used for Scientific Purposes (Law no. 199/2018). Also, the methods used in the meat quality tests were carried out in accordance with the current and commonly used methodology described in the “Material and methods” section.