# Bioactive Compounds in Breast Meat of Broiler Chickens Fed with Black Soldier Fly Wholesmeal
## 2 Materials and Methods
### Birds, Diets, and Experimental Design  
The study was conducted on 45 male broiler chickens (Ross 308), raised on the farm \"Quadrifoglio--Aziende agricole\", owned by [PERSON] (Picerno-Potenza, Basilicata, Italy). The birds were randomly divided into three groups: the control group (n = 15 birds; C) was fed a basal diet, the second group (n = 15 birds; HI5) was fed a basal diet plus 5% BSFL wholemeal flour; the third group (n = 15 birds; HI10) was fed a basal diet plus 10% BSFL wholemeal flour. The basal diet was designed to meet the age-specific nutritional requirements of broiler chickens. Each group of birds was divided into three replicates, with five birds per replicate, equipped with shared feeders and drinkers with teats. The litter in each pen consisted of approximately 8 cm of rice husks and wood shavings. Food and water were provided ad libitum during the experiment. A temperature of 32-34 \({}^{\circ}\)C was maintained for the first three days and then reduced to 22 \({}^{\circ}\)C at a rate of 2-3 \({}^{\circ}\)C per week. The light/dark cycle (14/10 h) and ventilation system of the house were automatically adjusted according to the growth stage of the chicks. The three groups of broilers (n = 45) were fed the respective diets (C, HI5, and HI10) for a period of two weeks until 35 days of age. The commercial basal diet included a grower ration (0-21 days) and a finishing ration (22-35 days), as shown in Table 1. All broiler chickens were slaughtered at 35 days of age, at a mean slaughter weight of 1.75 \(\pm\) 0.24 kg. The breast (Pectoralis Major Muscle) was removed from each bird, vacuum-packed, and stored at \(-\)20 \({}^{\circ}\)C until analysis. Wholemeal flour BSFL was supplied by Xflies s.r.l. (Potenza, Italy). After hatching, the newly hatched larvae were reared on the standard Gainesville diet, supplied by the animal feed factory Mangimi Losasso s.r.l.--Balvano (Potenza, Italy), composed of 20% corn, 30% alfalfa meal, and 50% wheat bran ([PERSON] et al. 2020) at 82% humidity, for 5 days, as described by [PERSON] et al. [21]. On the sixth day, BSFL were transferred to plastic boxes containing a standard diet. The larvae were cultured at 27 \(\pm\) 1 \({}^{\circ}\)C and 70 \(\pm\) 2% relative humidity. At the end of the feeding period, the larvae were separated from the residual material (larval excrete) using an industrial sieving machine (mesh size 4 mm) (Guangzhou Flysource Biotechnology Co., Ltd., Guangzhou, China), which removed most of the adherent material. They were stored at \(-\)20 \({}^{\circ}\)C for 24h and then dehydrated using an industrial microwave (MAX Industrial Microwave, Yantai, China) (as suggested by IPIFF--Guide on Good Hygiene Practices). To obtain fine particles and a homogeneous powder, the dried larvae were ground using a food mill (Tom Press Italia, Mantova, Italy). The ingredients and chemical composition (%) of the broiler grower and finisher diets are presented in Table 1. The fatty acid profile of the Black Soldier Fly (BSF) meal supplemented to the standard diet is presented in Table 2. Furthermore, in a previous study by [PERSON] et al. (2022) [1], the average amino acid profile and chitin content of the same BSFL flour used in this study were reported.