2.1. Animal housing and dietary treatments

The research enlisted a group of one hundred and twenty, one-day-old male Ross 308 broiler chicks acquired from Pinto Valouro (Bombarral, Portugal), each exhibiting an average initial weight of 39.3 ± 2.30 g. From day 1, they were randomly lodged in 40 wire-netted enclosures (56 cm height x 56 cm length x 50 cm width) for 35 days under regulated environmental conditions. Ambient temperature and airflow were persistently supervised as per the previously established protocols (2, 19). Adhering to the 3R’s doctrine (Reduce, Refine and Replace) to curtail the number of animals employed, every enclosure hosted three chicks, with 10 identical enclosures designated for each dietary experiment. The experimental trial was conducted in the School of Agriculture at the University of Lisbon (Lat: 38°42′27.5”N; Lng: 9°10′56.3”W). The Animal Welfare Committee of the School of Agriculture at the University of Lisbon (ORBEA/ISA) approved all the experimental methodologies involving animals, allocating the study a protocol code number 0421/000/000/2022.

In the initial fortnight, chicks were administered a staple diet formulated from corn and soybean meal sourced from Rações Veríssimo S. A. (Leiria, Portugal). Following this phase, from day 14 through day 35, they transitioned to one of four distinct experimental diets furnished ad libitum daily. These diets encompassed: (1) a standard diet predicated on corn and soybean meal (CTR); (2) a diet fortified with 15% Spirulina powder (procured from Allmicroalgae, Pataias, Portugal) (SP); (3) a diet containing 15% extruded Spirulina powder (SPE); and (4) a diet supplemented with 15% Spirulina powder alongside a tailored enzyme blend, constituted by 0.20% porcine pancreatin extract (sourced from Merck, Darmstadt, Germany) and 0.01% lysozyme extracted from chicken egg white (sourced from Sigma-Aldrich, Missouri, United States) (SPM). The microalga extrusion was performed by Sparos company (Olhão, Portugal), following detailed conditions: 340 mL of water addition per minute, at 34 bars and 118°C for the last extrusion barrel. This procedure occurred from 3 to 7 s. Then, the algal pellets were dried for 8 and 10 min at 120°C (12, 13). The encapsulated porcine pancreatin extract comprised 350 FIP-U/g of protease, 6,000 FIP-U/g of lipase, and 7,500 FIP-U/g of amylase. Lysozyme powder contained 70,000 U/mg protein. A detailed composition of the feed constituents and additives for each experimental diet is provided in Table 1. During the trial, a 2.5% mortality was observed (three animals of 120 in total).

Ingredient composition of broiler experimental diets from day 14 to day 35 (%, as fed basis).

CTR, corn and soybean meal-based diet; SP, 15% Spirulina; SPE, 15% extruded Spirulina; SPM, 15% Spirulina + 0.21% Enzyme mixture (0.20% porcine pancreatin + 0.01% lysozyme).

¹Premix provided the following per kilogram of diet: pantothenic acid 10 mg, vitamin D₃ 2,400 IU, cyanocobalamin 0.02 mg, folic acid 1 mg, vitamin K₃ 2 mg, nicotinic acid 25 mg; vitamin B₆ 2 mg, vitamin A 10000 UI, vitamin B₁ 2 mg, vitamin E 30 mg, vitamin B₂ 4 mg, Cu 8 mg, Fe 50 mg, I 0.7 mg, Mn 60 mg, Se 0.18 mg, Zn 40 mg.

Weekly assessments were conducted to record the weights of broilers and feeders, with daily feed provisioning to compute the parameters of body weight gain (BWG) (ascertained by the weekly weight differential divided by 7), average daily feed intake (ADFI) (calculated as weekly consumption per cage, divided by 7) and the feed conversion ratio (FCR) (the quotient of weekly consumption divided by 3 and weekly body weight gain).