The poultry experiments in HoloFood aimed at understanding how animal-microbiota interactions were modulated by feed additives, and how these interactions affected production indicators. The study consisted of three identical experiments (A, B, and C). The three trials were carried out in different seasons of the year: spring (trial A), summer (trial B) and autumn (trial C). An overview of the experimental conditions and set-up is shown in the following, while detailed information can be found in the following publication:

Tous N, Marcos S, Goodarzi Boroojeni F, Pérez de Rozas A, Zentek J, Estonba A, Sandvang D, Gilbert MTP, Esteve-Garcia E, Finn R, Alberdi A, Tarradas J. 2022.
Novel strategies to improve chicken performance and welfare by unveiling host-microbiota interactions through hologenomics. Frontiers in Physiology 13: 884925.

Experimental conditions

In each experiment, 24 pens were used to house a total of 960 day-old broiler chicks from two fast-growing genetic lines (Ross308® and Cobb500®) obtained from two hatcheries to increase genetic diversity. To eliminate the potential effect of parent stock, the chicks were distributed in a way that ensured each replicate had an equal number of birds from each hatchery tray. Each pen, which measured 2.25 m2, accommodated 40 birds and was equipped with one individual hopper feeder and two nipple drinkers. The barn was without windows but had automatic environmental control, including a gas heating system, screens for ventilation, and programmable lighting with TL tubes. The temperature was adjusted based on the standard farm program, with an increase from 32 to 34°C for the first two days and a gradual decrease by 3°C per week until it reached 21°C. The lighting program consisted of 24 hours of light for the first two days, followed by 18 hours of light until day 7, and 14 hours of light thereafter. The litter used was fresh wood shavings. All chicks were vaccinated against Avian Infectious Bronchitis and Gumboro diseases according to the hatchery’s standard vaccination program.

Experimental design

The study utilized a randomized complete block design, with a 2 × 2 × 3 factorial arrangement based on broiler line (Ross308® or Cobb500®), sex (male or female), and dietary treatment: 1) basal diet (BD), 2) BD supplemented with a probiotic additive (PR), and 3) BD supplemented with a phytobiotic additive (PH). Each treatment was randomly assigned to one pen in each block (two blocks per experiment), resulting in two replicates (pens) per treatment in each experiment (for a total of six), with 40 animals per pen (20 from each hatchery). The experimental design differed from conventional studies that test the effect of feed additives on performance, as it was designed to increase inter-individual genetic variability, thus improving the likelihood of identifying interactions between animal and microbial genomic features. To avoid observer bias in subsequent samplings, six animals per pen were randomly selected and marked on day 0 to be slaughtered at days 7, 21, and 35.

Diets, additives and feeding

To maximise the impact of feed additives, the basal diet (BD) was designed to be pro-inflammatory, utilising wheat (a cereal high in non-starch polysaccharides (NSP) with over 50% inclusion) and soybean meal as main ingredients, without the addition of enzymes, antibiotics, or coccidiostats. The diets were formulated according to bird requirements and commercial practices, and divided into three periods: starter (0-9 days), grower (10-23 days), and finisher (24-37 days). Feeds were presented as crumbles for the starter period and 3 mm pellets for later periods. The probiotic (PR) treatment included BD with a mixture of three strains, specifically Bacillus subtilis DSM 32324, B. subtilis DSM 32325, and B. amyloliquefaciens DSM 25840 (inclusion level of 0.75 g/kg feed, >3.20 × 109 CFU/g). The phytobiotic (PH) treatment used BD supplemented with a phytobiotic additive obtained from white grapes, containing 78% procyanidins and 22% polyphenols as active ingredients (inclusion level of 0.75 g/kg feed).

Animal sampling

Six animals per pen were selected randomly and individually weighed, evaluated for footpad dermatitis, and euthanized for sampling at days 7-8, 21-22, and 35-37 (multiple days were required due to workload). Euthanasia was performed in accordance with the ethical requirements established by RD 53/2013 (Spain). A total of 14 samples were collected from each animal after euthanasia to measure individual key performance indicators and generate multi-omic and complementary analyses. The measured KPIs were aimed at quantifying the quantity, quality, and safety of the produced food, the sustainability of the production process, and the welfare of the produced animals. Samples were obtained from sections of ileum and cecum, intestinal content from ileum and cecum, feathers, and blood. Different aliquots were distributed and stored appropriately for downstream analyses.