**2.1 Birds management and diet**

One-day-old chicks (Cobb 500) were obtained from a commercial hatchery and screened upon arrival to ensure that no abnormalities or symptoms of the disease were present. Throughout the experiment, standard operating procedures for

**Figure 1.** *Photograph of an open-sided poultry house used in the current experiment.*

*Microbial Diversity and Community Dynamics in the Intestines of Broiler Chicken Raised… DOI: http://dx.doi.org/10.5772/intechopen.103815*


#### **Table 1.**

*Composition of the conventional diet used in the experiment.*

broiler house management [14] were followed. Before the experiment, the Opensided house unit, cages, feeders, and drinks were fumigated to clean and disinfect them. In addition, strong cleanliness and biosecurity measures were implemented. The open-sided house was constructed from a galvanized iron shed with profiled steel shed roofing that was naturally ventilated. On all sides, chicken mesh panels and a one-meter-high block work protection were installed. It included four sets of electric wall fans to help circulate the air. Shade cloths were used to screen direct sun rays during midday (**Figure 1**). Six birds were randomly assigned to each of 15 suspended wire cages (62 × 62 × 37 cm) such that all cages had nearly a similar average initial weight. Feed was available ad libitum and the composition of the experimental diet is presented in **Table 1**. The house temperature was maintained at 33°C on day 1 and reduced by 3°C each week to reach a constant 22°C. The lighting program was 23 L—1D. There were 15 replicates with each replicate cage containing six birds (a total of 90 birds). Birds per replicate combinations were randomly allocated.

#### **2.2 Sample collection**

One bird per cage was chosen at random at the ages of 5, 15, 25, and 35 days. The birds were chosen, marked, and maintained in their cages until they were euthanized, based on the weight of their bodies. Each cage's birds with the closest body weight to the average were chosen, labeled, and euthanized accordingly. Then birds were injected intramuscularly with a xylazine-ketamine combination containing 5 mg xylazine (Ilium Xylazil-20-Xylazine 20 mg/mL, as hydrochloride) and 25 mg ketamine (Ketamine Injection-Ketamine 100 mg/mL as hydrochloride) to put the birds into a deep plan of sedation and anesthesia. Both xylazine–ketamine were supplied by Troy Laboratories Pty Limited, Glendenning, Australia. The bird was euthanized via cervical dislocation once it was totally immobilized. Then, at the

bottom of the breastbone, an incision was made and a huge V shape was carved toward the head. The abdominal cavity was opened at the apex of the V form, taking care not to burst the intestine below. The small intestine was carefully pushed out of the abdominal cavity till the ileal-caecal-colonic junction was observed once a large enough opening had been formed. The duodenum (from the gizzard to the bile and pancreatic ducts), jejunum (from the ducts to the yolk stalk), ileum (from the yolk stalk to the ileocecal junction), and caecum (two horns) were distinguished, separated, and cleaned with 70% alcohol wipes.

The mid portions of the duodenum, jejunum, ileum, and ceca were cut into 4 cm long sections (including digesta). After each bird, the dissecting instruments were cleaned with 70% ethanol. The entire procedure of collecting intestinal contents took less than 30 minutes and was completed on a thoroughly cleaned workbench.

The contents of the four segments of the intestine were collected and placed in a sterile 15-mL conical tube with labels. The samples were immediately placed on ice and transferred to the laboratory, where they were kept in an 80°C freezer until they were analyzed. The analysis of all samples began 2 weeks after the trial ended on day 35. All of the samples were taken under identical conditions.
