# Acoustic Detection of Vaccine Reactions in Hens for Assessing Anti-Inflammatory Product Efficacy
## 2 Material and Methods
### Material  
A Mediterranean chicken farm located in Jesus, southern Catalonia (Spain) was selected for the acoustic recording campaign. The farm is part of Avigan Terralta's production facilities for the breeding of future laying hens (www.avigan.es (accessed on 22 September 2023)). The rearing farm consisted of two identical houses (one on top of each other) with 45,000 semi-heavy layers with brown egg-laying ability, from the Hy-Line Brown genetics [11], as shown in Figure 1a. The hens were raised for 18 weeks, starting from the first day of life, and were exposed to artificial light daily from 5 a.m. to 10 p.m. At the tenth week, the birds were vaccinated with the ILT eye drop vaccine. The vaccination process, performed manually, lasted a total of three days.  
The objective of this study was to record the vocalization of birds before, during, and after the administration of the ILT vaccine, to monitor the changes in the vocalizations of the animals. The data collection campaign of this study took place from 28 November to 10 December 2022, and day zero is referred as the vaccination day.  
To certify measurement equivalence, two identical sensors, each placed in a farm house, were used to collect raw sound with non-invasive equipment. The distance and architectural barriers between the two sensors prevented possible interference between the calls of both experimental groups due to simultaneous recording. The farmhouses were built with brick and cement walls on both sides, with the ground floor and first floor being totally independent and separated by a roof made of beams and concrete, with a paved floor. The height of each floor was 3 m.  
Acoustic data was collected continuously from each house using a Superlux ECM999 measurement condenser microphone with a frequency response of 20-20 kHz and a sensitivity of \(-37\) dBV/Pa [12]. The sensor was placed in the center of the animal house, 0.5 m from the ceiling. The analog signal was connected to a Behringer U-Phoria UM2 digital audio interface [13], which digitized the data and allowed it to be recorded by a mini computer using a Python script [14]. Recording hardware was placed in an IP65 junction box hung from the ceiling, as shown in Figure 1b. The system was programmed to record 15 min files with a sampling rate of 22.05 kHz, which were archived with flac compression [15]. Similar recording systems have been implemented by the authors in previous studies [16; 17; 18] to characterize broiler calls in both hatcheries and farms, and to identify \(CO_{2}\)-related acoustic indicators.  
Figure 1: Sensor deployment in study hen farm. (**a**) Farm map and sensor deployment; (**b**) acoustic sensor deployment in control house.  
The characteristics of this herd, with two identical houses with the same environmental and management conditions, provide a suitable environment to validate the effectiveness of a product that reduces vaccination reactions. The hens were divided into two groups: a control group (CR) of vaccinated hens, located on the ground floor of the farm, and a treatment group (TX) of vaccinated hens with the anti-inflammatory product administered, which were located on the first floor. The complementary feed administered after vaccination in liquid form was composed of natural products, including _Camellia sinensis_, _Cynara scolymus_, cinnamaldehyde, carvacrol, citral, tannins, and _Capsicum oleoresin_.