The comparative effects of ventilation shutdown with heat (VSDH), relative humidity (VSDHRh), or CO2 (VSDCO2) on broiler electroencephalogram (EEG), blood chemistry and gene expression

This study consisted of two phases, Phase 1 (P1) and Phase 2 (P2), with both occurring in a total of four, 4.75 ft³ chambers made with Plexiglass®. The Plexiglass® was 0.25 in thick on each side, with three sides having 1-inch thick, closed cell foil backed insulation foam boards to prevent chamber heat loss. The front side was not covered which allowed for observations. The chambers were built to allow for attachments to collect data, such as carbon dioxide (CO₂) for measuring and injection of the gas, and attachments for relative humidity and temperature sensors. Each chamber was vented through a rigid barbed hose fitting (Grainger, Lake Forest, IL, USA; Model: 707020-0404) located on the top, back-right corner of each chamber. The venting allowed air to exit the box during the VSDCO₂ treatment as CO₂ was injected. Each chamber was equipped with a 100-watt incandescent light bulb. This provided a heat source for treatments with heat, or additional heat, as described in Table 1, but the light was not turned on for VSDCO₂ as to not provide additional heat. The chambers were located in a temperature-controlled, windowless room that was kept around 26°C±5 (78°F) to prevent heat loss to the environment. The door to the room was closed for the VSDH and VSDHRh treatments, but for human safety, the door was left open for VSDCO₂. P1 and P2 had three treatments that were evaluated which were ventilation shutdown plus heat (VSDH), ventilation shutdown plus heat and relative humidity (VSDHRh), and ventilation shutdown plus carbon dioxide (VSDCO₂). For P1, the CO₂ was injected into the chambers at 1 % CO₂ per minute to try and achieve ∼30 % CO₂ to correlate what would be seen in an industry setting.

Twelve, 42-day-old mixed sex broilers were used for P1, allowing for four replicates per treatment. Broilers used in P1 and P2 were raised at the Poultry Teaching Unit in the Prestage Department of Poultry Science and birds were randomly selected for each treatment. These birds were fed a standard broiler diet and were raised in floor pens. Treatment broilers were transported to the bird wing in Prestage Department of Poultry Science 1 day before the study began and were provided broiler finisher feed and water ad libitum. Treatment broilers were removed and blood was collected from the brachial vein and placed in 3 mL BD Vacutainer tubes with lithium heparin to prevent clotting. This allowed for a baseline analysis of corticosterone levels and blood chemistry prior to treatment. For blood chemistry, roughly 0.1 mL of heparinized blood was placed on the i-STAT® diagnostic system using the CG8+ cartridges (Abbott Park, IL, USA; Model: 03P8825) to be analyzed. The remaining blood in the tube was placed on a gentle rocker to be centrifuged later. After centrifugation, the plasma was poured off and frozen at 20°C for corticosterone analysis. Pre- and post-core body temperatures were collected before and after treatments for P1 and P2 using a SuperMeter® cloacal temperature probe (Stamford, CT, USA; Model: HHM290). Throughout P1, an electroencephalogram (EEG) was used to measure each bird’s brain electrical output in millivolts (mV). Before the EEG electrode placement, each bird was hobbled which allowed them to stand and move but prevented them from scratching their head resulting in the potential removal of the electrodes. The hobble, a rubber band, was put on the shanks and above the dewclaws. All electrodes were insulated, except for the tips that were 32-gauge needles. There was a total of three electrode colors: green, black, and red. The green electrode was the ground and was placed in between the waddles on the lower mandible. The red and black wires were placed on either side of the cranium, above the ear lobe and behind the comb. Once inserted, each electrode was secured using surgical adhesive and by means of the catheter taping method. Thereafter, these three leads were taped together and wrapped under the joint of the wing that provided an additional method to reduce the likelihood of the bird pulling out the inserted probes. Once the probes were secured, the birds were placed in their respective chambers, which were then sealed. The terminal end of the EEG electrodes were plugged into a pre-amplifier (AD Instruments, Colorado Springs, CO, USA), transferring the EEG readings to a laptop that was continuously monitored. All EEG data was recorded at 10-50 Hertz (Hz) and sampled at 100 Hz/channel and was recorded until time of death (TOD) was called by the veterinarian. Time of death for all birds in P1 and P2 was determined by cessation of movement as described in Table 2. Broiler behavior in P1 was observed and recorded in real time by a trained person and recorded at 2-minute intervals, with behaviors (described in Table 2) being evaluated based on observations by Krish (2018). The unconscious behaviors were determined after the broiler EEG readings dropped below 0.01 mV. Temperature and relative humidity of each chamber and each phase was recorded every minute with sensors (Aranet, Riga, Latvia; Model: SKU: TDSPT8U2; Accuracy: ±0.3°C and ±2 %) that were placed roughly 4 cm from the top of the inside of each chamber. Carbon dioxide was measured using two 0-100 % CO₂ monitors (CO₂Meter, Inc., Ormond Beach, FL; Model: CM-0003; Accuracy: ±70 ppm ± 5 %) and recorded every minute. Each chamber was equipped with a rigid barbed hose fitting (Grainger, Lake Forest, IL, USA; Model: 707020-0404) for a ¼-in inside diameter (ID) wide tube to be snugly fit over the fitting. The other end of the tube was attached to the CO₂ monitor. For VSDHRh, a fogger (AUAAQ, Seattle, WA, USA; Model: KLS-208) was utilized and turned on once start time was initiated and turned off when the Aranet sensors read 99 % relative humidity. The average flow rate of the fogger was 0.27 L/min. A 1-in diameter hole on the left side of each chamber allowed for the plastic tubing to be securely attached to the chamber with the other end being attached to the fogger. This hole was plugged for VSDH and VSDCO₂ treatments. To inject CO₂, each chamber was equipped with an attached ¼-in 250 pounds per square inch (PSI) air hose on the bottom right side. These hoses have a ¼-in body and hose fitting size with a PARKER Quick Connect Hose Coupling push-to-connect female attachment (Grainger, Lake Forest, IL, USA; Model: B33) at the end. An 11-gallon portable air tank (Torin Big Red Jacks, Ontario, CA, USA; Model: T88011), with a 125-PSI max, and equipped with a regulator, was charged with CO₂ from a tank to allow for easier control of flow and temper the CO₂ temperature. The portable air tank was also equipped with a ¼-in 250 pounds per square inch (PSI) hose with a ¼-in body and hose fitting size, PARKER Quick Connect Hose Coupling push-to-connect male attachment (Grainger, Lake Forest, IL, USA; Model: B32) allowing for attachment to the chamber. Each tank was equipped with an on/off ball valve with a lever attached to regulate the CO₂ flow into the chamber. The CO₂ was tempered to room temperature for P1 and P2. For P1, the CO₂ was injected into the chambers at 1 % CO₂ per minute and was verified by the CO₂ meter readings with a goal to achieve ∼30 % CO₂ to correlate what would be seen in an industry setting. Once TOD for a bird was called, the bird was removed, body temperature was measured, and blood was collected from the body cavity immediately. The blood was removed using a syringe and this method was used due to the immediate blood clotting issues observed, especially in VSDH and VSDHRh. The blood was then placed in a labeled BD Vacutainer tubes with lithium heparin and blood gas was evaluated again for post levels utilizing the same procedures as above for the i-STAT® diagnostic system using CG8+ cartridges and future corticosterone analysis. Birds were also necropsied to remove the brain which was placed in RNAlater® solution in a 25 mL conical tube. This tube was then placed in a refrigerator (4°C) for 24 hours and then moved to a freezer (-20°C) for later RNA extraction and analysis.

In P2 of this experiment, we aimed to determine the physiological aspects of the three treatments over time by examining the corticosterone, gene expression, and blood chemistry. Ten 42-day-old mixed sex broilers were used for each treatment allowing for two replications for each time point. To determine removal times in order to understand the changes over time, the four TOD from each treatment in P1 were averaged. The average was then quartered giving four removal times, with a total of five including the baseline. The calculated time points for this experiment are listed in Table 3. The flow rate of CO₂ for VSDCO₂ treatment was calculated similarly to the removal times with the final CO₂ for VSDCO₂ treatment only being summed up and quartered to determine the flow rate over time for this treatment. The flow rate of CO₂ for the VSDCO₂ treatment was 4 % CO₂ every five minutes. At time of removal, all birds were bled via the brachial vein and methods described above were followed for i-STAT® diagnostic system using CG8+ cartridges and corticosterone analysis. Broilers were then euthanized by a trained individual via cervical dislocation and necropsied with the brain tissue being collected and placed in RNAlater® solution following procedures described above.

An ELISA corticosterone kit (Cayman Chemical Company, Item: 501320, Ann Arbor MI, USA) was used to analyze corticosterone concentration levels in P1 and P2 following manufacturer guidelines. The concentration levels were evaluated using a standard curve of nanograms of corticosterone per milliliter of plasma. A 1-cm portion of brain tissues from P1 and P2 was cut and removed to isolate RNA. These pieces were placed into individually labeled screw cap homogenization vials and then 1 mL of TRI Reagent™ Solution. Each sample was homogenized using a bead-beater for 20 seconds and then incubated at room temperature for five minutes. Thereafter, 200 µL of chloroform was added to each sample and vortexed for a few seconds then left at room temperature for two minutes. These samples were then centrifuged at 13,000xg for 20 minutes. Afterwards, the RNA isolation was completed using an RNeasy kit (QIAGEN, Hilden Germany) following the manufacturer guidelines. A NanoDrop2000 (Thermo Fisher Scientific-Waltham, MA) was used to quantify the RNA levels and ensure the correct RNA dilution. The RNA was taken and transcribed into cDNA for quantitative polymerase chain reaction (qPCR) using a high-capacity synthesis kit (Applied Biosystems Waltham, Massachusetts, USA). All qPCR was completed on each sample in triplicate. In each of the wells there was 2.5 ng of samples, 500 nM of gene specific forward and reverse primers (Table 4), and 2X power SYBR green master mix (Applied Biosystems Waltham, Massachusetts, USA) and H₂O was added to a finalize the volume to 20 µL. The Applied Biosystems StepOnePlus real-time PCR system was used to perform qPCR. The results were then normalized to Beta Actin expression, a basic housekeeping gene. For ease of interpretation, the reciprocal was then taken using the formula: 1/(target gene cycle threshold/beta actin cycle threshold). All data presented as reciprocal cycle thresholds of CT^-1.