Evaluation of Auditory Brainstem Response in Chicken Hatchlings

The experiments described here were approved by Northwestern University's Institutional Animal Care and Use Committees (IACUC) and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

1. Chicken husbandry

1. Acquire fertilized white leghorn chicken eggs.
   NOTE: There are several chicken breeds used in scientific research, but the results shown here are from white leghorn chicken (Gallus gallus domesticus). While ABR variability between breeds is unknown, some differences have been found when comparing adult egg-laying chickens to meat-producing broiler chickens^24,25.
2. Incubate eggs at 38 °C, humidity at 50%, for 21 days before the desired testing date.
   NOTE: If eggs are not immediately incubated at 38 °C, they can be stored at 14 °C, humidity at 40%. However, the longer eggs are kept at 14 °C, the less likely they are to develop into viable hatchlings. After 7 days, egg viability can drop as low as 50% depending on how long eggs are kept at 14 °C. Egg viability will also drop in the winter months.
3. Periodically turn the eggs 2-3 times a day. Most incubators have a mechanism to perform this automatically.
4. If using a Styrofoam incubator or an incubator holding more than 6 eggs, transfer eggs to a small 38 °C incubator the day before hatch, embryonic day 20 (E20). Eggs should hatch 21 days (E21) after being set in the incubator.
   NOTE: In the hatching process, the animal will begin "pipping" out of the egg, making a small hole that eventually goes around the entire egg. If conditions are too dry, the egg can dry up, and the animal will be unable to hatch. The humidity should be kept around 50%, based on previous studies on egg hatching viability^26,27,28,29.
5. Determine the animal's age. If the hatch is not witnessed in person, the only indication of age is the 2-3 h it takes for the amniotic fluid to dry.
   ​NOTE: The hatchling incubator should be thoroughly cleaned daily with 70% isopropyl alcohol based on how many hatchlings are processed. Chicken hatchlings often leave excrement, feathers, and amniotic fluid in the incubator, which can contaminate conditions and air quality.

2. Drug preparation

1. Weigh the animal by placing it in a large weighing boat. With a gentle enough placement, the animal should not move.
   NOTE: Mass can range from 30-45 g. Younger animals are often heavier because of yolk reserves and not yet excreting waste. Older animals approaching 24 h of age and P2 usually weigh less.
2. Prepare an anesthetic cocktail of Ketamine (100 mg/mL) and Xylazine (20 mg/mL) such that the dosage is 50 mg/kg Ketamine and 16.68 mg/kg Xylazine based on animal weight.
   ​NOTE: This drug cocktail can be made with 1 mL of Ketamine (100 mg/mL), 1.5 mL of Xylazine (20 mg/mL), and 2.5 mL of H₂O. Anesthetic cocktail injections will range from 0.05-0.1 mL based on the 30-45 g range in animal weight.

3. Drug injection and animal prep

1. Hold the animal in one hand, making sure to hold the legs down.
2. Feel for the breastbone of the animal, the keel. On either side of the keel will be breast muscle.
3. Use a 29-G needle and syringe to penetrate 5 mm into the skin and inject the Ketamine/Xylazine cocktail into the breast muscle. Inject between 0.05-0.1 mL based on animal weight.
4. Place the animal back in the incubator after injection. Maintain animal body temperature for a few minutes as the anesthetic takes effect.
   1. Use forceps to pinch the toe of the animal and check if the neck is limp. If there is no reflex and a limp neck, the animal is unconscious.
5. Determine the sex of the chicken using its wing feathers. If the feathers are all the same length, the animal is male. If the feathers vary in length, the animal is female³⁰.
   NOTE: Another method of sexing the animal is venting. The male genitalia can be seen in the cloaca³¹. This method is very difficult and can harm the animal if not done correctly. It is recommended to use the wing feather method.
6. Apply depilatory cream with a cotton tip applicator to the head and neck area, especially near the ear opening for the bird.
7. Use 70% isopropyl alcohol wipes to wipe off feathers, any remaining depilatory cream, and the skin on the head and neck.
8. Use a 70% isopropyl alcohol wipe to sterilize the subdermal electrodes and rectal probe.
9. Place the animal in a sound isolation and electrically shielded chamber. Ensure that the environment has minimal electrical and acoustical noise for the best recordings.
   NOTE: The experiments here were made in a custom sound isolated enclosure measuring 24 x 24 x 25 inches. Any chamber or room that eliminates acoustical noise, as well as electrical noise from alternating electric current (60 Hz in the United States), is sufficient.
10. Use a heating pad or temperature control system to maintain animal body temperature.
11. Insert the lubricated rectal probe to ensure the animal's temperature is maintained between 37-41 °C (98.6-105 °F)^32,33.
    NOTE: If the probe is an incorrect size, the animal can lay on top of the temperature probe.
12. Fix the animal's head in place or rest the beak against an object to avoid unwanted movement. This can be done with modeling clay if breathing is not obstructed.
13. Administer a supplementary injection of anesthetic cocktail that is one-half the original dosage if the animal begins to regain consciousness during testing.
    ​NOTE: Any body movement or vocalization is a sign that a supplement dosage must be administered. Miniscule beak movements indicate breathing and are acceptable.

4. Electrode placement

1. Use three stainless steel, silver chloride needle electrodes with the following designations: the reference electrode, the active electrode, and the common ground electrode.
   NOTE: The reference electrode is also referred to as inverting or "-". The active electrode is also referred to as noninverting or "+".
2. Place each electrode sub-dermally 2-3 mm into the head, but not deep enough to penetrate the skull. Use electrodes that are 7 mm in length and 0.4 mm in diameter.
3. Poke the electrode out of the skin, exposing the tip. This helps to minimize contact with the skin and ensure consistent insertion depth across animals³⁴.
   NOTE: The electrode wire should have sufficient slack such that after placing the electrode, there is no tension that will pull it out or pull the skin taut.
4. For single-channel recording, place the active electrode above the skull at the midline, as far caudal as the ear canal.
   1. Place the reference electrode behind the ear where the stimulus will be delivered, and place the ground electrode behind the contralateral ear canal in the neck.
      NOTE: If performing surgery at the skull or ear canal of the animal, place the reference electrode in the neck at the midline of the animal. Both this and step 4.4.1 are considered horizontal electrode recording montages.
5. For two-channel recording, use two negative electrodes and a combined positive electrode that requires an adapter cable. Place the ground electrode subdermally in the neck and one reference electrode behind each ear canal.
6. Check the electrode impedance. Ensure that the overall electrode impedance does not exceed 5.0 kΩ. Maintain the interelectrode impedance below 3.0 kΩ.

5. ABR recording

1. Depending on acquisition hardware and software, be sure to perform calibration for correct sound levels across stimulus frequencies used.
   NOTE: Calibration techniques will vary based on equipment (see discussion). For some programs, sound attenuation can be edited within the software. Calibration procedures performed here involved using a 1/8-inch B&K 4138 condenser microphone to record frequency stimuli within a closed coupler system that approximated the chick ear canal (~5 mm). A chicken hatchling calibration table is provided as a Supplemental Table.
2. Move the sound transducer apparatus toward the active ear of the animal. Place the sound transducer at a shallow depth of 2 mm in the ear canal.
   NOTE: Depending on the sound transducer, a plastic speculum can be attached and inserted into the ear canal. The speculum placement is critical. If the sound is blocked by the canal wall or the ear canal is pinched shut, ABRs will be absent or resemble a ~40 dB shift in the threshold.
3. Check on the animal during testing if results look abnormal or absent. If they are, reposition the sound transducer in the ear canal.
   ​NOTE: Since the skin is loose and animal movement is possible, the speculum placement may shift during recording. However, with correct anesthetic injection and the animal fully unconscious, recording can go uninterrupted for 30-45 min.

6. Data acquisition

1. Utilize sufficient equipment/software to generate sound stimuli and record/acquire ABR recordings.
   NOTE: There are many commercially available or custom systems for ABR acquisition. For these experiments, the commercially available Intelligent Hearing Systems (IHS) SmartEP USB platform was used. The ability to manipulate recording parameters is critical; these include, but are not limited to stimulus intensity, stimulus length, stimulus frequency, stimulus presentation rate, high pass and low pass filter, artifact rejection, number of sweeps, sampling rate, envelope shape, and stimulus polarization.
2. Set the artifact rejection (AR) upper and lower limits to ±25 µV, such that animal movement or noise during a sweep will exclude that sweep from the analysis. Across the population tested, less than 1% of total sweeps were rejected due to artifacts.
3. Collect at least 1024 sweeps to obtain a grand averaged response. This can be done in two recordings of 512 sweeps each. This also ensures that the response is stimulus-evoked and repeatable.
4. Set the gain to 100,000, the low pass filter to 100 Hz, and the high pass filter to 3000 Hz.
   NOTE: The low and high pass filter settings were optimal for recordings using the IHS system. Therefore, these parameters are recommendations. ABR recordings in other avian species using the BIOSIG software filtered the signal between 30 and 3000 Hz ^{5,13,14,16,22}.
5. Set the stimulus presentation rate between 10 and 20 stimuli per second. High presentation rates will shift ABR peak latency, especially for later peaks¹³. Low presentation rates will increase the time required to acquire the ABR.
6. Set the time duration of the click stimulus to 100 µs.
   1. If using a tone burst stimulus, edit the frequency and duration of the stimulus based on the desired effect. A range of 100-4000 Hz was used for tone burst stimuli, although the range of behavioral hearing in adult chickens ranges from 2-9000 Hz³⁵.
      NOTE: In the IHS system, the rise and fall time of a tone burst stimulus can only be modified if the spectral envelope shape is a trapezoid. However, the cosine squared and Blackman envelopes provide a preset rise and fall time that is commonly used in animal ABR experiments. The IHS system can display the spectral envelope of a tone burst to ensure appropriate rise and fall times. The rise and fall time of a click stimulus cannot be edited in IHS.
7. Set the sampling rate to the highest value allowable (usually 40 kHz) for the best resolution data.
   NOTE: Some systems, including IHS, use a limited number of sampling points and will alter the length of the recording window. A 40 kHz (25 µs period) sampling rate may only allow for a 12 ms recording window, so to capture a tone burst ABR, a 20 kHz sampling rate (50 µs period) was used to allow for a 24 ms recording window. If directly comparing click and tone burst ABRs, keep the sampling rate constant to maintain the same resolution.
8. Set the stimulus polarization to alternating. This is done to eliminate the visualization of the cochlear microphonic from ABR recordings. To visualize the cochlear microphonic, use rarefaction or condensation for stimulus polarity.
   NOTE: Many settings can be changed when selecting stimuli. The gain and filter settings provided may not be optimal for other equipment setups. Factory defaults on most ABR machines are not set for recording in hatchling chicken.
9. If recording 512 sweeps, combine two separate tests to create a 1024 sweep average.
10. For a click or tone burst stimulus, acquire an ABR at a suprathreshold intensity.
11. Continue recording at lower and lower intensities until the evoked response can no longer be identified.
12. Define ABR threshold as the lowest stimulus intensity that elicits a detectable evoked response. Lower the stimulus intensity by steps of 5 dBSPL to find the lowest stimulus intensity that elicits a detectable peak.

7. Euthanasia and experiment end

1. Once ABRs are acquired, prepare an overdose (0.1 mL) of euthanasia solution (Pentobarbital Sodium 390 mg/mL Phenytoin Sodium 50 mg/mL).
2. After using a toe pinch to confirm that there is no reflex, inject the euthanasia solution in the breast muscle with a 29-G needle at a 5 mm depth. The injection technique is the same as the anesthetic injection.
   NOTE: The animal will expire after a few minutes. Do not manipulate or decapitate the animal until no movement is detected. An alternative euthanasia technique is to perform an intravenous injection in the brachial vein under the wing.
3. As soon as the animal is not reflexive and breathing and heartbeat have ceased, rapidly decapitate with sharp scissors or shears.
4. Clean the heating pad, rectal probe, and silver chloride electrodes with 70% isopropyl alcohol wipes.
5. Make sure all acquired traces have been saved. For further analysis, export files as .txt files which can be viewed in notepad or imported into a spreadsheet.