The auditory sensitivity of 18 harbour seals of varying age and sex (see Table 1) was tested on sandbanks in The Wash, on the east coast of the U.K., in January 2012. In February 2013, the auditory sensitivity of ten additional harbour seals, also of varying age and sex (see Table 1), was tested at the Zoo Duisburg and Tierpark Nordhorn in Germany. These animals had been either kept in these facilities for all of their adult lives or were born there.
Table 1 Location and date of auditory measurements and information on subjects tested
In The Wash, all animals were caught using hoop or seine nets. All procedures in the wild were carried out under Home Office Animals (Scientific Procedures) Act licence number 60/4009. Data on animal sex and weight were collected on site where ABR measurements were also taken. When possible, a tooth was extracted for aging purposes. Seals were aged by counting the growth layer groups in the cementum of an incisor tooth, using the method of Dietz et al. (1991). In the zoos, animals were moved to a veterinary lab for ABR measurements. Age was taken from zoo records.
Animals were given a premedication intramuscular injection of midazolam (Hypnovel®, females 0.09–0.13 mg/kg, males 0.09–0.38 mg/kg), after ~10 min they were anaesthetised with an injection of ketamine (Ketaset®, females 1.47–3.25 mg/kg, males 1.09–3.16 mg/kg) into the epidural sinus with a three and one half-inch spinal needle which was subsequently maintained in place. Additional intravenous doses of Ketaset® were administered to maintain the desired anaesthesia, and additional intravenous doses of midazolam were administered to control muscular tremors, a side effect of ketamine anaesthesia.
Acoustic stimulation
In-air hearing sensitivity was measured in sedated animals by measuring their ABRs. The hearing sensitivity was tested at 1.4, 2.0, and 2.8 kHz in all animals. Acoustic stimuli were presented binaurally via headphones (DT 48 A.0, Beyerdynamic GmbH and Co. KG) to the animals in trials of 512–2048 stimulus repetitions with a 5-dB step size in descending order. Short tone pips consisting of five cycles of cosine-gated sine waves were used as stimuli, with a duration between 5 ms (at 1 kHz) and 1.8 ms (at 2.8 kHz). Additional frequencies were tested (at half octave steps between 4 and 22.4 kHz) if time allowed or no reproducible results could be achieved at the initial test frequencies. At a repetition rate of 33.3 stimuli per second, the polarity of successive stimuli was inverted to avoid stimulus artefacts.
The signals emitted through the headphones were calibrated before the measurements using a frequency generator (Agilent, USA, type 33220A) for signal generation, an artificial ear (Brüel & Kjær, Denmark, type 4157) connected to a calibrated microphone (Brüel & Kjær, type 2669) and a conditioning amplifier (Brüel & Kjær, type NEXUS 2690) as receiver. The signals were calibrated in terms of sound pressure level (SPL rms) over the duration of the tone pips. All signals were visually inspected for spectral quality (distortion) on a digital oscilloscope (PeakTech Prüf- und Messtechnik GmbH, Germany, type 1205) over the tested frequency range. No signal distortion was documented for any of the frequencies and levels used in our study.
The starting sound pressure level for the first animal tested was chosen to be approximately 30 dB above the hearing threshold of harbour seals based on previous publications. In subsequent measurements on the remaining animals, levels were adjusted to start 30 dB above levels determined in the first animal. In addition, background electrical noise was measured in the absence of stimuli. In all trials (with and without acoustic stimulation), the headphones were held in place over the animal’s ear openings by one of the researchers. The opening of the outer ear canals was regularly checked and acoustic stimuli were only played when the outer ear canal was visibly open.
Background noise was measured as the equivalent continuous sound pressure level (L
Zeq, unweighted) using a Casella CEL-6X0 handheld sound level meter (Casella CEL Inc., Buffalo, NY, USA) (over a 5-min period in the frequency band of 6 Hz to 20 kHz (Fig. 1). In the laboratory settings, background noise was found to be 54 dB re 20 µPa (±2 dB). In the field, background noise was measured post hoc in the same weather conditions as when the initial auditory measurements were taken. On the sandbank, noise was mainly caused by wind and waves with an overall L
Zeq between 72 and 96 dB re 20 µPa (±2 dB) (1/3 octave band levels given in Fig. 1). In all experimental settings, the headphones provided 12-dB attenuation of the ambient noise (according to Beyerdynamic, technical specifications of DT 48.0A).
Stimulus generation and response acquisition
The hardware setup for measuring the ABRs differed between the laboratory and field setting. On the sandbanks, stimulus generation, transmissions and recording of the neuronal responses were conducted using the custom made EVREST system (Finneran 2009) which includes a data acquisition board (NI PCI-6251, National Instruments, Austin, TX, USA). The acoustic stimuli were digitally generated, converted to analog at a 1-MHz update rate and 16-bit resolution, low-pass filtered at 250 kHz (Krohn-Hite Corporation, Brockton, MA, USA) and attenuated (over a range of 0–70 dB) before being presented to the animals via headphones. Recorded neuronal responses (digitisation rate 20 kHz) were amplified (94 dB) and bandpass filtered between 300 Hz and 3 kHz.
The audiometric measurements at the two facilities in Germany were conducted using a Tucker-Davis Technologies Workstation System 3 [Tucker-Davis Technologies (TDT), Alachua, FL, USA]. The acoustic stimuli were generated using the TDT software SigGen at a digitisation rate of 50 kHz. The recorded electrode responses were amplified (TDT RA4L; 20 dB gain), passed through an anti-aliasing filter, and led to an A/D converter (TDT RA16). Subsequently, the response (digitisation rate 25 kHz) was digitally filtered (high pass 300 Hz, low pass 3 kHz), written to a memory buffer and tested for the presence of signal artefacts. We used the TDT software BioSig to average the resulting potentials to allow an assessment of artefacts that indicates successful reception of the signal.
In all animals, the neuronal signals were measured with subdermal needle electrodes (NIHON-Kohden, Tokyo, Japan; 30 gauge) which were placed along the dorsal midline of the head: the active electrode on the vertex, 2 cm in front of the line between both ear openings, the ground electrode in the nape of the neck (i.e., 10–15 cm behind the ear-line, depending on the animal’s size) and the reference electrode another 10–15 cm further back. The input impedance between the electrodes was 1 kΩ or below during all measurements.
Analysis
Neuronal waveforms were measured over a period of 10 ms after acoustic stimulation and averaged over the total number of presentations. The peaks of the recorded neuronal waveforms are numbered (I–VII) according to their succession (nomenclature of neuronal waves following Jewett and Williston 1971), with wave V being the most prominent wave which can also be identified more reliably at decreasing stimulus amplitude (under ideal conditions down to levels close to the hearing threshold). The amplitude of wave V of the response evoked by the tone pips was measured and used for threshold determination in this study.
In contrast to the EVREST system, the ‘TDT system 3’ provides no option for determining the threshold level based on the last positive identification of a neuronal response and the first miss. To allow for a comparative analysis of both data sets and reduce the influence of varying physiological noise levels between subjects and animal groups, a regression analysis of the wave V peak amplitudes was conducted after visual inspection of all recorded ABRs. A stimulus was considered as not perceived by the animal if an ABR was not detectable above the neuronal background noise level at each given frequency. Distorted measurements (due to technical reasons, strong movements of the animals, etc.) were not included into the regression analysis.
Comparison of threshold levels
The hearing thresholds measured by Wolski et al. (2003) represent the only other auditory data achieved for a harbour seal with the same methodology (ABR). Expressing thresholds in terms of the energy content of the entire stimulus over time (SEL) as done by Wolski et al. (2003) is, strictly speaking, not appropriate as ABRs are an onset response. To allow for comparison, the data reported by Wolski et al. (2003) were converted into SPL levels. This allows for direct comparison with the thresholds reported in this study as well as behavioural hearing thresholds measured by Reichmuth et al. (2013) (for our data see Table 2).
Table 2 Average aerial hearing sensitivity of captive (laboratory) and free-ranging harbour seals in this study determined by measuring the auditory brainstem response as a function of frequency