Matthew Wijers
email: matthew.wijers@gmail.com

Paul Trethowan

Byron Du Preez

Simon Chamaillé-Jammes

Andrew J Loveridge

David W Macdonald

Andrew Markham

Vocal discrimination of African lions and its potential for collar-free tracking

Keywords: African lion, fundamental frequency, passive acoustic monitoring, vocal individuality, vocalisation African lion, fundamental frequency, passive acoustic monitoring, vocalisation, vocal individuality

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Introduction

African lions (Panthera leo) are well known for their loud, characteristic roars, frequently audible from several kilometres away. Lions are, however, just one of many terrestrial mammalian species that are capable of communicating vocally over large distances. The basic function of long distance signalling is to aid individuals in searching for, locating and avoiding each other and therefore facilitating group cohesion and the maintenance of territorial boundaries [START_REF] Marler | Animal communication signals: We are beginning to understand how the structure of animal signals relates to the function they serve[END_REF][START_REF] Mitani | Contexts and social correlates of long-distance calling by male chimpanzees[END_REF][START_REF] Ramsauer | Acoustic communication in lions and its use in territoriality[END_REF]. For many species, a key component of the information conveyed to conspecifics through the use of long-distance calls is individual identity. Detection and decoding of this information are crucial for influencing the response of the receiver. For example, in the context of territoriality, identifying a call from a neighbouring animal in their usual location will likely elicit a different response to that of an unfamiliar animal [START_REF] Mcgregor | Signalling in territorial systems: a context for individual identification, ranging and eavesdropping[END_REF]. Several studies on terrestrial mammals such as elephants (Loxodonta africana; [START_REF] Clemins | Automatic classification and speaker identification of African elephant (Loxodonta africana) vocalizations[END_REF], orangutans (Pongo pygmaeua wurmbii; [START_REF] Spillmann | Who shall I say is calling? Validation of a caller recognition procedure in Bornean flanged male orangutan (Pongo pygmaeus wurmbii) long calls[END_REF], tigers (Panthera tigris; [START_REF] Ji | Discrimination of individual tigers (Panthera tigris) from long distance roars[END_REF] wild dogs (Lycaon pictus; [START_REF] Hartwig | Individual Acoustic Identification As a Non-Invasive Conservation Tool: an Approach To the Conservation of the African Wild Dog Lycaon Pictus (Temminck, 1820)[END_REF] and wolves (Canis lupus lycaon; [START_REF] Root-Gutteridge | Identifying individual wild Eastern grey wolves (Canis lupus lycaon) using fundamental frequency and amplitude of howls[END_REF] have shown that certain elements of vocal signals are unique to individuals and may convey information relating to caller identity. The production of these unique call features is likely related to small variations in the morphology of the vocal apparatus between individuals [START_REF] Fitch | Vocal tract length and formant frequency dispersion correlate with body size in rhesus macaques[END_REF][START_REF] Ey | Do age-and sex-related variations reliably reflect body size in non-human primate vocalizations? A review[END_REF].

Despite the ability of many animals to encode individually unique features within their calls, sound signals degrade progressively as they propagate through the environment. Higher frequencies are more rapidly absorbed through air and incur greater scattering by small objects (e.g. vegetation) compared to lower frequencies, resulting in changes to the spectral structure of the original signal [START_REF] Forrest | From Sender to Receiver: Propagation and Environmental Effects on Acoustic Signals[END_REF][START_REF] Wiley | Physical Constraints on Acoustic Communication in the Atmosphere: Implications for the Evolution of Animal Vocalizations[END_REF]. This degradation can impair the ability of distant receivers to extract information. Several studies on birds have shown that individuals responded more strongly to undegraded calls than to degraded calls [START_REF] Mcgregor | The Reaction of Great Tits (Parus major) to Playback of Degraded and Undegraded Songs: The Effect of Familiarity with the Stimulus Song Type[END_REF][START_REF] Mathevon | Reaction to conespecific degraded song by the wren Troglodites troglodytes: territorial response and choice of song post[END_REF]. Despite the loss of information incurred during signal propagation, some species are able to learn and accurately recognise the degraded call of a conspecific through experience [START_REF] Aubin | How to vocally identify kin in a crowd: the penguin model[END_REF][START_REF] Mouterde | Learning to cope with degraded sounds: female zebra finches can improve their expertise in discriminating between male voices at long distances[END_REF].

Knowledge of how species transmit information relating to individual identity is important for understanding species communication mechanisms. In some cases, vocal individuality can also be exploited by researchers and conservationists to monitor and survey species populations using individually distinct vocal features as a non-invasive marking method [START_REF] Terry | The role of vocal individuality in conservation[END_REF]. For example, [START_REF] Gilbert | Local annual survival of booming male Great Bittern Botaurus stellaris in Britain, in the period 1990-1999[END_REF] investigated the survival and movements of Great Bitterns (Botaurus stellaris) using spectrogram measures of vocalisations. Similarly, [START_REF] Delport | Vocal identification of individual African Wood Owls Strix woodfordii: A technique to monitor long-term adult turnover and residency[END_REF] In order to assess animal vocal individuality, acoustic features must first be extracted from the call recordings. Although this can be done automatically using state of the art deep learning techniques [START_REF] Stowell | Automatic acoustic detection of birds through deep learning: The first Bird Audio Detection challenge[END_REF], acoustic features are often extracted manually, whereby simple summary variables (e.g. min, max and mean) associated with the fundamental frequency and the harmonics are calculated [START_REF] Fan | Individuality in coo calls of adult male golden snub-nosed monkeys (Rhinopithecus roxellana) living in a multilevel society[END_REF][START_REF] Ji | Discrimination of individual tigers (Panthera tigris) from long distance roars[END_REF].

Another common approach involves the calculation of Mel-frequency cepstral coefficients (MFCCs) which effectively warps the frequency axis to the Mel-scale under the assumption that the species perceives frequencies on a logarithmic scale. The MFCC method is based the human auditory system and is widely used in human-speech recognition, however it is also becoming more popular for the identification of animal vocalisations [START_REF] Clemins | Automatic classification and speaker identification of African elephant (Loxodonta africana) vocalizations[END_REF][START_REF] Mielke | A method for automated individual, species and call type recognition in free-ranging animals[END_REF][START_REF] Spillmann | Who shall I say is calling? Validation of a caller recognition procedure in Bornean flanged male orangutan (Pongo pygmaeus wurmbii) long calls[END_REF]. In this work we adopt a different and more simple approach that uses short-time Fourier transformation to extract the fundamental frequency contour sequence which is used as the only acoustic feature. Following feature extraction, pattern recognition algorithms are implemented to 'learn' the acoustical features associated with each individual and subsequently classify 'unseen' calls. The recognition models are assessed based on their ability to classify the 'unseen' calls correctly. Several algorithms have been used for this purpose, including discriminant functions [START_REF] Blumstein | Individual, age and sex-specific information is contained in yellow-bellied marmot alarm calls[END_REF][START_REF] Fan | Individuality in coo calls of adult male golden snub-nosed monkeys (Rhinopithecus roxellana) living in a multilevel society[END_REF], artificial neural networks [START_REF] Mielke | A method for automated individual, species and call type recognition in free-ranging animals[END_REF][START_REF] Reby | Individuality in the groans of fallow deer (Dama dama) bucks[END_REF], gaussian mixture models [START_REF] Cheng | A call-independent and automatic acoustic system for the individual recognition of animals: A novel model using four passerines[END_REF]) and hidden Markov models [START_REF] Clemins | Automatic classification and speaker identification of African elephant (Loxodonta africana) vocalizations[END_REF][START_REF] Ji | Discrimination of individual tigers (Panthera tigris) from long distance roars[END_REF]. One of the main issues affecting the classification performance of vocal recognition models is the presence of background noise. Noise from any natural or anthropogenic source introduces additional sound information and thereby increases model variation and classification uncertainty [START_REF] Terry | A Comparison of Some Techniques Used To Assess Vocal Individuality[END_REF]. The majority of studies have dealt with this issue by removing samples with overlapping signals [START_REF] Clemins | Automatic classification and speaker identification of African elephant (Loxodonta africana) vocalizations[END_REF][START_REF] Reby | Individuality in the groans of fallow deer (Dama dama) bucks[END_REF].

Lions emit loud, low-pitched vocal signals referred to as 'roars'. A single roar is typically delivered in a bout consisting of one or two soft moans followed by several full-throated roars and a terminating sequence of short grunts [START_REF] Grinnell | Roaring and social communication in African lions: the limitations imposed by listeners[END_REF][START_REF] Mccomb | Roaring and numerical assessment in contests between groups of female lions, Panthera leo[END_REF]. The low fundamental frequency of the roar can be attributed to the long and heavy vocal folds, characteristic of the species' vocal anatomy [START_REF] Weissengruber | Hyoid apparatus and pharynx in the lion (Panthera leo), jaguar (Panthera onca), tiger (Panthera tigris), cheetah (Acinonyx jubatus) and domestic cat (Felis silvestris f. catus)[END_REF]. Both male and female members of a pride are known to roar and will do so to either maintain contact with distant companions, or to advertise territory ownership [START_REF] Grinnell | Roaring and social communication in African lions: the limitations imposed by listeners[END_REF][START_REF] Mccomb | Roaring and numerical assessment in contests between groups of female lions, Panthera leo[END_REF]. Lions also use roars to transmit information relating to group size. A series of overlapping roars emitted by three or fewer individuals provides an honest indicator of the number of individuals present [START_REF] Mccomb | Roaring and numerical assessment in contests between groups of female lions, Panthera leo[END_REF]. Receivers are then able to assess their chances of successfully deterring potential competitors. Several studies have reported evidence of vocal recognition in lions. For example, [START_REF] Mccomb | Female lions can identify potentially infanticidal males from their roars[END_REF] found that adult females are able to distinguish between playbacks of familiar, resident males and those of unfamiliar males. Similarly, individual lions have been shown to display stronger responses to situations where there is conflicting information (e.g. a particular call does not match the individual from which it is expected), suggesting that specific calls are associated with specific individuals [START_REF] Gilfillan | Cross-modal individual recognition in wild African lions[END_REF]. Other studies have examined the attributes of individual roars and have found differences in the temporal pattern and acoustic features between male and female roars, however, little is known about whether consistent differences exist between individuals [START_REF] Pfefferle | Do acoustic features of lion, Panthera leo, roars reflect sex and male condition?[END_REF][START_REF] Stander | Characteristics of lion roars in Etosha National Park[END_REF]. This knowledge gap is likely attributable to the difficulties associated with obtaining sufficient samples of roar recordings from known individuals in the wild and highlights the need for a new approach to acquire data on animal vocalisations.

Lions defend large territories, often in excess of 500 km 2 [START_REF] Tumenta | Changes in lion (Panthera leo) home range size in Waza National Park, Cameroon[END_REF][START_REF] Zehnder | Home ranges of lions in the Kalahari, Botswana exhibit vast sizes and high temporal variability[END_REF]. Given that the primary functions of the roar require receivers to differentiate between individuals in order to respond appropriately, it can be reasoned that the transmission of information relating to caller identity over long distances would be advantageous. It can therefore be expected that selection would favour animal calls that transmit information efficiently by minimizing degradation of the intended signal. Despite many studies being dedicated to lion vocal behaviour, little is known about how lions convey information relating to individual identity in their call structure. In this study, we investigate whether calls from individual animals might be differentiated based solely on the fundamental frequency of the full-throated roars; the frequency that is likely to suffer the least degradation over large distances. To achieve this, we use a novel acoustic -accelerometer biologger which facilitates the acquisition of a relatively large dataset of lion roar events. We also demonstrate the conservation implications of our findings by testing the application of acoustic recognition and localisation to lion tracking.

Materials and methods

Study site

The study took place in the Bubye Valley Conservancy (BVC), a privately-owned wildlife area located in southern Zimbabwe between latitudes 21.209 and 21.851 South, and longitudes 29.789 and 30.521 East. The BVC measures approximately 3400 km 2 in area and hosts a variety of indigenous megafauna including a high density of African lions [START_REF] Du Preez | Impact of risk on animal behaviour and habitat transition probabilities[END_REF]. Habitat within the Conservancy is dominated by mopane woodland savannah with riparian woodland occurring along several seasonal river lines. Annual rainfall is typically low, averaging 351 mm, and falling mostly in summer between November and March (du Preez et al. 2014). Maximum daily temperatures are high (> 40C) in the summer months and comparatively mild in winter.

Data collection

Animal-borne biologgers

Given that lions vocalise predominantly during the night and can be difficult to identify and observe, we used an on-animal audio recording approach to investigate lion vocal individuality. In November 2014 we fitted custom-designed biologgers to five male and three female lions as described in [START_REF] Wijers | Listening to Lions: Animal-Borne Acoustic Sensors Improve Biologger Calibration and Behaviour Classification Performance[END_REF]. Each device comprised a triaxial accelerometer and magnetometer sampling at 32 Hz per axis and a mono-electret microphone sampling audio at 16 Hz with 8-bit resolution (frequency response ~ 30 Hz -8 kHz; dynamic range ~ 40 dB). All components were encased in an epoxy resin reinforced housing with a hydrophobic vent provided for the microphone. Study animals were chemically immobilised using 75-100 mg Zoletil (Virbac RSA (Pty) Ltd, Halfway House, South Africa) combined with 5 mg medetomidine (Kyron Laboratories, Johannesburg, South Africa). Immobilisation drugs were delivered intramuscularly by 1 cc darts (Pneudart, Williamsport, Pennsylvania, USA) projected from a Dan-Inject CO 2 -pressurised dartgun (Dan-Inject, Børkop, Denmark).

After fitting the biologger, ~ 25 mg atipamazol (Antisedan, Pfizer Animal Health, Johannesburg, South Africa) was administered to reverse the effects of medetomidine allowing the animal to recover within 15 -90 mins. The biologgers recorded continuous audio (8 bit, 16 kHz mono) for between 4 and 10 days before the batteries were depleted.

Study animals were then recaptured and the data downloaded for processing and analysis.

Acoustic array

To explore the application of acoustic recognition and localisation for lion tracking, we set up a passive acoustic array system using CARACAL audio recorders [START_REF] Wijers | CARACAL: a versatile passive acoustic monitoring tool for wildlife research and conservation[END_REF]) within the home-range of a coalition of two male lions fitted with GPS collars (neither of which had been previously fitted with biologgers). Animals were captured using the same capture technique as described in the preceding section. The acoustic array consisted of 8 GPS-timesynchronised audio recording stations each separated by ~ 500 m from the nearest station.

When operational, each station recorded audio continuously (32 bit, 44.1 kHz, 4 channel).

The array was activated on selected nights between June and November 2018 when weather conditions were optimal (dry and low wind speed).

Vocal individuality

Roar extraction

All lion audio recordings were processed manually in Audacity 2.1.1 (Audacity Team 2015) by visually inspecting spectrograms and annotating the position of lion roar events. Labelled lion roars were then extracted and classified by individual identity. Roars could be reliably associated with the tagged individual simply by the roar amplitude as roars emitted by other individuals close by had distinctly lower amplitude. In addition, the concurrent accelerometer data provided a further means of validation as lions make consistent head and neck movements while vocalizing (Fig. 1). Roars containing interference (e.g. noise from natural or anthropogenic sources) or overlapping roars of nearby lions were excluded from the analyses. Because the aim of our study was to test for individual differences in the fullthroated (FT) roars for each study animal, we further annotated and extracted audio segments containing the first three FT roar units for each roar bout (Fig. 2). As described in several other studies, the FT roars are the high amplitude signals emitted after the initial soft moans and before the sequence of short grunts [START_REF] Grinnell | Roaring and social communication in African lions: the limitations imposed by listeners[END_REF][START_REF] Mccomb | Roaring and numerical assessment in contests between groups of female lions, Panthera leo[END_REF][START_REF] Stander | Characteristics of lion roars in Etosha National Park[END_REF].

Fundamental frequency extraction

For each FT roar unit, we first applied a 4 th order digital Butterworth bandpass filter function to focus further analyses on the fundamental frequency (f0) between 40 Hz and 230 Hz. We extracted the peak frequency contour for this bandwidth (the f0 contour) by computing the short-time Fourier transformation (STFT) for the signal using a 2048-point moving Hann window with 68% overlap and zero-padding to four times the window length. The above extraction process was conducted in python using the 'SciPy' library [START_REF] Jones | SciPy: Open Source Scientific Tools for Python[END_REF].

Plots of the extracted f0 contours are shown in figure S1 in the Supplementary Information.

Fundamental frequency summary features

Following the method used by [START_REF] Ji | Discrimination of individual tigers (Panthera tigris) from long distance roars[END_REF], we aimed to investigate whether four f0 summary features differed between individuals: maximum f0, minimum f0, mean f0 and roar duration. Because the data were non-normal, we used the Kruskal Wallis method to test the hypothesis that the median feature value of least one individual differed from that of at least one other individual. Post-hoc pairwise multiple comparisons were then conducted using Dunn's test with Bonferroni adjustment. In addition to the statistical analyses, we used the knearest neighbours (K-NN) algorithm to test the classification ability of models built using the four f0 features, individually and combined. The purpose of this test was to measure the discriminative ability of each f0 feature and to determine whether the combined feature set was sufficient to accurately discriminate between individuals. Each K-NN classifier was assessed using the overall accuracy metric (proportion of correctly classified data) produced from leave-one-out cross validation where the test sample always included FT roars from the same bout. This data splitting approach was necessary to ensure that the test set was independent of the training set and thus to avoid any bias resulting from the temporal autocorrelation between roars in a single bout. All statistical analyses were conducted in R (R Core Team 2019).

HMM classification of f0 contour

A Hidden Markov Model (HMM) is a statistical tool for representing the probability distributions over a sequence of observations and is therefore useful for modelling time series data [START_REF] Ghahramani | An introduction to hidden markov models and bayesian networks[END_REF]). An additional benefit of HMMs is their ability to model patterns of varying length as would be expected from variable duration lion roars. We used HMMs to model the temporal pattern of the f0 contour for each individual lion. The roar recognition experiment was implemented using the leave-one-out cross validation method whereby 10state Gaussian HMMs were trained for each individual using all but one sample of the f0 sequences. As in the K-NN validation, a sample consisted of FT roar f0 sequences from the same bout. The 'unseen' f0 sequences were then tested against each HMM by determining which model was most likely to produce the given sequence (using the log-likelihood metric). Classification performance was assessed by calculating overall accuracy, recall (proportion of data of a particular class that is classified correctly as positive) and precision (proportion of correctly predicted positive classifications for a particular class) metrics of classified sequences [START_REF] Sokolova | A systematic analysis of performance measures for classification tasks[END_REF][START_REF] Bidder | Love thy neighbour: automatic animal behavioural classification of acceleration data using the K-nearest neighbour algorithm[END_REF]. HMM classification was performed in python using the 'hmmlearn' library (hmmlearn development team 2019)

Lion tracking application

We used an acoustic array to record roars from two known male lions fitted with GPS tracking collars. Identified roars were manually matched across at least 4 stations with the time of arrival (ToA) of the signal recorded for each roar at each station. The difference in the ToA of the signal between stations allowed us to localise the source of the roar with an accuracy of ~ 100 m using custom-written functions in python. Estimated roar locations were then compared with GPS collar data from the two lions to determine the identity of the caller.

An example of the comparison between GPS collar positions and roar localisations is shown in figure 3. In this particular example, the second male lion was not near the array, allowing us to confidently identify the individual vocalizing. For the analysis, we excluded roars that contained noise from other sources (e.g. wind) as wells as roars for which the identity of the caller was uncertain (e.g. when lions roared while in close proximity to one another). To test roar recognition performance, we used audio recorded by the closest station to the lion. Raw 44.1 kHz audio was first down-sampled to 16 kHz, followed by FT roar and f0 extraction. We then trained 4-state Gaussian HMMs for each individual and carried out tests using the leaveone-out cross validation method as described previously.

Results

Vocal individuality

We recorded a total of 60 lion-days of audio from the eight lions fitted with biologgers.

Manual inspection of the audio revealed a total of 296 roaring bouts from the 5 male lions. The 3 female lions did not appear to roar and only uttered soft moans. Of the total number of bouts, only 78 were useable as the majority contained overlapping roars from conspecifics.

From the useable roar bouts, a total of 164 FT roars were extracted (Table 1).

Fundamental frequency summary features

Results of the Kruskal Wallis tests, produced p values below 0.001 for all f0 features indicating significant differences in max f0, min f0, mean f0 and duration between at least one pair of individuals. Post-hoc analyses using the Bonferroni-Dunn test revealed that f0 features did not differ significantly between all individuals (Table 2). Roars emitted by Lion A4, in particular, were found to have significantly lower maximum f0 (median = 162.1 Hz), mean f0 (median = 122.3 Hz) and duration (median = 1.01 s) compared to other lions. Consistent differences between lions A8, A9, A10 and A11 were not as apparent (Fig. 4).

The analysis of the classification ability of K-NN models built using each of the f0 features showed a performance range of between 24.4% (min f0) and 50.6% (max f0) when single features were used (Table 2) Overall classification accuracy increased to 70.7% when all four features were used together. Optimal performance was achieved using a k value of 7.

HMM classification of f0 contour

Visible differences in the shape of the f0 contour were evident in the FT roars for each lion (Fig. 5). Modelling the temporal pattern of individual f0 sequences resulted in high classification performance with an overall accuracy of 91.5% and average recall and precision of 91.0% and 91.7% respectively (Table 3). Four out of the five lions were classified with recall greater than 90%. Recall for lion A10 was found to be lower at 83%. We also investigated classification performance using training set sizes of between 40 (approx. 8 roars per individual) and 160 (approx. 32 roars per individual) roars. Performance was found to increase considerably from 40 to 120 roars and then more gradually beyond 120 roars (Fig. 6). Optimal performance is therefore likely to be achieved with datasets consisting of 24 or more roars per individual.

Lion tracking application

We extracted a total of 50 FT roars from 31 roaring bouts emitted by the two collared males while they were in the vicinity of the acoustic array. The FT roars were then localised using ToA differences and identity assigned according to corresponding collar GPS data. Results of the HMM classification showed that, using passively recorded audio, we could differentiate between the two individuals with an overall accuracy of 90.0%. Recall was slightly higher for lion B1 compared to lion B2 (Table 4). Vocalisations used for identification originated from distances ranging from 45 m to 1094 m with incorrectly classified calls distributed relatively evenly across the distance spectrum (Fig. 7).

Discussion

Previous research dedicated to African lion vocal communication focused on receiver responses to experimental playbacks with few studies exploring variations in the acoustic structure of calls [START_REF] Gilfillan | Cross-modal individual recognition in wild African lions[END_REF][START_REF] Grinnell | Roaring and social communication in African lions: the limitations imposed by listeners[END_REF][START_REF] Mccomb | Female lions can identify potentially infanticidal males from their roars[END_REF].

While playback-response experiments have undoubtedly provided important insight into the function of vocal signalling and the possible information content of the signal, knowledge of how this information is conveyed has been lacking. In this study, we have demonstrated that the temporal pattern of the fundamental frequency of lion roars is a possible mechanism for lion vocal identification and is useful for automated digital differentiation of individuals.

We acquired our lion roar dataset using a novel acoustic -accelerometer biologging method which made it easy to obtain a sufficient number of roar recordings from each individual and to be able to reliably associate roars with lion identity. When recording animal vocalisations manually, it can be difficult to tell which individual is vocalizing [START_REF] Clemins | Automatic classification and speaker identification of African elephant (Loxodonta africana) vocalizations[END_REF]. This is particularly true for identifying lions in the wild where the only clues to identity are subtle differences in whisker spot patterns or unique scars [START_REF] Pennycuick | A method of identifying individual lion with an analysis of the reliability of identification[END_REF].

Furthermore, lions roar mostly during the night which can make it even more difficult to accurately differentiate between individuals. Manual recording approaches are also costly and time consuming and can potentially influence animal behaviour as a result of observer presence.

Results of the statistical analysis exploring differences in roar duration and maximum, minimum and mean fundamental frequency between individuals showed that these factors were not consistently different between all individuals. This suggests that simple overall summary features that do not incorporate temporal variation may not provide optimum discriminatory power to differentiate between individual lions. This was confirmed by the lower classification accuracy of the K-NN model which classified 70.7% of calls correctly, although this is still better than random chance (20%) and suggests that these factors are relevant for acoustic monitoring of the species. Similar classification performance (69.9%) was reported by [START_REF] Ji | Discrimination of individual tigers (Panthera tigris) from long distance roars[END_REF] for tigers (Panthera tigris), using these same four features. The roars emitted by lion A4 appeared to be significantly shorter and have lower maximum and mean fundamental frequencies compared to the other four lions. Lion A4, although resident in the study area, was known to have originated from the Tuli Block in eastern Botswana from where it dispersed more than 60 km before breaking into the fenced Bubye Valley Conservancy. The differences observed for this lion alludes to the possible existence of geographical variation in lion roars. [START_REF] Stander | Characteristics of lion roars in Etosha National Park[END_REF] also reported that lions from Etosha National Park have shorter roars than lions in other parts of Africa. Geographical variations in vocalisations are known to exist among other species and may occur between different lion sub-populations [START_REF] Deecke | Dialect change in resident killer whales: Implications for vocal learning and cultural transmission[END_REF][START_REF] Martins | Vocal dialects and their implications for bird reintroductions[END_REF][START_REF] Mitani | Dialects in Wild Chimpanzees[END_REF].

Although the use of f0 summary features resulted in relatively poor classification performance, when we modelled the temporal pattern of the f0 sequence using HMMs, classification performance improved considerably to an overall accuracy of 91.5%. The overall shape of the f0 contour is therefore a defining characteristic that allows for better discrimination between individual lions. This finding is consistent with studies on other species which have found that the frequency distribution of vocalisations from each individual are similar but the temporal patterns of the signals are unique to each individual [START_REF] Clemins | Automatic classification and speaker identification of African elephant (Loxodonta africana) vocalizations[END_REF][START_REF] Ji | Discrimination of individual tigers (Panthera tigris) from long distance roars[END_REF]. The key novelty of our approach is in the use of the f0 sequence as the only feature for building individual HMM classifiers rather than the more common approach of using MFCC features derived from a range of frequencies which include the harmonics. As shown in figure 8, the higher frequencies of a full-throated lion roar degrade noticeably more than the fundamental frequency which is more consistent across space. Information contained in the fundamental frequency is therefore likely to be preserved over distance and received effectively by a listener. A study on long-distance communication between elephants also showed that the higher formant frequencies of a call are unable to carry information related to individual identity over long distance and therefore identity is more likely to be discerned from the lower frequency harmonics in the 115 Hz region [START_REF] Mccomb | Long-distance communication of acoustic cues to social identity in African elephants[END_REF]. Our results support this theory and thereby provide a likely explanation for the mechanism underpinning individual vocal recognition amongst African lions. We acknowledge, however, that it would be necessary to conduct playback experiments using spectrally modified calls in order to determine the extent to which lions are able to recognise conspecifics purely by the fundamental frequency of their calls. We therefore suggest this as a beneficial avenue for future research in lion communication as well as for other species. From a digital recognition perspective, the accuracy of roar classification models could also be improved by using fundamental frequency extraction algorithms that leverage cepstral transforms and autocorrelation as well as using additional predictive features such as contour slopes.

Understanding how lions are able to recognise conspecifics from their vocal signals is not only important for advancing our understanding of lion ecology, but also provides a useful feature by which lions could be identified using autonomous, acoustic recorders.

Traditionally, abundance estimates for lion populations have been achieved using call-in surveys [START_REF] Ferreira | Estimating lion population variables: Prey and disease effects in Kruger National Park, South Africa[END_REF], spoor transects [START_REF] Stander | Spoor counts as indices of large carnivore populations: The relationship between spoor frequency, sampling effort and true density[END_REF]) and camera trap surveys [START_REF] Cusack | Applying a random encounter model to estimate lion density from camera traps in Serengeti National Park, Tanzania[END_REF]. Vocal identification of lions may allow for the use of passive acoustic monitoring methods which have the potential to be fully automated using detection and recognition algorithms. Automation would facilitate longer term or even continuous surveys and significantly reduce the number of man-hours required for data management; a challenge that is inherent to camera trapping when individuals need to be manually identified. [START_REF] Hartwig | Individual Acoustic Identification As a Non-Invasive Conservation Tool: an Approach To the Conservation of the African Wild Dog Lycaon Pictus (Temminck, 1820)[END_REF] highlighted the value of individual vocal identification of African wild dogs as a supplement to radio telemetry and visual recognition. Similarly, one of the primary benefits of acoustic monitoring of lions would be its facilitation of collar-free tracking which is likely to provide movement information for a greater number of individuals at lower cost compared to animal-borne systems. Using an acoustic array and sound signal localisation techniques, we were able to obtain position estimates for several roars emitted by two resident male lions. The locations of the roars corresponded closely with collar GPS positions which provided reliable ground truth information for assigning identity labels to each roar in order to build individual recognition models. Models trained for each individual were able to accurately classify 'unseen' roars from varying distances. We therefore show that, with sufficient training data, it would be possible to locate and identify lion roars and thereby track individual movements across the landscape using passive acoustics as illustrated in figure 3. Furthermore, by integrating in-network data processing, real-time tracking would be achievable, particularly as the data content of the f0 contour is extremely small and therefore easily transmitted over the air. However, despite the roar classification performance being considerably better than chance, we acknowledge that this experiment was conducted for only two individuals, whereas in practice, discrimination between several different lions may be required including out-of-set (unknown) individuals. Obtaining a sufficient number of clean vocal samples from known lions without the use of animal-borne biologgers is a difficult task. As shown in our results, too few training samples can lead to reduced classification performance. This is especially true when attempting to distinguish between signals that have a greater degree of similarity [START_REF] Trifa | Automated species recognition of antbirds in a Mexican rainforest using hidden Markov models[END_REF]. Collecting such data is likely to present the biggest challenge to the development of large-scale, collar-free lion tracking systems. However, with the development of machine learning methods, particularly unsupervised clustering approaches, discriminating between individuals may be possible without the need for manual training [START_REF] Frasier | Automated classification of dolphin echolocation click types from the Gulf of Mexico[END_REF][START_REF] Stowell | Automatic acoustic identification of individuals in multiple species: Improving identification across recording conditions[END_REF][START_REF] Stowell | Automatic acoustic detection of birds through deep learning: The first Bird Audio Detection challenge[END_REF].

Although we have shown that lion FT roars are individually unique, it is necessary to highlight that fact that lion vocal behaviour can vary across space, time and social circumstances. None of the three tagged female lions in this study emitted full throated roars. This was unexpected, but could be related to the presence of small cubs in the pride (we acknowledge that further work would be required to confirm this assumption). Similarly, other research has shown that resident male lions tend to avoid roaring when outside of their own territories while nomadic males roar at greatly reduced rates or avoid roaring entirely until they have gained prides [START_REF] Grinnell | Roaring and social communication in African lions: the limitations imposed by listeners[END_REF]. Surveys that rely entirely on roaring may therefore be biased by the fact that not all lions vocalise consistently across the landscape or through time. In addition, communal roaring by several members of a pride is a common occurrence and can make it particularly difficult to dissociate individual roars, resulting in reduced classification performance of recognition algorithms. Another important consideration for long term surveys is the temporal consistency of individual calls. Agerelated variations in the calls of other species have been reported [START_REF] Ey | Do age-and sex-related variations reliably reflect body size in non-human primate vocalizations? A review[END_REF][START_REF] Ota | Age-dependent song changes in a closed-ended vocal learner: Elevation of song performance after song crystallization[END_REF]. Our study was conducted over a short period and therefore it remains uncertain as to whether the structure of an individual lions' roar varies with age. In addition to these biological caveats and uncertainties, we acknowledge that the hyperparameters used for the HMM and K-NN classification algorithms were selected to provide the best classification performance and could have led to overfitting, however, the leave-one-out cross-validation approach provides an effective measure of generalisation performance.

In this study, we have shown that the roar of an African lion contains an individually unique identifier in the form of the fundamental frequency contour. Although this finding has also been reported for other species [START_REF] Caldwell | Individualized Whistle Contours in Bottle-nosed Dolphines (Tusiops truncatus)[END_REF][START_REF] Lenhardt | Vocal contour cues in maternal recognition of goat kids[END_REF], our method of modelling the extracted f0 sequences using HMMs is unprecedented in the field of animal vocal recognition and is likely applicable to a number of species which emit loud, long distance calls. We have also demonstrated the conservation value of our findings through the use of a passive acoustic array to localise and identify individual lion roars. This work highlights the value of passive acoustic monitoring for African lion research, particularly as current GPS tracking methods, although reliable, are highly expensive and involve invasive capture procedures. With rapidly advancing technological innovations, especially opensource tools, monitoring vocally active wildlife species is likely to become more efficient and affordable, providing new avenues for research and conservation.

  relied on vocal identification of African wood owls (Strix woodfordii) to monitor territory turnover. However, despite the large number of studies that have explored vocal individuality, there are relatively few published examples of how acoustic monitoring of individuals can be applied in practice, particularly for mammalian species.

Other

  f0 features have been used in vocal identification studies on spotted hyaenas (Crocuta crocuta; Mathevon et al. 2010), monkeys (Rhinopithecus roxellana; Fan et al. 2019) and wolves (Canus lupus lycaon; Root-Gutteridge et al. 2013) with varying success (32%-88%).

Figure 1 .

 1 Figure 1. Concurrent audio (left) and accelerometer (right) data for two different roars

Figure 2 .

 2 Figure 2. Fundamental frequency extraction process.

Figure 3 .

 3 Figure 3. Comparison between collar GPS positions (left) and roar localisation (right) for a 4-

Figure 4 .

 4 Figure 4. Boxplots of the four f0 summary features for each lion.

Figure 5 .

 5 Figure 5. Examples of FT roar structure for each individual lion as recorded by the

Figure 6 .

 6 Figure 6. Classification accuracy using an increasing number of training set samples. Shaded

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Fundamental frequency extraction process.

Figure 3 .

 3 Figure 3. Comparison between collar GPS positions (left) and roar localisation (right) for a 4-

Figure 4 .

 4 Figure 4. Boxplots of the four f0 summary features for each lion.

Figure 5 .

 5 Figure 5. Examples of FT roar structure for each individual lion as recorded by the

Figure 6 .

 6 Figure 6. Classification accuracy using an increasing number of training set samples. Shaded

Figure 7 .

 7 Figure 7. Distance distribution of correct and incorrectly classified roars.

Figure 8 .

 8 Figure 8. Spectrograms of the same FT roar recorded at varying distances from the animal.

Table 1 .

 1 Number of bouts and FT roars for each individual lion.

	Individual	A4	A8	A9	A10	A11
	Bouts	17	18	16	13	14
	FT roars	38	35	40	24	27

Table 2 .

 2 Results of Kruskal Wallis tests (Chi-squared and p values) and overall accuracy of the K-NN classifiers for each of the four f0 features. Multiple comparisons were conducted using Dunn's test with Bonferroni adjusted p-values (significance threshold = 0.05).

	Feature K-NN	Kruskal Wallis	Pairs that were significantly different
		Classifier
		Accuracy	 2	P value
	Min f0	24.4	37.6	< 0.001 A9-A10; A10-A11; A10-A4; A9-A8
	Max f0	50.6	79.4	< 0.001 A9-A10; A9-A4; A10-A4; A11-A4; A9-A8; A8-A4
	Mean f0	42.1	98.2	< 0.001 A9-A10; A9-A11; A9-A4; A10-A4; A11-A4; A9-A8; A4-A8
	Duration	30.5	71.4	< 0.001 A9-A4; A10-A4; A11-A4; A4-A8

Table 3 .

 3 Confusion matrix of actual lion IDs (rows) vs predicted IDs (columns) for biologger recorded roars using HMMs based on FT roar f0 contours. Model training and testing were carried out on separate roar bouts.

	Individual A4	A8	A9	A10	A11	Recall	Precision
							%	%
	A4	36	0	2	0	0	94.7	100.0
	A8	0	33	0	0	2	94.3	84.6
	A9	0	2	36	1	1	90.0	92.3
	A10	0	2	1	20	1	83.3	95.2
	A11	0	2	0	0	25	92.6	86.2
	Average						91.0	91.7

Table 4 .

 4 Confusion matrix of actual lion IDs (rows) vs predicted IDs (columns) for passively recorded roars using HMMs based on FT roar f0 contours. Training and testing were carried out on separate roar bouts.

	Individual B1	B2	Recall	Precision
				%	%
	B1	25	2	92.6	89.3
	B2	3	20	87.0	90.9
	Average			89.8	90.1

Acknowledgements

We thank the John Fell Fund and The Beit Trust for funding this research. We are also grateful to the management staff of the Bubye Valley Conservancy for giving us access to their property. Finally, we acknowledge the assistance provided by Biotrack in manufacturing the biologgers and W. Gush in maintaining the CARACAL array.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author, MW, upon reasonable request.

Ethical Statement

This study was carried out in accordance with the recommendations of the Use of Animals in Research, ASAB/ABS, with the research protocol approved by the University of Oxford Animal Welfare and Ethical Review Board. Project members attended Zimbabwe's Physical and Chemical Capture of Wild Animals Course and held valid drugs licenses (Dangerous

Declaration of Interest

No conflict of interest is reported by the authors


Frédéric Clarençon

Eimad Shotar

Evelyne Cormier

Kevin Premat

Mehdi Drir

Ghizlene Lahlou

Veronique Morel

Jean-Philippe Spano

Raphaël Bonaccorsi

Federico Di Maria

Rémi Hervochon

Hugues Pascal-Mousselard

Jacques Chiras

Transoral Vertebroplasty for the C1 Lateral Mass: Single Center Experience

Keywords: AP: anteroposterior, DSP: dose-surface product, ICA: internal carotid artery, IRB: institutional review board, NRS: numeric rating scale, PMMA: polymethyl metacrylate, RFA: radiofrequency ablation, TOV: transoral vertebroplasty, VA: vertebral artery Vertebroplasty, transoral, C1, atlas ! 2

Background

Osteolytic lesions of the atlas (C1) are challenging to treat by vertebroplasty due to the vicinity of the vertebral artery (VA) and of the spinal cord. Our objective was to present our experience of transoral vertebroplasty (TOV) for osteolytic lesions of the lateral mass of the atlas.

Methods

Retrospective cases series involving 15 consecutive patients (9 males, 6 females, mean age: 63y) who underwent transoral vertebroplasty for the treatment of an osteolytic lesion of the lateral mass of the atlas. Among the osteolytic lesions, 10/15 (67%) were bone metastases from various cancers; 4/15 (27%) were lesions related to multiple myeloma and one lesion (6%) was an aggressive hemangioma.

All the transoral vertebroplasties were performed under general anesthesia and in most cases (10/15; 67%) in a hybrid angio-suite combining a C-arm flat panel and CT-scan. The remaining 5 patients were treated under biplane fluoroscopic guidance.

Results

Vertebroplasty of the lateral mass of C1 through a transoral route was feasible in all cases. Significant pain relief was obtained in most cases (1 month average decrease in numeric rating scale [NRS]: 4.9±4.1). No major complication was recorded. In 7/15 cases (47%), a cement leakage surrounding the C1 lateral mass was seen; none of these leakages had a significant clinical consequence. No additional spine surgery was required in any of the patients.

Conclusion

Our experience shows that TOV of osteolytic lesions of the lateral mass of the atlas is feasible and seems safe and effective in terms of pain relief and bone stabilization.

Introduction

The C1 vertebra (aka the atlas) has a specific anatomic configuration, compared to the other vertebrae of the human body. Indeed, the atlas presents a circle shape with 2 masses located laterally, which bear the skull through the joints with the occipital condyles. This bearing role of the lateral masses of the atlas explains the unstable characteristic of the osteolytic lesions involving this region of C1.

In addition to its specific anatomy, the atlas is surrounded by critical nervous and vascular components: the medulla oblongata-spinal cord junction, the C1 and C2 nerve roots, the distal aspect of the cervical internal carotid artery (ICA) and the vertebral artery (VA), which surrounds laterally and posteriorly each lateral mass of C1.

Transoral route has been described several years ago for vertebroplasties of C2 lesions. 1 2 Only scant data, mainly case reports 3 4 , are available in the literature on the feasibility, safety and effectiveness of vertebroplasty for C1 lesions, either by transoral 4 5 or alternative routes. 3 6 The purpose of this study was to report the authors' experience of transoral vertebroplasty (TOV) for lesions involving the lateral mass of C1 in terms of safety and pain relief.

! 3

Materials and Methods

Study design:

Retrospective monocentric case series.

Patients' demographic and lesions' characteristics:

From 2012 to 2019, all patients who underwent a TOV for a C1 lesion at a single Institution were included in a prospectively filled database. Clinical demographics (age, sex, underlying disease) as well as lesions' characteristics (pathological origin, osteolytic/mixed or osteoblastic pattern) were systematically recorded. All patients were treated for unstable lesions of C1 lateral mass. Instability was defined as an osteolysis involving more than 50% of the volume of the C1 lateral mass or cortical bone lysis or fracture of the C1 lateral mass. Two patients of this cohort have already been reported previously in an article describing the early experience of the authors in TOV of C1 lesions. [START_REF] Clarencon | Transoral approach for percutaneous vertebroplasty in the treatment of osteolytic tumor lesions of the lateral mass of the atlas: feasibility and initial experience in 2 patients[END_REF] Five patients of the present study have also been already reported through a study presenting the authors' experience in cervical vertebroplasty (from C1 to C7), reporting the safety and effectiveness of 140 cervical vertebroplasty procedures. [START_REF] Clarencon | Safety and effectiveness of cervical vertebroplasty: report of a large cohort and systematic review[END_REF]

Procedure descriptions:

All patients were treated under general anesthesia in supine position using an oral intubation with a reinforced oral tube. Ten out of the 15 patients (66.7%) were treated in a hybrid angiosuite, combining a flat panel C-arm and a 16-rows CT scan (Myabi, Siemens) (Fig. 1). The remaining 5 patients (33.3%) were treated under biplane fluoroscopic guidance (Artis Q, Siemens). All patients were treated according to a technique previously described by our team. [START_REF] Clarencon | Transoral approach for percutaneous vertebroplasty in the treatment of osteolytic tumor lesions of the lateral mass of the atlas: feasibility and initial experience in 2 patients[END_REF] Briefly, a Boyle-Davis mouth gag was positioned in order to expose the posterior aspect of the pharyngeal mucosa.

Disinfection with Betadine (povidone iodine, Meda Pharma, Soina, Sweden) oral antiseptic solution was then performed. After the induction of the anesthesia, 1 g intravenous cefazolin (or other broad spectrum antibiotic) was injected in order to reduce the infection risks. An 11G bone needle (Thiebaud, Margencel, France) was ! 4 positioned and advanced gently in the lateral mass of C1 to treat, under fluoroscopic guidance (Fig. 1). The satisfactory positioning of the bone needle was checked by a CT acquisition (in patients treated in a hybrid angio suite) or by a cone-beam CT acquisition (in patients treated in a by-plane angio-suite). In 73.3% of the cases (11/15), a compliant protection balloon was navigated in the V3 segment [START_REF] Fischer | Die Lageabweichungen der vorderen Hirnarterie imGefäßbild[END_REF] of the vertebral artery (VA) ipsilateral to the lesion from a 6F guiding catheter positioned in the VA, via a femoral access. This balloon protection was temporarily inflated under full IV anticoagulation to prevent retrograde cement migration in the VA ipsilateral to the lesion via the arterial feeders. Finally, low viscosity polymethyl metacrylate (PMMA) bone cement (Biomet V; Biomet UK Ltd, Bridgend, UK) was injected through the bone needle under anteroposterior (AP) and lateral fluoroscopic guidance.

In each case, the volume of cement injected in the lateral mass of C1 was recorded.

Total duration of the procedure as well as X-Ray doses (dose-surface product [DSP] in µGy.m 2 ) delivered to the patients were collected for patients treated only for a C1 lesion (patients treated during the same session for another location were excluded).

Safety:

Complications were systematically collected and graded using the CIRSE and SIR complication guidelines. [START_REF] Filippiadis | Cirse Quality Assurance Document and Standards for Classification of Complications: The Cirse Classification System[END_REF] Briefly, this classification is a 6 scales grading system evaluating the severity of the complication; grade 4 being a complication causing a permanent mild sequelae, grade 5 being a complication causing a permanent severe sequelae and grade 6 corresponding to death.

All patient underwent AP and lateral projection plain X-Ray at the end of the procedure, and an unenhanced CT. On post-procedure imaging, lesion's filling with the PMMA bone cement was evaluated. Bone cement leakages were systematically recorded as well, and were divided into 1) anterior soft tissue leakage, 2) vascular leakage, 3) C0-C1 joint leakage and 4) C1-C2 joint leakage.

Clinical outcome:

! 5 Clinical follow-up was evaluated 1 month after the C1 TOV trough a follow-up consultation with the physician who performed the procedure. Pain relief was evaluated on the Numeric Rating Scale (NRS). Occurrence of a delayed complication (i.e.: infection, delayed hematoma, delayed neurological complication,) was systematically recorded as well. In case of persistent pain, pain worsening or occurrence of neurological symptoms, new imaging work-up was performed (contrast enhanced CT and/or MRI) to depict any recurrence/progression of the treated lesion.

Late clinical follow-up, including NRS evaluation, was assessed by a phone interview.

Systematic review of the literature:

A systematic review of the literature focused on the safety and effectiveness on vertebroplasty for C1 lesions was performed according to the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines 10 . The different studies were analysed using combinations of terms in title, abstract, Animal studies, surgical series, non-relevant studies and non-English written series were then excluded (Fig. 2). Risk of bias was evaluated by both reviewers using the Newcastle-Ottawa Quality Assessment Form for Cohort and Case-control Studies. [START_REF] El-Ghanem | Expanding the treatment window for ischemic stroke through the application of novel system-based technology[END_REF] Ethical statement:

! 6
Our local institutional review board (IRB) approved the study protocol. The need for patients' informed consent for retrospective analyses of records and imaging data was waived by our IRB. This work adheres to the World Medical Association Declaration of Helsinki.

Statistical analysis:

Continuous variables were reported as means ± standard deviations (SD). The normality of the distribution was checked using a Shapiro-Wilk test. Comparison between pre and post-treatment NRS was performed using a Student-t test. All tests were calculated using Stata software (Stata/IC 13.1 for Mac; StataCorp LP, College Station, TX, USA); p values less than 0.05 were considered statistically significant.

! 7

Results

Patients' demographic and lesions' characteristics:

From 2012 to 2019, 15 consecutive patients (9 males, 6 females; mean age: 63±12.9, range: 38-84 y) underwent a transoral vertebroplasty for an osteolytic lesion of the lateral mass of C1 at a single Institution (Patients' demographics and lesions' characteristics are summarised in Table 1).

Most of the lesions (10/15 [67%]) were bone metastases from various cancers; 4 patients (27%) had a C1 lesion from a multiple myeloma; 1 patient (6%) had a biopsy proven aggressive hemangioma.

Seven lesions (46.7%) were located in the left lateral mass of C1; 8 lesions (53.3%) involved the right lateral mass of C1. In 3/15 cases (20%), cementoplasty of the ipsilateral occipital condyle was performed during the same procedure; using the same transoral route. In one case (6.7%) an additional vertebroplasty of the C2's dens was performed during the same procedure.

None of the patients had presurgical spine fixation.

Average cement volume injected was 1.5±0.9 ml. Average DSP was 5234.6 ± 4675.7 µGy.m 2 (range: 485-11968). Mean duration of the procedure was 117.2 min ± 38.7 min (range: 60-180).

Safety:

No major complication was recorded. One (6.7%) minor complication (grade 1 in the CIRSE-SIR grading scale [START_REF] Filippiadis | Cirse Quality Assurance Document and Standards for Classification of Complications: The Cirse Classification System[END_REF] ) was recorded in a patient (Patient # 13) who experienced a retropharyngeal mild hematoma after the procedure, responsible for a transient dysphagia. The symptoms spontaneously resolved without any specific treatment.

Bone cement leakages were depicted on immediate post-procedure CT acquisition in 7/15 cases (47%). In 3/7 cases (43%), the cement leakage was located in the retropharyngeal space; in 4/7 cases (57%) in the C1-C2 joint. One patient (Patient # 11) had cement leakage in both the retropharyngeal space and the C1-C2 joint. None of the patients had cement leakage in the C0-C1 joint. One ! 8 ! 9

! 10 patient (Patient # 7) had a cement peak released during the removal of the bone needle, which was removed manually from its proximal aspect in the oral cavity. In another patient (Patient # 5), a drop of cement was released in the uvula during the removal of the bone needle, which was removed under fluoroscopic guidance using a supple biopsy forceps (Fig. 1 and Video 1).

No delayed complication of any kind was reported. No infectious complication was recorded. No patient reported any dysphagia at follow-up.

Pain relief/follow-up:

Mean pretreatment NRS was 5.7±3. None of the patients required a surgical fixation in the aftermath of C1 TOV.

Systematic review:

! 11

The systematic review of the literature depicted 15 C1 vertebroplasty (28 in total, including the patients reported in the present study). 3-6 12-17 According to the Newcastle-Ottawa grading scale [START_REF] El-Ghanem | Expanding the treatment window for ischemic stroke through the application of novel system-based technology[END_REF] , the quality of the articles was poor for all the 11 series/case reports included in the systematic review. Two C1 vertebroplasties were performed via a posterior route (13.3%); 4 via a lateral route (26.7%); 1 via an anterior route (7.7%) and 8 via a transoral approach (53.3%) (21 [75%] including the patients of this study) (Table 2).

The underlying diseases responsible for the C1 osteolytic lesions were available in 14 It is noteworthy that one case of cement migration along the course of the V3 segment of the VA has been reported, without any neurological symptoms. [START_REF] Cianfoni | Percutaneous cement augmentation of a lytic lesion of C1 via posterolateral approach under CT guidance[END_REF] No other such vascular cement leakage was reported in the literature. Protection of the VA to prevent cement leakage was reported in 3/15 in the literature (with 2 patients from our cohort previously reported [START_REF] Clarencon | Transoral approach for percutaneous vertebroplasty in the treatment of osteolytic tumor lesions of the lateral mass of the atlas: feasibility and initial experience in 2 patients[END_REF] ). None of the patients previously reported out of our cohort had such use of protection balloon. In one case [START_REF] Wetzel | Painful osteolytic metastasis of the atlas: treatment with percutaneous vertebroplasty[END_REF] , to prevent the risk of cement leakage

cases
! 12
into the VA, due to its vicinity with the treated lesion, occlusion of the V3 segment of the VA by coils was performed; the patient having a contralateral VA with a satisfactory calibre.

All the cases of vertebroplasty for C1 lesions were associated with pain relief. No case of delayed fracture of the treated lesion was recorded, according to our systematic review of the literature.

! 13

Discussion

This series involving 15 consecutive patients showed the feasibility of TOV; indeed, no procedure failure was recorded. It also suggests the safety of TOV since no major and only one minor transient procedure-related complications were recorded.

Only scant data are available on the safety and effectiveness of TOV for C1 lesions.

Compared to open surgery (mainly tumour removal associated with occipitocervical fusion), which remains the criterion standard for the treatment of C1 osteolytic lesions [START_REF] Yang | Surgical treatment of upper cervical spine metastases: a retrospective study of 39 cases[END_REF] , the potential of percutaneous treatments is that they are less invasive, reducing the risk of major blood loss (especially in hypervascularized metastases or aggressive hemangiomas) and that they have a lower infectious risk. Different routes have been described for the treatment of C1's lateral mass lesions. The first case of C1 vertebroplasty has been published in 2002 by Wetzel SG et al. [START_REF] Wetzel | Painful osteolytic metastasis of the atlas: treatment with percutaneous vertebroplasty[END_REF] The authors performed a vertebroplasty for a C1 osteolytic metastatic lesion from a parotid adenocarcinoma. In this case report, postero-anterior route was chosen, under fluoroscopy guidance, with satisfactory results in terms of pain relief and bone stabilization. Afterwards, lateral 12 19 , then anterior [START_REF] Yang | Anterior retropharyngeal approach to C1 for percutaneous vertebroplasty under C-arm fluoroscopy[END_REF] approaches have been proposed

for cementoplasties of C1 lateral masses, with good results. The first TOVs for C1 lesions described in the literature were 3 cases, included in a series of 62 cervical VPs, reported by Masala S et al [START_REF] Masala | Percutaneous vertebroplasty relieves pain in metastatic cervical fractures[END_REF] in 2011. No technical issue was reported in these first 3 cases; satisfactory pain relief was obtained at follow-up in all cases.

In total, including the 15 cases reported in our study, 28 cementoplasties of C1 have been reported so far 3-6 12-14 16 17 19 20 , including 21 transoral vertebroplasties. No complication, either infectious or neurological has been reported with this technique.

Transoral route appears to be a safe approach since it decreases the risk of VA wound during the positioning of the bone needles. Indeed, the V3 segment of the VA has a course laterally, then posteriorly to the lateral mass of C1. In our opinion, lateral and postero-anterior approaches, even with cautious CT guidance, expose to a too high risk of VA wound during the bone needle positioning due to the course of the VA laterally, then posterior to the lateral mass of C1. We also believe that the anterior percutaneous approach is less convenient, since it requires a very accentuated

! 18
ascending course, which may not be feasible in patients with a short neck or with prominent clavicles. The only risk of the transoral approach regarding vascular wounds is the possibility of a retropharyngeal course of the ICA. [START_REF] De Regloix | Retropharyngeal course of the internal carotid artery[END_REF] Such variation of the cervical ICA course should thus be carefully studied on the pretreatment imaging work-up. We reported in our case series only one minor complication (6.7%), which consisted in a mild retropharyngeal hematoma that spontaneously resolved without any sequelae.

We acknowledge a potential risk of bone infection with the TOV, since bone needle is inserted through the oral cavity, which is known to contain a lot of bacteria. However, with oral cavity disinfection and by administrating an IV antibiotic with a broad spectrum, we think that this risk remains very low. It is noteworthy that, even reporting a disinfection of the oral cavity in all cases, no injection of antibiotics was reported in the previously published cases. IV antibiotics injection during vertebroplasty is part of our regular protocol and we think it may help in reducing infectious complications that, even very rare [START_REF] Park | Infection following percutaneous vertebral augmentation with polymethylmethacrylate[END_REF] , may be observed after PV.

Cement leakages were observed in 47% of the cases in our study. Most of them (57%)

were located in the C1-C2 joint. However, none of them were responsible for any clinical symptoms.

In our case series, we used a protection balloon in 73.3% of the cases. The use of protection balloon was left at the discretion of the operator. We used such protection balloon for two purposes. First to see the course of the vertebral artery under fluoroscopy due to the radiopaque characteristic of the microguide wire on which the balloon was navigated. Second, the balloon was inflated temporarily during the PMMA injection through the bone needle to prevent the risk of retrograde PMMA leakage from the lesion to the vertebral artery and thus to avoid the risk of cement embolism in the vertebrobasilar system.

Interestingly, one case of radiofrequency ablation (RFA) associated with cementoplasty has been reported, with satisfactory results in terms of safety and effectiveness. [START_REF] Bundy | Transoral Radiofrequency Ablation and Cementoplasty for Lateral Mass of the C1 Vertebral Body[END_REF] Such technique may be valuable to reduce the risk of local recurrence. However, it should be kept in mind the RFA may be challenging in such location, and is associated with a significant risk of nervous thermal lesions (nerve ! 19 root, spinal cord). [START_REF] Clarencon | Value of percutaneous radiofrequency ablation with or without percutaneous vertebroplasty for pain relief and functional recovery in painful bone metastases[END_REF] Additionally, even if the imaging follow-up was not available in all patients (available in 10/15 patients; 66.7%) we depicted only one local progression (1/10; 10%) in the patients treated in our study.

Clinical effectiveness (bone stabilization and pain relief) was observed in all cases in our series. In the literature, pain relief was observed in all cases as well. No secondary fracture of the treated lesion was recorded in the literature, as observed in our series.

However, it should be mentioned that a publication bias, inherent to case reports and short case series, may be responsible for an under-reporting of major complications and poor outcomes.

Limitations of the study

The main limitation of our case series is its monocentric and retrospective nature.

Additionally, the number of patients included in this work was relatively low (15 patients). However, it should be mentioned that C1 lateral mass is not a frequent location of bone metastases or multiple myeloma. Moreover, all the cases were performed by very experienced interventional radiologists, in modern angio-suite facilities (hybrid angio-suite or bi-plane angio-suite). Thus, these results may not be generizable. Finally, no comparison with a criterion standard (open surgery) was performed in this study.

! 20

Conclusion

Transoral vertebroplasty for the treatment of osteolytic lesions of the lateral mass of the atlas seems feasible, safe and effective in terms of pain relief and bone stabilization. Larger series are warranted to confirm these preliminary results on 15 patients.

! 21 For the Cochrane library, the search was organized as follows: 'vertebroplasty' AND 'cervical' OR 'cementoplasty'

! 24

! 25

  keywords and free text, until 1st September 2019. The selection was focused only on articles dealing with C1 vertebroplasty. The search was performed on MEDLINE via PubMed, Embase via Ovid and Cochrane central database via CENTRAL with advanced search builder. The following terms and synonyms were used: cervical, vertebroplasty, atlas and C1. Additionally, references from the publications obtained were checked to add relevant studies. Two investigators (*** and ***) performed this systematic review of the literature on C1 vertebroplasty.

3 .

 3 At 1 month, 4 patients (27%) were lost to the follow-up. In the 11 remaining patients (73%), mean NRS at 1-month clinical followup was 1.5±1. Mean NRS decrease between pretreatment and 1 month follow-up was 4.6±3.9 (p = 0.0002). Late clinical follow-up (i.e. > 1 month) was available in 53% of the patients (8/15); 4/7 (57.1%) patients with no late clinical follow-up were deceased from their tumour disease and 3/7 (42.9%) were lost to the follow-up. Average time interval between treatment and last clinical follow-up was 41.2±39.8 months. Average NRS at last follow-up was 1.1±1.46 and was not significantly different from the one at 1 month (p = 0.86).Imaging follow-up was available in 10/15 patients (66.7%), with an average time interval of 16±23.7 months. One case of post-TOV C1 lesion local progression was depicted (Patient# 12). No further local treatment on the C1 lesion was performed due to general progression of the lung cancer, that eventually lead to death 2 months after the C1 TOV.

Figure 1 .Video 1 .

 11 Figure 1. Patient with left C1's lateral mass metastatic osteolytic lesion from a lung cancer. A. and B. CT scan; axial slice (A) and coronal reconstruction (B) in bone windowing, showing the osteolytic lesion of the left lateral mass of C1 (asterisk). C. Periprocedural picture showing the positioning of the bone needle via a transoral route after exposing the posterior wall of the pharynx by means of a Boyle-Davis mouth gag. D. and E. Plain X-Rays in anteroposterior (AP) (D) and lateral (E) projections showing the satisfactory positioning of the bone needle (white arrow). Note the course of the protection balloon in the V3 segment of the left vertebral artery (VA) (double black arrow). F. Control unenhanced CT-scan in bone windowing, after the positioning of the bone needle. Satisfactory positioning of the bone needle's tip in the metastatic lesion is confirmed (white arrow).After the injection of the polymethyl metacrylate (PMMA) bone cement in the lesion, during the removal of the bone needle, a cement fragment released in the uvula was depicted. It was decided to remove it with a supple biopsy forceps. G. Lateral fluoroscopic view during the removal of the bone cement fragment located in the uvula (white arrow) with the biopsy forceps (double black arrow). H and I. Plain X-Rays after the cement fragment removal (H, AP and I, lateral projections). Satisfactory filling of the lesion is demonstrated, without residual cement leakage. J. Photograph of the removed cement fragment, side by side with a 10 cents coin.

Figure 2 .

 2 Figure 2. PRISMA flow chart.

Table 1 . Patients' demographics and lesions' characteristics Nb

 1

	Patient nb	Treated lesions	Si de	Lesion type	Underlying disease	PreTx NRS	Post-Tx NRS	Cement leakage
	1	C1	L	Osteolyt ic	Pancreatic Kc	9	NAv	0
	2	C1	L	Osteolyt ic	Hemangioma	10	0	0
	3	C1	L	Osteolyt ic	Multiple myeloma	4	3	0
	4	C1	L	Osteolyt ic	Thyroid Kc	10	0	0
	5	C1	L	Osteolyt ic	Lung Kc	5	NAv	Uvula
	6	C1	L	Osteolyt ic	Multiple myeloma	4	0	0
	7	C1 + occipital condyle	R	Osteolyt ic	Lung Kc	6	0	Anterior soft tissue
	8	C1 + occipital condyle + axis dens	R	Osteolyt ic	Breast Kc	6	2	C1-C2 joint
	9	C1	R	Osteolyt ic	Breast Kc	2	NAv	C1-C2 joint
	10	C1 + occipital condyle	R	Osteolyt ic	Multiple myeloma	0	0	0
	11	C1	R	Osteolyt ic	Lung Kc	0	0	C1-C2 joint and anterior soft tissue
	12	C1	R	Osteolyt ic	Lung Kc	8	NAv	Anterior soft tissue
	13	C1	R	Osteolyt ic	Multiple myeloma	9	0	0
	14	C1	L	Osteolyt ic	Lung Kc	5	4	C1-C2 joint
	15	C1	R	Osteolyt ic	Lung Kc	8	2	0

indicates number, F: female, M: male, L: left, R: right, Kc: cancer, PreTx: pre-treatment, Post-Tx: post-treatment, NRS: Numeric rating Scale, NAv: not available

  in our systematic review (27 cases including our study). Most of the treated

	lesions were bone metastases from various neoplasms (10/14 cases [71.4%]; 19/27
	cases [70.4%] including this study).

C1 vertebroplasties, regardless the route chosen, were feasible in all cases. Procedures were performed under fluoroscopic guidance only in 2/15 cases (13.3%) (2/28 cases; 7.1%, including our study); under CT guidance only in 5/15 cases (33.3%) (5/28; 17.9%, including our study); under both CT and fluoroscopic guidance in 6/15 cases (40%) (14/28 cases; 50%, including our study). It is noteworthy that in 10/14 cases (all in our centre) combined CT-fluoroscopic guidance was performed in a hybrid angio-suite, combining a CT and a C-arm flat panel. Finally, imaging guidance was fluoroscopy associated with cone-beam CT in 1/15 cases (6.7%) (6/28 cases; 21.4%, including our series). Neither major nor minor complication was reported in the literature. Including the present series, only one minor complication was recorded (1/28; 3.6%) (transient retropharyngeal hematoma). Cement leakages were observed in 4/15 cases (27%) in our systematic review of the literature (11/28 [39.3%] including this study). None of the cement leakage reported in the literature (and in our series as well) were responsible for any clinical symptoms.

Table 2 . Systematic review of the literature on C1 lesion vertebroplasty/cementoplasty

 2

	Authors	Yea r	Journ al	Nb of cas es	Lesion type	Route	Guidance	Clinica l compli cation	Pa f in re lie	Cement leakage	VA protection	Comment
	Wetzel SG et al 5	200 2	Spine	1	Metastasis	Percuta neous; posteroa nterior	Fluoroscopy	No	Ye s	Atlanto-l joint occipita	Occlusion / coils V3 segment	-
			Cardi
	Huegli RW et al 11	200 5	ovasc Interv ent Radio	1	Metastasis	Percuta neous, lateral	CT and fluoroscopy	No	Ye s	No	No	Tx of C4 during the same session
			l
	Anselme tti GC et al 14	200 9	J Vasc Interv Radio l	1	Metastasis	Percuta neous, lateral	CT	No	Ye s	No	No	-
			Clin
	Masala S et al 15	201 1	Ortho p Relat	3	Metastases	Transora l	CT and fluoroscopy	No	Ye s	NM	NM	-
			Res
	Cianfoni A et al 2	201 2	Spine J	1	Metastases	Percuta neous; posterol ateral	CT	No	Ye s	Yes, t VA along segmen V3	No	-
	Guo WH et al 12	201 2	Pain Physic ian	2	Metastasis and hemangiom a	Percuta neous, lateral	CT	No	Ye s	Little leakage extraoss reflux eous	No	-
	Clarenço n F et al 4	201 3	Spine	2	Metastasis a and hemangiom	Transora l	CT and angio suite) fluoroscopy (hybrid	No	Ye s	No	Protective (50%) balloon in V3 segment	-
	Yang JS et al 16	201 5	Spine J	1	Metastasis	Percuta neous anterior	Fluoroscopy	No	Ye s	No	No	-
	Kavakebi P et al 3	201 7	Acta Neuro chir	1	NM	Transora l	CT and fluoroscopy	No	N M	Minor, tissue lateral soft	No	-
	Wright CH et al 13	201 7	J Neuro surg Spine	1	Lymphangio matosis	Transora l	CT	No	Ye s	No	No	Tx clivus and C2 during same session
	Bundy JJ et al 18	201 8	J Vasc Interv Radio l	1	Multiple myeloma	Transora l	Fluoroscopy and cone-beam CT	No	Ye s	No	No	Associate d RFA
												! 15

-! 16 ! 17