ABSTRACT

Twenty-five edentate skull specimens were obtained from horses belonging to the Catalan Pyrenean
Horse breed, aged more than 8-12 months, and assessed by eruption of maxillary (M1): only M1
erupted (n=3), M1 and M2 erupted (n=4) and M3 (total molar series) erupted (n=18). Size and shape of
occlusal surface of M1 were evaluated by means of geometric morphometric methods using 6
landmarks. No differences in size were observed between age groups, but shape differences appeared,
mainly centred on the oral aspect. These results suggest that the occlusal outline pattern does not retain
its overall shape during the horse's life.
Keywords: Cavall Pirinenc Catal, cheek teeth, molar size/shape, Pyrenean horse, skeletochronology.
INTRODUCTION
Teeth are the hardest of tissues and are constantly
worn away by prolonged use [1]. The horse, like
other domestic mammals, has a heterogeneous
dentition that consists of incisors, canines,
premolars, and molars of which the latter two are
similar, and because of this they are referred to
simply as cheek teeth. The cheek teeth dentition
is characterized by cheek teeth being almost
hypsodont, possessing long crowns that are
completely covered by coronal cement at eruption
[2] and continuing to erupt in length after [3]. Such
teeth have to last the horse until death [4].
The crowns of equid cheek teeth are made up of
dentine, enamel and cementum [5]. This latter
surrounds the crown and acts as an attachment for
the periodontal ligament, securing the tooth to the
alveolar bone, while contributing too to the bulk of
the crown [2,6,7]. By progressive wear, cementum
will be worn away first, so different occlusal
appearances due to different cementum strips
between young and old animals can be expected. A
thin layer of hard enamel surrounds a core of soft
dentine [6]. Because enamel is generally associated
with the crowns of teeth, such cementum is
referred to as coronal cementum [2]. The coronal
cementum has been examined in horse by Jones
and Boyde [8] and Kilicet al. [9], among others.
The coronal cementum of horse cheek teeth is
quite different from that of other herbivorous
animals [2].
Compared to traditional morphometrics, geometric
morphometric methods (GMM) capture the form
of an object using Cartesian coordinates instead of
linear distances [10]. These coordinates, retrieved
from landmarks established on homologous points,

maximize the amount of biological information

captured. These configurations also provide a
measure of size, called centroid size (CS). The CS
provides a greater measure of scale than any of the
different ways used in traditional morphometrics to
define size (e.g. length, area, volume) [10].
Due probably in part to the large size of skulls and
the difficulty to collect specimens, very limited
studies have been performed on the change
structure of the coronal cementum of modern horse
cheek teeth [6]. In this current preliminary study,
left maxillary first molar teeth (M1, Triadan 209
the Triadan system provides a consistent method of
numbering teeth across different animal species.
This system is based on the permanent dentition of
the pig, which has 11 teeth in each quadrant-) of
diversely aged horses were selected and evaluated.
We used GMM, since they can capture form with
greater precision than traditional morphometrics
and can extract both the size and shape, allowing
investigation of co-variation with crucial factors
such as age [10]. The study specifically
investigates the effects of age (evaluated through
cheek teeth eruption and allometry (occlusal sizerelated change in occlusal shape) in M1 coronal
cementum form in a specific breed (CPC, Catalan
Pyrenean Horse). Authors are sure that the results
of this study will contribute to more morphological
information about natural tooth wear. Moreover,
as teeth tend to be a very well-preserved piece in
archaeological settings, our results can also act as
an aid for zooarchaeologists when considering ages
from studies on buried animals. To the authors
knowledge, no similar work has been performed on
any other horse breed to date.

Pars-Casanova and Morros

MATERIALS AND METHODS

Sampling
Twenty-five edentate skull specimens were
obtained from horses belonging to the CPC breed.
Skulls were obtained from different vulture feeding
points located in the Pyrenees. The origin of these
skulls represents dead farm animals of unknown
sex, but different ages. This is a local horse bred
for meat production in the North East part of the
Pyrenees along the Spanish-French border [11],
being compact and broad-built[12]. Genetic
analysis suggests that this small population (<4,600
individuals) [13] is closely related to the French
breeds Breton and Comtois [14]. The horses are
reared outdoors throughout the year. During spring
and summer they graze fresh high mountain
grasslands, below 3,000 m above sea level. The
animals do not receive any systematic clinical care
and live under extensive conditions (free
outdoors); animals are fed on grass, sometimes hay
during winter, with no concentrate food, but with
free access to salt blocks. So studies focused on
their dentition are representative of natural
grazing wear.
Geometric Morphometrics
M1 were photographed into their alveoli in an
occlusal view with a NikonD70 digital camera
(image resolution of 2240 1488 pixels) equipped
with a Nikon AF Nikkor28-200 mm telephoto
lens. The camera was placed on a tripod parallel to
the ground plane so the focal axis of the camera
was parallel to the horizontal and centred on the
ventral aspect of each skull and at a distance of
more than 1 m in order to minimize lens distortion
consistently. One picture was taken for each skull.
Variations in the form of landmark configurations
were examined using geometric morphometric
methods. A total of 6 landmarks symmetrically
distributed on the left first maxillary molar were
assessed (Fig. 1). These points were chosen to
ensure an adequate representation of the coronal
cementum surface. Enamel pattern (protocone,
hypocone, etc.) was excluded from the analysis
because of the poor precision for their localization
for all specimens collected (indeed, the occlusal
patterns of enamel foldings have been said to vary
with wear [15]. The software TpsUtility version

1.50[16] was used to prepare and organize the

images. Tooth pictures were digitized using
TpsDig version 2.16 [17]. As measurement error is
of particular concern, landmarks were digitized
twice (using the same image) in two separate
measurement sessions performed by the second
author (Morros).
The Procrustes analysis was performed to
eliminate differences in the size, location, and
orientation of the digitized images. This analysis
uses the centroid of the landmark configuration as
the starting point from which rotation and scaling
occur in order to eliminate differences due to
discrepancies in size. Procrustes distances among
landmark configurations relate to Kendalls shape
space [18], which is non-Euclidean. Subsequent
multivariate analyses used a projection of the
scatters of individuals from Kendalls space into a
Euclidean space tangent to the consensus. The
software TpsSmall version 1.20 [19] was used to
verify whether the distortion introduced by this
projection was significant. The correlation between
tangent-projected and Procrustes distances for both
replicas was large and significant (N=50,
r2=0.998), indicating that their variations were
sufficiently small for the tangent space to
approximate the shape space well in analyses.
Size and Shape Analysis
To test for M1 size differences, we used a
nonparametric multivariate analysis of covariance
(NPMANOVA) for size variable (CS) and a
discriminant analysis (also called discriminant
function analysis or Canonical Variate Analysis,
CVA) for shape variables.
Allometry
Occlusal CS was computed for each piece. The CS
can be defined as the square root of the summed
squared deviations of the coordinates from their
centroid (the mean of all the point coordinates
from the same object). Covariation size -expressed
as CS- and shape were investigated with
multivariate (Procrustes coordinates) regression.
The amount of shape variation explained by the
model is expressed as a percentage (proportion of
variation for which the regression accounts as a
percentage of the total variation) and the statistical
significance of the relationship was assessed with

Pars-Casanova and Morros

permutation tests (10,000 permutations). Natural

log transformation was applied in order to scale CS
relative to the mean properly.
MorphoJ v. 1.05 by Klingenberg [20] and PAST v.
3.01[21] were used for all analyses.
RESULTS
As no differences between replicas appeared
(p=0.903), ulterior analyses were performed with
the averaged values for each specimen. There were
no size differences (Figure 2). Shape was not
related to size (R2=0.025, Wilks =3.378) so size
can be discarded as a confounding factor for shape
differentiation. Plot of the Canonical Variate
Analyses for the age groups are presented in and
the Mahalanobis distances between all group pairs
for each view are given in Table 1.The CVA on
ages indicated no overlap for between grouped
young and immature and adult animals (p<0.001).
Changes with age were mainly centred on the oral
aspect of the tooth (landmarks 1 to 3), where the
cementum layer is thinner. As the lack of evidence
of dental pathology in the sample studied indicates
no pathological dental stress during eruption and
development, these results can be deduced as
natural grazing wear.
DISCUSSION
As this study has been performed on specimens
belonging to the same breed, which is managed
under quite similar conditions, the results obtained
in this research provide meaningful insights into
occlusal M1 form in the CPC horse, a breed that is
managed under extensive conditions, with no
dental care.
Dental wear refers to the macroscopic wear
patterns on herbivorous mammal teeth, specifically
with regard to the shape and relief of maxillary
premolar and molar cusps and it has been stated
that the continued occlusal and mesodistal wear
has a strong influence on size and shape variation.
Wear reflects the amounts of attritive (tooth-ontooth) wear and abrasive (food-on-tooth) wear.
This wear process is a complex phenomenon,
depending on the type of diet, individual and breed
variation in the hardness of dental tissue, the
ingestion of hard phytoliths (grits) in the diet, etc.
[22]. CPC breed can be viewed as a generalist that

may consume a variety of high- and low-abrasion

diets, with no age difference (food management is
the same except for animals destined for abattoir
and post-weaning growth, which were not
considered in this study). No explanation by
different diets according to age can therefore be
suspected.
We could find the explanation in cementum. In
older age, cementum layers continue to form, but
not every year [23]. Moreover, outer dental areas,
more exposed and being less protected by enamel,
would have more wear. Thus, changes in the
outermost layer of cementum would explain
different forms according to age. The shape change
detected could thus be a mere response to increased
dental wear associated with age, and more
concretely due to coronal cementum wearing.
It could now be interesting to carry out research in
breeds with different feeding ecologies. This could
be tested using the wear signature [24] to assess the
magnitude difference between different diets.
Investigation of size and shape variation at
different transverse sections down the crown of a
single tooth to the beginning of the roots would
moreover provide definitive data of how much
wear affects the size and shape of the occlusal
aspect in CPC horses. Further studies would also
be required for the rest of both upper and lower
cheek teeth to explore whether similar results
appear.
CONCLUSION
The results of present study demonstrated that
molar changes of Catalan Pyrenean Horse
according to age are mainly centred on the oral
aspect. The shape changes could be a mere
response to increased dental wear associated with
age, and more concretely due to coronal cementum
wearing.
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