Head Sexual Characterization of Sanmartinero Creole Bovine Breed Assessed by Geometric Morphometric Methods

Simple Summary

The study of sexual dimorphism of the head can provide information on natural selection and adaptation to different environments. The head sexual dimorphism of the Sanmartinero Creole bovine through geometric morphometric was studied. Research on sexual dimorphism of the head in Creole cattle is limited. In Colombia, the Sanmartinero Creole cattle are known, an animal adapted to the foothills and high plains of the Meta department (Colombia). The aim was to compare, using geometric morphometrics, the head shape (size + shape) of the Sanmartinero Creole breed bovine according to the animals’ sex. The heads of males and females differed in size and shape. Females had smaller heads. This study provides valuable insights into the morphological characteristics of a Creole bovine breed, contributing to veterinary anatomy, biology, and domestic breed phylogeny.

Abstract

Geometric morphometrics is performed on different species in different contexts. Here, the aim was to investigate morphological differences in the head of the Sanmartinero Creole bovine to examine head shape variations between sexes using geometric morphometric methods. A sample of cranial pictures of 43 animals (13 males and 30 females) was obtained, and form (size + shape) was studied by means of geometric morphometric techniques using a set of 14 landmarks. This approach eliminated potential dietary effects, ensuring that the observed shape variations were primarily due to intrinsic morphological differences. Sexual dimorphism was found in form (for both size and shape) of the head of the Sanmartinero Creole bovine breed. Males had significantly larger heads based on centroid size (U = 714, p = 0.0004), confirming true sexual size differences, and Principal Component Analysis revealed overlapping head shapes with sexual dimorphism concentrated at midline sagittal landmarks (between the most rostral and caudal orbit points) and paired lateral points, indicating that males have broader and longer heads. The two evaluated characters (head size and shape) are of special interest for the conservation of the breed, especially in those cases whose objectives are to maintain the uniqueness, distinctiveness, and uniformity of the populations. This study analyzed animals subjected to the same feeding program, ensuring the elimination of additional variables.

1. Introduction

Colombia is home to several distinctive cattle breeds, adapted to specific regional conditions and valued for their unique characteristics. A notable Creole breed is the Sanmartinero, which was adapted on the foothills of the Colombian Orinoquia plains, through natural selection, with the bovine introduced by the Spaniards in the 15th century. This Creole bovine has developed economically important traits under adverse environmental conditions and extensive management, including rusticity, resistance, and tolerance to ectoparasites and extreme conditions of temperature and humidity, as well as the ability to use fibrous forage. Thus, Sanmartinero constitutes a biological and economic heritage for the efficient provision of food (meat, milk), skins, and work [1].

Sanmartinero cattle are classified as mesolinean (medium size) and eumetric (medium weight) due to their conformation and size, respectively. The height (measured in the vertical of the withers) is 130 and 135 cm for females and males, respectively. Large and strong head with a straight profile; small, rounded ears with an abundant amount of hair. Strong and well-implanted horns with light colors at the root and darker tones at the tips. In males, horns are thick and directed forward in a crown shape, while in females the horns are lyre-shaped (twisted forward, backward, and upward) [1].

Morphometry can be defined as the study of shape variation and its covariation with other variables [2]. Its objective is to define the conformation of an individual and obtain a stable representation of physiological variations in size, and seeks to eliminate the variation in size that results from individual growth, which frequently has environmental causes, and differentiate this change in size from the change that results from evolutionary genetic causes [3].

Two types of morphometric techniques are recognized: traditional morphometry, based on distances between anatomical points, and geometric morphometrics, which uses the coordinates of these points [2,4]. Geometric morphometrics is more efficient than traditional morphometry because landmarks are digitized from scans or images and the spatial coordinates are automatically saved in special programs [4,5]. The advantage of the latter is that coordinates not only contain size information as distances, but also configuration and relative position. In addition, they allow the visualization of the conformation and its variation between individuals or groups. Unlike the point-to-point approach of traditional morphometry, geometric morphometrics allows the holistic characterization of the biological specimens under study and a graphical output of shape variation [6,7].

Different studies have observed variations in head morphology within the same species [8]. Shape analysis is an approach to explain the morphological diversity of biological structures [7,9]. Geometric morphometrics is a method used to quantitatively evaluate shape variation with biological foundations and sexual dimorphism, especially in scientific areas such as taxonomy and veterinary anatomy [10,11,12].

Various biological processes, mutations, and genetic developments cause differences in shape between individuals [7]. The evolutionary changes in body size that accompany the domestication process, and the morphological radiation of domestic breeds, imply a differentiation between sexes [13]. These phenotypic differences between females and males of the same species are observed throughout the animal kingdom and are manifested in morphological, behavioral, and life cycle traits [14].

Sexual dimorphism is the existence of phenotypic differences between individuals of the same species but of different sex [15]. Significant differences are manifested in body size, color, and morphology [16,17]. What is known about the patterns and causes of dimorphism comes from studies focusing on sexual dimorphism in body size [18]. It is not appropriate to study only sexual dimorphism in size since it is not clear whether selection (sexual or natural) operates on body size or on a specific part such as head size [18,19,20].

The degree of sexual dimorphism usually varies greatly from one species to another. In the case of domestic cattle, although not very pronounced [21], these differences may include the following [13,22]: size and weight (males are usually larger and heavier than females), and horns (in some breeds, males have larger, more prominent horns than females). The head is crucial in reflecting morphological features specific to species and breeds. In cattle, the skull is broad and short, with a square shape, and these characteristics may vary among different breeds. Another feature in some breeds is the presence of cornual processes, which form as epidermal elevations from the frontal bone. Identifying these differences provides more specific data for breed description and contributes to the body of knowledge in the zoo ethnological literature [23,24].

Most studies have been conducted for qualitative (size form of horn, prominence of hump) or quantitative traits assessed by traditional methods (e.g., lineal morphometry), but few studies have been conducted (in the Sanmartinero creole bovine) applying geometric morphometric methods, which, as said before, provide information on size and also on shape [6,25]. The study of sexual dimorphism of the head can be an indicator of adaptation to the environment, adaptation to types of forage, reproductive function, and evolutionary processes. The aim of this research was to use geometric morphometrics to compare the head form (size + shape) of the Sanmartinero Creole bovine breed, according to the animal’s sex. This study is based on the hypothesis that cattle possess distinctive morphological features in their skulls (besides the horns) according to sex.

2. Materials and Methods

2.1. Study Site

The research was carried out at the Libertad Research Centre of the Colombian Agricultural Research Corporation (with AGROSAVIA being its acronym in Spanish), Meta department, Colombia. It is in the municipality of Villavicencio, kilometer 17 of the road that leads to Puerto López, Meta (4.0574386, −73.4676386), with an average annual rainfall of 2.933 mm, average temperature of 26 °C, height above sea level of 336 m, and acidic and low-fertility soils. Its landscape consists of high, medium, and low terraces, typical of the “Piedemonte Llanero” sub region.

2.2. Sampling

A sample of 43 Sanmartinero Creole bovines (13 males and 30 females), aged 2 years for bulls, and from 2 to 4 years for cows, was studied. Animals were raised at the AGROSAVIA Research Centre. The sample was randomly taken from the 10 families that make up the Sanmartinero Creole bovine breed’s in vivo conservation animal germplasm bank, during 2024. A cranial digital photograph was taken of each animal using a Nikon P530 42X camera, Tokyo, Japan. The camera was positioned at a sufficient distance to ensure that the head occupied only a portion of the visual field, free from distortion. An inch ruler was included in the photograph. The images were stored in JPEG extension and then transferred to the computer. To facilitate the photograph, the animals were immobilized in livestock traps.

However, the ideal sample size can vary depending on the specific research question, the complexity of the shape variation and, effectively, the number of landmarks used. Our sample was of 42 animals. We know that the complexity of shape variation being studied was considered low. However, the results are relevant to this study in the Sanmartinero Creole bovine.

This study analyzed animals subjected to the same feeding program, to ensure a homogeneous sample, and avoiding potential dietary effects. This ensures that the observed shape variations were primarily due to intrinsic morphological differences.

2.3. Pictures

Once the photographs were collected, they were imported into the TPSdig program (“Thin-Plate Spline digitizer”) v. 1.40 [5], which made it possible to obtain and assemble a matrix of the coordinates of the landmarks or homologous points (Figure 1). Landmarks were located on classical corporal points used in ethnological research [26]. To ensure the accuracy of landmark placements, landmarking was performed by the same researcher (PMPC) with two independent replicas. The description of the reference points is shown in

Table 1. Characterization of reference points in the Sanmartinero Creole bovine.

1	Most Rostral Point of the Head (“Point of the Muzzle”)
2a and 2b	Narrowest points of the muzzle
3a and 3b	Most lateral margins of the orbits
4a and 4b	Narrowest points of the front
5	Most caudal point of the head (protuberantia intercornualis)
6	Midline between most rostral points of the orbitas
7	Midline between most caudal points of the orbitas
8a and 8b	Ventral point of horn base
9a and 9b	Dorsal point of horn base

.

2.4. Geometric Morphometric Analysis

A Procrustes ANOVA was used to determine the measurement error (whether the value of individuals mean squares was greater than the value of the error) to reduce the handling error of the researcher and statistically verify whether the digitization of the landmarks was suitable for further statistical analysis.

In geometric morphometrics, the isometric component of shape (i.e., the shape that changes in a 1:1 proportion with size) is removed from the dataset through the scaling procedure of the Procrustes superimposition. This step brings all specimens to the same size, producing “isometry-free” shape coordinates and a centroid size. Centroid size (CS) can be used subsequently as a proxy for specimen size. Allometric effect refers to how the size of the structure influences the percentage or rate of change in the shape. The effect of allometry was verified using the multivariate regression of shape (Procrustes coordinates) on size (log₁₀-transformed CS) which was treated here as a proxy for head size, while the information about the shape variation was extracted from the Procrustes superimposition. Comparison of allometries was performed with an ANCOVA.

Once these procedures were performed, a U Mann–Whitney test was applied to check the differences between sizes (expressed as centroid size). Principal Component Analysis (PCA) was then performed, to detect which changes are presented, related to the two first components. Following this, a discriminant function (DF) with 1000 runs was carried out in order to test for statistically significant differences in shape of the individuals.

The analysis was carried out with the statistical programs MorphoJ v. 1.07a [27] and PAST v. 4.03c [28], establishing the confidence level at 95%. All these computer programs are free to use.

3. Results

When carrying out image processing for geometric morphometric project, the Procrustes ANOVA established that the value of the mean square of individuals was greater than the error (MS Individual 0.0005194767 >> MS Error 0.0000097978) (

Table 2. Procrustes ANOVA to verify the intra-observer error and calculate the measurement error for the head dataset through the head shape of Sanmartinero Creole animals (n = 43).

Effect	Sum of Squares	Mean Square	Degrees of Freedom	F	p
Individual	0.305452	0.000519	588	4.84	<0.0001
Side	0.004745	0.000395	12	3.69	<0.0001
Individual * Side	0.063045	0.000107	588	10.94	<0.0001
Error	0.011757	9.8 × 10−6	1200

* Individual by size.

), meaning that landmark digitization was performed correctly.

3.1. Sexual Dimorphism in Size

The centroid size of the head significantly differed between the sexes, with males showing the higher value (U = 714; p = 0.0004).

3.2. Allometry

The allometric effect was 14.74% of shape variation explained by size. This represents the proportion of variation for which the regression accounts as a percentage of the total variation.

We tested for differences in allometric slopes between sexes and found no differences (F = 0.484; p = 0.488). This means that our tests on allometry in this study are not missing informative differences among the sexes related to differing allometric regression slopes. As allometry appeared to be significative, ulterior analyses were performed with residual regression.

3.3. Sexual Dimorphism in Shape

The PCA showed that the clusters formed by both sexes superimposed themselves on each other without a marked separation between the groups (Figure 2). The first two components explained a 58.89% of the total variation, each of them contributing 36.39% and 22.49%, respectively. This lack of differentiation between groups in PC1/PC2 space does not mean that the groups are undifferentiated. In fact, PCA does not assume a priori division and concentrates on the relation between variables and individuals of the same sample, reducing the dimensions of the original data to a new group of variables with axis or orthogonal components [21].

Sexual dimorphism is primarily related to variation at anatomical landmarks 6 and 7 (sagittal), and 2, 3, and 4 (with counterparts), so males tended to present more massive heads (wider head and longer cranium than females) (Figure 3).

In the discriminant function, the head shape differed significantly between males and females (Procrustes distance = 0.812; p < 0.01; Mahalanobis distance = 5.644; p < 0.001), with a correct classification percentage of 100% for both sexes (Figure 4).

4. Discussion

Geometric morphometrics has emerged as a powerful tool in recent research for elucidating the relationship between shape and size within samples and explaining variations between groups [6,10,29]. Unlike linear morphometry, which relies on length, depth, and width measurements, shape analysis focuses on the spatial relationships between landmarks, and while linear morphometry provides only limited information about the overall shape of a structure, often with overlapping measurements, geometric morphometry techniques offer a more comprehensive approach as they contain information related to form [11,25,29].

Sexual dimorphism is an important physiological characteristic for understanding animal adaptation to the natural and social environment [30]. Numerous studies have focused on it since Darwin’s theory of evolution [15,22,31,32,33], and it is considered to be a consequence of natural adaptation and sexual selection [31]. The head is the fundamental biological and skeletal structure for distinguishing morphological differences between species, breeds, and sexes, offering valuable information about their functional and environmental adaptations [24,34].

Sexual dimorphism in head size and shape was observed in the Sanmartinero Creole bovine, with a greater manifestation in size. Males have wider heads and longer skulls. Although sexual dimorphism in head size has been little studied by geometric morphometrics in domestic Creole bovines, a study conducted on the Casanare Creole bovine found that head length and width are greater in males [35], while in the Limonero Creole bovine, sexual dimorphism is evident due to greater head width in males [21]. In other animal species such as the Araucan Creole pig, males presented slightly greater head length and width, without any statistical differences [36].

The main mechanisms responsible for the evolution of sexual dimorphism in size and shape are sexual selection and natural selection [37,38], fertility, and intrasexual competition [39]. In several mammals, circulating testosterone concentrations are associated with cranial bone growth [40]. Studies in two species of lizards (Podarcis muralis and P. siculus) have shown that sexual dimorphism in head shape can arise from head segregation or competitive displacement in response to competition between the sexes [41]. In Canis lupus familiaris, males had a wider and longer head than females [42]; in Hynobius maoershanensis, sexual dimorphism is expressed in terms of head width with a bias towards males [17]. Other research carried out on several species of lizards (Reptilia: Squamata: Sauria) showed that males had longer heads [20]. In a population of domestic cats (Felis silvestris catus), males had a larger head circumference [43].

Studies report that sexual dimorphism in head width is explained by sexual selection, ecological selection, and fertility [17]. Sexual selection favors large-headed males, winning male competition and obtaining more mating opportunities [20,44]. A wide head facilitates the intake of greater amounts of food, providing more energy to carry out the corresponding activities [17]. In males, a large head may be a consequence of intrasexual selection, translating into the ability to carry out confrontations (fights) and aggressive encounters to maintain a territory [45,46]. In the case of the Sanmartinero, we have observed that Creole bovines are animals which become aggressive when their territory or herd is invaded.

The mechanisms of sexual selection modify the morphological properties of each sex; therefore, sexual dimorphism can be amplified by natural selection resulting in a natural segregation of both sexes [19,47]. Environmental adaptation, a complex multi-level structure, and a distinctive feeding system allow the evolution of sexual dimorphism [48]. These natural processes may be the cause of the sexual dimorphism expressed in the Sanmartinero Creole bovine, but the causes still need to be explained more deeply.

We found sexual dimorphism in form (for both size and shape) of the head of the Sanmartinero cattle breed, so not only was size bigger in males, as would be expected for the species, but they are also bigger in shape: bulls have bigger but shorter heads, with a wider forefront and a major base of the horns, compared to cows.

This variation is related to different reproductive functions and adaptations of the sexes and can be a consequence of different sexual strategies of the group, once the energy for growth is used from distinct shape in males and females [49]. Sexual dimorphism can be a direct consequence of sex-specific hormones that provide a distinct morphology related to the reproductive success of the species [50]. The variation in the shape of the head between the sexes occurs because the differentiated growth relative to head dimensions during ontogenesis, and this can be regulated by the presence or absence of androgen hormones [51]. In most mammals, anatomical differences between sexes are due to a trait associated with the action of sex hormones and, therefore, gonad maturation, which marks the beginning of sexual differentiation and the end of the period of sexual or pregonadal undifferentiation that precedes it. This differentiation would seem to follow a different “speed” in each sex [50,51].

Information on head dimorphism in bovines is limited. Potential studies are still lacking on the effect of environmental conditions, genetic factors, dietary habits, and housing type on the presence of sexual dimorphism in head size in the Sanmartinero Creole bovine. The breed’s uniqueness stems from various factors, including its origin, physical characteristics, and its temperament. Moreover, it is a rare breed due to limited populations and distribution, as well as its distinctive appearance. The consideration of all these evaluated characters is of special interest for the conservation of the breed, especially in those cases whose aims are to maintain the uniqueness, distinctiveness, and uniformity of the populations.

5. Conclusions

Sexual dimorphism in head size and shape was observed in the Sanmartinero Creole bovine. Males have wider heads and longer skulls. These results bring new character information on the form of the head of the Sanmartinero Creole bovine. In addition, the technique of geometric morphometry has proven to be efficient in recognizing cattle cephalic sexual dimorphism with refined detailing.