1. Introduction

The larynx is a part of the respiratory system [1] and protects the respiratory tract during swallowing, respiration, and voice production, and provides a mechanism for organisms to seal the airways, and hold their breath [1, 2]. The larynx is important for communication between members of the same species and evolutionary studies, as in the case of anuran species [2, 3]. Voice production occurs as a result of vibrations of the larynx [1, 4]. Although there are descriptions on the form and constitution of the cartilaginous muscle of the larynx of animals, there are few studies on signs of sexual dimorphism in the larynx in domestic or wild mammals [5]. Larynx vibration facilitates the production of sounds that exhibit reduced environmental attenuation [6]. “The size and composition of larynx” appears to contribute to species specific acoustic properties [7, 8]. Little is known about size and morphology of cattle larynx. Vocal organ morphology represents a “complex shape and the absence of homologous landmarks pose major challenges in its geometrical analysis” [9]. In older females, vocal cord contact increases, while in males it decreases with age [10, 11].

The association between larynx length, elasticity, and body size has been described in several mammal species, such as human, red deer, horses, and Rocky Mountain elk [4, 12]. Moreover, in humans the larynx is sexually dimorphic [13, 14] as well as in rats [14]. Despite this observed sexual dimorphism, little is known about the sexual dimorphism of the thyroarytenoid, the main muscle of the larynx [14]. The literature suggests that the number of muscle fibers decreases in the thyroarytenoid, and differences have been found between the thyroarytenoid muscles of male and female rats in old age [15]. But sex differences in larynx contact area during sound production have not been studied for any animal, at least in authors’ knowledge. We therefore hypothesized that there would be noticeable shape changes in the larynx associated with functional vocalizations differences among genders in cattle.

The aim of the present study was to assess anatomical asymmetry using GM techniques of the formalin-fixed larynx in “Bruna dels Pirineus” breed. A preprints.org has previously been published [18].

2. Materials and Methods

2.1. Sample

A sample of 14 larynges from calves belonging to “Bruna dels Pirineus” breed and its F1 crosses (cross with another meat breed) was obtained in an abattoir during the first semester of 2021. “Bruna dels Pirineus” is a breed from Catalonia (NE Spain) with meat purposes and managed under semiextensive conditions mainly on the Pyrenean and Prepyrenean area. Sample included 6 females and 8 males in an age range 335–625 days and a mean of 263.3 ± 53.26 kg for hot carcass weight (Figure 1), which is the commercial age demanded by the Catalan market. The age was calculated according to official individual documents (mandatory). No animal presented anomalies on antemortem inspection. Larynx samples were obtained from fresh carcasses. The samples included all the pieces and were never fragmented.

At the laboratory, fresh larynges were dissected and were preserved in buffered formaldehyde at least for a minimum of 72 h 4% until they were photographed. A transversal cut at the level of larynx was obtained and fixed in a standard plane and pictures were then taken using a digital camera (Nikon D1500) equipped with a lens (Nikon DX de 18–105 mm). A scale was included and placed parallel to the face plane (Figure 2).

3. Results

3.2. Allometry

The contribution of global size variation was 17.52% (p = 0.0008; 10,000 randomization rounds). A clear correlation between centroid size (Figure 3) and carcass weight was found (r² = 0.527; p = 0.0019, data log-transformed).

3.4. Shape Changes

Deformation grids helped visualize the shifts in aligned coordinates from one shape to another and it depicted a clear directional bias (toward left on the lateral parts and toward right on the ventral parts) (Figure 4). Significant differences in the morphometric profile were found between the right and left larynx, and between the sexes (p < 0.05).

4. Discussion and Conclusion

In this study, significant differences were found between the sexes and in the morphometric profile between the left and right part of the larynx. Laryngeal sexual dimorphism is recognized in humans [25] and reindeer [26] but has not been found in dogs [27], in horses [28] or pigs [29]. In the larynx of adult rats, researchers have also found sexual dimorphism in function and structure, with lower frequency of vocalization in males [14].

At the onset of normal larynx vibration, the larynx are adducted [30] (their displacement is away from the midline in symmetric movements), so our results signal an asymmetric vibration, with a possible lack of equivalence with the shape properties of the larynx. In fact, it has been presumed “that laryngeal asymmetry (either in mass or tension) causes irregular vibrations” [31]. The laryngeal tissue, in particular the cartilages is sensitive to testosterone leading to a larger overall growth in males, and this in turn leads to larynx size sexual dimorphism in some species [4], and this would explain the detected sexual dimorphism, as males were larger than females. Asymmetry was correlated with larynx size, which in turn appeared to be correlated with body weight (e.g., more asymmetry with body weight). Other studies report that the higher the body height, “the laryngeal measurements were statistically significant,” and higher for males [32]. Variations in laryngeal structure may explain much of the interpopulation signal differences [33], body size and phylogeny [34] and sexual differences. Efforts to quantify laryngeal morphology have been hampered by its complex structure [9], which poses challenges for comparative analyses [34]. However, imaging technology has made morphological reconstruction possible in such structures [4].

The main limitation of the present study is that the sample size was limited to 14 larynges. As formaldehyde fixation can induce shrinkage, the question remains how different a fixed larynx is from a fresh one. Our results do not necessarily correspond to the situation of live cattle, but we have just intended an approximation of comparison between sides, so detected asymmetry could be found in fresh structures as well.

In this study, calves were used because they are the ones that usually arrive at the slaughterhouse. Evidently, sampling in other factories could have enlarged the sample and the age range, but it was not possible due to sanitary restrictions. Likewise, there is an important lack of bovine larynx anatomy. This reinforces the importance and the novelty of our study.

This study can be considered the first to detect larynx asymmetries in bovine livestock. Further study involving a larger sample size is needed to validate this information in a larger population. Regarding other limitations, the present study has not sampled adult animals, so it would be worth investigating if results are similar among full-grown animals.

Ethics Statement

The study did not involve humans or animals. Animals were sacrificed for commercial purposes (meat production), and the inedible parts (larynx) were used postmortem.

Conflicts of Interest

The authors declare no conflicts of interest.

Author Contributions

Writing–original draft preparation, methodology, and formal analysis: P.M.P.-C. and A.S.-C. Writing–review and editing and writing–original draft preparation: A.S.-C., P.M.P.-C., and M.V.-T. Conceptualization: N.I.M.-O. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Universidad Cooperativa de Colombia.