4.1. Hair Morphological Analysis
Quadros and Monteiro-Filho [
9] stated that the cuticular scales with a transverse pattern are oriented transversely to the longitudinal axis of the hair. This type of cuticular pattern (transverse wavy pattern) cannot be used for species differentiation, since it is common to several orders of mammals [
16]. In the irregular pattern, the scales can be either transverse or oblique, and even longitudinally, towards the largest axis of the hair. In addition, the size of the scales may also vary [
9]. Although Keogh [
17] and Teerink [
18] described in their studies four types of ornamentation for species differentiation and, also, Quadros and Monteiro-Filho [
6] reported that the characteristic ornamentations may be interesting for this purpose, in the present work no ornamentation was observed and all breeds showed scales with smooth margins.
De Marinis and Asprea [
19] reported that the domestication process generated changes in several of the cuticular characteristics and its resulting homogeneity compromises the identification of the species. Some similarity between the characteristics studied for these local breeds was expected and such similarities were found between Curraleiro Pé-Duro and Bovino Pantaneiro. Nevertheless, similarities were unexpected between
Bos taurus taurus (Caracu) and
Bos taurus indicus (Nelore). One cannot affirm, but it is probable that the changes could have occurred with the cattle of this breed caused by selection programs and genetic improvement imposed on Caracu breeds to enhance characteristics related to meat and milk yields [
19].
In comparison with other studies, De Marinis and Asprea [
19], without citation of breeds, reported some characteristics similar to the patterns observed in this study. The authors stated that the scales are presented transversely with distant margin and irregular wave scale. Conversely Sari and Arpacik [
20] found irregular rippled scale patterns can be observed in all studied species except the Bovidae family and wild boar, and cited that this structure in the entire hair length of the Bovidae family and wild boar is regularly waved.
However, there are divergences in the nomenclature used by De Marinis and Asprea [
19] and Quadros and Monteiro-Filho [
9] in which this study is based on. For Quadros and Monteiro-Filho [
9], the scales orientation of guard hairs can be transverse or irregular, that is, the cuticle scales did not present the two patterns as suggested by De Marinis and Asprea [
19], unless this occurs in different portions of the hair (shaft and shield, for example).
Breed differentiation in two groups by scales assessment was only possible because Curraleiro Pé-Duro and Bovino Pantaneiro breeds presented a transverse pattern, whereas the group Caracu and Nelore an irregular pattern. It was not possible to conclude that this pattern is a more common characteristic for European bovine breeds since the study by De Marinis and Asprea [
19] did not specify the breed or breeds used to represent the bovine species. In fact, this was not the objective of the aforementioned authors but to distinguish wild ungulates from domestic ones by hair characteristics.
The cuticle morphological pattern studied here did not match the characteristics described in the literature [
21]. These authors described that the cuticle scales of
Bos taurus taurus are flat and deeply imbricated, arranged at an angle oblique to the hair longitudinal axis and with margins presenting a crenated appearance. Deedrick and Koch [
22] also described bovine cuticle scales as being imbricated and without protrusions on the hair shaft.
The medullary pattern observed in the guard hairs of Brazilian bovine breeds has been described before by other authors in studies for nomenclature proposals [
9,
18,
23,
24], in biological studies about eating habits of carnivores through fecal analysis [
16], in the identification of prey mammals and predators [
25], in studies of Brazilian felines [
26], in studies of species with forensic interest, in food quality control [
5], and for ecological studies [
27].
For Chernova [
27], the different patterns of medullary cells may be expressions of the evolution of the species. In addition, Perrin and Campbell [
28] and De Marinis and Asprea [
19] reported that biotic and abiotic ecological factors also played an important role in determining morphological patterns of hair.
Overall, the pattern for Caracu, Curraleiro Pé-Duro, Bovino Pantaneiro, and Nelore breeds was described as continuous, multiseriate, anastomosed, and trabecular with fimbriated margin. Literature is divergent about the morphological patterns of guardian hairs of species from the Bovidae family. This is because most of the literature found is focused on forensic research and not on differentiating and characterizing the bovine breeds. Gaudette [
29] reported that in the Bovidae family there may be individuals who do not present a medullary structure along the hair, whereas others present a continuous or discontinuous medullary pattern. Deedrick and Koch [
22] also described bovines hair without medulla or when present it was continuous.
It is difficult to compare the results of this study with those available in the literature since they only aimed to identify populations without specifying which gender or which breeds were used in these studies. Likewise, the lack of details on the portion of the hair used for observation compromises possible comparisons. For the same reasons, comparisons about hair width are uncertain.
In fact, Deedrick and Koch [
22] and Gaudette [
29] cited that this structure within a Family is relatively narrow. Conversely, De Marinis and Asprea [
19] described that the width of the cortex represents half or 1/3 of the width of the medulla. Given these points, it is possible that the medulla analyzed by these authors varied from intermediate to narrow, since this structure characterizes the innermost layer of the hair, and it is superimposed by the cuticle and the cortex.
In the current study, a narrow medulla was found in only 10% of the hair within the breeds evaluated. In 90% of the bovines analyzed, the predominant pattern was the width that corresponds to that described by literature [
12] for
Bos taurus taurus. De Marinis and Asprea [
19] report that domestic ungulates may have uniseriate and multiseriate medullary structures, whereas bovines have the latter one. The uniseriate pattern was not observed in the current study, that is, along the shield of the guard hair was observed a continuous medulla with multisserie rows of cells in its width with two or more rows of cells present.
The disposition of the cells inside the medulla in bovine breeds was described by Quadros and Monteiro-Filho [
6] as anastomosed because there were fusions between the cells forming cellular arrangements that can define spaces of the cortex with variations in shape and size. Regarding the way in which the cell can be present along the shield, all bovines were classified as trabecular, that is, the cells are flattened as septa or trabeculae and are arranged close and parallel to each other, but transversally to the largest axis, and with longitudinal anastomoses that ligate these parallel trabeculae [
6].
In all hairs, the margin ornamentation of the medulla along the shield was identified and described by Quadros and Monteiro-Filho [
6] as a fimbriae or fimbriated pattern, that is, the occurrence of many protrusions and narrow recesses, but with variable depths and irregular distribution along the margins, constituting what appears to be a fringe.
During the evaluations of the guard hairs (although all bovines had the same trabecular pattern), it was possible to visualize some circular or oval structures in three of the analyzed breeds, except for Nelore. These structures were called vacuoles and according to the quantity were defined in categories: Marked, moderate and absent. The vacuoles abundance was a determining characteristic for bovine breed differentiation. The same was previously described by De Marinis and Asprea [
19]; however, in this study, these cell structures were counted when they were present inside the medulla. The counting of these vacuoles enabled a better description of the medullary form of the breeds and improved the discriminatory power of the method. Thus, Caracu and Curraleiro Pé-Duro breeds had a moderate presence of vacuoles, Bovino Pantaneiro had a marked presence and Nelore was classified as absent.
Quadros and Monteiro-Filho [
25] reported that, in general, the cuticular characters are the most important for species differentiation, whereas medullary characters are relevant to describe Families and zoological Orders. It is noteworthy that these characteristics are also useful in studies related to the identification of domestic breeds as long as they are analyzed together.
Although the differentiation was possible, this type of analysis did not seem to be the most adequate to determine the microstructural patterns of both cuticles and bovine medulla due to its subjectivity. In addition, the structures forms were all very similar, besides the similarity with other mammalian species already described by other authors [
5,
9,
12,
18,
24,
25,
26,
30].
De Marinis e Agnelli [
31] mentioned that in microscopic morphology the use of medulla structure has priority over the cuticle pattern and the use of the cuticle pattern individually will cause confusion, but Sari & Arpacik [
20] reported that at lower levels, such as subfamily and genus, the medulla structure does not give the correct results in species identification due to its high resemblance, so the cuticle pattern (trichological analysis) was used for identification in all species.
4.3. Genetic Characterization
As in this study, in the genetic evaluation, the Caracu breed is easily distinguished from the others, because it possesses the lowest allelic richness, probably due to the genetic improvement by the breeders’ association [
34] and probably due to its formation and objective of selection (milk yield) since 1893 [
15,
37]. The results of this study can also be justified considering the history of formation of these populations from their introduction in Brazil by the colonizers, since the probable ancestor of the Curraleiro Pé-Duro and Bovino Pantaneiro breeds was
Bos taurus ibericus, whereas Caracu had as an ancestor
Bos taurus aquitanicus. Ginja et al. [
38] reported that historical information indicates that Creole breeds have their own identity and a fingerprint unique to this group. The genetic legacy of Iberian cattle is still represented in Creoles, but other influences can also be detected. The Nelore’s easy identification by different methods is completely justifiable and expected because they have Indian origin (
Bos taurus indicus).
The results for area and EC are consistent with those obtained in literature [
33] based on patterns of diversity and similarity obtained for the same bovine breeds, however using DNA markers (RAPD) and also with the study using microsatellites markers [
34]. Both studies mentioned that there was genetic introgression from zebu breeds in local cattle including Bovino Pantaneiro cattle. Considering population structures, it was possible to observe that the Curraleiro and Bovino Pantaneiro have a large number of shared alleles that place them in the same population.
After studying the information obtained by analyzing the morphological and morphometric data of guard hairs microstructures separately, the data were analyzed through multivariate methods. This decision was made based on the fact that the information obtained by univariate analyses can often be incomplete, especially when there is a correlation between the variables.
The results observed indicated that genetic and trichological techniques are similar in terms of individual classification within breed groups and because of that, the trichological analysis can be considered useful as a bovine cattle breed marker. Alberts et al. [
39] reported that, this allows more freedom in the choice of approaches, depending on the objective of the study. If one seeks more flexibility and lacks time and/or more elaborate laboratory resources, or needs a result when still in the field, one should choose trichology. If, on the other hand, one requires absolute accuracy and/or has the time and access to a genetics laboratory, the molecular method is the appropriate option.