Phenotypic and Genetic Study of the Presence of Hair Whorls in Pura Raza Español Horses

Citation: Encina, A.; Ligero, M.; Sánchez-Guerrero, M.J.; Rodríguez-Sainz de los Terreros, A.; Bartolomé, E.; Valera, M. Phenotypic and Genetic Study of the Presence of Hair Whorls in Pura Raza Español Horses. Animals 2023,13, 2943. https://doi.org/10.3390/ ani13182943 Received: 15 June 2023 Revised: 13 September 2023 Accepted: 14 September 2023 Published: 16 September 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). animals Article Phenotypic and Genetic Study of the Presence of Hair Whorls in Pura Raza Español Horses Ana Encina 1,* , Manuel Ligero 2, María JoséSánchez-Guerrero 2, Arancha Rodríguez-Sainz de los Terreros 1, Ester Bartolomé2and Mercedes Valera 2 1Real Asociación Nacional de Criadores de Caballos de Pura Raza Española (ANCCE), 41014 Sevilla, Spain; [email protected] 2Departamento de Agronomía, Escuela Técnica Superior de Ingeniaría Agronómica, Universidad de Sevilla, Carretera de Utrera Km 1, 41013 Sevilla, Spain; [email protected] (M.L.); [email protected] (M.J.S.-G.); [email protected] (E.B.); [email protected] (M.V.) *Correspondence: [email protected] Simple Summary: Hair whorls in horses are a hereditary trait that may be associated with various factors, including the temperament or the coat color of the animals. Hair whorls are described as changes in the hair pattern and may take various forms: circular whorls (spirals of hair with a round epicenter) and linear whorls (a line where the hairs span out on both sides from the center, producing an oval shape similar to a feather). The aim of this study is to estimate the frequency and genetic parameters of the number and position of circular and linear hair whorls (on head, body-neck and limbs) of the Pura Raza Española horse according to different factors such as gender, level of inbreeding, birth period and coat color. In this breed, circular whorls are more prevalent than linear whorls, with both showing a relevant symmetry. The laterality of hair whorls has been also evidenced and are most concentrated on the left-hand side. Most horses, particularly gray ones, showed circular hair whorls below the central line of the eyes; in a previous paper, this was associated with a calmer and more docile temperament. Hair whorls have medium-high heritability and can be included in a breeding program due to their relationship with behavior. Abstract: Hair whorls are a hereditary feature in horses that may be associated with temperament and coat color. Hair whorls are described as changes in the hair pattern and may take various forms, such as circular and linear whorls. We first carried out a frequency analysis of hair whorls (circular and linear). Next, a Generalized Non-Linear Model was computed to assess the significance of some potential influencing factors, and a genetic parameter estimation was performed. ENDOG software v4.8 was used to estimate the inbreeding coefficient of all the animals analyzed. It was more common to find horses with circular hair whorls than with linear whorls. The heritability ranges obtained were, in general, medium-high for both circular whorls (0.20 to 0.90) and linear whorls (0.44 to 0.84). High positive correlations were found on the between left and right positions, indicating a tendency to symmetry in certain locations. The laterality of hair whorls was also evidenced, with the biggest concentration on the left-hand side, particularly in gray horses, showing circular whorls below the central line of eyes, which has been associated in a previous paper with a calmer and more docile temperament. Keywords: behavior; heritability; equines; coat color; genetic models; temperament 1. Introduction Numerous papers have studied the relationship between body conformation, coat color or coat particularities and temperament traits in domestic animals [ 1 – 8 ]. Among the particularities which have been studied in greater depth are hair whorl patterns [5,9–12] . These hair features, also called trichoglyphs, are laid down between the 10th and the Animals 2023,13, 2943. https://doi.org/10.3390/ani13182943 https://www.mdpi.com/journal/animals Animals 2023,13, 2943 2 of 14 16th weeks in utero in humans and can act as an indicator of abnormal cerebral development [ 13 ]. It is widely known that hair whorls come from the same layer of cells in the embryo as the nervous system [ 14 ], and trichoglyphs have been linked to early fetal brain development. Further study of hair whorls may therefore provide useful insight into both behavioral and neurobiological development [ 15 ], having been related in humans to neuro developmental disorders such as schizophrenia. In addition, in dogs, relationships have been found between hair whorls and motor and sensory laterality [16,17]. In this context, the existence of an asymmetric connection between brain function and behavior has also been previously demonstrated [ 18 , 19 ]. In animals, the right hemisphere is related to flight response before new events, while the left hemisphere controls routine behavior when the animal is relaxed [16,20,21]. In horses, since ancient times, many superstitions have been related to the number and position of hair whorls, as pointed out by Hudayl [ 22 ]; for example, hair whorls that appear on the neck and in the interaxillary region are considered good omens, and those on the lower part of the thigh, and on the jaw, back and shoulders bring bad luck. Aguilar [ 23 ], writing in 1572, considered it a bad sign that hair whorls were located on the temples, jaws, back or heart. At the beginning of the 20th century, Suë [ 24 ] referred to the Arabian horse “Godolphin”, one of the founders of the thoroughbred horse, as an animal with a hair whorl on its chest that “boded ill for its owner”. However, nowadays, this association between the presence of hair whorls, mainly on the forehead, and equine temperament has been scientifically studied, and the results have been presented in different papers [9,12,25]. Certain genomic regions associated with hair traits in horses have now been identified [ 26 ]. The possible pleiotropic effect of these genes, whose metabolic activity is related to hair follicle growth, has been also highlighted, thus providing the first evidence of a genetic–biological association between hair whorls and temperament in horses [ 26 ]. In general, the phenotype of hair whorl traits is a good indicator of the genotype, since these traits have a large additive genetic variance. In addition, hair whorl traits in horses show high heritability [ 27 – 29 ] and are highly correlated with one another [ 27 ]. On the other hand, other particularities of whorls, such as linear whorls, have not been studied in depth before in horses. The Pura Raza Española (PRE) horse is the main native Spanish equine breed, with a census of 275,018 animals, with 26.23% of its census being distributed in 67 countries spread over the five continents. It is a breed that has contributed to the formation of numerous equine breeds [ 30 ], and the horses of this breed stand out for being balanced, docile and with a calm temperament. They also adapt with ease to different situations, such as working in the field or in equestrian competitions such as Dressage or High School Dressage [ 31 ]. They are horses that respond easily to the rider’s encouragement, which is why they are considered obedient, and they have a great capacity for learning. The aim of this study is to estimate the frequency and genetic parameters (heritability and genetics correlations) of the number and position of circular and linear hair whorls (on head, body–neck and limbs) of the PRE horse according to different factors such as gender, level of inbreeding, birth period and the horses’ coat color. 2. Materials and Methods 2.1. Description of the Traits and Database The database included records of 43,398 PRE horses with a range of ages from 6 to 12 months (15,782 stallions and 27,616 mares) taken as part of the veterinary identification process between 2000 and 2021. Veterinary identification is a mandatory condition for registering a horse in the Birth Registry of the PRE-Stud Book and involves carrying out a phenotype review of the animal by an authorized PRE vet. For this purpose, all the reviews from the PRE population are performed by 13 different authorized veterinarians, all of whom are familiar with the identification review routine for this breed. According to the principles of equine identification [ 32 ], hair whorls are described as changes in the hair Animals 2023,13, 2943 3 of 14 pattern and may take various forms. In this study, we are going to look at circular whorls (spirals of hair with a round epicenter [ 15 ]; Figure 1a) and linear whorls (a line where the hairs change their direction on both sides from the center, producing an oval shape similar to a feather). Animals 2023, 13, 2943 4 of 14 (a) (b) Figure 1. (a): circular hair whorl on the center of the head, above the eye line and (b) linear hair whorls on the ventral part of the neck. Yellow arrows indicate the location of the circular whorl (a) and the linear whorl (b). 2.2. Statistical Analyses First, we performed a frequency analysis of whorls (linear and circular). Next, a Generalized Linear Model (GLZ) was computed to assess the significance of some potential factors that could affect the number or distribution of these hair particularities over the horse body. The GLZ represents an extension of the general linear model designed to accommodate dependent variables with non-continuous or non-normally distributed values. A Generalized Linear Model of the binomial with a logit link function (glm (…, family = binomial (link = “logit”), …)) was used for counts of dichotomous traits; while, for discrete traits, GLZ for an ordinal multinomial distribution (a multinomial discrete distribution containing information on a rank scale) was used. The following factors included in the model were coat color (4 levels: grey, bay, black and chestnut); sex (2 levels: male and female); birth period (3 levels: animals born before 2001, between 2002 and 2011 and between 2012 and 2021); and the inbreeding coefficient (3 levels: <0.125; between 0.125 and 0.1875; and >0.1875), together with its influence on the number and position of hair whorls. The birth period focused on events in the last few decades, especially since the breeding program for the breed was initiated in 2002, and because, in recent years, breeders have selected their animals based on breeding values of morphological and functional traits related to Dressage, where the horse’s behavior is of utmost importance for the good performance of the routines. Additionally, in the last birth period, there has also been an increase in the prevalence of dark coat colors (bay, black, and chestnut [33]) in this breed. For this reason, the birth period helps us see what happened to the animals born in these last birth periods [34]. The inbreeding coefficient has been considered as a fixed factor, establishing a series of classes or levels, as in other studies in this breed [35]. Statistical analyses were performed using Statistics for Windows software v.11 [36]. 2.3. Genetic Model The estimation of the genetic parameters was carried out with a multivariate model (two to six traits carried out by each approach) animal model, with the number of hair whorls as linear discrete traits and the positions of hair whorls as dichotomic traits. The approaches included as systematic effects all the influencing factors that resulted statistically significant (p < 0.05) in the previous GLZ analysis: gender, the inbreeding coefficient, the coat color and the birth period. Additive genetic and residual effects were included as random factors. All the genetic models were analyzed using a Bayesian approach via Gibbs sampling with the GIBBSF90+ (http://nce.ads.uga.edu/wiki/doku.php?id=readme.gibbsf90plus#gibbsf90, accessed on 14 June 2023) module of the BLUPF90 software Figure 1. ( a ): circular hair whorl on the center of the head, above the eye line and ( b ) linear hair whorls on the ventral part of the neck. Yellow arrows indicate the location of the circular whorl (a) and the linear whorl (b). The position traits were analyzed on a dichotomic scale (with or without circular or linear whorls on a certain position) and on a linear discrete scale (number of whorls in the same location). All possible positions and number of hair whorls situated on head, bodyneck and limbs were considered (Table 1). Furthermore, in order to assess the symmetry in the appearance of these traits, we also evaluated the frequency of an animal with a hair whorl on the right side also having one on the left side, for all the locations considered. Table 1. Position and number of whorls and linear whorls located on the head, body–neck and on the limbs on Pura Raza Español horses. Location Position Number Head Animals 2023, 13, 2943 3 of 14 to the principles of equine identification [32], hair whorls are described as changes in the hair pattern and may take various forms. In this study, we are going to look at circular whorls (spirals of hair with a round epicenter [15]; Figure 1a) and linear whorls (a line where the hairs change their direction on both sides from the center, producing an oval shape similar to a feather). The position traits were analyzed on a dichotomic scale (with or without circular or linear whorls on a certain position) and on a linear discrete scale (number of whorls in the same location). All possible positions and number of hair whorls situated on head, bodyneck and limbs were considered (Table 1). Furthermore, in order to assess the symmetry in the appearance of these traits, we also evaluated the frequency of an animal with a hair whorl on the right side also having one on the left side, for all the locations considered. Table 1. Position and number of whorls and linear whorls located on the head, body–neck and on the limbs on Pura Raza Español horses. Location Position Number Circular whorls Linear whorls A: top right Class 1: 1 Class 1: 1 B: top center Class 2: 2 Class 2: 2 Head C: top left Class 3: 3 Class 3: ≥3 D: between the eyes Class 4: 4 E: lower right Class 5: ≥5 F: lower center G: lower left Class 1: 1–2 Class 2: 3–4 Body and Neck A: right Class 3: 5–6 B: center Class 4: 7–8 C: left Class 5: 9–10 Class 6: >10 Limbs A: right forelimb Class 1: 1 B: left forelimb C: right hindlimb Class 2: 2 D: left hindlimb Class 3: ≥ 3 A C B A B C A B A D C Circular whorls Linear whorls A: top right Class 1: 1 Class 1: 1 B: top center Class 2: 2 Class 2: 2 C: top left Class 3: 3 Class 3:≥3 D: between the eyes Class 4: 4 E: lower right Class 5:≥5 F: lower center G: lower left Body and Neck Animals 2023, 13, 2943 3 of 14 to the principles of equine identification [32], hair whorls are described as changes in the hair pattern and may take various forms. In this study, we are going to look at circular whorls (spirals of hair with a round epicenter [15]; Figure 1a) and linear whorls (a line where the hairs change their direction on both sides from the center, producing an oval shape similar to a feather). The position traits were analyzed on a dichotomic scale (with or without circular or linear whorls on a certain position) and on a linear discrete scale (number of whorls in the same location). All possible positions and number of hair whorls situated on head, bodyneck and limbs were considered (Table 1). Furthermore, in order to assess the symmetry in the appearance of these traits, we also evaluated the frequency of an animal with a hair whorl on the right side also having one on the left side, for all the locations considered. Table 1. Position and number of whorls and linear whorls located on the head, body–neck and on the limbs on Pura Raza Español horses. Location Position Number Circular whorls Linear whorls A: top right Class 1: 1 Class 1: 1 B: top center Class 2: 2 Class 2: 2 Head C: top left Class 3: 3 Class 3: ≥3 D: between the eyes Class 4: 4 E: lower right Class 5: ≥5 F: lower center G: lower left Class 1: 1–2 Class 2: 3–4 Body and Neck A: right Class 3: 5–6 B: center Class 4: 7–8 C: left Class 5: 9–10 Class 6: >10 Limbs A: right forelimb Class 1: 1 B: left forelimb C: right hindlimb Class 2: 2 D: left hindlimb Class 3: ≥ 3 A C B A B C A B A D C Class 1: 1–2 Class 2: 3–4 A: right Class 3: 5–6 B: center Class 4: 7–8 C: left Class 5: 9–10 Class 6: >10 Limbs Animals 2023, 13, 2943 3 of 14 to the principles of equine identification [32], hair whorls are described as changes in the hair pattern and may take various forms. In this study, we are going to look at circular whorls (spirals of hair with a round epicenter [15]; Figure 1a) and linear whorls (a line where the hairs change their direction on both sides from the center, producing an oval shape similar to a feather). The position traits were analyzed on a dichotomic scale (with or without circular or linear whorls on a certain position) and on a linear discrete scale (number of whorls in the same location). All possible positions and number of hair whorls situated on head, bodyneck and limbs were considered (Table 1). Furthermore, in order to assess the symmetry in the appearance of these traits, we also evaluated the frequency of an animal with a hair whorl on the right side also having one on the left side, for all the locations considered. Table 1. Position and number of whorls and linear whorls located on the head, body–neck and on the limbs on Pura Raza Español horses. Location Position Number Circular whorls Linear whorls A: top right Class 1: 1 Class 1: 1 B: top center Class 2: 2 Class 2: 2 Head C: top left Class 3: 3 Class 3: ≥3 D: between the eyes Class 4: 4 E: lower right Class 5: ≥5 F: lower center G: lower left Class 1: 1–2 Class 2: 3–4 Body and Neck A: right Class 3: 5–6 B: center Class 4: 7–8 C: left Class 5: 9–10 Class 6: >10 Limbs A: right forelimb Class 1: 1 B: left forelimb C: right hindlimb Class 2: 2 D: left hindlimb Class 3: ≥ 3 A C B A B C A B A D C A: right forelimb Class 1: 1 B: left forelimb C: right hindlimb Class 2: 2 D: left hindlimb Class 3:≥3 Animals 2023,13, 2943 4 of 14 2.2. Statistical Analyses First, we performed a frequency analysis of whorls (linear and circular). Next, a Generalized Linear Model (GLZ) was computed to assess the significance of some potential factors that could affect the number or distribution of these hair particularities over the horse body. The GLZ represents an extension of the general linear model designed to accommodate dependent variables with non-continuous or non-normally distributed values. A Generalized Linear Model of the binomial with a logit link function (glm ( . . . , family = binomial (link = “logit”), . . . )) was used for counts of dichotomous traits; while, for discrete traits, GLZ for an ordinal multinomial distribution (a multinomial discrete distribution containing information on a rank scale) was used. The following factors included in the model were coat color (4 levels: grey, bay, black and chestnut); sex (2 levels: male and female); birth period (3 levels: animals born before 2001, between 2002 and 2011 and between 2012 and 2021); and the inbreeding coefficient (3 levels: <0.125; between 0.125 and 0.1875; and >0.1875), together with its influence on the number and position of hair whorls. The birth period focused on events in the last few decades, especially since the breeding program for the breed was initiated in 2002, and because, in recent years, breeders have selected their animals based on breeding values of morphological and functional traits related to Dressage, where the horse’s behavior is of utmost importance for the good performance of the routines. Additionally, in the last birth period, there has also been an increase in the prevalence of dark coat colors (bay, black, and chestnut [ 33 ]) in this breed. For this reason, the birth period helps us see what happened to the animals born in these last birth periods [ 34 ]. The inbreeding coefficient has been considered as a fixed factor, establishing a series of classes or levels, as in other studies in this breed [35]. Statistical analyses were performed using Statistics for Windows software v.11 [36]. 2.3. Genetic Model The estimation of the genetic parameters was carried out with a multivariate model (two to six traits carried out by each approach) animal model, with the number of hair whorls as linear discrete traits and the positions of hair whorls as dichotomic traits. The approaches included as systematic effects all the influencing factors that resulted statistically significant (p< 0.05) in the previous GLZ analysis: gender, the inbreeding coefficient, the coat color and the birth period. Additive genetic and residual effects were included as random factors. All the genetic models were analyzed using a Bayesian approach via Gibbs sampling with the GIBBSF90+ (http://nce.ads.uga.edu/wiki/doku.php?id=readme.gibbsf90plus #gibbsf90, accessed on 14 June 2023) module of the BLUPF90 software [ 37 ]. The Gibbs sampler was run for 250,000 rounds, with the first 10,000 considered as burn-in; then, every 10th sample was saved for later analysis. Posterior means and standard deviations were calculated with the POSTGIBBSF90 software (version 3.15) [ 37 ] to obtain estimates of variance components. Convergence of the posterior parameters was assessed by visual inspection of the trace plots of posterior distributions generated by the Coda R package [ 38 ]. The equation in matrix notation for the model to be solved for a hypothetical trait considering all of the possible random effects was yi= xib+ziu + ei where y is the vector of observations, b are vectors of the systematic effects, u are vectors of the additive genetic effects, and x i and z i are incidence vectors for systematic and animal effects, respectively. The pedigree information for the genetic evaluation included 95,292 horses and was obtained from the official PRE studbook, reconstructing a minimum of 4 generations from each horse evaluated. ENDOG v4.8 software [ 39 ] was used to estimate the inbreeding coefficient of all the animals analyzed. Animals 2023,13, 2943 5 of 14 3. Results 3.1. Statistical Analysis We carried out a frequency analysis for both the position and number within the body location of hair whorls (circular and linear) in this breed. As regards the position of the whorls (Table 2), our results showed that it was more common to find horses with circular whorls on any part of the body than with linear whorls. The majority of PREs had circular whorls on the head and body–neck (95%; 41,570 and 41,379 PRE horses, respectively) in contrast with the horses with circular whorls on the limbs (16%; 4621 horses). The position with the highest percentage of PREs with circular whorls was the left side of the body–neck, with 38,757 PREs affected (89.30%). In fact, the left side always had more circular whorls than the right side, except in the hindlimbs. All the locations showed a high percentage of animals with symmetry between both sides, with 87% (20,972 horses) for the head, 99% (38,252 horses) for body–neck and 88% (3855 horses) for limbs. In addition, when the head position traits were grouped into the upper or lower part of the head, most horses showed hair circular whorls under the central line of the eyes. Table 2. Number (above) and percentage (below, in brackets) of the exact body location of circular and linear hair whorl phenotypes in Pura Raza Español horses. Location Without Top Right Top Center Top Left Between Eyes Low Right Low Center Low Left Symmetry Head Circular Whorls 1828 (4.21%) 993 (2.28%) 10,785 (24.85%) 1728 (3.98%) 23,396 (53.91%) 21,430 (49.38%) 8131 (18.74%) 22,308 (47.03%) 20,972 (87.25%) Linear Whorls 39,124 (90.15%) 40 (0.09%) 618 (1.42%) 78 (0.18%) 1156 (2.66%) 1501 (3.46%) 1569 (3.62%) 1546 (3.56%) 1422 (87.56%) Body–neck Without Right Center Left Symmetry Circular Whorls 2019 (4.65%) 38,712 (89.20%) 37,335 (86.03%) 38,757 (89.30%) 38,252 (98.70%) Linear Whorls 16,671 (38.41%) 18,910 (43.57%) 24,325 (56.05%) 18,906 (43.56%) 18,571 (98.21%) Limbs Without Right forelimb Left forelimb Right hindlimb Left hindlimb Symmetry Circular Whorls 38,777 (83.35%) 666 (1.53%) 696 (1.60%) 3723 (8.58%) 3699 (8.52%) 3885 (87.92%) Linear Whorls 39,898 (91.94%) 250 (0.58%) 271 (0.62%) 3128 (7.21%) 3100 (7.14%) 3056 (89.91%) The ‘symmetry’ column indicates that the same feature was found on the left and right side within the area studied. The percentage was calculated taking into consideration the number of horses with left + right whorls in the same location as a percentage of the maximum number affected in this location on the left or right side. On the other hand, there was a majority of PREs with circular whorls on the head and body–neck (95%), in contrast with the horses with circular whorls on their limbs (18%). The position with the highest percentage of PREs with circular whorls was the left side of the body–neck, with 38,757 PREs (89.30%). Again, the left side always had more circular whorls than the right side, except in the hindlimbs. All the locations again showed a high percentage of animals with symmetry between both sides, with 53% (22,687 horses) for the head, 88% (38,252 horses) for body–neck and 8% for limbs. As before, when the upper and lower head positions were considered together, most horses showed circular hair whorls above the central line of the eyes, with percentages between 30% (low center) and 47% (low left). Table 3shows the results for the number of circular and linear hair whorls a PRE horse has in the same location. Most PRE horses showed one (34%) or three (31%) circular hair whorls on the head, more than two on the body-neck (88%) and two on the limbs Animals 2023,13, 2943 6 of 14 ( 3705 PRE −8% ). However, most of the horses studied presented more than 10 linear whorls on the body–neck (1038 −2%). Table 3. Number (above) and percentage (below, in brackets) of linear and circular hair whorl phenotypes present in each location in Pura Raza Español horses. Without 1 2 3 4 ≥5 Head Circular Whorls 1828 (4.21%) 14,710 (33.89%) 5915 (13.62%) 13,645 (31.44%) 4218 (9.72%) 3086 (7.11%) Linear Whorls Without 1 2 ≥3 39,124 (90.15%) 2044 (4.71%) 1398 (3.22%) 319 (0.73%) Body–neck Without 1–2 3–4 5–6 7–8 9–10 >10 Circular Whorls 2019 (4.65%) 3287 (7.57%) 6907 (15.92%) 8751 (20.16%) 8120 (18.71%) 6002 (13.83%) 8312 (19.15%) Linear Whorls 16,671 (38.41%) 7390 (17.03%) 5386 (12.41%) 6736 (15.52%) 4397 (10.13%) 1780 (4.10%) 1038 (2.39%) Limbs Without 1 2 ≥3 Circular Whorls 38,777 (83.35%) 692 (1.59%) 3705 (8.54%) 224 (0.52%) Linear Whorls 39,898 (91.94%) 419 (0.97%) 2942 (6.78%) 139 (0.32%) The Generalized Non-Linear Model (GLZ) and percentage comparative analyses of the influencing factors related with the appearance of linear and circular hair whorls on PRE horses are shown in Tables S1 and S2. In general, all the factors studied (gender, inbreeding coefficient, coat color and birth period) were statistically significant (p< 0.05). The gender affected 10 positions of the circular (but not top of the head or center body– neck) and 7 linear whorls, with the stallions in general having more prevalence in both. The inbreeding coefficient affects circular and linear whorls in 5 positions and had a higher frequency in PREs with an inbreeding coefficient of over 0.1875. Coat color was a relevant effect for 12 circular and 10 linear whorl positions, with the gray PRE horse showing the highest frequency. The birth period was a relevant factor for 11 circular whorl and 10 linear whorl positions, with PREs born between 2002 and 2011 highest the prevalence. Regarding the number of whorls, the gender was relevant in animals for head and limbs in circular and body–neck and limbs in linear whorls, with the males having a higher number than the mares. The inbreeding coefficient affected the number of circular whorls in body–neck for circle and linear whorls traits, with, in general, a higher degree of inbreeding in PREs producing a greater number. Coat color was a significative factor in all circular and linear whorl number traits, with gray horses having the highest number of circular whorls in the three locations. The birth period was a relevant factor for all the circular and linear whorl traits, with PREs born after 2002 and 2011 being those with highest prevalence. 3.2. Genetic Parameters The genetic parameters of the models carried out in this study for body location and position of circular and linear hair whorls are presented in Table 4. The heritability ranges obtained were, in general, medium-high for circular whorls (0.20 to 0.90) and for linear hair whorls (0.44 to 0.84). Within the heritability values obtained for circular whorls, the highest were found on the top right (0.80) and top center (0.85) of the head, in all positions of body and neck (0.83 to 0.89) and on the left hindlimb (0.90). However, for linear whorls, the highest heritability values were found on the lower right-hand side (0.84) of the head, followed by the right hindlimb (0.83). Animals 2023,13, 2943 7 of 14 Table 4. Genetic variance ( σu ), residual variance ( σe ) and heritability (h 2 ) values for hair whorls according to body location and position. Location Position σuσe Mean Median HPD 95% Mean Median HPD 95% h2 Circular Whorls Head Top right 3.958 3.919 3.609–4.378 0.976 0.982 0.926–1.019 0.802 Top center 5.187 5.161 4.064–6.282 0.933 0.950 0.827–1.010 0.848 Top left 0.377 0.378 0.301–0.457 1.000 1.000 0.977–1.025 0.274 Between eyes 0.246 0.244 0.200–0.295 1.017 0.997 0.666–1.383 0.195 Low right 0.295 0.244 0.085–1.118 1.002 0.999 0.913–1.116 0.227 Low center 3.362 3.775 0.642–5.161 0.998 0.998 0.904–1.099 0.771 Low left 0.276 0.206 0.128–0.905 1.001 1.001 0.904–1.079 0.216 Body-neck Right 8.011 7.930 5.731–10.240 0.985 0.986 0.958–1.013 0.890 Center 7.793 7.674 5.733–10.110 0.986 0.986 0.959–1.012 0.880 Left 4.860 4.853 3.515–6.399 0.983 0.983 0.959–1.007 0.832 Limbs Right front 2.010 2.007 1.803–2.214 0.523 0.523 0.504–0.540 0.793 Left front 1.922 1.919 1.731–2.131 0.520 0.520 0.501–0.537 0.787 Right back 6.670 6.637 5.776–7.858 0.760 0.760 0.735–0.791 0.898 Left back 6.692 6.640 5.667–7.812 0.760 0.760 0.731–0.786 0.898 Linear Whorls Head Top right 0.200 0.204 0.165–0.228 0.189 0.189 0.180–0.198 0.515 Top center 0.866 0.864 0.765–0.975 0.758 0.757 0.711–0.805 0.533 Top left 0.402 0.407 0.337–0.457 0.270 0.270 0.258–0.287 0.598 Between eyes 1.841 1.833 1.558–2.195 0.720 0.721 0.668–0.782 0.718 Low right 3.991 4.094 2.419–5.175 0.748 0.742 0.453–1.055 0.842 Low center 2.487 2.476 2.157–2.860 0.767 0.768 0.709–0.821 0.764 Low left 3.834 3.828 3.452–4.213 0.804 0.804 0.752–0.849 0.827 Body–neck Right 0.136 0.136 0.131–0.140 0.079 0.077 0.075–0.081 0.636 Center 0.136 0.136 0.131–0.140 0.078 0.079 0.075–0.807 0.636 Left 0.102 0.102 0.097–0.107 0.128 0.129 0.125–0.132 0.442 Limbs Right forelimb 0.842 0.843 0.739–0.953 0.406 0.406 0.385–0.429 0.675 Left forelimb 0.883 0.883 0.796–0.973 0.396 0.396 0.379–0.415 0.690 Right hindlimb 4.876 4.876 4.391–5.499 0.983 0.983 0.960–1.005 0.832 Left hindlimb 4.798 4.822 3.983–5.442 0.981 0.981 0.958–1.004 0.830 HPD 95%: 95% Highest Probability Density interval. On the other hand, when considering heritability values for the number of whorls in each location (Table 5), circular hair whorls ranged from 0.32 for limbs to 0.51 for the body–neck, while linear whorls ranged from 0.26 for the head to 0.58 for the body–neck: the latter location showed the highest heritability values for both hair whorls (circular and linear). Table 6shows the genetic correlations among the different positions within a location. A total of 31% of the correlations have a medium-high magnitude ( ≥ 0.25), with 89% being positive and 11% being negative. High positive correlations were found on the head between the lower left and right positions for both linear (0.99) and circular whorls (0.81), indicating a tendency towards symmetry in these locations. In the same way, symmetry was also found on the limbs, due to the high correlations found between right and left forelimbs for circular (0.93) and for linear whorls (0.93); and the high correlations found between Animals 2023,13, 2943 8 of 14 right and left hindlimbs, for both circular (0.83) and linear whorls (0.99). If the laterality is studied, the linear whorls in the lower part of the head have medium correlations with the limbs on the same side, while in circular whorls, this laterality can be observed in close correlations on both sides between the lower part of the head and the body. Table 5. Genetic variance ( σu ), residual variance ( σe ) and heritability (h 2 ) values for hair whorls according to their frequency of appearance at head, body–neck and limb locations. σuσeh2 Location Particularity Mean Median HPD 95% Mean Median HPD 95% Head Circular whorls 0.646 0.646 0.604–0.683 1.013 1.012 0.982–1.044 0.389 Linear whorls 0.058 0.058 0.053–0.062 0.164 0.164 0.160–0.169 0.260 Body–neck Circular whorls 1.459 1.459 1.389–1.519 1.415 1.415 1.367–1.462 0.508 Linear whorls 1.500 1.500 1.445–1.555 1.092 1.092 1.054–1.131 0.579 Limbs Circular whorls 0.115 0.115 0.107–0.124 0.246 0.246 0.239–0.253 0.318 Linear whorls 0.126 0.126 0.120–0.133 0.158 0.158 0.153–0.163 0.444 HPD 95%: 95% Highest Probability Density interval. Table 6. Genetic correlations among linear hair whorl positions (above diagonal) and circular hair whorl positions (below diagonal). Location and Position Head Body-Neck Limbs Top Right Top Center Top Left Between Eyes Low Right Low Centre Low Left Right Centre Left Right Left Right Left Forelimbs Hindlimbs Head Top right − 0.081 − 0.184 0.377 0.141 0.300 0.148 0.047 0.058 0.047 0.032 0.108 0.104 0.108 Top center 0.005 0.437 0.123 0.280 0.720 0.286 0.250 0.238 0.251 − 0.037 − 0.054 0.156 0.152 Top left − 0.771 0.044 0.202 0.103 0.226 0.113 0.169 0.165 0.221 − 0.272 − 0.206 0.085 0.055 Between eyes 0.040 − 0.440 0.081 0.266 0.306 0.290 0.380 0.379 0.382 0.095 0.129 0.152 0.147 Low right − 0.426 0.164 0.578 −0.229 0.348 0.998 0.147 0.147 0.151 0.290 0.335 0.444 0.445 Low center − 0.048 − 0.040 − 0.095 −0.446 − 0.051 0.351 0.233 0.229 0.149 0.141 0.110 0.184 0.177 Low left − 0.078 0.101 0.270 −0.195 0.807 0.053 0.146 0.146 0.146 0.279 0.323 0.443 0.443 Body-neck Right 0.028 0.046 0.074 −0.021 0.998 − 0.120 0.998 0.999 0.773 0.060 0.038 0.096 0.096 Center 0.028 − 0.017 0.062 0.220 0.239 − 0.125 0.252 0.973 0.769 0.054 0.034 0.088 0.087 Left 0.018 0.053 − 0.005 0.109 0.998 − 0.121 0.998 0.792 0.900 0.084 0.034 0.104 0.101 Limbs Right forelimb − 0.172 0.007 − 0.081 0.01 0.027 0.066 0.033 0.056 0.048 0.058 0.932 0.375 0.372 Left forelimb − 0.252 0.001 0.164 0.031 0.026 0.054 0.024 0.068 0.065 0.062 0.926 0.417 0.419 Right hindlimb 0.028 0.035 − 0.024 0.030 0.038 − 0.065 0.060 0.176 0.998 0.190 0.082 0.071 0.998 Left hindlimb 0.016 0.032 − 0.027 0.034 0.038 − 0.055 0.048 0.998 0.998 0.189 0.096 0.061 0.828 Finally, Table 7shows the genetic correlations between the number of circular and linear hair whorls in different locations. The highest genetic correlations were found between the number of whorls located on the body–neck (0.43), with a genetic tendency to show the same number in both particularities. Finally, a close genetic correlation was found between the number of linear whorls located on the head and limbs (0.45) and linear hair whorls located on the limbs, which tended to be accompanied by linear hair whorls on the head. Animals 2023,13, 2943 9 of 14 Table 7. Genetic correlations between number of linear and circular whorls, according to their location on the horse. Location Head Body–Neck Limbs Linear Whorls Circular Whorls Linear Whorls Circular Whorls Linear Whorls Head Circular whorls 0.213 0.012 −0.004 0.138 0.054 Linear whorls 0.153 0.335 0.272 0.448 Body–neck Circular whorls 0.429 0.349 0.130 Linear whorls 0.183 0.125 Limbs Circular whorls 0.618 4. Discussion Hair normally follows a certain direction, namely from top to bottom, from front to back on the sides and in the opposite direction on the back and lower areas. However, every animal has certain peculiarities in the direction their hair grows that will remain with them for their lifetime. This, as well as the coat color, allows the animal to be correctly identified and aids the veterinary identification process. These particularities can be divided into three general groups: variation in hair direction, which can occur all over the animal’s body; the appearance of white hairs in depigmented skin areas; and primitive marks, which are defined according to the location described in the zootechnical breed standards [40]. With the domestication of the horse, artificial selection by men began fine-tuning the physiognomy and temperament of the modern horse. Those horses that stood out for their docile temperament and resistant conformation were given priority for reproduction. However, coat color was the attribute that attracted the most attention and was first selected by human beings, which led to an increased diversity in coat colors [ 31 ]. Horses were domesticated in this way, by giving mating priority to those whose coat colors were more showy [41]. The differences in behavior between animals of the same species and/or breed can be attributed to the temperament of each individual, in which genetics can play a major role. In human–horse relations, generic terms are attributed to horses, based on their owners’ daily experiences and according to the animal’s temperament and reactivity, such as being calm or nervous [ 25 ]. However, there are also descriptions of the relationship between phaneroptical characteristics and temperament, such as coat color and its relationship with reactivity [ 3 ] or the position of head whorls above or below the line of the eyes supposedly influencing personality characteristics of the animals as being more “calm” or “nervous” [ 5 , 9 , 12 , 15 , 42 , 43 ]. A number of different theories exist to explain the development of hair whorls, including genetics, molecular mechanisms and mechanical tension [44]. The relationship between the height of a hair whorl on the head and temperament has been previously explained due to the hair patterns in the fetus forming at the same time as the brain [ 13 ]. Our results showed a higher prevalence of whorls on the lower part of the head (below the center line of the eye) for PRE horses (Table 2), which agrees with the study by Lanier et al. [ 10 ] in cattle, who found that this location was related with a calmer and more noble temperament in the animal [ 10 ]. Previous studies have found differences in horse breeds according to temperament [ 45 , 46 ], with PRE horses being described as noble, calm and sound [ 47 , 48 ]. Curiously, animals with a grey coat color are the ones that have presented the highest number of whorls on the lower part of the head, both on the right and left sides (see Table S1). In this context, horses with a gray coat color of the breed Caballo de Deporte Español (CDE) presented lower infrared temperature values at rest, measured with infrared thermography, compared to chestnut, bay or black-coated CDE horses [ 3 ]. Other authors [ 1 , 4 , 49 ] have also reported differences in coat colors related to personality traits such as calmness, boldness or nervousness which, to some extent, are related with the way the animal perceives threat stimuli and reacts by showing stress [ 4 , 50 – 53 ]. Therefore, the position and the number of whorls on the head in PRE horses could be used as a