Although the external conformation of wild horse hooves has been proposed as an ideal model for domesticated modern horses, histological signs of laminitis have been reported among them. With the hypothesis that the lamellar tissue of horses of Iberian origin raised in semi-feral is healthier than those raised in an intensive management system (stall confinement and high-calorie diet intake), the objective was to compare their lamellar tissues. Lamellar tissue samples were taken from the forelimb hoof of eight domesticated Mangalarga Marchador (MM) horses and from six semi-feral Marajoara (MJ) and Puruca (MP) horses. Primary epidermal lamella (PEL) and secondary epidermal lamella (SEL) were measured (length and width) in several regions, and their shapes were morphologically classified into different types. Breed groups were compared using analysis of variance, followed by Tukey or Dunn tests (P<0.05). Early signs of laminitis such as abnormal keratinization of the abaxial primary dermal lamella, tapered tips of the axial PELs, pointed tips of the SEL, nuclei condensation, and abnormal spatial orientation of the secondary epidermal basal cells (SELBC), were seen only in MM horses. MP horses had a greater interface of epidermis/dermis contact than MJ horses and more rounded nuclei in the round SELBC than MM horses. In agreement with the study hypothesis, semi-feral MJ and MP horses' lamellae were classified as healthier than MM horses, which showed early signs of endocrinopathic laminitis.
The hoof of the horse is a unique structure with a complex anatomy [[
Marajoara (MJ) and Puruca (MP) breeds originated from horses brought by settlers 300 years ago from the Iberian Peninsula to the island of Marajó, Pará State, Brazil [[
In contrast, another domesticated Iberian Breed, Mangalarga Marchador (MM), undergo routine foot care and are known for their risk of developing laminitis. These horses have been highly bred, selecting for desirable show ring traits, but not necessarily for ideal hoof structure. Additionally, these horses are typically raised in partial stall confinement and on a diet rich in NSCs [[
The aim of this study was to compare the histology of hoof lamellae in Iberian breeds raised in modern domesticated conditions (MM) with those raised in a semi-feral state (MJ and MP). Our hypothesis was that the hoof tissue of horses raised in semi-feral conditions (i.e., exposed to daily exercise, limited access to nutrition, and no protection from environmental and pathogenic agents) would exhibit healthier lamellar tissue than horses of similar origin raised under intensive conditions (i.e., confined to stalls and fed high-energy diets rich in non-structural carbohydrates).
This study was approved by the Ethics Committee on Animal Use at the Universidade Federal de Minas Gerais (UFMG) (approval number: 205/2018). Lamellar biopsies were obtained from three different groups of horses and used for histological evaluation.
Twelve semi-feral horses, six each of MJs and MPs, were randomly selected from the herd of Perseverança Ranch, Marajó Island, Soure, Pará State, Brazil. The island is exposed to hot and humid weather, with a mean temperature of 27.0°C, and annually 2.900 mm of rain. The seasons are defined as extremely rainy (December—June) and less rainy, or "dry season" in the rest of the months, and the animal's diet consists only of native grasses, which grows in soils of poor quality [[
The MJ horses were comprised of four stallions and two geldings with mean (± standard deviation [SD]) weight of 300.83 ± 17.15 kg and age of 3.83 ± 0.98 years. The MP horses were comprised of two stallions and four geldings with mean (± SD) weight of 252.5 ± 21.79 kg and age of 7.75 ± 3.77 years. All horses in both groups had body condition scores between 5 and 6 [[
To compare animals with different environmental and management conditions, archived lamellar tissue samples from eight MM horses were used (four non-pregnant mares and four geldings), with an average weight of 402.77±58.31 kg and age of 4±0.41 years, all with a body condition score between 8 and 9 [[
The semi-feral horses were placed into a corral and restrained with halters and placed in clean and grassy locations. They were sedated with detomidine (Dormiun V, Agener Uniao) at 40 μg/kg IV; phenylbutazone (Equipalazone, Dechra) 4.4 mg/kg IV, sulfadiazine + trimethoprim (Tridiazin, Vansil) 15 mg/kg IM, and tetanus antitoxin (LemaInjex) 5000 IU IM were administered. A 14-gauge catheter was placed within the jugular vein; the horses were administered ketamine (Cetamin Syntec 10%) 2 mg/kg IV for anaesthesia induction. Total intravenous anaesthesia was maintained by administering a continuous rate infusion of guaifenesin (5%), ketamine (Cetamin Syntec 10%) 2 g, and xylazine 1 g (Equisedan 10%, J.A. Saude animal) diluted in 1 L of lactated ringer solution, at rate of 1.0–1.5 ml/kg/h. Hoof desensitisation was achieved with an abaxial nerve block using 60 mg lidocaine hydrochloride (Xylestesin, Cristalia) per site.
Aseptic preparation of the dorsal hoof wall was performed using 2% chlorhexidine and alcohol 70%. Lamellar biopsies were obtained from the dorsal portion of hoof capsule [[
The same biopsy technique was used on the MM horses; however, they were restrained into stocks and the procedure was performed in standing position under sedation (detomidine) and perineural anaesthesia (lidocaine) of the digital nerves.
After inclusion in paraffin, tissue samples from the three groups were cut into 5 μm slices and stained with periodic acid-Schiff and haematoxylin and eosin methods. The histological images were digitised at the Centro de Aquisição e Processamento de Imagem. Histological evaluation was performed using Panoramic Viewer 8.0 and Image J.
Histomorphometric assessment of lamellae was performed as previously described [[
Histological assessment of axial ends of the PEL were classified as standard, elongated, sharp, or bifurcated [[
The normality of the data distribution was checked using the Shapiro–Wilk test. Normally distributed variables were subjected to analysis of variance (ANOVA), followed by Tukey's test for comparison between groups. Non-normal data underwent logarithmic transformation, and normal data were analysed as described. When normality was not obtained by transformation, the data were analysed using the Kruskal-Wallis test, followed by Dunn's test. GraphPad Prism 7.0 (GraphPad Software, La Jolla, California, USA) was used for statistical analysis. Statistical significance was set at P<0.05.
As shown in Table 1, the length of the PEL was greater in MP and MM animals than in MJ animals (P = 0.0032 and P = 0.0001, respectively). Distance between the keratinized axis and axial tip of the PEL was greater in MJ horses than in MP horses (P = 0.0007), but there was no difference in relation to MM (P = 0.9999).
Graph
Table 1 Means (± SD) of the histological measurements of hoof lamellar tissue of the Marajoara (MJ), Puruca (MP) and Mangalarga Marchador (MM) breeds.
General measurements Breed MJ MP MM PEL length 2430±398.1b 2643±333.8a 2759±257.4a Kera-axial tip axis 556±204a 434.6±120.4b 517±119.8a Abaxial PEL width 248.9±38ab 299.5±43.08a 230.6±28.84b Interm. PEL width 211±21.38b 262±35.59a 168.6±33.41b Axial PEL width 190.4±19.07a 219±25.62a 140.1±28.05b Total PEL width 216.7±35.87b 260.2±47.52a 179.8±48.17c Abaxial angle PEL/SEL 40.11°±8.7b 41.81°±10.44b 59.16°±16.12a Interm. angle PEL/SEL 43.41±10.73a 44.29±12.93a 42.44±23.54a Axial angle PEL/SEL 81.95±21.63a 71.63±24.63ab 61.6±34.62b SEL Abaxial SEL length 127.7±26b 157.8±33.41a 95.64±23.04c Abaxial SEL width 17.65 ±3.54b 20.09±2.71a 18.91±3.68ab Interm. SEL length 142.8±42.64a 161.8±59.14a 110.9±56.36b Interm. SEL width 21.86±4.63b 25.43±4.39a 20.79±4.27b Axial SEL length 95.32±28.64a 96.9±55.72a 71.02±25.73b Axial SEL width 32.76±7.42a 33.25±7.19a 23.74±5.61b Tubules n°/area 22.17±2.78a 23.17±7.73a 22.13±4.94a SELBCs nuclei 16.05ab 19.8a 8.80b
1 PEL, primary epidermal lamina; SEL, secondary epidermal lamina; SELBCs, secondary epithelial lamellae basal cells; Interm, intermediate; Kera, keratinized; SD, standard deviation.
- 2
# Data submitted to the Kruskal-Wallis tests followed by Dunn. - 3 *Data submitted to the Tukey test.
- 4 Means followed by the same letters do not differ between lines (P <0.05).
Width of the PEL in the abaxial region was greater in MP than in MM (P = 0.0101). In the intermediate region, MP showed greater width than MJ (P = 0.0287) and MM (P = 0.0002). In the axial portion, the width was greater both in the MP (P≥0.0001) and in MJ (P = 0.0026) than in MM. Total assessment of the PEL width indicated that the three breeds differed from each other in three different regions. MPs had a wider PEL than MJ (P = 0.0134) and MM (P<0.0001), whereas MJ had a wider PEL than MM (P = 0.0325).
The angle formed between the PEL and SEL in the abaxial region of MM breed was greater than that of MP (P = 0.0001) and MJ (P≥0.0001). Angles were similar in intermediate regions. However, in the axial region, MJ horses had a significantly greater angle than MM horses (P = 0.0160).
Evaluation of SEL in the abaxial region revealed that the length was significantly different among the three breeds. SELs of MP were longer than those of MJ (P = 0.0002) and MM (P≤0.0001), and SELs of MJ were longer than those of MM (P<0.0001). SEL width in this region was greater in MP than in MJ (P = 0.0166). In the intermediate region, MM had shorter SEL length than MJ (P = 0.0142) and MP (P = 0.0003); SEL width was greater in MP than in MM (P = 0.0002) and MJ (P = 0.0132). SEL was shorter in the axial region in MM than in MJ (P = 0.0009) and MP (P = 0.0462); moreover, SEL width of MM was smaller than MJ (P≤0.0001) and MP (P≤0.0001).
Evaluation of the nuclei of the secondary epidermal lamellae basal cells (SELBC) revealed that MP (Fig 1B), had a higher rate of rounded nuclei than MM (P = 0.0039) (Fig 1A). MJs presented twice the percentage (16.5%) compared to MM (8.80%), but with P = 0.0552. Notably, in the axial region of the PEL, MM presented more condensed nuclei and disorientation regarding angulation with the basement membrane.
Graph: Fig 1 Photomicrographs of the equine hoof secondary epidermal lamellae (SEL) of the equine (20x magnification (100 μm), and periodic acid-Schiff staining).Note the predominance of SEL with a tapered shape and basal epithelial cells with condensed nuclei in the biopsy taken from a Mangalarga Marchador breed horse (A) compared with the predominance of rounded and not condensed nuclei in the basal epithelial in a biopsy taken from Puruca breed horse (B).
MM horses were the only ones that showed signs of fragility in lamellar tissue, characterised by the occurrence of abnormal keratinization of the PDL border (Fig 2B), discontinuity of PEL (Fig 2C and 2D), SEL with tapered shape (mainly in the axial region) (Fig 2A), and abnormal changes in SELBC, such as nuclei condensation and loss of perpendicular angle to the basal membrane (BM) (Fig 2A).
Graph: Fig 2 Photomicrograph of the hoof lamellar tissue from Mangalarga Marchador (MM) horses (10x magnification (200 μm), periodic acid-Schiff staining).(A) An axial portion of a primary epidermal lamina (PEL) is observed, with secondary epidermal lamellae (SEL) of tapered shape (arrows), with a predominance of basal epithelial cells with condensed nuclei and missing the perpendicular orientation to the basal membrane. (B) Note unusual proliferation and cornification of epidermal epithelial tissue at the abaxial border of a primary dermal lamina (arrow) (C and D) Areas of discontinuity are observed in the primary epidermal lamellae (arrows).
No significant difference existed in the morphology of the axial tip of the PEL between breeds. However, at the abaxial end of the PDL, MM horses showed a higher percentage of sharp shapes than MJ (P = 0.0426) and MP horses (P = 0.0426). MM was also the only one to show PDL in a keratinized format, different from MJ (P = 0.0150) and MP (P = 0.0150). Bifurcated shape was significantly higher in MJ than in MM (P = 0.0419), Table 2.
Graph
Table 2 Means (± SD) of the percentages of occurrence of different types of extremities in the primary epidermal layers (PEL) and primary dermal layers (PDL) as previously reported [
Morphology PEL (%) MJ MP MM Standard 71.6±9.8a 66.6±17.5a 54.28±19.8a Sharp 25±5.4a 21.66±7.5a 27.14±13.8a Tapered 1.6±4a 6.6±12.1a 17.14±14.9a Bifurcated 1.6±4a 5±8.3a 1.4±3.7a PDL Standard 86.66±15a 93.33±12.1a 83.33±12.7a Sharp 0b 0b 5±5.3a Bifurcated 13.33±15a 6.66±12.1ab 1.66±3.7b Keratinized 0b 0b 11.66±8.9a Proliferative 0a 0a 0a Separated 0a 0a 0a
- 5 SD, standard deviation.
- 6
# Data submitted to the Kruskal-Wallis tests followed by Dunn. - 7 *Data submitted to the Tukey test.
- 8 Means followed by the same letters do not differ between lines (P <0.05).
No difference existed in the lamellar morphology of the SEL between breeds in the abaxial region, with the standard format predominating. The standard format was predominant in the intermediate region; however, it was significantly smaller in MM than in MP (P = 0.0148). MM horses had a greater number of tapered shapes within the SEL than MP horses (P = 0.0251). In the axial portion of the PEL, only MM presented tapered SEL compared to MJ and MP (P = 0.0449). MJ horses showed a significantly greater number of club shape SELs than MM horses in the axial portion (P = 0.0450). Bifurcated format was more prevalent in MP than in MM (P = 0.0262), Table 3.
Graph
Table 3 Means (± SD) of the percentages of occurrence of different types of extremities in the secondary epidermal lamellae (SEL) of the lamellar tissue of horses, of the Marajoara (MJ), Puruca (MP) and Mangalarga Marchador (MM) breeds.
Morphology SEL Abaxial region (%) MJ MP MM Standard 85±6.5a 93±5.6a 80.33±12.3a Tapered 4.33±4.2a 3±6.4a 10±11.8a Club head 2.6±3.9a 0.6±1a 1±1.9a Hyperplasic SBL 0.33±0.8a 0a 0.33±0.7a Bifurcated 4.3±2.6a 3.3±3.2a 6±6.9 a Fused 0a 0a 0a Separated 0a 0a 0a Keratinized 0a 0a 0a Intermediate region (%) Standard 85±8.4ab 89±7.6a 62.66±19.4b Tapered 6.66±6.1ab 2±2.5b 23.42±20.a Club head 5.33 ±4.5a 4.33±7 a 3.66±4.7 a Hyperplasic SBL 1.66±2.6 a 3.66±4.2 a 4±6.9 a Bifurcated 1.33±2 a 1±1 a 4±5.5a Fused 0 a 0a 0 a Separated 0a 0a 0a Keratinized 0a 0a 0a Axial region (%) Standard 64.33±15.2a 63±8.4a 74.33±7.5a Tapered 0b 0b 7.71±7.5a Club head 12.33±7.7 a 8.33±5.5 ab 2.33±1.78b Hyperplasic SBL 20.33±11.8a 19.66±6.3a 14.66±6.1a Bifurcated 3.33±3.7 ab 9±6.2 a 1.66±2.3b Fused 0a 0a 0.66±1.5a Separated 0a 0a 0a Keratinized 0a 0a 0a
- 9 SBL, suprabasal layer; SD, standard deviation.
- 10
# Data submitted to the Kruskal-Wallis tests followed by Dunn. - 11 *Data submitted to the Tukey test.
- 12 Means followed by the same letters do not differ between lines (P <0.05).
The present study demonstrates that semi-feral horses, despite having histological variations related to the environment, nutrition, and hoof self-maintenance process, presented healthier lamellar tissue compared to horses raised in intensive systems.
The MM horses that received high concentrations of NSCs had lesions in the lamellar tissue, unsurprising since hormonal imbalances were previously identified [[
The length of SEL was shorter in MM in all regions of the PEL when compared with the other groups, contradicting the results found in natural cases of hyperinsulinaemia [[
Changes in the morphology of SEL are associated with early stages of laminitis [[
Induction of laminitis due to insulin infusion and oligofructose overload revealed a greater distance between the keratinized axis of the PEL and its axial tip in comparison to healthy animals [[
The marked occurrence of SELBC with round nuclei in semi-feral horses is worth noting. MP horses had a significantly higher percentage of nuclei with rounded shapes than MM. Also, although there were no statistically significant differences, the MJ group presented almost twice as many nuclei with rounded shapes as the MM group. According to Pollit [[
Changes in the appearance of the cell nuclei can occur pathologically when the cell suffers temporary or permanent damage that eventually induces cell death or physiologically, when the cell adapts to different causes, such as simple nutritional changes or changes in mechanical load [[
Other differences in the hoof lamella between feral and domesticated horses have previously been described, revealing that feral horses' fetuses had a higher density of PEL [[
It is important to consider several undetermined factors when interpreting the present results, including but not limited to the small sample size, differences in age, sex, and breed between the groups, as well as the distinct geographic conditions. However, this is the first study to demonstrate that semi-feral horses have lamellar histological characteristics that are very different from those observed in horses confined and excessively fed, which is a common practice in the modern Brazilian horse management system. Further studies with larger sample sizes are needed to describe the histological patterns of semi-feral horses.
Our preliminary findings suggest that Marajoara and Puruca horses from Marajó Island raised in semi-feral conditions, with daily exercise, limited access to nutrition and no protection to environmental and infectious factors, have healthier hoof lamellar characteristics than Mangalarga Marchador horses raised under modern domesticated conditions characterized by stall confinement and overfeeding practices. Despite their smaller stature, MP horses have greater length and width of their primary and secondary epidermal lamellae than other breeds. Conversely, several abnormal findings observed in MM horses indicate the detrimental effects of management practices on equine hoof health.
S1 Data.
(ZIP)
We would like to thank Perseverança Ranch, Hildegardo de Figueiredo Nunes and Henrique Lobato Nunes.
By Bruno Dondoni Malacarne; Rodrigo Ribeiro Martins; Cahuê Francisco Rosa Paz; João Victor Almeida Alves; Lucas Antunes Dias; Marina Alcantara Cavalcante; Alison Miranda Santos; André Guimarães Maciel Silva; Britta Sigrid Leise; Armando Mattos Carvalho and Rafael Resende Faleiros
Reported by Author; Author; Author; Author; Author; Author; Author; Author; Author; Author; Author