At birth, the hoof wall is covered and protected by soft horn (perionychium), which is rapidly worn away as a foal moves within its new environment. The hard outer pigmented part of the foot is gradually exposed to provide support for the newborn foal. Support tissues of the internal hoof wall include the stratum medium and stratum internum, with the latter consisting of the PEL and SEL around the wall perimeter.1–5,a The PEL has long been thought to form a homogeneous population of partially keratinized sheets on the inner surface of the hoof.1,2,4–6,a,b In adult horses, this inner hoof of the stratum internum has approximately 600 PEL distributed as single sheets around the perimeter of the hoof wall with an occasional branched one being interspersed among the laminae.1,5,a Each PEL has approximately 20 to 150 SEL around its surface.1,5,a,b These PEL enmeshed within the highly vascularized corium are joined to the distal phalanx to suspend the bone within the hoof capsule.1,2,4–6,a,b Microscopically, the commonality of shapes and sizes of the PEL and relative numbers of PEL among horses reinforces this notion of homogeneity of the structure of the stratum internum. Once formed at the coronet, the laminar structures move distally along the proximodistal length of the hoof wall.1–3,5,6–8 Although early morphologic evidence has suggested a rather more static structure of the hoof wall laminae during growth, more detailed findings suggest the development of greater architectural changes9,10,b as the overall length of the PEL increases,9,b as does the volume of SEL and associated dermal laminae in a proximodistal direction along the hoof wall dorsum.9
Interestingly, it is evident externally that the hoof wall lacks consistency of structural features from 1 horse to another because the feet within most populations vary greatly in size, shape, and conformation. The varying conformations can range from a rounded and symmetric foot to an asymmetric foot having an oval or elongated shape with a long toe and low heel, to an underrun heel, or to one with an upright hoof wall with high heels.1,11 In addition, a wide range of medial to lateral foot imbalances can be present, creating an asymmetric foot in each of these conformational conditions.12,13 The apparent disparity between the variations of external hoof conformations and the relative constancy of the inner hoof morphology of epidermal laminae and how they relate to each other are not known.
To identify potential factors capable of influencing or changing hoof wall structure is difficult because it is virtually impossible to isolate only a single variable capable of influencing the hoof, especially during its growth. Nutrition, foot disease, and certain husbandry practices, such as an adequate exercise program versus stall confinement, frequent or infrequent farrier care, and variable ground surface conditions, may potentially influence hoof structure. The birth of a foal may represent another situation in which one may be able to examine the hoof wall structure and determine whether certain factors, particularly external environmental influences, may affect the hoof wall morphology as the hoof wall passes from a non–weight-bearing state to a weight-bearing state within moments after birth. At birth, many organ systems undergo specific transformations, which enable the newborn to survive the transition from an in utero environment to a hostile external environment. In a foal, another requisite transition occurs as the precocious newborn rises to its feet and begins to support its weight to extricate itself from potential predators. The rapid transition of the feet from a non–weight-bearing state to full weight support shortly after birth indicates that the musculoskeletal system and the locomotor spinal circuitry are primed and ready to respond to these new environmental conditions. Although we assume that the hoof and foot tissues of a newborn foal do respond to these new weight-bearing conditions, we do not know what these potential changes might be. Thus, the purpose of the study reported here was to examine the inner hoof wall (stratum internum) of the fetus for evidence of structural alterations of the epidermal laminae during the transition period from fetus to newborn foal and compare morphologic changes to the structure of an older foal and yearling.
Materials and Methods
The forelimb and hind limb feet from near-term fetuses (n = 27; all gestation ages were within 3 weeks of the calculated foaling date), foals (19; newborn to 2 months old), and yearlings (8; 2 months to 9 months old) represented by several pleasure horse breeds, including Arabians, Quarter Horses, Thoroughbreds, Standardbreds, and mixed breeds and their crosses, were obtained from the Diagnostic Laboratory at Michigan State University. Near-term fetuses were stillborn within 3 weeks of the calculated foaling date, whereas newborn foals represented a group that was born alive and had lived for 1 or 2 days and up to 2 months of age and had conditions other than foot-related problems when they died or were euthanized. In this group, a range of the foal's ability to ambulate was evident by the solar surface wear of its feet from little wear in which the perionychium was only partially worn, but still present, to other feet in which the perionychium was worn away and the keratinized solar surface was visible. The third group of older horses (> 2 months to 9 months old) died because of other non–foot-related problems. The conformation of the hooves was examined visually by bisecting the hoof between the coronet and sole at the toe, with obvious differences between the 2 halves being classified as an asymmetric foot. In these 3 groups, hooves from fetuses and newborn foals were all determined to be reasonably symmetric on visual inspection, whereas in the third yearling group, 7 of 8 horses had asymmetric feet with the medial side being steeper than the more sloping lateral side of the hoof. The forelimb and hind limb feet were considered to be normal on the basis of the lack of deformation of tissues and lesions observed macroscopically or microscopically during histologic examination. Feet were frozen and then cut parallel to the ground surface into sections (4 to 6 mm thick) with a band saw, and each section was examined qualitatively by use of a dissecting microscope for overall shape and form of the stratum internum around the entire foot. Cut sections were nearly symmetric between the toe and the medial and lateral heels. Sections with obvious gross asymmetry because of sectioning with a band saw were discarded from the study. On the cut section, the overall hoof wall thickness of fetal hoof sections (n = 12 term fetuses; stratum externum and stratum medium) was measured macroscopically from the base of the PEL to the outside hoof wall by use of a dissecting microscope and photographs with a calibration measure. The entire full thickness of the hoof wall (stratum externum, stratum medium, and stratum internum) and that of the cortical bone of the distal phalanx of fetal and newborn horses were processed for histologic examination. The distal-most sections (1 to 2 sections/foot) from the hoof, but proximal to the white line, in which PEL were present around the entire wall including the bars, were used in the quantitative PEL density measurements. Because of differences in age and size of feet, hoof sections from the same fetal, newborn foal, and yearling groups were compared within their own groups at the toe and quarters of the hooves and at those sites on the ipsilateral forelimb and hind limb. In several fetuses (n = 7), proximal and distal sections were compared quantitatively for relative PEL density because the hoof shape differed from that of the newborn and older foal groups. Quantitative measurements of interlaminar distances of the PEL around the perimeter of the hoof wall at the interface of stratum internum (base) and the apical end of the primary dermal laminae were obtained by counting the PEL beginning at the toe. The midline of the cut section was determined by use of a dissecting microscope, and then 25 PEL were counted on each side of the midline point of the toe (zone 1; 50 PEL were counted at the dorsum of the foot). Groups, or bins, of 50 PEL were then counted medially and laterally from zone 1 at the dorsum to the quarters, with PEL at the quarters being measured over the distal phalanx and at a more palmar site of the lateral cartilage. As the hoof became larger from the fetus to the yearling, additional groups of 50 PEL were counted at the medial and lateral quarters and were included in the quantitative analyses. The demarcation between these bins of 50 PEL was marked at the stratum internum interface of the inner wall and at the apical ends of the dermal laminae, and the total length of each bin of 50 PEL was measured on a straight line by use of calipers to the nearest 0.025 mm. The bin size of 50 PEL was chosen because the hoof wall was reasonably straight for the distance occupied by this number and the effects of wall curvature could be ignored. This measurement provided an indication of interlaminar distance, or density, of the 50 PEL groups around the perimeter between the toe and quarters on each side of the midline. To ascertain the accuracy of the counts, repeat measurements of the perimeter distance of 50 laminae were performed as many as 5 to 6 times in each foot. Repeat measurements were consistently within 0.10 mm of the original measurement. The interlaminar distance of 50 PEL at the toe was compared with the same number measured at the quarters overlying the distal phalanx and at the lateral cartilage. Medial and lateral quarters over the distal phalanx and over the lateral cartilage at each zone were averaged as a single distance at these 2 sites in all 3 groups for comparisons of differences in the mean PEL density between the quarters and the toe as well as between the dorsum of the hoof and each of the medial and lateral sides of the hoof wall. When examining the sections with a dissecting microscope (30X magnification), locations and numbers of PEL having branched or Y shapes were noted and recorded. Branched PEL were counted as 2 laminae if each sheet of the branched PEL was observed. Tissue blocks from the middle and distal thirds of the hoof wall at the toe and the quarters of fetuses were removed and placed in sodium phosphate–buffered formalin, whereas only sections from the distal 2 sections of hooves from older foals were placed in fixative. The external portion of the hoof wall (the stratum externum and portions of the stratum medium) was removed from hooves of yearlings by use of a sharp instrument prior to submission for histologic preparation because of the difficulty of sectioning the keratinized hoof wall. Tissue blocks were embedded in paraffin and sectioned at 6 μm or placed on a freezing microtome and cut at 60 μm. Representative sections were stained with H&E stain, Masson trichrome stain, and Verhoeff-van Gieson stain for elastic fibers.
Statistical analysis—Numeric data were collected from distal-most hoof sections (1 to 2/hoof) from the 3 groups (fetuses, newborns, and yearlings) between the toe and mean distance at medial and lateral quarters and between the toe and individual quarters overlying the distal phalanx and lateral cartilage. Comparisons of the measured distances of 50 PEL were made in these various regions around the wall perimeter within each group as well as between the forefoot and hind foot of the same horse. Data were summarized and are given as mean ± SD. To determine significance within each group, comparisons of measurements were made by use of a 1-way ANOVA. A Scheffe post hoc test was used to indicate significant differences among the regions (toe, average of both quarters, and medial and lateral quarters) within the same group. Data on wall thickness were analyzed by comparing the measured structures on proximal and distal sections of hooves from term fetuses with 2-tailed paired t tests to determine whether there were consistent differences between the sections in individual hooves. For all analyses, values of P < 0.05 were considered significant.
Results
Examination of various regions of the hoof wall of stillborn fetuses revealed several general characteristic features in their gradual transition from term fetus to foal and yearling states. In the fetus, the stratum internum was consistent morphologically in the forelimb and hind limb specimens when examined with dissecting and light microscopes. The size, shape, and density of the PEL around the perimeter of the hoof wall were consistent within each hoof wall. After birth, the stratum internum and its epidermal laminae gradually changed as the PEL became more diverse morphologically both qualitatively and quantitatively as the age of foals increased to 2 months and to yearling. These changes included variations in shapes and sizes (width and length) of the PEL at the toe and quarters and the relative number of laminae along the perimeter of the hoof wall between the toe and quarters.
In fetal feet, the overall shape of the foot was visibly symmetrical when bisected between the coronet and sole and either the hoof walls were nearly straight with no outward flaring at the quarters or toe or the foot circumference was slightly greater proximally than distally, thereby resembling an upside down cone. Comparative laminar measurements of the hoof wall (n = 13 feet) at proximal (1 to 1.5 cm distal to coronet) and distal (at the level of the white line or slightly more proximal) levels were 13.4 ± 0.81 mm and 12.6 ± 0.78 mm, respectively, for 50 PEL (P < 0.05). Gross thickness of the dorsal hoof wall (stratum externum, stratum medium, and stratum internum) was significantly greater proximally than distally, with the distal sections being only 65% of the mean thickness of proximal sections in term fetuses (n = 12; P < 0.01). Mean hoof wall thickness at the most proximal section (1 to 1.5 cm distal to coronet) examined with formed lamina was 6.7 ± 0.9 mm, which decreased significantly in a stepwise manner in subsequent distal cut sections to 5.2 ± 0.7 mm; 4.5 ± 0.5 mm; and, finally, 3.5 mm (1 cm proximal to distal edge of wall), respectively. Microscopic examination of sections revealed that the PEL had a uniform shape around the perimeter of the hoof wall between the toe and the quarters and heel in forefeet and hind feet. The PEL of the forefeet were slightly more developed than the PEL of the hind feet when compared in the same horse, with the PEL of the forefoot being visibly longer and wider microscopically. The PEL extended as single sheets from the inner hoof wall and consisted of a central keratinized core with short SEL (range, 20 to 52 μm; Figure 1). The PEL had little evidence of branching at the toe. In measuring relative densities of 50 PEL along the wall perimeter, there was no significant difference between the toe and mean distances of the quarters (P = 0.569), between the toe and individual 50 laminae of the medial and lateral quarters (P = 0.311), or between the relative density of 50 laminae observed in the forelimbs and that of the hind limbs (P = 0.208; Table 1). Quantitatively, < 1 branched-shaped PEL (0.54 branched PEL/50 PEL) was observed in a histologic section at the toe of hooves from term fetuses, whereas branched-shaped laminae at the quarters and heel areas were observed less frequently.
Mean ± SD measured distances of 50 PEL in various regions (toe averages vs medial and lateral quarters averages, fore-limbs vs hind limbs, and toe vs individual medial and lateral quarters) around the wall perimeter of hooves from fetal, newborn, and yearling horses.
| Region | Fetus | Newborn | Yearling | |||
|---|---|---|---|---|---|---|
| No. of sections | Distance (mm) | No. of sections | Distance (mm) | No. of sections | Distance (mm) | |
| Toe averages | 22 | 12.38 ± 1.9 | 44 | 12.15 ± 1.3* | 16 | 13.44 ± 1.7† |
| Quarter averages | 42 | 11.78 ± 2.4 | 85 | 13.41 ± 1.4* | 31 | 15.43 ± 1.9† |
| Forelimbs | 8 | 12.60 ± 1.4 | 15 | 12.14 ± 1.4 | 5 | 12.34 ± 1.0 |
| Hind limbs | 7 | 13.79 ± 2.0 | 15 | 12.48 ± 1.2 | 5 | 13.64 ± 1.0 |
| Toe | 32 | 12.90 ± 1.4 | 77 | 12.09 ± 1.2* | 38 | 13.59 ± 1.6† |
| Lateral | 32 | 12.88 ± 1.8 | 77 | 13.90 ± 1.9* | 38 | 15.85 ± 2.7† |
| Medial | 32 | 13.49 ± 2.1 | 77 | 13.82 ± 2.1* | 38 | 16.64 ± 2.9† |
No significant differences were determined between toe averages and quarter averages, between individual measurements at the toe versus the medial and lateral quarters, or between the forelimb and hind limb in hooves from fetuses.
Significant (P < 0.05) differences were detected between the toe averages and quarter averages and between individual measurements at the toe versus the medial and lateral quarters, but not between the forelimb and hind limb.
Significant (P < 0.05) differences were detected between the toe averages and quarter averages and between the toe and medial and lateral quarters, with the medial quarter measurements being significantly different (thicker) from either the lateral quarter and toe measurements. No differences were detected between forelimbs and hind limbs.
Photomicrograph of a section of the PEL at the toe of a hoof from a late-term fetal horse. Notice the homogeneous appearance of the PEL. The population of PEL usually is of similar length and width, with shorter SEL extending around the perimeter of the PEL. H&E stain; bar = 250μm.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.1947
Photomicrographs of sections of the PEL at the toe of a hoof from newborn foals surviving up to 2 months. A— Notice the initial Y-shaped formation with dimpling at the apical end (arrows). The branched Y at the middle arrow as well as the branched Y (arrow on the left side of the figure) are interpreted as being more mature with longer branches and shorter stems than the other 2 Y branches in the photomicrograph. B—Notice several Y branches (arrows) in the PEL from a hoof of another foal (9 to 10 days old) and examples of branched PEL with a wider base or stem (arrowheads) than the adjacent single-sheeted PEL. H&E stain; bar = 500 μm.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.1947
In newborn foals surviving to 2 months of age, the overall shape of the hoof was reasonably symmetric on bisection of the hoof along the dorsum. The hoof wall in the more distal cut sections had a visibly greater circumference than more proximal cut gross hoof sections when superimposed on one another. However, the PEL were seen to change in terms of their basic microscopic morphology and distribution around the hoof wall (Figure 2). Qualitatively, morphology of the PEL at the toe of the newborn foal of several weeks varied more than in the fetus and had 2 basic shapes extending from the base of the laminae to the apex near the distal phalanx: the single-sheet laminae and Y-shaped or branched laminae interspersed between these single sheets of epidermal laminae. The single-sheet laminae accounted for most of the laminae detected along the perimeter of the hoof wall at the toe. These PEL were cornified from the base (thickness ranged from 25 to 50 μm) along the central three quarters of their length, whereas the apical ends were not. In addition, a considerable group of branched or Y-shaped PEL was detected at the toe. Microscopically, several branched PEL were seen to be interspersed among single-sheeted PEL. The configuration of the Y-shaped laminae varied. The PEL with a short branching pattern had an indentation at the apex with a long stem, whereas other Y-shaped PEL had 2 longer branches and a shorter stem (Figures 2 and 3). The width (width ranged from 75 to 150 μm) of the base or stem part of the Y-shaped laminae had a mean thickness of approximately 2 times the width of the adjacent laminae. The population of Y-shaped laminae represented 5 laminae/50 PEL (5 Y-shaped PEL/50 laminae) in those foals that survived for more than a few (> 4 to 5) days. Generally, the overall shape and size of the SEL were similar around each PEL with the exception of the SEL on branched PEL. The relative lengths of SEL along single-sheeted PEL varied, with those near the base generally being slightly shorter (mean, 38 μm) than those at the apical end of the laminae (mean, 46 μm). In Y-shaped PEL, the SEL associated with the branched laminae varied in their relative lengths depending on whether they were located on the inside (axial) or outside (abaxial) surface of the branch-shaped laminae. The SEL were shorter on the inside of the Y-shaped laminae, especially those at the vertex of the Y, or appeared as a layer of epidermal cells rather than as those SEL on the abaxial side of the laminae (Figure 4). The SEL became larger towards the apical end of the PEL than those at the basal end. Quantitatively, there were significant differences in the density of the 50 PEL around the hoof wall perimeter, with the 50 PEL at the toe having a greater density than the mean of both quarters (P < 0.05) as well as a greater density than the medial and lateral quarters (P < 0.05). No difference in distances of 50 PEL between forelimbs and hind limbs was detected.
In yearlings from 2 to 9 months old, hooves were more cone shaped than those in the other 2 groups of foals, and when bisected, 1 hoof was symmetrically shaped and 7 were asymmetrically shaped (n = 7/8 yearling feet were steeper on the medial side and more sloping on the lateral side of the foot). However, the morphology of the PEL appeared more varied than that in the newborn foal. Single-sheeted PEL were present and were more frequent than branched PEL. With the latter, the PEL with Y-shaped or branched features mainly consisted of short-stem and long branches or equal parts of branched and stem, rather than PEL having an initial indentation or dimple at its apex (Figure 5). Quantitatively, there was a significantly greater density of PEL at the toe than at the mean of both quarters (P < 0.05) and between the toe and the medial and lateral quarters (P < 0.05). The PEL densities of the forefoot and those of the hind foot were not significantly different. However, there was a significant (P < 0.05) difference between the PEL on the medial and lateral sides of the hoof wall. In 7 of the 8 yearling feet (ages in months, 2.5, 3 [n = 3], 4, 6, 7, and 9), a detectable asymmetry of the foot was evident on close inspection, with 1 side of the foot being slightly steeper (medial side was more upright) than the opposite or more sloping side (lateral side) of the foot. The interlaminar distances of the 50 PEL in these feet were not equally distributed around the perimeter of the hoof between the toe and quarter. The interlaminar density of the PEL on the steep side of the foot was significantly (P < 0.05) less (greater distance of the 50 PEL along the hoof wall perimeter) than those PEL at the toe and on the opposite or sloping side of the foot (n = 8).
Photomicrographs of sections of the PEL at the toe of a hoof from a newborn foal. A—Notice Y branching after initial dimpling effect (arrow). B—Notice a second Y branch (small PEL to the left of marked Y branch) has a short stem and the branched part is longer and is interpreted as being more mature. H&E stain; bar = 300 μm.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.1947
Photomicrograph of a section of the PEL at the toe of a hoof from a newborn foal. Notice the branching Y at the apical end of the PEL with a central dimpling effect (left arrow); the second Y (right arrow) is interpreted as being more mature with long branches. Secondary epidermal laminae of various lengths are distributed along the inside of the Y-shaped PEL. Near the vertex on the inside of the branching Y, the SEL are shorter (a) and gradually increase in length towards the apex and are also generally shorter than those SEL lengths on the exterior side of the PEL (b). Differences in SEL lengths may reflect differences in the dynamic state of keratinocytes within the SEL. H&E stain; bar = 140 μm.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.1947
Photomicrograph of a section of branching Y-shaped PEL observed in older foals and young yearlings (6 to 7 months). Notice branches of the Y-shaped PEL are longer than the stem. The dimpled Y branch was rarely observed. The SEL are shorter at the vertex (a), compared with the external (abaxial) side of the branched PEL and the apical end (b). The 2 branched PEL have a wider base than the adjacent single-sheeted PEL. H&E stain; bar = 180 μm.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.1947
Varying branching patterns of the PEL were observed in hooves from young foals (Figure 6). Although most of the PEL consisted of single sheets, there was a rather abrupt increase in the numbers of Y-shaped PEL, beginning with the appearance of an initial dimple followed by a larger Y-shaped PEL in hooves from older foals.
Our interpretation of the observed changes in the branching patterns is shown diagrammatically (Figure 7). In newborn foals, the PEL are nearly symmetrically distributed around the perimeter of the hoof and have few Y-shaped PEL. With the formation of Y-shaped PEL along the proximodistal length of the PEL hoof wall, additional PEL can be created (intermediate stage) as they form around the inner hoof wall, creating a larger circumference with similar interlaminar spacing in a well-balanced foot as that seen around the perimeter of the hoof wall of a young foal without the need for increased mitosis of keratinocytes along the dorsum of the inner hoof wall.
Illustration of several shapes of epidermal laminae detected microscopically in hooves from foals (newborn to 2 months old) and yearlings (2 to 9 months old). Single-sheet PEL (middle) are the most numerous type of PEL, whereas the initial dimpling seen at the apical end (right side of illustration and inset) was evident within the first few weeks after birth of a foal. Branching PEL (left side of illustration) is interpreted as being more mature with a short stem and relatively longer branches, which was more commonly detected in hooves from older foals and yearlings.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.1947
Illustration of the interpretative formation of the PEL as the hoof wall increases in circumference. Initially in a foal, the PEL (rod shapes) are equally distributed around the perimeter of the hoof (A). Then the PEL bifurcate to form Y-shape laminae (B). When bifurcation is completed with an increase in hoof size (C), interlaminae spacing around the PEL can remain the same even though there are more PEL around the perimeter of the hoof wall.
Citation: American Journal of Veterinary Research 67, 12; 10.2460/ajvr.67.12.1947
Discussion
Results of the study reported here indicate that the relative density and morphology of epidermal laminae around the perimeter of the hoof wall change from those of a near-term fetus to those detected in a new-born foal several weeks old and to those of a yearling. In the near-term fetus, the symmetric hoof either being straight walled or having a slight inverted cone shape has a laminar morphology and density that is rather homogeneous and not significantly different between the toe and the quarters. During the first few weeks to 2 months after birth, there is a gradual change in the morphology of the PEL as well as in PEL density because a significant increase in PEL occurs at the toe, compared with the quarters. This relative increase in PEL along the hoof wall perimeter coincides with the appearance and formation of branched or Y-shaped laminae. As the foal continues to grow to a yearling, the relative density pattern of the laminae remains significantly higher at the toe than at the quarters, whereas the relative branching of the PEL becomes qualitatively different. In the yearling group, most Y-shaped PEL had long branches and short stems rather than a dimpled appearance, which was commonly detected in newborn foals, suggesting the progression of a laminar bifurcation process from its apex to the base. In contrast to fetuses and newborns, which had nearly symmetric hooves, yearlings often had asymmetric hooves, with the medial side being steeper than the more lateral flared side of the hoof wall. In these instances, the relative density of epidermal laminae around the hoof wall perimeter changed, with a significantly greater density of PEL being associated with the sloping side of the foot, whereas the density of PEL is less on the steep side of the foot. We hypothesized that these morphologic and density changes in the PEL from the fetus to the newborn and yearling are the result of 2 processes: the normal growth of the foot as it increases in size (both in a proximodistal length and in circumference) and an adaptive response to loading of the foot.
Our descriptive study was designed to compare PEL densities along the hoof wall and examine the morphologic differences of laminae in the feet of immature horses to obtain potential insights into how the foot changes after birth as the foot becomes large and is exposed to external influences during growth. Hooves from near-term fetuses represented 1 group of growing feet that had had little or no exposure to the environment (ie, potential external loading forces), whereas hooves of older foals and yearlings were exposed to many environmental influences and underwent normal growth processes during their lives. Hooves of newborn foals represented an intermediate group that may or may not have been influenced by external factors and growth processes, depending on their length of life and ability to ambulate prior to death. Symmetry along the hoof wall at the quarters was determined visually by bisecting the dorsal hoof wall along a straight line between the midpoint of the dorsal hoof wall at the coronet and distal surface. Hooves of fetuses and newborns were determined to have been nearly symmetric, whereas only 1 of 8 hooves from yearlings was determined to be nearly symmetric. In the remaining hooves from this latter group, the lateral quarter of hooves from the forefeet had a more sloping surface along the wall between the coronet and ground surface than the steeper medial side of the hoof. In cut sections from the distal aspect of the hoof, distances between 50 PEL were measured with calipers and comparisons of PEL distances at the toe and quarters of the forelimbs and hind limbs of the same horse were made. By comparing distances of 50 laminae within each group, any potential problems associated with differences and variations in foot sizes between fetus, newborn, and yearling foals were obviated. Additionally, by measuring only PEL distances along those wall zones that were nearly straight, such as the toe and the midquarter area, other errors, including PEL having any arc to the inner hoof wall, were minimized. Such counts may have resulted in a greater number of PEL per unit of hoof wall length as the caliper measurements would have been obtained as a straight line between 2 points rather than along the long curvature of the hoof wall. Use of frozen sections and measuring distances enabled direct comparison of the PEL spacing to the outside of the foot and its overall conformation of the hoof. However, histologic comparisons of the morphologic features of the cellular architecture of PEL did involve embedding in paraffin and then staining of thinly cut sections. Although PEL from 3 groups were compared morphologically, any such quantitative comparisons of histologic findings among feet from horses of various ages or among frozen and histologic sections may have been misleading because there were not only differences in size of the hooves between fetuses and yearlings but also variation in the degree of shrinkage of tissues, including the keratinized hoof wall, in these various age groups because fetal and neonatal tissues undergo greater shrinkage than tissues from older animals when processed histologically.14,15
The general morphology of the hoof evolves from that of a term fetus to a newborn to a yearling foal as it changes from an inverted cone shape or a hoof with parallel walls at the quarters to a more upright coneshaped hoof. The inverted cone-shaped hoof may explain the quantitative measurements of similar PEL densities at the toe and quarters in hooves from fetuses and young newborns, but hoof shape alone does not explain changes observed in hooves from newborn foals, in which the density of PEL was increased at the toe, compared with the quarters seen in older foals. During this transition period, the shape of the hoof gradually becomes an upright cone-shaped hoof and, hence, has a greater circumference in the distal-most sections of the foot. Several concurrent changes during this transitional period may explain the findings detected in hooves from fetuses, newborns, and older foals. Hoof walls from fetal and newborn foals are thinner distally than proximally, as indicated in the study reported here, and contain a softer, more malleable horn16 than hoof walls from yearlings or adult horses. As a result, it is quite likely that the distal circumference of an immature hoof will expand and may become deformed to a greater degree when loaded than that of an older foal with a more hardened keratin. With the change to an upright cone-shaped hoof, relative numbers of PEL around the hoof would potentially decrease in density around the hoof wall or, at least, in certain areas because of wall expansion unless additional laminae were formed. The proposed bifurcation of PEL along the proximodistal direction of the hoof wall, particularly at the toe, represents a novel mechanism for an explanation for the relatively rapid increase in PEL density detected at the distal hoof wall toe observed in hooves from older newborn and yearling foals. Alternative mechanisms for the formation of additional PEL by increased mitosis of keratinocytes along the proximodistal length of the dorsal hoof wall or by additional laminae being formed at the coronet and growing distally are not supported in the literature.1,2,4–8,a,b However, results from several studies17,18 support the idea that some keratinocyte mitosis along the hoof wall may occur, although at a reduced rate when compared with that of the coronet and white line zone. Whether such mitosis can provide sufficient laminar tissue for increased PEL changes is not known.9,18 In addition to the bifurcation of PEL, keratinocytes lining the laminae may become mobile and migrate along the inner portion of the Y-shaped apical end of the forming PEL.10 Keratinocyte migration in cutaneous tissues is a well-established biological phenomenon and is controlled by several active growth factors, including epidermal growth factor, transforming growth factor, and fibroblast growth factor.19 In the immature foal, these factors may facilitate and contribute to changes in laminar density patterns, but their exact roles are not known. In other studies18,c of the fetal hoof wall, the laminar junction at the white line appears to be active with evidence of cellular replication, compared with other more proximal hoof wall growth. In human infants, during the transition period from fetus to newborn, dramatic changes in the epidermis are known to occur20 as the dermoepidermal junction and the stratum corneum of the premature infant undergo rapid changes as they become virtually identical to those in the newborn within days. These adaptive changes are believed to be facilitated by growth factors.19,20 Regardless of the factors or influences responsible for the increased PEL density in older foals and yearlings, such increased density at the toe remains into adulthood, presumably for load support via the distal phalanx,1,2,4–6,a,b as well as the sole.21
Other laminar changes along the dorsal hoof wall of adult horses are known to occur between the coronet and ground as the PEL increase in area distally, although not significantly.9 Linfordb also found an increase in the length of PEL in adult racehorses during examination of the descending hoof wall. However, morphologic changes in the SEL do occur along the descent, including bifurcation of SEL.9 These changes have been hypothesized to be attributable in part to cellular division, although at a much lower rate than that occurring at the coronet.9,18 As a result of PEL changes in morphology occurring in the distal hoof of adult horses, these architectural differences in laminae along the dorsum may create artifactual differences in laminar spacing or other morphologic features of fetal and newborn hooves if these morphologic differences in laminae occur in other wall areas. When feet are sectioned parallel to the ground, the PEL seen in hoof wall sections from the dorsum of the hoof are at a more distal level than PEL from the quarters, compared with when sectioning the hoof wall perpendicularly to the dorsal hoof wall. However, findings of the study reported here associated with differences in laminar spacing around the hoof wall or differences in laminar size (thickness or area) do not appear to be the result of such methodologic sectioning of hooves parallel to the ground for several reasons. First, in a previous study22 as well as in our laboratory, sectioning perpendicular to the dorsal hoof wall of adult horses results in a similar laminar spacing, with PEL density being greater at the toe than at the quarters. However, by sectioning hooves parallel to the ground surface, consistently thin, reproducible, and even sections (4 to 6 mm) can be obtained with a band saw, at least in our laboratory, and permits a greater detailed study of the palmar portions of the foot. Second, although laminar widths in adult horses at the dorsum vary proximally to distally,9 laminar widths at the quarters appear to be associated with the conformation of the hoof wall in our experience. Whether there are additional differences in PEL widths between the proximal and distal wall levels on each side of the hoof at the quarters is not known. Third, interestingly, in fetuses and newborn foals, mean hoof and laminar widths at the dorsum differ from that of adult horses in that the hoof wall and PEL are thicker proximally than distally, which is in contrast to that reported in adult horses.1,5,9 Sarratt and Hood9 and Linfordb hypothesized that the increased laminar changes distally are attributable in part to epidermal proliferation of keratinocytes, although at a lower mitotic index than that occurring at the coronet, as well as to some possible cellular movement. Furthermore, in sectioning of the hoof wall from the dorsal surface to the heels, there is often a gradual change in shape of the adult hoof wall (ie, varying degrees of long toes and flared and steep sides of the hoof to low or underrun heels, including the bars8,11,13), which will in all likelihood present varying angles and mean thicknesses of the hoof and laminae in each of the various regions of the hoof, regardless of whether the hoof was sectioned perpendicularly to the dorsum or parallel to the ground surface. Further exploration into this potential aspect of hoof wall morphology in various regions and conditions of the hoof for possible insights into differences in wall growth is warranted.
Findings of the study reported here indicating significant changes in PEL density around the hoof wall are in contrast to the long-held assumption that PEL are static structural entities in terms of their biology, spacing around the perimeter of the hoof wall, and having a relatively constant number per foot.1,2,5,a,b Other detailed studies9,10 have further reported laminar changes in the inner hoof wall as the hoof grows distally, indicating that the inner hoof wall does undergo morphologic changes during distal descent of the hoof wall.9,10 In a study by Douglas and Thomason,22 similar descriptions of the interlaminar spacing have been mentioned with a trend for a greater density of the PEL of the quarters in stillborn foals and newborns than in the toe. Furthermore, we detected a relatively thin distal wall, compared with the proximal hoof wall in fetuses and newborn foals, which gradually changes to a thick wall around the hoof wall perimeter during the first year of life. Whether additional differences in the mean thickness of the hoof wall (stratum medium and stratum externum) between the coronet and ground surface in varying regions of the toe, quarters, and heels of the hoof wall exist are not known. The increased density of PEL in the toes of foals and yearlings, compared with that of fetuses, may be attributable, at least in part, to the increased external forces impinging on this region of the hoof wall.23–26 In standing adult horses, loads are generally concentrated in the middle region of the frog of the foot23,24 and at palmar aspects of the distal phalanx as forces are being directed through the medial and collateral ligaments of the distal interphalangeal joint.24 The direction of loading of the PEL and the hoof wall when standing may vary, depending on the angles (45° to 52°) of the dorsal hoof wall and the conformation of the hoof wall with bending more caudally at the heels.12,26 Such a possibility is thought to explain the changes often detected at the white line juncture with apparent stretching or increased proliferation9,18,b of the epidermal laminae at the toe. Although biomechanical differences in loads between the toe and quarters may contribute to a portion of the interlaminar spacing differences at these sites, these spacing differences were also evident in hooves from young foals, suggesting that underlying tissues and their composition may also influence the laminar patterns around the hoof wall perimeter. Such a hypothesis is consistent with observations in the asymmetric foot and with the observed adaptive adjustments in laminar spacing patterns around the wall perimeter.
After a foal is born, the foot grows by increasing in length (proximodistal direction) and circumference. Presently, hoof wall growth of the tubular and intertubular horn has been reported as occurring from the coronet, explaining proximodistal growth of the hoof wall.1,2,4,8,11,b The coronet and proximal PEL represent a zone of high mitotic indexes, whereas distal to the coronet, mitotic divisions of cells of the stratum internum are considerably lower,8,9,18 suggesting a sterile bed as a zone with little mitotic activity5,6,7,18,a but not a zone of inactivity.9,10 However, the current belief that the hoof wall grows in a proximodistal direction does not adequately explain how keratin horn production at the coronet is capable of increasing the circumference of the foot. Such a growth process at the coronet should create an inverted cone-shaped foot, with the large portion of new growth zone detected proximally at the coronet and a smaller but older growth zone detected distally. During growth, circumference of the hoof wall becomes large with a gradual increase in its perimeter distance around the wall. The formation of branched PEL may potentially explain the increased circumference of the hoof during growth from the newborn foal to the yearling and adult horse. We hypothesize that with differential loading of the hoof wall, greater numbers of branched PEL will form, creating a differential laminar density around the hoof perimeter.
The wide range of variation in relative densities of the PEL around the hoof wall and in the shapes of the PEL may be a reflection of the external shape of the hoof during growth and other potential external factors. In a study by Douglas and Thomason,22 the orientation of the PEL was found to vary around the perimeter of the hoof wall of adult horses. At the dorsum, orientation of the PEL was consistently tangential to the outer wall, whereas at the quarters, the PEL were oriented with the dermal ends being drawn caudally (radial) to the hoof wall. This internal angulation of the PEL at the quarters may be a mechanism of dissipating the shearing forces between the inner hoof wall and the distal phalanx without compromising the laminae themselves. These findings suggest that the PEL are responsive to external (and internal) forces imposed on the hoof wall and will adapt to minimize such stresses on internal tissues of the foot. The morphologic variations of laminae may be another indication of these adaptive responses. Interestingly, the PEL may bifurcate in other hoof wall regions depending on physical forces affecting the hoof wall and on physical loading of the inner hoof and laminae, which creates a greater density of PEL in 1 region of the hoof wall than another. Such a notion would emphasize the importance to farriers or other professionals of balancing the hoof in nearly all planes when trimming the hoof wall to maintain symmetry.13
ABBREVIATIONS
| PEL | Primary epidermal laminae |
| SEL | Secondary epidermal laminae |
Leach DH. Structure and function of the equine hoof wall. PhD thesis, Department of Veterinary Anatomy, University of Saskatchewan, Saskatchewan, SK, Canada, 1980.
Linford RL. A radiographic, morphometric, histological and ultrastructural investigation of lamellar function, abnormality and the associated radiographic findings for sound and foot sore Thoroughbreds and horses with experimentally induced traumatic and alimentary laminitis. PhD thesis, Department of Comparative Pathology, University of California, Davis, Calif, 1987.
Kunsien L. Uber die Entwicklung des Hornhufes bei eingen Ungulaten. Diss, Dorpat University, Tartu, Estonia, 1882.
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