Histological structure of antlers in castrated male fallow deer (Dama dama)

Authors


Abstract

Antlers are periodically replaced cranial appendages that, except for the reindeer, are grown only by male deer. The annual antler cycle is controlled by seasonal fluctuations of sex steroid concentrations in the blood, and accordingly castration of male deer causes deviations from normal antler growth. The present study investigated antler histology of castrated fallow bucks (Dama dama). Castration in early spring was followed by casting of the hard antlers carried by the bucks and the growth of a new set of antlers, which remained in velvet permanently. In the following year, numerous bony protuberances developed from the original antler surface. Further growth of these protuberances, which were formed by subperiosteal intramembranous ossification, led to a marked increase in antler diameter in the affected areas. Compared to antlers of intact bucks, the antlers of the castrates showed histological signs of immaturity, suggestive of a reduced bone remodeling and an impairment of the mineralization process. These changes point to the dependence of the above processes on a stimulation by higher levels of sex steroids. Two years after castration, the antlers also developed integumental thickening and showed an initial formation of skin outgrowths. Cystic structures were present in the skin, which were often filled with a presumably sebaceous and/or keratinous material. Formation of intradermal bone or cartilage was not observed in the antlers of the castrated fallow bucks. The histological structure of the skin outgrowths suggested that they were caused by a hypertrophy of the dermal component of the velvet. Due to the localized bone overgrowth, resulting from the periosteal bone apposition onto the original antler surface, skin-lined infoldings originated, which reached deep into the newly formed bone. Our study revealed no indication of invasive/destructive bone growth in the antlers, i.e., of a penetration of the newly formed bone tissue into the pre-existing bone. The hypertrophic bone growth in the antlers of the castrates is compared with other forms of periosteally derived hypertrophic bone formation, including osteomas, in the mammalian skeleton. It is discussed whether the skin and bone outgrowths of the antlers of castrated fallow bucks may be classified as benign tumors. © 2004 Wiley-Liss, Inc.

Antlers are periodically cast and regenerated cranial appendages that develop on top of permanent processes (pedicles) of the frontal bones of deer. Except for the reindeer (Rangifer tarandus), in which both sexes carry antlers, these are normally grown only by male deer (Goss, 1983; Lincoln, 1992). Since the antler physiology of reindeer differs markedly from that in other deer species, this special case is not considered further and only the typical situation in male deer will be presented.

The annual antler cycle is controlled by seasonal fluctuations in the blood levels of sex steroids (Bubenik GA, 1983, 1990; Goss, 1983; Fennessy and Suttie, 1985; Rolf and Fischer, 1990; Sempéré, 1990). During antler growth, plasma testosterone concentrations are low. Increasing testosterone levels prior to the rutting period are associated with the formation of a densely structured antler cortex and an intense mineralization of the bone (Muir et al., 1988) as well as the shedding of the specialized type of skin (velvet) present in growing antlers. Casting of hard antlers and onset of antler regrowth occur when testosterone levels are again low after the rut.

The role of sex steroids for the timing of the basic events in the antler cycle was also demonstrated experimentally. Administration of testosterone or estrogens to intact males growing antlers or to castrated antlered males induces growth arrest of the antlers and velvet shedding (Blauel, 1935; Wislocki et al., 1947; Goss, 1968), with estradiol being a more potent growth inhibitor than testosterone (Goss, 1968). More recently, Price et al. (2000) reported that in red deer stags (Cervus elaphus), injection of the estrogen receptor antagonist ICI 182780 during antler growth increased the length of the antler growth period as well as total antler bone mass and area, cortical bone area, and cortical bone density.

Castration of male deer carrying hard antlers as well as the administration of substances interfering with testosterone production or inhibiting both testosterone production and its action at the receptor level causes premature antler casting (Wislocki et al., 1947; Lincoln, 1971; Muir et al., 1982; Jaczewski, 1985; Bubenik GA et al., 1987, 2002; Goss et al., 1992; Schams et al., 1992; Kierdorf U et al., 1993, 1995; Suttie et al., 1995). Administration of testosterone or estradiol to males in hard antler postpones antler casting beyond the normal date (Goss, 1968). When male deer carrying velvet antlers are castrated (Blauel, 1935; Wislocki et al., 1947; Goss, 1968) or treated with the antiandrogen cyproterone acetate (Bubenik GA et al., 1975; Kolle et al., 1993), the velvet is not shed. So far, it is a matter of debate whether or not antler growth itself requires (direct or indirect) stimulation by low levels of androgens (Bubenik GA, 1983, 1990; Schams et al., 1992; Kolle et al., 1993; Suttie et al., 1995; Bartoš et al., 2000; Sadighi et al., 2001).

The antlers of castrated male deer tend to develop conspicuous outgrowths of bone and skin. These structures are most pronounced in the roe deer (Capreolus capreolus) in which the amorphous antlers of castrates are referred to as perukes or wig antlers (von Raesfeld, 1919; Olt, 1921, 1927; Blauel, 1935; Bubenik AB, 1966; Bubenik GA, 1990). For fallow deer (Dama dama), Goss (1983) coined the term “antleromas” for the skin outgrowths developing on antlers of castrates. He later showed that the antleromas were composed of collagen fibers interspersed with scattered fibroblasts, but did not contain bone or cartilage (Goss, 1990). Contrary to fallow deer, intradermal bone has been described in wig antlers of castrated roe bucks (Olt, 1921, 1927) and in the velvet-covered antlers of a presumably hypogonadal male moose (Alces alces) (Bubenik AB et al., 1990).

Because the outgrowths from the antlers of castrated male deer resemble tumorous structures, the question of whether these outgrowths can be regarded as tumors has been repeatedly addressed (Olt, 1927; Blauel, 1935; Goss, 1983, 1990, 1995; Bubenik GA, 1990). To answer this question, a detailed knowledge of antler histology in castrated male deer is required (Goss, 1995). This article reports our findings on antler structure in castrated fallow bucks.

MATERIALS AND METHODS

The study was conducted on four fallow bucks that were approximately 33 months of age at the start of the experiment and carried their second set of antlers. From March 1992 to mid November 1993, the bucks were kept in an outdoor enclosure near Göttingen, Germany (51° 33′ northern latitude). They were then transferred to a heated barn to prevent freezing of their antlers, which had occurred during the winter of 1992/1993, and kept there until the end of the experiment in November 1994. In the barn, the animals were exposed to artificial light given at natural photoperiodic conditions in an on-off mode, i.e., without simulating dawn and dusk. The bucks were fed a commercial feed, hay, and grass (only summer) and had permanent access to water. The distal portions and the brow tines of the bucks' hard antlers had been removed prior to the rutting season of 1991.

Bilateral orchiectomy in the bucks was performed on 25 March 1992, about 4–5 weeks before the normal period of antler casting (late April/early May) by adult fallow bucks from our experimental herd. For surgery, the animals were sedated with a xylazine/ketamine mixture. After castration, the bucks were injected intramuscularly with a long-lasting antibiotic. Wound healing was uneventful in all bucks; for details of the procedure, see Kierdorf U et al. (1995).

Over the study period, the bucks were regularly inspected for antler growth and changes in the macroscopic appearance of their antlers. In December 1993, a tine was cut from the antlers of two of the bucks that had again been sedated with the xylazine/ketamine mixture. The antler pieces were fixed in 3.5% neutral-buffered formalin and subsequently cut into smaller full-diameter cross-sectional samples. Part of the material was decalcified in a saturated neutral-buffered aqueous EDTA solution. The specimens were then thoroughly rinsed in water, dehydrated in an ascending series of ethanol, embedded in paraffin wax, transversely sectioned at 12 μm, and stained with Azan dye. For the production of transverse thin ground sections, undecalcified block samples were dehydrated, embedded in epoxy resin (Biodur), and ground to a thickness of either 50 or 70 μm; for details of the method, see Schultz (1988, 2001).

In November 1994, the bucks were killed by shooting and their antlers removed. Some antlers were macerated to enable macroscopic inspection of the dry bone surface. Samples from the main beams of other antlers were fixed in 3.5% neutral-buffered formalin. Portions from these samples were either decalcified as described above, followed by embedding in paraffin wax, or embedded in methylmetacrylate (MMA) without prior decalcification using the method of Delling (1972). The material was then sectioned transversely or longitudinally at a thickness of 4 μm and stained with either hematoxylin-eosin (H&E; paraffin sections) or toluidine blue (MMA sections).

For comparison, we also studied the histological structure of the hard antlers (grown and cast on time) of an intact adult fallow buck. For that, transverse thin ground sections of 50 and 70 μm thickness were produced from these antlers as described above.

Low-power micrographs of the antler sections were obtained with a Leica MZ 8 stereo microscope (Leitz, Heerbrugg, Switzerland) equipped with an Olympus OM-2 camera using Ilford Pan F film. The negatives were digitized using an Epson filmscan 200 (Nagano, Japan) and imported as positives into Photoshop 7.0 (Adobe, San Jose, CA). For higher magnifications, antler sections were photographed with a photomicroscope (Axioskop 2 Plus; Zeiss, Jena, Germany) equipped with a Canon Powershot G2 digital camera using plain or polarized transmitted light. In the latter case, a hilfsobject red first order (quartz) was used as compensator; for details of the technique, see Schultz (1988, 2001). The acquired digital images were further processed using Adobe Photoshop 7.0 software.

RESULTS

Macroscopic Observations on Antlers of Castrates

Antler casting in the four bucks occurred between 12 and 26 days after castration. First signs of antler regrowth were already observed shortly before the old antlers were cast. The antlers kept elongating until August 1992, i.e., antler elongation ceased at approximately the normal time for the species. The shape of the antlers produced by the castrates was within the normal range for bucks of that age, with presence of brow and tray tines and a distal palm with backward projecting tines (spellers). Contrary to intact males, the velvet was not shed from the antlers. Instead, the antlers of the castrated bucks remained in velvet until the death of the animals. During a cold period in winter 1992/1993, the antlers suffered from frostbite and became partly necrotic. The necrotic antler parts were sequestered between January and March 1993, followed by wound healing at the separation sites; for details, see Kierdorf U et al. (1995).

In late April/early May 1993, limited antler regrowth started from the separation sites. Around the same time, numerous nodular bony protuberances began to develop from the antler surface. With time, these protuberances increased in size and in places got into close contact with each other. This led to a compression of the skin situated between them and to a marked increase in antler diameter (Fig. 1a). Inspection of macerated antlers obtained after the death of the animals revealed that the surface of the protuberances was typically of a coralloid structure. However, protuberances with a smooth surface were also observed rarely (Fig. 1b). The larger protuberances were pedicled, i.e., connected to the underlying bone by a stalk, and thus had an undercut outline (Fig. 2a). In places where no protuberances had developed, the surface of the macerated antlers showed numerous arterial impressions in the form of longitudinal furrows (Fig. 1b).

Figure 1.

Lateral views of antler surfaces of castrated fallow bucks. a: Velvet-covered antler, December 1993. Numerous bony protuberances have formed on the original antler surface (S). Due to their enlargement, many of the protuberances have come into close contact, leading to a marked increase in antler diameter. b: Macerated antler from a castrated fallow buck killed in November 1994. The antler surface shows numerous bony protuberances. Most of these possess a coralloid surface structure; however, one has a smooth surface (arrowhead). Note arterial impressions on the original antler surface.

Figure 2.

Transverse, undecalcified and unstained ground sections (70 μm) of antler tines obtained in December 1993, viewed in plain transmitted light. a: Section showing the antler formed in 1992 and two bony protuberances (P) that in 1993 developed from the antler surface. The outer bone zone (OZ) of the antler consists of tangentially orientated lamellar trabeculae. IZ, inner bone zone; S, skin (velvet); A, artery in the velvet. b: Same specimen as in a, showing two small bony protuberances (arrows) that are underlain by the tangentially orientated lamellar trabeculae of the outer bone zone. c: Bony protuberance connected to the inner bone zone by a stalk. A particularly large blood vessel (arrow) is located in the center of the bone stalk.

With ongoing enlargement of the bony protuberances, skin-lined infoldings reaching deep into the antlers originated as a result of the bone overgrowth (Figs. 2a and 3a). Furthermore, in 1994 the skin began to show prominent signs of local hypertrophy in the form of a thickening and the development of conspicuous outgrowths (Fig. 3a).

Figure 3.

Transverse, undecalcified and unstained ground sections (70 μm) of antler tines obtained in December 1993, viewed in plain transmitted light (b, c) or in polarized transmitted light using a hilfsobject red first order (quartz) as compensator (a, d–f). a: Loose system of tangentially orientated lamellar trabeculae in the outer bone zone (OZ) and cancellous bone in the inner bone zone (IZ). b: Peripheral portion of the inner zone with loosely arranged osseous trabeculae. The histological picture shows an early stage of lamellar bone formation with numerous areas of woven bone (arrowheads) within the trabeculae that have pronounced osteoid seams. c: Central portion of the inner zone, consisting of lamellar bone. Osteons (O) and interstitial lamellae (I) are present. d: Central portion of the inner zone with osteons in different stages of development. Presence of interstitial lamellae indicates remodeling activity. e: Connection (stalk region) between a bony protuberance (P) and the inner bone zone. Note tongues of skin (S) bordering on the bone stalk. f: Bending of the tangentially orientated lamellar trabeculae of the outer bone zone in an area where a large velvet artery (A) has been partly overgrown by bone.

Histological Observations on Antlers of Castrates

The osseous portion of the castrates' velvet antlers consisted of either lamellar or woven bone (Figs. 3–5). No intradermal bone or cartilage formation was observed.

Figure 4.

Transverse decalcified paraffin sections (12 μm) of antler biopsies obtained in December 1993, viewed in plain transmitted light. Azan. The images show details of the bony protuberances formed in 1993. a: Trabeculae of woven bone (B) within a protuberance. D, dermis; E, epidermis; P, periosteum. b: Surface zone of a protuberance with numerous islands of intramembranously forming woven bone. HF, hair follicle; SG, sebaceous gland. c: Higher magnification of the areas of forming woven bone. The bone is covered by active osteoblasts (Ob). Some cells have become incorporated into the newly formed bone as osteocytes (arrowheads). The zone of bone formation is covered by a periosteum consisting of a cambium layer (CL) and a fibrous layer (FL). d: Local resorption of newly formed woven bone by osteoclasts (arrows). Most of the bone surface is covered by active osteoblasts and the bone contains osteocytes (arrowheads).

Figure 5.

Undecalcified MMA (a, c, d) and decalcified paraffin (b) sections (4 μm) through antlers obtained in November 1994. a: Longitudinal section through distal antler zone. A densely structured lamellar inner bone portion (IB) is covered by a cancellous outer bone portion (OB). Note thickening of the skin (S) that in places has formed conspicuous pedicled outgrowths (SO) and tongue of compressed skin reaching deep into the outer bone portion (arrow). Large epidermis-lined cystic skin structures (arrowheads) are present in the outer bone portion. Toluidine blue. b: Longitudinal section through distal antler zone. A densely structured lamellar inner bone portion is covered by cancellous outer bone. Note blood vessel (V), which has become overgrown by the outer bone. H&E. c: Transverse antler section, showing loosely arranged osseous trabeculae in the outer bone portion that is overlain by the periosteum (P). Note hypertrophic dermis (D) showing hair follicles (arrows) with accompanying sebaceous glands (SG). E, epidermis; arrowhead, cystic structure whose lumen is presumably filled with sebum. Toluidine blue. d: Transverse antler section showing the periphery of the outer bone portion. Trabeculae of woven bone are lined by active osteoblasts (Ob) and have wide seams of osteoid (Od). A few cells have become incorporated into the bone as osteocytes (Oc). The presence of osteoclasts (Ocl) and of Howship's lacunae (HL) indicates remodeling activity. Toluidine blue.

Antler biopsies obtained in December 1993.

The osseous antler portion representing the growth of 1992 consisted entirely of cancellous bone. It could structurally be divided into an inner and an outer zone (Figs. 2a and b and 3a). The thin outer bone zone consisted of a few (2–4) tangentially orientated lamellar trabeculae with relatively wide intertrabecular spaces (Figs. 2a and b, 3a and f). In the inner bone zone, the overall thickness of the osseous trabeculae increased from the periphery toward the center (Fig. 2a and b). The more peripheral portion of the inner zone consisted of slender osseous trabeculae composed of a mixture of lamellar and woven bone (Fig. 3a and b). The trabeculae, which had pronounced osteoid seams (Fig. 3b), were apparently in a process of replacement of woven by lamellar bone.

The more central portion of the inner zone consisted entirely of lamellar bone (Fig. 3c and d). Systems of circular special lamellae were present that morphologically corresponded to the Haversian systems (osteons) of compact bone. These two terms will therefore also be used for the circular lamellar systems present in the antlers. The osteons were in different stages of development (Fig. 3c and d). Occurrence of interstitial lamellae, i.e., of remnants of destroyed lamellar systems (Fig. 3c and d), indicated remodeling activity in this bone area, which compared to the peripheral portions consisted of a more mature tissue.

In areas where larger (mushroom-shaped) bony protuberances had developed in 1993, the system of tangentially orientated lamellar trabeculae of the outer bone zone was discontinuous (Figs. 2a and 3e). The stalk regions of the protuberances consisted of slender trabeculae, which sometimes showed a preferential central-peripheral orientation (Figs. 2a and 3e). Except for these stalk region, whose trabeculae consisted of partly lamellar and partly woven bone, the protuberances were composed entirely of woven bone (Fig. 4). The protuberances were in a process of rapid enlargement, as was evidenced by the presence of numerous subperiosteally located islands of woven bone that were lined by plump active osteoblasts and undergoing appositional growth (Fig. 4b and c). The periosteum showed the typical subdivision into an outer fibrous layer and an inner cambium layer (Fig. 4c). Thus, the histological analysis revealed an intramembranous ossification by periosteally derived osteoblasts. The presence of numerous active osteoclasts indicated also marked bone resorption activity in the outer zone of the protuberances (Fig. 4d).

In addition to the large mushroom-shaped protuberances, smaller bony outgrowths were also observed along the antler surface (Fig. 2b). These small exostoses, which apparently represented earlier stages of protuberance development, were also in a process of rapid enlargement by intramembranous bone formation. Contrary to the larger ones, the small protuberances were underlain by the tangentially orientated lamellar trabeculae of the outer bone zone (Fig. 2b).

Larger vascular channels extending from the inner bone zone through the stalk regions into the larger bony protuberances were frequently observed (Fig. 2c). The protuberances themselves were richly vascularized (Fig. 2c). The presence of large velvet arteries, located in the integument between the large protuberances, was associated with deep infoldings of the bone surface. In these areas, the tangentially orientated lamellar trabeculae of the outer bone zone showed a pronounced bending (Figs. 2a and 3f).

The skin (velvet) covering the antler bone consisted of a well-vascularized dermis and a weakly cornified epidermis (Fig. 4a and b). Numerous hair follicles, each with an accompanying sebaceous gland, were present (Fig. 4b). The arteries of the dermis possessed the unusually thick walls and narrow lumina typical of velvet arteries (Figs. 2a and 3f). Tongues of skin, which also extended underneath the undercut surface of the large pedicled protuberances, were present between adjacent bony outgrowths (Figs. 2a and 3e).

Antlers obtained in November 1994.

On histological examination, the antler bone could be divided into two structurally different moieties (Fig. 5a and b). An inner, more densely structured bone portion was presumed to represent the antler growth of 1992 (corresponding to the zones IZ and OZ in Figs. 2 and 3), while the outer bone portion was thought to represent the bony outgrowths developing from the original antler surface in 1993/1994. This view was supported by the observation of several large blood vessels located directly external to the inner bone portion (Fig. 3b). According to our interpretation, these blood vessels, now situated deep within the bone, had originally been located along the antler surface and subsequently became overgrown by the apposition of new bone. Early stages of this process, i.e., blood vessels lying in deep depressions of the bone surface between two protuberances, were already visible in the antler biopsies obtained in December 1993 (Figs. 2a and 3f).

The inner antler portion consisted of rather dense lamellar bone with numerous osteons and relatively narrow vascular spaces (Fig. 5a and b). Remnants of woven bone were not observed in this zone. Compared to the antler biopsies obtained in 1993, the thickness of the osseous trabeculae was markedly increased.

The outer portion of the antlers consisted of cancellous bone (Fig. 5a and b). Its apposition onto the surface of the antlers formed in 1992 (represented by the inner bone portion) had in places led to more than a doubling of the original antler diameter (Fig. 5a). The inner trabeculae of the outer bone portion showed a lamellar structure, however, lacked typical Haversian systems. Toward the periphery, the trabeculae were increasingly composed of woven bone. The peripheral trabeculae of woven bone were densely covered by active osteoblasts and had wide osteoid seams (Fig. 5d). In places, osteoid-free trabecular surfaces were also observed that were either covered by active osteoclasts or revealed the typical scalloped concave outline of Howship's lacunae (Fig. 5d). As in the antler biopsies obtained in 1993, peripheral bone formation in the antlers occurred by subperiosteal intramembranous ossification (Fig. 5c).

The skin covering the antler surface was characterized by an epidermis of normal thickness and a thickened dermis. Numerous hair follicles with accompanying sebaceous glands were present (Fig. 5a and c). Sometimes, apparently sebum-filled cystic structures were observed deeper within the dermis (Fig. 5c). Where the integument had become overgrown and compressed by bone apposition, deep infoldings were present in the outer bone portion (Fig. 5a). In the skin adjoining these infoldings, epidermis-lined cystic structures were frequently observed that were often filled with what appeared to be larger masses of sebum and/or keratinous material (Fig. 5a and c). Supposedly the cysts developed in areas where sebum outflow was blocked and cyst formation was associated with degenerative changes in the sebaceous glands and hair follicles. In places, the skin showed conspicuous pedicled outgrowths, the bulk of which was formed by a hypertrophic dermis (Fig. 5a). No bone or cartilage was encountered in these skin outgrowths.

Histological Observations on Cast Antlers of an Intact Fallow Buck

The antlers of the intact buck possessed a cortical zone of dense osteonal bone. In the outer and central portions of this zone, the osteons were of a rather uniform width and possessed narrow Haversian canals (Fig. 6). Toward the center of the beam, the osteons of the cortical zone gradually increased in volume. The central portion of the antlers consisted of cancellous lamellar bone forming a secondary spongiosa.

Figure 6.

Transverse undecalcified and unstained ground section (50 μm) of hard antler from an intact fallow buck, viewed in plain transmitted light. The image shows the outer (cortical) antler zone, which consists of dense osteonal bone. The osteons (O) are of a rather uniform width and possess narrow Haversian canals.

DISCUSSION

The shape of the antlers produced by the castrated fallow bucks in 1992 was within the normal range for the species, and antler elongation in the castrates ceased at approximately the same time as in intact fallow bucks. Thus, during the first antler growth phase after castration, overall antler morphogenesis and antler elongation were apparently not significantly affected by the withdrawal of testicular sex steroids.

During normal antler growth in intact fallow bucks, the initially formed woven bone of the primary spongiosa is replaced by lamellar bone (Kierdorf H et al., 1995). Mature antlers of fallow deer are characterized by a compact cortical zone of osteonal bone that is built up during the later stages of antler growth (Fig. 6) (Kierdorf U et al., 1993; Rolf and Enderle, 1999). Formation of this compact cortical zone causes a progressive reduction of blood flow from the velvet to the antler core, eventually leading to ischemic necrosis of the velvet (Goss, 1983).

Compared with antlers of intact fallow bucks, the antlers of the castrates showed different signs of immaturity. The antler biopsies obtained from the castrates in December 1993 lacked a cortical zone of dense osteonal bone. Furthermore, the trabeculae in the peripheral areas of the inner antler portion still contained larger amounts of woven bone. Also in the antlers obtained in November 1994, the inner bone zone, which is regarded to represent the antler growth of 1992, showed an abnormal structure. It consisted of a rather dense system of lamellar bone without a clear distinction between a central and a peripheral portion. Compared with the histological structure of the antler biopsies obtained in December 1993, the bone appeared more mature (no woven bone left), indicating that it had undergone further remodeling.

The histological findings of the present study suggest a reduced intensity of bone remodeling in the antlers of the castrates compared to intact bucks. This impaired bone remodeling is hypothetically related to a lack of stimulation by higher levels of sex steroids in the castrates. Similar findings were reported by Bubenik G and Bubenik A (1978) for the antlers of intact white-tailed bucks (Odocoileus virginianus) treated with an antiestrogen (CI-628) during the antler growth period. The absence of a compact cortical antler zone in the castrated fallow bucks permitted a sufficient blood circulation in the antlers to be maintained, thereby allowing survival and further growth of both bone and skin.

The growth of the bony protuberances occurred via intramembranous ossification by osteoblasts derived from the periosteum. Subperiosteal intramembranous ossification is a regular feature also during normal antler growth. In this way, a sleeve of bone is laid down at the periphery of the growing antlers (Banks, 1974; Banks and Newbrey, 1983; Kierdorf H et al., 1995). The periosteally formed bone sleeve increases the mechanical stability of the developing antler and is responsible for its limited latitudinal growth. In contrast, antler elongation proceeds by the formation of a well-vascularized cartilage, which is subsequently replaced by bone. This process of bone development has been characterized as a modified form of endochondral ossification (Gruber, 1937; Banks and Newbrey, 1983; Li and Suttie, 1994; Kierdorf H et al., 1995; Price et al., 1996). Also, elsewhere in the mammalian skeleton, the ossification process of cartilage replacement bones regularly involves the formation of a sleeve of bone at the periphery of the cartilage by periosteal activity (Hall, 1978; Banks, 1981). This type of intramembranous bone formation is often referred to as perichondral ossification (Schultz, 2001), while Banks (1981) proposed the term “epichondral ossification” for the process.

Why periosteal bone apposition onto the original antler surface was limited to certain loci, causing the formation of pedicled protuberances, remains unclear. The deposition of extra bone did not initially lead to structural changes in the underlying bone, as was indicated by the observation of small protuberances underlain by tangentially orientated lamellar trabeculae. With ongoing expansion of the bony protuberances, the underlying bone was, however, restructured. Thus, the stalk regions connecting the central antler portions with the larger protuberances consisted of slender osseous trabeculae, which sometimes showed a preferential central-peripheral orientation. Furthermore, the stalks frequently possessed wide vascular channels. It is supposed that the main driving force behind these morphological alterations was the necessity to establish a sufficient vascular connection between the pre-existing bone and the enlarging protuberances.

While the bony protuberances in the antler biopsies from 1993 consisted entirely of woven bone, replacement of woven by lamellar bone had taken place in the more central portion of the outer bone zone of the antlers obtained in 1994. Due to lateral growth of the protuberances, locally a thick layer of bone was laid down external to the original bone surface, leading to a marked increase in antler diameter. As a consequence of this bone apposition, blood vessels originally situated along the antler surface became overgrown and located deep within the newly formed bone.

Formation of bony protuberances on the antlers of castrated male deer has been described in different deer species. In the 19th century, Owen (1868: p. 631) already noted that in castrated fallow bucks, the antlers “become thickened by irregular tuberculate masses of bone.” A sometimes massive overgrowth of exostoses in antlers of castrated or hypogonadal bucks is also observed in roe deer and white-tailed deer (von Raesfeld, 1919; Bubenik AB, 1966; Bubenik GA, 1990). However, the formation of these protuberances has apparently not been studied so far. We show that in fallow deer they develop by intramembranous ossification from the periosteum. Bubenik AB et al. (1990) described the subperiosteal formation of cartilaginous nodules in the velvet-covered antlers of a presumably hypogonadal male moose. According to these authors, the underlying nodules, which were originally not connected to the bone, later underwent ossification and fused to the bone surface. This endochondral mode of bone formation would differ markedly from the development of the bony protuberances in the antlers of the castrated fallow bucks.

The osseous trabeculae in the antlers of the castrated fallow bucks had wide osteoid seams, which is suggestive of an impaired mineralization process. There exists considerable evidence supporting the hypothesis that the terminal phase of antler mineralization is dependent on higher levels of sex steroids (Bubenik G and Bubenik A, 1978; Morris and Bubenik, 1983; Muir et al., 1988). Therefore, as in the case of the structural abnormalities of the bone, the impairment of antler mineralization observed in the present study can also be associated with the lack of gonadal sex steroids in the castrates.

The antlers of the castrated fallow bucks also produced conspicuous skin outgrowths. Their formation started, however, later than that of the bony protuberances. While the skin outgrowths began to form in 1994, growth of the bony protuberances commenced already in 1993. Our findings closely parallel earlier observations by Goss (1990), who also studied antler growth in castrated fallow bucks. According to him, the amorphous skin outgrowths from the antlers, which he termed “antleromas,” developed at the earliest during the second year after castration. This was also the case in the present study. Goss (1990) further describes that with time some of the antleromas became pendulous on elongating stalks of skin. Early stages of this process were seen in our study. Internally, the well-vascularized antleromas consisted of copious quantities of collagen fibers interspersed with scattered fibroblasts (Goss, 1990). Neither Goss (1990) nor we observed bone or cartilage in the skin outgrowths of the castrated fallow bucks. Based on his findings, Goss (1990, 1995) concluded that the antleromas were most likely of dermal origin. This view is supported by our histological findings.

In contrast to the observations in castrated fallow bucks, intradermal bone has been reported from wig antlers of roe deer (Olt, 1921, 1927) and from the antlers of a presumably hypogonadal male moose (Bubenik AB et al., 1990). This discrepancy suggests differences among deer species in the reaction pattern of the antler tissues to the withdrawal of sex steroids.

In his study, Goss (1990) does not describe the occurrence of epidermal cysts, which we found in the antler integument of the castrates. Such structures were, however, observed in the moose antlers studied by Bubenik AB et al. (1990). These authors likewise linked the formation of the cysts to a blocking of sebum outflow and subsequent degenerative changes of the sebaceous glands. Goss (1990) also does not mention the formation of bony protuberances in the antlers of the castrated fallow bucks studied by him. Since, according to our observations, the growth of the bony protuberances commences before the skin outgrowths start to develop, this is unlikely a matter of not enough time having elapsed after castration in the bucks studied by Goss (1990).

In conclusion, our study showed that the antlers of castrated fallow bucks develop abnormal bone and skin outgrowths. The bony protuberances were formed by intramembranous ossification from the periosteum, an osteogenetic process that is also part of normal antler growth in intact bucks. The development of the exostoses can therefore be characterized as a case of hypertrophic bone growth. The osseous component of the antlers in the castrates showed histological signs of immaturity, pointing to a reduced remodeling activity and an impairment of the mineralization process. With respect to integumental changes, the findings of the present study support the view of Goss (1990, 1995) that the conspicuous skin outgrowths (antleromas) developing in antlers of castrated fallow bucks result from hypertrophy of the dermal component of the velvet. Our study revealed a normal topographic relationship between the major tissue components of the antlers of the castrates. Furthermore, no indication of invasive/destructive bone growth, i.e., of penetration of the newly formed bone tissue into the pre-existing bone, was encountered. Heterotopic bone or cartilage formation was not observed in the skin of the antlers, and the replacement of woven by lamellar bone in the antlers took place in an apparently normal mode, but at a reduced rate.

In the mammalian skeleton, development of exostoses due to deposition of new bone by the periosteum is observed in a variety of skeletal disorders, including primary and secondary hypertrophic osteoarthropathy and osteomas (Resnick and Niwayama 1995; Adler, 1998). In primary hypertrophic osteoarthropathy (pachydermoperiostosis), the hyperostosis mostly goes along with pachydermia, and periosteal bone deposition affects tubular bones as well as axial skeletal sites (Resnick and Niwayama, 1995). Secondary hypertrophic osteoarthropathy typically develops as a sequela of a disease of the respiratory tract, such as bronchiogenic carcinoma (Resnick and Niwayama, 1995).

Osteomas, which are usually classified as benign bone tumors occurring in membrane bones, are also formed by periosteal activity (Resnick and Niwayama, 1995; Adler, 1998). An osteoma can be composed of either dense bone tissue with typical Haversian systems (osteoma eburneum) or spongious lamellar bone (osteoma spongiosum). The bone tissue in mature osteomas does not differ in cellular or histological structure from normal bone tissue (Adler, 1998), and Resnick and Niwayama (1995: p. 4400) state that an osteoma “represents no more than a localized exaggeration of intramembranous bone formation.”

Both the skin and the bone outgrowths developing on the antlers of castrated male deer constitute tumor-like structures. According to Goss (1990), the antleromas of castrated fallow bucks may be classified as benign tumors. Applying a general definition of the term “tumor,” e.g., an abnormal mass of tissue resulting from excessive mitotic activity, this view seems justified. In principle, the bony protuberances on the antlers of the castrates may also be classified as a type of benign tumor, especially when comparing their histogenesis with that of osteomas.

As was emphasized by Goss (1990, 1995), it is unclear if the growth of the antler protuberances in castrated male deer is a specific reaction to castration and the resulting pronounced lowering of androgen levels. It could also be that the indefinite maintenance of viable antlers (in the absence of higher levels of androgens) merely provides the extra time required for the development of the skin and bone protuberances. Goss (1995) further argues that in intact male deer, the short life span of the antlers precludes the development of antler tumors. This is, however, not generally true, as is illustrated by the case of a large bone tumor diagnosed as a solitary osteochondroma that had formed on a spike antler of an intact young fallow buck (Kierdorf H and Kierdorf U, 1985). Velvet was shed from both spike antlers at the regular time, which is indicative of a normal rise of androgen levels in the buck.

Information on the histological structure of the antlers in castrated males of deer species other than Dama dama is scarce. But even these limited data point to the existence of considerable variation among species, thereby precluding a generalization of the findings in the fallow deer. Further studies on antler histology of castrated males from various deer species addressing these species-specific differences are encouraged.

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