Cryptorchism, Hypogonadism and Antler Malformations in Black-Tailed Deer (Odocoileus Hemionus Sitkensis) of Kodiak Island1

I. ABHANDLUNGEN

University of Guelph

Cryptorchism, hypogonadism and antler malformations in black-tailed deer (Odocoileus hemionus sitkensis) of Kodiak island1

By G. A. BUBENIK, Guelph, J. P. JACOBSON, Kodiak, D. SCHAMS, Freising, and L. BARTOSˇ, Prague

1 Introduction Since the early nineteen nineties, an ever-increasing number of antler abnormalities were observed among males of Sitka black-tailed deer (Odocoileus hemionus sitkensis) of Kodiak Island, (Alaska, USA). These antler malformations included incomplete growth of points, serious distortion of antler form (asymmetric antlers), incomplete polishing of velvet, and extremely sharp antler points. Furthermore, some incompletely mineralized antlers were cast above the coronet exhibiting conspicuously concave seals (Fig. 2). Such casting and other malformations such as diagonal sequestration of antlers or „cauliflower-like“ antlers (Fig. 3), was associated with an excessive growth of the antler base which partly resembles the „helmet-perukes”. These wig-like structures are formed in castrates or hypogonadic deer by the proliferation of antler bone and velvet tissues without the regular mineraliza-

Fig. 1. Left side of a Sitka black-tailed deer mandible showing severe periodontal disease as indicated by deep periodontal pockets (arrow). Photos 1–9: J. JACOBSON

1 Eingesetzt wurde ein Druckkostenzuschuss des Hessischen Ministeriums für Umwelt, Landwirt- schaft und Forsten, für dessen Gewährung verbindlich gedankt wird. – Die Schriftleitung

U.S. Copyright Clearance Center Code Statement: 0044–2887/2001/4704–0241 $15.00/0 242 G. A. Bubenik, J. P. Jacobson, D. Schams, L. Bartoˇs

Fig. 2. Head and antlers (which dropped off when the deer was shot) of a bilateral cryptorchid. Note the severely concave seal (arrow) and remnants of old antlers. The presence of coronets and the remaining antler tissue can result in the for- mation of “helmet-peruke” – like antlers grown in the next antler cycle (see Fig. 4)

Fig. 3. Skull of the bilateral cryptorchid deer with the “cauliflower” antlers Cryptorchism, hypogonadism and antler malformations in black-tailed deer of Kodiak island 243 tion and casting (BUBENIK, A., 1966; HARTWIG, 1981). In addition, supernumerary pedicles with incomplete antlers and a severe periodontal disease were observed. Many of these and other physiological parameters (such as the lack of swollen neck during the rut and the female type of fat reserves) were indicative of a moderate or severe hypogonadism. An ade- quate seasonal concentration of male sex hormones (androgens) is essential for the normal development of deer antlers (BUBENIK, G., 1990). The lack of sex hormones could have also contributed to the fight-induced separation of frontal bones, a feature observed in several bucks. The suspicion of hypogonadism was strengthened when the culled bucks with malformed antlers were more closely examined. Many of these animals were lacking one or both testes in the scrotum and therefore a unilateral or bilateral cryptorchism was suspected. In order to elucidate the causes of the suspected hypogonadism, samples of blood and testes were taken from affected and control bucks. Serum concentrations of Luteinizing Hormone (LH) and testosterone (T) were determined by radioimmunoassay and the spermatogenic activity was investigated in histological section of the gonads. An extensive photographic documentation of antler and skull malformations was prepared and catalogued. We tested the hypothesis that serum concentrations of testosterone will be lower and LH concentrations higher in bilateral cryptorchids as compared to intact deer.

2 Materials and Methods

Blood and testes samples: Five to ten ml of blood was taken from the abdominal cavity or from the heart of bucks shot during the rut by a rifle. The samples were kept in a cool place until the serum separated from the coagulum. Then the serum samples were frozen at – 20 °C. Between October 23 and November 11 (the peak period of seasonal T concentrations in blood), blood samples were taken from nine bilateral cryptorchid bucks, two unilateral cryptorchid and nine control bucks. Testicular samples were taken from two intact and one unilaterally cryptorchid bucks. Hormone assays: Concentrations of LH were determined by a homologous bovine radioimmunoassay (RIA) with no cross reactivity to other pituitary hormones (SCHAMS et al., 1980). The reference preparation was bovine pituitary preparation (LH-DSA; biological activity 1.0 I.U. of NIH-bLH-S1). The sensitivity was 0.2 ng/ml; intra-assay coefficient of variation (CV) averaged 7.4%, and inter-assay CV was 9.2–13.8%. The assay was validat- ed for white-tailed deer (Odocoileus virginianus) by recovery studies. Testosterone was measured by a highly sensitive enzyme immunoassay after extraction with tertiary butylmethylether/petrolether 30/70 (V/V) (KARG et al., 1976). The sensitivity was 50 pg/ml. The inter-assay CV was 7.8–13.8%. Statistic: Since our data did not show a normal distribution, we applied a non-parametric Mann-Whitney test (Wilcoxon two sample tests, SAS). We calculated exact p-values because the asymptotic assumptions could not be met with our sample size. Standard asymptotic methods involve the assumption that the test statistic follows a particular distribution when the sample size is sufficiently large. When the sample size is not large, (such is our case) asymptotic results may not be valid, with the asymptotic p-values differing from the exact p-values. Histology: Cross-section of testes (around 10 mm thick) were taken shortly after death, fixed in buffered formaldehyde and later embedded in paraffin. The 10-micron sections were stained with Hematoxylin and Eosin and then observed in the optical microscope. The progress of spermatogenesis, the size of spermatogenic tubules and the proportion of interstitium and Leydig cells to tubules were compared. 244 G. A. Bubenik, J. P. Jacobson, D. Schams, L. Bartoˇs

Photographic documentation: Skulls and malformed antlers of intact and cryptorchid bucks were collected since 1994. The shape of antlers, number of points, presence of velvet in mature antlers, shape of the casting surface (the “seal”), coronet malformations, irregular casting lines, presence of periodontal disease and separation of nasal, frontal parietal skull bones were registered. Autopsies: Autopsies were performed on a number of bucks lacking external gonads, search- ing for the ectopic testes in the abdominal cavity or the inguinal canal.

3 Results

Periodontal disease, characterized by deep periodontal pockets, which is rare in other cervids of prime age, was present in about 35% of all cryptorchid bucks (Fig. 1). Moderate to severe antler malformations and the impairment of the antler cycle were indicative of hypogo-

Fig. 4. Antlers of a bilateral cryptorchid. Note the extremely thick antler base (arrow) and the very sharp antler tips. The antlers were still covered in velvet, when the buck was shot during the rut. The velvet was subsequently lost during the preparation of the skull and antlers Cryptorchism, hypogonadism and antler malformations in black-tailed deer of Kodiak island 245

Fig. 5. Skull and antlers of a bilateral cryptorchid buck. Except for the sharply pointed antler tips (arrow), the antlers appear normal nadism. Numerous signs of altered antlerogenesis were observed in some cryptorchid deer; these included: premature, concave (castration-like) separation of the antler beam from the pedicles (Fig. 2), (BUBENIK, A., 1990), osteofibroma-like proliferation of the suprapedicu- lar tissue, sometime called “cauliflower antlers” (Fig. 3) (BUBENIK, A., 1966), non-polished antlers with extremely sharp points and remarkably thick antler base reminding on “helmet-peruke” antlers (Fig. 4) (BUBENIK, A., 1966; HARTWIG, 1981). In addition, some antlers of bilaterally cryptorchid deer exhibited very sharp points, but otherwise looked normal (Fig. 5). Finally, several bilateral cryptorchid bucks had completely normal antlers (Fig. 6). Two bucks with unilateral cryptorchism also had normally looking antlers (Fig. 7). Despite a repeated search for intraabdominal or inguinal testes, none were found. The reason for the failure may be their small size and an increased amount of fat found in the abdominal cavity of hypogonadic deer. Several cryptorchid deer exhibited various degrees of impairment of sutures of nasal, frontal and parietal bones. These ranged from a complete separation (Fig. 8) to a visible re- fusion after separation (Fig. 9). 246 G. A. Bubenik, J. P. Jacobson, D. Schams, L. Bartoˇs

Fig. 6. A bilateral cryptorchid black-tailed buck. The antlers appear completely normal

Fig. 7. Two unilateral cryptorchids. Their antlers also appear normal Cryptorchism, hypogonadism and antler malformations in black-tailed deer of Kodiak island 247

Fig. 8. Frontal view of the skull of Sitka deer with the separated sutures of nasal, frontal and parietal bones. Note the almost complete closure of the wound by newly grown bony tissue, especially at the caudal (top) end of the separation gap

Because only two unilateral cryptorchids were found in our sample, we decided to compare only nine bilateral cryptorchids with eight control deer. As serum LH (15.7 and 6.6. ng/ml) and T (2.9 and 0.7 ng/ml) concentrations in unilateral cryptorchids, as well as antler forms were in the range of control bucks, we con- cluded that the unilateral cryptorchids were not hypogonadic and therefore they were exclud- ed from both groups. Histological analysis of testes found no difference between the intensity of spermatogenesis or in the size of interstitium or Leydig cells of controls and the unilateral cryptorchid.

Fig. 9. Skull of Sitka deer with an apparent re-fusion of the interfrontal suture (arrow) 248 G. A. Bubenik, J. P. Jacobson, D. Schams, L. Bartoˇs

Intact bucks Bilateral cryptorchids

3.00 25 2.70 2.40 20 2.10 1.80 15

ng/ml 1.50 1.20 10 0.90 0.60 5 0.30 0.00 0 LH testosterone Fig. 10. LH and testosterone concentrations (median, -lower quantile, +upper quantile) in serum of intact bucks and bilateral cryptorchids of Sitka black-tailed deer of Kodiak Island

Median serum levels of LH in bilateral cryptorchids (Fig. 10) were significantly higher (Mann-Whitney test, z = – 1.59, one side P = 0.05) than concentrations in intact deer. Con- versely, median T concentrations in intact bucks (Fig. 10) were almost ten times higher than T levels in bilateral cryptorchids (Mann-Whitney test, z = – 1.78, one sided P = 0.037). These results indicated hypogonadism and, as expected, a significant statistical difference was found between controls and deer with bilateral cryptorchism. Although about half of the bilaterally cryptorchid bucks exhibited some kind of antler malformation, there was no direct relationship between the severity of hypogonadic symptoms and the concentrations of LH or testosterone in the serum of the affected deer.

4 Discussion

Testicular atrophy as a cause of hypogonadism was reported in red deer (CARRASCO et al., 1997), mule deer (TILLER et al., 1997) and white-tailed deer (TAYLOR et al., 1964; MAR- BURGER et al., 1967; THOMAS et al., 1970). Cryptorchid deer have been also reported in elk (wapiti) (HUDSON, 1950), roe deer (EISSNER and REICHART, 1970), reindeer (LEADER- WILLIAMS, 1979) and sika deer (CHAPMAN et al., 1984). Although histological investiga- tions of affected gonads were performed in most studies, in only two papers testosterone (T) concentrations in blood were measured. In reindeer, T levels of male carrying two abdominal testes were much lower than of those having one ectopic and one scrotal testis. Surprisingly, although there was a slight delay in the antler cycle of unilateral cryptorchids, Cryptorchism, hypogonadism and antler malformations in black-tailed deer of Kodiak island 249 there was no delay in timing of the antler cycle in bilateral cryptorchids. However, the antlers of all cryptorchids were smaller than antlers of controls (LEADER-WILLIAMS, 1979). As expected, in mule deer with atrophic testes, T levels were significantly lower and gonadotropins were higher in affected bucks (TILLER et al., 1997). In most hypogonadic or cryptorchid cervids, the antlerogenesis was also significantly affected. In a bilateral elk cryptorchid no antler growth was observed (HUDSON, 1950) and a poorly calcified antlers with an incomplete velvet polishing were reported in a cryptorchid roe buck (EISSNER and REICHART, 1970). White-tailed deer with a severe hypogonadism exhibited asymmetric, poorly mineralized antlers retaining dry velvet (TAYLOR et al., 1964; MARBURGER et al., 1967). The causes of hypogonadism (which in turn may have caused the cryptorchism) are basi- cally unknown but several possible explanations exist: 1) Dietary components: A) A lack of certain minerals or essential elements may influence gonadal development (TAYLOR et al., 1964; ROBINSON et al., 1967). B) Phytoestrogens, produced in leguminous plants, such as Astragalus spp. and Meliotus spp. may also affect testicular development in deer (SALONIE- MI et al., 1995). C) Aflatoxins, produced naturally by some fungal species are known to affect early stages of spermatogenesis (FENSKE and FINK-GREMMELS, 1990). D) Organo- phosphates, such as DDT or PCB may act as xeno-estrogens, thus affecting gonadal dif- ferentiation and causing castration effect (ARNOLD et al., 1996). 2) Genetics: That some of these aberrations in gonadal development occur in only certain localized areas may indicate a genetic defect. In domestic animals testicular hypoplasia has been related to chromosomal alterations, such as XXY and XX/XY syndrome (BRUERE et al., 1969; BONGSO et al., 1981). In addition to chromosomal aberration, partial androgen receptor insensitiv- ity may also be the cause of suspected cryptorchism. Periodontal disease (Fig. 1), characterized by deep periodontal pockets may be con- nected to hypogonadism. The low concentrations of androgens impaired the immune sys- tem in Odocoileinae, such as white-tailed deer, resulting in infections and a premature death (BUBENIK, G., 1989). In several black-tailed bucks we observed a complete separation of frontal bones along the interfrontal suture. In one particular case the disunion was accompanied by a partial separation of frontoparietal and frontonasal sutures (Fig. 8). Similar separation was also observed in some roe deer and red deer males in Germany by U. HERBST (personal com- munication to J. JACOBSON). It has been speculated that the separation occurred as a result of the fight. The injured buck “locked in” antlers with another buck. When trying to free himself, he separated the incompletely fused skull suture. The injury seem to have occurred quite some time before the death of the buck. A substantial amount of bone tissue was formed between the frontal bones. The newly formed bone almost closed the space, especially at the caudal (top) end of the separation gap. In another case, a complete re-union of frontal bones was observed (Fig. 9). As the mineralization of bones and cartilages depends on the blood concentrations of androgens (WILLIAMS, 1974; BUBENIK, G., 1982), hypogonadism may contribute to the lack of cohesion of skull sutures, which are completely fused in bucks of prime age. Conversely, androgen concentrations in blood of the injured deer were sufficiently high to induce his participation in the rut. Testosterone concentrations were more than ten times higher in intact or unilateral cryptorchids than in the bilateral cryptorchids (Fig. 10), and statistically significant differences were detected between both groups (P = 0.037). Conversely, as expected, median LH concentrations were significantly higher in bilateral cryptorchids than in intact deer (P = 0.05). This compensatory increase of gonadotropins concentrations is usually observed in hypogonadic or post-prime bucks (BUBENIK, G. and SCHAMS, 1986; TILLER et al., 1997) as a result of a feedback response to the declining levels of androgens. Surprisingly, we found sever- 250 G. A. Bubenik, J. P. Jacobson, D. Schams, L. Bartoˇs al high concentrations of T in the serum of a few bilateral cryptorchids. These high T levels might have originated in the ectopic testes because the adrenal production of T in Odocoileus is only minimal (BUBENIK, G., 1982). Our findings may indicate that unde- scended testes of Odocoileus can produce a significant amount of T during the rut, which prevents a compensatory increase of LH. In addition it also appears that the severity of antler and skull malformation is not directly related to circulating levels of androgens. Conclusion: An incomplete hypogonadism, manifested in unilateral or bilateral cryptorchism and „castrate-like“ antler malformation has been studied. The normal levels of serum T found in some bilateral cryptorchids, the apparent lack of hypogonadism in unilateral cryptorchids and a typical “castrate-like” antler malformations observed only in about half of bilateral cryptorchids, presents an interesting scientific puzzle. In addition, the possibility of environmental contamination or genetic defects warrants a detailed scientific investigation. The Sitka deer is an important subsistence and personal use animal on the Kodiak Island. In addition, tourism is a major source of income in Alaska and the health of game animals is essential for the maintenance of the hunting industry. Finally the possibility of environmental contamination, even in the high Arctic regions, is gaining increasing attention because of its impact on human health. In that case, deer may be the prover- bial „canaries in the coal mine“ indicating the health of the environment (KIERDORF and KIERDORF, 2000).

Acknowledgments

We would like to thank E. STIRRUP and I. SMITH for the preparation of digital photographs, LORI FERGUSON for the clerical help and G. LODGE for the editorial comments. This study was partially supported by a grant from the Czech Grant Agency (524/00/0976) provided to L. BARTOSˇ.

Summary

Distinct antler malformations, (such as lack of points, distortion of antler forms, incomplete velvet shedding, casting above the coronet and a diagonal separation of cast antlers), were detected in Sitka deer (Odocoileus hemionus sitkensis) of the Kodiak Island (Alaska, USA). Many of these deer were also unilateral or bilateral cryptorchids and a few exhibited a moderate or severe separation of frontal skull bones. Radioimmunoassay analyses of serum revealed signs of hypogonadism: we found higher concentrations of LH (P = 0.05) and lower concentrations of T (P = 0.037) in bilateral cryptorchids as compared to intact bucks. There was no apparent impairment of spermatogenesis in the remaining testis of the unilateral cryptorchid deer. Unfortunately, no ectopic testes were so far recovered in cryptorchid deer. Surprisingly, only about half of deer with bilateral cryptorchism exhibited hypogonadic symptoms. In the other half of cryptorchid deer, the antlers appeared completely normal and the concentrations of LH or testosterone in the serum of several bucks were in the range of intact deer. It has been postulated that in some cryptorchid deer, ectopic testes can produce sufficient amount of testosterone, which prevents obvious antler and skull malformations, as well as reduces the compensatory elevation of LH. The possible causes of the hypogonadism resulting in cryptorchism and antler malformations are discussed.

Key words: Black-tailed deer, cryptorchism, hypogonadism, antler malformation, testosterone, LH, Kodiak Island Cryptorchism, hypogonadism and antler malformations in black-tailed deer of Kodiak island 251

Zusammenfassung Kryptorchismus, Hypogonadismus und Geweihmissbildungen bei Schwarzwedelhirschen (Odocoileus virginianus sitkensis) der Kodiak-Insel Bei Schwarzwedelhirschen (Odocoileus hemionus sitkensis) der Kodiak-Insel (Alaska, USA) wurden charakteristische Geweihmissbildungen (wie Endenarmut, abnorme Geweihform, unvollständiges Fe- gen, Abwerfen oberhalb der Rose, schräg verlaufende Abwurfflächen) beobachtet. Bei vielen dieser Hirschen wurde ein- oder beidseitiger Kryptorchismus festgestellt. Der Schädel einzelner Tiere wies zudem eine Zusammenhangstrennung der Ossa frontalia auf. Serum-Radioimmunoassays erbrachten Hinweise auf Hypogonadismus bei den Individuen. So wiesen beidseitig kryptorche Tiere höhere LH- Konzentrationen (P = 0,05) und niedrigere Testosteron-Konzentrationen (P = 0,037) auf als intakte Hirsche. Die Spermatogenese im skrotalen Hoden einseitig kryptorcher Hirsche war nicht erkennbar gestört. Die heterotopen Hoden kryptorcher Hirsche konnten bislang nicht entdeckt werden. Über- raschenderweise fanden sich nur bei etwa der Hälfte der Hirsche mit bilateralem Kryptorchismus Sym- ptome von Hypogonadismus. Die andere Hälfte besaß normale Geweihe, und die LH- und Testo- steronkonzentrationen vieler dieser Hirsche lagen im gleichen Bereich wie bei intakten Tieren. Ver- mutlich bilden bei einigen der kryptorchen Hirsche die heterotopen Hoden genügend Testosteron, um die Entstehung von Geweih- und Schädelmissbildungen sowie eine kompensatorische Erhöhung des LH-Spiegels zu verhindern. Die möglichen Ursachen des zu Kryptorchismus und Geweihmiss- bildungen führenden Hypogonadismus werden diskutiert. Schlüsselwörter: Schwarzwedelhirsch, Kryptorchismus, Hypogonadismus, Geweihmissbildungen, Testosteron, LH, Kodiak-Insel

Résumé Cryptorchidie, hypogonadisme et malformations des bois chez le Chevreuil à queue noire (Odocoileus hemionus sitkensis) dans l’Ile de Kodiak Des malformations caractérisées (telles que cors absents, bois déformés, dépouillement incomplet du velours, scission des bois au-dessus de la meule et plan de séparation diagonal des mues) ont été détec- tées chez le Chevreuil de Sitka (Odocoileus hemionus sitkensis) de l’Ile de Kodiak (Alaska, USA). Plu- sieurs de ces chevreuils manifestaient aussi de la cryptorchidie unilatérale ou bilatérale tandis que quelques autres présentaient une fragmentation plus ou moins sévère des os frontaux du crâne. Des examens radio-immunitaires de sérum révélèrent des signes d’hypogonadisme: nous avons ainsi trou- vé des concentrations plus fortes de LH (P = 0.05) et de plus faibles concentrations de T (P = 0.037) chez des cryptorchides bilatéraux comparés à des mâles indemnes. On n’a pas constaté de spermato- genèse réduite dans le testicule subsistant chez le chevreuil cryptorchide unilatéral. Malheureusement, jusqu’à présent, la récupération de testicules ectopiques n’est pas acquise chez des sujets cryptorchides. Curieusement, à peine la moitié des chevreuils avec une cryptorchidie bilatérale exhibait des symp- tômes hypogonadiques. Chez l’autre moitié, les bois apparaissaient tout à fait normaux et les concentrations de LH ou de testotérone dans le sérum de plusieurs broquarts atteignaient le niveau de chevreuils indemnes. On a postulé que, chez certains sujets cryptorchides, les testicules ectopiques peu- vent produire suffisamment de testotérone, laquelle prévient de toute évidence les malformations des bois et du crâne, tout en réduisant l’élévation du taux de LH. Les causes possibles de l’hypogonadisme donnant lieu à de la cryptorchidie et à des malformatons des bois font l’objet d’une discussion. Mots clefs: Chevreuil à queue noire, cryptorchidie, hypogonadisme, malfomation des bois, testosté- rone, hormone lutéinisante, Ile de Kodiak Trad.: S. A. DE CROMBRUGGHE

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Addresses of authors: Dr. G. A. BUBENIK, Department of Zoology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1, email: [email protected], Dr. J. P. JACOBSON, P.O. Box 1313, Kodiak, AK 99615, USA, Dr. D. Schams, Department of Phy- siology, Technical University Munich-Weihenstephan, D-85354 Freising, Ger- many, Dr. L. BARTOSˇ, Research Institute of Animal Production, CZ 104 00 Pra- gue-Uhrineves, Czech Republic.