Quality of Trophies in Game Ruminants: from the Study of Mechanical and Structural Properties, to the Mineral Composition and Characterization of the Trophy

UNIVERSIDAD DE CASTILLA-LA MANCHA

Escuela Técnica Superior de Ingenieros Agrónomos y de Montes Departamento de Ciencia y Tecnología Agroforestal y Genética

Quality of trophies in game ruminants: from the study of mechanical and structural properties, to the mineral composition and characterization of the trophy.

TESIS DOCTORAL

Jamil Cappelli Albacete, 2019

Departamento de Ciencia y Tecnología Agroforestal y Genética Universidad de Castilla-La Mancha

TESIS DOCTORAL

Quality of trophies in game ruminants: from the study of mechanical and structural properties, to the mineral composition and characterization of the trophy.

Memoria presentada por Jamil Cappelli para optar al Grado de Doctor en Ciencias Agrarias y Ambientales.

El Doctorando

______Jamil Cappelli

En Albacete, 2019

D. Andrés José García Díaz, D. Tomás Landete Castillejos y D. Laureano Gallego Martínez, Catedráticos de Universidad pertenecientes al Departamento de Ciencia y Tecnología Agroforestal y Genética, de la Escuela Técnica Superior de Ingenieros Agrónomos y de Montes de la Universidad de Castilla-La Mancha.

INFORMAN Que la presente memoria, titulada “Quality of trophies in game ruminants: from the study of mechanical and structural properties, to the mineral composition and characterization of the trophy”, de la que es autor el Ingeniero de montes Don Jamil Cappelli, con un máster en Ciencias y gestión de la fauna silvestre y recursos ambientales, ha sido realizada bajo nuestra dirección y cumple las condiciones exigidas para optar al grado de Doctor por la Universidad de Castilla- La Mancha (Programa de Doctorado en Ciencias Agrarias y Ambientales impartido por el Departamento de Ciencia y Tecnología Agroforestal y Genética, de la Escuela Técnica Superior de Ingenieros Agrónomos y de Montes de Albacete).

Fdo:

D. Andrés José García Díaz

Fdo:

D. Tomás Landete Castillejos

Fdo:

D. Laureano Gallego Martínez

Esta tesis ha sido posible gracias al proyecto cofinanciado por el Fondo Social Europeo para la formación predoctoral UCLM (ref.: 2015/4062) y al proyecto nacional financiado por el MINECO titulado “Factores que afectan a la rotura de las cuernas en España y en Europa: de la composición mineral al manejo” (ref.: AGL201238898). Durante la realización de esta tesis disfruté de las ayudas de FPI de la UCLM y ERASMUS+ para hacer varios periodos de estancia que suman más de 3 meses fuera de España en centro de investigación y universidades extranjeras; estas estancias en una institución de enseñanza superior o centro de investigación extranjero son un requisito necesario para la acreditación del doctorado internacional.

This thesis was made due to the project co-financed by the European Social Fund for UCLM pre-doctoral training (ref.: 2015/4062) and the national project funded by MINECO entitled "Factors affecting the breaking of the antlers in Spain and in Europe: from mineral composition to management "(ref.: AGL201238898). During the realization of this thesis I enjoyed an FPI grant from the UCLM and ERASMUS+ grant to make several periods of stay that total more than 3 months outside of Spain in foreign research center and universities; these stays in an institution of foreign higher education or research institution belong to the conditions necessary for the accreditation of the international doctorate (international PhD).

Acknowledgment Acknowledgment

En primer lugar, quisiera agradecer mis directores de tesis. Agradezco al Dr. Andrés José García Díaz, por guiarme y asesorarme de la mejor manera sobre mis actividades y por ayudarme con sus consejos. Gracias al Dr. Tomas Landete- Castillejos por sus ideas y dedicación invertido en mi formación, siempre con una perspectiva dirigida a nuevos desafíos. Y finalmente, gracias a Laureano Gallego, un pilar importante para el grupo de investigación y por su insustituible guía.

Gracias a todos los otros miembros del grupo de investigación, que me han acompañado en el pasado y que aun siguen trabajando duro en nuevos proyectos de investigación: Pablo Gambín, María López Quintanilla, Javier Pérez-Barbería, Martina Pérez Serrano, Francisco Ceacero. No quisiera olvidar tampoco José Maria, y todos los estudiantes que nos han ayudado con sus practicas en las tareas de campo y en los trabajos de laboratorio. Finalmente, gracias a todos los investigadores y profesores que conocí durante estos largos años de doctorado, solo por mencionar algunos: Alberto Stanislao Atzori, Marco Zaccaroni, Radim Kotrba, Martina Komárková.

Muchas gracias también a Ana Molina Casanova, que gracias a su contacto con la Universidad de Florencia me permitió llegar al campus de Albacete, inicialmente como estudiante del programa Erasmus, y desde este primer paso continué el camino en la investigación científica.

Gracias a Martina por apoyarme en este camino de la vida y por soportar las distancias y dificultades encontradas durante nuestro viaje.

Gracias a mi familia por su apoyo incondicional y por todo el cariño recibido, a lo largo de estos años.

The wild deer, wandering here and there Keeps the human soul from care.

(William Blake, Auguries of Innocence; 1863)

[…] el gran ciervo miraba y al mediodía su corona de cuernas brillaba cómo un altar en llamas.

(Pablo Neruda, Tercer libros de las odas; 1976)

Index General index Acknowledgment ...... 9 General index ...... 13 Index of figures ...... 15 Index of tables ...... 17 List of acronyms and abbreviations ...... 19 Summary ...... 21 Chapter 1. Introduction ...... 43 1.1 Economic aspects of hunting and game trophies ...... 45 1.2 Cranial appendages: antlers, horns, and much more ...... 50 1.2.1 Evolution of horns and antlers ...... 51 1.3 Antlers ...... 53 1.3.1 Structure ...... 54 1.3.2 Species as a study model: Red deer (Cervus elaphus spp.) ...... 60 1.3.2.1 Distribution and diffusion history ...... 60 1.3.2.2 Biology ...... 66 1.3.2.3 Ecology and reproduction ...... 68 1.3.2.4 Antler cycle ...... 70 1.3.3 Species as a study model: Roe deer (Capreolus capreolus) ...... 76 1.3.3.1 Distribution and diffusion history ...... 76 1.3.3.2 Biology ...... 78 1.3.3.3 Ecology and reproduction ...... 80 1.3.3.4 Antler cycle ...... 83 1.4 Horns ...... 87 1.4.1. Structure...... 87 1.4.2. Species as a study model: Common eland (Taurotragus oryx) . 91 1.4.2.1 Distribution and diffusion history ...... 91 1.4.2.2 Biology ...... 93 1.4.2.3 Ecology and reproduction ...... 94 1.4.2.4 Horn growth ...... 95 1.5. Factors affecting the growth of antlers and horns ...... 97 1.5.1 Influence of hormone secretion and environment ...... 97 1.5.2 Density of population and influence of the social environment ..... 101 1.5.3 Genetics and age ...... 103 1.5.4 Nutrition...... 106 Chapter 2. Justification ...... 111

Index Chapter 3. Objectives ...... 117 Chapter 4. Work plan, materials and methodology ...... 121 4.1 Material and geographical origin of animal’s samples ...... 123 4.1.1. Cervus elaphus spp...... 123 4.1.2. Capreolus capreolus ...... 126 4.1.3. Taurotragus oryx ...... 127 4.2. Measurement and scoring of the cranial appendages ...... 128 4.2.1. Cervus elaphus spp...... 128 4.2.2. Capreolus capreolus ...... 129 4.2.3. Taurotragus oryx ...... 130 4.3. Extraction and processing of the cranial appendages ...... 131 4.3.1. Antlers ...... 131 4.3.2. Horns’ bony core ...... 134 4.4. Analysis of bone tissue ...... 135 4.4.1. Moisture standardization of antlers sample ...... 135 4.4.2. Mechanical test ...... 136 4.4.2.1. Flexion test ...... 139 4.4.2.2. Impact test (charpy test)...... 142 4.4.3. Structure analysis ...... 143 4.4.4. Density and ash content...... 144 4.4.5. Mineral content analysis ...... 145 4.4.6. Histology analysis ...... 147 4.5. Statistical analysis ...... 147 Chapter 5. Results ...... 151 Article 5.1 Morphology, chemical composition, mechanical properties and structure in antler of Sardinian red deer (Cervus elaphus corsicanus)...... 155 Article 5.2 Smaller does not mean worse: variation of roe deer antlers from two distant populations in their mechanical and structural properties and mineral profile...... 165 Article 5.3 The bony horncore of the common eland (Taurotragus oryx): composition and mechanical properties of a spiral fighting structure...... 177 Chapter 6. Other studies ...... 189 Article 6.1 Manganese Supplementation in Deer under Balanced Diet Increases Impact Energy and Contents in Minerals of Antler Bone Tissue. 191 Chapter 7. General discussion ...... 205 Chapter 8. Conclusions ...... 227 Chapter 9. References ...... 239

Index Index of figures

Figure 1. Hunting bags of big game ungulates in Spain from 2001 to 2011 (modified from Garrido, 2012)...... 46 Figure 2. Example of red deer and roe deer antlers: in the image are described the parts composing the adult antlers of red deer and roe deer (image modified from Fandos & Burón, 2015)...... 55 Figure 3. In antler, macroscale arrangements involve both compact/cortical bone at the surface and spongy/trabecular bone (foam-like material with ~100μm thick struts) in the interior. Compact bone is composed of osteons and Haversian canals, which surround blood vessels. Osteons have a lamellar structure, with individual lamella consisting of fibres arranged in geometrical patterns. The fibres comprise several mineralized collagen fibrils, composed of collagen protein molecules (tropocollagen) formed from three chains of amino acids and nanocrystals of hydroxyapatite (HA), and linked by an organic phase to form fibril arrays (image modified from Chen et al, 2008 and Zimmerman et al, 2016)...... 56 Figure 4. Colonization routes of red deer in Europe and Asia based on cytochrome b sequences (image modified from Ludt et al., 2004)...... 60 Figure 5. The current distribution of Iberian red deer in the Iberian Peninsula. Dots indicate presence in UTM 10 x 10 km, (modified from Carranza, 2016)...... 64 Figure 6. The different stages of the population regression of the Cervus elaphus corsicanus in Sardinia (modified from Beccu, 1989) and the current distribution of Corsican red deer in the areas managed by EFS (modified from Murgia et al., 2015). Grey areas indicate natural or re-introduced areas of distribution: from a ubiquitous presence on the island (1900), to a sharp decline in population (1950), when only three areas of refuge remained in southern Sardinia (1=Montevecchio, 2=Sulcis, 3=Sarrabus). Later, even with reintroduction programs, the deer has spread (2015)...... 66 Figure 7. Photos showing the morphological differences between subspecies of red deer: Cervus elaphus corsicanus on the left and Cervus elaphus hispanicus on the right...... 67 Figure 8. Antler growing tip development and zones. (A) pedicle on the antler casting day and swollen rim presence around the edge, (B) formation of the growth centres and of the scab, approximately 10 days after hard antler casting, (C) formation of the main beam and the brow tine, approximately 30 days after hard antler casting. PS-pedicle skin, PP-pedicle periosteum, PCH-perichondrium, V-velvet, GT-’granulation’ tissue, UM- undiﬀerentiated mesenchyme, AP- antlerogenic periosteum, RM-reserve mesenchyme, PCHB-prechondroblasts, CART-cartilage (Adapted from Price et al.2005a)...... 71 Figure 9. Example of the development of red deer antlers: in the first years of life. From a simple shaft without branching (year 1), the structure becomes more complex with each passing year. In addition, the bone pedicle, in each growth cycle, is shortened in length (modified from Kowalski, 1981)...... 72 Figure 10. Timeline scheme of adult antlers’ development in Iberian red deer. In March-April, the new growth begins once the previous antlers are casted (A); then, there is a period of growth in which the antlers are covered with velvet, in which is present the blood circulation and are innervated (B); in August, thanks to a hormonal change, the blood supply starts to decrease and the antlers lose its characteristic velvet (C); after this phase, the antler persist on the head of the animal in a "hard" bone form until the spring of the following year when they will be cast (D). .. 74 Figure 11. Distribution of roe deer species in Europe and Asia. (Image modified from Fandos & Burón, 2015)...... 77 Figure 12. Reproductive cycles of European roe deer: for male with the phases of the antler cycle (external circle, blue words) and female (internal circle, black words)...... 83 Figure 13. Photos illustrating the growth cycle of the antlers of roe deer: closing of the scars after the fall of the antlers of the previous year (A); rapid growth of the new antlers and subsequent branching (B, C and D), at this stage the appendices are covered with velvet, with the presence of nerves and blood capillaries; cleaning from the velvet with gradual exposure of the bone tissue already largely mineralized (E); horns already clean formed of "dead bone”, ready to be used as a defense/offense weapon (F). Images modified from Sherer, 2009...... 84 Figure 14. Example of a growth scheme of the horn case and the cornual bone nucleus in Bovidae (Ovis canadensis). Horn grows at keratin sheath/bony core margin, pushing older sheath towards the tip (A); First and oldest keratin layer, deposited as a juvenile, is maintained at horn tip (B). The newer horn layers, deposited as an adult, growing under older layers (C). Modified from Goss,

1983...... 87 Figure 15. Hierarchical structure of bighorn sheep horn’s keratin sheath. The horns’ keratinous tissues are composed of elliptical tubules, embedded in a dense laminar structure. Each lamina has oriented keratin filaments interspersed in a protein-based matrix. These filaments are two- strand coiled-coil rope polypeptide chains (intermediate filament) helically wound to form ‘‘superhelical” ropes 7 nm in diameter. (modified form Tombolato et al., 2010)...... 88 Figure 16. Distribution of Common eland in its original natural habitat in Africa (modified from IUCN, 2004)...... 93 Figure 17. Geographical position of Experimental farm of University of Castilla-La Mancha (Albacete, Spain)...... 124 Figure 18. Procedure to obtain the Manganese gluconate solution for injections purpose. .... 125 Figure 19. From left to right: antlers cutting scheme in roe deer, in red deer spiker and in red deer adult...... 132 Figure 20. Example of cutting scheme applied on antler from distinct species. From the left to right: example of a cut antler of adult roe deer and a cut antler of adult red deer...... 132 Figure 21. Scheme of the protocol used for cutting cervid antler. In the bone cylinder, initially, two lateral cuts were made to detect the area to be cut (A); then with a further central cut (B) two bars could still be obtained in raw form (C). Later, with the use of a disk orbital polishing machine, it was possible to obtain pure cortical bone bars of the desired size (D). Red lines mark the cuts...... 133 Figure 22. An example of the steps to obtain cortical bone bars in red deer, starting from a cut cylinder: initially, longitudinal cuts were made in order to extract the raw bar, after which, by using a disk orbital polishing machine, the desired sizes are reached, eliminating the spongy bone tissue and cleaning the outer surface of the bars ...... 134 Figure 23. Scheme of the protocol used for cutting bovid horn’s core. Four cylinders sampling positions of about 5-6 cm of length were cut from each bony horn’s core (each cylinder was consecutive to the other, until the tip of the horn) (a); from each sampling position, two bone bars were extracted, one from the inner-medial face of the cylinder and the other one from the external- lateral face of the cylinder (b). In addition, from the position 1, two bone bars were cut from the bone spiral ridge (c), to study bone tissue features of this distinct structure...... 135 Figure 24. Types of forces involved during the performance of the mechanical tests: tensile stress (A), compressive stress (B) and shear stress (C)...... 138 Figure 25. Three-point bending test machine (Zwick/Roell 0.5 KN)...... 139 Figure 26. Three-point bending test with cortical bone sample during the application of the force...... 141 Figure 27. The curve that correlates the deformation (mm) and force (N) during a three-bending test on antler’s bone tissue...... 141 Figure 28. Machine used for the impact test (CEAST-IMPACTOR II) and functioning of the pendulum during an impact test...... 142 Figure 29. Effects of changing of dimensions of cortical bone layer on mechanical behaviour of antler bone (modified from Davison et al., 2006)...... 143 Figure 30. Measurements collected in the complete cross-sections for each sampling position along the main beam. ImageJ software was used to determine: total diameter and total perimeter (3 repetition), cortical bone thickness (6 repetition), external perimeter and total area, as well as of the spongy bone. TD=total diameter; CT= cortical thickness; CBA= cortical bone area; CBP= cortical bone perimeter; TBA= trabecular bone area; TBP= trabecular bone perimeter...... 144 Figure 31. Necessary steps to calculate the ash content in the bone samples: for this analysis was used a muffle-furnace at 480°C (1); before put the bone sample inside the hoven, the samples and the refractory cups they were weighted separately (2); then the refractory cup and the samples were put inside the muffle for 6 hours (3); finally, the bone samples were extracted from the muffle and were weighted together with the refractory cups, with a precision balance (4)...... 145

Index Index of tables

Table 1. Example of evaluation data sheet to collect measurements of red deer antler (modified from CIC, 1960)...... 129 Table 2. Example of evaluation data sheet to collect measurements of roe deer antler (modified from CIC, 1960)...... 130 Table 3. Example of evaluation data sheet to collect measurements of common eland bony horn core (modified from CIC, 1960 and SCI, 2016)...... 131 Table 4. Parameters of the three-point bending test...... 139

List of acronyms and abbreviations List of acronyms and abbreviations Abbreviations: AF= Fluctuating asymmetry ANOVA= Analysis of variance AP= antlerogenic periosteum AR= aspect ratio length to depth (for bone bar) B.Dm= total bone diameter of the section BS= Bending Strength BMD= Bone mineral density CART= cartilage CBA%= ratio area cortical/total, expressed in percentage CBD%= Average cortical thickness, expressed in percentage CBDcm= Average cortical thickness, expressed in cm CBP= cortical bone perimeter C.Dm= cancellous bone diameter CT= cortical thickness Ct.B.Ar= ratio between cortical and total area Ct.B.Wi= average cortical width, expressed in cm Ct.B.Wi%= the ratio between cortical bone width and total diameter of the section E= Young’s Modulus of elasticity GDP= gross domestic product GLM= General linear model GT=granulation tissue HA= nanocrystals of hydroxyapatite h2= Heritability index I= Second Moment of Area ICP-OES= Inductively coupled plasma - optical emission spectrometry PCH= perichondrium PCHB= pre-chondroblasts PIB= producto interno bruto

List of acronyms and abbreviations PP= pedicle periosteum PS= pedicle skin RM= reserve mesenchyme SHC= Antler-based selective-harvest criteria TBA= trabecular bone area TBP= trabecular bone perimeter TD=total diameter U= Impact energy UM= undiﬀerentiated mesenchyme V= velvet W= Work under the curve

Acronyms: CEBAS= Centro de Edafología y Biología Aplicada del Segura (Spain) CEEA= Comité de Ética en Experimentación Animal (UCLM, Spain) CIC= Conseil International de la Chasse CSIC= Consejo Superior de Investigaciones Científicas (Spain) EFS= Ente Foreste della Sardegna (Italy) FEDFA= Federation of European Deer Farmers Associations IDR= Instituto de Desarrollo Regional (Spain) IUCN= The International Union for Conservation of Nature MAPAMA= Ministerio de Agricultura y Pesca, Alimentación y Medio Ambiente (Spain) SCI= Safari Club International UCLM= University of Castilla-La Mancha (Spain)

Summary Summary

Summary

Nowadays, hunting plays a very important role in the conservation of biodiversity; and is considered a good tool to preserve the biological heritage and an important source of sustainable economic growth. Thus, hunting is a recreational activity which generates wealth and employments in depressed rural context. Moreover, the hunting sector seems to have a large turnover of money and it represents an important sector in the boundary of agrarian and wildlife related industry of European states. The Spanish example showed that the hunting sector employs more than 186,000 people and suppose a 0.3% of the GDP of Spain, and according to some estimates, a turnover equal to of 6,475 M€ that has a fiscal return of 614 M€. Moreover, the hunting permissions were 851,894 with an economic value of 20,5 M€; and only the red deer as species moves about 25,6 M€. Other authors estimate that in Spain, game estates have an economic impact which would range between 665 and 2,600 M€ (estimated based on USA study for economic impact for deer farm and per animal, respectively). In Spain the hunting estates are approximately 3,000, with an average surface of 1,000 ha, and the estimated total number of red deer is almost 650,000. As a matter of fact, European ungulate species represent an immense potential resource, not only in terms of biodiversity but also in economic terms. Economic estimates, based on economic impact per animal and per farm in USA study, assess an impact of 1120 M€ at the level of the EU farming industry (with 280,000 deer farmed in 10,000 farms, FEDFA data). At the present, trophy is the main objective of big game with a secondary use of meat (the economic estimation is 45 M€ for meat). In the commercial market, larger antlered males are more valuable to private landowners, and one of the most used criteria for selecting animals for future trophies is the length of the trophy's beam. Following these lines in the deer production, already in the past, our research group carried out various studies on wild and bred populations of red deer. The aim was to understand the effects of management and the effects of environmental factors on animal growth and the growth of their antlers. For this reason, a study protocol was developed using as parameters the quality of the trophy, intended as a tool for analysing the quality of a deer or, more generally, a herd of deer. This protocol provides for the study of the mechanical and structural characteristics of the trophy, as well as its

Summary mineral content. In this thesis we will examine these concepts in different subspecies of red deer, and moreover, for the first time, we will use this method of study on other species that play an important role in the world of hunting and wildlife production, at international level.

This thesis is focused on 4 studies completed on distinct species of ungulates included between Bovidae and Cervidae. The species included are: the red deer (with two subspecies: Cervus elaphus hispanicus and Cervus elaphus corsicanus), the roe deer (Capreolus capreolus) with two populations subject to different diets and habitats, and finally an African bovid (Taurotragus oryx), which it has considerable importance in the hunting world as well as for the production of meat on farms, especially in New Zeland, USA and South Africa; the latter country, only in the northern regions, has farms with about 30,000 animals. These species and subspecies have a strong faunistic-hunting interest; moreover, some of them are under the protection and management programs, in order to improve the coexistence between man and ungulates. The main aims of this thesis are the characterization of the trophies and the analysis of their quality; for this reason the mechanical properties (Young’s Modulus of elasticity= E; Bending Strength= BS; Work under the curve= W; Impact energy= U), structural characteristics of the bone tissue of the antlers and the mineral content were analysed, to determine: 1) the relationship between mineral composition, mechanical properties and function and type of growth (continuos or seasonal) of the Cervidae and Bovidae cranial appendages; 2) interspecific common patterns of differences; 3) the relationship between composition and mechanical properties in a wide frame of different species.

In the first research work, for the first time, the morphological characteristics and the mechanical and structural properties and the mineral content of the Sardinian deer's antlers were described (Chapter 5.1). This subspecies of the red deer is distributed on the islands of Sardinia and Corsica, and currently the population is increasing (4270 animals); since in the past they suffered from excessive hunting pressure, this subspecies is currently protected by Italian and European law. However, the protection status of the species is causing problems of interaction

Summary between the deer and the anthropic activities present in the area. For this reason, probably in a future it will be necessary to carry out a population management even through the controlled use of selective hunting. Given that this study is the first to assess the characteristics of the antlers of this species, initially the external morphological characteristics of the trophy were assessed, using antlers of 35 adult deer and 26 subadults. The antlers were collected within a protected area where the Sardinian deer is widely distributed ("Sette Fratelli" forest complex). The differences between subadults and adults were easily distinguishable: the adult trophy was heavier, wider and with more tines. Compared to other European populations, the Sardinian deer had a trophy with a lower weight and fewer tines (only 20% of the antler have three tines, while in red deer of the Italian peninsula three-tined trophies appear in more than 40% of cases). Compared to the Spanish deer, the Sardinian deer presented lower values for weight (-65%), length (-26%), burr perimeter (-30%) and length of the first tine (-45%). Subsequently, 12 antlers of adult deer were analysed in more depth, selected according to the morphology that characterizes the typical trophy of an adult Sardinian deer. On this set of antlers, the mechanical tests, the analysis of the internal structure and thet of the mineral profile were made; using cortical bone samples from 4 different positions along the vertical axis of trophy. The results showed that the bone tissue of the Sardinian deer has lower values for the average thickness of the cortical tissue, for the mechanical properties as stiffness (E) and work to fracture (W) and for the ash content; this was probably due to a lower quality diet that is reflected in a bone tissue with non-optimal characteristics compared to other sub-species of cervids. Even the mineral content turned out to be different compared to the Spanish deer. Probably the Sardinian deer follows the theory of the "phenotypic maintenance model", where phenotypic plasticity is driven through efficient growth models under the effect of restrictive ecological factors (as can be a Mediterranean environment). Moreover, the Sardinian deer may be subject to the effect of insular dwarfism, in which a species is smaller than other populations of the same species distributed on land. Finally, the discussion of this article also commented on the positive consequences of a selection hunt for this sub-species of deer, once its limit of spread on the island was reached, thus favouring also the economic growth of areas natural marginal.

Summary

Following the aim to compare different species, the second research (Chapter 5.2) concerned, for the first time, the study of two different roe deer populations subject to different habitat and management conditions. Sets of antlers from an extensive open-breeding farm near Prague (Czech Republic) and antlers from a closed-breeding farm of Murcia (Spain) were analysed. The animals of the Czech Republic lived in a vast area with mixed forest-cultivated cover; food supplementation was present only in a few cases, and the natural vegetation was covered by snow during the period of growth of the antlers for this species (winter). In contrast, the population of Iberian roe deer was managed more carefully, with a diet that provided food supplements throughout the year and a vegetative cover also continuous throughout the year. Therefore, the objective of this scientific study was to study differences in trophy characteristics (structural- mechanical qualities and mineral content of cortical bone tissue); in order to observe the effects of a very different management of two populations of the same species that lived in a very different habitat. A secondary aim was to compare the effects of an optimal/poor diet in the roe deer with similar studies in the antlers of red deer. From each analysed antler, samples were extracted from two different positions, along the trophy beam (at the base and near the upper tip). The results showed that the Czech’s antler set showed lower values for the size of the trophy: such as the length of the beam (-20%), the weight of the trophy (-130%), and therefore, lower score for the trophies (-24.3%). In contrast, the structural properties (cortical bone width = CBD% and cortical bone area = CBA%) are significantly higher in the Czech population. The two populations have different mechanical properties: the Spanish roe deer had a greater W and U (+19% and +72%, respectively), while the Czech roe deer offset its deficiencies with a greater cortical area and higher mechanical values for BS (+16%) and E (+12%). The mineral profile showed non-homogeneous differences: the roe deer of the Czech Republic had a higher value for ash content, Ca, K, P, Cr, Li, Sr and Zn; while the Spanish roe deer had higher values for Na, Mn, Tl and for the Ca/P ratio. In addition to the origin, the sampling position also has an effect on the observed variables; through the GLM it was possible to observe that for the values of the mechanical properties there was a variability of 27% for E, 36% for

Summary BS, 38% for W, 49% for U; for the structure the models explained a 55% of the CBDcm variability. For minerals, only Zinc was affected by the position (48% variability in GLM models); this mineral was also 25% higher in the antlers of the Czech Republic, perhaps due to a greater physiological stress during the growth of their trophy. In relation to the differences in the mineral profile between the two sampling positions, and the relationship with the physiological stress of the growth of the antler, there were no significant differences for both studied populations, probably because the roe deer's antlers is small, considering the body mass of this species. Since the group of Spanish animals was bred in optimal conditions with mineral supplements and continuous vegetative cover, while the Czech group grew in a poorer habitat, we concluded that these results support the hypothesis that a rich diet is reflected in the growth of the antlers, and that the antler can be considered an indicator of the quality of the environmental conditions.

In the third scientific study (Chapter 5.3) we aimed at comparing the previous studies and others in antlers with a similar study in the horn of a bovid, thus, for the first time, the bone nucleus belonging to the horn of the Common Eland was characterized, as example of bovid horn. The trophy of these African bovids has a spiral shape, with an evident thickening that, like a helix, rises from the base to the tip. Moreover, as characteristic for the bovid family, its trophy consists of a bony core (which remains moist), an outer keratin case and a membranous tissue interposed between these two components. In this case, samples of the core bone tissue were made in 4 sequential positions, starting from the base and climbing the vertical axis of the horn. Eight bony cores of different animals raised in an experimental breeding farm in Prague were used. The main objective was to observe the mechanical differences and the mineral content along the vertical axis of the horn, and for this, for each position, two bony bars were extracted for the mechanical tests and subsequent tests on mineral content, ash and bone density. Moreover, in the first position, at the base of the horn, the differences in the mechanical yields between the central standard bone and the bone tissue forming part of the characteristic spiral of the horn were studied. The results showed that the density and ash content decreased, from the base to the tip of

Summary the horn (-32% and -36%, respectively). Also, the mechanical properties achieved a similar decreasing gradient, but only the impact energy showed significant differences (U= -48%). This strong difference between horn areas was due to the different mechanical function and the characteristics of the material and microstructure. For the mineral profile, there are differences depending on the observed mineral: the concentrations of some of these significantly increased, rising from the base to the tip (Se= +4%, Cu= +84%, K= +16%), while other minerals decreased (Mg= -33%, Mn= -31%). This species, as in red deer, showed a similar dynamic of minerals: the content of Ca and Na decreased in the proximal-distal direction, while K showed an increase. Furthermore, our results followed the "gradient due to differential fighting stress within the horn" hypothesis; where the most stressed region of the horn (the one subject to bear more deformations) is that next to the skull of the animal; and this would explain why this region is best designed to support mechanical stress during thrusts and twists, in males fight. The mechanical and mineral profile variables, observed between the standard bone and the spiral horn region, showed no great differences, only some mechanical properties were weaker in the spiral bone tissue (W= -300%, BS= -153%, U= -118%). The most plausible explanation was that the weaker properties of the spiral region serve to deflect the transverse mechanical stresses suffered by the bone tissue during torsional collisions and pushes, with other males of the species. Moreover, another hypothesis was that a larger surface of the spiral could avoid slippage/rotation of the horny keratin sheath with respect to the internal bone nucleus, in the composite structure of the horns in Bovidae.

Finally, the fourth scientific study (Chapter 6.1), is part of a miscellaneous study, although in the line of this thesis, because it showed the effects in the diet of supplementation of an interesting mineral, manganese, in deer antler composition and mechanics. The study followed the steps already marked in previous studies of our group, in which it was possible to observe how the mineral content and the quality of the food are reflected on the composition and mechanical properties of the bone tissue of the antlers of adult and young Iberian deer (yearlings). This study confirmed the hypothesis that manganese

Summary supplementation also play an important role in the development of the antlers (a previous study assessed effects of Mn deficiency). The role of the Manganese was studied through a supplementation by injections of 4% Gluconate of Manganese. Nineteen adult and ten yearling animals were used, which were distributed between the control group and the group subjected to weekly injections; the experiment lasted from January to mid-August (the period of antler growth). A balanced diet was the same for all individuals, so the manganese supplementation may show effects on the growth of antlers of well-fed animal. The antlers, once cleaned and fully grown, were cut in September, and samples were taken in four positions along the main beam for adults and two positions for the young deer (base and tip of these smaller antlers). The results showed that young deer, compared to adults, have lower values for mechanical yields, structural features (less than the average thickness of cortical bone) and mineral content of bone tissue; in both groups the level of Mn increased in the groups of treated animals (2.5 times in the young and 2.3 times in the adults). The yearlings did not show significant results in the mineral content (only for Fe and Mn) between the control and the treated group, probably because these animals were subject to the strong constraint of the effort of their growth. Consistent with this hypothesis, there was a minor influence of supplementation in the rest of characteristics of the antlers (mechanical properties and structure). The treated adult deer, in contrast, showed that body weight increased by 10% less than the control group during the trial. In addition, treated adults showed higher content of Ca (+8%), P (+10%), Na (+14%), K (+47%), Se (+142%) and Cu (+29%); while Si was the only one decreasing. Regarding mechanical properties, adults showed a significant increase of +11.8% in U. To study the effects of animal weight and Mn supplementation, GLMs were used: it was observed that supplementation has an effect on Co, K, Mn, Se and Si; while only the weight of the animal has effect on the CBD, on antler’s length, the density of the bone, E, BS and on the content of the Zn. The difference between the base and the tip of the antlers, which shows the effort of the animal for the growth of the trophy, showed no significant differences in the mineral profile (only for the content of the Mn); in the distal position an increase of 16% of the W was observed, only for treated animals, after having checked for the effect of the weight of the animals. The results

Summary obtained in this study were very similar to a previous study carried out by our own group, assessing antlers grown in two years differing strongly in late winter frost (February). Thus, decrease in the content of ash, Ca, P, Na, Co, Se and an increase in Si content was observed in antler set grown by deer feeding on plants during exceptional frosts. The study concluded that a general antler breakage due to reduced cortical thickness, add change in mineral composition in the year of late winter frosts was actually caused by a reduction in Mn content in the plants, likely similar to Mn deficiency in deer diet. That is exactly the opposite to Mn supplementation, which resulted in logical opposite effects in the antler characteristics (Mn hypothesized deficiency vs. current Mn supplementation).

In conclusion, it can be said that the characterization of the trophies through the study of the mechanical properties, the structural characteristics and the mineral profile of the bone tissue of the trophies themselves can be an excellent tool for the management of both wild and bred populations. Moreover, with the studies carried out in this thesis, it is shown that this method of study can also be applied to Bovidae or other species of Cervidae.

Summary

Resumen

Summary

Hoy en día, la caza juega un papel muy importante en la conservación de la biodiversidad. Se considera una buena herramienta para preservar el patrimonio biológico y una fuente importante de crecimiento económico sostenible. Por lo tanto, la caza es una actividad recreativa que genera riqueza y empleos en un contexto rural empobrecido. Además, el sector de la caza obtiene una gran inversión de dinero y representa un sector importante en la industria agraria y relacionada con la vida silvestre de los estados europeos. El ejemplo de España mostró que el sector de la caza emplea a más de 186,000 personas y supone un 0.3% de su PIB, y según algunas estimaciones, una facturación igual a de 6,475 M € que tiene un rendimiento fiscal de 614 M €. Además, los permisos de caza fueron 851,894 con un valor económico de 20,5 M €; y solo el ciervo como especie mueve unos 25,6 M €. Otros autores estiman que, en España, las fincas de caza tienen un impacto económico que oscilaría entre 665 y 2,600 M € (estimado según el estudio de EE. UU. sobre el impacto económico por granjas de ciervos y por animal, respectivamente). En España, las fincas de caza son aproximadamente 2,000, con una superficie promedio de 1,000 ha, y el número estimado de ciervos es 650,000. De hecho, las especies unguladas europeas representan un inmenso recurso potencial, no solo en términos de biodiversidad sino también en términos económicos. Las estimaciones económicas, basadas en el impacto económico por animal y por granja en el estudio de EE. UU., evalúan un impacto de 1,120 M € a nivel de la industria agrícola de la UE (con 280,000 ciervos criados en 10,000 granjas, datos de FEDFA). En la actualidad, el trofeo es el principal objetivo de los grandes ungulados con un uso secundario de la carne (la estimación económica es de 45 M € para la carne). En el mercado comercial, los machos con astas más grandes son más valiosos para los propietarios privados, y uno de los criterios más utilizados para seleccionar animales para futuros trofeos es la longitud del asta. Siguiendo estas líneas en la producción de ciervos, ya en el pasado, nuestro grupo de investigación llevó a cabo varios estudios sobre poblaciones de ciervos salvajes y en cautividad. El objetivo era comprender los efectos de la gestión y de los factores ambientales en el crecimiento animal y el crecimiento de sus astas. Por esta razón, se desarrolló un protocolo de estudio utilizando como parámetro la calidad del

Summary trofeo, como una herramienta para analizar la calidad de un ciervo o, más generalmente, una manada de ciervos. Este protocolo contempla el estudio de las características mecánicas y estructurales del trofeo, así como su contenido mineral. En esta tesis, examinamos estos conceptos en diferentes subespecies de ciervos y, además, utilizamos por primera vez este método de estudio en otras especies que desempeñan un papel importante en el mundo de la caza y la producción de vida silvestre, a nivel internacional.

Esta tesis se centra en 4 estudios realizados en distintas especies de ungulados, Bovidae y Cervidae. Las especies incluidas son: el ciervo (con dos subespecies: Cervus elaphus hispanicus y Cervus elaphus corsicanus), el corzo (Capreolus capreolus) con dos poblaciones sujetas a diferentes dietas y hábitats, y un bóvido africano (Taurotragus oryx), el cual tiene una importancia considerable en el mundo de la caza y en la producción de carne en granjas, especialmente en Nueva Zelanda, EE. UU. y Sudáfrica. Este último país, solo en las regiones del norte, tiene granjas con aproximadamente 30,000 animales. Estas especies y subespecies tienen un fuerte interés de caza faunística; además, algunos de ellos están bajo los programas de protección y gestión, para mejorar la convivencia entre el hombre y los ungulados. Los objetivos principales de esta tesis son la caracterización de los trofeos y el análisis de su calidad; por esta razón se analizaron las propiedades mecánicas (Módulo de elasticidad de Young= E; Resistencia a la flexión= BS; Trabajo bajo la curva= W; Energía de impacto= U), las características estructurales del tejido óseo de las astas y el contenido mineral, para determinar: 1) la relación entre la composición mineral, las propiedades mecánicas y la función y el tipo de crecimiento (continuo o estacional) de los apéndices craneales en Cervidae y Bovidae; 2) patrones comunes de diferencias interespecíficas; 3) la relación entre la composición y las propiedades mecánicas en un marco amplio de diferentes especies.

En el primer trabajo de investigación, por primera vez, se describieron las características morfológicas y las propiedades mecánicas y estructurales y el contenido mineral de las astas de ciervo sardo (Capítulo 5.1). Esta subespecie del ciervo rojo se distribuye en las islas de Cerdeña y Córcega. Actualmente la

Summary población está aumentando (4270 animales), ya que en el pasado sufrió de una presión de caza excesiva, y está actualmente protegida por la legislación italiana y europea. Sin embargo, el estado de protección de la especie está causando problemas de interacción entre el ciervo y las actividades antrópicas presentes en el área. Por esta razón, probablemente en un futuro será necesario llevar a cabo una gestión de la población incluso a través del uso controlado de la caza selectiva. Dado que este estudio es el primero en evaluar las características de las astas de esta especie, inicialmente se evaluaron las características morfológicas externas del trofeo, utilizando astas de 35 adultos y 26 subadultos. Las astas se recolectaron dentro de un área protegida donde el ciervo sardo está ampliamente distribuido (complejo forestal “Sette Fratelli"). Las diferencias entre subadultos y adultos eran fácilmente distinguibles: el trofeo de adulto era más pesado, más ancho y con más puntas. En comparación con otras poblaciones europeas, el ciervo sardo tenía un trofeo con menor peso y menos puntas (solo el 20% del asta tiene tres puntas, mientras que en el ciervo de la península italiana aparecen trofeos de tres puntas en más del 40% de los casos). En comparación con los ciervos españoles, los ciervos sardos presentaron valores más bajos para el peso (-65%), la longitud (-26%), el perímetro de las rosetas (- 30%) y la longitud de la primera punta (-45%). Posteriormente, se analizaron 12 cuernas de ciervo adulto con mayor profundidad, seleccionadas de acuerdo con la morfología que caracteriza el trofeo típico de un ciervo adulto de Cerdeña. En este conjunto de astas, se realizaron las pruebas mecánicas, el análisis de la estructura interna y el perfil mineral; utilizando muestras de hueso cortical de 4 posiciones diferentes a lo largo del eje vertical del trofeo. Los resultados mostraron que el tejido óseo del ciervo sardo tiene valores más bajos para el grosor promedio del tejido cortical, para las propiedades mecánicas como la rigidez (E) y el trabajo de fractura (W) y para el contenido de cenizas; esto probablemente se debió a una dieta de menor calidad que se refleja en un tejido óseo con características no óptimas en comparación con otras subespecies de cérvidos. Incluso el contenido mineral resultó ser diferente en comparación con el ciervo español. Probablemente, el ciervo sardo sigue la teoría del "modelo de mantenimiento fenotípico", donde la plasticidad fenotípica es impulsada a través de modelos de crecimiento eficientes bajo el efecto de factores ecológicos

Summary restrictivos (como puede ser un entorno mediterráneo). Además, el ciervo sardo puede estar sujeto al efecto del enanismo insular, en el que una especie es más pequeña que otras poblaciones de la misma especie distribuidas en la península. Finalmente, la discusión de este artículo también comenta las consecuencias positivas de una caza de selección para esta subespecie de venados, una vez que se alcanza su límite de propagación en la isla, favoreciendo también el crecimiento económico de las áreas naturales marginales.

Siguiendo el objetivo de comparar diferentes especies, la segunda investigación (Capítulo 5.2) se refiere, por primera vez, al estudio de dos poblaciones diferentes de corzos sujetos a diferentes hábitats y condiciones de manejo. Se analizaron conjuntos de astas de una granja extensiva no vallada cerca de Praga (República Checa) y astas de una granja vallada de Murcia (España). Los animales de la República Checa vivían en una vasta área con cobertura de bosques mixtos; la suplementación alimenticia estuvo presente solo en unos pocos casos, y la vegetación natural fue cubierta por la nieve durante el período de crecimiento de las astas para esta especie (invierno). En contraste, la población de corzos ibéricos se manejó con más cuidado, con una dieta que proporcionaba complementos alimenticios y cobertura vegetal continua durante todo el año. Por lo tanto, el objetivo de este estudio científico fue estudiar las diferencias en las características de los trofeos (cualidades estructurales, mecánicas y contenido mineral del tejido óseo cortical) de dos poblaciones de la misma especie para observar los efectos de hábitats y de un manejo muy diferentes. Un objetivo secundario fue comparar los efectos de una dieta óptima/deficiente en el corzo con estudios similares del efecto de la dieta en las astas del ciervo. De cada cuerna analizada, se extrajeron muestras de dos posiciones diferentes, a lo largo del asta del trofeo (en la base y cerca de la punta superior). Los resultados mostraron que el conjunto de astas de la República Checa mostró valores más bajos para el tamaño del trofeo: como la longitud de la asta (-20%), el peso del trofeo (-130%) y, por lo tanto, una puntuación más baja para los trofeos (-24,3%). En contraste, las propiedades estructurales (ancho del hueso cortical = CDB% y área del hueso cortical = CBA%) son significativamente mayores en la población checa. Las dos poblaciones tienen

Summary diferentes propiedades mecánicas: el corzo español tenía una mayor W y U (+19% y +72%, respectivamente), mientras que el corzo checo compensó sus deficiencias con una mayor área cortical y valores mecánicos más altos para BS (+16%) y E (+12%). El perfil mineral mostró diferencias no homogéneas: el corzo de la República Checa tenía un valor más alto para el contenido de cenizas, Ca, K, P, Cr, Li, Sr y Zn; mientras que el corzo español tuvo valores más altos para Na, Mn, Tl y para la relación Ca/P. Además del origen, la posición de muestreo también tiene un efecto en las variables observadas; a través del GLM fue posible observar que para los valores de las propiedades mecánicas hubo una variabilidad de 27% para E, 36% para BS, 38% para W, 49% para U; para la estructura, los modelos explicaron un 55% de la variabilidad de CBDcm. Para los minerales, solo el zinc se vio afectado por la posición (48% de variabilidad en los modelos GLM); este mineral también fue un 25% más alto en las astas de la República Checa, tal vez debido a un mayor estrés fisiológico durante el crecimiento de su trofeo. En relación con las diferencias en el perfil mineral entre las dos posiciones de muestreo y la relación con el estrés fisiológico del crecimiento de la asta, no hubo diferencias significativas para ambas poblaciones estudiadas, probablemente porque las cornamentas del corzo son pequeñas, considerando la masa corporal de esta especie. Dado que el grupo de animales españoles se crió en condiciones óptimas con suplementos minerales y cobertura vegetal continua, mientras que el grupo checo creció en un hábitat más pobre, llegamos a la conclusión de que estos resultados apoyan la hipótesis de que una dieta rica se refleja en el crecimiento de las astas, y que el asta puede considerarse un indicador de la calidad de las condiciones ambientales.

En el tercer estudio (Capítulo 5.3), intentamos comparar los estudios previos y otros en astas con un estudio similar en el cuerno de un bóvido, por lo que, por primera vez, fue caracterizado el núcleo óseo del cuerno del Eland común. El trofeo de estos bóvidos africanos tiene una forma espiral, con un engrosamiento evidente que, como una hélice, se eleva desde la base hasta la punta. Además, como característica de la familia bovina, su trofeo consiste en un núcleo óseo (que permanece húmedo), una funda de queratina exterior y un tejido

Summary membranoso interpuesto entre estos dos componentes. En este caso, las muestras del tejido óseo del núcleo se realizaron en 4 posiciones secuenciales, comenzando desde la base y subiendo hacia el eje vertical del asta. Se utilizaron ocho núcleos óseos de diferentes animales criados en una granja de reproducción experimental de Praga. El objetivo principal fue observar las diferencias mecánicas y el contenido mineral a lo largo del eje vertical del cuerno, y para ello, para cada posición, se extrajeron dos barras óseas para las pruebas mecánicas y las pruebas subsiguientes sobre el contenido mineral, cenizas y densidad ósea. Además, en la primera posición, en la base del cuerno, se estudiaron las diferencias en los rendimientos mecánicos entre el hueso estándar central y el tejido óseo que forman parte de la espiral característica del cuerno. Los resultados mostraron que la densidad y el contenido de ceniza disminuyeron, desde la base hasta la punta del cuerno (-32% y -36%, respectivamente). Además, las propiedades mecánicas lograron un gradiente decreciente similar, pero solo la energía de impacto mostró diferencias significativas (U= -48%). Esta fuerte diferencia entre las áreas del cuerno se debió a la diferente función mecánica y las características del material y a la microestructura. Para el perfil mineral, existen diferencias según el mineral observado: las concentraciones de algunos de estos aumentaron significativamente, aumentando desde la base hasta la punta (Se= +4%, Cu= +84%, K= +16%), mientras que otros minerales disminuyeron (Mg= -33%, Mn= - 31%). Esta especie, como el ciervo, mostró una dinámica similar de los minerales: el contenido de Ca y Na disminuyó en la dirección proximal-distal, mientras que el K mostró un aumento. Además, nuestros resultados siguieron la hipótesis de "gradiente debido al estrés diferencial de combate dentro del cuerno"; donde la región más estresada del cuerno (la que debe soportar más deformaciones) es la que está junto al cráneo del animal; y esto explicaría por qué esta región está mejor diseñada para soportar el estrés mecánico durante los empujones y giros, en la lucha de los machos. Las variables de perfil mecánico y mineral, observadas entre el hueso estándar de la asta y la región de la espiral, no mostraron grandes diferencias, solo algunas propiedades mecánicas fueron más débiles en el tejido óseo de la espiral (W= -300%, BS= - 153%, U= - 118%). La explicación más plausible fue que las propiedades más

Summary débiles de la región espiral sirven para desviar las tensiones mecánicas transversales sufridas por el tejido óseo durante las colisiones torsionales y los empujes, con otros machos de la especie. Además, otra hipótesis era que una superficie más grande de la espiral podría evitar el deslizamiento/rotación de la funda de queratina córnea con respecto al núcleo interno del hueso, en la estructura compuesta de los cuernos en Bóvidos.

Finalmente, el cuarto estudio (Capítulo 6.1), es parte de un estudio misceláneo, aunque en la línea de esta tesis, porque mostró los efectos en la dieta de la suplementación de un mineral, el manganeso, en la composición y mecánica de las cuernas de los ciervos. El estudio siguió los pasos ya marcados en estudios previos de nuestro grupo, en los cuales fue posible observar cómo el contenido mineral y la calidad del alimento se reflejan en la composición y propiedades mecánicas del tejido óseo de las astas de adultos y jóvenes de ciervo ibérico (varetos). Este estudio confirmó la hipótesis de que la suplementación con manganeso también juega un papel importante en el desarrollo de las astas (un estudio anterior evaluó los efectos de la deficiencia de Mn). El papel del manganeso se estudió mediante una suplementación con inyecciones de gluconato de manganeso al 4%. Se utilizaron diecinueve animales adultos y diez varetos, que se distribuyeron entre el grupo control y el grupo sometido a inyecciones semanales; el experimento duró desde enero hasta mediados de agosto (el período de crecimiento de las astas). Todos los animales tuvieron la misma dieta equilibrada, por lo que la suplementación con manganeso puede mostrar efectos en el crecimiento de las cuernas en animales bien alimentados. Las cuernas, una vez limpias y completamente desarrolladas, se cortaron en septiembre, y se tomaron muestras en cuatro posiciones a lo largo de la vara principal para adultos y dos posiciones para el ciervo joven (base y punta de estas astas más pequeñas). Los resultados mostraron que los ciervos jóvenes, en comparación con los adultos, tienen valores más bajos para los rendimientos mecánicos, las características estructurales (menos el grosor promedio del hueso cortical) y el contenido mineral del tejido óseo; en ambos grupos, el nivel de Mn aumentó en los grupos de animales tratados (2.5 veces en los jóvenes y 2.3 veces en los adultos). Los varetos no mostraron resultados significativos en

Summary el contenido mineral (solo para Fe y Mn) entre el grupo control y el tratado, probablemente porque estos animales estaban sujetos a la fuerte restricción del esfuerzo de su crecimiento. De acuerdo con esta hipótesis, hubo una pequeña influencia de la suplementación en el resto de las características de las astas (propiedades mecánicas y estructura). En contraste, los ciervos adultos tratados, mostrarón que el peso corporal aumentó en un 10% menos que el grupo de control durante el ensayo. Además, los adultos tratados presentaron mayor contenido de Ca (+8%), P (+10%), Na (+14%), K (+47%), Se (+142%) y Cu (+29%); mientras que el Si fue el único decreciente. Con respecto a las propiedades mecánicas, los adultos mostraron un aumento significativo de +11.8% en U. Para estudiar los efectos del peso de los animales y la suplementación con Mn, se usaron GLM: se observó que la suplementación tiene un efecto sobre Co, K, Mn, Se y Si; mientras que solo el peso del animal tiene efecto en el CBD (espesor del hueso cortical), en la longitud de la asta, en la densidad del hueso, E, BS y en el contenido de la Zn. La diferencia entre la base y la punta de las astas, que muestra el esfuerzo del animal por el crecimiento del trofeo, no mostró diferencias significativas en el perfil mineral (solo por el contenido del Mn); en la posición distal se observó un aumento del 16% del W, solo para los animales tratados, después de haber comprobado el efecto del peso de los animales. Los resultados obtenidos en este estudio fueron muy similares a un estudio previo realizado por nuestro propio grupo, que evaluó las astas que crecieron en dos años y que difieren fuertemente en las heladas tardías de invierno (febrero). Por lo tanto, se observó una disminución en el contenido de ceniza, Ca, P, Na, Co, Se y un aumento en el contenido de Si en el conjunto de astas de ciervos que se alimentaron de las plantas durante las heladas excepcionales. El estudio concluyó que una rotura general de la cuerna debido a la reducción del grosor cortical y el cambio en la composición mineral en el año de las heladas tardías del invierno fue causada por una reducción en el contenido de Mn en las plantas, probablemente similar a la deficiencia de Mn en la dieta del ciervo. Eso es exactamente lo opuesto a la suplementación con Mn, que resultó en efectos lógicos opuestos en las características de la cuerna (deficiencia hipotética de Mn frente a la suplementación con Mn actual). En conclusión, se puede decir que la caracterización de los trofeos a través del

Summary estudio de las propiedades mecánicas, las características estructurales y el perfil mineral del tejido óseo de los mismos, puede ser una herramienta excelente para la gestión de poblaciones silvestres y cautivas. Además, con los estudios realizados en esta tesis, se muestra que este método de estudio también se puede aplicar a Bovidae u otras especies de Cervidae.

Summary

Introduction Chapter 1. Introduction

1. Introduction

Introduction

Introduction 1.1 Economic aspects of hunting and game trophies Nowadays, hunting plays a very important role in the conservation of biodiversity; and could be considered a good tool to preserve the biological heritage and an important source of sustainable economic growth (Rodriguez-Estival & Landete- Castllejos, 2010). Thus, hunting is a recreational activity which generates wealth and employments in depressed rural context (Farfán et al., 2004), something that is common to all countries from Spain to USA (Anderson et al., 2007). The hunting is an activity already present since ancient times, but the depletion of preys led on one hand to cattle farming about 9000 years ago and since records of history (e.g. Egypt 6000 years ago) to managing game species in royal preserves to ensure that pharaos, kings, emperors and nobles could enjoy hunting. Deer breeding in preserves or even in farms and parks of the middleage has been associated in western history to hunting, whereas in Asian countries has been associated to tradionational medicine for 2200 years (Serrano et al., 2019). The association between interest in trophy hunting of several species of deer and the consumption of their meat explains nowadays why most of the exports of deer meat from New Zealand (main farmed deer venison exporter with more than 2,500 farms and 833,000 deer at present) and Spain (main hunted deer venison exporter) are sent to Germany and surrounding countries (Austria, Czech Republic, Hungary). In the case of Spain, there was little management until 1973, when the Spanish hunting law was promulgated (to prevent poaching and restrict the control of predators), after this law the management of hunting became much more intense, even using territories fenced and private/public game estates (Carranza, 2007b). Nowadays, the hunting sector could move a large amount of money and represents an important sector in the boundary of agrarian and wildlife related industry of European states. The Spanish example showed that an average of 1,162,900 persons were included, during the 2015, in the agricultural, animal husbandry and hunting sector (MAPAMA, 2017), more specifically, the hunt sector employs more than 186,000 people and suppose a 0.3% of the GDP of Spain, and according to some estimates, a turnover equal to of 6,475 M€ that has a fiscal return of 614 M€ (Andueza et al., 2018). Moreover, the hunting permissions were 851,894 with an economic value of 20,5 M€; and only the red deer as species moves about 25,6 M€ (last data for 2014; MAPAMA, 2017); see

Introduction Figure 1 for details on the number of hunting bag for big game ungulates, in Spain that presents an upward trend, for several decades.

140000 Red deer Roe deer Fallow deer 120000 100000 80000 60000 40000 20000 0 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

10000 Ibex Mouflon Barbary sheep

8000

6000

4000

2000

0 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011

Figure 1. Hunting bags of big game ungulates in Spain from 2001 to 2011 (modified from Garrido, 2012).

Other economic study estimates that only the value brought to the producer for the meat from hunting animals is around 45 M€ (Serrano et al., 2019); in accordance with the presence of 650,000 deer in Spanish territory (Landete- Castillejos et al., 2010b). Although at European Union (EU) level, there are few economic studies that attempt to summarize the total number of farmed deer, the FEDFA estimated in 280,000 the number of deer farmed in 10,000 farms (Kotrba & Bartoš, 2010); and some authors registered an increment in the use of hunting resources (average numbers of ungulate hunters increase by 9% between 1996- 2006; Kenward & Putman, 2011). As a matter of fact, European ungulate species represent an immense potential resource, not only in terms of biodiversity but also in economic terms; on this topic, Apollonio et al. (2010b) described a

Introduction potential hunting revenue of several hundred million euros for more than 5.2 million of animal harvested each year. Serrano et al. (2019) estimates that the private management of deer population in fenced enclosures for the whole EU may be as high as 3,270 M€ (estimation based on deer farming impact study in the USA, see Anderson et al., 2007). There are also other less widespread game species in Europe in addition to native cervids and bovids (e.g. African wildlife or other introduced species in experimental farm). This species in their original habitat could have some hunting economic revenue. Thus, Safari Club International (SCI), based on 8 African countries study, claim that the overall economic benefit from their estimated 18,815 trophy hunter visits is between 132 and 426 million $USD, and that sustainable trophy hunting directly and indirectly supports from 15,000 to 53,000 jobs (Southwick, 2015; Murray, 2017) and in this line, Taurotragus oryx is described as “one of the great trophies of Africa” by SCI (2005). Normally for this kind of hunting activity, the price payed by hunter is affected likely by perceived rarity and size of the species (Johnson et al., 2010). South Africa (SA) is an example of the wildlife ungulate management for hunting and conservation; in this nation, game parks (ranches) were created starting from the 1960s and are 10-20 times more abundant than in other countries: 9000 in SA, 500 in Zimbabwe, 400 in Namibia (IDUBA, 2019). Moreover, economic research found that hunters (consumptive wildlife tourist) spend double and more the amount than non- consumptive wildlife tourists (bird watching, game viewing, etc.), with a contribution of trophy hunting close to 2 billion Rand (130.9 M$) to the South African economy in 2016/2017 season; this estimation, adding to that of the meat hunters (biltong), increases up to 13.6 billion Rand (909 M$), creating 31,500 jobs in the three provinces of the study (Van Der Merwe, 2018). Wildlife conservationists differ in their assessment of trophy hunting particularly is economic impact and potential for contributing to conservation (Lindsey et al., 2007; Johnson et al., 2010). With the existing information up to now, it can be said that the hunting regimes, if well organized and scientifically based, produce patterns of mortality specific for sex and age, similar to those that would occur in nature (Harris et al., 2002); and often generates revenues used for the conservation of biodiversity (Di Minin et al., 2016).

Introduction The hunting activity consists basically of the use of one or several wild species (utilization of wildlife renewable resources) in different types of game land: in hunting areas (which include public and private hunting grounds) and land in special hunting regime (in this typing there are wildlife refuges, game reserves, security zone, natural protect areas). Currently, the usual management measures, in the game farms, are aimed at achieving a good quantity and quality of trophies by: 1st) genetic improvement of animals, either by acquiring good stags, using different techniques of reproductive manipulation, or classical selection of best trophies; 2nd) improve nutrition to allow animals to express full genetic potential (reason why genetics is the 1st aim); 3rd) reducing impact of parasites and diseases. In game estates the 1st point is very difficult to achieve, as to a lesser extent the 3rd point (multiple game estate and farm owners; pers. com.). In addition to these measures, some authors point as important the conservation of the habitat (Peiró, 2003). Usually, in private hunting areas (fenced farm) there is a special attention on producing high quality trophies, with heavier male deer. Whereas another type of management involves the production of a large number of animals, but of average quality due to morphological and trophy characteristics. Often, these types of management involve the use of mineral supplements and food to improve production (Olguin, 2011). Another important hunting measure is the use of controlled breeding for restocking of populations, but also as a manner to improve fenced populations; this kind of practices are accepted for some authors (Montoya, 1999) but for other authors this practice could put genetic conservation at risk (Carranza, 2007b). Within the hunting farm of the red deer (one of the most important ungulate in Europe from an economic viewpoint; Tuckwell, 1998), two types can be differentiated based mainly on criteria of sex, age conditions or production targets of the hunting population: a first type of management (unordered hunting procedure) involves obtaining, the best trophy, taking animals in all age classes; a second type (active procedure) follows the priority criteria according to age or gender, decided by the managers of the administered hunting area (Coltman et al., 2003). Whereas, at international level, New Zealand is the largest producer of deer (almost 1 million, Serrano et al., 2019) mostly reared in fenced farm,

Introduction followed by China, Russia and Australia; in Europe we could found 280,000 deer, mainly fallow deer, in 10,000 farms (www.fedfa.org); and in Spain the game estates (mostly extensive ranch with an average area of 1,000 ha) are more than 3,000 with an average population of 300 deer per ha; most of the private big game estates (PBGE) are located in the regions of Andalucía, Castilla-La Mancha and Extremadura, which cover 40% of Spanish surface, this would mean that 4% of Spain is occupied by fenced game estates, adding up to 2.15 million hectares (Landete-Castillejos et al., 2010b). Considering a mean density of 0.3 deer/ha this means that there are almost 650,000 Iberian red deer. At the present, trophy is the main objective of big game with a secondary use of meat (Serrano et al., 2019). In the commercial market, larger antlered males are more valuable to private landowners, and one of the most used criteria for selecting animals for future trophies is the length of the trophy's beam (Montoya, 1999; Lockwood et al., 2007), which strongly influences CIC score. Nevertheless, international antler competition for deer use weight or have a double list of awards (weight and CIC score). As an example, form top European breeders (reaching trophies of 320 CIC and 23 kg) males of 3 years old are sold in the farm for 8,500 €, spikers for 2,500 € and females for 2,000 € (Tatschl & Schober, 2019). The best deer farm in Spain sells spikers for half that price (Lagunes managing director, pers. com.), other authors estimated that a gold metal deer could reach above 3,000 € (Garrido, 2012). Normally, extremely larger antlered males are sold as “breeders”, because their semen is used for the spread of the species, whereas males with less impressive antlers are sold as “shooter bucks” for shooting reserves (in white-tailed deer, Demarais et al., 2016). This criterion of selection of the males according to the quality of its antler/horns has proved to be valid, since the males with the larger cranial appendices also have a better index of sperm quality (Malo et al., 2005; Santiago-Moreno et al., 2007). Nevertheless, deliberate selection could have unintended consequences: from one hand the strong genetic association between the body and the antlers size permits that the culling of animals with small trophies favors the presence of more tines among the remaining breeding animals (in red deer; Hartl et al., 1995a); in other hand strong hunting pressure could give birth to a plastic response of male

Introduction horns/antlers, and this could give rise to evolutionary effects in large mammals where certain biological characteristics are favored by hunting regulations (Festa- Bianchet, 2017). Similar dynamics can also arise for the selection hunt in wild ungulate populations where the game activity is generally a non-random process (Festa-Bianchet, 2003), those some authors hypothesized that human-controlled selection could be consider as a step to semi-domestication of game ungulates (Mysterud, 2010). In this kind of activity, if hunters have a choice with no-strictly control, they select firstly for adult male animal that carry larger antlers/horns (Ramanzin & Sturaro, 2014; Balčiauskas et al., 2017), sometimes with subsequent problems on future trophies quality of population (lower dimension, Coltman et al., 2003; selective genetic changes, Allendorf et al., 2008); whereas, in well-designed management plans and under the strict control of managers, a better solution it is to hunt those animals who exhibit deformities or problems in the morphology of trophies (Apollonio et al., 2010a).

1.2 Cranial appendages: antlers, horns, and much more The cranial appendages represented by the horns or the antlers, as a trophy, is particularly important in Ruminants; in fact, in addition to being a memory for the hunter, the trophy has an undeniable value as an "indicator" of the vitality status of a population of wild animals (Vanpé et al., 2007; Ciuti & Apollonio, 2011) and therefore it is considered particularly useful for the management of the trophy exhibitions at the local level; on the other hand the national and international exhibitions in which the best trophies are exposed (evaluated according to official formulas recognized by C.I.C.; CIC, 1960). There has been talk of horns and antlers; it is indeed necessary to make a distinction: although all species of bovids have structures similar to the horns, with a bony core surrounded by a sheath of cornified epithelium, horns are exclusive of the family of bovidae (Goss, 1983). The horns are permanent, and they have a simple structure without ramifications, but their growth undergoes a slowing down, during the winter season, from November to March, due to various hormonal factors linked also to the reduction of avalaible food. The slowing down

Introduction or stunting in growth causes the appearance of junction circles, rings (accretion or age). In contrast, the antlers of cervids are a secondary sexual character, present only in males for most of the cervids; it is not a bone covered with a queratin sheath but a real bone tissue. These antlers are lost and re-grow each year; the cast is determined by the interruption of the blood circulation at the base of the antlers; the upper part of the osseous stems that depart from the frontal bone is modified considerably and present multiple points (García et al., 2010). Other family and species in the order Artiodactyla, in addition to cervids and bovids, present other types of trophies evaluated at international level; for example, wild boar (Sus scrofa) shows permanent tusks, which are valued for their length and width (CIC, 1960); and American antelope (Antilocapra Americana) bear an intermediate structure between horns and antlers: pronghorn. Each "pronghorn" is composed of a slender, laterally flattened bone that grows from the frontal bones of the skull, forming a permanent core cover with a keratinous sheath. Unlike the horns of the family Bovidae, the horn sheaths of the pronghorn are branched, and it is shed and regrown annually (Goss, 1983). For a detailed description of the development and the external morphological characteristics of the antlers and horns in the species considered in this thesis, please refer to the specific chapters for each species which can be found later in the text: Cervidae (red deer, chapter 1.3.2, roe deer, chapter 1.3.3) and Bovidae (common eland, chapter 1.4.2).

1.2.1 Evolution of horns and antlers Cranial appendages are conspicuous and diverse among artiodactyls species, they serve a diversity of functions for the species belonging to suborder of ruminants. Among these, there are existing four families which are characterized by possessing four different cranial appendages (also called headgear): antlers, horns, pronghorns and ossicones (Davis et al., 2011). Normally these appendices are found on head (frontal bones) and are symmetrical paired organs, usually there is a bony core covered by integument. Many studies have investigated the homology in ruminant headgear (Coope,1968; Webb & Taylor, 1980; Bubenik AB, 1990); while they varied in

Introduction detail, all suggest that there is a common history in the evolution of these tissues. Other researchers support the theory that consider the antler’s pedicle like homologous to ossicone, horns or pronghorns; accordingly, exposed bone of the antlers (that is renewed every year) could be seen as an evolutionary novelty (Lancaster, 1907; Pilgrim, 1941). Paleontological findings indicate that antlers developed first as soft non-mineralized permanent tissue serving more as display and scent-dispensing (Kužmová, 2011). The function of the headgear (antlers or horns) are various and there is some controversy in what function was the primary and which ones were the secondary (Clutton-Brock, 1982; Goss, 1983); surely, as characters related in a certain way to sexual selection they provide a benefit to their bearer in relation to reproduction (Darwin, 1871), not only because increasing the ability of their bearer to succeed in direct competition for mates, but also increase the attractiveness of males to members of the opposite sex. Generally, this headgear (as sexually selected character) are more developed in males (Andersson, 1994); and antler size plays a significant role in sexual selection as an indicator of individual quality (Kruuk et al., 2003). Particularly, the function of horn in bovid has long been debated; in the 19th century many biologists believed that horns functioned primarily as anti- predator weapons; however many bovids escape at the sight of the predator and do not often use the horns for this reason; in addition, in about 30% of the species the females do not have horns; therefore the need to use these structures as defence is probably not the first selective force in the evolution of the horns (Lundrigan, 1996). Thus, the evolution of the horns should be read especially in the context of sexual selection, as a weapon in male-male competitions. As observed by Clutton-Brock et al. (1982) in lifetime reproductive success in male of red deer, a role of the relationship between fighting ability and intra-specific combat in the evolution of antlers/horns is generally accepted (Geist, 1966; Packer, 1983). The annual renewal of antlers could be the compensation of frequent breakages after aggressive encounters or as the adaptation to temperate geographical zone (Coope, 1968). Hoem et al. (2007) have showed that male-male fights escalated more and were more complex when the difference in antler size between rival males was smaller. Furthermore, it seems that territorial males may evaluate the potential threat of yearlings, in terms of

Introduction mating competition, by using yearling antler size as an honest indicator (Strandgaard, 1972; Wahlström, 1995). In the same way, Vanpé et al. (2007) described that roe deer males may use antler size and body mass of rival males as a cue to assess the possibility of winning a fight. Other authors have hypothesized that antlers may act as a parabolic reflector and enable males to better locate calling female (in moose; Bubenik & Bubenik, 2008); whereas other have hypothesized a potential role of thermoregulation of the bovid horns (Picard et al., 2016). However, solving the origin of the ruminants’ headgear will requires more studies on the internal phylogenies for each family and through the study of the development processes for all types of headgear (an excellent example could be found in one study by Lister, 1982). Below are deepened the concepts about the antlers of cervids and horns of bovids, which were the subject of study in this doctoral thesis.

1.3 Antlers The antlers are an evolutive secondary sexual features of Cervidae family, and are characteristic of male individuals, with exceptions such as the Rangifer Tarandus (where they are present in male and female). These cranial appendages are normally branched and are completely and periodically regenerate in adults and the regeneration process are well studied (Li et al, 2005; Price et al, 2005a; Kierdorf & Kierdorf, 2010). Cervids develop the antler’s bone tissue from the lateral crest of the frontal bone (Li & Suttie, 1994) and during the longitudinal growth there is an active interaction between external and internal tissue (Li et al, 2008). The antlers of deer grow in an increasingly more complex form (with an increase of tines and increasing beam length) to reach their maximum at about 8-11 years in red deer (García et al., 2010) and 6 years for roe deer (Pelabon & Breukelen, 1998), and normally, when the male reaches senility, the antlers reflect these physiological changes in the external aspects with a more simplified structure or disappearance of some antlers characters. Following this observation, in a reverse procedure, some authors think that the size of the antlers can be considered a good indicator of the development and physical condition of the animal (Prieditis et al., 1998). As a matter of fact, the antlers of the cervids require a large amount of resources for their formation, they

Introduction represent the 5% of the total live weight of the animal (Huxley, 1931) and, what is more indicative of the investiment cost, 32% of the weight of the skeleton of males of 3 years or older (Gómez et al., 2012). Antlers grow relatively quickly (≈105 days of growth according to the latitudes with 0.33 cm/d in roe deer, Sempere 1990; ≈120 days with 0.67cm/d in red deer, Gómez et al. 2013), for this, there is a close relationship between abundance and food quality and the development of secondary sexual characteristics such as antlers (Estévez et al., 2008; 2009; Landete-Castillejos et al., 2012; 2013a).

1.3.1 Structure The description of the structure of the antlers will start from the most visible part of the antlers, the external shape and parts that forms the antler itself, and then it will describe the internal structure of the bone tissue, at the microstructural and macrostructural level. When describing the internal structure, the bone tissues and the antler’s internal structure, the red deer (Cervus elaphus) will be used as a model, given that the characteristics of the bone tissue in antlers of Cervidae are similar with a circannual cast and with regenerated apices of pedicles (Bubenik & Bubenik, 1990; Davis et al., 2011). The external structure presents some variations with a length, weight, circumference and different number of tines depending on the species considered. However, for a detailed description of the development phases and of the morphological characteristics of the antlers of the species under study, see the specific chapters for red deer (chapter 1.3.2) and roe deer (chapter 1.3.3), which are found later in the text. Using the antler of red deer (Cervus elaphus) as model, during the first year of life, usually the antler is formed by a long vertical shaft terminating in a tip (which gives its name to the young spikers). Later, during the growth, the animal develops a trophy more complex, and in adult male, generally, the antlers provides, along the main shaft development, the presence of a basal tine (brow tine), a second tine (bez tine), a central tine, and a crown (which can include the development of more tines). A similar process can also be observed in Capreolus capreolus, even if the mature trophy reaches a length and a lower number of tines, compared to that of red deer. See Figure 2 for the main parts constituting the mature antler in these two deer species.

Introduction

Figure 2. Example of red deer and roe deer antlers: in the image are described the parts composing the adult antlers of red deer and roe deer (image modified from Fandos & Burón, 2015).

At the base of the antlers can be found the pedicle, this anatomical part is a permanent outgrowth of the frontal bones where the antler starts growing after casting of the previous antler set (Goss, 1983). After some methods to estimate deer age were based on pedicle length (Morris, 1972) some authors have determined the negative correlation of pedicle length with age (Azorit et al., 2002a); the older the deer is, the shorter the length of the frontal process. Moreover, the first antler and its characteristics, generally, could be an important evaluation tool to know the future development of the future antler growth, examples are the circumference of the pedicle base (Goss, 1995) or the weight of the first antler, the latter was correlated with the weight of the five-successive trophy by Moore et al. (1988). The bone hierarchical structure (see Figure 3) provides the following levels of organization (Rho et al., 1998): 1- the macrostructure: cancellous and cortical bone 2- the microstructure: Havers systems’ channels, lamellar bone and osteons (10- 500 μm) 3- the sub-microstructure: lamellae (1-10 μm) 4- the nanostructure: fibrillar collagen and embedded minerals (<1 μm) in the form

Introduction of crystals, non-collagenic organic proteins.

Figure 3. In antler, macroscale arrangements involve both compact/cortical bone at the surface and spongy/trabecular bone (foam-like material with ~100μm thick struts) in the interior. Compact bone is composed of osteons and Haversian canals, which surround blood vessels. Osteons have a lamellar structure, with individual lamella consisting of fibres arranged in geometrical patterns. The fibres comprise several mineralized collagen fibrils, composed of collagen protein molecules (tropocollagen) formed from three chains of amino acids and nanocrystals of hydroxyapatite (HA), and linked by an organic phase to form fibril arrays (image modified from Chen et al, 2008 and Zimmerman et al, 2016).

At the macrostructural level, the internal structure of the antler consists of four different histological zones from the outer edge to the centre of the transversal section: the first layer is made up of osteoid, located just below the velvet; then a zone of osteonic bone, composed of lamellar compact bone; a third transition region between the osteonic bone and trabecular bone; and a fourth central zone, consisting of trabecular bone (Rolf & Enderle, 1999). The two external regions formed the compact bone, while the centre has got a structure formed by a spongy tissue. The latter consists of a network of trabeculae that are variously oriented and intertwined with numerous intercommunicating cavities; the trabeculae are arranged in space according to the lines of force that are exerted on the tissue (Rho et al., 1998). Generally, in the skeleton of mammals, about 80% of the skeletal mass is represented by the compact tissue, while the rest of the bone mass is spongy tissue (Bedini et al., 2009) and the antler can reach 32% of the weight of the skeletons of males aged 3 years and over (Gómez et al., 2012). The compactness/porosity of the antler bone cortex depends largely on the formation of the primary osteons that fill the pre-existing pores of a tubular bony framework (Gomez et al., 2013). At microstructural level, the cortical bone is made up of mineralized collagen

Introduction fibers organized in lamellae of 3-7 μm thick, the latter can form lamellar bone when they are organized in concentric layers within a central channel, thus forming the base unit called the osteon (Weiner & Addadi, 1997). The osteon is characterized by a cylindrical structure and is crossed along its entire length by the Havers canal, which allows the passage of blood vessels and the nervous system. There are two types of osteons in the bone tissue: the primary (Haversian system) and secondary osteons. The former, whose fibrils are oriented along the pores, are present in the initial phase of bone growth and improve antlers’ mechanical properties (Krauss et al., 2011), and the latter are a result of bone remodelling (Currey, 2002) and could intersect each other (digging a tubular path through the compact tissue and depositing new osteons layer by layer; Cowin & Doty, 2007). Nevertheless, other studies said that secondary osteons are scarce in antlers and most of the osteons present in antler bone are primary ones (Chen et al., 2009; Launey et al., 2010a; Gomez et al., 2013). The composition of the bone tissue of antler consists of 55%-65% of minerals and 35%-45% of a collagen matrix and proteins (Pathak et al., 2001; Currey et al., 2009a; Estévez, 2011); the former includes many of the macro minerals (Ca, K, Mg, Na and P) and micro minerals (as Al, Co, Cu, Fe, Mn, Se and more; Miller et al., 1985), whereas the latter is composed of about 90% collagen (Currey, 2002) and the inorganic-mineral matrix is composed of calcium phosphates in the form of crystals of hydroxyapatite (Field et al., 1974). Thus, the nanostructure includes the fibrillar collagen composed of collagen protein molecules (tropocollagen) formed from three chains of amino acids and nanocrystals of hydroxyapatite (HA), these packages of apatite crystal in collagen fibers were found, for the first time, by Landis et al. (1996) and they containing

Ca10(PO4)6(OH)2, but the crystal could be impure and 4-6% of carbonate could replace the phosphate groups and this carbonate substitution tend to reduce the crystallinity of the crystal (Ou-Yang et al., 2001 in Currey, 2002). Crystals’ average dimensions are 50x25 nm and their thickness is included between 2 and 3 nm (Ziv & Weiner, 1994; Landis, 1995). The HA crystal grow with a specific orientation, so that the main axis is parallel to the vertical axes of the fibrils; this nanostructure allows the bone tissue, subjected to monoaxial tensions, to absorb the forces applied to it, while the organic matrix absorbs the shear stresses, and

Introduction the crystals the tensile forces (Gao, 2006). Histologically, the primary antler could be divided in disto-proximal direction into eight different zones (dermis; perichondrium; zones of cartilage formation, hypertrophy, mineralization, and degeneration; primary spongiosa; secondary spongiosa). The histological results demonstrate that the elongation of the primary antler proceeded through a modified form of endochondral ossification, resembling that seen during formation of pedicles and secondary antlers (Szuwart et al., 1995). Another study by Kierdorf et al. (2013) showed that the outer cortex is formed late during antler growth by intramembranous ossification because of periosteal activity occurring in the more proximal antler portions, and this is in line with the fact that the formation of pearling take place during the last phase of growth of antler. These perlage is formed by woven bone that forms a trabecular framework incompletely filled with primary osteons. The formation of bone tissue in growing antlers is characterized by an initial formation of a scaffold of mineralized cartilage largely replaced by a trabecular scaffold of woven bone; in the tissue there are cylindrical pores which are oriented along the main antler axis (Gomez et al., 2013; Krauss et al., 2011). Subsequently to the deposit of lamellar bone, the longitudinal tubes are filled in and form primary osteons of the structure, the width of the honeycomb structure will determine the thickness of the cortical tissue of the antlers, and thus, it could affect the future mechanical performance of this biological structure (Picavet & Balligand, 2016). The filling process starts around the 70th day of growth and its development is organized from proximal to distal sequence. Formation of the primary osteons is mostly in charge of the compactness of the antler cortical tissue, which is determined in days 70–120 of antler growth (Gomez et al., 2013). As described before, since there are more primary osteons than secondary osteons (linked to a remodelling process, Chen et al., 2009), the antler that is growing can be said to show very limited remodelling activity; only some limited signs of remodelling activity were observed in the permanently viable antlers of castrated fallow bucks (Kierdorf et al. 2004a). Moreover, some authors (Skedros & Bloebaum, 1995) found a hyper mineralized lamellar structure situated around primary osteons that could play a role in the reinforcement of structure against propagation of microcracks. This structure would appear after bone deposition on trabecular surfaces

Introduction and could indicate resorption of the trabecular structure prior to infilling of the intertrabecular spaces (Kierdorf et al., 2013).

Introduction 1.3.2 Species as a study model: Red deer (Cervus elaphus spp.)

Kingdom: Animalia Phylum: Chordata (Bateson, 1885) Class: Mammalia (Linnaeus, 1758) Order: Artiodactyla (Owen, 1848) Suborder: Ruminantia (Scopoli, 1777) Family: Cervidae (Goldfuss, 1820) Subfamily: Cervinae Scientific name: Cervus elaphus (Linnaeus, 1758)

1.3.2.1 Distribution and diffusion history The red deer is the most widespread and diverse Old-world deer species, it has been introduced in many habitats, and is widely bred in many European, Asian and Oceania countries. Moreover, species of genus Cervus showing extensive morphological variability and wide ecological adaptability (Geist,1998); and actually, the Cervus elaphus was introduced in South-America and New Zealand for hunting and commercial purposes. The origin of the family Cervidae could be found in the Central Asia, more concretely in the current Mongolia, where in the Miocene there was a tropical climate, according to Gilbert et al. (2006). See Figure 4 for details.

Figure 4. Colonization routes of red deer in Europe and Asia based on cytochrome b sequences (image modified from Ludt et al., 2004).

Introduction The distributions and patterns of taxonomic differentiation of the red deer could have been largely shaped by natural post-glacial expansion of refugial populations (Sommer et al., 2008; Skog et al., 2009), with three lineages displayed a phylogeographical pattern dividing individuals into western European, eastern European and Mediterranean (Sardinia, Spain and Africa) groups, suggesting three separate refugia during the Pleistocene glaciation. A recent stusy hypothezied that red deer may have persisted in cryptic northern refugia in western Europe during the Last Glacial Maximum, most likely in southern France, southern Ireland, or in a region between them (continental shelf), and these regions were the source of individuals during the European re- colonization (Queirós et al., 2019). Other authors (Ludt et al., 2004) studying the mtDNA of cytochrome-B have emphasized that, the western European deer actually divided into various subspecies (e.g.: C.e.scotticus, C.e. hispanicus, C.e. atlanticus), should be considered as a single subspecies C.elaphus elaphus, which diverge from the eastern deer group (C.e.hippelaphus) and North-African red deer (C.e. barbarus). Most of these studies are based on the most appropriate tool to study phylogeny: DNAmt (Ludt et al., 2004; Skog et al., 2009). Some have used nuclear DNA trying to discern wether or not there were subspecies within each genetic A or C lines (Carranza et al., 2016; Zachos et al., 2016). However, having different mitochondrial lines does not equal to have subspecies separated spatially. A study of fossil deer bones from 25 individuals who died in the same sudden event in a cave in Spain 35,000 years ago (Liñares) found half of individuals belonged to western line A, and the other half to eastern European line C (Rey-Iglesia et al., 2017). Similarly, other studies have found Iberian haplotypes of mitDNA in natural populations of Hungary (Frank et al., 2017). It should be noted, when studying genetic samples of current populations, the recent consequences of human management and translocations, that might have led to genetic admixture and introgression with non-native red deer, which can make phylogenetic reconstruction more difficult (Zachos et al., 2016). Historically red deer was divided in subspecies by the rump patch, the body and tail sizes, body colour, the antler morphology (Dolan, 1988), and this external appearance and shapes vary noticeably according to its geographical distribution. Thus, actually we can distinguish varies subspecies, as the Central

Introduction European red deer (C.e. hippelaphus) that diverges from the smaller Spanish red deer (Cervus elaphus hispanicus) that is more greyish, while the Norwegian red deer (Cervus elaphus atlanticus) is smaller in size and paler in colour; other recognized subspecies are the North African Barbary stag (C.e. barbarus), the small short-legged Corsican deer (C.e. corsicanus) and the Scottish red deer (C.e. scoticus). However, as metioned earlier, the modern criterion to discriminate among subspecies is a molecular one, differences in DNAmt, and this technique proved there are no subspecies. During the last 50 years, red deer populations and harvest have shown a pattern of considerable increase regardless of the ecological condition, socio-cultural background or hunting system (Milner et al. 2006). In a recent study by Burbaitė & Csányi (2010) reported an increased red deer numbers (spring population) from 1.1 million to 1.7 million and hunting bag from 275,000 to 469,000 individuals (data collected from 1984 to the early 2000s), and the population size (density) and hunting bag for red deer increased over nearly all Europe, except for the south-eastern part of the continent. Thus, the over-abundance of deer species in Europe (red deer and roe deer) is becoming a complex management issue for the 21st century (Apollonio et al. 2010a).

The case of Iberian red deer The Iberian deer (C.e. hispanicus), subspecies belonging to the species elaphus, is well differentiated from the rest of the twelve subspecies of Cervus elaphus throughout a phenotypic adaptation to Mediterranean environment (Geist, 1998). Red deer could have been continuously present in the Iberian Peninsula from the late Pleistocene to the present (Sommer et al., 2008). Moving forward, in the end of the 19th century the species’ distribution was drastically reduced due to overhunting (Perez et al., 1998), and the population of red deer survived in two isolated geographical area: in the peninsular South-Western and central regions of Spain. Thus, some authors found two genetic lineages of the Iberian red deer in Spain, due to the study of mutations in the mitochondrial DNA in respect of other species distributed in Europe (Fernández-García et al, 2014); following this hypothesis, one of these lineages would be found exclusively in the south-west peninsular (C.e. hispanicus) and the other in the north and central peninsula, in

Introduction line with the subspecies (C.e.bolivari) postulated by Cabrera (1911) based on differences in size and morphology. However, these authors although pointed that the genetic distance between both populations was nearly that of two subspecies, rejected this conclusion based on analysis of nuclear DNA. In any case, the population from central and north Spain was closer to western Europe deer than to SW Spain based on mitDNA results of the authors. Moreover, inside Iberian red deer population, there is evidence of restocking using foreign stocks, also aimed to increase the size of antlers in native populations several centuries ago (Long, 2003; Nussey et al.,2006); this activity is still common in present populations of Spain (Carranza et al., 2003). Today, the Iberian population of red deer is present from South to North of the Iberian Peninsula, except small area of Galicia and East coast, and is found widespread in all types of habitats characterized by the presence of accessible pastures and forests (Carranza, 2007a). The Iberian individuals of red deer are expanding its distribution and its total number is estimated at around 500,000 (Montoya, 1999); however, there are sources that indicate that the deer population could reach 1 million (Dibu, 2009, personal communication). See Figure 5. Nevertheless, in Spain this species is widely bred and used for hunting purposes, thus many individuals live in fenced areas (private or public farm- estate) that make up the basic unit of wildlife-hunting management (in the 2014 were counted 43,864,720 ha destined to several types of hunting grounds; MAPAMA, 2017).

Introduction

Figure 5. The current distribution of Iberian red deer in the Iberian Peninsula. Dots indicate presence in UTM 10 x 10 km, (modified from Carranza, 2016).

The case of Corsican red deer The origin of the Sardinian-Corsican deer (Cervus elaphus corsicanus; Erxleben, 1777) still represents a story with some unclear points, there are authors who hypothesize the arrival of the species due to migration during the glaciations of the quaternary and a great regional differentiation of Cervus on Late Middle- Pleistocene (Di Stefano & Petronio, 2002), other authors highlighted that C.e. corsicanus could have African origins (Ludt et al., 2004; also looking at some common characteristics of the African and Sardinian antler, in Pitra et al., 2004), whereas others emphasize the artificial introduction by man in times not far away (Hmwe et al., 2006). According to the first hypothesis, the four glaciations of Gunz, Mindel, Riss and Würm or cold glacial phases, which characterized the European Quaternary, were separated by interglacial periods during which the climate was sometimes warmer than it is at present. During the glaciations, there were migrations of the fauna and had the opportunity to migrate from the continent to the islands through the mass of ice that connected to each other (Gibbard & van Kolfschoten, 2004).

Introduction Part of the Sardinian fauna has undoubtedly originated in relation to the glacial phases. So, it could possible that the first deer arrives in Sardinia during the second glaciation (of Mindel), which dates from 500000 to 250000 years ago, along with many representatives of the European fauna, driven in Italy by the advance of the glaciers (Beccu, 1989). The predecessor of the deer (genus Megaloceros) became extinct, however, according to the hypothesis today more accredited, due to the excessive hunting of which was the object of the Neolithic man, succeeded to the Paleolithic (Schüle, 1993). Moving forward in time, between the end of 1800 and especially the first decades of the 1900s, in conjunction with the strong deforestation and the intensification of the hunt, Sardinian deer has seen its distribution density and its range shrink considerably (Zachos & Hartl, 2011). Only 100–150 animals remained in the 1970s after the extinction of the Corsican population in 1970. In the 1980s and 1990s, 13 Sardinian deer were introduced to Corsica, and the present number on Corsica is estimated to be about 250 (Kidjo et al., 2007). Population size estimates for Sardinian deer differ among authors and Ecological Niche Factor Analysis for Sardinia has shown that suitable habitats are available throughout the island (Puddu et al., 2009). Presently, the C.e.corsicanus as well as being considered as “endangered” by the IUCN (IUCN, 2004), it is protected by Italian national and regional legislation (art. 2 L. 157/92 and art 5 L.R. 23/98, respectively) and is a target species in the Italian Natura 2000 network (under the European Directive "Habitat" n.92/43/EEC). The Sardinian population, target of our study, only survives in three mountainous areas in the southwest (Pula, Capoterra, Assemini, Uta, Sanrroch and Villa San Pietro), in the south-east (Sette Fratelli mountain) and in the west coast (between Monte Vecchio and Marina di Arbus). However, reintroductions are in progress to several areas of Sardinia and Corsica (Lovari et al., 2007), thanks also to a European project of the "Life" program (see Figure 6). The population estimated during the censuses of the Sardinian deer is approximately

Introduction of 4270 animals (Murgia et al., 2015).

Figure 6. The different stages of the population regression of the Cervus elaphus corsicanus in Sardinia (modified from Beccu, 1989) and the current distribution of Corsican red deer in the areas managed by EFS (modified from Murgia et al., 2015). Grey areas indicate natural or reintroduced areas of distribution: from a ubiquitous presence on the island (1900), to a sharp decline in population (1950), when only three areas of refuge remained in southern Sardinia (1=Montevecchio, 2=Sulcis, 3=Sarrabus). Later, even with reintroduction programs, the deer has spread (2015).

1.3.2.2 Biology The Cervidae family separated into two phylogenetic branches during the middle Miocene (circa 7 Mya; Pitra et al., 2004) and the two resulting subfamilies, the Cervinae and the Capreolinae subfamily, are distinguished by several characteristics such as: structure of the foot (plesyometacarpal in Cervinae), dentition (Cervinae without upper canines, excluding Muntiacus), glands (only in the feet) and growth of the antlers (Fandos & Burón, 2015). Observing the external morphology, red deer shows its upper parts brown coloured and the underparts are paler, with a prominent pale-colored patch on the rump and buttocks; the male have a long dense mane (Nowak, 1999). Compared to the other phenotypes of red deer (former subspecies) in Europe, the Iberian phenotype is smaller and of a more brownish colour, with less prominent black bands on the backside. Its weight could reach 130-150 kg and 80-100 kg in male and female, respectively; whereas the body length could be of 180-220 cm in male and 160-200 cm in female (Azorit et al., 2002b). The red deer shows a

Introduction remarkable sexual dimorphism; males are bigger and heavier, and, in addition, they have antlers; the latter characters is influenced by the environment, population density, nutrition and male weight (Huxley, 1931; Fierro et al., 2002). Antlers are made by solid bony core supported by permanent skin covered pedicle; these appendages of the skull follow an annual cycle of growing/casting. The antler become larger and with more tines in succeeding years, until the animal is mature (Nowak, 1999). More details on antlers could be found in the chapter 1.3.2.4. Usually, animals living in the north of Europe are taller than southern ones living in Mediterranean scrublands, for this reason the Iberian red deer are considered a smaller deer subspecies (Soriguer et al., 1994). However, this depends heavily on the habitat, as deer living in humid part of Spain rich in pastures all year round become larger than those living in dry areas with a much lower plant productivity (García A, pers. com.). For the case of the Sardinian deer it should be specified that: between the red deer distributed in Sardinia and the red deer that was once present in Corsica (before the translocation of new individuals from the Sardinian territories) there was a certain morphological community and are considered subspecies "corsicanus" of the Cervus elaphus. The first morphometric descriptions of this subspecies were already made in 1700s by Buffon and then Erxleben (see Beccu, 1989 for details); these two populations are clearly different from other European population of the genus Cervus: they have shorter legs and show in the antlers an identical structural simplification (see Figure 7).

Figure 7. Photos showing the morphological differences between subspecies of red deer: Cervus elaphus corsicanus on the left and Cervus elaphus hispanicus on the right.

Introduction

The red deer is a gregarious species with some differences in social behaviour depending on the season; for most of the year, and outside of the mating season, the sexes stay in separated herds of 4-7 members. Red deer hinds form small matriarchal groups throughout the year (dominant hind, her young daughters and dependent offspring; Clutton-Brock & Guinness, 1975) and young females disperse themselves when breeding for the first time, establishing home territories overlapping with that of their mothers (Clutton-Brock et al., 1988). Whereas male deer tend to joint in similar age groups after leaving matriarchal group when they reach puberty (Carranza, 2004). In this case, changes in population density, act directly on dispersal of young males (Loe et al., 2010). Contrary to hinds, which live associated more frequently with animals of the same matriline (Clutton-Brock et al., 1982b), males seem to associate with close- ranking deer instead of relatives (Appleby, 1983). There are hierarchical lines of line dominance in the male and female groups, which regulate access to food resources (Appleby, 1980). The growth conditions of the animal, the size and weight of the structure of the antlers have a strong impact on the intra-specific hierarchy, indeed the individuals who have heavier antlers, with greater number of tines and length, have a higher hierarchical position (Ludek et al., 1987). Males usually has greater home ranges than females (Georgii & Schröder, 1983); In Spain, the red deer could occupy 240-417 ha and 655-1185 ha for hinds and male respectively (Carranza et al., 1991). Daily activity often focuses at dusk and dawn; and, at least in Mediterranean environments, deer show more nocturnal activity than diurnal one (Carranza et al., 1991). Red deer employ up to two thirds of the time in feeding behaviours, and the other third in moving and resting (Peiró, 2003).

1.3.2.3 Ecology and reproduction Habitat selection by the target subspecies (Iberian population and Sardinian subspecies) ranges in most of the available habitats, being especially abundant in transition areas with open grassland is mainly influenced by human alterations (forest exploitation and hunting, Theuerkauf & Rouys, 2008). The red deer in the Mediterranean basin has co-evolved with Mediterranean scrublands and forests;

Introduction and in this kind of habitat red deer is a versatile feeder, mixing grazing and browsing: grass is the main feed from later autumn to early summer, whereas branches become important in the summer (Rodriguez-Berrocal, 1978); being summer the most limiting season for feeding of red deer in the Mediterranean habitat (Bugalho & Milne, 2003). According to the reproductive cycle, red deer is a polygynous ungulate. The male intends to copulate with as many females as possible, for this reason the male defends his own harem of females (almost 20 hinds); hinds invest more energy in parental care, and their success depends on the ability of rear calves (Landete- Castillejos et al., 2002). Generally, males could reach sexual maturity from a year and a half of life. However, most adult males (5-7 years old) get to reproduce successfully, due to the close hierarchy that is established during the breeding season (Montoya, 1999; Novak, 1999). Equally, the oldest males (>10 years old) have already little reproductive success, living often alone. The Iberian females of red deer are seasonal polyestrous of short days, which means that the animal has a series of estrous cycles at the time of the year when the days become shorter (Nowak, 1999). Deer females could reach sexual maturity already at 28 months, when the feeding is very favourable; and up to 50% of primiparous can get to give birth to two years of life, as long as in the previous reproductive season they reached 75% of their adult weight (Montoya, 1999). They adjust their reproductive season in order to get births in the favourable season with greatest food availability (spring; García, 2000). Gestation duration is almost 235 days, but some females may delay their oestrous cycle if they don’t reach a good body condition, having oestrous cycles every 18 days (Guinness et al., 1971; García et al., 2002). Gestation length is moderately variable in order to display a high synchrony of births (Scott et al., 2008). The rutting season lasts almost one month, from September to October, when the photoperiod shortens (Nowak, 1999; García et al., 2003) and could be delayed in years of bad nutrition; moreover, sometimes can take place a second rutting behaviour in November, especially when some hinds come late in heat or do not stay pregnant (Montoya, 1999). Thus, generally dominant male could defend better territories and larger harem, but if the rutting period lasts more than one-month, younger subordinate males may substitute the harem stag to mate. The female groups in the harem

Introduction could last about 20 days (Clutton-Brock et al., 1982) in territory defended by the deer male (geographical area where there is high food availability; Carranza et al., 1990). Roaring of rutting male constitute an honest signal advertising the fighting ability and hierarchy status (Clutton-Brock & Albon, 1979); in this manner males prevent dangerous combat and keep female united in the harem (Reby & McComb, 2003). There is usually a single offspring weighing 6-9 kg at birth (Landete-Castillejos et al., 2005), and lactation may last 4-7 months, sometimes longer; as already said before, the offspring will remain with the mother up to one year of age, later, if it is male, then it will be dispersed from the family nucleus, while if it is female it will remain with the matriarchal group.

1.3.2.4 Antler cycle The cycle of growth of the deer antler it is an annual cycle, and in adult animal immediately after the fall of the previous trophy begins the next cycle of development of another pair of antlers; while for the first year of life the growth of its first antlers have a slightly different cycle. The first antler is preceded by the appearance of bone buttons, while the animal reaches the first year of age, that will be its pedicle or stumps on which will develop the antler as solid bone appendages. This incipient pedicle develops from the lateral ridges of the frontal bone and differs from the surrounding skull; indeed, it is formed by columns of cartilage as extensions of the bony trabeculae and the entire structure is richly vascularized (Goss, 1983). Normally, the first antler is an unbranched main spike (for this reason are called “spikers”) and begins growing at puberty (Lincoln, 1971; Bubenik, 1982). Initially the growth-rate is slow and then it accelerates when reaching about 5 cm; this is the point when the growth of the “true antlers” begins (Fennessy & Suttie, 1985). The process of transformation from the pedicle to the first antler pass through an intramembranous to endochondral ossification at the distal zone of cranial appendages (Li & Suttie, 1994) and, during the growth of antlers, the ossification provides a constant formation of the enlargement of the spongy bone of the growth phase and a massive final increase in the deposition of bone tissue (Banks & Newbrey, 1982), with consequent formation of compact bone in the external cortical layer, just before completion of the development of the antlers

Introduction (Muir et al., 1987). Primary antler growth and annual re-growth are initiated from a stem cell niche localised in the pedicle periosteum (Rolf, 2006; Li et al., 2008). Prior to antler casting, stem cells form a swollen rim around the distal pedicle (Li et al., 2005); at his place the osteoclast activity starts the casting of antlers. After the casting, the exposed surface of the pedicle is rapidly covered by epidermis, the wound is healed, the scab is formed, and the formation of the new antler bud and future growth centres occur very rapidly (Goss, 1983). Antler growth occurs at the antler tip, the growing tip is divided into zones (Price et al.2005a) Under the velvet, the perichondrium is localised. This is followed by an intense proliferating progenitor cell layer responsible for growth in length; under this layer the prechondroblastic zone is situated, in this zone cells are richy vascularized and arranged into longitudinal columns. Further proximally, the chondrocytes undergo maturation ant the cartilage matrix is mineralized, later mineralized cartilage is resorbed and completely replaced by bone (Kierdorf et al., 2009). See Figure 8 for details.

Figure 8. Antler growing tip development and zones. (A) pedicle on the antler casting day and swollen rim presence around the edge, (B) formation of the growth centres and of the scab, approximately 10 days after hard antler casting, (C) formation of the main beam and the brow tine, approximately 30 days after hard antler casting. PS- pedicle skin, PP-pedicle periosteum, PCH-perichondrium, V-velvet, GT-’granulation’ tissue, UM- undiﬀerentiated mesenchyme, AP-antlerogenic periosteum, RM-reserve mesenchyme, PCHB-prechondroblasts, CART-cartilage (Adapted from Price et al.2005a).

This first antler grows for about 12-18 weeks (Gómez, 2004) with a growth rate of several centimetres per week (Lincoln 1992; Schmidt et al. 2001). Velvet cleaning occurs in late spring-summer, at the age of 10–15 months (Suttie and Kay 1982). There is a clear relationship between age and body weight at puberty and the quality of first antler growth (Lincoln et al., 1972; Gómez et al., 2006a);

Introduction and also, a relationship with maternal lactation, and therefore with weight differences at weaning; thus a greater milk supply helps calves to develop larger set of first antlers and it may initiate antler growth earlier (Landete-Castillejos et al., 2001; Gómez, 2004). After the casting of the first antler, the following trophies will be formed by an ever more complex structure, with increasing age of the animal (see Figure 9). The second antler is produced starting from the two years, with a structure that already has various tines and a well distinguishable burr. Afterwards, the third antler can already have a number of tines which may vary from 6 to 12 tines depending on the quality of the animal's trophy. The number of tines present in the deer's trophy is related to the length of the same main shaft (Azorit et al., 2002a). However, the increase in size of the antler, in the genus Cervus, with increasing age is not indefinite; thus, there is a quantitative-qualitative decline from 7° to 12° years of life, depending on the species and the diet (Huxley, 1926). As a matter of fact, antler growth appears to stabilize around 8 years of age and male shows a decline in some parameters starting 10 years of age, the tips number does not increase beyond the fifth age class, antler length and thickness increase until the sixth age class (Azorit et al., 2001b).

Figure 9. Example of the development of red deer antlers: in the first years of life. From a simple shaft without branching (year 1), the structure becomes more complex with each passing year. In addition, the bone pedicle, in each growth cycle, is shortened in length (modified from Kowalski, 1981).

The rate of antler’s bone growth, even if fast, does not follow the same speed during its development; generally, the grow rate is initially reduced and then accelerated in the first half of the growth period, and then slowly decreasing

Introduction again, approaching completion of the final structure of the antlers (forming a sigmoidal curve; Goss, 1983). The average value of growth could reach 0.67 cm/day (Gómez et al., 2013) and the duration of the growth is about 158 days to reach full adult structure (Gaspar-López et al., 2010), and 124 days for spikers or 88 days for the second antlers (Gómez, 2004). During the development of the antlers, the tissue is formed by mainly from cartilage with blood vessels and a hairy dermis (velvet); the latter is formed by tissue characterized by the absence of subcutaneous connective tissue and sweat glands, but with sebaceous glands. The growth of antlers takes place from the fall of the previous antlers (at the beginning of spring, e.g. March) until the beginning of the summer (July-August). Subsequently, the blood network is reduced, through a strict hormonal control, and the bone in the meantime is already mineralizing following the cranial-distal direction (from a ≈20% to almost ≈60-70% of mineral content when the bone tissue is fully mature; Bubenik & Bubenik, 1990). On this topic, Gomez et al. (2013) showed that initially a trabecular scaffold of woven bone is formed which largely replaces a pre-existing scaffold of mineralized cartilage. Lamellar bone is then deposited and from about day 70 onwards, primary osteons fill in the longitudinal tubes lined by the scaffold in a proximal to distal sequence. During the initial phases of primary osteon formation, the mineral apposition rate in is very high (average 2.15 μm/d); also, they suggest a peak in mineral demand around day 100 when the extent of mineralizing surfaces is maximal. At the end of August, deer males begin to lose the characteristic velvet that covers the external surface discovering the hard antlers, by scraping of the antlers’ surface on various substrates that they encounter in the environment (Li & Suttie, 2012); this activity is also useful for marking their territory, just before the start of the breeding season (rutting) (Clutton-Brock & Albon, 1979; Reby & McComb, 2003). See Figure 10 for details. Red deer at least shows a form of “reversible osteoporosis” in the skeleton. Because antlers formation imposes a high demand for bone mineral precursors that cannot be fully met from the diet, thus undergoing a great bone resorption of the skeleton to satisfy this demand (Baxter et al., 1999) this has been termed cyclic physiological osteoporosis (Borsy et al., 2009; Stéger et al., 2010). As a

Introduction curiosity genetic studies have shown gene expression similarities with human osteoporosis and pointed out potential interest for medicine of studying this process. In addition, a high correlation was observed between the development of the frontal bone (distance of the frontal apophyses) and certain characteristics of the trophy, such as the width of the burr and the length of the main shaft (Zedja & Babicka, 1983), this is in line with the concept that animals with good bone development have positive repercussions on the development of the future antlers.

Figure 10. Timeline scheme of adult antlers’ development in Iberian red deer. In March-April, the new growth begins once the previous antlers are casted (A); then, there is a period of growth in which the antlers are covered with velvet, in which is present the blood circulation and are innervated (B); in August, thanks to a hormonal change, the blood supply starts to decrease and the antlers lose its characteristic velvet (C); after this phase, the antler persist on the head of the animal in a "hard" bone form until the spring of the following year when they will be cast (D).

Generally, at the completion of antlers’ growth, the main beam made up approximately 71% of the total volume, the brow tine 7%, the trez tine 8%, and the crown 14%, and also, during this growth the more proximal tines complete their development before the more distal ones, while successive stages of growth may be taking place simultaneously in different branches of a growing antler (Fennessy et al., 1992). However, it is important to remember that certain trophy characteristics belong to a species but not to another, such as the presence of a trifurcation in the crown area, which is a characteristic of the European red deer while it is absent in the Wapiti (Goss, 1983). For the species studied in this thesis,

Introduction we must note a certain difference in the antler’s morphology between the European red deer and the red deer of Sardinia. Generally, in European red deer the well-developed antlers measure up to 80-90 cm in length, with a supernumerary tine (bez tine) above the brow tine and a central tine (Groves & Grubb, 2011). While the Sardinian deer has a more simplified structure with one central tine, one tine at the base of the main beam, and one/two tines forming the crown (if present two tines, they are aligned on the same plane) (Beccu, 1989).

Introduction 1.3.3 Species as a study model: Roe deer (Capreolus capreolus)

Kingdom: Animalia Phylum: Chordata (Bateson, 1885) Class: Mammalia (Linnaeus, 1758) Order: Artiodactyla (Owen, 1837) Suborder: Ruminantia (Scopoli, 1777) Family: Cervidae (Goldfuss, 1820) Subfamily: Capreolinae (Tribe: Capreolini, Brookes, 1828) Scientific name: Capreolus capreolus (Linnaeus, 1758)

1.3.3.1 Distribution and diffusion history The first person who defined the roe deer, under the taxonomic aspect, was Linnaeus in 1758 calling it "Cervus capreolus". Later Frisco, in 1775, name of it Capreolus and Gray in 1821, consolidated the generic name Capreolus. The European roe deer Capreolus capreolus was already present in Europe at least 600,000 years ago and it has been known from both glacial and interglacial phases since then. During the Middle and Late Weichselian Pleniglacial, the distribution of the roe deer was not restricted to the Mediterranean peninsulas but reached regions of central Europe; in contrast during the maximum extent of the ice during the last glacial period, roe deer population were largely confined to the Mediterranean peninsulas (Sommer et al., 2009). The combined pattern of genetic data and fossil records of European roe deer suggests several regions in the Iberian Peninsula, southern France, Italy and the Balkans as well as in the Carpathians or eastern Europe as glacial refugia (Fandos & Burón, 2015). Thus, roe deer might have recolonized most parts of central-northern Europe out of one eastern European or Carpathian refugia (Sommer et al., 2009). Other authors (Royo et al., 2007), highlighted the presence of two Iberian groups of roe deer, a central-southern group and a north-western group, and they inferred two Iberian glacial refugia. Similarly, Lorenzini & Lovari (2006) confirmed the presence of three roe deer lineages: an Iberian lineage, one from eastern and southern Europe (Romania, Poland, Lithuania, Italy, Turkey) and a third one from central

Introduction and northern Europe (France, Denmark and Sweden). The Italian Alps and Austria were somewhat intermediate between the south-eastern and the central- northern lineages. Currently, the roe deer species are spread all around Europe and Asia continent (Figure 11). As matter of fact, its distribution comprises most of Europe except for Ireland, some Mediterranean islands, northern Russia and the tundra regions. In Russia, its distribution is bordered by the western range of the Siberian roe deer Capreolus pygargus, whose species status has been supported by both morphological and genetic evidence (Danilkin, 1995; Lister et al., 1998). Genetic differences between European and Siberian Capreolus, suggest a prolonged period of divergent evolution that could be explained by the occurrence of natural barriers between Europe and Asia in the ancient glaciations (Semperé et al., 1996).

Figure 11. Distribution of roe deer species in Europe and Asia. (Image modified from Fandos & Burón, 2015).

The western roe deer (C.capreolus) seems to have extended its range at the expense of the C.pygargus in modern times (Groves & Grubb, 2011). Other studies (Randi et al., 2004; Lorenzini and Lovari, 2006) confirmed the existence of numerous subspecies, some example are: C.c.italicus (Festa, 1925); C.c.garganta (Geist, 1998), although whether this name may refer to the subspecies of roe deer in South Spain (Royo et al., 2007; Lorenzini et al., 2013);

Introduction C.c. capreolus (Linnaeus, 1758). The identification of C.c.caucasicus as the correct name for a large-sized subspecies north of the Caucasus Mountains is provisional (Sempéré et al., 1996; Lister et al. 1998). Animals in the Near East have been assigned to C.c.coxi (Harrison & Bates, 1991). However, this division into subspecies is debated and the existence of these subspecies is likely false for the same reasons that affects C.elaphus: 13,000 years ago, in the last glacial maximum (LGM), they were refuged in Iberia and Balkans, and 13,000 years is not considered enough time to evolve a subspecies after spreading form these refuges (80,000-90,000 years are required, see chapter 1.3.2.1) For more details on morphological differences of the two species (C.capreolus and C.pygargus) see the following chapter 1.3.3.2. on biology. During the late nineteeth and early twentieth century there was a serious decline in roe deer numbers and distribution in Europe, but its distribution range has been restored and even extended northward (Semperé et al., 1996; Thulin, 2006). On figures, the roe deer is the most widely distributed and abundant large herbivore in Europe (estimated 10 million roe deer; Linnell et al., 1998; Apollonio et al., 2010a), and due to its ecological plasticity the roe deer have adapted very well to the human-dominated landscape, preventing from being reduced to small isolated populations due to habitat fragmentation; nevertheless, populations could be affected in some genetic features by human settlements (Wang & Schreiber, 2001; Coulon et al., 2006). Roe deer, as well as red deer, is among the most important European game species, and hunting activities accounts in Europe for a great number of individuals culled during hunting period, with an important economic return on the areas where the hunt takes place. Examples of animals hunted annually are: in Germany at least 500,000 individuals (Novak, 1999); in Italy 70,170 individuals (for 2009-2010; Pelliccioni et al., 2013). In Spain there are 41,853 individuals (MAPAMA, 2017), although the species is expanding.

1.3.3.2 Biology The roe deer is an ungulate mammal and is characterized, as the rest of the taxonomic family, by stepping on the two central fingers of each extremity, protected by hooves and is part of the deer characterized by the telemetacarpal

Introduction structure of the leg (Capreolinae subfamily where are also the tribes of Alceini, Odocoileini; Pitra et al, 2004). With respect to these last two species, the roe deer presents, however, other divergent characteristics, as for the not well-developed glands of the eye canthus and the shed of the antlers in autumn, while in other telemetacarpalian species the antlers are shedded from January to April (Semperé et al., 1993). Even if the systematics of the genus Capreolus, especially the subspecies level, is still not clearly understood (Lorenzini et al., 1993), within the genus Capreolus have been recognized two species: Capreolus capreolus or European roe and Capreolus pygargus or Asian roe deer (Geist, 1998), the same result was found by Randi et al. (1998), who studied control region haplotypes of European and Siberian populations of both species and found them to be different. Moreover, C.capreolus and C.pygargus also differ in the number of chromosomes due to an extra B-chromosome in the latter (Danilkin, 1995). Similar to white-tailed deer in America, roe deer have a high genetic heterozygosity and may differ sharply across small geographic distances (Hartl and Reimoser, 1988) In general, all types of roe deer, at least externally, have similar characteristics: a uniform colour tending to greyish in winter (except the caudal white spot) and tending to red in summer; with fawn’s fur covered with small light spots. The pelage goes through a seasonal change of colour: the winter coat of the different populations of European roe deer is longer, it has a grayish-colored blot, while in summer the flanks of the body and the back are covered with shorter hair of reddish coloration; this colour change in addition to having camouflage motifs, could be based on thermoregulation, by selecting darker colours to absorb more heat (Johson & Hornby, 1975). All have front legs with a shorter morphological structure, compared to the posterior ones. The tail is rudimentary (2-3 cm) and the antlers, present only in the male, are small and not very branched. The antlers in the C.garganta (South Iberian subspecies) roe bucks are the same shape as those of European roe deer. Regarding to the general characteristics of roe deer, its external dimensions have a variable length between 100 and 145 cm and a weight between 18 and 59 kg, with low male-biased sexual size dimorphism (males are less than 10% heavier than females: Andersen et al. 1998). However, there are differences within this

Introduction morphological group, given that there is a certain important geographical variability, and for this reason different subspecies or "ecotypes" have been described in all the areas of its distribution (Fandos & Burón, 2015), this is a controversial point, because they differ mainly in size, not in external body characteristics as do red deer subspecies (Geist, 1998). The colour of the winter coat is greyish-brown with a large white caudal patch, whereas during summer its coloration is reddish-brown, metatarsal gland are brown and caudal patch is less marked. Antlers are short (about 230-280 mm long and 350 g of weight) and slightly roughened at the base with normally three tines, and with an annual cycle of growing-shedding (Bubenik AB, 1990); for more details on antlers see chapter 1.3.3.4.

1.3.3.3 Ecology and reproduction Although the roe deer is primarily a woodland species, adapted to exploit the initial stages of forest succession where their preferred food is most abundant (Wahlström, 1995), it occupies today a wide range of habitats, including coniferous and Mediterranean forests, shrublands, moorlands and marshes and may even adapt to open agricultural areas (Danilkin & Hewison,1996). Roe deer is classified as selective feeder (concentrate selector; Hofmann, 1989) preferring soft, energy-rich foods containing much water; due to the small volume of stomach and the relatively rapid rate of food transit in the digestive system, the roe deer requires frequent food intake. To be more precise, this feeding behaviour is present more in summer when they select highly digestible forage, but in winter they could switch to higher-fibered forage, (Holland & Staaland, 1991). The Roe deer live alone or in small groups, with a more accentuated seasonal gregarious behavior in the central European populations (Villarette et al., 2006). Aggregations of almost 100 animals could be found at favourable feeding site, especially during autumn-winter; although groups are unstable, the individuals in a given geographical area have a dominance hierarchy (Bresinski, 1982). Starting from January, animals begin to re-locate themselves, the adult males and mother-fawn groups occupy their territories (generally the same territory as the previous year), while the young males disperse in search of new empty territories, and if two animals of the same sex meet at such time, they may fight. Such

Introduction territories can reach 2-200 ha. for males and 3-180 ha. for females with overlaps between adjacent territories of females and males, and these areas represent only a portion of the total home range (Fruzinski et al., 1983). The Roe deer is essentially sedentary as every spring adult male (>3 years) hold the same territories. However, some populations of roe deer of northern Europe, males have been observed to migrate in order to return to occupy the areas of reproduction of the previous year (Mysterud, 1999). The reproduction activity of this species, compared to the red deer, follows a strategy closer to the "r strategy" (many descendants, and each with a low probability of survival; Fandos & Burón, 2015). Adult females show a fecundity level of 1.7 (Hewison, 1996) and pregnancy rate is generally estimated as 90% or more (Kaluzinski,1982). One of the regulatory elements of most reproduction processes is the biological clock associated with light, which can work in two ways: on the one hand the changes in the hours of light and darkness mark the circadian rhythms, while on the other hand, changes in the durability of periods of light mark the circannual rhythms. Natural light inhibits the production of melatonin in the pineal gland, while at night it activates its production; in the winter, with shorter days, there will be more melatonin circulating in the body, compared to the spring-summer days. Melatonin along with other hormones produced in the hypothalamic-pituitary axis (GnRH, FSH, LH), and through an adjustment of the amplitude and frequency in their release, regulating gonadal activity, and thus the production of testosterone (Roelants et al., 2002), the latter is one of the most important hormones that regulate the growth, mineralization and casting of the antlers, as well as regulate the breeding cycles of the species (Bubenik GA, 1990). During winter, when testosterone levels are low, males are non-territorial while their antlers grow, covered with velvet. As the secretion of testosterone increases during spring, male tolerance decreases, their antlers finish mineralization, and, in late spring, antlers finally shed their velvet. The increasing aggressiveness explains the onset of territorial behaviour, which commonly occurs before males get hard antlers (Johansson, 1996); full details on the antler cycle will be discussed in the next chapter (1.3.3.4). The reproductive process in the roe deer is associated with the long days, which means that the process is activated with the increase of the photoperiod and has

Introduction its maximum activity in spring-summer (Reganella-Peliccioni et al., 2007). For this reason, the roe deer mating season occurs from July to September (in Europe, and 1 month before in Spain) and sexual maturity is reached at 15 months of age for males; but males usually do not defend territories until three or four years of age, depending on population density (Liberg et al. 1998; Fandos & Burón, 2015). Females normally attain sexual maturity as yearlings, but under poor conditions first reproduction is often delayed one year until the age of 3 years old (Hewison 1996); whereas male bucks attain sexual maturity at one year, but they do not reach their full reproductive capacity until 3 years old (Semperé et al., 1996). Each male breeding territory is marked with urine, secretions from glands and vegetation destroyed by the antlers, and during the mating season a male usually fight other intruder males (Liberg et al. 1998); during the territorial period, the degree of overlap between adjacent adult male ranges is low, particularly at low male density (Danilkin & Hewison, 1996). Moreover, the roe deer’s rut lasts for 2- 5 days and males enter in a rutting inappetence during the chase and couplings with the females. The larger the territory occupied by a male, the greater the chance it will have to cover a greater number of females. During rut period, most antler contacts in roe bucks result in short benign fights in which the antlers could lock, allowing full-power wrestling matches, but damaging combats are rare (Hoem et al., 2007). Nevertheless, the level of fighting escalation is influenced by the presence or absence of a territorial-male (from 41% to 15%, Hoem et al., 2007). Generally, the subordinate male may break off the fight and then flee (Geist, 1998). A characteristic note concerns the females of the species, they have a birth synchronization (also as a survival strategy at the population level) and, even if there is not a strictly latitudinal component, the first births for European population are recorded in areas of southern Europe (Italy, Spain) while the last births take place in northern areas (Linnell & Andersen, 1998a). Moreover, females present a monoestrous cycle (Kozdrowski et al., 2005) with a duration of oestrus being 36 hours (Strandgaard, 1972). Females exhibit embryonic diapause, that means that does, after mating season, have a retarded implantation of the fertilized egg in uterus with a delay of about 5 months (Lambert, 2002) and in total, the gestation is almost 10 months. The fertilized egg

Introduction divide itself in a blastula within 14 days after fertilization, and then pauses in development and implants in the uterus till early January (Stubbe & Passarge, 1979), being the winter solstice the signal that causes the implantation. The gestation period from implantation to birth is short, 150 days, and births of fawn occur in the spring (April-May), with a number of fawns between one or three, more commonly there are two (the birth take place in quite place far from the offspring of the previous season). See Figure 12. The weight at birth is around 1.7 kg and lactation could last till 90 days (Nowak, 1999). The grow is fast and, normally, by autumn the 60-70% of the body mass of adult individuals is attained (Semperé et al., 1996).

Figure 12. Reproductive cycles of European roe deer: for male with the phases of the antler cycle (external circle, blue words) and female (internal circle, black words).

1.3.3.4 Antler cycle The roe's antlers have a very marked annual cycle; they normally begin to grow around November, once the previous antlers have fallen (for adult animal), whereas for young bucks before to develop the first antler, in their first year, a “button” on top of their pedicle is present. The growing antlers are covered with “velvet”, an innervated soft tissue with blood vessels which is very sensitive. The antlers are usually fully developed by March, when testosterone levels cause the blood supply to the new antlers to be cut off. The velvet then dries out and the bucks rub their antlers against trees and shrubs

Introduction to remove the velvet covering; this behaviour appears when the males delimit their territory, losing tolerance towards other conspecific males (Zejda & Bauerova, 1990). See Figure 12 for details on roe deer antler’s cycle and reproduction and Figure 13 for a photographic description of the cycle.

Figure 13. Photos illustrating the growth cycle of the antlers of roe deer: closing of the scars after the fall of the antlers of the previous year (A); rapid growth of the new antlers and subsequent branching (B, C and D), at this stage the appendices are covered with velvet, with the presence of nerves and blood capillaries; cleaning from the velvet with gradual exposure of the bone tissue already largely mineralized (E); horns already clean formed of "dead bone”, ready to be used as a defense/offense weapon (F). Images modified from Sherer, 2009.

Mainly, the growth of the antlers occurs through hormonal regulation (Semperé et al., 1992). As a matter of fact, the fall of testosterone levels that occur during the autumn-winter exerts a stimulating action of the growth of the antlers of the males, whereas the increase in the secretion of this hormone during its breeding season (late spring-summer), causes the transformation of the bone tissue (Semperé et al., 1992). However, there are other hormones that play a key role in the growth of the antlers, including gonadotropin and luteinizing hormone (LH),

Introduction and thanks to this complex endocrine regulation the trophy growth is very fast (up to 3 mm per day; Semperé & Boissin, 1981). Furthermore, there are additional factors that influence the growth of the antlers which, however, could delay or accelerate the phenological cycle of the antlers. An example is the population density, so that a greater number of animals correspond to an increase in asymmetries in the roe deer trophy (Pelabón & Van Breukelen, 1998). For this reason, these authors hypothesized that the development of the roe deer antlers represents a reliable signal of the quality of the individual. In a similar way as it happens in the red deer (section 1.3.2.4), Mateos-Quesada (2005) also points out that the antlers variations could be related in many cases with the type of feeding, the habitat, the relationship between the different males, or by genetic components (again, as it has been proved in the case of red deer). For more details see chapter 1.5.1. Typically, when fully develop, an adult roe deer male has three tines on each antler: a brow tine (frontal tine), a top tine (distal tine) and a back tine. The base is called the coronet or burr, which is formed on top of the pedicle on the roe deer’s skull. As the buck matures, the coronet may fuse together; sometimes the lower parts of the antler are covered with a pearling (small bony nodules of wrinkled appearance). The colour is generally dark reddish-brown or greyish- brown, fading slightly towards the tines. During the last years of life of the animal, in the senescence, the antlers begin to recede to simpler structures and lose their well-defined shape; they are then described as “going back” (Fandos & Burón, 2015). The same pattern was observed by Vanpé et al. (2007) who have found differences in the size of the antlers depending on the age classes. They observed a growth until the seventh year of life and a decrease in size after that age (senescence). In addition, same authors observed that the size of the trophy increased allometrically with body mass. This allowed them to hypothesize two reproductive strategies for senescent males: the older, heavier males invest in the growth of the antlers which, potentially, would allow them to compete for the territories; while the lighter old males develop smaller antlers, probably abandoning the protection of the territories. However, it may also happen that males attempt to grow the largest antlers possible and senescent males unable to mobilize bone material for antlers lose their territories as a conseguence (but

Introduction this would not be a specific strategy). Sometimes the presence of male supernumerary antler or the presence of pedicles in females have also been described in this species, mostly related to hormonal changes, undeveloped sexual organs, or traumas in the frontal bone (Mysterud & Østbye, 1999).

Introduction 1.4 Horns 1.4.1. Structure The horn is formed by an epidermal and hollow structure (keratin sheath) that covers the horn bony nucleus (cornual process). It presents a double layer of fused tissues interposed between these structures: the periosteum adjacent to the core and the corium adjacent to the sheath; these tissues produce annual additions to the sheath as well as to the core, and they are highly vascularized (Taylor, 1962), see Figure 14 for details. The bone core grows through intramembranous ossification and the keratin sheath is deposited in layers over the surface of the horn core, with each new layer adding to the accumulated keratin (Davis et al., 2011). Generally, the shape of the keratin sheath seems to correspond closely to that of the bony core in most taxa (Calamari & Fossum, 2017).

Figure 14. Example of a growth scheme of the horn case and the cornual bone nucleus in Bovidae (Ovis canadensis). Horn grows at keratin sheath/bony core margin, pushing older sheath towards the tip (A); First and oldest keratin layer, deposited as a juvenile, is maintained at horn tip (B). The newer horn layers, deposited as an adult, growing under older layers (C). Modified from Goss, 1983.

Basically, they differ from antlers in that they are non-deciduous and unbranched appendages; moreover, unlike the deer antlers, which are bone structures, the

Introduction horns of the Bovidae are formed from structures of dermal origin that subsequently merge with the frontal bone (from os cornu to processus cornualis), giving the appearance of a hypophysis (Santiago-Moreno et al. 2010). For more details on the growth process of horn and the factors affecting it, refer to the chapter 1.4.2.4 and chapter 1.5. The keratin sheath consisting of keratin lamellae periodically separated by tubules that extend the length of the horn. The structure formed a laminated composite arrangement that consists of fibrous keratin characterized by a porosity gradient across the thickness of the horn. This lamellar structure shows an elliptically shaped porosity (from 60-200 μm) interspersed between the lamellae of 2-5 μm thickness (McKittrick et al., 2012). It is composed primarily of α-keratin by polypeptide chains (Tombolato et al., 2010). See Figure 15 for details.

Figure 15. Hierarchical structure of bighorn sheep horn’s keratin sheath. The horns’ keratinous tissues are composed of elliptical tubules, embedded in a dense laminar structure. Each lamina has oriented keratin filaments interspersed in a protein-based matrix. These filaments are two-strand coiled-coil rope polypeptide chains (intermediate filament) helically wound to form ‘‘superhelical” ropes 7 nm in diameter. (modified form Tombolato et al., 2010).

Their mechanical features involved both the keratin sheath and bony core; as matter of fact, keratinized tissue plays an important role in the resistance and

Introduction stiffness of this structure, while the bone nucleus, as well as giving stability to the horn, serves to cushion the strokes suffered and reduce the forces transmitted on the cranium. Moreover, the bony core could undergo trough remodelling of its own tissue in case of damage, and it presents a porosity and a pore size variable decreasing from the distal to the proximal end near the skull (in cattle horns from 67% to 51%; Li et al., 2011). And the same shape of the horn promotes an optimal mechanical yield, according to its completeness and development (Drake et al., 2016). Its shape follows the structural design rule of “equal strength” (a superior load-bearing capacity with little material) and it also allows a uniform distribution of forces in the longitudinal direction. In this line, Kitchener (1985) hypothesized that changes in bending strength of horns (as indicated by the “second moment of area” of the base of the horns) would balance the bending stress suffered by horns during fights, and species with more forceful fighting (e.g.: sheep) have a larger diameter of base of the horn relative to body weight than those with weaker fighting styles (some antelope). The horns, like the antlers, have different functions: from that of playing important role of defence and attack weapons, to that of being used as a display for intraspecific communication, as courtship display (Geist, 1966) or recognition between animals of the same species (Coulon et al., 2007). Thus, the presence of broken horns could produce social disadvantage in the herd for the lack of self- protection ability (Leuthold, 1977). Moreover, the bearer of a trophy with the best symmetry and development, as well as having an advantageous physical situation, is an animal that reaches a higher hierarchical status (Moller et al., 1996) and, in the case of male sex, it can participate more actively in reproductive activities, with a greater number of females covered (Preston et al., 2003). On the same line, Côté & Festa-Bianchet (2001) suggested that secondary sexual characters, such as horns, exert a signal between the sexes as indicators of quality and genetic vigor, given that females select males with better development and symmetry of these characteristics. The morphology of horns reflects differences in fighting behaviour (Caro et al., 2003) and there is a strong correlation between horn size and the intensity of sexual selection within a species (Bro-Jørgensen, 2007). Thus, Geist (1966), and later by Lundrigan (1996), recognized three stage in the evolution of shape of the

Introduction horns: firstly species with small spike like horns and opponents stand parallel during fights and the attack are aimed at the opponent’s flank; the second stage is characterized by larger and more complex horns which are used mainly for defence; and the third stage included the “rammers” and the “wrestlers”, in which the head of the opponent it is the primary target of the fight. The common eland becomes part of this last group, where the fighters join in wresting with the horns locked with each other, and the movements are rotatory and pushing. Moreover, Calamari & Fossum (2017) related that horns’ bony cores are distinguishable in geometric morphometric analyses, extending the possibility of using geometric morphometrics to study the ecology and evolution of bovid horns, including the fossil record.

Introduction 1.4.2. Species as a study model: Common eland (Taurotragus oryx) Kingdom: Animalia Phylum: Chordata (Bateson, 1885) Class: Mammalia (Linnaeus, 1758) Order: Artiodactyla (Owen, 1837) Suborder: Ruminantia (Scopoli, 1777) Family: Bovidae (Gray, 1821) Subfamily: Bovinae (Gray, 1821) Scientific name: Taurotragus oryx (Pallas, 1766)

1.4.2.1 Distribution and diffusion history The majority of extant bovids occur in Africa (79 species), but wild bovids also are found in Europe, Asia and North America (Lundrigan, 1996). The natural habitat of this species is the savanna biome of sub-Saharan Africa harbours which are considered biogeographical regions with a great diversity of ungulates, due to the high level of spatial patchiness in the subregion (Owen-Smith & Cumming, 1993). From the study of fossil of African Bovidae (Vrba, 1995), it was observed that some oscillations between wetter and drier climates repeatedly promoted cycles of population’s range contraction and expansion in the late Pliocene (circa 3 Ma). During wetter climates, when forests replaced savanna habitat, population contact were reduced, and such isolated areas harbouring stable savanna habitat functioned as refugia and enabled the survival of species resistant to arid climates, promoting the divergence of populations (Hewitt, 2004). Thus, a hypothesis of Pleistocene refugia occurring in East and southern Africa was formulated, with the presence of a longer-standing population in the south and a mosaic of Pleistocene refugia in the east (Lorenzen et al., 2010). The first ancestor of Taurotragus genus, to which the Common eland belongs, first appeared in the fossil record discovered in Tanzania 0.96–0.6 Ma (Vrba, 1995). Currently, the Common eland is naturally widespread in African regions (Ethiopia and Southern Zaire to South Africa), but is often found in private or experimental breeding farms even outside the African continent given the docile and easily tame behaviour (Nowak, 1999); some examples are New Zeland, U.S.A. and

Introduction East Europe. In South Africa, only in the northern regions, could be found about 30,000 farmed animals (data from Van del Waal & Decker, 2000; these data are from 1994, this means that there could be many more today). There are two species of Eland (Genus Taurotragus): the Taurotragus oryx (or Common eland) and Taurotragus derbianus (or Derby eland), following Meester & Setzer (1977). Moreover, another author (Kingdon, 1997) recognized three subspecies based on differences in coat colour and number of stripes: Taurotragus oryx pattersoni in East Africa, Taurotragus oryx livingstonii in the central woodlands, and Taurotragus oryx oryx in southern and Southwest Africa. However, this latter theory requires investigation (Thouless 2013). Nowadays, Common eland diffusion includes a growing number of individuals in protected areas, and have been estimated at ca. 136000, about 50% of which occur in protected areas and 30% on private land (Bujis et al., 2016). Elands suffers poaching hunt to which must be added a wide natural habitat loss, these two causes have led to their disappearance from much of their original range, although Common elands are still widely distributed and well represented in national parks and are even semi- domesticated as exotics in several countries (Kingdon, 1997), with a population density between 0.4 and 1.0 individuals/km2 (East, 1999). Moreover, the Common eland have been reintroduced to a number of game ranches and private ranch-land, reflecting its value as a trophy animal, and this hunting interest has done much to increase the number of individuals of this species (Thouless, 2013). See Figure 16 for details.

Introduction

Figure 16. Distribution of Common eland in its original natural habitat in Africa (modified from IUCN, 2004).

1.4.2.2 Biology The head and body length could reach 1.8 to 3.45 m, shoulder height is 1-1.8 m, and the weight is included between 400 kg and 1000 kg, Eland males are much larger than females. Both species have white vertical stripes on the upper part of the body and its neck is black, the colour of Common eland is a fairly uniform greyish. A dewlap with longer hairs, thought to be an adaptation for heat dissipation, hangs from the throat and neck (Fahey, 1999). This species is characterized by massive rounded hooves, a hump on the withers and the presence of spiralled horns in both sexes. In males the spiralled horns are used like display organs and for pushes/twisting movements in clashes during confrontation in the reproductive periods. In contrast, in females, the horns are used more for the defence against predators, with short charges and attempts to stab the opponents. Every confrontation between males includes ritual movements: each male lowers its horns and eventually interlock them, pushing

Introduction forward and twisting their heads and neck until one of the bulls turns away. Sometimes, the sharp and spiralled horns of animals can inflict considerable damage (Nowak, 1999), and are very effective as weapons even in the sub-adult animals (Kiley-Worthington, 1978). The spiral ridges of the horn are used to guide the head down and bind the horns together to make the male-male fight less dangerous; thus, the presence of the spiral has been proposed to increase grip in wrestling between males (Geist, 1966; Caro et al. 2003). This hypothesis follows the line of Solounias (2007) that explained that wrestling was associated with twisted horns in polygynous bovids. However, in this species, no excessively aggressive behaviour was observed; this could be related to the fact that the Eland does not defend a territory in the strict sense, and often can tolerate the presence of other males in the group (Underwood, 1975).

1.4.2.3 Ecology and reproduction Common eland is one of the most adaptable ruminants, inhabiting sub-desert, savannah, woodland, and alpine moorlands until 4900 m asl. They are not found in deep forest, in true deserts, though they do occur in grassland with good herb cover (Thouless, 2013). This species is a browser of intermediate types (Hofmann, 1989); they feed on the leaves instead of lignified plant parts (Kerr & Roth, 1970), while selecting for the lowest fibre content forage types (Watson & Owen-Smith, 2002). The diet of elands consists of grasses, herbs, tree leaves, bushes. Water is consumed when available, but elands can abstain from drinking for prolonged periods, thus it is likely that they are able to meet much of their water requirements from their diet (Pappas, 2002). In private or experimental breeding farms, it’s demonstrated that the growth performances of domesticated eland may be improved by supplementary feeding (Treus & Kravchenko, 1968). In natural habitat, common eland uses the habitat with seasonal migration driven by changes in forage quality (Fabricius & Mentis, 1990), and the home range vary considerably depending on the sex and the season, with much overlap and no exclusive use of space between individuals. The herds could be formed from 25 to 700 individuals with cows, subadults and more large bulls, in any case larger groups were basically temporary aggregations of females and calves during wet season and no

Introduction enduring social bonds were observed except those between mothers and nursing offspring (Nowak, 1999). Breeding and calving seasons in natural habitat have not been clearly identified, and probably vary from one population to another (Kingdon, 1982). In Southern Africa females have been observed to give birth from August to October and to be joined by adult males from late October to January, but in other geographical region the calves are born in July-August (Wilson, 1969). The average estreous cycle is 23 days long, estrus lasts 1-3 days and gestation is an average of 255 days (Hayssen et al., 1993 in Novak, 1999). Normally, there is a single calf of 22- 36 kg; and lactation can lasts 4-5 months (Hillman, 1987). Sexual maturity is attained at about 3 years and 4 years buy females and males, respectively; but this can change in captive animals with a more extensive reproductive life (first birth at 22 months and last at 19 years; Treus & Lobanov, 1971). During the reproductive season, several adult males could be present in female herd, these males have got a strictly dominance hierarchy that governs access to oestrous females (Leuthold, 1977); thus, bulls can fight each other with violent clashes using their own horns.

1.4.2.4 Horn growth The growth of the horn occurs at the base level and persists throughout the animal's life. In a first phase, the specialized epithelial tissue, called the germinal epithelium (button), located in the frontal part of the skull, initiates an epidermal growth forming a cornified external sheath. This sheath that is hollow and fragile continues to grow, with annual differential velocity, in successive layers forming the keratin sheath (Santiago-Moreno et al., 2001). During this phase, any trauma can generate malformation or the stop of the horn development. In a second phase, the frontal bone initiates a proliferation of bone tissue (with a propagation of osteoblasts towards the inside of the horny envelope), forming the os cornu as an isolated bony element, that during later development fuses with the cranium, forming the processus cornualis of the frontal bone. The grow of the bony horncore follow this scheme: grows in length at the tip and appositional at the surface, leading to an increase in diameter, with a gradual deposition of compact bone from base to tip (with a continuous remodelling process; Davis et al. 2011).

Introduction However, the increase in diameter is also given by the keratinization of the germinative epithelium inside the horn. Although the horn grows throughout the life of the animal, the growth rate slows significantly as the animal ages (Lincoln, 1998). The target species of this thesis, the Common eland, follows the same pattern of growth described until now. Horns are present in male and female, in the former the cornual appendages are massive and more divergent, while in females they are slenderer and slightly longer. The growth rate decline gradually up to 20 to 22 months of age and the subsequent growth in length is very slow (Kerr & Roth, 1970). Generally, after 8-9 months of age the distinctive spiral rib becomes evident and horns curve inwards slightly; by the 15 months of age the first spiral rib (closest to skull) are already very prominent in male, then the horn becames progressively heavier, particularly at the base. Some authors hypnotised that the growth of bovid horns is often indicative of population characteristics and habitat quality (Geist, 1998). Thus, Wishart & Brochu (1982) highlighted the possibility to use the average pattern of annual horn sheath growth, with the body growth, to compare the populations and evaluate management options. This is a key point when the size limits of trophy hunting are evaluated often in terms of horn size and hence are influenced by horn growth (Feldhamer et al., 2003). Moreover, Loher et al. (2007) suggested that age and horn size were overall much better predictors of annual reproductive success for a ram. On the same line, Robinson et al. (2006) showed that the normal horn type and larger horn size, in Ovis aries, could be associated with greater annual breeding success but reduced longevity. The maximum annual horn growth varies among populations and bovid species, but generally the horn growth is mainly affected by the environmental quality and it initially greater in low density populations on good range (in Mountain sheep; Geist, 1971). For a more detailed description of the factors affecting horn growth see the following chapter 1.5.

Introduction 1.5. Factors affecting the growth of antlers and horns The development, size and quality of cranial appendages are influenced by numerous factors. Both in the Bovidae and in the cervids, these factors can be classified into two groups, the first is the group of species-specific hereditary characteristics (intrinsic) and the second one is the environmental effects (extrinsic factors). The first group includes earlier growth of bone component by the same individual (in bovid horn; Massei et aI., 1994 and Pérez-Barbería et aI., 1996), the role of genetics in ungulate mammals (Fitzsimmons et aI., 1995; Hartl et al., 1995a), the reproduction (Festa-Bianchet & Jorgenson, 1994) and the internal hormone balance during the antler growth (Lincoln et al., 1972; Bubenik GA, 1990); however, the influence of environmental factors on deer antler size is considered more important (Goss, 1983). Thus, some example of extrinsic factors, that could affect the horn growth, are the precipitation (Pérez-Barbería et aI., 1996), and geographic area (Foster, 1978; Fandos & Vigal, 1988). In the following chapter the effects of some of these factors are discussed in detail.

1.5.1 Influence of hormone secretion and environment It has long been established that the seasonality of the growth cycle at the antler is determined by the duration of the photoperiod (Jaczewski, 1954). Thus, this external factor affects the hormone secretion of the pituitary gland, which transforms the messages transmitted by the retina (which perceives the seasonal changes of the photoperiod), through the optic nerve, in signal for the production of melatonin, this hormone has a daily cyclical level in the organism of the mammal (Bubenik, 1982). These changes acts on the tight activity of the hypothalamus and hypophysis glands, causing changes in the secretion of androgen hormones, which are responsible for the reproductive activity and development of secondary sexual characteristics, such as deer antlers (Suttie et al., 1998). This close relationship between the photoperiod and hormonal regulation follow an endogenous rhythm of secretion, and the antlers are mainly affected by the secretion of testosterone in the male gonads (Chapman, 1975; Bubenik GA,

Introduction 1990). Testosterone is required for pedicle and primary antler development, indeed castration of a calf prevents pedicle and antler development, whereas, castration during velvet period will delay shedding and prevent full mineralization of the antlers (Price et al., 2005) or hypertrophic bone growth in the antlers of the castrates (Kierdorf et al., 2004), and roe deer reacts more massively than other species creating a benign tumour called “peruke” (Kužmová, 2011). The photoperiod-hormones relation was already studied in the past by Goss (1969), who, through experiments in exposing deer to artificial light cycles, observed that the growth cycle of the antlers adjusts to the cycles of light to which the animals were subjected (with a number of antler cycles as were the cycles of artificial light). In deer natural double trophy growth cycles were observed, only in case of an anticipation of puberty and of the extraordinary presence of testosterone peaks (for red deer, Meikle et al., 1992; Gómez et al., 2006a). Normally, red deer show changing pattern following the different seasons along the year: in the spring, when androgen hormones are low, the deer buck casts its antlers and starts the new cycle of development for a new trophy (its behaviour is gregarious and its appetite rate is normal), but from the middle of the summer the male circulating steroids start to increase and this mechanism stops the antler growth and causes the mineralization (Lincoln et al., 1972), at the same time the male reduces its food intake, lose weight, but start to fight for the control of females (Gaspar-López et al., 2010; Gómez et al., 2006a). For the horn growth rate was observed that was inversely correlated to seasonal levels in testosterone plasma concentration, and these results appear to support the hypothesis that high peripheral plasma levels of testosterone are linked with the seasonal arrest of horn growth during the rutting period: in September for the European mouflon and October-november for Spanish ibex (Toledano-Diaz et al., 2007). During antler re-growth, systemic levels of testosterone are at their minimum (Price, 2005). In this phase, based on several in vivo and in vitro studies, IGF-1 has become widely accepted as the stimulating hormone for antler (Price, 2005b; Leblanc, 2007). In the same way, for roe deer was shown in the past by Semperé et al (1992) that exist an internal rhythm for the luteinizing hormone (LH) and testosterone; during this experiment a group of roe deer males were exposed to 8 hours of light/day,

Introduction throughout the year, while other animals were maintained in natural condition. The former showed a double cycle of growth of antlers, whereas the latter showed the classical one cycle per year. Moreover, in the roe deer the growth and the mineralization of the antlers is associated with an indigenous hormone balance, through an increase of the osteoblast activity, stimulated by the parotid hormone (PTH) and vitamin D; in this process also alkaline phosphatase (ALP), and other metabolites such as calcitrol, come into play (Semperé & Boissin, 1981). There are other hormones that play an important role in the antler’s growth; between them we can find the luteinizing hormone (LH), the prolactine (PRL), the stimulating follicle hormone (FSH) and the thyroxine hormone that regulates basal metabolic rate and growth, which in turn is governed by a feedback mechanism for thyroid stimulating hormone (TSH) (Bubenik, 1982; Bubenik GA, 1990; Van Mourick & Stelmasiak, 1990; Semperé et al., 1992).

Although several studies have reported that weather and climate can influence the cranial appendage’s growth. Indeed, horns (as antlers) of males in polygynous herbivores offer a good opportunity to measure the influence of climate on individual performance (e.g.: for horn see Büntgen et al., 2014; for antlers see Landete-Castillejos et al., 2010), and their growth indicates greater ability to obtain fitness-enhancing resources (male ibex with greater horn in a given year have a higher survival; von Hardenberg et al., 2004). Moreover, studying the Svalbard reindeer population, Douhard et al. (2016c) hypothesized that the life-history responses to early-life conditions can buffer the delayed effects of weather on population dynamics. Other studies on Caprinae (Ovis canadensis, in Picton, 1994; Capra pyrenaica, in Fandos, 1995), showed that the precipitation quantity was correlated positively with horn growth, probably because it affected abundance and quality of food. Thus, if reported at the population level, horn growth is positively associated with the resources (forage) available (Festa- Bianchet et al., 2004), and at the individual level there is a trade-off between predation risk and foraging because more abundant forage is located further away from escape terrain (Bleich et al., 1996). A certain impact is also given, on a long-term scale, by the human activities that

Introduction with their influence could induce some effects on population structure and on secondary sexual characteristics, such as the cranial appendages (Milner et al., 2007); the latter are characters that are often used for hunting harvest management reasons in ungulate species with a permitted hunting regime. On this subject, studies of wild populations have suggested that phenotypic harvesting may result in an evolutionary response in horn growth rate (in Ovis canadensis, Coltman et al., 2003; or in Ovis gmelini musimon, Garel et al., 2007). Whereas, for other species as the Alpine Chamois (Rupicapra rupicapra), it was hypothesized that the selection by hunters was believed unlikely to have an evolutionary impact (Rughetti & Festa-Bianchet, 2011). From the same authors (Festa-Bianchet & Lee, 2009), however, it was observed that the trophy hunting of Ovis canadensis led to selective pressure for slow-growing horns, although it is unclear whether this had an impact on reproductive success. This hypothesis was also studied for trophies in species of cervids, as the Capreolus (Balčiauskas et al., 2017) and the Odocoileus virginianus, for which the effects of the implementation of antler-based selective-harvest criteria (SHC) in relation with the harvest rate were studied, to protect young males from intense hunting harvest (Strickland et al., 2001). The same human-activity impact was hypothesized in a recent study by Mysterud et al. (2006), who observed that foreign hunters selected the biggest roe deer antlers in Poland, while local hunters hunted animals with worse trophies. On the same line, Douhard et al. (2016b) showed that, in Stone's rams (Ovis dalli stonei) harvested over 37 years, horn length became shorter for a given base circumference, likely because horn base is not a direct target of hunter selection; in contrast, under relatively lower hunting pressure, there were no detectable temporal trends in early horn growth, number of males harvested, or horn length relative to base circumference. Finally, Ernande et al. (2004) described that management policies that cause harvest mortality to decrease with biomass (i.e. positively density-dependent harvest mortality) result in smaller evolutionary responses, and this sense, selective harvesting of mature as opposed to immature individuals is evolutionarily preferable.

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1.5.2 Density of population and influence of the social environment The density of the population is an important factor that could affect the growth of the animal (and so the development and quality of the secondary sexual traits), because a large population will have a reduced capacity (and more intra-specific competition) at the time of feeding on a given territory, compared to a smaller population size; and this mechanism is more incisive when there are low primary productivity and low natural predation (in Alces alces, Ferguson et al., 2000). Schmidt et al. (2001) found that the variation of antler length in red deer yearlings, between years, was best explained by variation in deer density, given that population density affects biomass availability for the individual. Thus, an excessive competition for environmental resources could allow a worst development of antler length (Clutton-Brock & Albon, 1989). On the same line, Azorit et al. (2002b) suggested an effect of animal density and farm management on antler quality, and the same was observed by Landete-Castillejos et al. (2013a) who analysed the effect of the population management on the quality of antlers features (bone mechanical and structural properties, and mineral content) between animals grown in wild natural environments (protected areas) and fenced areas (game estates and farms), with better trophies developed in private game estates and farms, given by a rational management of natural areas. For the roe deer, Pelabón & Van Breukelen (1998) showed that higher density increases the asymmetries in the roe deer trophy, and also the antler asymmetry decreases significantly with age and, at constant body mass, tended to decrease with the size of the antlers within each age class. Thus, their results show that the asymmetry in the size of the antlers could be considered as an indicator of the quality of the environmental conditions to which the populations are subject. In the same direction, Weladji et al. (2005) found that antler length, when corrected for the allometric effect of body mass, is positively influenced by increasing density and spring precipitation (in a wild population of reindeer), probably due to the effect of early summer weather and its influence on forage availability and quality as well as the level of parasitic insect harassment. Douhard et al. (2016a), studying from birth to three years of male bighorn sheep (Ovis canadensis), hypothesized that when environmental conditions

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Introduction deteriorated, males allocated relatively more resources to summer mass gain than to horn growth, suggesting a conservative strategy preferring maintenance of condition over allocation to secondary sexual characters. Moreover, horn growth was positively influenced by low population density and the density explained 27% of the variation in horn length. Another important factor, which can have effects on the growth of the antlers/horns in ruminants, is the influence of social relations and the structure of the herds. In the past it was observed that the antler size is related to dominance. In this sense, males with the highest position in the hierarchy can have better access and better control of resources during their lives, such as food or reproduction (Clutton-Brock et al., 1982; 1988). Dominant animals tend to cast and clean the antlers before the subordinates and they produce the larger antlers (Bubenik, 1982; Bartoš, 1990); and this mechanism also occurs through a close relationship with the hormonal level (Bartoš & Bubenik, 2011). An example can be found in the roe deer, in this species the possession of a territory is vital for reproduction. Indeed, the influence of social hierarchy, and therefore the possibility of having territories with better resources, has an important effect on the animal: away from his territory, roe deer bucks, change in appearance; their hair is depressed, and they look slim, like a juvenile buck (Goss, 1983). Moreover, Morina et al. (2018) demonstrated that, in white-tailed deer, females prefer males with large antlers to those with small antlers. Thus, because antler size is heritable in deer (Kruuk et al., 2002; Coltman et al., 2003), this female preference for larger antlers may be adaptive by increasing the reproductive success of her male offspring. Other authors have highlighted that an adverse social environment and with little possibility of reproduction (e.g. young males in the presence of dominant males/older males), animals with worse social status can opt to reduce the inversion in their antlers competing with other males of similar rank (Bartoš et al., 2004; Johnson et al., 2007). Recently, Ceacero et al. (personal communication) showed that the socio-sexual environment, particularly living continuously in close contact with females, promotes antler mineralization in Pampas deer (Ozotoceros bezoarticus), probably through modifications in testosterone secretion.

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Introduction 1.5.3 Genetics and age The effects of genetics on the properties of bone tissue have largely been studied in humans, especially in relation to degenerative diseases and bone mineral density, BMD (e.g.: Brown et al, 2005; Rocha-Braz & Ferraz de Sousa, 2016). For ungulate, the bone development of cranial appendages and skeletal system have a genetically-programmed size that can reach their maximum with adequate nutrition during critical growth period. The size and the shape of antlers and horns are hereditary (Williams et al., 1994), and the size of the appendages, more than shape, is much more dependent on age and nutrition (Scribner et al., 1989; Grasman & Hellgren, 1993; Michel et al., 2016a). In red deer, during the first 18 months of life, body growth takes precedence and most of the nutrients are used for the development of the internal skeleton (Estévez, 2011), later the animal slows its body-growth and more nutrients are available for antler development. Thus, most young males sometimes could not express their genetic potential due to limiting nutrients available. Nevertheless, the conformation of the antlers has an inherited component that causes its peculiar characteristics (determines the number of points, the presence of the crown or palm, etc.) and certain alleles can favour large trophies since early years and determine the tendency to a good bone development (Hartl et al., 1995a) or larger number of tines (Smith et al., 1982). According to Huxley (1926), the marked differences in trophies among animals born in the same year, and the repeated presence of these characteristics in the following years, is due to genetic influence. Thus, heritability (also called h2) provides a measure of evolutive capacity for a population (Brookfield, 2009). Moreover, studies on the heritability of characteristics of the antlers/horns have given different results. Some authors found a higher h2 index for some cranial appendices’ characteristics: Williams et al. (1994) found that the h2 of antler characteristics and body weight, in the white-tailed deer, is very high (0.8 and 0.71 for the basal circumference and weight, respectively); and, in the same species, Michel et al. (2016b) found that antler characteristics are moderately to highly heritable. For red deer, Ward et al. (2014) estimates high heritability for antler length in association of live-weight (h2=0.67–0.81) and same was found for bighorn sheep (h2=0.69; Coltman et al., 2003). Conversely, for other authors, the heritability is lower: in Sika deer was described a value around

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Introduction of 0.35 for antler weight (Zhou & Wu, 1979), and in red deer, it was found a heritability of 0.36 for velvet production (Ball, 1991), and antler mass and antler velvet mass were reported to be moderately heritable (Wang et al., 1999; Kruuk et al., 2002). For this reason, sometimes it is difficult to separate genetic factors from environmental factors (Chapman, 1975) and heritability estimation is difficult because non-genetic sources of variation may influence traits of interest (as maternal effects, Monteith et al., 2009; or habitat quality, Strickland & Demarais, 2008). In a first study, Scribner et al. (1984) showed that animal with lower heterozygosity bear a trophy smaller than 10-20% in respect of deer with greater genetic variability, within the same age group. Later, Scribner et al. (1989) showed that the heterozygosis is responsible of 10-15% of the differences observed in antler length and diameter; thus, the higher heterozygosity may influence metabolic efficiency resulting in enhanced nutritional condition, body size, and antler size that could affect potentially dominance status and reproductive success in white-tailed deer. Brown (1985), who observed a group of white-tailed deer, indicated that the importance of the nutrition of the offspring is less than that of genetics, in terms of its impact on the development of the first antler. At the population level, genetic variability is a very important parameter. Thus, populations with a high level of genetic variability and low levels of consanguinity are more likely to respond to climatic changes and less presence of genetic alterations (Falconer & Mackay, 1996; Frankham et al., 2002). In any case, many factors that affect genetic variability can be modulated by the management of the population, and its genetic flow (migration), in the wild/farmed population management (Slatkin, 1987; Castillo, 2010). On this subject, Carranza (1999) made an exhaustive description of the interaction between management and genetics of a deer population. Moreover, there are natural mechanisms during the reproduction of the polygynic species (such as the deer) that support the maintenance of genetic variability (dissimilar pairs, Carranza et al., 2009; heterozygous males, Pérez-González et al., 2009).

For the effect of age on the characteristics of the trophies, it should be remembered that in cervids, with the advancing age of the animal, normally a more complex structure of the antler is developed up to the senescence. In

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Introduction contrast, the bovids show a continuous growth of its cranial appendages, with small phases of slowing down due to the colder seasons, which, in certain species, form the characteristic annual growth rings. In the past, the age was considered as a limiting factor in comparison to the effects of habitat quality, which has been the dominant factor leading to large or small deer (Sauer, 1984). Bender et al. (1994) theorized that the deer antler is an incomplete indicator of age, although there is a certain relationship between the age and the number of tines developed along the trophy's beam (in the first 5 years, Huxley, 1931; Lincoln, 1992). An effect already studied in the past marks the different average growth rate of the antlers between adults and yearlings (3.6/3.8 cm/week and 2.0 cm/week, respectively; Gómez, 2004; Gaspar-López et al., 2008). Furthermore, Schröder (1983) showed that the size of the antlers increases more rapidly in the first 5 years, and after the rate of growth is reduced. As for the composition of the bone tissue, this changes with age, some authors indicate that there is an increase of the mineral content in the tissue (Boskey & Coleman, 2011), probably this should have effects also on the mechanical performance, that are correlated to the bone’s mineral content (Currey, 2002). In white-tailed deer, Blob & LaBarbera (2001) did not find a clear relationship between the reduction of bone stiffness and the mineral content, but in other studies a certain modification of the mineral composition due to age has been noticed (between yearlings, sub-adult and adult deer; Landete-Castillejos et al., 2007a, 2007c). On this line, we must remember that in human bones (much more studied for the effects of age), with advancing age, there is a process of decompensation between absorption and creation of bone tissue (Davison et al., 2006). Therefore, possibly, even in the deer (where old deer produce very small antlers for their body, and considering that antlers gorw mainly from the material extracted from the skeleton during their mineralization) there could be such a process of mineralization decompensation similar to that studied in humans. Heaney et al. (2000) described a reduction of bone mass due to the effect of age. On this topic, the most extensive review of the effects of age in humans and animal models is that of Boskey & Coleman (2011). Finally, some authors hypothesized that aging has a larger impact on the plastic properties of bone (determined by the organic phase) as compared with the elastic properties (McCalden et al., 1993). The age-related reduction in

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Introduction toughness may play a large role in fracture susceptibility with aging (Davison et al., 2006), and these authors indicated that the collagen denaturation is related to the age-dependent decrease in bone toughness (decrease of -35% in strength and -50% in toughness of the collagen network in whole bone; Wang et al., 2000).

1.5.4 Nutrition The effect of nutrition is observed from the first stages of the animal's life, such as the maternal environment of the uterus, and then during and after lactation (Dryden, 2016). Schmidt et al. (2001) found that red deer spikers antler length increased with birthweight. Moreover, Ceacero et al. (2010) described that red deer calves fed by lactating mothers, to which mineral supplements were given, increased their antler length. Gómez et al. (2006) found that high percentage of milk protein resulted in increased antler weight or length. Thus, nutrition level could influence the development, and variations, of the shape of the first antlers (from a single rod to the appearance of more tines; Blaxter, 1971). Early post- weaning nutrition influences both the time of initiation and growth of first antler (French et al., 1956; Puttoo et al., 1998); the growth period may also be prolonged in the case of reduced accessibility to food at the end of winter/early spring (in white-tailed deer; Long et al., 1959). Nevertheless, ingestion and the weight of the male deer changes with the seasons and is influenced by the photoperiod (Brown et al., 1990); however, the timing in which food is available is very important, indeed a greater food availability advances casting date (Muir & Sykes, 1988) and a lower food availability interacts with growth period of antlers (Landete-Castillejos et al., 2012; Michel et al., 2017). The animal can be fed to obtain an appropriate balance of nutrients, and the selection of certain plants can be a strategy to improve the contributions to the diet (Nielsen, 1999). As secondary sexual characters, antlers and horns, could indicate the quality of male, and thus they can reflect the body condition of the male and its level of food intake (Ditchoff et al., 2001; Landete-Castillejos et al., 2007a); and, broken antlers are more common in nutrionally stressed animals (McDonald et al., 2005; Landete-Castillejos et al., 2010). In deer bucks, the length and the diameter of the antler can also be an index of the nutritional condition of the herd, comparing animals of the same age class (Anderson and Medin, 1969).

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Introduction One of the important nutrients is protein content of the diet, and the latter influence the development of the antlers. Indeed, French et al. (1956) showed that a ratio of 13-16% of protein could be optimum for the development of antlers, and the same authors showed that, as the percentage of protein in the diet decreases (from 16.8% to 4.6%), this was reflected in the size of the deer antlers (lower length, animal with low-protein diet began antler’s growth from 2 to 4 weeks later and shed their velvet later); Suttie & Kay (1982) also observed that animals subjected to a restricted diet developed smaller, lighter and lesser number of tips. Moreover, this mechanism is evident from the early stages of growth, with the quality of mother milk, where protein received by milk influences antlers’ composition (Gómez et al., 2008). Equally, Ullrey (1982) discovered that an additional source of energy (oak acorn) or earlier availability of protein in the stages of growth of the antlers, in males of white-tailed deer, favoured the presence of bigger tines in the trophy. However, it may be erroneous to think that the need for protein for the antlers means that what is ingested, is metabolized and then fixed in the structure. In contrast, what is most important is a level of protein intake, which allows the animal to arrive at a maintenance metabolism in order to develop its genetic potential for body and antler growth (Brown, 1990; Michel et al., 2017), due to the close relationship between the body weight and the antlers. In this respect, the determination of the energy costs of maintenance and activity factors have been determined for many species: white-tailed deer (Moen, 1985; Michel et al., 2017), wapiti (Hudson et al., 1985), roe deer (Weiner, 1977), red deer (Fennessy et al., 1981). The most critical nutritional period for antler growth was from April to September, but also in the period between the end of the breeding season and antler cast, when it is important to obtain a good weight recovery to start the growth of the new antlers (Dryden, 2016). In deer calves, pedicle development was dependent on the onset of puberty, which in turn, depended on body weight (Fennessy & Suttie, 1985), having a threshold weight that deer must attain before pedicles can develop (Bubenik, 1982). This effect of nutrition on the quality of the antlers occurs both on the first set and on the following ones: a low level of nutrition, in addition to delaying and slowing the development, has an effect on the second and successive antlers, making them lighter and shorter (Suttie & Hamilton, 1983). Thus, the body condition and the

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Introduction body growth rate have a great effect on the final antler shape and its full development. As a matter of fact, the deer stags born at the beginning of the spring, with a greater weight at birth, and which have been feed with better quality and quantity of milk, could gain greater adult body weight; thus having a greater probability to have larger well-developed antlers (Landete-Castillejos et al., 2003; 2005; 2009). Body weight influences the antler characteristics of the hard antler (Huxley, 1931; Gaspar-López et al., 2010) and, generally, greater weight indicates that the animal has more energy available (or greater skeleton weight whose minerals can be resorbed to develop antlers). Moreover, Landete- Castillejos et al. (2012) found that a lower quality diet increased fivefold the porosity of the cortical layer of red deer antlers. Antlers are expensive to grow and mineral intake of the diet does not fully meet the needs during the growth of the antlers. Antlers are composed by 35-45% of organic matrix and 55-65% of inorganic salts (Ullrey, 1982; Estévez et al., 2009). Ullrey et al. (1982) hypothesized that for the development of antlers it is necessary from 0.5 to 1 g of Ca and from 0.25 to 0.5 g of P each day. An Antler weighing about 1.5 kg could contain about 350 g of Ca and 160 g of P (Scottish red deer; Huxley, 1931). Chapman (1975) showed differences in the composition of the antlers in various species (18.0% Ca in white-tailed deer, 18.8% Ca and 8.9% P in the fallow deer and 22.0 and 24.9% Ca in red deer and roe deer), and same have done Pathak et al. (2001) that extended to species-specific chemical composition of antlers. Food intake provides only 25-40% of Calcium (Muir et al., 1987) and, during antler growth, red deer could lose up to 200gr of Phosphorus from their skeleton (Robbins, 1993); and this sets in motion a mechanism for the reabsorption of minerals from the bones (Huxley, 1931), called physiological cyclic osteoporosis (Fowler, 1993; Baxter et al., 1999; Stéger et al., 2010). During antler growth, the physiological effort made to grow each part of the antler increases, and this can give rise to phenomena of mineral reabsorption from the skeleton (23% mineral from the ribs; Baxter et al., 1999); Gómez et al. (2012) found that antler investment increased from 6% in yearlings to 35% in 5-years- old deer. This is also reflected on the composition of minerals fixed in bone tissues; Muir et al. (1987) found that during the growth for antlers, almost 65% of mineral content is deposited in the last 10 weeks of development and the Ca

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Introduction concentration of the tip increased by 40% between 28-91 days after casting to 112 days after casting. Moreover Miller et al. (1985) noted that concentrations of Ca, P, K, Zn and Al tended to increase with the age of white-tailed deer stags. On this topic, Estévez et al. (2009) found that deer populations differing in feeding regimes also differ in antler chemical composition in the same particular minerals (greater content for protein, Na, K and Mg in antlers from high-quality diet group). Thus, in deer farm, normally is offered a salt supplementation during the male antlerogenesis period (in white-tailed deer; Atwood & Weeks, 2002), and the consumption of supplemented minerals by deer showed maximum level in spring and summer and moderate during autumn and winter (Estévez et al., 2010). Some authors have pointed that imperfect distribution of food disruptions in breeding could create an increase in clashes between males (red deer; Carranza, 1999). In mouflon, an excessively energetic and plentiful diet produces excessive speed of growing and with malformations (Uloth et al., 2002) and the same for unbalanced diets (Santiago-Moreno et al., 2003). Some minerals have given good results on the characteristics of the antlers. Manganese level intake and availability, probably induced by diet, may have marked effects in mechanical properties of bone (Landete-Castillejos et al., 2010). Copper may promote antler growth and tend to increase the cortical thickness of antlers (Gambín et al., 2017), however, the presence of antagonists (such as S, Mo, Zn or Fe) in feeding regime for deer can decrease the absorption and digestibility of Cu (Jacob et al., 1987). Always Copper and Potassium, in low concentrations, were hypothesized to be the cause of the higher rate of antlers’ breakage, in a population of Tule elk (Johnson et al., 2007). P and Mg, two important mineral components of the bone tissue, are correlated with bone density (Hyvarinen et al., 1977); while Zn is inversely related (possibly because Zn is related to osteoclast activity; Moonga & Dempster 1995). Landete-Castillejos et al. (2007a) found that Ca and P concentration at the base of red deer spike antler were higher than at the tip, inversely for Zn, K and Fe. The same authors found that the chemical composition of a few mineral could explain 77% of the variability of antler’s length and weight (Landete-Castillejos et al., 2007b) and at least Na, Mg, cortical thickness of antlers bone, could be an index of male quality, good management and other factors influencing antler composition (Landete-Castillejos et al., 2007c).

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Introduction However, there have recently been studies that attempted to summarize species- specific differences in the content of antlers minerals, and to describe a possible deer feeding management (Dryden, 2016). For wild population an important aspect in feeding regime is also the presence of toxic, harmful elements. Is the case of the heavy metal and pollutants that in some concentrations could be found in antlers, this reflect pasture contamination: Kierdorf et al. (2000) found that excess exposure of red deer to fluoride led to an extremely porous antler cortex and other bone-seeking pollutants may produce similar effects as they are readily trapped in antlers (Kierdorf & Kierdorf, 2005). Dobrowolska (2002) found highly variable Pb and Cd concentrations in the antlers od red deer. Thus, Pokorny et al. (2007) have found in Slovenia a relationship between Fluctuating Asymmetries (AF) of roe deer antlers and concentrations of various pollutants. These authors suggest that the AF of the antlers could be used as a good bioindicator. On the same line, Markusson & Folstad (1997) found that antlers may be exposed to directional selection in a visual signaler-receiver system and that information about parasite burden may be obtained from evaluation of asymmetry (AF) in antlers developed under exposure to a multitude of environmental stresses. In conclusion, the study of mineral profile of antlers opens the possibility of using antler composition as a diagnostic tool to assess population condition and nutrient deficiencies.

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Justification Chapter 2. Justification

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Nowadays, the hunting sector is able to move a thriving economy, and above all the hunting activity represents not only a recreational sport, but also an important economic source, especially in depressed rural areas. Hunting plays an important role in biodiversity conservation, as a tool to conserve the biological heritage, and as a sustainable economic source (Farfán et al., 2004; Rodriguez-Estival & Landete-Castillejos, 2010). Thus, the increasing importance of this sector has increased all economic activities of the hunting world, from an increase in wildlife- hunting companies and intensive and extensive farms, up to services related to the hunting activity (as licenses, suppliers of materials, taxation, etc.). At European level, the increment in the use of hunting resources was observed (Kenward & Putman, 2011); and it has been estimated that ungulate species represent an immense potential resource, not only in terms of biodiversity but also in economic terms with an estimated economic return of many hundreds of millions of euros for more than 5.2 million animals collected each year (Apollonio et al., 2010b). The impact of the breeding deer farm is estimated more than 3,000 M€ in the USA, and 25,000 M € for hunting (Anderson et al., 2007). One of the most widely exploited and most numerous ungulate species in hunting activity, in Europe as in Spain, is the deer (Milner et al., 2006). In Spain, the management of hunting sector became much more intense starting from 1973, even using territories fenced and private/public game estates (Carranza, 2007b). In the Spanish hunting activity are involved an average of 186000 persons and suppose a 0.3% of the GDP of the country; and only red deer could move about 25 M€ (MAPAMA, 2017). Through research, the knowledge and techniques on deer’s farms have increased in recent years, both worldwide and in Spain; in this last country the production is oriented more towards obtaining good trophies and in a minor part to produce meat or other products (Pearse, 1993; Carranza, 2004); and for this reason deer management involve the use of mineral supplements and food to improve production (Landete-Castillejos et al., 2010b). Trophy quality is influenced by few principal factors, between these there are: the gene pool (Hartl et al., 1995a), population density (Ferguson et al., 2000; Schmidt et al., 2001; Landete-Castillejos et a., 2013a), habitat characteristics (Goss, 1983; Pérez-

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Justification Barbería et aI., 1996; Landete-Castillejos et al., 2010), nutrition and mineral intake (Gómez et al., 2006; Landete-Castillejos et al., 2012). Moreover, for some authors, antlers are recognized as good signs of male quality (Vanpé, 2007), and their size reflects the reproductive capacity and sexual vigor of males (Möller, 1989; Wahlström, 1994). Deer antlers are easy to study because they are easy to collect (if the animal is hunted or the animal has cast the antlers), there is no need for complex surgical procedures to obtain them, and finally, since the antlers fall and regrow every year, there is the possibility to carry out studies on a wide time series for every individual. On this purpose, especially for the red deer, there are numerous studies that have tried to investigate the characteristics of the trophy in order to fully understand the factors that influence it and the biological rules that dictate its growth. In the antlers, unlike the horns of the bovids, during their growth, thanks to the bone tissue that has no bone remodelling, are an excellent tool for studying the mineralization of the bone tissue and the effects of the quality of the diet on the animal (Davis et al., 2011; Gómez et al., 2013). Precisely on this line of research, our research group has published numerous studies in the past, developing and improving a method that allowed to analyse the structural and mechanical characteristics, such as the mineral profile, of the bone tissue that forms part of the antlers of Cervidae (e.g.: see publications by Landete-Castillejos, Gómez, Estévez). Thus, in this thesis, this method of analysis is used for the first time in order to characterize not only the antlers of cervid, but also the horns of Bovidae. The aim is to understand the differences of the cranial appendages between the different species and between deer subspecies, as well as between populations of the same species subject to different habitats. Furthermore, this comparative characterization of trophies, between bovids and cervid, allows to understand also the relations between morphology, fighting behaviour and evolution of this distinctive appendages (Geist, 1966; Lundrigan, 1996; Caro et al., 2003; Davis et al., 2011). In addition to this, following the footsteps of the past article published by our own group (Landete-Castillejos et al., 2010), this thesis is enriched with a study on the role of micromineral in the characteristics of the cervidae antler. In this case we study the effect of supplementation of Manganese, which is a micro-mineral relevant to the deer and other ruminants (McDowell, 2003; Suttle, 2010), and its

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Justification deficiency could cause fragility in the antlers (Landete-Castillejos et al., 2010). Thus, a study was completed within the university experimental farm of Albacete, in order to understand the effects of this micromineral in a supplemented group, against a control group, when the diet is balanced.

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This thesis aims to broaden the applicability of a method to use antler bone mechanical properties, structure and mineral profile for management of other cervids and extend it to horn bone of bovids. In the past, this method has been proved to be useful as diagnostic tool in red deer (Landete-Castillejos et al., 2007a,b,c Currey et al., 2009a,b; Estévez et al., 2009; Landete-Castillejos et al., 2012; Gambín et al., 2017). Thus, this thesis includes a scientific article that follows those steps marked in the past regarding the role of Mn in antler growth, deepening the study of the role of Manganese on certain characteristics of the bone tissue and the size of the antlers. An important step to broaden this diagnostic tool was to test the applicability of these analyses for bovid species (such as the Taurotragus oryx). Moreover, useful data can be obtained to compare the qualities of antlers and horns comparing between intra and inter species; especially to be able to observe the effects of environmental features and management of the same species populations of ungulates (such as in roe deer). The same method could be used to understand the ecology and help the conservation of other species of ungulates in protected areas (such as the Sardinian red deer). Thus, for the reasons described above, the specific objectives are as follows: 1- Characterization and study of mechanical and structural properties and mineral content profile in bone tissue of trophies of red deer (Cervus elaphus spp.). 1.1- Characterization focused on the quality of the trophies of Sardinian red deer (Cervus elaphus corsicanus), for adult and subadult age class; application of the study of mechanical and structural properties and minerals content profile of antlers’ bone of a protected species (in adults deer), in order to understand the use of this scientific protocol for study the trophy bone characteristics in a species subject to conservation programs; see results in chapter 5.1. 1.2- Characterization focused on the quality of the antlers of Spanish red deer (Cervus elaphus hispanicus), trough out their mechanical and structural characteristics and mineral profile, in order to understand the effects of mineral supplementation (Manganese) in a regime of a balanced and controlled diet;

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Objectives in spikers and adult red deer under a balanced diet; see results in chapter 6.1.

2- Characterization and study of mechanical and structural properties and mineral profile in bone tissue of trophies of roe deer (Capreolus capreolus): differences between two population grown under different environmental characteristics and comparing with antlers of red deer, see results in chapter 5.2.

3- Characterization and study of mechanical and structural properties and mineral profile in bony horn-core tissue of horns of common eland (Taurotragus oryx): an example of the use of the scientific protocol for study the trophy bone characteristics on horn of bovid of hunting interests; to compare characteristics of different parts inside the spiral structure of the bony horn (core area VS spiral ridge of the horn), see results in chapter 5.3.

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Work plan, materials and methodology 4.1 Material and geographical origin of animal’s samples The material and samples collected for the studies reported in this thesis have different origins. For the study on antler bone of the red deer of Sardinia, the samples were collected in a protected area in the south of the island of Sardinia (Italy); while for the study of the effects of manganese supplementation on Iberian red deer antler bone properties, we used bone samples collected in the experimental farm of the University of Castilla-La Mancha (Albacete, Spain). For the study on the roe deer antler tissue, the samples came from an extensive open game estate near Prague (Czech Republic) and from a closed breeding fenced game estate in Murcia (Spain). For the study of horn bone tissue in bovids, common eland, the samples came from an experimental breeding farm located near Prague (Czech Republic). For further details on the area of origin of the samples and the populations used, see the next paragraphs of this chapter.

4.1.1. Cervus elaphus spp.

Cervus elaphus hispanicus For the research on Iberian red deer, in order to study the effects of manganese (Mn) supplementation on properties of antler bone, we used antlers collected in the experimental farm of University of Castilla-La Mancha in Albacete (south- eastern of Spain; 690 m altitude; see figure 17). In the same experimental farm, our research group regularly performs experiments on red deer biology, all experiments must be approved by the Committee of Ethics in Animal Experimentation (Comité de Ética en Experimentación Animal, CEEA) from the Universidad de Castilla-La Mancha (for this study, the authorisation number was 1002.04).

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Figure 17. Geographical position of Experimental farm of University of Castilla-La Mancha (Albacete, Spain).

Red deer were kept in a 10000 m2 open door enclosure on an irrigated mixed pasture. Deer were feed ad libitum with a diet of hay, lucerne, corn and fruits pulp. Diet ingredients were homogeneized and cut in small portions in a tractor-driven commercial mixer. All animals were adapted to routine management and maintained in good health and body condition during the experiment. Handling procedures and sampling frequency were designed to reduce stress and health risks for the animals (Ceacero et al., 2014). The animals were divided in two groups matched for body measurements such as weight and body condition. Rather than offering Mn mixed with salt, or with the food, which would not allow to control the exact amount of food given to each animal, and also it would mean that control animals had to be placed in another enclosure thus modifying the social environment, we decided to keep all animals together, and deliver the Mn by injections of an aqueous 4% of Manganese gluconate (C12H22MnO14, Fagron Ibérica S.A.U.) solution (5mL/100Kg live wt) in the treatment group, given every seven days from start of January to mid-August (see figure 18). The control group was injected a physiological saline solution. Each group counted animals with different ages: for the control group there were 8 adults, 2 subadults (2.5 years old) and 5 yearlings (1.5 years old), for the treatment group there were 8 adults,1 subadult and 5 yearlings. We decided to include subadults to increase group size of the adult class.

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Figure 18. Procedure to obtain the Manganese gluconate solution for injections purpose.

Antlers were cut off 1 cm above the burr for safety reasons when they were clean from velvet. We kept carrying out body measurements at the beginning of the trial, during and at the end of the experiment, to monitor potential changes in the animals. Before the antlers had been removed, they were analysed according to the standard method for the trophy evaluation in red deer, in order to find an evaluation score for each antler collected (CIC, 1960).

Cervus elaphus corsicanus The collection of antlers of Sardinian red deer take place inside a protected reserve, the forest complex Sette Fratelli. This natural area is located in the historical regions of Sarrabus and the Campidano, south-eastern Sardinia (39 14’N, 9 30’E, 210-500 m. of altitude), and it belongs to the climax of mesophilous holm oak forests, inside the phytoclimatic area of medium-hot lauretum (Bacchetta et al., 2009). The vegetation of the forest complex is quite varied appearing on a coastal strip with typical stain mastic (Pistacia lentiscus L.), with Phoenician juniper (Juniperus phoenicea L.), tree spurge (Euphorbia dendroides L.), sporadic specimens of wild olive (Olea europea L. var. sylvestris Brot.); while in the internal areas the forest is dominated by holm oak wood, that in different situations is associated with strawberry tree, phillyrea broadleaf and viburnum. At medium-low altitudes there are also cork oaks (Quercus suber). On the highest mountain ridges, there is vegetation with Genista corsica and thyme (Thymus

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Work plan, materials and methodology vulgaris), whereas along the river beds the riparian vegetation is formed by oleander (Nerium oleander), and willow (Salix spp.). Also important is the presence of reforestation Aleppo pine (Pinus halepensis; Bacchetta et al., 2009) performed during the last century. The climate is typically Mediterranean, distinctive of the southern Sardinia, with hot, dry and long summer that goes on for about four months, while the winter is mild and moderately rainy; nevertheless, concentrated precipitations of considerable erosive effect occur exceptionally. Sixty-one antlers, grown and casted in the same year, were collected in Sette Fratelli forest reserve, under the Italian State management. Deer antlers were casted directly inside the forest natural area. Given that they cannot be assigned to a particular male, and thus we are not certain which left and right antlers constitute a pair (antlers collected were divided only by class of age), every single antler has been treated individually in the initial process of characterization of the morphological features. Considering that in the area of collection of deer there are hundreds of individuals, the possibility that any two left and right antlers belong to one individual is very low. Based on their morphology, antlers were assigned as belonging to adults (35) and sub-adults (26). Antlers were classified by the presence of the following features: the first tine (brow tine), the central tine and a bifurcation in the distal part of the main beam (i.e. the presence of at least two tines in the crown). This criterion derives from morphological differences reported in one of the earliest studies (Beccu, 1989), about the characterization of the Sardinian deer antler. The antlers collected showed no sign of diagenetic alteration, no loss of colour due to sunlight or weathering and no cracks.

4.1.2. Capreolus capreolus Spanish antler set Thirteen Spanish roe deer antlers were collected inside a game ranch located in the south-eastern part of the country (near Caravaca de la Cruz, Spain, 685 m altitude); animals were grown on soil sown with selected herbs; in addition, animals had access, throughout the year, to additional food such as pellets, barley and oats. Each antler belongs to adult animals (>3 years). The climate conditions were characterized by a Mediterranean-semiarid climate, rainfall is very irregular. The daily thermal amplitude is moderate, but compared to other

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Work plan, materials and methodology regions of Europe, the annual range of temperatures is very narrow. The rainfall occurs mainly in spring and especially autumn, separated by a summer of severe drought.

Czech antler set For the Cezch roe deer, ten antlers were collected from a unfenced area of 1600 ha hunting ground around Vysoký Chlumec (Central District, Czech Republic; 600 m altitude) where hunting is organised between 16th May and 30th September always by stalking or watch of game from hide (high seat). Roe deer population density in this area supposed to be around 150 individuals per 1000 ha of all age categories, both sexes. Management is organised by private owner staff, i.e. game manager and his wardens consisted of census during spring and planning hunting bag based on that. Supplementary feeding is organised mainly during period from late Autumn early spring by meadow hay and oat distributed in 30 feeding sites. Habitat is a mosaic structure of forests mixed coniferous (spruce, pine) and broad-leaved (oak, beech, maple, ash, willow), crop fields (cereals- mainly barley, rye, oat, corn, rape, feed crop as trefoil and Leguminosae) and meadows. Animals harvested were not of prime trophy, but mainly adult (> 3 years). Live weight of animals was between 17-23 kg. Other cervid competitor for food resources are fallow deer, but there is just a few (10-20 individuals per 1000 ha).

4.1.3. Taurotragus oryx The horns of the common eland were collected in the experimental farm at Lány (Czech University of Life Sciences, Prague); eight animals were culled for purpose of meat production and their horns were used for the study of the properties of internal bone tissue. Slaughter procedure was approved and supervised by State Veterinary Authority of the Czech Republic (Act. no. SVS/WS22/2012-KVSS) described in Bartoň et al. (2014). Animals culled had an average age of 23 ± 2 months and an average weight of 232.8 ± 12.9 Kg. In that moment, the animals had a highly-advanced stage of horn’s growth (the growth rate begins to decline from 20-22 months: Kerr

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Work plan, materials and methodology & Roth, 1970; Jeffery & Hanks, 1981). This is the only European farm for this species. Eland farming began in 2006 and the average number of managed animals is around 50, they are fed by whole year by complete feed mixture diet consisted of 60% corn silage, 30% Lucerne haylage, 7% meadow hay, 3% barley straw presented ad libitum and access to grassy paddock of 2.5 hectares from April to November (Vadlejch et al., 2015). Animals had ad libitum access to mineral lick-block SOLSEL (European Salt Company, Hannover, Germany). This block contained Na (37%), Ca (1.1%), Mg (0.6%), Mn (0.1%), Zn (0.1%), Fe (0.07%), Cu (220 mg×kg-1), I (10 mg×kg-1), Co (20 mg×kg-1), Se (20 mg×kg-1). One horn from each pair was chosen for the destructive sampling procedure; later, the two structures composing the horn itself were separated: the horn sheath of keratin and its internal bone core. The internal bone core was preserved in freezer (-20°C) in Prague; subsequently, internal bone cores were transported to Spain (IDR-UCLM, University campus of Albacete), where were performed the mechanical tests and analyses of the mineral content.

4.2. Measurement and scoring of the cranial appendages 4.2.1. Cervus elaphus spp. For measure the antler of Cervus elaphus spp. was used the CIC method (CIC, 1960); for measuring the size of antlers, a folding rule (± 0.1 cm) was used. In the Sardinian red deer, the measurements recorded were: total curve length, first tine length, second tine length (if present), central tine length, burr perimeter, first half perimeter (between first tine and central tine), second half perimeter (between central tine and the crown), weight, and the final scoring. In the Spanish red deer antler set, measurements included: the total length of the main beam, lengths of all the tines, perimeters at three points along the main shaft (burr, between the first tine and the central tine, between the central tine and the crown), total weight of the antlers, number of tines, and beauty of antlers. For an example of data sheet used during the evaluation process, see table 1.

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Measure- ment Total Average Factor Points 1. Measurements 1.1 Length of main beam, left ….cm … …cm x 0,5 ….. Length of main beam, right ….cm 1.2 Length of brow tine, left ….cm … …cm x 0,25 ….. Length of brow tine, right ….cm 1.3 Length of tray tine, left ….cm … …cm x 0,25 ….. Length of tray tine, right ….cm 1.4 Circumference of coronet, left ….cm … …cm x 1 ….. Circumference of coronet, right ….cm 1.5 Circumference of lower beam, left ….cm x 1 ….. Circumference of lower beam, right ….cm x 1 ….. 1.6 Circumference of upper beam, left ….cm x 1 ….. Circumference of upper beam, right ….cm x 1 ….. 1.7 Weight (dry) of antlers .…kg x 2 …. 1.8 Inside span 0-3 points …. 1.9 Number if tine ends 1 tine end = 1 point .…+….= …. 2. Beauty and Penalty Points 2.1.1 Colour 0-2 points …. 2.1.2 Pearling 0-2 points …. 2.1.3 Tine ends 0-2 points …. 2.1.4 Bay tines 0-2 points …. 2.1.5 Crown tines 0-2 points …. Inside span ….cm Total …. 2.2 Penalty Points 0-3 points …. Final Score Table 1. Example of evaluation data sheet to collect measurements of red deer antler (modified from CIC, 1960).

4.2.2. Capreolus capreolus As it was made for red deer, also for roe deer antler was used the CIC method (CIC, 1960), using a folding rule (± 0.1 cm). In this case the measures collected were: total antler length, first tine length (brow tine), second tine length (back tine), burr perimeter, total weight, antler volume.

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Work plan, materials and methodology Some of these measures are necessary for the scoring of roe deer trophies, see table 2 for details.

Measure- ment Total Average Factor Points 1. Measurements 1.1 Length of main beam, left ….cm … …cm x 0,5 ….. Length of main beam, right ….cm 1.2 Weight (dry) of antlers .…g x 0,1 …. 1.3 Volume of antler ….cm3 x 0,3 …. 1.4 Inside span 0-4 points ….cm …. 2. Beauty and Penalty Points 2.1.1 Colour 0-4 points …. 2.1.2 Pearling 0-4 points …. 2.1.3 Coronets 0-4 points …. 2.1.4 Tines ends 0-2 points …. 2.1.5 Regularity and quality 0-5 points …. Total …. 2.2 Penalty Points 0-5 points …. Final Score Table 2. Example of evaluation data sheet to collect measurements of roe deer antler (modified from CIC, 1960).

4.2.3. Taurotragus oryx Based on the visual appearance, there are three factors that need to be considered in judging the quality of horns of the Eland: thickness, prominent ridges and length. For experimental reason, we collected only the total length and the circumference perimeter for each sampling position along the horn bony-core of Eland, before processing this bone tissue in order to obtain samples for mechanical test. Following part of the rules described by the method 2 for spiral- horned animal of SCI (2016) and CIC (1960). See table 3 for details.

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Measurement Average Factor Points

1. Measurements 1.1 Length of bony horn core, left …… cm …. cm X 1 …. Length of bony horn core, right …… cm 1.2 Circumference of the horn core, right …… cm …. cm X 1 …. Circumference of the horn core, left …… cm Repeat the measurements of the 1.2 for each sampling position obtained along the bony horn core. TOTAL …. Table 3. Example of evaluation data sheet to collect measurements of common eland bony horn core (modified from CIC, 1960 and SCI, 2016).

4.3. Extraction and processing of the cranial appendages 4.3.1. Antlers For cervid trophies the protocol used provided the cutting at different heights along the main shaft, to obtain from each position a circular transversal section (1 cm slice) and a cylinder of about 5-6 cm, from which to extract the bars for subsequent analyses. In adult red deer were extracted 4 positions along the main shaft: position 1 was above the first tine, position 2 in the first third of antler shaft (just below the central tine), position 3 after the central tine and position 4 below the crown or upper tines. For deer yearlings, antlers were sampled following a similar procedure but including only two sampling points due to the smaller size and different structure of the antlers: one position was close to the base, and the other one 5 cm below the tip (Landete-Castillejos et al., 2007a).

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Work plan, materials and methodology In roe deer, due the small dimension of its antler, the position extracted were only two: position 1 (directly above the burr) and position 2 (below the distal bifurcation or 5 cm under the upper tip). For more detail, see Figure 19 and Figure 20.

Figure 19. From left to right: antlers cutting scheme in roe deer, in red deer spiker and in red deer adult.

Figure 20. Example of cutting scheme applied on antler from distinct species. From the left to right: example of a cut antler of adult roe deer and a cut antler of adult red deer.

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Work plan, materials and methodology For both red deer and roe deer, a circular low-speed saw was used for the initial cutting of the antler. From each bone cylinder extracted, two bone bars were obtained by using an electric hand saw (PSA 700, Bosch), see Figure 21 for the scheme of the protocol.

Figure 21. Scheme of the protocol used for cutting cervid antler. In the bone cylinder, initially, two lateral cuts were made to detect the area to be cut (A); then with a further central cut (B) two bars could still be obtained in raw form (C). Later, with the use of a disk orbital polishing machine, it was possible to obtain pure cortical bone bars of the desired size (D). Red lines mark the cuts.

Then, the surfaces were abraded using a semiautomatic equipment for polishing (MetaServ® 250 Double, Buehler-Illinois Tool Works Inc., Lake Bluff, IL, USA) to get the right size of bars, to reach the right measurements was used a Silicon Carbide abrasive paper of 80 grains (SiC paper 80, Buehler, Lake Bluff, IL, USA). The final size was 4.5 mm wide, 2.5 mm deep, and a variable length but always allowing a gauge length of 40~45 mm, a length in which shear effects reducing on calculated Young’s Modulus of elasticity are very small (Spatz et al., 1996; Landete-Castillejos et al., 2010). Once the bone bars have been obtained, the latter were identified according to the animal identification and level in the main beam. The outer or periosteal side was also marked in order to test always with this side in tension, and to mark the end closer to the antler base (see Figure 22).

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Figure 22. An example of the steps to obtain cortical bone bars in red deer, starting from a cut cylinder: initially, longitudinal cuts were made in order to extract the raw bar, after which, by using a disk orbital polishing machine, the desired sizes are reached, eliminating the spongy bone tissue and cleaning the outer surface of the bars

4.3.2. Horns’ bony core Bovine horns’ bony core was sampled from various heights along the vertical axis of the horn and one position was consecutive to the other until the tip of the horn, to obtain cylinders from which to extract bone bars for analysis of the tissue. These cylinders were obtained by cutting the horny core with a circular low-speed saw, during this process the samples were kept constantly moist, using tap water, to prevent overheating of the bone tissue. In each sampling position two bone bars were obtained: one from the “medial” side and “lateral” side of the cylinder using a circular low-speed saw (see Figure 23 for details). In addition, only from the first sampling position (corresponding to the closest position to the base), two bone bars were obtained from the spiral ridge to investigate the properties of bone tissue in this area of the horny core. Since, the spiral of the horns seems to have a mechanical meaning especially for male fights, which often fight by pushing the rival with intertwined movements (Kiley-Worthington, 1978), and the use of the horn area of the spiral has been proposed to increase grip in wrestling fight (Caro et al., 2003).

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Figure 23. Scheme of the protocol used for cutting bovid horn’s core. Four cylinders sampling positions of about 5-6 cm of length were cut from each bony horn’s core (each cylinder was consecutive to the other, until the tip of the horn) (a); from each sampling position, two bone bars were extracted, one from the inner-medial face of the cylinder and the other one from the external-lateral face of the cylinder (b). In addition, from the position 1, two bone bars were cut from the bone spiral ridge (c), to study bone tissue features of this distinct structure.

After obtained the raw bone bars, as in the deer antlers (see chapter 4.3.1), the bar’s surfaces were abraded using a semiautomatic equipment for polishing to reach the right measurements, for this purpose a Silicon Carbide abrasive paper was used (80 grains paper). The final size was 4.5 mm wide, 2.5 mm deep, and a variable length but always allowing a gauge length of 40~45 mm, a length in which shear effects is reduced (Spatz et al., 1996). During all the process, bone bars were kept constantly moist.

4.4. Analysis of bone tissue 4.4.1. Moisture standardization of antlers sample The state of hydration of tissues, that form the antler and the horns, could change and can vary their response to mechanical stress; about that, was already observed that wet and dry antlers reacted differently in mechanical tests (Currey et al., 2009a,b). Trim et al. (2011) found that there were differences in the bighorn

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Work plan, materials and methodology sheep horn, that failed in a brittle manner in the dry condition, whereas wet horn was much more ductile. In that case, compressive failure occurred by microbuckling followed by delamination, in agreement with Tombolato et al. (2010). As with other keratin-based materials, the elastic and shear modulus decreased significantly with an increase in the moisture content (Kitchener & Vincent, 1987). In general, the linearly elastic behaviour at low stress levels is valid for bone tissue; however, at high levels of hydration the bone behaves visco- elastically, with greater elasticity (Cerrud er al., 2005). Therefore, in the case of this thesis, it was decided to mechanically test the bone tissues as they would be used by ungulates in vivo condition: moist condition for the bony core of the horn of the Bovidae, dry conditions for the antlers of the Cervidae. Prior to mechanical testing, the cervid antlers tissue samples passed through a process of moisture standardization (with a controlled moisturizing and drying process), while the samples of tissue extracted from the horns were kept moist until the mechanical tests (see chapter 5 for details). For the process of moisture standardization was used the Hanks' Balanced salt solution (Lonza BioWhittaker, Verviers, Belgium) which contains phosphate, was used to obviate the risk of small amounts of mineral leaching out of the bone (Currey et al., 2009a).

4.4.2. Mechanical test Mechanical tests and the study of mechanical properties really began to take off in the beginning of 1980, with three aspects of bone analysis: increasing precision and power of the mechanical test themselves, increasing realization of the importance of features on the testing method, and increasing ability to characterize the bone and to combine differences in bone structure and differences in mechanical properties (Currey, 2009a). From 1876 it is known that the mechanical properties of bone were very different depending on whether the bone was wet or dry, and a systematic approach to this was made by Evans & Lebow (1951). Moreover, was found that the "size effect" of specimen affect the results of the mechanical tests (Spatz et al., 1996; Taylor 1998, 2000). On this subject, Bigley et al. (2007) found that increasing the volume of specimens by a factor of four reduced the number of cycles to failure at a particular stress by two.

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Work plan, materials and methodology Until the 1990s not many studies have studied the spread of fractures and damage in the structure of the bone tissue (Zioupos & Currey, 1994). Recently studies devoted to microcracking are increased by the development of more and more detailed microscopy techniques (Currey, 2009), as the electron and transmission microscopy that allowed observation of the composition of the bony lamellae: it was possible to observe the apatite-crystal packages in the collagen fibers (Landis et al., 1996), or the arrangement of the same bony lamellae between them (Weiner & Addadi., 1997). In addition, many spectroscopy techniques have been used to study the characteristics of the bone tissue: Fourier transform infrared spectroscopy (FTIR) was used in bone to detect compositional differences in mineral/organic ratios (Boskey & Camacho, 2007) or NMR spectroscopy was used to measure the amount of water in the bone (Nyman et al., 2008). Moreover, nowadays it is possible to measure the mechanical properties of very small pieces of bone through analysis at the Nano level (Gupta et al., 2005; Fritsch & Hellmich, 2007). The mechanical behaviour of materials could be studied by loading a small sample (in our case the cortical bone bars) in a material testing machine. A stress inevitably causes a strain; the two are emphatically not the same thing, the former is how severely we impose forces on something, the latter is how much it is bent out of shape as a result (Vogel, 2003). There are several kinds of stresses, but those which came into play during our mechanical tests are: tensile stress (force per unit cross-sectional area, when a body is pulled by two equal and opposite forces, with the opposite direction), compressive stress (is just the same except that it pushes the material together) and shear stress (two forces applied to an object, with opposite directions but applied along lines of action that don’t coincide); see Figure 24.

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Figure 24. Types of forces involved during the performance of the mechanical tests: tensile stress (A), compressive stress (B) and shear stress (C).

The mechanical properties of bone depend on the physiological function of the tissue that may support weight (long bones) or impact absorption (antlers; Currey, 1979, 2010; Price et al., 2005b). The mechanical properties depend on architectural factors of the bone tissue (thickness of the compact layer or diameter) and intrinsic tissue factors that are independent of the diameter or size of the surface (Currey, 2002, 2007, 2004; Sharir et al., 2008); these two factors are independent of each other, in contributing to the resistance of the bone tissue (Davison et al., 2006). Therefore, both material and geometric properties are required to assess the structural integrity of a bone (Van der Meulen et al., 2001). The mineralization rate of cortical bone it is considered the responsible of the most important mechanical properties of the bone (Currey et al., 2004), and the resistance of the bone is related not only to the quantity of minerals fixed in its tissue (as percentage in ashes or Ca), but also for the size of hydroxyapatite crystals (greater crystal size, more rigidity; Davison et al., 2006). Moreover, other variables as porosity, histology and anisotropy also have an effect on properties of stressed bone (Currey, 2006). In the case of the cortical porosity, usually originates in the endosteal surface and progressively expands towards the periosteal surface. Thus, porosity in the inner diameter has less impact on bone strength than porosity in the outer diameter and periosteal surface (Davison et al, 2006). The following paragraphs explain the mechanical tests performed and the relative variations observed.

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4.4.2.1. Flexion test The three-point bending test was performed with a Zwick/Roell machine for universal quasi-static mechanical tests (with a preload of 500 Newton), see Figure 25.

Figure 25. Three-point bending test machine (Zwick/Roell 0.5 KN).

The main parameters of the three-point bending test (Spatz et al., 1996) are given in the following table 4:

Variables values Distance between supports 40 mm Effective range of the starting position 10 mm Pre-test speed 100 mm/min Pre-charge 0.5 N Pre-charge speed 2 mm/min Test speed 32 mm/min Threshold switching force 40% of max Force Determination method Regression Beginning of the determination of module E 4 N Ending of the determination of module E 10 N Table 4. Parameters of the three-point bending test.

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Work plan, materials and methodology This test allows to assess three mechanical characteristics of cortical bone tissue. The Young’s Modulus of elasticity (E) is dependent of the quality of the bone material, and it measures the deformation that occurs in the bone when a force is applied on it, without going beyond the elastic limit of the deformation/force curve (Ritchie et al., 2008); this variable is expressed in GPa, and the beginning and the ending of the determination of module E was taken in the plastic strain region (between 4N and 10N). 퐹 ∙ 퐿3 퐸 = 48 ∙ ∆푥 ∙ 퐼 Where: F = central load of the machine. L = separation between the supports of the machine Δx = vertical displacement due to bending with load F; corrected for the curve area included between 4 N and 10 N. I = second moment of area of the transversal section of the specimen; obtained with this formula [I=w·d3/12], where [w] is the width and [d] is the depth of the specimen.

The Bending Strength (BS) is the maximum force that the bone material can withstand before fracturing, generally is expressed in Megapascal or in Newton/meters2 (Currey, 2002; Ritchie et al., 2008). 퐵푀 퐵푆 = 퐼 Where: BM = Bending moment; that is, the maximum load when the sample is broken multiplied by [10 · sample depth/2]. I = second moment of area of the transversal section of the specimen; obtained with this formula [I=w·d3/12], where [w] is the width and [d] is the depth of the specimen.

The Work under the curve (W) it is the work, per unit area, necessary to break the bone sample and it is expressed in kJ/m2 (Ritchie et al., 2008). 퐴푟푒푎 푢푛푑푒푟 푡ℎ푒 푐푢푟푣푒 푊 = 퐶푟표푠푠 − 푠푒푐푡𝑖표푛 푎푟푒푎 Where: Area under the curve = area calculated below the force-deformation curve. Cross-section area = area calculated as width multiplied by depth, measures taken in the average point of the bone specimen.

See Figure 26 for the dynamics of the test and how the head of the machine exerts a continuous and constant force, up to the breaking of the bone sample.

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Figure 26. Three-point bending test with cortical bone sample during the application of the force.

The machine produced an output chart in the software TestXpert II (Zwick GmbH & Co, Ulm, Germany) with a curve that correlates the strain/deformation (mm) and force (N). See Figure 27.

Figure 27. The curve that correlates the deformation (mm) and force (N) during a three-bending test on antler’s bone tissue.

In the elastic region of the curve, the deformation is totally reversible, the tangent of the curve is the Young's modulus of elasticity (E). After a small transition phase, where the first non-homogeneous structural failures occur, there is the plastic region of the curve (Currey et al., 2007). In the latter the deformations suffered by the material are permanent and at each small increase of the force exerted by the machine a large deformation is observed (non-constant force-deformation relationship), this post-yield deformation, that bone undergone, is not a true “plasticity” but is damage to the bone that increases its compliance (Frost, 1960). At the end of the curve we can found the point of breakage, called maximum force or breaking point (BS). The work required to arrive at this point, represented by

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Work plan, materials and methodology the area under the curve, is the fracture work (W). Young's modulus is affected mainly by mineralization and porosity (Currey 1999), so, small differences in mineralization produce differences in stiffness (Currey, 1979). We can say that bone possesses the property of anisotropy, thus, it has different properties in different directions, and therefore, the Young's modulus (E) meets this property; this region is also called the pre-load region (Turner et al., 1993).

4.4.2.2. Impact test (charpy test) This mechanical test was conducted with a pendulum falling on a bone sample with the external side in tension, using a CEAST-IMPACTOR II testing machine (CEAST S.p.A., Pianezza, Italy) equipped with a hammer. The loss of kinetic energy of the pendulum is measured by the machine, and this is considered to be the energy required to break the sample (Currey et al., 2004; Landete- Castillejos et al., 2010). This energy is normalized by dividing by the cross- sectional area of the sample, producing the impact energy absorption or impact work (U), this variable is expressed in kJ/m2. See Figure 28 for details on the dynamics of the impact test.

Figure 28. Machine used for the impact test (CEAST-IMPACTOR II) and functioning of the pendulum during an impact test.

It should be noted that these tests, in which only the relationship between force and strain (or force and impact energy) is considered, are tests for standardized samples of bone. Therefore, to determine the characteristics of the bone as a whole, in addition to the mechanical tests for the intrinsic properties of the bone tissue, the geometry and architecture of the bone must be considered (Sharir et al., 2008).

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4.4.3. Structure analysis The structure could affect the mechanical behaviour of bone tissue in many ways due its complexity, therefore in order to evaluate the mechanical properties of the bone, it is important to consider both the mechanical properties of its components and the structural relationship between them (Landis, 1995; Weiner & Traub, 1992).

Figure 29. Effects of changing of dimensions of cortical bone layer on mechanical behaviour of antler bone (modified from Davison et al., 2006).

The diameter and the thickness of the cortical bone have an important impact on the biomechanical integrity of the bone (Turner, 2002). In long bones the material constituents of the cortical layer gain strength as they are moved away from the neutral axis of the bone, and the bending strength of a particular area of bone is proportional to the fourth power of its distance from the neutral axis (Davison et al., 2006). See Figure 29 for details on how cortical bone layer could affect the bone behaviour. The cortical wall is responsible for most of the mechanical properties (Davison et al., 2006), and therefore it is necessary to know its diameter and thickness, as well as the quality of the material with which it is made. A bone with a thicker wall has a higher resistance to flexion (Currey et al., 2004); moreover, the total diameter can be used as a predictor of bone resistance (in 55% of cases for long

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Work plan, materials and methodology bones; Amman et al., 1996). For this reason, the total diameter, the thickness of cortical layer, the total perimeters and areas of cortical and trabecular tissues, in each sampling positions and each antler collected, were studied. Particularly, the cortical bone thickness of the cross-sections was measured at six equally spaced points around the perimeter, and then the average thickness was calculated as the mean of these measurements (called Average cortical thickness or CBD). While, using the total areas of trabecular and cortical tissues, was calculated the ratio between these two tissues of the antler’s bone (termed ratio cortical/total or CBA%). See Figure 30 for details.

Figure 30. Measurements collected in the complete cross-sections for each sampling position along the main beam. ImageJ software was used to determine: total diameter and total perimeter (3 repetition), cortical bone thickness (6 repetition), external perimeter and total area, as well as of the spongy bone. TD=total diameter; CT= cortical thickness; CBA= cortical bone area; CBP= cortical bone perimeter; TBA= trabecular bone area; TBP= trabecular bone perimeter.

4.4.4. Density and ash content The degrees of mineralization of cortical and spongy bone are positively related, while decreasing one equally decreases the other (Boskey & Coleman, 2011). When mineralization increases, in the bone tissue, stiffness is increased and a greater effort to break is required; therefore, while increased mineralization is important for imparting stiffness to bones, too high a mineralization can introduce fragility through a decrease in the bone’s toughness (Davison et al., 2006). This means that the mineral content, which is normally measured as ash, calcium or bone density, is the main objective for understanding mechanical properties (Currey, 1984; Lam & Pearson, 2005). Hernandez et al. (2001) found that, in both cortical and trabecular bone, small changes in the mineral content of bone will

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Work plan, materials and methodology result in disproportionate losses in bone strength and stiffness. Therefore, to study the density of bone tissue, and its contents to ashes, two fragments of the bone bars were used (in cervid as well as in bovid species). For this reason, the fragments’ surfaces were abraded using a semiautomatic equipment for polishing to obtain regular shapes and to erase pencil marks or permanent marker. Moreover, before this analysis, these fragments were dried out fully in a controlled heating chamber for 72 hours at 60°C, to standardize the specimens. For the density of cortical bone tissue, each sample was weighed with a precision balance (±0.01 g) and measured with a precision calliper (±0.01 mm); thus, density was calculated dividing the weight by the volume (calculated as length x width x depth). Another fragment was used to measure the ash content in percentage: the samples were weighed with a precision balance (±0.01 g) to get the dry weight, and subsequently were placed in refractory cups (weighted separately from the samples) and then in a muffle furnace (HTC 1400, Carbolite, UK) for 6 hours at 480°C. Ash content was calculated as the value of ashes thus obtained divided by the dry weight, once excluded the weight of the refractory cups (see Figure 31).

Figure 31. Necessary steps to calculate the ash content in the bone samples: for this analysis was used a muffle-furnace at 480°C (1); before put the bone sample inside the hoven, the samples and the refractory cups they were weighted separately (2); then the refractory cup and the samples were put inside the muffle for 6 hours (3); finally, the bone samples were extracted from the muffle and were weighted together with the refractory cups, with a precision balance (4).

4.4.5. Mineral content analysis The determination of the mineral elements in the bone tissue was performed by

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Work plan, materials and methodology Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES), after the solid bone samples were passed through an acid digestion. This analysis was performed in the CEBAS-CSIC (Centro de Edafología y Biología Aplicada del Segura-Consejo Superior de Investigaciones Científicas) in Murcia. This method involves the measurement of atomic emission by means of an optical spectroscopy technique; the samples are nebulized, and the aerosol produced is transported to the plasma flame where the excitation of the electrons takes place. A radiofrequency inductively coupled plasma (ICP-OES) generates the spectra corresponding to the atomic emission lines. The light beams are dispersed by a diffraction grating spectrometer and the detectors are responsible for measuring the intensities of the lines; the signals originated in the detectors are processed and controlled by a computer system. The ambient temperature during the analysis was 20 ± 5 °C. For solid samples, such as the bar portions of compact bone tissue, 0.05-0.20 g of the sample are used in this analysis and they are added to 25 mL digestion tubes with 4 mL of concentrated nitric acid and 1 mL of 33% hydrogen peroxide. These tubes are placed in the microwave reactor for 30 minutes, where the temperature of 220 °C is reached through pressure increases of 10 bar/minute. In the same Teflon reactor, 300 mL of ultrapure water, 30 mL of 33% oxygenated water and 2 mL of concentrated sulfuric acid are added. After this cycle, the tubes are slowly cooled, and they are filled in with ultrapure water in a double-gauge tube of 25 mL. Finally, they are homogenized and allowed to settle before passing the measurement in the ICP-OES. For the case of samples in which we want to measure mineral traces, the filling after digestion instead of 25 mL, will be make it to 10 mL, which means that we do less dilution and lower the detection limits of the method. The concentration of the metal is read directly on the screen of the ICP instrument; in diluted samples, this concentration is multiplied by the dilution factor and considering the amount of sample that was weighed to perform the test. See the following equation:

푀푒푡푎푙 푐표푛푡푒푛푡 [푚𝑔/푘𝑔] = (퐶 ∙ 퐹)/푃 Where: C = concentration of the metal read directly from the instrument

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Work plan, materials and methodology F = dilution factor P = weight of the sample taken to carry out the analysis (in g)

The result could be expressed in [mg/kg], [mg/L] or [ppm], with two decimals. For each cycle of measurements was used a calibrated solution to assess the standard value for the ICP-OES machine, moreover a cleaning solution was intercalated during the sample’s analysis.

4.4.6. Histology analysis During the study of manganese supplementation effects in red deer bone tissue, was used the following technique to observe the differences in the basic units that form bone tissue: the osteon. Red deer adults’ antlers were sampled in position 1(directly above the burr) and 4 (below the crown or upper tines) were embedded in polymethyl-methacrylate. Mineralized sections (50 µm-thick) were prepared by grinding-polishing method, and sections were stained with toluidine blue/pyronin G for light microscopy examination (Landete-Castillejos et al., 2012). This allowed to study that primary osteons are complete in both cases. Moreover, the osteons of Mn-supplemented animals look normal, this means that there is no sign of toxicity due to an overdose of Manganese (osteomalacic syndrome).

4.5. Statistical analysis In this paragraph are described the statistical analyses performed during the study of the trophies of the target species. In general, for each statistical analysis performed, a P-value of less than 0.05 was considered significant, whereas P- values between 0.05 and 0.10 were considered as a trend. Moreover, all the results obtained from the ANOVAs are expressed with Mean ± Standard Error of the mean. The results obtained are reported in chapter 5.

Cervus elaphus hispanicus Subjects were 29 deer of different age classes (adult n=19, yearlings n=10) that were divided in a manganese injected group (n=14) and a control group (n=15). Antler content in ashes and minerals, intrinsic mechanical properties and cross

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Work plan, materials and methodology section structure were examined at 4 points along the antler beam. One-way ANOVAs were used to check for differences in body parameters between groups before being subjected to manganese treatment. The same statistical test was used to assess rough differences in each parameter assessed in whole antler and for the body weight, in this case animals were divided by age (spiker and adult red deer), and if the animal was injected (Mn treated) or not (Control) with manganese as nutrient supplement. Because data showed a very clear difference between yearlings and adults (with very few significant effect of the supplementation in the yearlings group, only for Mn and Fe), we performed a GLMs analysis, testing the effects of treatment and age class on each of the studied antler variables, and after results confirmed this pattern, the rest of the following statistical tests were carried out only for adults red deer. The second step of the analysis, used a series of linear models (GLMs) to assess the treatment effects on the same variables once the effects of body weight (which greatly influence antler size and characteristics, Landete-Castillejos et al., 2007a; Gaspar-López et al., 2010) was included in the models, for these analyses were used the average value for the four sampled positions in the adult deer antler. Then, was studied the proportional difference between the top and the base (Position 4 - Position 1 / Position 1; i.e. a decrease in values is shown as a negative difference in percent) using a one-way ANOVAs. Finally, the third analysis included only the fourth sampling position in the adult deer antler. For this sampling position were performed a one-way ANOVAs to describe the average values of variables studied in the bone tissue, and then was performed GLMs analysis for each variable where the influence of injections of Mn (as nutrient supplement) and body weight were included. All analyses were carried out with SPSS version 19 (SPSS Inc., Chicago, IL, USA).

Cervus elaphus corsicanus The statistical analysis for Sardinian red deer was performed with two steps at different depth. The first step was to make a descriptive analysis of the 61 antlers

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Work plan, materials and methodology collected for this sub-species through its external morphological characteristics. Like described before in the paragraph 4.1.1, since the antlers could not be assigned to a specific male, and thus we are not certain which left and right antlers constitute a pair, antlers collected were divided only by class of age (n= 26 for subadults and n= 35 for adults). Moreover, every single antler has been treated individually in the initial analysis of the morphological features. Statistical comparisons were conducted using a one-way analysis of variance (ANOVAs) between characteristics of the two age subgroups. Moreover, Pearson´s correlation coefficients were used to describe the relationship between the morphological variables of the antlers within each age class group. The second step was the study of mechanical properties, structural characteristics and mineral profile of bone tissue. For this purpose, were selected the most representative and well-developed 12 antlers, the rules to select the antlers were as follows: the first tine (brow tine), the central tine and a bifurcation in the distal part of the antler (the presence of at least two tines in the crown) (Beccu, 1989). The test selected was the one-way analysis of variance (ANOVAs) to study mineral composition, mechanical properties and bone structure in each sampling position, along the antler main beam. Also, were calculated the percentage differences of each variables between the basal position and the distal position of sampling (position 1 versus position 4), in order to understand the physiological effort done by the animal to grow the antler (Landete-Castillejos et al., 2007b). For the analysis were used SPSS version 19 (SPSS Inc., Chicago, IL, USA) and MINITAB 16. All data are expressed in terms of means ± standard error of the mean.

Capreolus capreolus For the roe deer, the first step was to obtain a comparative analysis of the morphological measurements, structural characteristics, mechanical properties and mineral profile of the two groups of roe deer antlers (Spain origin versus Prague origin), using a one-way ANOVA on the mean value per antler for each of the antler (Spain antler, n=13; Czech antler, n=10). Then, we performed a GLMs analysis on the antler characteristics to assess the influence of two factors: origin (geographical origin of antlers), and sampling position (position 1 versus position

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Work plan, materials and methodology 2); as well as the interaction between these two factors. In addition, the mean values for each sampling position of the antler characteristics, separately for each group of antlers, were compared using one-way ANOVAs analysis (position1 versus position 2, within each antler group). These values often show the variation between base (which reflects the condition of the animal at the beginning of growth) and top part of the antler (where many parameters highlight the effort made by the animal). The characteristics mentioned and subjected to analyses were: 1) the morphological measurements (total antler length, first tine length -brow tine-, second tine length -back tine-, burr perimeter, total weight and the antler score as trophy; 2) intrinsic mechanical properties (Young’s modulus of elasticity, E; bending strength, BS; work to peak force, W; and impact energy, U); 3) structural and physical characteristics (average cortical thickness expressed in cm, CBD; the same expressed as percentage, CBD%; ratio cortical to total antler section area of the bone slice in proportion, CBA; density of the cortical bone); 4) mineral composition (ash content in percentage; macro-minerals -Ca, K, Mg, Na, P, S and Ca/P ratio-; and micro-minerals -Al, Cr, Fe, Li, Mn, Sr, Tl and Zn-). All analyses were carried out with SPSS version 22 (SPSS Inc., Chicago, IL, USA).

Taurotragus oryx For this species were used ANOVAs and linear regression models (GLMs) throughout various steps. Initially, differences between means of each sampling position along the vertical axis of the horn were assessed through ANOVA analysis with Tukey test for groups’ comparison. ANOVA was also used to analyse differences between the spiral and core bone tissue in position 1. Then, a linear regression analysis for each observed variable was performed to assess clearly the trend of the values along the sampling position. Data of ANOVAs in tables are presented as means for all variables observed. The variables tested were: density of the bone; Young’s modulus of elasticity E; bending strength BS; work to peak force W; impact energy U; ash content; and that of minerals Al, Bi, Ca, Cr, Cu, Fe, K, Li, Mg, Mn, Na, P, Se, Sr, Tl and Zn. The statistical analyses were performed with SPSS version 22 (SPSS Inc., Chicago, IL, USA).

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5. RESULTS

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In this chapter, the studies carried out during the period of the doctorate are listed. The first three studies (5.1, 5.2, 5.3) aimed to characterize the trophies of species with hunting or conservation interests. In the following chapter 6, there is a study carried out on the characterization of red deer trophies, when this is subjected to mineral supplementation; in order to characterize the antlers of this species and to understand how certain micro-minerals can have effects on the quality of the deer's antlers. All studies have been published in indexed journals, except for chapter 5.2 which is in the process of writing and final submission. For each study carried out, the relative discussions of the results are extracted and can be found in the chapter of the General Discussion (chapter 7); part of them have been expanded and enriched with other considerations, compared to the original published version.

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Results Article 5.1 Morphology, chemical composition, mechanical properties and structure in antler of Sardinian red deer (Cervus elaphus corsicanus).

Morphology, chemical composition, mechanical properties and structure in antler of Sardinian red deer (Cervus elaphus corsicanus). Cappelli J, Atzori AS, Ceacero F, Landete-Castillejos T, Cannas A, Gallego L, Garcia AJ. Hystrix-The Italian Journal of Mammalogy 2017, 28(1):110–112.

ISI Journal Citation Reports Ranking: Q1, 38/167 in Zoology Impact factor: 1.862

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Abstract The deer population present in Sardinia and Corsica represents an endemic subspecies Cervus elaphus corsicanus. We describe for the first time the characteristics of cast antlers of Sardinian red deer from the forest complex Sette Fratelli, south-east part of the island. Moreover, we describe the material mechanical properties, the structural ones, and the mineral profile of antlers from adults, comparing them with the antler characteristics of the subspecies C. e. hispanicus examined exactly with the same methodology. Sixty-one deer casted antlers were collected and classified as belonging to adults (35) or sub-adults (26). A first part of the study described the common features of the antlers of subspecies C. e. corsicanus through the analysis of morphology in all deer antlers. Subsequently, a more detailed study used 12 adult deer antlers for a destructive analysis. Statistical comparisons were conducted using ANOVAs between characteristics of the two age subgroups and using Pearson´s correlation coefficients between the antler’s morphological variables. In general, morphological antler measures had greater values in adults than in sub-adults. In comparison with Iberian deer, Sardinian adult antlers have a more simple structure with lower values in morphological features, mechanical properties and structural characteristics.

Keywords: cervid, tines, wildlife characterization, Sardinia.

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Results The deer population present in Sardinia and Corsica represents an endemic subspecies Cervus elaphus corsicanus (Pitra et al., 2004). At present, there are approximately 4270 animals in Sardinia, but the subspecies is classified as "endangered" by the IUCN (IUCN, 2004). The Sardinian deer survived naturally only in a few mountain areas: one of this is the Sette Fratelli Mountain (Casula et al., 2013). This subspecies is slightly smaller and more slender than continental red deer. Its antlers differ from the European subspecies by weight, shape and by the number of tines, which is limited to 4-5, against the ordinary 8-10 tines of an adult deer in Central-Europe (Caboni et al., 2006). In the distal part of the beam tines are placed on the same plane. The bez or second tine (from base) can be found only in few trophies of this subspecies (Beccu, 1989). Studies on antler characteristics of Iberian deer (Estevez et al., 2008; Landete-Castillejos et al., 2007c) prove that morphological variables, composition and mechanical properties can be used to diagnose diet and management problems of a deer population. Thus, we describe for the first time the characteristics of cast antlers of Sardinian red deer from the forest complex Sette Fratelli, to provide reference values for further studies which may help the conservation of deer. The aims of this study are: to describe the antler morphometry, the mechanical properties, the structure and the mineral profile of antlers from adults and subadults of this subspecies, and to compare them with the well-studied C. e. hispanicus. The forest complex Sette Fratelli is located in southeastern Sardinia (39 14’N, 9 30’E, 210-500 m. of altitude). Sixty-one antlers, grown and casted in the same year, were collected inside the forest natural area. Antlers were assigned as belonging to adults (35) and sub-adults (26), according to the presence of the following features: the brow tine, the central tine and a bifurcation in the distal part of the main beam (Beccu, 1989). The antlers collected showed no sign of diagenetic alteration, no weathering (grey color) and no cracks. In order to describe their morphological characteristics, the following measurements were recorded: total curve length, first tine length, second tine length, central tine length, burr perimeter, first half perimeter (between first tine and central tine), second half perimeter (between central tine and the crown), weight, and scoring (CIC, 1960). Thereafter, 12 antlers of adult individuals were selected in order to obtain antlers that represent the characteristics of the adult antler, following these criteria: the greatest average total length of the main beam and the greatest number of tips. Since they cannot be assigned to a particular male, and thus we are not certain which left and right antlers constitute a pair, only left antlers were used in this part of the study. Samples were collected from four positions along the main antler beam (Fig.1). From each position, a 1 cm slice (complete transverse cross-section) and a cylinder about 5–6 cm (to prepare bars from the cortical wall) were obtained (Landete-Castillejos et al., 2010).

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Figure 1. Sampling technique for adult deer antlers: the antler bone was cut at various level along the vertical axis, in each sampling position were obtained a complete transverse cross-section (to study the structural characteristics) and a cylinder from which two cortical bone bars were extracted to study the mechanical properties and the mineral profile. Intrinsic mechanical properties (Cappelli et al., 2015) were measured in two ways: bending tests were carried out in a Zwick/Roell 500N machine and the following mechanical properties were measured: Young's Modulus of elasticity E, Bending strength BS, and the Work to peak force W; impact tests were carried out in a CEAST-IMPACTOR II machine (CEAST S.p.A., Pianezza, Italy), in order to obtain the impact energy absorption (U). Mineral content (Ca, Mg, K, P, B, Co, Cu, Fe, Na, Mn, S, Se, Si, Sr, Zn) was measured from cortical bone samples through spectrophotometry (Landete-Castillejos et al., 2007a). Transverse cross sections were used to measure the average cortical thickness and to calculate the ratio between cortical and total area, cortical/total ratio. Density of cortical bone was calculated dividing the weight by the volume of the bone bars fragments. Another fragment was used to measure ash content after burning in a muffle furnace (HTC 1400, Carbolite, UK). Statistical analyses were performed using MINITAB 16 and SPSS version 19 (SPSS Inc., Chicago, IL, USA); comparisons were conducted using ANOVAs between characteristics of the two age subgroups, and also using Pearson´s correlation coefficients to describe the relationship between the antler morphological variables. Descriptive statistics were also applied to the morphological, structural and chemical characteristics of a selected adult deer antler (N=12) for each sampling position along the antler main beam.

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Results The morphometric characterization of the antlers of adult and subadult C. e. corsicanus is shown in Fig. 2.

Figure 2. Descriptive statistics of antler morphological characteristics of Cervus elaphus corsicanus. Mean for antlers from subadult and adult deer (N=61). Probability calculated using ANOVA; levels p < 0.01 and p < 0.001 are indicated by ** and ***, respectively. Second half perimeter cannot be measured in antlers of subadult animals.

Generally, adult deer antlers were heavier (0.52±0.03 VS 0.34±0.03, p<0.001) and with higher scoring (66.7±1.8 VS 52.4±2.2, p<0.001). Pearson’s correlation analysis within each age class (Tab. 1) showed that in subadults the strength of association between the morphological variables was high and strongly significant. Afterwards, 12 adult deer antlers were selected to characterize the structural characteristics, mechanical properties and mineral profile. The means of each studied position (Tab. 2) showed that distal positions (3 and 4) had lowest values with a gradually decreasing trend form the base to the antler’s tip.

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Results Central Total First tine tine Burr First half Variables length length length perimeter perimeter

Subadult deer

First tine length 0.833***

Central tine 0.813*** 0.751*** length

Burr perimeter 0.749*** 0.732*** 0.663***

First half 0.782*** 0.636** 0.784*** 0.811*** perimeter

Antler weight 0.929*** 0.887*** 0.870*** 0.816*** 0.775***

Adult deer

First tine length 0.625**

Central tine 0.639*** 0.440** length

Burr perimeter 0.753*** 0.537*** 0.451**

First half 0.640*** 0.319 0.667*** 0.567*** perimeter

Second half -0.521 0.043 -0.798** 0.421 0.326 perimeter

Antler weight 0.761*** 0.545*** 0.775*** 0.666*** 0.791***

Table 1. Pearson’s correlation coefficients between measured morphological variables of subadult and adult deer antlers of Cervus elaphus corsicanus. Probability at levels p < 0.01 and p < 0.001 are indicated, respectively, by ** and ***.

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Variables Position 1 Position 2 Position 3 Position 4 Difference base/tip (%) Cortical/total ratio 0.54 ± 0.02A 0.37 ± 0.01C 0.43 ± 0.02B 0.42 ± 0.03BC -21.04±4.26 Average cortical thickness (mm) 4.48 ± 0.16A 3.27 ± 0.09B 3.39 ± 0.14B 3.27 ± 0.16B -26.54±3.36 A A B C Density of cortical bone (g/mL) 1.81 ± 0.02 1.77 ± 0.02 1.60 ± 0.04 1.50 ± 0.05 -17.63±2.38 Young’s modulus of elasticity (E), 14.72 ± 0.41A 13.93 ± 0.34A 12.22 ± 0.52B 10.74 ± 0.57C -26.53±3.57 (GPa)Bending Strength (BS), (MPa) 275.74 ± 16.38A 271.92 ± 5.00A 252.39 ± 8.19AB 225.66 ± 13.37B -22.26±5.02 -2 A A B B Work to peak force (W), (kJm ) 41.35 ± 2.37 39.03 ± 1.60 35.66 ± 2.17 32.07 ± 3.05 -22.68±7.20 Impact work (U), (kJm-2) 22.63 ± 1.16 21.64 ± 1.18 22.28 ± 0.94 20.87 ± 1.10 -6.10±5.44 Ashes (%) 59.09 ± 0.31 58.94 ± 0.52 58.75 ± 0.48 59.33 ± 0.52 0.40±0.59 Ca (wt%) 21.17 ± 0.36 20.99 ± 0.18 20.57 ± 0.26 20.63 ± 0.27 -2.40±1.24 Mg (wt%) 0.446 ± 0.007A 0.447 ± 0.006A 0.436±0.008AB 0.430 ± 0.007B -3.64±1.17 Na (wt%) 0.555 ± 0.009 0.556 ± 0.008 0.535 ± 0.010 0.534 ± 0.018 -3.30±4.28 P (wt%) 9.90 ± 0.18 9.94 ± 0.09 9.72 ± 0.14 9.62 ± 0.11 -2.63±1.25

B (ppm) 4.04 ± 0.12 4.10 ± 0.13 4.11 ± 0.14 4.05 ± 0.13 0.13±0.93 Co (ppm) 0.036 ± 0.010 0.058 ± 0.007 0.047 ± 0.010 0.034 ± 0.008 -15.09±29.04 Cu (ppm) 0.76 ± 0.05 0.72 ± 0.04 0.85 ± 0.04 0.87 ± 0.06 -17.25±7.41 Fe(ppm) 5.12 ± 1.95 12.49 ± 5.14 10.22 ± 1.26 9.38 ± 2.23 45.41±12.55 K (ppm) 360.48 ± 7.72A 341.16 ± 5.67AB 319.05 ± 7.07BC 304.03 ± 11.00C -15.18±3.72 Mn (ppm) 0.47 ± 0.05 0.42 ± 0.03 0.56 ± 0.06 0.61 ± 0.11 -3.78±6.45 S (ppm) 1279.49 ± 17.61B 1279.76 ± 18.22B 1376.97 ± 18.24A 1382.27 ± 12.45A 8.26±1.77 Se (ppm) 1.07 ± 0.10 0.82 ± 0.15 1.16 ± 0.12 1.23 ± 0.11 15.58±11.73 Si (ppm) 28.73 ± 3.84C 29.73 ± 4.04BC 40.25 ± 5.15A 38.90 ± 3.83AB 25.54±10.34 Sr (ppm) 240.28 ± 7.22 243.11 ± 7.47 237.71 ± 8.26 236.59 ± 8.55 -1.57±1.69 A AB AB B Zn (ppm) 68.73 ± 2.78 67.57 ± 2.66 66.71 ± 2.35 65.97 ± 2.11 -3.51±1.58 Table 2. Means (± S.E.) of the four studied positions for structural characteristics, mechanical properties and mineral profile of the adult deer of Cervus elaphus corsicanus (selected antlers, N=12). The last column shows mean (± S.E.) differences between the basal position (Position 1) and the distal position (Position 4) in percentage. ANOVA analyses with Tukey test show (in superscripts) differences among the positions.

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These findings show that antlers from adults have more tines, greater weight and longer total length. The comparison with C. e. italicus (Zachos et al., 2014; Mattioli & Ferretti, 2014) shows that Sardinian red deer has a quite similar number of tines per antler (2.11±0.16 in Sardinian deer VS 2.9±0.9 for C. e. italicus), but a lower average weight (0.52 kg in Sardinian red deer VS 0.9 kg in C. e. italicus). Furthermore, in the studied Sardinian subpopulation only a 20% of antlers have three-tined antlers; whereas the C. e. italicus recorded a 41.5%. This can be explained by the fact that antler size is strongly affected by ecological factors during their growth (Brown, 1990). The Sardinian deer antler morphology matches the maintenance phenotype model described by Geist (1998), according to which the phenotype plasticity is pushed to a model of efficiency.

Recent studies show that the composition of the antlers can tell us great information that may be useful to know the status of a wildlife population (Landete-Castillejos et al., 2013b); moreover, the monitoring of morphometrics could help to assess how individuals react to conservation measures (Mattioli & Ferretti, 2014). Generally, the mechanical and structural properties of Sardinian antler bone show a decrease in the observed values. This is coherent with the hypothesis that the decreasing trend shows the depletion of those minerals in the body (Landete-Castillejos et al., 2007c, 2010), thus reflecting the physiological exhaustion to grow the antlers. Comparing antler characteristics of C. e. corsicanus and C. e. hispanicus, the values in most traits are lower for the first subspecies. The average antler weight, first tine length, burr perimeter, and total length are lower by a 65%, 45%, 30% and a 26% respectively (data for C. e. hispanicus in Fierro et al., 2002). This could be due to its more slender body size. Animals with a smaller skeleton could invest less mineral bone resources for the growth of their antlers. Various relationships indicate that body morphometry characteristics were related to antler measurements (Ceacero, 2016). Regarding structure, the average cortical thickness values for Sardinian deer were much lower than populations of Iberian Red deer: -36% (data for C. e. hispanicus in Landete-Castillejos et al., 2010). The same happens for the mechanical properties: E of Sardinian deer is -17.8% than that of Iberian deer (12.89±0.33 VS 15.69±0.32 in C. e. hispanicus) whereas W is slightly lower (-3%, 38±1.5 in C. e. hispanicus). Also, BS and U show lower values (306.6±6.4 and 54.9±2.7 for C. e. hispanicus; Landete-Castillejos et al., 2010). The same trend exists for ash content, which showed a -5.2%. The reason may be that the conditions of the natural habitat in which the population is widespread could influence the diet, and a lower availability of resources may end up in antlers of worse quality (Scribner et al., 1989) and its observed differences in mechanical properties. However, it is possible that formal tests for tissue toughness, which were not specifically examined in this study, might be less degraded in our sample when compared to the results of impact and bending tests that we used herein. Additional studies would be required to test this hypothesis because various types of mechanical tests are required to fully assess the antler mechanical properties (Launey et al., 2010b; Skedros et al., 2014). Antler minerals play an important role in antler growth, especially for Ca and P (Ceacero, 2016). The comparison between Sardinian and Iberian red deer shows a variable trend: for Ca and P the difference is minimal (-1% and -3%, respectively; Landete-Castillejos et al., 2010), for other minerals the difference is greater (for Na -7.8%, for K -42.7%, and for Co -82.6%

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Results in C.e. corsicanus). In conclusion, adult and subadult Sardinian deer showed different morphological characteristics in antlers; this subspecies has a simplified antler structure compared to C.e. hispanicus and to other populations present in Italy (C.e. italicus). This research allows enriching the knowledge of this subspecies through an analysis of the structural characteristics, the chemical content and mechanical properties of antler bone material.

Acknowledgements

This work was supported by the pre-doctoral contract for the training of research personnel under the R+D+I Plan, co-financed by the European Social Fund (2015/4062; 2015). The authors thank to all the staff members of the UNISS and Fo.Re.STAS. Regional agency for the help in collecting the Corsican deer antlers.

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Results Article 5.2 Smaller does not mean worse: variation of roe deer antlers from two distant populations in their mechanical and structural properties and mineral profile.

Smaller does not mean worse: variation of roe deer (Capreolus capreolus) antlers from two distant populations in their mechanical and structural properties and mineral profile. Cappelli J, Ceacero F, Landete-Castillejos T, Gallego L, García AJ. Sent for revision to Journal of Zoology, 2019.

ISI Journal Citation Reports Ranking: Q2, 45/170 in Zoology Impact factor: 1.676

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Abstract The European roe deer (Capreolus capreolus) is the most abundant ungulate in Europe. Although a number of studies assessed distribution, behaviour and ecology, few studied antlers, and no one except the present study assessed how environmental conditions and diet affected their antler structure, mechanical properties of its bone material and its mineral profile. Antlers were collected from two game estates differing in location, climate and management: 10 specimens came from adult roe deer in the central-southern part of the Czech Republic and 13 from the south-east of Spain (both groups older than three years). After measuring whole-antler parameters, a destructive sampling was performed to obtain a full-transversal section and cortical bone samples from two sampling position along the main beam. Then bone structure, mechanical properties (three-point bending test and impact test) and the mineral profile were studied. Statistical comparisons were conducted using ANOVAs. Results showed that roe deer from Spanish population had bigger antlers (+118% for weight and +25% of total length) than Czech roe deer. They also differed in mechanical properties (greater impact energy and work to fracture, but lower Young’s Modulus and Bending Strength in Spanish vs. Czech roe deer), as well as in structure. We discuss that, in most cases, the differences found between populations may be caused by differences in habitat quality, including the diet in a similar way as reported for red deer. Thus, despite inter-species differences, this study shows general trends regarding how antler parameters change along its main axis, and how ecological factors affect them. Thus, may be useful to understand the role of management on roe deer population, using the antlers as an estimate of male physiological state.

Keywords: roe deer, antler, bone structure, minerals, mechanical properties, wildlife management.

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Results Introduction The European roe deer (Capreolus capreolus) is the most abundant ungulate species in Europe (Linnell and Zachos 2011). Commonly, roe deer is treated as a monotypic species in Europe with numerous variations that have been considered subspecies by some authors (Sempere et al. 1996; Aragón et al. 1998; Royo et al. 2007). Some studies have investigated the factors that determine its distribution: vegetation cover (Acevedo et al. 2005), food availability (Plard et al. 2014), climate (Cagnacci et al. 2011), or population density (Pettorelli et al. 2001). Other studies have investigated roe deer cranial morphology and habitat use (Milošević-Zlatanović et al. 2016) or biometric differences observed in skulls that could reflect minor morphological adaptations to different habitats (Fandos & Reig 1993). Other authors have studied deer behaviour assessing the role of antler in reproductive activity (Clutton-Brock 1982; Hoem et al. 2007) and the genetic components that are responsible for differences in antler dimensions (Hartl et al. 1995; Geist 1966). The main reasons for studying antlers are their rapid growth and annual regeneration which make them a good model for studying bone tissue and the possible factors that influence the bone growth process (Kierdorf et al. 2013). Generally, these factors, apart from the genetic characteristics, are populational density (Santiago-Moreno et al. 2001), climate (Mysterud et al. 2005; Landete- Castillejos et al. 2010), and the food quality (Brown 1990). The latter is a consequence of the previous two and influences the animal's weight and body condition and subsequently the antler growth (bigger antlers are grown by heavier animals, Gómez et al. 2006, 2012; Landete-Castillejos et al. 2007b; Ramanzin and Sturaro 2014). Moreover, the antler mass in C. capreolus represents approximately up to 23% of the dry skeleton mass and, due to the species’ small body size, in roe deer the investment in antler growth could be lower than in larger deer species, as has already been observed by Ceacero (2016). This author hypothesized that larger species are subjected to greater physiological constraints than smaller ones, and these constraints are mainly linked to skeleton size (skeleton mass in red deer could explain up to 72% of variability in antler investment, Gómez et al. 2012). In addition, secondary sexual traits such as antlers, are honest signals of male quality for reproduction and are expected to be costly to produce and maintain, particularly for males in poor condition (Vanpé et al. 2007; Ciuti and Apollonio 2011). Thus, well-developed antlers could be an indicator of a male with good nutrition (Landete-Castillejos et al 2007b, 2007c, 2012) or an indicator of allocation of resources and performance in adulthood (Lemaître et al. 2018). That means that antlers can be considered as a valuable tool for studying the performance of a population, and so far, the hypothesis that antlers constitute quality indicators has received little attention in roe deer (Pélabon and van Breukelen 1998; for other species: Solberg and Saether 1993; Landete- Castillejos et al. 2007c, 2013). Roe antler growth cycle starts in October-November, when the antlers of the previous year are cast (for adults). For animals in their first year of life, a bulge (button) is developed, from which the first antler will grow. The antlers grow protected by an epithelial tissue rich in blood vessels (velvet) and stop growing around March. Then, the loss of the external velvet (shedding) takes place

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Results (March-May). The animal remains with clean antlers during the mating season in summer (Sempere 1990). Since a qualitative and quantitative analysis of antler bone of roe deer is lacking, and there are no studies comparing these features in populations grown in habitats with different characteristics, this study has three aims: (I) to get an initial characterization of roe deer antlers; (II) to compare the antler bone structure characteristics, mechanical properties and mineral profile of two populations greatly differing in environmental conditions (mostly distinct diet and vegetation availability); and (III) to discuss parameters and trends in roe deer antlers with those reported in antlers of red deer.

Material and methods Two groups of roe deer antlers were collected: 10 pairs from Czech Republic and 13 individual antlers from Spain. The Czech antlers were collected from an open game estate around Vysoký Chlumec (Czech Republic; 600 m altitude); each pair of antlers were obtained from hunted adult animals (more than three years old). Animals were born and grown inside the area, where no mineral supplementation during winter was used, only meadow hay and oat. Habitat was a mosaic structure of mixed coniferous and broad-leaved forests, crop fields and meadows, where other cervid were present (fallow deer). The climate conditions were characterized by a temperate continental climate, with warm summers and cold, cloudy and snowy winters. The Spanish antlers were collected inside a game estate located in the south-east (near Caravaca de la Cruz, Spain, 685 m altitude); animals lived on soil sown with selected herbs; in addition, animals had access, throughout the year, to additional food such as pellets, barley and oats. All antlers belonged to adult animals (more than three years old). The climate conditions were characterized by a Mediterranean-semiarid climate with irregular rainfall. The daily thermal amplitude is moderate, but compared to other regions of Europe, the annual range of temperatures is very narrow. The rainfall occurs mainly in spring and especially autumn (but there is never snow or ice), separated by a summer of severe drought. Each trophy (pair of antlers of roe deer) was measured and evaluated using the CIC method (CIC 1960). For each roe deer, if possible, we selected only one antler, the right one, for the destructive analysis described below. Antler bone samples were extracted from two levels along the main vertical axis, in order to assess whether the properties of the bone tissue change at different stages of growth, thus reflecting the physiological effort made in growing the antler, as it happens in red deer (Landete-Castillejos et al. 2007b, 2007c; Estevez et al. 2008). The methodology to process these samples followed that previously used in studies on red deer antlers (Landete-Castillejos et al. 2010), adapted to the smaller size of the roe deer trophy. Thus, the sampling positions were: position 1 (directly above the burr) and position 2 (below the distal bifurcation or 5 cm under the tip), see Fig. 1. From each position a 1 cm-slice (complete transverse cross- section), if possible, and a cylinder of about 5–6 cm were extracted.

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Figure 1. Sampling technique for adult deer antlers of Capreolus capreolus: the antler bone was cut at two levels along the vertical axis, in each sampling position were obtained a complete transverse cross-section if possible and a cylinder from which two cortical bone bars were extracted to study the mechanical properties and the mineral profile. Firstly, to obtain raw bone bars, a circular low-speed saw was used. Then, the surfaces of bars were abraded using a semiautomatic equipment for polishing (MetaServ® 250 Double, Buehler-Illinois Tool Works Inc., Lake Bluff, IL, USA) to get the right size; the width and depth of each sample were recorded to the nearest 0.01 mm using a digital calliper prior to testing. Heating of the bone was controlled by keeping the specimens wet all the time during machining and sanding. The final size was 4.5 X 2.5 mm and a variable length allowing a gauge length of 40 mm (a length at which the shear effects are reduced thus minimizing errors in the estimate of the Young’s Modulus; Landete-Castillejos et al. 2010). Subsequently, to study the bone tissue structure, the transverse cross-sections were used. However, due to the small size of roe deer antlers, sometimes antler length was too short to get slices. In these cases, we estimated structural properties from transverse sections directly from the basal face of the cylinders of the position 1 and upper face of the cylinders in position 2 before the cylinders were cut to obtain the bars. Thus, for measures in transverse sections, the complete cross-sections were polished and then each slice was scanned on a flatbed scanner (ScanJet 4370 Photo Scanner, HP Inc., Palo Alto, USA) at 600 dpi. Each image was processed with an image analysis software (ImageJ); the cortical bone width was measured at six equally spaced points around the perimeter to obtain the average cortical width (Ct.B.Wi in cm) and the ratio between cortical bone width and total diameter of the section (Ct.B.Wi% in percentage). Then, we measured the total area of the section, and the area of cortical and cancellous bone tissues (to calculate the ratio between cortical and total area, Ct.B.Ar). Specimens for mechanical testing were first subjected to a procedure to standardize humidity content as described in Cappelli et al. (2015). This way, the

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Results bone bars had homogeneous humidity content when they were tested. Three- point bending test with the periosteal side in tension was carried out in a Zwick/Roell 500N machine and analysed with the software testXpert II (Zwick GmbH & Co, Ulm, Germany). Speed of the machine head was set at 32mm/min and the distance between supports was set at 40 mm (machine compliance was found to be negligible at this gauge length and sample depth, which has an aspect ratio length to depth (AR) of 16; Currey et al. 2009a; Spatz et al. 1996). In this first test the following mechanical features were observed: Young's Modulus of elasticity (E), an estimate of stiffness; Bending strength (BS), calculated from the maximum stress at the greatest load borne; and Work to peak force (W), the total work under the load-deformation curve up to the maximum load borne, divided by the cross-sectional area (Currey et al. 2009b). Moreover, the influence of structural stiffness of the antlers was calculate in terms of ExI, where I is the Second Moment of Area calculated on the transverse section (Currey, 2002; Burr & Turner, 2003), as an annulus or ring shaped object: I =π/4 (B.Dm4-C.Dm4); where B.Dm is the total bone diameter of the section and C.Dm is the cancellous bone diameter (Burr & Turner, 2003). The second mechanical test (charpy test) was performed using an oscillating pendulum which broke an un-notched sample with the periosteal side in tension. The loss of kinetic energy of the pendulum was measured, and this was the energy required to break the sample (Landete-Castillejos et al. 2010). This energy was normalized by dividing it by the cross-sectional area of the specimen, producing the impact work (U). Tests were carried out in a CEAST-IMPACTOR II testing machine (CEAST S.p.A., Pianezza, Italy). The fragments resulting after the mechanical tests were polished to achieve a regular shape and were used to study the antler bone density and the ash content. For such purpose, two fragments for each sampling position were placed in a controlled heating chamber (Memmert UN110, Memmert GmbH, Schwabach, Germany) for 72 hours at 60°C to dry them out fully. Afterwards, in order to calculate the cortical bone density (Ct.B.Dn), one fragment was weighed with a precision balance (±0.01 g) and measured with a precision scale (±0.01 mm); Ct.B.Dn was calculated dividing the weight by the volume. The same fragment was used to assess the antler mineral composition. Thus, samples were digested with HNO3-HCl and diluted with ultrapure deionized water. Then, total concentrations of minerals (Ca, P, Mg, Na, S, K, B, Cu, Fe, Mn, Sr, Zn, Al, Bi, Li, Cr) were quantified with optical emission spectroscopy (ICP-OES) using a Perkin- Elmer Optima 5300 DV (Shelton, CT, USA); for full details see Landete- Castillejos et al. (2010). The second fragment was used to measure ash content, by weighting the dried samples with a precision balance (±0.01 g) to get the dry weight, and, subsequently, they were placed in a muffle furnace (HTC 1400, Carbolite-Gero Ltd, Derbyshire, UK) for 6 hours at 480°C. Ash content was calculated as the value of ashes thus obtained divided by the dry weight. In the statistical analysis, the first step was to obtain a comparative analysis of the morphological measurements, structural and mechanical properties and mineral profile of the two groups of roe deer antlers, using a one-way ANOVA on the mean value per antler. Later, we performed a GLM analysis on the antler characteristics to assess the influence of two factors: origin (geographical origin of antlers), and sampling position (position 1 versus position 2); as well as the interaction between these two factors. In addition, the mean values for each

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Results sampling position, separately for each group of antlers, were compared using one-way ANOVAs. These values often show the variation between base and top part of the antler. Furthermore, a Pearson correlation analysis was performed to study the possible relationships between the observed mechanical and structural properties. The characteristics mentioned and subjected to analyses were: 1) the morphological measurements (total antler length, first tine length -brow tine-, second tine length -back tine-, burr perimeter, total weight and the antler score as trophy; 2) intrinsic mechanical properties (Young’s modulus of elasticity, E; bending strength, BS; work to peak force, W; and impact energy, U) and structural stiffness calculate as ExI; 3) structural and physical characteristics (average cortical tissue width expressed in cm, Ct.B.Wi; the same expressed as percentage of total diameter of the section, Ct.B.Wi%; ratio cortical to total antler section area of the bone slice in proportion, Ct.B.Ar; density of the cortical bone, Ct.B.Dn); 4) mineral composition and ash content in percentage. All analyses were carried out with SPSS version 22 (SPSS Inc., Chicago, IL, USA).

Results The two studied populations have slightly different characteristics in the morphology of their antlers. In general, roe deer in the Spanish population showed larger antlers with a longer length and greater weight. The average antler score was higher for Spanish antlers than for the Czech set (+14% in Spanish individuals). Almost all the antlers studied had three well-developed tines (length > 2 cm) with larger tines in the Spanish group (+62.7% and +63.8%, for first and second tine length respectively in these antlers). The mechanical and structural characteristics do not show differences in the same direction: Czech roe deer showed higher values for E and BS (+11% and +14% respectively), but lower for W (-21%) and U (-65%). The ExI was significantly higher for roe deer in the Spanish population; whereas, the Ct.B.Wi% and Ct.B.Ar were higher in antlers of the Czech population, only Ct.B.Ar difference achieved significance. Bone mineral content showed some significant differences between the two population: antlers in the Czech population showed higher values for Ca, K, P, Cr, Li, Sr and Zn (+11%, +15%, +16%, +35%, +39%, +19% and +16%, respectively); whereas antlers in the Spanish population showed higher values for Na (+13%), Mn (+19%), Tl (+84%) and for the Ca/P ratio (+24%). The Cu content was found, above the detectable limits, only in the Spanish population. See Table 1 for full results.

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Results Variables Czech Republic Spain p Burr perimeter (cm) 10.90 ± 0.31 17,41 ± 0.28 <0,001 1st tine length (cm) 4.170 ± 0.69 6.785 ± 0.45 <0.01 2nd tine length (cm) 2.680 ± 0.42 4.392 ± 0.42 <0.05 Antler curved length (cm) 18.06 ± 0.65 22.52 ± 0.50 <0.001 Antler weight (g) 52.71 ± 2.11 114.88 ± 3.71 <0.001 Antler score 34.26 ± 1.81 52.79 ± 3.39 <0.001 Young’s modulus of elasticity (E), Gpa 27.91 ± 1.00 24.88 ± 0.70 <0.05 Bending Strength (BS), Mpa 333.23 ± 6.35 286.63 ± 8.78 <0.001 Work to peak force (W), kJm-2 26.37 ± 2.28 31.85 ± 1.71 NS Impact work (U), kJm-2 10.92 ± 0.76 17.97 ± 1.34 <0.001 E x I 10.39 ± 1.76 28.06 ± 1.93 <0.001 Ct.B.Wi (cm) 0.40 ± 0.02 0.48 ± 0.02 <0.05 Ct.B.Wi%(%) 0.54 ± 0.04 0.45 ± 0.03 NS Ct.B.Ar (%) 0.84 ± 0.01 0.68 ± 0.03 <0.01 Ct.B.Dn (Kg/dm3) 1.62 ± 0.04 1.68 ± 0.02 NS Ashes (%) 57.35 ± 0.92 56.90 ± 0.59 NS Ca (g/100g) 17.10 ± 0.29 17.71 ± 1.07 NS K (g/100g) 0.0250 ± 0.0005 0.0212 ± 0.0008 <0.01 Mg (g/100g) 0.296 ± 0.008 0.347 ± 0.020 NS Na (g/100g) 0.472 ± 0.006 0.533 ± 0.013 <0.01 P (g/100g) 11.27 ± 0.21 9.44 ± 0.61 <0.05 S_g/100g 0.265 ± 0.003 0.250 ± 0.009 NS Al (mg/Kg) 17.17 ± 0.52 17.35 ± 1.37 NS Cu (mg/Kg) - 0.36 ± 0.04 - Cr (mg/Kg) 0.76 ± 0.02 0.49 ± 0.01 <0.001 Fe (mg/Kg) 18.91 ± 2.87 16.87 ± 2.43 NS Li (mg/Kg) 3.31 ± 0.15 2.03 ± 0.05 <0.001 Mn (mg/Kg) 19.50 ± 0.52 23.18 ± 0.89 <0.01 Sr_mg/Kg 172.88 ± 11.37 140.18 ± 20.42 NS Tl_mg/Kg 27.87 ± 0.72 51.20 ± 2.78 <0.001 Zn_mg/Kg 57.81 ± 2.17 48.41 ± 1.78 <0.01 Ca/P 1.52 ± 0.01 1.88 ± 0.02 <0.001 Table 1. Morphological and structural characteristics, mechanical properties and mineral profile for adult roe deer antlers from Spain (n=13) and Czech Republic (n=10). The p- value corresponds to one-way ANOVA on the mean (± SE) per antler of the two positions examined. The GLM analyses, assessing jointly origin and antler position effects (physiological effort during the antler grow), confirmed that antler features differed between the two origins, but also that position affected mechanical properties (E, BS and W), the ExI (R2= 0.81) and Ct.B.Wi (R2=0.45). Regarding mineral composition, origin of antlers affected almost all minerals, but only on the Zinc content showed also an effect due to the sampling position (R2=0.36, p=0.035). See Table 2 for details. Pearson’s correlation coefficients showed that Spanish group had stronger relationship between mechanical and structural properties of antler, especially for E and BS (Table 3).

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Factors in the model

Position Origin Position*Origin Variables R2 Intercept ± S.E. β ± S.E. Sig. β ± S.E. Sig. β ± S.E. Sig.

Young’s modulus of elasticity (E), Gpa 0.22 23.78±0.88 2.19±1.07 0.046 3.03±1.08 0.007 - - Bending Strength (BS), MPa 0.28 273.36±10.20 26.54±12.34 0.037 42.19±12.49 0.002 - - Work to peak force (W), kJ/m2 0.33 26.37±2.45 13.003±3.472 0.008 -0.86±3.72 0.018 -11.29±5.27 0.038 Impact work (U), kJ/m2 0.37 17.97±0.913 - - -7.05±1.38 <0.001 - -

E x I 0.81 3.47 ± 2.79 10.74 ± 4.06 <0.001 7.95 ± 3.78 <0.001 24.39 ± 5.38 <0.001

Ct.B.Wi (cm) 0.45 0.40±0.03 0.17±0.03 <0.001 -0.08±0.03 0.019 - -

Ct.B.Ar (%) 0.26 0.68±0.02 - - 0.14±0.04 <0.001 - - Ct.B.Dn (Kg/dm3) ------

Ashes (%) 0.15 56.90±0.39 - - 1.71±0.62 0.009 - - K (g/100g) 0.34 0.021±0.001 - - 0.004±0.001 <0.001 - -

Mg (g/100g) 0.14 0.34±0.11 - - -0.04±0.02 0.011 - - Na (g/100g) 0.31 0.53±0.09 - - -0.06±0.01 <0.001 - - P (g/100g) 0.24 9.34±0.35 - - 1.93±0.52 0.001 - - Cr (mg/Kg) 0.74 0.49±0.02 - - 0.27±0.03 <0.001 - -

Li (mg/Kg) 0.78 2.03±0.07 - - 1.28±0.10 <0.001 - -

Mn (mg/Kg) 0.30 23.14±0.56 - - -3.63±0.84 <0.001 - - Tl_mg/Kg 0.78 51.20±1.49 - - -23.33±2.06 <0.001 - -

Zn_mg/Kg 0.36 50.60±1.90 -4.89±2.24 0.035 9.65±2.26 <0.001 - - Ca/P 0.86 1.88±0.01 - - -0.36±0.02 <0.001 - -

Table 2. GLMs analyses showing the influence of antler position and population origin, on the composition, structure and mechanical properties of antlers in roe deer. The coefficient β (± S.E.) is related to the difference of the value observed in position 1 with respect to the position 2, and for the origin, is related to the difference of the value observed in Czech antlers in respect of the Spanish antlers. Dashes indicate coefficients that were not significant. For minerals, Ca and S, Al, Fe, Sr were not found any significantly suitable GLM models.

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Ct.B.Dn Ashes Ct.B.Wi Ct.B.Wi% Variables E (GPa) BS (MPa) W (kJ/m2) U (kJ/m2) (Kg/dm3) (%) (cm) (%) Spain antler set BS (MPa) 0.835** W (kJ/m2) 0.066 0.434 U (kJ/m2) -0.538 -0.736** -0.714** Ct.B.Dn (Kg/dm3) 0.346 0.104 -0.198 0.066 Ashes (%) 0.725** 0.714** 0.313 -0.571* 0.374 Ct.B.Wi (cm) 0.495 0.495 -0.148 -0.319 0.209 0.275 Ct.B.Wi%(%) 0.473 0.473 0.033 -0.412 -0.066 - 0.802** Ct.B.Ar (%) 0.582* 0.648* 0.137 -0.451 0.121 0.214 0.813** 0.923** Czech antler set BS (MPa) 0.745* W (kJ/m2) -0.030 0.224 U (kJ/m2) 0.442 0.745* 0.539 Ct.B.Dn (Kg/dm3) 0.721* 0.564 -0.139 0.406 Ashes (%) 0.103 -0.127 -0.600 -0.176 0.188 Ct.B.Wi (cm) 0.479 0.285 -0.539 -0.055 0.467 0.382 Ct.B.Wi%(%) 0.236 0.067 -0.770** -0.285 0.358 0.588 0.745* Ct.B.Ar (%) 0.055 0.212 -0.491 -0.030 0.236 0.394 0.588 0.770**

Table 3. Pearson’s correlation coefficients between mechanical and structural variables for adult roe deer antlers from Spain (n=13) and Czech Republic (n=10). Probability at levels p<0.05, p<0.01 and p<0.001 are indicated, respectively, by *, ** and ***.

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Results The structural characteristics, the mechanical properties and the mineral profile, according to the sampling positions, showed variable trends with few significant results. In roe deer antlers from Czech population, only U showed a decreasing tendency (position 1: 13.06±0.83 kJ/m2 and position 2: 10.98±0.62 kJ/m2, but marginally significant p=0.065). Structural characteristics showed significant values only for Ct.B.Wi (position 1:0.51±0.03 and position 2:0.30±0.02 cm, p<0.001) and Ct.B.Dn (position 1: 1.71±0.01 and position 2: 1.59±0.05, kg/dm3, p=0.045); whereas the ash content showed marginally significant results (58.93±0.43 vs 55.76±1.72 %, position 1 and position 2 respectively, p=0.091). For the mineral profile, only Zn increased significantly (53.83±2.78 vs 61.79±1.87 mg/kg, position 1 and position 2 respectively, p=0.029). Roe deer antlers in the Spanish population showed the same pattern for mechanical properties and structural characteristics observed in the Czech antlers. In position 1, we observed higher values only for BS (304.9±8.5 vs 268.3±15.7 MPa, p=0.05), W (39.4±2.5 vs 26.4±2.7 kJ/m2, p=0.002) and Ct.B.Wi (0.55±0.04 vs 0.41±0.03 cm, p=0.007). In contrast, the mineral profile did not show significant differences. The full list of non-significant results is not shown in tables for reasons of conciseness. Acknowledgements JC: sampling collection and execution of analysis, data analysis/interpretation, drafting of the manuscript; FC: study design, antler and sampling collection, critical revision; TLC: critical revision; LG: critical revision; AG: antler and sampling collection, critical revision. The authors thank to Pablo Gambín Ph.D. for sampling collection and critical revision, and Ing. Radim Kotrba, Ph.D. and all the staff members of the two ranches for the help in collecting the roe deer antlers.

Compliance with Ethical Standards Conflict of interest: The authors declare that they have no financial interests and no conflict of interest.

Funding: This work was supported by the pre-doctoral contract for the training of research personnel under the R+D+I Plan, co-financed by the European Social Fund (2015/4062; 2015); it was also funded by Spanish Ministry of Economy and Competitiveness (project INCYDEN RTC-2016-5327; 2016) and by IGA- 20175014 (Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Czech Republic). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Ethical approval: All applicable international, national and/or institutional guidelines for the care and use of animals were followed.

Data availability: The datasets generated and analysed during the current study are available from the corresponding author on reasonable request.

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Results Article 5.3 The bony horncore of the common eland (Taurotragus oryx): composition and mechanical properties of a spiral fighting structure.

The bony horncore of the common eland (Taurotragus oryx): composition and mechanical properties of a spiral fighting structure. Cappelli J, García A, Kotrba R, Gambín P, Landete-Castillejos T, Gallego L, Ceacero F. Journal of Anatomy 2018, 232(1): 72-79

ISI Journal Citation Reports Ranking: Q1, 4/21 in Anatomy and Morphology Impact factor: 2.479

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Abstract Horns are permanent structures projecting from the head of bovids, consisting of a bony horncore covered with one layer of skin and then a sheath of keratinous material showing variability of growth intensity based on nutrition. From the point of view of the horn’s mechanical properties, the keratin sheath has been widely- studied, but only few studies considered the complete structure of the horn and fewer studies focused on the bony horncore and its characteristics. The latter showed the important role of the bony core, when cranial appendages are subject to mechanical stress (as happens during fighting). The mechanical properties of bone material, along with its mineral profile, are also important, because they can show effects of different factors, such as nutrition and mineral deficiencies in diet. For this reason, eight horncores of captive common eland male were sampled at four positions along the vertical axis of the horn. The main aim was to study variation in mechanical properties and the mineral content along the vertical axis of the horncores. We further analysed whether the spiral bony ridge present on eland horncores differs in any of the studied properties from adjacent parts of the horncore. In other antelopes, spiral ridges on the horns have been proposed to increase grip during wrestling between males. Cross sections of the horncores were performed at four positions along the longitudinal axis and, for each position, two bone bars were extracted to be tested in impact and bending. Moreover, in the first sampling position (the closest position to the base) two bars were extracted from the spiralled bony area. The resulting fragments were used to measure ash content, bone density and mineral content. Results showed that horn bone decreased along the vertical axis, in ash (-36%), density (-32%), and in impact work “U” (marginally significant but large effect: -48%). The concentration of several minerals decreased significantly (Mg, Cr, Mn and Tl by - 33%, -25%, -31%, -43% respectively) between the basal and the uppermost sampling site. The bone tissue of the horncore spiral compared to non-spiral bone of the same position showed a lower ash content (53% vs 57%), Mg and Mn; in addition to showing approximately half values in work to peak force “W”, bending strength “BS” and “U”, but not in Young’s Modulus of elasticity “E”. In conclusion, similar to the results in a totally different fighting bony structure, the antlers, the horncore of eland shows better bone parameters corresponding to better bone quality in the base than in the tip, with higher values for mechanical properties, density and mineral profile. Moreover, the spiral bone tissue showed weakened material mechanical properties. Probably the spiral tissue of the horn may have a role in deflecting potential cross-sectional fractures during wrestling. In addition, it may serve to improve the grip during wrestling, and we propose that it may also prevent risk of rotation of sheath with respect to internal bone not only in this, but also in other straight bovid horns.

Keywords: Bovids, Processus cornualis, Horn, Minerals.

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Results Introduction Cranial appendages are common in artiodactyls, constituting distinctive attributes in four families of the suborder Rumantia. The three types of “headgear” occurring in ruminants are antlers in Cervidae, horns in Bovidae and Giraffidae, and pronghorns in Antilocapridae (Bubenik & Bubenik, 1990). Horns are permanent structures projecting from the head of bovids, consisting of a bony core covered with a sheath of keratinous material (Soloniuas, 2007), with an interposed layer of skin. Growth of the horn varies with nutrition (Monteith et al., 2013). From the point of view of the mechanical properties of the horn, keratin sheaths are a widely studied structure because of their nature and mechanical characteristics, in order to investigate on properties and deformation mechanisms of biological materials in several species (in Oryx sp., Kitchener & Vincent, 1987; in Ovis canadiensis, Tombolato et al. 2010; in Ovis aries, Zhu et al., 2016). Only few studies considered, at the same time, the complete structure of the horn (keratin sheath and bony horncore). Li et al. (2011) studied the mechanical properties of horns of Bos taurus and showed the complementary roles of the keratin sheath and the bony core: while the first ensures a high fracture toughness (for the structural integrity) the second can absorb energy and repair possible damage. In Drake et al. (2016) it was emphasized that the shape of horns also play an important role, by studying the relationship between the mechanical resistance with the presence/absence of a full horn structure in Ovis canadiensis. Even fewer studies focused on the bony horncore and its characteristics (mechanical and composition profile). It is important to understand that the mechanical performance of a structure depends on both structural factors (such as thickness of the cortical wall, diameter, etc.), and on those derived from the mechanical quality of the material, also called intrinsic mechanical properties of the bone (Currey, 2002). The mechanical properties of bone material, along with its mineral profile and even histology, are also important because they can show several effects such as physiological reduction of the capacity of the animal to supply the minerals during bone growth, which is then reflected by certain parameters in the cranial appendages (in antlers, Landete-Castillejos et al., 2007b). An interesting model to study horns is the common eland (Taurotragus oryx), which is one of the largest African antelopes. Males are larger than females (Kingdon, 1982). Both sexes have spiralled horns, but horns of males are shorter, thicker and have a much more pronounced spiral (Rowe-Rowe, 1983). Horns are used with attacking and defensive purposes: the short and thick horns of males are well designed for wrestling between males (Lundrigan, 1996; Martínez Valdeolivas, 2015). On the contrary, females use their long horns to deliver quick stabs to predators. Thus, the spiral of the horns seems to have a mechanical meaning especially for male fights. In fact, males often fight by pushing the rival with the contact of the horn area of the spiral, using intertwined movements (Kiley-Worthington, 1978). Spiral of eland horn has been proposed to increase grip in wrestling between males (Geist, 1966; Caro et al., 2003). This hypothesis received support from the fact that wrestling was associated with twisted horns in polygynous bovids (Caro et al., 2003; Soloniuas, 2007). For this reason, we hypothesized that the spiral zone of the horns may play an important role in understanding the mechanics and functionality-grip of the horn’s tissues in spiral- horned antelopes. Moreover, another hypothesis may also explain such spiral:

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Results our hypothesis in this respect is that a spiral in a composite structure consisting of a straight bone core covered with a sheath may serve to avoid rotation of the sheath relative to the internal bone. Thus, the objectives of this study are: i) to examine the bony horncore characteristics of common eland in terms of structural, mechanical properties and mineral content profiles; and ii) to compare characteristics of the spiral structure of elands’ horns to core part. Unfortunately, none of the previous hypotheses make a specific prediction whether the mechanical quality of the spiral ridge in the bone should be more than or equally resistant to surrounding bone material. In any case, it is not expected to be weaker.

Methods Eight horns of common eland males culled at the experimental farm at Lány (Czech University of Life Sciences Prague) for the purpose of meat production were used for this study. Slaughter procedure was approved and supervised by State Veterinary Authority of the Czech Republic (Act. no. SVS/WS22/2012- KVSS) described in Bartoň et al. (2014). Animals culled had an average age of 23 ± 2 months and an average weight of 232.8 ± 12.9 kg. At death, the animals showed an advanced stage of horn growth (in fact after 18 months the horns become progressively heavier, especially at the base, while the growth rate begins to decline from 20-22 months: Kerry & Roth, 1970; Jeffery & Hanks, 1981). This is the only European farm for this species. Eland farming began in 2006 and the average number of managed animals is around 50. The animals were fed year-round with a complete feed mixture diet consisting of 60% corn silage, 30% Lucerne haylage, 7% meadow hay, 3% barley straw presented ad libitum, and had access to grassy paddock of 2.5 hectares from April to November (Vadlejch et al., 2015). Animals had ad libitum access to mineral lick-block SOLSEL (European Salt Company, Hannover, Germany). This block contained Na (37%), Ca (1.1%), Mg (0.6%), Mn (0.1%), Zn (0.1%), Fe (0.07%), Cu (220 mg.kg-1), I (10 mg.kg-1), Co (20 mg.kg-1) and Se (20 mg.kg-1). One horn from each pair was chosen for the destructive sampling procedure after the horn sheath and the horn core were separated. The internal bone core was preserved in a freezer (-20°) in Prague; subsequently, bony horncores were transported to Spain (IDR-UCLM, University campus of Albacete), where samples were obtained in order to perform the mechanical tests and analyses of the mineral content; in this shipping process attention was paid to keep the internal bone in continuous hydration. The bony horncore was cut in four cross sections along the longitudinal axis of the horn, as shown in Figure 1; the cut included a cylinder for each sampling position (starting from the base of the horn, each position was consecutive to the previous). Sawing was performed under running tap water to avoid overheating of the bone tissue. For each position along the bony horncore, two bone bars were extracted from the medial face and lateral face of the cylinder (one bone bar from each side) using a circular low-speed saw. In addition, and only from the first sampling position (corresponding to the closest position to the base) two bars were extracted from the bony area of the spiral. The procedure, to get the standard size of bone bars (thickness 2.5 mm, width 4.5 mm, maximum length 40 mm),

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Results required that the external surfaces of bars were manually abraded using a polishing machine (MetaServ® 250 Double, Buehler, USA) equipped with an 80- grit silicon carbide grinding paper (SiC Paper, Struers Inc., USA). Care was taken to produce parallel surfaces, and the width and depth of each sample were recorded to the nearest 0.01 mm using a digital calliper prior to testing.

Figure 1. Sampling technique for horncore bone of common eland: horncore was cut at various levels along the horn’s vertical axis; in every position, it was obtained a cylinder (a); in each sampling position, two bone bars from inner (medial) and outer (lateral) faces of the cylinder were extracted (b); in the position 1 other samples from the spiral tissue were extracted (c).

Bone samples were kept hydrated during the sampling process and the performance of the mechanical tests (as described in Olguín et al., 2013), in order to observe the mechanical properties of bone tissue as it would occur under in vivo conditions. Hank's Buffered Salt Solution (HBSS, BioWhittaker) was used to maintain hydration of the bone bars. In addition to that, minerals in the dilution prevent bone dissolution of minerals. The bone samples were maintained at temperatures between 0 to 4 °C before the mechanical testing (in order to reduce further loss of minerals, which is temperature-dependent). During these tests the bone samples were at room temperature (≈20 ºC). The mechanical properties were determined by two mechanical tests: a three- point bending test and an impact test. Since mechanical properties of the bone differ depending upon the hydration state (Currey et al., 2009b), special care was taken to keep the specimen fully hydrated right up to the start of the mechanical testing. The bending test determined the stiffness (E), bending strength (BS), and work

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Results to fracture (W) of the bone tissue bars. A three-point bending test machine (Zwick/Roell 0.5 kN, Ulm, Germany) with a span length of the supports of 40 mm and speed of the cross head of 32 mm/min was used. The machine produced an output chart in the software TestXpert II (Zwick GmbH & Co, Ulm, Germany) with a curve that correlates the deformation (mm) and force (N). The impact test (charpy test) was performed with a pendulum falling on a bone sample with the external side in tension, using a CEAST-IMPACTOR II testing machine (CEAST S.p.A., Pianezza, Italy) equipped with a hammer. The loss of kinetic energy of the pendulum is measured by the machine and this is considered to be the energy required to break the sample (Landete-Castillejos et al., 2010). This energy is normalized by dividing by the cross-sectional area of the sample, giving the impact energy absorption or impact work (U). After carrying out mechanical tests, bone fragments from the bars were used to measure the ash content and the density and mineral composition of the bone tissue. Firstly, both bone fragments used to measure ash content and bone density were placed in a controlled heating chamber for 72 hours at 60°C to dry it out fully. Afterwards, bone fragments were weighed with a precision balance (±0.01 g) to obtain their dry weight and then ashed in a muffle furnace (HTC 1400, Carbolite, UK) for 6h at 480°C. Ash content was calculated as ash weight divided by dry weight. In order to calculate the bone density, another fragment was weighed with a precision balance (±0.01 g) to get the dry weight and measured with a precision calliper (±0.01 mm). Density was calculated dividing the weight by the volume for each bone sample. After this, the fragment was placed in a tube for mineral analysis at the Ionomic Laboratory (CEBAS-CSIC Centre; Murcia, Spain). In that laboratory, each sample was digested with hydrogen chloride, nitric acid and diluted with ultrapure deionized water. Total concentrations of each mineral were quantified with an inductively coupled plasma optical emission spectrometry (ICP-OES) using an ICAP 6500 DUO Spectrometer/IRIS INTR.EPID II XDL (Thermo Fisher Scientific Inc., Waltham, MA, USA) that generates spectral lines atomic emission for each mineral found in the sample. Each datum was taken as the mean of three measures recorded at 0.3 s intervals. Prior to the analysis of each element was prepared a blank with ultrapure deionized water, and different solutions, at known concentrations (certified standards of 1000 mg/kg; MERCK CertiPUR, Barcelona, Spain). These blank solutions were used to check the correlations between the certified values and those determined by the spectrophotometer, and they were run every 10 samples within each batch (more information in Landete-Castillejos et al., 2010).

The statistical analyses were performed with SPSS version 22 (SPSS Inc., Chicago, IL, USA); ANOVAs and linear regression models followed various steps. Initially, differences between means of each sampling position along the vertical axis of the horn were assessed through ANOVA analysis with Tukey tests for post- hoc comparisons of groups. ANOVA was also used to analyse differences between the spiral and core bone tissue in position 1. Then, a linear regression analysis for each observed variable was performed to assess the trend of the values along the four sampling positions. Data of ANOVAs in tables are presented as means for all the variables observed. For each statistical analysis performed, a P-value of less than 0.05 was considered significant, whereas P-values between 0.05 and 0.10 were considered as a trend. The variables tested were:

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Results density of the bone; Young’s modulus of elasticity E; bending strength BS; work to peak force W; impact energy U; ash content; and that of minerals Al, Bi, Ca, Cr, Cu, Fe, K, Li, Mg, Mn, Na, P, S, Se, Sr, Tl and Zn.

Results The horncore bone showed a decrease in ash content (-36%, P<0.001; last position vs. first position) and in density (-32% last position vs. first position, 2 regression function: Y(Density)=0.96-0.10·Position, r =0.20, P=0.018). Although most mechanical properties showed a decline from base to tip, only U showed a marginally significant trend (-48%, P=0.055; last position vs. first position; 2 regression function: Y(U)=6.60-0.72·Position, r =0.21, P=0.022). For the mineral profile, there were differences in content between the sampling positions along the vertical axis of the horn: concentration of a few minerals increased from the base to the tip (Se: +4%; Cu: +83%; K: +16%) while others decreased significantly (Mg: -33%, P=0.026 and Mn: -31%, P=0.019) or showed a decreasing tendency (Cr: -25%, P=0.082; S: -23%, P=0.088; Tl: -43%, P=0.053 2 and Li: -24% with a regression function of Y(Li)=6.75-0.40·Position, r =0.07, P=0.069). Values obtained with ANOVAs for each sampling position and linear regression’s models (using the sampling position as predictor) with their significant differences are shown in Table 1.

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Position 1 Position 2 Position 3 Position 4 P-value R2 Intercept±SEM Slope±SEM F P-value Density (Kg/dm3) 0.84±0.24 0.77±0.18 0.63±0.22 0.54±0.25 NS 0.20 0.96±0.10 -0.10±0.04 6.449 0.018 Ash content (%) 57.2±3.0a 58.3±2.9a 51.9±8.0a 39.0±3.2b <0.001 0.43 65.1±3.0 -5.1±1.2 17.469 <0.001 E (GPa) 3.3±2.0 2.4±2.2 2.6±1.8 - NS 0.02 3.38±1.34 -0.33±0.62 0.761 NS W (KJ/m2) 5.01±3.94 2.88±3.25 2.33±2.17 1.81±1.20 NS 0.12 5.74±1.53 -1.15±0.64 3.243 0.085 BS (MPa) 52.9±36.3 46.0±40.1 39.4±30.8 39.9±28.4 NS 0.02 57.64±16.62 -5.59±7.06 0.628 NS U (KJ/m2) 5.5±1.7a 5.6±0.8a 4.9±1.8ab 2.9±1.4b 0.055 0.21 6.60±0.75 -0.72±0.29 6.031 0.022 Ca (g/100g) 8.2±1.3 9.5±2.7 8.2±1.9 6.1±3.0 NS 0.07 9.06±1.06 -0.58±0.42 1.853 NS K (g/100g) 0.019±0.002 0.021±0.002 0.021±0.003 0.022±0.004 NS 0.09 0.019±0.001 0.0008±0.0005 2.466 NS Mg (g/100g) 0.13±0.02ab 0.16±0.04a 0.14±0.03ab 0.09±0.05b 0.026 0.10 0.16±0.02 -0.011±0.007 2.879 NS Na (g/100g) 0.35±0.03 0.38±0.04 0.36±0.05 0.35±0.02 NS 0.006 0.37±0.02 -0.003±0.007 0.168 NS P (g/100g) 5.35±0.78 6.23±1.69 5.36±1.27 4.03±1.99 NS 0.07 6.23±0.68 -0.37±0.27 1.851 NS S (g/100g) 0.15±0.02ab 0.17±0.04a 0.15±0.03ab 0.12±0.05b 0.088 0.08 0.173±0.016 -0.010±0.006 2.307 NS Al (mg/Kg) 11.9±3.1 13.3±5.9 13.5±8.6 14.6±12.0 NS 0.015 11.30±3.21 0.81±1.28 0.405 NS Bi (mg/Kg) 3.1±0.1 3.6±1.6 3.4±1.1 3.2±1.8 NS 0.002 3.2±0.6 0.05±0.26 0.036 NS Cr (mg/Kg) 0.67±0.07a 0.67±0.11a 0.58±0.13a 0.50±0.17a 0.082 0.21 0.75±0.05 -0.06±0.02 6.709 0.016 Cu (mg/Kg) 0.12±0.07 0.20±0.08 0.15±0.08 0.23±0.12 NS 0.06 0.120±0.04 0.022±0.017 1.712 NS Fe (mg/Kg) 3.9±1.9 4.8±3.0 4.4±1.1 8.2±6.1 NS 0.11 2.67±1.42 0.97±0.58 2.823 NS Li (mg/Kg) 5.8±0.9 6.7±1.8 5.8±1.3 4.4±2.1 NS 0.07 6.75±0.72 -0.40±0.29 1.929 0.069 Mn (mg/Kg) 9.2±1.2ab 10.6±2.2a 9.4±1.8a 6.3±3.0b 0.019 0.11 10.96±1.02 -0.74±0.41 3.251 0.083 Se (mg/Kg) 0.17±0.08 0.18±0.12 0.17±0.06 - NS 0.01 0.18±0.05 -0.006±0.023 0.075 NS Sr (mg/Kg) 52.7±10.8 61.9±20.2 53.0±11.9 39.4±15.9 NS 0.060 62.0±7.2 -3.72±2.87 1.673 NS Tl (mg/Kg) 11.7±2.0ab 13.1±3.8a 11.6±3.3ab 6.7±5.7b 0.053 0.114 14.25±1.73 -1.27±0.69 3.360 0.078 Zn (mg/Kg) 47.6±7.1 47.8±8.3 45.5±7.8 38.5±11.4 NS 0.098 51.50±3.75 -2.51±1.50 2.820 NS Table 1. Mineral composition and mechanical properties of horn core bone of common eland (Taurotragus oryx) in four positions along the horn. P-values correspond to one-way ANOVA on the mean (± SD) for the positions examined. Superscripts indicate homogeneous groups within each row (Tukey test, P≤0.05). In the second part of the table are shown linear regression models, using the sampling position as predictor. Dashes indicate the lack of data.

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There were also differences in the mineral content and mechanical properties between the core bone tissue and the spiral. There were significant differences for most of the mechanical properties: W (the value was three times higher between standard bone core and spiral, P=0.030), BS (+153% same comparison, P=0.036) and U (+118%, P=0.001). See Table 2. Mineral profile showed significant differences for Mg (-25% in the spiral, P=0.038) and Mn (-24% in the spiral, P=0.030), while other minerals showed a marginally significant tendency, like Ca (-20% in the spiral, P=0.081), K (+14% in the spiral, P=0.096), P (-18% in the spiral, P=0.097), S (-18.3% in the spiral, P=0.059), Fe (+ 42% in the spiral, P=0.056), Li (-18% in the spiral, P=0.073) and Tl (-29% in the spiral, P=0.086).

Spiral Position 1 P-value Density (Kg/dm3) 0.68±0.18 0.84±0.24 NS Ash content (%) 52.5±5.2 57.2±3.0 0.057 E (GPa) 1.3±0.7 3.3±2.0 NS W (KJ/m2) 1.26±0.87 5.01±3.94 0.030 BS (MPa) 20.9±14.3 52.9±36.3 0.036 U (KJ/m2) 2.5±0.9 5.5±1.7 0.001 Ca (g/100g) 6.8±1.6 8.2±1.3 0.081 K (g/100g) 0.022±0.003 0.019±0.002 0.096 Mg (g/100g) 0.11±0.03 0.13±0.02 0.038 Na (g/100g) 0.35±0.04 0.35±0.03 NS P (g/100g) 4.53±1.04 5.35±0.78 0.097 S (g/100g) 0.13±0.03 0.15±0.02 0.059 Al (mg/Kg) 10.7±4.1 11.9±3.1 NS Bi (mg/Kg) 3.0±0.3 3.1±0.1 NS Cr (mg/Kg) 0.59±0.12 0.67±0.07 NS Cu (mg/Kg) 0.18±0.10 0.12±0.07 NS Fe (mg/Kg) 6.6±2.8 3.9±1.9 0.056 Li (mg/Kg) 5.0±1.0 5.8±0.9 0.073 Mn (mg/Kg) 7.4±1.7 9.2±1.2 0.030 Se (mg/Kg) 0.13±0.03 0.17±0.08 NS Sr (mg/Kg) 44.2±12.0 52.7±10.8 NS Tl (mg/Kg) 9.1±3.5 11.7±2.0 0.086 Zn (mg/Kg) 44.1±10.2 47.6±7.1 NS

Table 2. Values observed (mean ± SD; one-way ANOVA) for the mineral composition and mechanical properties of the spiral’s tissue and the horn core bone tissue in the sampling position 1 of common eland horns.

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Results Acknowledgements This study was supported by the pre-doctoral contract for the training of research personnel under the R+D+I Plan, co-financed by the European Social Fund (2015/4062; 2015); by the grant IGA-20165008 (Faculty of Tropical AgriSciences, Czech Republic) and funded by MINECO project INCYDEN RTC-2016-5327-2 (University of Castilla-La Mancha, Spain). The authors declare no conflict of interest.

Author contributions J.C.: sampling collection, data analysis/interpretation, drafting of the manuscript; A.J.G.: sampling collection, critical revision; R.K.: study design, horn collection, critical revision; P.G.P.: acquisition of data, critical revision; T.L.C.: critical revision and adding hypothesis on function of spiral in horn core; L.G.: critical revision; F.C.: study design, horn collection, critical and statistical revision

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6. OTHER STUDIES

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Article 6.1 Manganese Supplementation in Deer under Balanced Diet Increases Impact Energy and Contents in Minerals of Antler Bone Tissue.

Manganese Supplementation in Deer under Balanced Diet Increases Impact Energy and Contents in Minerals of Antler Bone Tissue. Cappelli J, García A, Ceacero F, Gomez S, Luna S, Gallego L, Gambín P, Landete-Castillejos T. PLoS ONE 2015, 10(7): e0132738.

ISI Journal Citation Reports Ranking: Q1, 11/63 in Multidisciplinary Sciences Impact factor: 3.057

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Abstract Bone ash, collagen, Ca and P composition, are considered the main factors affecting mechanical properties in bones. However, a series of studies in bone and antler have shown that some trace minerals, such as manganese, may play a role whose importance exceeds what may be expected considering their low content. A previous study showed that a reduction in manganese in antlers during a year of late winter frosts led to generalized antler breakage in Spain, which included a reduction of 30% of cortical thickness, 27% reduction in impact energy, and 10% reduction in work to peak force. Starting for this observation, we experimentally studied the effects of manganese supplementation in adults and yearling (yearlings) red deer under a balanced diet. Subjects were 29 deer of different age classes (adult n=19, yearlings n=10) that were divided in a manganese injected group (n=14) and a control group (n=15). Antler content in ashes and minerals, intrinsic mechanical properties and cross section structure were examined at 4 points along the antler beam. A one-way ANOVA (mean per antler) showed that in yearlings, manganese supplementation only increased its content and that of Fe. However, in adults, Mn supplementation increased the mean content per antler of Ca, Na, P, B, Co, Cu, K, Mn, Ni, Se (while Si content was reduced), and impact work but not Young’s modulus of elasticity, bending strength or work to peak force. A GLM series on characteristics in the uppermost part examined in the antler, often showing physiological exhaustion and depletion of body stores, showed also a 16% increase in work to peak force in the antlers of the treated group. Thus, manganese supplementation altered mineral composition of antler and improved structure and some mechanical properties despite animals having a balanced diet.

Keywords: Antlers, deer, bone, mineral composition, mechanical properties, Mn, nutrition

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Results Introduction Antler, which is true bone despite being used when dry and having some unusual features such as the lowest ash content of all bones (Currey et al., 2009a), has attracted the attention of researchers in bone mechanics (Currey, 1979; Krauss et al., 2009; Landete-Castillejos et al., 2010) because, among mammalian bones, it has the highest work to peak force, that is, the amount of work needed to break specimens (Currey, 1979), and it is difficult to break in impact (Currey et al., 2009a; Currey et al., 2004). Furthermore, a series of studies (Landete-Castillejos et al., 2007a, b, c) have shown that antlers are an excellent model for studying bone biology: i) because they are easily accessible without surgery; ii) because they grow quickly (with an average of 0.67 cm/d (Gaspar-Lopez et al., 2008) in red deer) and demand a high mineral transfer from the skeleton (Davison et al., 2006). So that because this fast growth leaves little room for remodelling (Gomez et al., 2013), and thus shows more clearly nutrition or other effects than in internal bone, which is a mosaic of parts build in different times of the life of an animal. Previous studies by our group have shown that the composition, mechanical properties, and structure of deer antlers (and composition of internal bones too, Olguin et al., 2013), or even their histology (Gomez et al., 2013; Landete- Castillejos et al., 2012) could be influenced by diet, and natural factors likely affecting mineral composition of plants on which deer feed (Landete-Castillejos et al., 2010; Landete-Castillejos et al., 2007a, b; Estevez et al., 2009). Among the minerals that may affect the mechanical properties of antlers, at least one seems to have a disproportionate importance considering its small content in antlers: Mn. In 2010, a study by Landete-Castillejos et al. (2010) concluded that among the changes in composition produced in deer antlers by an event of extraordinary low temperatures at the onset of plant sprout, it was Mn decrease which produced a 27% reduction in impact energy, 10% reduction in work to peak force, 30% reduction in antler weight, and 18% reduction in cortical thickness. It was not the first time that Mn was shown to have an important role in bone. An early study by Strause et al. (1986) showed that a balanced diet with low Mn content kept rats in apparently good condition, but their bones had lower Ca content (however, no mechanical tests were performed) and moreover, a study by Leach et al. (1969) showed that the addition of Mn in chick cartilage in an ex vivo preparation enhanced significantly the synthesis of main cartilage constituents. However, so far, no study has tried to assess the effect of Mn supplementation on both composition, mechanical properties, structure and density of antler or other types of bone. Thus, we set out to assess the effect of manganese in deer antlers both in adults and in yearlings (which are under a greater growth constraint because they need Ca and other minerals to grow their skeleton in addition to growing their antlers – Gaspar-Lopez et al., 2008; Ceacero et al., 2010). The strength and stiffness of whole bones is the result of combination of the overall architecture, such as cortical thickness, and bone material properties (Davison et al., 2006). Histology was performed in order to assess if bone mineralization was impaired by Mn supplementation at doses used (Mn toxicity). Thus, we measured modulus of elasticity E, bending strength BS, work to peak force W, which is the work needed to reach the maximum force (Currey, 1979; Turner & Burr, 1993; Currey, 2002), and impact energy absorption U.

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Results Materials and methods Study site and antler collection This study was performed in the Experimental Farm of Universidad de Castilla– La Mancha in Albacete, south-eastern Spain (38º57’10’’N, 1º 47’00’’W, 690 m altitude) during 2010. Ours and other research groups regularly perform experiments in these premises (managed by our group) and no specific authorisation is needed to work here, although experiments have to be approved by the Committee of Ethics in Animal Experimentation (Comité de Ética en Experimentación Animal, CEEA) from the Universidad de Castilla-La Mancha (for our experiment, the authorisation number was 1002.04). Animals were kept in a 10,000 m2 open door enclosure on an irrigated mixed pasture. Deer were feed ad libitum with a diet of hay, lucerne, corn and orange pulp; to optimize management time in the experimental farm, all the diet's ingredients were homogeneized and cut in small portions in a tractor-driven commercial mixer. All animals were adapted to routine management and maintained in good health and body condition during the experiment. Handling procedures and sampling frequency were designed to reduce stress and health risks for the animals (Ceacero et al., 2014). The animals were divided in two groups matched for body measurements such as weight and body condition. Rather than offering Mn mixed with salt, or with the food, which would not allow to control the exact amount of food given to each animal, and also it would mean that control animals had to be placed in another enclosure thus modifying the social environment, we decided to keep all animals together, and deliver the Mn by injections of an aqueous 4% of Manganese 3 gluconate (C12H22MnO14) solution (5cm /100Kg live wt) in the treatment group, given every seven days from start of January to midAugust. . The control group was injected a physiological saline solution. Each group counted animals with different ages: for the control group there were 8 adults, 2 subadults (2.5 years old) and 5 yearlings (1.5 years old), for the treatment group there were 8 adults,1 subadult and 5 yearlings. We decided to include subadults to increase group size of the adult class. Thus, the statistical analysis examined yearlings and adults. Antlers were cut off 1 cm above the burr for safety reasons when they were clean from velvet. We kept carrying out body measurements at the beginning of the trial, during and at the end of the experiment, to monitor potential changes in the animals. Before the antlers had been removed, they were analysed according to the standard method for the trophy evaluation in red deer, in order to find a validation score for each antler collected (Gómez-Notario, 2002). Measurements included: the total length of the main beam, lengths of all the tines, perimeters at three points along the main shaft (burr, between the first tine and the central tine, between the central tine and the crown), total weight of the antlers, and number of tines.

Sample collection and preparation Because the deer had a balanced diet much better than deer kept in the wild without supplementation (Landete-Castillejos et al., 2007b, c; Estevez et al.,

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Results 2009), and because the studies performed so far in antler characteristics show a more marked effect of nutrition and other factors during the growing of the antlers main beam (Landete-Castillejos et al., 2007a, b, c, 2010, 2012; Gomez et al., 2013), we analysed, as in other studies (Landete-Castillejos et al., 2010), effects of Mn supplementation in four positions along the antler beam: position 1 is directly above the burr, position 2 referred to the first third of antler shaft, position 3 after the central tine and position 4 below the crown (upper tines). For yearlings, antlers were sampled following a similar procedure but including only two sampling points due to the smaller size and different structure of the antlers: one point was close to the base (yearlings have no burr), and the other one 5 cm below the tip (Landete-Castillejos et al., 2007a). From each position were obtained a 1 cm-slice (complete transverse cross- section) and a cylinder of about 5-6 cm (to obtain bars from the cortical wall). Bars were identified according to individual deer and level in the main beam. The outer or periosteal side was also marked in order to test always with this side in tension, and to mark the end closer to the antler base. A circular low-speed saw was used for the initial cutting of the antler. Then, the surfaces were abraded using a semiautomatic equipment for polishing (Struers LaboPol-21, Denmark) to get the right size of bars. Care was taken to produce parallel surfaces, and the width and depth of each sample were recorded to the nearest 0.01 mm using a digital calliper prior to testing. The final size was 4.5 mm wide, 2.5 mm deep, and a variable length but always allowing a gauge length of 40 mm, a length in which shear effects reducing calculated E are very small (Landete-Castillejos et al., 2010). In addition, antlers of adult groups were examined by histology. Antlers were sampled in position 1 and 4 were embedded in polymethyl-methacrylate. Mineralized sections (50 µm-thick) were prepared by grinding-polishing method, and sections were stained with toluidine blue/pyronin G for light microscopy examination (Landete-Castillejos et al., 2012). In order to increase the contrast between the trabecular and cortical bone to better measure the cortical thickness and derived variables, antler slices were coloured with a solution of ink/water in proportion of 1:4 (Pelikan ink 4001, Germany) and dried for 24 hours at room temperature. Then, the slices were polished to increase the contrast between cortical and trabecular bone. Once the cortical part had no remains of ink, each slice was scanned on a flatbed scanner (Ricoh aficio MP C2800) at 600 dpi and measured in an image analysis software (ImageJ), where cortical bone thickness of the cross-sections was measured at six equally spaced points around the perimeter. After this, we calculated the average thickness as the mean of these 6 measurement points. In addition, we measured the total area of the section, the area of cortical and trabecular bone. These areas were used to calculate, in turn, the ratio between cortical and total area (termed ratio cortical/total) (Landete- Castillejos et al., 2010).

Bone mechanical properties Specimens for mechanical testing were first subjected to a procedure to standardize humidity content. For this, they were first introduced in a buffer solution (BioWhittaker HBSS: Hank's balanced salt solution, Belgium) for 48 hours. This is a way to achieve maximum water content, but at the same time

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Results minimizing the risk that small amounts of mineral may leach out of the bone (Currey et al., 2009a). After this, the bars were dried in the laboratory, at a temperature of approximately 20ºC and relative humidity of 40% for a period of 72 hours. The bars that had thus homogeneous humidity content were tested in three-point bending with the periosteal side in tension. This was carried out in a Zwick/Roell 500N machine. The speed of the machine head was set to 32 mm/min. Gauge length or distance between supports was set to 40 mm. Machine compliance was tested and found to be negligible at this gauge length and sample depth. The mechanical properties measured were Young's Modulus of elasticity, an estimate of stiffness; Bending strength, calculated from the maximum stress at the greatest load borne following the procedure for this and the other intrinsic mechanical properties (Currey, 1979; Currey et al., 2009a; Landete-Castillejos et al., 2007c, 2010) and the total work under the load-deformation curve up to the maximum load borne, divided by cross-sectional area (Work to peak force). Total work normalized like this gives some idea of the toughness of the specimen. Although the software offers the mentioned mechanical properties, the key parameters for the formula used were extracted from the output chart in the software testXpert II (Zwick GmbH & Co, Ulm, Germany) and calculated in Excel following the formulae indicated, as our own studies have shown that there are differences between results calculated and derived from the machine, depending on gauge length, toe region excluded from the start of the curve in calculus of E, and others. The impact testing procedure consists, essentially, of a pendulum falling on, and breaking an un-notched sample with the periosteal side in tension. The loss of kinetic energy of the pendulum is measured by the machine, and this is considered to be the energy required to break the sample (Landete-Castillejos et al., 2010). This energy is normalized by dividing by the cross-sectional area of the specimen, producing the impact energy absorption or impact work. Tests were carried out in a CEAST-IMPACTOR II testing machine (CEAST S.p.A., Pianezza, Italy) with manual releasing device. A hammer with a potential energy of 1J was used for all tests.

Chemical analysis Breaking one antler bar in impact and one in bending test produced 4 similar-size fragments. One of these fragments was used to assess the content of different minerals. For this, the side of the fracture was polished to have a regular shape and also in areas where it may have any pen mark. Then, in order to calculate the specific density, the specimen was placed in a controlled heating chamber for 72 hours at 60°C to dry it out fully, and afterwards each sample was weighed with a precision balance (±0.01 g) and measured with a precision scale (±0.01 mm). Density was calculated dividing the weight by the volume (calculated as length x width x depth). After this, the segment of bar was placed in a tube for mineral analysis (details on the method used for mineral analysis are described in Landete-Castillejos et al., 2010). Another fragment was used to measure ash content. For this, the specimen was heated for 72 hours at 60°C. Then, the samples were weighed with a precision balance (±0.01 g) to get the dry weight, and subsequently was placed in a muffle

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Results furnace (HTC 1400, Carbolite, UK) for 6 hours at 480°C. Ash content was calculated as the value of ashes thus obtained divided by the dry weight.

Statistical analysis One-way ANOVAs were used to check for differences in body parameters between groups before being subjected to manganese treatment. The same statistical test was used to assess rough differences in body weight after the experiment by Mn treatment, also and in each parameter assessed in whole antler. We also examined the remaining antler variables by performing the ANOVA on the mean of the 4 antler levels. The antler variables tested were: cortical thickness; ratio cortical to total antler section area of the bone slice, in proportion; antler length; antler score as trophy; specific density/gravity of the cortical bone; Young’s modulus of elasticity E; bending strength B; work to peak force W; impact energy U; ash content and that of minerals Ca, P, Mg, Na, K, B, Co, Cu, Fe, Mn, Ni, S, Se, Si, Sr, and Zn. Because data showed a very clear difference between yearlings and adults, we performed a GLM, testing the effects of treatment and age class on each of the studied antler variables, and after results confirmed this pattern, the following statistical tests were carried out separately for yearlings and adults. The second part of the analyses involved a series of general linear models (GLMs) to assess the treatment effects on the same variables once the effects of body weight (which greatly influence antler size and characteristics) was included in the models. Three different sets of analyses examined: 1) the average value for the four sampled positions in the antler; 2) the proportional difference between the top and the base (Position 4 - Position 1 / Position 1; i.e. a decrease in values is shown as a negative difference in percent); 3) finally, the values at the fourth level sampled in the antler. GLMs 2 and 3 were carried out because values often show a variation between base (which shows the condition of the animal at the start of antler growth), and top part of the antler (when different value in most parameters shows the physiological effort made to grow the antler: Landete-Castillejos et al., 2007a, b, c). All analyses were carried out with SPSS version 19 (SPSS Inc., Chicago, IL, USA).

Ethical statement. This study was carried out in strict accordance with the Spanish legislation for the use of animals in research (law 6/2013 and 32/2007). The protocol was approved by the Committee of Ethics in Animal Experimentation (Comité de Ética en Experimentación Animal) from the Universidad de Castilla-La Mancha (Permit Number: 1002.04). Because antlers are dead when they are hard and clean of velvet (Currey et al., 2009a), antler removal produces no pain and no anaesthesia is needed. Nevertheless, a low dose of xylazine (0.3 mg/kg body mass) was used as tranquilizer to reduce stress and minimize suffering. Animals were restrained using a cushioned crush specifically designed to restrain deer movements, then the tranquilizer was delivered in an intravenous injection and, after one minute, antlers were cut using a low speed electrical saw. In general, this and all other experimental handling was designed to minimize stress and suffering of deer.

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Results Results Throughout the analyses, results from yearlings showed a very different pattern to that of adults. A set of GLMs including treatment and age class showed that they differ in all mechanical properties (greater values for adults), all antler structure and measurements (except average cortical thickness), and also in B, Cu, Fe, Mn, Ni, S, Si and Zn. Histology did not reveal any alteration in Mn- supplemented antlers. Primary osteons were completely formed both at position 1 and 4 (Figure 1), osteons in cortical bone did not show osteomalacic seams (which is considered a sign of Mn toxicity).

Figure 1. Histology of cortical bone in antlers from control and Mn-supplemented adult groups. Note that primary osteons are complete in both cases. Mn-supplemented osteons looks normal, osteomalacic seams —an indicator of Mn toxicity— are not observed (Bar scale = 200 µm).

When both age classes were tested separately in one-way ANOVAs, the only treatment effect which showed a common statistically significant effect was the increase in manganese content in the antler of the treatment group (Table 1). Manganese in antlers of animals injected was 2.5 times greater for yearlings (compared to the control group), and 2.3 times for antlers of adults. Yearlings only showed a 3-fold increase in antler content of Fe, but no other effect. As any other effect was found in any GLM in the group of yearlings, the rest of the analyses shown regard only to the adult’s group, for concision purposes.

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Results A first exam of results regarding Mn supplementation in adults’ group, showed that body weight increased by 10 % less in the supplemented group from start to end of the experiment than in the control group (Table 1). In contrast, Ca and P increased 8% and 10% respectively. Most macro minerals and trace elements also increased. Thus, Na increased 14%, K increased 47%, Se increased 142%, and Cu increased 29%. B, Co, and Ni also increased, whereas Si was the only mineral that decreased. No other minerals showed significant differences until effect of body weight were included in the GLMs. Among structural and mechanical properties Mn supplementation produced 11.8% increase in impact energy, but no other mechanical or structural property was affected. There was also an increase in impact energy in yearlings lower than that in adults (9%), but this was non-significant because of the large variability within each group.

Yearlings Adults Variables Mn- Control P Mn- Control P Treatment Treatment Young’s modulus of 12.0 ± 2.2 11.3 ± 1.8 Ns 14.4 ± 0.4 13.9 ± 0.8 Ns elasticity (E), Gpa Bending Strength (BS), 232.8 ± 42.0 202.3 ± 35.2 Ns 267.9 ± 6.1 266.7 ± 15.4 Ns Mpa Work to peak 35.2 ± 7.1 27.3 ± 6.8 Ns 44.0 ± 1.0 42.9 ± 1.3 Ns force (W), kJm-2 Impact work (U), 14.9 ± 2.2 13.7 ± 1.5 Ns 17.7 ± 0.6 15.8 ± 0.6 0.050 kJm-2 Body weight 54.4 ± 2.7 61.1 ± 4.2 Ns 19.8 ± 1.7 30.9 ± 2.5 0.003 difference (%) Cortical/total 0.8 ± 0.03 0.7 ± 0.1 Ns 0.5 ± 0.01 0.5 ± 0.03 Ns Ratio Average cortical 5.7 ± 0.6 5.1 ± 0.7 Ns 5.6 ± 0.3 6.2 ± 0.4 Ns thickness (mm) Antler length 52.0 ± 5.0 48.9 ± 3.7 Ns 83.2 ± 4.1 88.3 ± 3.7 Ns (cm) Antler score 56.8 ± 4.0 60.0 ± 1.8 Ns 147.1 ± 8.0 154.6 ± 7.2 Ns Specific gravity of cortical bone 1.6 ± 0.18 1.4 ± 0.2 Ns 1.8 ± 0.1 1.7 ± 0.5 Ns (g/mL) Ashes (%) 60.0 ± 6.8 52.8 ± 7.9 Ns 58.7 ± 5.7 51.0 ± 2.7 Ns Ca (wt%) 19.7 ± 2.2 17.4 ±2.6 Ns 21.9 ± 0.1 20.2 ± 0.5 0.013 Mg (wt%) 0.4 ± 0.5 0.4 ± 0.5 Ns 0.5 ± 0.01 0.5 ± 0.01 Ns Na (wt%) 0.5 ± 0.1 0.50 ± 0.1 Ns 0.6 ± 0.01 0.53 ± 0.01 <0.001 P (wt%) 9.5 ± 1.1 8.5 ± 1.3 Ns 10.4 ± 0.03 9.5 ± 0.2 0.002 B (ppm) 4.6 ± 0.6 4.0 ± 0.6 Ns 6.0 ± 0.1 4.2 ± 0.2 <0.001 Co (ppm) 0.5 ± 0.0 0.4 ± 0.1 Ns 0.6 ± 0.01 0.3 ± 0.05 <0.001 Cu (ppm) 0.7 ± 0.1 0.6 ± 0.1 Ns 1.0 ± 0.04 0.8 ± 0.03 <0.001 Fe (ppm) 12,0 ± 2.7 4.3 ± 1.2 0.045 3.8 ± 1.9 3.4 ± 0.9 Ns K (ppm) 655.6 ± 80.3 573.4 ± 113.3 Ns 635.6 ± 17.2 431.9 ± 38.8 <0.001

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Mn (ppm) 0.5 ± 0.1 0.2 ± 0.04 0.003 0.7 ± 0.4 0.3 ± 0.2 <0.001 Ni (ppm) 0.4 ± 0.1 0.3 ± 0.06 Ns 0.7 ± 0.5 0.4 ± 0.6 <0.001 1045.2 ± S (ppm) 935.5 ± 150.1 Ns 1177.3 ± 15.1 1126.1 ± 24.3 Ns 126.9 Se (ppm) 3.1 ± 0.4 2.9 ± 0.7 Ns 4.1 ± 0.2 1.7 ± 0.5 <0.001 Si (ppm) 27.4 ± 3.9 20.9 ± 4.1 Ns 32.7 ± 2.0 59.6 ± 11.5 0.044 1003.9 ± Sr (ppm) 725.2 ± 101.8 Ns 784.5 ± 54.3 754.3 ± 31.9 Ns 103.1 Zn (ppm) 63.6 ± 7.9 57.9 ± 7.5 Ns 84.3 ± 4.0 77.8 ± 4.8 Ns

Table 1. Antler characteristics of spiker and adult red deer injected (Mn treated) or not (Control) with manganese as nutrient supplement. P corresponds to one-way ANOVA on the mean (± SE) per antler of the four positions examined.

A more detailed set of analyses, controlling for the effect of weight, showed more clearly the effects of Mn in the antlers of adults, particularly in the upper sections of the antler where physiological exhaustion is more visible (Tables 2-4).

Factors in the model

Mn-Treatment Weight (Kg) Variables R2 Intercept ± S.E. β ± S.E. Sig. Β ± S.E. Sig. Young’s modulus of 0.50 6.8 ± 1.8 - - -0.04 ± 0.01 0.001 elasticity (E), Gpa Bending Strength (BS), 0.51 128.8 ± 33.5 - - -0.7 ± 0.2 0.001 Mpa Work to peak force (W), ------kJm-2 Impact Work (U), kJm-2 0.20 17.7 ± 0.6 1.9 ± 0.9 0.05 - - Cortical/total ratio ------Average cortical thickness 0.67 1.16 ± 0.8 - - -0.02 ± 0.004 <0.001 (mm) Antler length (cm) 0.55 39.0 ± 10.5 - - -0.2 ± 0.05 <0.001 Antler valuation score 0.61 55.6 ± 18.9 - - -0.5 ± 0.1 <0.001 Specific gravity of cortical 0.34 1.4 ± 0.1 - - -0.002 ± 0.001 0.009 bone (g/mL) Ashes (%) ------Ca (wt%) 0.31 21.9 ± 0.4 1.6 ± 0.6 0.013 - - Mg (wt%) ------Na (wt%) 0.62 0.6 ± 0,01 0.07 ± 0.01 <0.001 - - P (wt%) 0.44 10.4 ± 0.2 0.9 ± 0.3 0.002 - - B (ppm) 0.73 6.0 ± 0.2 1.7 ± 0.2 <0.001 - - Co (ppm) 0.71 0.9 ± 0.1 0.3 ± 0.05 <0.001 0.002 ± 0.001 0.034 Cu (ppm) 0.59 1.0 ± 0.03 0.2 ± 0.05 <0.001 - - Fe (ppm) ------K (ppm) 0.75 961.0 ± 94.7 194.0 ± 34.1 <0.001 1.7 ± 0.5 0.003

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Mn (ppm) 0.85 0.4 ± 0.1 0.4 ± 0.04 <0.001 -0.001 ± 0.001 0.045 Ni (ppm) 0.57 0.7 ± 0.1 0.4 ± 0.1 <0.001 - - S (ppm) ------Se (ppm) 0.67 7.4 ± 1.3 2.3 ± 0.5 <0.001 0.02 ± 0,01 0.019 Si (ppm) 0.46 -43.7 ± 29.4 -24.6 ± 10.6 0.034 -0.4 ± 0.1 0.016 Sr (ppm) ------Zn (ppm) 0.29 41.6 ± 15.2 - - -0.2 ± 0.1 0.018

Table 2. General Linear Model analyses showing the influence of treatment (injections of Manganese) and weight, on the composition, structure and mechanical properties of antlers in adult red deer. The coefficient β (± S.E.) is related to the difference of the value observed in animals that were injected with respect to the group not injected. Dashes indicate coefficients that were not significant.

Firstly, Table 2 shows the effects of Mn supplementation in the mean values of the four sections of the antlers which were tested. Controlling for body weight did not reveal any further effect as compared to the ANOVA, except that Co, K, Mn, Se and Si were both affected by Mn supplementation and deer body weight. In addition, body weight but not Mn supplementation, affected average values in cortical thickness, antlers length, specific density/gravity, Young’s modulus of elasticity, Bending strength, and Zn content. In the rest of the antler parameters, Mn treatment had only an effect on impact energy (U: Table 2), but in no other mechanical variable nor structural one.

A set of GLMs on the difference between tip and base of the antler (expressed as percentage), which shows the depletion of body mineral stores or physiological exhaustion, showed no effect of treatment except on Mn levels. The former did not decrease in the supplemented group as clearly as they decreased in the control group (the mean comparison of both groups is shown in Table 3, using ANOVAs).

Difference base/tip (%) Position 4 Mn- Mn- Variables Control P Control P Treatment Treatment Young’s modulus of -6.8 ± 0.4 -9.4 ± 4.0 Ns 13.9 ±0.9 13.2 ± 1.0 Ns elasticity (E), Gpa Bending Strength (BS), -5.2 ± 3.2 -5.7 ± 3.4 Ns 262.9 ±12.3 257.3 ± 17.6 Ns Mpa Work to peak force (W), 6.6 ± 7.7 -1.9 ± 6.6 Ns 46.9 ±1.9 42.3 ±2.9 Ns kJm-2 Impact work (U), kJm-2 -12.9 ± 6.2 -8.5 ± 54.7 Ns 15.0 ± 0.8 15.2 ± 1.1 Ns Cortical/total ratio (%) -32.8 ± 5.8 -28.7 ± 5.1 Ns 0.4 ± 0.02 0.4 ± 0.03 Ns Average cortical thickness -27.2 ± 5.4 -19.9 ± 4.9 Ns 4.7 ± 0.4 5.4 ± 0.3 ns (mm) Specific gravity of cortical -2.0 ± 0.7 -3.8 ± 1.4 Ns 1.7± 0.02 1.7 ± 0.3 Ns bone (g/mL) Ashes (%) -0.6 ± 0.3 -1.4 ± 1.6 Ns 63.9 ± 0.3 62.6 ± 0.6 Ns Ca (wt%) -1.3 ± 1.6 -1.6 ±1.2 Ns 21.6 ± 0.2 20.5 ± 0.1 <0.001

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Mg (wt%) 1.8 ± 3.2 -3.9 ± 1.1 Ns 0.5 ± 0.02 0.5 ± 0.01 Ns Na (wt%) -1.6 ± 3.1 -5.9 ± 1.7 Ns 0.6 ± 0.01 0.5 ± 0.01 0.002 P (wt%) -1.6 ± 0.8 -1.1 ± 0.8 Ns 10.3 ± 0.1 9.7 ± 0.1 <0.001 B (ppm) 6.5 ± 3.9 -0.2 ± 4.9 Ns 6.1 ± 0.2 4.5 ± 0.3 <0.001 Co (ppm) 9.8 ± 0.5 13.1 ± 7.1 Ns 0.6 ±0.02 0.3 ± 0.1 <0.001 Cu (ppm) 15.4 ± 7.9 5.7 ± 8.8 Ns 1.1 ± 0.1 0.9 ± 0.7 Ns Fe (ppm) 92.8 ± 72.9 83.3 ± 35.3 Ns 2.8 ± 1.6 7.5 ± 2.7 Ns K (ppm) 9.7 ± 3.6 0.4 ± 3.2 Ns 669.3 ± 22.7 472.8 ± 56.8 0.005 Mn (ppm) -0.6 ± 4.3 -10.0 ± 2.6 0.057 0.7 ± 0.05 0.4 ± 0.1 0.006 Ni (ppm) 21.8 ± 12.7 5.2 ± 11.9 Ns 0.8 ± 0.1 0.5 ± 0.1 Ns S (ppm) 7.9 ± 2.9 4.9 ± 3.0 Ns 1237.9 ± 19.1 1185.9 ± 37.6 Ns Se (ppm) 10.1 ± 8.3 15.4 ± 21.4 Ns 4.2 ± 0.3 2.3 ± 0.8 0.030 Si (ppm) 6.1 ± 20.3 52.4 ± 55.7 Ns 34.4 ± 3.8 58.6 ± 12.7 Ns Sr (ppm) 4.5 ± 1.2 3.3 ± 1.2 Ns 799.2 ± 51.9 760.8 ± 31.5 Ns Zn (ppm) 3.7 ± 2.3 1.8 ± 1.8 Ns 85.9 ± 4.0 83.6 ± 4.0 Ns

Table 3. Mean ± SE differences between the basal position (Position 1) and the distal position (Position 4) in percentage, in adult red deer injected (Mn-Treatment) or not (Control) with manganese as a nutrient supplement. A decrease from base to top is shown as a negative value. The right half of the table shows the mean ± SE for position 4 (the uppermost showing most clearly physiological exhaustion and depletion of body stores). Differences and probabilities are shown on the basis of one-way ANOVAs.

Some further effects were revealed in analyses regarding the distal position. Table 3 shows the means for the uppermost position examined in the antler (just below the crown). Although most minerals (except Cu) showed a similar effect than the analysis conducted on the mean antler content, ash content in the treated group was 13 percent units higher (Table 3) than in the control group. One of the most interesting differences regarded the effect on work to peak force, once the effect of body weight was controlled for. Thus, supplementation with Mn increased work to peak force (β for untreated group = -7.1±3.1; p=0.036; shown in Table 4).

Factors in the model Mn-Treatment Weight (Kg) Intercept ± Variables R2 β ± S.E. Sig. Β ± S.E. Sig. S.E. Young’s modulus of 0.50 6.8 ± 1.8 - - -0.04 ± 0.01 0.001 elasticity(E),Gpa Bending Strength 0.50 128.8 ± 33.5 - - -0.7 ± 0.2 0.001 (BS),Mpa Work to peak force 0.41 26.1 ± 8.3 7.1 ± 3.1 0.036 -0.1 ± 0.04 0.020 (W),kJm-2 Impact work (U),kJm------2 Cortical/total ratio(%) ------Average cortical 0.47 0.9 ± 1.1 - - -0.02 ± 0.005 0.001

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thickness(mm) Specific gravity of 0.42 1.4 ± 0.1 - - -0.001 ± 0.0004 0.004 cortical bone(g/mL) Ashes (%) ------Ca (wt%) 0.55 21.6 ± 0.2 1.1 ± 0.3 <0.001 - - Mg (wt%) ------Na (wt%) 0.47 0.6 ± 0.01 0.1 ± 0.02 0.002 - - P (wt%) 0.60 10.3 ± 0.1 0.6 ± 0.1 <0.001 - - B (ppm) 0.54 6.1 ± 0.3 1.7 ± 0.4 <0.001 - - Co (ppm) 0.56 0.6 ± 0.04 0.3 ± 0.1 <0.001 - - Cu (ppm) ------Fe (ppm) ------K (ppm) 0.39 669.3 ± 43.2 196.5 ± 61.2 0.005 - - Mn (ppm) 0.38 0.7 ± 0.1 0.3 ± 0.1 0.006 - - Ni (ppm) ------S (ppm) ------Se (ppm) 0.29 4.2 ± 0.5 1.9 ± 0.8 0.030 - - Si (ppm) ------Sr (ppm) ------Zn (ppm) ------

Table 4. Influence of injections of Mn (as nutrient supplement) and body weight, on the composition, structure and mechanical properties of antlers in the distal position (position 4), in adult red deer; the factor β is related to the difference of the value observed in animals that were injected with respect to the group not injected. Using GLMs analysis for each variable. Dashes indicate coefficients that were not significant.

Acknowledgments The members of the Animal Science Techniques Applied to Wildlife Management are the authors listed in this MS: LG, TLC and AG as permanent members, and JC and PG as PhD students.

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General discussion Chapter 7. General discussion

7. GENERAL DISCUSSION

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General discussion

In this chapter the results obtained in the different studies reported in chapter 5 will be discussed. The discussions were extracted from the published articles and partly expanded with further considerations.

7.1. Morphology, chemical composition, mechanical properties and structure in antler of Sardinian red deer (Cervus elaphus corsicanus). Results obtained in this study show that there is a difference between the values recorded in antlers of subadults with respect to those of adults. The latter, as expected, showed antlers with a higher number of tines, a greater weight and total length of the main beam. The comparison with C.e. italicus (Zachos et al., 2014; data for Italian red deer in Mattioli, 1993 and Mattioli & Ferretti, 2014), shows that Sardinian red deer has a quite similar number of tines per antler (average number of tines for Sardinian adults is 2.11±0.16 vs. 2.9±0.9 for C.e. italicus), but registered a lower average weight (0.52 Kg in Sardinian red deer VS 0.9 Kg in C.e. italicus). In addition, in the Sardinian studied subpopulation only a 20% of antlers have four-tined beam, and the same percentage is registered for the three-tined antlers; whereas the C.e. italicus recorded a 41.5% of adult deer with three tines. This can be explained by the fact that antler and tines size are strongly affected by ecological factors during their growth period such as food availability and quality (Brown 1990). Nevertheless, all the morphological values recorded in this study, resulted in the middle of the morphological data range observed in the past for antlers by Beccu (1989). In addition, our study shows results quite similar to those by Caboni et al. (2006) who examined morphometric traits of antlers from the three subpopulations surviving in southern Sardinia. In that study and our results, the bez tine is a rare characteristic (the frequency was 7.3%, and in our study is 6.5%), while the four-tined antlers structure recorded different percentages (52.2% in Caboni et al., 2006). The Sardinian antlers morphological characteristics match with the maintenance phenotype model described by Geist (1998), typically present in deer population living in environments with resource shortages (phenotype plasticity is pushed to a model of efficiency).

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General discussion This research allows to enrich and update the knowledge of this subspecies through an analysis of the structural characteristics, the chemical content and mechanical properties of antler bone material. Recent studies show that the composition of the antlers can tell us great information that may be useful to know the status of a wildlife population (Landete-Castillejos et al., 2013b), and furthermore, in population with conservation relevance, the monitoring of morphometrics could help to assess how individuals react to conservation measures (Mattioli & Ferretti, 2014). For this reason, from all antlers collected, 12 adult deer antlers were selected in order to obtain representative samples for well-developed antlers of adult of C.e. corsicanus. This allows the comparison of the properties and variables observed for the Sardinian antlers bone, with other subspecies that, in the past, have been characterized under the same scientific approach (mostly Iberian populations of C.elaphus, formely considered the subspecies C.e. hispanicus, since both subspecies live in similar habitats in the Mediterranean basin). Generally, the mechanical and structural properties of Sardinian antler bone show a decrease in the observed values, from the base towards the distal region of the main beam; the same mechanism holds for the content of minerals studied in the different positions along the main beam (only B, Cu, S and Se show a positive trend). This is coherent with the hypothesis that the decreasing trend shows the depletion of those minerals in the body (Landete-Castillejos et al., 2007b, c, 2010), thus reflecting the physiological exhaustion to grow the antlers, something also reflected in the decreasing trend of mechanical properties (Landete- Castillejos et al., 2007c). Comparing antler characteristics of the subspecies C.e. corsicanus and the Iberian populations of C.elaphus, the values in most traits are lower for the Sardinian red deer than for the Iberian one. The average antler weight, first tine length, burr perimeter, and main beam length are lower by a 65%, 44.6%, 30.3% and a 26,3% respectively (data for Iberian popoulations of C.elaphus in Fierro et al., 2002), and for the mean beam perimeter, our results show a value between - 10,4% and -24,2% lower than in Iberian red deer (using the values in Landete- Castillejos et al, 2007c and 2010, respectively). These studies include farmed deer, but our comparison is for the wild population studied there (with no

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General discussion supplement of feeding). This could be explained with the morphological characteristics of this subspecies that, normally, shows a slender body size than continental red deer (Beccu, 1989). Generally, vegetal and animal Sardinian subspecies show a smaller size than the European ones for directional selection common in small islands due to trophic resources shortages (Capula, 1994). Those animals with a smaller skeleton could invest less mineral bone resources, in quantitative terms, for the growth of their antlers annual cycle. Various relationships were found by Ceacero (2015) indicating that body morphometry characteristics were related to antler measurements. The author declared in his conclusions that a linear relationship exists between the body size of adult animals and the length of deer antler, and moreover the body mass is the best estimator, across cervid species, for the relationship between body size and the antler mass. However, there are other causes that influence the growth of a good antler which could be examined for this subspecies in further studies in the future. Besides the genetic characters (Scribner et al., 1989), the conditions of the natural habitat in which the population is widespread could influence the diet, and so, through an improved body condition, a better deer antler growth and its observed differences in mechanical properties. However, it is possible that formal tests for tissue toughness, which were not specifically examined in this study, might be less degraded in our sample when compared to the results of impact and bending tests that we used herein. Additional studies would be required to test this hypothesis because various types of mechanical tests are required to fully assess the antler mechanical properties (Launey et al., 2010b; Skedros et al., 2014). Regarding structure, the cortical/total ratio was similar to the values recorded in the past for other subspecies grown up in their natural state (0.44±0.01 VS 0.45±0.01 in Estevez et al., 2009). The average cortical thickness values for Sardinian deer were much lower than populations of Iberian Red deer: -36% respect to free ranging deer (data from Landete-Castillejos et al., 2010). Regarding material mechanical properties, again the comparison shows lower values for Sardinian deer antlers: the E of Sardinian deer is -17.8% than that of Iberian deer (12.89±0.33 VS 15.69±0.32 in Iberian C.elaphus) whereas W is slightly lower (-3%, 38±1.5 in Iberian C.elaphus). Also, BS and U show lower

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General discussion values (256.1±6.9 VS 306.6±6.4 and 21.86±0.81 VS 54.9±2.7 respectively, data for Iberian population of C.elaphus from Landete-Castillejos et al., 2010). The same trend exists for ash content, which showed a -5.2% in respect to Iberian red deer. We do not know the reason for these differences, but one possibility may be that Sardinian deer fight less often and with confrontations less powerful, and therefore, they can save resources by creating antlers with lower mechanical material properties than those of Iberian deer. Also, a poorer nutrition or a high density of the population may have effects on these observed differences in mechanical properties. Regarding antler minerals, they play an important role in antler growth, and usually are deficient in the ordinary diet of herbivore, especially for relevant minerals like Ca and P (Ceacero, 2016). A comparison with other past analysis in antlers from Iberian free ranging red deer was not fully possible because the methodology changed slightly including new minerals in our study, but discarding others (i.e. levels of B, Fe, Ni and S). The comparison in content of different minerals between Sardinian and Iberian red deer shows a variable trend: for Ca and P the difference is minimal (20.8±0.2 and 9.79±0.1 for C.e. corsicanus VS 21.0±0.2 and 10.1±0.1 for Iberian C.elaphus in Landete-Castillejos et al., 2010, respectively), for other minerals the difference is greater (for Na: -7.8%, for K: - 42.7%, and for Co: -82.6% in C.e. corsicanus respect to C.e. hispanicus). The difference in percentage between the first and fourth sampling positions shows values rather similar with respect to other studies in red deer antler mineral composition (Landete-Castillejos et al.,2010); for example, Ca and Na show - 2.40% and -3.30% in C.e. corsicanus against -2.81% and -7.95% in Iberian C.elaphus, respectively. In conclusion, adult and subadult Sardinian deer showed different morphological characteristics in antlers. Moreover, this subspecies has a simplified antler structure compared to Iberian population of C.elaphus. Compared to other populations present in Italy (C.e. italicus spp.), the red deer of Sardinia had a quite similar number of tines per antler but a slightly lower average weight, and lower percentage of four-tined antlers than Italian deer of Mesola. Nevertheless, it cannot be excluded that the cause of these differences was the high density of deer in the population examined in the present study. Whereas the material

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General discussion mechanical properties show in general, lower values than those of Iberian C.elaphus (as it happened with content of ash). Content in most minerals, as expected, shows a decreasing trend from the base to the tip of the deer antler similar to studies in Iberian deer. In general, the study offers, for the first time, interesting data on the mineral profile, mechanical properties and internal structure of Sardinian red deer, that should be borne in mind when designing special protection programs and reintroduction.

Present and future of the red deer management in Sardinia The Corsican-Sardinian subspecies is currently considered protected, because of its demographic conditions. In the last years the wild deer population are growing almost at the limits of the sustainability of the current habitat, and this generates movements of animals in anthropized areas (often present at the sides of the geographic areas of total protection). As previously mentioned, the population density is a factor that has an impact on the growth and morphological development of the deer and also on the quality of habitat; due to the fact that, with high densities there is excessive pressure on natural resources, that could endanger Mediterranean habitats. With this type of future scenario, various actions for managing populations within protected areas can be hypothesised, for a future in which this subspecies can be considered a resource to be protected, especially in marginal areas. The Corsican-Sardinian subspecies is currently considered protected and it is present in 13 distinct geographic locations; among these there are protected areas and wildlife breeding and restocking enclosures (Murgia et al, 2015). It is still strictly protected under Appendix II of the Bern Convention (entered into force in 1979, ratified by Italy in 1981) and Annexes II and IV of the EU Habitats Directive (no. 92/43/CEE), and in addition, the sub-species is protected by the regional law LR 23/98 and by national law 157/92. One problem found in the last years is that the growing wild population of deer is expanding in anthropized areas. A necessary environmental improvement is the increase of ecological corridors, in order to connect the populations living in different areas, especially for the three populations located in the south of Sardinia (Sulcis, Sarrabus, Arburense regions) that are completely isolated (Murgia et al, 2005); this would

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General discussion allow a natural gene improvement. One similar example, for conditions and possible negative drifts, could be found in Sprem et al. (2013), where the influence of habitat fragmentation on population structure of red deer in Croatia, and how the human land-use can lead to genetic substructuring and loss of genetic variability are described. The author gives appreciable advice on the managing of local red deer populations not as a single unit, divided by human infrastructure, but like a viable population that should be maintained in good conditions, using an appropriate land management. Moreover, the effects of isolation for a wild population were recorded in Zachos et al. (2007), where the inbreeding depression and the loss of heterozygosity of a small and isolated population of red deer in the northern Germany are described. Following the Italian national law 157/92, the following ungulate species can be hunted: Wild boar, Alpine chamois, Roe deer, Fallow deer, Red deer and Mouflon; although neither of these last two species may be hunted in Sardinia (Apollonio et al., 2010c). Ungulates cannot be hunted in protected areas in Italy by the general public (law n◦ 394/1992, Sardinian regional law n◦23/1998), but technically, they can be controlled by rangers or by hunters with special training and authorisation, under the control of rangers (culling programme for deer). During the hunting season could happen that wild populations (among ungulates there are deer, roe deer and wild boar), pushed from hunting activities, concentrate for short periods in the surrounding protected areas thereby creating damage from over-exploitation of the existing vegetation. Animal density is a factor that has an impact on the growth and morphological development of the deer and also on the quality of habitat; due to the fact that, with high densities there is excessive pressure on natural resources (Azorit et al., 2002), that could endanger Mediterranean habitats (Farris & Filigheddu, 2008; Pisanu et al., 2012). The density of the population plays an important role on the characteristics and properties of the deer antler, in fact, a high density has an impact on habitat quality, that could lead to changes in mineral profile of the diet that may produce a drastic effect in the composition and mechanical properties of antlers (Landete-Castillejos et al., 2013a). This suggests that these protected areas may play a very significant role in the overall dynamics of deer populations within the wider landscape area.

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General discussion If, nevertheless, the population of Sardinian deer has not reached critical numbers and density in the entire island (Lovari et al., 2007) and yet it is a protected subspecies for its conditions, in the near future, due to increasing animal residents, problems may arise related to high densities in relatively small areas, especially if there is not a comprehensive perspective between human development and land management. Some solutions on this topic, have been introduced by other authors (Cubeddu et al., 2006), and the proposals reported have been related to better management of protected areas (fences with appropriate materials) and better information for farmers who suffer damage by wild ungulates. A potential solution could be the introduction of a restricted selective hunting (controlled culling programme) of the ungulates, to avoid demographic problems in certain areas, and, from another point of view, this could generate a positive economy flourish in areas with low development. Since it would take a long time to take this controversial step (hunting of a subspecies with threatened status), due to the political-administrative debate that would arise, there are other complementary strategies to be undertaken with a landscape-level planning to ensure adequate habitat structures available for plants and animals, thereby reducing conflicts. It is also clear that management for limitation of damaging impacts must also be integrated with wider management objectives, like described in Reimoser & Putman (2011), which may include sustainable exploitation of those same ungulate population as a positive resource. Furthermore, it should be considered the possibility of establishing farms of wild ungulates, like described in Latte (1993); this could improve the conditions in marginal geographical areas and, from another point of view, it would encourage the creation of an economic sector linked to the exploitation of the species farmed. A further analysis on this subspecies would be useful in order to better understand the relationships between the wild population and the habitat that hosts it; also, to consider any possibility in the future to include this population in a strictly controlled hunting.

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General discussion 7.2. Smaller does not mean worse: variation of roe deer (Capreolus capreolus) antlers from two distant populations in their mechanical and structural properties and mineral profile.

This is the first study analysing the mineral profile and the mechanical and structural characteristics of roe deer antlers, comparing at the same time these features in two populations living in semi-natural environments with very different conditions; the results obtained show that the two roe deer populations, have marked differences in morphometric measurements, as well as in certain structural and mechanical properties, and in the bone mineral profile. Generally, cortical bone tissue shows lower values in antler tip for density, cortical width and mechanical properties, but not for the chemical composition (indicating almost no clear trends in physiological constraints for the completion of the mineralization phase of bone tissue). However, the two populations have different mechanical properties of bone material: roe deer antlers from Spanish population have higher W and U, while those from Czech population compensate the greater risk of fracture developing more cortical bone area although having higher E. One of the most interesting results of our analysis show how most antler properties covary in a consistent pattern in which antler indicate improved nutrition and greater bone quality of the Spanish population. This pattern is also recognized in the external structure and its characteristics. The greatest difference was the antler weight of Spanish deer (118%) compared to Czech individuals with lower availability of food resources. It is not surprising that quality of roe deer trophies is mostly indicated by their weight, because the differences in tine length were almost half that in weight (63% and 64% greater for Spanish vs. Czech antlers for second and first tine, respectively). The total length of the antler was 25% greater for roe deer with better diet. These trends could support the proposed hypothesis that a richer diet is also reflected in the development of antlers, since the Spanish animals were kept with food supplements and they were able to feed on a continuous presence of natural vegetation all year long, whereas the Czech set had poor vegetation availability during the winter period (when the roe deer antlers grow, Sempere 1990). This is consistent with Pélabon and van Breukelen (1998) who considered that secondary sex traits, as antlers,

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General discussion could be highly environmentally sensitive during the period of their growth. Similar consideration was theorized in the past by Pokorny et al. (2007), who considered roe deer antlers as an indicator of the quality of the environmental conditions, and by Mattioli et al. (2003), who observed a higher number of deer antler tines when feed is better. How such effect of better nutrition translates into differences in the rest of antler characteristics? The better feed Spanish population had a 64% increase in impact energy (U) with respect to the Czech one. The effect is more subtle in the W, which is 21% higher in the larger Spanish antlers. A similar greater environmental effect on U compared to W was found in red deer in the same population in years differing in the quality of the food (Landete-Castillejos et al. 2010). Greater resistance to impact or fracture is often achieved by increasing the protein content (which means a reduced ash content), which reduces the stiffness or E. In our results, populations did not differ in ash content, but the bulk of such content, Ca and P content, was significantly lower in the Spanish larger antlers by 11% and 16%, respectively. The effect of such reduction was likely a reduction of 11% of E and 14% for BS in antlers of the population with better diet. However, the roe deer with worse diet appeared to compensate by making a greater effort to develop a greater percentage of width occupied by cortical bone (Ct.B.Wi%, 16% more than better fed deer), and a similar trend was achieved in cortical bone area (Ct.B.Ar, +19%). Nevertheless, they appeared to be smaller in overall diameter (Ct.B.Wi in cm, -20%), which increases the risk of fracture (Davison et al. 2006). Because bone strength is determined by both its material and structural properties, and increases with increasing width of the cortical tissue and diameter of the bone (Currey 2002; Davison et al. 2006), this greater percentage of diameter invested in achieving a greater cortical bone may be an attempt to compensate for both the lower mechanical performance of the bone material and the reduced overall diameter. For the differences in values observed in the sampling position 1 and position 2, the bone mechanical and structural properties, on both antler groups, showed slightly negative or almost null trends. In the position 2, the cortical bone was characterized by less Ct.B.Dn, less average width and lower mechanical features (only W, BS in Spanish antlers, and U in Czech antlers). Probably, the differences are minimal for the dimensions of

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General discussion antlers, considering the body mass of roe deer species, as observed in the past by Ceacero (2016), who hypothesized that the antler mass reflect the existence of physiological constraints in the evolution of antlers (greater for larger sized species). Thus, our less pronounced results may be due to a less marked effect than those described in previous studies on red deer antlers (physiological stress of antler grow; Landete-Castillejos et al. 2010). Are these results consistent with studies in other deer antlers? The study of Landete-Castillejos et al. (2007c), assessing antlers from red deer with different diet showed that antlers grown under a better food quality had bone material with a greater W, greater percent of protein and greater average antler perimeter. In a subsequent study (Landete-Castillejos et al. 2013), assessing antlers from distinct game estates (public management with no supplementary food vs. private management including food supplements), antlers of deer with supplementary food were longer with an increased W at the cost of a reduced stiffness E, as in the present study with roe deer, although no difference in BS was found. Obviously, when comparing the characteristics of the deer antlers, we must also consider that their diversity is regarded as a multiple solution to the same problem, as for defence from enemies (Geist 1966). Comparative studies have concluded that also the habitat-specific changes in male fighting styles, resulting in part from evolutionary increases in overall body size, are likely to have contributed to evolution of larger male weapons (Emlen 2008), and large deer species have the biomechanical potential to carry particularly large antlers (Lemaître et al. 2014). Another factor to consider is the relationship between the shape of the antlers and the different use of this structure in the reproductive strategy (i.e.: the presence of a harem in red deer but not in roe deer, Baskin and Danell 2003). The relationship between antler size and reproductive success within species, and between antler size and degree of polygyny among species, offers hints on the function and evolution of antlers (Clutton-Brock et al. 1979, 1980). Plard et al. (2010) found that highly polygynous species have relatively longer antlers than less polygynous ones but the difference in the relative effect of breeding group size on relative antler length is weak. In roe deer, the access to females by adult males depends on possession of a territory, whereas that of red deer may be associated more to antler characteristics (Clutton-Brock 1982;

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General discussion Hartl et al. 1995) and their fencing behaviour in intraspecific fight (Caro et al. 2003). Thus, the roe deer is related to a low-risk low-gain strategy, where emphasis is placed on survival and multi-year tenure of a territory (Linnell and Andersen 1998) and few male display rituals can get very aggressive, indeed the level of fighting escalation is influenced by the presence/absence of a territorial- male (from 41% to 15%) or when rival individuals are similar in size (Hoem et al. 2007). So, possibly, behavioural aspects could also influence the presence of some traits of trophies and the bone tissue properties, as in the case of roe deer and red deer. What about the mineral profile of the antler? Making comparisons for the antler’s mineral profile with other deer species is not a simple procedure, but certain metabolic processes during the ossification of the antlers could be similar (Kierdorf et al. 2013). Moreover, in roe bucks, like other cervid, the minerals necessary for the formation of the antlers come also from a significant mobilization of mineral components resulting from mobilization of skeletal bone material (Baxter et al. 1999, Fandos and Burón 2015), and a reversible cyclic osteopenia occurs in internal bone during the antler cycle (Brockstedt- Rasmussen et al. 1987). For two main minerals of the bone tissue, Ca and P (Wilson, 1995; Currey, 2002), the values observed differed from those of other cervids. Ca is slightly lower, whereas P is quite similar or lightly lower according to the species (in C.elaphus: 21.0 and 10.1, Landete-Castillejos et al., 2010; in A.porcinus: 22.5 and 12.2, A.axis: 23.4 and 10.8, C.Duvaceli: 23.4 and 12.6, Pathak et al, 2001; respectively). An interesting point in this study is that the Ca/P ratio is considerably lower for the Czech roe deer population compared to Spanish one, the reason could be the high value of Phosphorus in this group of animals (+19.4%), which may be due to possible replacements of this mineral in 2− hydroxyapatite crystals (HPO4 -containing apatites have been called Ca- deficient apatites; Dorozhkin, 2009). Furthermore, the observed value for Ca/P ratio is much lower than the values observed in other species (2.21 in red deer antler, Kierdorf et al. 2014). Nevertheless, results regarding particularly Ca content should be carefully considered in future studies of antler mineral profile of roe deer, to confirm the effect or point our population as an abnormality. For Na, the values observed in Spanish roe deer are almost similar to other species

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General discussion of deer kept captive (0.56 in E. davidianus, 0.55 in C. canadensis; Ceacero et al., 2015). Whereas, Czech roe deer had a trend similar to that observed in red deer under poorer diet (Landete-Castillejos et al. 2007b, 2007c; 2013), with a lower content of Na in their antlers than those of roe deer with a better diet. In our study, the content of Mn was lower when diet was poorer, as in the study of Landete- Castillejos et al. (2010) for red deer. However, in the study by Landete-Castillejos et al. (2013) both private offered supplementary food and farmed red deer had a lower content in Mn (a lower content in Mn has been previously found in farmed deer compared to wild ones: Hyvarinen et al. 1977). Regarding Cu, it was found only for the Spanish roe deer but not in the Czech one; this difference may be attributable to the large range of factors influencing tissue Cu concentrations (Wilson and Grace 2002). Nevertheless, the levels of the Spanish population are similar to other captive deer (E. davidianus and C. nippon pseudoaxis, Ceacero et al., 2015) In general, there seem to be three minerals that usually indicate that antlers have grown under a more physiologically stressful situation: Fe, K and Zn. In red deer, Fe was found higher in public as compared to private management with food supplements (Landete-Castillejos et al. 2013), as it were compared free ranging deer to farmed deer (Landete-Castillejos et al. 2007c). However, in our study, although the content of Fe was higher in roe deer having a poorer diet, the trend was not statistically close to significance. K was 15% higher in roe deer with poorer diet from Czech Republic in the present study, as was found for red deer (variations of 10% in public vs. private management, in Landete-Castillejos et al. 2013). The last mineral is Zn, which shows the degree of mineralization of the antler and that, linked to the enzyme mineralizing the antler, alkaline phosphatase, disappears when the bone tissue is fully mature (Landete- Castillejos et al. 2012). In the present study the content is 16% higher in the antlers from Czech Republic, which probably did not have enough resources to mineralize antler bone tissue fully. Although our results are derived from a small sample of individuals, they show also in roe deer that the study of the antler characteristics can be useful to assess management practices of this species and confirm its use in another ungulate. Thus, differences in antler quality and morphology could estimate if a sustainable use of wildlife as a natural resource has been achieved. In conclusion, differences

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General discussion in habitat quality affecting the quality of the diet, likely affected the external characteristics of roe deer antlers, their internal structure, mechanical properties, and mineral content of the bone showing a number of effects similar to what has been published in red deer also differing in the quality of their diet. Despite inter- species differences, the present study shows general trends that may depend upon the cervid physiology mechanisms for growing antlers.

7.3. The bony horncore of the common eland (Taurotragus oryx): composition and mechanical properties of a spiral fighting structure. It is the first time that the horncore bone of common eland is characterized studying mineral content and mechanical properties. The results of this study showed that the lower positions sampled in the horn bone have significantly higher values than the upper ones. Also, ash content and bone density decreased in distal direction. Published research on horn bone found trends in mechanical properties along the horn, as we have observed in eland horn bone core tissue. Li et al. (2010) argued that the spatial variations of the mechanical properties of the cattle horn depend mainly on the mechanical function, in addition to materials/microstructures (α-keratin multilayered structure and bone spongy tissue). However, other factors, which may have effects in ungulate’s bone, are the environment (through the close relation among the season, light and hormones; in Tomlinson et al., 2004; in Toledano-Diaz et al., 2007 and in Davis et al., 2011) and nutrition (for antler bone: Estevez et al., 2009; Landete- Castillejos et al., 2010; for internal bones: Olguín et al., 2013). In a subsequent study, Li et al. (2011), found that the horn bone had higher values for E and Yield Strength in the proximal position compared to the upper part of the horncore. This is similar to what we have observed in E and BS in eland. Our results may be caused by the same “regional fighting function within the horn” hypothesis: the most stressed areas of the horn (those in direct contact with the horn of the opponent, but also those bearing greater bending moment) are the proximal portions that are located close to the skull roof and this would explain why they are better suited to absorb energy in males’ wrestling. In addition to trends in mechanical properties, those found in mineralization may be the proximate cause

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General discussion for explaining mechanical trends, since the mineral content of bone tissues is the most important factor affecting the bone mechanical yield (Currey et al., 1984). The eland mineral profile showed higher values in first and second sampling position with a gradual decrease in the distal part (Ca, Na, Mg, P, S and Sr show the same trend across the horn’s vertical axis). In this case, the tissue of the processus cornualis of the frontal bone with its continuous growth and remodelling seems to have the same behaviour of another bone tissue grown “at once” and without remodelling: the antler. Antlers and horns had a convergent evolution like weapons and guards (Geist, 1966). Thus, the compositional and mechanical parameters of horn bone is in most aspects better at the base, as it was observed in deer antlers: on one hand the mechanical properties are similar according to their trend (in deer: the physiological exhaustion of the body mineral stores makes deer younger tip region have a lower quality than the base, Landete-Castillejos et al., 2007b); on the other hand the eland has got a similar mineral profile also observed in cortical bone of the deer antler (content of Ca and Na decreased in a proximal-to-distal-direction, in contrast to the K content that showed an increased pattern, Landete-Castillejos et al., 2010; 2012, respectively). This is an interesting similarity, if we take into account that the horn bone grows differently compared to cervids. In fact, horncore grows in length at the tip and appositional at the surface, leading to an increase in diameter, with a gradual deposition of compact bone from base to tip (with a continuous remodelling process, Davis et al., 2011), while antler growth direction is orientated from the tip to the base (with a mineralization process similar to what was just described for the horn, but without remodelling processes; Gomez et al., 2013). The similarity in the compositional and mechanical parameters trends of bone tissues is given by the fact that both the red deer and the eland are creating a stronger bone material in order to withstand the loads incurred in the cranial region when using their headgear. The decrease in horn bone density may be explained by the fact that the bone tissue is characterized by the presence of porosity (micro and macro), that increases from the proximal to the distal end of the horn (Tombolato et al., 2010; Li et al., 2011). Compared to what has already been studied by Kitchener & Vincent (1987) about

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General discussion keratin, the mechanical response of eland horncore tissue has lower values. One explanation may be identified in the different strategies adopted by the two tissues that form the horn, in order to improve the energy absorption: the fracture resistance of the sheath was attributed to crack arrest mechanisms such as fiber delamination (McKittrick et al., 2012), while the horncore mainly plays a role of buffer and absorbs the collision energy (Zhu et al., 2016). An interesting finding in this study is that the bone tissue of the spiral does not seem to strengthen the structure, in fact its mechanical properties are significantly weaker than standard bone core tissue. The mechanical properties are significantly lower in the spiral (W, BS and U), as also happens in content of certain minerals (Ca, Mg, P, S, Li, Mn and Tl). So, what is the role of this spiral shaped structure in the horn bone? Caro et al. (2003) already pointed out that the form of intraspecific fighting and the degree to which individuals are expected to fight influence the shape of horns (wrestling was associated with twisted horns in polygynous bovids). This idea was already present in Geist (1966), who indicated that the presence of spiral antelope horns could allow the locking of males’ horns, avoiding more impact but allowing only pushes in fighting. Our results showed that the spiral ridge of bone core is weaker than surrounding bone. None of the hypothesis of why straight horns have spirals (increased grip or anti-rotational hypothesis) predicts such effect (in fact, they do not add any prediction regarding mechanical quality of horncore bone). The most likely explanation for us is that the weaker mechanical parameters may serve to deflect cross-sectional fractures along the ridge, thus dissipating the fracture energy not only into another direction, but also into a larger surface. In this way, it would end up fracturing part of the bone in longitudinal section but not with enough energy to split the bone in two. In any case, this is a hypothesis that our data cannot prove if it is the only reason for this weakened mechanical quality, or to what extent this could be effective. Our data cannot support or reject any of the hypothesis discussed (increased grip, or anti-rotational ridge). In conclusion, eland horncore bone has higher values for mechanical properties, density and mineral profile in areas proximal to the cranium. This pattern is similar to another analogue structure that grows in an opposite manner with respect to the horn: antlers. This is the consequence of the base of both structures having

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General discussion to withstand higher bending moments, which require greater mechanical performance. In addition, the spiral showed a weakened material mechanical property. The reason for the existence of a spiral is, in turn, hypothesized as base for the fighting system of the species. Another hypothesis is that spiral shape may serve to dissipate and deflect cross-sectional fractures into a fracture along the ridge, thus avoiding serious breakage. Certainly, further future studies are necessary on the mechanical properties associated with the shape of the horn.

7.4. Manganese Supplementation in Deer under Balanced Diet Increases Impact Energy and Contents in Minerals of Antler Bone Tissue. Results show clearly that manganese supplementation is reflected in antlers of both yearlings and adults. It is particularly interesting the double pattern found in the influence of manganese supplementation in mineral composition: whereas antlers of yearlings were not affected in any mineral except iron, those of adults, despite deer having the same balanced diet, Mn-treatment increased content in Ca, P, Na, K, Se, B, Co, Ni and Cu. As expected, Mn had also a positive effect increasing the impact energy and also the work to peak force, but in this latter case only after body weight effect was controlled for in GLM analysis. No other effects in mechanical properties nor structural variables were found. The fact that manganese supplementation was clearly reflected in antlers in both yearlings and adults follows matches previous results showing that antlers reflect the composition of the diet (Landete-Castillejos et al., 2007b, 2010; Estevez et al., 2009), as it happens also with trace elements in internal bones, at least for the case of deer (Olguin et al., 2013). A particular difference in our case is that manganese was not ingested orally but injected. This probably means that manganese raised its content in blood in the animals treated, although we have no measure on this. The study gives support to findings of a previous study which propose a change in Mn content of plants as a potential cause of breakage of many antlers in Spain: an effect of climate reduced work to peak force in antlers by 10%, impact energy by 27%, cortical thickness by 30%, and also affected content in ash, Ca, P, Mn, Si, Na, Co, Cu (in this case only with marginal significance), and Fe. Two characteristics are particularly interesting in this comparison: 1) both studies

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General discussion show nearly the same effects in antler composition, despite the difference between a manganese supplementation in deer under a balanced diet, and a climatic event affecting plant composition in the wild; 2) manganese affected most minerals in our farm study despite the animals being under a rich and balanced diet (which is only low in manganese, as in most other studies with deer fed wholemeal under a farming setup (Hyvarinen et al., 1977; Landete-Castillejos et al., 2010). However, with the doses used no signs of Manganese toxicity were appreciated by histology. So, the amount of Mn injected was safe. “Manganese rickets” (a disorder reported with excessive amounts of Mn in the diet – Svensson et al., 1987) was not observed. In the study mentioned above (Landete-Castillejos et al., 2010), it was hypothesized that the stress caused in plants by a period of extraordinary frosts produced an increase in Si (a generalized response of plants to stress: Ma, 2004; Ma & Yamaji, 2006; Liang et al., 2007), and this, in turn, produced the reduction in Mn, Na, and probably other minerals; then the effect of Mn possibly influenced reduction in Ca and P. Surprisingly, in the present study, even under a balanced diet, a supplementation with Mn increased ash content, that of Ca, P, Na, Co, Se and B, and reduced the content of Si, which are exactly the same effects shown in the 2010 study (except that Fe decreased with Mn induced deficiency by the climatic effect in that study, but there is no effect here, and K was not affected in that study but it is in the present one). As indicated in the second point, it is remarkable that such effects have been found in our study in animals under a balanced diet. This would explain why structural properties were not affected in the present case, as the control group is not feeding on plants growing in a dry year with exceptional frosts (Landete-Castillejos et al., 2010), but feeding on a rich protein, balanced mineral diet, as was the case in the treatment group. Before discussing the effect of Mn in adults, we would like to bring the attention of the reader to the fact that no effect is found in other minerals or mechanical properties of yearlings. The reason may be that these animals are under a strong constraint for growth, so that influences in antler characteristic are smaller than in adults. There are two lines of evidence supporting this hypothesis: 1st) most antler characteristics are different between yearlings and adults (except cortical thickness, all structural and mechanical variables, plus concentration of B, Cu,

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General discussion Fe, Mn, Ni, S, Si and Zn). 2nd) the increase produced by Mn injection as compared to the control is smaller in yearlings than in adults in mineral composition: Na (+13.7% in adults, +11.5% in yearlings), B (+40.9% adults,+14.6% yearlings), Co (+99.6% in adults,+12% yearlings), Cu (+29.5% adults, +25.2% yearlings), K (+47% adults, +14% in yearlings), Ni (+108% in adults,+26% in yearlings) and Se (+142.7% in adults, +7.5% in yearlings). The trend is not so clear in other antler characteristics. Why does Mn affect impact energy and mineral composition? Whereas it is not so clear why supplementation affects mineral composition of so many minerals, we have a relatively abundant literature that is suggestive of why Mn is particularly important in impact energy and work to peak force. It was shown long ago that Mn is found mainly in the mineral, and a small proportion is found in the organic matrix (Fore & Morton, 1952). Thus, because Mn2+ can substitute Ca2+ in the apatite lattice (Wopenga & Pasteris, 2005), at first glance one may think that the effect of Mn supplementation may be caused by its storing in the skeleton. However, despite the fact that 25% of Mn in human body is stored in this way (Kimlis-Zacas & Kalea, 2008), it is a coenzyme for glycosyltransferases, which create glycosaminoglycans (GAGs), and sulfonases, which sulphate these molecules in the final step to produce proteoglycans (Bolze et al., 1985; Kimlis- Zacas & Kalea, 2008). One of the main GAGs is chondroitin sulphate, the major constituent of cartilage, so that it is long known that Mn deficiency reduces cartilage growth by impairing chondroitin sulphate and other GAG biosynthesis (Leach et al., 1969; Bolze et al., 1985). In addition to the study by our group in 2010 (Landete-Castillejos et al., 2010), we do not know of other studies showing how this affects bone mechanical properties, but in arteries, Mn deficiency has been shown to affect mechanical properties by affecting GAG biosynthesis and sulfation (Kimlis-Zacas & Kalea, 2008; Yang et al., 1998). Thus, it is not surprising that the most important mechanical property influenced by Mn supplementation has been impact energy and, to a lesser extent, work to peak force. GAGs are highly polar and attract water, so that they affect particularly elastic properties and this is the reason why they are particularly important in arteries and why Mn affects cardiovascular disease in humans (Kimlis-Zacas & Kalea, 2008). However, the closest evidence to the effect of Mn in a mechanical property similar

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General discussion to impact energy is not found within humans or mammals, but in birds. In addition to the effect of Mn in chick cartilage, recent research has shown that Mn supplementation increases fracture toughness in eggs of laying hens (Xiao et al., 2014). Fracture toughness is similar to impact energy or work to peak force in antlers in that it measures the energy required to grow a thin crack in an egg shell. The study shows that the well-known effect of Mn supplementation in enhancing mechanical properties in eggshell is achieved through increasing the glycosaminoglycan synthesis. However, the situations are completely different and so may be the mechanisms by which in both cases energy required to grow a crack is increased by supplementation of Mn. In fact, for the case of eggs, the effect seems to be achieved by influencing the GAG contents in the eggshell membrane, rather than in the shell itself.

Why Mn increases the content of most minerals in antler? A very likely mechanism is, again, the increase in glycosaminoglycans. Cartilage, which precedes bone and antler mineralization (Gomez et al., 2013), is formed by an extracellular matrix consisting mostly of collagen II and proteoglycan, the latter attached chondroitin sulphate and other GAGs (Mobasheri, 1998). The author found that, because GAGs have a high fixed negative charge density, GAGs increased in content in cartilage, so did the content in Na+. Thus, it is very likely that as Mn increases chondroitin sulphate and GAGs in antlers of adult deer, the increased negative charge may have increased the content of all positive cations (most of these increased, although some, like Mg, Zn, Fe and Sr did not increased as a result of high variability, the rest showing a significantly higher content in antlers of deer injected with Mn, except Si). How could this be possible considering that antler is made of bone rather than cartilage? According to several studies (Kierdorf et al., 1995; Krauss et al., 2011; Gomez et al., 2013) antler is formed first as a scaffolding of cartilage leaving longitudinal tubes that at the end of the process are filled by osteons. Before the creation of osteons, the scaffolding of cartilage is first calcified and subsequently substituted by a bone. In bone, approximately 0.25% is made by non-collagenous proteins (Burr et al.,2014). One may think that such small remaining amount of GAGs and other non-collagenous proteins are unlikely to create the same effect as found in

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General discussion cartilage (Mobasheri, 1998). However, on the one hand the greater amount of cations brought by increased GAGs of antler may remain as increased contents in the bone scaffolding, and also they may influence the overall process in which antler bone is formed, and also in the way that cracks grow (Bertassoni & Swain, 2014; Launey et al., 2010a). Thus, it is particularly interesting that in the histological study, the staining of proteoglycans showed a different pattern between Mn-treated and control antlers: the treated having a more homogeneous distribution of proteoglycans. In conclusion, the present study is the first one to show that organic Mn injected in deer that otherwise had a balanced diet, affected content of most minerals and improved mechanical properties related with growth of fractures. In addition, Mn supplementation seemed to influence the distribution of GAGs assessed through histological staining. Thus, the present study may be a first step towards understanding effects in bone of supplementation with Mn in enhancing bone quality and some bone mechanical properties in situations where individuals are not deficient in them.

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Conclusions Chapter 8. Conclusions

8. CONCLUSIONS

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Conclusions

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Conclusions The conclusions of this thesis are divided following the different studies that make up the same.

Morphology, chemical composition, mechanical properties and structure in antler of Sardinian red deer (Cervus elaphus corsicanus). 1. The antlers of the adult Sardinian deer have more tines, greater weight and greater total length than those of the subadults. 2. The C.e. corsicanus presents antlers of smaller size, weight and branching than the C.e. italicus and the Spanish population of C.elaphus. 3. In addition, the Sardinian deer shows lower quality in terms of chemical composition (lower content of Na, K, Ca, P and Co) and several structural and mechanical parameters (E, W), compared to the Iberian deer.

Smaller does not mean worse: variation of roe deer antlers from two distant populations in their mechanical and structural properties and mineral profile. 4. The Spanish population of roe deer have larger antlers, with a longer length, greater weight and more tines, than those of the Czech population studied. 5. The two populations present different mechanical strategies: the Spanish roe deer has greater W (+19%) and U (+72%), while the Czechs compensate with more cortical bone area and greater E and BS (+12% and +16 %, respectively). 6. Antlers of the Czech population studied have higher levels of Ca, K, P, Cr, Li, Sr and Zn; while the trophies of the Spanish population studied have higher values of Na, Mn, Tl and the Ca/P ratio. 7. The sampling position mainly affects the mechanical properties (E, BS, W and U) and structure (CBD in cm), which were higher in the base. The antlers of both populations have higher levels of Zn at the tip than at the base, although the Zn content is higher by 25% in the Czechs than in the Spanish ones.

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The bony horncore of the common eland (Taurotragus oryx): composition and mechanical properties of a spiral fighting structure. 8. The density and ash content of the core bone of the horn decreases from the base to the tip, as well as most of the mechanical properties. 9. As for the mineral composition of the horns, there are elements that increase their concentrations from the base to the tip (Se, Cu, K) while others decreased significantly (Mg, Mn). 10. The mechanical properties correspond to a greater resistance to bone fracture at the base of the horn near the roof of the skull, which is logical based on the mineral profile found and on the type of fight of this species. 11. There are differences in mineral content and mechanical properties between the sampling point of the standard bone tissue and that of the spiral, so that the bone tissue of the spiral does not seem to strengthen the structure on the contrary it is weaker.

Manganese Supplementation in Deer under Balanced Diet Increases Impact Energy and Contents in Minerals of Antler Bone Tissue. 12. Supplementation with Mn had more effect on adults than on yearlings. Thus, treated adults suffered an increase in body weight (+10%) and most of the micro and macromineral (Ca, P, Na, K, Se, Cu), as well as an improvement in the mechanical properties of their antlers. 13. The antlers of the yearlings present a pattern very different from that of the adults. Thus, there are differences in all the mechanical properties (higher values for adults), structural and measures of the antler (except for the average cortical thickness) and the mineral profile. 14. The mineral composition of the antlers of the yearlings was higher in the case of Fe (3 times higher than in adults). However, the only effect of the treatment that showed a statistically significant effect common among the age classes was the increase in the concentration of Mn in the antler of the supplemented group (2.5 times in the yearlings and 2.3 times in the

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Conclusions adults), although in absolute terms, the concentration of Mn in the antlers, of the supplemented animals, is lower in yearlings.

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Conclusions

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Conclusions

CONCLUSIONES

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Conclusions

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Conclusions Las conclusiones de esta tesis se dividen siguiendo los diferentes estudios que conforman la misma.

Morphology, chemical composition, mechanical properties and structure in antler of Sardinian red deer (Cervus elaphus corsicanus). 1. Las cuernas de los ciervos sardos adultos tienen más puntas, mayor peso y mayor longitud total que las de los subadultos. 2. El C.e. corsicanus presenta cuernas de menor tamaño, peso y ramificación que el C.e. italicus y la población española de C. elaphus. 3. Ademas, el ciervo sardo muestra menor calidad en cuanto a composición química (menor contenido de Na, K, Ca, P y Co) y varios parámetros estructurales y mecánicos (E, W), respecto al ciervo ibérico.

Smaller does not mean worse: variation of roe deer antlers from two distant populations in their mechanical and structural properties and mineral profile. 4. Los corzos de la población española estudiada tienen cuernas más grandes, con una longitud más larga, mayor peso y más puntas que los de la población checa estudiada. 5. Las dos poblaciones presentan diferentes estrategias mecánicas: el corzo español tiene mayor W (+19%) y U (+72%), mientras que los checos compensan con más área de hueso cortical y mayor E y BS (+12% y +16%, respectivamente). 6. Las cuernas de la población checa estudiada presentan niveles más altos de Ca, K, P, Cr, Li, Sr y Zn; mientras que los trofeos de la población española estudiada tienen valores superiores de Na, Mn, Tl y de la ratio Ca/P. 7. La posición del muestreo afecta principalmente a las propiedades mecánicas (E, BS, W y U) y la estructura (CBD en cm), que fueron mayores en la base. Las cuernas de ambas las poblaciones presentan mayores niveles de Zn en la punta que en la base, aunque el contenido de Zn es superior en un 25% en las checas que en las españolas.

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The bony horncore of the common eland (Taurotragus oryx): composition and mechanical properties of a spiral fighting structure. 8. La densidad y el contenido en ceniza del hueso del núcleo del cuerno diminuye desde la base hasta la punta, así como la mayoría de las propiedades mecánicas. 9. En cuanto a la composición mineral de los cuernos, hay elementos que aumentan sus concentraciones de la base a la punta (Se, Cu, K) mientras que otros disminuyeron significativamente (Mg, Mn). 10. Las propiedades mecánicas corresponden a una mayor resistencia a la fractura osea en la base del cuerno cerca del techo del cráneo, lo cual es lógico en base al perfil mineral encontrado y al tipo de lucha de esta especie. 11. Existen diferencias en el contenido mineral y las propiedades mecánicas entre el punto de muestreo del tejido oseo estándar y el de la espiral, de modo que el tejido oseo de la espiral no parece fortalecer la estructura, sino que es más débil.

Manganese Supplementation in Deer under Balanced Diet Increases Impact Energy and Contents in Minerals of Antler Bone Tissue. 12. La suplementación con Mn tuvo más efecto en los adultos que en los varetos. Así, los adultos tratados sufrieron un aumento del peso corporal (+10%) y de la mayoría de los micro y marco minerales (Ca, P, Na, K, Se, Cu), así como una mejora de las propiedades mecánicas de sus cuernas. 13. Las cuernas de los varetos presentan un patrón muy diferente al de los adultos. Así, existen diferencias en todas las propiedades mecánicas (valores mayores para adultos), estructurales y medidas del asta (excepto del grosor cortical promedio) y del perfil mineral. 14. La composición mineral de las cuernas de los varetos fue superior en el caso del Fe (3 veces superior que en adultos). Pero, el único efecto del tratamiento que mostró un efecto estadísticamente significativo común

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Conclusions entre las clases de edades fue el aumento de la concentración de Mn en la cuerna del grupo suplementado (2.5 veces en los varetos y 2.3 veces en los adultos), aunque en términos absolutos, la concentración de Mn en las cuernas, de los animales suplementados, es menor en varetos.

237

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