4. Mineral Supplementation in Deer ______56 4.1

UNIVERSIDAD DE CASTILLA-LA MANCHA

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

Mineral supplementation in key periods of red deer: antler growth, gestation and lactation

Memoria presentada por Pablo Gambín Pozo para el grado de doctor, Albacete, 2018

VºBº de los directores:

Dra. Martina Pérez Serrano Dr. Andrés José García Díaz Dr. Laureano Gallego Martínez

A la memoria de mi Padre, con quien hubiera disfrutado mucho hablando de ciencia

Agradecimientos

En primer lugar, dar las gracias a mis directores y a mi tutor de tesis por haberme formado y guiado en el camino de la ciencia. Sobre todo, gracias a Andrés y a Tomás, por sus correcciones y sugerencias, sin ellos, esta tesis no hubiera sido posible. Agradecer a Laureano por su atención personal. Quisiera dar las gracias en especial a Martina por sus palabras de ánimo y brindarme la oportunidad de participar en los nuevos proyectos del grupo, pero sobre todo gracias por confiar en mí. Gracias. Agradecer también a Paco Ceacero y a Radim Ktorba por dejarme aprender de ellos en la estancia en Praga y por sus sugerencias como evaluadores externos. Gracias a Santiago Gómez por enseñarme los secretos de los tejidos duros en la Universidad de Cádiz.

Gracias a todos los miembros del Departamento (En especial a Almudena, Noemí, Javier, María y Jose Maria), sobre todo gracias a Jamil, de quien he aprendido mucho. Gracias por haberme mostrado tu calidad científica y a haberme apoyado en todo momento, de corazón, Gracias. Eres un científico de una altura intelectual increíble. Gracias a los alumnos de prácticas (Aníbal, Sara, Arianna, David, Valentina y Helena) y a los alumnos anónimos de la granja por haber colaborado de alguna manera esta Tesis. Gracias a Nicol, Olivia, David y Anna por las correcciones en el inglés. Gracias a Alba por el diseño de la portada de la tesis.

Gracias a mis compañeros y mis profesores (especialmente a Rocío y a Carlos) del Máster de Gestión de Fauna Silvestre por dejarme descubrir la ciencia en el medio natural. Gracias a todos mis compañeros que conocí en la carrera (Pedro, Joni, Rafa, Juanjo, Santi, Javi, Antonio, Vero, Blanca, Mar, Maria Paz, Ana y Deborah), de los que hoy, muchos son doctores, gracias por transmitirme la pasión de la ciencia. Gracias al Dr. Muñoz Rojo, con quien he compartido mi adolescencia y mi juventud, tienes parte de culpa de que haya decido hacer el doctorado. Gracias por tu apoyo. Ojalá publiquemos algo juntos.

Pero, sobre todo, muchas gracias a toda mi familia y a Rosa por llenar todo de felicidad. A mi hermano en especial por haber sido un pilar durante esta tesis en el que me he podido apoyar en los momentos difíciles. A mi madre, por todo su amor, su fuerza, su sonrisa, su apoyo, en definitiva, por todo, os quiero.

Por último, y haciendo un guiño a los novelistas gráficos Goscinny a Urdezo con quien tanto he disfrutado de niño, decir que esta Tesis ha costado 3285 cafés, 452 comidas en la cantina, 42 cenas en la facultad redactando la tesis, 4 tóner, 2 libretas, 8 bolígrafos y alguna que otra bebida fermentada con Juan.

INDEX

RESUMEN ______1 ______9 GRAPHICAL SUMMARY ______17 Chapter I. JUSTIFICATION ______21 Chapter II. INTRODUCTION ______27 1. General aspects of Iberian red deer ______29 2. Key periods in deer ______31 2.1. Key period in males ______31 2.1.1. Antler growth ______31 Antlers are sexual character exclusive to male deer (Lincoln, 1992). Therefore, current thesis will characterize external and internal morphology before describe antler growth and development. ____ 31 2.1.2. Rut ______36 2.2. Key periods in females ______37 2.2.1. Gestation ______37 2.2.1. Lactation and calf growth ______38 3. Nutritional components in the deer diet. The importance of minerals ______40 3.1. Energy ______40 3.2. Proteins______40 3.3. Vitamins ______41 3.4. Minerals ______41 3.4.1. Types of minerals ______42 3.4.2. Mineral requirements of deer ______48 3.4.3. Importance of minerals for antler ______49 3.4.4. Importance of minerals in serum ______51 3.4.5. Importance of minerals for gestation, lactation and calf growth ______52 3.4.6. Osteophagia and its relationship with minerals ______54 4. Mineral supplementation in deer ______56 4.1. Supplements providing short-term effects ______56 4.1.1. Injections ______56 4.1.2. Solidified products ______57 4.2. Supplements providing long-term effects ______58 4.2.1. Subcutaneous and intramuscular deposits injections of long term-effect______58 4.2.2. Intraruminal bolus ______58 4.2.3. Fertilized pastures with minerals ______59 Chapter III. OBJECTIVES AND HYPOTHESIS ______61 Chapter IV. METHODOLOGY ______65 1. Effect of Cu supplementation on antler properties and mineral composition of red deer male (trial 1) __ 69 1.1. Experimental design ______69 1.2. Body measurements ______70 1.3. Cu serum content ______71 1.4. Antler collection ______71 1.5. Antler cylinder processing ______73 1.6. Mechanical bone properties ______75 1.6.1. Mechanical properties ______75 1.6.2. Analysis of mechanical properties ______77 1.7. Antler bone cross section processing ______79 1.8. Bone structural properties ______79 1.9. Density ______80 1.10. Chemical analysis of antlers ______81 1.11. Statistical analyses ______83 2. Effect of Cu supplementation on mineral serum content of red deer male during antler growth and rut (trial 2) ______85 2.1. Experimental design ______85 2.2. Blood samples collection ______85 2.3. Antler collection ______86 2.1. Statistical analysis ______86 3. Effect of Mn and Cu supplementation on microhardness of antler (trial 3) ______88 3.1. Experimental design ______88 3.2. Antler collection ______88 3.3. Specimen preparation ______90 3.4. Microhardness ______90 3.4.1. Microhardness ______90 3.4.2. Analysis of microhardness ______91 3.5. Statistical analysis ______92 4. Effect of Cu supplementation of gestating and lactating hinds on calf growth and milk traits (trial 4) __ 94 4.1. Experimental design ______94 4.2. Body measurements ______95 4.3. Milk collection ______95

4.4. Milk composition ______96 4.5. Milk minerals ______96 4.6. Statistical analysis ______97 5. Effect of Mn supplementation of gestating and lactating hinds on milk traits and calf growth (trial 5) __ 98 5.1. Experimental design ______98 5.2. Body measurements ______99 5.3. Milk collection ______99 5.4. Milk composition and milk minerals ______99 5.5. Mn serum content ______99 5.7. Statistical analysis ______100 6. Osteophagia in key periods of red deer (trial 6) ______101 6.1. Experimental design ______101 6.2. Videotramps ______102 ______103 6.3. Statistical analysis ______103 Chapter V. RESULTS______105 1. Effect of Cu Supplementation on antler properties and mineral composition of red deer male (trial 1) _ 107 1.1. Effects of Cu supplementation on the average daily gain and body condition of deer ______107 1.2. Effects of Cu supplementation on the mechanical and structural properties and ash and mineral content of antlers ______108 1.3. Effect of Cu supplementation on the variation in characteristics and mineral composition between the base and top of antlers from yearling and adult deer ______110 2. Effect of Cu supplementation on mineral serum content of red deer male during antler growth and rut (trial 2) ______112 2.1. Effect of Cu supplementation on serum minerals ______112 2.2. Effect of antler growth and rut on serum minerals ______113 2.3. Correlations between serum and antler mineral ______115 3. Effect of Mn and Cu supplementation on microhardness of antler (trial 3) ______117 3.1. Influence of treatment (injections of Mn or Cu) and antler position on average of hardness Vickers in antler of adults and yearling of red deer ______117 3.2. Influence of Mn supplementation on hardness Vickers along main beam in adults and yearling of red deer ______118 3.3. Influence of Cu supplementation on hardness Vickers along main beam in adults and yearling of red deer ______119 3.4. Effect of Mn supplementation on the variation in hardness between base of crown and the burr from adult and yearling of red deer ______120 3.5. Effect of Cu supplementation on the variation in hardness between the base of crown and burr from adult and yearling of red deer ______120 4. Effect of Cu supplementation of gestating and lactating hinds on calf growth and milk traits (trial 4) _ 122 4.1. Effects of Cu supplementation to hinds on hind body weight and body condition and calf growth from birth to 18 weeks of lactation (forced weaning) ______122 4.2. Effects of Cu supplementation to hinds on milk production, milk composition, total yields and gross energy content of milk ______124 5. Effect of Mn supplementation of gestating and lactating hinds on milk traits and calf growth (trial 5) _ 126 5.1. Effects of Mn supplementation to hinds on hind body weight and body condition and calf growth from birth to 18 weeks of lactation (forced weaning) ______126 5.2. Effects of Mn supplementation to hinds on milk production, milk composition, total yields and gross energy content of milk ______127 6. Osteophagia in key periods of red deer (trial 6) ______130 Chapter VI. DISCUSSION ______133 1. Effect of Cu Supplementation on antler properties and mineral composition of red deer male (trial 1) _ 135 2. Effect of Cu Supplementation on mineral serum content of red deer male during antler growth and rut (trial 2) ______138 3. Effect of Mn and Cu supplementation on microhardness of antler (trial 3) ______143 4. Effect of Cu supplementation of gestating and lactating hinds on calf growth and milk traits (trial 4) _ 147 5. Effect of Mn supplementation of gestating and lactating hinds on milk traits and calf growth (trial 5) _ 151 6. Osteophagia in key periods of red deer (trial 6) ______155 Chapter VII. CONCLUSIONS ______159 FINANCIAL SUPPORT ______163 REFERENCES ______167

RESUMEN

Resumen

En España, el valor del ciervo reside en sus cuernas que, consideradas como trofeos, pueden alcanzar un valor de miles de euros cuando son de gran tamaño y están intactas. La calidad de éstas depende de factores genéticos y factores ambientales. Entre los factores ambientales destacan de los nutrientes recibidos durante el crecimiento de la cuerna en su etapa adulta y así como la calidad de la leche en su etapa lactante. Dado que, entre los componentes de la dieta que más influencia tienen en la calidad de las cuernas están los minerales, esta tesis estudió los efectos de la suplementación de Cu y Mn. La elección de estos minerales se debe, fundamentalmente, a sus efectos positivos sobre la osteogénesis (la cuerna es un tejido óseo) y sobre la lactación demostrados en la literatura. Los ensayos realizados en esta tesis se caracterizaron por una suplementación realizada en condiciones que superaban los niveles estándar establecidos para ciervos, pero sin llegar a niveles tóxicos. Para ello, se llevaron a cabo 5 ensayos que evaluaron la suplementación mineral en periodos clave del ciervo (crecimiento de la cuerna, gestación y lactación). Además de estos ensayos, se realizó un sexto estudio para evaluar si durante estos periodos clave de alta demanda nutricional los ciervos podían consumir cuernas (osteofagia) como fuente extra de minerales.

En el ensayo 1 se estudiaron los efectos de la suplementación con Cu sobre las características mecánicas y estructurales y el contenido mineral de las cuernas de varetos (1,5 años como promedio) y adultos (4 años como promedio) alimentados con una dieta equilibrada. Para ello, se inyectó Cu (0,83 mg Cu/kg de peso corporal) a 18 ciervos (11 varetos y 7 adultos) cada 42 días durante el crecimiento de la cuerna (grupo experimental), mientras que otros 17 ciervos (10 varetos y 7 adultos) recibieron con la misma periodicidad una inyección de solución salina

3 Resumen

fisiológica (grupo control). También se analizó la concentración de Cu en suero al comienzo del ensayo y en el día 84 después de la primera inyección. El efecto de la suplementación con Cu fue diferente en los varetos y en los adultos. En varetos, la suplementación no afectó a las propiedades de las cuernas. Sin embargo, la suplementación de Cu tendió (P = 0,06) a aumentar el espesor cortical de las cuernas en adultos.

En el ensayo 2 se investigó los efectos de la suplementación con Cu durante el crecimiento de la cuerna y durante la berrea sobre el perfil mineral de los minerales del suero. Para ello, se dividieron 18 machos (3 años de edad como promedio) en dos grupos homogéneos (n = 9) respecto a peso y edad. Además, se estudiaron las correlaciones entre el perfil mineral del suero durante el crecimiento de las cuernas y el contenido mineral de la cuerna a día 165 del incio crecimiento de la cuerna (ICC), cuando ya estaba descorreada. El grupo suplementado recibió Cu a razón de 0,83 mg Cu/kg de peso corporal cada 42 días desde el día -36 ICC al día 132 ICC, mientras que el grupo control recibió una solución salina fisiológica con la misma periodicidad. Los resultados mostraron que la suplementación con Cu produjo un aumento significativo en los contenidos de P (P = 0,03) y Cu (P = 0,04) en suero de un 7.6% (0.0085 g/100g vs. 0.0092 g/100g) y un 11.2% (0.0726 mg/kg vs. 0.8188 mg/kg), respectivamente. Los resultados también demostraron que los contenidos séricos de Ca, P, Mg, Na, S, Cu, Sr y Zn aumentaron (P < 0,05) mientras que los contenidos de K, Fe y Mn disminuyeron durante el crecimiento de la cuerna.

En el ensayo 3 se estudió el efecto de la suplementación con Cu y Mn (por separado) durante el crecimiento de la cuerna sobre la dureza Vickers (HV, por sus siglas en inglés, hardness Vickers) en las osteonas

4 Resumen

de la cuerna de varetos y adultos. Para este fin, se dividieron 20 varetos y 20 adultos en 4 grupos de 5 animales cada uno. Un grupo de varetos se trató con Mn y otro con Cu. Los adultos se trataron de la misma manera. Los microminerales (2 mg Mn/kg de peso corporal cada 7 días o 0,83 mg Cu/kg de peso corporal cada 42 días) se administraron durante el crecimiento de la cuerna a los grupos experimentales, mientras que los animales control fueron inyectados con una solución fisiológica salina con la misma periodicidad. La HV se midió en el centro del hueso cortical de la cuerna utilizando una carga controlada. Como promedio, la HV de los varetos tratados con Mn aumentó un 11,6% (P = 0,01) y la HV de los varetos tratados con Cu tendieron a aumentar un 10,3% (P = 0,08). Además, la dureza de Vickers del segundo tercio de la vara principal de los varetos fue significativamente mayor en los grupos tratado con Mn y Cu que en los grupos control (34,5 frente a 29,4 para el grupo tratado con Mn y control respectivamente; P < 0,001) y 37,8 frente a 32,6 para el grupo tratado con Cu y control respectivamente; (P = 0,03). Asimismo, la HV de las osteonas de la base a la corona fue mayor para los adultos tratados con Cu (HV = 29,4) que para el grupo control (HV = 25,3; P = 0,04) y el esfuerzo fisiológico fue menor en adultos tratados con Mn.

En el Ensayo 4 se determinaron los efectos de la suplementación con Cu en periodos clave de las ciervas (gestación y lactación) sobre el crecimiento de los gabatos lactantes, así como sobre los parámetros que definen la lactación. Se utilizó un total de 17 ciervas (n = 9 para el tratamiento con Cu y n = 8 para el control). Las ciervas experimentales recibieron 0,83 mg de Cu por kg de peso corporal cada 42 días desde el día 202 de la gestación (como media) hasta el final de la lactación (destete forzado a las 18 semanas de lactación). Las otras 8 ciervas

5 Resumen

actuaron como control y recibieron una dosis de solución salina fisiológica con la misma periodicidad. Todas las ciervas fueron ordeñadas desde la semana 2 de lactación hasta la semana 18. Las gabatas crecieron más rápido de la semana 14 a 18 de lactancia cuando sus madres fueron complementadas con Cu (0,319 vs. 0,231 kg/día; P = 0,047). Sin embargo, el rendimiento total de leche tendió (P = 0,07) a disminuir con la administración de Cu. Además, la leche de las hembras suplementadas con Cu tuvo un contenido más alto de K y Rb y un contenido más bajo de Al que la leche de las hembras control.

En el ensayo 5 se evaluó la suplementación con Mn en períodos clave de las ciervas (gestación y lactación) sobre el crecimiento de los gabatos lactantes, así como sobre los parámetros que definen la lactación. Para ello, se utilizó un total de 20 ciervas alimentadas con una dieta equilibrada, 12 de las cuales se inyectaron semanalmente con Mn (2 mg Mn/kg de peso corporal) a partir del día 140 de gestación (como promedio) hasta el final de la lactación (destete forzado a las 18 semanas). Las otras 8 ciervas se inyectaron con una solución salina fisiológica (grupo control) durante el mismo periodo y con la misma periodicidad. Las ciervas se ordeñaron desde la semana 2 a la semana 18 de lactación. El contenido sérico de Mn se evaluó antes de la primera inyección con Mn y en la semana 10 de lactación. Sin embargo, los gabatos cuyas madres fueron inyectadas con Mn tendieron a tener una ganancia de peso corporal relativa más alta durante la lactación en comparación con los gabatos de las ciervas control. Además, la suplementación con Mn aumentó la producción diaria de leche en un 10,2% (P < 0,05), el contenido de grasa en leche en un 11,2% (P < 0, 001) y el rendimiento total de grasa en un 18,2% (P < 0,05). La leche de las hembras suplementadas con Mn tuvo más contenido de Ca y P. Sin

6 Resumen

embargo, la suplementación con Mn no influyó sobre el contenido de Mn del suero en la semana 10 de lactación, pero aumentó su contenido en leche en un 18,3% (P < 0,001).

Finalmente, en el ensayo 6 y debido a que la osteofagia es un comportamiento común en ciervos, se analizó la evolución del consumo de cuernas en ciervos ibéricos durante los periodos clave de alta demanda fisiológica. En este sentido, se estudió la estacionalidad del consumo de cuernas del ciervo ibérico diferenciando entre clases de sexo y edad. Para ello, en una reserva de caza ubicada en el sureste de España, se ofrecieron cuernas a ciervos de vida libre y se registró su comportamiento de masticación mediante videotrampeo. Los resultados mostraron que los machos presentaron el mayor pico de consumo al final del crecimiento de la cuerna (P < 0,001), mientras que las hembras manifestaron el mayor pico al inicio de la lactación (P < 0,001).

En resumen, los resultados obtenidos en esta tesis sugieren que en ciervo ibérico el Cu y el Mn juegan un papel importante durante el crecimiento de la cuerna en los machos y en el período de lactación en las hembras, así como que esta especie puede consumir cuernas como fuente de minerales, durante estos periodos clave.

Summary

One of the main species of big game is deer. In Spain, deer is appreciated by its antlers which are prized as a trophy. Its quality depends largely on the nutrition received in both adult and calf stages. Previous studies have shown that minerals are needed primarily during the key periods of high physiological demand, i.e. during antler growth period in males and during the last third of gestation and lactation in females. Therefore, this thesis studied the effects of administration of the trace minerals, Cu and Mn, by subcutaneous injections (in order to ensure the necessary dose for each animal) and its effects on the quality of antlers in males, the quality of milk in hinds and growth in calves in five trials. Moreover, a supplementary trial was conducted to evaluate the consumption of a natural mineral source (antlers) during these key periods of high physiological demand in deer.

In trial 1 we studied the effects of Cu supplementation on the mechanical and structural characteristics, and mineral content of antlers from yearling and adult (4 years of age) red deer fed a balanced diet. Deer (n = 35) of different ages (21 yearlings and 14 adults) were studied. A total of 18 stags (11 yearlings and 7 adults) were injected with Cu (0.83 mg Cu/kg body weight) every 42 days, whereas the remaining 17 (10 yearlings and 7 adults) were injected with physiological saline solution (control group). The Cu content of serum was analysed at the beginning of the trial and 84 days after the first injection to assess whether the injected Cu was mobilized in blood. The effect of Cu supplementation was different in yearlings and adults. In yearlings, supplementation increased the Cu content of serum by 28%, but did not affect antler properties. However, in adults, Cu supplementation increased the Cu content of serum by 38% and tended to increase the cortical thickness of antlers (P = 0.06).

11 Summary

In trial 2 we investigated the effects of Cu supplementation of red deer fed with a balanced diet on during antler growth and rut. Eighteen 2.7- years-old (as average) male red deer were divided into two groups (control and Cu-supplemented). In addition, correlations between the mineral profile of serum and antler (after velvet cleaning at 165 of SAG) was studied. Control group was injected with a physiological saline solution from day -36 start antler growth (SAG) to day 132 SAG, while Cu supplemented group was injected with 0.83 mg Cu/kg of body weight, both every 42 days. Results showed that Cu supplementation increased the P (P = 0,03) and Cu (P = 0,04) contents of serum by 7.6% (0.0085 vs. 0.0092g/100g) and 11.2% (0.0726 vs. 0.8188 mg/kg), respectively. Also, serum contents of Ca, P, Mg, Na, S, Cu, Sr and Zn increased (P < 0,05) while K and Fe contents decreased during antler growth.

In trial 3 we studied the effect of Cu and Mn supplementation separately on Vickers hardness (HV) of osteons of antler from adults and yearling of red deer. Twenty adults and 20 yearlings were divided in 4 groups of 5 animals. One group of adults was treated with Mn (2 mg Mn/kg of body weight) and other with Cu (0.83 mg Cu/kg of body weight). Yearlings were treated in the same way. Microminerals (Cu or Mn) were administered during the antler growth period. Control animals were injected with a physiological saline solution. Microindentations were made in the middle of cortical bone of antler using a load controlled. As average, HV of yearling Mn treated raised the by 11.6% (P = 0,01) and HV of yearling Cu treated tended to increase by 10.3% (P = 0,08). Hardness Vickers of the second third of shaft of yearling was statistically higher in Mn and Cu treated groups than in their respective controls (34.5 vs. 29.34, for Mn treated and control group, P < 0.01 and

12 Summary

37.8 vs. 32.6 for Cu treated and control group; P = 0.03). Additionally, HV of osteons from base to crown was greater for adults Cu treated (HV = 29.4) than for control group (HV = 25.3; P = 0.04) physiological exhaustion of HV was lower in deer Mn treated than control deer.

Trial 4 addressed the hypothesis that Cu supplementation of gestating and lactating hinds could affect calf growth from birth to weaning and lactation traits (milk production and composition). A total of 17 hinds fed with a balanced diet was used to study the effects of Cu injections of hinds on offspring growth from birth to the end of lactation (forced weaning at 18 weeks of lactation), milk composition and milk yield. Experimental hinds (n = 9) were injected every 42 days from day 202 of gestation (as average) to the end of lactation with 0.83 mg Cu per kg body weight and compared to a control group (n = 8) injected with a physiological saline solution. All hinds were milked from week 2 to end of lactation and the effect of lactation week was also studied. Female calves grew faster from 14 to 18 weeks of lactation when their mothers were supplemented with Cu (0.319 vs. 0.231 kg/day; P = 0.047, for Cu supplemented and control group respectively). However, total milk yield tended to decrease (P = 0.07) with Cu supplementation. In addition, milk from hinds supplemented with Cu had higher content of K and Rb and lower content of Al than milk from control hinds.

In trial 5 we supplemented hinds to assess if Mn modified milk yield and composition and, in turn, influenced calf growth. A total of 20 Iberian red deer hinds fed with a balanced diet were used and milked between week 2 and 18 of lactation (forced weaning by physical separation). Experimental hinds (n = 12) were injected weekly with Mn (2 mg Mn/kg body weight) from day 140 of gestation (as average) until

13 Summary the end of lactation to compare with control hinds (n = 8) that were injected with a physiological saline solution. Serum Mn content of hinds was assessed just before the first Mn injection and at week 10 of lactation. Calves whose mothers were injected Mn tended (P = 0.07) to have higher relative average BW gain during lactation compared to calves from control hinds. In addition, Mn supplementation increased daily milk production by 10.2% (P < 0.05), milk fat content by 11.2% (P < 0.001) and total fat yield by 18.2% (P < 0.05). Milk from hinds supplemented with Mn had more Ca and P but lower Na content than milk from control hinds. Manganese supplementation did not influence Mn serum content at week 10 of lactation, but increased its content in milk by 18.3% (P < 0.001).

Finally, in trial 6, due to osteophagia (bone consumption) is a common behavior among deer, we evaluated and analysed the evolution of antler consumption among the Iberian deer during key periods. In this sense, we aimed to study the seasonality in antler consumption of Iberian red deer and to assess the differences among sex/age classes. For this, in a game reserve located in South- Eastern Spain, antlers were offered to free-ranging animals, and their chewing behavior was recorded with camera traps. Males showed greatest peak of consumption at the end of antler growth (P < 0.001); females showed the greatest peak at the beginning of the lactation (P < 0.001) and calves after delivery of the newborn (P < 0.001).

In summary, the results obtained in this thesis suggest that in red deer supplementation with Cu and Mn play an important role during antler growth in the males and during lactation period in the hinds. In

14 Summary addition, antlers are consumed as natural mineral resources supplementing, mainly Ca and P, during key periods of deer.

GRAPHICAL SUMMARY

Graphical Summary

Artificial mineral supplementation: Positive effects

Adults

No effects in this thesis

Yearling

Growth Milk

Gestating- + Growth Lactating hinds

Natural mineral supplementation (osteophagia): Consume of antler

Males Hinds Calves

Antler mineralization Gestation Milk depletion

Ad hoc

Anonymus

Chapter I JUSTIFICATION

Chapter I

One of the main species of big game not only in Spain (MAPAMA, 2017), but rest of Europe as well, is the deer (Milner et al., 2006; Apollonio et al., 2010). Particularly, in Castilla-La Mancha (Spain), hunting activity has a great relevance and is not limited to only a sport. In fact, generates business activities which have a great socioeconomic impact on the region. According to data from the Junta de Comunidades de Castilla La Mancha (2016), hunting generates more than 6,500 of direct jobs and a total of 1.7 million indirect of jobs annually, with a revenue of 600 million euros per year.

Although in some countries deer is reared for its meat (Apollonio et al., 2010), the main value of the deer in Spain resides in its antlers which are prized as a trophy when deer are hunted. Antler quality is influenced by the mineral nutrition received in both adult and lactating calf stages (Gómez et al., 2006, 2008, Landete-Castillejos et al., 2010a), but also by mineral profile of antler (Landete-Castillejos et al., 2007a,b, 2010a). Therefore, it is important to specify the influence of specific minerals on calf growth and antler quality (mechanical and histological properties) when these minerals are increased in deer diet. Moreover, due to red deer males require not only dietary minerals, but also mobilization of minerals from the skeleton via blood during the antler growth (Baxter et al., 1999) it is also interesting to evaluate the behaviour of serum minerals respect to an increment of minerals in deer diet.

In deer, minerals are needed primarily during the key periods of high physiological demand (called in this thesis key periods), i.e. antler growth in males, the last third of gestation lactation in females and calf growth (Oftedal, 1985; Atwood and Weeks, 2003; NRC, 2007). Therefore, mineral supplementation during these periods might be

23 Chapter I

beneficious for deer, decreasing physiological effort. Despite rut period is also a critical period for deer males (Yoccoz et al., 2002; McElligott et al., 2003; Forsyth et al., 2005), this thesis did not supplement any mineral during rut because it could be a risk for our group since deer present high aggressiveness during this period (Lincoln et al., 1972). The minerals most studied in deer, respect to key periods are calcium (Ca) and phosphorus (P; Banks et al., 1968; Baxter et al., 1999; Gallego et al., 2006; NRC, 2007). However, trace minerals such as copper (Cu) and manganese (Mn) are also indispensable for ruminants as deer (McDowell, 2003) and their study may be interesting for key periods of deer.

The choice of these two minerals is due to its effects on osteogenesis (related to antler growth) and lactation demonstrated in the literature. In this sense, Cu is important in deer because it contributes to maturation of collagen (Prohaska, 2006), which is 34-45% of the antler (Ullrey, 1983; Pathak et al., 2001) and its deficiency causes bone disorders such as osteochondrosis (Wilson et al., 1979; Thompson et al., 1994; Audigé et al., 1995) and bone fractures (Johnson et al., 2007). Copper also take part of antioxidant enzymes and stimulates the immune system (Suttle, 2010). To date, studies on Cu supplementation in deer have mainly aimed to increase this mineral in the serum or liver of these animals in order to avoid the consequences of their deficiency (Harrison et al., 1989; Grace and Wilson, 2002, Grace et al., 2005, Handeland et al., 2008). Furthermore, in other ruminants as bovines, it has been shown that Cu supplementation results in an increase of milk protein (Osorio et al., 2016) and, therefore, better growth for calves (Martin, 1984). However, there is no previous evidence of the effects of Cu

24 Chapter I

supplementation on the composition of the mature hard antler quality or on the quality of milk in hinds.

On the other hand, Mn is very relevant for deer and other ruminants since it is responsible for the maintenance of cartilage, lipid metabolism (Suttle, 2010) and its deficiency causes damage in antlers (Landete-Castillejos et al., 2010a). Moreover, it has been reported that supplementation with Mn in red deer males produces an increase in antler quality (Cappelli et al., 2015). Just like the Cu supplementation, there is a background of Mn supplementation involving increased milk production in bovids (Hackbart et al., 2010), which could increase the offsprings growth rate (Martin, 1984). Although the effects of Mn supplementation on the antler quality have already been evaluated by Cappelli et al. (2015), the effects on the hardness of osteons and milk quality in deer is unknown, and this is the reason why Mn supplementation is studied in this thesis.

Thus, this thesis studied the effects of parenteral administration of Cu or Mn by subcutaneous injections (to ensure the necessary dose for each animal) and its effects on the quality of antlers and serum in males, the quality of milk in hinds and growth in calves, feed with a balanced diet. The use of balanced diet is justified by the study of L'abbé et al. (2003). These authors suggested that, at least in humans, a contribution of micro-nutrients above established standard levels could provide benefits beyond expectations due to their low concentration. Based on this, we expected that Cu and Mn supplementation may increase quality of antler and quality of milk as well calf growth.

25 Chapter I

In addition to this, the current thesis also aimed to evaluate and analyse the evolution of cast antler consumption (osteophagia) for the Iberian deer during key periods (antler growth, gestation and lactation). This research was due to a controversy found in literature respect to the origin of this common behaviour in deer. While Krausman and Bissonette (1977), Bowyer (1983) and Cáceres et al. (2011) maintained the hypothesis that this behaviour was the result of a lack of phosphorus and calcium in diet, other authors (Sutcliffe, 1977, Wika, 1982; Barrette, 1985) suggested that this behaviour was due to increased mineral needs during lactation, gestation and antler growth. However, any study has been carried in deer. Therefore, we designed a trial by videotramp to monitoring antler composition and clarify the origin of this behaviour in Iberian red deer.

Posside sapientiam, quia auro melior est

Proverbia 16:16, Vulgata

Antler Growth

Chapter II INTRODUCTION

Chapter II

1. General aspects of Iberian red deer

The Iberian red deer (Cervus elaphus hispanicus, Hilzheimer 1909) belongs to the kingdom Animalia, filo Chordata, class Mammalia, subclass Theria, order Artiodactylia, suborder Ruminantia, family Cervidae, sufamilia Cervinae, genus Cervus, species Cervus elaphus and, apparently, (there is controversy on its real existence) to the subspecies Cervus elaphus hispanicus. This subspecies, would be formed by deer originating specifically from the Iberian Peninsula which, according to some authors, is different from the rest of deer in Eurasia and North Africa (Geist, 1998). Basically, it is distinguished by its smaller size, brown coloration and smaller skull (Bützler, 1986; Carranza, 2002). Moreover, Carranza et al. (2016) based on mitochondrial DNA, establish the existence of two linages of Iberian red deer (western Spanish population and central Spanish population) in Iberian Peninsula, differenced from other red deer subspecies. Nevertheless, other authors do not consider Iberian red deer as a linage genetically different from European wester red deer population (Ludt et al., 2004; Sommer et al., 2008; Zachos and Harlt, 2010) or from Sardinian/Corsica red deer population (Skog et al., 2009).

The Iberian red deer has a large sexual dimorphism (Whitehead, 1993) with respect to weight (Delibes, 1990; Soriguer et al., 1994; Gaspar-López et al., 2009), with males being heavier and larger than females. In addition, as in almost all cervidae (except for the reindeer, Rangifer tarundus Linnaeus, 1758), males are characterized by the presence of antlers that are renewed annually (Gaspar-López et al., 2008; García et al., 2010). With regards to social behaviour, they organize themselves into groups separated by sex for much of the year. However,

29 Chapter II during reproduction season, heterosexual groups are formed because of the approach of males to the areas of where the females are, and they form harems (Recuerda, 1984). The primary source of feed of wild deer feed are pastures and consumption of low-scrub vegetation (Rodríguez- Berrocal, 1978; Patón et al., 1999; Garín et al., 2001; Fernández-Olalla et al., 2006), which varies according to their seasonal appetite (Blaxter et al., 1974; Estévez et al., 2010).

30 Chapter II

2. Key periods in deer

In general, during the life cycle of the animals, periods with high nutritional requirements occur because of high physiological demands (Clark, 1934; Moen, 1978; NRC, 2007). In the case of deer males, there are two critical periods that require physiological efforts. One of them is the annual cycle of growth of the antlers (Muir et al., 1987; Baxter et al., 1999; Gómez et al., 2012; Ceacero et al., 2016), and the other is rut during matting season (Yoccoz et al., 2002; McElligott et al., 2003; Forsyth et al., 2005).

Females have two periods of great investment: gestation and lactation (Clutton-Brock et al., 1982b; Festa-Bianchet et al., 1998). In fact, the nutritional requirements of pregnant and lactating deer increase between 17-32% and 65-215%, respectively, compared to non-breeding females (NRC, 2007). Similarly, the offspring, also called calves (Clutton-Brock et al., 1982a,b), require a lot of energy for growth (Wilmshurst et al., 1995; Fisher, 1997; Villalba and Provenza, 1999), which is positively related to survival (Blaxter and Hamilton, 1980; Milne et al., 1987) and thus, with the possibility of reaching adulthood and developing the antlers.

2.1. Key period in males

2.1.1. Antler growth

Antlers are sexual character exclusive to male deer (Lincoln, 1992). Therefore, current thesis will characterize external and internal morphology before describe antler growth and development.

31 Chapter II

-Structure: externally, the morphology of the adult deer antler (Cervus elaphus) presents different parts. According to Brown (1990), from the base to the apex, we can differentiate the following structure (Figure 1):

Crown

Trez tine Main beam

Bez tine

Burr Brow tine

Figure 1. External morphology of antler from an adult red deer (source: own elaboration).

- Burr: thickening in the basal area of the antler.

- Main beam: is the main axis of the antler, from the base to the apical area.

- Brow tine: it is the first tip that protrudes from the main beam.

- Bez tine or second tip: point that is just above brow tine.

- Trez tine, tine in the middle of the length of the main beam.

- Crown: is in the most apical area and is characterized by a branch of the main branch.

As antlers are bone formations (Currey et al., 2009), have different internals structural levels (Figure 2). At first glance, the antler is composed by a peripheral zone of compact bone, also called cortical

32 Chapter II bone, and by a spongy internal zone called trabecular bone (Rolf and Enderle, 1999; Price et al., 2005; Kierdof et al., 2013).

Antler 30cm

Cortical Trabecular Bone Bone

2 cm (N/ 1 mm 2 cm (N/ 2 Osteon m Hydroxyapatite (≈50 x 25 x 2 nm) OsteincStr Boneess ) Collagen Collagen 100-200 µm Fiber Molecule (≈5 µm) (≈ 300 x 1,5 nm) 1mm

Figure 2. Schema showing the internal structure of the antler from an adult deer. On the surface, antler is formed mainly by cortical bone in the external zone and trabecular bone in the inner zone. In microscopic view, the cortical bone consists of osteons that are structures of concentric tubes formed by collagen fibres and hydroxyapatite crystals aligned with collagen molecules (source: modified from Chen et al., 2008).

At the microscopic level, (Figure 2) in the cortical bone, osteons are differentiated, which are concentric laminar tubes along the longitudinal direction of the antler where there are large vascular channels (Currey, 1982). These concentric rings have hydroxyapatite

33 Chapter II

crystals, Ca10(PO4)6(OH)2, embedded in the collagen fibers (Rho et al., 1998; Weiner and Wagner, 1998).

- Development of the antler: follows an annual cycle and requires an enormous physiological effort (Linclon, 1992; Baxter et al., 1999; Gómez et al., 2012). This cycle can be divided into three stages: growth, mature stage (when it can be used as a weapon) and drop of antler.

Antler growth begins immediately after antler casting, usually mid-March in the northern hemisphere and it is due to hormonal changes (Suttie et al., 1992) induced by photoperiod (Bubenik et al., 1991; Sempere et al., 1992; Price et al., 2005). This growth begins with permanent protrusions of the skull that are called pedicles (Ullrey, 1983; Kierdorf et al., 2013). In Iberian red deer, the first antler begins to develop between the seventh (Gómez et al., 2006) and tenth month of life and consists of a simple beam with a single peak of about 40cm in length (Gaspar-López et al., 2008). These animals are called spiker or yearling (Bronw, 1990).

During its growth, antlers are a live tissue that contains low amounts of mineral and high amount of protein (Ullrey, 1983; Sunwoo et al., 1995), mainly collagen (Jeon et al., 2011, 2012). The antler growth is characterized by a highly vascularized cartilaginous tissue full of nerve endings, covered by what is called a velvet (which is a hairy epidermis), beneath which the minerals are deposited (Goss, 1983). Once the antler has completed its mineralization, growth stops as consequence of increase of testosterone (Price et al., 2005). In the Iberian red deer, the mineralization lasts approximately 135 days (Gómez et al., 2013).

34 Chapter II

Mineralization of the antler occurs at two levels: (i) in length, through endochondral ossification and (ii) in thickness, by intramembranous ossification (Banks et al., 1974; Muir et al., 1988; Brown, 1990; Li et al., 2005; Price et al., 2005). In the endochondral ossification, cartilage and then bone tissue are formed firstly by the deposit of mineralized material within the cartilaginous matrix and not by osteoclastic resorption (Price et al., 2005), whereas in intramembranous ossification, the bone of the antler is formed directly by osteoblasts (Rolf and Enderle, 1999). In the Iberian red deer, their antler has different phases of mineralization (Gómez et al., 2013). In the first phase, from day 28 to day 70 of antler growth, the scaffold and the lamellar bones are mineralized. Later, between days 70 and 130 after the growth of the antler, the osteons are mineralized (with a maximum of mineralization at day 100).

Once the growth is complete, the males rub their antlers against trees and/or shrubs to loosen and get rid of the skin surrounding the bone formation. In the case of the Iberian red deer, this occurs between days 135 and 163 of growth (Gómez et al., 2013). This process is called velvet shedding (Brown, 1990) which happens between August and September for adults of 3 to 8 years old, confirming the established average of Gaspar-López et al. (2010) at 160 days of duration for the growth of the antler in Iberian red deer.

The end of velvet shedding gives place to the phase of the mature antler, in which the antlers are completely developed and without velvet (Ullrey, 1983). For the Iberian, it usually lasts from the beginning of September until the middle of March (Gómez et al., 2013). During this phase, the antlers are considered as inert bony formations (Currey et al.,

35 Chapter II

2009) and their composition is more mineral than protein (Pathak et al., 2001; Jeon et al., 2011, 2012).

The last phase of the cycle of the antler is characterized by the natural drop of the antler, usually known as antler casting, which is produced by the decrease of testosterone synthesis (Linclon et al., 1992). The fall of testosterone activates the last layer of living osteoclasts that destroys the bone in a horizontal layer (Li et al., 2005). According to Gaspar-López et al. (2010), testosterone is lowest during antler growth and increases when mating period approaches, interrupting the growth of the antler and inducing its ossification and cleaning of the velvet. In addition, a drop of testosterone during the mating season positively affects both the weight in the recovery during the following spring and the size of the antler of the next year. Once the antler has been detached, it is called the cast antler (Brwon, 1990). In the case of adult Iberian red deer, the antlers are usually cast in the middle of March (Gómez et al., 2013). From this moment, the antler begins a new cycle of growth.

2.1.2. Rut

The approach of mating season (rut) in deer males is associated with a weight gain that increases the ability of fighting against other males (Clutton-Brock, 1982). Therefore, antlers have a relevant role during the mating season (September-November, Carranza, 2011). During this time, the males also emit a sound (bellowing) that can intimidate other males to avoid constant fights (Clutton-Brock and Albon, 1979). However, two males may decide to face each other and test their strength by knocking at each other’s antlers. In this case, males with the larger antlers will be more successful in combat and, therefore,

36 Chapter II may have greater access to females and the acquisition of resources (Clutton-Brock et al., 1979; Barrette and Vandal, 1990). In rut, deer males experience a strong decrease in food intake (Rhind et al., 1998) that force them to mobilize their body reserves, causing an intense weight loss of almost 25% (McMahon, 1994), which is traduced in a physiological effort during matting season (Yoccoz et al., 2002; McElligott et al., 2003; Forsyth et al., 2005). Rut period in Iberian red deer is concomitant to an increase of testosterone levels in serum (Goss 1968; Suttie et al., 1984; Gaspar-López et al., 2009) that increase the aggressiveness in the individuals (Lincoln et al., 1972).

2.2. Key periods in females

2.2.1. Gestation

Gestation period is defined as the time from conception to parturition (Scott et al., 2015). Fetal growth occurs by the transfer of nutrients from the mother throughout the placenta (Rosso, 1975). In the Iberian red deer, it lasts approximately 230-240 days (García et al., 2006) and parturition usually occurs between May and June months (Soriguer et al., 1994), coinciding with the most adequate period of survival for offspring (Lincoln et al., 1980; Loudon et al., 1984).

Gestation is governed by hormonal factors such as estrogen and progesterone (Hutchins et al., 1996). However, other factors such as nutrition (Asher et al., 2005) and body condition (Henshaw, 1988) also have influence on gestation. In fact, gestating hinds that during the costliest period during gestation, (the third third of gestation, Oftedal, 1985), have nutritional deficits, lengthen gestation without compromising birth weight, reducing the rate of fetal growth (Asher et

37 Chapter II al., 2005; Scott et al., 2013), whereas reabsorb of fetus is probably in hinds that do not reach a good body condition at the end of winter (Henshaw, 1988). Therefore, having optimal conditions during gestation is essential for reproductive success in deer (Kelly and More, 1977; Brelurut et al., 1990, White, 1992; Stewart et al., 2005). In fact, a good fetal is reflected in a good birth body weight, increasing the calves’ survival (Gaillard et al., 1997; Audigé et al., 1998; Bowyer et al., 1998; Keech et al., 1999; Barten et al., 2001).

2.2.1. Lactation and calf growth

Milk satisfies all nutritional requirements of offspring in addition to promoting immune competence and endocrine maturation in the newborn (Goldman, 2002; Bösze, 2008). In red deer, lactation period usually lasts about 16-18 weeks (Clutton-Brock et al., 1982 a, b). However, if mothers and calves are kept together and they are not mated again, it has been shown that lactation can reach at least 34 weeks (Landete-Castillejos et al., 2000a).

According to Robbins et al. (1987) and Landete-Castillejos et al., (2000a) milk of deer is composed mainly of fat (6-11%), protein (6- 10%), lactose (3-6%), and the percentage of dry matter is 15-27%. Among factor affecting the composition and production of deer milk, nutrition is key (Clutton-Brock et al., 1982a; Oftedal, 1985; White, 1992; Landete-Castillejos et al., 2003a), due to females produce milk mainly from daily ingestion and not from body reserves (Sadleir, 1987). However, fat reserves can be mobilized to milk to compensate food deficiencies (Landete-Castillejos et al., 2003a). Other factors affecting the composition and production of deer milk are the date of calving

38 Chapter II

(Landete-Castillejos et al., 2000a), the sex of calves (Landete-Castillejos et al., 2005; Gallego et al., 2006), the age of females (Landete-Castillejos et al., 2009), the weight of females (Landete-Castillejos et al., 2005) and their social status (Landete-Castillejos et al.,2010b).

Lactation is critical for the physiological development of calves (Matiello, 2009). In fact, milk is particularly important for calf growth, even when calf eats other foods in addition to milk (Arman, 1974; White, 1992). Among the components of milk, protein is the most influential for growth as it is directly involved in calf growth (Landete-Castillejos et al., 2001; Asher et al., 2011), as well in the growth of future antlers (Gómez et al., 2006).

39 Chapter II

3. Nutritional components in the deer diet. The importance of minerals

To successfully overcome these key periods mentioned before, animals must meet all nutritional requirements, i.e. meet the needs of energy, protein, vitamins and minerals (Wilmshurst et al., 1995, 1999, Allen and Oftedal, 1996; Nicol and Brookes, 2007; NRC, 2007). Therefore, in this section we will describe the main components of nutrition with special emphasis on minerals.

3.1. Energy

The main energy components that can be found in the diet are carbohydrates (in green plants and fruits) and fats (seeds). Fats are mainly in the form of triglycerides whereas carbohydrates are in the form of simple sugars (glucose, fructose), complex sugars (disaccharides and oligosaccharides) or complex polymerized compounds (cellulose and hemicellulose). The latter one is the fiber which is fermented by the bacterial flora of ruminants producing volatile fatty acids, which enter into the energetic metabolism of ruminants (Allen and Oftedal, 1996; NRC, 2007). Metabolizable energy requirements vary to weight, sex and physiological state. Thus, for a 100 kg male deer during antler growth requires 6.9 Mcal/day, while a female of the same weight would need 8.7 Mcal/day (NRC, 2007).

3.2. Proteins

Proteins are mainly composed by amino acids. In mammals, most of the protein requirements are due to the requirements of essential amino acids that cannot be synthesized. In the case of ruminants such as

40 Chapter II deer, essential amino acids are obtained from the ruminal flora (Allen and Oftedal, 1996). Nonetheless, deer need consume proteins for maintenance that involves losses of N by feces and production that include body growth, antler growth and lactation and pregnancy (NRC, 2007).

3.3. Vitamins

Vitamins perform essential and specific functions for animal maintenance and health. They are divided in two categories: water soluble and liposoluble. Among the water-soluble vitamins are thiamine

(B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), folic acid (B9), cyanocobalamine (B12) and ascorbatic acid (C) while among liposoluble vitamins are retinol (A), cholecalciferol (D3), tocopherol (E) and phylloquinone (K). Rumen bacteria can synthesize vitamin K and all water-soluble vitamins except vitamin C (Allen and Oftedal, 1996). In deer, retinol seems to be key to regenerate antler (Kierdorf and Bartos, 1999; Allen et al., 2002).

3.4. Minerals

Minerals are chemical elements that cannot be synthesized by deer, thus must be consumed in the diet. Minerals can be divided into two categories depending on their concentration in the tissues and/or the amounts required in the diet. In this sense, macrominerals are found in the body at amounts greater than 5 g and are required at concentrations greater than 100 ppm while trace minerals or microminerals are below 5 g in body and are needed in diet below 100 ppm (McDowel, 2003).

41 Chapter II

3.4.1. Types of minerals a) Macrominerals

- Calcium (Ca): Ca is the most abundant mineral in the body of vertebrates and 99% is fixed in the skeleton (Suttle, 2010). In deer, it has been shown to be key both for antler development in males (Muir et al., 1987; Dryden, 2016) and during lactation in females as Ca is the most abundant mineral in hind milk (Gallego et al., 2006, 2009). As milk minerals of hinds are especially important, they are treated separately in the apart 3.5.5.

- Phosphorus (P): P is the second most abundant mineral in the animal body and approximately 80% is found in the bones as hydroxyapatite (Suttle, 2010). In deer, P is present mainly as hydroxyapatite crystals that form in antlers (Estévez et al., 2009; Dryden, 2016) and is the second most abundant mineral in hind milk (Gallego et al., 2006, 2009).

- Magnesium (Mg): Most Mg of animal body (60 to 70%) is present in the skeleton (Suttle, 2010). However, only 3% of the skeleton is made up by Mg (Doyle, 1979), influencing mechanical properties (Robey and Boskey, 2003). Despite its importance, it has been shown that red deer prefer to consume plants with low amounts of Mg (Ceacero et al., 2015b).

- Potassium (K): K is found as intracellular ion in tissues. It contributes mainly to the regulation of the acid-base balance muscles, contraction and breathing regulation throughout the change with Cl ions (Suttle, 2010). Also, K supplementation may improve bone metabolism

42 Chapter II in mammals by increment bone mass (He et al., 2010). In deer, K is really important for antler since can account for 40% of the weight of the antler (Landete-Castillejos et al., 2007a).

- Sodium (Na): Na is essential for the maintenance of osmotic pressure, regulates the acid-base balance and controls the balance of corporal moisture (Suttle, 2010). In deer, during lactation, 48% of Na intake is required for milk production, thus a large amount (72 g) are recommended during this period (Pletscher, 1987). In deer, its deficiency can be detected by examining the mineral content of the antler (Landete- Castillejos et al., 2007a).

- Sulfur (S): S is present in molecules that provide the spatial configuration of polypeptide chains, takes part of the glutathione that protects the tissues from oxidants, and is present as sulfate (SO4), forming complexes with chondroitin and heparin (Suttle, 2010). In deer, it can become toxic, so they select diets avoiding foods rich in S (Ceacero et al., 2015b). b) Trace minerals

- Aluminum (Al): Al is considered as a toxic element that can cause osteomalacia when renal absorption or excretion is affected (Riihimäki and Aitio, 2012; Han et al., 2013; Willhite et al., 2014). Although there is an absence of literature regarding the toxic effects of Al on deer, Zimmerman et al. (2008) proposed that concentrations of Al of 0.46-1.41 mg/kg in the liver can be considered as normal quantities for males and females of red deer.

43 Chapter II

- Borom (B): B improves the mechanical properties of bones (Armstrong et al., 2002) and may influence the metabolism of regulation of Ca, P and Mg (McDowell, 2003). In the studies of Landete-Castillejos et al., (2010a) and Cappelli et al. (2015) studied the concentration of B in red deer antlers as well as their behavior regarding the concentration of Mn in the diet. However, no clear relationship between B and the mechanical properties of the antler has been established.

- Cobalt (Co): Co is not directly needed by ruminants but it is required by rumen bacteria for vitamin B12 synthesis (Kennedy et al., 1996). In fact, in deer, the role of Co has been studied mostly in relation to vitamin B12. In the studies of Beatson et al. (1999), a linear relationship was established between serum Co and liver Co measured as vitamin B12 concentration in males. Clark et al. (1986) determined that

Co supplementation as vitamin B12 in deer did not lead to weight gains despite its increment in serum.

- Copper (Cu): Cu is very important for bone tissue formation, since it is a cofactor of lysil-oxidase enzyme (Suttle 2010) that forms the collagen (Prohaska, 2006) of the connective tissues, such as bone and cartilage (Ham et al., 1975). Studies carried out in deer showed that Cu deficiency might cause bone disorders (Thompson et al., 1994), ataxia (Mackintosh et al., 1986) and broken antlers (Johnson et al., 2007) and its supplementation increase body weight 6–10 kg (Harrison and Familton, 1992; Ellison, 1995). On the other hand, Cu takes part of antioxidant enzymes such as Superoxide Dismutase I that is found in the cytosol and improves the immune system (Tian et al., 2002), therefore its deficiency also affects antibody production and phagocytosis (Boyne and Arthur, 1981; Suttle and Jones, 1986). In consequence, Cu

44 Chapter II deficiency in deer may produce a higher incidence of infectious diseases (Grace and Wilson, 2002). In addition to this, it is known that Cu is distributed among body tissues and the liver is an important storage organ (Booth et al., 1989). In this sense, the study of Reid et al. (1980) showed that liver Cu concentrations in the foetus/ neonate of deer are much greater than those of the hind (5650 vs 166 μmol/kg fresh tissue). In fact, Cu is transported across the placenta and increases foetal liver Cu stores, which results in Cu concentrations of the foetus/ neonate being much higher than those of their dams (Grace and Wilson, 2002). Occasionally, according to Davis and Mertz (1987), Cu absorption can be reduced as a consequence of high levels of S and molybdenum (Mo) in diet, thus, a Cu:Mo ratio of less than 4: 1 can generate problems in Cu absorption (Miller et al., 1988).

-Iron (Fe): By 60% is in hemoglobin, but also is necessary for catalase, that is the major antioxidant enzyme (Suttle, 2010). Although there is little information about the role of Fe in deer, according to Gutowska et al. (2009), in roe deer, it seems that Fe is responsible for the accumulation of other minerals such as Cu and Zn in the antler.

- Iodine (I): I is mainly related to the synthesis of thyroid hormones (Behne and Kyriakopoulos, 2001). Iodine deficiency produces goiter in deer calves (Grace and Wilson, 2002), causes a decrease in milk production in hinds, and, can affect the growth of the antlers in stags (Bubenik et al., 1987). Despite the importance of I in deer, this mineral has not been included in neither the study of the antlers nor of the milk as the types of analysis used for the determination of I could not be obtained.

45 Chapter II

- Manganese (Mn): Mn is a cofactor of several enzymes, including manganese superoxide dismutase II (MnSOD), which is the most powerful antioxidant enzyme in cells that neutralizes free radicals generated by environmental toxins and metabolism in mitochondria, in which it is found (Suttle, 2010). In addition, Mn is responsible for the maintenance of cartilage, pancreas, liver and reproductive functions. It also regulates the metabolism of carbohydrates and lipids (Li et al., 2011). Previous studies in deer male have been focused on the positive effect on the antlers of Mn supplementation (Cappelli et al., 2015; Bao et al., 2017). Although the reduction of Mn content of the antler bone increase frequency of antler breakage in red deer (Landete-Castillejos et al., 2010a). In the case of pedicles, Kierdorf et al. (2014) suggested that lower content of Mn in pedicles compare to antlers is related to higher degree of mineralization and lower proportion of organic matrix in pedicles and thus, pedicles and antlers behaving differently respect to Mn content. Additionally, Mn is also required for gestation of ruminants as deer and contributes in the health of the fetus (Suttle, 1979). Although Mn seem to be important to deer toxic effects of Mn on deer has been described. The studied carried out by Wolfe et al. (2010) showed a positive association between liver Mn and probability of infection of chronic wasting disease (CWD) status in mule deer. In this sense, Lavelle et al. (2014) suggested also in deer that soil consumption with high concentrations of Mn could increase infection of CWD.

- Selenium (Se): This element takes part of the selenoproteins that protect against oxidizing effects (Köhrle et al., 2000). In male deer, it has been demonstrated that Se supplementation did not result in weight gain (Mackintosh et al., 1989; Grace et al., 2000), and in hind, it has been

46 Chapter II reported that Se is secreted in milk during lactation, in direct relation to its intake (Grace and Wilson, 2002).

- Silicon (Si). The role of this mineral is unknown accurately (NRC, 2007), but has been shown to be involved in bone calcification (Seaborn and Nielsen, 2002; Nielsen and Poellot, 2004). In deer, Cappelli et al. (2015), injecting Mn to stags fed with a balanced diet, showed a reduction in Si concentration as a consequence of Mn supplementation.

- Strontium (Sr): Although the role of Sr in the organism is not exactly known, it seems to have a beneficial effect on bones. In fact, Sr supplementation increases the number of osteoblasts (Canalis et al., 1996) improves bone strength (Ammann et al., 2004). In cervids, the role of Sr is not very clear, and studies involving Sr and deer are related to the use of antler as biomonitors of Sr-90 contamination (Schönhofer et al., 1994; Tiller and Poston, 1999).

- Zinc (Zn): Zn is essential for transcription factors (Beattie and Kwun, 2004; Cousins et al., 2006), alkaline phosphatase, enzyme key during antler mineralization (Maki et al., 2002; Robey and Boskey, 2003) and the Superoxide Dismutase I (Tian et al., 2002). In red deer, it has been observed that antler Zn concentration is related to its porosity and, therefore, with its mechanical properties (Landete-Castillejos et al., 2012c).

47 Chapter II

3.4.2. Mineral requirements of deer

The knowledge about the mineral requirements for deer in Mediterranean environments is scarce (Ceacero, 2010). However, studies for the most important minerals carried out by Blaxter et al. (1988) in C. e. scoticus in Scotland, by Brelurut et al. (1990), in C. e. elaphus in France and by Grace and Wilson (2002) in New Zealand determined some roles important in the deer diets (as shown in Table 1). Ceacero et al. (2009), the only the study in Mediterranean conditions corroborated that the deficiencies established by Blaxter et al. (1988) for deer in Scotland could be applied for Mediterranean environments too. Since no studies under Mediterranean conditions have been carried out to establish the maximum levels of tolerance and deficiency for most minerals, recommended levels for sheep and goats are frequently suggested for cervids (Ceacero, 2010).

Table 1. Mineral requirements in deer diet (modified from Ceacero, 2010) Mineral Maximun Marginally Element requirements in tolerance level Deficient level deficient level total diet a in total dietb Ca (g/kg) 5.0-7.7 20 <3.0c 3.0-4.9 P (g/kg) 3.5-5.0 6 <2.5c 2.5-2.9 Mg (g/kg) 2.0 5 <1.5c 1.5-1.9 K (g/kg) 6.5 30 <6.0c 6.0-6.4 Na (g/kg) 0.7 35 <0.6c 0.6-0.7 Cu (mg/kg) 15 20 <4b 4.0-14.9 Fe (mg/kg) 50 500 <30c 30.0-49.9 Mn (mg/kg) 35 1000 <20c 20.0-34.9 Se (mg/kg) 0.3 4 <0.06c 0.10-0.29 Zn (mg/kg) 35 750 <25b 25.0-34.9 aRecommended mineral requirements by Blaxter et al. (1988), Brelurut et al. (1990) and Grace and Wilson (2002).bSince information is not available in deer, recommended levels for sheep are suggested (McDowell, 2003). cCritical levels for forage mineral content based on the needs of ruminants (McDowell, 2003 and references therein).

48 Chapter II

Mineral requirements vary throughout the life cycle of deer, increases occur in the development of skeletal growth (Robbins, 1993) or due to key periods mentioned previously, i.e antler growth (Ullrey, 1983) or gestation and lactation (NRC, 2007). Due to these periods of high mineral demands, individuals may suffer deficiencies when the amount or balance of minerals in the diet is not adequate (ARC, 1980; Davis and Metz, 1987; Miller et al., 1988; Fontenot et al., 1989). In this sense, during antler growth, it has been established that deer need 1.3- 5.0 g of Ca (Dryden, 2016) between 1.4 and 3.2 g of P, between 1.5 and 1.9 g of Mg, between 5.0-6.5 g of K, between 0.7 and 1.2 g of Na and between 1.5 and 2.5 g of S per kilogram of food consumed (Blaxter et al., 1974; Adam, 1994; Sinclair, 1999). For deer is essential to satisfy these nutritional demands since it has been determined that there is a correlation between the mineral composition of the diet and the mineral composition of the antler, which may have implications in the mechanical properties (Estévez et al., 2009).

3.4.3. Importance of minerals for antler a) Antler mineral content

Minerals are essential for antlers (Landete-Castillejos et al., 2012c). In fact, more than half of the antler (55-65%) are minerals, (Currey, 1990; Pathak et al., 2001; Landete-Castillejos et al., 2007a,b), mainly in the form of hydroxyapatite (Szuwar et al., 1998). The distribution of minerals in the antler is complex (Landete-Castillejos et al., 2007ab). Among macrominerals, the content of Ca and P decreases in the antlers from the base to the top, reflecting, thus, a physiological

49 Chapter II effort (Landete-Castillejos et al., 2007ab, 2012c). These two minerals are extraordinarily important since they account for 37% of the variability in length and weight of the antler and, surprisingly, K alone accounts for 40% of this variability (Landete-Castillejos et al., 2007a).

Another relevant microminerals for the mineral content of antler is Mn. In fact, a deficiency in Mn decreases the fixation of Ca from the skeleton to the antler (Landete-Castillejos et al., 2010a) while Mn supplementation in males of Iberian red deer increases the content of Ca, Na, P, B, Co, Cu, K, Mn and Se, and decrease the Si content in antlers (Cappelli et al., 2015). b) Minerals and mechanical properties of antler

Traditionally, mechanical properties of antler have been associated with macrominerales such as Ca (Wang et al., 1998; Heaney, 2003) and Na (Atwood and Weeks, 2003). However, trace minerals such as B, Si, Mn and Zn can also influence its mechanical or architectural properties in bone tissues (Armstrong et al., 2002; McDowell, 2003; Nielsen and Milne, 2004; Huttunen et al., 2006).

Among trace minerals, Mn seems to be particularly important. On the one hand, a Mn deficiency in the diet produces a reduction of 27% in impact work and of 10% in work peak force (Landete-Castillejos et al., 2010a). On the other hand, Mn supplementation increases the impact work of antlers by 16% (Cappelli et al., 2015). Other important trace minerals are Cu and Zn. In this sense, Cu deficiency increases the rate of broken antlers (Johnson et al., 2007), whereas Zn is associated with porosity of the antlers (Landete-Castillejos et al., 2012c), which

50 Chapter II directly influences in the mechanical properties of bone tissues (Davison et al., 2006). c) Flow of minerals: from the bones to the antlers

Antler growth is of 2-4 cm/day in the common deer (Goss, 1983) and 0.7 cm/day in the Iberian subspecies as average (based on Gómez et al., 2013). The rapid growth of deer antlers, requires a partial demineralization of skeleton, due to the fact that their diet cannot supply the high amount of minerals needed (Meiste, 1956; Muir et al., 1987). This movement of minerals from the bones to the rest of the organism produces osteoporosis (decrease in bone density of the skeleton) that is repeated annually with each growth of the antler, which was demonstrated by Banks et al. (1968) in mule deer (Odocoileus hemionus, Rafineske 1817) and by Baxter et al. (1999) in red deer.

3.4.4. Importance of minerals in serum

Blood serum is the fluid remaining after blood clotting (Randal and Burggren, 1998). Since serum is loaded with minerals (Randal and Burggren, 1998), a decrease in their concentrations could directly affect enzymatic reactions, affecting in turn, a great variety of other biological processes (Carlson and Bruss, 1997) such as antler growth or lactation in deer. Thus, evaluation of trace mineral concentrations can be an important component of assessing deer health (Rosef et al., 2004; Roug et al., 2015). For this reason, the study of serum minerals in deer has been used mainly to diagnose mineral deficiencies (Wilson, 1989; Grace and Wilson, 2002) or to evaluate the utility of mineral serum (including Mn and Cu) as predictors of trace mineral concentrations in other tissues in deer (Roug et al., 2015). In this section and according to literature

51 Chapter II existence we propose reference levels of the mineral concentration serum from red deer in Table 2.

Table 2. Reference concentrations of minerals in serum from red deer.

Mineral Concentration Reference Ca (mmol/L) 1.8-3.2 P (mmol/L) 0.7-3.7 Kucer et al., 2013 Mg (mmol/L) 0.5-1.2 K (mmol/ L) 3-6 Na (mmol/L) 136-150 Cu (µmol/L) 8 Grace and Wilson, 2002 Fe (µmol/L) 0.52-13 Roug et al., 20151 Mn (µmol/L) 0.005* Grace et al., 2008 Se (µmol/L) 46-74** Wilson and Grace, 2001 Zn (µmol/L) 0.05* Grace et al., 2008 1In mule deer (Odocoileus hemionus). *In plasma. ** In blood.

In addition to this, the study carried out by Kuba et al. (2015) shows that serum minerals change according a circannual cycle. These authors suggest that the increases in the concentrations of Ca, P and Mg in deer serum in spring are related to the elevated bioavailability of these compounds in the food and reflect the complementation of the mineral reserves depleted during winter. They also suggest that the decrease in the concentrations of these macroelements between summer and autumn is probably related to the process of antler mineralization.

3.4.5. Importance of minerals for gestation, lactation and calf growth

Gestation and lactation are interesting stages to study the influence of minerals. According to NRC (2007), pregnant and lactating deer need between 1.5 and 2 times more minerals than non-breeding deer. The high demands of minerals during these periods (Okah et al., 1996; Suttle, 2010) produce a bone resorption of minerals

52 Chapter II

(fundamentally, Ca and P) from the mothers bones (Taylor et al., 2009; Eliondo-Salazar et al., 2013). In fact, during pregnancy, a transplacental mineral transfer occurs from the mother to fetus, which, in the case of Ca reaches 150 mg/kg fetal weight (Mughal, 1992). During lactation transference of milk minerals from the mother to the offspring take place (McDowell, 2003; Cashman, 2006), leading a transient osteoporosis (Cross et al., 1995). In fact, Ca, P and Mg needed in ungulate lactation do not depend on feed intake, as it is primarily met by bone resorption (Wysolmerski, 2002). Therefore, production of milk minerals is remarkably costly to mothers (Pletscher 1987). Nevertheless, microminerals seem to be constrained by daily intake (Loudon & Kay, 1984), and thus, the content of trace minerals as Cu and Mn in the diet may influence on their own concentration in milk or calostrum.

. Minerals perform different functions in milk. For example, Na, K and Cl provide constant maintenance of osmotic pressure in milk (Holt, 2011), while Ca, P and Mg take part of casein, which is the main milk protein (Choi et al., 2007; Bijl et al., 2013; Holt et al., 2013; Malacarne et al., 2014). Literature on milk mineral content in red deer is scarce, and published results shows a wide variation among them as is showed in Table 3. Among factors affecting mineral content of milk it has been described the stage of lactation, milk accumulation, feed, genetic variance, birth delay and sex of the offspring (Peaker, 1977; Vegarud et al., 2000; Vergara et al., 2003; Gallego et al., 2006, 2009). In Iberian red deer, milk minerals (except Mg) vary during lactation period but do not change in content greatly with lactation stage except iron and zinc that are increase at the end of lactation (Gallego et al., 2006).

53 Chapter II

Table 3. Milk mineral composition in mg/100 g of common deer according to previous studies (source: modified from Malacarne et al., 2015). Ca P Mg Na K Cl Fe Zn Reference 2201 1801 241 411 1301 711 Arman et al., 1974 269-305 191-208 18-37 23-31 65-141 Krzywinski et al., 1980 262 177 16 45 155 55-89 Csapó et al., 1987 172 90 14 23 86 0,96 Vergara et al., 2003 233 64 14 38 110 0.07 1,25 Gallego et al., 2006 256 191 13.3 39.5 121 0.75 1,30 Gallego et al., 2009 1mg/100 ml

Milk minerals are notably important for calves since mineral requirements are maximum during early growth in lactation (Holt, 1997 Abdelrahman, 2008). This has been showed by Gallego et al. (2009) that found that Ca and P explain 27% of variability in calf growth. In addition, minerals continue being important after weaning. Studies in other ruminants have shown that during growth after weaning, a dietary mineral composition must be increased about 10 times than that reported for adults (in sheep and cattle, Marston, 1970; Kirchgessner et al., 1998).

3.4.6. Osteophagia and its relationship with minerals

Free-ranging deer consume minerals throughout plants or throughout licking rocks rich in essential minerals (Ammerman et al., 1995). Deer can in fact discriminate between the minerals in the diet, therefore they can select the mineral content. According to studies carried out by Ceacero et al. (2010 a, b, c), this selection is shaped by internal physiological efforts affected by nutritional requirements. However, in mineral deficient environments, deer may consume products that, although not common to their diet, provide a large number of these nutrients. For example, it has been documented that deer are able to consume algae (Ceacero et al., 2014), rocks (Lavelle et al., 2014) and bone tissues (Cáceres et al., 2011) to meet their mineral needs. This last

54 Chapter II

feeding behavior is called osteophagia (Table 4), which may occurs as a consequence of the deficiency of P in the body as suggested Denton et al. (1986) for cows.

2017a).

et al., et

. Publish record of osteophagia by cervids species (Gambíncervids species by . osteophagia Publish of record

4 Table Table

55 Chapter II

4. Mineral supplementation in deer

Studies about mineral supplementation in deer can be classified fundamentally into two types: those that study to alleviate the deficiencies in the diet and avoiding associated pathologies, such as the study of Handeland et al. (2008), and those that study the possible benefits of supplementation, such as those performed by Ceacero et al. (2010c) and Cappelli et al. (2015).

In this context, supplementation of minerals can be a challenge in free-ranging animals, such as deer. In order to facilitate the administration of minerals, there are different types of supplements that are released in the short- and long-term (Grace and Knowles, 2012).

4.1. Supplements providing short-term effects

These supplements are the most suitable for farm animals that are frequently handled since should be administered continuously over an extended period of time. In famed animals, the most commonly supplements used are given throughout oral injections by solidified products of free choice (Grace and Knowles, 2012).

4.1.1. Injections

These supplements are usually chemically simple, soluble in water, and absorbed quickly. In addition, the dosage can be controlled in each animal (i.e, no more mineral than food ingested, as in the case of food supplementation). However, this type of supplementation needs special handling as it is better suited for deer on farms with handling vessels, being unpractical for free-ranging animals (Grace and Knowles,

56 Chapter II

2012). In the case of deer, highlighted in the study of Cappelli et al. (2015), it was shown that weekly subcutaneous injections of Mn (200 mg/100 kg body weight) in adult males of Iberian red deer feed with a balanced and non-deficient diet affected positively mechanical properties, mainly impact work and mineral content of the antler (Ca, Na, P, B, Co, Cu, K, Mn, Ni and Se), as well as osteons structure.

4.1.2. Solidified products

These products are usually salt blocks. They are a simple and complete method of providing microminerals as salts of different mineral content (Grace and Knowles, 2012). However, these blocks may serve as a source of contagion for deer (Lavelle et al., 2014). Its has been demonstrated in other ruminants that behavioral factors influence the interest of their consumption, resulting in not all animals receiving an adequate intake (Money et al., 1986). Largely, the underlying principle in the use of these blocks is that deer can detect their own needs and consume minerals accordingly to satisfy them, as demonstrated by Ceacero et al. (2010a,b). In the case of multi-mineral blocks, the ruminants appetite for NaCl) is what would make the animal consume a quantity of other minerals that the manufacturer considers appropriate (Villalba et al., 2008). Another way to supply minerals is in pre-mixes that are mixed with diet. In this case, mineral dose is calculated based on the average daily feed intake of percentage of premixed included in diet. from the amount of food that the deer ingests and the percentage of premix of vitamins and minerals. This type of supplementation has recently been used in the studies of Kun et al. (2016) and Bao et al.

(2017) for Zn and Mn, respectively in Cervus nippon (Temminck 1838).

57 Chapter II

4.2. Supplements providing long-term effects

They are the most recommended for free-ranging animals. Since products act in a prolonged way, there is no necessary to administer the dose repeatedly. In consequence, labour costs and problems arising from frequent handling of animals are reduced. These products tend to be chemically sophisticated and more expensive than fast-acting versions (Grace and Knowles, 2012). In the case of deer, the following have been described.

4.2.1. Subcutaneous and intramuscular deposits injections of long term-effect

These products are usually injected into the anterior region so there is little risk of crawling in the canal. They provide controlled release of trace elements or other micronutrients throughout water- insoluble chemical forms or encapsulation in liposomes or solidified resins (Grace and Knowles, 2012). In deer, only the study by Harrison et al. (1989) evaluated this kind of supplementation, using injections of Cu- EDTA (Ethylene-Diamino-Tetra Acetate). According to their results, these injections provide a rapid increase of Cu concentration, of liver during 12 weeks that could serve to avoid problems associated to Cu deficiency.

4.2.2. Intraruminal bolus

Intraruminal bolus (sometimes called pellets, bullets or capsules) are devices designed to be administered orally and remain in the reticulorumen until their completely disolution. They can be solid metals or resins containing one or more trace elements. In the market, different sizes can be found for different types of livestock or species (Millar and

58 Chapter II

Meads, 1988; Hemingway et al., 2001; Sprinkle et al., 2006; in Grace and Knowles, 2012). In deer, there are several studies that show the effects of mineral supplementation throughout intraruminal boluses on serum and liver (Booth et al., 1989; Harrison and Familton, 1992; Wilson and Audigé, 1998; Beatson et al., 2000; Walker et al., 2002).

4.2.3. Fertilized pastures with minerals

The use of programmed inputs of fertilizer in the pastures can be a vehicle to supplement minerals. Although this practice is extensively in New Zealand with Cu and Se, it is not common in Europe. When animals graze in enriched prairies for 3-4 months, the minerals are absorbed and retained in the body for later use. This type of fertilizer can prevent deficiencies of Cu and Se for, at least, 12 months. However, it presents a number of drawbacks, such as sedimentation and accumulation of minerals in the soil, so animals cannot use these treated pastures during this time (Grace and Knowles, 2012). In deer, only the study by Grace et al. (2005) used this type of mineral supplementation on hinds. The authors supplemented clovergrass pastures with copper sulfate pentahydrate (CuSO4∙5 H2O), showing that the Cu content of serum and liver increased, as well as the weight of these females can be used to avoid problems associated with its deficiency.

Current thesis studied the effects of parenteral administration of Cu by subcutaneous injection of long term effects and Mn by subcutaneous injections of short term effects in to ensure the necessary dose for each animal.

Sapere aude

Horacio

Chapter III OBJECTIVES AND HYPOTHESIS

Chapter III

Based on the importance of minerals and their supplementation above standard reference levels, which could produce benefits antler properties and reproductive traits in deer (Chapters I and II), the first hypothesis of this thesis was as follows:

The supplementation with trace minerals (Cu and Mn) of males and females of Iberian red deer, fed with balanced diet, should positively affect antler quality, the serum mineral composition, the milk quality and the calf growth.

Additionally, since it has been documented that deer can consume antlers (which are rich in minerals) and that the consumption of minerals depends on the physiological needs (Chapters I and II), the second hypothesis of this thesis was that:

The pattern of consumption of a natural source of minerals (antler bone tissue) during key periods (antler growth, gestation and lactation) should increase.

Therefore, to test these hypotheses and to extend the current knowledge about the effects of mineral supplementation and the pattern of consumption in relation to a source of natural mineral supplementation in Iberian red deer in Mediterranean ambience, the following objectives were proposed:

1. To evaluate the effects of Cu supplementation on the structure and mechanical properties and mineral content of the Iberian deer antler bone tissue, both in yearling and adults feed with balanced diet.

63 Chapter III

2. To determine the effects of Cu supplementation on the mineral composition of serum during the antler growth of Iberian deer adults fed with balanced diet.

3. To study the effects of Cu and Mn supplementation on the hardness of Iberian deer antler osteons, both in yearling and adults fed with balanced diet.

4. To evaluate the effects of Cu supplementation of gestating and lactating Iberian red deer hinds fed with balanced diet, on the production and composition of milk as well as its effect on the calf growth.

5. To determine the effects of Mn supplementation of gestating and lactating Iberian red deer hinds fed with balanced diet, on the production and composition of milk as well as its effect on the calf growth.

6. To study the pattern of consumption of cast antler, understood as a natural source of minerals, during the antler growth, gestation, lactation and calf growth in Iberian red deer.

These objectives will allow to determine the extent of the benefits produced by subcutaneous mineral supplementation (Cu and Mn) in Iberian red deer, even if these minerals are found in deer above the established standard levels, as well as reveal the pattern of consumption of a natural supplementation source (antler) in males, females and calves.

Quis, quid, ubi, quibus, auxiliis, cur, quomodo, quando?

Marco Fabio Quintiliano

Chapter IV METHODOLOGY

Chapter IV

All trials from this PhD were carried out in accordance with Spanish legislation regarding the use of animals in research (Boletín Oficial del Estado, 2013) and with the approval of the Ethical Committee in Animal Experimentation at the Universidad de Castilla-La Mancha (UCLM, Permit Number: 1002.04). Handling and sampling were designed to minimize stress, health risks and suffering of deer. Males, hinds and calves were examined daily by farm personnel and weekly by an experienced veterinarian. To develop the six objectives mentioned in chapter III, six trials were conducted in the current thesis.

The first five trials were performed at the UCLM (Universidad Castilla-La Mancha) experimental farm in Albacete, south-eastern Spain (38°57´10´´, N, 1°47´00´´W, altitude 690 m). In these trials, animals feed and water were offered ad libitum throughout whole experimental procedure. The feeding program (diet and unifeed ration) was the same for all deer and met or exceeded the nutrient requirements for deer (NRC, 2007). The ingredients and determined nutrient content (AOAC, 2000) of the diet and unifeed ration are shown in Table 1.

The trial 6 was conducted in a hunting area (3,931 ha) located at an approximate altitude of 1,000 m, between the municipalities of Alpera, Alatoz and Carcelén (Albacete), in Southeast Spain (38°59' N, 1 °15'W). called "Las Dehesas". This zone has a population of 300 Iberian deer, which are fed naturally, without any supplementation and without any antiparasitic treatment. They share the habitat with wild boar (Sus scrofa) and other small herbivores, which compete for food. The vegetation is characterized by the natural pastures, shrubbery and wooden areas as was described in Estévez et al. (2010).

67 Chapter IV

Table 1. Ingredient composition (% dry matter1) and determined nutrient content (% dry matter1) of diet and unifeed ration.

Composition Diet Unifeed ration Ingredient composition Barley 31.3 - Wheat 6.8 - Corn gluten feed, 21% Crude Protein 11.3 - Corn DDGs, 26% Crude Protein 6.8 - Wheat bran, 20% starch 11.3 - Rice bran, 14% ether extract 11.3 - Sunflower meal, 28% Crude Protein 12.4 - Rapeseed meal 00, 34% Crude Protein 5.6 - Molasses, sugarcane 6.8 - Palm kernel expeller 5.6 - Calcium carbonate 2.6 - Salt 0.6 - Vitamin and mineral premix2 0.5 - Oat - 27.6 Alfalfa meal, dehydrated - 48.4 Cereal straw, from barley - 20.8 Citrus pulp, from orange - 13.5 Determined nutrient content Moisture 9.9 30.1 Crude protein 19.4 15.6 Ether extract 3.5 1.2 Crude fibre 12.0 34.9 Ash 11.7 10.1 Ca 2.0 1.6 P 0.9 0.2 Cu (ppm) 28.0 11.8 Mn (ppm) 122.6 30.5 1 Unless otherwise indicated. 2 Supplied per kg of diet: vitamin A (trans-retinyl acetate), 10 000 I.U.; vitamin D3 (cholecalciferol), 2 000 I.U.; vitamin E (all-rac-tocopherol-acetate), 15 I.U.; Mn (MnSO4·H2O), 75 mg; Fe (FeCO3), 50 mg; Zn (ZnSO4·H2O), 115 mg; Cu (CuSO4·5H2O), 7.5 mg; I (KI), 2 mg; Co (2CoCO3·3Co(OH)2·H2O), 0.83 mg; Se (Na2SeO3), 0.22 mg; ethoxiquin, 0.025 mg; BHT (butilhidrotoluen), 0.18 mg; BHA (butylhydroxyanisole), 0.016 mg; sepiolite, 950 mg.

68 Chapter IV

1. Effect of Cu supplementation on antler properties and mineral composition of red deer male (trial 1)

1.1. Experimental design

At the beginning of the trial, deer from experimental farm of UCLM were divided in two groups matched for BW (mean BWs of yearlings and adults were 72.9 and 147.4 kg, respectively) and body condition (means of 3.5 and 3.8 for yearlings and adults, respectively). Copper was administered by subcutaneous injection of Glypondin (König S.A., Argentina) solution (1 cm3/30 kg live BW) containing 0.83 mg of Cu per kg BW in the treatment group. Injections were administered every 42 days during the whole antler growth period. The control group was injected with a physiological saline solution. Each group comprised animals of different ages: there were 10 yearlings and seven adults in the control group, and 11 yearlings and seven adults in the treatment group. As average, yearlings were 1.5 years old (range from 17 to 18 months) and adults were 4 years (range from 2 to 6 years). Diet and unifeed ration mineral contents were analysed by inductively coupled optical emission spectrometry (ICP-OES) at the Ionomic Laboratory (IL), Centro de Edafología y Biología Aplicada del Segura- Consejo Superior de Investigaciones Científicas (CEBAS-CSIC, Murcia, Spain), detailed in section 1.9 from this chapter. Unifeed rations were homogenized and cut into small portions in a tractor-driven commercialmixer. Average daily feed intake of diet and unifeed ration was calculated from the amount used to replenish the feeders daily.

69 Chapter IV

1.2. Body measurements

Body weight and body condition were measured every 42 days throughout the trial, at the same time that Cu injections were administered. To obtain the weight adults and yearling an electronic scale (Mettler Toledo ID 1 Plus, Barcelona, Spain) with ± 0.01 g precision was placed in a small restriction cage of 2 m x 2 m x 0.5 m that prevents movement of the animal to reduce the risk of error. However, when males develop antlers they cannot access this restriction cage, so they were weighed on another scale (Tru-Test-702, Dewey, OK, USA) located in a Heenan Work Room, a hydraulic immobilizer (Figure 1). The Heenan Work Room allows animal immobilization by bars and soft padded walls of 10 cm in thickness, which only leaves the head of the animal free. These bars and walls were controlled by hydraulic systems to safely handle deer. Average daily gain (ADG) was calculated from BW data for yearlings and adults.

a) b)

Figure 1. a) Image of a hydraulic immobilizer Heenan (modified from Farmquip Cor. (Http://www.farmquip.co/) b) Adult male trapped in the immobilizer belonging to the UCLM farm during a weight control.

Body condition was measured as indicated by Carrión et al. (2008), which, in turn, is based on the method of Russel et al. (1969) for

70 Chapter IV

sheep and adapted by Audigé et al. (1998) for deer. In short, it is a palpation of the lumbosacral region to evaluate the amount of muscle mass, lipid reserves and bone relief. Depending on the prominence and the thickness of the tissues between the spinous and transverse processes, a value between 1 (very poor body condition) and 5 (very good body condition) was taken, with increments of 0.25 units.

1.3. Cu serum content

Blood samples were taken from resting deer after an overnight fast on the first day of the trial and on the 84th day after the first Cu injection. Analyses were carried out as is described in section 1.10 from this chapter.

1.4. Antler collection

According to Currey et al. (2009), antlers can be considered as inert tissue when the velvet is absent. Velvet shedding is completed at the end of August and September, therefore cut were made in this period. The cut was made approximately 1 cm above the burr with a low speed electric saw. In order to perform this procedure without risk of hurting the animals and farm personnel, deer were anesthetized using a combination of xylazine (at 0.5 mg/kg body weight) and ketamine (1 mg/kg body weight). Once the antlers were obtained, the anaesthesia was reversed with yohimbine at a rate of 0.25 mg/kg body mass. To identify the orientation of the antlers for subsequent tests, the medial face and the inner face of the antlers were marked with an indelible marker, indicating the animal's number, the cut-off date as well as its laterality. Only one antler was used to avoid axial difference

71 Chapter IV

The effects of Cu supplementation were analysed at four positions along the antler beam in adults, as indicated by Landete- Castillejos et al. (2010): position one, close to the base (burr); position two, the first third of the antler shaft; position three, the second third of the shaft; and position four, close to the base of the crown. Measurements were taken ~2 cm above the burr, 2 cm below the main tip, 2 cm above the main tip and 2 cm below the crown. Antlers from yearlings were sampled following a similar procedure to that for adults; however, only three sampling points (positions one, two and three) were included, due to the smaller size and different structure of the antlers. The last position sampled in the antler indicates more clearly the effects of diet and other factors (Landete-Castillejos et al., 2007a,b,c). From each position, a 1 cm cross section and a cylinder of 5-6 cm were obtained (Figure 2).

a) b)

Position 4

Position 3 Position 3

Position 2 Position 2

Position 1 Position 1

Adult Yearling Cylinder Cross section

Figure 2. Schema showing the cut of antlers in the experimental farm of the UCLM. a) Antler from adult male. b) Antler from yearling red deer (source: own elaboration).

72 Chapter IV

1.5. Antler cylinder processing

Using a saber saw, 3 longitudinal cuts were made on the medial face of each cylinder, obtaining two irregular prisms with a quasi triangular base composed of trabecular and cortical bone. The longitudinal axis of these prisms coincided with the vertical axis of the main beam/shaft and the cross section coincided with the largest measurement in the tangential direction. The irregular prisms composed of trabecular and cortical bone were molded by a polisher (Meta Serv ® 250 Doble, Buehler, IL, USA) forming regular prisms with a rectangular base, with dimensions of 45 mm × 2.5 mm × 4.5 mm, hereinafter referred to as sticks. These sticks consisted exclusively of cortical bone as mechanical properties depending fundamentally on the cortical bone tissue (Turner and Burr, 1993; Davison et al., 2006).

An 80-grit piece of sandpaper was used to mold the irregular prisms into sticks by rotating them on the polisher, concomitant with water flow to avoid generating heat, damaging the samples and working more efficiently. The irregular prism was held by the fingers and pressure was exerted along the prism. First, the external face of the prism was polished (easily identifiable by its colour and texture) until obtaining a flat surface. The trabecular part was then sanded until reaching the cortical layer producing a rectangular shape, avoiding the appearance of wedges. Subsequently, in order to achieve the measurements of 45 mm × 2.5 mm × 4.5 mm, an equal amount of pressure was carefully exerted, continuously checking the measurements with a digital caliper measuring ± 0.01 mm (Powerfix ®, Berdfordshire, United Kingdom). From each cylinder, two sticks were obtained. One of them was used to perform mechanical bending test, in which Young's modulus, bending

73 Chapter IV

strength and work peak force were determined. The other stick was used on the impact test to obtain the impact work.

Once measurements of 45 mm × 2.5 mm × 4.5 mm were obtained, the sticks were placed in a container with HBSS (Hank's Balanced Salt Solution, BioWhittaker, Belgium) and held for 48 hours at room temperature (20°C). After the 48 hours, the sticks were removed from the Hank's solution with a pair of tweezers and allowed to dry in the laboratory at 20ºC and 40% relative humidity for 72 hours. Using this method, the same degree of humidity is obtained on all sticks. (Figure 3). After the bending test, the stick was fractured in approximately 2 equal parts. Next, the edges produced by the fracture were polished until there were two regular prisms with rectangular bases with approximate measurements of 20 mm × 2.5 mm × 4.5 mm. One of these parts was used to determine the density and ash content and the other to determine the mineral content (Figure 3). Saw cutting

Antler external side

External cortical bone side 50 50 mm Cortical bone polished

Trabecular bone of antler Cylinder 50 mm Quasi triangular base prism 10 mm Polished

2,5 mm Rectangular base prism (Stick) 45 mm

IMPACT TEST BENDING TEST

2,5 mm 20 mm

DENSITY AND ASH MINERAL STORAGE CONTENT CONTENT Figure 3. Schema showing the preparation of the antler cylinders to establish the intrinsic mechanical properties through the impact and bending test, as well as to know the composition in ash, minerals and density (source: own elaboration).

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1.6. Mechanical bone properties

1.6.1. Mechanical properties

Mechanical properties of antlers are subjected to mechanical forces as the consequence of shocks produced during the rutting period (Rajaram and Ramanathan, 1982; Zioupos, 1996; Currey, 2002). There is a relation between the applied force and the elongation/deformation of the antler that determine the mechanical properties. Since antlers are bone tissue, factors affecting the mechanical properties of bones will also affect the antlers. Therefore, its mechanical properties depend mainly on two factors: (i) the internal structure, which determines the structural mechanical properties and (ii) the composition, from which the intrinsic mechanical properties are derived (Davison et al., 2006).

a) Structural properties are related to the size of the cortical bone, which is the mainly responsible for the mechanical properties in toto (Turner and Burr, 1993; Davison et al., 2006; Wallace, 2013). The most studied variables in previous studies in antlers have been the thickness, area, and the diameter of the cortical bone (Landete- Castillejos et al., 2010a, Cappelli et al., 2015, Ceacero et al., 2015a).

b) Intrinsic properties are related to the mineral content (Currey, 1979a; Currey et al., 2004; Lam and Pearson, 2005; Wallace, 2013), the size of the hydroxyapatite crystals (Davidson et al., 2006), and the amount of the organic matrix, essentially protein (Burr, 2002; Currey, 2003; Wallace, 2013), and are independent of the sample size (Keaveny and Hayes, 1993; Davison et al., 2006). The intrinsic properties most commonly evaluated in the antler in previous studies have been Young's modulus of elasticity, bending strength, work peak force, and impact

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work (Currey et al., 2009; Landete-Castillejos et al., 2010a, 2012a,b; Cappelli et al., 2015; Ceacero et al., 2015a). Due to the fact these properties depend on the material, the mechanical properties of the antlers and the rest of the materials usually undergo a normalization, expressing the strength or energy respect to cross section. Thus, stress is defined as force per unit area and deformation is understood as elongation per length unit (Wallace, 2013). In this sense, we will define the intrinsic mechanical properties in the following way:

• Young's modulus of elasticity (E): deformation of the 1% that occurs in the sample when applying a force per unit over a continuous area and that does not produce a permanent deformation that is, without crossing its elasticity limit (Erickson et al., 2002; Ritchie et al., 2008). Its value is obtained in GPa based on the slope of the stress-elongation curve in Figure 3 (Currey, 2002; Wallace, 2013). The antlers are not highly mineralized, therefore have a very high final deformation, but a low Young's modulus, which makes the antler extremely difficult to break (Currey, 1990). Thus, they avoid fracture during struggles with other males (Currey, 1979a, 2002, 2009).

• Bending strength (BS): maximum force per unit area that the sample can withstand before fracturing (Figure 3). It is expressed in MPa or N/m2 (Currey, 1999, 2002; Ritchie et al., 2008) and is obtained from the stress-elongation curve (Currey, 2002; Wallace, 2013).

• Work to the maximum peak force (W): amount of energy that the sample absorbs per unit of area before it fractures through continuous effort (Figure 3). It is expressed in kJ/m2 (Currey, 1979b; Ritchie et al.,

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2008) and is calculated as the area under the stress-elongation curve (Currey, 2002; Wallace, 2013).

+ Break BS

)

2 W

Stress (N/m Stress

Elastic Region Plastic Region + Deformation Figure 3. Schema showing the evolution of the deformation of a sample when subjected to an effort. The slope of the elastic region (the one where the deformation is fully reversible) determines the modulus of elasticity of Young (E). After the reversible deformation, a permanent deformation of the sample occurs at greater forces called the plastic region. Finally, the sample is broken at a given force which establishes the bending strength, (BS). The area under the curve determines the work done to break the sample, i.e. work peak force or W (source: modified from Wallace, 2013).

• Impact work (U): energy required to separate by fracture in two a unit of the section surface and is expressed in kJ/m2 (Currey, 1979b).

1.6.2. Analysis of mechanical properties

The three-point bending test was performed by placing the cortical bone of the stick on a specific machine (Z 0.5, Zwick / Roell, Ulm, Germany). Samples were placed on metal supports 40 mm apart. The outer side of the antler was face down so that the pressure load exerted by the moving head was directed on the inner side of the cortical bone of the antler. The speed of the moving head was set at 32 mm/min.

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This head was connected to a sensor that associates its deformation to the deformation of the sample. The data was processed by management software (II testXpert, Zwick GmbH & Co., Ulm, Germany). The software created real-life graphs during the test, where the abscissa corresponded to the deformation (mm) of the antler using length (mm) and the ordinate corresponded with force (N) exerted by the moving head using the area of the sample (mm2). The mechanical properties of antler bone samples were measured as previously described (Landete- Castillejos et al., 2007a,c and 2010; Currey et al., 2009) and included Young’s modulus of elasticity (E), an estimate of stiffness; bending strength (BS), calculated from the maximum load borne and the work to peak force (W), determined by total work done on the specimen up to the greatest load borne divided by the central cross-sectional areaThrough this test, Young's modulus, bending strength, and peak force work were obtained. The impact work (U) testing procedure measures the energy used to break an un-notched specimen by a falling pendulum. This value of U was normalized by dividing by the cross-sectional area of the specimen (Cappelli et al., 2015). For the impact test, A CEAST- IMPACTER II machine (CEAST S.P.A., Pianezza, Italy) was used exclusively for this type of analysis. This machine was equipped with a standard pendulum that could exert up to 1 J of maximum force. Basically, it is the free fall of a pendulum on the midpoint of the inner face of the cortical bone of the stick, which keeps the external side under pressure. The starting angle of the pendulum was set at 150°. Through software paired to the machine (with predefined parameters of humidity, temperature and the type of hammer), the loss of energy of the pendulum when breaking the stick was measured. As the energy of the pendulum

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was distributed over the cross section of the stick, the impact energy work was expressed in kJ/m2.

1.7. Antler bone cross section processing

The study of the structure was made from 1 cm thick cross sections. The surfaces of these sections were polished using a polisher (Meta Serv ® 250 Double, Buehler, IL, USA), which was paired with water flow and with 80 and 180- grit paper. Once polished, they were left to dry for 24 hours in the laboratory, at approximately 20°C and 40% relative humidity. The cross sections were then digitized for further analysis.

1.8. Bone structural properties

Each 1 cm thick antler cross section was scanned using a scanner (CanoScan LiDE 200, Canon Inc., Tokyo, Japan) at a resolution of 600 dpi. Later, the digital image of the cross-section was processed with the image software IMAGEJ (Schneider et al., 2012) obtaining the variables that determine the internal structure of the antler (Figure 4).

2 2 1 3 1 3

CB TB

6 4 5 Diameter Cortical thickness Cortical area Trabecular area Figure 4. Schema showing the measures to obtain bone structural properties of cross section of antler. CB= Cortical Bone, TB= Trabecular bone (source: own elaboration).

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• Diameter of cross section: average value of 3 diameter equidistant in mm

• Cortical thickness: average of 6 thicknesses equidistant in mm of cortical bone.

• Relative cortical thickness: quotient between cortical thickness and cross-sectional diameter. The result was multiplied by 100 to express it in %.

• Trabecular area: surface in mm2 corresponding to the trabecular bone.

• Cortical area: surface in mm2 of cortical bone in the cross section, excluding the trabecular bone. It was obtained from the difference between total and trabecular area.

1.9. Density

The breakage of stick by bending test produced two similar sized fragments (20 mm x 2.5 mm x 4.5 mm). One of these fragments was used to determine density in this way. Fragment were placed in a stove (Memmert ®, model UN 110, Schwabach, Germany) at 60°C for 72 hours. After that time, all fragments underwent the same percentage of humidity. Afterwards they were weighed quickly on an electronic scale of ± 0.001 g precision, (Gram Precision ®, ST Series, Barcelona, Spain) avoiding the absorption of the ambient humidity. The length and size (average of two measure of the fragment) were then measured with a digital caliper with an accuracy of ± 0.01 mm (Powerfix ®, Berdfordshire, UK). Since the fragments were regular prisms with a rectangular base and knowing the length and the width, the volume could

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be obtained by geometric procedures that, together with the mass of the fragments allowed the calculation of the density.

1.10. Chemical analysis of antlers

From the sample used to calculate the density, the ash content was calculated as a percentage. To this, the sample was cremated in a muffle (HTC 1400, Carbolite-Gero Ltd, Derbyshire, UK) at 480°C for 6 hours. The ash percentage was determined by the weight difference of the sample, before and after the incineration with an electronic balance of ± 0.001 g precision (Gram Precision ®, ST Series, Barcelona, Spain). In order to ensure the absence of moisture in the pre-incineration weighing, the samples were placed in the oven at 60ºC for 48 hours.

The other fragments from the breakage of stick by impact test were used to determine mineral composition of antler. These analyses were performed by means of Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES,), which is part of the Ionomic Laboratory located at CEBAS-CSIC (Centro de Edafología y Biología Aplicada del Segura-Consejo Superior de Investigaciones Científicas) in Murcia.

Antler fragments were subjected to digestion with high pressure and temperature before mineral determination. To do this, fragments with dimensions of approximately 20 mm × 2.5 mm × 4.5 mm (from the bending test and not used for the calculation of density or ash) were deposited in a digestion tube of 25 ml with 4 ml of concentrated nitric acid and 1 ml of 33% hydrogen peroxide. All of this was placed in a high temperature microwave for digestion. Then, 300 ml of ultrapure water, 30 ml of 33% hydrogen peroxide and 2 ml of concentrated sulfuric acid

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were added to the Teflon reactor. Subsequently, the tubes were placed in the microwave reactor. The reactor’s starting pressure was 40 bar and was rising 10 bar per minute for 30 minutes. The temperature gradually increased to 220°C where it was maintained for 20 minutes. After cooling the reactor and releasing all the pressure, the tubes were removed and were flushed to 25 ml with ultrapure water being ready to be analyzed in the ICP.

Once the ICP was calibrated, the sampling sequence (consisting of the antler samples and cleaning solutions intercalated) was placed. Then, the concentrations of the minerals were calculated directly by the software, taking into account the amount of antlers that had been weighed.

푚푔 표푓 푚푖푛푒푟푎푙/푘푔 표푓 푠푎푚푝푙푒 = 퐶/푃

Where:

C = Concentration read directly on the instrument, in mg / l.

P = Weight of the sample in g extracted for analysis.

In general, the microminerals were expressed in mg / kg, i.e., ppm. While the macrominerals were transformed into g / 100 g as follows: g of mineral/100 g = (mg/kg of mineral)/10,000

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1.11. Statistical analyses

A GLM test was performed to determine the effects of treatment and age class on each of the studied antler variables. Interaction between treatment and age was not significant for any trait studied. However, preliminary analysis indicated a very clear difference between yearlings and adults. Consequently, all subsequent statistical tests were carried out separately for yearlings and adults. Deer were weighed, and their body condition was measured at the beginning of the trial (before being subjected to Cu treatment). Deer were assigned to the experimental treatments so that the average BW (72.9 v. 72.9 kg for yearlings and 144.4 v. 147.4 kg for adults) and body condition (3.5 v. 3.5 for yearlings and 3.8 v. 3.8 for adults) in the control and Cu groups, respectively, were not different (P >0.10). A GLM procedure (GLMM model) was used to study the effect of Cu supplementation on ADG and body condition at the beginning of the trial and every 42 days, coinciding with each Cu injection, until the end of the experiment. In addition, antler data were analysed using the GLMM model; the model included the main effects of Cu supplementation and antler position, as well as the interaction between these variables. When the model indicated a significant difference, a Tukey’s test was used to make pairwise comparisons of treatment means.

As values derived from samples from the base (which indicate the condition of the animal at the start of antler growth) and top part (which, for the majority of parameters, indicates the physiological effort made to grow the antler, as described by Landete-Castillejos et al., 2007a and 2007b) of antlers often differed from one another, a GLM analysis was performed to study the proportional difference between the top or

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last position and the base or position one [(Last position−Position one)/Position one]. A decrease in values from the base relative to the top of antlers was indicated by a negative percentage difference. All statistical analyses were carried out using SPSS version 19 (SPSS Inc., Chicago, IL, USA).

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2. Effect of Cu supplementation on mineral serum content of red deer male during antler growth and rut (trial 2)

2.1. Experimental design

Eighteen deer from UCLM experimental farm (Albacete, south- eastern Spain), fed with a balanced diet (Table 1) were divided in two groups (control and experimental) matched for BW, 114.9 ± 53.6 and 104.4 ± 43.7 kg as average respectively and body condition; 3.6 as average for both control and for supplemented groups. As average, control group was 2.8 years old (range from 18 months to 6 years old) and Cu-treated group was 2.6 years old ranged (from 18 months to 5 years old). Copper was administered by subcutaneous injections of Glypondin (König S.A., Buenos Aires, Argentina) solution (0.03 cm3/kg BW) containing 0.83 mg of Cu per kg BW to the animals of the treatment group (n = 9). Injections were administered every 42 days, following the manufacturer recommendations from day -36 start of antler growth (SAG) to day 132 SAG. The control group (n = 9) received an injection of a physiological saline solution of the same volume and with the same periodicity that Cu supplemented group.

2.2. Blood samples collection

Blood samples were drawn by jugular venepuncture using tubes with activators Vacutest Clot ® (Vacutest Kima s.r.l., Arzergrande, Italy) at four different dates: at day -36 SAG (considered as the basal level and coinciding with the beginning of circannual period of increase of body condition that prepare the stags for rut, Gaspar-López et al., 2009), at day 54 SAG (middle of antler growth), at day 132 SAG (when antler

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cortical bone growth stops; Gómez et al., 2013) and at day 197 SAG (antler cleaning, when deer rub to strip the velvet from the antlers, Gaspar-López et al., 2010). Serum was separated using a centrifugal separator at 2,848 g at 4ºC for 15 min (CR23-22, Jouan; Thermo Fisher Scientific Inc., Waltham, MA, USA). Blood samples were kept in a freezer at -20°C until analyses, which were carried out as is described in section 1.10 from this chapter.

2.3. Antler collection

Antler were cut and at day 165 SAG at same way as is showed in 1.4 from this chapter. Mineral analyse of antler was carried out as is showed in 1.10 from this chapter.

2.4. Statistical analysis

In order to assess the effect of Cu supplementation on mineral content of serum at different stages of antler growth, linear mixed-effect regression models were used. The main two factors were fitted in the models, as fixed effects: Cu supplementation treatment and antler growth at day 54 and 132 SAG, as well as during rut at day 197 SAG. The serum concentration of Cu before supplementation (at day -36 SAG) and BW were included in the model as covariates to control the differences between animals in their Cu concentration and BW at the beginning of the experiment. Animal was used as a random effect. Body weight was fitted in all models as a linear and orthogonal polynomials of degree 2, but no significant quadratic effects were found. In consequence, BW was retained in the model as a linear term only when it was significant. The full model was simplified using backward elimination by removing one at a time, the non-significant fixed effect terms, following the principle

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of marginality. This model selection procedure was carried out on the basis of P-values, rather than information criteria, as the objective was to identify the main drivers of the dependent variables, rather than creating predictive models. A model was fitted for each mineral. When effects were significant, confidence interval (CI) at 95% was used to make pairwise comparisons between treatment means. Pearson correlations coefficients (r) between serum mineral content during the antler growth (days 54 and 132 of SAG) and antler mineral content at day 165 of SAG in control and Cu supplemented group were calculated. This analysis was conducted to determine if mineral serum content during antler growth served as an indicator of mineral content of mature antler and also to check if Cu supplementation was implicated. All analyses were conducted in ‘R’ software (R Development Core Team 2015) mainly using “lmer” package (Bates et al., 2015).

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3. Effect of Mn and Cu supplementation on microhardness of antler (trial 3)

3.1. Experimental design

Twenty adults and twenty yearling red deer males from UCLM experimental farm feed with a balanced diet (Table 1) were used. Both, adults and yearling were divided in 4 groups of 5 animals (two control groups, one group treated with Mn and one group treated with Cu). Groups were homogeneous respect to age and weight (as average adults were 4.6 years and 148.5 kg while yearling were 1.5 years and 70.1 kg).

Manganese was supplied subcutaneously by injections of 5 cm3/100 Kg live BW of an aqueous 4% of Manganese gluconate solution

(C12H22MnO14, 99.86% purity Fagron®) every seven days from January to mid-August. Other group of adults was Cu treated by subcutaneous injections of Glypondin (König S.A., Argentina) solution (1 cm3/30 kg live BW) containing 0.83 mg of Cu per kg BW. Injections were administered every 42 days following the recommendations from the product manufacturer (first injection at mid-February and last injection at the end of July). In all cases, micromineral supplementation comprised the whole antler growth period and control groups were injected with a physiological saline solution during the same period. Yearling were treated in the same way.

3.2. Antler collection

This procedure was already described in section 1.4 from this Chapter. However, antlers from yearlings were sampled following a

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similar in section 1.3 but including only 2 sampling, from the base to the top due to their smaller size as proposed Cappelli et al. (2015).

3.3. Specimen preparation

From each of the 1 cm cross-sections that had previously been digitized, a circular sector with an angle of 20-40o was obtained through a manual multipurpose tool (Dremel ® 4000, Breda, The Netherlands, Figure 5). These circular sectors were polished with 80-grit paper to remove the trabecular layer and leave only the cortical bone. We then formed a rectangular base prism of approximately 20 mm x 5 mm x 8 mm and once polished, they were placed and glued into 20 ml plastic cans with a solution of poly (methyl methacrylate). The solution 10% in polymethyl (Aldrich ®, Saint Louis, MO, USA) of methyl methacrylate (Prolabo ® VWR chemical, Fontenay-Sous-Bois, France) at was performed 24 hours earlier. The solution needed 24 hours of magnetic stirring in order to ensure the correct dissolution of the products. After 24 hours, 1% benzoyl peroxide (AppliChem ® GmbH, Darmstadt, Germany) was added as the catalyst. The entire process was carried out under the laminar flow hood because the poly-methyl emits toxic vapors. The plastic cans containing the sample and the polymethyl methacrylate (PMMA) solution were placed at a distance of 15 cm from a black light bulb (404 nm) for 7 days. The black light favored the polymerization of the PMMA and therefore it solidified, enclosing the antler in its interior. The block was polished using a polisher (Meta Serv ® 250 Double, Buehler, IL, USA) and increasing the grit sandpaper to 80, 180, and 240- grit until the block had a thickness of 5 mm. 500, 1200 and 4000- grit sandpaper was then used to reveal osteons of the cortical bone. Manually, the block was polished using a 1 μm alumina slurry

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(Al2O3, MicroPolish ™ II, Alumina, Buehler, IL, USA) and a 1 μm diamond paste (MetaDi® Supreme, Buehler, IL, USA). The entire polishing process was performed under wet conditions. In order to obtain a quality polished surface, (ie the observation of osteons without abrasions) the surface was evaluated throughout the process by means of a microscope and a microindentator (IndentaMet Series 11100, Buehler, IL, USA).

Saw Cross section cutting

10 mm PMMA 20 mm Poliseh 8 mm 5 mm External cortical bone side 20 mm Cortical bone polished Trabecular bone of antler HARDNESS Figure 5. Schema showing the preparation of 1 cm antler cross-sections to determine the hardness of osteons (source: own elaboration). PMMA= Poly methyl methacrylate.

3.4. Microhardness

3.4.1. Microhardness

There is an intrinsic mechanical property: the hardness, referring to the resistance of the surface of a sample to the deformation (Evans et al., 1990; Zysset et al., 1999; Oyen, 2006). The hardness of the osteones (micro-hardness) depends on the relationship between the structure and the mechanical characteristics of the sample on a microstructural scale (Zioupos et al., 2000). Although there is only one study that studies the hardness of osteons in the antlers (Evans et al., 1990), it has been shown

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that this hardness is a key factor in the mechanical properties of bone tissues (Zysset, 2009). In fact, there is evidence that the hardness of antlers osteones is related to Young's modulus (Evans et al., 1990). Since the hardness has no units, it is expressed by a scale. One of the most used in bone tissues is the Vickers hardness scale (Boivin et al., 2008; Dell'Ara et al., 2011, 2012).

3.4.2. Analysis of microhardness

The hardness of the osteons is characterized by the opposition to the penetration offered by the bone tissue of the antlers at microscopic level. The hardness was determined with a micro-indenter (IndentaMet Series 11100, Buehler, IL, USA) in the cortical bone along the main beam of the antler. To obtain the hardness of the osteons, we followed the suggestion of Bala et al. (2010), performing micro-indentations in the middle region of the cortical bone. Micro-indentations were performed on primary osteons, avoiding the Havers systems and the scaffold substance. The average hardness of the osteons was 10 equidistant measures. The Vickers hardness was determined by an indentation of two cross-linked diagonals. This imprint is a consequence of the force applied by a tiny quadrangular pyramid diamond head with a 136° angle, the base of which is paired to a controlled load of 25 g for 10 seconds (Figure 6). The micro-indenter was paired to an internal software that automatically determined the Vickers hardness using the following formula:

휃 퐹 2퐹 sin 퐹 퐻푉 = 0,102 = 0,102 2 = 0,1891 푆 푑2 푑2

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Where: HV = Hardness Vickers. F = Strength of the test (N). S = Indentation surface area (mm2), determined by the length of the two diagonals. θ= Face angle of diamond indenter (136°). D = Average length of the two diagonals (mm).

a b ) ) Base of crown TB

nd 2 third of shaft CB

st nd 2 third of shaft 1 third of shaft

O Burr Burr

200µ Figure 6. a) Scheme showing the cross-section taken from different antler positions of yearling and adult red deer. b) Scheme of cross-section showing cortical and trabecular bone as well as the indentations on each cross-section in medial region. TB = Trabecular bone. CB = Cortical bone. O = Osteon. Black arrow indicates the place of indentation. = Indentation.

3.5. Statistical analysis

The experimental unit was the animal with 40 deer of different ages (10 yearlings and 10 adults for Mn supplementation trial and 10 yearlings and 10 adults for Cu supplementation trial). ANOVA analysis was used to compare average of age and BW per treatment. In addition, a general linear mixed procedure (GLMM analysis) was performed to study the effect of mineral supplementation (Mn or Cu) on the microhardness along main beam. Model included mineral supplementation and position of antler as main effects as well as their interaction. When models were significant, Tukey test was used to make

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pairwise comparisons among treatment means. Values derived of percentual difference between base of crown and burr, calculated as [100 x (HV in base of the crown – HV in burr) / HV in burr)] were analysed to estimate the physiological effort along the antler growth process (Landete-Castillejos et al., 2007b) by GLM. All analyses were carried out with SPSS version 20 (SPSS Inc., Chicago, IL, USA).

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4. Effect of Cu supplementation of gestating and lactating hinds on calf growth and milk traits (trial 4)

4.1. Experimental design

At the beginning of the trial (day of the first Cu injection), in the UCLM experimental farm there were 20 Iberian red deer hinds, 10 hinds were allocated to the control group and 10 hinds to the Cu treatment group. However, three animals were removed because two calves from the control group died for causes not related with the experiment and one calf from Cu supplemented group because the hind did not bore. Therefore, the number of experimental replicates at the time of lactation was reduced to eight for control group and nine for Cu experimental group. Hinds were assigned to the treatments so the average, age (10.8 ± 1.65 and 9.8 ± 1.68 years of age for hinds from control and Cu group as average, respectively), BW and body condition per treatment were the same. Hinds and calves had free access to diet and unified (Table 1).

Copper was injected every 42 days from day 202 of gestation (as average) to week 18 of lactation (forced weaning by physical separation) to hinds from the treatment group by subcutaneous injections of Glypondin (König S.A., 1868 Buenos Aires, Argentina) containing 0.83 mg of Cu per kg BW (1 cm3 per 30 kg live BW). Control group (n = 8) was injected with a physiological saline solution of the same volume and with the same periodicity than the experimental group.

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4.2. Body measurements

BW and body condition of hinds were measured 3.5 days before (as average) and after calving (as average). Also, hinds were individually weighed (± 50 g) at weeks 2, 4, 6, 10, 14 and 18 of lactation. BW and body condition score of hinds were evaluated as was detailed in section 1.2. from current chapter.

Calving took place during the month of May. Progeny was composed by four males and four females in the case of the control group and by four males and five females in the case of the Cu supplemented group. Calves were individually weighed within 12 h of life (birth BW) and marked with ear tags (76 by 57 mm, Felixcan, GTLF1, 02080 Albacete, Spain). In addition, calves were individually weighed at 2, 4, 6, 10, 14 and 18 weeks of lactation. These data were used to calculate the average daily gain (ADG) for male and for female calves throughout the lactation and the weaning BW of calves.

4.3. Milk collection

Milking was conducted once a day after 6 hours of separation between mother and calf in weeks 2, 4, 6, 10, 14 and 18 (end of the trial) of lactation following the procedure described by Landete-Castillejos et al. (2000a, b). As hinds were isolated six hours before each milking to avoid breastfeeding, estimating therefore that the amount of milk collected during milking was produced for 6 hours (one quarter of a day). Accordingly, daily milk production was determined by multiplying the milk yield by 4.

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A machine milking set-up to 50/50 massage/milking ratio and 44 kPa of vacuum was used. Previously to milking, anaesthesia was injected in the right jugular vein (0.5 mg/kg of xylazine and 1 mg/kg of ketamine reverted with 0.25 mg/kg of yohimbine). Once anaesthesia was induced, 10 i.u. of oxytocin were injected in the right jugular vein 1 min before milking to induce milk let down.

4.4. Milk composition

Two 30 mL milk samples were collected from each hind to assess milk composition (fat, protein, lactose and dry matter concentrations) in duplicate from each milking. These analyses were carried out in an automatic milk analyzer Milkoscan FT+ (FOSS Analytical A/S, 3400 HillerØd, Denmark) based on infrared spectrophotometry by the reference laboratory LILCAM (Laboratorio Interprofesional Lácteo de Castilla La Mancha, Talavera de la Reina, Spain). Data obtained were used to calculate protein to fat ratio (PFR) and total production of milk (total milk yield or TMY), fat (total fat yield or TFY), protein (total protein yield or TPY), lactose (total lactose yield or TLY) and dry matter (total dry matter yield or TDMY) during lactation. Milk gross energy content (MGEC) of fresh milk was calculated as described by Landete- Castillejos et al. (2003b).

4.5. Milk minerals

An amount of 2 ml of milk samples collected at weeks 2, 4, 14 and 18 of lactation were colleted in tubes (Eppendorf ®, Hamburg, Germany) and were stored frozen at -20°C until mineral analysis. Mineral content in milk was analysed by inductively coupled optical emission spectrometry (ICP-OES) at Ionomic Laboratory (IL) placed at

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Centro de Edafología y Biología Aplicada del Segura of Consejo Superior de Investigaciones Científicas (CEBAS-CSIC, Murcia, Spain), detailed in section 1.10 from this chapter.

4.6. Statistical analysis

A general linear model (GLM) analysis was performed to study the effects of Cu supplementation on hind BW and body condition (day of the first Cu injection and 3.5 days before and after calving), BW of calves at birth and at weaning, ADG of calves (males and females separately) throughout lactation and TMY, TFY, TPY, TLY, TDMY. On the other hand, a general linear mixed model (GLMM) was used to study the effects of Cu supplementation and lactation week on BW of hind throughout the lactation, daily milk production, milk composition (fat, protein, PFR, lactose, dry matter and minerals) and MGEC. Model included Cu supplementation and lactation week as well as their interaction. When the model was significant for lactation week, a Tukey test was used to make pairwise comparison among week means. In all cases, the replicate was the animal. Data in tables are presented as means for all traits and analyses were carried out with SPSS version 19 (SPSS Inc., Chicago, IL, USA). A P-value ≤ 0.05 was considered significant and P-values between 0.05 and 0.10 were considered a tendency.

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5. Effect of Mn supplementation of gestating and lactating hinds on milk traits and calf growth (trial 5)

5.1. Experimental design

At the beginning of the trial (day of the first Mn injection),in the UCLM experimental farm there were 23 Iberian red deer hinds that were divided in this way: 12 hinds for Mn treatment group and 11 hinds for control group. However, three animals were removed from the control group because one hind did not bore, one calf died for causes not related with the experiment, and the other hind was sick and, in consequence, could not be milked. Therefore, the number of experimental replicates at the time of lactation was reduced to 8 for control group.

Manganese was injected weekly from day 140 of gestation (as average) to week 18 of lactation (forced weaning by physical separation) to hinds from the treatment group (n = 12) by subcutaneous injections of manganese gluconate (C12H22MnO14, 99.86% purity Fagron®) in aqueous solution at 8% (2.5 cm3 per 100 kg of body weight, BW) containing 2 mg of Mn per kg BW following the recommendations from the product manufacturer used. Control group (n = 8) was injected with a physiological saline solution of the same volume and with the same periodicity than the experimental group.

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and control groups, respectively ranged from 2 to 18 years in both cases) per treatment were the same. Hinds and calves had free access to diet and unified (Table 1).

5.2. Body measurements

BW and body condition of hinds were measured 3.5 days before and after calving (as average) and at weeks 2, 4, 6, 10, 14, 16 and 18 throughout lactation. All calving of experimental hinds took place during the month of May. Progeny was composed by 7 males and 5 females in the case of the Mn supplemented group and by 4 males and 4 females in the case of the control group. In all cases, hinds and calves were weighed using the same method described in section 1.2 and section 4.2 from this chapter, respectively. Data of calf BW were used to calculate the relative BW gain of calves as follows: [100 × (final BW – initial BW)/initial BW] for each period studied.

5.3. Milk collection

Milking was conducted in the same way that was detailed in section 4.3 from this chapter.

5.4. Milk composition and milk minerals

Milk composition and minerals were obtained as was described in section 4.4. and 4.5 from this chapter, respectively.

5.5. Mn serum content

Blood samples were taken from resting hinds after an overnight fast just before the first injection with Mn (basal level) and at week 10

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of lactation (as average). Blood samples were drawn, without sedation, by jugular venipuncture (Vacutest Clot activators, Kima S.A., Arzergrande, Italy) and were coagulated in the tubes. Serum was separated using a centrifugal separator (3,500 rpm at 4ºC for 15 min) and samples were kept in a freezer at -20°C until later analysis. Manganese serum content was analysed by ICP-OES at IL placed at CEBAS-CSIC (Murcia, Spain).

5.6. Statistical analysis

A general linear model (GLM) analysis was performed to study the effects of Mn supplementation on hind BW and hind body condition (day of the first Mn injection and 3.5 days before and after calving), BW of calves at birth, relative BW gain throughout lactation and TMY, TFY, TPY, TLY and TDMY. On the other hand, a general linear mixed model (GLMM model) was used to study the effects of Mn supplementation and lactation week on lactation hind BW, daily milk production, milk composition (fat, protein, PFR, lactose, dry matter and mineral content) and MGEC. Model included Mn supplementation and lactation week as well as their interaction. When the model was significant for lactation week, a Tukey test was used to make pairwise comparison among week means. In addition, Pearson correlation coefficients were computed among performance traits studied and milk composition for pooled data, Mn treatment group and control group. In all cases, the replicate was the animal. Data in tables are presented as means for all traits and analyses were carried out with SPSS version 19 (SPSS Inc., Chicago, IL, USA). A P-value ≤ 0.05 was considered significant and P-values between 0.05 and 0.10 were considered a tendency.

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6. Osteophagia in key periods of red deer (trial 6)

6.1. Experimental design

The study was conducted in “Zona de Caza Controlada Las Dehesas”, a 3931-ha game reserve located in the municipalities of Alpera, Alatoz and Carcelén (Albacete), south-eastern Spain (38°59′ N, 1°15′ W). The antlers used in this study were collected in the same area following the casting period (end of February). Immediately after collection, some antlers were offered to the free ranging animals under controlled conditions. The experiment started as soon as 15 antlers from adult animals were collected by the keepers of the estate. However, exhaustive collection of cast antlers continued for some more weeks (common practice, as these antlers are usually sold reaching several thousand euro for the whole harvest of 1 year). Thus, the only antlers available to wildlife for chewing during the study period were those used in our setting.

Anyway, still, some antlers may remain in the area, and skeletons from dead animals are also available as bone sources. Antlers used in the experiment ranged from 745 to 1145 g. The mineral composition of these antlers can be found in Estévez et al. (2008, 2009). The experiment started at the beginning of March and lasted until the end of September when it had to be stopped because of management reasons. Three antlers were drilled in their base and held together in an open area with easy accessibility for animals. The camera trap was placed in a tree separated approximately 5 m from the antlers (Figure 7). This setting was repeated in five locations covering the whole estate. The antlers used in the experiment were measured and weighed at the beginning and the end of

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the experiment. Nevertheless, as consumption was not as strong as expected, only differences in weight are shown.

6.2. Videotramps

The activity and behaviour of the wildlife interacting with the antlers were recorded by camera traps working day and night following the recommendations of O’Connell et al. (2011). We used Ecotone HE- 30 (Warsaw, Poland), with 3.1 MP camera, infrared flash and four batteries which were recharged by solar panels, and 2 GB SD card. All the system was protected by a waterproof camouflage box. Cameras were placed approximately 2 m high, to avoid damage from wildlife. Care was taken not to have branches between the camera and the antlers, as their movement can be detected by the double infrared sensor. When some movement was detected, short videos of 30 s were recorded, illuminated by daylight or by infrared flash. Camera traps might produce a reaction in animals due to infrared-light wavelengths (Newbold and King 2009; Meek et al., 2012; Glen et al., 2013; Cohen et al., 2014). However, it is unlikely that our deer responded because animals were recorded chewing antlers without any reaction to the camera traps and without influence in their behavioural pattern of activity described by Clutton-Brock et al. (1982). Date and time were recorded on each video. Wildlife ranging in the area includes two ungulates (red deer, Cervus elaphus; and wild boar, Sus scrofa) and all the common species in Mediterranean ecosystems (Palomo et al., 2007). For red deer, we recorded males, females and calves. Calves were considered as such until breeding season started, which happened next May. From that moment onwards, previous year calves were coded as females or males.

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Figure 7. Disposition of videotrap cameras in respect to the antler, which acted as a natural source of mineral supplementation in the controlled hunting zone Las Dehesas (photos made by Francisco Ceacero, 2012).

6.3. Statistical analysis

For the statistical analysis, SPSS version 20 (SPSS Inc., Chicago, IL, USA) was used. Chi-squared test was used to examine monthly differences in the frequency with which females, males and calves appeared in the videos and also the frequency with which they were observed chewing the antlers. Kruskal-Wallis tests were used to highlight differences in the mean monthly occurrence of events (number of females, males and calves observed eating antlers) among three periods of the day: dusk (18:00 to 02:00), dawn (02:00 to 10:00) and day (10:00 to 18:00). These day periods were defined using 02:00 as the middle of the shorter night of the year; thus, dusk and dawn include half night each plus a variable amount of the beginning and the end of the daylight.

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Anonymus

Chapter V RESULTS

Chapter V

1. Effect of Cu Supplementation on antler properties and mineral composition of red deer male (trial 1) 1.1. Effects of Cu supplementation on the average daily gain and body condition of deer

Copper supplementation led to an increase in the serum content of this mineral in yearlings from 0.68 to 0.94 ppm (P = 0.02) and in adults from 0.59 to 0.95 ppm (P = 0.02) compared with control groups. The magnitude of the observed effect was greater in adults than yearlings (38% vs. 28%). In spite that, Cu supplementation did not influence ADG neither body condition of yearlings or adult deer at any time point (Table 1) nor the antler mineral content as is described in section 1.2 and 1.3 from this chapter. Average daily intakes of dry matter diet and unified ration were, as average, 2.1 and 1.4 kg, respectively.

Table 1. Effects of Cu supplementation on average daily gain (ADG, g/day) and body condition measured every 42 days1 through the trial in yearling and adult red deer.

Variable Yearlings Adults Control Cu- SEM P-value Control Cu- SEM P-value injection (n=21)2 injection (n=14)2 ADG 0 to 42 days 206 192 10.9 0.56 331 395 85.5 0.73 42 to 84 days 233 255 11.5 0.36 312 400 47.9 0.38 84 to 126 days 207 217 12.5 0.69 328 388 70.3 0.70 126 to 168 days 145 176 27.3 0.59 318 632 128.2 0.31 0 to 168 days 199 210 10.7 0.84 322 454 50.7 0.27 Body condition

Day 0 3.5 3.5 0.04 0.58 3.8 3.8 0.06 0.66

Day 42 3.7 3.7 0.05 0.95 4.0 4.1 0.11 0.53 Day 84 3.8 3.8 0.04 0.60 4.3 4.4 0.11 0.55 Day 126 3.8 3.9 0.05 0.38 4.5 4.6 0.08 0.61 Day 168 3.7 3.7 0.05 0.48 4.2 4.2 0.10 0.29 1 Coinciding with each Cu injection.2 The experimental unit was the animal.

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1.2. Effects of Cu supplementation on the mechanical and structural properties and ash and mineral content of antlers

No significant interactions between Cu supplementation and antler position were detected for any traits studied; therefore, only main effects are shown for yearlings and adults. Supplementation with Cu did not influence any antler traits studied in yearlings (Table 2).

Table 2. Effects of Cu supplementation (CS) and antler position (AP) on mechanical and structural properties of antlers from yearling and adult red deer.

CS AP P-value 1 Variable Cu- 1st third of 2nd third of Base of SEM Control Burr CS AP injection shaft shaft crown

Yearlings Young’s modulus of 18.3 18.2 18.8a 14.3b 19.2a - 0.65 0.95 0.03 elasticity, E (GPa)

Bending strength, 253.6 272.4 259.6B 278.1A 263.1AB - 7.29 0.29 0.001 BS (MPa) Work to peak force, 35.1 37.7 34.5 41.8 36.7 - 1.28 0.76 0.18 W (kJ/m2) Impact work, U 20.0 21.5 19.3b 28.0a 19.7b - 1.10 0.72 0.03 (kJ/m2)

Density (kg/dm3) 1.61 1.65 1.61 1.74 1.62 - 0.021 0.32 0.15 Average cortical 5.18 4.83 5.96A 6.20A 3.51B - 0.311 0.56 0.009 thickness (mm) Average cortical 0.41 0.46 0.50A 0.51A 0.36B - 0.026 0.94 0.04 thickness (%) Average cortical 0.65 0.68 0.74a 0.72a 0.56b - 0.027 0.99 0.03 area (%)

Adults Young’s modulus of 17.1 18.0 18.7A 19.1A 17.4B 15.3C 0.81 0.78 0.002 elasticity, E (GPa) Bending strength, 254.2 272.1 284.7A 281.4A 258.3AC 233.2BC 6.01 0.09 0.005 BS (MPa) Work to peak force, 40.5 41.7 43.9 42.9 39.9 38.2 0.99 0.67 0.26 W (kJ/m2) Impact work, U 21.8 21.2 19.7 22.6 21.1 22.5 0.70 0.80 0.28 (kJ/m2) Density (kg/dm3) 1.55 1.59 1.62 1.62 1.54 1.49 0.023 0.43 0.12 Average cortical 5.13 5.35 7.45A 5.03B 5.29B 3.25C 0.276 0.06 < 0.001 thickness (mm)

Average cortical 0.24 0.24 0.35A 0.22C 0.28B 0.12D 0.014 0.99 < 0.001 thickness (%)

Average cortical 0.43 0.44 0.59A 0.42B 0.48B 0.25C 0.021 0.90 < 0.001 area (%) 1 The experimental unit was the animal (21 and 14 yearlings and adults, respectively). a,b Values within a row with different superscripts differ significantly at P ≤ 0.05. A,B,C,D Values within a row with different superscripts differ significantly at P ≤ 0.01. Interaction of Cu supplementation × antler position was not significant (P > 0.10) for any trait studied; therefore, only the main effects are presented.

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However, average cortical thickness tended to increase by a mean of 4.1% in adults receiving Cu supplementation (P = 0.06). In addition, cortical thickness correlated positively with increased Cu serum before and after treatment in the supplemented group, supporting an effect of Cu in increasing cortical thickness; however, the high correlation coefficients (r = 0.73 and 0.80 for yearlings and adults, respectively) did not achieve statistical significance (P > 0.10), likely due to the small sample size. No effect on the Cu content of antlers was observed in either yearlings or adult deer (Tables 3 and 4). Nevertheless, adult Cu supplemented tended to increase the Sr content (P = 0.06) and significantly increased the Zn content (P = 0.02) of antlers.

Table 3. Effects of Cu supplementation (CS) and antler position (AP) on ash and mineral content of antlers from yearling red deer.

CS AP P-value SEM Variable Cu- 1st third of 2nd third of 1 Control Burr (n=21) CS AP injection shaft shaft Ashes(%) 59.5 58.9 58.2b 60.3a 59.8a 0.41 0.96 0.40 Ca(%) 20.2 19.9 20.0 19.2 20.4 0.16 0.56 0.22 P(%) 11.3 11.6 11.2 12.5 11.4 0.17 0.71 0.28 Ca/P 1.80 1.74 1.80A 1.56B 1.81A 0.026 0.57 0.003 Mg(%) 0.50 0.50 0.50 0.61 0.59 0.006 0.57 0.27 Na(%) 0.60 0.60 0.60 0.61 0.59 0.005 0.55 0.79 S(%) 0.26 0.25 0.25 0.23 0.26 0.003 0.49 0.05 Cu(ppm) 0.39 0.41 0.43 0.19 0.47 0.043 0.71 0.28 Mn(ppm) 21.7 21.2 21.4 21.0 21.6 0.25 0.52 0.34 Sr(ppm) 472.8 445.5 473.8 380.2 470.7 12.30 0.50 0.52 Zn(ppm) 63.1 58.4 57.6B 55.9C 64.9A 1.34 0.38 <0.001 Al(ppm) 18.5 17.3 17.7ab 14.0b 19.4a 0.72 0.59 0.02 Li(ppm) 5.76 5.14 5.89a 2.13b 6.18a 0.419 0.86 <0.001 Cr(ppm) 1.46 1.49 1.49a 1.39b 1.49a 0.014 0.71 0.03 1 The experimental unit was the animal. a,b Values within a row with different superscripts differ significantly at P ≤ 0.05. A,B,C Values within a row with different superscripts differ significantly at P ≤ 0.01. The interaction of Cu supplementation × antler position was not significant (P > 0.10) for any trait studied; therefore, only main effects are presented.

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Table 4. Effects of Cu supplementation (CS) and antler position (AP) on ash and mineral content of antlers from adult red deer.

Variable CS AP SEM P-value (n=14)1 Control Cu- Burr 1st third 2nd third Base of CS AP injection of shaft of shaft crown Ashes(%) 59.7 59.5 61.3A 60.1A 58.9AB 58.0B 0.48 0.92 <0.001 Ca (%) 19.6 19.5 19.6 19.7 19.2 19.7 0.31 0.90 0.48 P (%) 11.7 11.7 12.3 11.7 11.4 11.3 0.23 0.96 0.24 Ca/P 1.73 1.71 1.65 1.72 1.72 1.78 0.048 0.94 0.09 Mg (%) 0.54 0.59 0.58 0.57 0.57 0.56 0.014 0.33 0.18 Na (%) 0.62 0.63 0.64 0.63 0.62 0.62 0.013 0.79 0.15 S (%) 0.22 0.20 0.21 0.21 0.21 0.21 0.009 0.62 0.73 K (%) 0.03 0.03 0.03 0.03 0.03 0.04 0.001 0.80 0.21 B (ppm) 1.83 2.03 1.99 1.97 1.92 1.89 0.056 0.35 0.53 Cu (ppm) 0.38 0.34 0.39 0.32 0.32 0.39 0.020 0.25 0.56 Fe (ppm) 3.57 4.35 3.44b 3.24b 4.98a 4.45a 0.320 0.15 0.05 Mn 26.0 26.7 26.9 26.5 28.0 23.8 1.23 0.58 0.60 (ppm) Sr (ppm) 344.3 378.4 374.8 371.1 354.2 351.2 8.46 0.06 0.56 Zn (ppm) 63.6 71.7 69.3 67.9 68.3 67.3 1.46 0.02 0.73 Al (ppm) 16.5 17.5 17.9 16.1 18.2 16.2 0.83 0.63 0.85 Li (ppm) 4.11 4.17 4.14 4.13 4.09 4.21 0.354 0.97 0.65 Cr (ppm) 1.43 1.45 1.42 1.50 1.48 1.38 0.027 0.73 0.15 B (ppm) 1.83 2.03 1.99 1.97 1.92 1.89 0.056 0.35 0.53 1 The experimental unit was the animal. a,b Values within a row with different superscripts differ significantly at P ≤ 0.05. A,B Values within a row with different superscripts differ significantly at P ≤ 0.01. The interaction of Cu supplementation × antler position was not significant (P > 0.10) for any trait studied; therefore, only main effects are presented.

1.3. Effect of Cu supplementation on the variation in characteristics and mineral composition between the base and top of antlers from yearling and adult deer

In yearlings, the difference between the top and the base of the antler (expressed as a percentage), was not affected by Cu supplementation, other than the characteristics U and Ca/P proportion. In this age group, U increased in the Cu supplemented group, while it decreased in the control group (20.4 % vs. −9.6 %; P = 0.04). The ratio of Ca to P tended to decrease in deer administered Cu by injection, whereas this was not the case in control deer (−1.6 % vs. 1.5%; P = 0.06).

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In adults, the average cortical fact, Cu supplementation had little influence on the ash or mineral content of yearling or adult antlers. The only effect observed in antlers of yearling deer was a significant interaction between Cu supplementation and antler position for K, B and Fe content (Figure 1). In adults, Cu supplementation tended to increase the Sr content (P = 0.06) and significantly increased the Zn content (P = 0.02) of antlers.

Figure 1. Interaction effect between Cu supplementation and antler position (burr, first third of shaft and second third of shaft) on K content (%), B content (ppm) and Fe content (ppm) of antlers from yearling red deer. The experimental unit was the animal, with a total of 21 deer. a, b Different alphabetic superscripts denote mean values which are significantly different at P < 0.05. A, B Different alphabetic superscripts denote mean values which are significantly different at P < 0.01.

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2. Effect of Cu supplementation on mineral serum content of red deer male during antler growth and rut (trial 2)

2.1. Effect of Cu supplementation on serum minerals

Data showed that means for P (0.0092 vs 0.0085 g/100g; SEM = 0.00022; P < 0.05) and Cu (0.8188 vs. 0.7269 mg/kg, SEM = 0.03291; P < 0.05) in the Cu treated group were 7.6% and 11.2%, respectively higher than in the control group. However, Cu supplementation did not affect the other minerals studied (Table 5).

Table 5. Means minerals in serum as response of Cu supplementation (CuS) in red deer males.

CuS SEM (n=18)3 P-value Control CuS CuS Macrominerals (g/100 g)

Ca 0.0070 0.0071 0.00009 0.71 P 0.0085 0.0092 0.00022 0.03 Mg 0.0021 0.0021 0.00013 0.54 K 0.0261 0.0256 0.00122 0.54 Na 0.2928 0.2957 0.00284 0.46 S 0.0843 0.0838 0.00163 0.75 Trace minerals(mg/kg) Al 0.1979 0.2599 0.02209 0.14 B 0.1685 0.1871 0.00678 0.16 Cu 0.7269 0.8188 0.03291 0.04 Fe 3.5102 3.3992 0.22326 0.12 Mn 0.1367 0.1359 0.00279 0.87 Sr 0.1767 0.1883 0.00483 0.33 Zn 0.6601 0.6580 0.02162 0.51 1 Days before beginning of antler growth.2 Days after beginning of antler growth. 3 The replicate was the animal.

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2.2. Effect of antler growth and rut on serum minerals

Concentrations of all minerals of serum were influenced by antler growth and rut in a complex pattern. Calcium and P of serum content obtained the highest values at day 54 SAG (0.0079 and 0.0098 g/100g for Ca and P, respectively; P < 0.01, Figure 2). Magnesium content increased from day -36 SAG to day 54 SAG (P < 0.001) and after that it was maintained during the whole antler growth period and rut.

Figure 2. Effects of antler growth at days 54 and 132 of start of antler growth (SAG) and rut at day 197 SAG on serum content of macrominerals respect to serum basal level at day -36 SAG.a,b,c Different alphabetic superscripts denote mean values significantly different at 95 % of confidence interval.

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Among trace minerals, Cu and Zn content of serum reached their highest values during rut at day 197 SAG (0.9738 mg/kg and 0.7672 mg/kg for Cu and Zn respectively; P < 0.001, Figure 3).

Figure 3. Effects of antler growth at days 54 and 132 of start antler growth (SAG) and rut at day 197 SAG on serum content of trace minerals respect to serum basal level at day -36 SAG a,b,c Different alphabetic superscripts denote mean values significantly different at 95 % of confidence interval.

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2.3. Correlations between serum and antler mineral

Regarding to correlations, only Fe content of serum at day 132 of SAG was positively correlated with the Fe content of antler in the control group (r = 0.853; P < 0.05, Table 7).

Table 6. Pearson correlations between minerals of serum at day 54 of start antler growth (SAG) and antler at day 165 of SAG from red deer males of control and Cu supplemented (CuS) groupsA (only significant results are shown).

Control serum minerals CuS serum minerals Item Cu Mg Ca P Zn Antler minerals Control K -0.897* P 0.947* CuS Ca -0.792* -0.793* Cu 0.747* Mg -0.733* Na -0.846** Zn 0.744* AThe replicate was the animal in all cases (n = 9). *P < 0.05 ** P < 0.01.

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Table 7. Pearson correlations between minerals of serum at day 132 of start antler growth (SAG) and antler at day 165 SAG from red deer males of control and Cu supplemented (CuS) groupsA (only significant results are shown).

Control serum minerals CuS serum minerals

Item Ca Cu Fe P Zn Cu K Mg Mn S Sr

Antler

minerals

Control Fe 0.853* -0.911* K -0.885* -0.867* P 0.873* S -0.843* CuS K 0.751* Mg 0.841* 0.883** 0.877** 0.786* Mn 0.798* 0.866** 0.720* P 0.833* 0.795* 0.807* 0.772* Sr 0.769* Zn 0.798* 0.827* AThe replicate was the animal in all cases (n = 9). *P < 0.05** P < 0.01.

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3. Effect of Mn and Cu supplementation on microhardness of antler (trial 3)

3.1. Influence of treatment (injections of Mn or Cu) and antler position on average of hardness Vickers in antler of adults and yearling of red deer

Adults from both trial (Mn and Cu supplementation) were not directly influenced by mineral supplementation. However, yearling Mn treated raised the HV in an 11.6% (P = 0.01), whereas yearling Cu treated showed a trend in the increment of HV by 10.3% (P = 0.08). In addition, we studied the effect of antler position on HV. In Mn trial, HV of antler from adults showed a trend (P = 0.06), burr was 16.6% higher than HV of base of crown while yearling showed differences significates (P = 0.02) between burr and second third of the main beam, being a 5.8% greater in burr. Vickers hardness of antler of adults from Cu trial was also influenced by antler position being a 20.9% greater in the burr than the top of antler. However, yearlings from Cu trial did not showed the difference significant between the top and the base of the antler (Table 8).

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Table 8. Lineal mix model analyses showing the influence of mineral supplementation(S), Cu or Mn and antler position on Vickers hardness (HV) of osteons from cortical bone of red deer antler.

Supplementation Antler position (AP) SEM P-value (n=10) Control(S) Treated Burr 1st third 2nd third of Base of S AP of shaft shaft crown Mn-Trial

Adults 33.58 34.86 36.77a 34.30a 33.17 a 32.65 a 0.985 0.709 0.064 Yearlings 31.04 34.64 33.77 31.92 0.579 0.012 0.016

Cu-Trial Adults 29.13 30.28 33.11a 28.99b 29.35 abc 27.38cbd 0.763 0.601 0.031 Yearlings 33.24 36.67 34.68 35.23 0.780 0.083 0.602 1The experimental unit was the animal. a,b,c,d Values within a row with different superscripts differ significantly at P ≤ 0.05.

3.2. Influence of Mn supplementation on hardness Vickers along main beam in adults and yearling of red deer

There was not a significant interaction between Mn supplementation and antlers position in adults. Regardless, a significant interaction (P = 0.04) between Mn supplementation and antler position was detected for yearling. Control group decreased HV along main beam while Mn treated group remained stable along antler. Additionally, HV of the second third of shaft in control group of yearlings was lower than in Mn treated group (29.36 vs. 34.46; P < 0.001; Figure 4).

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Figure 4. Hardness values of osteons along main beam of antler of adults (a) and yearling (b) from red deer in control (n = 5) and Mn treated group (n = 5). No interaction was found between antler position and Mn supplementation (P > 0.10) in adults. In yearling an interaction was found between antler position and Mn supplementation (P = 0.04) a,b Different superscripts differ significantly at P ≤ 0.05. *** Indicates difference at P ≤ 0.001 between control and Mn treated group.

3.3. Influence of Cu supplementation on hardness Vickers along main beam in adults and yearling of red deer

A significant interaction between Cu supplementation and antler position was detected for adults (P = 0.04). Hardness Vickers from control decreased along main beam while adults Cu treated remained stable along main beam of antler. Additionally, HV of osteons from base to crown of control adults was statistically lower that Cu treated adults (25.34 vs. 29.42; P = 0.03). In yearlings, HV of the second third of shaft in Cu treated group was also higher than in control group (37.82 vs. 32.63; P = 0.03; Figure 5).

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Figure 5. Hardness values of osteons along main beam of antler of adults (a) and yearling (b) from red deer in control (n = 5) and Cu treated group (n = 5). A interaction was found between antler position and Cu supplementation (P = 0.04) in adults, but in yearling no interaction was found between antler position and Cu supplementation (P < 0.10). a,b Different superscripts differ significantly at P ≤ 0.05. *Indicates difference at P ≤ 0.05 between control and Cu treated group in base of crown.

3.4. Effect of Mn supplementation on the variation in hardness between base of crown and the burr from adult and yearling of red deer

In both, adults and yearlings the difference of microhardness between the top and the base of the antler (expressed as a percentage), was affected by Mn supplementation. In adults, microhardness decreased in control and Mn treated group, but this decrease was lower in Mn treated (−18.9% vs. −1.6%; P = 0.03). In yearling, microhardness also decreased in control and Mn treated group, with greater decrement in control group (−9.9% vs. –0.9%; P = 0.03).

3.5. Effect of Cu supplementation on the variation in hardness between the base of crown and burr from adult and yearling of red deer

In spite of in both adults and yearlings shown a great difference of microhardness between the top and the base of the antler, was not

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significate (−24.9% vs. −9.8%; P = 0.171, for control and Cu treated group from adults and −3.7% vs. 7.3%; P = 0.11, for control and Cu treated group from yearling).

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4. Effect of Cu supplementation of gestating and lactating hinds on calf growth and milk traits (trial 4)

4.1. Effects of Cu supplementation to hinds on hind body weight and body condition and calf growth from birth to 18 weeks of lactation (forced weaning)

At the beginning of the trial (day of the first Cu injection at day 202 of gestation as average), hinds were assigned to the treatments so the average BW (109.4 and 105.6 kg for hinds from control and Cu groups, respectively) and body condition (3.8 and 3.7 for hinds from control and Cu groups, respectively) per treatment were the same (P = 0.362, Table 9). Copper supplementation did not influence BW or body condition of hinds at any stage studied or BW of calves at birth or weaning. However, differences for growth rate of progeny were observed between treatments according to sex. In fact, ADG of female calves from hinds injected with Cu grew more from 14 to 18 weeks of lactation than those from hinds of control group (0.319 vs. 0.231 kg/day; P = 0.047). In contrast, no differences were observed for ADG of male calves at any age studied.

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Table 9. Effects of Cu supplementation of gestating and lactating hinds on body weight (BW) and body condition of hinds and offspring growth performance from birth to 18 weeks of lactation (forced weaning) of red deer.

Trait Control Cu injection SEM† P-value Hind BW (kg) First Cu injection‡ 109.4 105.6 1.81 0.362 Before calving§ 124.6 121.7 1.81 0.443 After calving§ 109.9 107.0 2.15 0.531 Average lactation¶ 109.2 107.3 0.85 0.275 Hind body condition First Cu injection‡ 3.8 3.7 0.08 0.596 Before calving§ 3.9 4.0 0.05 0.736 After calving§ 3.9 3.9 0.07 0.944 Calf birth BW (kg) Males 9.3 8.4 0.45 0.382 Females 7.9 7.8 0.37 0.971 Calves lactation average daily gain (kg/day) Males Birth to 2 weeks 0.551 0.459 0.0410 0.295 2 to 4 weeks 0.313 0.366 0.0281 0.384 4 to 6 weeks 0.326 0.367 0.0224 0.391 6 to 10 weeks 0.380 0.344 0.0317 0.617 10 to 14 weeks 0.388 0.418 0.0354 0.704 14 to 18 weeks 0.304 0.269 0.0633 0.809 Birth to 18 weeks 0.370 0.362 0.0238 0.875 Females Birth to 2 weeks 0.500 0.471 0.0412 0.744 2 to 4 weeks 0.304 0.292 0.0249 0.822 4 to 6 weeks 0.305 0.279 0.0159 0.454 6 to 10 weeks 0.326 0.351 0.0282 0.688 10 to 14 weeks 0.304 0.313 0.0180 0.819 14 to 18 weeks 0.231 0.319 0.0229 0.047 Birth to 18 weeks 0.315 0.334 0.00756 0.220 Calf weaning BW (kg) Males 55.9 54.0 2.98 0.775 Females 47.5 49.9 0.92 0.208 †The experimental unit was the animal (n = 8 and 9 for control and Cu treatment groups, respectively) ‡ Beginning of the trial at day 202 of gestation as average § As average, BW and body condition of hinds were measured 3.5 days before and 3.5 days after calving ¶ Average at 2, 4, 6, 10, 14 and 18 weeks of lactation.

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4.2. Effects of Cu supplementation to hinds on milk production, milk composition, total yields and gross energy content of milk

Supplementation with Cu did not influence daily milk production or milk content of fat, protein, lactose and dry matter. Moreover, no differences were observed between groups for TFY, TPY, TLY, TDMY neither for MGEC. However, TMY tended to decrease with Cu supplementation (P = 0.074). In addition, milk from hinds supplemented with Cu showed a trend of higher content of K (P = 0.068) and a significantly higher content of Rb (P < 0.05) and lower of Al (P < 0.05) than milk from control hinds. However, supplementation did not influence the Cu content of milk (Table 10).

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Table 10. Effects of Cu supplementation of gestating and lactating hinds on daily milk production, milk composition, total yields and milk gross energy content (MGEC) of red deer.

Trait Control Cu injection SEM† P-value Daily milk production (mL/d) 2,535 2,303 94.8 0.494 Milk composition Fat (g/100g) 9.8 10.0 0.21 0.357 Protein (g/100g) 7.0 7.0 0.06 0.414 Protein to fat ratio 0.73 0.73 0.013 0.564 Lactose (g/100g) 4.3 4.4 0.04 0.230 Dry matter (g/100g) 23.4 23.7 0.22 0.155 Milk macromineral composition (g/100g) Ca 0.12 0.13 0.004 0.314 K 0.07 0.08 0.002 0.068 Mg 0.009 0.009 0.0003 0.585 Na 0.02 0.02 0.0008 0.433 P 0.098 0.102 0.0029 0.324 S 0.03 0.03 0.0009 0.320 Milk trace mineral composition (mg/kg) Al 1.24 1.07 0.125 0.047

B 0.96 1.03 0.090 0.512 Cr 0.04 0.04 0.002 0.631 Cu 0.18 0.18 0.015 0.578 Fe 1.16 0.96 0.089 0.431 Li 0.03 0.03 0.001 0.255 Mn 0.54 0.55 0.014 0.764 Rb 0.48 0.56 0.020 0.045 Sr 1.42 1.57 0.069 0.267 Zn 5.79 5.77 0.296 0.309 Total yields Milk (L) 315.7 290.8 6.95 0.074 Fat (kg) 31.0 29.8 0.67 0.360 Protein (kg) 22.5 21.1 0.51 0.160 Lactose (kg) 14.0 13.2 0.32 0.247 Dry matter (kg) 75.5 71.1 4.07 0.608 MGEC (kcal/kg) 1,492 1,516 18.5 0.337 †The experimental unit was the animal (n = 8 and 9 for control and for Cu treatment groups, respectively).

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5. Effect of Mn supplementation of gestating and lactating hinds on milk traits and calf growth (trial 5)

Manganese content of diet came from raw materials and from vitamin and mineral premix that was included at 0.5% of diet (as dry matter basis) providing 75 mg of Mn per kg of diet as MnSO4·H2O. Analyzed Mn content was of 122.6 and 30.5 ppm for diet and unifeed ration, respectively.

5.1. Effects of Mn supplementation to hinds on hind body weight and body condition and calf growth from birth to 18 weeks of lactation (forced weaning)

At the beginning of the trial (first Mn injection at day 140 of gestation as average), hinds were assigned to the treatments so the average BW (109.5 and 106.3 kg for hinds from Mn and control groups, respectively) and body condition (4.0 and 4.1 for hinds from Mn and control groups, respectively) per treatment were the same (P ˃ 0.10; Table 11). Manganese supplementation did not influence BW or body condition of hinds at any stage studied, nor BW of calves at birth. However, calves from hinds that were supplemented with Mn, compared to control ones, tended to have higher relative growth rate from 4 to 6 weeks of lactation (P = 0.06), from 10 to 14 weeks of lactation (P = 0.06) and from birth to forced weaning at 18 weeks of age (P = 0.07).

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Table 11. Effects of Mn supplementation to hinds on hind body weight (BW) and body condition and calf growth from birth to 18 weeks of lactation (forced weaning) of red deer.

Mn supplementation Mn treatment Control SEMa P-value Hind BW (kg) First Mn injectionb 109.5 106.3 2.51 ns Before calvingc 122.6 121.6 0.90 ns After calvingc 106.7 104.1 0.91 ns Average lactationd 107.6 105.0 0.89 ns Hind body condition First Mn injectionb 4.0 4.1 0.07 ns Before calvingc 3.9 4.1 0.03 ns After calvingc 3.8 4.0 0.03 ns Calf birth BW (kg) 8.6 8.8 0.83 ns Calves lactation relative body weight gain (%) From birth to 2 weeks 48.7 55.7 3.86 ns From 2 to 4 weeks 38.9 34.3 1.90 ns From 4 to 6 weeks 27.1 23.9 0.83 0.06 From 6 to 10 weeks 40.0 38.1 2.70 ns From 10 to 14 weeks 30.1 22.5 1.99 0.06 From 14 to 18 weeks 15.8 14.9 1.20 ns From birth to 18 weeks 448.2 398.7 13.42 0.07 a The experimental unit was the animal (n = 12 for Mn treatment and n=8 for control group). b Beginning of the trial at day 140 of gestation. c As average, BW and body condition of hinds were measured 3.5 days before and 3.5 days after calving. d Average at 2, 4, 6, 10, 14 and 18 weeks of lactation.

5.2. Effects of Mn supplementation to hinds on milk production, milk composition, total yields and gross energy content of milk

No significant interactions between Mn supplementation and lactation week were detected for any traits studied. Therefore, only main effects are shown. Supplementation with Mn increased 10.2% daily milk production (P < 0.05; Table 12). In addition, milk produced by hinds injected with Mn had 11.2% more fat (P < 0.001) and 4.2% more dry matter (P < 0.001) than milk produced by hinds from control group. However, Mn supplementation did not influence protein or

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lactose content of milk. In consequence, PFR was higher for milk from control hinds than for milk from Mn supplemented hinds (P < 0.001). Manganese supplementation increased 18.2% the TFY of milk (P < 0.05), but no differences between treatments were observed for TMY, TPY, TLY or TDMY. Manganese supplementation increased the MGEC of fresh milk (P < 0.001).

Supplementation with Mn increased the milk content of Ca (P < 0.001), P (P < 0.05), Mn (P < 0.001), Sr (P < 0.001) and marginally Rb (P = 0.07). However, milk from hinds supplemented with Mn had less Na content (P < 0.05) and marginally less Cu (P = 0.06) than milk from hinds of control group. No differences were observed for the Mn content of serum at basal level measured the same day of the first injection with Mn (0.15 ppm for Mn injected group and for control group). Also, no differences were observed either at week 10 of lactation (0.16 and 0.15 ppm for Mn injected group and for control group, respectively).

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Table 12. Effects of Mn supplementation to hinds on milk production, milk composition, total yields and gross energy content of milk of red deer.

Mn treatment Control SEMa P-value Daily milk production (mLday-1) 2,475 2,223 69.0 * Milk composition Fat (%) 9.8 8.7 0.19 *** Protein (%) 7.0 7.0 0.05 ns Protein to fat ratio 0.75 0.83 0.012 *** Lactose (%) 4.9 4.9 0.04 ns Dry matter (%) 23.6 22.6 0.18 *** Total yields Milk (L) 310.8 283.9 13.00 ns Fat (kg) 29.7 24.3 1.33 * Protein (kg) 22.5 20.4 0.92 ns Lactose (kg) 16.0 14.5 0.73 ns Dry matter (kg) 74.6 65.4 3.12 ns Milk gross energy content (kcalkg-1) 1,497 1,409 16.8 *** Milk mineral composition Ca (%) 0.22 0.20 0.002 *** P (%) 0.20 0.19 0.002 * Mg (%) 0.017 0.016 0.0002 * Na (%) 0.031 0.033 0.0004 * S (%) 0.05 0.05 0.0008 ns K (%) 0.15 0.14 0.002 ns B (ppm) 0.23 0.23 0.004 ns Cu (ppm) 0.16 0.18 0.0096 0.06 Fe (ppm) 0.46 0.43 0.016 ns Mn (ppm) 1.04 0.85 0.013 *** Sr (ppm) 3.4 3.0 0.06 *** Al (ppm) 0.81 0.82 0.024 ns Li (ppm) 0.03 0.02 0.0008 ns Cr (ppm) 0.03 0.02 0.0004 0.05 Rb (ppm) 0.83 0.77 0.015 0.07 Zn (ppm) 11.6 13.0 0.29 * Serum Mn content (ppm) Basal levelb 0.15 0.15 0.0027 ns Week 10 of lactationc 0.16 0.15 0.0053 ns a The experimental unit was the animal (n=12 for Mn treatment and n=8 for control group). b Day of first injection with Mn. c 168 days after the first Mn injection.

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6. Osteophagia in key periods of red deer (trial 6)

During the 210 days that cameras stayed working, we recorded 811 videos where wildlife was observed. A total of 1030 animals were seen in the videos, corresponding mainly to red deer (90.5%), wild boar (9.2%) and three foxes (0.3%). No fox or wild boar was observed to interact with the antlers.

Wild boars were curious about the antlers during the first month, when sniffing was frequent. Thereafter, they were just observed crossing the area with low or no interaction with the antlers. Thus, all consumption of antlers can be assigned to red deer, as 47.7%of deer in the videos were observed consuming the antlers.

The frequency of appearance of deer in the videos was similar for males and females (35.3 vs. 36.3%), but lower for calves (17.1%). The remaining (11.4%) were deer which could not be assigned to any sex/age category since the head was not visible in the video (obviously, these animals were not chewing the antlers). Chi-squared test showed that, in general, deer visited antlers mainly in May–June and September, with a sharp decrease in July and August (Table 11). The three sex/age groups showed different patterns of consumption during the study period (the percentage of animals chewing antlers is shown in Table 13). Overall, 53.9% of females were observed eating antlers. Chi-squared tests showed differences among months: March and April showed lower values, May and September intermediate values, while June, July and August showed the highest values. For males, 66.3% were observed chewing the antlers, with an important peak in June.

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Table 13. Summary of monthly observations of red deer observed in camera-trap videos in an experimental setting were antlers were offered for studying osteoghagia. Results show the total number of animals observed, the number of animals eating the offered antlers, and the percentage of animals observed which ate the antlers. Chi- squared test showed the existence of overall monthly differences in the observations. Highlighted in bold appear those months when the observation of animals (total and eating) was 50% greater than expected in an homogeneous monthly distribution.

2 Mar. Apr. May Jun. Jul. Aug. Sep. Χ 6 P-value

Total number 17 16 67 35 13 19 122 236.0 <0.001 of females Females 5 7 36 25 10 14 70 134.4 <0.001 eating antlers

% Females 26.5 37.5 51.5 71.3 69.9 64.9 56.1 31.7 <0.001 eating antlers

Total number 2 13 57 73 13 29 97 188.5 <0.001 of males

Males eating 2 7 26 69 8 19 50 144.4 <0.001 antlers

% Males 100.0 53.9 43.9 92.0 61.5 62.1 50.9 41.2 <0.001 eating antlers

Total number 17 22 25 5 6 6 50 82.5 <0.001 of calves

Calves eating 8 18 15 0 4 4 8 30.1 <0.001 antlers

% Calves 47.1 75.0 57.5 0.0 66.7 66.7 14.3 105.8 <0.001 eating antlers

Finally, 46.2% of calves were observed chewing antlers, but consumption was lower from June to August with respect to the other months. Kruskal-Wallis tests showed differences in antler chewing behavior during dusk (18:00 to 02:00), dawn (02:00 to 10:00) and day (10:00 to 18:00) in females, calves and males of Iberian red deer, respectively. Significant differences among the three periods of day were found for males and females (P < 0.05), with higher values in the dusk, intermediate values in the dawn and lower during the day. Calves only showed a trend for greater activity during the dusk (P = 0.06; Figure 7).

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Figure 7. Mean monthly occurrence of red deer (females, males and calves) eating antlers at three periods of the day: Dusk (18h00 to 02h00), Dawn (02h00 to 10h00) and Day (10h00 to 18h00). Within each group, Kruskal-Wallis test show differences in the mean occurrence among those day periods.

All the observations accounted for the consumption of 966 g of antler bone material in all the 15 antlers displayed (i.e., 80.7 ± 26.8 g per antler), which means 8.7 ± 2.1% of each whole mature antler. Deer always started to chew the antlers by the tines.

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Felix qui potuit rerum cognoscere causas

Virgilio

Chapter VI DISCUSSION

Chapter VI

1. Effect of Cu Supplementation on antler properties and mineral composition of red deer male (trial 1)

Our results indicated a limited effect of Cu supplementation on antler characteristics and mineral composition. There may be a number of factors underlying these data. First, and importantly, animals were fed a balanced diet that exceeded their nutrient requirements (NRC, 2007). Second, the dose of Cu used for supplementation was relatively small, because excess of Cu is toxic for some ruminants, including sheep (Winge and Mehra, 1990) and deer (Fairley, 2008).

Wilson and Grace (2001) proposed reference criteria for deer serum Cu content: values <0.32 ppm were considered to indicate deficiency; values between 0.32 and 0.50 ppm were considered marginal; and values >0.50 ppm were considered adequate. In the current trial, the average serum Cu content from the yearling and adult control groups were 0.68 and 0.59 ppm, respectively. Therefore, according to the criteria of Wilson and Grace (2001), even deer in the control group had adequate serum Cu levels in this study. In early studies, Strause et al. (1986) showed that either Mn or Cu deficiency led to reduced Ca and P content in the bones of rats, and the animals did not appear to suffer from any other problems. In the current trial, there was no general effect of Cu supplementation on Ca or P levels or the Ca/P ratio in either yearling or adult deer. It is possible that, if this study had been conducted using deer suffering from Cu deficiency, the results would have been very different from those reported here. Different patterns of serum Cu levels were observed in yearling and adult deer during this study. The percentage increase of serum Cu

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content was higher in adults than in yearlings (38% vs. 28%). One reason for this may be that the blood Cu content in the control yearling group was much higher than that of adult controls. The fact that the percentage increase of Cu content in adult serum was higher than that in yearlings may account for the greater effect of Cu supplementation on the antlers of adults than yearlings. These results indicate that Cu may be more highly mobilized in adults than yearlings. Given that the same dose of Cu was administered to each group, one possible explanation for such an effect could be that the higher metabolism of yearlings and their greater need for minerals (and for nutrients in general) may have led to a faster use of the injected Cu in these animals, and a consequent reduction in their serum levels. In general, plasma mineral levels vary with deer age (Kucer et al., 2013). In fact, in a previous Mn supplementation experiment carried out by our group (Cappelli et al., 2015), adults showed a wide range of effects, whereas yearlings only exhibited increases in Mn and Fe antler content. However, in the current study, the variation of serum Cu level with age was higher than expected. Our data include more blood samples taken at different dates to assess the influence of Cu supplementation on the evolution of serum mineral content, comparing both yearling and adult deer. Although Cu injections did not influence antler characteristics as extensively as expected, supplementation did influence some of the traits investigated. The most interesting effects of Cu supplementation were the trend towards increased cortical thickness and the attenuation of reduction in cortical thickness from the base to the top of antlers, both of which were observed in adults. The increase in cortical thickness may increase the structural resistance of bone to fracture. The mechanical performance of structures depends on two types of factors (Landete-

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Castillejos et al., 2012a): those depending on structure, such as cortical thickness, diameter or area, and those derived from the mechanical quality of the material, such as E, BS, W or U. In fact, diameter and cortical thickness account for 55% of the variation in the mechanical performance of bones (Davidson et al., 2006). Moreover, given that an increase in cortical thickness influences antler weight, supplementation with Cu, even when animals are receiving a balanced diet, may affect the trophy value of antlers, as weight is an influential measure in the International Council for Game and Wildlife Conservation method of trophy valuation. In the current trial, which is the first to investigate mineral content in both serum and antlers, the increase in serum Cu content did not result in an increase in the content of this mineral in antlers, demonstrating the limitations of using serum Cu content to assess bone Cu status. These results may have several contributing factors. It is possible that increases in serum Cu are not reflected in antlers. Alternatively, serum Cu may be captured by the liver, or other organs with higher priority for uptake than bone tissue. Indeed, the liver is an organ where Cu is stored, while the amounts of this mineral in bone are very small (Suttle, 2010).

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2. Effect of Cu Supplementation on mineral serum content of red deer male during antler growth and rut (trial 2)

In the present study, we provide evidence that Cu supplementation (0.83mg/kg BW) in red deer feed with a diet and unifieed (28 mg/kg and 11.8 mg/kg of Cu according with Gambin et al., 2017b), above standard requirements of Cu (8 mg/kg) stablished by Grace et al. (2008), increase serum level of P and Cu, without affect mineral profile of antler. In addition, results showed that, in general, mineral content of serum during antler growth cannot be used to predict mineral profile of antler, except for Fe. Finally, serum mineral profile varied throughout antler growth and rut as consequence of numerous physiological changes that take place during these processes.

L´abbe et al. (2003) proposed that micro nutrient supplementation above standard level of reference could produce beneficial unexpected effects (i.e. folic acid supplementation decrease neural tube defects in embryogenesis in mammals). In this sense, in the current trial, Cu is a micronutrient and its supplementation via intramuscular injections increased by 7.6% P content of serum. This mineral is key for antler growth (as hydroxyapatite), being the main mineral of antler bone (Estévez et al., 2009). Also, it has been demonstrated that high content of P in serum increases bone mineral density in while-tailed deer (Grasman and Hellgren, 1993). Based on the positive relationship between Cu supplementation and P deposition in bone that has been demonstrated in pigs by Davin et al. (2016), we expected an increment of P in antlers in deer Cu supplemented. Nevertheless, no increment of P antler content mediated by Cu

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supplementation was observed. The cause of difference are unknown but might be due to pigs diets, which were also supplemented with phytase that improve P availability while in current trial only Cu was supplemented.

Additionally, in the current research, Cu supplementation increased serum Cu content by 11.2% although showed lower values to those reported by Walker et al. (2002) who found that Cu supplementation (20 g of copper-oxide wire particles) to 2-years old red deer stags during antler growth increased Cu of serum by mean of 21%. The difference among results from current trial and those from Walker et al. (2002) could be attributed to kind of supplementation (subcutaneous injection vs intrarruminal particules). In this sense, copper-oxide wire particles used by Walker et al. (2002) seem to be more effectives than subcutaneous Cu injections used in current trial. The increment of Cu serum content could enhance the quality of antler by increase of antler cortical bone as consequence of stimulation of chondrogenesis, as it was showed by Gambín et al. (2017b). Current research studied if serum mineral profile during antler growth and rut could serve as an indicator of mineral profile of antler at day 165 of SAG because minerals are transported via blood to antler during its development. In consequence, the objective was can predict the mineral content of antlers from mineral content of serum and, therefore, prevent mineral deficiency that could impair antler quality. In fact, only Fe content of antler at day 165 of SAG was positively correlated with Fe content of serum at day 132 of SAG in control group (but not in Cu supplemented stags). These results imply that Fe antler content at day 165 of SAG might be predict by Fe content of serum at day 132 of SAG.

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Nonetheless, no correlation was found for the other minerals. Lack of correlations between mineral content in serum during antler growth and this in antler at day 165 of SAG in the current study might be due to homeostasis of minerals and systems of excretion (Berndt and Kumar, 2009), which implies a constant regulation of minerals, even when they are mobilized during antler growth

During antler growth period, some minerals (such as Ca, P and Mg) are need for mineralization (Dryden, 2016). This process not follows a constant velocity neither starts at the same place of antler at the same time according with Gómez et al. (2013). Firstly, mineralization of antler lamellar bone takes place from day 28 to 74 SAG. Then, mineralization of cortical bone is produced from day 70 to 135 with a peak of mineralization around day 110 SAG. In consequence, the highest values of Ca, P and Mg of serum observed at day 54 SAG in the current study might be due to the mineralization of antler lamellar bone while antler mineralization is already completed at day 132 (Gómez et al., 2013). However, Kuba et al. (2015) suggested that during antler mineralization Ca, P and Mg may decrease in serum due to they are sequestered by antler.

Results also showed that growth periods affected Cu content of antler. In our study, the levels of Cu in serum showed the greatest values at day 54 SAG that corresponds to mid-May month. This value could be explained by the high activity of lysil-oxidase which need Cu as cofactor to form collagen during antler growth (Price et al., 1996; Hyun et al., 2004; Suttle, 2010). These results agree with those from Jeon et al. (2011) who found the highest antler collagen content in the middle of antler growth around day 65 SAG in elk.

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Rut period occurs when antler finishes its growth and development, coinciding with day 197 SAG. In this period, stags suffer an important physiologic effort (Mysterud et al., 2008). In the current trial, the second peak of serum Cu content occurred during rut. This result might be explained by an increment in superoxide dismutase (SOD), that is a potent enzyme that protects from oxidants and requires Cu as cofactor (Suttle, 2010). In fact, Koziorowska-Gilun et al. (2015) found an increment of SOD in testicular and epidydimal tissues during matting season in roe deer. The reason for the peak of Cu in serum may lie to reproduction functions since, in the current study, day 197 coincides with the month of October, i.e. rut period. Although we are discussing the importance of serum minerals in relation with antler, we should not disregard that content of minerals in serum may respond to the demand of some other organs or systems.

Values observed for Cu serum (0.77 mg/kg as average) were in accordance with those found by Wilson and Grace (2001) and below than toxic effects levels (1.3-1.7 mg/kg) suggested by Laven and Wilson (2011). Other trace element that reached a peak in serum during rut period was Zn. This mineral form part of the alkaline phosphatase, which is needed for the mineralization of bone. In fact, its content in the antler increases during mineralization and disappears later (Landete-Castillejos et al., 2012c). Nevertheless, antler mineralization is supported by mobilization of minerals from bone. In consequence, a period after end of the mineralization of antlers is needed to recover the losses of minerals in bone. Thus, Zn would still be needed after antlers have been mineralized but in support of remineralization of the skeleton rather than the antler (Baxter et al., 1999). In fact, these authors found that rib porosity was a 52% higher at day 123 SAG than at day 170 SAG

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indicating that the skeleton requires recovering its mineral status when antler growth has finished. The fact that the lowest content of Zn observed in serum was during the antler growth (days 54 and 132 SAG) may be consequence of that antlers up taking as much serum Zn as they can support mineralization. Therefore, Zn might be uptaken from serum for alkaline phosphatase during antler mineralization, and upon completion of this, and might be used to remineralize the skeleton. In addition, results for Zn serum content (0.66 mg/kg as average) were within 0.51-3.40 mg/kg ranged by Roug et al. (2015) in mule deer and did not exceed the recommended values 0.50-1.00 mg/kg published by Puls (1994) for ruminants.

Overall, these results mentioned agree with Kuba et al. (2015) who suggest that changes in serum mineral content may be explained by physiological changes that take place during antler growth and rut.

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3. Effect of Mn and Cu supplementation on microhardness of antler (trial 3)

The main purpose of this study was to investigate if Mn or Cu supplementation produced differences in HV on osteons of antler from red deer in both adults and yearling. Interestingly, current research presents first time the evidence of Mn and Cu supplementation enhances microhardness in antler bone tissue. According with our findings, Cu and Mn supplementation affected positively HV of osteons of antler, especially in yearling. Apparently, there is no other study determining the effect of Mn or Cu supplementation on HV of antler, therefore authors from the current trial could not compare results obtained with other that assed the influence of Mn or Cu subcutaneous injections on HV of osteons of antlers or bones.

In present study, hardness was always obtained in medial region avoiding heterogeneity in results. This procedure was in accordance with Ferreño et al. (2017) who observed that osteons of femur from rat showed greater hardness in the anterior region than in the posterior region. They suggested that difference in the behavior of the bone tissue based on their location is due to stress state in the bone under in different conditions (produced by daily life of the animal) involved the alteration of the pattern bone remodeling. The latter is consistent with the idea that the bone form reflects in some way its mechanical loading history during life, this has been supported by the theory first call "wolff's law" and then recently "bone functional adaptation" (Huiskes et al., 2000; Ruff et al., 2006). Additionally, our data suggested that microhardness of antler did not increase with age of animal. Furthermore, it seems that microhardness is greater in yearling than in adults (although any analyses

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were performed because there was not the objective of current study), contradicting the finding of significant age-dependant increase of microhardness in animal cortical bone by Chittenden et al. (2015). This may be due to the existence of bone remodeling (Smith et al., 2010) in antler from red deer is scarce compared to internal bone (Gomez et al., 2013).

Mechanical properties of bones, such as hardness, are a consequence of interaction among mineral and collagen contents and a small change in the amount of mineral can have drastic effect in bone structure or mechanical properties (Evans et al., 1990). In this sense, the only study concerning mineral supplementation on HV was carried out by Hakki et al. (2015) and concluded that borom supplementation in diet did not affect HV of osteons of teeth. Nevertheless, Cappelli et al. (2015) reported the importance of other minerals as Mn in antler bone properties. In this sense, our results are in agreement with these authors since data showed the improvement of HV in yearling Mn treated. This increment in HV could be due to antler content of Fe is increased twofold in yearlings treated with Mn compared with control group (Cappelli et al., 2015). In fact, a recent study in vitro, showed that HV of artificial hydroxyapatite structure (main substance of bones and antler) was increased when Fe3+ was added to hydroxyapatite (Alshemary et al., 2017).

Apart from Mn, Cu has also been proposed as a relevant mineral in antler quality. This may be supported by essential role of Cu in the normal maturation of collagen (Prohaska, 2006), which is implicated in the chondrogenesis of growing antler in red deer (Price et al., 1996). Furthermore, according Opsahl et al. (1982) Cu deficiency increased

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fragility of bone. In this sense, our finding are in consonance and confirm the importance of Cu in antlers and bones. A possible reason of this improvement of hardness in both, adults and yearling Cu treated might be due to an increase in bone mineral density. In fact, it is known the existence of a positive correlation between bone mineral density and bone hardness (in bovids, Houde et al., 1995) and the existence of a relationship between serum Cu content and bone mineral density (Alghadir et al., 2016). Furthermore, it has been demonstrated that Cu deficiency reduce bone mineral density (Sierpinska et al., 2014). In addition, the improvement of HV due to Cu supplementation is in concordance with Gambín et al. (2017b), who suggested that Cu supplementation could have potential implications for humans bones.

Data from current trail showed that HV of base of antler was by 16-20 % harder than HV of base of crown in adults, while yearling had by 5.8 % greater in base of antler than second third of the main beam. This may be caused by physiological effort made during the antler grow period. In fact, Landete-Castillejos et al. (2007a) suggested that differences between mineral composition from the burr to the base of crown might be caused by physiological exhaustion, which indicates depletion of body mineral stores or physiological exhaustion and affects mechanical properties of antler. In this sense, results from current trial showed that Mn supplementation reduced the effect of physiological exhaustion on HV of osteons. This is in concordance with Cappelli et al. (2015) who showed the increment of Ca and P content of antler and also an improvement of impact work at the top of antler (showing most clearly physiological exhaustion) in adults deer treated with Mn. Results also showed that physiological exhaustion (expressed as a percentage), was not influenced by Cu supplementation. Nevertheless, direct

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measures showed that HV of osteons at the top of antler analyzed of Cu treated group was statistically higher than in control group (in both adults and yearling). This suggest that results obtained by direct measures of HV at the top of antler not always coincide when data are studied by indirect measured (percentage), which indicates physiological effort or physiological exhaustion. Moreover, it is known that microindentation might be correlated with the overall mechanical property of bone tissue (Grover et al., 2016). In this sense, in spite that our finding showed clearly the improvement in HV due to Mn and Cu supplementation in yearling, previous works did not detect enhance in mechanical properties in yearling from red deer (Cappelli et al 2015; Gambín et al., 2017b). The positive effect found in yearling in current trial could be explain to the ability of microindentation to detect the minimal changes in mechanical properties of bones (Dall'Ara et al., 2012).

Due to antler are an interesting model for research in bone biology (Landete-Castillejos et al., 2007a,2012a) and that antler have been used as a model of osteoporosis (Landete-Castillejos et al., 2012b), result from current trial could be applied to field of osteoporosis. Indeed, a previous study have showed that patients with osteoporosis have osteons less hard and with less bone density than controls (Boivin et al., 2008). Therefore, based on results from present trial, we proposed that Mn and Cu supplementation could be useful in patients with osteoporosis because increment HV of osteon in cortical bone. Other data carried out in antler that could support this proposal are that Mn supplementation increase content of Ca, P and Fe of antler bone tissue (Cappelli et al., 2015), and Cu supplementation increment mineral content of Sr and Zn (Gambín et al., 2017b), which could increase mineral bone density.

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4. Effect of Cu supplementation of gestating and lactating hinds on calf growth and milk traits (trial 4)

In the current research, injections with Cu were administered to hinds from day 202 of gestation (as average) to the end of lactation at 18 weeks of age (forced weaning) and ADG of offspring was calculated. Copper supplementation was provided during part of the last third of gestation since this is the period during which the higher offspring growth takes place. However, any effect of Cu supplementation of gestating and lactating hinds on birth or weaning BW of calves was observed. The only effect observed was an increase of ADG of female calves from 14 to 18 weeks of lactation when their mothers were supplemented with Cu.

Authors have not found any paper that studies the influence of Cu subcutaneous injections of gestating and lactating hinds on birth BW or growth performance of calves during lactation to compare with the current results. The cause of the lack of influence of Cu supplementation of gestating and lactating hinds on growth rate of calves during lactation was unknown but might be due to some factors such as the Cu content of diet consumed by hinds at the time of supplementation. In this sense, Cu injections of hinds might be useful to improve growth rate of progeny during lactation (Grace and Knowles, 2012) and to affect the Cu status of their fawns (Grace et al., 2003) when reproductive hinds are deficient in this mineral but not when its needs are fulfilled as occurred in the current paper where hinds were fed with a balanced diet that met or exceeded the nutrient requirements of deer (NRC, 2007). Results obtained in the current trial agreed with those of Bartoskewitz et al.

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(2007) who conclude that Cu supplementation has not significant effects on male BW providing diets with higher levels of Cu than in the current paper (200–236 ppm Cu vs. 28.0 and 11.8 ppm of Cu provided by the diet and the unifeed, respectively) directly to mature (> 4 years old) male deer over an 18 months period and to 1∙5 years old (as average) male deer over a 1-year period. In addition, Grace et al. (2003) conclude that serum Cu concentrations must be very low (< 1 µmol/L) and remain so for several months for a BW gain response to Cu supplementation in deer. In the current trial, the serum Cu content was higher than this value in all cases (0.46 and 0.64 mg/kg at 7 days after the first Cu injection for control and Cu supplemented groups, respectively; data not shown). In consequence, BW gain would be a relative insensitive monitor of Cu status (Nicol et al., 2003) when Cu is not deficient in deer.

In the current research, Cu supplementation of gestating and lactating hinds decreased the TMY but no effects were observed for daily milk production, milk composition and total nutrient production. However, no studies about the influence of Cu injections of hinds on lactating traits are available on deer. Contrary, information available about the influence of Cu supplementation is extensive for cattle (Chase et al., 2000; Engle et al., 2001; Sinclair et al., 2013). For instance, Sinclair et al. (2013) find a positive trend in BW gain for lactating cows treated with organic Cu. In addition, these authors observe that supplementation of cows with organic Cu increase the milk fat content and decrease the milk yield, in agreement with results from the current trial for milk yield. Also, Rabiee et al. (2010) observe that supplementation with organic trace minerals, including Cu, increases daily milk production by 0.93 kg, milk fat by 0.04 kg and milk protein by 0.03 kg, in contrast to results observed in the current trial. The

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discrepancies between authors are unknown but might be due to that Rabiee et al. (2010) provide a mix of trace minerals while in the current paper only Cu was supplemented.

Copper supplementation did not influence milk Cu content. Copper secretion in milk is reduced when the dietary Cu supply is inadequate (Whitelaw et al., 1983) but cannot be increased by supplementing a diet with an adequate Cu level (Suttle, 2010). Grace et al. (2005) suggest that, when balanced diets are used as in the current trial, increase of the Cu status of calves could only be achieved by Cu supplementation of their dams prior to mid-gestation (instead of to last third of gestation, as in the current trial) or possibly by direct supplementation of calves (instead of their mothers, as in the current trial). In addition, milk from hinds supplemented with Cu presented higher content of K and Rb and lower of Al than milk from control hinds. However, authors have not found any research studying the effect of Cu supplementation of gestating and lactating hinds on mineral profile of milk from deer.

Based on results from our own group with stags (Gambín et al., 2017b) and those from the current trial with hinds, the management of Cu supplementation should be different for stags and hinds in deer farms. The effects of Cu supplementation in red deer stags, in particular for antler characteristics, have been previously studied (Walker et al., 2002; Gambín et al., 2017b). In general, Cu supplementation may increase growth of cervids (Handeland et al., 2008) as well an improve of disease resistance (Bartoskewitz et al., 2007). Also, Gambín et al. (2017b) observe an increase in antler cortical thickness when Cu is supplemented as long-term injections to males fed with a balanced diet. The higher

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cortical thickness makes antlers both heavier and less likely to break which increases their value as trophy. In consequence, Cu supplementation as long-term injections is recommended for adult males fed with a balanced diet during the growth period of antlers.

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5. Effect of Mn supplementation of gestating and lactating hinds on milk traits and calf growth (trial 5)

Studies about the effects of mineral supplementation in red deer are focused mainly on antler development and antler bone tissue characteristics (Cappelli et al., 2015; Gambin et al., 2017b) or internal bones (Olguin et al., 2013). Also, previous authors assessed on red deer the influence of mineral supplementation supplied as salt licks on milk production and nutrient composition (Ceacero et al., 2009; Malacarne et al., 2015) and its effect on calf performance (Gallego et al., 2009). However, to our knowledge there is no any study in the literature involving a fixed dose of one injected mineral (Mn as in this case) in late-gestating and lactating hinds to compare with the current results.

Results found in this study showed that supplementation of late- gestating and lactating hinds with Mn did not affect BW of calves at birth but tended to produce calves with higher increase of relative BW from birth to weaning at 18 weeks of age compared to control hinds. Regarding the effect during gestation, the lack of effect for calf birth BW is coherent with the findings reported by Sprinkle et al. (2006) who supplemented late-gestating beef cows with trace minerals (in organic or inorganic form) and found that this did not impact calf birth BW, despite the fact that many minerals, like Mn are transported across the placenta. Perhaps the period of gestation in which we supplied Mn is not enough, or effects were so subtle that could not be shown with our small sample size (which, furthermore, was split in two by sex of the calf). However, some positive effect should be 336 expected since Mn plays an important

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role on the animal growth, particularly in the synthesis of protein and cartilage mucopolysaccharide and in the metabolism related with the energy, lipids and vitamins (Suttle, 2010). In fact, we found a positive effect of Mn on the higher relative BW gains from birth to weaning. The effect achieves only marginal significance because we used a small sample size, and this effect could be further obscured because each group was split in two by calf sex, so that it could only be revealed when controlling for calf birth BW (higher for males) by expressing gains in percentage of birth BW. Such effect may have two causes that are not mutually exclusive: 1) a direct effect of the greater content of Mn in milk from supplemented mothers and 2) and indirect effect because supplementing with Mn improves the production of milk nutrients (fat, Ca, and P), and these, in turn, usually affect calf growth. Thus, milk from hinds supplemented with Mn had 11.2% more fat (17.8% higher TFY) than milk from control hinds. The increase of fat content of milk from Mn supplemented hinds could be through its role as cofactor of the acetyl-coA carboxylase phosphatase enzyme that is implicated in the lipid biosynthetic pathway (Thampy and Wakil, 1985).

In addition, milk from hinds supplemented with Mn had higher content of some minerals such as Ca, P (both take part of skeleton) and Mn than milk obtained from control hinds. Gallego et al. (2009) concluded that milk minerals (such as Ca, P and Fe) explained more than a third of the total variability explained by the model for calf growth. Therefore, the increase of fat, Ca and P content of milk observed in the current trial could be the cause for the higher growth rate of calves from Mn supplemented hinds. One of the mechanisms of the observed effect may be that Mn may increase the growth of some tissues of the body of calves (e.g. skeleton) which, in turn, would prompt the calves to demand

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higher milk production by the mother, and this would end up in changes in milk production (in fact, it increased 10.2% with Mn supplementation in the current trial) and fat content. Fat is the milk component that can be more easily mobilized in support of lactation (Landete-Castillejos et al., 2003b) in contrast to protein (Kaufmann, 1979; Landete-Castillejos et al., 2003b). Current results agree with the increase of TFY observed in cows after trace mineral supplementation including Mn (Ballantine et al., 2002; Griffiths et al., 2007) without any effect on TPY (Griffiths et al., 2007; Siciliano-Jones et al., 2008).

The increment in Ca and P content of milk from the supplemented group was not expected since, in general, the content of macrominerals in milk remains rather stable. Calcium content of milk might be near maximum, and its concentration is important maintaining the transfer of Ca to calf (Gallego et al., 2006). The Ca supply is very important because a reduction in transfer may affect Ca content in calf bones, and small changes in such content can produce large changes in bone mechanical properties (Currey, 2003). Thus, it is very surprising that such concentration in milk varies with Mn supplementation. However, authors previously observed that Mn supplementation increased the content of Ca and P on antlers from adult deer (Cappelli et al., 2015). Bone turnover is increased during lactation resulting in a mobilization of 5-10% of Ca from bone to milk (Kovacs, 2005). Therefore, the increase of Ca and P content of milk with Mn supplementation could be the result of an increase of the bone turnover to deliver the increased content of these minerals in milk.

The increase of Mn content in milk after its supplementation, as occurred in the current trial, has been observed previously in milk from

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humans (Vuori et al., 1980) and cows (Archibald, 1958). In fact, Vuori et al. (1980) found a positive correlation between Mn consumption and the content of this mineral in milk. Transition element cations (such as Cu, Zn or Mn) have concentrations in blood tissues and milk that are largely independent on their intake (Pechova et al., 2008). In fact, their concentrations are related with the regulation of the gut absorption and vary with the metabolic demands (Windisch, 2002). Therefore, supplementation by injected minerals should result in their increase in milk content, as we found in the current study. However, no differences were observed on serum for Mn content at week 10 of lactation. In consequence, serum concentration of Mn did not reflect the level of Mn supplementation in accordance with Legleiter et al. (2005). The reason could be that Mn is captured quickly from blood by liver. In fact, liver excretion can be increased up to 200 times in response to a high level of Mn serum in cows (Hall and Symonds, 1981). Thus, the increase of Mn excreted by milk of supplemented hinds would be achieved by fast transfer from the liver or tissue storing Mn achieved at a constant blood serum.

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6. Osteophagia in key periods of red deer (trial 6)

Results showed that females and males consumed cast antlers mainly in May–June and September, while calves consumed them mainly in April–May. Besides, females and males showed a similar daily antler chewing behavior: higher values at dusk, intermediate values at dawn and lower during the day. On the contrary, calves showed greater values during dusk and lower for dawn and day.

According to Denton et al. (1986), osteophagia would be a consequence of phosphorous deficiency levels in diet. In fact, the natural vegetation in our study site has a concentration of P below the deficiency levels suggested for deer by McDowell (2003) all year round, and especially during summer (Estévez et al., 2010). However, the consumption of antlers did not increase during summer as would be expected. On the contrary, peaks of consumption matching those periods of increased mineral demands were observed: the end of gestation and the beginning of lactation in females (Oftedal, 1985), the antler mineralization peak in males (Gómez et al., 2013) and late growth for calves. Thus, we may conclude that red deer consumed antlers due to increased mineral needs in these periods. The number of females chewing antlers showed a peak in May, likely due to nutritional needs of the last third of gestation (Oftedal, 1985). Despite the number of females chewing antlers being lower during most part of the lactation period (June–July–August), the percentage of visits was higher in those months, which correspond with lactation (i.e., most of the hinds observed in the videos during summer were motivated for the consumption of antlers).

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This period demands a great amount of minerals (Atwood and Weeks, 2002), and thus, females may use any available source of minerals including antlers.

The number of males chewing antlers peaked in June and September. The increase in June might be explained by antler growth: According to mineral needs establish by Ullrey (1983) for deer, osteophagia may be a good source for the Ca and P needed during the antler growth period. Nevertheless, although mineralization of the antler occurs in July (Atwood and Weeks, 2002), the number of males that consumed antlers was lower in July than in June, which agrees with other studies that showed the same pattern for other mineral-consumption behaviors (Atwood and Weeks, 2002; Ping et al., 2011). It may be argued that males store minerals in their bones in June, in order to use them in July during the mineralization period, since antler mineralization cannot be fulfilled from the diet and requires certain demineralization of the skeleton (Banks et al., 1968; Muir et al., 1987; Baxter et al., 1999).

The number of males consuming antlers in September was also high and it could be interpreted as a way for males to recover the minerals removed from the skeleton during the mineralization of the antlers (Baxter et al., 1999). That means that it is bone depletion more than actual daily needs that drives consumption by males. For calves, we found high consumption in April and May (i.e., calves from the previous year) but low from June to September (newborn calves). Although the percentage of calves chewing antlers was 67% during July and August, it is necessary to indicate that the number of newborns that were recorded was on average 6 per month, compared to 25 observed in May (Table

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11). That reflects scaling mineral demands with weight in calves, together with the depletion of milk as mineral source.

Our results also show that all sex/age classes prefer to chew antlers during the dusk. This agrees with observations of Ping et al. (2011), which show the same pattern of use of salts licks by sika deer. This is not surprising because deer are more active during twilight and during the night than the rest of the day (Carranza et al., 1991). However, although the mean number of females eating antlers during dawn and day was almost the same, they were classified into two different subsets in Kruskall-Wallis test. This is because during the day the number of females chewing antlers was 0 in every month, but September, when it raised to 47 (the highest number observed in any combination of sex/time/month).

Sibbald (1994) also observed high values of food intake during daylight in September, but there is no satisfactory answer for that change in their behavior. In general, seasonal consumption of antlers observed in our study may be explained by the hypothesis of seasonally increased requirements suggested by Ceacero et al. (2014). Although antlers are available during the whole year, they seem to be mainly consumed by each sex/age category in the period with their greatest requirements and greatest relative benefits, such as lactation, weaning and growth of antlers. The only drawback for the support this hypothesis is that consumption was quite low, only 8.7% of each antler, which suggests that antlers in the wild may take several years before being totally consumed. However, the fact that antlers were artificially placed and held together could have a negative effect on consumption.

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Finally, although this study concerns to red deer, we also detected wild boar interacting with the antlers, but they did not consume them. This suggests that it is not a common behaviour in this species, in contrast to that reported by Greenfield (1988) and Fillios (2016) for bone consumption. This suggests that future investigation of additional factors influencing bone consumption by wild boar is necessary. Fat may be the key, since hard antler has very low-fat content (<5%; Pathak et al., 2001), while bones have a remaining grease coating on the medullary surface of broken shaft specimens (Domínguez-Solera and Domínguez-Rodrigo, 2009) and very high fat percentage in the bone marrow (>50%; Murden et al., 2016).

Results, considered all together, suggest that microminerals (Cu and Mn) play important roles in red deer during the antler growth in males and lactation period in females. In addition, antlers are consumed as natural mineral resource supplementing mainly Ca and P during these key periods in red deer.

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Antler Serum Hinds Osteophagia

Chapter VII CONCLUSIONS

Chapter VII

Copper

I. Supplementation with Cu (0.83mg/kg of body weight) increases the cortical thickness of antler bone in adult deer, even when deer are fed with a balanced diet.

II. In subadults and adult male red deer, Cu injection can be used to increase contents of P and Cu in serum even when deer are fed with a mineral balanced diet.

III. Serum mineral profile changes from day -36 of starting to antler growth (SAG) to day 197 SAG, thus serum mineral changes with stage of antler growth.

IV. At the osteon level, hardness of Vickers (HV) is affected positively by Cu supplementation in yearlings and adults of red deer male. Likewise, Cu supplementation reduces the effects of physiological effort on antlers bone tissue that is reflected on HV of antlers osteons in both yearlings and adults.

V. In females fed with a balanced diet, Cu supplementation with subcutaneous injections is not interesting from day 202 of gestation (as average) to the end of lactation at 18 weeks to improve growth performance of offspring from birth to weaning and lactation traits (milk production and composition).

Manganese

VI. Supplementation with Mn (2mg/kg of body weight) increases HV at the osteon level in yearlings of red deer. Likewise, Mn supplementation

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reduces the effect of physiological effort on antlers bone tissue that is reflected on HV of antlers osteons in yearlings.

VII. Supplementation with Mn in hinds is advantageous in deer farming, even when animals are fed with balanced diets, because increases daily milk production and content of fat, Ca and P of milk, which, in turn, improve offspring growth from birth to weaning at week 18 of lactation.

Osteophagia

VIII. Osteophagia in red deer is consequence of increment of mineral requirements in key periods: antler mineralization for males, end of gestation in hinds and during the parturition period for yearlings to compensate the increasing needs together with the forced weaning due to the newborns.

Final Conclusion

Based on the results obtained in this thesis, I recommended to deer farmer, even when deer are fed with a balanced diet, the use of artificial mineral supplementation by subcutaneous injection. For red deer males, I recommend supplementation of Cu (0.83mg/kg of BW) and Mn (2mg/kg BW) during antler growth. For hinds I recommend supplementation of Mn (2mg/kg BW) during last third of gestation and lactation. This practice would improve antler quality in males and calf growth and milk quality in case of hinds. Moreover, I recommended to the wild deer managers in Mediterranean basins to provide solid mineral resources from April to September to satisfy minerals needs in males, females and calves.

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Pecunia regina mundi

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FINANCIAL SUPPORT

Financial Support

This Thesis has been supported by Consejería de Educación, Cultura y Deportes de Castilla-La Mancha within Predoctoral Fellowship Program with reference numbers 2014/10620 and 2016/12998 and also by a grant of the University of Castilla La Mancha co funded by the European Union with number 2015/13630. Research of this thesis has been funded by the Spanish Ministry of Economy (Ministerio de Economía, Industria y Competitividad) within the Estate program of Research, Innovation and Development oriented towards challenges of society, within the framework plan of scientific, technical research, and innovation 2013-2016 and co-funded by the European Union with the reference number RTC-2016-5327-2, by the project No. PEII-2014-004-P (Consejería de Educación, Cultura y Deportes de Castilla-La Mancha) and by the grant IGA-20172007 (Faculty of Tropical AgriSciences, Czech Republic).

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Litterarum radices amaras, fructus dulces

Ciceron

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