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Body weight, early growth and antler size influence antler bone mineral composition of Iberian Red Deer (Cervus elaphus hispanicus)
Bone 40 (2007) 230 – 235 www.elsevier.com/locate/bone
Body weight, early growth and antler size influence antler bone mineral composition of Iberian Red Deer (Cervus elaphus hispanicus) T. Landete-Castillejos a,b,c,⁎, A. Garcia a,b,c , L. Gallego a,c a
Departamento de Ciencia y Tecnología Agroforestal, ETSIA, Universidad de Castilla-La Mancha (UCLM), 02071 Albacete, Spain Instituto de Investigación en Recursos Cinegéticos, IREC (CSIC, UCLM, JCCM), Campus Universitario s/n, 02071 Albacete, Spain Grupo de Recursos Cinegéticos. Instituto de Desarrollo Regional (IDR). Universidad de Castilla-La Mancha (UCLM). 02071 Albacete, Spain b
c
Received 1 March 2006; revised 22 June 2006; accepted 12 July 2006 Available online 1 September 2006
Abstract Researchers have devoted little attention to the possibility that the chemical composition of bone might be variable under normal nutrition conditions. This study assessed antler bone composition of 25 one-year old deer (spikes). Antler content of ash, Ca, P, K, Na, Mg, Fe and Zn was assessed in base and tine, and the mean composition or the difference in composition between tine and base was used to explain variability in antler length, weight and perimeter. In turn, mean composition and difference in concentration of each mineral were related to body measures at 1 year of age, weight at birth, weight at 1 year of age and weight gains during lactation, or between weaning and year of age. Chemical composition differed between base and tine in ash, Ca, P, K, Zn and Fe, but not in Na or Mg. Composition explained a mean variability of 77% in antler length and weight. Body weight and size, in turn, influenced mineral composition. The greatest body effect was that of gains during lactation on principal components analysis factor related to Ca, P and other major minerals such as Na, K or Mg. Antler bone composition is variable in normal conditions and such variability may play a role in biomechanical properties of the antler, but it is also likely to show the nutritional status or physiological effort to grow antlers. Assessing bone composition may emerge as a new useful tool to obtain information regarding bone biology and its bearer in other species including ours. © 2006 Elsevier Inc. All rights reserved. Keywords: Antler; Bone mineral composition; Iberian red deer; Early growth
Introduction Antlers are unique among animal bones in that they grow and are cast every year. This allows for basic research in bone biology without the interference of surgical procedures and their adverse effects in animals where samples are obtained. In addition, antlers also allow for the gathering of a large amount of samples from different populations to assess nutritional and ecological effects on bone composition and structure. According to prevailing theories on costly secondary sexual characters such as antlers, these indicate the quality of the male, and thus they are likely to reflect body condition and food intake ⁎ Corresponding author. Instituto de Investigación en Recursos Cinegéticos (IREC, CSIC-UCLM-JCCM), Sec. Albacete (IDR), Universidad de Castilla-La Mancha, 02071 Albacete, Spain. Fax: +34 967 599233. E-mail address: [email protected] (T. Landete-Castillejos). 8756-3282/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2006.07.009
(1; in deer antlers: 2,3). Thus, it is long known that antlers constitute 1 to 5% of body weight [4]. Another fact suggesting that body condition influences antler size is that food processing cannot supply the mineral needs required for antler growth and thus, males must resorb minerals from their own skeleton for antler growth [5,6]. Detailed studies have shown that daily food intake provides between 25 and 40% of calcium needed for antler mineralization, which results in temporary skeleton demineralization [6]. Conversely, nutrition affects also antler development and thus, food availability influences antler length in spikes [7], or volume, diameter of the beam, length and number of tines [8]. A corollary of the former theory should be that antlers, and possibly other bones, might reflect in their chemical composition the quality that is conveyed by their weight and shape. Very few studies have considered that bone chemical composition might be variable. Early studies showed that bone chemical
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composition varied between domestic species of poultry and cattle [9,10], and thus considered such composition as constant and particular of each species. This research has been recently extended to species-specific chemical composition of antlers [11]. Some authors considered normal composition as constant but postulated that contaminant trace elements (such as lead or mercury) would be incorporated into the bone and they would serve as indices of contamination [9; in antlers: 12]. Other authors have suggested the use of the levels of microminerals in bones as an index of assimilation in nutrition studies [11,13,14]. To our knowledge, no study has considered the hypothesis that antler bone composition in elements other than Ca and P might be variable within standard conditions and that such composition might indicate the size, weight and structural characteristics of the antler, or that bone composition might be influenced by body condition, weight or size of the bearer. This study aimed at assessing whether composition of antler bone varies in standard conditions and whether such variation is influenced by antler region, whether it is associated to bone external characteristics and influenced by body size and growth. For this, antler bone chemical composition was examined in content of macrominerals calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na), potassium (K), and the two most essential microminerals, iron (Fe) and zinc (Zn) in two parts of the spike antlers differing in their mechanical function: base and tine. We examined whether antlers differing in structural development (i.e., size, weight, base or mid beam perimeter) differed in chemical composition. Finally, we tested whether the deer body weight at birth, weight gained during lactation, or weight and height at the end of the first year of life affected the mean concentration of each mineral or the difference between concentration at the base, and that at the tine. Materials and methods Subjects were 25 Iberian red deer males whose antlers were sawed at the base at one year of age (spikes). Animals were kept from birth in a 10,000 m2 open door enclosure on an irrigated pasture including tall fescue (Festuca arundinacea, 52.4%), cocksfoot (Dactylis glomerata, 28.6%), lucerne (Medicago sativa, 14.3%) and white clover (Trifolium repens, 4.8%). Both during gestation and throughout lactation, the mothers of the calves were fed ad libitum with diets based on suggestions by Brelurut et al. [15], using barley straw and meal from barley, alfalfa, oat and sugar beets (16% CP). Calves could access whole meal feed intended for hinds, although they were not observed to feed on it during lactation. Calves were weaned at 18 weeks by separating them from their mothers. No record of individual intake of feed was attempted at any stage. Animals were weighed weekly as a handling routine, although body weight included in this study is that at birth, at weaning (18 weeks of life), and that at the end of the first year of age. Body measures included shoulder height and thoracic perimeter. These, together with antler length, weight, circumference of the antler base and that at midpoint between base and tine were measured as explained in Gomez et al. [16]. Antlers in our farm are ordinarily cut about 1 cm above the burr for safety reasons on the first week of September (when antlers are dead bone and cleaned of velvet skin remaining). Antlers were filed with a metal file (made of Fe, Cr, Mo and V, and wearing away less than 0.1 mg in the whole test) to obtain 0.2 grams of cortical bone per sample (one from the base and one from the tip of the tine). Mineral analysis was performed drying antler samples for 3 h at 102°C, and then for 24 h at 130°C. After this period, they were incinerated at 520°C for 12 h. Ashes were then dissolved with 10 mL of 3 N HCl and then
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heated until the dilution emitted white smoke (this and following process similar to details explained in 17). After cooling, samples were filtered with paper ALBET REF.1300 (Filalbert, Barcelona, Spain) in a 50 mL volumetric flask and the process was repeated in the capsule that contained the ashes to dissolve any remains. The volume was completed with Ultrapure water (i.e., water having a resistivity of 18.2 MΩ/cm at 25°C and at pH 7.0) so that a 1/1000 dilution resulted for Ca analysis, 1/200 for Na, Mg and K, and 1/10 for Fe and Zn to adjust concentrations to calibration lines. Ash samples were examined with an atomic absorption spectrophotometer (Perkin-Elmer 2280, Boston, MA). The concentration of Ca, Mg, Fe and Zn was analyzed with atomic absorption spectrophotometry, while K and Na were examined using atomic emission (using the same equipment without the hollow cathode lamp used above). In the case of Ca, 0.2% lanthanum trichloride was used to prevent interference from other elements. The spectrum lines for Ca, Mg, Na, K, Zn and Fe were, respectively, 422.7, 285.2, 589.0, 766.5, 213.9 and 248.3. Absorbance was measured at 2 s intervals. Each datum was the mean of 5 measures recorded at the interval mentioned, after checking that their variation coefficient was smaller than 2%. For P determination, UV visible spectrophotometry was used according to the colorimetric method, analyzing it as phosphomolybdic acid according to Osborne and Voogt [18]. The equipment used was a Shimadzu UV 1230 spectrophotometer at 650 nm wave length (Shimadzu Co., Kyoto, Japan). Statistical differences in composition between base and tine were examined using a one-way ANOVA. A general linear model (GLM) procedure examined the effect of antler bone composition on the variability in antler weight, antler length, perimeter at the base or that at midpoint between base and tip. To prevent problems of overfitting and as a result of the reduced sample size included in this study, only models including 5 significant factors or less were included in the results. Because assessing the influence of body weight and measures on antler composition would require 7 GLM for mean antler composition, and another 7 for difference in antler composition, and because some of these minerals covary together such as Ca and P, a principal components analysis was performed to reduce the number of variables. Eigenvalues were set to 0.5 so that only factors above them would be selected to be subjected to GLMs. The resulting factors did not covary with each other and were related to original composition variables as shown in Table 3. The scores of each factor for each individual were then used as new variables to be subjected to GLM analysis which assessed on them the effect of birth weight, weight gains during lactation (weight at week 18 minus weight at birth), post-weaning weight gains (weight at one year of age minus that at week 18), weight at the end of the first year of age, height at shoulders and thoracic perimeter. Factors were extracted independently for mean antler composition and difference in antler composition between tine and base.
Results Table 1 shows the mean composition and significant differences between base and tine of the antler. The base had greater ash content, more Ca and P, less K, Zn and Fe. No differences were found for Na or Mg.
Table 1 Differences in chemical composition of macro and microminerals between the base and the tine in 25 spikes (1 year old) Iberian deer (Cervus elaphus hispanicus)
Ash (%) Ca (%) P (%) K (mg/kg) Na (mg/kg) Mg (mg/kg) Zn (mg/kg) Fe (mg/kg)
Antler base
Antler tine
P
55.7 ± 0.6 17.3 ± 0.2 8.0 ± 0.1 280 ± 20 7950 ± 70 4600 ± 60 70 ± 2 50 ± 3
49.9 ± 1.2 15.5 ± 0.5 7.0 ± 0.2 980 ± 80 7560 ± 270 4780 ± 140 99 ± 4 123 ± 19
0.001 0.001 0.001 0.001 > 0.1 > 0.1 0.001 0.001
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Table 2 Variability in antler characteristics explained by composition of macrominerals and essential microminerals (Fe and Zn) Model
Antler length
Antler weight a
R2 Intercept Ca P K Na Mg Zn Fe Dif Ca Dif P Dif K Dif Na Dif Mg Dif Zn Dif Fe
76.4% 220 ± 40 – −0.0013 ± 0.0003*** – −0.010 ± 0.003** – – – (4 ± 0.5) × 104*** – – – – – −0.08 ± 0.02***
77.4% 1410 ± 290 – – − 1.2 ± 0.2*** – − 0.15 ± 0.05* – 1.6 ± 0.5* – − 0.007 ± 0.002** – – – 4 ± 1** –
Factors included in the General Linear Model are mean composition and difference in composition between the tine and base (tine minus base). Probability at 0.5, 0.01 and 0.001 is indicated, respectively, by *, ** and ***. Dashes indicate coefficients that were not significant. a The simplest model in this case, which included only K, explained a surprisingly large variability of R2 = 40.7% (coefficient for K = − 0.4 ± 0.1).
Mineral composition not only differed between points of the antler differing in function, but it also differed among antlers of different characteristics. Concentration or difference in concentration explained 76.4% variability in antler length with only 4 factors, and 77.4% in antler weight with only 5 (Table 2). In this latter case, it is strongly suggesting that 40% of the weight variability is explained by K concentration alone. Base perimeter and antler mid beam perimeter produced problems
of overfitting at high number of factors and no significant model with less than 5 factors. Principal components analysis and General linear models also showed that body weight at different stages of growth or body measures, in turn, influenced antler composition (Table 3). PCA analysis on mean composition extracted 4 factors above 0.5 eigenvalues threshold which absorbed 87.8% of the variance, and in the case of difference in composition between base and tine, the same number of factors above 0.5 absorbed 90.0% of the variance. Out of these factors, only factors first and third in the ranking of variability absorbed for mean composition, and first, second and fourth for difference in composition yielded significant GLM models. The GLM models showed that body weight, body measures or body growth at different stages affected composition PCA factors. Although results are somewhat difficult to interpret, factors most related to Ca, P and other major minerals such as Na, K and Mg were greatly influenced by weight gain during lactation in some cases with preference to growth in stages closer to antler development, or weight near time of antler growth completion. This is more easily interpreted when GLMs on direct mean composition are examined. Thus, just as an example, the GLMs examining ash and Ca content showed that out of the variability explained by the model (R2 = 51.9% for both), 46% for ash and 45% for Ca were explained by gain during lactation, 24% and 25%, respectively, were explained by gains from lactation to end of the first year, and 30% in both cases was explained by body weight at one year old. A greater proportion of variability was influenced by gains during lactation in the case of P (model R2 = 51.9%; 69% of the variability explained by factors corresponded to lactation gains). Models in similar proportion to that of Ca were found for K and Na, but not for Mg, Zn or Fe.
Table 3 Influence of body weight and measures on antler composition in Iberian red deer spikes (Cervus elaphus hispanicus) PCA
CF1
CF3
DCF1
DCF2
DCF4
VEF (%) Ca P K Na Mg Zn Fe Model R2 Intercept WGLac a PostWG WYear HeightY ThoraxPY
43.5 0.86 0.82 − 0.45 − 0.74 0.80 0.50 0.05 47.1% − 2.7 ± 1.4 0.36 ± 0.10** 0.30 ± 0.11* − 0.25 ± 0.10* – –
14.9 − 0.11 − 0.16 0.48 − 0.05 0.43 0.19 − 0.75 19.2% 10.0 ± 4.3 – – – − 0.11 ± 0.05* –
42.6 − 0.87 − 0.59 0.92 0.08 − 0.54 0.60 0.60 62.8% − 6.7 ± 3.8 – 0.18 ± 0.04*** – – – 0.12 ± 0.04*
24.5 0.14 0.18 0.08 − 0.83 0.67 0.32 0.65 39.6% − 15.5 ± 6.7 0.44 ± 0.19* 0.53 ± 0.21* − 0.53 ± 0.20* – 0.22 ± 0.10*
8.2 0.26 −0.38 0.00 0.26 0.32 0.41 −0.15 57.3% 0.16 ± 0.04 – – 0.16 ± 0.04*** – −0.30 ± 0.06***
The first part (up to Fe) shows a principal components analysis (PCA) which reduced Ca, P, K, Na, Mg, Zn and Fe mean antler concentration variables to 4 factors (CF1 to CF4) or difference in composition between the tine and base (tine minus base) to another 4 factors (DCF1 to DCF4). In contrast to covarying minerals, factor variables are independent of each other. The data represent the correlation between factors and minerals. The second part of the table (below Fe) is a general linear model testing the effects that on the previous PCA factors exerted birth weight, weight gain during lactation, post-weaning weight gain up to 1 year of age, and weight, height and thorax perimeter at 1 year of age. VEF indicates the variability in mineral composition explained by each PCA factor. Probability at 0.5, 0.01 and 0.001 are indicated, respectively, by *, ** and ***. Dashes indicate coefficients that were not significant. PCA factors CF2, CF4 and DCF3 did not produce any significant model, and calf birth weight was not significant in any GLM either. a WGLac stands for weight gain during lactation; PostWG stands for post weaning weight gain; WYear, HeightY and ThoraxPY stand for weight, height and thorax perimeter at 1 year of age, respectively.
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Discussion Antler bone composition is variable within standard conditions and such variability appears to convey information about the bearer: growth trajectory, structural development and possibly function at different parts of the antler, and degree of physiological exhaustion to grow it. This will be further discussed below. Antler bone composition was different for base and tine in most minerals studied except Na and Mg. Although one study [19] showed a difference in mineral content between the base and tine of antler similar to our effect in ash content, this is the first evidence showing that, within minerals, bone composition between different parts of the antler is variable in standard range of conditions. Why should the composition of antlers change from one part to another? One possible explanation might be that different parts of the antler have a different function. If so, they should change in biomechanical properties. One of the leading researchers in mechanical properties of bones, Currey [19], noted that “these different [mechanical] properties seem to vary regionally over the antler, and I am not sure of the significance of this variation”. Much of his work and that of other researchers showed that mechanical properties between different mammal bones depend mainly on the degree of mineralization [19–25]. Because other researchers found that phylogeny did not influence these mechanical properties [26], differences in mechanical properties are likely to reflect different working conditions or functions. Thus, Currey [19] found that, as in our case, mineral content in antlers are higher in the base than in the tip. At least this gross composition difference between base and tip appears to be linked to changes in mechanical properties. Antlers called the attraction of Currey and other researchers because, among mammalian bones, they have the highest work of fracture, i.e., the amount of work needed to break it [21], and they are almost insensitive to breaking by impact [25]. This might depend upon having the lowest mineral content of all mammalian bones [25], as work of fracture reaches a maximum at 59% mineral content (that of antlers) and then falls rapidly [20]. This percentage is close to that found in the antler base in our study (56%), and means that the work of fracture is greatest in the base than in the tine (49%). The base is more likely to bend by impact than any other part of the antler and thus, this would explain why the mineral content is close to the maximum of the highest work of fracture. Thus, at least the ash content of antler base might be attributed to an attempt to reach optimum mechanical properties. But, why should the composition change in minerals other than Ca and P? Currey [25] also noticed that bone mechanical properties “are related to Ca content, although there is a considerable amount of scatter in general, and in antlers in particular”. The size of the statistical regressions obtained ranged from 78% in some mechanical properties (Young's modulus of elasticity vs. Ca content) to 28–46% in others (log yield stress vs. log Ca content: 25). The “unexplained” variability might be caused, at least in part, by the variability in some other minerals. Thus, silicon has been shown to be
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involved in bone hardness [14], and in fact, Si substituted hydroxyapatite phase grows at greater speeds than phase pure hydroxyapatite [27]. Mg and Sr can be directly incorporated into bone, substituting Ca in the crystal lattice, and yielding mineral crystals smaller and less perfect, which influences mechanical properties [28]. Moreover, 3% of the bone is constituted by magnesium phosphate [9]. Another interesting possibility is that minerals such as Na and Mg and other minor minerals in bone can be included in apatite phases different to the most common hydroxyapatite [29]. In the case of Zn, it has been shown to affect increase bone density because it is a constituent of enzymes such as alkaline phosphatase [30], which is one of the most important enzymes increasing rate and extent of mineralization [28]. It is very likely that such different phases or size of crystals have different mechanical properties, and thus mechanical properties might vary with the concentration of minor minerals without change in overall Ca and P (i.e., concentration of minor minerals could explain variability in mechanical properties that, according to biomechanic theory, should behave similarly). However, it is not clear why should the tine have less ash content than the base. The different function or need to withstand different mechanical properties might not be the only possible explanation for such variability in bone composition. Another possible explanation might be that bone composition reflected the mineral status of the animal (i.e., the circulating or reserve levels for at least some minerals). This would explain lowest levels of some minerals in the tine of the antler. Thus, perhaps spike deer attempt to build the antler at Ca and P contents near that of the maximum work of fracture in all parts of the antler, but a depletion of Ca reserves would render the last part to be built, the tine, with contents lower than the optimum. In fact, physiological exhaustion might explain also the high importance of K in explaining antler weight variability, and the greater percent of K and Zn in the tine: K intake reduces Ca excretion in urine [31] and, as mentioned above, Zn, through its effects on alkaline phosphatase increases rate and extent of mineralization [28,30]. Thus, deer might ingest increasing amounts of K and Zn as Ca from skeleton is depleted during the process of antler growth in order to increase the efficiency of circulating levels of Ca in blood. It should be expected that Ca and P intake was also increased, but this is unlikely to balance the Ca and P depletion from skeleton because 60–75% of Ca is obtained from bone resorption [6]. Thus, physiological exhaustion would also explain why variability of structural properties such as antler weight, length and others can be explained at such a high degree in terms of its composition: animals in better body condition might develop large antlers, and their good body condition would be shown in high mineral reserves and circulating levels close to the optimum (or lack of need for greater intake of K or Zn). This is further supported by the fact that content of each mineral is influenced by body weight and size. If physiological exhaustion can explain variability in bone chemical composition, minerals involved in such exhaustion should differ increasingly from base to top end of the antler in comparisons between well fed individuals and animals with restricted food availability.
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The hypothesis that bone chemical composition reflects the physiological status of the animal also receives support from the literature. On one hand, extreme mineral deficiency results in less developed bones and more fragile ones (reviewed in 14), while the single addition of other minerals such as copper can result in increased growth in domestic pigs [32]. Detailed studies in animal science have shown that increasing the level of some minerals such as P increased bone strength and ash content within standard nutrition plane [33], or that concentration of some minerals in bone such as manganese can be raised or lowered by adding this mineral to a standard diet [9]. However, the studies conducted in deer showed that antler size and composition, and bone composition did not vary by diet (diets differing in P content: [34]; diets differing in protein content: [35]), although ecological conditions affecting forage availability did influence antler characteristics [3,7]. This is more striking considering that, apart from the effect of availability of nutrients studied in healthy domestic animals, wild deer have to cope with the effect of parasites and pathogens. Nematodes and other parasites can produce disturbances in protein metabolism and result in reduced absorption or retention of minerals, especially P, [36]. Some pathogens such as red blood infecting protozoans also require minerals (iron in this case) to survive, and must take these from the host [37]. Thus, subclinical pathogen infections in cattle [38], and platyhelminthes infection that do not result in weight losses in deer [39], result in reduced concentration of some minerals in serum or body organs such as the liver. It is likely that some of these parasites and pathogens affect bone composition, and an interesting hypothesis to test in future studies should be examining antler and other bone composition with respect to parasite or pathogen load. Finally, a striking effect is that, among body weight at different stages or body measures, most of the variability in mean concentration of Ca, P, K and Na was explained by gains during lactation, and this effect was also found in PCA factors mostly related to these minerals. This is even more striking when considering that antlers start growing at almost one year of age. Early nutrition, particularly during the period of maternal care, is of profound importance as it can affect subsequent life trajectory of animals by altering growth rates [40,41]. Such maternal effects are more likely to be shown in lactation because this is the most energetic expensive stage of reproduction [42], and thus in Iberian deer adverse effects affecting lactation result in reduced growth that last further than the first year of life [16,43–49]. However, it is striking that lactation gains were more important than body size or weight at the age of antler growth precisely in minerals contributing to most of the mineral content in antlers, but not in that of microminerals. A possible explanation might be that the growth trajectory of the body and, in particular, that of the skeleton was set during lactation. If so, this should result in a great influence on antler size and composition because most of the major minerals (e.g., Ca and P) used for growing the antler are taken from bone resorption [6].
Because, as mentioned above, mechanical properties of bone do not depend on phylogeny, and because Ca deposition (and possibly physiological effort) for antler growth is similar to that of lactation in most mammals including humans [6,50], some of the effects found in this study and, in general, this technique, might be extended to other mammals and possibly our own species. In conclusion, bone composition (at least that of antlers) is variable within standard conditions. It is likely that at least part of such variation plays a role in bone mechanical properties. Bone composition also provides information about the bearer. This opens an interesting area of research in bone biology because the information that could be obtained from bone composition may range from early growth to possibly mineral nutrition status, or even be an index for some diseases. This technique might prove useful in other species including humans, but deer appear to be a fantastic model for bone studies because it allows carrying out destructive studies on bone without the need of invasive techniques or surgery. Acknowledgments This study was supported by projects AGL2003-08547 (MCYT), PAC06-01304298 (JCCM) and PBI 05-040 (JCCM). The authors wish to thank Jose Angel Gómez Nieto and Pedro Jesús Sánchez for help in data collection, Fulgencio Cebrián and Isidoro Cambronero for help in handling the animals and two anonymous referees.
References [1] Harvey PH, Bradbury JW. Sexual selection. In: Krebs JR, Davies NB, editors. Behavioural Ecology: An Evolutionary Approach. Oxford, UK: Blackwell Scientific; 1991. p. 203–33. [2] Ditchkoff SS, Lochmiller RL, Masters RE, Hoofer SR, Van Den Busshe RA. Major-histocompatibility-complex-associated variation in secondary sexual traits of white-tailed deer (Odoicoleus virginianus): evidence for good-genes advertisement. Evolution 2001;55:616–25. [3] Weladji RB, Holand O, Steinheim G, Colman JE, Gjostein H, Kosmo A. Sexual dimorphism and intercohort variation in reindeer calf antler length is associated with density and weather. Oecologia 2005;145:549–55. [4] Huxley J. The relative size of antlers of deer. Proc Zool Soc London 1931;72:819–64. [5] Meister W. Changes in biological structure of the long bones of whitetailed deer during the growth of antlers. Anat Rec 1956;124:709–21. [6] Muir PD, Sykes AR, Barrell GK. Calcium metabolism in red deer (Cervus elaphus) offered herbages during antlerogenesis: kinetic and stable balance studies. J Agric Sci Camb 1987;109:357–64. [7] Schmidt KT, Stien A, Albon SD, Guinness FE. Antler length of yearling red deer is determined by population density, weather and early lifehistory. Oecologia 2001;127:191–7. [8] Ullrey DE. Nutrition and antler development in white-tailed deer. In: Brown RD, editor. Antler Development in Cervidae. Kingsville, TX, USA: Caesar Kleberg Wildlife Research Institute; 1983. p. 49–59. [9] Doyle JJ. Toxic and essential elements in bone—A review. J Anim Sci 1979;49:482–97. [10] Field RA, Riley ML, Mello FC, Corbridge MH, Kotula AW. Bone composition in cattle, pigs, sheep and poultry. J Anim Sci 1974;39:493–9. [11] Pathak NN, Pattanaik AK, Patra RC, Arora BM. Mineral composition of antlers of three deer species reared in captivity. Small Rumin Res 2001;42:61–5.
T. Landete-Castillejos et al. / Bone 40 (2007) 230–235 [12] Purdey M. Elevated silver, barium and strontium in antlers, vegetation and soils sourced from CWD cluster areas: do Ag/Ba/Sr piezoelectric crystals represent the transmissible pathogenic agent in TSEs? Medical Hypotheses 2004;63:211–25. [13] Brown RD. Nutrition and antler development. In: Bubenik GA, Bubenik AB, editors. Horns, Pronghorns and Antlers. New York, NY, USA: Springer-Verlag; 1990. p. 427–41. [14] McDowell LR. Minerals in Animal and Human Nutrition. Amsterdam, Netherlands: Elsevier; 2003. [15] Brelerut A, Pingard A, Thériez M. Le Cerf et Son Élevage. Paris, France: INRA; 1990. [16] J.A. Gómez, T. Landete-Castillejos, A. Garcia, L. Gallego, Importance of lactation on posterior growth and antler development in the Iberian red deer (Cervus elaphus hispanicus). A longitudinal study from birth to 2.5 years of age. Livestock Prod Sci, in press. [17] Gallego L, Landete-Castillejos T, Garcia A, Sánchez PJ. Seasonal effects and lactational changes in mineral composition of milk from iberian red deer (Cervus elaphus hispanicus). J Dairy Sci 2006;89:589–95. [18] Osborne DR, Voogt P. The Analysis of Nutrients in Foods. London, UK: Academic Press; 1978. [19] Currey JD. The design of mineralized hard tissues for their mechanical functions. J Exp Biol 1999;202:3285–94. [20] Currey JD. Comparative mechanical properties and histology of bone. Am Zool 1984;24:5–12. [21] Currey JD. Mechanical properties of bone tissues with greatly differing functions. J Biomechanics 1979;12:313–9. [22] Currey JD. Tensile yield in compact bone is determined by strain, post yield behaviour by mineral content. J Biomechanics 2004;37:549–56. [23] Currey JD, Brear K. Hardness, Young's modulus and yield stress in mammalian mineralized tissues. J Mater Sci: Mater Med 1990;1:14–20. [24] Currey JD, Brear K. Ontogenetic changes in the mechanical properties of the femur of the polar bear Ursus maritimus. J Zool (London) 1990;222: 49–58. [25] Currey JD, Brear K, Zioupos P. Notch sensitivity of mammalian mineralized tissues in impact. Proc R Soc London, Ser B Biol Sci 2004; 271:517–22. [26] Erickson GM, Catanese J, Keaveny TM. Evolution of the biomechanical properties of the femur. Anat Rec 2002;268:114–24. [27] Patel N, Best SM, Bonfield W, Gibson IR, Hing KA, Damien E, et al. A comparative study on the in vivo behavior of hydroxyapatite and silicon substituted hydroxyapatite granules. J Mater Sci: Mater Med 2002; 13:1199–206. [28] Robey PG, Boskey AL. Extracellular matrix and biomineralization of bone. In: Favus MJ, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. Washington, DC, USA: American Society for Bone and Mineral Research; 2003. p. 38–46. [29] Driessens FCM, Verbeeck RMH. The dynamics of bone mineral in some vertebrates. Z Naturforsch 1986;41c:468–71. [30] Maki K, Nishioka T, Nishida I, Ushijima S, Kimura M. Effect of zinc on rat mandibles during growth. Detofacial Orthop 2002;122:410–3. [31] Rafferty K, Davies KM, Heaney RP. Potassium intake and the calcium economy. J Am Coll Nutr 2005;24:99–106. [32] Paik I. Application of chelated minerals in animal production. AsianAustralian J Anim Sci 2001;14:191–8.
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[33] Traylor SL, Cromwell GL, Lindemann MD. Bioavailability of phosphorus in meat and bone meal for swine. J Anim Sci 2005; 83:1054–61. [34] Grasman BT, Hellgren E. Phosphorus nutrition in white-tailed deer: nutrient balance, physiological responses, and antler growth. Ecology 1993;74:2279–96. [35] Asleson MA, Hellgren EC, Varner LW. Effects of seasonal protein restriction on antlerogenesis and body mass in adult male white-tailed deer. J Wildl Manage 1997;61:1098–107. [36] Coop RL, Kyriazakis I. Influence of host nutrition on the development and consequences of nematode parasitism in ruminants. Trends in Parasitology 2001;17:325–30. [37] Rivera MT, Pereira De Souza A, Cremonini Araujo-Jorge T, Lisboa De Castro S, Vanderpas J. Trace elements, innate immune response and parasites. Clin Chem Lab Med 2003;41:1020–5. [38] Naresh R, Dwivedi SK, Dey S, Swarup D. Zinc, copper and cobalt concentrations in blood during inflammation of the mammary gland in dairy cows. Asia-Australian J Anim Sci 2001;14:564–6. [39] Vengušt G, Klinkon M, Bidovec A, Vengušt A. Fasciola hepática: effects on blood constituents and liver minerals in fallow deer (Dama dama). Vet Parasitol 2003;112:51–61. [40] Bernardo J. Maternal effects in animal ecology. Am Zool 1996;36:83–105. [41] Metcalfe NB, Monaghan P. Compensation for a bad start: grow now, pay later. TREE 2001;16:254–60. [42] Oftedal OT. Pregnancy and lactation. In: Hudson R, White RG, editors. Bioenergetics of Wild Herbivores. Boca Raton, FL, USA: CRC Press; 1985. p. 215–38. [43] Landete-Castillejos T, Garcia A, Molina P, Vergara H, Garde J, Gallego L. Milk production and composition in captive Iberian red deer (Cervus elaphus hispanicus): effect of birth date. J Anim Sci 2000;78: 2771–2777. [44] Landete-Castillejos T, Garcia A, Gallego L. Calf growth in captive Iberian red deer (Cervus elaphus hispanicus): effect of birth date and hind milk production and composition. J Anim Sci 2001;79:1085–92. [45] Landete-Castillejos T, Garcia A, Gómez JA, Laborda J, Gallego L. Effects of nutritional stress during lactation on immunity costs and indices of future reproduction in Iberian red deer (Cervus elaphus hispanicus). Biol Reprod 2002;67:1613–20. [46] Landete-Castillejos T, Garcia A, Gómez JA, Gallego L. Lactation under food constraints in Iberian red deer (Cervus elaphus hispanicus). Wildl Biol 2003;9:131–9. [47] Landete-Castillejos T, Garcia A, Gómez JA, Berruga MI, Gallego L. Lactation under induced five-month delay reproduction in Iberian red deer (Cervus elaphus hispanicus). J Exp Zool 2004;301A:261–5. [48] Landete-Castillejos T, Garcia A, Gomez JA, Berruga MI, Gallego L. Effects of birth date and order in lactation performance of iberian red deer (Cervus elaphus hispanicus). J Dairy Sci 2005;88:154–8. [49] Landete-Castillejos T, Garcia A, López-Serrano FR, Gallego L. Maternal quality and differences in milk production and composition for male and female Iberian red deer calves (Cervus elaphus hispanicus). Behav Ecol Sociobiol 2005;57:267–74. [50] Wysolmerski JJ. The evolutionary origins of maternal calcium and bone metabolism during lactation. J Mammary Gland Biol Neoplasia 2002;7: 267–276.
Body weight, early growth and antler size influence antler bone mineral composition of Iberian Red Deer (Cervus elaphus hispanicus) T. Landete-Castillejos a,b,c,⁎, A. Garcia a,b,c , L. Gallego a,c a
Departamento de Ciencia y Tecnología Agroforestal, ETSIA, Universidad de Castilla-La Mancha (UCLM), 02071 Albacete, Spain Instituto de Investigación en Recursos Cinegéticos, IREC (CSIC, UCLM, JCCM), Campus Universitario s/n, 02071 Albacete, Spain Grupo de Recursos Cinegéticos. Instituto de Desarrollo Regional (IDR). Universidad de Castilla-La Mancha (UCLM). 02071 Albacete, Spain b
c
Received 1 March 2006; revised 22 June 2006; accepted 12 July 2006 Available online 1 September 2006
Abstract Researchers have devoted little attention to the possibility that the chemical composition of bone might be variable under normal nutrition conditions. This study assessed antler bone composition of 25 one-year old deer (spikes). Antler content of ash, Ca, P, K, Na, Mg, Fe and Zn was assessed in base and tine, and the mean composition or the difference in composition between tine and base was used to explain variability in antler length, weight and perimeter. In turn, mean composition and difference in concentration of each mineral were related to body measures at 1 year of age, weight at birth, weight at 1 year of age and weight gains during lactation, or between weaning and year of age. Chemical composition differed between base and tine in ash, Ca, P, K, Zn and Fe, but not in Na or Mg. Composition explained a mean variability of 77% in antler length and weight. Body weight and size, in turn, influenced mineral composition. The greatest body effect was that of gains during lactation on principal components analysis factor related to Ca, P and other major minerals such as Na, K or Mg. Antler bone composition is variable in normal conditions and such variability may play a role in biomechanical properties of the antler, but it is also likely to show the nutritional status or physiological effort to grow antlers. Assessing bone composition may emerge as a new useful tool to obtain information regarding bone biology and its bearer in other species including ours. © 2006 Elsevier Inc. All rights reserved. Keywords: Antler; Bone mineral composition; Iberian red deer; Early growth
Introduction Antlers are unique among animal bones in that they grow and are cast every year. This allows for basic research in bone biology without the interference of surgical procedures and their adverse effects in animals where samples are obtained. In addition, antlers also allow for the gathering of a large amount of samples from different populations to assess nutritional and ecological effects on bone composition and structure. According to prevailing theories on costly secondary sexual characters such as antlers, these indicate the quality of the male, and thus they are likely to reflect body condition and food intake ⁎ Corresponding author. Instituto de Investigación en Recursos Cinegéticos (IREC, CSIC-UCLM-JCCM), Sec. Albacete (IDR), Universidad de Castilla-La Mancha, 02071 Albacete, Spain. Fax: +34 967 599233. E-mail address: [email protected] (T. Landete-Castillejos). 8756-3282/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2006.07.009
(1; in deer antlers: 2,3). Thus, it is long known that antlers constitute 1 to 5% of body weight [4]. Another fact suggesting that body condition influences antler size is that food processing cannot supply the mineral needs required for antler growth and thus, males must resorb minerals from their own skeleton for antler growth [5,6]. Detailed studies have shown that daily food intake provides between 25 and 40% of calcium needed for antler mineralization, which results in temporary skeleton demineralization [6]. Conversely, nutrition affects also antler development and thus, food availability influences antler length in spikes [7], or volume, diameter of the beam, length and number of tines [8]. A corollary of the former theory should be that antlers, and possibly other bones, might reflect in their chemical composition the quality that is conveyed by their weight and shape. Very few studies have considered that bone chemical composition might be variable. Early studies showed that bone chemical
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composition varied between domestic species of poultry and cattle [9,10], and thus considered such composition as constant and particular of each species. This research has been recently extended to species-specific chemical composition of antlers [11]. Some authors considered normal composition as constant but postulated that contaminant trace elements (such as lead or mercury) would be incorporated into the bone and they would serve as indices of contamination [9; in antlers: 12]. Other authors have suggested the use of the levels of microminerals in bones as an index of assimilation in nutrition studies [11,13,14]. To our knowledge, no study has considered the hypothesis that antler bone composition in elements other than Ca and P might be variable within standard conditions and that such composition might indicate the size, weight and structural characteristics of the antler, or that bone composition might be influenced by body condition, weight or size of the bearer. This study aimed at assessing whether composition of antler bone varies in standard conditions and whether such variation is influenced by antler region, whether it is associated to bone external characteristics and influenced by body size and growth. For this, antler bone chemical composition was examined in content of macrominerals calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na), potassium (K), and the two most essential microminerals, iron (Fe) and zinc (Zn) in two parts of the spike antlers differing in their mechanical function: base and tine. We examined whether antlers differing in structural development (i.e., size, weight, base or mid beam perimeter) differed in chemical composition. Finally, we tested whether the deer body weight at birth, weight gained during lactation, or weight and height at the end of the first year of life affected the mean concentration of each mineral or the difference between concentration at the base, and that at the tine. Materials and methods Subjects were 25 Iberian red deer males whose antlers were sawed at the base at one year of age (spikes). Animals were kept from birth in a 10,000 m2 open door enclosure on an irrigated pasture including tall fescue (Festuca arundinacea, 52.4%), cocksfoot (Dactylis glomerata, 28.6%), lucerne (Medicago sativa, 14.3%) and white clover (Trifolium repens, 4.8%). Both during gestation and throughout lactation, the mothers of the calves were fed ad libitum with diets based on suggestions by Brelurut et al. [15], using barley straw and meal from barley, alfalfa, oat and sugar beets (16% CP). Calves could access whole meal feed intended for hinds, although they were not observed to feed on it during lactation. Calves were weaned at 18 weeks by separating them from their mothers. No record of individual intake of feed was attempted at any stage. Animals were weighed weekly as a handling routine, although body weight included in this study is that at birth, at weaning (18 weeks of life), and that at the end of the first year of age. Body measures included shoulder height and thoracic perimeter. These, together with antler length, weight, circumference of the antler base and that at midpoint between base and tine were measured as explained in Gomez et al. [16]. Antlers in our farm are ordinarily cut about 1 cm above the burr for safety reasons on the first week of September (when antlers are dead bone and cleaned of velvet skin remaining). Antlers were filed with a metal file (made of Fe, Cr, Mo and V, and wearing away less than 0.1 mg in the whole test) to obtain 0.2 grams of cortical bone per sample (one from the base and one from the tip of the tine). Mineral analysis was performed drying antler samples for 3 h at 102°C, and then for 24 h at 130°C. After this period, they were incinerated at 520°C for 12 h. Ashes were then dissolved with 10 mL of 3 N HCl and then
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heated until the dilution emitted white smoke (this and following process similar to details explained in 17). After cooling, samples were filtered with paper ALBET REF.1300 (Filalbert, Barcelona, Spain) in a 50 mL volumetric flask and the process was repeated in the capsule that contained the ashes to dissolve any remains. The volume was completed with Ultrapure water (i.e., water having a resistivity of 18.2 MΩ/cm at 25°C and at pH 7.0) so that a 1/1000 dilution resulted for Ca analysis, 1/200 for Na, Mg and K, and 1/10 for Fe and Zn to adjust concentrations to calibration lines. Ash samples were examined with an atomic absorption spectrophotometer (Perkin-Elmer 2280, Boston, MA). The concentration of Ca, Mg, Fe and Zn was analyzed with atomic absorption spectrophotometry, while K and Na were examined using atomic emission (using the same equipment without the hollow cathode lamp used above). In the case of Ca, 0.2% lanthanum trichloride was used to prevent interference from other elements. The spectrum lines for Ca, Mg, Na, K, Zn and Fe were, respectively, 422.7, 285.2, 589.0, 766.5, 213.9 and 248.3. Absorbance was measured at 2 s intervals. Each datum was the mean of 5 measures recorded at the interval mentioned, after checking that their variation coefficient was smaller than 2%. For P determination, UV visible spectrophotometry was used according to the colorimetric method, analyzing it as phosphomolybdic acid according to Osborne and Voogt [18]. The equipment used was a Shimadzu UV 1230 spectrophotometer at 650 nm wave length (Shimadzu Co., Kyoto, Japan). Statistical differences in composition between base and tine were examined using a one-way ANOVA. A general linear model (GLM) procedure examined the effect of antler bone composition on the variability in antler weight, antler length, perimeter at the base or that at midpoint between base and tip. To prevent problems of overfitting and as a result of the reduced sample size included in this study, only models including 5 significant factors or less were included in the results. Because assessing the influence of body weight and measures on antler composition would require 7 GLM for mean antler composition, and another 7 for difference in antler composition, and because some of these minerals covary together such as Ca and P, a principal components analysis was performed to reduce the number of variables. Eigenvalues were set to 0.5 so that only factors above them would be selected to be subjected to GLMs. The resulting factors did not covary with each other and were related to original composition variables as shown in Table 3. The scores of each factor for each individual were then used as new variables to be subjected to GLM analysis which assessed on them the effect of birth weight, weight gains during lactation (weight at week 18 minus weight at birth), post-weaning weight gains (weight at one year of age minus that at week 18), weight at the end of the first year of age, height at shoulders and thoracic perimeter. Factors were extracted independently for mean antler composition and difference in antler composition between tine and base.
Results Table 1 shows the mean composition and significant differences between base and tine of the antler. The base had greater ash content, more Ca and P, less K, Zn and Fe. No differences were found for Na or Mg.
Table 1 Differences in chemical composition of macro and microminerals between the base and the tine in 25 spikes (1 year old) Iberian deer (Cervus elaphus hispanicus)
Ash (%) Ca (%) P (%) K (mg/kg) Na (mg/kg) Mg (mg/kg) Zn (mg/kg) Fe (mg/kg)
Antler base
Antler tine
P
55.7 ± 0.6 17.3 ± 0.2 8.0 ± 0.1 280 ± 20 7950 ± 70 4600 ± 60 70 ± 2 50 ± 3
49.9 ± 1.2 15.5 ± 0.5 7.0 ± 0.2 980 ± 80 7560 ± 270 4780 ± 140 99 ± 4 123 ± 19
0.001 0.001 0.001 0.001 > 0.1 > 0.1 0.001 0.001
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Table 2 Variability in antler characteristics explained by composition of macrominerals and essential microminerals (Fe and Zn) Model
Antler length
Antler weight a
R2 Intercept Ca P K Na Mg Zn Fe Dif Ca Dif P Dif K Dif Na Dif Mg Dif Zn Dif Fe
76.4% 220 ± 40 – −0.0013 ± 0.0003*** – −0.010 ± 0.003** – – – (4 ± 0.5) × 104*** – – – – – −0.08 ± 0.02***
77.4% 1410 ± 290 – – − 1.2 ± 0.2*** – − 0.15 ± 0.05* – 1.6 ± 0.5* – − 0.007 ± 0.002** – – – 4 ± 1** –
Factors included in the General Linear Model are mean composition and difference in composition between the tine and base (tine minus base). Probability at 0.5, 0.01 and 0.001 is indicated, respectively, by *, ** and ***. Dashes indicate coefficients that were not significant. a The simplest model in this case, which included only K, explained a surprisingly large variability of R2 = 40.7% (coefficient for K = − 0.4 ± 0.1).
Mineral composition not only differed between points of the antler differing in function, but it also differed among antlers of different characteristics. Concentration or difference in concentration explained 76.4% variability in antler length with only 4 factors, and 77.4% in antler weight with only 5 (Table 2). In this latter case, it is strongly suggesting that 40% of the weight variability is explained by K concentration alone. Base perimeter and antler mid beam perimeter produced problems
of overfitting at high number of factors and no significant model with less than 5 factors. Principal components analysis and General linear models also showed that body weight at different stages of growth or body measures, in turn, influenced antler composition (Table 3). PCA analysis on mean composition extracted 4 factors above 0.5 eigenvalues threshold which absorbed 87.8% of the variance, and in the case of difference in composition between base and tine, the same number of factors above 0.5 absorbed 90.0% of the variance. Out of these factors, only factors first and third in the ranking of variability absorbed for mean composition, and first, second and fourth for difference in composition yielded significant GLM models. The GLM models showed that body weight, body measures or body growth at different stages affected composition PCA factors. Although results are somewhat difficult to interpret, factors most related to Ca, P and other major minerals such as Na, K and Mg were greatly influenced by weight gain during lactation in some cases with preference to growth in stages closer to antler development, or weight near time of antler growth completion. This is more easily interpreted when GLMs on direct mean composition are examined. Thus, just as an example, the GLMs examining ash and Ca content showed that out of the variability explained by the model (R2 = 51.9% for both), 46% for ash and 45% for Ca were explained by gain during lactation, 24% and 25%, respectively, were explained by gains from lactation to end of the first year, and 30% in both cases was explained by body weight at one year old. A greater proportion of variability was influenced by gains during lactation in the case of P (model R2 = 51.9%; 69% of the variability explained by factors corresponded to lactation gains). Models in similar proportion to that of Ca were found for K and Na, but not for Mg, Zn or Fe.
Table 3 Influence of body weight and measures on antler composition in Iberian red deer spikes (Cervus elaphus hispanicus) PCA
CF1
CF3
DCF1
DCF2
DCF4
VEF (%) Ca P K Na Mg Zn Fe Model R2 Intercept WGLac a PostWG WYear HeightY ThoraxPY
43.5 0.86 0.82 − 0.45 − 0.74 0.80 0.50 0.05 47.1% − 2.7 ± 1.4 0.36 ± 0.10** 0.30 ± 0.11* − 0.25 ± 0.10* – –
14.9 − 0.11 − 0.16 0.48 − 0.05 0.43 0.19 − 0.75 19.2% 10.0 ± 4.3 – – – − 0.11 ± 0.05* –
42.6 − 0.87 − 0.59 0.92 0.08 − 0.54 0.60 0.60 62.8% − 6.7 ± 3.8 – 0.18 ± 0.04*** – – – 0.12 ± 0.04*
24.5 0.14 0.18 0.08 − 0.83 0.67 0.32 0.65 39.6% − 15.5 ± 6.7 0.44 ± 0.19* 0.53 ± 0.21* − 0.53 ± 0.20* – 0.22 ± 0.10*
8.2 0.26 −0.38 0.00 0.26 0.32 0.41 −0.15 57.3% 0.16 ± 0.04 – – 0.16 ± 0.04*** – −0.30 ± 0.06***
The first part (up to Fe) shows a principal components analysis (PCA) which reduced Ca, P, K, Na, Mg, Zn and Fe mean antler concentration variables to 4 factors (CF1 to CF4) or difference in composition between the tine and base (tine minus base) to another 4 factors (DCF1 to DCF4). In contrast to covarying minerals, factor variables are independent of each other. The data represent the correlation between factors and minerals. The second part of the table (below Fe) is a general linear model testing the effects that on the previous PCA factors exerted birth weight, weight gain during lactation, post-weaning weight gain up to 1 year of age, and weight, height and thorax perimeter at 1 year of age. VEF indicates the variability in mineral composition explained by each PCA factor. Probability at 0.5, 0.01 and 0.001 are indicated, respectively, by *, ** and ***. Dashes indicate coefficients that were not significant. PCA factors CF2, CF4 and DCF3 did not produce any significant model, and calf birth weight was not significant in any GLM either. a WGLac stands for weight gain during lactation; PostWG stands for post weaning weight gain; WYear, HeightY and ThoraxPY stand for weight, height and thorax perimeter at 1 year of age, respectively.
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Discussion Antler bone composition is variable within standard conditions and such variability appears to convey information about the bearer: growth trajectory, structural development and possibly function at different parts of the antler, and degree of physiological exhaustion to grow it. This will be further discussed below. Antler bone composition was different for base and tine in most minerals studied except Na and Mg. Although one study [19] showed a difference in mineral content between the base and tine of antler similar to our effect in ash content, this is the first evidence showing that, within minerals, bone composition between different parts of the antler is variable in standard range of conditions. Why should the composition of antlers change from one part to another? One possible explanation might be that different parts of the antler have a different function. If so, they should change in biomechanical properties. One of the leading researchers in mechanical properties of bones, Currey [19], noted that “these different [mechanical] properties seem to vary regionally over the antler, and I am not sure of the significance of this variation”. Much of his work and that of other researchers showed that mechanical properties between different mammal bones depend mainly on the degree of mineralization [19–25]. Because other researchers found that phylogeny did not influence these mechanical properties [26], differences in mechanical properties are likely to reflect different working conditions or functions. Thus, Currey [19] found that, as in our case, mineral content in antlers are higher in the base than in the tip. At least this gross composition difference between base and tip appears to be linked to changes in mechanical properties. Antlers called the attraction of Currey and other researchers because, among mammalian bones, they have the highest work of fracture, i.e., the amount of work needed to break it [21], and they are almost insensitive to breaking by impact [25]. This might depend upon having the lowest mineral content of all mammalian bones [25], as work of fracture reaches a maximum at 59% mineral content (that of antlers) and then falls rapidly [20]. This percentage is close to that found in the antler base in our study (56%), and means that the work of fracture is greatest in the base than in the tine (49%). The base is more likely to bend by impact than any other part of the antler and thus, this would explain why the mineral content is close to the maximum of the highest work of fracture. Thus, at least the ash content of antler base might be attributed to an attempt to reach optimum mechanical properties. But, why should the composition change in minerals other than Ca and P? Currey [25] also noticed that bone mechanical properties “are related to Ca content, although there is a considerable amount of scatter in general, and in antlers in particular”. The size of the statistical regressions obtained ranged from 78% in some mechanical properties (Young's modulus of elasticity vs. Ca content) to 28–46% in others (log yield stress vs. log Ca content: 25). The “unexplained” variability might be caused, at least in part, by the variability in some other minerals. Thus, silicon has been shown to be
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involved in bone hardness [14], and in fact, Si substituted hydroxyapatite phase grows at greater speeds than phase pure hydroxyapatite [27]. Mg and Sr can be directly incorporated into bone, substituting Ca in the crystal lattice, and yielding mineral crystals smaller and less perfect, which influences mechanical properties [28]. Moreover, 3% of the bone is constituted by magnesium phosphate [9]. Another interesting possibility is that minerals such as Na and Mg and other minor minerals in bone can be included in apatite phases different to the most common hydroxyapatite [29]. In the case of Zn, it has been shown to affect increase bone density because it is a constituent of enzymes such as alkaline phosphatase [30], which is one of the most important enzymes increasing rate and extent of mineralization [28]. It is very likely that such different phases or size of crystals have different mechanical properties, and thus mechanical properties might vary with the concentration of minor minerals without change in overall Ca and P (i.e., concentration of minor minerals could explain variability in mechanical properties that, according to biomechanic theory, should behave similarly). However, it is not clear why should the tine have less ash content than the base. The different function or need to withstand different mechanical properties might not be the only possible explanation for such variability in bone composition. Another possible explanation might be that bone composition reflected the mineral status of the animal (i.e., the circulating or reserve levels for at least some minerals). This would explain lowest levels of some minerals in the tine of the antler. Thus, perhaps spike deer attempt to build the antler at Ca and P contents near that of the maximum work of fracture in all parts of the antler, but a depletion of Ca reserves would render the last part to be built, the tine, with contents lower than the optimum. In fact, physiological exhaustion might explain also the high importance of K in explaining antler weight variability, and the greater percent of K and Zn in the tine: K intake reduces Ca excretion in urine [31] and, as mentioned above, Zn, through its effects on alkaline phosphatase increases rate and extent of mineralization [28,30]. Thus, deer might ingest increasing amounts of K and Zn as Ca from skeleton is depleted during the process of antler growth in order to increase the efficiency of circulating levels of Ca in blood. It should be expected that Ca and P intake was also increased, but this is unlikely to balance the Ca and P depletion from skeleton because 60–75% of Ca is obtained from bone resorption [6]. Thus, physiological exhaustion would also explain why variability of structural properties such as antler weight, length and others can be explained at such a high degree in terms of its composition: animals in better body condition might develop large antlers, and their good body condition would be shown in high mineral reserves and circulating levels close to the optimum (or lack of need for greater intake of K or Zn). This is further supported by the fact that content of each mineral is influenced by body weight and size. If physiological exhaustion can explain variability in bone chemical composition, minerals involved in such exhaustion should differ increasingly from base to top end of the antler in comparisons between well fed individuals and animals with restricted food availability.
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The hypothesis that bone chemical composition reflects the physiological status of the animal also receives support from the literature. On one hand, extreme mineral deficiency results in less developed bones and more fragile ones (reviewed in 14), while the single addition of other minerals such as copper can result in increased growth in domestic pigs [32]. Detailed studies in animal science have shown that increasing the level of some minerals such as P increased bone strength and ash content within standard nutrition plane [33], or that concentration of some minerals in bone such as manganese can be raised or lowered by adding this mineral to a standard diet [9]. However, the studies conducted in deer showed that antler size and composition, and bone composition did not vary by diet (diets differing in P content: [34]; diets differing in protein content: [35]), although ecological conditions affecting forage availability did influence antler characteristics [3,7]. This is more striking considering that, apart from the effect of availability of nutrients studied in healthy domestic animals, wild deer have to cope with the effect of parasites and pathogens. Nematodes and other parasites can produce disturbances in protein metabolism and result in reduced absorption or retention of minerals, especially P, [36]. Some pathogens such as red blood infecting protozoans also require minerals (iron in this case) to survive, and must take these from the host [37]. Thus, subclinical pathogen infections in cattle [38], and platyhelminthes infection that do not result in weight losses in deer [39], result in reduced concentration of some minerals in serum or body organs such as the liver. It is likely that some of these parasites and pathogens affect bone composition, and an interesting hypothesis to test in future studies should be examining antler and other bone composition with respect to parasite or pathogen load. Finally, a striking effect is that, among body weight at different stages or body measures, most of the variability in mean concentration of Ca, P, K and Na was explained by gains during lactation, and this effect was also found in PCA factors mostly related to these minerals. This is even more striking when considering that antlers start growing at almost one year of age. Early nutrition, particularly during the period of maternal care, is of profound importance as it can affect subsequent life trajectory of animals by altering growth rates [40,41]. Such maternal effects are more likely to be shown in lactation because this is the most energetic expensive stage of reproduction [42], and thus in Iberian deer adverse effects affecting lactation result in reduced growth that last further than the first year of life [16,43–49]. However, it is striking that lactation gains were more important than body size or weight at the age of antler growth precisely in minerals contributing to most of the mineral content in antlers, but not in that of microminerals. A possible explanation might be that the growth trajectory of the body and, in particular, that of the skeleton was set during lactation. If so, this should result in a great influence on antler size and composition because most of the major minerals (e.g., Ca and P) used for growing the antler are taken from bone resorption [6].
Because, as mentioned above, mechanical properties of bone do not depend on phylogeny, and because Ca deposition (and possibly physiological effort) for antler growth is similar to that of lactation in most mammals including humans [6,50], some of the effects found in this study and, in general, this technique, might be extended to other mammals and possibly our own species. In conclusion, bone composition (at least that of antlers) is variable within standard conditions. It is likely that at least part of such variation plays a role in bone mechanical properties. Bone composition also provides information about the bearer. This opens an interesting area of research in bone biology because the information that could be obtained from bone composition may range from early growth to possibly mineral nutrition status, or even be an index for some diseases. This technique might prove useful in other species including humans, but deer appear to be a fantastic model for bone studies because it allows carrying out destructive studies on bone without the need of invasive techniques or surgery. Acknowledgments This study was supported by projects AGL2003-08547 (MCYT), PAC06-01304298 (JCCM) and PBI 05-040 (JCCM). The authors wish to thank Jose Angel Gómez Nieto and Pedro Jesús Sánchez for help in data collection, Fulgencio Cebrián and Isidoro Cambronero for help in handling the animals and two anonymous referees.
References [1] Harvey PH, Bradbury JW. Sexual selection. In: Krebs JR, Davies NB, editors. Behavioural Ecology: An Evolutionary Approach. Oxford, UK: Blackwell Scientific; 1991. p. 203–33. [2] Ditchkoff SS, Lochmiller RL, Masters RE, Hoofer SR, Van Den Busshe RA. Major-histocompatibility-complex-associated variation in secondary sexual traits of white-tailed deer (Odoicoleus virginianus): evidence for good-genes advertisement. Evolution 2001;55:616–25. [3] Weladji RB, Holand O, Steinheim G, Colman JE, Gjostein H, Kosmo A. Sexual dimorphism and intercohort variation in reindeer calf antler length is associated with density and weather. Oecologia 2005;145:549–55. [4] Huxley J. The relative size of antlers of deer. Proc Zool Soc London 1931;72:819–64. [5] Meister W. Changes in biological structure of the long bones of whitetailed deer during the growth of antlers. Anat Rec 1956;124:709–21. [6] Muir PD, Sykes AR, Barrell GK. Calcium metabolism in red deer (Cervus elaphus) offered herbages during antlerogenesis: kinetic and stable balance studies. J Agric Sci Camb 1987;109:357–64. [7] Schmidt KT, Stien A, Albon SD, Guinness FE. Antler length of yearling red deer is determined by population density, weather and early lifehistory. Oecologia 2001;127:191–7. [8] Ullrey DE. Nutrition and antler development in white-tailed deer. In: Brown RD, editor. Antler Development in Cervidae. Kingsville, TX, USA: Caesar Kleberg Wildlife Research Institute; 1983. p. 49–59. [9] Doyle JJ. Toxic and essential elements in bone—A review. J Anim Sci 1979;49:482–97. [10] Field RA, Riley ML, Mello FC, Corbridge MH, Kotula AW. Bone composition in cattle, pigs, sheep and poultry. J Anim Sci 1974;39:493–9. [11] Pathak NN, Pattanaik AK, Patra RC, Arora BM. Mineral composition of antlers of three deer species reared in captivity. Small Rumin Res 2001;42:61–5.
T. Landete-Castillejos et al. / Bone 40 (2007) 230–235 [12] Purdey M. Elevated silver, barium and strontium in antlers, vegetation and soils sourced from CWD cluster areas: do Ag/Ba/Sr piezoelectric crystals represent the transmissible pathogenic agent in TSEs? Medical Hypotheses 2004;63:211–25. [13] Brown RD. Nutrition and antler development. In: Bubenik GA, Bubenik AB, editors. Horns, Pronghorns and Antlers. New York, NY, USA: Springer-Verlag; 1990. p. 427–41. [14] McDowell LR. Minerals in Animal and Human Nutrition. Amsterdam, Netherlands: Elsevier; 2003. [15] Brelerut A, Pingard A, Thériez M. Le Cerf et Son Élevage. Paris, France: INRA; 1990. [16] J.A. Gómez, T. Landete-Castillejos, A. Garcia, L. Gallego, Importance of lactation on posterior growth and antler development in the Iberian red deer (Cervus elaphus hispanicus). A longitudinal study from birth to 2.5 years of age. Livestock Prod Sci, in press. [17] Gallego L, Landete-Castillejos T, Garcia A, Sánchez PJ. Seasonal effects and lactational changes in mineral composition of milk from iberian red deer (Cervus elaphus hispanicus). J Dairy Sci 2006;89:589–95. [18] Osborne DR, Voogt P. The Analysis of Nutrients in Foods. London, UK: Academic Press; 1978. [19] Currey JD. The design of mineralized hard tissues for their mechanical functions. J Exp Biol 1999;202:3285–94. [20] Currey JD. Comparative mechanical properties and histology of bone. Am Zool 1984;24:5–12. [21] Currey JD. Mechanical properties of bone tissues with greatly differing functions. J Biomechanics 1979;12:313–9. [22] Currey JD. Tensile yield in compact bone is determined by strain, post yield behaviour by mineral content. J Biomechanics 2004;37:549–56. [23] Currey JD, Brear K. Hardness, Young's modulus and yield stress in mammalian mineralized tissues. J Mater Sci: Mater Med 1990;1:14–20. [24] Currey JD, Brear K. Ontogenetic changes in the mechanical properties of the femur of the polar bear Ursus maritimus. J Zool (London) 1990;222: 49–58. [25] Currey JD, Brear K, Zioupos P. Notch sensitivity of mammalian mineralized tissues in impact. Proc R Soc London, Ser B Biol Sci 2004; 271:517–22. [26] Erickson GM, Catanese J, Keaveny TM. Evolution of the biomechanical properties of the femur. Anat Rec 2002;268:114–24. [27] Patel N, Best SM, Bonfield W, Gibson IR, Hing KA, Damien E, et al. A comparative study on the in vivo behavior of hydroxyapatite and silicon substituted hydroxyapatite granules. J Mater Sci: Mater Med 2002; 13:1199–206. [28] Robey PG, Boskey AL. Extracellular matrix and biomineralization of bone. In: Favus MJ, editor. Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. Washington, DC, USA: American Society for Bone and Mineral Research; 2003. p. 38–46. [29] Driessens FCM, Verbeeck RMH. The dynamics of bone mineral in some vertebrates. Z Naturforsch 1986;41c:468–71. [30] Maki K, Nishioka T, Nishida I, Ushijima S, Kimura M. Effect of zinc on rat mandibles during growth. Detofacial Orthop 2002;122:410–3. [31] Rafferty K, Davies KM, Heaney RP. Potassium intake and the calcium economy. J Am Coll Nutr 2005;24:99–106. [32] Paik I. Application of chelated minerals in animal production. AsianAustralian J Anim Sci 2001;14:191–8.
235
[33] Traylor SL, Cromwell GL, Lindemann MD. Bioavailability of phosphorus in meat and bone meal for swine. J Anim Sci 2005; 83:1054–61. [34] Grasman BT, Hellgren E. Phosphorus nutrition in white-tailed deer: nutrient balance, physiological responses, and antler growth. Ecology 1993;74:2279–96. [35] Asleson MA, Hellgren EC, Varner LW. Effects of seasonal protein restriction on antlerogenesis and body mass in adult male white-tailed deer. J Wildl Manage 1997;61:1098–107. [36] Coop RL, Kyriazakis I. Influence of host nutrition on the development and consequences of nematode parasitism in ruminants. Trends in Parasitology 2001;17:325–30. [37] Rivera MT, Pereira De Souza A, Cremonini Araujo-Jorge T, Lisboa De Castro S, Vanderpas J. Trace elements, innate immune response and parasites. Clin Chem Lab Med 2003;41:1020–5. [38] Naresh R, Dwivedi SK, Dey S, Swarup D. Zinc, copper and cobalt concentrations in blood during inflammation of the mammary gland in dairy cows. Asia-Australian J Anim Sci 2001;14:564–6. [39] Vengušt G, Klinkon M, Bidovec A, Vengušt A. Fasciola hepática: effects on blood constituents and liver minerals in fallow deer (Dama dama). Vet Parasitol 2003;112:51–61. [40] Bernardo J. Maternal effects in animal ecology. Am Zool 1996;36:83–105. [41] Metcalfe NB, Monaghan P. Compensation for a bad start: grow now, pay later. TREE 2001;16:254–60. [42] Oftedal OT. Pregnancy and lactation. In: Hudson R, White RG, editors. Bioenergetics of Wild Herbivores. Boca Raton, FL, USA: CRC Press; 1985. p. 215–38. [43] Landete-Castillejos T, Garcia A, Molina P, Vergara H, Garde J, Gallego L. Milk production and composition in captive Iberian red deer (Cervus elaphus hispanicus): effect of birth date. J Anim Sci 2000;78: 2771–2777. [44] Landete-Castillejos T, Garcia A, Gallego L. Calf growth in captive Iberian red deer (Cervus elaphus hispanicus): effect of birth date and hind milk production and composition. J Anim Sci 2001;79:1085–92. [45] Landete-Castillejos T, Garcia A, Gómez JA, Laborda J, Gallego L. Effects of nutritional stress during lactation on immunity costs and indices of future reproduction in Iberian red deer (Cervus elaphus hispanicus). Biol Reprod 2002;67:1613–20. [46] Landete-Castillejos T, Garcia A, Gómez JA, Gallego L. Lactation under food constraints in Iberian red deer (Cervus elaphus hispanicus). Wildl Biol 2003;9:131–9. [47] Landete-Castillejos T, Garcia A, Gómez JA, Berruga MI, Gallego L. Lactation under induced five-month delay reproduction in Iberian red deer (Cervus elaphus hispanicus). J Exp Zool 2004;301A:261–5. [48] Landete-Castillejos T, Garcia A, Gomez JA, Berruga MI, Gallego L. Effects of birth date and order in lactation performance of iberian red deer (Cervus elaphus hispanicus). J Dairy Sci 2005;88:154–8. [49] Landete-Castillejos T, Garcia A, López-Serrano FR, Gallego L. Maternal quality and differences in milk production and composition for male and female Iberian red deer calves (Cervus elaphus hispanicus). Behav Ecol Sociobiol 2005;57:267–74. [50] Wysolmerski JJ. The evolutionary origins of maternal calcium and bone metabolism during lactation. J Mammary Gland Biol Neoplasia 2002;7: 267–276.