General Direct Anticancer Effects of Deer Growing Antler Extract in Several Tumour Cell Lines, and Immune System-Mediated Effects in Xenograft Glioblastoma.

Deer antlers are the fastest growing tissue. Because they are based on proto-oncogenes, to avoid the risk of cancer, antlers evolved strong anticancer mechanisms, and thus their extract (DVA) is effective also against the few human tumours studied so far. We assessed whether DVA is a general anticancer compound by testing the direct effects in cells of different tumours: glioblastoma (GBM; lines U87MG and U251), colorectal (CRC; lines DLD-1, HT-29, SW480, and SW620), breast cancer (BRCA; lines MCF7, SKBR3, and PA00), and leukaemia (THP-1). DVA reduced the viability of tumours but not healthy cells (NHC; lines 293T and HaCaT). Mobility decreased at least for the longest test (72 h). Intraperitoneal/oral 200 mg DVA/kg administration in GBM xenograft mice for 28 d reduced tumour weight by 66.3% and 61.4% respectively, and it also reduced spleen weight (43.8%). In addition, tumours treated with DVA showed symptoms of liquefactive necrosis. Serum cytokines showed DVA up-regulated factors related to tumour fighting and down-regulated those related to inducing immune tolerance to the tumour. DVA shows general anticancer effects in the lines tested and, in GBM mice, also strong indirect effects apparently mediated by the immune system. DVA may contain a future anticancer medicine without secondary effects.

Traces of Human-Mediated Selection in the Gene Pool of Red Deer Populations.

In this study, we analysed the effect of human-mediated selection on the gene pool of wild and farmed red deer populations based on genotyping-by-sequencing data. The farmed red deer sample covered populations spread across seven countries and two continents (France, Germany, Hungary, Latvia, New Zealand, Poland, and Slovakia). The Slovak and Spain wild red deer populations (the latter one in a large game estate) were used as control outgroups. The gene flow intensity, relationship and admixture among populations were tested by the Bayesian approach and discriminant analysis of principal components (DAPC). The highest gene diversity (He = 0.19) and the lowest genomic inbreeding (FHOM = 0.04) found in Slovak wild population confirmed our hypothesis that artificial selection accompanied by bottlenecks has led to the increase in overall genomic homozygosity. The Bayesian approach and DAPC consistently identified three separate genetic groups. As expected, the farmed populations were clustered together, while the Slovak and Spanish populations formed two separate clusters. Identified traces of genetic admixture in the gene pool of farmed populations reflected a strong contemporary migration rate between them. This study suggests that even if the history of deer farming has been shorter than traditional livestock species, it may leave significant traces in the genome structure.

Stem cells drive antler regeneration.

Understanding the rapid growth of deer antlers could have applications in medicine.

Pelt Biting as a Practical Indicator of Social and Environment Stress in Farmed Red Deer.

Agonistic behavioural interactions play a decisive role in the competition for food, space, mating opportunities, and establishing social rank. We used pelt biting (number of bites on an animal's body) as a proxy for assessing the intensity of agonistic animal interactions and how it responded to social, population, and heat stress factors. We modelled a 14-year time series of pelt biting records and observational data of agonistic interactions on a population of captive Iberian red deer (Cervus elaphus). We found that (i) the higher the social rank of deer, the lower the number of pelt bites received; (ii) increasing heat stress conditions caused deer to suffer more pelt bites; (iii) males received more bites than females; (iv) the heavier the deer, the lower the number of bites on their bodies; (v) the bigger the group, the more bites exhibited on its members; (vi) deer 5-6 years old suffered greater rate of pelt biting than younger or older deer; and (vii) hinds that gave birth earlier in the parturition period suffered less pelt biting than those that gave birth around the peak of the parturition season (p < 0.01 for all effects). Pelt biting is useful to predict management situations in which deer welfare could be at stake.

SWATH-MS Quantitative Proteomic Analysis of Deer Antler from Two Regenerating and Mineralizing Sections.

Antlers are the only organ in the mammalian body that regenerates each year. They can reach growth rates of 1-3 cm/day in length and create more than 20 cm2/day of skin in the antler tips (their growth centers). Previous proteomic studies regarding antlers have focused on antler growth centers (tips) compared to the standard bone to detect the proteins involved in tissue growth. However, proteins of cell differentiation and regeneration will be more accurately detected considering more growing tissues. Thus, we set out to compare proteins expressed in antler tips (the highest metabolism rate and cell differentiation) vs. middle sections (moderate cell growth involving bone calcification), using ribs as controls. Samples were obtained in mid-June with antlers' phenology corresponding to the middle of their growth period. Quantitative proteomic analysis identified 259 differentially abundant proteins mainly associated with antioxidant metabolic mechanisms, protein formation and Wnt signalling pathway, meanwhile, the mid antler section was linked to blood proteins. The high metabolic rate and subsequent risk of oxidative stress also seem to have resulted in strong antioxidant mechanisms. These results suggest that redox regulation of proteins is a key factor in the model of deer antler regeneration.

Anti-tumour activity of deer growing antlers and its potential applications in the treatment of malignant gliomas.

A recent study showed that antlers have evolved a high rate of growth due to the expression of proto-oncogenes and that they have also evolved to express several tumour suppressor genes to control the risk of cancer. This may explain why deer antler velvet (DAV) extract shows anti-tumour activity. The fast growth of antler innervation through the velvet in close association to blood vessels provides a unique environment to study the fast but non-cancerous proliferation of heterogeneous cell populations. We set out to study the anti-cancer effect of DAV in glioblastoma (GB) cell lines in comparison with temozolomide, a chemotherapeutic drug used to treat high-grade brain tumours. Here we report, for the first time, that DAV extract from the tip, but not from mid-parts of the antler, exhibits an anti-tumour effect in GB cell lines (T98G and A172) while being non-toxic in non-cancerous cell lines (HEK293 and HACAT). In T98G cells, DAV treatment showed reduced proliferation (37.5%) and colony-formation capacity (84%), inhibited migration (39%), induced changes in cell cycle progression, and promoted apoptosis. The anticancer activity of DAV extract as demonstrated by these results may provide a new therapeutic strategy for GB treatment.

Quality of main types of hunted red deer meat obtained in Spain compared to farmed venison from New Zealand.

Deer venison is increasingly valued as a natural meat. This study examines the three main sources of venison: farmed venison from New Zealand (NZ), the world's leading producer, and wild deer from Spain (SP), the second largest producer, which mainly supplies venison from traditional autumn-winter driven hunts (monteria), involving packs of dogs, and a smaller proportion culled through summer selective stalking. Meat from NZ contained more protein, lower shear force and lower n-6/n-3 ratio (P < 0.01). Spanish meat had a greater content of total, essential and non-essential amino acids (P < 0.01). Meat from deer culled in winter had lower intramuscular fat and saturated fatty acids (FA) but higher polyunsaturated FA (P < 0.01) and pH (P < 0.001) than meat from summer stalked deer. Therefore, venison presents differences by country of origin for composition, FA and texture that are likely to affect its health characteristics. Anomalies observed in meat between the winter driven hunt and non-stressful summer stalking may be attributed to the level of death stress in the case of variables such as pH. However, the effect on fat and mineral composition seems to be seasonal, depending respectively on wild diet or cyclic osteoporosis in males.

Roe deer as a bioindicator: preliminary data on the impact of the geothermal power plants on the mineral profile in internal and bone tissues in Tuscany (Italy).

The European roe deer (Capreolus capreolus) is one of the most abundant ungulate species in Europe. Many studies have investigated its distribution, behavior, and ecology, but few have focused on its role as bioindicators for pollutants, particularly regarding antlers, which has been shown to indicate also deer physiology. The presence of geothermal power plants can induce accumulation of potentially polluting elements (such as Tl, S, and Pb). Thus, we collected roe deer samples from areas of Tuscany (Italy) where power plants are present. They were divided according to whether their home range included areas close or far from geothermal power plants. We analyzed the body measurements and the profile of the minerals in the liver and antlers tissues using the ICP-OES technique. Results showed that livers from roe deer close to power plant accumulated higher quantity of Bi, Co, Ni, Tl, and S compared to controls. Males culled close to geothermal power plants had significantly lower values for weight and chest circumference, and also, the antlers showed higher values for Li and Sr in the first sampling position. Thus, despite the small sample size in this preliminary study, antlers and livers of roe deer seem to be a bioindicator of industrial impact on the environment.

Parenteral Cu Supplementation of Late-Gestating and Lactating Iberian Red Deer Hinds Fed a Balanced Diet Reduces Somatic Cell Count and Modifies Mineral Profile of Milk.

: This study describes the effects that Cu supplementation of late-gestating and lactating females (hinds) of Iberian red deer fed a balanced diet have on milk production, composition, and somatic cell count (SCC). Experimental hinds (n = 9) were subcutaneously injected every 42 days with Cu (0.83 mg Cu/kg body weight) from day 202 of gestation until the end of lactation (week 18). Control hinds (n = 8) were injected with a physiological saline solution with the same volume and at the same frequency as the experimental group. Copper supplementation decreased the SCC from 1.64 to 1.36 log 10/mL (p = 0.003) and modified the milk mineral profile. In particular, milk from hinds supplemented with Cu had more Ca (p = 0.02), Mg (p = 0.06), and K (p = 0.03) than milk from control hinds. However, Cu supplementation did not influence the Cu content of milk. Therefore, it can be concluded that Cu supplementation of hinds fed a balanced diet from late-gestation to the end of lactation could be a good strategy to reduce SCC and modify the mineral profile of milk increasing its Ca content.

A proteomic-based approach for the search of biomarkers in Iberian wild deer (Cervus elaphus) as indicators of meat quality.

The positive perception of deer meat among consumers is growing. Consequently, further efforts are needed to control its meat quality. Specifically, the aim of this study was to search protein biomarkers in connection with tenderness and intramuscular fat (IMF) content of Iberian wild red deer (Cervus elaphus) meat. Twelve venison samples classified as tender, intermediate and tough meat, as well as with different IMF were studied, using quantitative proteomic by SWATH-MS combined with bioinformatic analyses. The study of muscle proteome showed significant differences among these three groups of meat samples, since five differentially abundant proteins (IVD, LAMB1, MYL3, SDHC and SDHA) and six (FABP4, IVD, LAMB1, MYL3, CRYZ and SERPINB6) were strongly correlated with tenderness and IMF, respectively. The proteomic approach provides protein biomarkers linked to venison meat quality and marked changes in proteins related to oxidoreductase complex in skeletal muscle. SIGNIFICANCE: Meat tenderness and IMF are major concerns in game meat. The proteomic approach demonstrated a strong correlation between proteins and these quality traits, despite the complexity of samples (wild animals hunted at different ages). Our results suggest that several proteins can be biomarkers and provide a molecular insight.

Carcass and meat quality characteristics from Iberian wild red deer (Cervus elaphus) hunted at different ages.

BACKGROUND: Deer-meat consumption is increasing in several countries based on claims that it is a healthy red meat. This study assessed the effect of slaughter age (low - 26 months and younger; medium - from 27 months to younger than 42 months; and high - 42 months and older) on carcass characteristics and meat physicochemical properties of 150 Iberian wild red deer. RESULTS: Whole carcass yields of neck, backbone and flank increased (P < 0.001) with age, while yields of shoulder, tenderloin and leg decreased (P < 0.05) with age. The pH measured at 48 h post mortem at 9th rib level was not affected by age, while pH measured at 72 h post mortem at the longissimus thoracis et lumborum muscle decreased (P < 0.05) with age. Meat chemical composition was not affected by slaughter age, except for intramuscular fat (IMF), which increased (P < 0.05) with slaughter age. Finally, cooking loss and shear force were higher (P < 0.05) in meat from older deer. CONCLUSION: Most characteristics of meat from wild red deer depend on age at slaughter. The greater IMF of meat from older animals may render it more palatable. However, meat shear force also increased with age. Additional studies testing palatability seem to be necessary to evaluate the effects of slaughter age on the sensorial quality of meat from Iberian wild red deer. © 2018 Society of Chemical Industry.

Effect of age on nutritional properties of Iberian wild red deer meat.

BACKGROUND: This study assessed the effect of age (L, less than 27 months old; M, from 27 to 42 months old; H, 43 months and older) on the fatty acid profile, cholesterol content, amino acid composition and mineral content of 150 Iberian wild red deer meat samples. RESULTS: Intramuscular fat content increased (P < 0.05) with age (0.05 vs 0.12 vs 0.34% for L, M and H groups respectively), while cholesterol content decreased (P < 0.05) as the slaughter age increased (52.78 vs 48.72 vs 45.34 mg per 100 g meat for L, M and H groups respectively). The slaughter age showed differences among groups for saturated fatty acids, with the highest content in older animals (30.41 vs 34.55 vs 38.21% for L, M and H groups respectively), whereas younger deer displayed the highest polyunsaturated fatty acid levels (50.05 vs 45.24 vs 37.55% for L, M and H groups respectively). The n-6/n-3 ratio was more favorable (P < 0.05) for young and medium ages compared with that for older animals. In contrast, amino acid profile and mineral content were only slightly affected by age. CONCLUSION: As a general conclusion, wild red deer meat could be considered a good alternative to red meats for human consumption. © 2018 Society of Chemical Industry.

Between-year vocal aging in female red deer (Cervus elaphus).

OBJECTIVES: Studying animal vocal aging has potential implication in the field of animal welfare and for modeling human voice aging. The objective was to examine, using a repeated measures approach, the between-year changes of weight, social discomfort score (bites of other hinds on hind pelt), body condition score (fat reserves) and acoustic variables of the nasal (closed-mouth) and the oral (open-mouth) contact calls produced by farmed red deer hinds (Cervus elaphus) toward their young. RESULTS: Repeated measures ANOVA revealed that with an increase of hind age for 1 year, the acoustic variables of their nasal contact calls (the beginning and maximum fundamental frequencies, the depth of frequency modulation and the peak frequency) decreased, whereas in their oral contact calls only the end fundamental frequency decreased. Duration and power quartiles did not change in any call type. Body weight and body condition score increased between years, whereas discomfort score decreased. Results of this study revealed directly the short-term effects of aging on the acoustics of the nasal contact calls in the same hinds. This study also confirmed that elevated emotional arousal during emission of the oral contact masks the effects of aging on vocalization in female red deer.

Social rank affects the haematologic profile in red deer hinds.

We studied the effects of social rank on the haematologic profile in a herd of 24 female Iberian red deer hinds. Social rank hierarchy was determined and blood samples were taken and analysed. After adjusting for age and body mass, dominance ranking showed a significant negative effect (ie, lower values in dominant hinds) on white blood cell (WBC) count, haemoglobin and haematocrit. Our results are similar to those reported for stressed individuals due to physical immobilisation, but do not support the predicted enhanced erythropoiesis due to higher levels of androgens. The results for WBC numbers may also reflect that subordinate hinds must allocate a higher amount of resources to immunity as a result of injuries incurred from dominant hinds, while simultaneously facing restricted access to food sources. For red blood cell (RBC) counts, the results may be due to subordinate hinds likely needing increased haematocrit and haemoglobin levels for fast flight responses. Our data show that social rank influences haematologic profile, and thus it should be considered when correctly interpreting blood analyses in social cervid species.

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

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

The Many Axes of Deer Lactation.

In undomesticated animals information about the production and composition of milk over time is still scarce. In general, for most mammals it is known that milk composition changes across lactation, is different for male and female offspring, and even that marsupials, such as kangaroos, can simultaneously produce milk of different compositions for young of different ages. Such parallel milk production of differing compositions has not yet been studied in single-offspring placental mammals, but may help to explain behavioural processes like allosuckling (feeding the young of other adults) and lateralized suckling preferences. In this study we analysed the production and composition of milk in red deer throughout the lactation period and now confirm for the first time that there are axial differences present. The front teats, which are the favoured suckling positions of the deer's offspring, produce milk with a greater protein-to-fat ratio. Also, from the beginning of lactation the yield is greater on the left side, the side preferred by calves in all of the studied species, both at population and individual level. The links between milk production and calf behaviour in deer deserve further study.

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

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

Avoiding toxic levels of essential minerals: a forgotten factor in deer diet preferences.

Ungulates select diets with high energy, protein, and sodium contents. However, it is scarcely known the influence of essential minerals other than Na in diet preferences. Moreover, almost no information is available about the possible influence of toxic levels of essential minerals on avoidance of certain plant species. The aim of this research was to test the relative importance of mineral content of plants in diet selection by red deer (Cervus elaphus) in an annual basis. We determined mineral, protein and ash content in 35 common Mediterranean plant species (the most common ones in the study area). These plant species were previously classified as preferred and non-preferred. We found that deer preferred plants with low contents of Ca, Mg, K, P, S, Cu, Sr and Zn. The model obtained was greatly accurate identifying the preferred plant species (91.3% of correct assignments). After a detailed analysis of these minerals (considering deficiencies and toxicity levels both in preferred and non-preferred plants) we suggest that the avoidance of excessive sulphur in diet (i.e., selection for plants with low sulphur content) seems to override the maximization for other nutrients. Low sulphur content seems to be a forgotten factor with certain relevance for explaining diet selection in deer. Recent studies in livestock support this conclusion, which is highlighted here for the first time in diet selection by a wild large herbivore. Our results suggest that future studies should also take into account the toxicity levels of minerals as potential drivers of preferences.

The power of oral and nasal calls to discriminate individual mothers and offspring in red deer, Cervus elaphus.

BACKGROUND: In most species, acoustical cues are crucial for mother-offspring recognition. Studies of a few species of ungulates showed that potential for individual recognition may differ between nasal and oral contact calls. RESULTS: Vocalizations of 28 hinds and 31 calves of farmed Iberian red deer (Cervus elaphus hispanicus) were examined with discriminant function analyses (DFA) to determine whether acoustic structure of their oral and nasal contact calls encodes information about the caller's identity. Contact calls were elicited by brief separation of individually identified animals by a distance over 10 m or by a bar fence. Both oral and nasal calls of both hinds and calves showed high potential to discriminate individuals. In hinds, individuality was significantly higher in the oral than in the nasal calls, whereas in calves, individuality was equally well expressed in both oral and nasal calls. For calves, the maximum fundamental frequency was higher and the duration was longer in oral calls than in nasal calls. For hinds, the maximum fundamental frequency and the duration were indistinguishable between oral and nasal calls. Compared to the pooled sample of oral and nasal calls, separate oral or nasal call samples provided better classifying accuracy to individual in either hinds or calves. Nevertheless, in both hinds and calves, even in the pooled sample of oral and nasal calls, the degree of individual identity was 2-3 times greater than expected by chance. For hinds that provided calls in both years, cross-validation of calls collected in 2012 with discriminant functions created with calls from 2011 showed a strong decrease of classifying accuracy to individual. CONCLUSIONS: These results suggest different potentials of nasal and oral calls to allow the discrimination of individuals among hinds, but not among red deer calves. The high potential of individual recognition even with the pooled sample of oral and nasal calls allows mother and young to remember only one set of acoustic variables for mutual vocal recognition. Poor between-year stability of individual characteristics of hind oral and nasal calls would require updating keys to individual recognition each calving season.

Why do cervids feed on aquatic vegetation?

Consumption of aquatic plants is rare among cervids, despite the common occurrence of this form of vegetation. However, the paucity of literature reporting on this feeding behaviour suggests that Na (but also other minerals), protein, and the ubiquitous availability of aquatic vegetation may play a role in its consumption. We present results quantifying those factors that regulate the consumption of aquatic plants in the Iberian red deer. We focussed our study primarily on two questions: (i) what nutritional values are red deer seeking in the aquatic plants?; and (ii) why do red deer primarily use aquatic plants during the summer? A comparison of the seasonal variations in Na content between terrestrial vs. aquatic vegetation did not fully support the hypothesis that aquatic plants are being consumed more in summer because of any seasonal variation in Na availability. The Na content in the aquatic vegetation was adequate all the year-round; whereas, the Na content in the terrestrial vegetation was consistently deficient. However, a greater summer content of essential minerals and protein in the aquatic vegetation may be the cause for their consumption exclusively during the summer. We suggest that seasonal variations in the consumption of aquatic vegetation by cervids is primarily driven by temporal variations in the nutrient content, combined with seasonal variations in the physiological demands for these nutrients.