cf2

Research Paper

Metal Analysis of Deer Hooves in South Alabama: Sentinels of Deer Health, Nutrition, and the Environment

Aubree Archie, Emily K. Fike, and Maureen K Murphy*

Department of Chemistry & Biochemistry, Huntingdon College, Montgomery, Alabama 36106, USA, *Corresponding author: Maureen K. Murphy, Email: maureenm@hawks.huntingdon.edu

Received April 17, 2017, revised August 27, 2017, accepted August 29, 2017

Publication Date (Web): August 29, 2017

© Frontiers in Science, Technology, Engineering and Mathematics

Abstract

Metal presence in deer may be a consequence of deer nutrition/health, deer baiting, contamination via lead or metal ammunition, and/or environmental factors. We sought to determine a method to examine the extent of metal content ( Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, V, and Zn) in hooves of deer killed in three counties of south Alabama. After the 2016 hunting season opened, a total of 32 deer hooves from deer were obtained from deer processing companies and hunters in central Alabama. Samples were weighed, identified by county, sex, and deer size, and completely dissolved and digested in concentrated nitric acid, followed by a 100-fold dilution in 5% nitric acid.  All samples were analyzed for ten metals using inductively-couple plasma optical emission spectroscopy (ICP-OES) against standards used in industrial protocol. Lead content in samples of central Alabama deer hooves ranged from 0.3 to 1.4% by mass. Based upon the amount of ten metals determined in our studies, the concentration of metals determined in the deer hooves samples as measured by ICP-OES showed that the comparative levels of metals present were: Zn > Fe > Cu > Ni > Mn  > Pb > Cr > Cd, with V not detected and Co detected in hooves from only two deer in our studies.  The metals detected in deer hooves from three counties in Alabama reflect deer nutrition, health, and environment, including added metals from mineral supplements. Our metal analysis of deer hooves demonstrates that deer hooves may be used as sentinels of the environment, health, and nutrition of deer in a specific region.

Keywords

Keratin, ICP-OES, Atomic emission, Plasma, Calibration, Digestion, Metals, Deer nutrition, Environmental metals, Deer hooves

Introduction and Background

Interest in the chemistry and biochemistry of wild game is high in the southeastern United States. Miller and researchers at The Deer Laboratory at the University of Georgia studied the chemical composition, including some minerals, of female deer serum (Miller et al 1989). Hooves contain the protein keratin, the same protein found in human fingernails and toenails. The structure of keratin revealed a protein rich in sulfur-containing amino acids and disulfide bonds (Wang et al 2016). Human nails have been analyzed for minerals and metals, and have been found to store metal ions ingested from food or the environment (Abdulrahman et.al 2012). A University of Georgia study (Miller et al 1985) detected 11 different minerals in a whitetail’s antlers. In addition to calcium (19 percent) and phosphorus (10 percent), the next most common elements were magnesium (1 percent) and sodium (0.5 percent). Lesser amounts of other minerals were found including potassium, barium, iron, aluminum, zinc, strontium and manganese. There are reports of selected metal content measured in other ruminants, including cattle and elk (Hidirogou and Williams 1986). There has not been a comprehensive analysis of metal content in deer hooves.

Materials and Methods

All chemicals were obtained from Sigma-Aldrich Chemical Co. and all ICP-OES standard samples were used as received from Inorganic Ventures, Inc. Mineral free nitric acid (HNO3) was purchased from Ultra-high purity argon gas (UHP) used for the plasma was obtained from Air Gas, Inc.

Preparation and Digestion of Deer Hooves Samples. The deer hooves were removed from each deer just after killing the deer and were immediately frozen after assigning a sample number to each hoof.  For analysis, each hoof was cut 1.0 cm above the keratin hoof to remove non-hoof material, dried, and weighed. When ready for analysis, the trimmed hooves were allowed to defrost, and were dissolved completely in concentrated nitric acid and kept covered in a hood overnight to prevent excessive foaming, while digesting. After filtering each sample solution through fast filter paper, exactly 1.0 mL of each sample was removed from the hooves-nitric acid mixture and was diluted volumetrically to 100 mL with 5% nitric acid.

Elemental Analysis of Samples. Determination of Cu, Zn, Co, Mn, Fe, Cr, Cd, Ni, V and Pb was made directly on each final solution using an inductively coupled plasma optical emission spectrometer (Varian VISTA ICP-OES). Standard solutions of each sample Cu, Zn, Co, Mn, Fe, Cr, Cd, As, Ni and Pb were prepared according to Varian manufacturer procedures for ICP-OES to be used. Known standards containing 26 elements/metal ions were purchased from Inorganic Ventures, Inc. at various concentrations, including 1000 mg/L and 5000 mg/L. Standards were diluted to 500 ppb, 1000 ppb, and 5000 ppb for use as calibration standards. A calibration blank using the same solvent as was used to analyze our sample was used as well for the blank on each calibration curve. All metal ions selected passed the calibration test of the ICP-OEP software. All calibration curves (based upon standards with 0, 500, 100, and 5000 ppb of each element measured) were linear with correlation coefficients (R2) greater than or equal to 0.9990. All samples and standards were analyzed in triplicate in the same run and under the exact same instrumental settings. Prior to loading each measured sample into the auto-sampler of the ICP-OES, each sample was filtered through a syringe filter into a polycarbonate sample tube. Each sample made it through the analysis via ICP-OES without errors. The sample preparation and analysis methods used in this research duplicate those of other researchers (Hidirogou and Williams 1986; Abdulrahman et al 2012) in which percent recovery tests were conducted to certify the ICP-OES protocol used in this paper.

Table 1. Mass of deer hoove samples analyzed.

Table 2. Summary of trace metal analysis of fresh deer hooves from study sites in central Alabama countiesa,b.

aMarengo County, Coffee County, and Geneva County.

   bICP standards were obtained from Inorganic Ventures, Christiansburg, VA. www.inorganicventures.com

   cCorrected for dilution factor of samples. V not detected in any of the samples.

Figure 1. (a) Deer hoove samples prior to sample preparation. (b) Alabama counties where deer hooves were collected:  Marengo , Coffee , and Geneva .          

Results

Our results are shown in Tables 1-2 and in Figure 1. Table 1 one shows the mass of each deer hoove sample for the eight deer studied.  Figure 1 displays a photo of the deer hooves prior to sample preparation. Table 2 contains all of the data for deer hooves samples used in this study along with the county in which each deer was killed. 

Discussion

In general, the concentration of metals determined in the deer hooves samples as measured by ICP-OES showed that the comparative levels of metals amongst all samples was  Zn > Fe > Cu > Ni > Mn > Pb > Cr > Cd, with V not detected and Co detected in hooves from two of eight deer samples. The concentration of Fe measured in all samples was in the range of 29.473-40.170 ppm, with a mean of 34.310 ppm. The most massive of the deer hooves samples (Deer #8) did not contain the highest concentration of the metals studied.   Specifically, we noted some county-specific differences in metal analyses from the deer hooves studied as follows:

Hooves from Deer #1- #3 (Coffee  County)

Deer #1 hooves displayed the highest amount of Cu and Zn present in our metals tests, consistent with location of Deer #1 being killed in an area where Cu/Zn mineral blocks were used to supplement keratin levels and antler growth. Deer# 1 samples also displayed the highest levels of Zn, Cu, Cd, Fe, and Pb of all samples. All samples from this group had highest Zn levels (9.0691-11.083 ppm), and Pb levels from 1.5565-4.0699 ppm. Deer hooves from Deer #1 contained from 3-8 times the concentration of Pb as did hooves from other deer samples in our study. All were in the range of 0.5-4.0 ppm Pb. Water analysis of streams in all three counties detected Pb in the ground water at the level of 0.1-3 ppm due to  corrosion of  plumbing and/or erosion of natural deposits  The U.S. Geological Survey (USGS) has been collecting soil and stream sediment samples across the nation over the last several decades . Analysis of these samples has been aggregated at the county level. The average lead level in Coffee County soil and sediment was measured by the USGS to be  13.688 parts per million, which is below the EPA 400-ppm hazard limit (See: http://environmental-health.healthgrove.com/l/67/Coffee-County-AL) . The major industries in Coffee County include: poultry, auto parts manufacturing, and tractor-trailer manufacturing.

Hooves from Deer #4 - #6 (Geneva County)

Deer #4 sample was one of only two in which Co was detected at 2.7635 ppm. Deer #5 sample had the highest measured Mn level at 9.9357 ppm. The mean for all samples from all counties was 3.8847 ppm Mn. Recently, researchers have determined relatively high content of Mn in peanut shells by ICP-AE analysis (Anike et al 2016). Low Cu, Zn, and Pb levels were measured for Geneva County samples compared to other counties. Major industries in Geneva County include: manufacture of aluminum grills, peanut shell processing, and wholesale food production.

Hooves from Deer #7 - #8 (Marengo County)

Deer hooves from Deer #7 and Deer #8 contained the highest measured values of nickel (22.219 and 20.032 ppm, respectively). The mean Ni content measured for all samples in this study was 12.464 ppm. Deer hooves from Marengo County had the second-highest Pb and Cu levels of three counties. Copper-leaching from wood preservatives and nickel from erosion of ores may also contribute to metal ions in deer hooves. Major industries in Marengo County include: paper production, wood products, veneer products, and cement production.

The role of Zn and Cu in animal hooves is critical. Zinc is found in high concentrations in animal hooves, with one form being the zinc finger proteins, rich in the amino acid cysteine. As part of the zinc finger proteins, zinc is needed for cell multiplication and the assembly of keratin. Zinc proteins incorporated into keratin are also responsible for the helical structure that gives hooves their strength. One study showed that horses with insufficient hoof horn strength had less zinc in the hoof horn and plasma than did horses with no hoof horn damage (Osborn et al 2004). Many enzymes that are responsible for a multitude of cellular processes require zinc, such as binding calcium in keratin. In humans, more than 100 different enzymes depend on zinc for their ability to catalyze vital chemical reactions (Burt at al 2008).

The synthesis of the harder type of keratin is linked with copper as well as formation of connective tissue. In horses, one study found a deficiency in both zinc and copper increased the incidence of seedy toe in performance horses (Kincaid 1999). In cattle, a copper deficiency is known to be a cause of poor hoof condition, greater incidence of foot rot, heel cracks and sole abscesses (Hidirouglou et al 1986).

Conclusion

Our research demonstrates that a reliable method has been developed for the ICP-OES metal analysis of fresh deer hooves, and that results from this research can be used to gauge the health, nutrition, and environment of deer in the state.  Future research will be expanded to include deer hoove samples from each county in the state, with GPS location of each deer killing site incorporated into our research, and expansion of the number of  metals studied to include aluminum.

Acknowledgements

We are grateful to Steve Rodopoulos and the Montgomery Water Works (MWW) for the donation of the Varian VISTA ICP-OES instrument, supplies, and training. We also acknowledge Karen Blake of MWW, for training faculty and students in the use and maintenance of the instrument, and Chad Commander of Agilent Technologies, Inc. for getting the instrument operational and software optimized.

References

Abdulrahman, FI, Akan, JC, Chellube, ZM,and Waziri, M (2012)  Levels of heavy metals in human hair and nail samples from Maiduguri Metropolis, Borno State, Nigeria, World Environ., 2, 81-89 .

Anike FN, Yusif M, and Isikhuemhen OS (2016) Co-substrates of peanut shells with cornstalks enhances biodegradability by Pleurotus ostreatus, J. Bioremediat. Biodegrad. 7, 1-7.

Burt A, and Trivers R, “Genes in conflict: The biology of selfish genetic elements,” Harvard University Press, 2008

Hidiroglou M., and Williams CJ (1986) Mineral and amino acid composition of beef cattle hooves, Am. J. Vet. Res., 47, 301–303.

Kincaid RL. 1999. Assessment of trace mineral status of ruminants: A review, J. Anim. Sci., 2000, 1–10.

Miller, KV, Marchinton, RL, Beckwith. JR, and Bush, PB (1985) Variations in density and chemical composition of white-tailed deer antlers, J  Mammalogy, 66, 693-701.

Miller, KV, Knox, WM, Marchinton, RL, and Bush, PB, “Serum chemistry of female white-tailed deer,” American Society of Mammalogists Meeting, Fairbanks, Alaska, 1989.

Miller, KV (1994) Deer hooves and deer tracks, In “Deer,” D. Gerlach Ed., pp. 50-52, Stackpole Books.

Osborn, DA, Bubenik, G, and Miller, KV (2004) Doppelkopf and other antler abnormalities, Quality Whitetails, 11, 26-30.

Wang, B, Yang, W, McKittrick, J, Meyers, MA (2016) Keratin: Structure, mechanical properties, occurrence in biological organisms, and efforts at bioinspiration, Progress in Materials Sci., 76, 229–318.

Citation:

Aubree Archie, Emily K. Fike, and Maureen K. Murphy (2017) Metal analysis of deer hooves in south Alabama: Sentinels of deer health, nutrition, and the environment, Frontiers in Science, Technology, Engineering and Mathematics, Volume 1, Issue 1, 5-9