Hemoglobins of elk (Cervus canadensis), moose (Alces alces), pronghorn antelope (Antilocapra americana), mule deer (Odocoileus hemionus), and desert bighorn sheep (Ovis canadensis) were analyzed by analytical polyacrylamide gel isoelectric focusing (APGIF). Hemoglobin patterns of fresh, frozen, and air-dried hemolysates were characteristic for each species. Moose and elk patterns were similar, but not identical. Hemoglobin patterns of wild ungulates were compared to those of domestic sheep, cattle, and swine. With the exception of wild and domestic sheep, banding positions and densities were species specific. APGIF is a reliable, consistent, rapid, and inexpensive technique that may be used to identify animal species from fresh, frozen, and air-dried blood samples. J. WILDL. MANAGE. 40(3):517-522 Identification of animal proteins has proven to be an important forensic tool in wildlife law enforcement. As early as 1914, the use of an antiserum to precipitate venison protein was considered valid evidence for meat identification (Clarke 1914). Additional techniques have been developed since to identify animal proteins of suspect origin. Jackson (1958) demonstrated various methods for identifying the meat of white-tailed deer (Odocoileus virginianus) by analysis of muscle tissue. Later, he described an improved method to identify muscle tissue with paper chromatography (Jackson 1962). From chromatographic patterns of amino acids, Brunetti (1965) identified subspecies of deer from California. More recently, Dilworth and McKenzie (1970) reported protein differences between three types of wild cervids and three of domestic animals, using starch-gel electrophoresis of muscle extracts. Blood protein analyses, particularly the identification of hemoglobins, provide another important method for identifying animal proteins to species. The two most common techniques used to separate blood proteins have been ion exchange chromatography and gel electrophoresis (Drysdale et al. 1971). Ion exchange chromatography, although superior to gel electrophoresis, can be costly, cumbersome, and time-consuming. Many prefer starch-gel or polyacrylamide electrophoresis, because it is relatively fast, simple, and convenient. However, because of difficulties associated with detecting small charge differences in the physiochemical nature of blood proteins, no single conventional electrophoretic system has proven adequate, and resolution often has been inadequate for critical evaluation. Isoelectric focusing, a newer and modified form of electrophoresis, separates proteins on the basis of surface charge and, under ideal conditions, can resolve isoelectric point differences that vary as little as 0.02 pH unit (Vesterberg and Svensson 1966). Drysdale et al. (1971) found that isoelectric focusing provided a significant advance in high resolution separations of hemoglobins. By this method, Jeppsson and Berglund (1972) were able to detect human hemoglobin variants that would not have been detected by conventional electrophoretic methods. Because of the relative ease with which blood samples generally may be acquired J. Wildl. Manage. 40(3):1976 517 This content downloaded from 207.46.13.97 on Sun, 22 May 2016 05:48:10 UTC All use subject to http://about.jstor.org/terms 518 IDENTIFICATION OF UNGULATE HEMOGLOBINSBunch et al. at or near the site of a kill or from meat, and because of the relatively stable nature of the hemoglobin molecule, this investigation was undertaken to evaluate the possible forensic application of isoelectric focusing by (1) identifying hemoglobin types and patterns of Utah's major big game species by analytical polyacrylamide gel isoelectric focusing (APGIF), and (2) comparing these patterns with those of domestic livestock animals. We wish to thank the Utah Division of Wildlife Resources for assistance and cooperation in collecting blood samples from big game species. MATERIALS AND METHODS Blood samples were collected in heparinized Vacutainer (Becton-Dickinson Co., Rutherford, N.J.) tubes from the jugular vein of live animals and from harvested animals at checking stations. Hemolysates were prepared as described by Drabkin (1946). Red cells were washed thoroughly and packed by successive mixing and centrifuging: 2 times with a mixture consisting of 0.9 percent sodium chloride and 0.01 M potassium cyanide and 3 times with a mixture composed of 1.2 percent sodium chloride, 0.01 M potassium cyanide, and 0.0025 M aluminum chloride. Potassium cyanide was used in the washing process to nullify the effects of auto-oxidation (Drysdale et al. 1971). A stroma-free solution was obtained by lysing the cells with a 1.5 volume of distilled H20, thoroughly mixing with an 0.5 volume of toluene, storing at 4 C for 10-15 hours, centrifuging at 5,000 rpm for 10 minutes, and then siphoning off the reddish hemoglobin layer. Hemoglobin was of the order of 8-10 mM/1 and was sufficient for analysis without further concentration. From each processed blood sample, an aliquot was frozen at -30 C and another was air-dried at room temperature. Both frozen and air-dried hemolysates were analyzed by APGIF quarterly for 1 year to assess the stability of the hemoglobin molecule under long-term storage conditions. All fresh hemolysates were analyzed 4-5 days after collection. Samples were collected from 24 elk, 25 moose, 10 pronghorn antelope, 36 mule deer, and 12 desert bighorn sheep. Hemoglobin patterns of 38 Rambouillett, Columbia, and Dorset domestic sheep; 20 Holstein and Hereford cows; and 12 Hampshire domestic pigs were compared with hemoglobin patterns of wild species. Patterns of fresh, frozen, and air-dried hemolysates were compared. Hemoglobins were analyzed by acrylamide electrofocusing as described by Wrigley (1968). The acrylamide (Eastman electrophoresis grade) and ampholyte (LKB distributors-California branch, pH 6-8, 40 percent wt/vol) were diluted to final concentrations of 4.0 and 1.8 percent, respectively. Hemolysates were diluted 30fold with distilled H20 and then further diluted 150-fold upon incorporation into the acrylamide-ampholyte solution. The solution was degassed under vacuum, then gel polymerization was initiated by the addition of ammonium persulfate. Polyacrylate tubes, 12 x 2.5 cm (i.d.) sealed at one end with parafilm, were filled to within 0.2 cm of the top and carefully layered with H20 to provide a flat surface. Gel electrofocusing was conducted with an apparatus similar to those commonly used for disc electrophoresis. The lower two-thirds of the tubes were contained in a water-jacketed chamber and maintained at 4-5 C. Solutions of 1 percent phosphoric acid (upper chamber) and 1 percent ethylenediamine (lower chamber) were used as anolyte and catholyte, respectively. The current of 2 mA/tube was established until J. Wildl. Manage. 40(3):1976 This content downloaded from 207.46.13.97 on Sun, 22 May 2016 05:48:10 UTC All use subject to http://about.jstor.org/terms IDENTIFICATION OF UNGULATE HEMOGLOBINS Bunch et al. 519