25 

 and blood samples were obtained via jugular venipuncture on d 

 1 before animals were administered the concentrate and 

 thereafter on d 3, 14 and biweekly. To obtain serum, samples 

 were centrifuged at 700 x g for 25 min, supernatant decanted 

 and frozen until analyzed for Zn and Cu levels. Erythrocytes 

 were harvested from centrifuged (700 x g for 25 min) whole 

 blood from which plasma had been removed and had been washed 

 twice with 9 M cold saline solution. An erythrocyte lyase was 

 prepared by combining 1 ml erythrocytes with 1 . 5 ml deionized 

 water and frozen for storage. At the end of the experiment on 

 d 84, animals were stunned with a captive bolt shot and 

 euthanized by exsanguination . 



Liver, pancreas, kidney, bone and bone marrow 

 (metacarpus), skin, hair, hoof, neck muscle (sterno 

 mandibularis) , and eye were excised and frozen for further 

 mineral analyses. Zinc and Cu in tissues, blood constituents 

 and diet samples collected were determined by air acetylene 

 flame atomic absorption spectrophotometry on a Perkin-Elmer 

 Model 5000 with AS-50. 



Metallothionein was measured in liver, pancreas and 

 kidney by the 10Q Cd J+ -hemoglobin affinity assay (Eaton and Toal, 

 1982) . For this procedure .2 g of the tissue was homogenized 

 with 4 volumes of cold lOmM Tris-HCl buffer (pH 7.4), with a 

 Potter-Elvehjem glass-teflon tissue grinder, and centrifuged 

 (40,000 x g; 10 min; 4°C) . The supernatant was then heated 

 (100°C; 5 min) and centrifuged (10,000 x g; 5 min) . Next, 200 



