Master of Science / Grain Science and Industry / Greg Aldrich / Processing pet food can be beneficial, but can also have adverse effects on shelf-life and nutrient survival. Most affected are supplemental vitamins and essential fatty acids (EFA). Pet food complicates this relative to human foods by combining all elements into the product before processing and requiring an extensive shelf-life (up to 2 years). The objective of this research was to determine the effects of processing, diet, and storage conditions on vitamin (vitamin A, vitamin D₃, vitamin E, folic acid and thiamine) and omega-3 fatty acid (with an emphasis on eicosapentaenoic acid; EPA 20:5n3, and docosahexaenoic acid; DHA; 22:6n3) retention. The research was conducted in two separate experiments. Each experimental diet was produced on a single-screw extruder and triple-pass dryer. Target nutrients were evaluated in premixes in tandem to extruded diets. The vitamin study was conducted as a 3 X 2 X 2 factorial arrangement of treatments with 3 levels of dietary crude protein (CP), 2 screw speeds in the extruder, and 2 levels of time X temperature combinations in the dryer. Vitamins were added at 10 times normal levels to aid in analysis. The EFA study was conducted as a 3 X 3 factorial arrangement of treatments with 3 levels of dietary protein and 3 different omega-3 sources: fish oil, fish meal, or purpose-grown algae rich in DHA. In the vitamin premix study, the quantity of vitamins declined by approximately 50% over 6 months storage in ambient conditions (AMB; 20C, 50%RH), and all except folic acid were lost to some degree in stressed shelf life testing (SSLT; 50C, 70% RH) over 6 weeks. In all cases, the concentration of vitamins in food exiting the extruder and dryer were lower than target levels. As CP increased, the retention was higher (P ≤ 0.05) for vitamins A, E, and folic acid off the extruder (e.g. 225,352 vs. 219,184 and 206,249 IU/kg of vitamin A for high vs. medium and low CP, respectively), and vitamin D₃, E, and folic acid off the dryer (e.g. 9,047 vs. 7,473 and 6,945 IU/kg of vitamin D₃ for high vs. medium and low CP, respectively). During storage of finished pet food in AMB, vitamins A and D₃ were lost (P < 0.05) to the greatest degree (49 and 22%, respectively). The total retention following both processing and AMB storage was 27, 68, 78% for vitamins A, D₃, and E, respectively, while folic acid and thiamine were relatively stable. In SSLT storage, all vitamins except vitamin E were depleted more than 60% (P < 0.05) by 24 weeks, whereas total retention following both processing and SSLT storage was 3, 59, 43, 33, and 7% for vitamins A, D₃, and E, folic acid, and thiamine, respectively. This would suggest that beyond processing losses, the vitamins are relatively stable in premixes and foods if stored in AMB conditions. In the study to evaluate fatty acid stability within a vitamin premix, EPA, DHA, and total omega-3 fatty acids were relatively stable during storage over 6 weeks with losses no greater than 12% in stressed shelf life testing (SSLT; 40C, 70% RH). While in ambient conditions (23C, 50% RH) over 3 months, there was a total loss of EPA, DHA and total fatty acids by 17, 9, and 11%, respectively. Exiting the extruder and dryer, EPA and DHA were not affected by CP level or Omega-3 source. As SSLT storage of finished pet food increased through 24 weeks, EPA, DHA, and total fatty acids declined slightly (P < 0.05; 125, 82 mg/kg for EPA and 77, 60 mg/kg for DHA, and 418, 476 mg/kg for total fatty acids at 0 vs. 24 wk. As time in ambient storage reached 24 months, EPA, DHA, and total fatty acids declined slightly (P < 0.05; 125 vs. 78 mg/kg for EPA and 77 vs. 50 mg/kg for DHA, and 387 vs. 373 for total fatty acids at 0 vs. 24 mo.) Algal-DHA appears to be a stable source of DHA when compared to fish oil and fishmeal. During processing retention of fat soluble vitamins was less than water soluble vitamins, and the omega-3 fatty acids were relatively unaffected. Whereas, vitamins appeared to be more sensitive to temperature during storage and the omega 3 fatty acids more affected by time.