© 2009 American Registry of Professional Animal Scientists. The use of cooled and frozen semen offers many advantages to breeders. However, many stallions produce spermatozoa that are unable to endure the stresses associated with these preservation processes. Improving the quality and viability of equine spermatozoa via appropriate dietary alterations may render spermatozoa from these stallions commercially viable for preservation. One way to improve sperm quality may be through increasing dietary intake of PUFA, specifically docosahexaenoic acid (22:3n-3), to enhance the plasma membrane of the sperm cell. To evaluate this possibility, along with analyzing varying supplemental sources of n-3 fatty acids that are high in docosahexaenoic acid, stallions were placed into 1 of 2 treatment groups and fed either an algae- and flaxseed-based or a fish-based dietary supplement. Semen was collected every 2 wk for 112 d, 28 d before and 84 d during PUFA supplementation. Spermatozoa motion characteristics and membrane integrity and morphology of fresh, cooled, and stored (24 and 48 h), and frozen and thawed semen samples were analyzed. When spermatozoa obtained from stallions in each treatment were compared, no overall improvements were found (P > 0.05) in motility, percentage of membrane-intact spermatozoa, and percentage of morphologically normal spermatozoa regardless of supplementation or source of supplementation.