Problem statement: Two experiments were conducted to determine the effect of the prostatic fluid from the first and third fractions of the ejaculates on the freezability of poodle dog sperm. Approach: The ejaculates from 2 trained poodle dogs were collected. Effect of prostatic fluid from the first and third fractions of ejaculates on cryosurvival of dog sperm was examined. SDS-PAGE analyses were performed to verify whether there were any modifications in the protein patterns of the first and third fractions. Results: When second fractions of Dog A sperm were combined with the prostatic fluid from the first and third fractions of the ejaculates, sperm motility index was decreased compared to second fractions were collected alone. The post-thaw motility of Dog A sperm was significantly reduced when prostatic fluid from the first and third fractions of the ejaculates was added. The motility of frozen-thawed Dog B sperm did not appear to be altered when it was diluted with either of the prostatic fluids. The major bands present in both prostatic fluids corresponded to proteins with molecular masses of 30 and 75 kDa. The amount of proteins in the prostatic fluid from the third fraction was twice-three times as much protein present compared to the first fraction. Conclusion: Collection of the sperm-rich fraction along with either the prostatic fluid from the 1st and 3rd fractions of the ejaculates during sperm processing has varying detrimental effects on the motility characteristics of frozen-thawed sperm. © 2009 Science Publications.