Fig. 3. Partial purification of the sperm chemoattractant by RP-HPLC. The active fraction from egg-conditioned sea water (ESW) was eluted from a Sep-Pak cartridge in 25 % methanol, concentrated, and injected onto a C₁₈ column. Compounds were eluted over a 30 min run using a gradient from 5 % to 50 % methanol in 0.1 % TFA. Fractions (0.5 ml) were pooled as indicated, lyophilized, and bioassayed for sperm motility and orientation. (A) Absorbance spectrum at 220 nm. (B) Sperm swimming response to isolated fractions. Fractions were diluted to the appropriate starting volume of sea water and tested in the sperm motility assay; swim speeds were measured using computer-assisted video motion analysis. Values are means + S.E.M. Fresh ESW and filtered sea water (FSW) were used as positive and negative controls, respectively. ^*Means significantly greater than negative controls (P<0.01). (C) Chemotactic response of abalone sperm to isolated fractions, represented as cell counts in a flat capillary bioassay. Values are means + S.E.M.