I have read with much interest the MS entitled ‘Ovarian fluid proteome variation associates with sperm swimming speed in an externally fertilising fish’. This paper presents the results of a study in which the author/s investigated the differential effect of the ovarian fluid on sperm swimming performance in an externally fertilizing salmonid fish. The protein composition of the same ovarian fluid used in the in vitro sperm analyses was subsequently quantified using proteomic tools and the covariation between OF-sperm interaction and OF protein composition allowed to identify putative proteins implicated in regulating sperm activity and, possibly, influencing sperm competition success in case of polyandrous mating (which, in this species, are very frequent).
The MS is well written, results presented clearly and I think it represents an important contribution to the literature of the field. Indeed, evidence that OF affects postmating sexual selection are rapidly growing, yet we know very little (I would say nearly nothing) about the mechanisms.

I am not expert of proteomic analysis. Therefore, I will not comment on that and my suggestions are more general.

Results about sperm velocity experiment. I would suggest the authors to present first the data regarding the sperm velocity assays. Since they used a nearly complete factorial male x female analysis, I think this information should be provided. In particular it would be interesting to know whether there was a significant male x female interaction (see Rosengrave et al. 2008). A significant interaction would indicate that OF effect depends on the identity of male and female. This would slightly different from what has been shown from the results of the present analysis, which suggests that the OF of some females had a stronger effect on sperm velocity than the OF of other females, irrespective of male identity. The two results are not incompatible, as there may be a significant female effect (which is the one revealed indirectly by the proteomic analyses), even in the presence of a significant male x female interaction. It is important to note, however, that OF proteins, to have an role on CFC, need to have a different effect on different individual males (non-directional CFC) or on different male phenotype (directional CFC – e.g. on sperm of males with different alternative reproductive tactics in case of salmons). It seems to me that there are two main results from the present study: it identified specific proteins that affect sperm velocity (and hence may potentially be involved in CFC), and it demonstrated that OF composition differs significantly across females. Both results are relevant, and worth publishing! The involvement of these proteins in CFC, in contrast, is only a possibility.

My second (minor) point regards the effect of abundant proteins, whose concentration varies across females and that are associated (when abundant) to a reduced sperm velocity. This effect may be due to a simple effect of OF viscosity, rather than to a specific effect of the protein identity. This is in contrast with the effect of rare proteins, that enhance sperm velocity and whose effect is independent of total protein content (as PC1 and PC2 should not be correlated one with the other). I think that it is known that OF protein content is positively related to viscosity and negatively affects sperm velocity. If this is correct, emphasis in the discussion should be given to the rare proteins that affect sperm velocity. This would reduce a little bit the results and the discussion, that are perhaps a bit lengthy.

Finally, I have found at the end of the MS the same list of peptides that is also given in the SEM. Not sure whether this was the intention of the author/s, but it should be removed.