In the present study, Umair et al used multiparametric flow cytometry approaches to investigate changes occurring during the conservation of equine spermatozoa. This paper is proof of the utility of flow cytometry in the field of spermatology. I think this is an interesting paper, that after thorough and careful revision can be published.

Although I consider this is a paper of merit, there are two major points to clarify.

The first one is the terminology, the term “capacitation like” should be avoided or at least used with caution. I will suggest that the authors should focus on what is really changing, merocyanine staining patterns, and Ca2+. The term “capacitation-like” is vague and, in any case, reflects a process of sperm deterioration, that conventional analysis is unable to detect. This approach adds value to the work of the authors and should underline the value of flow cytometry approaches to disclosing this kind of sperm damage. In addition, changes in membrane fluidity/permeability and intracellular Ca2+ are not exclusive of capacitation and appear in different forms of cell death

There are major technical concerns, is very surprising that in a paper submitted to Cytometry, there is not a single representative cytogram. I can´t imagine a paper given data on WB analysis without presenting the actual western blots. I see that the authors uploaded the FCS files to a repository, but this is not sufficient. In relation to this, there are some “odd” probes combinations; for example, in page 9, lines 180 onwards the authors claim to have used the combination Mitosox red, and dihydroethidium to determine mitochondrial and total superoxide. Mitosox red has an excitation-emission maxima of 510/580 nm while Dihydroethidim has an excitation-emission maxima of 518/605 nm, this is nearly the same. Separation of the spectra of these probes will require a spectral flow cytometer, but the authors have used a conventional one, and with the filters that the authors indicate, seems that both probes have been detected using a 585/42 BP. So, this point must be clarified, do these assays were conducted independently? Otherwise, is nearly impossible to differentiate both probes under the conditions described by the authors.

In addition, although a NAPH oxidase may be present in the sperm membrane, most of the production of superoxide is mitochondrial in the spermatozoa, so probably the use of dihydroethidium is redundant

Other minor technical questions

How debris and doublets/ clumps were separated?

In sum, to be further considered authors should

1.- provide representative cytograms of all the multiparametric assays used, clearly showing the gating strategy used and the population studied

2.- provide an adequate explanation of the overlap in the Ex/Em between Mitosox red and dihydroethidium

3.- Provide an updated terminology regarding the “aging” that the spermatozoa experiences under storage

The authors have done remarkable work revising their manuscript. They have provided accurate explanations to all the questions. Obviously, the combination of probes can always be further discussed, and some concerns regarding some very popular assays in veterinary andrology can be raised. But there is only one important technical detail to be addressed before publication:

In the dot plots, there is no indication if the fluorescence is collected as area or high, this is particularly important to identify and remove doublets, clumps etc…please specify in figure 1 (and in the correspondent supplementary figures) that forward scatter is collected as area