I’ve reviewed the article “Exposure to female olfactory cues hastens reproductive ageing and increases mortality when mating in male mice.” The article evaluates the impact of olfactory cues from female mice on the timing of reproductive senescence and longevity in male mice. Utilizing male house mice as the model, the authors found that males exposed to olfactory cues from unfamiliar females displayed increased mortality and reduced fertility later in life. Interestingly, this decline in fertility did not appear to be associated with changes in sperm, testicular function, body mass, or production of major urinary protein when compared to those not exposed to additional odor. This is a novel finding, as the impacts of olfactory cues from females on male performance had only been described in invertebrates previously, and this work suggests that this phenomenon may be more ubiquitous. The article falls within the biological scope of Proceedings B and aligns with the aim to publish research that is relevant to fundamental biological principles and processes.

Comments
Overall, I thought this was a fascinating study, which suggested that olfactory cues from females can impact a male’s physiology in a manner that influences their life history trajectory. I have several suggestions that provide more clarity and grammatical and rephrasing suggestions, which I believe would improve the final manuscript.

This investigation uses wild-derived mice as a model.
I think this was a strong choice for this study, but there is no emphasis on why this model was used. This is important to emphasize.

The authors nicely describe how exposure to olfactory cues from females could alter a male’s energy expenditure and other mechanisms. In this case, there would indeed be an energetic cost to anticipated reproduction. I think the use of ‘cost of reproduction’ should be more precise throughout the paper, and I encourage the authors to carefully check each use of cost of reproduction for precision. For example, in lines 230-232, ‘the costs of reproduction may be driven by olfactory cues in mice…’, other than a change in lifespan, there is no evidence that there was a greater cost to their reproductive effort when males both had mates and were exposed to the scent of additional females. I would suggest that this is a response to an anticipation that there will be additional mating opportunities, and the physiological changes associated with this anticipation could trade off with lifespan. Reproduction never occurred. I recommend that the authors carefully look at each usage of ‘costs of reproduction’ and modify that wording as necessary for accuracy.

There were several points in the methods that could use clarification. First, when were the males first paired for mating? Was this at 8 months? It is noted that the males were given new young females 8 months after the start of the study when they were 16 mo. – implying the males were 8 months? I questioned it because 8 mo. is relatively old to start a breeding study – it’s not a problem, but it did make me question the accuracy of the description – stating the age when males were first paired will clarify this. Second, the methods note that the reproductive performance of males was quantified between 24 and 26 months. Could you be more specific, stating the number of days or weeks data was collected? Depending on the day of introducing mates and sacrifice, this could be 2 or 3 months. In the discussion, it is noted that the males had 2 months to mate. I was surprised how many males sired 6 litters in 2 months, with ~18d gestation and mating 24h later, 3 litters per female is possible, but these are old males. I’m surprised how many quickly took advantage of available opportunities. Third, please clarify what you mean by soiled bedding – is this from the nest that has odors that primarily come from that which has accumulated from the female’s integument, or is it contact bedding that has been urinated on? Fourth, I assume at dissection – the testes from each individual were divided into analysis for sperm count and analysis for pathology – please clarify.

Minor comments:
Abstract
● The abstract needs a transition between the description of finding in insects vs your work in mice. Even just changing the wording in the next sentence to clarify that it is not known if the phenomenon described in insect also occurs in vertebrates.
● Line 17, page 2 - “and that rewards associated with mating alleviate these effects.” - this sentence could be more explicit, i.e., what reword associated with mating, what effects.

Introduction:
● Line 41, page 3 - There a space before the citation here. The inclusion of a space before the citation is not consistent throughout the paper. Please check throughout.
● Line 42, page 3 - “producing gametes” add comma
● Line 46-49, page 3-4 – this sentence should be worded for clarity and specificity.
● Line 49, page 4 - comma after melanogaster
● Line 57, page 4 – comma after species, change negative to negatively
● Line 60, page 4, - specific of olfactory cues are from males or females
● Line 64, page 4 - “In male mice” add comma after

Results
● Line 87, page 5 - comma after “age”
● Line 87-91, page 5 - Long sentence that becomes confusing. Add comma after “age”.
● Line 93, page 6 - comma after “age”
● Line 95, page 6 - comma after citation
● Line 99-100, page 6 – the use of ‘factor’ is not needed. Consider more explicate language – perhaps – significant interaction between housing with females and added odor exposure.
● Line 106, page 6 - comma after “study”
● Line 111, page 6 - comma after “age”
● Line 144, page 8 - add d to “increase”

Discussion

● Schmidt et al given a different style citation throughout. Follow journal format consistently.
● Line 207-211, page 11 - Run on sentence that becomes hard to follow “They have a different genetic background…”
● Line 211-212, page 11 – Add citation. “The previous effects…” – be specific for clarity.
● Line 234, page 12 - “The cause of faster aging… “ sentence could be stronger. Perhaps – The mechanisms responsible for reduced longevity in males exposed to female odor remains to be established.
● Line 248 - 252, page 12-13 – This is a long, wordy, sentence, consider making this more concise.
● Line 255, page 13 - “females’” add apostrophe to females
● Line 259 - 262, page 13 – Split this into 2 sentences: “A decline in the fertilization…”. Clarify what is meant by fertility differences. This sentence spells fertilization with a “z” and the rest are with an “s”
● Line 269-270, page 15 - tense issues with “engaging in”

Methods
● Throughout, review for consistency of typing out numbers versus writing the numerical value
● Line 277, page 15 - Check spacing of “progeny of a”
● Line 336, page 16 – Check spacing between 1 and min.
● Line 346, page 17 – Spell out H&E;
● Line 348, page 17 - 8-bit
● Line 355, page 17 - “the start of the start” should be rephrased.
● Line 358-360, page 17-18 – rephrase sentence “After the trail scent marks…” – consider, ‘…under UV, an experimenter…’
● Line 362-363, page 18 - reword sentence “urine samples were collected…” – as worded implies that 13 and 24 months were prior to the experiment.
● Line 363, page 18 - remove comma after cage
● Line 364, page 18 – remove space after 20 in 20oC
● Line 368, page 18 - check tense and wording of “assessing and divided protein levels”
● Line 373, page 18 - 48h inconsistent with time data from above
● Line 380, page 19 - remove comma after “tests”
● Line 385, page 19 - remove comma after “time”
● Line 391, page 19 - add comma after “number”