I appreciate the effort and time you the authors spent to conducting these experiments. The topic of seminal fluid effect on female and offspring they investigate is very important and timely, with significant implications for several fields in broad. However, I have serious concerns regarding the clarity and structure of the writing, statistical reports and organising data. Improving the manuscript’s organization and presentation would greatly enhance its readability and impact. Below I listed some of my concerns and suggestions.
1) Introduction first sentence: The wording at the beginning of the sentence, “pregnancy in mammals,” is unclear and should be revised for clarity.
2) The reference list needs to be checked for consistency in formatting, including issues with italics and other citation elements.
3) The acronym "IVF" should be introduced in full as "In vitro fertilization" when it first appears.
4) Check the reference list, the format of citations is not consistent, there are italic issues etc.
5) IVF represents what in words (In vitro fertilization) should have been introduced
6) Intro last paragraph: The word "this" is vague. The hypothesis should be introduced before this point, and the sentence needs reordering for clarity.
7) I suggest the authors include a few examples from other taxa, such as telegony in flies (Crean et al., 2014, Revisiting Telegony, or see Patlar review, 2022 On the Role of Seminal Fluid Proteins for additional examples). This would strengthen the hypothesis by suggesting it could be a more widespread and significant phenomenon in nature.
8) The authors do not introduce the evolutionary significance of the male seminal fluid’s impact on females and offspring, even though the introduction mentions adaptations. For instance, transgenerational paternal effects are a relevant and popular topic currently, which could make the manuscript more appealing to a broader audience.
9) Introduction paragraph 3. The sentence is unclear. It seems that subsequent fertility, depending on the genetic background of earlier mates, was not measured. The genetic background differed between the subsequent males, not the first ones the females mated with.
10) Introduction last paragraph. This is not the appropriate place to report results.
11) Figure S1 legend: Clarify what "AV" stands for. The legend is difficult to link with what the figure displays. Additionally, references cited in the supplementary methods (e.g., Bromfield, 2014 #117; Tremellen, 1998 #208) could not be found in the main manuscript.
12) Figure 1: The legend needs revision; some commas and parentheses are missing.
13) The supplementary tables are not adequately described. It is difficult to follow which statistics correspond to which experiments, and variable names seem arbitrary. These appear to be raw files from statistical software and need to be better organized.
14) There is no mention of Experiment 1 in the titles, yet Experiment 2 is mentioned in the results section.
15) There are several issues with the reporting of statistics and presentation of results. I’ve commented on these issues regarding the first set of results, but similar problems occur throughout the manuscript.
Experiment 1 Results:
The results on female weight should be reorganized to better highlight the key findings, specifically that mating with vas or vas-svx males affects female weight compared to the control group. For instance, it may not be necessary to display p-values between vas and vas-svx males, as this obscures the more important finding that housing with vas and vas-svx males has different effects from the control (black) group on female weight.
Clarify what “weight change” on the y-axis represents. Did you measure the absolute weight of females at different ages? If so, the y-axis should indicate absolute weight. If you calculated weight differences between different age points (e.g., Age 0, Age 1 month, Age 2 months), it’s interesting that females appear to gain more weight with each passing month. Please specify the measurement.
The supplementary tables showing the statistical analyses are missing. For instance, you mention an interaction between time and treatment, but time is not included in the analyses presented in the supplementary files. If they are present, they are not referenced in the main text, and the supplementary tables lack legends to explain their contents, appearing to be raw outputs from SPSS.
Figure 1B and 1C show the effect of the first mating partner’s treatment on female weight for each genotype of intact males. However, one genotype-treatment combination is missing in Figure 1C. How was the interaction between treatment and male genotype analyzed? If the data was subsetted, this should be mentioned, and an interaction figure would be helpful (e.g., solid lines for females mated with one genotype, dashed lines for the other).
Why does Figure 1A begin at the first month, while other figures start at the second month?
I couldn’t find Table S1. You mention no interaction between male genotype and early treatment, yet significant differences in total pups born between male genotypes are observed (Figure 2A). Please clarify.
Experiment 1 Methods:
What are the "batches"? Were the first half of the females run in one round and the second half later? If so, batch should be a fixed factor in the analysis since measurements were taken at two different time points with males of different ages. Time should not be treated as random either.
Why were females allocated equally into treatment groups? The Vas + SVX group has a low sample size, especially considering the two batches.
The discussion could be better structured. Not all results are discussed, for instance, why female weight differs based on seminal fluid receipt. The authors only discuss immune-related responses, but other fitness factors may also be involved. For example, in insects, seminal fluid increases female appetite, leading to more egg production, which benefits males in sperm competition. This might also explain why the effect on body size is smaller when the seminal vesicle is removed, as the seminal vesicle often produces key sperm competition proteins, such as mating plugs, in mammals. A conclusion can be beneficial summarizing the key findings.

See the attached file.

Dear authors,

Thank you for clarifications and improvements. I believe your findings will contribute significantly to the seminal fluid literature. Thanks for your effort and cooperation.