This interesting article reports results that suggest that prior mating and exposure to seminal fluids can promote litter size in subsequent mating in mice. In a nice design, the authors housed female mice in one of four conditions, and then compared litter sizes of these females after mating to a subsequent male. The four conditions were being housed with (a) normal (sham-operated) males, (b) vasectomized males (can mate and transfer seminal fluid but not sperm, (c) vasectomized males with seminal vesicles removed (can mate but transfer neither sperm nor seminal fluid, and (d) females (“control”). Experiments were done in duplicate, with reasonable sample sizes, and used females of reproductive age; where the authors had enough mice, effects of genetic background were tested also. The authors examined weight after the 4 housing conditions and, in the females after subsequent mating, litter size, progeny surviving to weaning, placental size and characteristics.

By comparison to “control” females that had been housed with other females, the authors observed that females that had been housed with vasectomized males gave birth to larger litters. In contrast, females housed with vasectomized males lacking seminal vesicles birthed litters of sizes more like those of control females. These data suggest that prior receipt of seminal fluid by a female could “prime” her reproductive tract to improve fertility, a phenomenon that has been reported in invertebrates. These results in mice are interesting and important, although how the prior exposure causes the difference in sizes of litters at birth is not addressed here. The effect could be due to a higher chance of fertilization or to later effects such as a greater likelihood of implantation or greater survival of the embryos. Dissecting these will be fruitful areas for future study. The authors also noted that smaller placental labyrinthine area in the context of normal fetal weights in the litters from group b suggested that seminal fluid may promote better intraplacental transport. This reasonable hypothesis also will be fruitful to investigate in the future.

Interestingly, by the times the pups are weaned, differences in litter size between the groups have disappeared. Thus, more pups from the females pre-mated with vasectomized males don’t survive to weaning. Whether this is due to some intrinsic problem with those pups that does not show up till later, or to limits on how many pups can be successfully nursed by any female is unclear.

Some items need attention prior to publication:
1. Do Vas and Vas+SVX males mate as often with the females as each other and as intact males?
2. Condition a (mating with intact males) results in pregnancies so interpreting differences between females from that condition and females from conditions b and c, upon mating with the subsequent C57BL/6J male is not simple. As the authors note in the discussion, pregnancy permanently alter female physiology and energy reserves. It would have been interesting to include a class of males that produce sperm but are infertile due to mutation; this would remove the complication of prior pregnancy in the tests of litter size by group a females after mating with the C57BL/6J male. I recognize the work involved in this and do not require this experiment for acceptance of the paper.
3. Are there differences between the willingness of the females from conditions a-d to mate with the C57BL/6J male introduced after the 5 month housing?
4. The differences in birth-to-weaning numbers in the different groups were interesting but also concerning, as noted in the review above.
5. I found lines 137-139 confusing in light of lines 121-123. Lines 137-139 say that after mating with a C57BL/6J male, the number of pups birthed by females housed with Vas+SVX males “did not differ from control females or female previously co-housed with Vas males”. Yet lines 121-123 say that these two groups of females (control, housed with Vas) differ; the latter birthed more pups per litter than the former. Figure 2A was not helpful in resolving this; all groups looked similar. I see the differences between the groups discussed in lines 121-123 in the BALB/C part of that figure, but for technical reasons the Vas+SVX-housed females, which are the most important ones for the paper’s conclusion, were not tested in the BALB/C experiments. So it is not completely clear what to conclude.
6. Line 165 would be easier to understand if reworded to “…previous mating experience”
7. Although I don’t disagree with the authors’ conjecture that hybrid vigour could cause larger litter sizes with BALB/C males, there are other explanations. I suggest the auth

The authors’ revisions addressed my original concerns, including the one that was truncated on the review website. Their revisions or justifications in response to other review comments also seem appropriate, to the extent that I can judge.
I have only one request: in line 198, please add “in mice” because otherwise it could be interpreted as being in humans, given the previous sentence.