Reproductive output of old males is limited by seminal fluid, not sperm number.

Male reproductive senescence is typically characterized by a decline in the number of sperm produced and transferred by old males, a phenomenon that may be exacerbated in polygynous species where males mate multiply. However, males also transfer seminal fluid to females, and little is known about its role in modulating male reproductive senescence. Here, we explore the contributions of sperm and seminal fluid towards male reproductive senescence in a series of sequential matings, using Drosophila melanogaster. As expected, old males produce fewer offspring than young males. However, this pattern is not driven by sperm limitation: old males have more sperm and transfer similar numbers to females, compared to young males. Instead, females storing fewer sperm of old males compared to that of young males, over a long term, drives male reproductive senescence. We are able to mitigate the age-related decline in male reproductive output by supplementing females with the seminal fluid of a young male, before she mates with an old male. Similarly, we alleviate the reduction in reproductive output across sequential matings by supplementing females with seminal fluid. Our findings highlight that seminal fluid, rather than sperm number, limits reproductive success in old or multiply mating males, highlighting its underappreciated role in reproductive aging.