Female novelty and male status dynamically modulate ejaculate expenditure and seminal fluid proteome over successive matings in red junglefowl.

Theory predicts that males will strategically invest in ejaculates according to the value of mating opportunities. While strategic sperm allocation has been studied extensively, little is known about concomitant changes in seminal fluid (SF) and its molecular composition, despite increasing evidence that SF proteins (SFPs) are fundamental in fertility and sperm competition. Here, we show that in male red junglefowl, Gallus gallus, along with changes in sperm numbers and SF investment, SF composition changed dynamically over successive matings with a first female, immediately followed by mating with a second, sexually novel female. The SF proteome exhibited a pattern of both protein depletion and enrichment over successive matings, including progressive increases in immunity and plasma proteins. Ejaculates allocated to the second female had distinct proteomic profiles, where depletion of many SFPs was compensated by increased investment in others. This response was partly modulated by male social status: when mating with the second, novel female, subdominants (but not dominants) preferentially invested in SFPs associated with sperm composition, which may reflect status-specific differences in mating rates, sperm maturation and sperm competition. Global proteomic SF analysis thus reveals that successive matings trigger rapid, dynamic SFP changes driven by a combination of depletion and strategic allocation.

The Seminal fluid proteome of the polyandrous Red junglefowl offers insights into the molecular basis of fertility, reproductive ageing and domestication.

Seminal fluid proteins (SFPs) are emerging as fundamental contributors to sexual selection given their role in post-mating reproductive events, particularly in polyandrous species where the ejaculates of different males compete for fertilisation. SFP identification however remains taxonomically limited and little is known about avian SFPs, despite extensive work on sexual selection in birds. We characterize the SF proteome of the polyandrous Red junglefowl, Gallus gallus, the wild species that gave rise to the domestic chicken. We identify 1,141 SFPs, including proteins involved in immunity and antimicrobial defences, sperm maturation, and fertilisation, revealing a functionally complex SF proteome. This includes a predominant contribution of blood plasma proteins that is conserved with human SF. By comparing the proteome of young and old males with fast or slow sperm velocity in a balanced design, we identify proteins associated with ageing and sperm velocity, and show that old males that retain high sperm velocity have distinct proteome characteristics. SFP comparisons with domestic chickens revealed both qualitative and quantitative differences likely associated with domestication and artificial selection. Collectively, these results shed light onto the functional complexity of avian SF, and provide a platform for molecular studies of fertility, reproductive ageing, and domestication.