Extreme fertilisation bias towards freshly inseminated sperm in a species exhibiting prolonged female sperm storage

The storage of sperm by females across successive reproductive cycles is well documented in internal fertilisers, yet the fate of stored sperm when they compete with ‘new’ sperm to fertilise a female’s eggs has rarely been considered. This gap in our understanding is likely due to the logistical difficulties of controlling behavioural interactions during or after mating, which in turn may influence how many sperm are inseminated and how stored sperm are ultimately utilised during successive bouts of sperm competition with freshly inseminated sperm. Here, we use artificial insemination (AI) in guppies (Poecilia reticulata), a polyandrous livebearing poeciliid fish exhibiting prolonged sperm storage by females, to overcome these challenges. The use of AI enables us to control potential differential maternal effects (e.g. behaviourally mediated cryptic female choice) and specifically test for post-copulatory paternity biases that favour either stored or fresh sperm when they compete to fer...