Final Activity Report Summary - OPOSSUM SPERM COMP (Sperm competition in the grey short-tailed opossum (Monodelphis domestica) and the post-copulatory role of sperm storage) The objective of this project was to investigate the mating system of the grey short-tailed opossum, monodelphis domestica. In particular, the role of sperm pairing and oviductal sperm storage crypts in sperm competition and cryptic female choice within the female reproductive tract were investigated. Extensive training and research licences were obtained in order to undertake the necessary work. A reverse light and time-lapse video system was established and software-assisted tools were developed to select breeding pairs and monitor mating behaviour in opossums. Pairs were monitored continuously by time-lapse video and reproductive tracts from 24 females were dissected between 0.5 and 24 h after mating. The lower tract, right uterus and right oviduct were further dissected into major anatomical sections, then flushed and counted for sperm and embryos. The left uterus and left oviduct were fixed and processed for histology. Sperm counts from flushes and detailed histological analysis of sections were used to describe the pattern of paired and single sperm colonisation of the oviduct around the time of ovulation in the opossum. Results revealed that extremely low numbers of sperm, approximately 2 million, were ejaculated in this species, being 250 times fewer than in the case of the rabbit. However, sperm migrated extremely efficiently, almost 1 in 300 which corresponded to 35 times more than the rabbit, to the oviduct, which was the site of fertilisation in the female tract. Sperm rapidly reached the oviduct within 0.5 h of mating where they remained in the isthmus until ovulation which occurred from 18 h after mating. Among males the proportion of paired sperm that were ejaculated varied greatly, ranging from 4 to 60 %. Surprisingly however, in contrast to single sperm, paired sperm migrated almost exclusively to the oviducts shortly after mating. Further histological analysis of sections of the opossum oviduct revealed the presence of sperm in the lumen. Nevertheless, in contrast to the closely related Virginia opossum, sub-luminal crypts containing sperm were not observed and thus did not appear to occur in this species. The adaptive significance of sperm cooperation, including sperm pairing in the opossum, was poorly understood. This was the first in vivo evidence of a functional benefit conferred by sperm cooperation in any mammal. This provided new insight into an alternative evolutionary strategy for efficient migration and fertility in the female tract. If sperm pairing conferred an advantage in migration efficiency, then variation in the proportion of paired sperm ejaculated by males might form the basis for individual male advantage in sperm competition. These findings might redefine the theory that intense sperm competition selection pressure always resulted in large testes and increased sperm production in species. In light of the above findings, research was subsequently directed at further understanding the mechanism and functional significance of sperm cooperation in the female reproductive tract of the opossum. Further work was underway by the time of the project completion to optimise and develop assisted reproductive technologies including semen collection and artificial insemination, which would be used as tools to directly test the adaptive significance of this unique reproduction strategy. Once optimised, these tools might also be of considerable value in the conservation and genetic management of this and other more endangered species of opossum.