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Simple Summary: Analysis of sperm performance under in vitro conditions provides a good indication of fertilizing potential. Parameters such as motility, swimming kinetics, acrosome integrity, or ATP content are thus examined in efforts to characterize such potential. Hamster species are a good model to study sperm parameters that are key determinants of fertilizing capacity because these species are at the higher end of the diversity of mammalian sperm morphology and performance. In vitro functional studies demand that sperm remain viable during a long period of time under conditions that resemble those in the female tract. Sperm from certain species require supplementation of the incubation medium with factors that stimulate viability and swimming, or that promote acquisition of fertilizing capacity. Molecules important for sperm performance in hamsters have been identified, namely D-penicillamine, hypotaurine and epinephrine (PHE). In the present study, we investigated the effect of PHE on spermatozoa from five hamster species incubated for up to 4 h. Our results revealed that PHE maintains sperm performance in the golden hamster, whereas it improves sperm quality in the Chinese hamster. In contrast, it does not seem to have any effect on sperm from the Siberian (Djungarian), Roborovski and Campbell's dwarf hamsters. These results are valuable to understand the different regulatory mechanisms of sperm motility and survival in different species. Assessments of sperm performance are valuable tools for the analysis of sperm fertilizing potential and to understand determinants of male fertility. Hamster species constitute important animal models because they produce sperm cells in high quantities and of high quality. Sexual selection over evolutionary time in these species seems to have resulted in the largest mammalian spermatozoa, and high swimming and bioenergetic performances. Earlier studies showed that golden hamster sperm requires motility factors such as D-penicillamine, hypotaurine and epinephrine (PHE) to sustain survival over time, but it is unknown how they affect swimming kinetics or ATP levels and if other hamster species also require them. The objective of the present study was to examine the effect of PHE on spermatozoa of five hamster species (Mesocricetus auratus, Cricetulus griseus, Phodopus campbelli, P. sungorus, P. roborovskii). In sperm incubated for up to 4 h without or with PHE, we assessed motility, viability, acrosome integrity, sperm velocity and trajectory, and ATP content. The results showed differences in the effect of PHE among species. They had a significant positive effect on the maintenance of sperm quality in M. auratus and C. griseus, whereas there was no consistent effect on spermatozoa of the Phodopus species. Differences between species may be the result of varying underlying regulatory mechanisms of sperm performance and may be important to understand how they relate to successful fertilization. [ABSTRACT FROM AUTHOR]