A new approach to sperm preservation based on bioenergetic theory. 
To date, attempts to preserve chicken sperm have been based on a trial-and-error experimental approach.  The present work outlines the development of an alternative approach based on empiricism and bioenergetic theory.  In previous work, we found fowl sperm motility to be dependent on mitochondrial calcium cycling, phospholipase A(2), and long-chain fatty acids as an endogenous energy source.  It is noteworthy that fowl sperm reside within the sperm storage tubules (SST) of the oviduct over an interval of days to weeks after insemination.  In this regard, a model for in vivo sperm storage was developed and tested in additional previous research.  Sperm penetration of the SST, sperm residence within the SST, and sperm egress from the SST can be explained in terms mitochondrial function.  Understanding sperm function and longevity in terms of bioenergetics presented the possibility that sperm could be inactivated by disrupting mitochondrial calcium cycling and could thereby be preserved.  However, this possibility also posed a problem: maintenance of the inner membrane potential of the mitochondrion within inactivated sperm.  This report describes a series of experiments in which fowl sperm were inactivated by treatment with the calcium chelator tetrasodium 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, and then reactivated by treatment with calcium ions.  The effect of tetrasodium 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid on mitochondrial calcium cycling was confirmed by flow cytometry and confocal microscopy.  When treated sperm were cooled to 10 degrees C, inactivated sperm could be reactivated throughout a 5-h storage interval.  When stored sperm were held for 3 h before reactivation and insemination, fertility was 88% of the control.  Storage did not affect hatchability.  In summary, short-term storage was realized by manipulating mitochondrial function.  We propose that 1) complex V consumes ATP within inactivated sperm and, by doing so, maintains the inner membrane potential of the mitochondrion, 2) ATP is regenerated within inactivated sperm by the action of creatine kinase on phosphocreatine, and 3) necrosis follows depletion of intracellular phosphocreatine.  Therefore, future attempts to preserve chicken sperm can be based on a theory that encompasses regulation of energy production, a biological context in which sperm cells are motile, and the consequences of mitochondrial failure.