Energy metabolism during long-term storage and subsequent thermal stress in liquid preserved boar spermatozoa

In major pork producing countries, artificial insemination is used in more than 90 % of breeding sows. Maintenance of good sperm quality during semen preservation is mandatory to ensure high fertility. Boar spermatozoa are highly sensitive to cold shock, and therefore are stored in unfrozen stage. With currently marketed extenders, the ideal temperature to store liquid preserved boar semen for up to five days is between 16 and 18°C (Johnson et al. 2000, Riesenbeck 2011). Storage at lower temperature would be advantageous for prolonging semen shelf live, decreasing bacterial growth and slow- down of chemical reactions (Johnson et al. 2000, Schmid et al. 2013). During hypothermic storage, sperm metabolism is suppressed to save energy for essential sperm functions on the route of fertilization. Spermatozoa specifically utilize ATP as the energy source for cellular activities such as motility, capacitation and acrosome reaction (du Plessis et al. 2015). Similarly to the situation in somatic cells, sperm function relies on a balanced level between ATP, ADP and AMP, which is expressed in the adenylate energy charge (Ford and Leach 1998). Spermatozoa are able to generate ATP through mitochondrial respiration and through glycolysis in the fibrous sheet and in the sperm head. The relevance of mitochondrial activity for ATP-production and sperm function is controversially debated and seems to vary between species (Rodriguez-Gil 2013). Depending on storage length and temperature, preservation of boar semen may cause deficits in energy metabolism and thereby affect sperm function. In the presence of metabolic substrates, activation of in hypothermically stored spermatozoa by re-warming to body temperature might partially or fully restore the energy metabolism. However, under the condition of both natural and artificial insemination, sperm need to survive in the female reproductive tract for up to 24 h until oocytes are released into the oviduct. It is well known that motility decreases when spermatozoa are exposed to prolonged thermal stress in vitro, particularly with a preceding longer preservation period. Whether in vitro storage and subsequent thermal stress leads to exhaustion of sperm energy metabolism and whether this is causative for the loss of motility is yet not clear. The aim of the present study was to examine the effect of prolonged storage at different temperatures and subsequent thermal stress on the energy metabolism and sperm quality in liquid preserved boar spermatozoa. Therefore, protocols for ATP extraction were revised to ensure sensitive and precise measurements of adenine nucleotides by the luciferin-luciferase reaction. The role of mitochondrial membrane potential for ATP production and adenylate energy charge as well as the relation between motility and energy measures were elucidated.