Liquid preservation of boar semen: Insights into the sperm’s energy budget

As many fertilization-relevant sperm functions rely on the availability of ATP, semen preservation should maintain a well-balanced energy budget. Our aim was to shed light on the spermatozoa’s energy balance during long-term storage at 17°C, which is the conventional storage temperature of boar semen, and under hypothermic condition at 5°C, as recently introduced into swine insemination for prevention of microbial growth. First, improved ATP and adenylate energy charge (EC) assay protocols were validated for the use in spermatozoa. The assays were then applied to semen samples (n=7 boars) stored at 17°C in Beltsville Thawing Solution for up to seven days. Basic ATP levels and EC values remained stable throughout storage (p>0.05). However, re-establishing of sperm motility upon rewarming to 38°C revealed an increased (p<0.05) net investment of ATP with ongoing storage time, reaching 25% of the preserved ATP level (452±57 pmol/105 sperm) after five days. Experimental inhibition of glycolysis demonstrated that re-establishment of motility and mitochondrial function was fully dependent on functional glycolysis. EC levels below 0.4 were associated with decreased viability and motility (<70%) in the stored samples. In samples (n=7 boars) stored at 5°C, cold shock resulted in loss of sperm motility and viability (p<0.05), and a concomitant increase of adenine nucleotides in the semen extender. At the same time, the spermatozoa’s energy budget, i.e. the samples ATP level, was lower (182±84 pmol/105 sperm) compared to semen stored at 17 °C (319±129 pmol/105 sperm) after 24 h (p<0.05). A similar situation was observed at the level of the individual, viable spermatozoon (5°C: 2.4±1.1 fmol ATP/sperm; 17°C: 3.5±1.4 fmol ATP/sperm; p=0.063). The reduced ATP levels were accompanied by a higher ratio of spermatozoa with slow movement and low beat cross frequency at 5°C as identified by cluster analysis of data from computer assisted sperm analysis (p<0.05). In conclusion, the viable spermatozoa’s energy budget and thus, sperm function is at risk during extended storage at 17°C or chilling to 5°C. Analyses on sperm metabolomics may provide further insights in how the spermatozoa’s energy metabolism can be supported and thereby improve liquid boar semen preservation.