In vitro storage of boar spermatozoa increases the demand of adenosine triphosphate for reactivation of motility

Background
Prolonging the shelf-life of liquid-preserved semen without compromising its fertilizing capacity may increase the efficiency of artificial insemination in pigs. Many fertilization-relevant processes are ATP dependent. The impact of semen storage and rewarming to body temperature on the energy status of sperm are as yet unknown.

Objectives
To investigate the energy status of boar spermatozoa during storage and subsequent rewarming, and to reveal the potential role of mitochondrial function for reactivation and maintenance of sperm motility.

Materials and Methods
Extended semen samples (n = 7 boars) were used. Spermatozoa were challenged by storage at 17°C for seven days and incubation at 38°C for 180 minutes. The ATP concentration and energy charge (EC) in semen samples and lactate concentration in the extracellular medium were assessed. Viability and mitochondrial activity were determined by flow cytometry, and clustered single cell analysis of motility parameters were performed.

Results
The energy status was not affected by semen storage (p>0.05). Rewarming resulted in a net reduction in ATP concentration which increased with storage time (maximum Day 5: -24.2±10.3 %), but was not accompanied by a loss in viability, motility or mitochondrial activity. Blocking glycolysis with 2-Deoxy-D-glucose prevented re-establishing of motility and mitochondrial activity after rewarming. Mitochondrial activity gradually subsided in virtually all spermatozoa during incubation at 38°C, while ATP and EC remained high. Concomitantly, extracellular lactate levels rose and sperm populations with lower velocity, increased linearity, and low lateral head-displacement grew larger. Size changes for major sperm subpopulations correlated with the percentage of viable sperm with high mitochondrial activity (r = 0.44 to 0.70 for individual subpopulations, p<0.01).

Conclusion
Storage of boar spermatozoa increases the demand of ATP for reactivation of sperm towards fast, non-linear and hyperactivation-like motility patterns upon rewarming. Maintenance of glycolysis seems to be decisive for sperm function after long-term storage in vitro.