# Processing of boar spermatozoa with phosphate-buffered saline at 4˚C induces an increase in 32 kDa tyrosine-phosphorylated protein (p32)

## TL;DR

Cooling boar sperm with phosphate-buffered saline increases a specific protein linked to dead sperm, which can be blocked by a protease inhibitor.

## Contribution

The study identifies protease activity in dead sperm as a novel factor contributing to p32 increase during cold PBS processing.

## Key findings

- Cooled PBS washing increases p32 levels and acrosomal damage in boar sperm.
- Adding AEBSF inhibits the increase in p32 during cooled PBS treatment.
- The p32 increase is linked to protease activity in dead spermatozoa.

## Abstract

We aimed to investigate the impact of processing boar spermatozoa with phosphate-buffered saline (PBS) at 4 ˚C on acrosomal integrity and increase in 32 kDa tyrosine-phosphorylated protein (p32). Following cooled PBS washing, we observed a significant increase in p32 levels and in the proportion of dead spermatozoa with compromised acrosomal integrity compared to sperm washing using PBS at room temperature. Interestingly, this increase in p32 was effectively inhibited when cooled PBS was supplemented with 1 mM AEBSF, a serine protease inhibitor. Our findings suggest that the increase of p32 in response to cooled PBS washing in boar spermatozoa is associated with enhanced protease activity in dead spermatozoa.