Proteases play an important role during mammalian fertilization. Their function is frequently investigated using specific inhibitors. We analyzed four
serine protease inhibitors [4-(2-aminoethyl)
benzene sulfonyl fluoride hydrochloride (
AEBSF), soybean
trypsin inhibitor from glycine max (
STI), Nα-tosyl-
L-lysine-chloromethyl
ketone hydrochloride (
TLCK) and N(p)-tosyl-
L-phenylalanine-chloromethyl
ketone (
TPCK)] for their in vitro effect on fertilization and sperm quality in pigs. Inhibitor concentrations were chosen based on the reduction of fertilization rate during preliminary dose-response experiments with cryopreserved epididymal spermatozoa. The inhibitor effects on in vitro fertilization (IVF) and sperm parameters (membrane and acrosomal integrity, motility and mitochondrial membrane potential -
MMP) were evaluated using diluted fresh semen.
AEBSF (100 μM),
TLCK (100 μM) and
TPCK (100 μM) decreased total fertilization and polyspermy rates by at least 50%.
STI (5 μM) lowered total fertilization rates but not the level of polyspermy.
AEBSF and
TPCK reduced fertilization parameters to a similar degree using cryopreserved epididymal spermatozoa (dose-response experiment) or diluted fresh semen. Inhibition by
STI was more pronounced using cryopreserved epididymal spermatozoa, whereas
TLCK inhibited IVF only with diluted fresh semen.
AEBSF and
STI had no effect on sperm parameters, and
TLCK significantly reduced motility.
TPCK diminished
MMP and motility and affected membrane and acrosomal integrity in a negative way. In summary,
serine protease inhibitors differed in the way they reduce the fertilization rate. These results emphasize the necessity of inhibitor testing before they can be applied in fertilization studies.
AEBSF and
STI can be used in the future IVF studies without compromising sperm quality.