Abstract
ABSTRACT Seminal plasma contains serine proteases and serine protease inhibitor, which are involved in mammalian fertilization, and the inhibitors can be applied to prevent cold-induced sperm capacitation. The effects of different concentrations of two serine protease inhibitors were analyzed, Plasminogen activator inhibitor 1 - PAI-1 (70ƞg, 140ƞg and 210 ƞg) and Antipain (10µg, 50µg and 100µg) as supplementation to bovine semen cryopreservation extender. The effects of the inhibitors on the sperm parameters (sperm kinetics - CASA, acrosome integrity, plasma membrane integrity, mitochondrial membrane potential, sperm defects and acrosome reaction rate) were evaluated in the post-thaw semen. Cryopreservation of sperm with Antipain decreased post-thaw kinetic parameters of MP, VSL, LIN, SRT and the percentage of hyper-activated sperm while PAI-1 (210 ƞg) decreased VSL and LIN. Antipain and PAI-1 had no effect on the integrity parameters of the plasma membrane, mitochondrial membrane potential and sperm defects. Sperm cryopreserved in the presence of Antipain and PAI-1 (70 and 140 ƞg) preserved acrosome integrity, as they were able to complete the in vitro acrosome reaction. In conclusion, the serine protease inhibitors, Antipain and PAI-1 (70 and 140ƞg) are able to preserve the acrosome integrity of cryopreserved bovine sperm.
Highlights
Over the last decades, bovine semen cryopreservation has been used to preserve genetic material from breeds at risk of extinction and to propagate animals with superior genetic characteristics by artificial insemination (AI) (Gurupriya et al, 2014)
The spermatozoa cryopreserved under extender containing different concentrations of Antipain showed lower values (P
The plasminogen activator inhibitor 1 (PAI-1) at a concentration of 210 ƞg had a negative effect on the VSL and LIN parameters
Summary
Bovine semen cryopreservation has been used to preserve genetic material from breeds at risk of extinction and to propagate animals with superior genetic characteristics by artificial insemination (AI) (Gurupriya et al, 2014). Some changes observed in in vivo sperm capacitation are similar to cryo-capacitation and include an increase in plasma membrane permeability, intracellular increase in calcium ion concentrations (Ca2 +) and cyclic adenosine monophosphate (cAMP) and tyrosine phosphorylation (Pommer and Meyers, 2002; Kumar and Atreja, 2011; Singh et al, 2012) In response to these changes, there is destabilization of proteins from the serine protease family present both in the acrosome and the sperm membrane (Cesari et al, 2010), leading to exocytosis of hyaluronidase and acrosin, which are important enzymes that digest the zona pellucida matrix, compromising the penetration of spermatozoa into the oocyte (Adham et al, 1997). Previous studies have shown that sperm cell, oocyte and their cumulus cells, contain and/or segregate several proteases (Tulsiani et al, 1998; Iwamoto et al, 1999; Shimada et al, 2004; Burkart et al, 2012)
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