Tyrode's Albumin Lactate Pyruvate Research Articles

Attempts to remove bovine virus diarrhoea virus (BVDV) from the semen of persistently infected bull prepared for in vitro fertilization (IVF)

The effect of peroxynitrite (ONOO−) on the capacitation rates of cryopreserved bull spermatozoa and the participation of protein kinases in the capacitation process were evaluated. A pool of spermatozoa from five bulls was incubated in Tyrode's albumin lactate pyruvate (TALP) medium in the presence of heparin (10 IU/ml), sodium nitroprusside (SNP, 50 nM), a nitric oxide donor or 3-morpholinosydnonimine (SIN-1, 1–20 μM), a ONOO− donor. The participation of ONOO− was evaluated at 15, 30 and 45 min and confirmed by using a specific scavenger, uric acid (2–20 mM). Spermatozoa capacitated with SIN-1 were incubated with ovarian follicular fluid of cattle to evaluate their ability to undergo acrosome reaction. The role of ONOO− during capacitation induced by heparin or nitric oxide was evaluated by the addition of uric acid. The participation of protein kinase A (PKA), protein kinase C (PKC) and protein tyrosine kinase (PTK) in capacitation induced by ONOO− was evaluated by incubation with specific inhibitors (50 μM H-89, 0.1 μM bisindolylmaleimide I, and 3 μM genistein, respectively). Capacitation percentages were determined by the fluorescence technique with chlortetracycline (CTC) and true acrosome reaction was determined by trypan blue and Differential-Interferential Contrast (DIC). SIN-1 concentrations employed had no effect on progressive motility or sperm viability. Capacitation values of 10 μM SIN-1 treatment (23 ± 2%) were significantly greater with respect to the control (4.6 ± 1.62%). At 15 min of incubation the greatest capacitation was observed (P < 0.05), reaching a plateau between 15 and 45 min. Follicular fluid induced acrosome reaction in spermatozoa previously capacitated with 10 μM SIN-1 (P < 0.05). Uric acid prevented SIN-1-induced capacitation and significantly diminished capacitation induced by heparin or SNP. The addition of PKA and PKC inhibitors failed to modify the capacitation induced by SIN-1 (27.4 ± 3.85 and 24.8 ± 4.75, respectively). Genistein, a PTK inhibitor, produced a significant capacitation decrease (8.6 ± 5.5%). These results indicate that endogenous ONOO− may be generated during heparin- or SNP-induced capacitation. Exogenous ONOO− acts as a capacitation inducer and involves the participation of PTK, as part of the intracellular mechanisms that lead to capacitation in cryopreserved bovine spermatozoa.