Nitric Oxide and Superoxide Anion Production During Heparin‐Induced Capacitation in Cryopreserved Bovine Spermatozoa

Contents The aim of this work was to quantify NO, O 2 − and ONOO − production during heparin‐induced capacitation of cryopreserved bovine spermatozoa. A time dependent hyperbolic increase was observed for heparin‐dependent capacitation, O 2 uptake, and NO production. Conversely, O 2 − production was increased during the first 15 min of incubation, showing a decrease from this time until 45 min. At 15 min of heparin incubation, a threefold increase in O 2 consumption (5.9 ± 0.6 nmol/min × 10 7  cells), an enhancement in NO release (1.1 ± 0.2 nmol/min × 10 7  cells), and a five‐fold increase in O 2 − production (1.3 ± 0.07 nmol/min × 10 7 cells), were observed. Peroxynitrite production rate was estimated taking into account NO and O 2 − generation and the second‐order rate constant of the reaction between these species. To conclude, heparin‐induced capacitation of cryopreserved bovine spermatozoa activates (i) mitochondrial O 2 uptake by high ADP levels due to increased energy requirements, (ii) NO production by a constitutive NOS and (iii) O 2 − production by a membrane‐bound NAD(P)H oxidase. The products of both enzymes are released to the extracellular space and could be involved in the process of sperm capacitation.