In vitro sperm capacitation with l-Arginine and heparin in the absence of cumulus-oocyte complexes and its impact on embryo production

The aim of this study was to evaluate the effects of L-arginine (L-arg, a precursor of NO synthesis) in the quality of the in vitro capacitation of cryopreserved bovine sperm induced by heparin and its effects on in vitro embryo production. All reagents used in the capacitation and sperm tests were purchased from Sigma Chemical Co. Aldrick (St Louis, USA). The medium base used in the sperm tests was modified Tyrodes (TALP-sp). The wash medium used in sperm capacitation was Tyrodes (Chamberland, Theriogenology, vol. 55, p. 823-835, 2001). The medium used in the process of in vitro embryo production were obtained from Progest Biotechnology in Animal Reproduction Co. (Botucatu, Brazil). In experiment 1, the experimental groups were: control 0h without pre capacitation, and capacitated for 30 min in the absence of COCs with heparin (control 30 min); L-arg and L-arg + heparin. The sperm capacitation and acrosome reaction (AR) were evaluated by chlortetracycline test, and the integrity of the plasma membrane (PM) and acrosome (AM) by the association of three fluorescent probes (PI; Hoechst, FITC-PSA). The sperm capacitation with L-arg + heparin increased the percentage of capacitated sperm, compared to control 0h and the group capacitated with heparin (61,1 vs 18.24 e 47.01%, respectively), and decreased the AR (19.6 vs 25.20%) compared to the group capacitated with heparin (P 0.05). In experiment 2, the sperm was incubated with COCs in the presence of heparin (control), or previously incubated in the absence of COCs for 30 min with heparin, L-Arg or L-Arg + heparin and then washed and transferred to the IVF droplets without heparin. The sperm quality was evaluated by the in vitro production rate of blastocysts. There was no significant difference in cleavage rate between treatments (P> 0.05), however the group capacitated with L-arg + heparin increased the blastocyst rate at 31.8% compared to the control group, capacitated with heparin in the presence of COCs (53.71 vs 40.76%, P<0.05). The results allow us to conclude that: 1) the addition of L-arg to the capacitation medium containing heparin increases the number of in vitro capacitated spermatozoa with 30 min of cultive; 2) maintains a low percentage of spermatozoa with damaged in the plasma membrane and acrosomal membrane and 3) the addition of L-arg to the capacitation medium with heparin, in the absence of COCs, was the most effective method in the blastocysts production.