Association of L-arginine with heparin on the sperm capacitation improves in vitro embryo production in bovine.

Gester Breda Aguiar,Valter Luiz Maciel Junior,Maria Clara Caldas-Bussiere,Cláudio Luiz Melo De Souza,Carla_Sobrinho Paes De Carvalho

doi:10.21451/1984-3143-ar2019-0022

Abstract

We aimed to evaluate the effects of L-arginine (L-arg) in the quality of in vitro heparin-induced capacitation of cryopreserved bovine spermatozoa and its effects on IVP. The experimental groups were: Control 0 hour without pre-capacitation, and groups of sperm capacitated for 30 min in the absence of COC with heparin (Control 30 min), with 1 mM L-arg and with 1 mM L-arg + heparin. The capacitation pattern was evaluated by chlortetracycline assay and the integrity of the plasma membrane (PM) and acrosome membrane (AM) by the association of Hoescht 33342 and propidium iodide. Further, we assess the sperm quality by the rate of in vitro blastocysts production. Treatment with 1 mM L-arg + heparin increased the percentage of capacitated sperm when compared to Control 0 hour and the treatment with heparin (61.1 vs. 18.2 and 47.0%, respectively, P<0.05). The addition of 1 mM L-arg to the medium has capacitated the spermatozoa (26.2 ± 3.8) but was less effective than heparin (47.0 ± 4.0) (P<0.05). There was no difference in the percentage of sperm with intact PM between treatments when compared to Control 0 hour (P>0.05). The group capacitated with 1 mM L-arg + heparin for 30 min increased the blastocyst rate compared to Control IVF (53.7 vs. 40.8%, P<0.05). We conclude that the addition of L-arg with heparin increases the number of capacitated spermatozoa in vitro with 30 min of pre-incubation in the absence of COC not altering the integrity of plasma and acrosomal membrane. This treatment in the absence of COC was the most effective method for blastocysts production, and the method of pre-incubation could be used to assess the role of other substances in the sperm capacitation and its effect on IVP.

Highlights

Sperm capacitation process can be defined as the biochemical, biophysical, molecular, and metabolic changes of the sperm cell that confers the ability to fertilize an oocyte in vivo or in vitro (Bailey, 2010)
Assessment of the quality of sperm capacitation by the in vitro blastocysts production There was no statistically significant difference between the percentage of cleaved embryos of Control In Vitro Fertilization (IVF) when the sperm were capacitated with heparin in the presence of COC (78.0 ± 3.2%) and the treatments that were capacitated in the absence of COC with L-arg + Hep (82.2 ± 3.5%) or Hep (79.3 ± 4.2%, P>0.05)
Nitric oxide plays a fundamental role in the sperm capacitation process (O’Flaherty et al, 2004; Rodriguez et al, 2005; Leal et al, 2009)

Summary

Introduction

Sperm capacitation process can be defined as the biochemical, biophysical, molecular, and metabolic changes of the sperm cell that confers the ability to fertilize an oocyte in vivo or in vitro (Bailey, 2010). L-arg plays a significant role in sperm motility, capacitation process, and induces acrosome reaction in cattle (O’Flaherty et al, 2004; Leal et al, 2009; Silva et al, 2014). Such effects have been linked to the synthesis of nitric oxide (NO) from L-arg by the sperm cell (O’Flaherty et al, 2004; Leal et al, 2009). Common IVF medium is enriched to stimulate sperm capacitation, in this model, L-arg could influence both sperm and COCs

Objectives

Methods

Results

Conclusion