Effects of chelating calcium in cryopreservation extender on frozen-thawed dog semen

T Deco-Souza,L.R.B Carazo,L.C.F Bergo,G.R Araujo,M.C.C Silva,G.S.C Vasconcelos,T.A.R Paula

doi:10.1590/1678-4162-10890

Abstract

ABSTRACT We evaluated the effect of reducing free calcium in the cryopreservation medium, using the calcium chelator ethylene diamine tetracetic acid (EDTA) at 0.3% and 0.5% concentrations. Three male mixed breed dogs were subjected to semen collection by digital manipulation (n=16). Each ejaculate was divided in three aliquots, and each one was diluted in TRIS-glucose-egg yolk extender with 6% glycerol and 0.5% Equex STM Paste® (TGE, control); and added with 0.3% EDTA (EDTA 0.3) or 0.5% EDTA (EDTA 0.5). Calcium concentration reduced in EDTA 0.3 and all the calcium ions were chelated in EDTA 0.5. The EDTA addition did not affect sperm morphology or plasma membrane integrity; however, by removing all free calcium (EDTA 0.5), the sperm motility reduced (64.7% in TGE and 45% in EDTA 0.5; p<0.05). Acrosome integrity and sperm binding ability were not improved by calcium chelation. The failure to prevent the premature AR may explain why sperm longevity was not affected by calcium removal. Thus, the partial or complete calcium removal, through EDTA addition, is not able to prevent acrosomal damage or premature acrosomal reaction, and therefore does not improve the dog sperm binding ability.

Highlights

Sperm cryopreservation, especially during cooling and freezing, may damage the sperm and reduce its viability (Hallap et al, 2006; Luvoni et al, 2003)
An option to reduce premature acrosome reaction (AR) in cryopreserved sperm is reducing the availability of ionized calcium in the extender, which can be achieved by adding a calcium chelating substance in the freezing extender, such as ethylene diamine tetraacetic acid (EDTA)
Braud et al (2016) observed that the concentrations of 0.1% and 0.25% EDTA could reduce (p0.05) on sperm with intact acrosome when compared to the control

Summary

Introduction

Especially during cooling and freezing, may damage the sperm and reduce its viability (Hallap et al, 2006; Luvoni et al, 2003). There are indications that calcium influx from sperm environment and from its intracellular reservoirs plays an important role in capacitation and acrosome reaction (AR) induced by cryopreservation (cryocapacitation and premature acrosome reaction) (Száz et al, 2000; Alhaider and Watson, 2009). In this regard, an option to reduce premature AR in cryopreserved sperm is reducing the availability of ionized calcium in the extender, which can be achieved by adding a calcium chelating substance in the freezing extender, such as ethylene diamine tetraacetic acid (EDTA). The EDTA concentrations (0.1% and 0.25%) probably did not reduce calcium concentrations below the ideal concentration in which AR occurs

Methods

Results

Conclusion