ANALYSIS OF SPERMATOZOA QUALITY USING PERCOLL DENSITY GRADIENT CENTRIFUGATION THROUGH THE ADMINISTRATION OF PHOSPHOLIPID + EGTA

Rooije Rumende,Laya Rares,Eva Baideng,Fredine Rares

doi:10.46909//journalalse-2021-026

Abstract

The Percoll density gradient centrifugation (PDGC) method is frequently used in the sexing of spermatozoa. However, this method causes damage to the spermatozoa membranes, resulting in a decreased quality of spermatozoa. We analysed the impacts of phospholipid PC (phosphatidylcholine) and EGTA (ethylene glycol bis (β-aminoethyl ether) N,N,N',N'-tetraacetic acid) Ca2+ free buffer on the quality of bovine spermatozoa after the PDGC process, using semen from Friesian Holstein (FH) bulls aged 5 - 8 years. The following variables were observed: spermatozoa motility, spermatozoa viability, spermatozoa membrane integrity, spermatozoa that have not experienced capacitation, spermatozoa that have experienced capacitation and spermatozoa that have undergone acrosomal reaction. The results showed that the administration of phospholipid PC + EGTA Ca2+ free buffer to spermatozoa, followed by the PDGC process, could maintain or further improve the values of all variables. In the PDGC process, phospholipid PC 10% + EGTA Ca2+ free buffer at 1 mM was most suitable.

Highlights

Separation of the X and Y chromosomes of spermatozoa can be carried out using the Percoll density gradient centrifugation (PDGC) method
We analysed the impacts of phospholipid PC and EGTA (ethylene glycol bis (β-aminoethyl ether) N,N,N',N'-tetraacetic acid) Ca2+ free buffer on the quality of bovine spermatozoa after the PDGC process, using semen from Friesian Holstein (FH) bulls aged 5 - 8 years
The results showed that the administration of phospholipid PC + EGTA Ca2+ free buffer to spermatozoa, followed by the PDGC process, could maintain or further improve the values of all variables

Summary

Introduction

Separation of the X and Y chromosomes of spermatozoa can be carried out using the Percoll density gradient centrifugation (PDGC) method. This approach is easy in its implementation and application and a valid and inexpensive method. Damage to the spermatozoa membrane after the PDGC process is caused by three factors, namely loss of seminal plasma, the presence of chemical factors characterised by an increase in free radicals, as well as physical factors that occur due to ANALYSIS OF SPERMATOZOA QUALITY USING PERCOLL DENSITY GRADIENT CENTRIFUGATION friction/collision between spermatozoa. To fix the physical factor, the integrity of the spermatozoa membrane, which is composed of lipids, proteins, carbohydrates and other substances, is crucial (Tucker and Jansen, 2002)

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