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Freeze‐dried stallion spermatozoa: evaluation of two chelating agents and comparative analysis of three sperm DNA damage assays
Author(s) -
Olaciregui M.,
Luño V.,
Martí J. I.,
Aramayona J.,
Gil L.
Publication year - 2016
Publication title -
andrologia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.633
H-Index - 59
eISSN - 1439-0272
pISSN - 0303-4569
DOI - 10.1111/and.12530
Subject(s) - dna fragmentation , sperm , acridine orange , semen , stain , andrology , dna , dna damage , egta , biology , microbiology and biotechnology , chemistry , staining , biochemistry , genetics , medicine , apoptosis , organic chemistry , programmed cell death , calcium
Summary During the freeze‐drying procedure, sperm DNA might become damaged by both freezing and drying stresses. Sperm DNA status can be detected using well‐established assays; however, most techniques are expensive and involve elaborate protocols and equipment. Indirect assessments can provide alternative strategies. The objective of this study was to compare a simple test of DNA status using Diff‐Quik ( DQ ) with two established procedures: acridine orange test ( AOT ) and sperm chromatin dispersion ( SCD ) on freeze‐dried ( FD ) stallion spermatozoa. Ejaculated spermatozoa from three stallions were freeze‐dried in basic medium supplemented with two different chelating agents: EGTA or EDTA . After rehydration, the spermatozoa were subjected to DNA damage detection using a SCDt , AOT and DQ stain simultaneously. The results showed that the DNA damage levels in the EGTA group were significantly lower than those in the EDTA group. AOT detected a significantly higher proportion of spermatozoa with fragmented DNA than DQ and SCD . The results of the SCD test and DQ stain exhibited a significant positive correlation for DNA fragmentation ( r = 0.528), whereas a negative correlation was observed between SCD , DQ and AOT ( r = −0.134 and r = −0.332 respectively). The present study shows that both the SCD test and DQ assay are effective methods for detecting FD stallion sperm DNA fragmentation, whereas using of AOT is questionable.