Freeze‐dried stallion spermatozoa: evaluation of two chelating agents and comparative analysis of three sperm DNA damage assays |
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Authors: | M Olaciregui V Luño J I Martí J Aramayona L Gil |
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Institution: | 1. Reproduction and Obstetric Area, Departamento de Patología Animal, Universidad de Zaragoza, Zaragoza, Spain;2. Pharmacology and Physiology Area, Facultad de Veterinaria, Universidad de Zaragoza, Zaragoza, Spain |
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Abstract: | 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. |
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Keywords: | Denaturation diff‐quik DNA fragmentation freeze‐drying halomax |
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