Chromatin dispertion test in equine spermatozoa

CARRETERO, MI.; ARRAZTOA, CC.; CALDEVILLA, M.; FERRANTE, A.; LOMBARDO, DM.; NEILD, D.

Veterinary Research (Invet – Investigación Veterinaria)

BM Press

Lugar: Buenos Aires; Año: 2010 vol. 12 p. 249 - 250

1514-6634

There are different methods to evaluate sperm DNA. The Sperm Chromatin Dispersion assay (SCD) is based on the loss of proteins from sperm nuclei after exposition to lysing solutions, producing or not a halo depending on DNA fragmentation. The Toluidine Blue (TB) stain evaluates the degree of sperm chromatin decondensation by detecting disulfi de bond absence. The objective of the study was to use the SCD test on equine sperm, to determine a positive control for the test, to establish the patterns observed and to correlate them with TB patterns. Semen samples were obtained from 4 stallions (n=4: r=2). Three positive controls for SCD were evaluated: a) semen exposure to 100° C for 30 minutes, b) incubation for 30 minutes in 0.3M NaOH at room temperature and c) 2 hs UV exposure. Three concentrations of 2-mercaptoethanol (ME) (2.5; 5 and 10%) were tested as SCD lysing solutions. An image analyzer was used to capture 600 images/ejaculate (200/solution) and both the halo and the residual nuclear structure or “core” were measured. The experimental design considered the treatment as a fi xed factor and the ejaculate nested in the male. The SCD and TB patterns were correlated using Spearman’s test. Signifi cant differences (p=0.04) were observed in halo size between the 2.5% (60.4 ± 23.2 μm) and 10% (71.9 ± 26.6 μm) ME solutions, but not with the 5% (63.9 ± 23.1 μm) solution (mean ± SD). The patterns observed were the same as those reported for humans: (i) large dispersion halos, (ii) intermediate halos, (iii) small halos and (iv) no halo. All positive controls proved effective. No correlation was observed between SCD and TB patterns. To conclude, the 5% ME solution was selected, because no differences in halo size were observed between this and the 2.5% ME solution. The absence of correlation between SCD and TB could be due to evaluating different DNA alterations. The use of the SCD test to evaluate equine sperm DNA fragmentation was simpler and less expensive than other methods such as TUNEL, SCSA and Comet.