view - Department of Reproduction, Obstetrics and Herd Health

CHAPTER 1.1
The sperm chromatin structure assay (SCSA) is perhaps the most frequently used test to
analyze the DNA content of sperm. This assay measures the susceptibility of DNA to denaturation
when exposed to a low pH. The SCSA test was introduced by Evenson in 1980 (Evenson et al., 1980)
and can be used for analyzing a number of species, including horses (Evenson et al., 1995). The test is
based on the metachromatic properties of acridine orange (AO), which is a fluorescent dye that shifts
fluorescence from green to red when associated with double or single stranded DNA, respectively
(Evenson and Jost, 2000). Normally, AO does not penetrate well into the sperm chromatin, so
nucleoproteins must be decondensated first using a low pH solution (Schlegel and Paduch, 2005).
The chromatin susceptibility to denaturation, as measured with the SCSA, is correlated with the level
of actual DNA strands breaks (Evenson et al., 1995). These lesions may induce post-fertilization
embryonic failure (Fatehi et al., 2006), which explains the clinical relevance since this represents a
potential noncompensible defect (Varner, 2008). This means that for spermatozoa with
noncompensible defects increasing the insemination dose will not improve pregnancy rate since the
percentage of noncompensable defects will remain proportionally equal in the increased
insemination dose.
Besides the SCSA, the terminal deoxynucleotidyl transferases dUTP end labeling (TUNEL)
assay is frequently used to assess the DNA integrity of sperm. This assay analyses DNA breaks directly
and the original technique has been used in human and veterinary medicine to analyze sperm from
different species (Waterhouse et al., 2006; Filliers et al., 2008; Purdy, 2008). However, recent work
from the group of Dr. Aitken (Mitchell et al., 2011) proved that the original TUNEL assay is not
suitable for analyzing DNA strand breaks in (human) spermatozoa. They found that the assay was
insensitive and unresponsive to DNA fragmentation induced in spermatozoa when exposed to
Fenton reagents (H2O2 and Fe 2+ ). Additionally, they demonstrated that it is important to assess the
viability of sperm simultaneously when analyzing DNA breaks. Moreover, they proved that DNA
fragmentation can appear as a cause as well as a consequence of cell death. Based on these findings,
the protocol for TUNEL assay of sperm has been modified to the new sperm TUNEL assay which
combines a vitality stain (LIVE/DEAD Fixable Dead Cell Stain (far red) from Molecular Probes) (Fig. 8)
with a 45 min exposure to 2mM dithiothreitol, allowing for a correct assessment of DNA damage in
live cells (Mitchell et al., 2011). So far, this adapted TUNEL protocol has not been used to analyze
equine sperm. An additional advantage of this assay is the possibility to combine it with either a
flowcytometer as well as with fluorescence microscopy.
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