view - Department of Reproduction, Obstetrics and Herd Health
view - Department of Reproduction, Obstetrics and Herd Health
view - Department of Reproduction, Obstetrics and Herd Health
Create successful ePaper yourself
Turn your PDF publications into a flip-book with our unique Google optimized e-Paper software.
CHAPTER 1.1<br />
The sperm chromatin structure assay (SCSA) is perhaps the most frequently used test to<br />
analyze the DNA content <strong>of</strong> sperm. This assay measures the susceptibility <strong>of</strong> DNA to denaturation<br />
when exposed to a low pH. The SCSA test was introduced by Evenson in 1980 (Evenson et al., 1980)<br />
<strong>and</strong> can be used for analyzing a number <strong>of</strong> species, including horses (Evenson et al., 1995). The test is<br />
based on the metachromatic properties <strong>of</strong> acridine orange (AO), which is a fluorescent dye that shifts<br />
fluorescence from green to red when associated with double or single str<strong>and</strong>ed DNA, respectively<br />
(Evenson <strong>and</strong> Jost, 2000). Normally, AO does not penetrate well into the sperm chromatin, so<br />
nucleoproteins must be decondensated first using a low pH solution (Schlegel <strong>and</strong> Paduch, 2005).<br />
The chromatin susceptibility to denaturation, as measured with the SCSA, is correlated with the level<br />
<strong>of</strong> actual DNA str<strong>and</strong>s breaks (Evenson et al., 1995). These lesions may induce post-fertilization<br />
embryonic failure (Fatehi et al., 2006), which explains the clinical relevance since this represents a<br />
potential noncompensible defect (Varner, 2008). This means that for spermatozoa with<br />
noncompensible defects increasing the insemination dose will not improve pregnancy rate since the<br />
percentage <strong>of</strong> noncompensable defects will remain proportionally equal in the increased<br />
insemination dose.<br />
Besides the SCSA, the terminal deoxynucleotidyl transferases dUTP end labeling (TUNEL)<br />
assay is frequently used to assess the DNA integrity <strong>of</strong> sperm. This assay analyses DNA breaks directly<br />
<strong>and</strong> the original technique has been used in human <strong>and</strong> veterinary medicine to analyze sperm from<br />
different species (Waterhouse et al., 2006; Filliers et al., 2008; Purdy, 2008). However, recent work<br />
from the group <strong>of</strong> Dr. Aitken (Mitchell et al., 2011) proved that the original TUNEL assay is not<br />
suitable for analyzing DNA str<strong>and</strong> breaks in (human) spermatozoa. They found that the assay was<br />
insensitive <strong>and</strong> unresponsive to DNA fragmentation induced in spermatozoa when exposed to<br />
Fenton reagents (H2O2 <strong>and</strong> Fe 2+ ). Additionally, they demonstrated that it is important to assess the<br />
viability <strong>of</strong> sperm simultaneously when analyzing DNA breaks. Moreover, they proved that DNA<br />
fragmentation can appear as a cause as well as a consequence <strong>of</strong> cell death. Based on these findings,<br />
the protocol for TUNEL assay <strong>of</strong> sperm has been modified to the new sperm TUNEL assay which<br />
combines a vitality stain (LIVE/DEAD Fixable Dead Cell Stain (far red) from Molecular Probes) (Fig. 8)<br />
with a 45 min exposure to 2mM dithiothreitol, allowing for a correct assessment <strong>of</strong> DNA damage in<br />
live cells (Mitchell et al., 2011). So far, this adapted TUNEL protocol has not been used to analyze<br />
equine sperm. An additional advantage <strong>of</strong> this assay is the possibility to combine it with either a<br />
flowcytometer as well as with fluorescence microscopy.<br />
25