Assessment of DNA fragmentation of feline epididymal spermatozoa : comparison between Sperm-Halomax® and TUNEL assay

The integrity of the paternal DNA is of crucial importance for the embryo development (1) and a relationship between DNA damage and infertility has been demonstrated in humans. Spermatozoa with severe DNA damage remain functionally intact, with normal fertilizing ability, but a high index of DNA fragmentation (DFI) results in a significant decrease in pregnancy rates (2). Feline species is affected by teratozoospermia, but sperm morphology per se is not a reliable predictor of DNA alterations (3). Among different techniques aimed at analyzing sperm DNA status, terminal deoxynucleotidyl transferase mediated nick end labelling (TUNEL) has been applied to spermatozoa of different species including cats (4). However, this technique is either labour intensive and requires expensive instrumentations. Recently, a kit (Sperm-Halomax®) specifically developed for boar (5), bull (6), stallion (7) and dog semen (8), but not for cat semen, became commercially available. This kit is based on the sperm chromatin dispersion (SCD) test. Spermatozoa are immersed in an agarose matrix on a slide and briefly incubated in a lysing solution to remove membranes and proteins. DNA fragmentation produces large halos, whereas those sperm with low levels of fragmentation show small halos and the evaluation can be performed by light microscopy. The aim of the present study was to verify whether the Sperm-Halomax® assay could be used for the evaluation of DNA status of epididymal cat spermatozoa. For this purpose, DNA fragmentation indexes obtained with Sperm-Halomax® and TUNEL were compared. Materials and methods. Epididymal spermatozoa were collected from 10 tomcats subjected to routine orchiectomy. Epididymides and vasa deferentia were dissected and squeezed to collect epididymal fluid in a warmed (37°C) PBS without Calcium and Magnesium. Samples were divided in two aliquots and DNA status was evaluated by Sperm-Halomax® for canine spermatozoa (Halotech DNA SL, Madrid, Spain) and TUNEL (Calbiochem® FragEL™ DNA fragmentation detection kit, Fluorescent – TdT Enzyme; EMD Millipore Billerica, MA, USA) according to manufacturer's instructions. Values are presented as mean ± standard deviation (SD). Significant differences (P<0.05) were determined by Student’s t-test. Results. No differences were observed in DFI obtained with Sperm-Halomax® and TUNEL (4.34 ± 0.93% vs. 4.26 ± 0.83%; P = 0.84). These results show that Sperm-Halomax® for canine spermatozoa gives a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. Conclusion. Sperm-Halomax® assay is rapid and easy to perform compared to TUNEL and the assessment of DFI might be included in the standard semen analysis. It might significantly contribute to the evaluation of semen quality and may be an effective diagnostic and prognostic tool to evaluate male factor infertility. Future investigations should be focused on the setting of DFI threshold limit values for feline semen. For this purpose, the sperm DNA status should be correlated with fertility in terms of embryo quality, implantation, and pregnancy.