313 ASSESSMENT OF SPERM DNA FRAGMENTATION IN CANINE EJACULATES USING THE Sperm-Halomax^® KIT: PRELIMINARY RESULTS

Abstract

Recently, a new procedure for the analysis of sperm DNA fragmentation has been developed for humans and different mammalian species, using a commercial kit based on the sperm chromatin dispersion (SCD) test; however, a descriptive study in canine semen has not been performed. The aim of this work was to assess the sperm DNA fragmentation in canine ejaculates using the SCD test and 2 different staining techniques. For this purpose, ejaculates were collectedby digital manipulation from4 healthy dogs of different breeds (1 German Pointer, 2 Spanish Greyhounds, and 1 Crossbreed). After collection, the sperm-rich fraction of the ejaculates from 3 dogs were pooled each time (n = 4) and then extended in Dulbecco’s phosphate buffered saline. All the pooled semen samples presented physiological values concerning routine semen parameters (motility, morphology, and sperm concentration). The sperm DNA fragmentation was assessed using the Sperm-Halomax^® commercial kit specifically developed for canine semen (Halotech DNA SL, Madrid, Spain). Two semen aliquots of the diluted pooled semen samples were processed on each pre-treated slide provided in the kit following the manufacturer’s instructions. The last step was the staining technique. We stained each slide with 2 different staining procedures. The first half of the slide was stained with propidium iodide (Sigma-Aldrich, St. Louis, MO, USA) mixed in a proportion 1 : 1 with an antifading solution. The second half of the slide was stained for 15 min in Wright solution (1.01383.0500, Merck, Whitehouse Station, NJ, USA) 1 :1 in Phosphate Buffer pH 6.88 (1.07294.1000, Merck). The stained slides were observed using fluorescence and light microscopy, respectively. Five hundred sperm per slide were counted. Spermatozoa with fragmented DNA showed a large and spotty halo of chromatin dispersion. Unfragmented sperm only showed a small and compact halo. Statistical analyses were performed using the Statistical Package for Social Science version 12.0 (SPSS Inc., Chicago, IL, USA). The sperm DNA fragmentation index was compared between Wright and fluorescence staining methods by ANOVA. Results were expressed as mean ± standard error of the mean. The first report of the sperm DNA fragmentation index in canine ejaculates was 2.26 ± 0.53% for Wright staining and 1.99 ± 0.10% for fluorescence technique. No differences were found between staining procedures. In conclusion, it was possible to assess the sperm DNA fragmentation of canine ejaculates using 2 different staining procedures, expecting that continuous research could be useful in defining the role of DNA fragmentation SCD test in canine semen evaluation and cryopreservation.