ALTERNATIVE APPROACH TO FELINE EPIDIDYMAL SPERMATOZOA CHARACTERIZATION.

The cauda of the epididymis is a reservoir of spermatozoa which can be used in assisted reproductive technologies (ARTs). This might significantly contribute to the preservation of genetic material and to the generation of offspring from individuals of high genetic or emotional value that die unexpectedly, undergo orchiectomy for medical reasons or that cannot mate or ejaculate semen. Epididymal samples should be evaluated prior to the use in ARTs. Routinary semen analysis is based on the conventional evaluation, and, in some cases, on the assessment of other functional integrity parameters. Other aspects can be evaluated to extend the characterization of spermatozoa. Among these, the assessment of sperm ubiquitination, DNA fragmentation and morphometry represent the alternative approach to feline epididymal spermatozoa characterization described in the present study. Ubiquitin is a 8.5 kDa peptide that tags other proteins for proteasomal degradation, and it is also involved in the regulation of protein function. It might be responsible of the elimination of defective spermatozoa during transit through epididymis but the exact biological function of this peptide in seminal plasma has not yet been clarified. Magnetic cell separation techniques, based on the use of antibodies or proteins-coated magnetic beads, as magnetic ubiquitin beads, may allow the selective capture of ubiquitinated spermatozoa from semen, thus contributing to the identification of a potential correlation between semen quality, spermatozoa maturation and ubiquitination process. The general aim of the first study was to understand whether ubiquitin could be considered a biomarker of quality of epididymal feline semen, and to characterize by a proteomic approach all the proteins ubiquitinated in the spermatozoa retrieved in the three regions of the epididymis. Results demonstrate the presence of ubiquitinated proteins in cat epididymal semen. However, a correlation between semen quality and ubiquitination process has not been found. Thus, ubiquitin cannot be considered as a biomarker of quality, in terms of morphological and acrosomal patterns, of epididymal feline spermatozoa. Identification of all the ubiquitinated proteins of cat spermatozoa collected from different epididymal regions would contribute to give a better understanding of the ubiquitin role in feline sperm maturation. Sperm DNA fragmentation is an important parameter to assess sperm quality and can be a putative fertility predictor. Since the sperm head consists almost entirely of DNA, subtle differences in sperm head morphometry might be related to DNA status. Several techniques are available to analyze sperm DNA fragmentation, but are labor-intensive and require expensive instrumentations. Recently, a kit (Sperm-Halomax®) based on the sperm chromatin dispersion (SCD) test and specifically developed for spermatozoa of different species, but not for cat spermatozoa, became commercially available. To enhance the evaluation of feline epididymal spermatozoa used in ART the first aim of the second study was to verify the suitability of Sperm-Halomax® specifically developed for canine semen, for the evaluation of DNA status of epididymal cat spermatozoa. For this purpose baseline values of DNA fragmentation obtained with Sperm-Halomax® and TUNEL were compared. The second aim of this study was to investigate whether a correlation between DNA status, sperm head morphology and morphometry assessed by Computer Assisted Semen Analysis (CASA) exists in cat epididymal spermatozoa. No differences were observed in baseline values of DNA fragmentation index obtained with Sperm-Halomax® and TUNEL. This result indicates that Sperm-Halomax® assay provides a reliable evaluation of DNA fragmentation of epididymal feline spermatozoa. The DFI seems to be independent from all the measured variables of sperm head morphology and morphometry. Thus, the evaluation of the DNA status of spermatozoa could effectively contribute to the completion of the standard analysis of semen used in ARTs. In conclusion, the alternative approach to spermatozoa characterization based on the assessment of sperm ubiquitination, DNA fragmentation and morphometry demonstrates for the first time some peculiar aspects of cat epididymal spermatozoa.