DYNAMIC OF THE SPERM DNA INTEGRITY IN FROZWN-THAWED SEMEN FROM ENDANGERED GREY STEPPE BREED BULLS
Sabina Andra Neculai-Văleanu, D.L. Dascălu, A.M. Ariton, M. Davidescu, Elena Ruginosu, G. Nacu, Şt. Creangă
Aim of the study The purpose of this study was to evaluate the dynamic of the sperm DNA integrity of spermatozoa from endangered Grey Steppe cattle breed bulls in order to establish the potential use of sperm DNA fragmentation (SDF) assay for improving the routine fertility screening in the conservation program.
Material and methods Cryopreserved semen doses from two Grey Steppe bulls were thawed by placing the straws into a water bath at 38°C for 30 seconds.The dynamic of the sperm DNA integrity was analyzed by determining the sperm DNA fragmentation index using the Toluidine blue stain at T0 moment and subsequentely after 3, respectively 6 hours of incubation at 38°C, in a incubator. Briefly, wet smears were fixed in acetone:ethanol, washed, hydrolized in HCl and stained with Toluidine Blue.
Results Variability of percentages of spermatozoa with fragmented DNA was assessed in 2 Grey Steppe bulls. In the case of both bulls, the sperm DNA fragmentation index (DFI%) increased progresively during the 6 hours incubation, when the hightest DNA fragmentation index (DFI) was observed, 7.5% in bull 2, respectively, 4.5% in bull 1. At T0, the DFI% was greater for Bull 2, a value of 3.5% being registered as compared to bull 1, in which the DFI% was 2.5%.
Conclusions The semen from the two Grey Steppe bulls analyzed in this study presented moderate DNA fragmentation index and may be used for AI and IVF in conservation program of the Grey Steppe breed.
Key words: sperm chromatin, Grey Steppe, bull, spermatozoa