br Materials and methods br Discussion To date only

Materials and methods
Discussion To date, only a fraction of studies pertaining to analysis of avian semen have focused on the study of DNA, with the vast majority being associated with the effects of cryopreservation on sperm DNA integrity (Kotłowska et al., 2007, Madeddu et al., 2009, Partyka et al., 2010, Gliozzi et al., 2011, Partyka et al., 2011, Shanmugam et al., 2016). Increasing evidence from mammalian species suggest, however, that sperm PSB 1115 fragmentation (induced by heat stress, age, oxidative stress, etc.) may be related to male infertility, embryo development failure and spontaneous recurrent abortion (Ahmadi and Ng, 1999, Virro et al., 2004, Fatehi et al., 2006, Tamburrino et al., 2012, Simoes et al., 2013). Age-related increases in sperm chromatin abnormalities have been previously described in roosters, with older males (60 wk of age) having more chromatin alterations than younger and more fertile roosters (35 wk of age) even though the older roosters have fewer morphologically abnormal sperm (Soares and Beletti, 2006). This raises questions about the importance of sperm DNA integrity for fertility and embryo survival in poultry, and, therefore, the need for validating a simple and reliable assessment technique for the occurrence of DNA damage in rooster spermatozoa. Results of the present study provide evidence that TB staining was effective in detecting DNA fragmentation in rooster semen smears, and its applicability has been recently confirmed in research to determine the effects of free radicals on different organelles of rooster spermatozoon (Rui et al., 2017). Soares and Beletti (2006) previously considered TB inadequate to assess chromatin condensation in rooster sperm. Inconsistent findings in this previous and the present study may be related to differences in fixation, hydrolysis and staining techniques. Additionally, in the present study there was a high correlation between the data accumulated with TB and SCD measurements, and thus these technologies may constitute an economical alternative for sperm DNA integrity assessment in rooster spermatozoa as compared with the current methods being used (e.g., SCSA and comet assay). Regarding the determination of the acrosomal status, estimates based on FG-RB staining were associated with the expected percentages of acrosome-intact spermatozoa and FITC-PSA readings, thereby corroborating the effectiveness of this dye in the quantification of acrosomal damage in avian spermatozoa. In the present study, intact acrosomes had a purple-blue color and conical shapes whereas reacted acrosomes appeared as rounded edges (Fig. 2). Such morphological patterns may be attributed to the complete detachment of the acrosomal cap during the process of acrosome reaction, a phenomenon meticulously depicted by Ahammad et al. (2013). Findings from this previous research and unlike mammalian spermatozoa, the acrosome reaction in rooster spermatozoa occurs through acrosomal cap shedding from an opening formed at the posterior region of the acrosome (i.e., detached acrosome caps remain intact with no detectable vesiculation in the apical region). Hence, it seems reasonable to assume that FG-RB staining could be an excellent choice for those situations in which technical and structural constraints preclude the use of procedures formerly adopted for assessing the acrosome reaction in rooster sperm, such as fluorescently labeled lectins (e.g., PNA and PSA) in conjunction with fluorescence microscopy or flow cytometry (Ashizawa et al., 2006, Celeghini et al., 2007, Lemoine et al., 2008, Partyka et al., 2010, Partyka et al., 2011, Ahammad et al., 2013).