br Materials and methods br Discussion To date
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 OSI-930 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). Among several variables that have been assessed to determine spermatozoa quality, mitochondrial function has drawn attention because of its important role in sustaining spermatozoa motility, and, therefore, some investigators collect mitochondrial function data as additional information for conventional semen analysis. To that end, methodological approaches in poultry have focused primarily on measurements of mitochondrial membrane potential (MMP − via Rhodamine 123, JC-1 and MitoTracker Green FM) and ATP concentration (Bakst and Cecil, 1997, Long and Guthrie, 2006, Celeghini et al., 2007, Partyka et al., 2010, Partyka et al., 2011, Singh et al., 2015, Chen et al., 2016;). The MMP technique can be used to assess the capacity of the mitochondria to pump protons across the membrane of this organelle, which may not always be the most desirable variable because research with uncouplers of oxidative phosphorylation indicates that mitochondria with low MMP continue to retain or increase the functions of the electron transport chain (Terada, 1990). Consistent with these findings, ATP quantification does not ascertain the contribution of glycolysis and mitochondrial respiration to total ATP production, leading to a measurement bias with respect to mitochondrial activity. The DAB technique results in selective cytochemical staining for cytochrome oxidase activity in cristae of mitochondria, making it a reliable indicator of oxidative phosphorylation and, consequently, mitochondrial activity of spermatozoa (Hrudka, 1987, Blumer et al., 2012, Fariello et al., 2012). Furthermore, the techniques used in the present research have the advantage of being useful for classifying spermatozoa in four different categories according to the number of active mitochondria. These procedures can, therefore, be considered useful as a supplementary assay for elucidating mitochondrial function in spermatozoa of poultry.