The present work shows that normal

The present work shows that normal climatic conditions are not critical for the long time survival of DNA in untreated blood stains. This information may be useful for laboratories who wish to store reference blood samples in a simple way, while saving the costs of commercial substrates that are treated with DNA stabilizing agents. The amplicon size of standard forensic STR’s are <500bp, and <300bp for STRs tailored for degraded DNA (e.g., Minifiler®, Applied Biosystems, Foster City, CA), and for SNP-based systems the amplicon size is <150 bp. We believe that the present findings, using a 1600bp fragment that is much larger than any STR amplicon, and a 245bp fragment larger than any SNP amplicon, provide an adequate assessment of the forensic applicability of DNA after long time storage of blood stains.
Introduction Performance in poultry breeding flocks, routinely measured by fertility and hatchability of eggs, is dependent on genetic and non-genetic components related to males and females, as well as the synergy of these two components (Wolc et al., 2009). These contributions of both sexes to egg fertilization and CEP-37440 development enhance the need for selection of females and males simultaneously (Wolc et al., 2009). In males, reproductive efficiency may be affected by behavioral and semen variables, which seemingly have low and moderate-to-high heritability, respectively (Soller et al., 1965, Dunnington and Siegel, 1983, Ansah et al., 1985, Barbato, 1999, Kabir et al., 2007, Hu et al., 2013). Semen analysis in poultry breeding programs is, therefore, considered a useful technique, not only for sire selection (i.e., prior establishment of the breeding flock), but also for monitoring adult males throughout chick production (Donoghue, 1999, Parker and McDaniel, 2002). Various features can be assessed to estimate quality and function of poultry spermatozoa including concentration, motility, morphology, viability, metabolism, lipid peroxidation, and ability of spermatozoa to bind and penetrate the perivitelline layer (Bakst and Cecil, 1997). Nevertheless, many techniques for semen evaluation are time-consuming, laborious, entail technical expertise and expensive equipment, limiting the in situ application to breeding flocks (Donoghue, 1999). These constraints could be overcome by implementing new approaches of evaluating poultry spermatozoa characteristics with rapid, easy to use and low-cost techniques. In this regard, brightfield microscopy of stained semen smears is considered a simple, reliable and inexpensive method, which can be simply adapted to breeder management procedures (e.g., repeated disinfection/fumigation of materials and equipment). Besides, depending on the stain, cytochemical assays allow the examination of different spermatozoon structures such as DNA, mitochondria, acrosome, plasma membrane and flagellum (Hrudka, 1987, Pope et al., 1991, Bjorndahl et al., 2003, Kim et al., 2013). Spermatozoa DNA integrity is an important variable that has been associated with fertility and early embryo development, but it has only been measured in chicken spermatozoa by the comet assay and flow cytometry (Lopes et al., 1998, Evenson et al., 1999, Erenpreiss et al., 2001, Partyka et al., 2010, Gliozzi et al., 2011, Simoes et al., 2013). Alternatively, toluidine blue (TB) is a basic thiazine metachromatic dye that stains phosphate residues of spermatozoa DNA with fragmented ends and lysine-rich regions of histones, having been applied in humans due to its simplicity and high correlation of findings with those obtained using other acknowledged assays (e.g., acridine orange, aniline blue, and TUNEL; Erenpreiss et al., 2001, Erenpreiss et al., 2004, Kim et al., 2013, Esteves et al., 2015). To our knowledge, however, the TB technology has not yet been validated for use in avian spermatozoa. Likewise, no studies have addressed the utilization of single-step staining technique with Fast Green-Rose Bengal (FG-RB) solution to detect acrosome status in poultry spermatozoa, even though it has wide application in mammalian species (Pope et al., 1991, Olson et al., 2003, Angrimani et al., 2016). Another cytochemical assay that can be employed in male breeders on commercial farms is the monitoring of mitochondrial activity through oxidation of 3,3′-diaminobenzidine (DAB; Hrudka, 1987, Blumer et al., 2012). This method is based on oxidation of DAB by Cytochrome c complex (including Cytochrome c oxidase), in a chain reaction in which the reagent is polymerized and deposited in the mitochondria (Hrudka, 1987). Considering the previous results, the purpose of the present study was to validate these practical and accurate staining methods to assess DNA fragmentation, acrosomal integrity, and mitochondrial activity in rooster spermatozoa.