Comparative Study of Four Different Sperm Washing Methods Using Apoptotic Markers in Ram Spermatozoa

Abstract

The accurate measurement of semen fertilizing potential is of great importance in determining the acceptability of processed semen for breeding purposes. A good sperm preparation technique results in a sample with high viability and motility and also takes into account other parameters such as the capacitation and apoptotic state which could compromise the ability to fertilize an oocyte. In this study, we investigate the effects of 4 sperm preparation techniques (a dextran/swim-up procedure, discontinuous Percoll density gradient centrifugation, sucrose washing, and filtration) on ram sperm quality parameters. Besides the evaluation of viability and the capacitation state, we also analyzed the apoptotic status of the sperm samples by assessing the phosphatidylserine translocation and caspase-3 and -7 activities. This is the first report, to our knowledge, that evidences the presence of active caspases in ram sperm. The results confirm the better ability of the dextran/swimup procedure to select nonapoptotic spermatozoa, in addition to viable and noncapacitated sperm, compared with other sperm preparation methods. This should be considered to enhance results of artificial insemination techniques in ovine reproduction.

Viability Tests, Active Caspase‐3 and −7, and Chromatin Structure in Ram Sperm Selected Using the Swim‐Up Procedure

“…Because sperm motility is an indicator of cell viability, it is assumed that SU-processed sperm are of good quality. Some studies have demonstrated that membrane integrity and mitochondrial function are high, whereas DNA fragmentation and caspase expression decrease after SU processing (Sakkas et al, 2000;Martí et al, 2006Martí et al, , 2008Kotwicka et al, 2008). We evaluated in neat and SU samples cytoplasmic esterases and mitochondrial dehydrogenases as indicators of cytoplasmic membrane integrity and mitochondrial function by using calcein AM and resazurin, respectively.…”

“…Some ejaculated sperm express markers of apoptosis (Kotwicka et al, 2008;Martí et al, 2008), which are negatively correlated with semen quality (Weng et al, 2002;Almeida et al, 2005) and fertility (Fatehi et al, 2006). Using SU to sort sperm reduces the number of sperm expressing caspases (Martí et al, 2006(Martí et al, , 2008.…”

“…However, DNA fragmentation may originate from processes other than apoptosis (Sakkas et al, 1999), and because intrinsic and extrinsic paths of apoptosis converge in caspase activation, it is a more reliable marker of cell death. Caspase activity has been detected in the sperm of humans (Weng et al, 2002;Kotwicka et al, 2008) and rams (Martí et al, 2006(Martí et al, , 2008, and both procaspases and caspases have been immunolocated in the ram (Martí et al, 2008); however, the specific location of active caspases is unknown (Martí et al, 2006(Martí et al, , 2008. Previous studies have reported that ejaculates with a high number of sperm positive for caspases are negatively correlated with fertilization rate after in vitro fertilization (Marchetti et al, 2004), but this relationship has not been described following intracytoplasmic sperm injection (Said et al, 2006).…”

Flow cytometry for the assessment of animal sperm integrity and functionality: state of the art

“…Apoptosis markers have been detected in spermatozoa of many species, but the role of these markers is probably not yet elucidated completely. 49,[119][120][121][122] Changes of sperm membrane permeability have been considered as a typical event of apoptosis in many studies. Since cryopreservation, freezing and thawing cause cryodamage, and presumably even apoptotic-like changes, 123 apoptotic markers can be good tools for forecasting semen freezability and cellular damage occur during cryopreservation, albeit not necessarily indicting apoptosis really occurs.…”

Impact of caspase activation in human spermatozoa

“…The investigations were performed in sperm derived from healthy male donors (Grunewald et al, in press), infertility patients (Brum et al, 2008;Martí et al, 2006) and animals (ram and equine spermatozoa) (Blanc-Layrac et al, 2000). In detail, double density gradient centrifugation is able to reduce the number of sperm showing caspase-3 activation to 15-20% in neat donor semen samples and to 43.5% in semen specimens from infertility patients.…”