Quality characteristics and fertilizing ability of ram sperm subpopulations separated by partition in an aqueous two-phase system

Centrifugal countercurrent distribution (CCCD) in an aqueous two-phase system (TPS) is a resolute technique revealing sperm heterogeneity and for the estimation of the fertilizing potential of a given semen sample. However, separated sperm subpopulations have never been tested for their fertilizing ability yet. Here, we have compared sperm quality parameters and the fertilizing ability of sperm subpopulations separated by the CCCD process from ram semen samples maintained at 20 degrees C or cooled down to 5 degrees C. Total and progressive sperm motility was evaluated by computer-assisted analysis using a CASA system and membrane integrity was evaluated by flow cytometry by staining with CFDA/Pl. The capacitation state, staining with chlortetracycline, and apoptosis-related markers, such as phosphatidylserine (PS) translocation detected with Annexin V. and DNA damage detected by the TUNEL assay, were determined by fluorescence microscopy. Additionally, the fertilizing ability of the fractionated subpopulations was comparative assessed by zona binding assay (ZBA). CCCD analysis revealed that the number of spermatozoa displaying membrane and DNA alterations was higher in samples chilled at 5 degrees C than at 20 degrees C. which can be reflected in the displacement to the left of the CCCD profiles. The spermatozoa located in the central and right chambers (more hydrophobic) presented higher values (P<0.01) of membrane integrity, lower PS translocation (P<0.05) and DNA damage (P<0.001) than those in the left part of the profile, where apoptotic markers were significantly increased and the proportion of viable non-capacitated sperm was reduced. We have developed a new protocol to recover spermatozoa from the CCCD fractions and we proved that these differences were related with the fertilizing ability determined by ZBA, because we found that the number of spermatozoa attached per oocyte was significantly higher for spermatozoa recovered from the central and right chambers, in both types of samples. This is the first time, to our knowledge that sperm recovered from a two-phase partition procedure are used for fertilization assays. These results open up new possibilities for using specific subpopulations of sperm for artificial insemination or in vitro fertilization, not only regarding better sperm quality but also certain characteristics such as subpopulations enriched in spermatozoa bearing X or Y chromosome that we have already isolated or any other feature. (C) 2011 Elsevier B.V. All rights reserved.

Heparin and penicillamine–hypotaurine–epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development