The present study was conducted to detect sperm apoptosis in fresh and frozen semen and to determine its relationship with bull fertility. Three ejaculates were collected from five breeding bulls with different fertility levels and were cryopreserved using standard methods. Two flow cytometric methods were employed to measure apoptosis: an assay for phosphatidylserine (PS) translocation across the plasma membranes using fluorescein-labeled Annexin V and propidium iodide (PI), and an assay for nicked DNA using bromodeoxyuridine (BrdU), terminal deoxynucleotidyl transferase, and fluorescein-labeled anti-BrdU monoclonal antibody. Both assays showed that fresh sperm contained 10%-20% apoptotic sperm. Significant differences in the percentage of apoptotic sperm were observed among the bulls. Cryopreservation induced translocation of PS to the outer leaflet of the plasma membrane and caused most of the necrotic cells in fresh sperm to disintegrate. Bull fertility was significantly related to the percentage of necrotic or viable sperm in fresh semen as detected by the Annexin V/PI assay, to the number of apoptotic sperm in fresh semen as detected by the TUNEL assay, and to the level of chromatin or DNA condensation as detected by PI staining. The present study suggests that the presence of apoptotic spermatozoa in fresh semen could be one of the reasons for poor fertility in breeding bulls.

Fresh boar sperm were incubated with small unilamellar liposomes composed of either the total lipids extracted from head plasma membranes (HPM) of fresh boar sperm or selected lipids (SL) of five defined phospholipids with specific acyl chains. To optimize fusion, liposomes with 2 mol% octadecyl rhodamine fluorophore in Beltsville Thawing Solution +/- 1 mM CaCl(2) were incubated at 35 degrees C with 1;ts 10(7) or 10(8) spermatozoa/ml and monitored over 60 min, using flow cytometry and fluorescence microscopy. The HPM fused to both sperm concentrations faster than SL but was equivalent by 30 min (10(8) sperm/ml) or 60 min (10(7) sperm/ml; 57.5 +/- 3% and 67.1 +/- 8% sperm fused to HPM and SL, respectively) +/- Ca(2+). Neither HPM nor SL affected onset of capacitation or spontaneous or ionophore-induced acrosome reactions at 0 or 3 h (chlortetracycline and fluorescein isothiocyanate-Pisum sativum agglutinin; n = 3). During cooling and after cryopreservation (n = 4 ejaculates), SL but not HPM significantly improved sperm motility and viability (Sybr14/propidium iodide staining) +/- 20% egg yolk, but egg yolk alone was more effective than SL alone. Liposomes of complex composition can fuse to boar sperm without harming in vitro capacitation or acrosome reaction and reduce sperm chilling sensitivity.

Background Liposomes are used to carry pharmaceutical agents and to alter the lipid composition of cell membranes. This study compared resonance energy transfer (RET), fluorescence dequenching, and flow cytometry as monitors and quantifiers of fusion between liposomes and mammalian spermatozoa. Methods Preliminary experiments used RET to determine the optimum sperm concentration for fusion of DL‐α‐phosphatidylcholine dipalmitoyl (PC)/DL‐α‐phosphatidylethanolamine dipalmitoyl (PE) liposomes at 35°C ± 5 mM Ca2+. Microscopy confirmed the fusion of liposomes, not just adhesion (n = 3). Dequenching tested the time‐dependent fusion of liposomes of two different lipid compositions to sperm, both, (n = 3) ± 1 mM Ca2+ and (n = 3) without Ca2+ at two sperm concentrations. Finally, flow cytometry absolutely quantified the percentage of sperm fusing to liposomes at different liposome‐to‐sperm ratios (n = 4) and with sperm from different donors (n = 3). Results RET detected fusion of liposomes with sperm and microscopy confirmed the interaction to be true fusion. Dequenching detected more fusion of liposomes with sperm at 100 × 106 sperm per milliliter than at lower concentrations (P < 0.05). Fusion dynamics differed with lipid composition but Ca2+ had no effect. Flow cytometry reliably quantified the percentage of sperm fusing with liposomes, which varied from bull to bull (P < 0.05). Conclusion Liposome fusion with mammalian sperm membranes can be quantified cytometrically and varies with lipid composition, sperm‐to‐liposome ratio, and individual animals. Cytometry 49:22–27, 2002. © 2002 Wiley‐Liss, Inc.