Assessment of Sperm Apoptosis in Cryopreserved Bull Semen After Swim-up Treatment: A Flow Cytometric Study


Author's address (for correspondence): A. Chaveiro, Animal Reproduction, Department of Agrarian Sciences, University of the Azores, 9700-851 Angra do Heroismo, Portugal. E-mail:


The techniques used to prepare bovine spermatozoa for in vitro fertilization, to enhance the percentage of motile sperm cells include the swim-up (SU) method, among others. The objective of the present study was to evaluate the phosphatidylserine (PS) translocation and plasma membrane integrity as the indicator of apoptosis and necrosis in post-thaw bull sperm after SU treatment using annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) assay. A flow cytometric method was employed to measure apoptosis levels on frozen-thawed bull spermatozoa. The assay detects PS translocation across the plasma membrane using a fluorescein-labelled annexin-V and PI. By using the annexin V/PI assay four different subpopulations of sperm were observed: (i) a population of apoptotic sperm, labelled with annexin V–FITC but not with PI; (ii) a population of early necrotic spermatozoa, sperm labelled with annexin–FITC and PI; (iii) a population of necrotic sperm, labelled with PI but not with annexin–FITC; and (iv) a population of fully viable sperm cells, sperm not labelled with annexin V–FITC and without PI. Results clearly indicated that SU technique itself could have an adverse effect on the spermatozoa membrane stability. It has also been found, significant differences between bulls in the levels of apoptotic sperm, after SU treatment.