Submission Number: MUH-4-1-23

Abstract Number: 73

FLOW CYTOMETRIC DETECTION OF EARLY APOPTOSIS IN BULL SPERM USING FLUORESCEIN-LABELED ANNEXIN V.

Muhammad Anzar ¹, He Liwei ², Mary M Buhr* ¹, Tom Kroetsch ³ and Karl P Pauls ²

Department of Animal and Poultry Science, University of Guelph, Guelph, ON, Canada ¹
Department of Plant Science, University of Guelph, Guelph, ON, Canada ²
Gencor, Guelph, ON, Canada. ³

Abstract:
Apoptosis or programmed cell death can be induced by physiological activators, heat shock, oncogenes, chemotherapeutic drugs, ultraviolet and gamma radiation. This study hypothesized that cryopreservation induces early apoptosis in bull sperm. Each of 9 ejaculates (4 bulls) provided fresh and frozen semen. Sperm were cryopreserved in liquid nitrogen using Tris-citric acid-egg yolk-glycerol extender following standard procedures, and thawed at 37C for 50 sec. All sperm were washed twice with cold PBS, resuspended in Annexin V Binding Buffer (0.01 M HEPES/NaOH, pH 7.4; 0.14 mM NaCl; 2.5 mM CaCl₂) at 110⁶ sperm/ml and received either 0 or 10 mM camptothecin. Camptothecin is a known stimulator of apoptosis. Annexin V-FITC (5 l) and Propidium Iodide (PI, 10 l) were added to 110⁵ (100 l) sperm, incubated (15 min, room temperature, dark) and 400 l Binding Buffer was added. In early apoptotic cells, phosphatidylserine translocates from the inner to outer leaflet of plasma membrane, where it is bound by the green fluorescent Annexin V dye. Necrotic sperm (PI-positive, red stain) were excluded from the early apoptotic sperm population. Flow cytometry assayed sperm for size and colour. The data were analyzed by ANOVA using GLM procedures. Semen from all bulls had a significant percentage of early apoptotic sperm (Annexin-positive, PI-negative) which was stable over 60 min, and camptothecin significantly increased this population in fresh sperm. The percentage of early apoptotic sperm in fresh semen (11.40.7) varied among bulls (5.90.7 to 15.11.5, P<0.001) and was increased after cryopreservation (34.00.9, P <0.001 versus fresh; range: 30.82.3 to 37.71.6). In conclusion, we here validate for the first time the use of flow cytometry with Annexin V for assessing early apoptosis in bull sperm. Cryopreservation increases the percentage of early apoptotic cells in bull semen. The molecular basis of early apoptosis in sperm and its relationship with bull fertility are currently being studied. Supported by NSERCC and The Semex Alliance.

Keywords: Apoptosis, Sperm, Bull, Cryopreservation

This abstract is being presented at: 8:00 AM in session:
Apoptosis