Acta Vet. Brno 2014, 83: 13-18

Elimination of apoptotic boar spermatozoa using magnetic activated cell sorting

Janko Mrkun1, Tamara Dolenšek2, Tanja Knific2, Anja Pišlar3, Marjan Kosec1, Janko Kos3,4, Petra Zrimšek1

1University of Ljubljana, Veterinary Faculty, Clinic for Reproduction and Horses, Slovenia
2Students of the final year at University of Ljubljana, Veterinary Faculty, Slovenia
3Faculty of Pharmacy, Ljubljana, Slovenia
4Institute Jožef Stefan, Department of Biotechnology, Ljubljana, Slovenia

One of the features of apoptosis is the externalization of phosphatidylserine which could be used to remove apoptotic cells from semen preparations. Magnetic-activated cell sorting using annexin V-conjugated microbeads which bind to phosphatidylserine could be used to enhance semen quality. Twelve boar semen samples after 3 days of liquid storage at 16­­–17 °C were subjected to magnetic-activated cell sorting. Bound and unbound fractions and control samples were subjected to flow cytometry following the staining of spermatozoa with Annexin V conjugated with Alexa Fluor 488 and propidium iodide. Four subpopulations were obtained: live, early apoptotic live, late apoptotic, early necrotic dead and late necrotic dead. The frequency of early apoptotic and late necrotic spermatozoa was significantly higher (P < 0.05) in bound (14.1 ± 10.6% and 24.1 ± 10.2%, respectively) than in unbound fractions (3.4 ± 2.1% and 12.7 ± 3.1%) and control (3.5 ± 1.6% and 12.0 ± 5.0%). The lowest concentration of live spermatozoa was found in the bound fraction (10.6 ± 8.0 %), which differed significantly (P < 0.05) from the control. In unbound fractions there was a significantly higher concentration (P < 0.05) of morphologically normal spermatozoa (31.8 ± 12.6%) compared to bound ones (5.9 ± 7.3%). A significantly (P < 0.05) lower proportion of morphologically normal spermatozoa was observed in both fractions compared to control (67.2 ± 17.0%). Boar spermatozoa were separated by the above method for the first time, however, the results showed this method to be inappropriate for boar semen separation under the tested conditions.


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