Quantification of Y-bearing spermatozoa of beef cattle throught ciscontinuous Percoll gradient centrifugation using fluorescent in situ hybridization

Abstract

An appropriated procedure for proportion of Y-bearing spermatozoa is needed, especially to monitor sperm separation techniques. In this study was aimed to determine the Y-bearing spermatozoa proportion in bull semen through pre and post 6 layers discontinuous Percoll centrifugation and by using fluorescent in situ hybriddization (FISH) technique with digoxigenin (Dig)-labeled bovine Y-specific probe, which prepared by polymerase chain reaction (PCR) using BC1.2 primers. This results fould that the metaphase male lymphocyte was clearly shown green-yellow fluorescence spot on the short arm of Y-chromosome, whereas an interphase male lymphocyte showed this signal within nuclei, in conversely, both interphase and metaphase female lymphocytes were not shown this signal. An average interphase male lymphocyte obviously showed the signal 96.8%. The proportion of Y-bearing spermatozoa in semen pre and post discontinuous Percoll centrifugation were 48.78% and 41.68%, respectively (P>0.05). These results indicated that FISH protocol with Dig-labeled bovine Y-specific probe could be used to mornitor the proportion of Y-bearing spermatozoa through discontinuous Percoll centrifugation in bull semen which after sperm separation, the proportion of Y-chromosome trend to be not significantly reduced.

Keywords

• Y-specific probe
• bovine spermatozoa
• FISH
• Percoll