Technical note Adaptation of ubiquitin-PNA based sperm quality assay for semen evaluation by a conventional flow cytometer and a dedicated platform for flow cytometric semen analysis J.F. Odhiambo a,1 , M. Sutovsky a , J.M. DeJarnette b , C. Marshall b , P. Sutovsky a,c, * a Division of Animal Sciences, University of Missouri, Columbia, Missouri, USA c Departments of Obstetrics, and Gynecology and Women’s Health, University of Missouri, Columbia, Missouri, USA b Select Sires, Inc., Plain City, Ohio, USA Received 7 February 2011; received in revised form 25 April 2011; accepted 6 May 2011 Abstract The purpose of semen quality evaluation is to predict the fertility potential of the sample in an objective, rapid and inexpensive manner. However, utilization of sperm quality biomarkers such as ubiquitin and lectin Arachis hypogaea agglutinin (PNA) for flow cytometric semen evaluation might eliminate the need for visual assessment by microscopy. Herein, we demonstrate a robust ubiquitin and PNA-based semen evaluation conducted on a simple, easy to operate, dedicated sperm flow cytometer, EasyCyte Plus (IMV Technologies, L’Aigle, France). Semen samples were collected periodically from two dairy bulls, which were subjected to temporary scrotal insults to induce variable semen quality. Samples were labeled with fluorescently-conjugated anti-ubiquitin antibodies (bind exclusively to the surface of defective sperm) and lectin PNA (binds to acrosomal surface in prematurely capacitated and acrosome-damaged sperm). Fluorescent properties of the samples were measured with a conventional flow cytometer (Becton Dickinson FACScan; Becton Dickinson Corp., Franklin Lakes, NJ, USA) and by the EasyCyte (IMV Technologies) instrument. Data from the two flow cytometers were positively correlated for the percentage of PNA-positive sperm with a damaged acrosome (r = 0.47; P  0.001) and the percentage of ubiquitin-positive, defective sperm (r = 0.68; P  0.001). Relative intensities of ubiquitin-induced fluorescence in cells with high ubiquitin levels were also positively correlated (r = 0.90). The proportion of sperm with abnormal morphology was positively correlated with ubiquitin-induced fluorescence measured by EasyCyte (IMV Technologies) (r = 0.63; P  0.001). These observations provided a rationale for the adaptation of a dual ubiquitin-PNA sperm quality assay for flow cytometric semen evaluation. © 2011 Elsevier Inc. All rights reserved. Key words: Sperm; Flow cytometry; Ubiquitin; Lectin PNA; Semen evaluation 1. Introduction Traditional methods of semen quality assessment involve evaluation of percent motile sperm (either sub- jectively or computer-assisted), percent sperm with normal morphology, and concentration in an insemina- tion dose [1]. The goal of semen analysis is to identify and discard subfertile samples as accurately as possible in an objective, rapid, and inexpensive manner [2,3]. The discovery of fluorochromes and compounds con- jugated to fluorescent probes facilitated functional anal- yses with fluorescent microscopy. However, only a small number of sperm can be analyzed by microscopy. Hence, the process can be time-consuming and subjec- 1 Present address: Department of Agricultural, Food and Nutri- tional Science, University of Alberta, Edmonton, AB T6G 2P5, Canada. * Corresponding author. Tel.: +1 573 882 3329; fax: +1 573 884 5540. E-mail address: SutovskyP@missouri.edu (P. Sutovsky). Available online at www.sciencedirect.com Theriogenology 76 (2011) 1168 –1176 www.theriojournal.com 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2011.05.009