Two-dimensional polyacrylamide gel electrophoresis of bovine semen after cryopreservation in half- milliliter straws

Abstract

One of the problems encountered with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the streaking of proteins so that individual spot identification is compromised. This study was conducted to determine whether a low loading dose (50μg) of protein would permit resolution of more discrete protein spots using megapixel camera technology, and if so, to present a nomenclature for future comparisons of the identified proteins. If the major proteins could be identified in a 50-μg sample we aimed to determine whether they could be identified in the supernatant (seminal plasma plus extender) of cryopreserved semen. Two ejaculates were obtained from each of 6 bulls and bovine seminal plasma (BSP) protein concentration was standardized to 50μg/10μl. Isoelectric points (pI) and molecular weights (MWt) of BSP proteins were determined by measuring spot mobility on 2-D PAGE (15% polyacrylamide). Three distinct protein spot constellations (a,b,c) could be readily seen by the naked eye and a faintly stained constellation “d” was identified by the megapixel camera. The image analysis software located 6 protein spots in both constellation “a” (MWt 26 kDa; pI 4.2 to 4.8) and “b” ( MWt 27 kDa; pI 6.6 to 8.0). Constellation “c” contained 13 protein spots distributed in a right-angled triangle with its base towards the acidic end of the gel (MWt 14.7 to 18.8 kDa; pI 5.3 to 7.4). Only spots c 2, c 3, c 5, c 8, and c 13 were present in all 12 samples. Streaking can be eliminated by using 50μg protein for 2-D PAGE, and the major protein spots are readily identified by megapixel camera technology. Protein spots c 3, c 5, c 13 and constellation “a” appear to correspond with Manjunath's proteins (BSP-A 1, -A 2; -A 3; -30kDa). Killian's 2 low fertility proteins may lie in the “c” constellation, and 1 of the high fertility proteins may lie in the “b” constellation. The 3 major BSP proteins can be visualized in the supernatant of cryopreserved semen. We believe that the technique may prove useful for retrospective analysis of processed semen batches that achieve less than satisfactory results in the field.