Characterization of proteins in cryopreserved and non-cryopreserved seminal plasma of dairy bulls of dif-fering fertility

Seminal plasma is composed of secretions from accessory sex glands, which are mixed with sperm at ejaculation and contribute the majority of semen volume. Seminal plasma is considered a transport and support medium for sperm in the female reproductive tract. Because seminal plasma is not required for fertilization, the importance of its constituents to the establishment of normal pregnancy has been overlooked. Four seminal plasma proteins, Osteopontin, Sper-madhesin Z13, BSP 30 kDa and Phospholipase A2, have been identified as markers of fertility in dairy bulls (Cancel et al., 1997; Moura et al., 2006, 2007). The objective of the present study was to characterize the expression patterns of these proteins and other proteins found to be of interest in seminal plasma of cryopreserved and non-cryopreserved bull semen. Seminal plasma samples were obtained from 16 mature Hol-stein-Friesian bulls at Select Sires Inc. Samples were divided into two groups based on assigned fertility score expressed as the percentage point deviation (PD) of the bull’s non-return rate (NRR) from the average NRR of all bulls in the Select Sires Inc. reproductive management program. Group 1 (high fertility bulls, n = 8) 1.9 ≤ PD ≤ 2.7%, and group 2 (low fertility bulls, n = 8) -6.5 ≤ PD ≤ 1.8 %. Additionally, the samples were categorized as processed (cryopreserved) or unprocessed (non-cryopreserved) for protein analysis. Protein expression was analyzed by 2-D fluorescence difference gel electrophoresis (2D-DIGETM). Protein spots were picked from a reference gel, analyzed by mass spectrometry and, subsequently identified by MS/MS ion searches performed on the SwissProt database. Protein expression did not differ (P > 0.05) with fertility grouping but displayed two distinct patterns among the processing groups: majority of the functional proteins were highly expressed in seminal plasma of non-cryopreserved semen while the cryopreserved semen contained mainly structural/extender derived proteins. Functional proteins identified included Spermadhesin Z13, BSP A1/2, BSP 30 kDa, Nucleobindin-1 and metalloproteinase inhibitor 2. Some of these proteins have been identified as anti-fertility or fertility enhancing agents in males. Whether this alteration in protein expression after processing might affect semen fertility is worthy of further evaluation