Seminal plasma proteins after testicular insulation in Brangus bulls

Seminal plasma is comprised of secretions originating from the accessory sex glands, epididymis, and the testis, that contribute to reproductive performance and helps to prevent premature capacitation and to protect sperm against oxidative stress for normal spermatogenesis. Detection of changes in these mechanisms will serve to determine possible causes of the decrease in the seminal production. The objectives of the present study were to identify seminal plasma proteins using 2D-GE and to determine the correlation between individual proteins and semen characteristics among insulated and non-insulated Brangus bulls. We evaluated seminal plasma proteome using 2DGE among insulated and non-insulated Brangus bulls. All procedures were approved by the Ethical Committee for Care and Use of Experimental Animals (Project 26250, CEUA/UFRGS). Nine Brangus bulls were randomly allocated into two groups: scrotal insulated bulls for 72 hours (Insulated Group; n=6) and non-insulated bulls (Control Group; n=3). Semen collections were performed using eletroejaculator on the day of insulation (D0) and weekly for the following 13 weeks (W1 to W13). In order to verify the effect of insulation on seminal plasma protein composition, samples were analyzed by 2D SDS-PAGE on D0, W4 and W13. Semen samples were centrifuged at 700 × g for 10 min (at 4°C) to separate seminal plasma from sperm. Then, the supernatant was centrifuged at 10,000 × g for 60 min, at 4°C, divided into aliquots and kept at −80 °C. Total protein concentration was determinate using a BCA kit (Pierce) using BSA as standard. 2D SDS-PAGE was carried out as described by Moura et al. [Journal of Andrology, 27:201-211, 2006; Journal of Proteomics, 73:2006-2020, 2010] and images were analyzed using PDQuest software. Seminal analyses and optical density spots were normalized and statistically compared between groups (at insulation, post-insulation and recovery) by means of Student’s t-test or ANOVA followed by Tukey test, assuming significance when P<0.05. Overall, sperm motility was greater in the control group than in the insulation group (76.7 vs. 52.0%, respectively; P<0.05). The proportion of total sperm defects in insulated group was higher than control group from W2 to W7 (P<0.05). PDQuest software in 30 seminal plasma gels constructed within the 3–10 pH range detected an average of 65±28 spots. No difference in the number of spots was observed between groups tested: D0 61±27, W4 72±18 and W13 52±18 spots. The analysis of 2D gel by PDQuest software allowed the quantification and identification of protein spots differently present at D0, 4, and 13 weeks after insulation when sperm parameters were normalized. We observed ten spots that were differentially expressed, where eight had a higher optical density in post insulation group and two where more abundant in recovery group. This variation in sperm function after insulation could be explained by variation in the protein composition of seminal plasma. This study confirms that multiple proteins collected from Brangus bulls are associated with semen parameter after scrotal insulation. We conclude that interactions among several proteins explain a significant proportion of the variation during scrotal heat stress induction in Brangus bulls. These findings emphasize that further studies at the molecular level, e.g. using tandem mass spectrometry, are needed to characterize and to understand different semen handling procedures in bovine.