Sperm parameters and seminal plasma proteome of rams undergoing intermittent testicular heat stress

The present study evaluated the effects of testis heat stress on rams by means of intermittent scrotal insulation (SI). Six adult and reproductively sound Morada Nova rams had their testis insulated during four consecutive nights (6:00 p.m. to 6:00 a.m.). Semen was obtained by electroejaculation 14 days before and weekly for 120 days after the last night of SI. Scrotal circumference (SC), sperm motility and morphology were evaluated, as well as sperm DNA integrity, using the sperm chromatin dispersion test. Day 0 was the first day of insulation. Then, seminal plasma was obtained on days -14, 0, 7, 18, 28, 48, 77 and 120 and analyzed by 2D-SDS-PAGE. Gels were stained with Coomassie G-250 and evaluated using PDQuest software (Bio-Rad, USA). Proteins were identified by tandem mass spectrometry (ESI-Q-ToF). Data obtained over time in relationship to SI was evaluated by LSM (P≤0.05) using SAS software (v. 9.0; 2002) procedures. In silico analysis of protein-protein networks were evaluated by STRING v. 10.0. SC averaged 30.5 cm at pre-insulation, increased to 31.8 cm at the end of insulation (day 4), reached 27.9 cm on day 28 and returned to normal values on day 57 (30.6 cm). Sperm motility decreased from 75% to 33% between days 0 and 4, respectively. Motile sperm were undetectable in the ejaculates on days 23 and 28. However, sperm motility was 10% on day 35 and increased to 52% on day 57, returning to normal (67%) on day 77. The percentage of normal sperm decreased from 96% on day 0 to 77% and 6% on days 7 and 35, respectively, returning to normal on day 91 (87%). Sperm DNA integrity decreased from 86.5% (day 0) to 52.3% on day 7, reached the lowest scores between days 11 and 63 (10.7%) and returned to normal on day 120 (80.4%). Considering that spermatogenesis and the sperm transit into the epididymis takes close to 60 days, it is necessary two sperm cycles to for the reestablishment of the normal sperm DNA integrity. After SI (day 7), the amount of seminal plasma proteins identified as carboxypeptidase Q precursor (CPQ) and superoxide dismutase [Cu-Zn] isoform X1 (SOD) decreased while albumin precursor (ALB) increased. Protein CPQ had the lowest expression from day 7 to 48, returning to normal on day 77, coinciding with the return of normal sperm motility. Protein CPQ interacts with natural resistance-associated macrophage protein 1, which controls resistance to pathogens through mechanisms that involves sequestration of Fe2+ and Mn2+, cofactors of catalases and superoxide dismutases. Protein CPQ may act to protect macrophages against their own generation of reactive oxygen species (ROS). The decrease of CPQ could explain the reduction of SOD expression; in addition to the reduction in sperm motility after SI. SOD had the lowest expression on day 18. This protein destroys ROS produced by cells, which is toxic to biological systems. Additionally, SOD interacts with peroxiredoxin-6 and thioredoxin. Peroxiredoxin-6 is an antioxidant with a protective effect against oxidative stress on DNA and cells. Studies shows thioredoxin regulates caspase-3 activity, thus reduced levels this protein is associated with high CASP3 activity which in turn is related to higher sperm DNA fragmentation. In our investigation, high DNA fragmentation was observed when there was lower CPQ and SOD expression in the seminal plasma of rams (from days 18 to 48). Albumin is characterized as a non-enzymatic antioxidant and it cloud bind to ROS and protect sperm from lipid peroxidation. Seminal plasma ALB had higher expression after SI (day 7), showing higher values on day 28 (no sperm motility) and returning to normal when seminal parameters were reestablished. Thus, intermittent testicular heat stress caused significant changes in sperm motility, DNA integrity and seminal plasma proteome of rams. Proteins with changed expressions during the study were likely involved in protective processes against oxidative stress on sperm cells.