Evaluation of metabolomic profiling and oxidative stress markers of bull seminal plasma and their relationship with sperm motility before and after thawing

Mechanisms underlying differentiation of good or poor freezability ejaculates in bull are still poorly understood. This study aims at investigating oxidative stress parameters and metabolomic profiling of seminal plasma (SP) in relation to bull semen motility before and after cryopreservation. Semen samples were collected by artificial vagina from 20 bulls aged from 10 to 12 mo. An aliquot of semen was centrifuged to obtain SP (stored at -80°C), another was diluted 1:1 with a pre-warmed extender and cryopreserved. Motility assessment (CASA) was performed on fresh and thawed semen (0 and 3 h after thawing). Semen samples were divided into good freezers (GF) and bad freezers (BF) groups according to thawed semen motility, with cutoff values as follows: TM ≥ 60% and PM ≥ 30%. SP was used for oxidative stress markers analysis by spectroscopy techniques and metabolomic profile by nuclear magnetic resonance (NMR). The relationship between NMR spectra and semen quality was evaluated by the PCA/PLS-DA analysis. General linear models and mixed linear models were used to evaluate the relationship between seminal plasma oxidative stress parameters and semen quality. A clear separation between GF and BF ejaculates was observed on the first two principal components of the variance (PC1 vs PC2) of NMR spectra of SP. The increase of advanced oxidative protein products (AOPP) and thiols concentrations on SP were significantly related with higher straightness (STR), beat cross frequency (BCF) and linearity (LIN) and the decrease of amplitude of lateral head displacement (ALH) of fresh semen. Thiols and AOPP concentrations on SP were positively related to semen freezability. In addition, higher AOPP concentration was significantly related to higher TM, PM and LIN of thawed spermatozoa, whereas higher levels of thiols in SP concentration were positively related to the increment of sperm PM after thawing. These findings suggested that SP composition can modulate bull semen freezabilty. Further analyses could elucidate which metabolites and oxidative stress parameters could be used as biomarkers of good semen quality after cryopreservation