Association of sperm oxidative stress, proteomics and fatty acid profiles in nellore bulls at sexual rest

Abstract

Association of sperm oxidative stress, proteomics and fatty acid profiles in nellore bulls at sexual rest. Adviser: Simone Eliza Facioni Guimarães. This study was carried out with the objective of determining the effect of sexual rest on the oxidative status of spermatozoa from adult Nellore bulls and its effect on the fatty acid composition and protein profile. Six bulls were used, which were submitted to Breeding Soundness Evaluation and consecutive semen collection by the electroejaculation method. For each semen collection, the physical and morphological characteristics of the ejaculates were evaluated using conventional optical microscopy and phase contrast microscopy. After analyzing the semen qualitative parameters, the first and last ejaculate of each were centrifuged to separate the spermatozoa from the seminal plasma. From sperm aliquots, the enzymatic activity of Super Oxido Dismutase, Catalase and Glutathione S-Transferase was determined by colorimetric methods. The markers of lipid peroxidation, malondialdehyde and nitric oxide were also determined. Using gas chromatography, the fatty acids of sperm from the first and last ejaculate were identified. For proteomic analysis, total sperm proteins were extracted and precipitated in a 4% CHAPS solution, then quantified by the Bradford method and subsequently analyzed by monodimensional short-run electrophoresis and mass spectrometry (LC-MS/MS). Proteins were identified using PEAKS software version 7.0. Proteins were functionally classified by the RPSBLAST tool of BLAST version 2.13.0 into categories from the KOG database. For the label-free quantitative proteomics analysis, proteins identified in at least three technical replicates of each group with a fold-change greater than 1.5 times were included. Data were analyzed using GraphPad Prisma software version 9.1. Four semen collections were necessary so that the bulls at sexual rest presented sperm parameters compatible with reproductive fitness. Differences (p<0.05) were observed between all physical and morphological characteristics of the first and last ejaculate. A total of 974 proteins were identified, of which 735 were common between the two groups and 123 and 116 proteins were unique to spermatozoa from the first and last ejaculate, respectively. Functional classification analysis revealed that KOG categories with the highest number of classified proteins were Posttranslational modification, protein turnover, chaperones and Cytoskeleton and Signal transduction mechanisms. The protein abundance profile showed that 6 proteins were differentially abundant in spermatozoa from the first ejaculate and 30 proteins weredifferentially abundant in spermatozoa from the last ejaculate. In addition, gas chromatography analysis allowed the identification of 19 fatty acids, 47.3% of the saturated type and 52.7% of the unsaturated type. The C12:0 and C18:0 fatty acids showed difference between the two groups, being more abundant in the spermatozoa of the last ejaculate. The results indicate that oxidative stress may be related to sperm characteristics displayed by bulls at sexual rest, since the accumulation of by-products of lipid peroxidation can cause alterations in the metabolic pathways used by spermatozoa to generate energy used for motility and alter fluidity. of the membrane causing sperm abnormalities. Keywords: Bovine. Semen. Antioxidants. Lipids. Proteins.