142 Metabolomic analysis of fresh and frozen bovine seminal plasma: A preliminary study

Abstract

Sperm cryopreservation is essential for assisted reproduction; however, freezing induces biochemical and physical damage to the sperm membrane structures, negatively affecting sperm fertilising ability (Castro et al. 2006 J. Anim. Sci. Biotechnol. 5, 1-9). Metabolomics is the study of small molecules, commonly known as metabolites, which play essential roles in biological systems. Emerging research in the field of metabolomics showed a potential role in identifying male fertility biomarkers (Bieniek et al. 2016 Asian J. Androl. 18, 426-433). Metabolites present in the seminal plasma play several roles related to sperm motility, sperm membrane protection from oxidative stress, and regulation of metabolic activity (Therien et al. 1995 Biol. Reprod. 52, 1372-1379). However, the extent of metabolite changes in seminal plasma during cryopreservation is still not well known. Therefore, the aim of this study was to evaluate the differences of the seminal plasma metabolites between fresh and frozen bovine sperm. Four ejaculates from eight Holstein (Bos taurus) bulls (4-6 years age) maintained at an authorised national semen collection center (Centro Tori Chiacchierini, Civitella D’Arna, Italy) under uniform management conditions were collected weekly using an artificial vagina (IMV Technologies). Each ejaculate was split into two aliquots (one fresh and one frozen) at −20°C for at least 2 h. Seminal plasma from fresh (immediately) and frozen-thawed semen was separated from sperm by centrifugation (1600 × g for 10 min), and supernatants were then transferred to a 2-mL tube and stored at −80°C until use. After methanol extraction of metabolites, samples were centrifuged at 6400 × g for 30 min at 4°C, and supernatants were analysed using a liquid chromatography-mass spectrometry (LC-MS; Agilent 1260 HPLC; Agilent Technologies) system with an autosampler and a binary pump coupled to an Agilent Q-TOF 6540 (Agilent Technologies). Univariate analyses of the data were performed using bioinformatics approaches. The differences between fresh and frozen seminal plasma samples were analysed using a paired Student's t-test. Twenty-three metabolites were found in both fresh and frozen seminal plasma samples, out of which eight were significantly different (P < 0.05) between the two groups; among these, hypotaurine, ceramide, and isoindoline were identified. In particular, hypotaurine and ceramide were downregulated, whereas isoindoline was upregulated in fresh samples compared with frozen samples. To the best of our knowledge, this is the first study to identify seminal plasma metabolites in fresh and frozen samples by using LC-MS. In order to ensure the reliability and accuracy of results, the validation of the method used in the analysis of biosamples is still in progress, as is the evaluation of the metabolic profile in sperm cells. In conclusion, the results of this study suggest that LC-MS can be a promising screening tool to detect metabolites potentially associated with bull fertility.