80 Energetic substrate availability affects the metabolome profile in bovine sperm
Authors: da Rocha H. C. , dos Santos E. C. , Feitosa W. B. , De Martino E. , de Lima C. B. , Ispada J. , Martins A. F. J. , Alcantara J. V. S. , Gasparrini B. , Milazzotto M. P. 
Published: January 2021

Bovine spermatozoa are specialised cells that require high ATP production for flagellar movement and other physiological events necessary for fertilization. Glycolysis and oxidative phosphorylation (OXPHOS) are the most studied energy pathways in sperm cells and involve metabolites such as glucose, pyruvate, and lactate. Although glycolysis has been described as the preferential pathway for ATP generation in bovine spermatozoa, other metabolites may also be used, leading to distinct metabolome profiles. Thus, the objective of this work was to characterise the metabolome profile of culture media derived from sperm cells incubated in the presence of different energy substrates for ATP production. For that, a semen straw from one bull (n=3) previously tested for IVF was thawed and motile sperm were separated by Percoll gradient, washed and resuspended in FertTalp medium (FT) without capacitator agents (Parrish et al. 1989 Biol. Reprod. 41, 683–699) to a final concentration of 30×106cells mL−1. Then, samples were centrifuged and resuspended in 5 different groups: positive control (PC, FT supplemented with 2mM glucose, 0.2mM pyruvate, and 11mM lactate); negative control (NC, FT without energy substrates); Glu (FT and 3.5mM glucose); Pyr (FT and 0.11mM pyruvate), and Lac (FT and 5.5mM lactate). Samples were incubated at 38.5°C, 5% CO2 in high humidity for 15 and 45min. After incubation, samples were centrifuged and supernatant was collected and analysed by Raman spectroscopy as previously described (Santos et al. 2015 Biomed. Opt. Express 6, 2830–2839; https://doi.org/10.1364/BOE.6.002830). Data were preprocessed and submitted to principal component (PCA) and loading plot analysis (LP) by using Minitab software (Minitab Ltd.). The results showed that after 15min of incubation, the metabolic profiles were similar for the PC, Glu, and Lac groups, suggesting that they present similar metabolic activity. NC and Pyr were a separate cluster, indicating that pyruvate is not metabolized through OXPHOS in this phase. LP analysis comparing Glu and Pyr groups indicated phosphatidylserine, phenylalanine, DNA/RNA, and lipids as the most distinct metabolites. After 45min, PC and Pyr had a similar metabolome profile, whereas NC, Glu, and Lac clustered together, suggesting that for long-period incubation OXPHOS takes place as the preferential pathway for energy production in bovine sperm cells. At this time, the comparison of Glu versus Pyr revealed phosphatidylserine, proline, phenylalanine, carboxylic acid, DNA/RNA, proteins, and lipids as the most different metabolites between groups. Based on these results, we hypothesised that the glycolysis to OXPHOS transition may be a consequence of the depletion of glycolytic enzymes, leading the sperm cells to use distinct pathways for long-term maintenance of ATP production. In conclusion, our data showed that the metabolome profile of bovine spermatozoa varies according to the period of incubation and substrates availability for energy production. However, more studies are necessary to characterise the ability of these metabolites to maintain sperm motility and viability.
This research was funded by FAPESP 2017/18384-0.