Atlantic halibut, Hypoglossus hypoglossus , is a marine flatfish farmed in Iceland, UK and Norway. Lack of control over stocks’ reproduction, use of wild stocks, inconsis tence supply of gametes and larvae, are constraints that need to be addressed to optimise th is species’ aquaculture . Supplementation of feeds with natural antioxidants might be the approach to improve gamete and offspring quality . Extracellular vesicles (EVs) are cell-derived membrane-surrounded vesicles that carry bioactive molecules and deliver them to recipient cells. These EVs are very important for cell-to-cell communication and are transported in the different body fluids, being specific to each one. EVs and their cargo are considered reliable biomarkers of sperm quality and progeny development. This study aimed to observe the effects of an algal blend supplementation in sperm traits and their correlation with EV-protein cargo in seminal plasma.
Five halibut males from Sogn Aqua Juveniles, Norway, were fed with commercial dry feed during 2022, which was further supplemented with an algal blend in 2023. In the reproductive season, sperm was collected and different quality parameters ( i.e., motility, viability, DNA fragmentation, lipid peroxidation and antioxidant status) were assessed. Seminal plasma was obtained and EVs were isolated employing size-exclusion chromatography, where all the fractions containing EVs were collected . EVs were characterised (size and concentration) using the tunable resistive pulse sensing (TRPS) method. EV-proteins were extracted using RIPA buffer followed by freeze-thaw cycles. Tryptic peptides were separated and analysed by LC-MS/MS. Proteins were identified using Fragpipe software at an FDR < 1%.
Significant d ifferences (paired Student’s t -test p < 0.05) were observed in sperm lipid peroxidation, DNA fragmentation, oxidative enzymes (GSR and GPX ) between males fed with control and algal diet . There were no differences between treatments in terms of EVs size and concentration. Regarding proteomics, 2146 proteins were identified in EV fractions. A canonical correlation analysis (Pearson; r>0.9) between the proteome and the different sperm traits revealed that pathways associated with DNA fragmentation, curvilinear velocity , total motility and oxidative enzymes , like apoptosis, autophagy and cellular response to oxidative stress, were downregulated, while vesicle-trafficking was upregulated , in diet-supplemented fish. These results suggest that algal diet improved the antioxidant capacity of the sperm cells in halibut.
This work was funded by EEA Grants (BREEDFLAT: PT-80-INNOVATION) and national funds from FCT (UIDB/04326/2020, UIDP/04326/2020 and LA/P/0101/2020) . E.F. and C.M. were supported by FCT through 2020.04181.CEECIND and 2023.00304.BDANA.