Atlantic salmon aquaculture production has seen declines of more than 35% in the past two decades due, in part, to decreased embryo survival rates. In response to a need for the rapid assessment of brookstock reproductive performance, NIST is developing new broodstock quality assessment tools for aquaculture managers based on metabolite markers from salmon biofluids . T he utilization of Nuclear Magnetic Resonance (NMR)-based metabolomics for the identification of small metabolites in organismal tissues and biofluids has proven to be a powerful and sensitive molecular technology for biomarker discovery . Changes in fish reproductive function are reflected in metabolic changes to sustain this energy-demanding process in different stages of gonadal development or growth. T hese changes alter t he biochemical composition of biofluids. Metabolomic profiles of mucus, ovarian fluid and plasma of high- and low-yield broodstock are being assessed using NMR metabolomics to identify bio markers of broodstock reproductive performance . Among different biofluids, o varian fluid plays a crucial role in the preservation of egg viability and in promoting fertilization, thus providing a promising biological matrix for the identification of putative biomarkers of reproductive success in female broodstock.
Currently, the processing of fish ovarian fluid for NMR-based metabolomic analysis has yet to be investigated. Therefore, w e evaluated the performance of four ovarian fluid preparation methods 1 ) methanol precipitation , 2) ultrafiltration, 3) lyophilization , 4) dilution, in terms of spectral reproducibility , NMR spectral quality, and signal-to-noise ratio. Additionally, the effects of blood contamination on the metabolite profiles were assessed by comparing two distinct pools of ovarian fluid with different degrees of suspected hemolysis (low vs. high). In collaboration with University of Maine and USDA, ovarian fluid was collected from broodstock female Atlantic salmon at the USDA-ARS National Cold Water Marine Aquaculture Center (NCWMAC) in Franklin, ME. P olar metabolites across the different methods were analyzed by NMR spectroscopy at the NIST laboratories at Hollings Marine Laboratory in Charleston, SC .
Our results show that ultrafiltration allowed efficient removal of macromolecules from the samples with significant improvement in overall spectral quality . The addition of a concentration step to the ultrafiltration protocol provided enhanced signal-to-noise ratio. Additionally, the degree of hemolysis of samples could confound the analysis for reproductive quality, as we observed significant differences in metabolite profiles of high and low hemolysis: thus, sample hemolysis should be carefully evaluated prior to conducting metabolomic analysis of ovarian fluid samples . The identification of putative biomarkers of reproductive success in broodstock Atlantic salmon will be used for the development of on-site screening tools for farm managers to guide the selection of the best fish to use for breeding, while providing new insights into biochemical factors underlying reproductive performance, and could improve the economic viability of the Atlantic salmon aquaculture industry.