Aquaculture America 2024

February 18 - 21, 2024

San Antonio, Texas

DEVELOPMENT OF OVARIAN FLUID METABOLOMIC ANALYSIS FOR REPRODUCTIVE BIOMARKER DISCOVERY IN ATLANTIC SALMON

Fabio Casu* , Tracey Schock , Amanda Bayless and Ashley Boggs

 

National Institute of Standards and Technology

Chemical Sciences Division

Charleston, SC 29412, USA

fabio.n.casu@nist.gov

 



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.