Scientists Improve the Way they Measure Fish Fat to Accelerate Understanding of Fish Population Health

Scientists at the Alaska Fisheries Science Center have streamlined a method used to determine the amount of energy-rich fat in fish. As a result, the number of fish samples they are now able to analyze has substantially increased.

Scientists have long regarded lipids (or fat) as the most important energy reserve for animals in marine ecosystems. They are more calorie-dense than proteins and sugars and are used to fuel growth. Fish with lower lipid values have slower growth and lower reproductive potential, and are weaker overall. Measuring the total lipid content of fish is widely used as a metric to understand the overall health and condition of a population. Moreover, prey species with higher lipid content contribute to the overall health of the predators that eat them.

Traditional methods for analyzing lipids take a lot of time and produce relatively few results. On average, a technician can produce results from 15 samples in an 8-hour day using gravimetric analysis. Using the new method, a single technician can produce results from 69 samples in an 8-hour day. The new method drastically increases productivity while maintaining very high accuracy and precision. More data generated on a faster turnaround is important for decision-makers to inform fish stock assessments and fisheries management on tighter time scales.

Optimizing the Method for Efficient Data Delivery

"Good data about fish stocks requires large sample sizes," remarks Cody Pinger, lead author and analytical chemist with the Alaska Fisheries Science Center. "And in my experience of measuring fish lipids, laboratory methods are usually slow and tedious. For nearly a decade, our group has been working to optimize the sulfo-phospho-vanillin assay, and I'm thrilled to say that we have increased our productivity many-fold while maintaining good accuracy and precision."

The sulfo-phospho-vanillin (SPV) assay has been long studied as a rapid alternative to traditional methods for lipid analysis. "We ramped up productivity by adapting the assay to modern chemistry instrumentation, and then validated these measurements on important Alaska fish stocks," continues Pinger.

The sample processing starts with a fish. For some species, the entire fish is blended into a uniform mixture; for others, we only process the muscle tissue. The sample is extracted into organic solvent. The following steps include heat and concentrated sulfuric acid followed by a reaction with vanillin in the presence of phosphoric acid-hence the name sulfo-phospho-vanillin. This process generates a final product that is a clear liquid with a pink tint. The darker the pink equates to more lipid in the sample.

From there the sample is passed through a spectrophotometer, which measures the amount of light absorbed by the sample. This fine-tuned instrument can quantify the amount of lipid in each sample. The 96-well format allows the team to analyze many more samples at once, which streamlines the process and is more efficient than traditional methods.

Calibrating and Applying the Data

Prior research studies show that achieving high accuracy with this method depends on proper calibration so that it can be applied to a variety of species.

The chemical reaction between lipids, acid, and vanillin is dependent on the specific molecular composition of the fat, which varies between species of fish. Fat from a salmon filet is chemically different from fat from a Pacific cod. In a perfect world, all samples could be calibrated using a widely available fish oil. In Pinger and his team's case, they used menhaden oil for that purpose. However, this does introduce some error when you're analyzing many different species with different lipid profiles.

To fine-tune the results of the SPV assay using menhaden oil for calibration, the team developed calibration models for each species analyzed. This studylooked at:

• Pacific herring
• Cod, pollock
• Capelin
• All five species of Alaska Pacific salmon (Chinook, coho, pink, sockeye, and chum)

The results produced data that matched what traditional methods produced. This validated the SPV method and certified a novel way to measure lipids in Alaska marine fish.

After many years in the making, Cody Pinger and his team are thrilled with the results. Pinger says, "This has been a long time in the works, and we are all thrilled to be able to provide high quality data in such an efficient manner. The more information we can provide about the annual status of our ecosystems and fish stocks, the better for management of these commercially and ecologically important marine resources."

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