Zebrafish Live and Dry Feeds: The Tradeoff between Food Sterility and Micronutrients
By Dr. Terry Snell
Chair Emeritus, Georgia Institute of Technology Biology Department
Are you testing pharmaceutical efficacy against a strong or weak innate and adaptive immune system?
This article examines the inevitable tradeoffs between sterilizing food in zebrafish labs and the resulting nutritional tradeoffs that compromise the animal’s ability to grow and regulate their innate and adaptive immune systems. We present a series of design choices for labs to consider recognizing that sterilizing feeds comes at the cost of slower growth rates and compromised immune systems.
Zebrafish play a key role in pharmaceutical research as they share over 70% of human genes (including 84% of the genes associated with human disease), have transparent embryos that enable easy observation from fertilization to adulthood, genes are easy to manipulate. Zebrafish lay many eggs enabling many replicates and have a innate and adaptive immune systems that mimic the human immune system.
The adaptive and innate immune systems in animals evolved over 100s of millions of years using macro and micronutrients to protect themselves from pathogens.
Any well-designed lab experiment will control for variables in live/dry feeds to limit the risk of pathogens contaminating the results of the experiment.
Replicating the macro & micronutrients present in animal diets in live and dry feeds is not a simple task, further complicated by feed sterilization requirements. We present methods, procedures and tradeoffs that we have made in designing our live and dry zebrafish feeds and present options to zebrafish labs to make their own choices to sterilize feeds or optimize bioavailability of micronutrients.
Live Feeds- Rotifers
Rotifers are indiscriminate filter feeders with their own microbiome.
Cysts: We offer 15 varieties of resting rotifer cysts which are easily stored in the freezer for years and used on demand to start rotifer cultures. Our cysts are very clean but not sterile. Zebrafish labs can easily decapsulate our rotifer cysts with chlorine bleach which cleans the hatching rotifers of bacteria.
Algae: After hatching, most labs feed rotifers algae such as Tetraselmis suecica which is not sterile. We offer five algae starter cultures guaranteed to be single-species maintained a clean room and amplified using aseptic techniques.
Labs seeking higher levels of sterility have two common choices, neither one perfect.
Option A: Autoclave. Sterilization can be easily accomplished using heat, however that same process kills the algae and denatures the micronutrients that the rotifer takes up and passes on to the zebrafish larvae.
Option B. Filter sterilization. To retain the micronutrients, you can remove most bacteria using a 0.2 µM filter using a hard vacuum. However, fungi and some resistant bacteria are known to survive this filtration process.
We recommend all rotifer cultures be raised in enclosed bioreactors to minimize risk of contamination by bacterial pathogens or ciliates. The algae in these closed systems outcompete bacteria and ciliates and secrete compounds that suppress their invasion. We offer lab scale closed bioreactors that can reliably produce millions of rotifers as needed.
During harvest, we recommend rinsing rotifers with filtered sterilized seawater prior to feeding.
Rotifer Dry Feeds. Sustainable Nutrition has developed Amplifeed Replete Rotifer & Artemia feed.
Most dry feeds are extruded into pellets at temperatures of 130°C and up, which like autoclaving, sterilizes the feed. Unfortunately, exposing the feeds to high temperature denatures the micronutrients required by animals for proper growth and immune system development. Denaturing of some compounds starts as low as 90°C with most gone by 115°C.
Amplifeed Replete is manufactured at low temperature and therefore is not sterile, but it has all necessary micronutrients intact.
Amplifeed Replete can be used as an algae extender in rotifer mass cultures to accelerate population growth and to enrich the nutritional quality of rotifers the day before harvest and feeding to zebrafish. Feeding enriched rotifers to fish larvae improves their survival, accelerates their growth and maturation, and increases their resistance to stressors.
Amplifeed Replete does not contain any antibiotics or probiotics and is derived from non GMO algae and yeast. In addition, Amplifeed Replete contains the 3S, 3’S isomer of astaxanthin which is known to act both as a strong antimicrobial agent as well as a strong antioxidant.
Astaxanthin mitigates mitochondrial dysfunction originating from oxygen singlets and reactive oxygen species from the mitochondria’s oxidative phosphorylation process.
Dry Zebrafish Feeds
We offer a product called Hatchery Diet at .125mm and .5mm sizes which is suitable as a first feed for zebrafish larvae. It can be used in lieu of rotifers or as the first dry feed after weaning larvae off live rotifers. Our Hatchery Diet provides all macro and micronutrients required by zebrafish to optimize their growth rate and develop a strong immune system.
Our zebrafish Hatchery Diet is not sterile and like Amplifeed Replete, we do not recommend autoclaving because this degrades its micronutrients.
Micronutrients and the Innate/Adaptive Immune System
Our sister company, Sustainable Aquatics, operates a marine ornamental fish hatchery raising over 200 species of marine ornamentals and salmonoids. Because of the excellent diet, this hatchery never needs to use antibiotics, vaccines or probiotics.
To produce these yields, the company abandoned the use of commercial hatchery extruded feeds and manufactured our own feeds with highly bioavailable micronutrients. Sampling their RAS systems revealed that every natural pathogen is present in the hatchery, yet their hatch-to-harvest yields are >98% with <<2% deformities. For further information on how micronutrients impact the innate and adaptive immune system see our series on covering the Four Pillars of the Immune System.