Supplemental Information

Effects of dietary selenomethionine on cutthroat trout (Oncorhynchus clarki bouvieri) growth and reproductive performance over a life-cycle

Ronald W. Hardy · Libbie L. Oram · Gregory Möller


Additional Materials and Methods

Experimental fish. Eyed cutthroat trout eggs from a pooled spawning of 12 females, fertilized with 12 males were obtained from Henry’s Lake Hatchery, Idaho Department of Fish and Game, and transported to the University of Idaho’s Hagerman Fish Culture Experiment Station (HFCES), where they were placed in Heath tray incubators supplied with 8.0 to 8.9°C recirculating water. Fish hatched 14 days later (560 temperature units) and reached 95% yolk-absorption after an additional 10-11 days. After enumeration of dead and deformed fry and tempering to warmer water, fry were stocked into 120 L fiberglass tanks supplied with fresh spring water (14.5°C), and fed commercial trout feed (Silver Cup Steelhead, Nelson & Sons, Murray, UT). The fish were reared until they reached approximately 1g in average weight, at which time they were stocked into experimental tanks to begin the feeding trial.

For the feeding trial, water flow to each tank was kept low when the fish were small, and was increased as the fish grew. Photoperiod was held constant at 14 h day, 10 h night.

Experimental Diets. The six experimental diets were produced at the HFCES by cold extrusion. The formulation and proximate composition of the diets was similar to commercial trout diets (Table S1). The only difference among diets was the amount of selenomethionine added to each dietary treatment. Selenomethionine (Sigma, St. Louis, MO) was dissolved in distilled water to produce a stock solution (100 mg L-1 Se). Appropriate volumes of stock solution were diluted with water and then added to each batch of feed to facilitate pelleting. Thus, the target concentration of Se was obtained through use of a stock solution and the dilution procedure to ensure dispersion in the feed during mixing prior to pelleting. After pelleting, feeds were dried to <10% moisture in a forced-air pellet dryer and stored in plastic buckets until used. Samples of feed were taken for proximate and Se analysis as described from each batch of feed (feed was made fresh approximately every two months when fish were small and feed pellet size was increased to match fish requirements). During the course of the study, 14 batches of feed were made. The trial was designed as a completely randomized design for statistical evaluation of data, and, as mentioned, diets were assigned to replicate tanks of fish in a completely randomized design.

Proximate and Selenium Analysis. Proximate analysis was conducted using standard methods: moisture by oven drying at 105°C overnight, protein by nitrogen determination using a LECO FP 428 nitrogen analyzer, crude lipid by extraction in a Goldfisch apparatus with methylene chloride, and ash by incineration at 550°C in a muffle furnace. Selenium analysis was conducted on dry samples by hydride-generation inductively-coupled argon plasma spectrophotometry (ICP) after high temperature nitric, perchloric, and sulfuric acid digestion followed by HCl reduction (Tracy and Möller 1990). Laboratory method detection limits were 0.5 mg g-1 total Se in tissue, and recovery limits of QC reference samples was greater than 80%. All selenium analyses were conducted under a rigorous QA/QC program that included chain of custody, the use of certified traceable standards, the use of control materials, and the use of certified standard reference materials by the University of Idaho Analytical Sciences Laboratory (UIASL). UIASL is a certified USEPA laboratory compliant with auditable Good Laboratory Practice Standards and it is the state-wide veterinary diagnostic toxicology laboratory for Idaho and Washington State, U.S.

Additional Results

Concentrations of protein and lipids in diets were always within target concentration ranges. Details on the composition of the experimental diets are included in Table S1.

For the depuration study, selenium concentrations and body burdens decreased in all groups, but the higher the initial Se concentration and body burden, the greater the change in total body burden and Se concentration. Table S2 includes fish tissue Se concentrations and total Se body burden before and after the 32 week depuration study and change in Se concentration.

Additional References

Tracy ML, Möller G (1990) Continuous flow vapor generation for inductively coupled argon plasma spectrometric analysis. Part I: selenium. J Assoc Off Anal Chem 73: 404-410


TABLE S1. Composition of experimental diets (g kg-1 diet) fed to cutthroat trout and proximate composition. All diets were mixed as one batch, divided into six equal batches, and Se was supplemented.

Ingredient Concentration (g kg-1 diet)

Fish meal (LT Icelandic capelin) 600

Wheat middlings 254

Fish oil 123

Ascorbic acid (phosphate ester) 1

Choline Chloride (70% liquid) 6

Trace mineral premix 1

Vitamin premix 15

Calculated Proximate Composition (% as-is basis)

Moisture 6.3

Crude protein 45.0

Crude fat 16.0

Ash 10.3

TABLE S2. Average whole body Se concentration and body burden of cutthroat trout at the beginning and end of the 32-week Se depuration study, change in total body burden, and rate of Se loss. Dietary treatments are the calculated levels of Se in each diet and refer to the treatment group from which fish were taken. Initial values are means ± SD, with two replicate tanks per diet, and five fish pooled and sampled for Se level per tank. Final values are means ± SD (n = 10 fish for diets 4 and 6, n = 9 fish for diet 5, and n = 6 fish for diet 3).

Dietary Treatment / Whole Body Se
(µg Se g-1 dw) / Total Body Burden
(μg Se fish-1) / Change
(μg Se fish-1) / Rate
(μg Se lost g-1gain)
Initial / Final / Initial / Final
5.2 µg g-1Se (Diet 3) / 6.80 ± 0.14 / 5.30 ± 1.43 / 336.28 ± 17.43 / 237 ± 100 / -98 ± 100 / 1.01
7.2 µg g-1Se (Diet 4) / 10.00 ± 0.00 / 3.06 ± 0.62 / 493.80 ± 32.00 / 218 ± 101 / -275 ± 101 / 2.84
9.2 µg g-1Se (Diet 5) / 12.00 ± 1.41 / 2.67 ± 0.60 / 595.60 ± 102.51 / 167 ± 74 / -429 ± 74 / 4.42
11.2 µg g-1Se (Diet 6) / 12.50 ± 0.71 / 1.82 ± 0.42 / 600.66 ± 51.97 / 171 ± 49 / -429 ± 47 / 4.42