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North American Journal of Aquaculture
ISSN: 1522-2055 (Print) 1548-8454 (Online) Journal homepage: http://www.tandfonline.com/loi/unaj20
Dietary Protein Source and Level Affects Growth in
Neon Tetras
Wendy M. Sealey , Frederic T. Barrows , Mike Casten & Ronald W. Hardy
To cite this article: Wendy M. Sealey , Frederic T. Barrows , Mike Casten & Ronald W. Hardy
(2009) Dietary Protein Source and Level Affects Growth in Neon Tetras, North American Journal
of Aquaculture, 71:4, 320-324, DOI: 10.1577/A08-017.1
To link to this article: http://dx.doi.org/10.1577/A08-017.1
Published online: 09 Jan 2011.
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Dietary Protein Source and Level Affects Growth in Neon Tetras
WENDY M. SEALEY*
University of Idaho, Hagerman Fish Culture Experiment Station,
3059F National Fish Hatchery Road, Hagerman, Idaho 83332, USA
FREDERIC T. BARROWS
U.S. Department of Agriculture, Agricultural Research Service, Trout Grains Project, Hagerman Fish
Culture Experiment Station, 3059F National Fish Hatchery Road, Hagerman, Idaho 83332, USA
MIKE CASTEN AND RONALD W. HARDY
University of Idaho, Hagerman Fish Culture Experiment Station,
3059F National Fish Hatchery Road, Hagerman, Idaho 83332, USA
Abstract.—Nutritional studies for aquarium fishes like the neon tetra Paracheirodon innesi are sparse in
comparison with those for food fish. To determine the optimum dietary protein level and source for growth of
neon tetras, diets were formulated to contain 25, 35, 45, and 55% dietary protein from either marine animal
protein or plant protein sources in a 4 3 2 factorial treatment design. Neon tetras (initial weight, approximately
0.12 g) were reared in 5-L fiberglass tanks (25 fish/tank, 3 tanks/diet) in a freshwater recirculating system.
Fish were hand-fed the experimental diets three times per day for 12 weeks. Average weight gain of neon
tetras fed diets with marine protein sources was significantly higher than that for fish fed diets based on plant
proteins. Fish fed diets containing 45% or 55 % crude protein had significantly greater weight gain than did
fish fed 25% crude protein from either protein source. Fish fed 25% crude protein from either source had a
significantly higher feed conversion ratio than did those fed 45% or 55% crude protein. Survival ranged from
71% to 84% and was not significantly altered by dietary protein source or level. No significant interactions
between dietary protein source and level were found for any of the response variables. As the price of fish
meal continues to increase, the formulations of feeds for food fish will probably contain lower amounts of fish
meal and higher amounts of plant protein products. If a similar trend occurs for ornamental fish diets, further
refinement of nutritional requirements and assessment of palatability of feed ingredients for neon tetras and
other aquarium species will be required.
Keeping an aquarium is a hobby that engages an
estimated 10–20 million enthusiasts, who own more
that 90 million tropical fish with a retail value of
approximately US$1.5 billion (Chapman 2000). Japan
and the USA are the primary markets for aquarium
fishes and account for over half of the world’s
ornamental fish trade (Holt 2000). In a 2000 survey,
the American Pet Products Manufacturers Association
found that a growing number of U.S. households own
fish as pets (12.9% in 2000 versus 9.2% in 1998).
Continued growth of the ornamental fish industry is
anticipated based, in part, on the increasing popularity
of keeping fish as a hobby and the increasing
restrictions on collecting fish from the wild.
The neon tetra Paracheirodon innesi is one of the
most popular ornamental fishes kept in U.S. house-
holds, with an average of 1.8 million neon tetras sold
during a single month in the USA (Chapman et al.
1997). Most neon tetras available in the United States
are imported (Chapman et al. 1998). Imported
ornamental fish are usually in poor health, and losses
of 50–70% are often reported (Conroy 1975); for neon
tetras and cardinal tetras P. axelrodi specifically,
interviews with commercial fish importers indicate
that losses average 80% (Chapman et al. 1998).
Published data on nutrient requirements for orna-
mental fish are limited. Most ornamental fish farmers
buy their feed from commercial manufacturers (Royes
and Chapman 2003). The top-selling commercially
available tropical fish feeds reported by the Florida
Tropical Fish Farmers Association (Wallat et al. 2005)
were Arkat Minnow Meal (Arkat Feeds, Dumas,
Arkansas), BioKyowa Series C-700 (Kyowa Hakko
Kogo, Tokyo, Japan), and Silver Cup Color Enhancing
Blend (Nelson and Sons, Murray, Utah). Manufactured
guaranteed protein analysis for these feeds list protein
levels of 42, 55, and 3 5% protein, respectively.
However, in addition to the wide variations in protein
content, these diets also differ substantially in protein
sources. For these reasons, we conducted a preliminary
* Corresponding author: wsealey@uidaho.edu
Received April 10, 2008; accepted December 18, 2008
Published online July 9, 2009
320
North American Journal of Aquaculture 71:320–324, 2009
Ó Copyright by the American Fisheries Society 2009
DOI: 10.1577/A08-017.1
[Article]
Downloaded by [US Fish & Wildlife Service] at 14:44 25 September 2015
study to assess the effects of dietary protein source and
level on growth performance of neon tetras.
Methods
Experimental approach.—A practical-type aquacul-
ture diet (NRC 1993; Hardy 2002) was formulated to
contain 25, 35, 45, or 55% dietary protein from either
marine animal (marine) or terrestrial plant (plant)
sources in a 4 3 2 factorial design (Table 1). Krill
and squid meals were included in addition to fish meal
for the marine animal protein diets to simulate
premium, commercially available marine ornamental
formulations, while the diets based on plant proteins
included ingredients that are typically used in less-
expensive formulations for freshwater ornamental
species. Diets were manufactured as flake feeds at the
U.S. Fish and Wildlife S ervice’s Bozeman Fish
Technology Center, Bozeman, Montana. Approximate-
ly 800 juvenile neon tetras were obtained from a
commercial source and stocked into eight 38-L tanks at
the University of Idaho’s Hagerman Fish Culture
Experiment Station. Water temperature was maintained
at approximately 258C with aquarium heaters. Fish
were fed a commercially available ornamental fish diet
for 1 week before initiation of the experimental feeding
trial. To begin the trial, fish were counted into groups
of 25 fish each, group weighed, and stocked into
twenty-four 5-L tanks with three replicate tanks per diet
(average 6 SE initial tank weight, 2.92 6 0.3 g).
Tanks were randomly assigned to one of eight
experimental diets. Fish in each tank were hand-fed
all that they would consume in 20 min; such feedings
were administered three times per day, 6 d/week for 12
weeks. A constant photoperiod was followed (14 h of
daylight) using fluorescent lights controlled by a timer.
Fish in the trials were bulk-weighed and counted every
3 weeks, and fish growth rates and feed conversion
ratios (FCRs) were calculated. All fish handling and
experimental protocols were approved by and con-
ducted in accordance with the guidelines of the
University of Idaho’s Animal Care and Use Commit-
tee.
Chemical analyses.—Dry matter and ash analysis of
diets was performed according to standard methods
(AOAC 1995). Cr ude protein (N 3 6.25) was
determined by the Dumas method (AOAC 1995) on
a LECO nitr ogen analyzer (TruSpec N; LECO
Corporation, St. Joseph, Michigan). Lipid was deter-
mined using a Foss Tecator Soxtec Model HT6 solvent
extractor (Foss Tecator, Ho¨gana¨s, Sweden). Total
energy was determined by adiabatic bomb calorimetry
TABLE 1.—Ingredients and proximate composition of eight experimental diets fed to neon tetras during a growth performance
study.
a
Ingredients or
component
Marine protein (%) Plant protein (%)
25 35 45 55 25 35 45 55
Atlantic menhaden
(Brevoortia tyrannus) meal
7.27 14.02 20.76 27.51
Krill meal 8.42 16.24 24.07 31.90
Squid meal 6.61 12.73 18.85 24.98
Soy concentrate 9.27 17.69 26.11 34.54
Wheat gluten 2.21 4.22 6.23 8.24
Rice protein 5.12 9.77 14.43 19.08
Barley concentrate 6.21 11.86 17.50 23.14
Starch 55.68 38.89 20.01 0.91 50.79 30.05 9.31
Wheat flour 13.00 13.00 13.00 13.00 13.00 13.00 13.00 1.56
Fish oil 5.39 3.42 1.61 0.0 7.72 7.95 8.18 8.42
Dicalcium phosphate 1.93 3.98 3.76 3.54 3.32
Vitamin premix
b
0.80 0.80 0.80 0.80 0.80 0.80 0.80 0.80
Choline chloride 0.60 0.60 0.60 0.60 0.60 0.60 0.60 0.60
Ascorbic acid 0.20 0.20 0.20 0.20 0.20 0.20 0.20 0.20
Trace mineral premix
c
0.10 0.10 0.10 0.10 0.10 0.10 0.10 0.10
Analyzed composition
d
Crude protein (%) 19.6 31.8 43.9 56.9 19.0 31.3 43.8 56.2
Lipid (%) 7.5 9.2 10.3 11.6 4.2 5.2 6.0 6.7
Gross energy (kcal/g) 4,668 4,923 5,079 5,264 4,567 4,822 5,031 5,297
Ash (%) 4.5 5.0 7.2 9.2 4.8 5.5 5.8 6.0
Moisture (%) 7.4 6.8 6.4 6.2 7.3 6.6 6.0 5.5
a
Diets were formulated on an as-fed basis.
b
Contributed per kilogram of diet: vitamin A (as retinol palmitate), 10,000 international units (IU); vitamin D
3
, 720 IU; vitamin E (as DL-a-
tocopheryl-acetate), 530 IU; niacin, 330 mg; calcium pantothenate, 160 mg; riboflavin, 80 mg; thiamin mononitrate, 50 mg; pyridoxine
hydrochloride, 45 mg; menadione sodium bisulfate, 25 mg; folacin, 13 mg; biotin, 1 mg; and vitamin B
12
,30lg.
c
Contributed per kilogram of diet: zinc, 37 mg; manganese, 10 mg; iodine, 5 mg; and copper, 3mg.
d
Means of two replicate samples per diet on an as-fed basis.
DIETARY PRO TEIN A LTERS NEON TETRA GROWTH 321
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(Parr 6300; Parr Instrument Company, Inc., Moline,
Illinois).
Statistical analyses.—The MIXED procedure in the
Statistical Analysis System version 7.00 (SAS Institute
1990) was used to conduct a 2 3 4 factorial analysis of
variance for a mixed-effects model (Ott 1977) in which
protein source (marine or plant) and protein level (25,
35, 45, and 55%) were defined as a fixed effects and
tank within treatments was defined as a random effect.
Binomial data were transformed using the arcsine
transformat ion before analysis. Differences among
treatment means were determined using the Tukey’s
procedure for pairwise comparisons. Treatment effects
in all statistical analyses in this project were considered
different when probabilities for a greater F-value were
less than 0.05.
Results
Dietary protein level and source significantly altered
growth performance of neon tetras (Table 2, Figure 1).
TABLE 2.—Growth performance of neon tetras fed experimental diets for 12 weeks. Means of three replicate tanks (25 fish/
tank) are shown. Within columns, values with different letters differ significantly (P 0.05) based on Tukey’s multiple range
test.
Dietary
protein source
Protein
level (%)
Survival
(%)
Weight gain
a
(% increase)
FCR
b
(g feed/g gain)
Marine 25 75 89.9 y 5.48 z
35 81 114.5 yz 4.41 yz
45 68 163.0 z 3.24 y
55 79 162.2 z 2.70 y
Plant 25 77 60.9 y 6.75 z
35 71 109.7 yz 5.24 yz
45 77 108.2 z 4.11 y
55 84 108.1 z 4.23 y
Pooled SE 7.8 22.7 0.67
Analysis of variance P . F
c
0.7027 0.0216 0.0171
Protein source 0.6787 0.0162
Marine . Plant
0.0372
Plant . Marine
Protein level 0.6056 0.0170 0.0046
Source 3 level 0.4878 0.5019 0.9495
a
Percent increase ¼ [(average fish weight initial average fish weight)/initial fish weight] 3 100; 25 fish/tank.
b
FCR ¼ feed conversion ratio (g dry feed/g wet gain).
c
Significance probability associated with the F-statistic.
FIGURE 1.—Effect of dietary protein level (25, 35, 45, or 55%) and source (marine or plant) on average weight of neon tetras
after 3, 6, 9, and 12 weeks of feeding.
322
SEALEY ET AL.
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As early as 6 weeks postfeeding and throughout the
remainder of the 12-week feeding trial, average weight
gain of neon tetras fed diets with marine protein
sources was significantly higher than that of fish fed
the plant-protein-based diets (Figure 1). At the
conclusion of the 12-week trial, fish fed 45% or 55%
crude protein had significantly greater weight gain than
fish fed 25% crude protein from either protein source.
Feed conversion ratio followed the same trend as the
growth data; FCR in fish fed plant-based protein
sources was significantly elevated when compared with
that in fish fed the marine-based protein sources (Table
2). Fish fed 25% crude protein from either source had a
significantly higher FCR than those fed either 45% or
55% crude protein.
Survival ranged from 71% to 84% and was not
significantly altered by dietary protein source or level
(Table 2). No significant interactions between dietary
protein source and level were observed for any of the
examined response variables.
Discussion
Nutrition of ornamental fish is primarily based on
extrapolation of results from research conducted on
food fish species under intensive farming conditions
(Sales and Janssens 2003) as only a limited amount of
research on nutrient requirements of o rnamental
species has been conducted. Of the limited data that
are available, published protein requirements have
varied from 29% dietary protein for growing omniv-
orous goldfish Carassius auratus (Lochmann and
Phillips 1994) to 50% for the carnivorous discus
Symphysodon aequifasciata (Ch ong et al. 2000) .
Results of the present study indicate that neon tetras
performed best when diets contained at least 45% crude
protein, a level closer to the value reported for the
discus. While these levels are considerably higher than
the 29% reported for juvenile goldfish, other research-
ers have observed that goldfish larvae required 53%
crude protein (Fiogbe´ and Kestemont 1995). The
difference in goldfish protein requirements between
these two studies has been attributed to the age of the
fish used; however, the growth rate of the juvenile neon
tetras in this study suggests that the high dietary protein
needs are probably related to species-specific differ-
ences as opposed to ontogenetic stage effects. In
agreement with the results from the present study, other
species, including the tinfoil barb Barbonymus schwa-
nenfeldii (Elangovan and Shim 1997), the redhead
cichlid Vieja synspila (Olvera-Novoa et al. 1996), and
the green swordtail Xiphophorus hellerii (Kruger et al.
2001), have been reported as having protein require-
ments of 41, 41, and 45% crude protein, respectively,
although the protein sources used varied somewhat
among those studies.
Although little research has addressed protein source
preferences of ornamental fish, it is generally recom-
mended that fish meal should be the major protein
source in ornamental fish diets (Francis-Floyd 2002).
The conventional wisdom behind this recommendation
is that fish meal is a highly digestible protein source
with an appropriate amino acid profile. Results from
the current study support this assertion in that fish fed
marine-based protein grew faster and larger than those
fish fed the diets based on plant proteins. Despite the
negative effect of plant protein sources on neon tetra
growth in the present study, Elangovan and Shim
(2000) demonstrated that soybean meal could replace
33% of the fish meal protein in the diet of juvenile
tinfoil barbs without reducing performance. However,
in the present study the plant protein diets were not
supplemented with lysine and methionine, which were
previously shown to be necessary for rainbow trout
Oncorhynchus mykiss fed these ingredients (Barrows et
al. 2007). Thus, amino acid deficiencies may have
contributed to the poor performance of fish fed these
diets. In contrast, Elangovan and Shim (2000) found
that the remaining fish meal in diets fed to tinfoil barbs
provided the necessary levels of these amino acids.
Alternatively, analyzed dietary lipid values differed
somewhat from the targeted 10% level; thus, we cannot
rule out the possibility that the reduced lipid content of
the 45% and 55% plant protein diets could have
contributed to observed results.
A further limitation of the present study is the
potential differences in palatability of diets formulated
with plant-based ingredients versus marine protein
sources. A number of studies have shown that
carnivorous fish species may find plant-based ingredi-
ents unpalatable; therefore, consumption and growth
rates will be suboptimal even if the diets are
nutritionally complete. We attempted to minimize the
palatability concerns by using refined plant ingredients
that have been demonstrated as palatable for rainbow
trout (Gaylord et al. 2006), but palatability was not
addressed directly in the present trial. Also, the feeding
strategy employed (using flake feeds) precluded
feeding to apparent satiation; therefore, excess feed
was given, as indicated by the high FCR.
From this experiment, it appears that approximately
45% crude protein from marine sources will support
good growth of neon tetras. As the price of marine
proteins, including fish,krill,andsquidmeals,
continues to increase, the composition of aquaculture
feeds will change, with decreased amounts of marine
proteins and increased amounts of protein products
from plants. If a similar trend occurs for ornamental
DIETARY PRO TEIN A LTERS NEON TETRA GROWTH 323
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fish diets, development of plant-b ased feeds f or
ornamental fishes will require further evaluation of
nutritional requirements and assessment of feed
palatability.
Acknowledgments
We thank the staff at the Hagerman Fish Culture
Experiment Station for their contributions. Funding for
the study was provided, in part, by the Morris Animal
Foundation. Mention of trade names or commercial
products in this article is solely for the purpose of
providing specific information and does not imply
recommendation by the U.S. Department of Agricul-
ture or the University of Idaho.
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