Effect of Dietary
Phaffia rhodozyma on Atlantic Salmon Performance
June 22, 1998
A Final Report Provided
to Igene Biotechnology, Inc And Maryland Industrial Partnerships
Submitted by:
Reginal M. Harrell,
University of Maryland Center for Environmental Science, Horn
Point Laboratory, Steven G. Hughes, University of Maryland Eastern
Shore, Renate Reimschuessel, Aquatic Pathobiology Center University
of Maryland, Baltimore
Background
The Atlantic salmon
(Salmo salar) aquaculture industry is one of the fastest growing
segments of fish farming on a global basis, and one factor that
determines consumer acceptance of a farm-raised product is its
outward flesh appearance. Atlantic salmon naturally have a pinkish-colored
flesh that is imparted by the consumption of crustaceans and other
organisms rich in a carotenoid compounds. Farm-raised salmon have
historically been pale in color due to the lack of the color enhancement
pigments found in artificially formulated diets. To date, the
color enhancement pigment astaxanthin (the pigment primarily responsible
for pink flesh coloration) has been approved by the U.S. Food
and Drug Administration (FDA) to be incorporated into salmon diets.
However, the only commercially available source to date is a synthetic
compound produced by Hoffmann-LaRouche, a German based company,
which supplies about 95% of the global demand.
Carotenoids have long
been recognized as holding important nutritional and physiological
roles in aquatic animals (reviewed by Torrissen 1989). Among the
many roles accredited to these compounds are those of vitamin
A precursor, components of chromatophores, immunological enhancers,
visual pigments, and as antioxidants. These pigments have also
been shown to have a significant impact on the sensory qualities
(particularly color) of seafood in the marketplace (Simpson 1982).
The distinctive red color attributed to astaxanthin is particularly
important to the marketability of certain salmonids, particularly
Atlantic salmon (Torrissen et. al. 1989).
Many dietary sources
of carotenoids have been researched for salmonids (Torrissen et
al. 1989; Meyers and Chen 1983), but most of the natural sources
have proven too expensive for practical incorporation into cost-effective
feeds. Research has indicated, however, that certain strains of
the yeast Phaffia rhodozyma can contain very high concentrations
(up to 1% of dry matter) of astaxanthin isomers (Johnson et al.
1978; Haard 1988, An et al. 1989), which are readily available
to fish (Johnson et al. 1980, Okagube and Lewis 1983, Gentles
and Haard 1991). Recent advances have made the production of astaxanthin
from this source economically comparable to the use of synthetic
astaxanthins, but with the added value of incorporating additional
nitrogen into the fishes diet.
A Maryland based company,
Igene Biotechnology, Inc. (Columbia, MD) has perfected a yeast,
Phaffia rhodozyma, fermentation process whereby the pigment can
be naturally produced in commercial quantities that should be
more appealing to consumers. The company has an enzyme that cracks
the cell wall of the yeast making the pigment more bioavailable
to the salmon. Their process and patented approach will permit
more efficient colorization of the fish flesh at a lower concentration
in the feed. The significance of this project is related to the
global market for salmon feed, which is approximately $150 million
annually. To date, over 90% of the pigment used to enhance flesh
color is purchased by a handful of feed manufacturers. These customers
are EWOS, TROUW, Aquaculture, Fulmar, and Moore and Clark.
Unfortunately, while
FDA has approved the use of astaxanthin as a feed additive in
salmon diets, they have not approved the use of yeast-laced sources
of astaxanthin. Thus for Igene to be competitive, they must prove
to FDA that their compound is safe to the fish. Preliminary studies
by Igene have already demonstrated the efficacy of the yeast product
to impart an acceptable coloration in salmon flesh. This project
was devoted to evaluating the effect naturally-derived astaxanthin
had on on-grown Atlantic salmon as a food additive, and consisted
of an 18 month effort that concentrated on feeding fish differing
levels of astaxanthin from the juvenile (ca 100g) through market-sized
(ca 700g) fish.
Objective
The project had two
objectives: 1) to evaluate the growth and performance of Atlantic
salmon fed varying levels of dietary astaxanthin, and 2) to determine
the effect of dietary astaxanthin on the well-being of the fish.
Methods
Fish Collection - Atlantic
salmon utilized in this study were obtained from a single brood
spawned at the Craig Brook Hatchery in Penobscot (ATS-95-P-CB-S).
On October 24, 1996, at 20-40 grams, fish were transported from
Pennsylvania to the University System of Maryland Center for Environmental
Sciences Horn Point Laboratory (HPL) and maintained in 800 liter
circular tanks on fresh water and fed a commercial salmon diet
(Perdue, ASD). In February 1997, 408 salmon were randomly selected
for experimental studies in HPL's recirculating system. Diet Formulation
and Feeding- The basal (control) diet for this study was a modified
standard fish meal and soy ration (U.S. Fish and Wildlife Service
open-formula salmon diet ASD2-30; Table 1). The modification requires
the elimination of shrimp meal so as to avoid any uncontrolled
addition of astaxanthin in the ingredients. Three experimental
diets were formulated by adding varying levels of dried Phaffia
rhodozyma to the control formulation. This particular strain of
yeast when dried generally contains 4000-5000 ppm. of total carotenoids
of which 70% is astaxanthin. Based on a proposed titer of approximately
10,000 ppm of astaxanthin in the dried yeast product (analyzed
values given by IGENE Biotechnology, Inc.) the experimental diets
were formulated to contain approximately 80, 400, and 800 ppm
yeast astaxanthin (which represents 1, 5, and 10 times the anticipated
levels to be used in commercial feeds) by substituting 0.8, 4,
and 8% yeast into the diet at the expense of soybean meal. Though
the 8% level of inclusion would lead to excessively high levels
of dietary nucleic acids for most monogastric species, data for
other salmonids indicated that there should not be any nutritional
problems with this formulation (Rumsey et al. 1991 a,b).
All diets were mixed
and pelleted using standard cold extrusion practices (e.g., Rumsey
and Ketola 1975) at the University of Maryland Eastern Shore using
a California Pellet Mill CPL-3 Laboratory Scale Pellet Mill. All
diet ingredients were obtained from Perdue Specialty Feeds, Catawissa,
PA, and were kept frozen at -12ˇC until the day of use. The ingredients
were mixed in the bowl of a Hobart mixer for two minutes at speed
I and water (15% w/w) was added to facilitate the pelleting process.
This mixture was then mixed for at least 10 minutes at speed 2
to insure adequate distribution of all components throughout the
mixture. The pelleted feeds were then placed in a drying oven
for 1.5 hours at 50ˇC to reduce the final moisture content below
12%. Each diet was mixed in 10 kg batches as needed, delivered
to HPL within 24 hours of pelleting, and kept frozen (-2ˇC) to
maintain freshness and to minimize any negative effects of storage
on the completed diets.
Feeding rates were
determined using a US Fish and Wildlife software program, which
allows for incremental increase in feeding rates as the fish grew.
During the experiment, the amount of food fed was initially based
on a hatchery constant of 5.5, calculated according to the method
of Buterbaugh and Willoughby (1967). However, these suggested
constants were insufficient for optimal growth and thus were increased
during the course of the study period.
HPL's Recirculating
System- The water-reuse system at HPL contains 10,000 gallons
of water, which is recirculated 16 times daily. Makeup water averaged
approximately 7% per day with water coming from a local freshwater
well. Filtration was accomplished by a Hydrotech¨ drum filter,
and a Bio-Ball¨ down flow biofilter, with a final polish through
two rapid-sand filters before returning to the production tanks.
All experimental fish
were grown in twelve 605 liter (160 gallon) semi-square polytanks.
Temperature was maintained at 16 - 18 'C by separate titanium
heat exchangers and chiller units controlled by pneumatic mixing
valves. Each mixing valve controlled four tanks (one block), although
temperature was maintained equal in all tanks. Oxygen was supplied
to each tank separately by an air blower and diffusers. In addition,
critical factors (i.e., oxygen pressure, temperature, water level,
and power) were remotely monitored by a Sense phone alarming and
automatic dialing unit.
Experimental Design
- Fish were stocked at an initial density of 34 fish per tank
with treatments randomly assigned within each block. Treatments
for this study consisted of the four diets (control and the three
differing levels of astaxanthin). All diets were stored at -2
'C until fed and administered via automatic feeders approximately
8 times per day.
Total population measurements
were taken monthly for weight and length determination and feeding
rates were adjusted accordingly. At scheduled intervals, five
fish per tank were removed for histological evaluation. This work
was performed by the University of Maryland, Baltimore. The experiment
was terminated after one year of growth when fish averaged over
900 grams (Table 2). Production variables included comparative
growth in terms of weight and length, food conversion, and condition
factor.
Statistical Analyses-
Although temperature among the three blocks was maintained at
the same level, both block and treatment effects were analyzed
using a two-way ANOVA (Proc GLM, SAS V. 6.07) for ending weight,
length, condition factor, and food conversion. Least square means
procedures were utilized for further examination of treatment
differences at the 0.05 level of significance. Starting weights
and lengths were examined with a simple ANOVA since blocks were
of no consequence at this time.
Tissue and Organ Analysis-
A total of 245 fish were examined by Dr. Renate Reimschuessel
of UMAB for gross abnormalities and histological analysis. Five
fish were taken at random from the general population before the
feeding trials begin and 60 fish each quarter (3, 6, 9, and 12months
into the study) of the study duration thereafter. Each quarter
sample included five fish from each treatment including the control.
Fish were delivered live to the University of Maryland, Baltimore.
Necropsy exams were
performed immediately after bleeding. Any abnormalities were noted
on the gross necropsy sheets. Histological examinations were made
on three organs, liver, kidney, and lower intestines. Liver, posterior
intestine, and kidney sections were placed into cassettes and
submerged into 10% phosphate buffered formalin. Any gross lesions
were also sampled and placed into separate cassettes or containers.
Muscle sections were saved from each fish and have been submitted
to IGENE for color comparison determinations against industry
standards. Photographs of muscle were taken. For histological
analyses, 6 µm sections were stained with hematoxylin and eosin.
These sections were examined by Dr. Reimschuessel for lesions
and the amount of liver glycogen/fat was rated on a scale of 1-5.
All the slides were read and. data placed onto data sheets. Data
was transferred to an Excel spreadsheet and statistical analysis
was performed using Statistix for Windows (Analytical Software,
Tallahassee, FL).
Reporting Format- Each
fish was given a unique identification accession number. This
number was placed on all record sheets, sample containers, paraffin
blocks, and slides. All data regarding the fish, normal and abnormal,
is contained in this final report. Original handwritten data sheets,
blocks and slides will be archived at the Aquatic Pathobiology
Center for five years.
Results
Water Quality - Daily
water quality measurements (Table 3) were taken for oxygen and
temperature in each tank using a YSl Model 55 oxygen meter (Yellow
Springs Instruments, Yellow Springs, 0.1-1 % accuracy ± 0.3 mg/L).
Dissolved oxygen concentrations were maintained at 10 mg/L with
three exceptions in April 1997 where D.O. dropped below 9 mg/L.
Ammonia nitrogen, nitrite-nitrogen, and pH were monitored daily
with a LaMotte Smart Colorimeter (LaMotte Company, Chestertown,
MD USA, stability ± 0.2%, Wavelength Accuracy ± 1 nanometer, Photometric
Accuracy 0.5 % ). Mean concentrations of ionized ammonia were
0.23 ± .011 mg/L, nitrite = 0.09 ± .004 mg/l, and pH = 8.25 ±.007.
Nitrate was monitored weekly colorimetrically and averaged 8.15
mg/l. Weekly hardness and alkalinity measurements were determined
with LaMotte kits. A YSI Model 30 salinity/conductivity meter
was used to monitor salinity (Yellow Springs Instruments, Yellow
Springs, OH accuracy ± 0. 1 ppt.). Mean salinity was 0.74 ± 0.189
(SD) ppt, total calcium hardness = 202.82 ± 76.42 (SD) mg/l, and
alkalinity = 228.88 ± 29.34 (SD) mg/L. Calcium chloride was added
to the system whenever total hardness fell below 150 mg/L.
Growth - Mean starting
weight was 115.05 grams and was not significantly different among
treatments (P = 0.95). Growth to 900 grams was similar among all
treatments (Figure 1) during the 12 month growth period and responded
similarly in all treatments to changes in the hatchery constant.
Ending weights were not significantly different (P = 0.96) suggesting
that varying dietary levels of astaxanthin have no effect on Atlantic
salmon weight gain. Starting and ending lengths were not significantly
different (p = 0.99 and 0.57) and averaged 23.45 ± 0.04 cm and
399.36 ± 2.79 cm respectively.
Condition factor -
Beginning condition factor was not significantly different among
treatments or blocks (p = 0.10 trt and 0.79 blk) and averaged
0.867 ± 0.002. Ending condition factor averaged 1.41 ± 0.02 and
was not significantly different among treatments (p = 0.97, Figure
2) but was among blocks (p = 0.004). Block one condition factor
was lower in all treatments than the others (Figure 3). It is
unclear whether this is an artifact of random removal of fish
for histopathology, the fact that this block was closest in proximity
to where daily movement for water quality analyses and weighing
feed rations was conducted, thereby instituting a constant stressor
of people movement, or a true block effect. In either case it
was not biologically significant with regard to growth.
Food Conversion - Food
conversion was not significantly different among treatments (p
= 0.68) with no block effect (p = 0.72). Overall, mean food conversion
was 1.81 ± 0.04 and generally increased with fish size. Detailed
information on mean weight gain, rations fed, and food conversion
are presented in Table 4.
Histopathology Analyses
- This component of the study was to determine if there were any
effects of dietary Phaffia rhodozyma on hematological parameters
such as pcv, total protein and red and white blood cell morphology,
gross physical alterations, or histopathologic alterations in
liver, kidney or posterior gut.
Gross lesions:
A number of gross lesions
were noted in the fish. No lesions were specific to any particular
treatment group. The most commonly encountered lesions were loss
of fins - Fig. 4,
The second most common
lesion was injury to the eye, usually the left eye - Fig. 6.
Both of these lesions
are most likely due to trauma incurred in tanks and during handling.
Other lesions were
nodules on or necrosis of the gills - Fig. 7. During the final
study period nodules were noted on liver, spleen, kidney or testis
of a small number of fish (including controls).
Muscle sections from
control fish were all quite pale. As the fish grew the muscle
from the treatment groups became pink, especially those in the
400 and 800 groups. By the fourth sampling period all the treatment
groups were noticeably more pink than controls. Fig. 8. Even the
gross appearance of these fish (through the skin) was much pinker,
with fins appearing pinkish also. Figs. 4 and 5.
Hematology:
Blood Counts and PCV/TP
- no significant differences were seen in the blood counts or
PCV. Total protein means ranged from 7.8 to 7.4, with group 400
being significantly different from 80 and control. This range
in total protein, although statistically significant, is not clinically
relevant. See enclosed statistical analysis for summaries of the
means in each group. Representative photomicrographs have been
submitted previously to demonstrate typical cell morphology.
Histopathology Lesions:
Liver:
Inflammatory changes
in the liver were the most predominant lesions. During the two
early time periods these lesions consisted of small, multifocal
areas of lymphocytic and macrophagic infiltrates. During the last
time periods the inflammatory lesions contained giant cells and
granuloma surrounded by fibrous connective tissue. Fig. 9. There
were no parasitic or infectious organisms visible in these lesions
by light microscopy. In several fish, the fibrous connective tissue
proliferated to a marked degree, causing grossly visible lesions.
Figure 4. Gross - Body
5-97- F 197 800 period 4
Figure 5. Gross - Body
5-97- F 187 0 period 4
Figure 6. Gross - Eye
5-97- F 235 0 period 4
Figure 7. Gross - Gills
5-97- F 186 0 period 4
Figure 8. Gross -
Muscle 5-97- F 186 0 period 4
5-97- F 229 80 period
4
5-97- F 236 400 period
4
5-97- F 199 800 period
4
Figure 9. Histo - Liver
5-97- F 183 0 period 3 The liver glycogen within the hepatocytes
was graded on a scale of 1-5, with 1= minimal amount, 2 = mild,
3 = moderate, 4 = marked, 5 = severe.
Kidney:
Inflammatory lesions
were also the most commonly observed lesions in the kidneys. The
type of inflammation was similar to what was seen in the liver.
Rarely, lesions such as hyline droplets within tubules or mesangial
thickening were observed. These occurred only in a handful of
fish, and were present in control as well as treated fish.
New nephron development
was coded on a scale of 1-5. New nephrons develop in rapidly growing
fish as the kidney grows. Their increase can also indicate previous
renal tubular damage. There was no significant difference between
control and treated new nephron incidence.
There were very few
lesions noted in the intestines. Several foci of calcification
found (3 fish) and one focus of inflammation.
Other organs:
Eyes:
The
eye lesions consisted of inflammation and scar tissue. These histologic
changes were consistent with repair of trauma induced injury.
Gills:
Gills samples were
included in randomly chosen fish from different tanks to help
assess water quality throughout the experiment. Almost all gills
were in excellent condition. Five fish had grossly visible gill
lesions. Histologic changes in these gills were either necrosis,
granulomata or chronic inflammatory changes. These lesions were
present in treated and control animals. Figs. 10 and 11.
Spleen:
Inflammatory lesions
were observed in the spleens of several fish. The type of inflammation
was similar to what was seen in the liver and kidney.
Figure 10.
Histo -- Gills 5-97-F136
800 period 3
Histo --Gills 5-97-
F 136 800 period 3 high mag.
Summary
A detailed spreadsheet
itemizing the hematology, lesions and severity is appended. Extra
organs that were periodically examined are listed in comments
section, as are descriptions of the lesions.
There were no lesions
identified in the treated animals, which did not occur in a similar
frequency or severity in the controls. The cause of the granulomatous
inflammation has not been identified. Acid fast stains and silver
stains have identified no organisms in these lesions, although
some type of infectious agent is quite likely. Further investigation
into the etiology of these lesions is beyond the scope of this
study.
Conclusions
It appears from the
growth and histopathological data that increased levels of Phaffia-laced
yeast incorporated in the diet of Atlantic salmon up to 10 times
the amount projected to be required to elicit a flesh color change
did not have a negative impact. While individual fish had certain
lesions or growth differences present no one group or replicate,
as a whole, were negatively affected in comparison to the controls.
With the exception of one block of fish having a lower ending
condition factor, which did not appear to be biologically significant,
there were no other aberrations from the control fish performance
noted.
All original data sheets
and information is archived at either Horn Point Laboratory or
the Aquatic Pathobiology Center and are available for examination
if requested.
