Food Requirements and Lipid Accumulation in Captive Yellowfin
Tuna: Thunnus albacares
Bianca Perla, May 1995
ABSTRAC
Through the months of December 1994-March 1995 captive yellowfin tuna kept at the Tuna
Research and Conservation Center in Monterey, CA. displayed behavioral and physiological signs
of high whole body lipid levels. Autopsies revealed lipid accumulation in the heart, gills and
muscles, and 4 sudden deaths occurred that may have been due to cardiac arrest or lipid
accumulation in the blood. The fish acted sluggish and over-satiated during feedings.
Possible high lipid levels in the diet were thought to be a contributing factor to increased lipid
storage in the tissue. To test this hypothesis, diet was cut for the months of April and May from 2
times a day every day to once a day every other day. Individual tuna in each tank were followed
during the feedings and data were recorded on type of food eaten, amount of food eaten and
activity level before and during feeding. Food amounts obtained from these observations were
analyzed for fat content and total caloric content. Old and new diets were compared on this basis.
In addition histological techniques were used to compare fat levels in red and white muscle
tissue of TRCC tuna-fed a diet of squid, anchovies and sardines, with Kewalo basin captive tuna
- fed a diet of 100% squid.
Activity of the tuna on the reduced diet increased dramatically. Analysis of fat content and
caloric content of both old and new diets revealed that a reduction in total calories as well as a
reduction in fat content on the new diet may have contributed to the increased activity and overall
health of the TRCC tuna. It was also found that it may be possible to decrease total fat content of
the diet while keeping total caloric intake from falling. Histological staining of tissues revealed
that TRCC tuna contain higher levels of lipid, than Kewalo basin tuna, in both red and white
muscle tissues.
FOOD REQUIREMENTS AND LIPID ACCUMULATION IN CAPTIVE
YELLOWFIN TUNA : Thunnus albacares
Bianca Perla May, 1995
INTRODUCTION
The Tuna Research and Conservation Center (TRCC) at Hopkins Marine Station in Monterey
California houses one of the few captive yellowfin tuna populations in the world. The tuna are
separated by size and age into three tanks. Tank 1 (T1), the largest tank, is 89,000 gallons and
contains 2-3 year old yellowfin tuna that have been in captivity for 1.7 years. Tanks two and three
(T2, T3) are both 29,000 gallons and hold yellowfin tuna from 1-2 years of age that have been in
captivity 8 months.
Recently, TRCC tuna have shown signs of high lipid levels. Autopsies on mortalities during
the months of December through March revealed evidence for excess lipid deposits around the
heart and gills and in the muscles of TRCC tuna (Block Lab pers. comm., pers. obs.). Four of
these mortalities were sudden deaths suggestive of cardiac arrest. All deaths from heart attacks
occurred in TI which contains the fish who have been in captivity the longest. Cardiac arrest is
commonly caused by high lipid levels.
Three hypotheses for the cause of these high lipid levels have come to the forefront. One is
cool temperature. Water temperatures in all tanks range from 19.5-21 degrees Celsius. Wild
vellowfin tuna are usually found in waters around 25 degrees Celsius (Sund et al., 1981). Low
temperature slows down metabolism and hence has the capacity to increase fat storage. Secondly,
TRCC tuna may have a lower activity level than wild yellowfin tuna. Lack of activity decreases the
amount of energy metabolized and may lead to increased lipid storage. This study concentrates on
the third possible contributor to high fat levels- diet. During February and March TRCC fish acted
sluggish and over-satiated at feedings. Diet is the source of all energy used for metabolic
processes. Presumably by controlling input of fat through the diet one can control the amount of
lipid available to store in the first place.
Food requirements, food preferences and total energy budget have been studied for many
species of wild tunas (Graham and Laurs 1982, Blackburn 1968, Olson and Boggs 1986). King
and Ikehara (1956) studied the food preferences of wild yellowfin tuna in the Pacific and
concluded that squid and fish were the major components of the diet while crustaceans and
molluscs were secondary. Olson and Boggs (1986) concurred that fish were the primary source of
food for yellowfin where as cephalopods were secondary. Estimates of daily food requirements
of wild tunas have been derived from analysis of stomach contents, and models estimating
metabolic rate. These estimates range widely from as low as 3% to as high as 30% of body weight
per day (Sund et al. 1981). There is little consensus of optimum dietary requirements of wild
tunas and even less is known about how these food requirements change in captivity.
Research conducted on other species of captive fish, like salmon (Shearer 1993), and the
Caribbean Jack (Walters et al. 1995) indicate that fish in captivity, fed commercial pelleted diets,
contained higher levels of whole body lipid than their wild equivalents. Davies (1989) found that
increasing dietary lipid in Rainbow trout increased whole body lipid stores. Olson and Boggs
(1986) showed that prey higher in fat was evacuated from wild yellowfin tuna stomachs at a much
slower rate than prey containing less fat. Lipid is a high energy food component-containing 9.4
dietary calories per gram as opposed to carbohydrates and protein which contain approximately 4
calories for 1 g (Schmidt-Nielson 1990). Accordingly, it takes longer to metabolize fat than other
food components such as carbohydrates and protein.
A diet high in lipid could be contributing to the high lipid levels and sluggish behavior of the
TRCC tuna. By monitoring how much and what types of food TRCC captive tuna eat, total
calories consumed and fat calories consumed can be calculated. Furthermore, histological
techniques can be used to compare fat levels of Kewalo basin captive tuna (fed a 100% squid diet
and kept at water temperatures of 25 degrees Celsius) and TRCC tuna (fed a diet of squid,
anchovies, and sardines and kept at 19.5-21 degrees Celsius) to see how diet affects fat levels.
MATERIALS AND METHODS
Feeding Observation and Analysis
To study the effects of a reduced diet on the health and behavior of the fish, feedings were cut
from twice a day everyday to once a day every other day. Composition of the diet stayed the same-
squid, sardines, anchovies. However, anchovies were cut out in May. Samples of squid,
sardines, and anchovies were weighed three times throughout the months of April and May and
separated into large and small categories to reduce standard deviation around the mean weight.
Four specimens from each large and small category of food items (sardines, squid and anchovies)
were sent to Michelson laboratories for bomb calorimetery tests which gave caloric content, percent
fat, percent protein and percent carbohydrate (see Table 1).
Before each feeding food was seperated by type and by size category. During each feeding
individual fish were watched and the quantity and type of food that they ate was recorded.
Morphological factors (like distinctive shapes of the fins) were used for identification. The tuna
Tip (Tank 1), RN (Tank 2), and Notch (Tank 3) were followed in a previous study from December
1994-March 1995 (Marcinek, unpublished). I continued following these fish so I could compare
the old diet with the new reduced diet.
Behavior and level of activity during each feeding was noted for the tank and for the individuals
followed. Lunging for the food, competing with other fish for food and anticipatory behavior
before feeding were noted when they occurred.
Data for December 1994-March 1995 (Marcinek-unpublished) and data collected from the new
diet (April 21-June 2 1995) were used to calculate total caloric content, percent fat and fat calories
eaten per day. Caloric content and fat content of foods, determined by the bomb calorimetery tests,
were used in calculations. Mean weights found at the beginning of the study were used as well
(Table 2). The Monterey Bay Aquarium provided data on the amount of food fed to each tank as a
whole from the months of December through May (Chuck Farwell, pers. comm.). This data was
analyzed to calculate an expected amount of calories per fish per day. Expected values were
obtained by dividing the total amount fed to the tank per day by the number of fish in each tank.
These values were compared to observed values to see if individual feeding habits could be
extrapolated to other fish in the tank.
Histology
To discover if lipid levels in TRCC tuna were indeed high, red and white muscle tissue samples
of TRCC tuna, wild tuna, and Kewalo basin captive tuna were qualitatively analyzed for lipid
content. Histology for the Kewalo basin tuna and for the TRCC captive tuna was done by Bay
Histology. The stain used was hematoxylin which stained muscle cells pink and left lipid white.
Muscle tissues of wild tuna and TRCC tuna were also stained for intra-cellular lipid. Samples
of red and white muscle, fixed in 10% formalin were frozen, cut at -20 degrees Celsius on a
cryostat, and stained for lipid with Oil Red O (Bancroft, 1975). TRCC tuna tissue broke easily
when cut. To increase strength of the tissue it was cryoprotected by soaking it in SM and IM
sucrose solutions prior to freeze clamping them. Because of the high lipid content, TRCC tuna
tissue did not adhere to the slides well and fell off during the staining process. To combat this
problem I used Cetyl-Acetylene glue to stick the tissue to the slide for staining (Perla, pers.
comm.).
Äfter staining, the slides were photographed with a zeiss axioplot microscope and lipid contents
of each test tissue were qualitatively compared.
Results
Diet
Bomb calorimetery tests revealed that anchovies had the highest percent lipid of all the food
eaten (Table 1). Mean weights for small and large food items stayed constant throughout the
study (Table 2).
Caloric intake per day on the new diet decreased to 75% for TI, and T2 between December and
May. T3 caloric intake decrease by about 25% between December and May (see figure 1). T-tests
revealed that there was a significant difference between caloric content of the old and new diets (TI
p—.0048, T2 p-2.7e-8, T3 p-.000108).
As shown in figures 2,3,and 4, fat calories eaten per day decreased significantly for all tanks
from the old to the new diet (T1 p-.0024, T2 p-.013, T3 p-3.83e-6). Between April and May
calories increased slightly for TI and T2 and stayed constant for T3 while fat calories decreased in
all tanks. The decrease in fat calories was only significant for T2 (p-3.83e-4) yet it was a trend for
all tanks. While percent fat did not seem to be greatly affected by the amount of squid and
sardines eaten per feeding (Figures 5,6,7) percent fat eaten per feeding did correlate closely with
percent anchovies eaten per feeding for each individual fish observed (see Figures 8,9,10).
In TI observed calories eaten per day for Tip were not significantly different than the
calories/day expected for both old and new diets (old diet p-.68, new diet p-.48). T2 observed
and expected values were significantly different in March (p-.02) and April (2.8e-8) but not for the
other months observed (Feb p-.280, May p=.81). The observed values for Notch in T3 were not
significantly different than expected values for the old diet (p-.287) but were significantly different
for the months of April and May (p-.005).
Behavior
Behavior during feedings is drastically different on the new diet. Fish activity increased
dramatically. For example, fish surge and sprint toward the food thrown into the tank. They even
leap out of the water at times and make sharp turns to orient themselves toward the food source.
Now the tunas compete for food. Food is swiped from the mouths of some individuals by others
and nipping at another tuna was observed once in T2 (May 25 1995). Fish show what appears to
be anticipatory behavior before feeding in all tanks- especially Tank 1. Fish congregate toward the
feeding platform whenever some one stands on it.
Histology
Qualitative analysis revealed that TRCC tuna red and white muscle tissues contained more lipid
between muscle fibers than Kewalo basin tuna tissues (see figures 11- 14). As shown in figures
11-14, the hematoxylin stain used by Bay Histology for the Kewalo basin tuna and the TRCC
captive tuna, stained muscle fibers dark pink or red and left lipid unstained (white). The lipid
apparent in these stains was extracellular lipid- lipid between muscle cells. Ösmium stains and Oil
Red O stains indicated intracellular lipid as small circular droplets within individual muscle cells.
An osmium stain, by Bay Histology, revealed possible high levels of intracellular lipid in TRCC
captive tuna red muscle. However, Oil Red O stains for intracellular lipid in wild and TRCC
specimens were not perfected to a level appropriate for comparison.
Discussion
Diet
Switching to a diet significantly lower in fat created positive changes in tuna behavior and
health: activity is high and no deaths from cardiac arrest have occurred since the new diet.
However, since this was correlated with a significant decrease in total calories on the new diet,
decrease in total energy intake must not be ignored as contributing to these behavorial changes.
Since the fish were acting over- satiated and sluggish at feedings, and are no longer doing so after
a cut in total calories and fat calories, it seems strongly possible that both the total calories and fat
calories on the old diet were too high.
Judging from the trend in all tanks between April and May decreasing calories from fat without
decreasing total calories consumed is possible to some extent. This was accomplished by
eliminating high fat foods, specifically anchovies, from the diet. If total fat content can be
decreased with out decreasing total calories consumed enough energy can be provided to the tuna
with out having to feed high energy, fatty foods that are more easily stored as lipid in the tissues.
Although the decrease in fat calories between April and May was only statistically significant for
12 the fact that it was present in all tanks suggests that it is possible to maintain a steady caloric
intake while decreasing fat. Feeding a diet of 100 percent squid may reduce fat calories while
maintaining a reasonable total caloric intake.
Because expected and observed values were determined to be similar in TI for all months, data
from one fish in TI can be extrapolated to the whole tank. Two exceptions to this could be the
smallest yellowfin in the tank and one skipjack tuna. Data are currently being taken on the smallest
yellowfin-in TI, to discover if observations on Tip can be applied. T2 and T3 expected and
observed values were less consistent. The lack of consistency with expected values could have to
do with age, or size. Perhaps younger yellowfin tuna do not eat as consistently as older yellowfin.
Because the fish are younger, there is a greater size range in T2 and T3. The bigger fish may take
more than their share. A recent mortality in T3 (May 21) supports this assumption. The fish that
died was fairly large compared to the others in the tank and stomach contents from its last feeding
revealed that the tuna had eaten two more sardines than the expected value. This points to the need
to increase the number of individual fish followed at each feeding- especially for T2 and T3.
Observations on another set of fish has already been started but their data were not included
because they were not followed for long enough to make accurate comparisons.
This study succeeded in determining that caloric levels and fat levels were too high on the old
diet. However, this was a qualitative analysis of caloric intake and fat content. Is the new diet too
much of a decrease or not enough? Continuing observations on the new diet and testing tissues of
future mortalities will reveal how efficient the diet change has been on reducing lipid levels. For
even more accuracy, the data from this study can be used, along with growth curves, to develop a
mass specific measurement for what tuna eat. By coupling this mass specific food measurement
with data on total energy expenditure an overall energy budget for tuna can be developed. This
overall energy budget can be used to find the optimum, maximum, and minimum amount of
calories and fat that the TRCC tuna diet should contain.
Histology
Red muscle in tunas is used for aerobic activity whereas white muscle is used for powering
sprinting behaviors. White muscle usually contains less fat than red muscle tissue, however,
staining revealed that TRCC tuna have high levels of lipid in their white muscle indicating
accumulation. One possible explanation is that TRCC captive tuna are not using their white muscle
as much. Possibly the increased sprinting and leaping behavior seen recently during feedings
could exercise the white muscle more and lead to a decrease in lipid storage.
Results showed that lipid content in TRCC tuna tissue was higher, in both red and white
muscle, than captive tuna fed lower fat diets. Are there any other factors contributing to this
difference? The effect of temperature should be studied as another factor in lipid accumulation. At
lower temperatures metabolic processes decrease and lipid is burn more slowly (Shearer, Karl D.
1993). Kewalo basin tuna were kept at 25 degrees C where as TRCC tuna are kept at 19.5-21
degrees C. This could be another reason besides diet that TRCC tuna have higher fat levels than
Kewalo basin tuna.
Quantitative analysis of intra-cellular stains can improve accuracy in comparing fat content of
muscle tissues. By over-lying a grid system on the prints of stained tissue, individual lipid
droplets can be counted and a percentage of fat accumulation in the tissue can be determined.
Counting the actual lipid droplets would provide a more accurate basis for comparison of tissues
than the qualitative estimation performed in this study. Oil Red O will be a valuable stain to use to
determine the percentage of intracellular lipid, and to compare wild and captive tuna lipid levels but
problems of over-staining must be overcome before Oil Red O can be used with confidence.
There is little information of great accuracy available on food requirements, on metabolic rate
and on many other physiological and behavioral aspects of yellowfin tuna because the open ocean
environment makes tuna difficult to follow and observe for long periods of time. Even less is
known about the effects of captivity; for example, how diet changes in captivity as compared to
the wild. Open ocean fish by nature, it is believed that tuna spend much time in low productivity
environments. As a consequence they may go without food for long periods of time and then
gorge when they come to an oasis of food in the ocean or when they reach higher productivity
areas near shore (Sund et al. 1981). Olson and Boggs (1986) found that Yellowfin tuna fed to
satiation ate more than most other species of fish. They suggested that this could be because of
their feast and famine life style; when tunas swim into a feeding area they may need to take full
advantage of the food source as fast as possible before they move on to the sparse habitat of the
Pacific Ocean basin. No data could be found on the periodicity of this fasting and gorging lifestyle
in the wild. Is this habit of feeding constant year round or does it only coincide with long
migrations? This problem of feeding to satiation is not encountered by the tuna in captivity. No
one knows the effect, if any that this has. Much more research on the comparison of wild and
captive tuna must be done.
Studying tuna in captivity allows research to be conducted that is not feasible in the field. For
example, observing the amount and type of food individuals ate at every feeding provided data on
the feeding habits of yellowfin tuna for a longer period of time than would be possible in a field
experiment. This data can be used, along with measurements of activity to develop a mass specific
energy budget for tuna. Through individual observation it is possible to monitor the exact amount
of food that a tuna eats. By converting the total amount eaten per day into measurements of
calories and fat, models can be made that determine how a change in diet will affect the energy
intake of yellowfin tuna (Appendices 1,2). If the optimum range of fat content and total caloric
intake is known, these models can be used to determine the best feeding strategy. Knowledge of
captive tuna food requirements not only adds to knowledge about tuna; it also will enable
researchers to keep tuna healthy and alive in captivity for future study.
Acknowledgements
I would like to thank Professor Barbara Block, Dave Marcinek, and Chuck Farwell of MBA.
for guiding me and encouraging me throughout this project and Carolyn for helping to coordinate
the feedings, and for sharing her compassion for the tuna we fed. I would also like to thank Scotty
and Manny of MBA for their help at feedings and the rest of the Block lab for making me feel part
of the lab and for helping me on many occasions- especially John and Rich for battling with
computers with me, and Tara for assisting me with some of the feedings.Thanks also goes out to
my father who created the new histological technique of sticking tissue to slides with crazy glue.
Thank you all,
Bianca
Large Sardine
Small Sardine
Large Squid
Small Squid
Anchovies
TABLE 1
Percent Fat
3.56%
2.54%
54%
54%
14.46%
Calories/100g
120
98
76
77
193
Large Sardine
Small Sardine
Large Squid
Small Squid
Anchovies
TABLE 2
Mean Weight
69.7 g
38.18 g
49.2g
30.57 g
28.04 g
Standard Deviation
9.7
5.1
4.8
5.3
5.1
Figure Legend
Figure 1: Average calories eaten per fish per day calculated from expected values over 6 months.
Compares the old diet (December-March 1995) with the new diet (April- May 1995).
Figure 2: Average calories observed eaten per day by Tip in TI. Average fat calories eaten per day
are shown as well. Tip was observed Jan-May 1995.
Figure 3: Average calories observed eaten per day by RN in T2. Average fat calories eaten per
day shown as well. RN was observed February 1995-May 1995.
igure 4: Average calories observed eaten per day by Notch in T3. Average fat calories eaten per
day shown as well. Notch was observed December- May 1995.
Figure 5: %fat calories eaten per day by Tip for 6 feeding days in April. This is compared to the
% of sardines, anchovies and squid that Tip ate at each meal. Notice how fat levels rise and fall
most drastically with % anchovies eaten out of total meal.
Figure 6: Same as Figure 5 for RN in T2.
Figure 7: Same as Figure 6 for Notch in T3.
Figure 8: %fat in calories of total meal compared to % of anchovies eaten out of total meal for 6
feeding days in April 1995. Tip-TI.
Figure 9: Same as figure 8 for RN, T2.
Figure 10: same as Figure 8 for Notch, T3.
Figure 11: White muscle of TRCC captive tuna stained with hematoxylin. Light colored or white
patches are lipid in between muscle bundles. Dark patches are muscle bundles. 20 magnification.
Figure 12: Red muscle of TRCC tuna stained with hematoxylin. Light colored patches are lipid,
dark colored patches are muscle. 20 magnification.
Figure 13: White muscle of Kewalo basin tuna stained with hematoxylin. 20 magnification. Light
color=lipid between muscle bundles. Darkcolor- muscle.
Figure 14: Red muscle of Kewalo basin tuna stained with hematoxylin. 20 magnification. Light
color = lipid. Dark color- muscle.
1000
900 -
800
700
600
500
400 -
300
200 -
100
T1
Figure 1
Dec Jan Feb Ma
Months
72
April May
73
1000 -
900
800
700
600
500
400
300
200
100
Jan Feb
Avg cal/day
Figure 2
I

E

Mar April May
Months
Avg fat cal/day
400
300 -
200
£ 100
0
Feb
Avg cal/day
Mar
Figure 3
April
Months
Avg fat cal/day
—
May
400
300
5 200
100 -
Figure 4


Dec Jan Feb Mar April May
Months
Avg fat cal/day
Avg cal/day
Figure 5
70
60
50
8 40

30
20
10
April 21 April 23 April 25 April 27 April 29 May
Feeding day
fat calsquid T1 H%anch
%sar
Figure 6
100
80
60
40
20
April 21
April 25
April 29
Feeding day
H%fat cal%squid T2 +%anch
%sar
Figure
100
80
60
40
20
April 21 April 23 April 25 April 27 April 29 #
feeding day
—%fat calsquid T3 H %anch
%sar
40
30
20
10
Figure 8
mka aa-
4/21 4/23 4/25 4/27 4/29 5/1
Feeding day
%fat cal  %anch
70
60
50
§ 40

30
20
10
0
Fig
ure 9



4/21 4/23 4/25
4/27 4/29 5/1
Feeding day

- %fat cal %anch
40
30
20
10
421
Figure 10
4/23
4/25
4/27
Feeding day
- %fat cal %anch
4/29 «
o
FIGURE 11
FIGURE 12
FIGURE 13
FIGURE 14
REFERENCES
Bancroft, John D. 1975. Histochemical techniques. 2nd edition. London Press.
Blackburn, M. 1968. Micronekton of the easter tropical pacific. Marine Biology 72:1-6.
Brill, R.W. 1979. The effect of body size on the standard metabolic rate of skipjack tuna,
Katsuwonus pelamis. Fish. Bull. US 77:494-498.
Davis, G.E. 1971. Methods for assessment of fish production. I.B.P Handbook No. 3.
Gooding R.M., W.H. Neill, and A.E. Dizon. 1981. Respiration rates and low oxygen tolerance
limits in skipjack tuna, Katsuwonus pelami. Fish Bull. US 79:31-48.
Graham J.B., and R.M. laurs. 1982. Metabolic rate of the albacore tuna-Thunnus alalunga.
Marine Biology. 72:1-6.
Olson, R.T. and C.H. Boggs. 1986. Apex predation by yellowfin tuna (Thunnus albacares):
independent estimates from gastric evacuation and stomach contents, bioenergetics and cesium
concentrations. Canadian Journal of Fisheries Aquatic Science. 43:1760-177.
Schmidt-Neilson, K. 1990. Animal Physiology 3rd edition.
Shearer, Karl D. 1994. Factors affecting the proximate composition of cultured fishes with
emphasis on salmonids. Aquaculture. 119:63-88.
Sund, Paul, Blackburn M., Williams, F. 1981. Tunas and their environment in the Pacific
Ocean: A Review. Oceanogr. Mar. Biol. Ann. Rev. 19: 443-512.
Walters, C.A., K.A. Brown, W.M. Cole, J.F. Battery, D. Noorhasen. 1995. Lipid levels and
fatty acid profiles of the Caribbean Jack, Trachinotus goodei (Pisces, Carangidae) raised in
mariculture. ASLO Abstract.
ENDICES
AI
Appendix 1: Model of feeding including feeding to satiation twicw a month
Appendix 2: Model of feeding for all tanks increasing total calories per day
slightly from May levels.
APPENDIX 1
Generic month T2/ Feast and Famine Model #1 Fat/day-1.607%in
Squid lb Sarlbs Total lbs Squi kcal Sar kcal Tot kcal
Day
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
8
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
178
37.5 7.441016 7.781307 15.22232
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
178
37.5 7.441016 7.781307 15.22232
20
30
7.5 1.860254 1.111615 2.971869
67.5 Total kcallmonth 69.07895
105 totsarib.
tot sgib.
Generic month T1 Feast and Famine Model #1 fat/day-1.987% in
Day
Squid lb Sar lbs Total lbs Squi kcal Sar kcal Tot kcal
10 1.860254 2.22323 4.083485
10 1.860254 2.22323 4.083485
10 1.860254 2.22323 4.083485
10 1.860254 2.22323 4.083485
10 1.860254 2.22323 4.083485
10 1.860254 2.22323 4.083485
40 7.441016 8.892922 16.33394
0
10 1.860254 2.22323 4.083485
10 1.860254 2.22323 4.083485
10 1.860254 2.22323 4.083485
10 1.860254 2.22323 4.083485
10 1.860254 2.22323 4.083485
10 1.860254 2.22323 4.083485
40 7.441016 8.892922 16.33394
29
10 1.860254 2.22323 4.083485
tot sqib.
105 totsarib.
105 Total kcallmonth 85.75318
APPENDIX 2
Generic month T2, T3
Easy does it #1
Fatiday=2.15% in 9
Squid lb Sar lbs
Total lbs Squi kcal Sar kcal Tot kcal
Day
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971868
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
1.860254 1.111615 2.971869
1.860254 1.111615 2.971868
1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971868
1.111615 2.971869
7.5 1.860254
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
7.5 1.860254 1.111615 2.971869
77.5 Total kcallmonth
92.12795
155 tot sar lb
tot sq ib
Generic month T1
Easy Does it Model #1
Fat.day=2.93% in g
Day
Squid lb Sar lbs
Total ibs Squi kcal Sar kcal
Tot kcal
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
30
10 1.860254
2.22323 4.083485
10 1.860254
2.22323 4.083485
tot sqib.
155 totsarib
155 Total kcallmonth
126.588