THE EFFECT OF PROTEIN DIGESTION ON ZEBRAFISH METABOLIC RATE INTRODUCTION protein form one of the primary nutrients that are used in the body for cellular structures building (Acheson 525). the process of protein digestion involves the breakdown of food substances we eat into soluble solutions that can be absorbed in the bloodstream. protein is broken down to form amino-acids within the digestive systems and the amino acids are absorbed into the hepatic portal system.   the proteins are the building blocks in the body and they include food substances such as eggs, meat, milk as well as beans and nuts among many other food products (Acheson 527).   the digestion of proteins mainly begins in the stomach and continues in the small intestine (Acheson 530).   digestion of proteins is facilitated by pepsin enzymes that promotes the digestion of the large proteins molecules into amino acids.   the digestion of proteins in the body also includes the use of hydrochloric acid (Acheson 526).   zebra-fish is preferred in research studies since it is cheap, have similar organs as human beings and produces a lot of offspring. zebra-fish also has a short life-span of about two years and are easy to take care of. Therefore, the primary objective of this research paper is to evaluate the effects that protein provide on the fish metabolic rate especially after three to four hours. STATEMENT OF THE PROBLEM In most cases, protein digestion takes place in both the stomach and in the small intestine in the body of human beings. However, in Zebra fish, protein digestion involves the use of associated organs. the dietary ingredients, and nutrients as well as anti-nutritional remain important factors that affect Zebra fish growth and development.  However, there lacks proper nutritional control due to the absence of standardized reference diet (Boyle et al.,2008). Moreover, according to epidemiological studies by Richard et al. (2015), prenatal conditions are important in the growth and development of Zebra fish. As a way of providing a standardized dietary framework, a need emerges for the provision of specific dietary and nutritional standard aimed at improving the growth and development of Zebra fish. Thus, this project aim to evaluate how the level of? protein in commercial fish foods affects the metabolic rate of Zebra fish (Danio rerio). BACKGROUND The consumption of proteins could have significant effects on body metabolism. proteins commonly require more calories for breakdown than carbohydrates and this could have significant effects on various metabolic aspects, including an increase in the metabolic rate of organisms (Williams et al., 2014). Due to the strong bond making the protein molecules, a lot of energy is required to break down the high protein that has been consumed. about 30% of the calories in proteins are burned in the process of digesting proteins(Acheson et al. 528). The heat generated in the process increases the general body heat output h ...

1. THE EFFECT OF PROTEIN DIGESTION ON ZEBRAFISH
METABOLIC RATE
INTRODUCTION
protein form one of the primary nutrients that are used in the
body for cellular structures building (Acheson 525). the process
of protein digestion involves the breakdown of food substances
we eat into soluble solutions that can be absorbed in the
bloodstream. protein is broken down to form amino-acids within
the digestive systems and the amino acids are absorbed into the

2. hepatic portal system. the proteins are the building blocks in
the body and they include food substances such as eggs, meat,
milk as well as beans and nuts among many other food products
(Acheson 527). the digestion of proteins mainly begins in the
stomach and continues in the small intestine (Acheson 530).
digestion of proteins is facilitated by pepsin enzymes that
promotes the digestion of the large proteins molecules into
amino acids. the digestion of proteins in the body also
includes the use of hydrochloric acid (Acheson 526). zebra-
fish is preferred in research studies since it is cheap, have
similar organs as human beings and produces a lot of offspring.
zebra-fish also has a short life-span of about two years and are
easy to take care of.
Therefore, the primary objective of this research paper is to
evaluate the effects that protein provide on the fish metabolic
rate especially after three to four hours.
STATEMENT OF THE PROBLEM
In most cases, protein digestion takes place in both the stomach
and in the small intestine in the body of human beings.
However, in Zebra fish, protein digestion involves the use of
associated organs. the dietary ingredients, and nutrients as well
as anti-nutritional remain important factors that affect Zebra
fish growth and development. However, there lacks proper
nutritional control due to the absence of standardized reference
diet (Boyle et al.,2008). Moreover, according to
epidemiological studies by Richard et al. (2015), prenatal
conditions are important in the growth and development of
Zebra fish. As a way of providing a standardized dietary
framework, a need emerges for the provision of specific dietary
and nutritional standard aimed at improving the growth and
development of Zebra fish. Thus, this project aim to evaluate
how the level of? protein in commercial fish foods affects the

3. metabolic rate of Zebra fish (Danio rerio).
BACKGROUND
The consumption of proteins could have significant effects on
body metabolism. proteins commonly require more calories for
breakdown than carbohydrates and this could have significant
effects on various metabolic aspects, including an increase in
the metabolic rate of organisms (Williams et al., 2014). Due to
the strong bond making the protein molecules, a lot of energy is
required to break down the high protein that has been
consumed. about 30% of the calories in proteins are burned in
the process of digesting proteins(Acheson et al. 528). The heat
generated in the process increases the general body heat output
hence influencing metabolism.
The consumption of protein in diet has been established as a
factor that increases the metabolic rate in human beings(Howard
et al. 117). The process of protein synthesis in the body causes
the amino acid structures of the proteins consumed to begin the
process of building body muscles. The construction of these
tissues in the body consumes high amounts of energy hence the
body requires increased energy. Nevertheless, it is important
that the researcher provide enough energy in order to maintain
the high metabolic rate of Zebra Fish. The provision of enough
energy will also promote the breaking down amino acids to form
muscle units.
HYPOTHESES, GOALS, AND OBJECTIVES
Furthermore, the study also aims to establish, the metabolism
rate of Zebra fish when fed with different commercial diets The
evaluation of the relationship between protein level in food and
metabolic rate in Zebra-fish.
HYPOTHESIS
it should be expected that the proteins are important body
building components and their consumption in a diet could have
a significant impact on the metabolic rate of organisms.
Moreover, it would be expected that would present a positive
impact on increasing the metabolic rate of the Zebra-fish as

4. well.
GOALS
The goal of the experiment includes testing the metabolic rate
following the application of 2 different protein levels available
in commercial diet by measuring the oxygen level of water in
Zebra fish
METHODOLODY
Zebra-fish (Daniorerio) are a small shoaling fish which belongs
to the minnow family of Cyprinadae and order cypriniformes. It
is a tropical fish that resides in fresh water conditions and is a
native species of the Himalayan region. Due to its unique
characteristics it possesses, the Zebra-fish remain vastly
utilized as a vertebrate model organism in scientific research.
These include cancer prevention, cardio-vascular diseases as
well as investigating immune systems and other drug discovery
systems
Furthermore, because of their omnivorous characteristics, they
are have been extensively utilized in environmental monitoring
activities as a way of preventing water pollution by estrogen
(Howard et al. 1173). Based on past literatures and research
studies investigating the growth and metabolic rate of Zebra-
fish (Daniorerio), they are imperative in conducting studies in
the development, genetics as well as the human disease
conditions(Williams, Mirbahai, and Chipman 157)
The experiment will be conducted using two groups of Zebra-
fish placed in provided with two different types of commercial
feeds with the same levels of fat and fiber but varying protein
content. The available commercial feeds have formed the
guideline for undertaking the research since the levels of
protein and other nutrients are established by the manufacturers.
Each of the two groups will be provided with a food that has a
specified level of fat and fiber with varying protein level. All
nutrients play a part in the metabolism process, and although
the research seeks to determine the impact of protein, the
effects of the other nutrients present in the food cannot be
ignored.

5. The tables below indicate the groups of organisms and the type
of feed provided to them
Name
Protein
Fat
Fiber
New Life Spectrum Premium
48%
5%
4%
Dainichi Veggie Deluxe
28%
5%
4%
In this study, a total of four water tanks will be utilized and 20
male Zebra-fish. Each tank will contain 5 Zebra-fish and they
will be provided with two different commercial types of feeds
the tanks will be fed with two different foods on two separate
days, one of the food contains 48% protein, and the other
contains 28% protein. The fish will be transferred to small
measuring tubes, to take control measurements of their oxygen
level, for more accurate results to be accrued.to do this, the
closed flow respirometry system will used. When the fish will
be transferred to the tubes, their oxygen level will be measured
and recorded before and after the fish would be added to the
tanks.
On the first day at 1.00 pm, all the equipment will be set up and
tested to be in good conditions. The oxygen level, the pH and

6. the temperature of the water, will be measured.4 zebra-fish will
be added to each tank, after which the pH and the temperature
will be checked again. Using Vernier? optical probe, the oxygen
level will also be measured. On the same day at 5.00 pm, the
water oxygen level will be measured again. For tanks 1 and 2,
they will be fed with type A food two times in a day. For tanks
3 and 4, they will be fed with type B food once in a day. The pH
and temperature will be checked y. Oxygen level will also be
measured before adding the fish to the tubes, and also for every
hour, for four hours during the time when digestion will be
taking place.
On day 2, oxygen level, pH, and temperature, were checked.
The observer will make sure that all the food is digested. No
residue should be left in the tanks.
On Day 3, all other procedures will resemble those of Day
1(5.00 pm).However, tanks 1 and 2 will be fed with type B
food, twice a day, while tanks 3 and 4 will be fed with type A
food.
There are significant discrepancies in activities of Zebra-fish in
terms of behaviors based on the gender. These behaviors include
swimming and movements which are sex-specific characteristics
that can be utilized to assess the fish (Lee, Green, and Tyler
128). , same sex fishes will be utilized in order to minimize the
possibility of sex-related behaviors affecting the findings.
Female Zebra-fish have been found to have higher glucose
utilization that males, when put in the same
environment(Ampatzis and Dermon 387).
The study specimen who will be provided within each of the
tanks will be of similar physical traits in order to cater for
difference in metabolism that might be caused by differences in
physical characteristics like size and age. Similarities in these
traits which also affect the metabolism rate will ensure that
these physical attributes do not affect the outcome of the
research findings(Augustine et al. 277). The researcher will also
measure the mass specific metabolic rate of the Zebra Fish
The use of LED and direct lighting will provide an equal light

7. to dark photoperiod of 12 hours. Temperature within the
experimental environment will also be monitored daily using a
thermometer (Fisher Scientific, Pittsburgh, PA). The levels of
Ammonia-nitrogen, pH and NO2-N will be assessed weekly
using a standard aquarium pharmaceuticals test kit (Aquarium
Pharmaceuticals, Chalfon, PA). Each tank will contain one
cylindrical plastic mesh cage (0.25 m height by 0.09 m
diameter) that will be covered with nylon mesh (Fashion Knee
Hi; American Corporation, Henderson, NC). Temperature must
be maintained at 28°C (±0.5°C), pH between 8.0 and 8.4, and
ammonia-nitrogen and NO2-N, contained below visual detection
limits.
Metabolism is a process that is affected by a myriad of external
factors other than the nutrition and bodily constituents in terms
of muscle, age, body size and others. Temperature of the
environment in which the organisms are residing should be
similar to ensure that the differences observed are not as a
result of the other factors.
While the organisms are maintained in similarity, the
environment of the experiment should also be similar in seeking
to minimize the effect of environment induced metabolic
differences. The levels of oxygen can also be affected by the
external environment like the presence of other gases within the
environment hence the tanks in which the experiment will be
conducted must be maintained in an enclosed state(Vergauwen
et al. 153).
The experiment will include the following procedures and will
cover three days and three days replication of the same process.
Figure 1
Figure 1 shows number of tanks and fish that will be used in
this experiment, and type of food that will be giving to each
group of Zebra-fish within the three days of the experiment
(Lee et al., 2015, p. 124).

8. Oxygen measurements
A Vernier optical probe will be utilized in the assessment of the
DO values. This probe provides a quick and accurate test for
assessment of dissolved oxygen content.
Significance and impact
This research experiment seeks to determine the impacts of high
protein diet on the metabolic rate of Zebra-fish. While many of
the nutrients available in food have significant -impact in the
metabolism process, proteins have a complex structure which
causes them to require high amounts of energy to be processed.
It is presumed that the high protein compared to low one in
diets increase the metabolic rate of Zebra-fish. This research
will provide significant information to the numerous individuals
who are involved in the rearing of Zebra-fish for different uses.
The knowledge gained will be critical in aiding them to
understand the kind of food that they can provide to the fish to
achieve maximum metabolism, which is critical in providing
necessary energy for the growth(Sisman et al. 794).
The research adds to the existing body of knowledge on the
metabolism of Zebra-fish. These organisms have been
extensively studies and utilized in many medical experiments.
They thus, form an important part of scientific research and
through the results of this research, the knowledge available
will be expanded(Varshney and Burgess).The broader ecosystem
requirements of the fish will also be understood. The research
forms the basis upon which other components of the diet like
carbohydrates can be studied for the impact on the metabolic
rate to be determined.
STATISTICS
The study will involve the use of Nested ANOVA Tests and it
will be used to measure the metabolic rate of Zebra Fish as well
as the temperature and the oxygen levels in water. While using

9. the Nested ANOVA, the attribute variables are nested in order
to allow only one value of the higher-level on the combination.
Furthermore, it is evident that the top-level attribute variable
may be either Model I or Model II. The lower level attribute
variables are mostly Model II. It should also be noted that
Nested ANOVA is used where we have one measurement
variable and more than one nominal variable. The nominal
variables are nested in order to form subgroups within the
group. Therefore, it should be used where there is significant
variation in means among groups as well as among subgroups
within the groups. However, the assumptions made while using
Nested ANOVA tests is that the observation within each
subgroups are normally distributed and also have equal standard
deviations. The other major basic assumptions used in ANOVA
include the view that the expected values of the errors are zero;
the variances of all errors are equal to each other as well as the
idea that all the errors are independent and normally distributed.
A four level Nested ANOVA test has been carried out to
distinguish any existing differences between the treatments
applied (control, no food, veggie deluxe & life spectrum day 1
and veggie deluxe & life spectrum day 3), the four different
tanks, each of the five fishes on the different tanks and the time
interval passed from the moment they were fed and the
measured of oxygen dissolved levels. As can be observed in the
ANOVA table presented below, there is a significant difference
between the results obtained for the different treatments and the
different time elapsed from the moment the fish were fed up, as
indicated by the low p value obtained in the among treatments
and among time:
Source of variation
Sum of squares
Degrees of freedom
Mean sum of squares
F

10. p-value
Among treatments
14.73
3
4.911
186.25
6.61*10-4
Among tanks
0.08
3
0.026
0.32
0.813
Among fish
0.33
4
0.083
3.20*10-3
1.000
Among time
77.81
3
25.937
60.26
1.22*10-30
Within data
131.71
306
0.430
This result indicates that the main sources of variation in the
obtained results include not only the time elapsed from the
moment the fish were fed up, but most importantly, the type of
food used in feeding them. Hence, having a look at the obtained

11. results, we find the following trends:
throughout the 4 hours in all tanks and for all the fish
throughout the 4 hours in all tanks and for all the fish
vel increases after feeding the fish with
Veggie deluxe & life spectrum, both on day 1 and day 3.
Generally, the maximum concentration of oxygen is reached one
hour after feeding the fish with Veggie deluxe & life spectrum
indicating a higher metabolic rate during this time frame. The
concentration of oxygen then slowly decays after this initial
hour.
These trends are summarized in the following graph:
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