FIX MY REPORT – READ THE PROFESSOR COMMENTS

FIX MY REPORT – READ THE PROFESSOR COMMENTS

THE EFFECT OF PROTEIN DIGESTION ON ZEBRAFISH METABOLIC RATE

ABSTRACT

Proteins are important components required for manufacturing all essential bodily structures including organelles, organs and muscles. The consumption of different amounts of protein in a diet could have significant impact on the metabolic rate of organisms. 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. Dietary ingredients as well as specific key nutrients remain important factors that affect Zebra fish growth and development.  However, there lacks a proper nutritional control due to the absence of standardized reference diet. Moreover, according to epidemiological studies 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. Increase in protein content has been known to be a major factor in increasing the metabolic rate to build essential organs and muscles necessary for survival. Earlier, it was also shown that increase in metabolic activity in fishes directly correlated with increased oxygen consumption. Thus, this project aims to evaluate how the level of protein content in commercial fish foods affects the metabolic rate of Zebra fish (Danio rerio). Here, we show that increasing contents of protein will increase their metabolic activity thereby increasing oxygen consumption in our model organism Zebra fish. Comment by Author: This is vague. Also, are there no associated organs involved in other animals’ digestion? Is that unique to zebrafish? Comment by Author: I can’t follow what you are trying to communicate here, or understand how it is relevant to your study. I suggest you consider removing much or all of this and stay focused on your topic more specifically. That will also help keep your abstract short. Comment by Author: Other than the two comments above, I think you have good material here that will flow well with the suggested revisions and maybe a little more revision/tweaking of phrasing. Much improved.

 

 

INTRODUCTION

Protein is one of the six primary nutrients necessary for the body to synthesize various cellular structures (Acheson et al., 2011). The process of protein digestion involves the breakdown of complex peptide bonds into soluble amino acid solutions that can be absorbed by the bloodstream from our intestines. Absorbed amino-acids within the digestive system is transported directly into the hepatic portal system. Food substances such as eggs, meat, milk as well as beans and nuts constitutes various important sources of proteins (Acheson et al., 2011).   The process of digestion is facilitated by enzymes known as pepsin which promotes the disintegration of large protein molecules into amino acids. The consumption of proteins could have significant effects on body metabolism. Protein breakdown commonly requires more calories 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). About 30% of the calories in proteins are burned in the process of digestion (Acheson et al. 2011). The heat generated in the process increases the general body heat output hence influencing metabolism. Comment by Author: Check your grammar for switching between plural and singular forms. Check throughout the paper, since I have noticed it in several places but only commented here. Comment by Author: This sentence sounds like you are saying that heat output is what causes an increase in metabolic rate. Actually, it is the opposite- higher metabolic rate increases body heat generation. However, I don’t see how this is very relevant for your focus on oxygen consumption.

The consumption of protein in diet has been established as a factor that increases the metabolic rate in human beings (Howard et al. 2015). 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. In a seminal review article by Jobling., 1981, it was shown that metabolic activity in fishes increased with increasing consumption of oxygen. In the same article, the author also provided evidence that consumption of protein had the greatest effect in increasing this metabolic rate (Jobling, 1981). Similar results were obtained in the early studies with mammalian species (Ganong, 1969; Avidson & Passmore, 1969) where this phenomenon have been explained based on heat loss which occurs when ATP is made from amino acids rather than from carbohydrate or fatty acids (Krebs, 1964). The basis of this theory came from Brody., 1942 following his observations of the association between metabolic rate and growth rate in young rats. Gawecki et al., 1978 determined the increase in metabolic rate of rats after feeding meals of varying composition and showed that the increase was greatest following the ingestion of a meal containing protein of high quality. These observations are particularly interesting when considered in conjunction with work relating to protein synthesis. Therefore, post-prandial metabolic rate varies with dietary composition and diets most favorable to growth induce the greatest effect. Comment by Author: This is a good time to break to a new paragraph about protein digestion affect on metabolism. Comment by Author: You are primarily interested in protein breakdown, so don’t drift into a discussion of protein synthesis. Maybe you can go there in the discussion, as another way that metabolism can be affected. Comment by Author: Put in parentheses…same for the next citation. Comment by Author: Drifting into synthesis of proteins again…

Zebrafish have many features that make them an attractive research tool. A fundamental advantage is that they share a considerable amount of genetic identity with humans, and several zebrafish organ systems are remarkably like those in humans (Seth et al., 2015). Additionally, zebrafish are relatively inexpensive to maintain, produce large numbers of offspring and undergo rapid development. These advantages make this organism an excellent model to study complex phenomena such as metabolic activity (Williams et al., 2014). Comment by Author: THIS PARAGRAPH IS ALMOST ENTIRELY COPIED AND PASTED FROM THIS WEBSITE: http://dmm.biologists.org/content/6/5/1080THAT WILL CAUSE YOU TO FAIL THIS COURSE IF YOU INCLUDE SOMETHING LIKE THIS IN YOUR FINAL DRAFT.

Metabolism is a physiological process reflecting the energy expenditure of living organisms and hence their food requirements in heterotrophs. The metabolic rate of fish is usually measured by their rate of respiration, i.e., their rate of oxygen consumption (Seibel et al., 2007). Therefore, the primary objective of this research paper is to evaluate the effects that protein provide on the fish metabolic rate within the first three to four hours. 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 (Boyle et al.,2008). Thus, this project aims to evaluate how the level of protein content in commercial fish foods that affects the metabolic rate of Zebra fish (Danio rerio). In this study, we hypothesize that increasing contents of protein will increase their metabolic activity thereby increasing oxygen consumption in our model organism Zebra fish. To prove our point, we used two different commercial fish foods containing varying protein levels and measured the dissolved oxygen (DO) of water after the consumption of each of those food. We expect to observe a decrease in the level of dissolved oxygen in the surrounding water environment with increasing protein concentration in food. Comment by Author: Necessary here? Comment by Author: This should have been established in the paragraph that discussed metabolic rate. Comment by Author: Vague. Be more precise with the time frame…and hours after what? Comment by Author: Unnecessary and hard to follow Comment by Author: In general, you have provided a lot of good information in this intro. There are some parts that get off track and should be removed, while others need clarification. Work on organizing your intro into 3-4 clear paragraphs, each with a key topic that is being discussed (e.g. protein digestion effects on metabolism), then tie them together to have a clean line of thought throughout.

MATERIALS & METHODS

 

The experiment was conducted using two groups of Zebra-fish who were given two different types of commercial feeds containing the same levels of fat and fiber but having different 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.

The table below indicates the two different commercially available fish food which were given to the two groups.

Name

Protein Fat Fiber
New Life Spectrum Premium 48% 5% 4%
Dainichi Veggie Deluxe 28% 5% 4%

 

Table 1. Commercially available fish food with varying percentage of protein content.

In this study, a total of four water tanks were utilized each harboring five Zebra fish at any time. Therefore, total of twenty Zebra-fish (all males to avoid gender biasness) were used for the entire experiment. On Day 1, fishes from Tank 1 & 2 was provided with New Life Spectrum Premium food whereas Tank 3 & 4 received Dainichi Veggie Deluxe. This routine was reversed on Day 3 when Tank 1 & 2 received Danichi Veggie Deluxe and Tank 3 & 4 received New Life Spectrum Premium food. This whole process was carried out in a way to make sure that each fish gets to eat their own share of food to capture the correct metabolic activity of each fish. Specifically, the fish was transferred to small measuring tubes, food was provided and dissolved oxygen levels in the water were measured. A Vernier optical probe was used to measure the dissolved oxygen (DO). For accurate measurements, oxygen level was measured and recorded in these small tubes before and after the fish were returned to the tanks. In order to avoid any interference of other variables, pH and temperature was measured and kept constant with a thermostat and pH buffer respectively. Comment by Author: Any time you mention some part of your apparatus, be sure to be very specific. Here, you should give the tank volume in liters. Check for other occurrences throughout the methods section. Comment by Author: How did you differentiate? Comment by Author: Vague…how much, how often? Comment by Author: I suggest you use sub-headings to break your methods into sections that address different aspects of the experiment (treatments and rearing, metabolic tests, data analysis, etc.) this is a good place to break to the metabolic test section. Comment by Author: They were fed in the tubes? What size tubes? How much water? More details necessary… Comment by Author: Need to give specific item number (look on the box or online) Comment by Author: More details…did you really use a thermostat and buffer?

Control measurements of pH, Temperature and DO levels were performed in all the tubes before the introduction of any type of food on Day 1. Food was given in each tube as described before for Day 1 & 3. No food was introduced on Day 2 so that the fish could complete their digestion of food from Day 1. DO measurements were made for each small tube at time t=0, then at t=1h, t=2h and t=3h. Special care was taken to make sure the fish specimens used within each tank were of similar physical traits in order to cater for differences in metabolism that might be caused by differences in physical characteristics like size and age. Similarities in these traits ensured that these physical attributes did not affect the outcome of the research findings (Augustine et al., 2011; Vergauwen et al., 2010). It is important to note here is that due to the consistency in the DO measurements in all the tanks during control readings as well as no food readings on Day 2, both the data has been combined to represent one single control group and one single No food group respectively. In contrast, the data has been divided into two groups of veggie Deluxe Day 1 and Life Spectrum Day 1 and the averages of the groups from each of those two tanks were taken as one single reading to build the graph (Fig. 2). The average of the raw readings is provided in Appendix. Comment by Author: What probes? Comment by Author: Probably should have measured mass…too late now, but you might want to include that in your discussion. Comment by Author: What does that mean? “No food group” Comment by Author: I can’t follow what you are trying to say here.

Temperature within the experimental environment was monitored daily using a thermometer (Fisher Scientific, Pittsburgh, PA). The levels of Ammonia-nitrogen, pH and NO2-N were assessed weekly using a standard aquarium pharmaceuticals test kit (Aquarium Pharmaceuticals, Chalfon, PA). Temperature was constantly 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. A visual representation of the experiment is given below in Figure 1.

 

Figure 1

Total of four tanks were used harboring five fish in each tank. Control measurements were taken as described in text above from all the four tanks before introducing food in each of them. Two types of commercially available diet were given. No food was given to any of the tanks on Day 2. Reversal of food type was done on Day 3. Comment by Author: The methods section: You have begun to describe things, but there are many aspects that are unclear at this point. I culd not possibly replicate your study based upon what you’ve included here. Address the comments above and try to evaluate if someone who is unfamiliar with the study would know how you did everything.

 

 

RESULTS

Increased metabolic activity is indicated by increased uptake of oxygen from the surrounding environment. In this study, we measured the amount of DO in each tube before and after the fish were fed with a specific type of commercial fish food with varying percentage of protein content. Our results indicate that there was a general trend in which the level of DO decreased with the progression of time. This was more pronounced in those fishes which received the Life Spectrum feed with the higher protein content (Fig 2). Although, the actual percentage of reduction was in fractions, the results validated our hypothesis upto a certain extent. In both cases, where the fish had consumed the Life Spectrum feed, there was an overall reduction of 0.37% of DO in the surrounding water as measured by the probe. In contrast, the Veggie Deluxe fed had a lower reduction percentage of 0.24% for Day 3. Fish who were fed Veggie Deluxe feed on Day 1 started with a lower DO which apparently did not reduce till our final time point at t=3h (Fig 2). The control measurements as well as the day (Day 2) with no food had much lower DO to start with and had almost no changes with time. This proved that indeed intake of food increased oxygen consumption from the surrounding water. Similarly, increase in protein content in diet also increases the consumption of oxygen from the surrounding environment and thus decreases the overall DO in water. Comment by Author: This is interpretations of the results and should be in the discussion section Comment by Author: No need to summarize methods that the reader just read. Remove this. Comment by Author: Interpretation discussion section Comment by Author: Interpretation discussion section. BUT, you don’t PROVE things in science. You data might support the conclusion that increased proteins results in increased oxygen consumption. Sa Comment by Author: How can you tell that from your data?

 

 

Figure 2 Comment by Author: Caption should also mention study species…it needs to be completely understandable on its own, without reading the text.

The graph measures the concentration of dissolved oxygen in the water with various food types. The readings were taken at every hour starting from time t=0h till t=3h. At least three measurements were obtained from each time point. Standard error of the mean (+/- SEM) was plotted from the average of at least three or more of those readings.

A nested ANOVA was performed to determine the significance between different groups. In this case, the test was to establish whether there is any statistical significant difference between the mean of the oxygen level and food protein level. From the results obtained (Appendix), the p value is less that the alpha value of 0.05 used at a 95% significance level. Since the p value is less than the alpha value, the null hypotheses is rejected and the conclusion is that the means of the groups of data are significantly different. The ANOVA test does not specify the groups whose means are significantly different hence requires further analysis by running the pro hoc test to determine the groups that have different Means. In conclusion, the means of the oxygen level and food protein level groups have significant statistical difference as per the ANOVA test. Comment by Author: I don’t know how you did this with your data, or which data you used. Let’s talk about this in our next meeting, because I’m almost certain it is incorrect. Also, did you ever measure metabolism of a fish that hadn’t been fed anything? A negative control?

ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 11.99025 1 11.99025 1420.401 7.23E-81 3.900989
Within Groups 1.33375 158 0.008441
Total 13.324 159        

Table 2. Statistical analysis using ANOVA.

DISCUSSION

This research 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 content foods compared to lower ones 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 & development (Sisman et al. 2013). From the study, it was concluded that the protein content in the diet increases the metabolic activities of the Zebra fish. So, the consumption of protein has a crucial role in the body metabolism. Proteins require more calories for breakdown, and therefore increases the metabolic rate of the fish. Comment by Author: Don’t put this at the beginning of the discussion. You want to start narrow (specifics about your conclusions), then go broader. Maybe this can fit in later.

Although, our results indicate that there was a general trend in the lowering of the DO with time in either of the diets, the feed with the higher protein content (Life Spectrum) had a greater reduction of DO than the one with the lower protein content (Veggie Deluxe). This is in accordance to our hypothesis which validates that protein plays a key role in fish metabolism. Our overall measurements of DO from either of these commercial diets were significantly higher than the control or the day with no food given on Day 2 (Fig.2). To keep our variables to the minimum, the pH, temperature and gender of the fish (male) were always kept constant in each tank and on different days (Appendix). Comment by Author: Unnecessary to include stuff like this here. It distracts from the line of thought about interpreting your data and relating that data to the bigger concepts.

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. In our study, the actual difference of the measurements observed at t=0 with t=3 with either of these commercial diets were not significantly different and had a much smaller difference than expected. This could be due to several reasons. One reason could be the amount of food given and the time for which the measurements were taken. Additionally, the DO was measured with one fish at a time kept in a small tube. It can be predicted that measurements taken at further time points up to 24h could give us data with increased differences in the DO when compared with t=0. Comment by Author: You didn’t test for significance of the change over time, so you can’t say this. However, you are correct in general because there was very little change over time.

In contrast to mice and humans, zebrafish are poikilothermic (body temperature varies with the ambient temperature), and brown adipose tissue (BAT) depots have not been identified. Therefore, the study of pathways that are activated by non-shivering thermogenesis, for example the β-adrenergic system, will be more limited in zebrafish than in mice. On the other hand, most standard animal facilities house small rodents at an ambient temperature substantially below the temperature associated with thermoneutrality in mice; thus, metabolic analysis is routinely carried out using animals that are chronically challenged by cold stress. This condition induces a range of physiological responses – such as increased food intake, metabolic rate and sympathetic activity – that could mask the metabolic phenotype under analysis (Overton, 2010). Moreover, the small size of the zebrafish does hamper some types of metabolic investigation. The current techniques to measure food intake (Volkoff and Peter, 2006) and energy expenditure (Makky et al., 2008) in the zebrafish are not as sophisticated as those in rodents, meaning that subtle metabolic phenotypes might not be detectable. These limitations should be considered when planning metabolic studies in zebrafish. Genetic variations or any possible mutations in the experimental fishes can also not be ruled out. Another point to consider is the high genetic diversity observed between individual zebrafish genomes, even within fish of the same strain (Guryev et al., 2006). Comment by Author: Not really relevant, and doesn’t include a citation until the last sentence. This is certainly not your original thought throughout, right? Comment by Author: ALMOST THIS ENTIRE PARAGRAPH IS COPIED DIRECTLY FROM THIS PAPER: THAT IS PLAGIARISM!http://dmm.biologists.org/content/6/5/1080YOU CANNOT COPY AND PASTE INTO A PAPER, EVEN IF YOU INCLUDE A CITATION! I WILL GIVE A ZERO IF I CATCH ANYTHING LIKE THIS IN THE FINAL DRAFT.

Now, it would be beneficial to mention that though protein content determines a major bulk of the metabolic activity in most organisms, the contribution of fat and carbohydrates cannot be ignored. Proteins are harder to digest than carbohydrates. Similarly, it is easy to obtain energy faster from easily digestible foods such as carbohydrates. Indeed, carbohydrates are the choice of food for almost every organism including fishes for obtaining energy for various metabolic activities (Krebs., 1964). Therefore, it will be tough to determine whether the carbohydrate content of the food given was utilized first to jumpstart the metabolic activity in Zebra fish. This could only be determined when the commercial feed will contain no carbohydrate or fats to rule out their participation in determining the metabolic rates and hence oxygen consumption. Energy can be obtained via two ways from carbohydrates- aerobic oxidative phosphorylation and anerobic glycolysis. In this case, it is hard to determine whether the Zebra fish were obtaining its energy and hence metabolism from aerobic or anerobic mechanisms. All these factors could contribute to the small differences observed during our experiments and needs to be addressed for future experiments. Comment by Author: Discussion brings up a few good topics. Try to revise to keep it better focused on things that are relevant to your study, and keep improving on the flow of ideas. If you can cut the length a little (by wording things more concisely), that would be helpful.

 

 

 

REFERENCES

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Ampatzis, Konstantino, and Catherine R. Dermon. Sexual Dimorphisms in Swimming behavior, Cerebral Metabolic Activity and Adrenoceptors in Adult Zebrafish (Danio rerio). Behavioral Brain Research 312 (2016): 385–393.

Augustine, S, Gagnaire BFloriani MAdam-Guillermin C, Kooijman SA. Developmental Energetics of Zebrafish, Danio rerio. Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 159.3 (2011): 275–283.

Brody, E. Litter size, growth rate and heat production of suckling rats. Am. J. Physiol. (1942). 138, 180-183.

Davidson, S. & Passmore, R. Human Nutrition and Dietetics. (1969). Edinburgh: E. S. Livingstone.

Ganong, W. F. Review of Medical Physiology. (1969). California: Lange Medical Publications.

Gawecki, J., Jeszka, J., Urbanowicz, M. & Gabrys-Modrzynska, M. Studies on the Specific Dynamic Effect of Proteins of Different Nutritional Value. Acta Physiol.pol. (1978). 29,319-383.

Guryev, V., Koudijs, M. J., Berezikov, E., Johnson, S. L., Plasterk, R. H., van Eeden, F. J. and Cuppen, E. Genetic variation in the zebrafish. Genome Res. (2006). 16, 491–497

Howard, R.D., Rohrer KLiu YMuir WM. Mate Competition And Evolutionary Outcomes In Genetically Modified Zebrafish (Danio rerio). Evolution 69.5 (2015): 1143–1157.

Jobling, M. The influences of feeding on the metabolic rate of fishes: a short review. J. Fish Biol(1981). 18, 385–400.

Kim, M. J, Choi JKim NHan GC. Behavioral Changes Of Zebrafish According To Cisplatin-Induced Toxicity Of The Balance System. Human & Experimental Toxicology 33.11 (2014): 1167–1175.

Krebs, H. A. The Metabolic Fate of Amino Acids. In Mammalian Protein Metabolism, (1964). (H. N. Munro & J. B. Allison, eds). London: Academic Press.

Overton, J. M. Phenotyping small animals as models for the human metabolic syndrome: thermoneutrality matters. Int. J Obes. (2010). 34 Suppl. 2, S53–S58

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Sisman, T., Özlem, G. Özlem Gür

1Department of Biology, Faculty of Science, Atatürk University, Erzurum, Turkey See all articles by this author

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, Nesli, D., Nesli Doğan

1Department of Biology, Faculty of Science, Atatürk University, Erzurum, Turkey See all articles by this author

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APPENDIX

 Food Given Tank 1 Tank 2 Tank 3 Tank 4
Day-1 Life Spectrum Life Spectrum Veggie Deluxe Veggie Deluxe
Day-2 No Food No Food No Food No Food
Day-3 Veggie Deluxe Veggie Deluxe Life Spectrum Life Spectrum

Table representing the design of experiment and the tanks receiving commercial fish food

 Average pH Tank 1 Tank 2 Tank 3 Tank 4
Control (Day 1) 8.2 8.2 8.2 8.2
Day-1 8.2 8.2 8.2 8.2
Day-2 8.2 8.2 8.2 8.2
Day-3 8.2 8.2 8.2 8.2

Table representing the average pH.

Average Temperature (0F) Tank 1 Tank 2 Tank 3 Tank 4
Control (Day 1) 70 70 70 70
Day-1 70 70 70 70
Day-2 70 70 70 70
Day-3 70 70 70 70

Table representing the average temperature measurements for all the tanks on all days.

Table representing the average DO measurements in control for all the tanks on Day 1 Comment by Author: Did you measure 02 in the tanks, or in individual tubes?

Table representing the average DO measurements on Day 1 in all the tanks

Table representing the average DO measurements with no food in all the tanks on Day 2

Table representing the average DO measurements on Day 3 in all the tanks.

13

Time (h)t=0t=1t=2t=3t=0t=1t=2t=3t=0t=1t=2t=3t=0t=1t=2t=3

Control8.028.028.028.028.028.028.028.028.028.028.028.028.028.028.028.02

TANK 3 TANK 4 TANK 1 TANK 2

Time (h)t=0t=1t=2t=3t=0t=1t=2t=3t=0t=1t=2t=3t=0t=1t=2t=3

Day 18.098.088.078.068.098.088.078.068.078.088.088.088.078.088.088.08

TANK 1 TANK 2 TANK 3 TANK 4

Time (h)t=0t=1t=2t=3t=0t=1t=2t=3t=0t=1t=2t=3t=0t=1t=2t=3

Day 2 (No Food)8.008.018.018.018.018.018.018.018.008.018.018.018.008.018.018.01

TANK 1 TANK 2 TANK 3 TANK 4

Time (h)t=0t=1t=2t=3t=0t=1t=2t=3t=0t=1t=2t=3t=0t=1t=2t=3

Day 38.098.098.098.078.098.098.098.078.118.118.108.088.118.118.108.08

TANK 1 TANK 2 TANK 3 TANK 4

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