6 hours assignment/biology Asap

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 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 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.

The tables below indicate the groups of organisms and the type of feed provided to them

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 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 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).

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 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:

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 results, we find the following trends:

The oxygen level in the control group remains constant throughout the 4 hours in all tanks and for all the fish

The oxygen level in the “no food” group remains constant throughout the 4 hours in all tanks and for all the fish

The oxygen level 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:

References

Acheson, Kevin J. et al. “Protein Choices Targeting Thermogenesis and Metabolism.” The American journal of clinical nutrition 93.3 (2011): 525–534. Print.

Ampatzis, Konstantinos, and Catherine R. Dermon. “Sexual Dimorphisms in Swimming Behavior, Cerebral Metabolic Activity and Adrenoceptors in Adult Zebrafish (Danio Rerio).” Behavioural Brain Research 312 (2016): 385–393. Print.

Augustine, Starrlight et al. “Developmental Energetics of Zebrafish, Danio Rerio.” Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 159.3 (2011): 275–283. Print.

Boyle, David et al. “Natural Arsenic Contaminated Diets Perturb Reproduction in Fish.” Environmental science & technology 42.14 (2008): . Print.

Howard, Richard D. et al. “Mate Competition And Evolutionary Outcomes In Genetically Modified Zebrafish( DanioRerio).” Evolution 69.5 (2015): 1143–1157. Print.

Kim, M. J. et al. “Behavioral Changes Of Zebrafish According To Cisplatin-Induced Toxicity Of The Balance System.” Human & Experimental Toxicology 33.11 (2014): 1167–1175. Print.

Lee, Okhyun, Jon M. Green, and Charles R. Tyler. “Transgenic Fish Systems And Their Application In Ecotoxicology.” Critical Reviews In Toxicology 45.2 (2015): 124–141. Print.

Sisman, Turgay et al. “Single-Cell Protein as an Alternative Food for Zebrafish, Danio Rerio: A Toxicological Assessment.” Toxicology and industrial health 29.9 (2013): 792–799. Print.

Varshney, Gaurav Kumar, and Shawn Michael Burgess. “Mutagenesis AndPhenotyping Resources In Zebrafish For Studying Development And Human Disease.” Briefings In Functional Genomics 13.2 (2014): 82–94. Print.

Vergauwen, Lucia et al. “Long-Term Warm or Cold Acclimation Elicits a Specific Transcriptional Response and Affects Energy Metabolism in Zebrafish.” Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology 157.2 (2010): 149–157. Print.

Williams, Tim D., Leda Mirbahai, and J. Kevin Chipman. “The Toxicological Application OfTranscriptomics And Epigenomics In Zebrafish And Other Teleosts.” Briefings In Functional Genomics 13.2 (2014): 157–171. Print.