Make me a conclusion for my research paper on The Effect of Different Temperatures on Planaria Regeneration following these instructions: To write a conclusion, you must address 7 major questions:

1. What was the purpose of the experiment?

2. What were the major findings, including the inferential statistics test results?

3. Was the hypothesis supported by the data? You NEVER prove your hypothesis- just if it is supported or not supported.

4. How did your findings compare with other researchers? Use your lit reviews and the information you have found.

5. What possible explanation can you offer for the findings? Why were the results significant or not significant?

6.How could this experiment have been improved? What were the sources of error and how could they have been fixed for future experimentation?

7. What recommendations do you have for further study?

Items to remember when writing the conclusion:

1. Use the present and past tense, third person and complete sentences

2. Times New Roman, 12 point font, 1 inch margins (top, bottom, left & right) and double spaced

3. Incorporate a minimum of 3 different references to prior research (use the lit reviews you have been doing and refer to the research you have discovered)

Example of a Conclusion: (this is an example and can use improvement)

The purpose of this experiment was to determine the effects of different fertilizers on plant growth. Pea plants were exposed to 3 different levels of the independent variable: no fertilizer, fertilizer A and fertilizer B and after 20 days, their growth was measured. A research hypothesis was formulated that if fertilizer B was used on a plant, it would grow more than fertilizer A or no fertilizer at all. It was found that fertilizer A had a greater impact on plant growth than fertilizer B and no fertilizer. Due to these results, the research hypothesis was not supported by the data but did show that fertilizer did have an impact on plant growth.

A t-test was performed on the data to determine if the data was significant. The data for the fertilizer having an effect on plant growth is both statistically and not statistically significant. The data for Fertilizer A vs. the control implies that the data is not statistically significant. The results that were obtained for Fertilizer A were most likely due to chance and error versus the independent variable. The data for Fertilizer B vs. the control implies that the data is statistically significant and the results were due to the independent variable and Fertilizer B does have an affect on plant growth.

Other researchers have investigated fertilizer A and fertilizer B. Smith and Anderson found that fertilizer A had a dramatic affect on spinach plants. They found that spinach plants treated with fertilizer A had a 22% increase in growth and mass than plants not treated with fertilizer (Smith & Anderson, 2006). Other studies have shown that fertilizer B can increase plant growth. One such study found that peanut plants treated with a 20% solution of fertilizer B produced twice as many peanuts as the plants not treated with fertilizer (Jones, 2005). The findings from this study were more similar to Smith and Anderson since the pea plants treated with fertilizer A had more growth than fertilizer B and no fertilizer.

There are a number of possible explanations for the results of this experiment. Although the data from fertilizer A vs. the control was not considered significant, there was a difference in the plant growth between fertilizer A and fertilizer B. Fertilizer A consists of a mixture of nitrogen and minerals and gives soil the ability to hold twice the amount of water than it normally would (Johnson, 2004). These factors create an ideal environment for pea plants which thrive on watery soil that is rich with nitrogen. A study which was performed by Cooper and Marsh found that pea plants treated with an abundance of nitrogen and water were 32% more likely to grow over 7 cm (Cooper & Marsh, 2005). While fertilizer A creates an ideal environment for the pea plants, fertilizer B may not. Fertilizer B consisted mainly of natural chemicals, such as manure and decomposed organic matter (Jones, 2005). While pea plants can grow in these conditions, they do not prefer decomposed organic matter as much as they prefer nitrogen and an abundance of water.

There were sources of error in the this experiment and the experiment could have been improved a number of ways. All of the plants were not exposed to the same amount of sunlight each day which could have affected the results and could have skewed the data to favor fertilizer A. In addition to the amount of sunlight, the plants were only given 20

days to grow. Fertilizer B may be a slow acting fertilizer and may have needed more time to affect the plants. Lastly, the directions for using fertilizer B indicated that each plant needed 5g but a constant was established that each plant would be treated with either 3g of fertilizer A or 3g of fertilizer B. The plants treated with fertilizer B may have grown more if they were given the recommended dosage. For continued study, the optimum amount of fertilizer A should be investigated. In this study, 3 g was used but the optimum dosage may be more or less than 3g. In addition to the optimum amount of fertilizer A, the effects of fertilizer A on other legume plants should be studied.

ALSO USE THESE: The Effect of Different Temperatures with Vitamin A on Planaria Regeneration

Abstract

This study aimed to elucidate the effect of temperature and Vitamin A on the regenerative abilities of planaria, emphasizing their resemblance to human stem cells. The hypothesis proposed that planarians subjected to 23°C and supplemented with Vitamin A would exhibit the most regeneration. The experiment involved three temperature conditions, 3°C, 23°C (control), and 37°C, with a constant Vitamin A concentration of 4%. The measurement technique was the length of planarian regeneration in millimeters after 14 days. Safety protocols for handling planarians and Vitamin A were adhered to in the experiment. Groups of 25 planarians were bisected through the middle, and regeneration of the anterior parts of the planaria were measured. Each group was placed in the specified temperature conditions for 14 days, with regeneration length measured using an electronic caliper. Statistical analysis included t-tests and calculation of variance and standard deviation. Findings revealed the most substantial regeneration at the control temperature of 23°C, showcasing an average regrowth of 3.3 millimeters, as opposed to 1.0 millimeters at 3°C and a notably lower mean of 0.4 millimeters at 37°C, confirming the research hypothesis. The statistical significance was supported by all t-values exceeding the critical t-value, rejecting the null hypothesis across all temperature comparisons. Consequently, the study concludes that temperature, with Vitamin A supplementation, significantly impacts planarian regeneration, with room temperature proving most conducive for regenerative processes for planaria. These observations suggest that environmental and nutritional factors intertwine to significantly influence regenerative outcomes in planaria using neoblasts and humans using stem cells.

Introduction

The process of regeneration in planarians attracts researchers due to planarians’ extraordinary ability to regrow lost parts under various conditions (Seebeck et al, 2017). This current study investigates how different temperatures with Vitamin A impact planarian regeneration. Including different temperature ranges and constant levels of Vitamin A in the experimental design is chosen to simulate various environmental conditions and constant nutrient supplementation scenarios (Deochand et al, 2018). Consequently, the research explores how these variables affect the self-renewal ability of pluripotent stem cells using neoblasts (Lai et al, 2017). Planaria serve as model systems for embryonic stem cells in humans, driving the choice of this study. It contains real-world implications and explores a topic that assists planaria to become better models for human stem cells (Ermakova et al, 2009).

The potential implications of this research promise broader societal implications (Deochand et al, 2018). The findings may reveal information applicable in regenerative medicine, tissue engineering, and developmental biology (Sarkar et al, 2019). Insights from this research may prove beneficial to human health, for instance, in providing crucial knowledge for the development of regenerative therapies & regenerative medicine. Furthermore, this research holds the potential to assist advances in regenerative medicine by uncovering the fundamental mechanisms/functions managing and aiding regeneration in planarians (Ermakova et al, 2009).

The independent variable in this study encompasses understanding different temperatures and varying Vitamin A concentrations (Sarkar et al, 2019). This includes the impact of different temperatures (3, 20, & 37 degrees Celsius) on planarian survival and how Vitamin A potentially influences cellular processes, primarily around tissue and stem cell regeneration (Sarkar et al, 2019). Vitamin A, an external factor added to the planaria, is usually already present in the human body due to a daily diet containing a certain amount of Vitamin A. Vitamin A improves and increases vision, growth, cell division, reproduction and immunity. Vitamin A is used for cell differentiation and embryonic development. It also decreases the risk of heart disease, cancer, and skin diseases (Marie et al, 2021). Adding Vitamin A to planaria assists the planaria to become better models for regeneration applications in humans and the real world.

Understanding planarian regeneration, tissue regeneration, and neurogenesis becomes crucial for the dependent variable (DV) (Sarkar et al, 2019). This knowledge aids in understanding the processes involved in planarian tissue repair and the role of neoblasts in

pluripotent stem cells in regeneration. Pluripotent stem cells means that the stem cell can divide into any cell type. Neoblasts, which are adult stem cells that can divide, have the role of dividing and transforming into any cell type such as red blood cells, white blood, cells, platelets, and more (Lai et al, 2017). There are neoblasts spread across the bodies of planaria, in fact, they make up about 25%-30% of all the cells in planaria (Ge et al, 2022). In other words, neoblasts are a type of pluripotent stem cells. Planaria also show better regeneration than other animals that exhibit regeneration such as zebrafish, axolotls, and spiny mice because they contain plenty more neoblasts and pluripotent stem cells than other organisms (Deochand et al, 2018).

The purpose of this study is to understand the impact of different temperatures with Vitamin A on planarian regeneration (Ermakova et al, 2009). By observing the regenerative capacity of planarians under varying experimental conditions, the research seeks to conclude on the possible effects on tissue organization, neurogenesis, and stem cell populations (Seebeck et

al, 2017). It is hypothesized that the variations in temperature, combined with Vitamin A supplementation, will have different effects on planarian regeneration (Ermakova et al, 2009).

The temperature of 23 degrees Celsius with a 4% concentration of Vitamin A is thought to increase the regeneration of the planaria the greatest. This is because planaria cultures, which are ideal habitats for growing planaria, are maintained at optimal temperatures around 23 degrees Celsius or room temperature. Also, many studies observe and utilize room temperature, typically around 23 degrees Celsius as the optimum temperature for planaria growth (Sarkar et al, 2019). 

Specifically, conditions of 23 degrees Celsius supplemented with Vitamin A are anticipated to be ideal for planaria and to support pluripotent stem cells' self-renewal ability using neoblasts, potentially resulting in the most effective planarian regeneration. This hypothesis is backed by previous studies highlighting the crucial role of environmental factors such as high humidity, low light, and a 6.0 - 7.0 (slightly acidic) pH (Ge et al, 2022). The hypothesis is also supported by nutrient supplementation play, such as glycogen and dextrose, provided by hard-boiled egg yolk, pieces of earthworm, or beef liver (Abel et al, 2022). These factors influence the developmental and regenerative processes in various organisms, including planarians (Deochand et al, 2018). It is also supported by studies indicating that planaria grow best around room temperature in a dark environment (Sarkar et al, 2019).

The independent variable in this study comprises the different temperature conditions (3, 20, and 37 degrees Celsius), with a constant Vitamin A concentration (4%). These temperatures were chosen to test how planarian regeneration and cellular processes would increase or decrease due to drastic differences in temperature of environment of the planaria; with the addition Vitamin A. This concentration of Vitamin A was chosen because it is known to not be too toxic to the planaria, but it was not too little to not have any effect on the planaria. The control group is set at 23 degrees Celsius because it is room temperature, and the temperature of the environment isn't changed. The dependent variable is planarian regeneration, determined through the length of regeneration of planaria, measured in millimeters (mm). Different temperatures with a constant concentration of Vitamin A can also affect tissue organization, neurogenesis, and the self-renewal ability of pluripotent stem cells since the change of temperature is an environmental factor change and the addition of Vitamin A is a nutritional/supplemental factor change. (Lai et al, 2017).

The purpose of this experiment is to determine if different temperatures with an addition of Vitamin A have an effect on planaria regeneration. By exploring several temperatures, the study aims to provide a comprehensive understanding of how these factors affect planarian regeneration, subsequently offering valuable insights into possible mechanisms or processes involved (Deochand et al, 2018). This study involves measuring planarian regeneration in millimeters, as planarians are ideal sources to explore regeneration processes (Ermakova et al, 2009). There will be 3 different levels of the independent variable; planaria grown in an environment at 3 degrees Celsius with an addition of Vitamin A, planaria grown in an environment at 23 degrees Celsius with an addition of Vitamin A, and planaria grown in an environment at 37 degrees Celsius with an addition of Vitamin A. The planaria grown at 23 degrees Celsius with an addition of Vitamin A will be the control group of this experiment. These temperatures were selected because they are very different temperatures and could have a drastic effect on planaria regeneration, the self-renewal of neoblasts, tissue organization, neurogenesis (the growth and development of nerve tissue), and other cellular processes (Karge et al, 2020). 3 degrees Celsius is very close to freezing temperature, 23 degrees Celsius is very close to room temperature, and 37 degrees Celsius is very close to body temperature for humans. The dependent variable of planaria regeneration was measured in millimeters (mm). It is thought that planaria grown in an environment consisting of the temperature at 23 degrees Celsius (around room temperature) will result in the greatest growth of planaria. This is based on previous studies using a temperature close to room temperature in their experiments; room temperature is considered to be the optimal temperature for planaria regeneration (Sarkar et al, 2019). Most experiments use room temperature with another variable. Other temperatures haven’t been experimented with nearly as much, and their effect is not known with the addition of different variables on planaria regeneration.

Procedure

Safety equipment like gloves, safety goggles, and a pipette were prepared and safety regulations for Vitamin A were read, such as using safety goggles and not ingesting Vitamin A. Also, safety and care rules for planarians were read, such as wearing gloves and pointing knife away when cutting planaria. Safety equipment was used and worn, safety regulations for Vitamin A and planarian care were followed, loose clothing was secured, and hands were sanitized. Brown planaria were used in the experiment. A 4% concentration of Vitamin A was used in the experiment. This Vitamin A concentration was chosen because each location will contain one pill grinded and evenly distributed into each container (snack cup with lid), which is not too little to not affect planarian regeneration or too much to kill the planarians. A concentration of 4% of Vitamin A was diluted and added by grinding Vitamin A pills and mixing it to 25 mL of pond water. Then, 24 mL of pond water was put into each container, and 1 mL of diluted Vitamin A with pond water was put in each container. Then, the planaria were split into three equal groups of 25, with each group getting its own clean container. Each container was put in a temperature (23, 37, or 3 degrees Celsius) using an incubator, a fridge, or just placing the planaria containers in a garage. Carefully, 25 planarians were cut in half using an “X-Acto” knife. The bottom halves of the planaria were kept in a separate container; the top halves were used to measure regeneration. Carefully, 25 head halves of the planaria were each put in their respective containers. The initial length of the planaria, measured in millimeters, and when the experiment started were recorded. Then, the planaria were put in their groups of 3, 23, and 37 degrees Celsius. The temperature was maintained and kept steady for 14 days. The planaria were fed with pieces of earthworm every 7 days. The planaria were left for 14 days to regenerate in their own containers.

After 14 days, one planarian was slowly taken out of its container. The size of the planaria after their regeneration period was measured, in millimeters, with an electronic caliper. The process for measuring the planarians was duplicated for each planarian in each container and group. This procedure was rerun for 75 trials in total. Then, the data for the measurements of the planarians, the conditions the planarians were put in, and the finished date for the experiment was recorded. After the experiment was finished and the data was documented, the planaria were frozen for 48 hours and disposed of in a designated waste disposal plant. The Vitamin A was also disposed of in a waste disposal plant.

Results

This experiment explored the effect of different temperatures, combined with a constant concentration of vitamin A (4%), on planaria regeneration. The results of the statistical analysis were presented in table 2. The research hypothesis proposed that planaria placed in 23°C would regenerate the most. Quantitative data was collected, measuring the regeneration in millimeters at 3°C, 23°C (control), and 37°C. A statistical analysis of the average for each group revealed that the mean regeneration was higher at the control temperature of 23°C (3.3 mm) than at 3°C (1.0 mm) or 37°C (0.4 mm). This demonstrated that planaria regeneration is influenced by both temperature and vitamin A. Based on the results, the research hypothesis was supported, and it was found that planaria given a constant concentration of Vitamin A would regenerate most at 23°C.

The variance and standard deviation were calculated for each of the independent variable levels. The data showed that the 23°C control condition demonstrated the highest variance and standard deviation, indicating the most variation in planaria regeneration among all the independent variable groups. The results contained one outlier, which was trial 21 of the 23°C independent variable group, as a 6.2 millimeter regeneration of the planarian, found in table 1. This measurement of the regeneration of the planarian was outside of the 2 SD range of the 23°C independent variable group, which was 0.514 to 6.086, found in table 2.

A t-test was performed for the data, using a significance level of 0.001 and 48 degrees of freedom. This data had a null hypothesis, which stated that the effect of different temperatures with vitamin A had no significant difference on planaria regeneration. The calculated t-values for the 3°C vs. control (t=7.913), 37°C vs. control (t=10.233), and 3°C vs. 37°C (t=6.149) comparisons were all greater than the critical t-value found in the t-table (3.551). Consequently, the null hypothesis was rejected for all independent variable comparisons, indicating that the temperature did influence planaria regeneration when combined with vitamin A. The resulting probability values of the results being based on chance (p<0.001) helped the rejection of the null hypothesis, suggesting a less than 0.001/1 in 1000 chance of these results occurring by chance. This implied that the results were most likely due to the independent variable. Overall, this research revealed that different temperatures with a constant concentration of Vitamin A having an effect on planaria regeneration is statistically significant, and that 23°C/room temperature (control) helps planaria grow the most with a constant concentration of Vitamin A.

USE THESE: Works Cited:

Peer Reviewed

Deochand, N., Costello, M., & Deochand, Michelle. (2018, November 9). Behavioral Research with Planaria. Perspectives on Behavior Science, 41(2), 447-464. Retrieved October 17, 2023, from doi: 10.1007/40614-018-00176-w

Ermakova, O. N., Ermakov, A. M., Tiras, K. P., & Lednev V. V. (2009, December 3). Retinoic acid as a regulator of planarian morphogenesis. Russian Journal of Developmental Biology, 40(6), 449-455. Retrieved October 3, 2023, from

https://doi.org/10.1134/S106236040906006X

Lai, A., Kosaka, N., Abnave, P., Sahu, S., & Aboobaker, A. (2017). The abrogation of

condensin function provides independent evidence for defining the self-renewing population of pluripotent stem cells. Developmental Biology, 433(2), 218-226. Retrieved November 13, 2023, from doi: 10.1016/j.ydbio.2017.07.023

Sarkar, A Mukundan, N., Sowndarya, S., Dubey, V., Babu, R., Lakshmanan, V., Rangiah, K.,

Panicker, M., Palakodeti, D., Subramanian, S., & Subramanian, R. (2019). Serotonin synthesized by phenylalanine hydroxylase in the planarian eye is essential for eye regeneration. FEBS Letters, 593(22), 3198-3209. Retrieved October 23, 2023, from https://doi.org/10.1002/1873-3468.13607

Seebeck, F., März, M., Meyer, A. W., Reuter, H., Vogg, M. C., Stehling, M., Mildner, K.,

Zeuschner, D., Rabert, F., & Bartscherer, K. (2017). Integrins are required for tissue organization and restriction of neurogenesis in regenerating planarians. Development, 144(5), 795-807. Retrieved October 30, 2023, from https://doi.org/10.1242/dev.139774

Abel, C., Powers, K., Gurung, G., & Pellettieri, J. (2022). Defined diets for freshwater

planarians. Developmental dynamics: an official publication of the American Association of Anatomists, 251(2), 390–402. Retrieved December 30, 2023, from

https://doi.org/10.1002/dvdy.400

Karge, A., Bonar, N. A., Wood, S., & Petersen, C. P. (2020). tec-1 kinase negatively regulates regenerative neurogenesis in planarians. eLife, 9, e47293. Retrieved December 30, 2023, from https://doi.org/10.7554/eLife.47293

Non Peer Reviewed

Marie, A., Darricau, M., Touyarot, K., Parr-Brownlie, L. C., & Bosch-Bouju, C. (2021). Role

and Mechanism of Vitamin A Metabolism in the Pathophysiology of Parkinson's Disease. Journal of Parkinson's disease, 11(3), 949–970. Retrieved December 30, 2023, from https://doi.org/10.3233/JPD-212671

Ge, X. Y., Han, X., Zhao, Y. L., Cui, G. S., & Yang, Y. G. (2022). An insight into planarian regeneration. Cell proliferation, 55(9), e13276. Retrieved December 30, 2023, from https://doi.org/10.1111/cpr.13276

MANY PARAGRAPHS. Make me a conclusion for my research paper on The Effect of Different Temperatures on Planaria Regeneration following these instructions: To write a conclusion, you must address 7 major questions:

1. What was the purpose of the experiment?

2. What were the major findings, including the inferential statistics test results?

3. Was the hypothesis supported by the data? You NEVER prove your hypothesis- just if it is supported or not supported.

4. How did your findings compare with other researchers? Use your lit reviews and the information you have found.

5. What possible explanation can you offer for the findings? Why were the results significant or not significant?

6.How could this experiment have been improved? What were the sources of error and how could they have been fixed for future experimentation?

7. What recommendations do you have for further study?

Items to remember when writing the conclusion:

1. Use the present and past tense, third person and complete sentences

2. Times New Roman, 12 point font, 1 inch margins (top, bottom, left & right) and double spaced

3. Incorporate a minimum of 3 different references to prior research (use the lit reviews you have been doing and refer to the research you have discovered)

Example of a Conclusion: (this is an example and can use improvement)

The purpose of this experiment was to determine the effects of different fertilizers on plant growth. Pea plants were exposed to 3 different levels of the independent variable: no fertilizer, fertilizer A and fertilizer B and after 20 days, their growth was measured. A research hypothesis was formulated that if fertilizer B was used on a plant, it would grow more than fertilizer A or no fertilizer at all. It was found that fertilizer A had a greater impact on plant growth than fertilizer B and no fertilizer. Due to these results, the research hypothesis was not supported by the data but did show that fertilizer did have an impact on plant growth.

A t-test was performed on the data to determine if the data was significant. The data for the fertilizer having an effect on plant growth is both statistically and not statistically significant. The data for Fertilizer A vs. the control implies that the data is not statistically significant. The results that were obtained for Fertilizer A were most likely due to chance and error versus the independent variable. The data for Fertilizer B vs. the control implies that the data is statistically significant and the results were due to the independent variable and Fertilizer B does have an affect on plant growth.

Other researchers have investigated fertilizer A and fertilizer B. Smith and Anderson found that fertilizer A had a dramatic affect on spinach plants. They found that spinach plants treated with fertilizer A had a 22% increase in growth and mass than plants not treated with fertilizer (Smith & Anderson, 2006). Other studies have shown that fertilizer B can increase plant growth. One such study found that peanut plants treated with a 20% solution of fertilizer B produced twice as many peanuts as the plants not treated with fertilizer (Jones, 2005). The findings from this study were more similar to Smith and Anderson since the pea plants treated with fertilizer A had more growth than fertilizer B and no fertilizer.

There are a number of possible explanations for the results of this experiment. Although the data from fertilizer A vs. the control was not considered significant, there was a difference in the plant growth between fertilizer A and fertilizer B. Fertilizer A consists of a mixture of nitrogen and minerals and gives soil the ability to hold twice the amount of water than it normally would (Johnson, 2004). These factors create an ideal environment for pea plants which thrive on watery soil that is rich with nitrogen. A study which was performed by Cooper and Marsh found that pea plants treated with an abundance of nitrogen and water were 32% more likely to grow over 7 cm (Cooper & Marsh, 2005). While fertilizer A creates an ideal environment for the pea plants, fertilizer B may not. Fertilizer B consisted mainly of natural chemicals, such as manure and decomposed organic matter (Jones, 2005). While pea plants can grow in these conditions, they do not prefer decomposed organic matter as much as they prefer nitrogen and an abundance of water.

There were sources of error in the this experiment and the experiment could have been improved a number of ways. All of the plants were not exposed to the same amount of sunlight each day which could have affected the results and could have skewed the data to favor fertilizer A. In addition to the amount of sunlight, the plants were only given 20

days to grow. Fertilizer B may be a slow acting fertilizer and may have needed more time to affect the plants. Lastly, the directions for using fertilizer B indicated that each plant needed 5g but a constant was established that each plant would be treated with either 3g of fertilizer A or 3g of fertilizer B. The plants treated with fertilizer B may have grown more if they were given the recommended dosage. For continued study, the optimum amount of fertilizer A should be investigated. In this study, 3 g was used but the optimum dosage may be more or less than 3g. In addition to the optimum amount of fertilizer A, the effects of fertilizer A on other legume plants should be studied.

ALSO USE THESE: The Effect of Different Temperatures with Vitamin A on Planaria Regeneration

Abstract

This study aimed to elucidate the effect of temperature and Vitamin A on the regenerative abilities of planaria, emphasizing their resemblance to human stem cells. The hypothesis proposed that planarians subjected to 23°C and supplemented with Vitamin A would exhibit the most regeneration. The experiment involved three temperature conditions, 3°C, 23°C (control), and 37°C, with a constant Vitamin A concentration of 4%. The measurement technique was the length of planarian regeneration in millimeters after 14 days. Safety protocols for handling planarians and Vitamin A were adhered to in the experiment. Groups of 25 planarians were bisected through the middle, and regeneration of the anterior parts of the planaria were measured. Each group was placed in the specified temperature conditions for 14 days, with regeneration length measured using an electronic caliper. Statistical analysis included t-tests and calculation of variance and standard deviation. Findings revealed the most substantial regeneration at the control temperature of 23°C, showcasing an average regrowth of 3.3 millimeters, as opposed to 1.0 millimeters at 3°C and a notably lower mean of 0.4 millimeters at 37°C, confirming the research hypothesis. The statistical significance was supported by all t-values exceeding the critical t-value, rejecting the null hypothesis across all temperature comparisons. Consequently, the study concludes that temperature, with Vitamin A supplementation, significantly impacts planarian regeneration, with room temperature proving most conducive for regenerative processes for planaria. These observations suggest that environmental and nutritional factors intertwine to significantly influence regenerative outcomes in planaria using neoblasts and humans using stem cells.

Introduction

The process of regeneration in planarians attracts researchers due to planarians’ extraordinary ability to regrow lost parts under various conditions (Seebeck et al, 2017). This current study investigates how different temperatures with Vitamin A impact planarian regeneration. Including different temperature ranges and constant levels of Vitamin A in the experimental design is chosen to simulate various environmental conditions and constant nutrient supplementation scenarios (Deochand et al, 2018). Consequently, the research explores how these variables affect the self-renewal ability of pluripotent stem cells using neoblasts (Lai et al, 2017). Planaria serve as model systems for embryonic stem cells in humans, driving the choice of this study. It contains real-world implications and explores a topic that assists planaria to become better models for human stem cells (Ermakova et al, 2009).

The potential implications of this research promise broader societal implications (Deochand et al, 2018). The findings may reveal information applicable in regenerative medicine, tissue engineering, and developmental biology (Sarkar et al, 2019). Insights from this research may prove beneficial to human health, for instance, in providing crucial knowledge for the development of regenerative therapies & regenerative medicine. Furthermore, this research holds the potential to assist advances in regenerative medicine by uncovering the fundamental mechanisms/functions managing and aiding regeneration in planarians (Ermakova et al, 2009).

The independent variable in this study encompasses understanding different temperatures and varying Vitamin A concentrations (Sarkar et al, 2019). This includes the impact of different temperatures (3, 20, & 37 degrees Celsius) on planarian survival and how Vitamin A potentially influences cellular processes, primarily around tissue and stem cell regeneration (Sarkar et al, 2019). Vitamin A, an external factor added to the planaria, is usually already present in the human body due to a daily diet containing a certain amount of Vitamin A. Vitamin A improves and increases vision, growth, cell division, reproduction and immunity. Vitamin A is used for cell differentiation and embryonic development. It also decreases the risk of heart disease, cancer, and skin diseases (Marie et al, 2021). Adding Vitamin A to planaria assists the planaria to become better models for regeneration applications in humans and the real world.

Understanding planarian regeneration, tissue regeneration, and neurogenesis becomes crucial for the dependent variable (DV) (Sarkar et al, 2019). This knowledge aids in understanding the processes involved in planarian tissue repair and the role of neoblasts in

pluripotent stem cells in regeneration. Pluripotent stem cells means that the stem cell can divide into any cell type. Neoblasts, which are adult stem cells that can divide, have the role of dividing and transforming into any cell type such as red blood cells, white blood, cells, platelets, and more (Lai et al, 2017). There are neoblasts spread across the bodies of planaria, in fact, they make up about 25%-30% of all the cells in planaria (Ge et al, 2022). In other words, neoblasts are a type of pluripotent stem cells. Planaria also show better regeneration than other animals that exhibit regeneration such as zebrafish, axolotls, and spiny mice because they contain plenty more neoblasts and pluripotent stem cells than other organisms (Deochand et al, 2018).

The purpose of this study is to understand the impact of different temperatures with Vitamin A on planarian regeneration (Ermakova et al, 2009). By observing the regenerative capacity of planarians under varying experimental conditions, the research seeks to conclude on the possible effects on tissue organization, neurogenesis, and stem cell populations (Seebeck et

al, 2017). It is hypothesized that the variations in temperature, combined with Vitamin A supplementation, will have different effects on planarian regeneration (Ermakova et al, 2009).

The temperature of 23 degrees Celsius with a 4% concentration of Vitamin A is thought to increase the regeneration of the planaria the greatest. This is because planaria cultures, which are ideal habitats for growing planaria, are maintained at optimal temperatures around 23 degrees Celsius or room temperature. Also, many studies observe and utilize room temperature, typically around 23 degrees Celsius as the optimum temperature for planaria growth (Sarkar et al, 2019). 

Specifically, conditions of 23 degrees Celsius supplemented with Vitamin A are anticipated to be ideal for planaria and to support pluripotent stem cells' self-renewal ability using neoblasts, potentially resulting in the most effective planarian regeneration. This hypothesis is backed by previous studies highlighting the crucial role of environmental factors such as high humidity, low light, and a 6.0 - 7.0 (slightly acidic) pH (Ge et al, 2022). The hypothesis is also supported by nutrient supplementation play, such as glycogen and dextrose, provided by hard-boiled egg yolk, pieces of earthworm, or beef liver (Abel et al, 2022). These factors influence the developmental and regenerative processes in various organisms, including planarians (Deochand et al, 2018). It is also supported by studies indicating that planaria grow best around room temperature in a dark environment (Sarkar et al, 2019).

The independent variable in this study comprises the different temperature conditions (3, 20, and 37 degrees Celsius), with a constant Vitamin A concentration (4%). These temperatures were chosen to test how planarian regeneration and cellular processes would increase or decrease due to drastic differences in temperature of environment of the planaria; with the addition Vitamin A. This concentration of Vitamin A was chosen because it is known to not be too toxic to the planaria, but it was not too little to not have any effect on the planaria. The control group is set at 23 degrees Celsius because it is room temperature, and the temperature of the environment isn't changed. The dependent variable is planarian regeneration, determined through the length of regeneration of planaria, measured in millimeters (mm). Different temperatures with a constant concentration of Vitamin A can also affect tissue organization, neurogenesis, and the self-renewal ability of pluripotent stem cells since the change of temperature is an environmental factor change and the addition of Vitamin A is a nutritional/supplemental factor change. (Lai et al, 2017).

The purpose of this experiment is to determine if different temperatures with an addition of Vitamin A have an effect on planaria regeneration. By exploring several temperatures, the study aims to provide a comprehensive understanding of how these factors affect planarian regeneration, subsequently offering valuable insights into possible mechanisms or processes involved (Deochand et al, 2018). This study involves measuring planarian regeneration in millimeters, as planarians are ideal sources to explore regeneration processes (Ermakova et al, 2009). There will be 3 different levels of the independent variable; planaria grown in an environment at 3 degrees Celsius with an addition of Vitamin A, planaria grown in an environment at 23 degrees Celsius with an addition of Vitamin A, and planaria grown in an environment at 37 degrees Celsius with an addition of Vitamin A. The planaria grown at 23 degrees Celsius with an addition of Vitamin A will be the control group of this experiment. These temperatures were selected because they are very different temperatures and could have a drastic effect on planaria regeneration, the self-renewal of neoblasts, tissue organization, neurogenesis (the growth and development of nerve tissue), and other cellular processes (Karge et al, 2020). 3 degrees Celsius is very close to freezing temperature, 23 degrees Celsius is very close to room temperature, and 37 degrees Celsius is very close to body temperature for humans. The dependent variable of planaria regeneration was measured in millimeters (mm). It is thought that planaria grown in an environment consisting of the temperature at 23 degrees Celsius (around room temperature) will result in the greatest growth of planaria. This is based on previous studies using a temperature close to room temperature in their experiments; room temperature is considered to be the optimal temperature for planaria regeneration (Sarkar et al, 2019). Most experiments use room temperature with another variable. Other temperatures haven’t been experimented with nearly as much, and their effect is not known with the addition of different variables on planaria regeneration.

Procedure

Safety equipment like gloves, safety goggles, and a pipette were prepared and safety regulations for Vitamin A were read, such as using safety goggles and not ingesting Vitamin A. Also, safety and care rules for planarians were read, such as wearing gloves and pointing knife away when cutting planaria. Safety equipment was used and worn, safety regulations for Vitamin A and planarian care were followed, loose clothing was secured, and hands were sanitized. Brown planaria were used in the experiment. A 4% concentration of Vitamin A was used in the experiment. This Vitamin A concentration was chosen because each location will contain one pill grinded and evenly distributed into each container (snack cup with lid), which is not too little to not affect planarian regeneration or too much to kill the planarians. A concentration of 4% of Vitamin A was diluted and added by grinding Vitamin A pills and mixing it to 25 mL of pond water. Then, 24 mL of pond water was put into each container, and 1 mL of diluted Vitamin A with pond water was put in each container. Then, the planaria were split into three equal groups of 25, with each group getting its own clean container. Each container was put in a temperature (23, 37, or 3 degrees Celsius) using an incubator, a fridge, or just placing the planaria containers in a garage. Carefully, 25 planarians were cut in half using an “X-Acto” knife. The bottom halves of the planaria were kept in a separate container; the top halves were used to measure regeneration. Carefully, 25 head halves of the planaria were each put in their respective containers. The initial length of the planaria, measured in millimeters, and when the experiment started were recorded. Then, the planaria were put in their groups of 3, 23, and 37 degrees Celsius. The temperature was maintained and kept steady for 14 days. The planaria were fed with pieces of earthworm every 7 days. The planaria were left for 14 days to regenerate in their own containers.

After 14 days, one planarian was slowly taken out of its container. The size of the planaria after their regeneration period was measured, in millimeters, with an electronic caliper. The process for measuring the planarians was duplicated for each planarian in each container and group. This procedure was rerun for 75 trials in total. Then, the data for the measurements of the planarians, the conditions the planarians were put in, and the finished date for the experiment was recorded. After the experiment was finished and the data was documented, the planaria were frozen for 48 hours and disposed of in a designated waste disposal plant. The Vitamin A was also disposed of in a waste disposal plant.

Results

This experiment explored the effect of different temperatures, combined with a constant concentration of vitamin A (4%), on planaria regeneration. The results of the statistical analysis were presented in table 2. The research hypothesis proposed that planaria placed in 23°C would regenerate the most. Quantitative data was collected, measuring the regeneration in millimeters at 3°C, 23°C (control), and 37°C. A statistical analysis of the average for each group revealed that the mean regeneration was higher at the control temperature of 23°C (3.3 mm) than at 3°C (1.0 mm) or 37°C (0.4 mm). This demonstrated that planaria regeneration is influenced by both temperature and vitamin A. Based on the results, the research hypothesis was supported, and it was found that planaria given a constant concentration of Vitamin A would regenerate most at 23°C.

The variance and standard deviation were calculated for each of the independent variable levels. The data showed that the 23°C control condition demonstrated the highest variance and standard deviation, indicating the most variation in planaria regeneration among all the independent variable groups. The results contained one outlier, which was trial 21 of the 23°C independent variable group, as a 6.2 millimeter regeneration of the planarian, found in table 1. This measurement of the regeneration of the planarian was outside of the 2 SD range of the 23°C independent variable group, which was 0.514 to 6.086, found in table 2.

A t-test was performed for the data, using a significance level of 0.001 and 48 degrees of freedom. This data had a null hypothesis, which stated that the effect of different temperatures with vitamin A had no significant difference on planaria regeneration. The calculated t-values for the 3°C vs. control (t=7.913), 37°C vs. control (t=10.233), and 3°C vs. 37°C (t=6.149) comparisons were all greater than the critical t-value found in the t-table (3.551). Consequently, the null hypothesis was rejected for all independent variable comparisons, indicating that the temperature did influence planaria regeneration when combined with vitamin A. The resulting probability values of the results being based on chance (p<0.001) helped the rejection of the null hypothesis, suggesting a less than 0.001/1 in 1000 chance of these results occurring by chance. This implied that the results were most likely due to the independent variable. Overall, this research revealed that different temperatures with a constant concentration of Vitamin A having an effect on planaria regeneration is statistically significant, and that 23°C/room temperature (control) helps planaria grow the most with a constant concentration of Vitamin A.

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