Fish on the Atypical Antipsychotic Seroquel and their Performance on an Avoidance Test

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Fish on the Atypical Antipsychotic Seroquel and th eir Performance on an Avoidance Test By Jessica E. Burgan A thesis Submitted to the division of natural sciences New College of Florida In partial Fulfillment of the requirements for the degree Bachelor of Arts under the sponsorship of Dr. Alfred Beulig Jr. Sarasota, Florida May, 2010

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Fish on Seroquel 2 Acknowledgements Without the help and guidance from my baccalaureate committee this thesis would not have been possible so I would like to thank Dr. Alfred Beulig, Dr. Leo Demski, and Dr. Gordon Bauer. I would like to thank my parents for their continual support and inspiration. Thank you to my three amazing sisters Julie, Michelle and Lynn for always being there I would also like to thank my two closest friends Brittany and Karin for always being supportive and understanding.

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Fish on Seroquel 3 Table of Content Dedications and Acknowledgements Abstract Introduction Stress Stress and animal learning 7 Animal Paradigms in the study of drug effects 9 Stress and cognition 11 Cortisol and the connection between the endocrine, immune, and nervous system .12 Seroquel ...16 Methods 20 Subjects 20 Housing 20 Aquaria 22 Feeding 23 Shuttle box 23 24 Results 28 Discussion 32 References 36 Appendix: Avoids 40

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Fish on Seroquel 4 Appendix:Escapes 4 2 Appendix: Avoid latencies 4 4 Appendix: Escape latencies 4 6 Appendix: Fails .4 8 Appendix: Fish weight and length 5 0

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Fish on Seroquel 5 Fish on the Atypical Antipsychotic Seroquel and th eir Performance on an Avoidance Test Jessica Burgan New College of Florida, 2010 Abstract Seroquel is an antipsycho tic drug that may have a positive effect on cognition. The shuttlebox avoidance task is a way to measure cognition. The goal of this experiment is to determine whether the drug Seroquel has any effect on shuttlebox avoidance A possible mode of action of Seroquel is the reducti on of stress and cons equent enhancement of cognitive ability and may have clinical significance. In this study chronic stress was produced by the injection of cortisol. In this study sixty four subjects were randomly assigned to four groups of 16 fish: 1) fish given Seroquel in aquarium water and injected with cortisol; 2) fish given Seroquel only and injected with water; 3) fish injected with cortisol; 4) control group injected only with water. The hypothesis was that the experimental fish given the Seroquel would perform a greater number of avoids than fish not given Seroquel The results showed no significant difference in performance between groups given Seroquel and groups not given Seroquel However, the fish injected with cortisol performed significantly better than the control group on Seroquel with water. These results may indicate that the goldfish used in this experiment were less affected by cortisol in producing stress due to being do mesticated this may have provide d an optimal amount of stress for avoidance performance Dr. Alfred Beulig Jr. Division of Natural Sciences

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Fish on Seroquel 6 Introduction It has been demonstrated that stress has a negative effect on learning and performance in learning and memory tasks (Mendl, 1999). However, the various stressful conditions may have different effects on learning and there may be means to reduce the effects of stress on learning by stress reduction techniques. There are many relaxation techniques that have been suggested and many have been successful (Carrington, 1980). However, some individuals do not respond well to these approaches and there may be alternative means to address their needs. A characterization of stress mechanisms is needed in order to provide a background for potential evaluation of stress reducing approaches that ma y have clinical significance. Stress In 1940 the Adaptation Syndrome and the Alarm Reaction were relatively new concepts. The proposal that stress could be defined as a general, quantifiable reaction found in all organisms was not well received by many scientists, of the time (Selye, 1946). Stress was defined as the interaction between d amage and defense. Every stress or is also responsible for producing certain specific actions. General Adaptation S yndrome (GAS) is the defense response of the body or th e psyche to injury or prolonged stress. It consists of an initial stage of shock or Alarm Reaction, followed by the Resistance Phase in which adaptation occurs by means of the various defense mechanisms of the body or mind, and culminates in a state of adj ustment and healing or if these mechanisms are overcome, a state of exhausti on and

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Fish on Seroquel 7 disintegration. General Adaptation S yndrome integrates a number of different observations that seem unrelated into a single biologic system (Selye, 1950). This is a holistic view of the importance of maintaining homeostasis. When connections are made between physiological and pathological events then there is a better understanding of how to treat disease (Selye, 1950). Stress and Animal Learning A study done by Mendl (1999 ) found that Stressors can cause shifts, lapses and narrowing of attention. Stress can also influence decision speed, which is important to performance under stress. The hypothalamic pituitary adrenal (HPA) axis mediates responses that are considered part of the GAS and also play a role in memory formation (Mendl, 1999). It has been proven that many animals (pigs, sheep, cattle and deer), when agitated do not make definite choices in preference or avoidance tests (Mendl, 1999) A study showed that mice aft er a passive shock avoidance test showed impaired recall of the avoidance response 24 hours later (Mendl, 1999). Pigs also have shown disturbances in spatial memory performance when exposed to stressors like social isolation (Mendl, 1999). There is a limit to how many complex tasks an animal can perform efficiently at one time. The high levels of attention needed to do so cannot be maintained indefinitely, and under conditions of stress, exhaustion comes sooner (Mendl, 1999). Later in this

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Fish on Seroquel 8 paper it will be shown that Seroquel is able to help people focus on one stimulus, which could help performance of complex tasks, and could postpone the onset of exhaustion. Another experiment carried out by Arnsten (1998) examined the effect of noise induced stress on pr efrontal cortical function in monkeys. The basis of this experiment is that previous studies have shown that stress can increase the symptoms of many different disorders, especially psychiatric disorders, and many of the psychiat ric disorders are highly lo caliz ed in the prefrontal cortex (Arnsten, 1998). Spatial working memory tasks that depend on prefrontal cortex were combined with a loud noise (105 db). Under the same conditions, a reference memory task was also administered which requires visual pattern discrimination and depends on the inferior temporal cortex. Agents that decrease dopamine receptor stimulation were used to determine the role dopamine mechanisms may have in the stress response. The exposure to noise stress in monkeys significantly impai red delayed response performance. However, stress did not impair performance on the no delay control trials and did not alter visual pattern discrimination performance. The results led to the overall conclusion that stress impairs prefrontal cortex functio n (Arsten, 1998). Cognitive function was negatively affected by a hyperdopaminergic mechanism that acted to disrupt habitual responses mediated by posterior cortical and subcortical structures. The facilitative function of the prefrontal cortex was interru pted (Arnsten, 1998).

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Fish on Seroquel 9 Animal Paradigms in the Study of Drug Effects Animal paradigms have been used in previous studies to test how well atypical antipsychotics restore diminished prepulse inhibition (PPI). PPI is a measure of sensorimotor gating PPI occurs when a relatively weak sensory event is presented 30 500 ms before a strong startle inducing stimulus and reduces the magnitude of the startle response A st udy by Geyer (2001) used rats in their experimental paradigm. They mimicked the signs of s chizophrenia by recreating a symptom of schizophrenia that would diminish PPI (Geyer, 2001). One major symptom that patients with schizophrenia experience is the inability to properly gate sensorimotor stimuli which cause diminished PPI This causes a floo ding effect and the patient cannot filter and inhibit the overflow of stimuli, which people with regular levels of PPI can. This causes an increase in the startle response (Geyer, 2001). This study selected a measure sensitive to one aspect of a diagnosti c feature of schizophrenia tha t could be reliably quantified (Geyer, 2001). An animal paradigm was used by Mansbach et al. (1998), which led to a breakthrough in research on schizophrenia. They were the first researchers who used systematic administratio ns of apomorphine, a dopamine agonist, and amphetamine in intact rats. This did lead to disruptions in prepulse inhibition and with this technique a new gro up of atypical antipsychotics (Clonapine, Abilify, Zyprexa, R isperidone, and Seroquel ) could be compared with the older gr oup of typical antipsychotics (H aldol). The Mansbach et al. study (1998) was based on evidence that dopamine D 4 receptors play a role in schizophrenia due to their antagonist actions. Antipsychotics have D 4 dopamine

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Fish on Seroquel 10 receptor antag onist properties and could be assessed with their regard to their ability to restore diminished PPI in an animal paradigm (Mansbach, 1998). The success of this animal paradigm was the basis of the rationale for the Geyer (2001) experiment in which they dec ided to look at the direct effect that atypical antipsychotics may have had on PPI. Previous studies have mostly focused on the antipsychotics were administered directly into th e ventral pallidal region of rats and this resulted in a more robust increase in PPI. This study resulted in two important findings, that animal models are useful in studying antipsychotic drug effects, and that the atypical antipsychotics were the most ef ficient category of drugs able to restore PPI (Geyer, 2001). Newer research has further verified these results. In rats, PPI is reduced or eliminated by the psychotomimetic non competitive glutamate antagonist phencyclidine (PCP). The effects of PCP are n ot reversed by antipsychotics such as haloperidol but are reversed by atypical agents such as clonazipine, olanzapine, and quetiapine (Goldstein, 1999 ). In rats PPI is disrupted by systemic administration of dopamine agonists, serotonin agonists, or glutam ate antagonists and by a variety of surgical or pharmacological manipulations of neural circuitry linking the limbic cortex, striatum, pallidum, and pontine reticular formation (Swerdlow, 1998).These same systems are involved in cognitive learning and memo ry. Furthermore, they are involved in arousal, emotional behavior, and stress response.

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Fish on Seroquel 11 Stress and cognition Cognition is affected by stress in a number of ways, acting rapidly via catecholamines and more slowly via glucocorticoids. Brief periods of stress can potentiate memory formation. However, more severe or prolonged stressors can have deleterious effects upon broad aspects of cognition. Irreversible loss of hippocampal neurons can result from truly prolonged exposure to stress. The loss of hippo campal neurons in correlation to prolonged stress may be relevant to the cognitive deficits seen in many aged individuals (McEwen, 1995) function under stress. The corticosteroids divert energy supply t o challenged tissues and control excitability of the neuronal networks that are fundamental to learning and memory processes. Corticosteroids promote the interpretation and the storage of novel information while mediating the extinction of behavior that is no longer relevant. However, corticosteroids can turn maladaptive when actions via the two corticosteroid receptor types are imbalanced for too long. In humans, corticosteroids have been mostly found to disrupt memory formation (Kloet,1999). In prior rese arch rainbow trout did worse on memory formation tasks when given cortisol (Barreto, 2006). The shuttle box avoida nce test measures the ability of an organism to form associations. Since stress is known to lower cognition, performance on a cognitive test can be used as an indicator of stress. Therefore an increase in performance on the shuttle box avoidance test could be an indicator that stress had been reduced.

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Fish on Seroquel 12 Cortisol and the connection between the endocrine, immune, and nervous system In fish, the primary response to acute stress is an elevated level of cortisol in plasma. It is not clear if cortisol acts directly or indirectly on immune cells but what is known is that it causes lymphocytopenia and neutrophilia (Espelid, 1996). James Harris and Davi d Bird (2000) looked at the effect that cortisol, reproductive hormones, growth hormones and prolactin, and some proopiomelancortin derived peptides had on the immune endocrine interaction. Stimulatory (+) and inhibitory ( ) effects of various hormones o n immune responses in teleost fish were documented: Table 1 ______________________________________________________________________ Hormone Phagocytosis Respiratory burst Mitogenesis Cortisol GH + + + PRL + + + 11 Ketotestosterone Oestradiol + MSH + + +/ MCH + + + EP + +

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Fish on Seroquel 13 ACTH + In humans the link between the immune system and stress has been studied. As part of the stress response the hypothalamic pituitary adrenal axis is activated, which over time can impair the immune system and can be a contributing factor to the development and progression of certain cancers (Vissoci, 2004). Several cellular and molecular immunological factors are compromised when there is chronic stress and depression (Vissoci, 2004). Both stressors and depression are associated with decreased cytotoxic T ce ll and natural killer cell activities that affect immune processes including the immune surveillance of tumors and with the events that modulate the development and accumulation of somatic mutations and genomic instability (Vissoci, 2004). The neuroendocr ine and immune system share common signal mediators and receptors that suggests that the brain has an immunoregulatory role and the immune system has a sensory function (Vissoci, 2004). The interaction between emotions and immune functions may play a part in increased susceptibility to infectious diseases or malignant tumor. It would then make sense that diseases that greatly activate the immune system, such as trauma, sepsis, and autoimmune disorders, can include psychopathological manifestations (Vissoci, 2004). Over the past 15 years the notion of functional autonomy of the regulatory functions of the immune, endocrine, and nervous system have been successfully challenged (Ader, 1995). Each system has evolved so that it can r espond to specific stimuli th at originate from the internal or external environment. The ultimate conclusion is that the immunoregulatory processes are part of an integrated system o f defense (Ader,

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Fish on Seroquel 14 1995). This le d to the development of a new area of study: psychoneuroimmunology, the study of the behavioral neural endocrine immune system interaction. A clinical implication of these findings is that when studying a disease or illness it canno t be written off as a disorder a ffecting just on e body system (Ader, 1995). A ll these systems mu st be taken into account when treating an illness (Ader, 1995). In 2004 a meta analysis review was done on 208 laboratory studies on acute psychological stressors (Dickerson, 2004). This was to test a theoretical model delineating conditions capable of eli citing cortisol responses. Consistent with most articles, this study also c oncluded that cortisol levels rise whenever there are psychological stressors (Dickerson,2004). Logically enough they also found that different stressors elicited different cortisol reactions (Dickerson, 2004). The tasks that were associated with the largest cortisol and adrenocorticotropin hormone changes and had the longest recovery times, were tasks that included both uncontrollable and social evaluative components. The conclusion reinforced the belief that uncontrollable social threats had but contradicted the belief that all types of stressors had an effect on cortisol production (Dickerson, 2004). The literature re viewed were studies of both animals and humans and specifically f ocused on articles that discussed how psychological factors can influence the HPA axis. Of all the studies done, only two concrete conclusions emerged (Dickerson, 2004). The first of which is that physical stressors, like electrical shock and prolonged exe rcise, psychological stressors are capable of activating the HPA axis. Second of which is that

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Fish on Seroquel 15 the effects of psychological stressors on the physiological system are highly variable (Dickerson, 2004). In animals, research has supported the assertion that there could be stressor specific pathways to cortisol activation (Dickerson, 2004). However, this has not been supported in human studies. An example is that systemic stressors have been differentiated from psychological stressors when it comes to their ne ural correlates and downstream physiological effects, including the activation of components of the HPA system (Dickerson, 2004). It has also been found that distinctive physiological correlates have been found for different stress relevant behavioral patt erns in animals. The behaviors include fighting, fleeing, and submitting. However, in humans such distinctions in differentiation is not clear. There is a sequence of events that make psychological stressors so important to study. A psychological stressor may activate specific cognitive and affective processes in c ertain areas of the brain that m ay have far reaching effects (Dickerson, 2004). Brain regions important in these processes are the frontal lobes and the thalamus where the integration of sensory information starts. At this point the thalamus and frontal lobes evaluate or appraise the significance or the meaning of environmental stimuli (Dickerson, 2004). This potentially leads to emotional responses made possible by the connections of the prefront al cortex and limbic system. The structures of the limbic system that are connected to the hypothalamus are the primary pathways for the activation of the HPA axis.

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Fish on Seroquel 16 The HPA axis is crucial for normal physiological functions and regulating other systems. C ortisol plays a role in metabolism by mobilizing energy resources. This is accomplished by elevating blood glucose levels. The elevation of glucose levels is done by stimulating the conversion of amino acids and other substances to glucose in the liver, al so by promoting the breakdown of protein and fat storages in the tissue. This is how adequate metabolic functions can be carried out with the help of energy reserves being released (Dickerson, 2004) The amount of research conducted on the links and commu nication between different body systems is a testament to how complicated they are and how important they are to discover. Use of a fish model system has implications that go far beyond the mere maintenance of humane conditions for farmed fish, but also ar e important in the study of disease prevention and treatment in humans With so many bod il y systems that can potentially cause a detrimental domino effect to multiple other systems just from the exposure to stress or physical trauma, it is crucial to conti nue this line of research. That was the inspiration for my thesis. Seroquel There are six prescription medications in the class of second generation atypical antipsychotics: Zyprexa, Seroquel Abilify, Geodon, Clozaril, and Risperdal. There were certain r equirements of the medication to be included in this experiment: (1) it had to be water soluble so that i t could be given to the goldfish (2) the percentage absorbed ha d to be reliably predictable, (3 ) and the drug had to be safe for the fish.

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Fish on Seroquel 17 Clozaril was ruled out because there were too many intervening variables that could not be controlled for, which include the requirement of a normal EKG and initial blood cell count before it can be prescribed in addition to blood cell counts every week for the fir st six months of treatment. Clozaril has a high risk of causing agranulocytosis which is a severe reduction in the number of white cells (basophils, eosinophils, and neutrophils). Neutropenia results, whereby the body is severely depleted in its ability to defend itself against infection (Anderson, 2002). Geodon was also ru led out because there was no method of consistently predicting how much of the drug was absorbed since it depends on the amount of food consumed. The next two drugs, Zyprexa and R isperdal were eliminated because they are not water soluble. In order to administer the medication it would have to be dissolved in water. This left two possible dr ugs, A bilify and Seroquel Since a fish is considerably smaller than a human, it was vitally import ant to find a drug that has the smallest chance of resulting in a fatality in the case of an overdose. Seroquel (Quetiapine fumarate), when compared to Abilify, requires a considerably larger dosage to cause adverse side effects. Seroquel is a white to of f white crystalline powder which is moderately soluble in water. To introduce this medication into the fish it must first be crushed to be able to measure the significantly smaller dose than would be appropriate for a fish when compared to the dosage appro priate for human consumption. The mul tiple dose pharmacokinetics of Seroquel are dose proportional within the proposed clinical dose range, and Seroquel accumulation is predictable upon multiple dosing (Quetiapine, 2007).

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Fish on Seroquel 18 The highest recommended dose in h umans is 800 mgs per day, but it has been known to be effective at 300 mgs per day (Quetiapine, 2007). There is no dosage difference between genders or different weight ranges. So the dose used does not have to be precise because the fish will not die from the level given, and the weight difference of the fish does not factor into the efficacy of the dose given as long as the same dose is given to each fish (Quetiapine, 2007). There have been reported overdoses of up to 30 grams without fatality with full r ecovery in humans Due to relative safety of high doses it can be somewhat saf ely assumed that administering Seroquel to a fish will not result in death even considering the significant weight differences between fishes and humans. However this is not abso lutely known, but as mentioned before it is the safest one to be consumed in large dosages (Quetiapine, 2007). Elimination of quetiapine is mainly via hepatic metabolism with a mean terminal half life of about 6 hours within the proposed clinical dose rang e. Steady state concentrations are expected to be achieved within two days of dosing. Quetiapine fumarate is rapidly absorbed after oral administration, reaching peak plasma concentrations in 1.5 hours (Quetiapine, 2007). The tablet formulation is 100% bio available relative to solution. Quetiapine is widely distributed throughout the body with an apparent volume of distribution of 10 plus or minus 4 L/kg. Following a single oral dose of quitapine, less than 1% of the administered dose was excreted as unchan ged drug, indicating that quetiapine is highly metabolized. Quetiapine is extensively metabolized by the liver (Quetiapine, 2007). The major metabolic pathways are sulfoxidation to the sulfoxide metabolite and oxidation to the parent acid metabolite; both metabolites are pharmacologically inactive. In vitro studies using human liver

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Fish on Seroquel 19 microsomes revealed that the cytochrome P450 3A4 isoenzyme is involved in the metabolism of quetiapine to its major, but inactive, sulfoxide metabolite (Quetiapine, 2007). The organ distribution of microsomal cytochrome P 450 isoenzymes (P 450), microsomal epoxide hydrolase (EH) and cytosolic glutathione S transferases was investigated by immunoblotting (Waziers, 1990) and enzyme measurements in rats and humans. In rats, P 450 IA1 was detected only in the duodenum, and P 450 IA2 and IIC11 were detected only in the liver. The highest concentrations of P 450 IIB1/B2 were found in the lung and in the duodenum; pentoxyresorufin O dealkylase activity was closely correlated with the amounts of P 450 IIB1/B2 in the different organs. P 450 IIE1 was present in liver, kidney and lung, whereas EH was found in liver, intestine and kidney (Waziers, 1990). This is important in order to take the results found in the animal model and apply it t o humans. If a population of patients who are in an almost constant state of emotional stress are administered second generation atypical antipsychotics and the stress is not alleviated, then it may be suggested that a different class of prescription medic ation may be more successful For example, anxiolytic ( Alprazolam/Xanax; Chlordiazepoxide/Librium; Clonazepam/Klonopin; Diazepam/Valium; Lorazepam/Ativan) drugs or mood stabilizers (Carbamazepine/Tegretol; Lamotrigine/Lamictal; Valproic acid/Depakene). B ased on the assumption that fish perform cognitive functions worse while stressed it was expected that Seroquel could help an organism function under stress and therefore improve performance on a cognitive test. In contrast the expected outcome for

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Fish on Seroquel 20 the two groups of fish given cortisol was to experience a higher level of stress, which would cause lower performance on a cognitive task since prior research has shown that cortisol negatively a ffects cognition. Furthermore, it was expected that the group gi ven cortisol with Seroquel would do better than the group of fish given cortisol since prior research has shown that Seroquel restored cognitive function in rats with diminished PPI and the parts of the brain involved with PPI are involved with memory. M ethods Subjects This experiment was conducted with a total of 64 carassius auratus specifically comets and better known as goldfish Half were used for the first 16 days of testing and the other half were used in the second 16 days of testing. Before test ing on the fish began each one was quarantined. To make sure that all the fish had the same level of health, testing started a week after receiving the fish to make sure that all the fish used were eating and healthy. There was one death in both the NSNC ( no Seroquel no cortisol) and the YSNC (yes Seroquel no cortisol) groups. The fish were purcha sed from Seascape Aquarium and Pet C enter. Housing There is debate over how much fish can feel. One popular view is that fish have not evolved the salient biological characteristics that are believed to be necessary in order

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Fish on Seroquel 21 to have sentience (Chandroo, 2004). The prevalence of this view is evident in the lack of a level of animal welfare of farmed fish. The contradicting view is that due to anatomical, pharmacological and behavioral data there is reason to believe that there is a legitimate reason to be concerned with the lack of a standard of care when it comes to farmed fish (Chandroo, 2004). The aforementioned data goes as far to be a basis to the conclusion that the affective states of pain, fear and stress are likely to be experienced by fish in similar ways as in tetrapods (Chandroo, 2004). This gives a different insight when it comes to answering one important question, can fish suffer? Every precaution to maintain water quality and the overall health of the fish was taken, but they were still exposed to stressful testing. The article (Chandroo, 2004 ) suggesting that there is a level of pain perception in fish does bring up the problem that there is not enough research to conclusively prove their hypothesis. Preliminary examination of neurological, pharmacological and behavioral evidence supporting po ssible pain perception at the very least warrants further research on the topic. This topic has been greatly overlooked when it comes to research (Chandroo, 2004). Granted my experiment does not involve farmed fish, it does have relevance to the aspect of stress in my experiment potentially being psychological in addition to biological. It does however add credence to my hypothesis that fish given Seroquel will do better during testing that is stressful since Seroquel deals more with the psychological stre ss that is associated with the presence of a mental disorder. Since it is not certain how much psychological stress fish can experience, for the purpose of this experiment stress is defined as a purely biological event induced by stress

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Fish on Seroquel 22 hormones to ensure consistency. These subjects were handled in conformity with IACUC protocol #345 9 University of South Florida, Division of Research Compliance and I ntegrity, Tampa, Florida. Aquaria The goldfish were held in twelve 20 gallon tanks. Each tank had two partit ions with holes in them small enough so that the fishes could not pass through the holes but they were big enough so that the fish were not isolated. With the partitions three fish could be maintained in each tank. Each tank had a biofilter and a heater. W ith the heaters, each tank was kept a t 74 degrees. The fish were maintained on a regular day night cycle by turning on the lights in the morning and turning them off when the sun was going down. Each of the tanks were treated for ammonia with proline befor e the beginning of the experiment and again half way through the experiment. When treating the water 10 gallons from each tank was siphoned out. The water left was treated with a mixture of 2 tablespoons of proline dissolved into 4.5 liters of reverse osmo sis treated water. 250ml of the 4.5 liters was poured into each tanks then each tank was refilled with 10 gallons of tap water. There was a 24 hour waiting period between each water change and testing. Procedure This experiment was conducted in two stages with 32 fish in both stages because there were 8 shuttle boxes and the laboratory could house up to 45 fish. The first sixteen days were experimenting on two conditions with sixteen subjects in each condition. The first two conditions tested were the cont rol and the Seroquel groups. The subjects were

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Fish on Seroquel 23 named after the tank they were in (A L), the section of the tank (1 3), and the condition they were in ( ( NSNC (Control) YSNC (Seroquel) YCNS ( cortisol ) YCYS (Seroquel+cortisol) ). So the fish in tank a, secti on 1, and condition NSNC was named A1NSNC. Feeding The fish were all fed after they were done testing for the day. This was to make sure that the slight differences in amounts of fish food given did not interfere with the testing. Each fish was given a pinch of the fish food TetraMin Tropical Flakes. The IACUC requirements were met under protocol #3459. Shuttle box The avoidance training device used was a shuttle box chamber that was filled with 4.5 liters of water inside of an isolation enclosure. The dimensions of the shuttle box chamber was 11.5 cm x 40.5 cm x 16.5cm. In the midline of the chamber there was an inf rared photoelectric beam above a hurdle so that the fish would have to swim over the hurdle in order to break the photoelectric beam. The performance level was determined based on number of avoids, number of escapes, avoid latencies, escape latencies, and number of failures. Using Habitest Universal Linc modules and Graphic State Notation software the stimuli, a light and a shock, was controlled and the performance of each fish was recorded. A protocol was made with the Graphic State Notation software that cycled four stages (SO, S1, S2 and S3) 15 times. A 28 volt light bulb was used for the

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Fish on Seroquel 24 conditioned stimulus and a 3.8 volt shock was used as the unconditioned stimulus. The cycle started with S0 where there was no shock and no light which lasted for a rand omized time interval. When S1 began a light came on and this stage lasted for ten seconds. If the fish crossed over the hurdle and broke the infrared photoelectric beam then the fish would not receive a shock and the cycle would start again at S0. Breaking the photoelectric beam in S1 was recorded by the computer as an avoid. The computer also recorded how many seconds the light was on before the goldfish crossed the photoelectric beam and that was the avoid latency. If the goldfish did not break the photoe lectric beam in S1 then S2 would begin. In S2 the light would stay on but the fish would also receive a 3.8 volt shock with a 500ms duration once per second for ten seconds. If the fish broke the photoelectric beam in that ten seconds the shock would stop and it was recorded by the computer as an escape and the cycle would begin again with S0. If the fish crossed the photoelectric beam in S2 the computer recorded how many seconds the fish was shocked before it crossed the photoelectric beam which was the es cape latency. If the fish did not break the photoelectric beam in S2 then it would be shocked once per second for the entire ten seconds and then would proceed to S3 which ended the cycle. If any fish got to S3 it was recorded as a fail and then the cycle would start over again. Drugs The first 16 days of this experiment tested two conditions. The first condition only received injections of water with a m icro syringe (NSNC). Each fish was weighed and er is a control for the stress of

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Fish on Seroquel 25 handling and receiving an injection so the amount injected was determined the same way the cortisol was. There is no research done on the effects of Seroquel on fish so the safety of the drugs had to be assumed from studi es with Seroquel done on rats and dogs. Before testing began preliminary studies were done to find a safe dosage to give the fish. 200mgs was dissolved in a tank with two healthy fish and within 24 hours they both died. The next dose experimented with was 150mgs with three healthy fish and they were still alive after 24 hours. A more conservative dosage of 100mgs was used since it was only known that fish would be able to survive a few days at 150 but it was not known if they could survive the entire 16 day s of testing with a higher dose. The second condition tested in the first run was given 100 mgs of Seroquel along with the injections of water (YSNC). The water used in both conditions was first treated with reverse osmosis. The Seroquel was supplied in 2 00mg pills so they were first ground up and then dissolved into 200mls of water. The six tanks that housed the 16 fish in the YSNC group received 100mls of the Seroquel solution. 10 gallons from each of the 20 gallons tanks were siphoned out on the fourth day to remove waste and they were treated with an ammonia, filtrite, filtrate, chlorine, and chloramine removing powder. Since Seroquel is water soluble is was assumed that with each water change half of the initial dose was removed from the tank. Since th e goal was to keep a consistent amount over the 16 days each tank housing the Seroquel condition had 50 mgs of Seroquel added. Like the initial dose, a 200mg pill was crushed up and dissolved in 200mls of reverse osmosis

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Fish on Seroquel 26 treated water. However each tank on ly received 50mls to raise the level back to 100mgs per tank. To administer the cortisol to groups YSYC and NSYC it was first mixed with cocoa butter. 20ml of cocoa butter was heated in a 50ml beaker on a heating plate set to 1.5. After there was 20ml of cocoa butter heated to a liquid, 800mgs of cortisol was measured out and added to the heated cocoa butter The mixture was covered with P arafilm and then micellated for several minutes then returned to the hot plate. After trying to inject the solution of cocoa butter and cortisol it was obvious that it would not liquefy enough to be injected. This is why coconut oil was used for the experiment instead of cocoa butter. The coconut oil was prepared in the exact same way as the cocoa butter with successful re the fish. The cortisol was injected into the body cavity against the grain of the scales. In order to keep which fish were used in each condition random the tanks that would house each condit ion were predetermined. When the fish arrived at the Pritzker m arine lab oratory they were placed into the tanks by people who did not know anything about the experiment including which tanks would house each condition. For the weeks before the experiment 10 gallons of each 20 gallon tank was replaced every four da ys. The water was treated with P roline to remove nitrites, nitrates, ammonia, chlorine, and chloramines every time that the water was changed. This was to stabilize the previously mentioned ions and chemical compounds. All the levels were stabilized before the experiment started so the water would only have to be treated every eight days instead of four during the course of the experiment. This was important to

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Fish on Seroquel 27 make sure that a healthy level of ba cteria that the fish need to stay healthy could grow on the biofilter. Temperature was also stabilized at 74 degrees Fahrenheit before the experiment started by using a water heaters. Procedure Before testing started each day every shuttle box was tested with a voltmeter in a zap test mode to make sure that they were all working and receiving a 3.8 volt shock. 4.5 liters of room temperature water was poured into each shuttle box chamber. After each shuttle box chamber had water in it a soft net was used to transport the first row of 8 fish into the tanks. Each subject was given two minutes to adjust to being in the shuttle box before testing started. The net was washed with hot water every time it was used in different tanks in case if any of the fish got a parasite that could be transferred from one tank to another. As soon as the testing was done on a fish it was put back into its original tank. After testing the first 8 fish all the water in each shuttle box chamber was drained out and replaced with new w ater. The water in each chamber was not used on more than one fish due to the possibility of the fish giving off fright pheromones. The water used in testing was reused after passing through a carbon filter. After the first row of fish was tested and the w ater was changed then the second row was tested. This was repeated until every row was tested.

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Fish on Seroquel 28 Results Each condition had some goldfish die. The NSNC and the YSNC groups both had one fish die. The NSYC groups had five deaths and YSYC had two deaths. To determine if there was a significant difference between the four groups a two way ANOVA with repeated measures was performed using the statistical software SAS. When looking at the data for each fish, the scores were div ided into four blocks. Each block consisted of four days. The two way ANOVA was used to determine if there was any significant difference between the conditions, the blocks of time, and interaction between block and condition. In order to perform a re peate d measures test, the number of subjects in each group was balanced. Each fish that died was represented by the average score from that group so that each N would equal the original 16. The interaction was not significant. The main effect for blo ck was sign ific ant. The main effect for group wa s close to being significant (p=.0525). A post there were significant differences between the groups that had a difference of mean higher than 5.985. MSe df Q crit HSD 655.66 60 3.74 5.985 The differences in means were: Control Sero = 2.800 Sero Cort = 37.109 Cort Cor +Sero =4.266

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Fish on Seroquel 29 Control Cort =39.909 Control Cort+Sero =35.643 Sero Cort+Sero =32.843 *Denotes a significant difference D the significant differences were between: Cortisol and Seroquel Control and cortisol Seroquel and Seroquel + cortisol Control and Seroquel + cortisol There was a significant difference between conditions; the determining factor was the presence of cortisol. The fish that were administered cortisol had a significantly higher number of avoids than the goldfish that were injected with just water, and therefore did better. The mean amount of fails for NSNC was 1.23 (295/240). The average amount of fails for YSNC was .76 (183/240). The average amount of fails for NSYC was .65 (125/192). The average amount of fails for YSYC was .41 (92/224).

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Fish on Seroquel 30 Figure 1 Mean Frequency of Avoids of Treatment Groups in blocks of daily sessions compared over days 1 4 Figure 2 Mean Frequency of Avoids of Treatment Groups in blocks of daily sessions compared over days 5 8.

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Fish on Seroquel 31 Figure 3 Mean Frequency of Avoids of Treatment Groups in blocks of daily sessions compared over days 9 12. Figure 4 Mean Frequency of Avoids of Treatment Groups in blocks of daily sessions compared over days 13 16.

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Fish on Seroquel 32 Table 2 Anova of mean frequency of avoids per blocks of four daily sessions compared among treatment groups over days. group block 1 block 2 block 3 block 4 Control 2.13 5.04 4.73 5.83 ad Sero 1.69 5.29 5.45 6.02 bc Cort 3.51 7.7 7.75 8.75 ac Sero+Cort 3.64 7.27 7.16 8.59 cd Means Discussion The hypothesis with which this study b egan, first projected on page 17 was complex. It involved questioning the way fish would react to Seroquel cortisol, and a combina tion of Seroquel and cortisol. The hypothesis predicted that subjects given cortisol would have the least amount of avoids. The subjects given Seroquel were expected to do slightly better than the control group. Lastly the subjects given Seroquel and cortisol were expected to do significantly better than the subjects given cortisol. However, results differed substantially from this educated guess. There was no statistically significant difference between subjects given Seroquel and subjects not g iven Seroquel There was a significant difference between the groups administered cortisol and not administered cortisol. What was surprising is that the cortisol groups were expected to have significantly less avoids than the groups not given cortisol whe n the results indicated that the cortisol groups had significantly higher amount of avoids than

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Fish on Seroquel 33 the groups not given cortisol. In sum, cortisol enhanced performance on a cognitive task while Seroquel had no statistically significant effect The results ra ised some important questions. The first question is what role did cortisol and Seroquel have in this study? Were there any differences between subjects in prior research and the current study that would account for the unexpected results? How did the role of stress affect cognition? There was not mu ch known on how Seroquel would a ffect stress. The hypothesis that Seroquel would lower stress was based on the fact that Seroquel is known to restore diminished prepulse inhibition in rats and the same parts of the brain that control PPI are important in memory formation. There also was not enough prior research to estimate an effective and safe dose for fish. In the preliminary study conducted, 200 mgs dissolved in a 20 gallon tank proved to be fatal to two fis h after a few hours. Since the goldfish could not tolerate 200mgs of Seroquel a more conservative dosage was used to try to minimize fatality from Seroquel It is not clear if a therapeutic level could be met due to this limitation. Prior research with Ser oquel was done on rats which greatly differ from fish. The expected outcome was that the goldfish given Seroquel would be significantly better than the goldfish not given Seroquel The rationale behind expecting the Seroquel group to do better is that it w ould help the goldfish cope with stress and as a result would have prevented burnout and exhaustion from GAS. The cortisol groups were expected to do worse than the groups not administered cortisol. In prior research fish did worse on memory formation task s when given cortisol (Barreto, 2006).

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Fish on Seroquel 34 However, prior studies were performed on a different species of fish. Since the goldfish for the current experiment have been domesticated for centuries they could have become less susceptible to stress leaving them with lower motivation to avoid the shocks. Mowrer ( 1956 ) has suggested that fear conditioning as a precursor to the development of the avoidance response would enhance crossing of the hurdle. In this study the cortisol could have made the sh ocks salient enough to encourage avoidance learning. It was previously mentioned that brief periods of stress could potentiate memory formation (McEwen, 1995) The fact that the cortisol groups did significantly better on a cognitive task could indicate th at the level of stress induced by cortisol was not high enough to cause negative effects but was high enough to enhance memory formation. Domestication There are distinct differences between domesticated and wild fish. In the present experiment, domesticated gold fish were used while wild fish were used in past research. Domesticated fish exhibit less fright than wild fish. Wild fish also could stand a greater concentration of accumulated metabolites and could endure higher water temperatures tha n domesticated fish. Domestic fish also lacked a desire to conceal themselves in comparison to wild fish. Wild fish also had higher stamina in swimming test when compared to domesticated fish (Vincent, 1960). Wild fish also have more extreme changes in p lasma cortisol, glucose, and chloride in response to stress. This was verified in a past experiment that confined trout to a net and subjected the trout to electroshock ( Woodward 1987). The differences in how domestic and wild fish behave may explain why the results were not consistent with past experiments.

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Fish on Seroquel 35 Two factor learning Habits are learned on the basis of positive reinforcement provided by reward or drive reduction while fears are learned on the basis of contiguous occurrence of a signal and punish ment. The two factor theory incorporates what were believed to be distinct and different processes (Mowrer, 1956) When a subject is shocked after some kind of stimuli the subject will become afraid of the stimuli alone and learn to do whatever will reduce fear. In this experiment the stimuli was a light that stayed on for ten seconds before a 3.8 shock. The learned behavior to reduce fear was to swim across a hurdle to break a photoelectric beam tha t would prevent the shock. Fear is an inherent component of total reaction or drive state. It is a form of conditioning since once the fear is connected to a stimuli, any response which reduces the fear will be reinforced. The results indicate that in the current study the cortisol groups were more motivated by fear and put more effort into reducing fear. In conclusion, how an organism will respond to stress seems to be dependent on different factors. These factors include the species and amount of stress along with other factors such as domestication. The subjects in this study had a positive reaction to stress which is consistent with Kloet (1999) and McEwen (1995) in that a small amount of stress was beneficial to memory formation

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Fish on Seroquel 36 References Ader, Robert, Cohen, Nicholas & Felten, David. (1995). Psychoneuroimmunology: interactions between the nervous system and the immune system. The Lancet, 347 99 103. Nursing, and Allied Heath Mosby United States edition. Arnsten, Amy F. T. PhD, & Goldman Rakic, Patricia S. Ph D. (1998). Nois e stress impairs prefrontal cortical cognitive function in monkeys: E vidence for a hyperdopaminergic mechanism. Archive of general psychiatry, 55: 362 368. Barreto, R.E., Volpato, G.L. & Pottinger T.G. (2006). The ef fect of elevated blood cortisol levels on the extinction of a conditioned stress response in rainbow trout Hormones and Behavior, 50: 484 488 Carring ton, Patricia Collings, Gilbeart H. Jr. M.D. M.P.H.; Benson, Herbert M.D.; Robinson, Harry Sc.D.; Wood, Loring W. M.D.; Lehrer, Paul M. Ph.D.; Woolfolk, Robert L. Ph.D.; Cole, Jean W (1980). The use of meditation relaxation techniques for the management of stress in a working population Journal of Occupational and Environmental Medicine, 22 206 287 Chandroo, K.P., Duncan, I.J.H. & Moccia, R.D. (2004). C an fish suffer?: perspective on sentience, pain and stress. Applied Animal Behaviour Science, 68, 225 250.

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Fish on Seroquel 37 Dickerson, Sally S. & Kemeny, Margaret E. (2004). Acute stre ssors and cortisol responses: a theoretical integration synthesis of laboratory research. Ps ychological Bulletin:130, 3, 355 391 Espelid, Sigrun, Lokken, Gunn Berit, Steiro, Kari & Bogwa ld, Jarl. (1996) Effects of cortisol and stress on the immune system in Atlantic Salmon (Sa lmo salar L.). Fish & Shellfish Immunology, 6, 95 110. Geyer, Mark A., Krebs Thomson, Kirsten, Braff, David L., & Swerdlow, Neal R.(2001). Pharmalogical studies of prepulse inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review. Psychopharamocology: 156: 117 154 Goldstein, JM (1999). Quetiapine fumarate ( Seroquel ): a new atypical antipsychotic. Drugs today 3, 193 210 Harris, James & Bird, David J. (2000). Modulation of the fish immune system by hormones. Veterinary Im munology and Immunopathology, 7, 163 176. Kloet, E. Ron de, Oitzl, Melly S. & Joels, Marian. (1999). Stress and cognition: are corticosteroids good or bad guys? Trends in Neurosciences, 22, 422 426 Mansbach RS, Brooks EW, Sanner MA, Zorn SH (1998) Selective dopamine D4 receptor antagonists reverse apomorphine induced blockade of prepulse inhibition. Psychopharmacology:135, 194 200

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Fish on Seroquel 38 McEwen, Bruc e S. & Sapolsky, Robert M. (1995). Stress a nd cognitive function. Current Opinion in Neurobiology, 5, 205 216 pressure: stress and cognitive Applied Animal Behavior Science 65: 221 244. Mowrer, O.H. (1956). Two factor learning theory reconsidered, with special reference to secondary reinforcement and the c oncept of habit. Psychological R eview, 63:114 128 Quetiapine (2007) Drug prescribing information. The drug information s heet released by AstraZenec a Selye, Hans M.D. (1946). The general adaptati on syndrome and the diseases of adaptation. The Journal of Clinical Endocrinology :6, 117 230 Selye, Hans M.D. (1950). Stress and the general adaptation syndrome. British Medical Journal: 1383 1392 Swerdlow, N.R., Bakshi, V. & Geyer, M.A. (1996). Seroquel restores sensorimotor gating in phencyclidine treated rats The Journal of Pharmacology and Experimental Therapeutics 279: 1290 1299 Vincent, Robe rt E (1960) Some influences of domestication upon t hree s tocks of b rook t rout (Salvelinus fontinalis Mitchill) Transactions of the American Fisheries Society [Trans. Am. Fish. Soc.]. Vol. 89, no. 1, pp. 35 52

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Fish on Seroquel 39 Vissoci Reiche, Edna Maria, Odebrecht Vargas Nunes, Sandr a, & Morimoto, Kelena Kaminami. (2004). Stress, depression, the immune system, and cancer Lancet Oncology, 5 617 625. Wazier, I de, Cugnec, PH, Yang, CS, Leroux, JP, & Bea une PH (1990). Cytochrome P 450 isoenzymes, epoxide hydrolase and glutathione transferas es in rat and human hepatic and extrahepatic tissues. American Society for Pharmacology and Experimental Therapeutics: 253. Woodward, Colleen Caldwell & Strange, Richard J. (1987). Physiological stress responses in wild and hatchery reared Rainbow Trout. Transactions of the A merican Fisheries Society, 116:574 579

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Avoids GROUPA ID GROUP DAY_1 DAY_2 DAY_3 DAY_4 DAY_5 DAY_6 DAY_7 DAY_8 DAY_9 A A1NSNC 1 0 0 0 1 5 3 6 7 6 A A2NSNC 1 0 0 5 0 4 7 7 7 8 A B1NSNC 1 0 3 3 6 3 7 4 5 3 A B2NSNC 1 0 1 6 10 7 9 4 6 7 A B3NSNC 1 0 0 0 0 0 0 0 0 1 A C1NSNC 1 0 0 0 0 1 2 3 5 8 A C2NSNC 1 2 2 4 3 8 7 9 7 8 A D1NSNC 1 0 1 0 0 2 1 0 0 3 A D2NSNC 1 2 0 10 3 9 11 13 13 12 A D3NSNC 1 15 8 13 15 15 15 13 14 15 A E1NSNC 1 0 0 0 0 0 3 1 3 1 A E2NSNC 1 0 0 0 1 1 0 1 0 0 A E3NSNC 1 1 0 0 0 2 2 1 2 1 A F1NSNC 1 1 0 1 0 1 1 2 3 1 A F2NSNC 1 0 0 4 7 12 10 10 9 10 A F3NSNC 1 1.4 1 3.066667 3.066667 4.666667 5.2 4.933333 5.4 5.6 B G1YSNC 2 1 1 0 6 6 5 3 3 3 B G2YSNC 2 0 0 5 1 5 10 12 12 13 B G3YSNC 2 2 15 14 15 13 15 15 13 15 B H1YSNC 2 0 0 0 1 0 3 1 3 2 B H2YSNC 2 0 0 0 0 0 0 0 1 0 B H3YSNC 2 0 0 0 0 3 6 5 9 4 B I1YSNC 2 0 0 1 7 7 9 9 11 14 B I2YSNC 2 0 1 5 4 10 8 8 10 7 B J1YSNC 2 0 0 0 9 11 6 9 9 8 B J2YSNC 2 0 0 0 0 0 0 3 5 2 B K1YSNC 2 0 0 0 0 0 1 0 0 3 B K2YSNC 2 0 0 1 0 1 0 1 1 0 B K3YSNC 2 0 0 0 1 1 3 4 4 0 B L1YSNC 2 0 0 5 5 8 12 9 9 8 B L2YSNC 2 0 0 0 1 2 1 1 2 3 B L3YSNC 2 0.2 1.133333 2.066667 3.333333 4.466667 5.266667 5.333333 6.133333 5.466667 C A1NSYC 3 0 0 1 2 8 11 10 10 12 C A2NSYC 3 0 0 0 0 0 0 0 0 0 C A3NSYC 3 8 10 15 14 14 14 15 15 15 C B2NSYC 3 0 2 3 2 0 4 7 5 7 C C2NSYC 3 0 1 1 5 5 6 5 8 8 C D1NSYC 3 0 1 0 1 8 6 9 11 9 C D2NSYC 3 0 3 6 3 1 5 14 9 6 C D3NSYC 3 1 10 13 14 13 14 14 15 13 C E1NSYC 3 0 0 0 3 2 4 10 5 9 C F1NSYC 3 0 5 10 3 6 8 7 8 8 C F2NSYC 3 1 3 7 7 10 4 9 10 10 C F3NSYC 3 0.909091 3.181818 5.090909 4.909091 6.090909 6.909091 9.090909 8.727273 8.818182 C F4NSYC 3 0.909091 3.181818 5.090909 4.909091 6.090909 6.909091 9.090909 8.727273 8.818182 C F5NSYC 3 0.909091 3.181818 5.090909 4.909091 6.090909 6.909091 9.090909 8.727273 8.818182 C F6NSYC 3 0.909091 3.181818 5.090909 4.909091 6.090909 6.909091 9.090909 8.727273 8.818182 C F7NSYC 3 0.909091 3.181818 5.090909 4.909091 6.090909 6.909091 9.090909 8.727273 8.818182 D G1YSYC 4 0 0 4 6 9 8 9 6 8 D G2YSYC 4 0 2 1 9 2 5 3 3 4 D G3YSYC 4 2 13 15 15 14 15 15 13 15 D H1YSYC 4 0 0 2 9 4 4 9 4 7 D H2YSYC 4 0 1 3 8 4 6 8 10 9 D I1YSYC 4 0 0 0 0 0 1 3 4 3 D I2YSYC 4 0 0 1 5 4 7 4 6 8 D J2YSYC 4 0 3 1 6 6 6 6 7 8 D J3YSYC 4 1 7 14 15 14 14 15 10 13 D K1YSYC 4 0 0 0 1 1 3 2 3 1 D K2YSYC 4 0 2 8 5 9 8 9 7 9 D K3YSYC 4 0 5 6 8 6 7 12 7 4 D L1YSYC 4 0 0 0 2 3 3 3 11 6 D L2YSYC 4 0 3 7 14 14 15 13 13 13 D L3YSYC 4 0.214286 2.571429 4.428571 7.357143 6.428571 7.285714 7.928571 7.428571 7.714286 D L4YSYC 4 0.214286 2.571429 4.428571 7.357143 6.428571 7.285714 7.928571 7.428571 7.714286

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Avoids GROUPA ID A A1NSNC A A2NSNC A B1NSNC A B2NSNC A B3NSNC A C1NSNC A C2NSNC A D1NSNC A D2NSNC A D3NSNC A E1NSNC A E2NSNC A E3NSNC A F1NSNC A F2NSNC A F3NSNC B G1YSNC B G2YSNC B G3YSNC B H1YSNC B H2YSNC B H3YSNC B I1YSNC B I2YSNC B J1YSNC B J2YSNC B K1YSNC B K2YSNC B K3YSNC B L1YSNC B L2YSNC B L3YSNC C A1NSYC C A2NSYC C A3NSYC C B2NSYC C C2NSYC C D1NSYC C D2NSYC C D3NSYC C E1NSYC C F1NSYC C F2NSYC C F3NSYC C F4NSYC C F5NSYC C F6NSYC C F7NSYC D G1YSYC D G2YSYC D G3YSYC D H1YSYC D H2YSYC D I1YSYC D I2YSYC D J2YSYC D J3YSYC D K1YSYC D K2YSYC D K3YSYC D L1YSYC D L2YSYC D L3YSYC D L4YSYC DAY_10 DAY_11 DAY_12 DAY_13 DAY_14 DAY_15 DAY_16 5 4 1 2 7 7 8 7 9 5 12 8 8 8 3 6 3 5 7 5 7 4 3 2 2 4 1 6 0 0 1 0 0 0 0 7 8 4 4 8 10 12 4 6 5 6 9 8 12 0 0 0 0 0 1 4 7 14 9 13 7 10 10 15 13 14 15 13 13 15 2 2 1 3 2 5 2 0 0 0 0 0 0 0 4 0 4 2 3 5 0 2 1 4 10 5 3 7 9 7 5 8 10 7 11 4.6 4.866667 3.866667 5.466667 5.533333 5.533333333 6.8 3 4 1 1 4 3 6 10 7 8 8 12 10 12 15 15 15 15 15 15 14 3 4 3 9 5 8 6 1 0 0 1 3 1 0 8 8 8 9 2 2 1 12 11 11 12 13 13 11 11 11 10 12 10 8 10 9 7 1 2 10 8 6 3 2 4 4 3 2 3 2 1 3 1 2 4 1 1 1 1 2 1 0 0 2 1 2 2 0 1 0 6 8 7 11 8 13 11 1 2 2 5 4 5 1 5.8 5.466667 5.066667 6.266667 6.133333 6.2 5.466667 9 10 8 13 10 10 10 1 0 0 0 0 0 0 0 9 0 4 8 11 11 7 7 6 8 6 7 4 12 14 10 13 10 12 14 6 6 7 6 6 9 9 8 12 8 8 7 7 10 0 14 0 13 4 15 11 9 12 11 13 10 14 9 7 8 8 8 11 6 10 11 13 11 11 13 12 12 6.363636 9.545455 6.272727 8.818182 7.727273 9.363636364 9.090909 6.363636 9.545455 6.272727 8.818182 7.727273 9.363636364 9.090909 6.363636 9.545455 6.272727 8.818182 7.727273 9.363636364 9.090909 6.363636 9.545455 6.272727 8.818182 7.727273 9.363636364 9.090909 6.363636 9.545455 6.272727 8.818182 7.727273 9.363636364 9.090909 5 5 10 6 7 5 7 7 3 9 3 6 2 6 0 15 1 15 12 15 13 4 3 6 8 3 7 5 9 5 7 10 11 9 9 5 9 1 5 2 4 8 7 10 7 13 10 11 10 2 7 8 2 4 7 8 0 10 13 15 13 12 14 2 2 2 2 1 3 3 8 8 8 8 11 12 10 11 9 10 10 14 14 14 6 9 9 7 7 10 5 14 14 13 11 14 14 14 5.714286 7.785714 7.428571 8.214286 8.214286 8.928571429 9 5.714286 7.785714 7.428571 8.214286 8.214286 8.928571429 9

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Escapes DAY_1 DAY_2 DAY_3 DAY_4 DAY_5 DAY_6 DAY_7 DAY_8 DAY_9 DAY_10 A1NSNC 13 14 8 14 10 12 9 8 9 10 A2NSNC 1 14 10 14 11 8 8 8 7 8 B1NSNC 13 11 12 9 12 8 11 10 12 12 B2NSNC 14 14 9 5 7 6 11 9 8 11 B3NSNC 8 3 2 2 9 5 4 8 3 3 C1NSNC 14 14 14 14 14 13 12 10 7 8 C2NSNC 13 13 11 12 7 8 6 8 7 11 D1NSNC 13 14 9 15 13 14 15 15 12 15 D2NSNC 9 15 4 12 6 4 2 2 3 8 D3NSNC 0 7 2 0 0 0 2 1 0 0 E1NSNC 15 15 15 15 15 12 14 12 14 13 E2NSNC 13 14 15 10 13 13 12 14 14 14 E3NSNC 13 15 15 14 13 13 14 13 13 11 F1NSNC 9 13 12 15 14 13 13 10 12 12 F2NSNC 14 14 11 8 3 5 5 6 5 6 G1YSNC 14 14 8 9 9 10 12 12 12 12 G2YSNC 1 14 10 13 10 4 3 3 2 5 G3YSNC 8 0 1 0 2 0 0 2 0 0 H1YSNC 13 14 15 14 15 12 13 12 13 12 H2YSNC 15 13 14 15 14 15 15 14 15 13 H3YSNC 0 14 15 15 12 9 10 6 11 7 I1YSNC 12 13 13 8 8 6 6 4 1 3 I2YSNC 14 14 8 11 5 7 7 5 8 4 J1YSNC 14 10 5 6 4 9 6 6 7 6 J2YSNC 14 15 14 15 15 15 12 10 13 12 K1YSNC 10 5 11 10 10 10 8 12 9 12 K2YSNC 12 14 13 14 13 14 12 14 14 13 K3YSNC 15 15 13 14 14 12 11 11 15 13 L1YSNC 15 15 10 10 7 3 6 6 7 9 L2YSNC 2 14 14 14 12 13 14 13 12 14 A1NSYC 15 15 14 13 7 4 5 5 3 6 A2NSYC 15 15 14 15 15 15 15 15 15 14 A3NSYC 7 5 0 1 1 0 0 0 0 0 B2NSYC 12 13 12 13 15 11 8 10 8 8 C2NSYC 15 14 13 9 10 8 10 7 7 3 D1NSYC 15 14 15 13 7 9 6 4 5 9 D2NSYC 15 12 9 12 14 10 1 6 9 7 D3NSYC 13 5 2 1 2 0 1 0 2 0 E1NSYC 15 15 15 12 13 11 5 5 6 6 F1NSYC 15 10 5 11 9 7 8 7 7 8 F2NSYC 14 12 8 8 5 11 6 5 5 4 G1YSYC 15 15 11 9 5 7 6 9 7 10 G2YSYC 15 13 14 6 13 10 12 12 11 8 G3YSYC 13 2 0 0 1 0 0 0 0 0 H1YSYC 15 15 13 6 11 11 6 11 8 11 H2YSYC 15 14 12 7 11 9 7 5 6 6 I1YSYC 14 15 15 15 15 14 11 11 12 10 I2YSYC 15 15 14 10 11 8 9 9 7 8 J2YSYC 15 12 14 9 9 9 9 8 7 13 J3YSYC 14 8 1 0 1 1 0 0 2 0 K1YSYC 15 13 12 14 13 11 13 12 14 13 K2YSYC 15 13 7 10 6 7 6 8 6 7 K3YSYC 13 10 9 7 9 7 3 8 10 4 L1YSYC 11 13 12 13 12 11 12 4 9 9 L2YSYC 14 12 8 1 1 0 2 2 2 1

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Escapes A1NSNC A2NSNC B1NSNC B2NSNC B3NSNC C1NSNC C2NSNC D1NSNC D2NSNC D3NSNC E1NSNC E2NSNC E3NSNC F1NSNC F2NSNC G1YSNC G2YSNC G3YSNC H1YSNC H2YSNC H3YSNC I1YSNC I2YSNC J1YSNC J2YSNC K1YSNC K2YSNC K3YSNC L1YSNC L2YSNC A1NSYC A2NSYC A3NSYC B2NSYC C2NSYC D1NSYC D2NSYC D3NSYC E1NSYC F1NSYC F2NSYC G1YSYC G2YSYC G3YSYC H1YSYC H2YSYC I1YSYC I2YSYC J2YSYC J3YSYC K1YSYC K2YSYC K3YSYC L1YSYC L2YSYC DAY_11 DAY_12 DAY_13 DAY_14 DAY_15 DAY_16 11 14 13 8 8 7 6 10 3 7 7 7 9 12 10 8 10 8 12 13 13 10 14 8 3 1 0 0 2 2 7 11 11 7 5 3 9 10 9 6 7 3 15 14 15 15 14 11 1 6 2 8 5 5 2 1 0 2 2 0 13 14 12 13 10 12 14 15 13 12 13 13 15 11 13 12 10 15 14 10 5 9 12 8 8 9 7 5 8 4 11 14 14 11 12 9 8 7 7 3 5 3 0 0 0 0 0 1 11 12 6 10 7 9 15 15 14 12 12 15 7 7 6 13 13 14 4 4 3 2 2 4 4 4 3 5 7 5 8 14 13 5 7 9 13 11 11 12 13 12 13 11 13 12 11 14 13 13 13 14 14 11 7 12 13 15 14 15 7 8 4 7 2 4 13 12 10 11 10 13 5 7 2 5 5 5 15 15 15 15 15 15 1 0 0 1 1 0 8 9 7 9 8 11 1 5 2 5 3 1 9 8 9 9 6 6 3 7 7 8 8 5 0 0 0 0 0 0 3 4 2 5 1 6 7 7 7 4 9 5 2 4 4 2 3 3 10 5 9 8 10 8 12 6 12 9 13 9 0 0 0 1 0 0 12 9 6 11 8 10 10 8 5 4 6 6 6 14 10 13 11 7 5 8 2 5 4 5 7 7 13 11 8 7 2 1 0 0 2 0 13 13 13 14 12 12 7 7 7 4 3 5 6 5 4 1 1 1 6 6 8 8 5 10 1 2 4 1 1 1

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Avoid Latencies 1 2 3 4 5 6 7 8 9 10 A1NSNC N N N 7 7 3.33 4.17 5.43 1.83 4.6 A2NSNC N N 4.2 N 4.75 5.14 3.29 6 3.75 4.29 B1NSNC N 5 5.67 6.67 8.33 5.57 7.25 7.6 6 6.67 B2NSNC N 8 6.17 4.6 5.71 6.11 3.75 6.67 5 6.25 B3NSNC N N N N N N N N 9 N C1NSNC N N N N 7 6.5 6 3.6 6.5 5 C2NSNC 4 8 9.75 4 4.63 4.43 7.56 4.29 7.5 8.5 D1NSNC N 8 N N 7 7 N N 6.67 N D2NSNC 7 N 6 7.67 4.78 6.45 4.77 4.62 5.5 4.86 D3NSNC 5.53 5.88 4.92 3.6 3.2 2.47 3.46 2.86 2.87 3.93 E1NSNC N N N N N 5.67 9 5.33 10 6 E2NSNC N N N 3 8 N 3 N N N E3NSNC 3 N N N 8 5.5 6 8.5 5 6.25 F1NSNC 6 N 10 N 10 7 6 8.33 7 6 F2NSNC N N 5 4.14 5 4.6 4.3 6.11 5.2 5 G1YSNC 4 8 N 4.33 4 4.8 7 5 7.67 7.33 G2YSNC N N 5.4 9 6.4 5.5 4.75 4.08 4.77 5.9 G3YSNC 7.5 4.6 4.29 3.33 4.23 3 4.13 4.31 3.93 4.33 H1YSNC N N N 4 N 7.33 7 8 9.5 8.67 H2YSNC N N N N N N N 3 N 10 H3YSNC N N N N 6.33 5.5 6.8 6 7.25 4.63 I1YSNC N N 3 2.86 5 4.22 3.22 4.27 5.14 5.5 I2YSNC N 8 4.4 5.5 3.5 6.13 5.88 5.2 6.43 6.55 J1YSNC N N N 5.67 5.64 6.67 7.44 6.67 6.88 6.22 J2YSNC N N N N N N 4 6 4.5 6.33 K1YSNC N N N N N 4 N N 4.33 4.5 K2YSNC N N 4 N 7 N 5 9 N 8 K3YSNC N N N 8 5 7.33 6.5 5.75 N 5 L1YSNC N N 6.6 5.6 5.13 5 5.89 5.67 8 5 L2YSNC N N N 3 4 9 5 6.5 6 3 A1NSYC N N 5 8 5.13 3 4.4 4.8 4.58 3.89 A2NSYC N N N N N N N N N 8 A3NSYC 6.13 5.2 4.4 4.29 3.57 3.14 2.53 2.93 3.8 N B2NSYC N 6.5 5.3 4.5 N 8 7 5.8 6.71 6.29 C2NSYC N 9 6 5.4 3.8 5.17 7.8 5.75 4.13 5.67 D1NSYC N 7 N 7 4.25 6 5 5.82 7.22 7.17 D2NSYC N 5.67 5 3 9 3.6 4.07 4.11 4.83 3.75 D3NSYC 8 6.1 6.08 5.07 4.31 4.57 3 3.27 3.31 N E1NSYC N N N 6.33 6.5 7.25 6 5.6 6.67 5.89 F1NSYC N 5.4 5.6 7.33 7.83 5.63 5.71 4.63 4.5 4.43 F2NSYC 2 6 4 5.29 3.8 6.25 3.89 6.3 4.6 5.45 G1YSYC N N 5.75 5.83 3.89 4.75 4.78 6.17 6.5 4.4 G2YSYC N 9 9 5.11 6.5 5.2 7.33 8 5.75 6.71 G3YSYC 8 4.31 3.27 3.87 3.64 3.4 3.67 3.92 3 N H1YSYC N N 9 7.56 6.75 7.5 7.56 5.75 8.43 7.5 H2YSYC N 10 5.67 5.88 7.25 6.17 5.13 4.9 5.22 7.44 I1YSYC N N N N N 4 9.33 8.5 8.67 7.8 I2YSYC N N 8 2.6 5.75 6 7 5 5.63 6 J2YSYC N 5.67 6 4.33 5.83 5.83 5.17 5.14 4 4 J3YSYC 2 7.14 6 5.33 5.36 4.5 4.47 6.9 5.54 N K1YSYC N N N 4 7 6.67 8 6.67 9 7 K2YSYC N 6 4.5 3 4.78 6 3.56 5.29 3.89 3.63 K3YSYC N 5.2 4.17 4.13 4.5 5.86 4.58 4.86 4 3.55 L1YSYC N N N 5.5 6.67 4 8 5.82 4.17 6.5 L2YSYC N 6 4.29 4 4.79 4.93 4.23 4 3.23 4.64

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Avoid Latencies A1NSNC A2NSNC B1NSNC B2NSNC B3NSNC C1NSNC C2NSNC D1NSNC D2NSNC D3NSNC E1NSNC E2NSNC E3NSNC F1NSNC F2NSNC G1YSNC G2YSNC G3YSNC H1YSNC H2YSNC H3YSNC I1YSNC I2YSNC J1YSNC J2YSNC K1YSNC K2YSNC K3YSNC L1YSNC L2YSNC A1NSYC A2NSYC A3NSYC B2NSYC C2NSYC D1NSYC D2NSYC D3NSYC E1NSYC F1NSYC F2NSYC G1YSYC G2YSYC G3YSYC H1YSYC H2YSYC I1YSYC I2YSYC J2YSYC J3YSYC K1YSYC K2YSYC K3YSYC L1YSYC L2YSYC 11 12 13 14 15 16 3 3 6 3.29 5.14 5.88 3.89 2.6 4.17 3.25 4.25 2.63 7.5 7 7.8 7.43 6 8.57 9.33 9 7 9.5 7 5.17 N 6 N N N N 7.38 8.75 8.75 5.75 6.3 4.67 5.33 7.8 6.67 7.11 6.13 6.25 N N N N 8 7.25 4.93 6 6.92 7.57 6.4 7.1 4.15 3.57 2.8 2.92 4.08 3.6 2 8 7.67 8.5 8.6 8 N N N N N N N 8.5 9 9.33 8.8 N 7 6.75 6.6 7.6 8 7.29 4.71 5.2 4.25 5.7 5.86 3 7.25 8 8 6.75 7.33 6.33 4.86 6 6.75 6.25 7.5 5.75 3.93 4.87 3.73 4.33 3.93 3.71 7 5.67 6.56 8.8 7.75 7.67 N N 7 8.67 8 N 4.75 5.5 6.11 8.5 4 3 3.36 5.36 3.92 5.92 4.46 4.82 3.55 5.4 4.33 8.1 7.13 5 7 10 9 5.8 5.38 7.33 8 6.5 9 8.67 5 4.67 8 3.67 8 3.5 3.75 3 4 9 9.5 9 N N 4 5 9.5 N 4 N 7 5.14 4.91 5.5 4.77 4.55 6 3 4.6 4.75 3.8 7 3.2 4.13 4.92 4 4.8 5.4 N N N N N N 4.22 N 3.75 4.75 4 4.09 5.57 5.5 4.13 4.5 6.57 7 5.5 6.5 5.92 4.5 3.92 5.57 6.5 7.43 6.17 6 6 5.78 4.5 5.88 3.5 4.86 4.14 3.7 4.29 N 4.62 3.75 4.33 4.45 5.58 4.09 4.54 5.8 5.64 6.56 5.38 4.13 3.75 6.27 4.67 4 5.69 5.82 6.36 4.46 5.33 6.17 7.6 6.3 6.5 6.86 5.8 6.71 7 7.33 9.67 7.83 9.5 7.33 3.73 7 3.67 4.92 4.07 4.77 9.67 7.33 7.13 7.67 8.57 9.2 5.8 5.29 5.1 7 8 6.78 6.56 10 7.2 10 8.25 7.75 5.6 N 4.85 5.2 6.64 5.9 4.71 5.75 3.5 7.75 7.29 5.13 6 6.15 4.8 5.69 5.83 4.79 8 6 7 5 6.67 6 4.63 4.38 4 3.36 4.17 3.6 4.44 4.3 4.8 3.43 3.93 4.93 8.22 7.33 6 4.71 5.1 5.6 5.07 4.92 4.09 5.79 5.07 4.86

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Escape Latencies DAY_1 DAY_2 DAY_3 DAY_4 DAY_5 DAY_6 DAY_7 DAY_8 DAY_9 DAY_10 A1NSNC 2.15 1.64 2.63 1.71 1.5 2.08 1.67 1.89 1.78 2 A2NSNC 1 1.64 1.9 1.86 2.09 1.63 1.75 2 1.71 1.38 B1NSNC 1.53 1.55 2.08 1.56 1.92 1.38 1.27 1.9 1.17 1.5 B2NSNC 1.36 1.5 1.56 1.2 2 1.17 1.36 1.22 1 1.64 B3NSNC 2.75 2 3.5 2.5 3.22 3.2 2.75 2.25 4 3 C1NSNC 2.43 2.07 2.29 2.07 1.86 2.31 2.25 1.6 2.14 2 C2NSNC 1.54 1.31 1.82 1.58 1.86 1.38 1.67 1.23 1.43 1.36 D1NSNC 1.77 2.21 3.11 1.2 1.46 2.29 1.53 1.93 1.67 1.33 D2NSNC 2.67 1.93 2.5 2.17 1.33 2.5 1.5 1.5 2.67 1.63 D3NSNC N 2 2.5 N N N 1 1 N N E1NSNC 1.8 1.47 1.27 1.67 1.6 1.42 1.43 1.42 1.14 1.54 E2NSNC 1.69 2.21 2.27 2.4 1.77 1.85 2.5 1.79 2 2.86 E3NSNC 1.92 2.2 2.07 2.07 2.38 1.54 2 1.77 2 2.09 F1NSNC 2.33 2.69 2.33 2.87 1.93 1.77 2.08 1.2 2.58 1.42 F2NSNC 2.07 1.5 1.64 1 1.67 2.6 1.4 1.33 1.4 1.5 G1YSNC 1.5 1.57 2.25 1.22 1.22 2 1.25 1.25 1.58 1.58 G2YSNC 2 1.71 1.6 1.77 1.9 2.5 1.67 1.67 3 2.6 G3YSNC 2.13 N 1 N 1 N N 1 N N H1YSNC 1.85 1.71 2.4 2 2.13 1.83 1.77 1.83 1.77 1.67 H2YSNC 1.93 2.46 2.21 2.33 1.79 1.67 2.27 2.07 1.87 1.85 H3YSNC N 1.64 1.53 1.87 2 1.56 1.6 1.5 2.36 1.71 I1YSNC 1.5 1.69 1.31 1.63 1.75 2.5 1.67 1.25 1 1.67 I2YSNC 2.43 1.93 1.75 1.64 2.2 1.57 2.14 1.2 1.63 2 J1YSNC 1.93 1.7 2.8 1.67 2 1.56 1.33 2.17 1.57 2 J2YSNC 1.43 2.07 1.79 1.4 2 1.67 1.75 1.7 1.38 1.5 K1YSNC 3 2.2 2.82 2.6 3.4 3.1 1.75 3.25 2.33 2.08 K2YSNC 2.33 1.36 2.31 2.86 2.15 2.07 2.67 2.07 2.14 1.85 K3YSNC 2 1.27 1.69 1.86 2.5 1.92 1.64 2.18 1.47 1.31 L1YSNC 2 2.07 1.9 1.5 1.29 2.67 2 1.17 1.71 2.33 L2YSNC 2 2.14 2.07 1.86 2.58 2.15 2.07 2.08 1.92 1.64 A1NSYC 1.47 1.4 1.71 1.54 2.86 1.25 1.6 1.6 1.33 1.33 A2NSYC 1.73 1.6 1.93 1.67 1.87 1.67 1.53 1.87 1.2 1.43 A3NSYC 1 1.4 N 1 1 N N N N N B2NSYC 1.83 2 1.67 1.38 1.6 1.45 2 1.5 1.5 1.13 C2NSYC 1.53 1.43 1.23 1.22 1.6 2 1.2 2.29 1.43 2.67 D1NSYC 1.73 1.71 1.53 1.77 2.14 2 1.33 1.5 2.4 1.78 D2NSYC 1.6 1.92 2.44 2.25 1.86 1.9 2 2 2 1.14 D3NSYC 1.85 2.2 2 1 2 N 2 N 2 N E1NSYC 1.47 1.8 1.47 1.83 1.85 1.64 1.4 1.2 1.5 2 F1NSYC 1.87 1.9 1.6 1.82 1.56 1.86 1.63 2 1.71 1.13 F2NSYC 1.64 1 1.25 1.13 1.2 1.27 1.16 2 1 1 G1YSYC 1.4 1.47 1.55 1.56 2.6 1.71 1.67 2.44 1.57 1.8 G2YSYC 1.73 1.54 1.5 1.5 1.46 1.5 1.33 1.75 1.64 1.5 G3YSYC 1.69 1.5 N N 1 N N N N N H1YSYC 1.4 2.33 1.69 1.67 2 1.55 1.83 1.64 1.75 1.91 H2YSYC 1.47 1.64 1.25 1.29 1.18 1.56 1.43 1.2 1.67 1.17 I1YSYC 2.79 2 2 1.13 1.93 2 2.18 1.91 1.83 1.3 I2YSYC 1.67 1.4 1.36 1.4 1 1.88 1.45 2 1.29 1.63 J2YSYC 1.73 1.42 2.07 2.22 1.78 1.22 1.67 1.75 1.29 1.69 J3YSYC 1.36 1.13 1 N 1 2 N N 1 N K1YSYC 1.67 1.92 2.58 1.71 2.23 1.82 2.15 2 2.07 1.92 K2YSYC 1.33 1.69 1.57 1.3 1.5 1.43 2.17 1.88 1.83 1.71 K3YSYC 1.77 1.8 1.56 1.57 1.78 2.14 2 1.25 2 1.25 L1YSYC 1.73 1.92 2.17 2.23 2.25 1.91 2.17 2 1.78 2 L2YSYC 2 1.83 1.88 1 1 N 1.5 1 2 1

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Escape Latencies A1NSNC A2NSNC B1NSNC B2NSNC B3NSNC C1NSNC C2NSNC D1NSNC D2NSNC D3NSNC E1NSNC E2NSNC E3NSNC F1NSNC F2NSNC G1YSNC G2YSNC G3YSNC H1YSNC H2YSNC H3YSNC I1YSNC I2YSNC J1YSNC J2YSNC K1YSNC K2YSNC K3YSNC L1YSNC L2YSNC A1NSYC A2NSYC A3NSYC B2NSYC C2NSYC D1NSYC D2NSYC D3NSYC E1NSYC F1NSYC F2NSYC G1YSYC G2YSYC G3YSYC H1YSYC H2YSYC I1YSYC I2YSYC J2YSYC J3YSYC K1YSYC K2YSYC K3YSYC L1YSYC L2YSYC DAY_11 DAY_12 DAY_13 DAY_14 DAY_15 DAY_16 1.45 1.79 2 1.63 1.75 1.43 1.33 1.7 2 1.43 1.57 1.71 1.22 1.58 1.5 2.13 1.6 1.38 1.83 1.54 1.85 1.7 1.64 1.5 2 2 N N 2.5 3 1.43 1.36 1.45 1.86 1.8 2.67 1.11 1.6 1.44 2 1.14 1 1.4 2.14 1.8 2 1.71 1 1.83 2.5 1.88 2.6 2 2 N 1.5 1.5 1.38 1.79 2 1.77 1.6 1.71 2.33 2.38 1.83 1.62 1.47 2.09 2.15 1.75 2 1.71 2.1 1 1.67 1.58 1.38 1.11 1.57 2.4 1 1.64 1.57 1.43 1.36 1.42 1.22 1.38 1.43 1.57 1.33 1.8 1.67 N N N N N 1 1.73 1.83 2 1.9 1.71 1.33 1.6 1.67 1.93 1.42 1.5 1.4 1.43 1.86 1.33 1.69 1.85 2.14 1.25 1 1.67 1 1 2 1.25 1.75 1.33 1.8 1.86 1.8 2.75 2.07 2.62 2.2 2.29 2.11 1.46 1.55 1.64 1.5 1.92 1.58 2.23 2 2.92 2.42 2.45 2.29 2.77 2 2.23 2.07 1.79 2.27 1.86 1.08 1.31 1.93 1.71 1.73 1.86 1.38 1.5 1.71 1.5 1.75 1.85 2.33 1.8 2.09 2 1.54 1.2 1.43 2.5 1 1.6 1.6 1.2 1.13 1.4 1.33 1.6 1.53 5 N N 5 1 N 1.25 1.44 1.86 1.67 1.25 1 2 1.6 2 1.4 1.33 1 1.33 1.63 1.33 1.44 1.33 1.33 2.32 1.43 1.57 1.88 1.88 1 N N N N N N 2 1.5 2 1.6 2 1.33 1.14 1.43 1.29 1.5 1.44 1.2 1 1.25 1.25 1 3 1.33 1.5 1.2 1.56 1.75 1.2 1.25 1.33 1.17 1.33 1.33 1.31 1.56 N N N 5 N N 1.42 1.44 1.33 1.73 1.75 1.7 1.4 1 1.4 1 1.33 1.5 1.83 1.29 1.7 1.62 1.27 1.57 1.4 1.75 2.5 1.8 1.25 1.8 1.57 1.14 1.54 2 1.13 1 2 1 N N 3 N 1.92 1.85 2.23 1.29 1.92 1.67 1.29 1.43 1.43 1.75 1.67 1.4 2.17 1.6 1 1 4 2 2 1.67 1.38 1.75 2.2 1.9 1 1 2.25 1 1 1

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Fails ID DAY_1 DAY_2 DAY_3 DAY_4 DAY_5 DAY_6 DAY_7 DAY_8 DAY_9 DAY_10 A1NSNC 2 1 7 0 0 0 0 0 0 0 A2NSNC 14 1 0 1 0 0 0 0 0 0 B1NSNC 13 1 0 0 0 0 0 0 0 0 B2NSNC 1 0 0 0 1 0 0 0 0 0 B3NSNC 7 12 13 13 6 10 11 7 11 12 C1NSNC 1 1 1 1 0 0 0 0 0 0 C2NSNC 0 0 0 0 0 0 0 0 0 0 D1NSNC 2 1 6 0 0 0 0 0 0 0 D2NSNC 4 0 1 0 0 0 0 0 0 0 D3NSNC 0 0 0 0 0 0 0 0 0 0 E1NSNC 0 0 0 0 0 0 0 0 0 0 E2NSNC 2 1 0 4 1 2 2 1 1 1 E3NSNC 1 0 0 1 0 0 0 0 1 0 F1NSNC 5 2 2 0 0 1 0 2 2 1 F2NSNC 1 1 0 0 0 0 0 0 0 0 G1YSNC 0 0 7 0 0 0 0 0 0 0 G2YSNC 14 1 0 1 0 1 0 0 0 0 G3YSNC 5 0 0 0 0 0 0 0 0 0 H1YSNC 2 1 0 0 0 0 1 0 0 0 H2YSNC 0 2 1 0 1 0 0 0 0 1 H3YSNC 15 1 0 0 0 0 0 0 0 0 I1YSNC 3 2 1 0 0 0 0 0 0 0 I2YSNC 1 0 2 0 0 0 0 0 0 0 J1YSNC 1 5 10 0 0 0 0 0 0 0 J2YSNC 1 0 1 0 0 0 0 0 0 0 K1YSNC 5 10 4 5 5 4 7 3 3 1 K2YSNC 3 1 1 1 1 1 2 0 1 1 K3YSNC 0 0 2 0 0 0 0 0 0 0 L1YSNC 0 0 0 0 0 0 0 0 0 0 L2YSNC 13 1 1 0 1 1 0 0 0 0 A1NSYC 0 0 0 0 0 0 0 0 0 0 A2NSYC 0 0 1 0 0 0 0 0 0 0 A3NSYC 0 0 0 0 0 1 0 0 0 15 B2NSYC 3 0 0 0 0 0 0 0 0 0 C2NSYC 0 0 1 1 0 1 0 0 0 0 D1NSYC 0 0 0 1 0 0 0 0 1 0 D2NSYC 0 0 0 0 0 0 0 0 0 0 D3NSYC 1 0 0 0 0 1 0 0 0 15 E1NSYC 0 0 0 0 0 0 0 5 0 0 F1NSYC 0 0 0 1 0 0 0 0 0 0 F2NSYC 0 0 0 0 0 0 0 0 0 0 G1YSYC 0 0 0 0 1 0 0 0 0 0 G2YSYC 0 0 0 0 0 0 0 0 0 0 G3YSYC 0 0 0 0 0 0 0 2 0 15 H1YSYC 0 0 0 0 0 0 0 0 0 0 H2YSYC 0 0 0 0 0 0 0 0 0 0 I1YSYC 1 0 0 0 0 0 1 0 0 0 I2YSYC 0 0 0 0 0 0 0 0 0 0 J2YSYC 0 0 0 0 0 0 0 0 0 0 J3YSYC 0 0 0 0 0 0 0 5 0 15 K1YSYC 0 2 3 0 1 1 0 0 0 0 K2YSYC 0 0 0 0 0 0 0 0 0 0 K3YSYC 2 0 0 0 0 1 0 0 1 0 L1YSYC 4 2 3 0 0 1 0 0 0 0 L2YSYC 1 0 0 0 0 0 0 0 0 0

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Fails ID A1NSNC A2NSNC B1NSNC B2NSNC B3NSNC C1NSNC C2NSNC D1NSNC D2NSNC D3NSNC E1NSNC E2NSNC E3NSNC F1NSNC F2NSNC G1YSNC G2YSNC G3YSNC H1YSNC H2YSNC H3YSNC I1YSNC I2YSNC J1YSNC J2YSNC K1YSNC K2YSNC K3YSNC L1YSNC L2YSNC A1NSYC A2NSYC A3NSYC B2NSYC C2NSYC D1NSYC D2NSYC D3NSYC E1NSYC F1NSYC F2NSYC G1YSYC G2YSYC G3YSYC H1YSYC H2YSYC I1YSYC I2YSYC J2YSYC J3YSYC K1YSYC K2YSYC K3YSYC L1YSYC L2YSYC DAY_11 DAY_12 DAY_13 DAY_14 DAY_15 DAY_16 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 12 13 15 15 13 13 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 2 3 2 2 0 0 0 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 1 1 0 0 1 4 7 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 5 15 11 6 3 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 15 2 11 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14 0 2 0 2 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 3 1 0 2 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

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Fish Weight (g) Length (cm) Notes A1NSNC 5.6 6.1 A2NSNC 15.68 8.4 A3NSNC* 4.77 5.2 Died B1NSNC 5.88 5.7 B2NSNC 9.62 6.4 B3NSNC 4.27 5.1 C1NSNC 12.83 7.2 C2NSNC 12.57 6.9 D1NSNC 11.85 7.5 D2NSNC 10.47 7 D3NSNC 13.59 7.3 E1NSNC 5.54 5.4 E2NSNC 6.72 5.3 E3NSNC 12.91 6.9 F1NSNC 5.04 4.4 F2NSNC 8.46 6.3 G1YSNC 8.44 5.9 G2YSNC 12.29 6.4 G3YSNC 8.2 4.8 H1YSNC 7.46 5.7 H2YSNC 7.08 5.8 H3YSNC 11.24 6.5 I1YSNC 7 5.9 I2YSNC 8.64 6.3 J1YSNC 9.37 7.2 J2YSNC 7.24 5.3 J3YSNC* 9.32 6.5 Died K1YSNC 8.69 7.4 K2YSNC 8.85 6.9 K3YSNC 9.36 6.6 L1YSNC 5.76 5.3 L2YSNC 4.81 5.2 A1NSYC 8.2 6 A2NSYC 11.5 7.2 A3NSYC 11.8 7.1 B1NSYC* 11.4 6.8 Died B2NSYC 9.16 6.4 B3NSYC* 13.1 7.5 Died C1NSYC* 9.82 6.5 Died C2NSYC 10.81 6.4 D1NSYC 15.49 7.6 D2NSYC 10.3 6.7 D3NSYC 12.15 7.4 E1NSYC 10.76 7 E2NSYC* 7.58 6.8 Died E3NSYC* 11.85 6.9 Died F1NSYC 11.18 6.7 F2NSYC 7.76 6.4 G1YSYC 13.36 6.9 G2YSYC 8.37 6.2 G3YSYC 9.59 7 H1YSYC 11.4 7.2 H2YSYC 7.72 6.4 H3YSYC* 8.1 6.2 Died I1YSYC 9.74 6.9 I2YSYC 8.86 6.4 J1YSYC* 9.08 6.6 Died J2YSYC 9.12 5.7 J3YSYC 8.48 6.5 K1YSYC 8.23 6.6 K2YSYC 14.16 7.1 K3YSYC 4.81 5.4 L1YSYC 13.76 7.6 L2YSYC 7.72 6.1