Section 1

For as long as I can remember, I have always had an immense love for animals. When I was little, around 6 or 7 years old, all I wanted was a puppy. Every moment of every day, that’s all I would talk about, constantly asking my parents to please buy me a puppy. Finally after either seeing my side or not being able to stand my nagging, my parents gave in and said that I could get a puppy. Naturally I had the big decisions of what breed of dog, its name, where it would sleep and buying all of the supplies. Eventually I had my heart set on an English Springer Spaniel because my grandfather had had five and said they were wonderful family dogs. So, we found a breeder and on June 7th, 1997, we picked up our black and white English Springer Spaniel. All I can remember was never wanting to put it down and constantly want to pet it and just love it. She would be named Carnation after my mother’s favorite flower, and to this day she is my beautiful dog, almost like a daughter to me. As time went on, I became very interested in Carnation’s veterinarian appointments and made it a priority to become friendly with my veterinarian. In going to many appointments, I found a deep respect for veterinarians and have decided that this would be my life’s work. Even to this day, I am still an active part in Carnation’s physical health and am still good friends with the veterinarian. So from this point on, every time in the veterinarian’s office I am amazed at all of the instruments and I get to learn something new every time I go.

When I announced to my family that I wanted to become a veterinarian, my grandfather felt that it would be a great idea to take me up to Cornell University where he is an undergraduate alumni, and show me Cornell’s College of Veterinary Medicine. During the summer of second grade, my grandfather and I went and visited. This experience really helped solidify my dream of becoming a veterinarian. Today my dream still lives on, and my goal is to go to Cornell’s College of Veterinary Medicine.

When I first heard about the Authentic Science Research Course offered at my high school, I knew that this was most definitely something I would be able to help further my love of animals and my dream of becoming a veterinarian. If my memory serves me correctly, I believe that I was the first person to return my preliminary information for the course, because I was so excited about enrolling.

Through the 9 or so years I’ve wanted to be a veterinarian, the actual patients I want to work on have changed. For much of the time, I was very interested in opening a small animal clinic and actually had a layout of my office and a friend of mine said she wanted to be my receptionist. Then I was interested in horseback riding, so I figured I wanted to be an equine veterinarian. For about the past two years, I have been fascinated my marine mammals, and have made it a priority to see the local aquarium whenever my family and I are on vacation. So this was where my topic for my research project was born.

Now that I knew I wanted to work on a project involving marine mammals, I started looking at different animals and eventually narrowed it down to three. I was interested in sea otters, walruses and manatees. After doing some preliminary researching on these three mammals, I think that the amazing enthusiasm, the extreme playfulness and all around cuteness of the sea otters was what drew me to them.

Once deciding on the sea otters, I now had to figure out what aspect of the sea otters that I wanted to research. I remember thinking about a plethora of ideas, but for some reason pathology and the study of infections and diseases really drew me in. I guess, going along with the veterinarian theme, I wanted to help the sea otters. I wanted to make sure that my experiment would be beneficial to help save some of the most beautiful creatures of this world.

I continued to pursue sea otter pathology and came across some researchers in California. I was able to obtain their articles and eventually decided to make contact with these researchers. They were very kind to me, allowing me to ask questions and providing me with whatever information I needed. I continued to build a relationship with these researchers and eventually started to narrow down my topic to a certain sea otter disease known as Toxoplasma gondii. In speaking with the researchers, I started to see that research in this area would be very beneficial and eventually I decided on a research question. In the articles that I read, it is mentioned that Toxoplasma gondii is in oocysts which are in cat feces. Looking at different places like agricultural, city sewers, etc., would be helpful to see where the highest concentration of the Toxoplasma gondii oocysts are coming from. What also might be an added piece would be to compare cities along the southern California coastline. Hopefully, this way I would be able to come up with the most potent and highly concentrated areas for the Toxoplasma gondii oocysts.

It was at this time when I was very much settled on a project and thrilled with the prospect of doing research in California that I came to the unfortunate and tragic realization that conducting research in California would not be feasible due to the distance. Feeling very anxious to find a new topic quickly, I pursued otters but decided to change otter species when an incredible opportunity presented itself at the Norwalk Aquarium in Connecticut. I was given the opportunity to become an intern at this aquarium and in doing so I would be able to work with their two North American River Otters.

In making a small switch from sea otters to river otters, I began a search to see who was researching river otters and what experiments were being conducted. I came across a professor from Frostburg State University in Maryland who is a river otter researcher. When I contacted him, he put me in touch with one of his graduate students, who very willingly sent me a copy of her thesis proposal. Her experiment was to measure the stress levels in river otters that were going to be reintroduced into the wild in the Pennsylvania River Otter Reintroduction Program. First she had to validate an Adrenocorticotropic hormone (ACTH) challenge to show that stress could accurately be measured in fecal samples of river otters through the release of glucocorticoids, or steroids released during stress from the adrenal cortex. From this proposal was where the idea came from for my very own authentic research experiment.

In just one and a half short years, I had already dealt with many obstacles. As I would later find out, that is a big part of the research process. I was shattered when I realized that my project that was basically already completely set up would no longer be able to be completed. However, I was glad for the people including my family and research teacher who pushed me to not give up and continue to pursue research that interested me, as hard as it was to leave the old project behind. In doing so, I found a project that I created completely on my own and it is one of the biggest accomplishments that I have pride in as I look back on my high school years. This project was not able to be done without a mentor, though, and it is with extreme happiness that I had three mentors. These three individuals were and are phenomenal teachers and friends. I am so blessed to have met them and to have had the opportunity to work with them. From tragedy and devastation come hope and a future. Don’t let go of your dream, find a way to make it happen regardless of how long or how much work it takes to get there. After my initial project was aborted, I had many contemplations of leaving the research class at my high school. Now, looking back, staying in that class was the best decision I could have ever made.

Section 2

While much information does come from wild river otter populations, a great deal has been recorded by researchers through the observation and experiments done on captive river otters in zoos and aquariums throughout the world. Caged environments can be extremely stressful for captive animals, especially wild animals in zoos. Stereotypical behavior such as pacing and weaving is exhibited by captive river otters, and these stereotyped behavior patterns are considered symptomatic of boredom and unhappiness in the animal caused by captivity. These occurrences are seen as a result of the river otter spending long periods in captivity, the majority of whom spend their entire lives there. Behavioral/environmental enrichment has been introduced in many facilities to increase the welfare of the animals by incorporating a stimulating environment. Enrichment goals include reducing stereotyped behaviors and increasing more natural behaviors such as hunting, foraging and mating techniques observed in the wild. Interaction with trainers, commands and tasks required for execution before feedings, use of varied scents and play items, like objects or toys, are all common forms of environmental enrichment.

While enrichment is currently widespread, there is still concern about the stress in captive animals. In studying the stress response, the area that is focused upon during the response is the hypothalamic-pituitary-adrenocortical axis (HPA). Stress activation of the HPA leads to the secretion of corticotropin-releasing hormone (CRH) from the neurosecretory cells of the hypothalamus. The primary function of CRH is to stimulate the secretion of adrenocorticotropic hormone (ACTH) from the pituitary gland. ACTH stimulates the synthesis and secretion of glucocorticosteriods, like cortisol, from the adrenal cortex. Cortisol and corticosterone are glucocorticoid (GC) hormones, with cortisol being the primary GC in humans and mammals such as river otters. Maintaining a sufficient concentration of GCs is imperative in order to maintain homeostasis in the body.

GCs can be measured through extraction from blood, urine, scat or saliva. In order to collect blood, urine, or saliva the animal must be handled. However, handling an animal has been shown to elevate stress levels, as it is a stressor in itself. Measuring GCs extracted from fecal samples is a relatively new procedure in free ranging animals and unlike blood, urine and saliva samples, collection of fecal samples is a non-invasive method and does not involve contact with the animal. GCs can be measured through enzyme immunoassays and each individual species must have the use of these assays validated. Recently this procedure was validated for use in the North American River Otter by demonstrating there were no significant differences between the slopes of a serially diluted standard and a pooled scat sample from captive river otters.

The purpose of my research study was to evaluate whether non-edible behavioral enrichment, in the form of objects of artificial or natural composition intended for mental and physical stimulation, reduces the stress levels in captive North American river otters (Lontra canadensis). While varying the frequency of non-edible behavioral enrichment in zoo/aquarium river otter exhibits, measurement of stress via corticosterone levels were evaluated to see if this change in frequency reduced stress levels. The research hypothesis was that as the frequency of non-edible behavioral enrichment increases, the stress levels of the captive river otters will decrease.

Included in the study were one male and one female from the Maritime Aquarium of Norwalk, CT, and two males from the Seneca Zoo of Rochester, NY. Each river otter was given a unique identification code for later identification of scat samples.

An 18 day experiment was conducted at both facilities from May 17, 2006 to June 3, 2006. The Seneca Zoo of Rochester, NY, began by including non-edible behavioral enrichment in the exhibit every day for the first 6 days (May 17, 2006 to May 22, 2006), then non-edible behavioral enrichment was included in the exhibit every other day for the following 6 days (May 23, 2006 to May 28, 2006) and finally no non-edible behavioral enrichment was included in the exhibit for the last 6 days (May 29, 2006 to June 3, 2006).

The Maritime Aquarium of Norwalk, CT, began with no non-edible behavioral enrichment included in the exhibit for the first 6 days (May 17, 2006 to May 22, 2006), then non-edible behavioral enrichment was included in the exhibit every other day for the following 6 days (May 23, 2006 to May 28, 2006), and finally non-edible behavioral enrichment was included in the exhibit every day for the last 6 days (May 29, 2006 to June 3, 2006).

The amount and specific non-edible objects/toys were determined by the facility staff based upon the amount and kind of non-edible behavioral enrichment objects/toys available at the given time. The objects/toys that were included in the exhibit were usually objects that had not been in the exhibit recently or on a frequent basis. All other procedures normally followed by each facility, including edible enrichment schedule, feeding times, cleaning, trainer interactment, etc., remained unchanged during the 18-day experiment. Beginning with day 1 of the experiment, fecal samples were collected every other day from each river otter in the mornings prior to the first feeding. The fecal samples were labeled with identification numbers and stored at -20˚C. By the end of the experiment, nine fecal samples were collected for each river otter. Information including diet, feeding times, and training behaviors of the river otters were also recorded. In addition, medication and veterinarian examinations also were recorded by zoo keepers.

The Correlate-EIA Corticosterone Enzyme Immunoassay Kit was used to determine the glucocorticoid values in the river otter fecal samples. The directions included in the kit were followed precisely, without any modifications.

Results of Correlate-EIA Corticosterone Enzyme Immunoassay Kit

Corticosterone Levels for Otters A1, A3, C1, and C2 for May 17, 2006 to June 3, 2006

Time	Corticosterone Levels (ng/ml)
Point	Otter
	A1	A3	C1	C2
17-May	16.785	9.955	16.270	15.804
19-May	84.698	9.721	16.508	19.112
21-May	20.935	16.764	31.979	20.820
23-May	85.146	18.840	13.983	75.801
25-May	27.157	29.555	13.708	28.788
27-May	19.346	10.616	22.999	18.613
29-May	21.627	18.875	15.140	24.710
31-May	17.094	8.966	11.053	16.964
2-Jun	31.536	14.217	21.616	23.541

The lowest average GC values occurred during the period where non-edible behavioral enrichment was included in the exhibit every other day, with the exception of Otter A3 who exhibited only a 1.8727 ng/ml average difference between non-edible behavioral enrichment every day and no non-edible behavioral enrichment. Average GC levels during the periods of no non-edible behavioral enrichment and non-edible enrichment every day were lower then every other day, however the lowest GC levels when non-edible behavioral enrichment was provided every day. All 4 river otters had increased average GC values when given non-edible behavioral enrichment every other day compared to when given non-edible behavioral enrichment every day. Additionally, three of the four (75%) river otters in this study exhibited the lowest GC levels during periods of non-edible behavioral enrichment consistently every day. These GC levels were the lowest when non-edible behavioral enrichment was provided every day in the river otter exhibits. This supports the idea that non-edible behavioral enrichment provided every day is an ideal protocol for non-edible behavioral enrichment. Periods of on again-off again non-edible behavioral enrichment are not ideal in a captive setting because it is a bigger stressor then having no non-edible behavioral enrichment at all.

The results of this experiment show that my hypothesis was supported in that stress levels were the lowest when non-edible behavioral enrichment was occurring every day. However, the highest stress levels were actually found to be during periods of non-edible behavioral enrichment every other day versus no non-edible behavioral enrichment at all. This could possibly be because of the irregularity of the frequency of the non-edible behavioral enrichment. When the river otters are unaware of whether or not non-edible behavioral enrichment will be provided it potentially elevates stress levels. When non-edible behavioral enrichment is included in the exhibit every day, the river otters are expecting non-edible behavioral enrichment because it is on a fixed schedule; hence this is when the lowest GC values are observed.

The uses of fecal GC assays have tremendous and far reaching practical applications. This procedure could greatly benefit captive wildlife management in enabling zoos/aquariums to see what sources of stressors there are for their captive river otters. Fecal GC assays could be used to see if other parts of a captive environment, like edible behavioral enrichment, land/water ratio, outdoor vs. indoor enclosure, etc., are stressors and then could design an ideal exhibit bearing these stressors in mind.

• Biology