Ferrets ( Mustela putorius furo) under the influence of Ginkgo biloba

1. Figure 2: Mazes for each part of this study had different goal boxes in which the treat was placed. A fan was situated at the entrance of the maze to insure the subjects were not following the treats’ scent to maneuver the maze. Setup designated B, was used for the first, third, and fifth mazes. Setup A, was used for maze numbers two, four, and six. Figure #3: The effect of treatment on average maze running time . Bars represent standard error. Letters designate times that are significantly different from one another (p < 0.05). Figure 4: Age of subjects at 6 months, 7 months and 9 months affects the average maze times of subjects. Bars represent standard error. Letters designate ages that are significantly different from one another (P-value<0.05). Ferrets (Mustela putorius furo) under the influence of Ginkgo biloba By: Tracy WilliamsAdvisors: Dr. McShaffrey and Dr. Lustofin Introduction Background: Ginkgo biloba has been used homeopathically for years as a memory enhancer. Ginkgo biloba extract has been shown to significantly increase the neurological impulses of mice (Williams et al., 2004, 958). In 2003, a study found that short term memory was increased by Ginkgo biloba treatments; rats dosed with Ginkgo biloba were found to navigate water mazes faster than undosed rats (Hoffman et al., 2003, 538). ). In a review by DeFeudis and Drieu, it was found that Ginkgo biloba reduce s neurological disruptions and is currently being studied as a treatment for Alzheimer’s disease (DeFeuris & Drieu, 2000, 31). Although drugs are frequently tested on rats and mice, ferrets (Mustela putorius furo) are becoming a more common subject for infection and pathogen studies in the medical field . Drug development companies are also utilizing ferrets. Ferrets are also commonly used for studies of cancer, respiratory disease and pathogens, gastrointestinal disease, cardiovascular disease, renal disease, and nutritional research (Ball, 2006, 349). Use of ferrets as a model organism is increasing, in part because ferrets are physiologically and morphologically more similar to humans than mice (Li & Engelhardt, 2003, 2). Given this similarity, it makes sense to test the effects of Ginkgo on ferrets. Hypothesis When ferrets are subjected to mazes, the ones that are dosed with Ginkgo will be faster and more accurate in their maze running than ferrets dosed with sucrose alone. Materials and methods Four jill ferrets were obtained from Marshall Ferrets. The ferrets resided in a community setting. This setup helped the ferrets to maintain social and behavioral normalcy and reduce separation anxiety; ferrets are social animals and this setup was to insure that undue stress was reduced. The ferrets were handled regularly to minimize fear and subsequent poor data from apprehensive subjects. Subjects were six months old at beginning of trial. To insure subject differentiation, photographs (see Figure #1) and detailed physical descriptions were recorded. Dosing was modeled after an experiment conducted on rats by Hoffman et al. (2004). Ferrets in this study received 10 milligrams of Ginkgo biloba per kilogram body weight suspended in a 10% sucrose solution; placebo of a 10% sucrose solution was utilized as a control. Treatment was administered 30 days prior to maze introduction and continued throughout duration of experiment. Administration of treatment and placebo alike was given orally by un-needled syringe. Experiment #1 consisted of Subjects A and B receiving treatment of Ginkgo, whilst Subjects C and D received placebo only, as a control. After a dosing time of 30 days, the ferrets were introduced to a maze. Maze was had a different goal box, Figure 2 shows the treat locations. Ferrets were encouraged by use of treats to maneuver the maze, individually. Time to complete the maze and the path of the ferret were recorded. Three sessions were conducted daily. After several days of maze #1, a new maze, designated maze #2, was constructed and runs continued. Time lapse between experiment #1 and experiment #2 was 30 days to allow treatment to clear from the subjects’ systems. Experiment #2 consisted of Subjects C and D receiving the treatment animals while Subjects A and B received placebo. The procedure for Experiment #2 was otherwise identical to Experiment #1. New mazes were also utilized and designated maze #3 and maze #4. Experiment #1 was repeated to insure that age of subject was not affecting the data. Two weeks was allowed for treatments to clear from subjects’ systems. During this time, Subjects A& B were reintroduced to Ginkgo biloba. For this part of the experiment, protocol was followed just as in Experiment #1. New mazes were constructed and named maze #5 and maze #6. Effects of subject on time and treatment versus control for all experiments were statistically analyzed using one way-ANOVA. Results In first experiment, time taken to navigate the maze varied from 1 to 300 seconds. The difference in amount of time it took each subject to run the maze was not significantly different , regardless of treatment status (P-value= 0.068). In the second experiment, maze completion times per subject varied from 1 to 176 seconds. Statistically though, there was not a significant difference between subjects whether on Ginkgo biloba or not (P-value = 0.618). In the third experiment, time taken to complete the maze was 1 to 38 seconds. The difference in the amount of time elapsed for maze completion was not significantly different between the untreated and treated subjects (P-value = 0.277). Overall, there was no significant effect with the treatment of Ginkgo biloba on maze running times (P-value = 0.479). This is shown in Figure 3. There was also no significant effect of treatment on the number of mistakes made by each individual (P-value = 0.106). Subjects compared to each other were significant in the difference in time they spent in the maze. As seen in Figure 3, Subject A was significantly different from Subject B (P-value = 0.000). Subject A to Subject D was also statistically significant (P-value = 0.025). Subject B to Subject C was significant (p-value= 0.000). Subject C to Subject D was also significant (P-value = 0.022). Only Subjects A and C were not significantly different from each other (P-value = 0.968). There was a significant effect of the subject on maze times. Figure 4 shows the variation of maze times between the ferrets at 6, 7, and 9 months old. In addition to significant differences between the ages, at each age the subjects were significantly different from each other: at 6 months the P-value = 0.003; at 7 months the P-value = 0.000; however at nine months old, the subjects’ age had no significant effect (P-value = 0.430). Conclusions There is not enough sample evidence to conclude that ferrets dosed with Ginkgo biloba ran faster or more accurately in mazes than placebo dosed ferrets. Though Ginkgo biloba was not found to be statistically significant for this study, it does not necessarily mean that this treatment is not beneficial for the promotion of short term memory. The variance between the subjects as well as the variability that age causes could have affected the data. The age of ferrets significantly affected the maze completion times; however, at nine months, age was no longer a significant factor. Future studies should consider using subjects nine months old or older to eliminate this variable. A concentration of 10mg/kg Ginkgo evidently is not an optimum level for ferrets. This study used the concentration found to be most beneficial for rats (Hoffman et al., 2004, 535). Future studies should determine the ideal level of Ginkgo needed to increase short term memory in ferrets. Literature Cited Ball RS. 2006. Issues to consider for preparing ferrets as research subjects in the laboratory. ILAR. 47: 248-357. DeFeudis FV and Drieu K. 2000. Ginkgo biloba extract (Egb 761) and CNS functions: basic studies and clinical applications. Current Drug Targets 1: 25-58. Hoffman JR, Donato A, Robbins SJ. 2004. Ginkgo biloba promotes short-term retention of spatial memory in rats. Pharmacology Biochemistry and Behavior. 77: 533-539. Li Z and Engelhardt JF. 2003. Process toward generating a ferret model of cystic fibrosis by somatic cell nuclear transfer. Reproductive Biology and Endocrinology 1: 1-8. Williams B, Watanabe CMH, Schultz PG, Rimbach G, Krucker T. 2004. Age-related effects of Ginkgo biloba extract on synaptic plasticity and excitability. Neurobiology 25: 955-962. Acknowledgments Special thanks to my advisors: Dr. McShaffrey and Dr. Lustofin for countless hours of assistance. Dr. Brown is also appreciated for his valuable input. The Marietta College Biology Department is acknowledged for the financial support provided for my research. The graduating class of 2011 is given thanks for beneficial feedback. A. B. b b c a c a a c Figure 1: Test Subjects from left to right -- Subject A, Subject B, Subject C, and Subject D.