Abstract
Manatee cold stress syndrome is a cascade of events that predisposes manatees to opportunistic pathogens involving multiple organ systems. Affected manatees are forced to make dietary changes, which alter their gut flora. Manatees depend on gut microbes for digestion. The affect of cold stress syndrome on microbial populations in the gut is unknown, and thus creates challenging decisions for manatee conservation and health. The objectives of this study were to establish in vitro conditions to culture manatee gut microbes, compare and contrast manatee and rumen microbial fermentations, and simulate cold stress conditions in vitro to identify the effects of fasting and subsequent recovery by manatee gut microbes.
Since the Florida manatee is considered a hindgut fermentor, the general knowledge of domestic terrestrial herbivorous animals' gastrointestinal anatomy, physiology, and microbial ecology, and the techniques used to culture gut contents of herbivorous animals can be employed to closely mimic the manatee microbial ecosystem. In July 2003, manatee carcasses transported within 24 hours of death to the Marine Mammal Pathobiology Laboratory (St. Petersburg, FL) for necropsy were considered for the fermentation studies. During each necropsy, the entire GI tract from the stomach to the rectum was removed and tightly tied with a string to isolate sampling areas. The sampling and batch culture fermentation protocols followed for this study were similar to rumen microbial in vitro fermentation studies.1 Colon microbial fluid was obtained for culture. Anaerobic batch culture fermentations were incubated for either 24h or 48h and included the following treatments: no substrate with delayed feeding, or 0.4 g of freeze-dried sea grass (Thalassia testudinum). After 24 h or 48 h of incubation, the serum bottles were refrigerated to inhibit fermentation. The bottles were transported on dry ice to North Carolina State University for subsequent analyses. Rumen fluid from a cannulated non-lactating dairy cow fed a predominantly forage diet was collected for rumen batch culture experiments using the same protocol as described above. Methane, culture pH, and volatile short chain fatty acids (SCFA) were determined for both manatee & rumen fermentations. Data was analyzed using a general linear model, which included the effect of animal (manatee vs. ruminant), substrate (none vs. seagrass), time (24 hour fermentation vs. 48 hour fermentation) and residual error. The SAS statistical program was used and significance was reported at P < 0.05.
Incubation of manatee microbial cultures based on in vitro fermentation techniques used for rumen contents proved successful. Overall seagrass incubated with manatee cultures produced significantly higher concentration of total SCFA, lower concentrations of methane and resulted in a lower pH than rumen fluid fed the same substrate. Furthermore, manatee cultures resulted in a higher concentration of acetate when compared to rumen microbes fed the same substrate. A higher acetate, lower methane fermentation pattern suggests that manatee microflora were more efficient in reducing the potential loss of carbon energy in the form of methane. Delaying feeding for 24 hr resulted in significant reductions in overall fermentation. Microbial cessation was not seen. The effect of starvation on microbial growth may be more pronounced at intervals greater than 24 or 48 h. Further manatee microbial fermentation work is needed to provide better conservation and health protocols for captive and rehabilitating manatees.
Acknowledgements
Funding was provided by Geraldine R. Dodge Foundation "Frontiers for Veterinary Medicine" Fellowship. This research made possible in a variety of ways by the following people: Sarah McLeod, Dr. C. Edward Stevens, Dr. Michael Stoskopf, & Dr. Felica Nutter (North Carolina State University), Dr. Scott Martin (University of Georgia), and Dr. Todd Bishop (Cornell University). Manatee necropsy, sample collection, and fermentation odor tolerance was provided by the follow people: Butch Rommel, Andrew Garrett, Alex Costidis, Ken Arrison, Tom Pitchford, Katie Tripp (Florida Fish and Wildlife Conservation Commission-Marine Mammal Pathobiology Lab).
References
1. Lynch HA, SA Martin. 2002. Saccharomyces cerevisiae culture and Saccharomyces cerevisiae live cells on in vitro mixed ruminal microorganism fermentation. Journal of Dairy Science. 85:2603-2608