Developing a Cryopreservation Protocol for Embryos of Xenopus laevis , the South African Clawed Frog
The development of a cryopreservation protocol for Xenopus laevis embryos was investigated to increase the utility of X. laevis for research and educational purposes. This project evaluated the developmental effects of cryoprotectants, freezing and thawing procedures, and low temperature (exposure to 4°C) on early stage embryos, using the Frog Embryo Teratogenesis Assay-Xenopus (FETAX) protocol. These aspects have shown great importance in the cryopreservation of bovine, fish, mouse, and human embryos. X. laevis, the South African clawed frog, was selected for this research as it has historically been an important model organism used in developmental biology and molecular biology research. X. laevis embryos are easily manipulated and their developmental stages well established. Embryos were exposed to cryoprotectants, including DMSO, PVP, Galactose, and cryoprotectant cocktail. Exposed embryos were either cryopreserved in a Mr. FrostyTM freezing container or placed in an incubator for FETAX. Frozen embryos were thawed by various methods. For FETAX experiments, mortality, malformation, and length measurements were recorded for each experiment. The 96-hour LC50 (concentration to kill 50% of a population), 96-hour EC50 (concentration to cause malformations in 50% of a population), and Teratogenic Index (TI=LC50/EC50) were calculated using Bonferroni’s multiple comparison test via Systat 13. For cryopreservation experiments, the percentage of embryos that remained structurally intact after thawing were recorded. Based on these results, an improved protocol utilizing cryoprotectant mixtures with low temperature exposure and a modified thawing procedure was developed.
1 Dept of Biology, Jacksonville State University, Jacksonville, AL; 2 Dept of Biology, Jacksonville State University, Jacksonville, AL
80 • W. Walker Stinnette1, Jordan M. Ellington1, Peter Van den Hurk2, Dennis C. Haney1
Relationships Between Land Cover, Water Chemistry, Biomarkers, and Fish Diversity in the Piedmont Region of South Carolina, USA
Row crops, livestock pastures, and forested areas are common in the Piedmont region of South Carolina. To determine the biological effects of exposure to agricultural runoff associated with these land covers, fish, and water samples were collected from streams in the Savannah and Broad River Basins in the summers of 2011 through 2013. Sampling sites were selected based on varying percentages of land cover devoted to pasture, forest, and row crop determined using land cover data generated by the United States Dept of Agriculture. Using a backpack electrofisher, seine, and dip nets, fish were collected, preserved, and identified to species in order to determine species richness, species abundance, and Simpson’s diversity at each site. Preliminary measurements suggest fish populations were not significantly different between farmland, pasture, and forested streams. For biomarker analyses, previously visited sites, four in the Broad and nine in the Savannah River Basin, were revisited to collect live fish (genus Lepomis). Lepomis individuals were transported alive to the laboratory where they were anesthetized and tissue samples (liver, blood, and gall bladders) were removed for later analyses of glutathione-S-transferase and acetylcholinesterase activity as indicators of anthropogenic exposure. Water samples were tested for concentrations of nutrient chemicals (nitrate and phosphate) and turbidity. The biological effects, species information, and water chemistry were examined with respect to the varying percentages of pasture, forest, and row crop land covers to potentially uncover the presence of a relationship between the biomarkers, water quality, species diversity, and land cover activity.
81 • Jordan M. Ellington, W. Walker Stinnette, Dennis C. Haney
The Effect of Agricultural, Pastured, and Forested Land Covers on Freshwater Stream Fish in the Piedmont of South Carolina
Land covers are affected differentially by anthropogenic influences. Two major anthropogenic problems found in streams are (1) channelization that removes the natural run, riffle, and pool patterns that many fish species depend on for survival, and (2) the runoff of excess sediment, fertilizer, pesticides, and other human-produced chemicals that can negatively influence biodiversity and organismal abundance. This study was conducted to examine the effects of pastured, forested, and agricultural land covers on stream fish assemblages and life history characteristics, including age class structures of abundant fish species. We sampled from rural streams in close proximity to row crop agriculture, pastures, and forests in both the Saluda and Savannah River basins in South Carolina and hypothesized that areas with higher human usage (e.g., agricultural areas) would have lower fish abundance, diversity, and fewer age classes present. In conjunction with fish sampling, performed using a backpack electrofisher, seine, and dip-nets for 480 seconds of electrofishing time at each site, we also performed a grain size analysis of each stream site and collected water samples that were analyzed for temperature, pH, dissolved oxygen, and all major anions and cations. Fish were identified to the species level, measured for standard length, and weighed. Agricultural and pasture sites were found to consist of mostly coarse sand and the forested sites consisted mostly of coarse to medium size sand. Despite anthropogenic affects of farmland on streams, measurements of fish populations were not significantly different between farmland, pasture, and rural streams.
