Gametes galore!

By Cecilia Bueno, Lewis & Clark College

I am conducting my experiment in two parts: the first looks at how increased salinity affects fertilization success, the other looks at how salinity affects sperm function.

The fertilization experiments start with watching the weather- squirrel treefrogs mate on nights when there has been a lot of rain. We collected frogs from dixie plantation on nights when there had been 0.5 inches of rain accumulation or more. Since squirrel treefrogs mate at night, we would go out around 10:30pm and stay often until 3:00am when we found pairs.

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Grassy wetland at Dixie Plantation where several pairs of squirrel treefrogs were found

The frog pairs were taken back to the lab where they were placed in individual tubs with water at 6ppt salinity. When the pair had laid around 200 eggs, we removed them from the tubs and placed them with a new partner. This repeated until the frogs no longer went into amplexus- running from 4:30am to 8:00am.

After the eggs had been laid and the frogs stopped going into amplexus, the eggs were transferred from the large tubs to smaller weigh dishes. These weigh dishes with eggs were placed under the microscope and photographed for counting. I then counted how many eggs in each dish were fertilized. These counts- total eggs and number fertilized- will be used to calculate fertilization success of each of the pairs.

The second part of the experiment focused on sperm function of the frogs. Males from the initial pairs were kept for this experiment, while females and fertilized eggs were released. We created a sperm concentrate using the testes of each male. This sperm concentrate was then added separately to different tubes containing water at different salinity levels. We tested sperm at salinity levels which had previously been shown to have an effect on sperm function- 4ppt, 5ppt, 6ppt, 7ppt and 8ppt- as well as a control of 0.39ppt.

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Screenshot of a video of sperm from a squirrel treefrog male

Once the sperm concentrate has been added to a test tube, the sperm is activated so I have to act quickly. The diluted sperm solution is placed under a microscope and video taped.

After the videos have all been taken, they will be analyzing them with the CASA (Computer Assisted Sperm Analysis) software developed by Wilson and Leedy. This software, run on ImageJ, tracks motile sperm and calculates percent motility and average velocity of the motile sperm.

When I have counts on percent motility and average velocity of sperm in different treatments, I will be able to compare the results I get from the sperm experiments to the results from the fertility experiments.

I would like to thank the National Science Foundation for funding this REU program, and the Grice Marine Lab of the College of Charleston for hosting us. In particular, I would like to thank my mentor Dr. Allison Welch for her help and support.

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