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Flow-Cytometric Analyses of Viability Biomarkers in Pesticide-Exposed Sperm of Three Aquatic Invertebrates

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Abstract

Toxicity studies on sperm often use fertilization success as the end point. This type of assay can be affected by sperm density, egg quality, and sperm–egg compatibility. Testing sperm viability biomarkers with flow cytometry is a fast, high-throughput technique for seminal analysis. In this study, we detected sperm viability biomarkers with several fluorescent reporter dyes using flow cytometry in three aquatic invertebrates (Crassostrea virginica, Dreissena polymorpha, and Lytechinus variegatus) after exposure to a pesticide and herbicide. The pesticide, Bayluscide, appeared to affect mitochondrial membrane potential in the sperm of all three species, as measured with MitoTracker Red CMXRos®. A decrease in the percentage of sperm stained with SYBR®-14 (indicating uncompromised plasma membrane) was observed in C. virginica and D. polymorpha sperm exposed to Bayluscide, but propidium iodide staining (indicating compromised plasma membranes) appeared to be inhibited by Bayluscide. Acrosome-reacted sperm, as measured by FITC-PNA, decreased after Bayluscide exposure in C. virginica and D. polymorpha sperm. The herbicide, Roundup Ready To-Use-Plus®, did not affect the overall percentages of sperm stained with MitoTracker but did cause an increase in MitoTracker fluorescence intensity at 16 mg/L in D. polymorpha. Roundup also caused significant decreases in SYBR-14 staining, significant increases in propidium iodide staining, and significant increases in FITC-PNA staining in D. polymorpha sperm. By not having to rely on egg availability and optimal sperm density, sperm toxicity can be more accurately assessed with flow cytometry as being directly correlated to sperm viability rather than the possibility of altered toxicity results due to sperm-to-egg compatibility.

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Acknowledgments

We would like to thank Shana Garrett for assisting in the flow-cytometry experiments and Dr. Steven Watts for providing the sea urchin feed. Collection and research on the sea urchins were conducted pursuant to a Special Activity License (08SR-1078) issued by the Florida Fish & Wildlife Conservation Commission. We are also grateful to the anonymous reviewers for their comments on the article.

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  1. Department of Biological Sciences, Louisiana State University, 202 Life Sciences Bldg, Baton Rouge, LA, 70803, USA

    Karen P. Favret & John W. Lynn

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  1. Karen P. Favret
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  2. John W. Lynn
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Correspondence to John W. Lynn.

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Favret, K.P., Lynn, J.W. Flow-Cytometric Analyses of Viability Biomarkers in Pesticide-Exposed Sperm of Three Aquatic Invertebrates. Arch Environ Contam Toxicol 58, 973–984 (2010). https://doi.org/10.1007/s00244-009-9410-z

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  • DOI: https://doi.org/10.1007/s00244-009-9410-z

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