All extant groups of Elasmobranches have internal fertilization and the structure of the male reproductive organs is very specific: sperm passes from the internal organs via the cloaca, but the male copulating organ (clasper) is distant from the cloaca. This suggests that sperm can contact the surrounding medium before fertilization. Because of this involvement with the environment, external signaling in sperm motility activation could occur in these species even though their fertilization mode is internal. In this case, spermatozoa of Elasmobranches should hypothetically possess a specific structure and membrane lipid composition which supports physiological functions of the sperm associated with environmental tonicity changes occurring at fertilization. Additionally, sperm motility properties in these taxa are poorly understood. The current study examined sperm lipid composition and motility under different environmental conditions for the ocellate river stingray, Potamotrygon motoro, an endemic South America freshwater species. Sperm samples were collected from six mature males during the natural spawning period. Sperm motility was examined in seminal fluid and fresh water by light video microscopy. Helical flagellar motion was observed in seminal fluid and resulted in spermatozoon progression; however, when diluted in fresh water, spermatozoa were immotile and had compromised structure. Lipid class and fatty acid (FA) composition of spermatozoa was analyzed by thin layer and gas chromatography. Spermatozoa FAs consisted of 33 ± 1% saturated FAs, 28 ± 1% monounsaturated FAs (MUFAs), and 41 ± 1% polyunsaturated FAs (PUFAs), and a high content of n-6 FAs (32 ± 2%) was measured. These results allowed us to conclude that sperm transfer from P. motoro male into female should occur without coming into contact with the hypotonic environment so as to preserve potent motility. In addition, this unusual reproductive strategy is associated with specific spermatozoa structure and lipid composition. Low level of docosahexaenoic acid and relatively low PUFA/MUFA ratio probably account for the relatively low fluidity of freshwater stingray membrane and can be the main reason for its low tolerance to hypotonicity.

Biology Centre of Czech Academy of Sciences Institute of Parasitology Branišovská 31 370 05 České Budějovice Czech Republic

São Paulo State University Ilha Solteira Faculty of Engineering Department of Biology and Zootechny Neotropical Ichthyology Laboratory LINEO Monção Street 226 15385 000 Ilha Solteira SP Brazil

Swedish University of Agricultural Sciences Department of Molecular Sciences PO Box 7015 75007 Uppsala Sweden

University of South Bohemia in České Budějovice Faculty of Fisheries and Protection of Waters Bohemian Research Center of Aquaculture and Biodiversity of Hydrocenoses Zátiší 728 2 389 25 Vodňany Czech Republic

University of South Bohemia in České Budějovice Faculty of Science Institute of Chemistry Branišovská 1760 370 05 České Budějovice Czech Republic

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