Validation of manual semen collection methods and sperm evaluation in living freshwater stingrays (Potamotrygon falkneri) kept in ex situ conditions

The Potamotrygon from the Potamotrygonidae family is a freshwater stingray that is native to and distributed throughout South American watersheds. The degradation suffered by the environment in recent years, particularly in aquatic habitats, directly affects native fish species. Due to anthropic actions, some species of the genus Potamotrygon are threatened with extinction, encouraging a study on captive breeding procedures. Six Potamotrygon falkneri adult males, kept under the care of the São Paulo Aquarium, Brazil, were divided into groups to compare two distinct semen collection protocols: physical restraint (PR) and chemical restraint (CR – 3 mg / kg propofol, directly into the gills). The protocols were alternated between the two groups in a 50-day interval. For both restraining protocols, after positioning the animal on a moistened foam in dorsal recumbency, semen collection was performed by placing a collecting tube at the cloaca opening while performing a gentle massage of the vas deferens. Sperm evaluation was performed at the moment of collection (time 0) by means of conventional semen analysis. There was no significant difference between the restraining methods examined for any parameter evaluated. The results suggest that physical restraint followed by manual massage is an effective approach to harvest semen in stingrays and should be considered the first choice of method, as it not only demonstrated to be a faster procedure but more importantly eliminated anaesthetic-associated risks for the animal. To our knowledge, this is the first study that validates the semen collection technique in live Potamotrygon falkneri rays and is extremely relevant for the development of future reproductive biotechniques in Potamotrygon .