While the conservation status of the West Indian manatee (Trichechus manatus) has been downgraded from ‘endangered’ to ‘threatened’, the continuation of the population still remains at risk. In addition to both natural and anthropogenic threats that result in high numbers of injury and mortality on an annual basis, the West Indian manatee faces potential challenges to reproduction related to the moderate level of inbreeding occurring within the population. In a first effort to mitigate the effects of inbreeding within this population, a pilot study was conducted to determine the best methods for liquid storage and cryopreservation of West Indian manatee semen. As part of this, the current study aims to understand the morphometric changes associated with liquid storage and cryopreservation on the spermatozoa of West Indian manatees. The objectives of this project are to compare the influence of different semen extenders, determine the effects of cryopreservation, and compare the influence of different parameters, such as storage temperature, storage time, and presence/absence of seminal plasma on the morphometric parameters of West Indian manatee spermatozoa.
Whole semen was collected from a wild-born, adult West Indian manatee under managed care at the Puerto Rico Manatee Conservation Center in Bayamón, PR as part of a prior research project. Semen samples were diluted in one of four liquid extenders under a variety of parameters including storage temperature (room temperature vs. 4°C), storage time (initial, 6hrs, 12hrs, 24hrs), and presence/absence of seminal plasma. In addition to liquid storage, two samples were successfully cryopreserved. For each of the liquid storage and cryopreservation samples, sperm smears were made and stained with SpermBlueÒ for morphometric analysis. 200 sperm per sample will be randomly analyzed for the following morphometric parameters: head length (µm), head width (µm), head area (µm2), head perimeter (µm), head ellipticity (length/width), head elongation ((length – width) / (length + width)), head roughness (4p(area/perimeter2), and head regularity (p(length*width/4*area)). Only fully intact sperm will be analyzed and all morphometric parameters will be analyzed at 400x total magnification by automated sperm morphometry analysis (ASMA) using a Microptic SCA® computer-aided sperm analysis (CASA) system with the SCA® morphology module. All analyzed sperm will be visually assessed to ensure no over-or underestimation of morphometric parameters occur through the automated analysis. Improperly measured sperm will be removed from analysis.