Cryopreservation of Rhesus Monkey (Macaca mulatta) Epididymal Spermatozoa Before and After Refrigerated Storage

Abstract

Recently, there has been an increased interest in preservation of epididymal sperm as a potential source of material for genetic resource banking; however, cryopreservation of epididymal sperm from the rhesus monkey has not been explored. This study evaluated the effect of prolonged refrigerated storage of the intact cauda epididymides at various conditions on the postthaw motility of rhesus monkey epididymal spermatozoa, and also tested whether altering cryoprotectants and cooling methods could improve post-thaw motility for epididymal sperm after refrigerated storage. Motility before freezing decreased significantly after refrigerated storage (0 degrees C) for a period of 24 or 48 hours. Although postthaw motility was not significantly different after 24 hours of refrigerated storage, epididymides stored at a higher temperature (4 degrees C-10 degrees C) yielded better results, but postthaw motility still decreased significantly after 48 hours of refrigerated storage at 4 degrees C. Comparisons of glycerol and ethylene glycol at 3% and 6% revealed similar postthaw motility. However, consistently high postthaw motility was obtained with 3% glycerol throughout all freezing trials regardless of whether samples were collected fresh or after refrigerated storage for 24 or 48 hours. Cooling at a higher rate of 220 degrees C/min was found to yield better postthaw motility than the slower rate of 29 degrees C/min. Thawing time duration was evaluated, and a minimum of 30 seconds was required for thawing 0.25-mL straws containing 50-microL semen samples. An overall average of 42% postthaw motility was obtained for rhesus monkey epididymal sperm packed in 3% glycerol and cooled after 24 or 48 hours refrigerated storage. These postthaw motility results for epididymal sperm indicate that this method should be practical for use in preserving epididymal sperm, even if tissue must be shipped from sites remote from the cryopreservation laboratory.