Fresh and frozen-thawed spermatozoa suspension concentration and motility from 4-month-old C57BL/6NTac proven breeders.

aConcentration of spermatozoa suspension per ml used for IVF was determined via a hemocytometer under light microscopy at 100× magnification, and calculated as dilution factor *count in 5 squares *0.05×106, where the dilution factor was 25 [20]. Ten µl of spermatozoa suspension (approximately 3 million spermatozoa) was added to each 200 µl IVF drop of HTF medium (15 million spermatozoa/ml). At 100× magnification, the first 100 spermatozoa manually visualized were counted to establish % progressively motile, % non-progressively motile and % immotile. Based on visual observation, progressive motility was defined as spermatozoa swimming in a forward manner [21].