The effect of swim-up purification and incubation of cells on sperm viability in dogs of different ages

The influence of selected semen extenders on the motility of frozen-thawed dog spermatozoa has been clearly demonstrated in several studies, although there are no reports indicating the effect of swim-up purification on sperm viability in this species of mammals. Therefore, this study was aimed at investigating phosphatidylserine (PS) externalization and necrosis in sperm after variable lengths of time of in vitro incubation after swim-up purification. Dog semen samples were collected from (i) ten dogs aged six months to 1.5 year, (ii) ten dogs aged six to eight years, and (iii) ten dogs aged 11 to 13 years. A flow cytometric method was employed to evaluate dog sperm viability in animals of different age groups after employment of a swim-up (SU) purification technique. After SU spermatozoa were incubated for 15, 30, 45 and 60 min in Sperm-TALP medium. We observed an increase in the number of viable sperm (double negatives) after 15 min of incubation compared to sperm undergoing PS externalization and late necrotic sperms (P < 0.001) in each group of dogs. We also found a higher number of early necrotic sperm after 60 min of in vitro incubation (P < 0.001). The amounts of late necrotic sperm and cells with PS externalization were similar among animals of different age groups. We show for the first time that most viable sperm are recovered after an in vitro incubation step of 15 min (control samples in this study) because as the time of incubation increases so does the number of degenerated or damaged cells. The higher number of early necrotic cells after 60 min of in vitro incubation may be a special feature of this species and may result from the induction of necrosis in the sperm. This knowledge may be used in future experiments for the preparation of spermatozoa following in vitro fertilization in dogs.