Isolation and viability of presumptive spermatids collected from bull testes by Percoll density gradient

The objective of the present study was to develop a procedure for isolating pure populations of round spermatid(s) (RS) by Percoll density gradient from bull testes. Bull testes were de-capsulated and testicular tissues were dissociated enzymatically to recover RS. After being filtered through a 20 μm nylon mesh, the cells were centrifuged at 650 × g for 25 min through the discontinuous Percoll density gradients (20, 35, 40, 45 and 90% Percoll solution). Isolated cells were analyzed by microscopic observation for survivability and apoptosis. In Experiment 1, both microscopic observation and DNA analysis by flow cytometry showed that ∼40% of cells collected from 35% Percoll gradient were presumptive RS, whereas in 40% Percoll gradient, mostly primary spermatocytes were observed. Experiment 2 compared the effect of 35% Percoll density isolation on the incidence of apoptosis and necrosis in fresh and frozen–thawed cells to those of untreated cells. The percentage (mean ± S.E.M.) of necrosis in cells collected from 35% Percoll gradient was less (P < 0.05) than in untreated and frozen–thawed cells from 35% Percoll gradient (11.7 ± 3.1% compared with 26.3 ± 2.0% and 53.5 ± 1.3%, respectively), but the rate of apoptosis did not differ (1.2 ± 0.49% compared with 2.5 ± 0.8% and 0.9 ± 0.04%, respectively). The proportional data (mean ± S.E.M.) of live cells in Percoll treated group were greater (P < 0.05) than in untreated and frozen–thawed cells from the 35% Percoll gradient (86.7 ± 3.26% compared with 70.8 ± 2.73% and 41.9 ± 1.69%, respectively). Experiment 3 compared the development rates of embryos injected with RS isolated from fresh and frozen–thawed cells collected with the 35% Percoll gradient to those of untreated cells, and parthenotes as control. There were no significant (P > 0.05) differences in the rates of cleavage and blastocyst development between untreated fresh cells and fresh cells collected from the 35% Percoll gradient (75.4 and 10.5% compared with 82.4 and 12.8%). However, there were lesser (P < 0.05) cleavage and blastocyst rates in frozen–thawed cells from the 35% Percoll gradient (51.6 and 6.3%) and parthenotes (60.7 and 4.1%) were observed. These results suggest that isolation of presumptive RS by 35% Percoll density gradient is effective in eliminating apoptotic and early necrotic cells. However, the use of RS in improving the developmental potential of embryos merits further studies.