Flow cytometric sorting of frozen - thawed spermatozoa in sheep and non-human primates

Abstract

Research was conducted in sheep to determine an effective preparation method for high-purity sorting of frozen–thawed spermatozoa. The efficacy of sorting frozen–thawed spermatozoa was then investigated in several non-human primate species. An aliquot of each ejaculate (three rams, three ejaculates per ram) was processed as a fresh control (FRESH). Frozen spermatozoa were thawed and prepared for sorting by no further processing (FT-NEAT), washing (FT-WASH) or gradient centrifugation (FT-GRADIENT) and evaluated for motility at 1 h post-staining and motility and acrosomal status at 0 and 4 h post-sorting. Samples were analysed using a high-speed cell sorter. High levels of purity for X- and Y-enriched samples were achieved for all treatments (85–92%). The percentage of motile spermatozoa before sorting was lower (P < 0.05) for frozen–thawed samples (FT-NEAT: 32.7 ± 2.5%; FT-WASH: 32.2 ± 3.3%; FT-GRADIENT: 73.9 ± 3.7%) compared with FRESH (83.3 ± 1.2%). Post-sorting, the percentage of motile spermatozoa before and after incubation for FT-NEAT (60.0 ± 5.1% and 27.2 ± 6.1% for 0 and 4 h, respectively) was lower than that for FRESH (87.8 ± 0.9% and 83.3 ± 1.2% for 0 and 4 h, respectively; P < 0.05), FT-WASH (80.0 ± 2.4% and 71.7 ± 3.6% for 0 and 4 h, respectively; P < 0.05) and FT-GRADIENT (84.4 ± 1.3% and 77.2 ± 1.7% for 0 and 4 h, respectively; P < 0.05). Vanguard sperm migration distance through artificial cervical mucus was lower (P < 0.05) for FT-NEAT (17.7 ± 1.7 mm) compared with FT-WASH (29.1 ± 3.8 mm) and FT-GRADIENT (28.4 ± 2.0 mm) and similar (P < 0.05) to FRESH (23.7 ± 1.8 mm). Sample preparation using a modified wash method enabled high-purity sorting (range 86–97% purity) of frozen–thawed epididymal spermatozoa in the baboon (Papio hamadryas), common marmoset (Callithrix jacchus) and common chimpanzee (Pan troglodytes). For all non-human primate species, sorted spermatozoa were progressively motile (marmoset: 20.5 ± 5.5%; baboon: 37.5 ± 2.5%; chimpanzee: 73.0 ± 2.0%), acrosome intact (marmoset: 68.5 ± 7.5%; baboon: 89.5 ± 1.5%; chimpanzee: 84.0 ± 1.0%) and able to penetrate an artificial cervical mucus. In summary, high-purity sorting of frozen–thawed ram and non-human primate spermatozoa with recovery of progressively motile, acrosome-intact spermatozoa was possible after processing to remove cryodiluent.