Flow cytometric sorting of frozen–thawed spermatozoa in sheep and non-human primates
J. K. O’Brien A C , F. K. Hollinshead A , K. M. Evans B , G. Evans A and W. M. C. Maxwell AA Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, The University of Sydney, NSW 2006, Australia.
B XY Inc., Fort Collins, CO, USA 80523.
C To whom correspondence should be addressed. email: justineo@vetsci.usyd.edu.au
Reproduction, Fertility and Development 15(7) 367-375 https://doi.org/10.1071/RD03065
Submitted: 5 September 2003 Accepted: 10 November 2003 Published: 10 November 2003
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.
Extra keywords: cryopreservation
Acknowledgements
This research was supported by XY, Inc. (Fort Collins, CO, USA), the Zoological Parks Board of NSW and the Australian Research Council. The authors thank L. He and R. Wadley (University of Sydney) for assistance with sorting, S. Heffernan, Dr S. Thompson (Royal Prince Alfred Hospital), Dr P. R. Martin, S. G. Solomon (University of Sydney) and Dr L. Vogelnest (Taronga Zoo) for assistance with collection of post-mortem tissue and Bioniche (Armidale, NSW, Australia) for donation of sodium hyaluronate.
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