title: Flow cytometric sorting of fresh and frozen-thawed spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla).
abstract: We adapted flow cytometry technology for high-purity sorting of X chromosome-bearing spermatozoa in the western lowland gorilla (Gorilla gorilla gorilla). Our objectives were to develop methodologies for liquid storage of semen prior to sorting, sorting of liquid-stored and frozen-thawed spermatozoa, and assessment of sorting accuracy. In study 1, the in vitro sperm characteristics of gorilla ejaculates from one male were unchanged (P > 0.05) after 8 hr of liquid storage at 15 degrees C in a non-egg yolk diluent (HEPES-buffered modified Tyrode's medium). In study 2, we examined the efficacy of sorting fresh and frozen-thawed spermatozoa using human spermatozoa as a model for gorilla spermatozoa. Ejaculates from one male were split into fresh and frozen aliquots. X-enriched samples derived from both fresh and frozen-thawed human semen were of high purity, as determined by fluorescence in situ hybridization (FISH; 90.7%+/-2.3%, overall), and contained a high proportion of morphologically normal spermatozoa (86.0%+/-1.0%, overall). In study 3, we processed liquid-stored semen from two gorillas for sorting using a modification of methods for human spermatozoa. The sort rate for enrichment of X-bearing spermatozoa was 7.3+/-2.5 spermatozoa per second. The X-enriched samples were of high purity (single-sperm PCR: 83.7%) and normal morphology (79.0%+/-3.9%). In study 4 we examined frozen-thawed gorilla semen, and the sort rate (8.3+/-2.9 X-bearing sperm/sec), purity (89.7%), and normal morphology (81.4%+/-3.4%) were comparable to those of liquid-stored semen. Depending on the male and the type of sample used (fresh or frozen-thawed), 0.8-2.2% of gorilla spermatozoa in the processed ejaculate were present in the X-enriched sample. These results demonstrate that fresh or frozen-thawed gorilla spermatozoa can be flow cytometrically sorted into samples enriched for X-bearing spermatozoa.
authors.affiliation: Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, University of Sydney, Sydney, Australia. justineo@vetsci.usyd.edu.au
authors.forename: J K
authors.initials: JK
authors.lastname: O'Brien
authors.forename: T
authors.initials: T
authors.lastname: Stojanov
authors.forename: E G
authors.initials: EG
authors.lastname: Crichton
authors.forename: K M
authors.initials: KM
authors.lastname: Evans
authors.forename: D
authors.initials: D
authors.lastname: Leigh
authors.forename: W M C
authors.initials: WM
authors.lastname: Maxwell
authors.forename: G
authors.initials: G
authors.lastname: Evans
authors.forename: N M
authors.initials: NM
authors.lastname: Loskutoff
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000836:Animals, Zoo; D015925:Cryopreservation; D005434:Flow Cytometry / Q000379:methods*; D006071:Gorilla gorilla; D008297:Male; D013094:Spermatozoa / Q000166:cytology*; D014960:X Chromosome