Flow Cytometric Sorting of Fresh and Frozen-Thawed Spermatozoa in the Western Lowland Gorilla (Gorilla gorilla gorilla)

• Published in: American journal of primatology Vol. 66; no. 4; pp. 297 - 315
• Main Authors: O'Brien, J.K., Stojanov, T., Crichton, E.G., Evans, K.M., Leigh, D., Maxwell, W.M.C., Evans, G., Loskutoff, N.M.
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01-08-2005; Wiley-Liss
• Subjects: Analysis of Variance; Animals; Animals, Zoo; Biological and medical sciences; Biological anthropology; captive management; Cryopreservation; Flow Cytometry - methods; Fundamental and applied biological sciences. Psychology; Gorilla gorilla; Gorillas; Male; Mammalia; Primate biology; Primate genetics; Primates; Primatology; Sex; sex predetermination; sperm sexing; sperm sorting; Spermatozoa - cytology; Vertebrates: general zoology, morphology, phylogeny, systematics, cytogenetics, geographical distribution; X Chromosome; sex predetermination; Flow cytometry; Spermatozoa; sperm sexing; Methodology; Sex; captive management; Gorilla gorilla; Vertebrata; Mammalia; Cryopreservation; Sexing; Primates; Simioidea; sperm sorting; Captivity
• ISSN: 0275-2565; 1098-2345
• DOI: 10.1002/ajp.20158

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°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. Am. J. Primatol. 66:297–315, 2005. © 2005 Wiley‐Liss, Inc.