Help with determination of sex

Jim Cummins cummins at possum.murdoch.edu.au
Fri Jan 28 00:53:27 EST 1994


Patrick Lamastus writes:

>t his technique is based upon the different densities of sperm (or other cells) and uses centrifugation to seperate cell type (those w/X and those w/Y). This is termed flow cytometry.

Wrong, sorry. There have been a number of attempts to separate X and Y
sperm by differential centrifugation.  None have been repeatable.

Flow cytometry relies on separation of sperm based on fluorescence
staining.  This can be a specific in situ hybridisation stain (eg for a
region of the Y or X chromosome) or a generalised DNA stain that gives
information about the total DNA content.  Theoretically  X and Y sperm
differ in DNA content by about 2% but as this is less than the
within-gender variation you can see that there are big problems in
successful sorting.   The following refernces may help
Auger J, Leonce S, Jouannet P, Ronot X (1993): Flow cytometric sorting
of living, highly motile human spermatozoa based on evaluation of their
mitochondrial activity. J Histochem Cytochem 41:1247-51.
Cran DG, Johnson LA, Miller NG, Cochrane D, Polge C (1993): Production
of bovine calves following separation of X- and Y-chromosome    
bearing sperm and in vitro fertilisation. Vet Rec 132:40-1.
Dresser DW, Atkins CJ, Pinder A, Morrell JM (1993): Analyses of DNA
content of living spermatozoa using flow cytometry techniques. J Reprod
Fertil 98:357-365.
Johnson LA, Clarke RN (1988): Flow sorting of X and Y
chromosome-bearing mammalian sperm: activation and pronuclear
development of sorted bull, boar and ram sperm microinjected into
hamster oocytes. Gamete Res 21:335-343.
Johnson LA, Welch GR, Keyvanfar K, Dorfmann A, Fugger EF, Schulman JD
(1993): Gender preselection in humans? Flow cytometric separation of X
and Y spermatozoa for the prevention of X-linked diseases. Human Reprod
8:1733-1739.
Morrell JM (1991): Applications of flow cytometry to artificial
insemination: a review. Vet Rec 129:375-8.
Pasteur X, Maubon I, Sabido O, Cottier M, Laurent JL (1992): Comparison
of the chromatin stainability of human spermatozoa separated by
discontinuous Percoll gradient centrifugation. A flow cytometric
contribution. Anal Quant Cytol Histol 14:96-104.
Pasteur X, Sabido O, Maubon I, Perrin CM, Laurent JL (1991):
Quantitative assessment of chromatin stability alteration in human
spermatozoa induced by freezing and thawing. A flow cytometric study.
Anal Quant Cytol Histol 13:383-90.
Peter AT, Jones PP, Robinson JP (1993): Fractionation of bovine
spermatozoa for sex selection - a rapid immunomagnetic technique to
remove spermatozoa that contain the H Y antigen. Theriogenology
40:1177-1185.
Seligman J, Shalgi R, Oschry Y, Kosower NS (1991): Sperm analysis by
flow cytometry using the fluorescent thiol labeling agent
monobromobimane. Mol Reprod Devel 29:276-281.
Spano M, Evenson DP (1993): Flow cytometric analysis for reproductive
biology. Biology of the Cell 78:53-62.
Tao J, Du JY, Critser ES, Critser JK (1993): Assessment of the
acrosomal status and viability of human spermatozoa simultaneously
using flow cytometry. Human Reprod 8:1879-1885.


Jim Cummins                   
School of Veterinary Studies
Murdoch University
Western Australia 6150  Tel +61-9-360 2668 Fax +61-9-310 4144
"An inordinate fondness for Beetles"



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