Selection and separation of X‐ and Y‐ chromosome‐bearing mammalian sperm

Abstract

Preselection of the gender of offspring is a subject that has held man's attention since the beginning of recorded history. Most scientific hypotheses for producing the desired sex of offspring address separation of X-and Y-bearing sperm, and most have had limited, if any success. Eight of these hypotheses and their experimental verifications are discussed here. Three hypotheses are based on physical characteristics of sperm, one on supposed differences in size and shape, another on differences in density, and a third on differences in surface charge. There has been no experimental verification of differences based on size and shape, and the results from attempts to verify separation of X-and Y-bearing sperm based on density have been mixed. Electrophoresis may provide a method for separating Xand Y-bearing sperm, but it is currently unproven and would be of little practical utility, since sperm motility is lost. A fourth hypothesis employs H-Y antigen to select preimplantation embryos. This method reliably produces female offspring, but does not permit the selection of male offspring and does not work on sperm. There are two applications of the theory that X-and Y-bearing sperm should be separable by flow fractionation. Flow fractionation using thermal convection, counter-streaming sedimentation, and galvanization is highly promoted by its originator but has not gained wide acceptance due to lack of independent confirmation. Flow fractionation by laminar flow is said to provide up to 80% enrichment of both X-and Y-bearing sperm; however, this method also has not been confirmed by other workers or tested in breeding trials. The sixth theory discussed is that of separation through Sephadex gel filtration. This method may provide enrichment of X-bearing sperm, but, again, other experimenters have not been able to adequately confirm the enrichment. The best-known approach to sperm separation is that employing albumin centrifugation, yet even with this method, not all researchers have been able to confirm a final fraction rich in Y sperm, and trials in animals have given contradictory results. The most reliable method for separating X-and Y-bearing sperm is use of flow cytometric and flow sorting techniques. These techniques routinely separate fractions with a purity greater than 80% and can be above 90%. Unfortunately, these methods do not always work for human samples. Furthermore, as with electrophoretic approaches, the methods identify and separate only chemically fixed sperm and provide limited biological applications.Generally accepted experimental laboratory procedures for verification of proportions of X-and Y-bearing sperm are lacking. Staining of sperm with the fluorochrome dye quinacrine will identify a structure known as the "F-body" in human sperm and sperm from 378Gledhill a few primates. The dye does not work with other mammalian sperm. Its validity as a measure of sperm genotype is the topic of debate. We have used two methods to verify claims of separation of sperm, flow cytometry, and in vitro fus...

Quantification of Y chromosome bearing spermatozoa of cattle using in situ hybridization

“…Although recent advances in recombinant DNA technology have made it possible to develop more accurate and rapid techniques for sexing of embryos by Y chromosomal DNA sequences (e.g., Leonard et al, 1987;Bondioli et al, 1989;Schroder et al, 1990a), the method of choice for preselection of the gender of offspring would be insemination with a homogeneous population of either X or Y chromosome-bearing sperm. Several physical, biochemical, and immunological methods of semen treatment have been investigated in various laboratories for altering the sex ratio of offspring, but no technique seems to be easily reproducible in other laboratories (see Amann, 1989;Blottner et al, 1990;Gledhill, 1988, Johnson et al, 1989. To some extent the lack of progress might be due to the lack of appropriate methods to monitor the success of sperm separation procedures.…”

Comparison of different chromatin staining techniques for bull sperm

“…Spermatozoa are haploid cells, and the chromosomes contained in them are monochromatid structures. Owing to differences in mass between cells that contain X or Y sex chromosomes, male gametes can be separated in order to select the sex of the offspring (Johnson et al 1987, Gledhill 1988 Semen analysis is a multi-staged process. The first step is evaluation of motility, the number of sperm and the proportions of live and dead sperm (Guzick et al 2001, Hidalgo et al 2006.…”

Sex predetermination by separation of X and Y chromosome-bearing sperm: a review

“…None of the many separation techniques proposed to date for use in livestock has proved to be consistently reproducible (reviewed by Gledhill 1988;Amann 1989). A possible exception is cell-sorting by flow cytometry (FCM).…”