The Beltsville sperm sexing technology: high-speed sperm sorting gives improved sperm output for in vitro fertilization and AI

Abstract

The Beltsville sperm sexing technology is currently the only effective means of altering the sex ratio of offspring in livestock. The method is based on the flow-cytometric separation of X- and Y-chromosome-bearing sperm based on X/Y DNA content difference. It is an effective means of producing progeny of predetermined sex in cattle, swine, sheep, and laboratory animals. The method involves treating sperm with a DNA-binding fluorochrome, Hoechst 33342, and flow-cytometrically sorting them into separate X and Y populations that can subsequently be used for surgical intratubal or intrauterine insemination, deep-uterine insemination, regular artificial insemination in some cases, in vitro fertilization to produce sexed embryos for transfer, and intracytoplasmic sperm injection of ova. Skewed sex ratios of 85 to 95% of one sex or the other have been repeatably achieved in most species. The method has been used worldwide to produce several hundred morphologically normal animal offspring of the predicted sex. It has also been validated in the laboratory using DNA reanalysis of the sorted sperm populations and by fluorescence in situ hybridization and PCR of individual sperm. We developed a new orienting nozzle that we have fitted to both conventional and high-speed cell sorters that have been modified for sperm sorting. Recently we completed the adaptation of the new orienting nozzle to a Cytomation MoFlo high-speed cell sorter modified for sperm. This adaptation of the nozzle has increased the overall production rate of sorted X and Y sperm from about .35 million/h to 5 or 6 million sperm/h (each population). Calves have been born from cows artificially inseminated using conventional technique and sexed sperm. In addition, numerous litters of pigs have been born after transfer of embryos produced from X or Y sorted sperm.

Sex Preselection in Bovine by Separation of X- and Y-Chromosome Bearing Spermatozoa

“…The tip of the flow cell was remolded to provide 2-3 times the orientation capability of the needle system. This novel nozzle resulted in a greater orienting percentage [35]. Rens et al [45] a n d J o h n s o n e t a l .…”

“…Application of these paint to a FISH protocol for bull sperm resulted in clear identification of more than 90% of sperm. When the fluorescence-labeled X-and Y-specific paints were tested in samples of bull sperm sorted using the Beltsville sperm sexing technology [35], the results indicated that the FISH procedure is robust, reproducible, and simple with high efficiency for evaluation of the sex of sorted sperm [53].…”

“…Although sorting results in elimination of dead and damaged sperm, those cells surviving the sperm sorting process tend to degenerate faster than unsorted control gametes from bulls and boars [46]. Recently, Johnson et al [35,36] developed a commercial high-speed sorter and stated that it can sort up to 2 million sperm per hour with a standard beveled needle and 6 million sperm per hour with a orienting nozzle. There are three manufacturers offering high-speed sorters that can be adapted for sperm sorting if modified as described above [36].…”

“…Recently, Johnson et al [35,36] recommended that 1 µl of food coloring, FD&C #40, be mixed into sperm suspension stained with Hoechst 33342 in order to stain dead sperm. Live sperm membranes are highly permeable to Hoechst 33342 when incubated at 35 C [34], whereas the food coloring penetrates the membranes of dead sperm and quenches the intensity of fluorescence, thus resulting in eliminating of dead sperm from the viable sperm population [35][36][37]. In general, propidium iodide is also used for the same purpose but has the drawback of being an intercalating dye.…”

Impact of Biotechnology on Animal Breeding and Genetic Progress

“…Sexing of semen and of embryos provides in species and production circumstances where one sex is preferred, a way to produce the wanted type of animal (Betteridge et al, 1981;Johnson et al, 1998). Sexing of embryos is a technique that is rather reliable nowadays and currently used on a rather large extent.…”