New Method Offers Improved Sex Sorting for Livestock

A new adaptation of a system for separating X- and Y-chromosome bearing spermatozoa has been developed by researchers at the US Department of Agriculture Germplasm and Gamete Physiology Laboratory in Beltsville, Maryland, USA. The ability to separate the X- and Y-bearing spermatozoa enables farmers to predetermine the sex of livestock when the sorted sample is used for insemination.

The new technique, developed by Lawrence Johnson and colleagues, builds on the patented Beltsville Sperm Sexing Technology that was patented in 1989. This method relied on separating sperm according to DNA content and was licensed to several firms for commercial development for livestock reproduction and in human medicine. The original technique could be used in rabbits, pigs, and cattle, but had the limitation that only 1.5-2 million sperm could be sorted per day and accuracy varied within 75-90%. Recent research by the team has enabled the technique to be modified and its effectiveness has already been tested in sheep, laboratory animals and other species.

The technique relies on the use of a fluorescent dye that sticks to DNA. The dye binds to the sperm based on how much DNA the X and Y chromosomes in the sperm are carrying. Female-producing X sperm contain 2.8 to 7.5% more DNA than male-producing Y sperm, depending on the species. The dyed sperm cells are analysed and sorted using a flow cytometer/cell sorter. When a laser beam illuminates the dye, each sperm gives off light proportional to its DNA content and is separated into different tubes, depending on the amount of light emitted. The X sperm always glows brighter because of the greater amount of DNA. Johnson and colleagues, Glenn R. Welch and Wilem Rens, have made three significant enhancements to the Beltsville technology that dramatically increase the practicality and chances that the animal industry will adopt the technology.

The first adaptation involves the sperm processing procedures and enables the sperm’s exposure to excess dye to be reduced. The second improvement relates to the production of a new nozzle for cells sorting machines that is more suitable for the shape and size of mammalian spermatozoa. Cell counters, or cytometers, are usually optimized for cells such as blood cells. The new nozzle, which is awaiting a patent, allows 70% of the sperm, compared with only 30% for the older version, to be properly aligned to the laser beam and sorted into the correct tube. The development of a new commercial high-speed cell sorter adapted for the new nozzle and for sperm-sorting activity, constituted the third improvement to the separation technique.

The combination of these improvements has led to a 15- to 20-fold increase in production rates of sorted sperm populations. Now, 35 to 40 million of both X and Y sperm can be sorted in an average 8-hour day, compared with 1 to 2 million previously. The accuracy of the technique depends on the DNA difference between the X and Y sperm, which varies with the species being sorted. For example, pigs carry about 3.6% percent more DNA in their X sperm than in their Y sperm; cattle, 3.8% more; and humans, 2.8%. The wider the difference, the easier it is to sort with greater accuracy.

Once the sperm has been sorted, it can be used with both conventional and deep-uterine artificial insemination in cattle. With conventional artificial insemination, sperm are placed just inside the cow's uterus, a procedure that requires about 5 million sexed sperm and an hour of sorting time. With deep-uterine insemination, only about 300,000 sperm need to be placed to achieve pregnancy. As pigs require much larger doses of semen for artificial insemination, an in vitro fertilization system has been developed using sexed sperm. The resulting embryo is then implanted in a surrogate sow.

In a recent experiment to demonstrate the effectiveness of the sperm- sexing technology, eight litters of pigs were born at Beltsville using sorted X-chromosome sperm. Ninety-eight percent of the pigs in the 8 litters were female. Three control litters produced at the same time with unsexed sperm resulted in equal numbers of male and female offspring.

To date, more than 500 animals have been born using sexed semen, and all have been healthy and normal. The technology has the potential to save the cattle industry millions of dollars annually, according to Johnson. "Sexing sperm could help farmers get the required numbers of the desired sex without producing the other sex," says Johnson. "This speeds the rate at which they can achieve genetic improvement while reducing production costs." The best example of this improvement can be seen in dairy cows. Under normal circumstances, a farmer would use the top 40% of his herd to reproduce enough female replacement calves. With sexed semen, a farmer would only need to use the top 20%. Another benefit of sex-sorting sperm may be its efficiency in conserving genetic resources. When storing rare germplasm for use by later generations, conservators could maintain only the sperm needed to produce sexed offspring desired for particular situations. According to Johnson, "Sex preselection provides an opportunity to improve management flexibility using this technology." he says.

The new sperm-sexing machine is being perfected for commercial use via collaborations between Johnson and several scientists around the world.

Contact: Lawrence A. Johnson, Agricultural Research Service Germplasm and Gamete Physiology Laboratory, Bldg. 200, Rm. 124, 10300 Baltimore Ave., Beltsville, MD 20705-2350, USA.
Tel: +1 (301) 504 8545
Fax: +1 (301) 504 5123

James Reecy, Animal Science, 515-294-9269
Danelle Baker-Miller, Office of Biotechnology, 515-294-7356

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