Research dealing with sex predetermination in swine and other livestock, to provide the producer with faster genetic progress and greater flexibility, has been a priority of Agricultural Research Service (ARS) scientists at the U.S. Department of Agriculture's Germplasm and Gamete Physiology Laboratory, Beltsville, MD, for more than a decade.

After several years of preliminary research, an effective method of separating X- from Y-chromosome-bearing sperm was developed and reported in 1989. The method uses the greater DNA content of the X-bearing sperm (3.6% in the pig) to differentiate it from the Y-bearing sperm. This method is known as the Beltsville Sperm Sexing Technology. The method has been applied to most farm animals with a resultant shift in the sex ratio so that 90% or more of the offspring are of the predetermined sex.

The technology relies on the use of flow cytometric analysis and cell sorting (See Fig. 1). Each sperm is identified for the amount of DNA it contains as it passes a laser beam. The sperm are then directed to collecting tubes, one tube for X sperm and one tube for Y sperm. The sperm are differentiated according to how much light is detected from the fluorescent dye that is proportionally bound to the sperm's DNA. Consequently, X-bearing sperm with more total DNA glow brighter than the Y-bearing sperm when hit by the laser beam.

According to Larry Johnson, ARS researcher, a drawback of the original method was its low efficiency. Less than 10% of the original sperm were recovered in the tubes containing X and Y sperm with that method. "In the past year or more, we have found a way to overcome some of that inefficiency and raise the recovery rate to more than 30%, while at the same time increasing the production rate of X and of Y sperm by 10 to 15 fold," Johnson says. That translates into the following improvements: an increase from 0.4 million of both X and Y sperm per hour, to 5 to 6 million per hour of both X and Y sperm.

The dramatic improvement came about through two developments. First, the ARS scientists developed a new nozzle that improved how sperm were oriented to the laser beam (orientation is essential for accurate differentiation between X and Y sperm). Instead of only 20-30% of the sperm being oriented properly, the oriented population was changed to 65-70% of the total. This improvement alone increased the sorting speed from less than 0.4 million per hour to 0.8 to 1.0 million sperm per hour using a standard speed sperm sorter.

Next, the new orienting nozzle was adapted to fit the newest technology in the cell-sorting field, the high-speed cell sorter. This adaptation to high-speed sorting was successful in increasing the speed again by several fold. The current rate of X-bearing sperm production is 5 to 6 million sperm per hour. The Y sperm hourly production rate is about the same.

"Our earliest report (1991) with sexed sperm and intra-tubal insemination by surgical intervention in the pig produced litters with significantly skewed sex ratios," Johnson says. "In the past couple years, we have combined the use of sexed sperm with in vitro fertilization (IVF) using eggs recovered from slaughterhouse ovaries."

The researchers were able to produce litters of piglets with highly skewed sex ratios from embryos produced from sexed sperm and subsequently transferred to recipient gilts in several studies. The first study combining IVF and sorted sperm produced two litters of pigs from embryos that were transferred into recipient females after being produced from X-sorted sperm. All the pigs were female.

Ninety-seven percent of the piglets from six litters of pigs were female (33 of 34 pigs) in a more extensive recent study. Control gilts into which embryos from non-sexed sperm were transferred produced 25 pigs in three litters. The sex balance of these offspring was about equal (52% males and 48% females).

Other experiments have been conducted in which sperm has been sorted at Beltsville, MD, and shipped by air to the University of Missouri for use in their IVF system. Results from these experiments were also very encouraging, showing a 97% female proportion in five litters produced from X-sorted sperm and 100% males in three other litters produced from Y-sorted sperm.

Improvements in sorting efficiency decreases the number of sperm that are needed, but more importantly it broadens the applications for sexed semen usage in the pork industry, Johnson relates.

Sorted, sexed sperm can be used for intra-tubal insemination, either by surgery or laparoscopy, to produce offspring, or they can be used for IVF.

Sexed sperm may also have application for modified artificial(AI) approaches with improved methods of sperm delivery to the site of fertilization that will allow lower sperm numbers to be used per dose. "At the present time, its use within conventional AI is doubtful, however, due to the current high numbers of sperm required per insemination," Johnson explains. "If the development of low sperm number insemination techniques for the pig are successful, sexed semen on the farm has definite potential. Reduced sperm numbers per dose would be as helpful to the use of sexed sperm as it would also be to the efficiency of conventional AI, now being used so extensively in the United States as well as other countries of the world."

Researchers: Larry A Johnson, Wim Rens, Glenn Welch, Charles Long, Detlef Rath and John Dobrinsky, Beltsville Agricultural Research Center, ARS, USDA, Beltsville, MD. Phone Johnson at (301) 504-8009, or email: LAJohnsn@Lpsi.barc.usda.gov.