Producer question: What new developments can we expect in the world of swine reproduction?

Singleton/Krisher response: Predicting the future is always difficult. As the pork industry becomes more specialized, technologies that may seem too complicated or impractical may be adapted more rapidly than expected.

Let’s take a look at what’s on the horizon.

Sexed semen. USDA, Beltsville Sperm Sexing Technology uses a technique that separates stained X and Y spermatozoa. Each cell is stained and encased in a droplet, which is given a positive or negative charge based upon DNA content. Charged droplets pass through an electrostatic field where individual X and Y spermatozoa are deflected into collection tubes. This can be done because DNA content of X-bearing spermatozoa is about 3.6 percent greater than the Y-bearing spermatozoa.

Commercial application is limited by the relatively slow speed of sorting the cells, as well as the cost and laboratory expertise required. USDA’s technology can produce several million cells per hour. A typical boar ejaculate contains 30 billion to 80 billion spermatozoa, and most commercially produced semen contains 3 billion to 4 billion cells per dose. However, when combined with in vitro fertilization requiring only a few hundred cells or surgical insemination (requiring only 200,000 to 400,000 cells), the technology could soon be applied at the nucleus seedstock level. The high-speed cell sorter costs about $200,000 and requires trained technicians.

Real-time ultrasound. This is a practical diagnostic tool for early pregnancy (22 to 28 days).

Studies are underway to use real-time ultrasound to more accurately determine ovulation timing that could lead to adequate reproductive performance with single-dose inseminations. It also could allow the use of low-sperm dosages, sex-sorted sperm or in harvesting immature follicles for in vitro maturation and fertilization. Ultrasound also provides a means of intervention when abnormal conditions such as cystic ovarian follicles are observed.

Low-sperm dosage. The standard semen dose contains 2.5 billion to 4 billion sperm cells. Reducing the cells per dose could reduce costs. One approach has been to develop a more accurate method to assess the number of viable cells in the boar ejaculate with computer aided sperm analysis.

Other approaches have been to develop systems that would allow for semen deposition closer to the site of fertilization. The idea is to deposit semen beyond the sow’s cervix up into the uterine horn or even near the utero-tubual junction where fewer sperm cells would be required for fertilization.

Non-surgical embryo transfer. Pig embryos have been successfully transferred for several years. Until recently, transferring embryos to the recipient animal required surgery. University of Missouri researchers have developed a trans-cervical cannula that is able to deliver embryos into the uterine lumen without surgery. With further development, this technology may become practical to transport cultured or frozen embryos from nucleus herds to commercial production units. It also may be possible that embryos from females infected with certain pathogens could be transferred without risk of disease.

One limitation is the ability to recover in vivo developed embryos non-surgically. It is not now possible to flush embryos from a sow. Thus, surgery is an inherent part of embryo transfer in pigs, even though the transfer is non-surgical. This increases expense and time and limits the prospect of an on-farm procedure.

To overcome this, several techniques may be used. But, all have limitations. First, embryos may be surgically recovered at a central location and frozen, then held until transport to the farm for transfer into a recipient. However, the freezability of pig embryos at stages appropriate for transfer is low, and many embryos must be transferred to produce a pregnancy.

Second, in vitro produced embryos may be used. Embryos can be produced in the laboratory from oocytes collected at slaughter or by laparoscopy, then fertilized and cultured to an appropriate stage for transfer. These embryos would also have to be frozen for transport to the farm, and have even lower survival rates.

Embryo freezing. Porcine embryos appear to be extremely sensitive to intracellular chilling injury before the peri-hatching blastocyst stage. The development stage is an important consideration for embryo freezing because they should be frozen at or before the stage when they can be transferred back into the uterine. Typically, this is the early blastocyst stage. One possible reason for the pig embryos’ difficulties is their high lipid content relative to other domestic species.

Vitrification, on the other hand, appears to be more successful for porcine embryo cryopreservation. Vitrification is a fast-freezing process (two minutes in liquid nitrogen vapor followed by plunging in liquid nitrogen) that inhibits ice crystal formation within the embryonic cells. This holds the embryo in a glass-like state during storage in liquid nitrogen.

There are still some restrictions on the stage of embryo. Day-five embryos, which are pre-expansion blastocyst stage embryos, did not survive vitrification. Porcine embryos, particularly the more advanced stages, can be successfully vitrified and thawed as measured by continued in vitro development and pigs born alive.

Another method uses a technique known as delipation. Embryos are centrifuged at high speed to polarize intracellular lipid, which is then removed by micromanipulation to avoid injuries. Removing the lipid increases the freezability of early stage porcine embryos using standard slow cooling protocols.

Lastly, a new technique known as the open-pulled-straw method improves embryo freezability. This is an efficient vitrification method in which the embryo, after exposure to cryoprotectants, is taken up in a small-diameter, plastic straw in a small amount of medium, then plunged into liquid nitrogen. The limited medium and small-diameter straw let the embryo freeze rapidly, hopefully before damaging ice crystals can form. This appears promising in vitro, leading to excellent post-thaw survival. However, no live piglets have been produced.

In vitro maturation/-fertilization. These techniques have been developed after years of unsuccessful attempts. There are three stages of in vitro embryo production that must be considered.

First, the oocyte must be matured, meaning it must progress from a stage of meiotic arrest to a point where it is ready to be fertilized. This has been difficult in pigs. Not only must the oocyte nucleus progress through meiosis and extrude the first polar body before fertilization can occur, but the cytoplasm of the oocyte must undergo important changes to support early embryonic development. We are just beginning to understand these mechanisms.

Fertilization of in vitro matured oocytes has been another stumbling block. A common problem encountered is polyspermic fertilization, where more than one sperm successfully fertilize the oocyte. In the live animal, if insemination is done after ovulation, polyspermy also can occur. However, it rarely occurs if insemination takes place before ovulation.

Pig oocytes and sperm interact differently than other species. Often, sperm will be bound to the zona pellucida of the embryo days after both in vitro and in vivo fertilization. Polyspermy may be a reflection of inadequate maturation conditions in vitro or aging of the oocyte in vivo, such that the oocyte cannot adequately block subsequent sperm after the first fertilizing sperm has entered the cell. It also may be a result oftoo many sperm arriving at the oocyte simultaneously.

Regardless, in vitro fertilization can now be accomplished in pigs with either fresh or frozen sperm. These embryos appear viable and live piglets have been produced, although many embryos are typically transferred to produce a litter.

Polyspermy rates still run 20 percent to 40 percent. We must learn how to reduce these before in vitro fertilization can become commercially feasible.

Porcine embryo culture has proven to be more difficult than in other domestic species. We are still learning about the requirements of pre-implantation stage embryos. Once these requirements have been identified, we can develop a medium to support high embryo development. Only about 20 percent of oocytes matured and fertilized in vitro are able to develop to the blastocyst stage. More work is needed if we are to apply advanced reproductive technologies to pigs.

Wayne Singleton is an animal science professor at Purdue University in West Lafayette, Ind. Rebecca Krisher is an assistant professor of animal science. Both specialize in swine reproductive physiology.