Oocyte Aspiration for In Vitro Embryo Production in Farm Animals

Robert A. Godke  |  4/26/2006 8:08:14 PM

This is the first calf in the world produced from an in vitro fertilized embryo where the egg was harvested from a live pregnant cow. The Brangus donor female remained pregnant and produced her own calf from artificial insemination after a normal-length gestation. This IVF calf was carried by a surrogate female and was born a little over a month later than the AI calf born to his biological dam. (Photo by Marius Meintjes)

LSU Ag Center scientists began work in the early 1980s on embryo and gamete biotechnologies using a variety of farm animals, including goats. This photo was taken at that time at the research farm near St. Gabriel. Robert A. Godke was and still is the director of reproductive physiology research. (Photo by Jim Zietz)

Transvaginal Oocyte Recovery Procedure

Joel A. Carter, Marius Meintjes, M. Shane Bellow, Richard Cochran, Kay Graff and Robert A. Godke

Males of an animal species have an advantage over females in the propagation of their genes. For example, in wild populations, it is common for males of many of the hoofstock species to mate with seasonal female breeding groups, as long as the male is strong enough to ward off subordinate males. Also, sperm cells can be produced by the testes throughout the male’s life in almost unlimited amounts.

In contrast, the female is born with all the oocytes stored in her ovaries she will ever have. For example, a beef heifer at birth may have 200,000 primordial follicles containing oocytes in her ovaries. Since a beef cow might have only 10 calves in her lifetime, what happens to the remaining 199,990 oocytes? First, nonpregnant cows exhibit estrus and ovulate, with this sequence of events occurring again 20 to 22 days later (the estrous cycle). Follicles in cattle develop in waves of four to 12 follicles at a time, and a cow may have two to three waves of these growing follicles during a 21-day estrous cycle. Over this estrous cycle, 12 to 36 follicles develop to varying degrees and most then regress during this interval, with usually only one follicle ovulating. Thus, only one oocyte has a chance to become fertilized each cycle.

Valuable female genetic material
This difference in male and female physiology presents the problem of how to maximize genetic material (gametes) from valuable females. Embryo transfer (ET) was the first procedure to enhance the female’s reproductive potential. With ET, females are superovulated with hormones so that their ovaries release more than one egg before fertilization. Over the years, embryo transfer technology has been used to increase the number of possible offspring from selected females.

This approach has a few drawbacks, however. For example, some females do not respond or stop responding to the stimulatory agents or develop physiological conditions that make it difficult to retrieve the embryos. One of the major concerns with the overall ET procedure, however, is that cows are most often kept “open” so they may be hormone-stimulated for subsequent embryo collections. Cows that are superovulated and collected may take 60 days or longer to become pregnant either with artificial insemination or natural mating. A small percentage of hormone-treated donors also may develop cystic follicles on their ovaries.

The advent of in vitro fertilization (IVF), or fertilization in a test tube, changed animal reproductive management. This technique was first successfully used to produce offspring in rabbits (1959) and a healthy bull calf (1982). Even after years of research, IVF methodology is still being tested and finetuned for farm animals.

Slaughterhouse ovaries used initially
Initial in vitro embryo production (IVP) used oocytes from slaughterhouse ovaries. This worked well during early experimentation when large numbers of immature eggs (oocytes) were needed to develop procedures. In the 1980s, it was proposed that the application of IVP in animals would likely be its use in rare exotic animals and in genetically valuable seedstock. Early attempts at retrieving oocytes from potential donor cattle included surgical and less invasive laparoscopic procedures, but there was a limit to how many procedures could be performed without causing injury. At this stage, there was essentially no safe, repeatable method of harvesting the oocytes from live farm animals.

Then in the late 1980s, a method was developed in humans for retrieving oocytes using ultrasonography to visualize the ovary while a needle was guided transvaginally into the follicle. The oocyte could be aspirated from the follicle and subjected to in vitro maturation, fertilization and then culture procedures. These efforts paved the way for the new reproductive technology now available for farm animals. Currently, transvaginal ultrasound-guided oocyte aspiration (TUGA), also known as ovum pick-up (OPU) in human medicine, is now used in cows, goats, mares and more recently in pigs and exotic hoofstock species.

Horses present unique problem
The horse has presented a unique problem for researchers working in the assisted reproductive technology area. Even though embryo collection and transfer are relatively simple in the mare, attempts to superovulate horses have produced poor results. Because of the unique anatomical structure of the horse ovary, only one ovum usually ovulates at the appropriate time during each estrous cycle. Also, for some yet unknown reason, typical in vitro procedures have not worked consistently in the horse. One of the goals of the LSU Agricultural Center’s Embryo Biotechnology Laboratory has been to develop assisted reproductive technologies to increase the number of offspring from genetically valuable mares.

Even though only one follicle normally matures and ovulates during an estrous cycle, mares also are thought to have one or two waves of multiple follicles during that cycle. Once again, this developing follicle population makes it possible to use transvaginal ultrasound-guided aspiration to collect oocytes from live mares. The first foals produced from aspiration of live mares were produced at the Embryo Biotechnology Laboratory in 1998, using a procedure called intracytoplasmic sperm injection (ICSI), where a single sperm cell is injected into the oocyte. After ICSI, embryos are surgically transferred at the 2- to 4-cell stage into the oviducts of suitable recipients, since the culture of IVF-derived equine embryos has still not been perfected.

Goats are another farm animal species in which in vitro embryo production has proved successful. Transvaginal aspirations have been performed successfully on cyclic and noncyclic adult goats by Ag Center scientists.

IV success with pregnant cows
Another problem with the larger farm animals is that their gestation periods are considered to be long in comparison with those of dogs (62 days) and cats (63 days) and that the animals are out of embryo production during their gestation. Cows have 9 ½-month and mares have 11-month pregnancies, but both are known to continue follicle wave development during early to mid-gestation. Another goal of the AgCenter laboratory has been to take advantage of these developing ovarian follicles and attempt to produce IVF-derived offspring fromoocytes harvested from females during pregnancy.

The main concern was whether the oocyte aspiration procedure would affect the ongoing pregnancies. This oocyte aspiration approach proved not to be a problem, and pregnant donors were found to consistently produce more oocytes per collection than similar nonpregnant, cyclic females. The first offspring produced from oocytes collected by transvaginal ultrasound-guided aspiration from pregnant donor animals resulted from cows and horses at this Ag Center laboratory.

Ultrasound-guided follicle aspiration also has been used successfully in other animals, with modifications made primarily to account for anatomical differences of the donor animals. This aspiration technique has been used successfully to harvest oocytes from adult pigs, as long as rectal manipulation of the ovaries was possible within the donor females. Transvaginal ultrasound-guided oocyte aspiration has also been used successfully to harvest oocytes in llamas. This procedure, the same used for other exotic hoofstock, has been used successfully by members of this AgCenter laboratory team to obtain oocytes in the rare Bongo antelope and also the African eland.

There is still much to be learned in the use of assisted reproductive technologies to maximize reproductive potential in genetically valuable animals. Now that repeatable oocyte retrieval methods are being fine-tuned, it is likely these procedures will become routinely used to obtain oocytes for further gamete and embryo research and also by seedstock producers for in vitro embryo production from farm animals in the commercial sector.

Transvaginal ultrasound-guided oocyte aspiration is now used to harvest valuable oocytes from minor farm animal breeds, from domestic females representing rare bloodlines, clinically infertile females and cows too old to become pregnant. Research continues in our laboratory to find applications for this technology, including harvesting oocytes from injured females, young prepubertal heifers and early postpartum beef cows for in vitro embryo production. We also plan to use ultrasound-guided oocyte aspiration to obtain oocytes for in vitro embryo production to help preserve germplasm of endangered exotic species.

(This article was published in the winter 2000 issue of Louisiana Agriculture.)

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