Robert A. Godke, Denniston, Richard S.
Robert A. Godke, Richard S. Denniston and Brett Reggio
Recent developments in cell biology, molecular biology, immunology and genetic engineering have given new dimensions to research and application of biotechnology to farm animals. Historically, artificial insemination, one of the early reproductive technologies, has provided excellent opportunities to expand the superior genetics of selected animals in planned breeding programs. With the development of applied aspects of embryo transfer technology (nonsurgical collection and transfer methods for cattle) in the mid 1970s, animal reproduction again entered a new age of technical advancement. Although beef cattle prices, industry promotion and producer interest enhanced the use of this technology in the late 1970s and early 1980s, embryo transplantation is more often used today by dairy producers.
Embryo transfer methodologies in the future will likely be conducted using unique or laboratory-derived specialized embryos. In the years to come, the embryos for transfer will be produced with frozen sperm from genetically valuable males and oocytes (eggs) harvested from cows in the producer’s own herd, evaluated for gender and likely tested for valuable genetic traits before the embryos are transferred to the recipient females. On the horizon, cloned embryos will be produced from cells from valuable males and females, or even produced with foreign genes introduced into the genetic makeup of the embryo before the cloning procedure.
Selecting the Sex of Embryos
Various studies have reported that male embryos develop at a faster rate than female embryos in the early stages of embryonic development. The faster development of male embryos has been described for mouse, pig, cow and human embryos, and has been attributed to the presence of the Y chromosome in male embryos. An alternative hypothesis is that before X chromosome inactiva-tion, the activity of two X chromosomes subsequently hinders the growth of female embryos. Knowing that male embryos generally grow faster, and those are at a later stage of development than female embryos, one could increase the chances of producing a male offspring by selecting the most developed cattle embryos from an embryo collection to transfer to recipient females.
Microsurgical methods have been developed to extract individual cells (called blastomeres) from early stage embryos. The cells remaining in the biopsied embryo generally survive and develop into a viable offspring. The efficiency of embryo production from early stage embryos, however, tends to lessen in farm animals because there are fewer cells remaining in the embryo. The individual cells removed from both early- and later-stage embryos can be used to determine the sex of each embryo before transfer to a recipient female. Embryo sexing using the DNA (deoxyribonucleic acid) amplification procedure known as the polymerase chain reaction (PCR), with specific Y-chromosome DNA probes, is remarkably accurate for sexing cattle embryos. This method has recently been made user-friendly and can be complet-ed within 2.5 to 6 hours after the embryos are harvested from donor animals. Sexing later-stage embryos at 6, 7 or 8 days of age before transplanta-tion is now available at most embryo transplant stations. There are at least two commercial companies operating in North America that distribute a complete cattle embryo-sexing kit for in-field use. The capability of sexing embryos would give the producers the option of selecting bull or heifer calves for market and reproductive manage-ment purposes.
Animal Genetic Testing
After removing individual cells from the embryo (using microsurgery) just before transfer, genetic markers can now be used to identify genes causing genetic diseases in farm animals, for example bovine leukocyte adhesion deficiency, more commonly known as BLAD, and economically important quantitative trait loci (QTLs) can be identified in the embryo.
Once a defective gene or a specific QTL is identified in the embryo, the producer would have the option to either transfer or discard an embryo. An embryo with a proper combination of alleles for myostatin, thyroglobulin and calcium-activated neural protease, for example, could have an increased market value because of its potential for enhanced growth rate, improved marbling and increased tenderness. Cells of the embryo could also be tested to verify parentage or to enable selection for or against a phenotypic trait, such as red coat color in cattle. Selecting embryos with specific animal produc-tion traits (for example, high milk production or increased feed efficiency) would give the producer an advantage in efficiency over other producers not using this molecular technology. This powerful new biotechnology tool has the potential to greatly enhance the live-stock industry in the years to come.
Monoclonal Antibodies To Detect Disease
Antibodies that aid in diagnosing and treating disease can now be produced through animal biotechnology. Laboratory animals (for example, rabbits) are injected, usually two or more times over several months, with a foreign protein and an adjuvant to trigger the animal to respond by producing antibodies to the foreign protein in their blood stream. After a laboratory cell fusion step, hybrid cells called hybridomas, under specific laboratory culture conditions, will produce antibodies to the original foreign protein injected into the animal. These highly specific antibodies are referred to as monoclonal antibodies.
These hybridoma cells are considered to be immortal and have the capacity to produce large quantities of antibodies under industrial conditions. Each antibody is uniquely specific for a selected protein. The specificity of the monoclonal antibodies makes these laboratory-produced proteins useful in live-animal diagnostic tests for various infectious agents and for immunological treatment of infectious diseases in farm animals.
The value of this new technology for producers will be in detecting diseases in farm animals. Immuno-diagnostic kits, each with a specific monoclonal antibody to a causative disease agent, are now being used on farms and ranches for rapid in-field identification of specific diseases. These kits can help producers identify a disease before it ravages their herd.
Recombinant DNA Products
Using recombinant DNA technology, a specific gene can be identified, removed from the cell nucleus with an enzyme, placed in a suitable vector and transferred into a host microorganism, such as fast-growing bacteria. While in culture, these microorganisms can reproduce into a large population containing many copies of the gene originally introduced into the micro-organism via the DNA-loaded vector. Multiple copies of the gene then synthesize a highly specific peptide or protein product for commercial use.
Biotechnology companies now produce new recombinant DNA proteins to treat various viral diseases in farm animals. An example is a class of natural antiviral proteins called inter-ferons used to treat calf scours and various respiratory diseases, such as shipping fever in cattle.
In recent years, researchers have isolated the genes for specific viral proteins by recombinant DNA technol-ogy. Once isolated, these genes can be used in conjunction with recombinant DNA technology to produce large quantities of the viral protein for use in animal immunization programs. The major advantage of this approach over previous vaccine production methods is that the viruses are not present in the vaccine. This eliminates the potential risk of disease outbreaks in the producer’s herd following immuniza-tion. Also, production costs for these new vaccines are generally lower than the conventional manufactured vaccines.
In the early to mid 1980s, procedures were developed to produce genetically identical twin offspring (clones) by bisecting or splitting individual sheep, goat, swine, cattle and horse embryos 5 to 8 days of age. A fine glass needle or a razor blade chip was used to bisect the embryo. The pregnancy rates (45 percent to 70 percent) in cattle after the transfer of half of a bisected embryo (known as “half” embryo or demi-embryo) are similar to those of intact embryos from the same donor female. Embryo collection, embryo microsurgery and transfer of demi-embryos to recipient females can be completed in an hour with this new reproductive technology. For optimal success rates, each embryo should not be bisected more than once. The technology has been continually refined, and today methods are available to bisect farm animal embryos with a glass microscope slide and a hand-held razor blade. This low-cost microsurgery procedure is simple, effective and relatively easy to learn.
Embryo bisection offers the potential of doubling the number of viable embryo transplant offspring produced from valuable donor females. For example, 100 good quality intact cattle embryos may result in 65 transplant offspring born; whereas, 100 similar quality embryos divided into halves would yield 200 demi-embryos, which then may result in 130 split-embryo transplant calves born or a 130 percent pregnancy rate from 100 genetically superior embryos. Using this procedure on beef cattle embryos has been averaging 1 to 1.2 calves per donor embryo bisected. Twin calves produced by microsurgery will result in genet-ically identical offspring of the same sex. This would remove the concern for bovine reemartinism, if both demi-embryos are transferred to the same recipient female. A freemartin is an infertile female calf born as co-twin to
Research efforts are under way to improve methods for freezing and storing demi-embryos in liquid nitrogen to subsequently produce twin calves anytime during any calving season. With cryopreservation, the possibility exists that one demi-embryo of the set could be transferred to a recipient animal and the remaining demi-embryo of the pair could be frozen for transfer later. If the offspring derived from the first “half” embryo was of the genetic quality the owner desired, the remaining demi-embryo could then be thawed and transferred to another recipient animal to produce the second twin offspring. The second demi-embryo (a clone) also could be marketed as a sexed embryo clone of established genetic quality.
A new method of cloning called nuclear transfer (separating and transferring individual, undifferentiated embryonic cells to enucleated oocytes) emerged in the mid to late 1980s. Multiple nuclear transfer-derived offspring from individual blastomeres from a single prehatched embryo have been produced in several farm animal species (sheep, cattle). Nuclear transfer-derived offspring have been produced four generations from a single cattle embryo. Unexpectedly, some of the nuclear transplant calves have extended gestation length, and there are reports of abnormally large term offspring (calves, lambs), which often need assistance at birth. The reason for these problems is not clear, although laboratory culture conditions have been implicated as a potential cause. Even though improve-ments in the nuclear transfer methodol-ogy are still needed, this approach has a great deal of potential for seedstock procedures in the future.
Somatic Cell Cloning
More recently, there has been a major breakthrough in the animal nuclear transfer procedure. With “Dolly,” the famous sheep, cells for cloning were harvested from the mammary gland of a mature ewe. These mammary cells were incubated in the laboratory to produce a larger popula-tion of similar type cells for the nuclear transfer procedure. The production of cloned sheep in Scotland was important because it was the first mammal produced in the world from an adult differentiated body cell (somatic cell).
The cloning of adult sheep (Dolly and her sisters reported in 1997) stimulated interest in nuclear transfer technology by the livestock industry. To construct cloned embryos with this new approach (Figure 1), take a somatic cell from a developing fetus or an adult animal (male or female) and microsurg-ically transfer it to an unfertilized oocyte from which the female nuclear DNA has been microsurgically removed (called enucleation). The enucleated oocyte with the newly introduced foreign somatic cell becomes activated (as though it had been naturally fertilized) and the reprogrammed nucleus directs embryonic cell develop-ment into a cloned embryo for subse-quent transfer to a recipient female. Once the donor somatic cell population has been prepared, hundreds of cloned embryos can be produced in the laboratory on a weekly basis using oocytes extracted from abattoir ovaries.
Use of this new biotechnology has tremendous potential. Somatic cell clones have been produced in mice, rabbits, cats, sheep, goats, swine, domestic beef and dairy cattle, exotic cattle and exotic sheep. Most recently, cloned mules, a cloned horse and cloned exotic cats have been produced. Cloning would provide the cattle producer an opportunity to reproduce genetically valuable seedstock animals, clone animals that have suffered a severe injury such as a fractured leg and can no longer reproduce, or clone males that had been prematurely castrated, such as a prize-winning show steer. Cloned calves have now been produced from frozen adult cattle tissue stored in a standard deep freezer for years. It has been proposed that sloughed off somatic cells from milk and semen could be used to produce cloned farm animals. A healthy cloned calf has recently been produced from somatic cells extracted from the milk (fresh colostrum) shortly after calving in a dairy cow.
Cloning technology would also provide livestock producers with ready access to production-tested breeding stock, thus increasing the accuracy of selection in their breeding herds. It has been proposed that cloning F1 terminal-breed males to produce males for market steers might be the ultimate beef production management system. With this scenario, fewer cows would be needed to produce annual replacement heifers, so more F1 recipient females could be available to produce the cloned F1 males for use as steers. This scenario, however, assumes that the new cloning methodology becomes more efficient and economically feasible for cattle producers.
DNA is the genetic template stored in a highly compacted nucleus and is needed for the replication of living cells. This DNA material is stored in strands as small nucleotide subunits (called genes) along the chromosomes that reside in the nucleus or in the mitochon-dria residing in the cytoplasm of the cell. Individual genes are responsible for the production of specific proteins in the cell. Some genes are expressed at higher levels than others in cells at various times, resulting in production of larger amounts of the corresponding protein at various stages of life.
Advances made during the last decade in molecular biology have enabled scientists to identify and isolate specific genes within the chromosomes of animal cells. In recent years, much effort has been directed toward incorpo-rating foreign DNA (genes) into the nuclear material of oocytes, termed gene transfer, and more recently into cells of early-stage embryos. The basic approach to gene insertion involves the transfer of DNA via microinjection techniques into the male pronuclei of recently sperm-activated ova. Following the fertilization process, the resulting genetically engineered embryos are then transplant-ed into recipient females. The embryo transplant offspring would then have the potential for gene expression (transgenic animals with genetically altered capabilities) throughout various parts or all of their lives.
The first experiments using microinjection of DNA into the pronucleus of a sperm-exposed oocyte introduced hemoglobin genes and growth hormone genes of other mammals (rabbits, humans) into mouse ova. Microinjected genes then insert into the genetic material during the fertilization process, resulting in incorporation into the nucleus of the resulting mouse embryos. These embryos were transplanted into mouse recipient females, which then produced live, transgenic offspring. As these young transgenic offspring started growing, copies of their inserted gene became activated resulting in a foreign animal protein being produced in the mouse.
The mice that had the inserted growth hormone gene produced elevated levels of growth hormone in their body resulting in larger animals than their nontransgenic littermates. In addition, these introduced genes were ultimately transmitted to the offspring of the mice that had originally received the gene. Recent refinements in microinjection techniques have led to successful introduction of growth hormone genes into sheep, pig and cow embryos, although the efficiency of gene incorporation was low and variable in expression among the different animals.
This methodology does show promise for genetic alteration (engineering) of farm animals but more efficiency is needed to be economically feasible. With the original gene injection procedure, it was difficult to control the amount of the foreign DNA incorpo-rated into the nuclear material of the resulting embryo and, furthermore, where the DNA was incorporated in the chromosomes. This generally results in variable levels of gene expression in the progeny, with a portion of the animals producing low levels of the foreign protein and others producing elevated levels of the protein. Researchers subsequently have worked on developing different methods for incorporating DNA into embryonic and somatic cells and on using targeted gene expression of the foreign gene in specific body tissues, such as the muscle.
One of the most obvious ways in which biotechnology can affect sheep, swine and beef production is by increasing growth efficiency in market animals. Human growth hormone genes have already been introduced into some farm animals but the animals produced with this incorporated gene generally did not have a growth advantage over those not receiving the gene. Efforts have been made to isolate the specific growth hormone gene of pigs and cattle and to use the gene appropriate for that species to make transgenic animals. Alternative newer methods for DNA incorporation into ooctyes and embryos (sperm-mediated gene transfer, retrovirus vectors, electroporation, nuclear transfer) should improve the efficiency of this methodology. Although the current focus for trans-genic farm animal research is for biomedical purposes, recent efforts have been directed toward the improvement of animal health, such as the lysostaphin gene to reduce mastitis in dairy cattle.
The regulation of animal growth and development is a complex physio-logical process involving a multitude of genes in addition to the growth hormone gene. A recent example is the Callipage gene identified in sheep that can increase the efficiency and the amount of muscle in growing lambs. The inactivation of the myostatin gene in beef cattle has been shown to dramatic-ally increase the amount of muscle in animals compared with those without the inactivated gene. It is anticipated that the other genes involved in growth and production efficiency will also be isolated and their role in growth regulation identified in the near future. Inserting multiple genes into an embryo may ultimately improve the growth and feed efficiency in meat-producing farm animals.
Transgenic pigs have been produced that carry extra copies of the alpha-lactoalbumin gene to increase milk production during lactation. Once these and other genes that enhance milk secretion are isolated, they could be used to make transgenic founder animals to transmit these milk production capabilities to their offspring. The ultimate use of transgenic technology for producing farm animals in the future will likely be from using transgenic males with genes of interest to transmit specific traits to their offspring.
Gene farming (termed “pharming”) refers to the concept of using farm animals as biological factories to manufacture commercially valuable products in their milk. It was first reported that transgenic mice with genes incorporated in their mammary glands were able to produce high levels of human growth hormone in their milk. Since then, genes with site-directed promoters have been produced that can secrete human pharmaceutical peptides and proteins in the milk of mice, rabbits, sheep, goats, swine and cattle. These transgenic animals are then mated and when their offspring are produced, the females are allowed to nurse their offspring. The females are then milked to obtain the human pharmaceutical proteins from their milk. The human proteins are then extracted from the milk and purified. Production of human pharmaceutical products in this way has been shown to be cheaper and more efficient than by conventional industrial procedures. This technology opens a new potential for the use of farm animals.
This new biotechnology using transgenic farm animals to produce biopharmaceutical products in their milk (human anti-thrombin III, human serum albumin and human blood clotting factors VII, VIII and IX) has resulted in remarkable achievements in recent years. The best example is anti-thrombin III, a blood anticoagulant used for heart patients and during surgery in humans, which will be the first of these pharmaceutical products expected to reach the commercial market early in 2004.
Advances in assisted reproductive technologies during the last decade have been occurring at such a rapid rate that even scientists themselves are amazed. The potential for use of these new biotechnology procedures for animal production extend almost as far as one can imagine. Tremendous progress has been made in the development and application of these technologies. Although the availability and cost effectiveness of some of these new technologies still remain in question, there is little doubt about their potential impact on livestock production. Many of these applications will require more research and in-field testing before they reach the marketplace. It is obvious that use of these emerging technologies will require more intensive management by the livestock producer. These new technologies, if economically practical, will provide the producers with the opportunity to change the genetic potential of farm animals at a faster rate than is possible by the conventional methods presently in use.
It is predicted that marker-assisted selection for both single and multiple gene traits will become a potent assisted reproductive technology for embryos, newborn offspring and young adult animals. The challenge to the industry comes in identifying those traits that merit the application of these new assisted reproductive technologies.
There is little doubt that biotechnology will contribute to the improvement of human health and medical treatments, improved production of animal food sources and development of new commercial products. Biotechnology companies are using these new methodologies to improve animal production efficiency, increase animal disease resistance and to alter genetic traits in food animals.
In the future, biotechnology products and new assisted reproductive technologies will likely become a larger part of the livestock producer’s tools in embryo production and in the produc-tion of herd replacements. The research approach in our laboratory and that of others is to develop new assisted reproductive technologies that have economic, agricultural and biomedical applications.
(This article appeared in the fall 2003 issue of Louisiana Agriculture.)