Chapter II: Technology Overview: Somatic Cell Nuclear Transfer and Other Assisted Reproductive Technologies
Since the beginnings of livestock agriculture, selection criteria have been applied to foster the propagation of animals with traits more desirable to humans. The expansion of herds with desirable traits has been limited, however, by the reproductive capacity of the species or breed and the prevalence of particular versions of genes (or sets of genes) responsible for those traits in the available gene pool. (The gene pool can be considered all of the animals available for breeding.) The female contribution to reproductive success, for example, is limited by species-specific characteristics such as average litter size, frequency of estrus, and gestation length. In natural breeding, male contributions are restricted by the degree of proximity to fertile females and the ability to inseminate females with a sufficient number of normal sperm. Finally, individuals of both sexes are limited to the extent that they may carry the desired versions of genes or combination of genes.
To help overcome some of these complications, various forms of assisted reproductive technologies (ARTs) have been adopted in animal agriculture for over a century, and at least one (artificial insemination) has been used for several hundred years. These technologies form a continuum that ranges from the fairly minimal assistance provided to animals engaged in natural service through those containing components of significant in vitro manipulation such as in vitro fertilization and embryo splitting, to the more recent development of somatic cell nuclear transfer (SCNT), or what is colloquially referred to as “cloning”8 (Faber et al. 2004; Sakai 2005; Oback and Wells 2007b). Beginning with the development and application of modern artificial insemination (AI) methodologies in the first half of the 20th century, ARTs have aided in the genetic improvement of domestic species, including selection of phenotypes such as behavioral and production traits in domesticated animals (Youngquist 1997, Faber and Ferre 2004). By accelerating the rate at which selective breeding goals can be met, improved genotypes have expanded rapidly into national herds in the United States and other countries (Faber et al. 2004; Wells 2005). In turn, this has resulted in lower costs for livestock producers and retail consumers, while simultaneously maintaining or improving the quality and consistency of foods of animal origin.
Reproductive technology advances have also proven to be powerful tools in curbing the spread of vertically transmitted diseases (i.e., those that are passed from the dam to her offspring during the period immediately before and after birth, either across the placenta or in the dam’s milk) (Youngquist 1997). For example, embryo transfer (ET) (see subsequent discussion for a description of this ART) has been used to prevent vertical transmission of Neospora caninum in cattle (Baillargeon et al. 2001; Landmann et al. 2002), scrapie in sheep (Wang et al. 2001), Bovine Virus Diarrhea (BVD) in cattle (Smith and Grimmer 2000), and Brucella abortus in an American bison (Robison et al. 1998). Embryo transfer is commonly used in laboratory animal research to re-derive valuable strains of gnotobiotic (i.e., animals in which all of the bacterial species are known) or specific pathogen-free research animals when colonies become infected with undesirable disease agents that cannot be controlled through more conservative means.
The following chapter begins with a brief overview of what cloning is, followed by an overview of the continuum of other ARTs commonly in use in current US agricultural practice, placing nuclear transfer technology into context of these breeding practices. Appendix B provides additional details on overall reproductive efficiency observed in current agricultural practice in the US, and Appendix C provides a comprehensive summary of the outcomes observed in ARTs, with particular emphasis on those technologies that contain a significant in vitro culturing component. Although all of these technologies are currently in practice, all are continually undergoing development and refinements with the goal of improving efficiencies. A reasonable expectation then, is that success rates (defined as the rate of production of healthy animals) will improve as expertise increases.
A. What is Cloning?
Cloning, or somatic cell nuclear transfer, is a process by which animals are reproduced asexually (embryo splitting and blastomere nuclear transfer are other ways of reproducing animals asexually and are discussed later in this chapter). In cloning, a differentiated somatic cell (a non-germ line cell from an existing animal) is introduced to an oöcyte (a cell that is the immediate precursor of a mature egg) that has had its nucleus (and thus its genome) removed, and then, following some manipulations, is induced to start replicating. If all goes well, the dividing cell is implanted into a female animal (dam), continues to develop normally, and is delivered just as any newborn.
Since the first report of a clone produced by SCNT (Wilmut et al. 1997), several other species have been cloned (Table II-1), although in some cases (e.g., companion animals) only a limited number of animals have been generated. The reasons for this are multi-fold, but are largely driven by the relative difficulty in producing clones, and the various drivers, economic and technical, that affect the expansion of the technology. For example, the use of clones in expanding elite breeding stock in domestic livestock is perceived to have benefit for breeders and consumers. This risk assessment does not attempt to address those issues, however, and instead concentrates on those domestic livestock clones commonly consumed as food (e.g., cattle, swine, sheep, and goats).
Table II-1: Species of Animals that Have Been Cloned Using SCNT (Resulting in Live Birth)
|Sheep||Wilmut et al. (1997)|
|Mouse||Wakayama et al. (1998)|
|Gaur||Lanza et al (2000)|
|Pig||Polejaeva et al. (2000)|
|Mouflon Sheep||Loi et al. (2001)|
|Cat||Shin et al. (2002)|
|Cow||Forsberg et al. (2002)|
|Goat||Keefer et al. (2002)|
|Rabbit||Chesne et al. (2002)|
|Deer||Texas A&M announcement (2003)|
|Horse||Galli et al. (2003a)|
|Mule||Woods et al. (2003)|
|Rat||hou et al. (2003)|
|Wildcat||Gomez et al. (2003)|
|Dog||Lee et al. (2005)|
|Banteng||Sansinena et al. (2005)|
|Ferret||Li Z et al. (2006b)|
|Swamp Buffalo||Suteevun et al. (2006)|
|Gray Wolf||Kim et al. (2007)|
B. Continuum of Reproductive Technologies
Although many people who are not involved in intensive animal agriculture assume that most breeding occurs "naturally" 9 (e.g., a male animal mates with receptive female), in fact, human intervention is the industry standard for many livestock operations (Youngquist 1997). In the US dairy industry, for example, most reproduction involves some technological component, and swine producers rarely use natural mating for their production of offspring. Conversely, in the beef industry most reproduction occurs by natural service, and most of the world’s sheep and goat production occurs under free range conditions and depends on natural mating.
Humans have assisted animals in natural mating by monitoring the reproductive status of females, introducing receptive females to the same location (e.g., field, corral, or pen) as the male, and allowing nature to take its course. When this process does not result in sufficient offspring of the desired phenotype, or is otherwise compromised, assisted reproductive technologies can be called into play.
Artificial Insemination and Synchronized Estrus
The first ART developed was artificial insemination (AI), which in its simplest form involves the collection of semen from males and its subsequent human-assisted introduction into a physiologically receptive female. It is an important technique for the genetic improvement of animals, as a few select males can produce sufficient sperm to inseminate thousands of females per year, while natural service would provide for the insemination of only a fraction of those animals.
Reports of AI in horses as part of breeding programs have been traced to the Arabian Peninsula in the 14th century (Bearden and Fuquay 2000). AI of a beagle dog was first described by Spallanzani in 1780 (Hafez and Hafez 2000). In 1899, the Russian Czar Nicholas II commissioned I.I. Ivanov to develop an AI program for horses, and by 1933 Ivanov had developed methods for collecting semen and inseminating horses, cows, sheep, and pigs (Foote 2001). In 1931, 19,800 cows were bred by AI in Russia. By 1936, Denmark had established an AI cooperative association, and by 1939, the use of AI had spread to the United States. In 1970, it was estimated that 7,344,420 dairy cows were bred using AI (Webb 2003).
Although there are several methods for collecting semen, most involve training males to ejaculate into an artificial vagina. Semen is then diluted to maximize the number of services that one male can provide. A normal ejaculate from a dairy bull usually contains between 5 and 10 billion sperm; good conception rates generally require about 12-20 million sperm to be introduced. The diluting solution contains factors that help to stabilize and preserve the sperm, as well as antibiotics to inhibit bacterial growth and reduce the danger of spreading any potential disease or contamination. Most collected semen is stored in glass ampoules or plastic straws, and is generally stored either in dry ice and alcohol (-100oF) or liquid nitrogen (-320oF). To date, there appears to be no limit on the amount of time that bovine sperm can remain frozen and regain viability upon appropriate thawing. Since 1997, use of AI in swine breeding has increased dramatically. A survey of swine producers conducted by the National Pork Board in 2003 indicated that even among small producers (1,000 to 3,000 swine marketed annually) as many as 60 percent of litters were sired by AI in 2003, while for large producers (> 50,000 swine marketed annually) 98 to 100 percent of litters were sired by AI.10 Rams (male sheep) and bucks (male goats) can also be donors for artificial insemination.
In the US, AI of the female is usually performed either by trained technicians employed by breeding companies or large farms or by the producers themselves. The most common technique employed today for dairy cows involves the use of sterile, disposable catheters that are inserted vaginally and extended through the cervix into the body of the uterus of the recipient cow (whose estrous cycle has been documented). Thawed semen is warmed to the appropriate temperature, and sperm are deposited in the uterine/cervical regions.
The primary advantages of AI to farmers include the ability to use semen from bulls anywhere in the world rather than those that are more geographically proximate, and thus to have desirable genetics available for propagation. It also allows the farmer to use multiple sires in a herd without the attendant costs of maintaining animals that are often difficult to handle and in multiple breeding pastures. AI tends to be less expensive than natural service (a straw of semen generally costs less than transporting a female to the sire and the stud fee) and avoids the potential physical risks to either sire or dam as part of the mating process. The disadvantages of AI include the need to train personnel engaged in the breeding operations on how to detect estrus in females (see subsequent discussion of estrous synchronization), and training or retaining individuals to perform the insemination. Further, care needs to be taken not to rely excessively on a few apparently superior sires so as not to reduce the genetic diversity of the resulting herds.
Sperm collection and AI were further improved by the advent of sperm sexing, or selection of sperm carrying an X (female) or Y (male) chromosome.11 Development of an effective and simple method for producing animals of the desired sex is economically desirable for livestock producers; sperm sexing is currently being used when available and economically feasible (Foote 2001; Faber and Ferre 2004). For example, in the dairy industry, females are desired because males do not produce milk; and excess males often become veal. In the beef industry, however, males are desired because they grow faster. Females can be the desired sex in the swine industry where leaner animals generally receive higher prices; young female pigs (gilts) tend to be leaner than castrated male pigs (barrows) when they arrive at market.
One method that shows the most promise for predetermining the sex of offspring is sexing semen using flow cytometry. This technique is based on the observation that in livestock species, sperm with X chromosomes have about 3 percent more DNA than those with Y chromosomes. Collected semen is diluted, and single sperm are passed through a laser beam that allows for the determination of the amount of DNA in each individual sperm. Based on their relative DNA content, sperm are sorted into "heavier" (female producing) and "lighter" (male producing) fractions. Another method sexes early embryos by removing one or two of the cells from the early embryo, arresting the further growth of the embryo by freezing, and identifying genes found only on the Y chromosome using the polymerase chain reaction (PCR) in the selected cells (Youngquist 1997). Semen sexing is more rapid, less invasive, and more economical, while embryo sexing is impractical at this time, as it is invasive, time intensive, and quite expensive. Further, the potential to damage the embryo by piercing the protective layer around it (zona pellucida), removing cells, and freezing the remaining cells in the embryo is quite high.
Estrous synchronization, or the timed induction of estrus (commonly referred to as heat), further permits the efficient use of artificial insemination (Hafez and Hafez 2000). Estrous synchronization was first practiced in the US in the 1960s and allows for the insemination of large groups of animals in a specified window of time when the female is capable of conceiving a pregnancy. The alternative to estrous synchronization is the time- and labor-intensive method of observing females’ behavior to detect estrus, and then arranging insemination at the appropriate time on an animal-by-animal basis. Estrous synchronization is achieved by treating females with hormones (Hafez and Hafez 2000). These synchronization regimens mimic the natural hormonal patterns leading to estrus and ovulation, and allow the producer to control the timing of these patterns. Several FDA-approved hormones are available for estrous synchronization, such as progesterone and prostaglandin F2a.12 These hormones are commonly used for conventional breeding in cattle throughout the U.S. According to a recent survey of large U.S. commercial dairies, 87 percent of herds use hormonal estrous synchronization methods (Caraviello et al. 2006). The advantage of linking AI to estrous synchronization lays in the ability of contained agricultural practices to operate on a more predictable schedule. For example, cattle breeders can avoid the reduced conception rates that occur during summer’s heat by breeding animals during the cooler spring season. Predictability can benefit farmers by allowing them to allocate resources (e.g., farm labor, veterinary visits) more efficiently, thus lowering production costs.
It is impossible for a fertile female mammal to bear all of her potential offspring. Litter size, gestation time, and post-partum decreases in fertility all limit the potential number of progeny that she can produce. When the female animal reaches the end of her reproductive period, any remaining unfertilized eggs represent potential offspring that have been lost. One solution to this dilemma is to transfer embryos of genetically superior female animals to multiple surrogate dams. This technique, called "embryo transfer," is particularly useful in species in which a low number of progeny are produced per gestation.13 In concept, then, embryo transfer (ET) is analogous to AI in that the total yield of offspring from a genetically superior, in this case, female animal can be increased (Youngquist 1997).
In 1890, rabbit embryos were first transferred from a donor female to surrogate rabbits. The experiment demonstrated that the surrogate’s genetics would not influence the transferred embryo’s genetics or development. In 1951, a successful live bovine ET was accomplished, but non-surgical methods of embryo collection did not succeed until the late 1960s (Hafez and Hafez 2000).
Currently, it is possible to flush large numbers of viable embryos from a superovulated cow with minimal stress to the animal (Hafez and Hafez 2000). Superovulation of the donor animal is generally accomplished by injecting the animal with follicle stimulating hormone or other exogenous gonadotropins before she enters estrus. The hormones induce production of a large quantity of ovarian follicles containing mature, preovulatory oöcytes. Insemination is performed at appropriate times relative to ovulation depending on the species and breed. Using standard synchronization methods, the estrous cycle in recipient surrogate mothers is synchronized in parallel with the cycle of the donor to be ready to accept embryos for implantation and gestation. When embryos are about a week old, they may be flushed out of the donor dam’s uterus nonsurgically14 by guiding a catheter through the cervix and into the uterus. Saline solution is injected into the uterus via the catheter and then flushed, along with the embryos, back out of catheter into a collection vessel. Embryos are isolated from the flushing solution, and examined microscopically to determine whether they are of sufficiently quality to implant. If they meet the criteria for further use, embryos can be transferred immediately to a waiting synchronized recipient animal using the same non-surgical technique that was used to collect the embryos. Alternatively, embryos can be frozen for later use or split into halves (see embryo splitting discussion below). Fresh or thawed embryos are inserted into surrogate mothers, where they attach to the lining of the uterus. Following transfer of embryos, no additional hormones or special treatments are required and the pregnancy is allowed to progress normally.
This technique, referred to as MOET (multiple ovulation and embryo transfer), is often used in relatively intensive cattle breeding programs, but is less developed in other livestock species. Similar to fertilizing many females with sperm from one superior male, MOET provides the breeder the ability to expand genetic traits exhibited in superior females. Further, the ability to freeze embryos allows for the preservation of "genetic stock" to be used at a later time. Its prevalence in livestock breeding, however, is much lower than AI, as it is considerably more expensive (Wilmut et al. 2002).
The International Embryo Transfer Society (IETS), a professional society whose membership includes breeders and researchers, estimates that a total of approximately 612,178 in vivo derived bovine embryos were transferred worldwide in 2005 (Thibier 2006). This is an increase of approximately 62,000 embryos from the previous year. Most of those transfers occurred in North America (45.2 percent), with the rest taking place in South America (20.5 percent), Asia (18.9 percent), and Europe (~14 percent).
In vitro Fertilization
The first in vitro fertilized (IVF) offspring was a rabbit born in 1959 (Chang 1959a). Since that time, IVF offspring have been born to mice, rats, hamsters, cats, guinea pigs, squirrels, pigs, cows, monkeys, and humans (Bearden and Fuquay 2000). IVF allows for the production of offspring from animals where other ART methods fail due to difficulties with either the female (blocked oviducts, non-responsive ovaries) or male (marginal semen quality and/or quantity), or where disease is present. In cattle, it is also used for the production of embryos from sexed semen because of the low sperm counts resulting from current sexing protocols, and for the further extension of the semen of superior sires due to the relatively low level of sperm required for in vitro fertilization. (IVF procedures are also used to assist human couples with limited fertility.)
The overall technique for IVF is similar among species, and involves significant manipulations in vitro, or outside the body of animals. In livestock species, oöcytes are collected from the ovaries of either living or deceased animals whose genetic potential is desirable (Goodhand et al. 1999). Ovaries can be obtained by transvaginal aspiration from live animals, or from a deceased animal at time of slaughter. Slaughterhouse ovaries are cross-sectioned and the contents of all of the follicles are collected; mature oöcytes are collected, evaluated for quality, and used for fertilization. Immature oöcytes must be allowed to continue to develop in a maturation medium.
Either fresh or frozen-thawed semen can be used for fertilization. Sperm need to be capacitated in vitro in order to penetrate the zona pellucida and fuse with the ovum or to undergo the same maturation process that they would normally undergo in the female reproductive tract. Capacitation involves a series of cellular changes to the sperm including increased motility, calcium uptake and protein binding (binding to proteins produced by the female reproductive tract). In vitro capacitation is accomplished by creating a medium designed to simulate the female reproductive tract and allowing the sperm to incubate in it for a period of time. Sperm are then added to ova, incubated in culture medium for approximately 8-22 hours, and the resulting fertilized ova, called zygotes, are washed, examined for appropriate development, and allowed to continue to divide for up to seven days, again in culture. At that time, if embryos appear normal, they may either be frozen for future use or inserted into the uterus of a reproductively competent female.
The IETS reported that the number of in vitro embryos transferred in 2005 increased by 11 percent over 2004 numbers to more than 265,000. Almost all of these transfers were performed in South America (particularly in Brazil) and Asia (especially Korea and China).
The numbers of in vivo and in vitro embryo transfers for other species (sheep and goats) were considerably lower, with 34,458 sheep embryos transferred, mostly in Australia, New Zealand and South Africa, and approximately 5,000 goat embryos transferred, mostly in South Africa and Asia. According to IETS statistics, approximately 66,000 swine embryos were transferred in 2005 (a huge increase over the previous year’s number of 16,016), with almost all occurring in Vietnam and Korea (Thibier 2006).
Genetically identical individuals derived from a sole embryonic source can arise naturally, as in the case of spontaneous monozygotic twinning, or in vitro via the manual separation (splitting) of early stage embryos. Embryo splitting may be considered the first true "cloning" procedure involving human intervention, and was first described by Willadsen and Polge in 1981, when monozygotic twin calves were produced.
Embryo splitting, or the mechanical separation of cells,15 can be used in very early embryos. Briefly, two-cell embryos derived from either in vitro fertilization, or embryo rescue following in vivo fertilization (as described for embryo transfer) are held in place with micropipettes under a microscope. The zona pellucida (the clear layer of protein surrounding the oöcyte and fertilized ovum) of these embryos is opened, and the two-celled embryo is then split into individual cells with a finely drawn needle or pipette. One of the cells is left in the original zona pellucida and the other is either placed into an empty zona pellucida or allowed to develop without a zona pellucida. These so-called demi-embryos can be cultured in vitro for a few days, inspected for appropriate growth and then transferred directly to synchronized recipient dams or frozen for future use. Similar procedures can be used to multiply embryos that have developed beyond the 2-cell stage (Willadsen 1980). Each of the individual cells or blastomeres from a single early embryo is totipotent. That is, each cell retains the ability to generate a fully functional individual identical to the other individual(s) derived from the other cells of the original embryo.
Although commercial applications of embryo splitting have been tracked by breeders’ associations, the technology has never gained significant market penetration for several reasons. It is a very expensive and time consuming procedure that has not provided the yield initially anticipated for the technology. For example, actual calf yield from blastomere splits is approximately 105 calves per 100 embryos, while direct transfer of intact embryos yields approximately 60 calves per 100 embryos (Wilmut et al. 2000). Unless embryos are sexed at the time of splitting, however, breeders may end up with half of their animals being of the undesired gender, thus incurring twice the cost for the desired offspring. In addition, even if the resulting calves are of the desired gender, their production potential is not known, making the procedure an expensive gamble.
Blastomere Nuclear Transfer
The next evolution of ART evolved from additional manipulations of the blastomere cell, and involved its fusion with an enucleated oöcyte. This method expands on the relatively simple early stage embryo cell separation procedure described previously by allowing the use of cells from later stage embryos. In this case, embryos of the eight to sixteen-cell stage, compact morulae, and the inner cell mass from blastocysts can be used as donor nuclei (First and Prather 1991a). Fusion of these later stage blastomere cells, which have lost their totipotency, with enucleated oöcytes, reprograms the blastomere nuclei to allow them to develop as zygotes. Blastomeres from bovine embryos up to the 64-cell stage can be fused with enucleated freshly fertilized oöcytes and cultured to develop into genetically identical individuals (Keefer et al. 1994). Cell nuclei derived from the inner cell mass of expanded blastocysts transferred into enucleated host cells are also capable of development resulting in offspring (Sims and First 1994).
This technology, which may be considered the true antecedent of somatic cell nuclear transfer, had limited commercial applicability for the same reasons as embryo splitting: high cost, high loss rate, and the inability to predict phenotypic performance or the gender of the resulting offspring.
Somatic Cell Nuclear Transfer (SCNT)
In 1962, biologist John Gurdon of Oxford University pioneered the method of the two step "nuclear transfer" process in frogs: the enucleation of a recipient oöcyte and the subsequent transfer of a differentiated somatic cell nucleus to that oöcyte. Gurdon’s experiments showed that despite the differentiated status of the donor nucleus, reconstituted cells appeared to reprogram, or dedifferentiate, the nucleus and enable it to function much as a naturally produced zygote. These zygotes successfully developed into viable embryos that hatched and grew into tadpoles. Because the tadpoles had all come from the gut cells of the same adult frog, they all had the same genetic material and thus were all clones. However, Gurdon’s nuclear transfer tadpoles clones failed to metamorphose into frogs. When scientists attempted to apply this technology to other species such as mice, cattle, or other mammals, the developmental program could not be reset (Gurdon and Uehlinger 1966; Byrne et al. 2002).
Scientists continued to tackle the problem and in 1986, Randall Prather and colleagues, then working in Neal First’s laboratory at the University of Wisconsin-Madison, cloned a cow from early embryonic cells using nuclear transfer (Prather et al. 1987). Although this was an example of blastomere nuclear transfer, it effectively set the stage for Dolly’s birth a decade later, on July 5, 1996. Dolly the sheep, the first organism ever to be cloned from adult cells, was created by Ian Wilmut and Keith Campbell using a technique similar to that used to create the first sheep from differentiated embryo cells (i.e., a blastomere clone) in 1995 (Wilmut et al. 1997).16
In July 1998, Ryuzo Yanagimachi, Toni Perry, and Teruhiko Wakayama of the University of Hawaii announced that they had cloned fifty mice from adult cells using the "Honolulu technique" (Wakayama et al. 1998). This was particularly significant because mouse embryos begin to divide almost immediately after the ovum is fertilized, and scientists had believed that this would not allow sufficient time for reprogramming to occur. Sheep, on the other hand, because their ova do not divide for several hours after fertilization, were thought to be an "easier" species to clone, as the natural delay between fertilization and division might be replicated in SCNT, possibly giving the oöcyte time to reprogram its new nucleus.
SCNT is a relatively new technology described by many as complex, technically demanding and inefficient, that continues to be developed and improved. As such, there is no set "method" that is universally employed, although the basic steps outlined below are common to most SCNT procedures at the time that this overview was written.
For species in which the cloning process has been relatively well developed, the first step is identifying the animal to use as a nuclear donor. Animals to be used for breeding purposes are selected because they have been shown to be genetically superior to herd mates for the trait(s) to be propagated. Somatic cells can be collected from the ear (hole punch) or skin (surgical incision or needle aspiration), although many other cell sources have been used. Multiple factors may influence success or failure of the nuclear transfer process. Coordination of the cell-cycle stage of the donor nucleus and the recipient egg cytoplasm appears to be important for successful development of embryos. In general, the selection of a cell type for commercial cloning from an adult animal has evolved to choosing a collection method that is relatively noninvasive and minimizes stress to the live animal donor.
Several characteristics have been identified as contributors to the degree to which any given donor cell or type of cell will likely result in a successful cloning event (Sung et al. 2006; Oback and Wells 2007a). One example is the "replicative state" of the donor cell. In general, cells in culture accumulate nutrients, grow, and when they reach certain conditions, divide. Cells that adhere to a solid substrate, such as the bottom of a tissue culture dish tend to grow until there are so many of them that they begin to touch each other. Once that happens, they generally stop dividing, and go into a "resting state" with respect to replication (referred to as G0). Cells can also be directed into G0 by depleting the nutrients in their growth medium. Some laboratories have concluded that cells in G0 are the most effective donors (Wilmut and Campbell 1998, De Sousa et al. 2002). Conversely, other laboratories have found that actively dividing cells make good donors (Cibelli et al. 1998a, Lanza et al. 2001). Some laboratories find that cells from embryos or fetuses are the best donors (Batchelder 2005), while others are successful at cloning cells from aged or even deceased animals (Hill et al. 2000a, Tian et al. 2001). Another characteristic that has been shown to influence the degree to which cells make good donors is how "inbred" the donor animal is (Rideout et al. 2000). These researchers have determined that "hybrid vigor" is important for the success rate of animal cloning and the more inbred the donor animal, the less likely it is that cloning will occur successfully. Further, some species appear to be more amenable to cloning than others (e.g., goats compared with cattle, see Chapter V), and some species have not been cloned at all. At this time, the best conclusion that can be drawn with respect to the degree to which a cell (or animal) will serve as a "good" donor is that the technology is not sufficiently mature to predict with certainty which set of conditions will optimize cloning efficiency.
Once a cell has been isolated from culture, depending on the laboratory, either the entire cell or just its nucleus is transferred under the zona pellucida of the enucleated oöcyte using a very thin glass micropipette (Solter 2000) to await fusion.
The cell type used as the recipient for the donor cell to be cloned is the mature oöcyte, the version of the ovum that participates in fertilization during sexual reproduction. The oöcyte contains all of the non-nuclear cellular components required for the early development of an embryo. Oöcytes can be obtained from ovaries collected at slaughterhouses or from live animals using aspiration techniques (see previous discussion of in vitro fertilization). Because the oöcyte donates only its cytoplasm (the oöplast), it must be enucleated prior to fusion with the donor. The nucleus is generally removed by microaspiration, using a finely honed needle (PIFB 200317).
In order to begin the development process, the membranes separating the oöplast and the donor nucleus (or cell) must be fused. This can be accomplished in two ways: (1) by the administration of a brief electrical pulse, or (2) chemical fusion. Electrical stimulation appears to be the more commonly used technique and involves the application of one to several microbursts of a mild electrical current in the vicinity of the cells. This induces the formation of pores between the somatic donor cell and oöplast which functionally makes the two cells one. This process also stimulates embryonic development,18 which if successful, results in the development of blastocysts that are transplanted into surrogate mothers (Cervera et al. 2002).
Technical modifications aimed at increasing the success rate of cloning by improving the efficiency of the enucleation and fusion approaches are steadily evolving. For example, Oback et al. (2003) have developed a method that removes the zona pellucida from the oöcyte, aligns the donor cells with enucleated oöplasts, and uses electrofusion and chemicals to activate the cells to begin dividing. The results of this technique seem to show similar success rates for generating cattle clones as the cloning techniques more commonly used, with the advantage of being faster to perform (in the authors’ hands), and requiring less expensive equipment. Peura (2003) has also described a modified technique for preparing fused donor/oöplasts in which sheep oöcytes whose zona pellucidae had been removed were enucleated after fusion with donor cells, reversing the order in which those steps are usually performed. This technique appears to provide a higher rate of development of the blastocyst stage, implying that some factors present near the oöcyte chromosomes may be of assistance. Other factors that may influence the success rate of SCNT are the timing and method of embryo activation relative to fusion. For example, Sung et al. (2007) found that simultaneous electrical fusion and chemical activation of SCNT bovine embryos resulted in a higher rate of development to the blastocyst stage compared to embryos that were activated four hours postfusion. Compared to bovine SCNT embryos that were chemically activated, Schurmann et al. (2006) reported that postimplantation development of SCNT embryos was improved by using a non-chemical, more physiological method of activation (by transferring donor nuclei into enucleated oöcytes that had been fertilized in vitro).
Over the next few years other technical refinements may be developed, some based on improved technical practice, and others on increased knowledge about basic molecular mechanisms involved in the developmental process. These should increase the success rate of cloning, and decrease the potential for adverse events to occur.
Transfer to recipient
Just as the case for other ARTs with an in vitro phase, the developing clone is transferred at the blastocyst stage into a surrogate dam in which the estrous cycle has been synchronized using standard methods. After transfer of the embryo clone(s), the pregnancy is allowed to proceed normally; no additional hormones or special treatments are required to establish or maintain pregnancy.
In cloning’s earliest days, the surrogate mother was often chosen to be distinctively different from the donor animal with respect to some clearly visible trait. For example, Dolly’s donor animal was a Finn Dorset sheep, a breed with white faces. Dolly’s surrogate mother, however, was chosen to be a black-faced sheep, so that if a white-faced sheep were born, it would be clear that it was not a genetic relative of the surrogate mother. In addition to choosing a distinctively different embryo recipient, Dolly’s identity was also confirmed by DNA fingerprint analysis of the donor cell line from which she was derived (Wilmut et al. 1997). DNA fingerprint analysis enables definitive confirmation that an animal clone was indeed derived from a specific cell type, and is now the method of choice for confirming genetic parentage of animal clones (First et al. 1994).
C. Critical Biological Events in SCNT
Although SCNT is an inefficient process with a relatively low success rate, the extraordinary nature of the technology and the demands that it places on the biological system being manipulated should not be overlooked. Unlike the fertilized egg or early embryonic cells that may be considered totipotent (capable of becoming any cell in an organism) or pluripotent (capable of become many cells in an organism) "generalists," donor cells tend to be specialists. That is, they have differentiated to such a degree that their genomes have been "reconfigured" in ways that are, as yet, not fully understood in order to carry out the particular function for which they have been destined by their particular developmental fate. Kidney cells, therefore, do not transcribe the milk producing instructions of the mammary gland, yet they continue to carry those genes. The question then, is how to "reprogram" the full set of instructions contained in the genome such that "normal" development can occur. The following is a general overview of the events that are thought to occur during the SCNT process. There are several excellent reviews of the overall process or individual components that interested readers can reference for more details, and Chapter IV deals with some of these issues in more detail (Kikyo and Wolffe 2000; Sinclair et al. 2000; Solter 2000; Young and Fairburn 2000; Fulka et al. 2001; Rideout et al. 2001; Novak and Sirard 2002; NAS 2002a, b; Colman 2002; Dean et al. 2003, Santos et al. 2003; Morgan et al. 2005; Vajta and Gjerris, 2006; Meissner and Jaenisch, 2006; Oback and Wells, 2007a).
In principle, SCNT has demonstrated that cell differentiation can be reversed. Genetic reprogramming, the process of altering the gene expression pattern associated with the differentiated cell to one that is appropriate or early embryonic development, is normally carried out at two stages in the development of fertilization-derived embryos: after fertilization, and during the development of gametes (the sperm and ovum). The actual molecular events involved in reprogramming are not fully understood, although they may be categorized into a few overall steps. These include altering the way in which the chromosomes are packaged by changing the chemical nature of the proteins involved, and changing the chemical structure of the DNA in portions of the molecule that are not responsible for base-pairing (NAS 2002 a,b). A more complete description of these processes is found in Chapter IV.
The nucleus of a cell contains a complete copy of all of the genes required for life. This information is encoded in genes. Physically, genes are the linked nucleotides that comprise DNA, or the "master molecule" of biology. The total genetic material of an organism is referred to as its "genome," and consists of long strands of DNA packaged in chromosomes, which come in pairs except for those specifying the sex of the resulting organism. The number of pairs of chromosomes differs among species. Cattle, for example, have 30 pairs, pigs have 19, sheep 27, and goats 30. (Humans have 23 pairs of chromosomes.)
Chromosomes can exist in different "conformations" depending on the stage of the cell cycle. When DNA needs to be moved, as in when a cell divides, or when a sperm needs to deliver the male genome, chromosomes are tightly condensed. During the rest of the cell’s life cycle, chromosomes tend to exist in less tightly coiled conformations so the information encoded in the DNA is more accessible for processing. Specific proteins are responsible for holding chromosomes in different conformations. In all cells but sperm, these proteins are called histones; in sperm chromosomes are packaged by proteins referred to as protamines.
When an ovum is fertilized by a sperm, a complex series of molecular events ensues that is referred to as "chromatin remodeling" (chromatin is another term for the protein:DNA complexes that make up chromosomes.) Although the exact steps are not known, the overall process involves stripping away the protamines packaging the paternal DNA, removing histones from the ovum’s DNA, and allowing the newly associated DNA molecules to reform chromatin in a way that allows the fertilized ovum and early embryonic cells to replicate and be "totipotent"(capable of developing into a complete organism). Many proteins are involved in this, only a few of which have been identified, and it is likely that there are chemical markers on the DNA bases that are altered (such as methylation). Chromatin remodeling is likely very different in SCNT. Disassembly of the tightly condensed sperm chromatids and the subsequent removal of protamines do not occur because there is no sperm present. Instead the oöplast must decondense and repackage the chromosomes of the donor somatic nucleus.
In order to perform the functions of life, cells have to convert the information in the DNA to ribonucleic acid (RNA) (a process referred to as transcription), and then to translate that RNA into proteins, which are the molecules that carry out life’s functions. This coordinated set of activities is referred to as gene expression. Alterations in the expression of a given set of genes are often referred to as "epigenetic effects" (or "around gene effects") because they do not require changes in the base-pairing properties of the DNA that comprise genes. Instead, they reflect changes in the structure of the chromosome around the gene (such as control regions), or on the nucleotides, but outside the portion of the molecule involved in coding. (See Chapter IV for a more complete discussion). A classic example of the manifestations of epigenetic effects is the different fingerprint or freckle patterns observed in human twins. These individuals have exactly the same coding regions, but small changes in the non-coding regions of the DNA result in different phenotypes. Other examples of epigenetic control of gene expression include the coat color or color patterns of many mammals.
D. Outcomes Observed in ARTs
As this risk assessment is being prepared, biologists are just beginning to understand the highly complex interactions that must occur to choreograph the millions of molecular interactions that signal the expression or silencing of genes in a particular cell or at any point in its life cycle. Although the exact mechanisms by which these effects occur are not fully understood, in all forms of reproduction, ranging from natural mating to SCNT, these processes may go awry in early development. Although most of the animals born following ARTs with significant in vitro components appear to be completely "normal," some of the outcomes are not so successful. In particular, some of the adverse outcomes noted in these "high in vitro component ARTs" appear to have common defects in gene expression, particularly in the overgrowth outcomes (Humpherys et al. 2002).
Published studies involving cattle, sheep, and mice demonstrate that embryos produced using in vitro systems may differ in morphology and developmental potential compared to embryos produced in vivo (Kruip and den Daas 1997; Young et al. 1998; Farin et al. 2004; Farin et al. 2006; see Appendix C for a more detailed discussion and additional references). For example, common abnormalities have been noted in fetuses (Farin and Farin 1995) and calves (Behboodi et al.1995; Sinclair 1999) associated with the transfer of bovine embryos produced using in vitro maturation (IVM), in vitro fertilization (IVF), in vitro culture (IVC) systems, and SCNT (Hill et al. 2000b). One set of reported adverse outcomes following transfer of embryos from cloning or in vitro production systems is often referred to as Large Offspring Syndrome (LOS). These include lowered pregnancy rates, increased rates of abortion, production of oversized calves, musculoskeletal deformities and disproportionalities, as well as hydroallantois (abnormal accumulation of fluid in the placenta) and other abnormalities of placental development.
The phenomenon of "large calves" was first described by Willadsen et al. (1991). The syndrome has also been identified in fetal and newborn lambs and in mice where the embryos were cultured in vitro (Eggan et al. 2001). Offspring with LOS tend to exhibit difficulties with placentation (Farin et al. 2004; Bertolini et al. 2004; Lee et al. 2004; Batchelder 2005). In cattle and sheep, the placentae of developing fetuses with LOS are unusually large for their species, and tend to have abnormal development of placentomes (the sites of attachment between fetal and maternally derived tissues of the placenta). LOS fetuses tend to have longer than usual gestation lengths, and often labor in the dams must be induced followed by Caesarian section deliveries. The newborns tend to be large for their breeds, and often have abnormal or poorly developed lungs, hearts, or other affected internal organs (liver and kidney), which makes it difficult for them to breathe or maintain normal circulation and metabolism. LOS newborns may appear to be edematous (fluid filled), and if they are to survive, often require significant veterinary intervention. Problems have also been noted in muscle and skeletal development of animals with LOS. These animals also often have difficulty regulating body temperature. (For a more detailed discussion of LOS, see Chapter V).
Although the cause of LOS is not known with certainty, it is likely be related to changes in gene expression (i.e., epigenetic changes) that result from the in vitro manipulation and culturing of embryos. A review by Young et al. (1998) suggests that in vitro culture alone is adequate to perturb the embryo. This hypothesis is supported by data from Sinclair et al. (2000) where in vivo matured and fertilized eggs recovered from superovulated sheep donors, cultured in vitro for 6 days, showed an 18-36 percent increase in mean birth weight at day 125 of gestation, depending on the culture system used (Sinclair et al. 2000, Young et al. 1998). Table C-1 (Appendix C) provides a more comprehensive summary of adverse outcomes noted in different ARTs.
This is an area of extensive research in the cloning and developmental biology communities. It is likely that advances in the understanding of these mechanisms will lead to significant improvements in the rates of successful outcomes of all ARTs that include a significant in vitro component, including cloning.
E. Future of Reproductive Technologies in Modern Agricultural Practice
Modern agricultural practices will likely continue to employ all of the reproductive modalities described in this overview. The factors that may influence which practices are used will likely be a function of the breeder/farmer/ranchers’ needs and opportunities. Seidel (2006), in a foreword to a symposium on ARTs, emphasized that current differences in the reproductive management of cattle in different parts of the world are driven by multiple considerations. Some of these have to do with the nature of the differences in the husbandry of beef (mostly pasture based) and dairy (mostly intensively housed in the US, more pasture based in Australia and New Zealand) cattle. Some are economic (ARTs are much more expensive than natural matings), some are practical (the fertility of dairy cattle has declined significantly in the last 20 years, making ARTs more attractive; using ARTs in beef cattle is not practical for ranchers who look for replacement by natural coverage), and some are technological (the ability to choose genetics more precisely versus the developmental problems associated with ARTs with significant in vitro components).
Technological issues will be addressed by continued research and development in this field. To that end, several professional and scientific societies (e.g., the International Embryo Transfer Society, various animal science organizations and breeding associations) have been actively involved as clearing-houses for information and interaction.
SCNT has the potential to impact animal breeding in as fundamental a manner as artificial insemination. Given its current high costs (approximately $20,000 for a live calf) and relatively low success rates (< 10 percent), SCNT will likely be used to improve production characteristics of food producing animals by providing breeding animals, just as any breeding program would select the most elite animals for breeding, and not as production animals. In this way, cloning does not differ from any of the other ARTs that have been described in this chapter. Cloning has the relative advantage of allowing for the propagation of animals with known phenotypes to serve as additional breeding animals. This is critically important in breeding programs, especially when it may take years to "prove" the merit of a sire or dam. Second, it allows the propagation of animals whose reproductive function may be impaired. It has already been used to increase the available genotype of a particular dairy cow with low fertility; her clones appear to be exhibiting normal fertility (PIFB 2003). Third, it allows the propagation of valuable deceased animals from which tissue samples have been appropriately collected or preserved, which may have profound implications for species or breeds nearing extinction. Finally, for the first time, cloning allows for the careful study of the "nature-nurture" interactions that influence breeding programs by allowing a large enough sample of genetically identical animals to be raised in different environments, or with different diets. Such studies have been impossible to perform prior to the advent of SCNT and are likely to yield important information for developing livestock species to live in areas that have, until this time, been marginal for food animal production. This is of particular importance to the developing world, where even slightly increased wealth generally favors the incorporation of animal-based agriculture.
Regardless of the degree to which cloning may be adopted in animal breeding programs, FDA’s role in performing this risk assessment is clear: the agency’s responsibility is to determine whether cloning poses any risk to animals involved in the cloning process, and whether the consumption of food products from clones or their progeny poses any additional risk compared with food from conventionally produced animals. This Risk Assessment presents the method by which we evaluated data on clones and their progeny, the data themselves, and the agency’s conclusions, including discussions of uncertainty.
8 The term "clone" originated before the late 1990s. The British biologist J.B.S. Haldane, in a speech entitled "Biological Possibilities for the Human Species of the Next Ten-Thousand Years," used the term in 1963. The Merriam-Webster dictionary, however, dates its use in a biological context to 1903.
9 The process of non-assisted mating is referred to as natural “mating,” “coverage” or “service.”
11 In normal mammalian sexual reproduction, the female always donates an “X” chromosome, and the male can donate either an “X” or a “Y” chromosome. XX yields a female animal; XY produces a male.
12 See http://pewagbiotech.org/events/0924/proceedings2.pdf
18 This step is also sometimes referred to as activation. During sexual reproduction, physiological activation of the oöcyte is stimulated by fertilization. Since sperm-mediated fertilization is subverted in SCNT, activation must be stimulated artificially in vitro by electrical current; addition of chemicals that increase intracellular concentrations of calcium (calcium ionophores) or inhibit protein synthesis; or a combination of electrical and chemical stimuli.