Chapter V: Animal Health Risks
A. Potential Hazards and Risks to Animals Involved in Cloning
This analysis identifies hazards and characterizes risks to animals involved in somatic cell nuclear transfer (SCNT) in the context of other assisted reproductive technologies (ARTs) in use in current US agricultural practice. Although hazards have been identified in the literature, a systematic assessment of potential risks is difficult, due to the relative newness of the technology, and the variability in outcomes among laboratories and species cloned. This section reviews the publicly available information and applies existing knowledge of animal biology and agricultural practices to cast that information in a risk context.
In addition to characterizing hazards, this chapter identifies information gaps that when filled may provide a more complete understanding of the risks to animals associated with SCNT technology. It is not intended to be an overview of all the techniques used to produce clones, nor does it review studies that attempt to optimize the early stages of clone embryo production. Rather, this analysis focuses on those studies relevant to the overall objective of this risk assessment, namely, identifying the biological risks that cloning poses to animal health
Because of the diversity of approaches in the peer-reviewed studies, CVM has relied on various ARTs including an earlier type of “cloning” called blastomere nuclear transfer (BNT) for context. Current agricultural statistics also are used to provide readers with a frame of reference for these technologies (see Appendix B). Outcomes for various ARTs are located in Appendix C. Peer-reviewed reports of primary findings were used as references for SCNT, while some recent reviews of artificial insemination (AI), embryo transfer (ET), and in vitro produced embryos (IVP), as well as primary data reports, were employed as references for the older ARTs.
Most of the studies on SCNT and other ARTs that are of utility for identifying and assessing risk to animals, and that make up the subject of this Risk Assessment are in ruminants.33 Cattle studies are the most abundant, followed by sheep, swine (a non-ruminant species) and goats. Peer-reviewed research reports on these four species, with supplemental data from studies in mice, primarily have been used as the basis for this assessment. Additionally, CVM evaluated veterinary records, blood clinical chemistry and hematology, and urinalysis provided by two private firms: (1) Cyagra, Inc. provided data on 134 individual cattle clones ranging from birth to approximately one and a half years of age (Appendix E); and (2) ViaGen, Inc. provided data on 11 swine clones and 402 progeny of swine clones through slaughter age (Appendix F). Additional unpublished data were provided by several sources, in the form of veterinary records, blood chemistry and hematology, and reproductive performance on small groups of cattle and swine clones (Appendix G).
Publications from peer-reviewed journals were searched for information relating to health of surrogate dams, animal clones, and clone progeny. Whenever possible, data on contemporary comparators have been used to provide reference rates for purposes of comparison. Where comparisons were not made within a study, the historical literature and other available databases (e.g., USDA National Agricultural Statistics Service (NASS34 ) or National Animal Health Monitoring Service (NAHMS35 )) were searched for applicable comparative information. For example, Table V-1 (Survival Rates of Live-Born Bovine Clones and Comparators) presents data on survival rates of clones and comparators, drawn from both contemporaneous comparators and historical datasets. Descriptions of how other data were analyzed are described in Appendix E (Cyagra Data), Appendix F (ViaGen Data), and Appendix H (Comprehensive Veterinary Exam and Its Interpretation).
B. The Critical Biological Systems Approach to the Analysis of Clone Animal Health: Cattle, Swine, Sheep, and Goats
To provide an assessment of animal health risks that was as comprehensive as possible, the CBSA was applied at all five developmental nodes at multiple levels of observation and analysis. In addition to macroscopic anomalies observed in surrogate dams and clones themselves, the information in this chapter includes available evidence to indicate whether subtle hazards exist that might affect the health of clones. For the purposes of this risk assessment, subtle hazards may be thought of as physiological anomalies that may be present in apparently normal animals, but are not obvious because their identification requires analysis of physiological parameters in blood and tissues (e.g., clinical chemistry, hematological measurements, hormone levels).
1. Pregnancy and Parturition (Developmental Node 1)
Pregnancy is a remarkable time in mammalian development. A carefully orchestrated and incompletely understood sequence of changes in both the pregnant female and developing embryo/fetus must occur to produce a successful outcome: a healthy newborn and mother. Despite this complexity, most pregnancies in domestic livestock proceed normally and result in healthy offspring.
Criticisms of cloning point to the “inefficiency” of the process, which is often translated to mean that successful outcomes are relatively uncommon (Wilmut 2002). Reports of early pregnancy loss or later-term spontaneous abortion of embryonic and fetal clones are frequently cited in the literature (Le Bouhris et al. 1998; Kishi et al. 2000; Chavatte-Palmer et al. 2002; Lee RS et al. 2004). Loss due to defects in the embryo or failure to implant in the uterus of the surrogate dam does not pose a hazard to the dam at this early stage. Rather, the female simply resorbs any embryonic tissues and returns to cycling (Merck Veterinary Manual Online 200536 ). Mid- and late-term spontaneous abortions may be hazardous to surrogates if they are unable to expel the fetus and its associated membranes, possibly resulting in metritis (uterine infection), retained fetal membranes (in which the placenta is not expelled), or a mummified (dead, desiccated) fetus. Other complications can occur during pregnancy and labor that may pose a risk to both the pregnant female and the fetus. Developmental Node 1 examines the causes and frequency of pregnancy complications, and the relative risks to both the female and fetus, using other ARTs for comparison where such data are available.
It is important to note that external factors unrelated to breeding method such as animal management and environment that can influence pregnancy outcomes. In evaluating any ART, including cloning, the potential impact of these external influences should be considered before assigning the cause of pregnancy loss to the technology itself. For example, stress is an important risk factor in the loss of any pregnancy, particularly in the preimplantation phase (before the embryo attaches to the uterine lining). Disease, under-nutrition, and severe environmental conditions (e.g., high ambient temperature) are stressors known to interfere with animal fertility and embryo survival (Lucy 2001, Merck Veterinary Manual Online 2005). In these cases, the risk to the pregnancy is directly related to those stress factors, not the technology used, and must be mitigated in order for normal reproduction to resume.
Another factor to consider is the methodology used in the SCNT process. A review of the literature suggests limiting in vitro manipulation of the embryo or changing the culture conditions to more closely resemble oviduct/uterine conditions may improve the chances for successful pregnancy outcomes. Many of the abnormalities reported in cattle and sheep pregnancies have not been noted in goats or swine carrying SCNT clones. Of the reports reviewed for this assessment, goat embryos were only cultured through the first or second cleavage stage (less than one day in culture) before transfer to the recipients (Keefer et al. 2002), compared with sheep and cattle, whose embryos were generally cultured to the blastocyst stage (seven to eight days in culture) prior to transfer. Walker et al. (2002) reported success after only brief in vitro culture of swine embryos (1-3 hours after activation) before transferring to recipients. Onishi et al. (2000) also reported the successful birth of SCNT pigs following culture to the 2 to 8 cell stage (one or two days in culture), while none of the embryos cultured to the blastocyst stage developed to term. In contrast, ViaGen, Inc. has indicated that they have had greater success recently transferring swine clone blastocysts (5 days in vitro culture) into surrogate dams (see CVM Memorandum II at www.fda.gov/cvm/cloning.htm). Lagutina et al. (2006) reported the birth of pigs following culture of swine embryos for six days in synthetic oviductal fluid (SOF) supplemented with amino acids, rather than supplementation with serum.
Abnormalities in cattle and sheep clones may result from incomplete reprogramming of the donor nucleus. As noted in Chapter IV, epigenetic reprogramming occurs at different times in embryos in different species, possibly in relation to gestation length. Despite that observation, it is interesting to note that although goats and sheep have the same gestation length (about five months), abnormal pregnancy outcomes are frequently reported with SCNT sheep, whereas SCNT goats have had relatively few problems reported (Wells et al. 1998a; Young et al. 1998; Baguisi et al. 1999; Reggio et al. 2001; Keefer et al. 2002; Ptak et al. 2002). It is important to note that epigenetic remodeling has been studied primarily in mice, swine, and cattle, and that very little is known about the timing and extent of reprogramming in small ruminants.
The biology of placental attachment also may account for differences among pregnancy outcomes in the species evaluated in this risk assessment. In contrast to ruminants with a “cotyledonary” (cotyledon37 ) type attachment via placentomes (see discussion below on this type of fetal attachment to the uterine lining), swine have what is classified as a “diffuse” type of placenta where fetal attachment occurs over the entire surface of the placenta and uterine lining (Hafez and Hafez 2000). This gross morphologic difference in fetal attachment may influence outcomes of clone pregnancies in the ruminant vs. swine species.
2. Perinatal Period (Developmental Node 2)
The perinatal period (from initiation of labor through approximately one week post partum) is one of the most critical times in the lives of all young animals. Several studies (reviewed by Moore et al. 2002) noted that 75 percent of mortality from all causes for naturally produced and AI beef calves occurred within the first seven days of life.
The process of labor and birth can be as stressful on the neonate as it is on the dam, particularly if complications arise during the process. The newborn must begin breathing almost immediately after birth, either spontaneously or with stimulation from the mother or human attendant. For ruminant animals, as for other herbivores, it is instinctive for the newborn to attempt to stand within the first 5-15 minutes after birth, and to suckle shortly thereafter. Swine are less mature at birth than most other farm livestock, and although they are able to walk and nurse almost immediately after birth, they are not able to control their body temperature (known as thermoregulation) for the first 10 to 14 days of life, and generally require supplemental heat.
In mammals, neonates have little endogenous immune protection from disease during the first few weeks to months of life. Young mammals are dependent on antibodies transmitted from their dams either through the placenta or by consumption of colostrum (the antibody- and nutrient-rich first fluid secreted by the mammary glands after birth preceding the production of true milk). The process of providing immunity to the offspring in this manner is called passive transfer of immunity. In ruminants and swine, the principal means of this transfer is through colostrum. In species where this form of transfer predominates, the neonate must consume colostrum as soon after birth as possible to insure intestinal absorption of functional immunoglobulins, large proteins which contain antibodies (Merck Veterinary Manual Online 200538 ). Within approximately 48 hours after birth (although this may vary among species), the neonatal intestine loses the ability to absorb large, functional proteins, (a process known as “gut closure”) and the opportunity for this method of immune transfer is lost (Donovan 1992).
3. Juvenile Developmental Node (Developmental Node 3)
Another critical period in the lives of young mammals is immediately post-weaning to approximately six months of age. In general, health and survival of any young animal post-weaning is dependent on management conditions. Relatively little information has been published in the peer-reviewed literature on health and survival of animal clones during this developmental node. As previously discussed, one clone producer has supplied data (Cyagra, Inc.), including health records and laboratory measurements that have been evaluated along with the published literature; these may be found in Appendix E.
Age at weaning varies among species, breeds, and individual farm management. Swine are typically weaned at about 21 days of age, but may be weaned as early as 10 to 14 days. Sheep and goats may be weaned between 8 and 12 weeks of age. Dairy calves typically receive milk replacer (after colostrum consumption is complete) until 28 to 60 days, when they are weaned to solid feed. Beef calves may remain with their dams and continue to nurse for four months or longer.
Weaning is a period of stress for all developing animals. Weight loss is common during weaning as the young animal must compensate for the loss of a primary source of nutrition and adapt to what previously may only have been offered as a supplement. Changing diet can induce scours (diarrhea), particularly if it is done abruptly. Diarrhea is a common ailment in all young mammals, and can be serious, resulting in dehydration and death if not treated in a timely manner (Merck Veterinary Manual Online 200539 ). In addition, between two and six months of age in ruminants, or as early as 21 days in swine, maternally derived immunity wanes, and the young animal must depend on its own immune system. In some animals, such as beef cattle, this may occur concurrently with transportation stress when they are sold to feedlots or stocker operations, resulting in relatively high losses.
4. Reproductive Development and Function Node (Developmental Node 4)
Due to the complexity of the reproductive system, careful attention was directed to reports of puberty and reproductive function in clones in order to determine whether cloning had perturbed this delicately balanced system. Data from this stage of development in animal clones are sparse, however.
In conventional cattle, inappropriate intrinsic, nutritional, and environmental factors have been shown to adversely influence reproduction in both male and female conventional animals. Under- and over-nutrition can influence the age at puberty and, particularly in the case of under-nutrition, can disrupt the normal estrous cycle. Environmental stressors such as extreme heat or cold can also suppress normal cycling and estrous behavior in females and reduce fertility and libido in males (Lucy 2001). Derangements in metabolic pathways, such as hypothyroidism, genomic disorders manifesting as freemartins40 and hermaphrodites,41 as well as congenital anomalies such as hypospadias42 can also result in reproductive failure (Merck Veterinary Manual Online 2005).
Considerable differences exist among species and even among breeds within a species for age at puberty. In cattle, puberty is related to body weight, and a heifer will achieve her first estrus when she reaches approximately 65 percent of her adult body weight. Depending on management, then, heifers will typically begin cycling between 10 and 13 months of age. Goats and sheep mature at a younger age, with first estrus typically occurring between seven and eight months. Dwarf goat and sheep varieties may mature at a much younger age. Nigerian Dwarf goats, such as those used in the Keefer et al. (2001a) study, mature as early as four months (Pugh 2002). Swine also mature sexually at a relatively young age, and gilts typically begin cycling between 6 and 8 months of age. Male animals generally reach sexual maturity at similar ages to females of the same breed and species.
In female animals of agricultural species, the estrous cycle is typically 21 days in length, although some variation exists among species. For example, the estrous cycle in sheep is only 17 days. In cattle, both males and females are fertile year round, although fertility may be decreased during parts of the year in regions with hot, humid climates. Sheep and goats originating in temperate zones are seasonal breeders, becoming fertile in response to decreasing day length. Breeds of sheep and goats that originated in the tropics are less sensitive to day length, and some are fertile year round. Swine, like cattle, are year-round breeders. A cow’s gestation is approximately nine months, with some breeds having slightly shorter and others having slightly longer gestations. Sheep and goats have gestations lasting approximately five months, with less variation among breeds. In swine, gestation is approximately four months.
With the exception of parturition, the reproductive period is characterized as low risk for the general population of healthy, properly managed agricultural animals. By this point in the animals’ growth the immune system is fully developed, and typically assisted by vaccination and parasite control practices. As previously noted, however, heifers are at greater risk of dystocia compared to older cows, largely because they are less than mature size at the time of their first calving. Although it is common practice to select sires with records of producing low birth weight calves (“calving ease”), dystocia continues to be a hazard for heifers. Dystocia is less of a concern in animals that typically bear multiple young, such as swine, as individual fetuses in multiple-fetus pregnancies are usually small compared to single births.
5. Post-Pubertal Maturation and Aging (Developmental Node 5)
Maturity and aging in food animal clones have not been studied extensively due to the relatively short time that cloning has been practiced. Common practice among conventional animals kept for breeding stock indicates that males may be kept to a later age than females, as they generally continue to be fertile for a longer period. Thus, highly valued males would continue in the herd as long as adequate quality semen was still being collected. When fertility of females declines, they are typically sold for slaughter, regardless of age. This decline in fertility generally occurs well before the animal shows other signs of aging or age-related disease.
a. Telomere Length as an Indicator of Aging
Studies have suggested that telomeres, long strands of repetitive DNA that “cap” the ends of chromosomes, are the “biological clock” that controls aging (Lanza et al. 2000, Betts et al. 2001). In all eukaryotic43 cells, the terminal ends of chromosomes are capped by short, repetitive sequences of noncoding DNA that are repeated up to many kilobases in length, in conjunction with specific binding proteins. Telomeres play a role in chromosome stability, protecting DNA from digestion by exonucleases (enzymes that attack the ends of chromosomes), facilitating attachment of chromosome ends to the nuclear envelope, ensuring proper segregation of chromosomes during replication, and ensuring the full replication of coding DNA during cellular divisions (Kuhholzer-Cabot and Brem 2002).
Although the DNA in chromosomes is generally double stranded along its length, the end of the chromosome, or the telomere, differs in that it consists of a single-stranded overhang (called a lagging strand) of variable length that forms a loop. Conventional DNA polymerases (enzymes that replicate DNA) cannot replicate the extreme 5' ends of chromosomes. Instead, these lagging strands are replicated in a series of fragments, rather than as a continuous strand. Each fragment is “primed” by a short sequence of RNA and the gaps between fragments are filled in by DNA polymerase. However, when the RNA primer at the furthest end of the lagging strand is removed, a small gap of un-copied DNA is left that is not filled in by the DNA polymerase. This leads to the loss of 50 to 200 base pairs each time the cell divides. For this reason, telomeres have been proposed to act as “mitotic clocks” that limit the capacity of cells to replicate through the single stranded region, which is interpreted as a DNA damage signal. The net effect is that at some critical telomere length, cell cycle progression is halted, and the cell becomes “replicatively senescent” or incapable of further division. Senescent cells remain viable and metabolically active for very long periods of time with minimal cell death (Schaetzlein and Rudolph 2005).
Telomeres appear to be longest in the nuclei of early stage embryos, and begin to decrease in length starting in the embryonic period. Early stage embryos and immortalized cells in culture appear to have the capacity to rebuild telomeres through the action of an enzyme known as telomerase (Betts et al. 2001, Xu and Yang 2001). Telomerase, the enzyme responsible for telomere replication and elongation, is active during embryogenesis, suppressed postnatally in most somatic tissues, but remains active in germ cells, tumor cells, and in a subset of stem/progenitor cells (as reviewed by Xu and Yang 2003, Schaetzlein and Rudolph 2005). The activation of telomerase appears to occur about the time when the genome becomes activated in the embryo: at approximately the 2-cell stage in mice, or the 8 to 16 cell stage in cattle (Betts and King 2001). The ability of SCNT embryos to rebuild telomeres may depend on species, the source of the donor nucleus, and culture conditions for early stage embryos (Betts and King 2001, Miyashita et al. 2002).
Concerns over genetic age and potential longevity of SCNT-derived animal clones were first raised after a report by Shiels and coworkers (1999) who noted that telomeres of “Dolly,” the first SCNT clone, were 10-20 percent shorter at one year of age than age-matched conventionally bred sheep (Shiels et al. 1999). Since that report, studies in animal clones have examined the effects of the nuclear transfer process on telomere length and telomerase activity to determine whether the SCNT process “resets” telomere length. Some early studies in cattle suggested that the SCNT process may influence cellular age and senescence. For example, Betts et al. (2001) noted reprogramming abnormalities affected telomerase activity in some early bovine SCNT embryos. In contrast, Cibelli et al. (1998a) cloned from a late-passage cell line (after 30 passages in vitro; the lifespan of cells in vitro is approximately 31-33 passages). At 40 days gestation, the fetus was harvested and a fibroblast cell line established. These fibroblasts appeared to have an extended lifespan compared to the original donor cells, and underwent another 31-33 passages in vitro.
Other studies suggest that reduction in telomere length may be more related to animal species, type of cells used to derive the donor cell line, or duration of time in culture (Shiels et al. 1999, Kuhholzer-Cabot and Brem 2002, Miyashita et al. 2002, Betts et al. 2005). Although telomere shortening may have led to a premature aging phenotype in telomerase-knockout mice (Blasco et al. 1997, Rudolph et al. 1999), convincing data on clones addressing the issue of premature aging are not currently available.
Telomere length variation has not been observed consistently across cloning studies or species. The group that produced “Dolly” stated that her telomeres were of the same length as the cultured mammary gland cells (from a six year old ewe) from which she was generated (Shiels et al. 1999). Dolly’s reduced telomere length was not associated with any other measurable signs of premature aging.44 Betts et al. (2001) noted that SCNT sheep generated from cultured embryonic or fetal cells had telomeres 10 -15 percent shorter than age-matched controls. Studies in cattle clones indicated that telomere lengths differ among tissues within an animal, and that DNA from some tissues were more amenable to telomere rebuilding, while DNA of nuclei from other tissues yielded clones with substantially shorter telomeres. For example, Miyashita et al. (2002) have reported that although clones derived from epithelial cells of a 13-year-old cow and clones derived from the oviductal epithelial cells of a six-year-old cow had telomeres shorter than age-matched controls, clones derived from muscle cells of a 12-year-old bull were similar to age-matched controls. Similarly, Kato et al. (2000) noted that telomere lengths in ear fibroblasts of a calf clone were similar to that of the 10-year-old nuclear donor bull, but telomeres in white blood cells of the same clone were similar to those of an age-matched control.
The telomere length of goat clones derived from fetal fibroblast donor cells were shorter than in those from age-matched control animals (Betts et al. 2005). These authors also noted that progeny from goat clones were found to have shorter telomere length in testicular biopsies compared to conventionally derived animals, and the telomere lengths were intermediate to the values obtained for their clone fathers’ and age-matched control testes (Betts et al. 2005). This suggests that there was incomplete telomere elongation in the offspring of clones, although as mentioned above it is uncertain whether telomere length is a predictor of longevity.
By contrast, the telomere length of sheep clones (Clark et al. 2003) and cattle derived from adult or fetal fibroblasts were comparable to conventionally bred cattle (Tian et al. 2000, Betts et al. 2001, Jiang et al. 2004) or even slightly increased when near senescent bovine fibroblasts were used for cloning (Lanza et al. 2000).
Using a slightly different technique for measuring telomere length, Meerdo et al. (2005) found no significant difference between blastocysts derived from adult bovine fibroblast cell lines and in vitro fertilization-produced blastocysts, but the clone blastocysts had longer telomeres than the two donor cell lines. They also noted detectable telomerase activity in oöcytes and a dramatic increase in telomerase activity at the morula stage. A second study in cattle and one in mice also demonstrated telomere elongation during the transition from morula to blastocyst in clone embryos (Schaetzlein et al. 2004). Cellular aging in tissue culture is also reflected by telomere shortening, and its reversal during SCNT was evident in a study by Clark and coworkers by the partial restoration of telomere length after nuclear transfer from late-passage cells (Clark et al. 2003). This and several other studies suggest that gametes have telomerase activity sufficient to lengthen the telomeres through the maturation process (Xu and Yang 2000, Betts et al. 2001, Meerdo et al. 2005).
Wakayama et al. (2000) evaluated successive generations of mouse clones for signs of premature aging and changes in telomere length in chromosomes from peripheral blood lymphocytes. Female mice were reiteratively recloned to six generations (i.e., Mouse G1 was derived from a somatic cell, Mouse G2 was cloned from a cell from Mouse G1, etc. for 6 generations) and four generations in two independent lines. The mouse clones (n = 35) showed no physical signs of increased aging, and behaved normally relative to age-matched controls as measured by tests of learning ability, strength, and agility. There also was no evidence of shortening of telomeres, as had been reported in some studies of livestock clones. In contrast, telomere length increased with successive cloning, although this finding may be confounded by age-related contributions or by characteristics of the donor cells (the cumulus cells used to produce the clones were found to express telomerase, suggesting that these cells may have long telomeres at the outset). They concluded that “telomere shortening is not a necessary outcome of the cloning process,” and suggested the possibility that the differences among the results observed in various species may be due to the selection of cells of longer or shorter telomere length in the different SCNT protocols. Clark et al. (2003) noted that fibroblast cell lines derived from fetal sheep clones had the same capacity to proliferate and the same rate of telomere shortening as the donor cell line from which the fetuses were cloned. This observation led King et al. (2006) to hypothesize that replicative senescence was under genetic control, and not triggered by a pre-determined telomere length.
Recently, Yonai et al. (2005) reported on the growth and production characteristics of six Holstein and four Jersey clones (described in detail in Chapter VI). These clones were derived from oviduct epithelial cells and had shorter telomeres than those observed in conventionally bred old cows (Miyashita et al. 2002). The overall success rate in terms of calf survival beyond the perinatal period was 4.8 percent for the Holstein group and 10.8 percent for the Jersey group. At the time of publication of their article all of these remaining clones had produced two calves and were artificially inseminated and had conceived for a third time. The authors concluded that “reduced telomere length did not influence productivity between birth and 3 years of age.”
Thus, although there have been reports of different telomere length outcomes in clones, at this time it is not possible to determine what the exact mechanism for telomere shortening is in clones, as studies have demonstrated that clones do have sufficient telomerase activity to return the shorter telomere lengths of the donor cells to lengths appropriate for normally developing embryos. Further, although some studies indicate that clones have shorter telomere lengths than would be expected, other clones have age-appropriate telomere lengths, and some appear to have longer telomeres. The most detailed study of clones with shortened telomeres indicates that the animals appear to be healthy and function normally. Finally, at this time, because most clones have not been alive for the full “natural” lifespan of their species, it is not possible to predict whether clones with shortened telomeres will exhibit premature aging.
C. Data on Animal Health by Species
1. Cattle
As mentioned above, the majority of available data on health of animal clones and their surrogate dams are derived from studies in cattle. Survival of live-born bovine clones from various studies is summarized in Table V-1. Because relatively few studies included contemporary comparators, historical data from various references and data bases were also incorporated into the table to provide context. For additional information on health and survival of calves derived by other ARTs, see Appendix C.
| Table V-1: Survival Rates of Live-Born Bovine Clones and Comparators | ||||
| Reference | Transgenic Status (proportion of animals in study) | Surviving/Total Live-Born Clones (fraction)1 | Surviving /Total Live- Born Comparators (fraction) | Comments |
|---|---|---|---|---|
| Batchelder 2005 | None | 2/8 (0.25) | 6/6 ET 3/3 AI (1.00) | |
| Chavatte-Palmer et al. 2002 | None | 21/21 (1.00) | 20/20 IVF 176/176 AI (1.00) | Described in Chapter VI |
| Chavatte-Palmer et al. 2004 | None | 36/58 (0.62) | NP | Update on animals generated since 1998, includes some animals from the 2002 publication |
| Cyagra 2003: Appendix E | None | 104/134 (0.78) | NP2 | Data from complete comparator birth cohort (animals surviving vs. animals born) not available |
| Edwards (unpublished) | None | 12/27 (0.44) | NP | See Appendix G for complete data |
| Gibbons et al. 2002 | None | 8/9 (0.89) | NP | |
| Gong et al. 2004b | None | 12/27 (0.44) | NP | |
| Green et al. 2007 | None | 9/9 (1.00) | NP | |
| Heyman et al. 2002 | None | 11/15 (0.73) | 20/25 (0.80) | IVF derived contemporary comparators |
| Heyman et al. 2004 | None | 35/50 (0.70) | 65/68 (0.93) | AI derived contemporary comparators |
| Hill et al. 1999 | All | 6/8 (0.75) | NP | |
| Hill et al. 2000a, 2001a | All | 1/2 (0.50) | NP | |
| Ideta et al. 2005 | None | 0/1 (0.00) | NP | |
| Kato et al. (1998, 2000) | None | 13/24 (0.54) | NP | An additional clone died between the perinatal period and 117 days of age (12/24 or 0.50 overall survival) |
| Kishi et al. 2000 | None | 3/4 (0.75) | NP | |
| Kubota et al. 2000 | None | 4/6 (0.67) | NP | |
| Lacham-Kaplan et al. 2000 | None | 2/2 (1.00) | NP | |
| Lanza et al. 2000 | All | 6/6 (1.00) | 5/5 (1.00) | IVF and ET derived comparators |
| Lanza et al. 2001 | All | 24/30 (0.80) | NP | |
| Lawrence et al. 2005 | None | 1/3 (0.33) | NP | The surviving calf died at 9 months due to clostridial infection (subcohort of Edwards above) |
| Matsuzaki and Shiga 2002 | None | 8/13 (0.62) | 7/7 (1.00) | IVF and AI derived comparators |
| Meirelles et al. 2001 | None | 1/1 (1.00) | NP | |
| Mello et al. 2003 | None | 1/1 (1.00) | NP | |
| Pace et al. 2002 | None | 82/106 (0.78) | NP | |
| Panarace et al. 2007 | None | 225/317 (0.71) | NP | Results reported for Cyagra operations in U.S., Argentina, and Brazil from 2000 to 2005; includes data from Cyagra 2003: Appendix E |
| Powell et al. 2004 | All | 5/8 (0.63) | NP | |
| Renard et al. 1999 | None | 0/1 (0.00) | NP | Case study on a clone of clone |
| Shiga et al. 2005 | None | 4/8 (0.50) | NP | One death associated with Akabane virus |
| Schurmann et al. 2006 | None | 6/9 (0.67) IVF-NT 3/4 (0.75) NT | 8/8 (1.00) | AI was used as a negative control for this study |
| Urakawa et al. 2004 | None | 8/9 (0.89) | NP | |
| Wells et al. 2004 | None | 104/133 (0.78) | 37/52 (0.71) | Table reflects survival to 3 months, due to unexplained differences in numbers at the beginning of later periods. Reports on number of calves delivered; unclear how many were stillborn. Comparators are progeny of clones |
| Wells et al. 2003b | Some | 22/31 (0.71) 11/24 (0.46) | NP | Non-transgenic (31 calves born alive) and transgenic (24 calves born alive) listed separately |
| Zakhartchenko et al. 1999b | None | 1/2 (0.50) | NP | |
| Hasler et al. 1995 | NA | NA | 361/428 (0.84) | Historical data on IVF derived beef calves in a commercial operation |
| Lombard et al. 2007 | NA | NA | 0.91 | Survey of three large dairies in Colorado; based on calves alive 24 hrs after birth |
| Nix et al. 1998 | NA | NA | 0.96 | Historical comparison from a university herd of beef cattle using AI |
| Schmidt et al. 1996 | NA | NA | 13/18 (0.72) | Calves produced by IVF. Two embryos transferred to each recipient, yielding 11 live-born twins and 7 singles; 4 twins dead by 14 days. One singleton dead by 14 days. |
| USDA/NAHMS 1997 (12/96 - 2/97) 6 | NA6 | NA | 0.97 | Historical data from beef cattle produced through AI and natural mating in commercial operations |
| USDA/NAHMS 2002 (1/02 - 12/02) 7 | NA | NA | 0.98 | Historical data from dairy cattle produced through AI and natural mating in commercial operations |
| Xu et al. 2006 | NA | NA | 457/458 (1.00) | Used sex-sorted, vitrified, IVF embryos |
| 1 Survivors through the Juvenile Period/Live births 2 NP = not provided; data not available 3 Beef calves; 4 Dairy heifers 5 NA = not applicable 6 http://nahms.aphis.usda.gov/ 7 Ibid Transgenic Status: All = All of the clones cited in the publication are derived from transgenic donor cells, Some = Some of the clones cited in the publication are derived from transgenic donor cells, None = None of the clones cited in the publication were derived from transgenic donor cells. IVF = in vitro fertilization AI = artificial insemination ET = embryo transfer | ||||
a. Developmental Node 1: Pregnancy and Parturition
i. Pregnancy
Most abortions in natural service and AI pregnancies in cattle remain undiagnosed due to the expense of laboratory work and the low profit margin in both the beef and dairy industry. Producers and veterinarians become concerned when the rate of abortion exceeds 3-5 percent in a herd. Many causative factors, both infectious (e.g., bacterial, protozoal, viral, fungal) and non-infectious (e.g., genetics, nutrition, stress, toxicity), have been identified (Merck Veterinary Manual Online 200545 ). Fetal losses later in pregnancy may be more common in goats and swine compared to cattle (Engeland et al. 1997, van der Lende and van Rens 2003, Vonnahme et al. 2002), and are not necessarily associated with disease (Engeland et al. 1997).
Farin et al. (2001) stated that up to 40 percent of pregnancy losses in cattle occur between days 8 and 18 of gestation. A recent study (Silke et al. 2002) indicated that most pregnancies are lost during the same period in dairy cattle, while a smaller percentage of pregnancies are lost between days 16 and 42 of pregnancy (late embryonic period). Total pregnancy loss in moderate to high yielding dairy cattle may be as high as 40 percent (Silke et al. 2002). Losses at later stages of pregnancy in cattle bred by AI are estimated to be less than 5 percent (Thompson et al. 1998). A study of beef heifers indicated that losses in the first days following embryo transfer are the most common (Dunne et al. 2000), with similar pregnancy rates at days 14 and 30 days of pregnancy, and at term (68 percent, 76 percent, and 71.8 percent, respectively). These early losses do not pose a hazard to the surrogate dam, and the net result is typically a longer than normal estrous cycle (Merck Veterinary Manual Online 200546 ).
Early embryo loss in other forms of ART may be related to in vitro culture conditions that may cause abnormal development and early embryo/fetal death. In a review of studies of in vitro produced (IVP) and clone bovine embryos, Farin et al. (2004) reported lowered pregnancy rates and increased rates of abortion associated with IVP.
Similar to other ART, by far the greatest loss of pregnancies resulting from SCNT embryos occurs prior to 60 days gestation in cattle (Le Bouhris et al. 1998, Hill et al. 1999 with transgenic clones, Kishi et al. 2000, Lanza et al. 2000 with transgenic clones, Chavatte-Palmer et al. 2002, Pace et al. 2002 using mixed transgenic and non-transgenic clones). High pregnancy losses during the time of placental formation suggest that embryonic death may be a consequence of faulty placentation, possibly due to a delay in chorioallantoic development, as proposed by Hill et al. (2000b) and Bertolini et al. (2004). Abnormal placentation may lead to a build up of wastes in the fetus and associated membranes, or inadequate transfer of nutrients and oxygen from the dam to the fetus.
Unlike AI and ET, IVP and SCNT pregnancy losses occur at all stages of gestation in cattle, although later term spontaneous abortion is more common in SCNT than IVP (Farin et al. 2006). Clone pregnancies have been lost during the second and third trimesters and have been accompanied by reports of hydrops (discussed in more detail in section 1.a.ii.), enlarged umbilicus, and abnormal placentae (Batchelder, 2005). Indeed, a major factor contributing to mid- and late-term spontaneous abortion of clones of both embryonic and somatic cell origin is abnormal development of the placenta (Wells et al. 1999, Farin et al. 2001, Chavatte-Palmer et al. 2002). Normal placental development is essential to ensure proper exchange of nutrients and gases between mother and fetus (Farin et al. 2001, Bertolini et al. 2004). Placental insufficiency has been cited as a possible cause of fetal loss in cattle, goats and swine bred by AI or natural mating (Lucy 2001, Engeland et al. 1997, Vonnahme et al. 2002). Studies have reported too few and/or abnormal cotyledons present in the placentae of sheep and cattle clones (Farin et al. 2001, Chavatte-Palmer et al. 2002, Heyman et al. 2002a, Lee RS et al. 2004, Batchelder 2005). Although fewer in number, these abnormal placentomes are found to be larger, weigh more, and comprise a greater surface area for exchange than “normal” placentomes. Enlarged placental surface area in IVP suggests an increase in substrate uptake and transport capacity (Bertolini et al. 2004).
Lee RS et al. (2004) noted pregnancy rates were similar between NT, AI, and IVP at 50 days gestation (65 vs. 67 and 58 percent, respectively), but from that point onward NT pregnancies were continually lost. By day 150, only 40 percent of NT embryo recipients were still pregnant. There were no losses during this time period for either AI or IVP pregnancies. Mean fetal weights at 100 days gestation were not different between the three groups; however, the authors noted that more NT fetuses were two standard deviations above the mean weight of AI fetuses (283 ± 2 g) compared to IVP fetuses (5/6 vs. 1/4). A similar trend was noted among fetuses examined at day 150. Fetal livers and kidneys were larger among NT fetuses compared to AI or IVP fetuses, and one liver and the kidneys from three NT fetuses were noted to have fatty infiltrations. Fatty liver was also diagnosed on post-mortem of one neonatal calf in the recent study by Chavatte-Palmer et al. (2004).
(a). Placental development
Few detailed descriptions of placentae of cattle clones exist. Lee RS et al. (2004) examined placentae of developing SCNT fetuses at 50, 100 and 150 days of gestation. These time periods roughly correspond to the periods before placentome formation is complete (50 days), shortly after complete placentome formation (100 days), and the period when hydrops may first be detected (150 days). The authors noted that at day 50, fetal cotyledon formation and vascularization initiated normally in NT fetuses, but fewer cotyledons successfully formed placentomes compared to AI and IVP control pregnancies. At day 50, 5/10 NT fetuses were noted to have very good vascularization of the cotyledons, compared to 2/5 AI and none of the IVP fetuses, which were said to have pale cotyledons. However, at day 100, the mean number of caruncles among NT pregnancies was lower than for either AI or IVP groups (58 ± 9 vs. 103 ± 15 and 99 ± 16, respectively). Although numbers of cotyledons were reduced in the NT group, total weight of caruncles was significantly higher in NT fetuses compared to the other groups at day 100, suggesting an attempt to compensate for lower numbers. The authors described NT placentomes as larger than AI or IVP placentomes, and having thicker, fist-shaped structures compared to AI or IVP placentomes, which were typically flat and discoid in shape.
Batchelder (2005) conducted a systematic histological exam of placentae collected at birth from seven cattle clones. She noted all clone placentae exhibited one or more abnormalities of varying severity: moderate to severe edema, enlarged vessels, adventitious placentation, and large areas devoid of placentomes. No abnormalities were noted for the comparator placentae collected (n=9). In general, clones had fewer (67.4 vs. 98.3) and larger placentomes (6.05 vs. 3.84 kg) compared to the pooled means for AI and ET comparators, and surface area of placentomes was greater and more variable in placentae of clones vs. comparators. The placenta of one clone contained two masses comprised of fatty and connective tissue with hair, but exhibiting no bone or organ development. These may have derived from embryos that failed to undergo complete differentiation, likely due to failure to completely reprogram the donor nucleus to a totipotent (able to become any tissue type) state (See Chapter IV). These may pose a potential hazard (metritis) to the dam if the fetal membranes are not completely expelled at termination of the pregnancy. In this study, all clones were delivered by planned C-section, and the placentae were manually removed.
Constant et al. (2006) noted that placental overgrowth preceded fetal overgrowth in bovine SCNT pregnancies with developing hydrallantois, and that the fetal to placental weight in SCNT pregnancies was lower than for AI or IVF pregnancies after day 220 of gestation. Constant et al. selected 18 SCNT pregnant surrogate cows demonstrating signs of hydroallantois for their study, and compared them with 10 normal AI and six normal IVF pregnancies. Pregnant cows were slaughtered between 180 and 280 days gestation, and fetuses and placentae removed and weighed. Recovered placentae were examined macroscopically and histologically. Differences between SCNT and control pregnancies were noted mainly in placentae and fetal tissues after gestation day 220 in this study. Although placentome number remained constant in AI and IVF pregnancies, placentome number in SCNT pregnancies tended to increase with gestational age. Within the SCNT group, Constant et al. identified two distinct populations: one in which placentome weight and number were within the range considered normal; and one group in which mean placentome weight was heavier than expected given the number of placentomes. Within placentomes from SCNT placentae, histologic examination revealed flattened uterine epithelia with small nuclei, rather than the expected raised caruncular tissue. Fetal connective tissue (the cotyledons) was enlarged even without evidence of edema, with reduced cell density and vessel dilation in some SCNT placentomes. Where edema was observed, it occurred around, but not within, placentomes. In this study, placental abnormalities and hydroallantois were linked to gestational age; however, there did not appear to be a direct relationship between hydrallantois and other placental abnormalities. It is important to note that only placentae and fetuses from pregnancies demonstrating hydrallantois were used in this study. Tissues from apparently normal SCNT pregnancies were not examined.
Failure of epigenetic reprogramming has been cited in numerous studies as a likely cause of early embryo failure and abnormal placental development for SCNT (see Chapter IV). Recent collaborations across laboratories suggest that improper epigenetic reprogramming may also lead to immune-mediated rejection of the developing SCNT fetus during early pregnancy (Hill et al. 2002, Davies et al. 2004). In conventional bovine pregnancies, placental trophoblast cells (cells that interface with the uterus) express antigenic proteins, referred to as major histocompatability complex (MHC) class I, during the third trimester of pregnancy, and may be involved in the process that leads to the normal expulsion of the placenta following birth of the calf. Work by Davies et al. (2004) indicated that the trophoblast cells of SCNT embryos expressed MHC class I proteins during the first trimester, between 34 and 63 days gestation. These workers also noted accumulation of lymphocytes within the uterine lining, indicating an inflammatory response, in pregnancies where embryos that were immunologically incompatible with their surrogate dams were aborted. Conversely, embryos that were compatible with their surrogates’ immune systems survived to term.
Hashizume et al. (2002) noted differences in the expression of placental lactogen (PL)47 and pregnancy-associated glycoprotein (PAG)48 between Japanese Black cows bred by AI vs. cows carrying SCNT pregnancies at 60 days of gestation. Pregnancy was diagnosed by ultrasound between days 30 and 60 of gestation, and fetal viability confirmed by presence of a heartbeat. Confirmed pregnant cows were slaughtered around day 60 of gestation, and uteri and fetal tissues removed for examination. The differences in gene expression were most notable among cows with immotile NT fetuses, which had low or non-existent expression of these pregnancy related genes. It is likely that these pregnancies would have failed spontaneously if allowed to proceed. These SCNT placentae also exhibited poor caruncular development as well as cotyledonary development with respect to both shape and number of these structures compared to placentae from AI derived pregnancies. In addition, the cotyledonary area was pale and flattened in SCNT placentae compared to AI placentae, which had the expected thickened cotyledons with well developed villi (finger-like structures which interlock and increase the surface area across which fluids and gases flow). The number of SCNT cotyledons was approximately half that of AI placentae in the Hashizume et al. study.
Similarly, Chavatte-Palmer et al. (2006) noted reduced fetal crown-rump length and placentome width in clones compared to AI and IVP controls at all stages of pregnancy. Concentrations of PAG were significantly lower in clones with early pregnancy loss (between days 35 and 90 of gestation) compared to clones with pregnancies that survived to a later date, although there was no difference in PAG levels between clones that died later in gestation versus those that were born alive.
In contrast to the Hashizume et al. study, Bertolini et al. (2006) found increased levels of PL and pregnancy-specific protein B (homologous with PAG-1) in placentomes from IVP fetuses compared to in vivo ET fetuses in pregnancies terminated at 180 days gestation. Interestingly, for the production of IVP embryos, Bertolini et al. utilized a culture system intended to induce LOS pregnancies, which contained 10 percent fetal calf serum and a mixture of follicle-stimulating hormone, luteinizing hormone, and estradiol 17ß.
The underlying cause(s) of the higher rate of pregnancy failure and placental abnormalities in SCNT compared to IVP may be related to a number of factors or combination of factors, including the culture media (Thompson 1997; van Wagtendonk-de Leeuw 2000; Gardner and Lane 2005), selection of the oöcyte (Greeve and Calleson 2005, Roberts et al. 2006), and selection of the donor cell for nuclear transfer (Wells et al. 2003b, Ideta et al. 2005, Lawrence et al. 2005). For both IVP and SCNT, oöcytes are generally subjected to a period of maturation in vitro, which by-passes the natural developmental processes in vivo49 , and may result in the selection of oöcytes that are not capable of sustaining normal embryo/fetal development (Greve and Callesen 2005). Even for non-nuclear transfer embryos, Farin et al. (2004) noted substantial differences in developmental competence of in vivo vs. in vitro matured oöcytes, with blastocyst formation ranging from 50 to 80 percent for in vivo matured versus 15 to 40 percent for in vitro matured oöcytes.
(b). Cell selection and cell culture
Wells et al. (2003b) noted that survival rates to term differed depending on cell cycle of the nuclear donor cells. Putative G0 cells (cells that apparently were not dividing) used for nuclear transfer had high early pregnancy losses, but no losses after 120 days of gestation, and no reported hydrops. Cells that had begun to divide (G1 phase) had higher losses to term (21/43 pregnancies lost after 120 days gestation) and higher incidence of hydrops (18/43 (42 percent) of pregnancies), but higher post natal survival than clones from G0 cells.
Similar to Wells et al. (2003b) findings, Lawrence et al. (2005), found that when somatic cells were “starved” into quiescence by serum deprivation prior to nuclear transfer, more surrogate heifers were confirmed pregnant compared to heifers that received embryos from the serum-supplemented group. However, in contrast with the findings of Wells et al., embryo loss in the Lawrence et al. study was higher in the serum-starved group compared to the serum-supplemented group between 25 and 50 days post-transfer of embryos. Eight of nine pregnancies in the serum-starved group failed during this early period of development, with the ninth pregnancy failing later in gestation. Although early pregnancy losses were lower in the serum-fed group, (4/11 embryos failed during the period up to 50 days post transfer), later gestational losses beginning on day 174 of gestation resulted in only one viable Jersey calf from this group. Six of the remaining pregnancies resulted in hydroallantois, with fetal deaths occurring between 174 and 255 days. The surviving clone was reported as healthy until nine months of age; two pregnancies were terminated by emergency C-section at 260 and 271 days, but these calves did not survive beyond one week of age. The surviving calf died at nine months of age (see Section 2.c.i).
Fetal muscle cells were serum-starved for one, four, or five days prior to NT to induce different stages of differentiation in a recent study by Green et al. (2007). Three muscle cell types developed in vitro were frozen, thawed, and serum-starved for an additional five days prior to NT. Nine apparently healthy calves were born: four from myogenic precursor cells, two from myotubes, and three from muscle fibroblasts. All calves were euthanized prior to weaning, and no additional data is provided on them. The reason for euthanasia was not provided. The authors only state that the calves were euthanized for “unrelated reasons.”
In contrast to the Wells et al. study, Urakawa et al. (2004) reported success using fetal fibroblast donor cells in the G1 phase. Two cell lines were used, derived from fetuses with the same dam but two different bulls. All embryos that survived to = 6 cells (day 3) continued to develop to the morula/blastocyst stage by day 6. Ten of these blastocysts were transferred into ten recipients, resulting in nine live calves. According to the authors, calving was “uneventful.” Differences were noted between cell lines, in that three calves resulting from one of the lines tended to be heavier at birth than the six calves of the other cell line used (actual birth weights not provided). One of these three heavy-weight calves died after two days without standing. The authors do not report on the health or survival of the remaining eight calves beyond the first six days of life.
Similarly, Ideta et al. (2005) compared development of embryos constructed with G1 or M phase (the period in the cell cycle when cell division takes place) fetal fibroblasts, and noted that G1 SCNT embryos had higher rates of development to blastocyst than M phase cells (31 vs. 16 percent). Although these results are considerably lower than those noted in the Urakawa et al. study, the numbers are calculated based on total number of embryos cultured prior to first cleavage, whereas the Urakawa et al. study calculated development based on embryos surviving the first three days in culture. Only five surrogate cows received embryos in the Ideta et al. study, of which three were diagnosed pregnant on day 30 of gestation, and one live calf was delivered. All of the transferred embryos were developed from G1-phase somatic cells. The single calf died two days after birth. Health of the surrogate dams, method of delivery, and birth weight of the single calf was not reported in this study.
Another study (Schurmann et al. 2006) investigated whether enucleation after IVF would enhance the survival of SCNT derived embryos. In this study, male and female pronuclei and the zona pelucida of the single-cell zygote were removed approximately four hours after IVF, and nuclear transfer performed using adult ear skin fibroblasts. In vitro development to blastocyst was similar between the controls, derived from unfertilized oöcytes, and the early zygote group. Post-implantation development was higher in the group derived from early zygotes or “sperm activated” embryos. More transferred blastocysts survived to establish pregnancies at 35 days gestation in the sperm-activated group compared to controls (30/49 vs. 17/41), and numerically more pregnancies resulted in live calves at birth in the sperm activated group (9/30 vs. 4/17), although this difference was not significant. Survival of calves to weaning was low for both groups, and did not appear to be different between groups (6/9 vs. 3/4).
ii. Parturition
(a) Hydrops
The set of conditions generally termed hydrops refers to abnormal fluid accumulation (edema) in one or more compartments of the placenta and/or the fetus itself, and are variously referred to as hydroallantois, hydrallantois, hydramnios or hydrops fetalis, depending on where the edema occurs (Heyman et al. 2002a,b, Merck Veterinary Manual Online 200550 , Pace et al. 2002 (including transgenic clones)). Hydrops is estimated to occur in 1 in 7,500 pregnancies in the general population of cattle (Hasler et al.1995). The incidence is higher in cattle and sheep recipients of IVP embryos, with one study estimating a rate of approximately 1 in 200 in IVP pregnancies in cattle, or 0.05 percent (Hasler et al. 1995). However, a more recent but smaller study (Block et al. 2003) reported 1/28 (four percent) IVP pregnancies resulted in hydrops. Reports of hydrops in SCNT pregnancies are highly variable, and range from 13 to 40 percent (Table V-2). One research center (AgResearch, NZ), reported that on average 42 percent of SCNT-bearing surrogate cows pregnant at 120 days gestation will experience pregnancy failure, and that 58 percent of these mid- to late-term failures may be attributed to hydrops. This indicates an average hydrops incidence of 24 percent for this laboratory (Forsyth and Wells 2006).
Table V-2 presents a summary of reports of hydrops in cattle from the peer-reviewed literature for clone, IVP, ET, and AI pregnancies. Survival rates of dams developing hydrops generally were not reported. Most studies that discussed outcomes indicated that dams developing hydrops were euthanized.
| Table V-2: Incidence of Hydrops in Cattle Surrogate Dams | ||||
| Study | Transgenic Status | Incidence (fraction) in clone pregnancies | Incidence (fraction) in comparator pregnancies | Comments |
|---|---|---|---|---|
| Batchelder 2005 | None | 1/8 (0.13) | 0/6 (0.00) | Comparators were ET (n=6) and AI (n=3) |
| Block et al. 2003 | None | NA | 1/28 (0.04) | Study used frozen/thawed IVP embryos |
| Edwards (unpublished) | None | 27/46 (0.59) | NP | |
| Hasler et al. 1995 | NA | NA | 1/200 (0.005) | Study based on commercial IVP operation |
| Heyman et al. 2002a,b | None | 3/20 (0.15) 5/21 (0.24) | 0/24 (0.00) | IVP comparators |
| Hill et al. 1999 | All | 2/8 (0.25) | NP | |
| Lawrence et al. 2005 | None | 6/11 (0.55) | NP | Sub-cohort of Edwards |
| Lee RS et al. 2004 | None | 2/8 (0.25) | 0/9 (0.00) | 4 IVP and 5 AI comparators. A third clone fetus was suspected of developing hydrops. All pregnancies terminated at gd 150. |
| Matsuzaki and Shiga 2002 | None | 2/13 (0.15) | 0/7 (0.00) | 2 IVP and 5 AI comparators |
| Mello et al. 2003 | None | 1/3 (0.33) | NP | |
| Pace et al. 2002 | Some | 30/178 (0.17) | NP | Pregnancies lasting beyond 60 days |
| Panarace et al. 2007 | None | 24/37 (0.65) | 0/13 (0.00) 0/10 (0.00) | 13 ET and 10 IVP comparator pregnancies |
| Wells et al. 2003b | Some | 18/43 (0.42) 1/6 (0.17) | NP | Pregnancies lasting beyond 120 days. Non-transgenic (n=43) and transgenic (n=6) listed separately |
| Zakhartchenko et al. 1999a | None | 2/5 (0.40) | NP | |
| NA = not applicable NP = not provided; data not available Gd = gestation day or day of pregnancy | ||||
Not all cases of hydrops in clone-bearing pregnancies develop into a significant complication or threat. In an interview with CVM staff (see CVM Memorandum I at http://www.fda.gov/cvm/CloningRA_Memorandum_I.htm), U.S. clone producers indicated that many pregnancies result in some excess fluid accumulation in the fetal membranes and tissues.
In most cases this accumulation is mild or moderate, and does not threaten the surrogate dam or calf. The producers interviewed for this assessment indicated that they monitor surrogate dams closely, beginning as early as 150 days of gestation, for any signs of developing hydrops. They indicated that if the veterinarian determines that hydrops is sufficiently severe to threaten the surrogate, the pregnancy is terminated. In contrast, Forsyth and Wells (2006) indicated that in their experience at AgResearch in New Zealand, only the mildest cases of hydrops result in normal calves, and it is now their practice to terminate any pregnancy as soon as hydrops is diagnosed.
A few studies have directly compared cloning procedures with other ART under the same conditions. These studies are limited, with few clones and often fewer comparators from alternative technologies (Heyman et al. 2002a,b, Matsuzaki and Shiga 2002, Lee RS et al. 2004, Batchelder 2005). In one such study, Matsuzaki and Shiga (2002) compared 13 SCNT clones with five AI and two IVP-derived calves used as controls. Five of the 13 clones required delivery by Caesarian section (C-section), while all seven controls were delivered without assistance. Two cows carrying clones had to be induced at 250 days gestation due to rapidly expanding hydroallantois, and the calves were delivered by C-section.
Batchelder (2005) indicated that the largest clone in that study (weighing 71.0 kg at birth) exhibited edema at birth, particularly in the head and neck, suggesting that it suffered from mild hydrops fetalis. This calf was successfully delivered at term by planned C-section, although it died three days after birth. This calf’s surrogate dam apparently was unharmed by the complication, although another surrogate dam was euthanized at 211 days gestation due to severe hydrops.
In one of the largest cattle cloning studies reported, Pace et al. (2002) estimated that approximately 6 percent (30/535) of all pregnancies established with SCNT embryos resulted in hydrops, but among pregnancies with clones that lasted beyond 60 days, the incidence of hydrops was 17 percent (30/178). An important consideration in interpreting these outcomes, however, is that approximately 75 percent of the embryo clones in this study were transgenic. Heyman et al. (2002) observed that 3 of 20 (15 percent) recipients of fetal and adult SCNT embryos (non-transgenic) developed severe hydroallantois during the time from approximately six months of gestation to term. In another trial reported in the same paper, five cases of late
abnormal pregnancies were detected among 21 SCNT recipients (24 percent) by repeated ultrasonography, and the recipients were euthanized between day 155 and 233 of gestation. Severe hydroallantois was confirmed at necropsy and the size of the placentomes from these pregnancies was measured (142.3 ± 61.7 g vs. 46.7 ± 22.7 g for controls). No abnormalities were reported among the IVF-derived pregnancies in the Heyman et al. 2002 study.
Similarly, a study by Wells et al. (2003b) reported a high rate of pregnancy loss of non-transgenic bovine fetal fibroblast clones after 120 days gestation, with hydrops cited as the cause of pregnancy loss in 86 percent (18/21 losses) of the cases.
Panarace et al. (2007) monitored 37 clone-bearing, 13 ET, and 10 IVP pregnancies by ultrasound, and reported a high incidence of hydrops fetalis (13/37 pregnancies), placental edema (21/37 pregnancies), and hydroallantois (5/37 pregnancies) among clone-bearing pregnancies. Most of the hydrops fetalis cases (12/13) also developed placental edema. Most of these complications resulted in loss of the pregnancy. Only one clone experiencing hydrops fetalis and three clones with placental edema were born alive in this study; only two of these four calves survived. The authors do not report the outcome for the surrogate dams. None of the ET or IVP comparators developed hydrops in this study.
Lee RS et al. (2004) examined survival and development of AI, IVP and SCNT fetuses at 50, 100 and 150 days of gestation. Although there were no significant differences in fluid volume of fetal membranes at day 50 or 100, total fetal membrane fluid volume was significantly higher in SCNT (n = 8) fetuses compared to IVP (n = 4) fetuses (8033 ± 1800 ml vs. 5088 ± 698 ml) at 150 days gestation. For AI fetuses, mean fetal membrane fluid volume was 6500 ± 444 ml. The study noted the high variability in membrane weights and fluid volume among clone fetuses, and stated that 2/8 SCNT fetuses examined had particularly high allantoic fluid volumes (20 and 12 L), which were largely responsible for the high mean fluid volume among clones. The authors stated that these two cases indicated developing hydrops. The authors suspected a third SCNT fetus was developing hydrops, but did not provide data on this case. Fluid volumes were less variable among membranes of AI and IVF fetuses.
In contrast, hydrops has only been detected in one or two cows out of 250 to 300 transgenic clone-bearing surrogate cows, as reported in discussions with U.S. clone producers, suggesting that these results vary considerably among labs performing animal cloning (see CVM Memorandum I at http://www.fda.gov/cvm/CloningRA_Memorandum_I.htm). The producers also noted that hydrops occurred in IVP-derived pregnancies, but less frequently than with clone-bearing pregnancies, although no actual numbers were available.
The causes of hydrops in conventional animals are unclear. Liu and Wintour (2005) stated that hydramnios can result from abnormalities in fetal renal function, and that the large volume of dilute urine produced by the fetal kidney is essential for the maintenance of normal amniotic and, in some species, allantoic fluid volume. Conversely, Constant et al. (2006) concluded fetal abnormalities noted in NT pregnancies developing hydrops were the consequence of placental dysfunction. Although actual cause and effect is not yet clear, it appears that placental function is integral to the development of LOS and its related clinical signs, particularly hydrops and congenital kidney defects. Lee RS et al. (2004) also suggested that the association between excessive fetal fluid accumulation and renal and placental growth deregulation may indicate impairment of renal and placental function. “Although the placenta is the major organ regulating the fetal environment, the fetal kidney also plays an important role in the regulation of fetal arterial pressure, fluid and electrolyte homeostasis, acid base balance, and hormone synthesis. In ruminants, fetal urine contributes to the allantoic and amniotic fluid. Reports have appeared of kidney defects and impaired renal function in cloned offspring as well as impaired liver function in cloned mice…”
Although it is likely related to placental insufficiency, not all abnormal placentae develop hydrops (Hill et al. 2001b). In SCNT, incomplete or improper epigenetic reprogramming and subsequent inappropriate gene expression may be an important factor in placental development and hydrops (see Chapter IV).
(b) Dystocia
Dystocia, o difficult labor, is an identified hazard for any pregnancy that goes to term. A common cause of dystocia is incompatibility between the size of the fetus and the pelvic opening through which it must pass. Although oversized offspring occur in all species, it is more common in animals that typically produce only one or two offspring per pregnancy. Other causes of parturition difficulty include malpresentation of an individual fetus (e.g., breech birth, head or leg out of position), or simultaneous presentation of multiple fetuses in the birth canal. Severe dystocia may increase the risk of retained fetal membranes and metritis (uterine infection), and cause damage to the reproductive tract, including uterine adhesions, uterine rupture and uterine prolapse, and nerve and musculo-skeletal damage (Merck Veterinary Manual Online 200551 ). Such complications could compromise future reproductive capability and result in culling of the dam. Another risk is that dystocia may lead to an emergency C-section. Complications of emergency C-section surgery may include uterine tearing, peritonitis, infected suture line, incisional hernia, and respiratory and circulatory compromise from anesthesia and recumbancy. Stress of labor is also a complicating factor in the case of emergency C-section.
Previous estimates of dystocia in natural and AI-derived bovine pregnancies range between 4 and 6 percent. Nix et al. (1998), in a large study of 2,191 births of natural and AI bred beef cattle at Clemson University reported that 6 percent of births required assistance. Calf birth weight and parity of dam (number of times she had given birth) were the major factors in the incidence of dystocia. Calves heavier than 40 kg were associated with greater calving difficulty. Heifers were more likely to experience dystocia, despite the common practice of selecting sires known to produce smaller calves. Dystocia contributed to the increased neonatal mortality of the calves and decreased reproductive performance of the dams in this study. In another large study that evaluated dairy cattle, 6.3 percent (1,749/27,713) of pregnant cows experienced dystocia (Lucy 2001). USDA estimates the mean dystocia risk in the general cattle population at 4 percent of pregnancies (USDA/NAHMS 199752 ).
A more recent study of three large dairies (herds ranging between 1,000 and 5,000 cows) in Colorado indicated the overall incidence of severe dystocia (requiring assistance) at 10.9 percent for all cows on these dairies (Lombard et al. 2007). Again, calf birth weight and parity of dam were important factors in incidence of dystocia, with incidence among heifers (18.9 percent) higher than for multiparous cows (6.9 percent). This study indicated that incidence of dystocia, stillbirth and calf mortality associated with dystocia appeared to be increasing compared to previous reports. However, the report also suggested that large herd size and other management factors, such as training of farm personnel, were also factors in the increased incidence of stillbirth and later calf mortality.
Rates of dystocia in surrogate dams carrying clone pregnancies are difficult to determine as clone producers have often elected to deliver clones via planned C-section as part of their animal care protocol (Wells et al. 1999, Lanza et al. 2000 using transgenic clones, Gibbons et al. 2002, Batchelder 2005). Planned C-section deliveries are associated with decreased parturition risk, and in most cases the surrogate dam recovers without ill effects. Although this does not eliminate the risk associated with giving birth, particularly in the event of hydrops, very few surrogate dams are lost, and most recover normally. The relationship between cloning, large offspring syndrome (LOS), and dystocia will be discussed in greater detail in the next section and in section 2.b.i.a.
(c) Large Offspring Syndrome
Large Offspring Syndrome (LOS) (Table V-3) has been described as occurring at a relatively high frequency in clone-bearing pregnancies, and at a lower frequency in cattle derived from IVP and ET pregnancies, and in some cases may be related to the development of hydrops (Kruip and den Daas 1997; Chavatte-Palmer et al. 2002). This syndrome will be discussed in greater detail in Section 2. b. i. a., as it also has implications for the health and survival of the newborn animal. For the surrogate dam, LOS increases the likelihood of dystocia, frequently requiring human intervention to remove the calf vaginally, or by C-section, due to the inability of the dam to expel the calf without assistance. Reported incidences of LOS in peer-reviewed publications on cattle clones have ranged from as low as 1/12 (8.3 percent) (Miyashita et al. 2002) to as high as 12/24 (50 percent) (Kato et al. 2000). Average birth weight of clones (some transgenic) of various cattle breeds (Holstein, Brown Swiss, Angus and Holstein x Jersey crossbreds) in the Pace et al. (2002) study was 51 ± 11 kg, with 54/106 (51 percent) live-born calves weighing more than 50 kg at birth. Given the inability to distinguish between transgenic and “just clone” pregnancies in the Pace et al. study, it is difficult to put these numbers into context with other studies of non-transgenic clones. Average birth weight of calves produced by AI or natural service varies depending on breed, and may range from 30 kg in small breed cattle to 45 kg or more in large breed cattle (NAS 1996b).
(d) Other complications
Although other complications associated with SCNT pregnancies have been noted, potential interactions with transgenic manipulation of the donor cell and predisposing conditions in the surrogate dam make it difficult to ascribe the complications exclusively to the cloning process. For example, the ketonuria53 and fatty liver associated with ketosis and “fat cow syndrome”54 described by Hill et al. (1999) are not only confounded by the existing obesity of the surrogate dams at the time of diagnosis, but also by the transgenic nature of the fetal clones. Cows that are obese at calving are most likely to develop fatty liver, and cows that develop fatty liver at calving are most susceptible to ketosis. Fatty liver can occur whenever there is a decrease in feed intake and may be secondary to the onset of another disorder. Obesity in late-gestation cattle is a commonly reported problem resulting in anorexia (due to reduced gut capacity), ketosis, fatty liver deposits, and hepatic insufficiency in pregnant cattle (Merck Veterinary Manual Online 200555 ).
Wells et al. (1999) noted weak or non-existent uterine contractions, poor mammary development and failure to lactate in cattle carrying fetal clones. Actual incidence of these complications is not known, but all have been reported in sheep (Ptak et al. 2002) and failure to lactate was noted in swine surrogate dams (http://www.fda.gov/cvm/CloningRA_Memorandum_I.htm).
iii. Unpublished Data
J.L. Edwards’ laboratory at the University of Tennessee submitted a three page table to CVM summarizing the outcomes of cloning studies in Jersey cattle conducted between 2000 and 2003 (Appendix G.) The table provides data on all five developmental nodes for clones and their surrogate dams, and summarizes the outcomes of 47 late second- and third-trimester pregnancies.
Twenty-five pregnancies (53 percent) were delivered by C-section, and five surrogate dams (11 percent) required assistance during vaginal delivery. Twenty-seven pregnancies (58 percent) resulted in hydrops, with 10 surrogate dams (37 percent of hydrops cases) euthanized as a result of this condition. The reasons for the unusually high incidence of hydrops in these surrogate dams have not been identified. In a teleconference with Dr. Edwards, she indicated that most of the C-sections were planned as part of a timed labor induction protocol introduced after several surrogate dams developed rapid onset of hydrops.
b. Developmental Node 2: Perinatal Period
i. Peer-Reviewed Publications
One factor that makes analysis of survival data on clones difficult is the difference in how stillbirth is interpreted between studies of clones compared to studies in the general population of cattle. Studies on cattle cloning reviewed for this risk assessment defined stillbirth as calves born dead. Conversely, studies on conventionally bred dairy cattle typically define stillbirth as death within 24, and sometimes 48, hours of birth (Lombard et al. 2007). This may be attributed to different methods of management between clone producers and conventional cattle operations, especially beef cattle. Due to the expense of generating clones, surrogates bearing clones are likely more closely monitored than dams in commercial cattle breeding operations. Also, in most of the studies on clones reviewed for this risk assessment, labor was induced labor and, in many cases, clones were delivered by C-section. Thus, personnel were present when clones were born, and status at time of birth could be recorded. Under more conventional breeding practices, onset of labor is spontaneous, and neonates are frequently delivered unaided and unobserved.
In the general population of cattle and sheep, neonatal death rates are typically low. Overall, the estimated death rate of beef calves within 24 hours of birth (including stillbirths) was 3.4 percent (USDA/NAHMS, 199756 ). Nix et al. (1998) found that dystocia affected calf mortality within the first 24 hours, with mortality rates increasing with increasing severity of dystocia. Overall calf mortality attributed to dystocia was 4.5 percent of all calvings in this study (2,191 births). A more recent study (Lombard et al. 2007) indicated that calf mortality among dairy heifer calves, including stillbirths, was increasing (13 percent) compared to earlier studies. Dystocia was the most influential factor on calf mortality in these studies, due to trauma of difficult labor and emergency C-section. Dystocia was also associated with high calf morbidity (illness) in a study of 2,490 beef cattle herds (Sanderson and Dargatz 2000), and in the Lombard et al. study of three large Colorado dairies.
Among dairy replacement heifers, the highest losses occur during the first week of life (1.8 ± 0.3 percent deaths for all heifer calves born alive). In these heifers, the most commonly reported illnesses were due to respiratory problems and scours (diarrhea), with incidence of these illnesses peaking during the first two weeks of life (USDA/NAHMS 199457 ).
Because the number of animal clones available to study is small, it is difficult to draw conclusions on rates of morbidity and mortality of live-born clones. However, some trends appear to be common across most of the studies reviewed. Early reports, beginning in 1998, of clone mortality rates were 50 to 80 percent (reviewed by Solter 2000). Survival rates have improved in some recent studies, with mortality during the first month of life of approximately 18 percent (21/117; Pace et al. 2002 for a cohort of mixed transgenic and non-transgenic clones) and 20 percent (6/30; Lanza et al. 2001 for a cohort of transgenic cattle), with most of the deaths occurring during the first 48 hours postpartum. Similarly, data supplied by Cyagra, Inc. (2003) indicate 22 percent mortality in the first 48 hours (30/134) among non-transgenic clone calves born between 2001 and 2003. A more recent study from this group (Panarace et al. 2007) which covered Cyagra operations in the US (including data submitted directly to CVM), Argentina, and Brazil between 2000 and 2005, indicated a 12 percent mortality rate among live-born calves in the first 24 hours after birth. (For a summary of survival rates among live-born bovine clones, see Table V-1.)
(a) Large Offspring Syndrome
Large Offspring Syndrome (LOS) has been described in calves and lambs produced by ET, IVP, BNT, and SCNT. References describing this syndrome in the following section include descriptions of abnormalities noted for any of these ARTs. As the name indicates, the most readily recognized sign is oversized fetus or newborn, characterized as having a birth weight greater than 20 percent above the average birth weight for that species, breed, and sex. Dystocia and related morbidity and mortality of the young animals are common in cases of LOS when C-sections are not planned. Mortality rates for LOS calves can be high (Behboodi et al. 1995, Farin et al. 2001, Farin et al. 2004, Lee RS et al. 2004). A description of clinical signs associated with LOS is provided in Table V-3. A summary of incidence and survival rates of calves born with LOS and related clinical signs are in Table V-4. Survival of LOS calves is highly variable, and appears to depend on severity of the clinical signs and neonatal management practices. Studies that included such data indicated that survival ranged from 0 to 88 percent of calves diagnosed with LOS.
| Table V-3: Clinical Signs Associated with Calves Displaying Large Offspring Syndrome (LOS) |
| Fetal size > 20% above average for species/breed Slow to stand Inability to thermoregulate Weak or absent suckle reflex Large umbilicus with patent blood vessels Deformities of limbs (tendon contracture) and /or head Disproportionate or immature organ development Increased susceptibility to infection Respiratory signs: insufficient lung surfactant, failure of lungs to inflate Cardiovascular signs: patent ductus arteriosis, enlarged heart /ventricle, septal defects Kidney defects (hydronephrosis) Hepatic congestion/fatty liver Hydrops |
Stress associated with dystocia, prolonged labor and emergency C-section birth is a risk factor for large calves, regardless of how they are produced (Kato et al. 1998, Kubota et al. 2000, Lombard et al. 2007, Panarace et al. 2007). Lombard et al. (2007) noted that calves born to conventionally bred dams with mild or severe dystocia were at increased risk of hypothermia, respiratory and digestive problems, failure of passive transfer of immunity, and death. Matsuzaki and Shiga (2002) reported that SCNT clone calves born by emergency C-section had a higher mortality rate (4/5) compared to clone calves that were delivered vaginally (1/8). It is not clear whether the higher mortality is entirely due to the emergency surgery or whether adverse factors in the clones themselves contributed to the mortality.
Congenital abnormalities that may be related to fetal oversize include deformities of limbs and head, and may be a function of crowding in the uterus (Meirelles et al. 2001, Zakhartchenko et al. 1999a, Hill et al. 1999 with transgenic clones, Garry et al. 1996 with BNT clones). Intrauterine infections may also be responsible for some of these abnormalities (Kato et al. 2000, Kubota et al. 2000). LOS includes a large number of abnormalities, only some of which may be directly related to dystocia and congenital effects of unusually large size.
Some clinical signs associated with LOS also may be the result of perinatal hypoxia (oxygen deprivation) and metabolic acidosis (blood pH < 7.4) due to dystocia (Vaala and House 2002). Hypoxia during dystocia is due to premature separation of the umbilicus or other interruption in flow of oxygenated blood from the dam to the fetus. Hypoxia increases the production of lactic acid, which results in metabolic acidosis. Mild acidosis is expected in neonates, and is usually transient, but dystocia and the accompanying hypoxia result in prolonged and pronounced acidosis. The associated clinical signs include delayed time to stand; weak suckle reflex; delayed transition from fetal to neonatal circulation; pulmonary hypertension; inadequate lung surfactant production; hypothermia; impaired hepatic function; altered glucose metabolism; and poor absorption of immunoglobulins from colostrum. These clinical signs are directly associated with dystocia, and occur in conventionally bred calves as well as calves resulting from ART and clones, when delivery is difficult or prolonged (Vaala and House 2002).
Other abnormalities reported to coincide with LOS include respiratory, cardiac, hepatic, renal, umbilical, and immunologic problems, and may occur even among animals with birth weights within the normal range for their breed. These abnormalities may result from dysregulation of developmentally important genes rather than the uterine environment (see Chapter IV), or may be the result of placental abnormalities, as discussed in Section 2.C.i.a. Systemic abnormalities including organ dysfunction result in morbidity and often result in high mortality. Pulmonary abnormalities include immature lung development, insufficient lung surfactant, and failure of the lungs to inflate. Cardiovascular abnormalities include patent ductus arteriosus and ventricular defects (Table V-3).
i. Potential Sources of LOS
In vitro culture conditions are suspected to contribute to development of LOS in IVP-derived embryos (Farin and Farin 1995, Farin et al. 2001, Bertolini et al. 2002a,b). Various culture systems used in different laboratories often use slightly different media ingredients,58 such as fetal calf serum (FCS), bovine serum albumin (BSA), and serum derived from cows in estrous (ECS), and may expose developing embryos to hormones and growth factors that may not be in appropriate concentrations for the stage of development, possibly contributing to gene dysregulation (Sinclair et al. 1999, Gardner and Lane 2005).
Lazzari et al. (2002) noted differences in the expression of several developmentally important genes between IVF-derived blastocysts cultured with BSA and blastocysts recovered from cows bred by AI, including the IGF-I and –II receptors, fibroblast growth factor, and several genes important for the transport of glucose.
Farin and Farin (1995) compared bovine IVP embryos cultured in mixed media containing 10 percent ECS and other hormones for seven to eight days with embryos fertilized in vivo and collected and transferred on the same day via embryo transfer (ET). Pregnancy rates 53 days after transfer were higher for heifers (a cow that has not yet produced her first calf) receiving ET (15/19 embryos transferred; 79 percent) compared with IVP embryos (7/19 embryos transferred; 37 percent).
Behboodi et al. (1995) reported that birth weights were not significantly different between calves produced by AI and IVP-derived calves when embryos were cultured to the blastocyst stage in sheep oviducts; however, birth weights of calves born from embryos that developed into blastocysts in vitro were higher than those for calves from embryos that developed in the sheep oviduct or from AI. In this study, 7/8 calves produced from embryos cultured in vitro died within 48 hours of birth, compared to 1/8 calves from embryos cultured in the sheep oviduct after fertilization. Hasler et al. (1995) noted that approximately seven percent of clients purchasing cows carrying IVP-derived calves reported high birth weights. In this study, of 428 IVP calves born, 67 died at birth (15.6 percent). Block et al. (2003) reported 5/28 IVF calves were stillborn (17.9 percent), one of which suffered hydrops, and another hydrocephalus. In contrast, a recent study (Xu et al. 2006) reported that out of 458 calves born from vitrified IVF embryos, two calves were born with unspecified physical defects, one of which died within an hour of birth.
| Table V-4: Incidence of LOS and related clinical signs and survival rates of neonatal calves produced with ARTs 1 | ||||||
| Study | Transgenic Status | Clone LOS incidence | Survival of LOS clones | Comparator LOS incidence | Survival of comparators | Comments |
|---|---|---|---|---|---|---|
| Batchelder 2005 | None | 8/8 (1.00) | 2/8 (0.25) | 2/9 (0.22) | 9/9 (1.00) | Comparators were ET (n=6) and AI (n=3). See Table V-5 for clinical signs. |
| Behboodi et al. 1995 | Some | NP | NP | 4/8 (0.50) 0/72 (0.00) | NP | 8 IVF calves compared to 72 AI calves |
| Block et al. 2003 | None | NP | NP | 3/28 (0.11) | NP | All calves derived by IVP |
| Cyagra 2003: Appendix E | None | 73/1232 (0.59) | 56/733 (0.77) | NP | NA | Clinical signs: contracture; septicemia; nephritis; failure to thrive; umbilical, gastrointestinal, cardiac-circulatory anomalies |
| Edwards (unpublished) | None | 14/27 (0.52) | 11/14 (0.77) | NP | NA | See Appendix G. Multiple internal organ defects, gastro-intestinal problems |
| Garry et al. 1996 | None | 34/40 (0.85) | 26/34 (0.77) | 0/26 (0.00) | NA | BNT clones, AI comparators. Clinical signs: respiratory and musculo-skeletal |
| Gibbons et al. 2002 | None | 8/9 (0.88) | 7/8 (0.88) | NP | NA | Clinical signs: respiratory, umbilical, septicemia, hydrocephalus, GI problems |
| Gong et al. 2004b | None | 7/27 (0.26) | 0/27 (0.00) | NP | NA | |
| Hasler et al. 1995 | NA | NP | NA | 23/343 (0.07) | NP | Data gathered from owners of IVF-pregnant cows |
| Hill et al. 1999 | All | 4/8 (0.50) | 2/4 (0.50) | NP | NA | Clinical signs: respiratory, umbilical, cardiac, hepatic anomalies; contracture, acidosis, weak suckling reflex |
| Heyman et al. 2004 | None | 7/50 (0.14) | NP | NP | NP | Birth weights of AI comparators used to set range for determining LOS in clones |
| Kato et al. 2000 | None | 6/17 (0.35) | 3/6 (0.50) | NP | NA | Clinical signs (may be result of Akabane virus): musculo-skeletal, kidney abnormalities |
| Kubota et al. 2000 | None | 6/6 (1.00) | 4/6 (0.67) | NP | NA | Clinical signs: respiratory, polyuria and polydypsia Akabane virus |
| Lanza et al. 2001 | Some | 14/30 (0.46) | 8/14 (0.57) | NP | NA | |
| Miyashita et al. 2002 | None | 1/12 (0.08) | 0/1 (0.00) | NP | NA | |
| Pace et al. 2002 | Some | 70/106 (0.66) | 59/70 (0.84) | NP | NA | Clinical signs: umbilical, respiratory, cardiac, musculo-skeletal, GI; hydrocephalus, bacterial infection |
| Xu et al. 2006 | None | NP | NP | 2/458 (0.0004) | 457/458 (1.00) | Studied sex-sorted, vitrified IVF embryos |
| Zakhartchenko et al. 1999a | None | 1/2 (0.50) | 0/1 (0.00) | NP | NA | Clinical signs: musculo- skeletal and hepatic abnormalities |
|
1 Data on live-born calves | ||||||
Sire selection may also contribute to the large calves resulting from ET and IVP. Knight et al. (2001) indicated that one of the sires used in a two year study had a tendency to produce large ET calves. High birth weights in this study may have contributed to low survival rates in a previous study in the same herd. In cattle, sires may be selected based on their IVP and ET calf birth weight records (Knight et al. 2001). A similar relationship between high birth weights of calves was noted in two other studies of IVP (Thompson et al. 1998, Block et al. 2003).
In a large study comparing birth weights, dystocia incidence, and neonatal death rates in AI, ET, IVP, and BNT produced calves of various beef and dairy breeds from labs in several countries, Kruip and den Daas (1997) noted that on average 31.7 percent of IVP calves (n=308) weighed more than 50 kg at birth, compared to 10 percent for AI (based on 495,000 calf records from the Netherlands). Interestingly, only 15 percent (n=126) of calves produced by BNT had birth weights greater than 50 kg in this study. For one breed (Holstein-Friesian), perinatal losses were similar between AI (n=1,160) and ET (n=45) calves (6.1 ± 0.6 and 6.6 ± 0.6 percent), but loss was higher for IVP calves (14.4 ± 2.3 percent; n=251). Perinatal death loss was higher (11.6 vs. 2.3 percent) for IVP (n=308) compared with BNT calves (n=126) for the six breeds studied (Holstein-Friesian, Belgian Blue, Simmental/Fleckvieh, Limosin, Piedmontese, and Alentejano).
More recent studies in which IVP and SCNT embryos were produced under the same culture conditions reported considerably higher incidences of LOS in fetal and adult cell SCNT-derived calves compared to IVP (Heyman et al. 2002, Chavatte-Palmer et al. 2002, Matsuzaki and Shiga 2002), indicating that culture conditions may not be the only factor influencing the development of LOS in cattle clones. Average birth weight of adult-cell SCNT clones was significantly higher than IVP-derived calves (53.1 ± 2.0 kg vs. 44.5 ± 2.1 kg) in the Heyman et al. (2002) study. Chavatte-Palmer et al. (2002) found considerable variability in organ development among calf clones, and reported that one apparently normal clone fetus had small kidneys for its size and stage of development. Also in this study, Chavatte-Palmer et al. noted differences in body temperature, plasma leptin, thyroxine (T4) and insulin-like growth factor-II (IGF-II) in surviving clones compared to IVP and AI controls during the first week to 15 days after birth, although the clones appeared normal and healthy. Differences between clones and controls resolved by 50 days of age (see Chapter VI for a more complete discussion of this study). The differences in outcomes between SCNT and IVP pregnancies observed in these studies suggest that some additional factor(s) may be at least partially responsible for the higher rate of abnormalities in animal clones compared to IVP calves, and not solely due to culture conditions. One possible explanation for this increase in abnormalities is incomplete epigenetic reprogramming (see Chapter IV).
In a later study by this same group (Chavatte-Palmer et al. 2004), an additional cohort of 58 live-born calves were followed through maturity. Clone survival after the first week following birth was 76 percent (44/58). Clinical signs and necropsy findings for nine clones that died during the perinatal period included hyperthermia, umbilical hernia, respiratory problems, ascites (abnormal fluid accumulation) in the chest and abdomen, fatty liver, limb deformities, various digestive tract problems, and abnormal or degenerating kidneys.
Culture media requirements may differ between SCNT and IVP embryos. Mastromonaco et al. (2004) compared development to blastocyst for IVP and SCNT embryos using different media ingredients at different stages of the in vitro process (oöcyte maturation and embryo culture stages). Although IVP embryos had similar rates of development to blastocyst and hatched blastocyst regardless of culture media used, development to blastocyst was greater among SCNT embryos cultured in synthetic oviductal fluid with 2 percent steer serum. Unfortunately, this study only looked at development through day 9 of embryo culture, and did not examine in vivo embryo development or subsequent calving outcomes. It is possible, however, that culture conditions impact epigenetic reprogramming, and this may be related to differences in outcomes observed in the Heyman et al., Chavatte-Palmer et al., and Matsuzaki and Shiga studies.
In a recent study comparing SCNT (n=8) to ET (n=6) and AI (n=3), Batchelder (2005) noted large birth weights among three Hereford clones (n=3), ranging from 50.0 to 71.0 kg. By comparison, ET comparator Hereford calves ranged from 31.5 to 48.0 kg (n=3). Curiously, the mean weight for Holstein clones (n=5) was similar to contemporary ET comparators (n=3) (37.1 vs. 39.4 kg), and within the average range for Holstein heifer calves. Neonatal clones in this study had lower RBC (6.8 x 106 vs. 8.6 x 106 cells/µl) and hematocrit at birth than their comparators, and remained low for the first hour after birth, but were similar to comparators thereafter. White blood cell counts (WBC) and differential patterns were similar between clones and comparators. Clones exhibited lower blood glucose and lactate levels during the first 24 hours after birth than comparators, but were similar to comparators by 48 hours.
To explore the possible relationship between epigenetic dysregulation and organ abnormalities associated with increased risk of mortality in SCNT-derived neonatal calves, Li et al. (2005) investigated the expression (defined in this experiment as relative levels of messenger RNA (mRNA))59 of eight developmentally important genes (PCAF, Xist, FGFR2, PDGFRa, FGF10, BMP4, Hsp70.1, and VEGF) in nine clones that died within the first two days after birth. At necropsy, seven calves were found to have multiple cardiac, hepatic, and pulmonary defects; two calves had underdeveloped kidneys; two calves had hemorrhagic spleens; and three calves had brain lesions or underdeveloped brain.60 Compared to organs from healthy AI-derived calves (n = 3), levels of mRNA encoding several genes were altered in heart, liver, spleen, kidney, lung, and brain tissues from the deceased clones. The magnitude of differences in mRNA levels seemed to be the greatest (two- to five-fold) in heart tissue, in which expression of five of the eight genes studied (PCAF, Xist, FGF10, BMP4, and VEGF) was up-regulated, and least in kidney, in which expression of two genes (FGF10 and PDGFRa) was down-regulated by approximately 60 percent. Across all of the tissues studied, levels of mRNA encoding VEGF, BMP4, PCAF, and Hsp70 demonstrated the greatest changes (either increased or decreased). The products of these genes are involved in regulation of blood vessel formation, regulation of lung and heart development, transcriptional coactivation, and protection against apoptosis,61 respectively. These observations support the idea that in some clones, aberrant epigenetic reprogramming may alter the expression patterns of genes that control embryo development and organogenesis. In this study, the level of dysregulation was apparently sufficient to result in congenital organ defects that were not compatible with life outside the uterus. It is not known whether congenital organ defects in animals derived by other ARTs are also associated with altered patterns of gene expression.
Batchelder (2005) also noted several clinical signs often associated with LOS in both Holstein and Hereford clones, including delayed time to suckle and stand, hypoglycemia, forelimb flexor tendon contracture, enlarged umbilicus, patent urachus, and respiratory distress. These clinical signs were not always associated with high birth weight. Interestingly, a small number of comparators exhibited some of these same clinical signs. Table V-5 is partly reproduced from Batchelder 2005.
| Table V-5: Clinical signs observed in neonatal clones and comparators for Batchelder 2005. | ||
| Clinical Sign | Clones | Comparators |
|---|---|---|
| Time to nurse (> 3hrs) | 5/8 | 0/9 |
| Time to stand (>3 hrs) | 5/8 | 1/9 |
| Hypoglycemia (? 50mg/dl) | 3/8 | 2/9 |
| Respiratory distress | 3/8 | 1/9 |
| Flexor tendon contracture | 4/8 | 0/9 |
| Enlarged umbilical vessels | 8/8 | 2/9 |
| Patent urachus | 5/8 | 1/9 |
All calves in this study survived the first 48 hours; however, one clone died at 72 hours after birth, and another at six days after birth. The first clone, a Hereford heifer, was the largest at birth (71.0 kg), and at necropsy was diagnosed with pulmonary hypertension and multiple severe organ abnormalities including diffuse fibrosis of the liver, dysplasia of the biliary system, right ventricle hypertrophy, and patent ductus arteriosus. The second clone died at six days of age after suffering bloat and various other clinical signs involving the heart and lungs.
(b) Other complications
In discussing health and mortality among clones it is often difficult to distinguish between defects resulting from the uterine environment, placentation, and/or difficulties during delivery, and epigenetic factors intrinsic to the clone that impede normal development of the fetus and adaptation following birth. Dystocia, for example, can result in premature separation of the placenta, causing inhalation of amniotic fluid prior to birth, predisposing the neonate to pneumonia in both conventional calves (Moore et al. 2002) and clones (Kato et al. 1998). Respiratory failure is one of the most commonly reported clinical signs in neonatal clones (Table V-3), and appears to result from numerous causes, including inadequate surfactant and failure of the lungs to inflate, as well as pneumonia arising from various causes (Garry et al. 1996, Hill et al. 1999, Chavatte-Palmer et al. 2002). Pneumonia may result from dystocia in natural pregnancies as well as those derived by ART (Moore et al. 2002). However, many of the respiratory conditions reported to occur in association with LOS (failure to inflate) have not been reported for calves from natural service or AI, and may be peculiar to ARTs that involve more extensive in vitro manipulation of the embryo (i.e., IVF and cloning), or may be related to labor-induction protocols (Batchelder 2005).
Calves exhibiting LOS may also show prolonged time to stand and poor or late-developing suckling behavior (Chavatte-Palmer et al. 2002, Pace et al. 2002 (mixed transgenic and non-transgenic clones), Batchelder 2005). Poor suckling may preclude immune transfer in colostrum-dependent species, resulting in decreased ability to respond to immune challenge. Most of these studies, however, indicate that colostrum was administered by tube-feeding if the animal failed to suckle within one to two hours postpartum (Garry et al. 1996 (BNT clones), Hill et al. 1999 (transgenic clones), Gibbons et al. 2002, Batchelder 2005). Poor immune response in such cases may be due to a number of causes: inability of the neonate to absorb immunoglobulins; colostrum that is inadequate in immunoglobulin content; excessive or overwhelming stress; or high levels of pathogens in the neonatal environment. Clone producers have indicated that some calves are born with large umbilici, often with patent (open) blood vessels. This factor may increase the risk of bacterial infection, and clone producers indicated that surgery was generally performed on the enlarged umbilici of calves to reduce the risk of infection (see CVM Memorandum I at www.fda.gov/cvm/cloning.htm; also Appendix E and Batchelder 2005).
Most studies that reported supplemental colostrum feeding did not indicate the source of the colostrum or whether tests of its adequacy (gravimetric density or IgG concentration) had been performed. Two studies reported testing colostrum of surrogate dams for adequacy or blood tests of neonates to determine immunoglobulin status (Hill et al. 1999, Pace et al. 2002). In the study of transgenic clones by Hill et al., several of the surrogate dams were judged to have adequate colostrum. Transgenic calf clones that failed to suckle were administered colostrum by tube and fostered to other cows as needed. Pace et al. (2002) reported testing plasma IgG of calves 12 hours after birth, followed by plasma infusion if plasma IgG concentrations were less than 1,200 mg/dL. Calf clones in this study were reported to have normal serum IgG levels 24 hours after birth. As noted throughout this report, the data derived from clones that are transgenic are extremely difficult to extrapolate to “just clones” (the only subject of this risk assessment) because of the inability to determine the relative contributions of the transgenic modification and the cloning process to the observations.
In Batchelder 2005, clones were provided 2 liters colostrum (either by bottle or esophageal tube) within three hours of birth as well as supplemental plasma by I.V. over 40 minutes. At 24 hours after birth, clones and comparators had similar levels of serum IgG. However, one clone had sub-normal IgG (435 mg/dl IgG), and was classified as having failure of passive transfer of immunity, and a second clone was classified as marginally protected (1500 mg/dl IgG). Batchelder related failure of passive immune transfer to poor metabolic status and respiratory distress.
ii. Cyagra Data: Perinatal Cohort
A complete discussion of the Cyagra dataset, including how it was analyzed and the context in which results should be interpreted, is presented in detail in Appendix E. Briefly, the Cyagra dataset provided information on the overall health status and laboratory tests (clinical chemistry and blood cell parameters (hemograms)) for a group of SCNT-derived cattle clones and their approximately age- and breed-matched comparators. Among 10 neonates, four liver-related analytes were lower in clones than comparators: AST, GGT, cholesterol, and bile acids. Except for the values from one calf that did not survive, all red blood cell analytes were within the comparator group range. Three calves, all of which were infected with rotavirus, had low lymphocyte counts (lymphopenia).
Of the 134 clone calves in the Cyagra cohort, 11 were stillborn. Birth weights were available for 34 of the 123 live-born clones, and ranged from 19.5 kg (a twin calf) to 76.8 kg. Eighteen of the 34 (53 percent) birth weights were at least 20 percent above the average for their breed. Most oversized calves (13/18 (72 percent)) survived the critical first 48 hours after calving. Six of the oversized calves did not exhibit any other clinical signs associated with LOS. Fifty-five additional calves that were not oversized at birth, or for which birth weights were not available, showed clinical signs often associated with LOS; 43 of these animals survived the first 48 hours after calving. The most common clinical sign was umbilical problems (41 cases), followed by tendon contracture (15 cases), ranging from mild to severe. There were also four animals with respiratory signs, five with cardio-vascular signs, three with thermoregulatory problems, two with renal or nephric signs, and five animals listed as having “abnormal development.” Some of the calves exhibited more than one sign, often umbilical problems with contracture, cardiac or respiratory signs.
iii. Unpublished data
Two small datasets on perinatal bovine clones were also submitted to CVM. These are presented in their entirety in Appendix G, along with a more complete analysis. In brief, the body temperatures, pulse and respiration rates of 19 clones of unidentified breeds for the first 72 hours after birth averaged 39.4ºC, 95.2 beats/minute, and 57.4 breaths/minute. The second set of data submitted were birth records and veterinary observations for two Holstein heifer calves, indicating the animals were generally in good health, showed normal increases in plasma glucose in response to feeding, and total protein and glucose values similar to non-clone neonatal calves in the Cyagra dataset.
According to the data submitted by J.L. Edwards’ laboratory at the University of Tennessee (Appendix G), 19/27 (70 percent) live-born Jersey clones survived the perinatal period. Causes of death included placental abnormalities, suspected detached umbilicus, and hepatic, cardiac and renal lesions or defects. Average birth weight for these calves was 31.6 kg, and ranged from a very low birth weight calf (6.3 kg) to a high of 45 kg. A study at North Carolina State University indicated average birth weight for female Jerseys was 23.64 kg (Washburn et al. undated circular).
c. Developmental Node 3: Juvenile Development
i. Peer-Reviewed Publications
From weaning to puberty, the rates of morbidity and mortality in AI-produced and naturally bred cattle are generally very low. According to USDA statistics (USDA/NAHMS 1996), mortality of dairy replacement heifers (from weaning to calving) was 2.4 percent. In beef replacement heifers, the most common cause of morbidity was pinkeye (1.9 percent) followed by scours (i.e., diarrhea, 1.0 percent; USDA/NAHMS 199762 ).
Compared to the perinatal period, there is less detailed information in the literature on the health of bovine clones during the juvenile period. Most studies merely report that animals surviving the first 30 to 60 days postpartum are “healthy and normal” (Campbell et al. 1996, Lanza et al. 2000 with transgenic clones, Heyman et al. 2002). Kubota et al. (2000) reported that veterinary exams, growth curves and blood clinical chemistry were used to determine the health of six clone calves, and that no differences were noted between clones and age matched controls.
In a study of the effects of fetal bovine serum on the development of clones, Lawrence et al. (2005) reported the death of the single surviving calf (from the serum fed group) at nine months of age due to enterotoxemia, attributed to gastrointestinal infection with Type A Clostridium perfringens. Various gastrointestinal and immunological problems have been reported in cattle clones, including bloat, poor motility, and poor nutrient absorption (Cyagra 2003: Appendix E, Wells et al. 2004, Batchelder 2005). Some studies of clones have also reported reduced immune responsiveness, although this has mostly been noted in neonatal clones (Renard et al. 1999, Chavatte-Palmer et al. 2004, Wells et al. 2004). Type A C. perfringens is a common soil and feed-borne bacteria. Enterotoxemia caused by proliferation of Type A C. perfringens and release of toxins in the gut is normally prevented by vaccination and good feeding management. The report of this study does not state whether this calf was vaccinated, nor does it provide details as to the possible source of the infection. The lack of detail in this study makes it difficult to determine whether the death of this calf was related to cloning, or if it was due to management practices.
Heyman et al. (2007) reported that between one quarter to one third of calves produced at the INRA laboratory died by six months of age, but calves surviving beyond that time had normal physiological status. It has been suggested that clones may have different sensitivity to stress compared to conventional animals. Stress affects a number of physiological systems throughout the body, and is often associated with increased secretion of cortisol and catecholamine metabolites (epinephrine and norepinephrine) by the adrenal gland (Minton 1994). These hormones result in physiological changes that allow the animal to adapt to stressful situations. Stress can have a negative impact on immune function and can make animals more susceptible to infection. Therefore, Heyman et al. (2007) postulated that chronic stress in bovine clones might be the basis for the sudden unexplained death of clones reported in some studies (e.g., Chavatte-Palmer et al. 2004; Wells et al. 2004). To test this postulate, Heyman et al. (2007) measured plasma concentrations of cortisol and urinary concentrations of epinephrine and norepinephrine in clones and comparators every two months from 4 to 36 months of age. The variability of measurements was greater in clones, but no differences were found between clones and comparators in concentrations of blood cortisol or catecholamine metabolites, suggesting that apparently healthy clones were not suffering from chronic stress.
Heyman et al. (2007) noted some differences between clones and AI-derived calves up to two months of age, even among clones that were clinically healthy. As an example, these authors note that although hematological parameters were within the normal range, hemoglobin and hematocrit of clones were lower than those of their age-matched AI comparators. The authors do not describe the reference range used in this study. As discussed in Appendix F on the analysis of Cyagra data, it is important to use a range that is relevant to the animals of interest. Thus, the authors’ use of an age-matched comparator group raised under the same conditions is appropriate, and their finding that these young cattle clones may hav
