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Chapter VI: Food Consumption Risks

A. Potential Hazards and Food Consumption Risks Associated with Food Products from Animal Clones and their Progeny

1. Assumptions

This Chapter of the Risk Assessment is focused on food safety concerns, and assumes that any clones or their products would be subject to the same local, state, and federal laws and regulations as conventional food animals or their products. These assumptions exclude animals with gross anomalies that would not enter the human food supply (although they might be rendered). It also assumes that any hazards arising from the consumption of products derived from animal clones would result from epigenetic dysregulation of the genome of the developing animal, as described in Chapter IV.

Because much of the focus of this analysis is the identification of subtle hazards in otherwise healthy-appearing animals, the Critical Biological Systems Approach (CBSA) evaluates animal health data on as fine a level of resolution as possible. This includes individual animals or even individual analytes per animal in order to have a sensitive screen for adverse outcomes (and thus food consumption risks). Thus, although some of the data in this chapter reprises information previously addressed in Chapters IV and V, the methods by which the data were evaluated differed. Because the emphasis in the Chapter is on subtle hazards, the focus of Chapter VI is to evaluate adverse outcomes observed in animals to see if they can provide insight into identifying food consumption hazards, and not the actual risks to the animals themselves, which have been discussed in Chapter V. Chapter VI also includes all of the information that we could identify on the composition of meat or milk from clones or their progeny. Much of this information has been published or made available in 2005, and tends to evaluate very similar compositional components; much of it is on animals for which physiological data are also available.

2. Critical Biological Systems Approach to Animal Clones of Cattle, Swine, Sheep, and Goats

Chapter V and VI review the health outcomes reported for clones of cattle, swine, sheep, and goats. Over 1,700 references were identified in our literature searches; closer examination revealed that approximately 350-400 papers were useful to the understanding of the subject, and a smaller fraction of those actually cited papers were cited for information on the health of clones or the composition of their food products. Many of these reports are on the same cohorts of animals, but concentrate on different measurements or life stages. Several are reviews of adverse outcomes that have been observed in individual animals or cohorts of animals, but do not provide new data. As indicated previously within the Risk Assessment and detailed in Appendix D, some of the animals on which reports are provided are somatic cell nuclear transfers of transgenic cells, thereby actually being reports on transgenic animal clones. These have been included in the food consumption risk assessment when they provide corroborative information, and the transgenic status of the animals has been indicated when that information is available.

The following section reviews the available information on animal cloning by species, sorting the information into developmental node-specific groupings. This approach was most applicable to bovine clones, where there is significantly more information compared to other species. For those species where information is very limited, such as sheep, the available information is presented as a single unit.

a. Bovine Clones

The largest number of publicly available publications and data sources address clones of dairy and beef cattle. Many reports on effects noted in the Cell Fusion/Reprogramming, Embryo/Fetal, and Perinatal periods tend to come from the early cloning experiments. Others test hypotheses regarding some component of the SCNT process (e.g., cell cycle, cell source, culture conditions, epigenetic reprogramming (see Chapter IV)), and either do not result in live births, or result in very few live births. Very few systematically evaluate the health of the animals, many simply state that “animals appear normal and healthy” or that “no differences were observed between clones and controls.” CVM has extracted as much information as possible from these studies, and has incorporated its findings into the appropriate Developmental Nodes.

During the course of preparing this risk assessment, clone producers shared information on various cloning outcomes with CVM. The most comprehensive dataset was generated in response to preliminary presentations of the risk assessment methodology by FDA staff at various scientific meetings. In particular, one clone producer, Cyagra, Inc., has attempted to gather information on all of the cattle clones that it has produced, including animals that did not survive or that were culled for various reasons. In some cases, this has proved impracticable due to the dispersal of clones to their ultimate owners. The Cyagra dataset is the most comprehensive survey of the health status of cattle clones that has been assembled, and this information has been incorporated into this Risk Assessment. Details on the animals, the methods used to collect and interpret the data, and the actual data themselves can be found in Appendix E. Cyagra also collected data on the composition of meat from several clones; these data are also in the Appendix ¹.

   ¹ Viagen, Inc. has also developed an extensive dataset on the health and composition of swine clones and their progeny. This is the most comprehensive dataset on the health of swine clone progeny and the composition of their meat. Similar to the Cyagra dataset, these data and their detailed analyses are found in Appendix G, and are summarized within the text of this Chapter.

The information provided by Cyagra differs from that presented in the peer-reviewed literature for several reasons:

• The data were collected specifically to address issues raised in this risk assessment, and thus are not part of a hypothesis-testing study, or written to provide examples of novel or unusual events;

• They have not been peer-reviewed outside CVM (to the Center’s knowledge);

• They include individual animal data; and

• They are far more extensive with respect to the number of clearly non-transgenic animals evaluated (n=78 surviving and tracked animals), and the number of observations on individual animals than any other study or series of studies from a particular laboratory.

i. Cell Fusion, Nuclear Reprogramming, and Embryonic and Fetal Development in Bovine Clones² (Developmental Node 1)

SCNT is a relatively inefficient process. “Successful” event estimates can be based on the number of fused cells, implanted blastocysts, or pregnancies confirmed at some day of gestation, estimates range from one in one thousand (usually based on fused cells) to one in four (confirmed pregnancy at gestation day 60). The former estimates include the earliest reports of SCNT, as well as studies testing various methodological variables, and reflect the “technology development” nature of the reports. When measured from the detection of an established pregnancy in the surrogate dam, the success rate can be considerably higher, and can range from 1-2 percent (as reviewed in NAS 2002b) to approximately 20-25 percent as related to CVM by commercial cloning ventures.

Lack of success at the cell fusion stage is likely due to several factors, the most significant of which are technological (e.g., damage to the oöcyte or donor cells) or biological (e.g., incorrect reprogramming of the genome of the donor cells (Chapter IV) or possible lack of synchrony between donor cell and oöcyte). An alternative justification proposed by Hochedlinger and Jaenisch (2002) among others, is that the extremely low success frequency is a reflection of the inability of all but “stem cells” of various degrees of pluripotency to be reprogrammed, and the serendipitous outgrowth of such cells selected at random for use as donor cells. Regardless of the explanation, few fused donor/oöcyte pairs survive to divide or to become established as pregnancies in surrogate dams.

The following overview of methods that may affect success rates of SCNT are included to allow the reader to understand that there are many different components that may influence cloning efficiency. It is important to remember, however, that the goal of this chapter of the risk assessment is to identify and characterize potential subtle hazards in clones and to determine whether they pose food consumption risks.

(a) Peer-reviewed Publications

² This Chapter emphasizes the morphological changes observed in this Developmental Node, unlike Chapter IV, that summarized molecular findings.

The following section provides summaries of studies that contribute to identifying some of the factors that may contribute to successful nuclear transfer at the earliest developmental node. It is intended to be illustrative, and not comprehensive.

Effect of the Zona Pellucida. The importance of the zona pellucida in embryo development is not clear, and there are conflicting outcomes in different studies evaluating its role. Dinnyes et al. (2000) compared developmental rates of cattle oöcytes subjected to SCNT, parthenogenetic activation, or in vitro fertilization. For the oöcytes undergoing SCNT (n=106), 74 percent fused, 90 percent of fused embryos cleaved by Day 2, and 29 percent of cleaved embryos developed to blastocysts. Eighty-one percent of parthenotes³ (early embryos arising from parthenogenetic activation) (n=47) incubated in 5 percent CO2 in air cleaved by Day 2 of the experiment, but only 17 percent developed into blastocysts. Parthenotes (n=98) incubated in 5 percent O2, 5 percent CO2 and 90 percent N2 had a 79 percent cleavage rate on Day 2, and a 32 percent survival to blastocyst stage. By comparison, in vitro fertilized oöcytes (n=98) had a 69 percent cleavage rate by Day 2, and 35 percent developed to blastocysts. Because parthenotes are “clones” that have not undergone nuclear transfer, the zona pellucida of the embryo is not disrupted. This disruption has been hypothesized to be a possible cause of early embryo failure in nuclear transfer (NT) embryos. The lack of difference in development to blastocyst between SCNT, parthenotes and IVF embryos cultured under the same conditions suggests that disruption of the zona pellucida may not be an important factor in early loss of SCNT embryos. Conversely, Ribas et al. (2006) noted no difference in development to blastocyst in zona-free vs. zona-intact IVF mouse embryos, although the authors stated that zona-free blastocysts were smaller and more irregular than zona-intact embryos. None of the embryos in this study were transferred to recipients for gestation, however, so further development could not be assessed. In another study involving IVF-derived sheep embryos, Ritchie et al. (2005) transferred eight zona-free embryos to four surrogate ewes. One of these pregnancies progressed to term and resulted in a live lamb.

   ³ A form of reproduction in which an unfertilized egg develops into a new individual, which occurs among crustaceans and certain other arthropods.  Parthenotes, unlike somatic cells, do not need to be reprogrammed, as they are already in an undifferentiated state.

Cell Culture Conditions. Several laboratories have attempted to optimize culture conditions to improve cloning efficiency (Kubota et al. 2000; Li et al. 2004; Park et al. 2004b; Du et al. 2005). These manipulations have included addition of various compounds to culture media, co-culture with “feeder cells,” and serum starvation. Results of these studies have been mixed, as described below.

In order to study the influence of culture conditions of donor cells used for SCNT, Kubota et al. (2000) used fibroblasts derived from a skin biopsy obtained from a 17 year old Japanese Black beef bull. Donor cells for nuclear transfer were obtained from cultures that had undergone 5 (n=570), 10 (n = 269), or 15 (n = 264) passages.⁴ All cultures were serum starved prior to nuclear transfer, except that cells from passage number five were divided into two groups, one of which was serum-starved (n=288), and the other was not (n=282). There were no differences among groups for fusion or cleavage rates, but development to blastocyst stage was lower in cells from Passage 5, relative to cells from the higher passage rates, regardless of whether or not the cells were serum starved. A total of 54 blastocysts were transferred to 36 recipient cows. Fifteen cows were diagnosed pregnant, of which nine spontaneously aborted between 39 and 123 days of pregnancy. All three of the pregnancies derived from Passage 5 cell cultures spontaneously aborted. Six calves derived from the two more extensively passaged cultures were delivered at term, two from cultures that had undergone 15 passages, the other four from cells that had undergone 10 passages. Two calves derived from Passage 10 donor cells died shortly after birth. In this study it appears that cells that have been more extensively passaged make better donors than less extensively passaged cells. The biological basis for this is not clear, unless cells that have been passaged more extensively in culture somehow become more amenable to epigenetic reprogramming.

In another study of culture conditions, Li et al. (2004) compared development of SCNT embryos co-cultured with bovine cumulus cells or with one of two different types of serum (fetal calf serum (FCS) or bovine serum albumin (BSA)) for seven days. The rates of cleavage, morula and blastocyst formation were similar across treatment groups. Fewer blastocysts in the FCS group exhibited normal chromosomal ploidy compared to the BSA group (24/41 or 58.5 percent vs. 24/35 or 68.6 percent), but both of the serum supplemented groups performed poorly compared to the cumulus cell co-culture group, in which 34/42 (80.9 percent) of blastocysts had normal ploidy.

Park et al. (2004b) noted that although not effective in improving embryo development alone, the combination of ß-mercaptoethanol (ME) and hemoglobin (Hb) enhanced the rate of development of NT embryos to the morula stage compared to unsupplemented media (19/57 vs. 55/85). Development to blastocyst, however, was similar between untreated controls and either the combined treatments or ME or Hb supplementation alone (16/57 vs. 18/99, 15/95, and 40/104 for control, Hb, Me and Hb + ME, respectively). Similarly, Du et al. (2005) found no beneficial effect of adding phytohemagglutinin-L (PHA) to culture media for survival, cleavage or blastocyst formation of NT embryos. From a total of 324 fused embryos, three live calves were born: two from the PHA group and one from the untreated group.

   ⁴A passage is a cell culture process in which culture vessels that are full of cells are diluted to lower cell densities. This allows the cells to overcome the growth inhibition that comes with limited space. Each dilution is referred to as a passage, so that a culture that has been passaged five times has started with low cell density, grown up to high cell density, been diluted, and had that process repeated four more times.

Heterogeneity of Fusion Components. Hiendleder et al (2004) studied how differences between nuclear and oöplasm sources can influence SCNT outcomes by using three breeds of cattle (Brown Swiss, Dwarf Zebu, and two varieties of Simmental) as oöcyte sources and granulosa cells from a Brown Swiss cow as the source for somatic cells. Four groups of SCNT embryos were produced. All pregnancies were terminated at 80 days gestation and uterine contents collected to determine the number of viable fetuses. Details on individual fetuses were not discussed, but the authors noted that SCNT fetuses in general were heavier, had a larger thorax circumference, and a reduced crown rump length: thorax ratio (a standard measure of body size) compared to AI fetuses. The proportion of viable fetuses was significantly affected by source of oöplasm, and was higher for fetuses produced using Dwarf Zebu oöplasts than the other three sources. The lowest viability was noted for one, but not both, of the Simmental sources. Interestingly, the difference between the two Simmental sources for viability was significantly different. No details regarding the oöcyte donors, other than breed, were provided, so there is no way to determine if other factors (e.g., age of the oöcyte donor cows, nutritional status, health history, or size of follicles collected) might have influenced fetal viability. The authors also compared mitochondrial DNA sequences between the two Simmental oöcyte sources, and noted extensive polymorphism in coding and non-coding regions of the two mitochondrial genomes. Although there has been speculation that mitochondrial dimorphism may affect development of SCNT embryos, only one study was identified that looked specifically at mitochondrial effects on embryo development (Takeda et al. 2005). Also of interest, when fetal morphology was compared in the Hiendleder et al. study, hybrid fetuses (reconstructed using either Zebu or Simmental oöplasm) were not significantly different in size compared to AI fetuses of the same gestational age; however, fetuses produced using the same breed as source of both oöplasm and nucleus (Brown Swiss) exhibited fetal overgrowth. The Brown Swiss cows that were used as sources of oöcytes were different individuals from the Brown Swiss donor of the nuclear DNA. The authors do not report whether they compared mitochondrial DNA of the nuclear donor with that of any of the Brown Swiss oöcyte donors.  

Source of Donor or Recipient Cells. Tissue source of nuclear donor cells can also affect development and survival of NT embryos. Galli et al. (1999) used bovine blood lymphocytes as nuclear donors. Lymphocytes, involved in the immune system, must undergo rearrangement of their DNA in order to produce immunoglobulins. Panelli et al. (2004) examined tissues of four aborted NT fetuses and the chondrocytes of the single surviving clone from the Galli et al. experiments. The results were compared to chondrocytes from three non-clone bulls (how the comparator bulls were generated is not described). The aborted fetuses exhibited DNA rearrangement in brain cells that was typical of terminally differentiated lymphocytes, but the surviving clone showed no rearrangement in chondrocytes isolated from his sperm, similar to chondrocytes collected from non-clone bulls. Based on this small dataset, the authors suggested that although terminally differentiated cells can sustain development through the late fetal stage, cells more amenable to reprogramming (dedifferentiation), such as stem cells, were more likely to result in live clones.

Xue et al. (2002) reported on the relative success rates associated with generating clone embryos from three different tissues collected from a 13 year old Holstein cow. In their hands, ovarian cumulus cells had the highest rate of development to blastocyst (57 percent, n=92), compared to skin fibroblast cells (34 percent, n=110) and mammary epithelial cells (23 percent, n=96). Six term pregnancies resulted following transfer of ovarian cumulus nuclear transfer (NT) embryos to recipient cows (5.5 percent, n=109), and four (7 percent, n=57) term pregnancies resulted from skin fibroblast NT embryos. None of the embryos generated from mammary epithelial cells resulted in a term pregnancy when transferred to recipient cows (n=34). The expression of X-chromosome linked genes in various tissues from deceased animals and conventional controls, and from the placentae of surviving clones was also investigated. Results indicated that X-chromosome inactivation occurred normally in the surviving female clones, but was incomplete in the clones that died. Embryo samples were taken to determine if there were differences in cell counts in embryos from parthenotes and SCNT-derived embryos at the same stage of development. Cell numbers for NT embryos were lower compared to parthenotes at all stages examined (Day 5 morula: 35.1 ± 1.1, n=48 for NT vs. 43.5 ± 1.5, n=58 for parthenotes; Day 7 blastocyst: 81.0 ± 3.7, n=46 for NT vs. 93.8 ± 5.6, n=48 for parthenotes). The importance of differences in cell numbers is not clear from this study, as mammalian parthenotes generally do not develop to term. Cell counts of IVF embryos, which would have been a more informative comparison, were not provided.

Gong et al. (2004) compared granulosa cells from adult cattle of two different breeds (Holstein and Chinese red-breed yellow cattle), skin fibroblasts from two individual Holsteins and a Holstein fetus, and oviductal cells from a Holstein fetus for development and survival through the birth of clones. The rate of blastocyst formation was lowest for one of the two adult skin fibroblast sources (253/906 blastocysts/fused couplets or 27.9 percent), although the other adult fibroblast cell line was comparable to the fetal fibroblast cell line (52/132 or 39.4 percent vs. 1294/3412 or 37.9 percent). Fetal oviductal cells had the highest rate of blastocyst formation in this experiment (456/1098 or 41.5 percent). A total of 346 Day 7 blastocysts were transferred to 171 recipients. Pregnancy rate at day 60 was 34.5 percent (59/171), with 25 surrogates carrying 27 calves to term. Because of the small numbers of calves delivered at term, no differences could be detected among donor cell sources for live birth. Of the 27 calves born, eight died during the perinatal period, and another seven died at later stages. Seven of the calves died of causes associated with LOS (hepatic, cardiac, or gastro-intestinal defects, respiratory distress), and eight animals apparently died due to management errors. It is not clear what portion of the perinatal deaths were due to birth defects/respiratory failure or management errors. Birth weights of calves were not reported.

Some authors have suggested that the stage of the cell cycle may also influence cloning outcomes. However, results in different laboratories (Wells et al. 2003; Urakawa et al. 2004; Ideta et al. 2005) using cells in different stages have been mixed. Wells et al. (2003) compared putative G0 cells (cells that apparently were not dividing) to G₁ phase (cells that had begun dividing) cells for SCNT. They noted high early pregnancy losses, but no losses after 120 days of gestation, and no reported hydrops in the G0 group. In contrast, G₁ phase cells 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. In contrast to the Wells et al. study, Urakawa et al. (2004) reported success using fetal fibroblast donor cells in the G₁ phase. However, it should be noted that Urakawa et al. used only G₁ phase cells, and did not compare to other stages of development. Two cell lines were used, derived from fetuses with the same dam but two different bulls. Ten 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. Ideta et al. (2005) compared development of embryos constructed with G₁ or M phase fetal fibroblasts, and noted that G₁ SCNT embryos had higher rates of development to blastocyst than M phase cells (31 vs. 16 percent). 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.

Based on these studies, two of which used only embryos developed from G₁ phase cells, at this time it is not possible to determine the influence of the stage of the donor cell cycle on subsequent development of the embryo/fetus.

Embryo and Fetal Development. Early pregnancy failures in bovine clones are thought to be a function of incorrect reprogramming of the donor cell that manifest as lethal developmental defects (see Chapter IV). Some of those developmental defects may manifest as difficulties in placentation. For example, Hill et al. (2000b) noted that placentae from gestation day 40-50 clone embryos were hypoplastic (low cell density), and had poorly developed cotyledons (Hill et al. 2000b). (In ruminants, the cotyledon is the fetal part of the junction between the maternal and fetal sides of the placenta where nutrients and wastes are exchanged.) Additional placental anomalies in first trimester aborted fetal clones may include decreased numbers of placentomes (the junction of maternal and fetal components of the ruminant placenta that serve to transport nutrients into and waste out of the fetal environment), and poor formation of blood vessels in the placenta. In contrast, Lee et al. (2004) noted that although fewer cotyledons were present in SCNT placentae compared to AI and IVF placentae at day 50 of gestation, vascularization was very good, and appeared more developed in SCNT compared to AI or IVF placentae. Edwards et al. (2003) also studied this phenomenon in transgenic and non-transgenic bovine clones and observed that approximately 50 percent of transferred embryo clones established a pregnancy when measured by the presence of a heart beat between gestational days 29-32. This rate was compared favorably to that observed for non-clone IVF embryos. Edwards et al. (2003) noted that 50-100 percent of embryo clones spontaneously aborted between 30–60 days of pregnancy. Dindot et al. (2004) have noted more than 80 percent of hybrid bovine clone pregnancies (Bos gaurus X Bos taurus) were lost between gestational days 30 and 60. Evaluation of the early placental structures at gestational day 40 indicated an absence of cotyledons in each clone pregnancy, while the control (AI) fetuses had between 4 and 25 cotyledons per pregnancy). Pace et al. (2002), in a study that included transgenic clones, noted that the fetal abortion rate prior to gestational day 60 was 67 percent. A comparison of the crown-rump length of calved and aborted clone fetuses with AI-generated fetuses from gestational day 25 to gestational day 70 indicated that prior to abortion fetuses grew at the same rate.

Later pregnancy failures are thought to be a function of developmental defects, including placentation abnormalities. Heyman et al. (2002), for example, compared pregnancy loss between gestation day 90 and calving among clones derived from adult somatic cells, fetal somatic cells, blastomere nuclear transfer (BNT), and in vitro fertilization (IVF) animals. They noted that the somatic cell clones showed a pregnancy loss incidence of approximately 44 percent and 33 percent, while BNT clones were lost in only 4 percent of the pregnancies, and the IVF control group lost no pregnancies.

Abnormal placentation can, however, result in the birth of a viable clone (Hill et al. 2000b). In this case, one of six transgenic fetal clones detected at 40 days of gestation continued to develop to term, and when delivered vaginally weighed 37.7 kg, within the normal weight range for Holstein calves (35 to 45 kg). The calf was considered normal based on physical examination at birth. It suckled normally, and at the time of publication, was two years old and considered to be normal. The placenta was similar in weight for term Holsteins (4.3 kg vs. mean expected weight of 5.6 kg). Its structure, however, was highly abnormal, with only 26 cotyledons present, of which only 12 were judged to have been functional. These were enlarged, and the authors hypothesized that this increased size allowed the normal development of the calf. The authors also note that pregnancies resulting from IVF have also been reported to contain fewer placentomes⁵ than those of conventional cattle. As discussed in Chapter III, the role of transgenesis in the development of this pregnancy cannot be determined. Batchelder (2005), however, working with non-transgenic clones, also noted fewer and larger placentomes in placentae of eight live-born clones compared to AI and ET comparators.

   ⁵The structures involved in connecting the fetal and maternal tissues consisting of a cotyledon and a caruncle in the cotyledonary placenta. The cotyledons or chorionic villi are of fetal origin and "plug into" the caruncles or receptacles in the maternal uterine wall.

(b) Summary for the Embryonic/Fetal Developmental Node in Bovine Clones (Developmental Node 1)
This period manifests the highest degree of risk for the developing clone. The probability of an SCNT-embryo implanting, and the subsequent likelihood of an implanted clone embryo surviving and continuing to develop are low. Various investigators have attempted to understand the role of various components of the donor/recipient/cell culture system that comprises the “cloning unit” to improve efficiency with different sources of nuclear or oöcyte donors or by manipulating the culture conditions. These studies have been met with mixed results. Lack of success can be attributed to failure of the genome to be reprogrammed (Chapter IV), including failure of the embryo to begin dividing and implant in the uterus, and failure of development in the first trimester (likely due to defects in reprogramming that manifest as poor placentation or other defects that do not allow the fetus to develop), or physical damage to the early embryo. Difficulties that may persist in later pregnancy are largely associated with placentation anomalies that may co-develop with Large Offspring Syndrome (LOS) (see Chapter V). Nonetheless, some of these early embryos do divide, implant, develop, and give rise to live animals, as discussed in the subsequent Developmental Nodes.

ii. Perinatal Development in Bovine Clones (Developmental Node 2)

In the early studies of the technology, relatively high perinatal losses were reported. Deaths generally resulted from phenomena associated with LOS, including poor development of the respiratory and cardiovascular systems. (For a more complete description, refer to Chapter V.) In general, animals with LOS tend to have high birth weights (ranging from 20-50 percent greater than breed averages), poorly developed and sometimes edematous (fluid-filled) lungs and other tissues, and heart malformations and malfunctions. These animals may also have kidney and liver anomalies, and may initially exhibit difficulties in maintaining homeostatic functions such as body temperature and glucose metabolism. The latter are discussed in more detail later in this section. As the expertise develops, however, more animals are either born with no apparent defects, or have supportive care perinatally and survive to grow into healthy cattle.

(a) Peer-reviewed Publications

Most of the adverse outcomes that have been reported result in loss of the fetus before birth, although there is another period of loss after delivery, usually within the first few days of life. Reproducible sets of adverse outcomes have been observed, including LOS and gross morphological abnormalities that may result in pregnancy loss either early in gestation or late in gestation. For example, contracture of tendons has been noted in some clones. None of the abnormalities noted in animal clones are unique to animals derived by SCNT; all have been observed in natural reproduction, as well as in ARTs such as AI and IVF (reviewed by Cibelli et al. 2002 and Pace et al. 2002, and in Chapter V).

Despite the initial frequency of publications describing adverse outcomes of SCNT, two classes of successful outcomes actually predominate at birth. The first includes animals that may require assistance with delivery and immediate post-natal support in maintaining oxygenation and body temperature. Among others, Cibelli et al. (2002) noted that adverse effects associated with abnormal placental functions in the birth of a group of transgenic clones can be mitigated by intensive veterinary care immediately following birth. One bull clone described by Hill et al. (2000a) required considerable veterinary support immediately after birth due to respiratory problems (immature lungs and pulmonary hypertension), lack of suckling reflex, apparent Type I diabetes, and other health problems. According to this report, the calf improved rapidly, and the diabetes resolved (the calf was able to maintain normal blood glucose and insulin levels) by two months of age. This animal has fully recovered, and is reported to be a vigorous and healthy bull (PIFB 2003).

The second set of successful outcomes consists of those animals born with relatively little assistance (due to the high cost of developing animal clones, most are delivered via planned C section, and may require more supportive care than animals derived from more conventional breeding techniques), and appear to be normal and healthy (see especially the Cyagra database (Appendix E)). Although many reviews attribute the difference in birth weight to various degrees of LOS, higher birth weights may also be due to the greater care afforded surrogate dams carrying animal clones relative to standard husbandry of conventional animals. Alternatively, birth weight may be related to genetics of the nuclear donor. No data were found on birth weights of nuclear donors, but studies indicate that birth weight is heritable (Knight et al. 2001; Chapter V).

Forsberg et al. (2002) reported the production of 103 cattle clones, of which 47 were produced from non-transgenic cells and 56 from transgenic cells, including a Holstein bull calf generated by recloning an embryo derived from genital ridge cells. Of five pregnancies initiated from that recloning, two aborted prior to gestational day 30, one pregnancy was terminated at gestational day 203 due to hydrops, one set of twins died at birth due to the surrogate dam’s ketosis, and the fifth gave rise to “Gene,” the first cattle clone not produced from an embryonic cell line.  ⁶Little further information on Gene’s birth status, growth, or development is found in the peer-reviewed literature, except that as of the end of 2001, when the Forsberg et al. manuscript was accepted for publication, Gene had matured into “a healthy, fertile bull.” In a separate recloning trial described in this report, fibroblast cell lines derived from another fetal clone were used as donors to generate 28 blastocysts that were then transferred into 14 surrogate dams. Nine pregnancies were initiated. Four of those pregnancies went to term, and five calves (three singletons and one set of twins) were produced.

   ⁶The first publication describing the production of cattle SCNT clones appeared in Science in 1998 (Cibelli et al. 1998).  Gene’s gestation overlapped with Dolly’s and due to species differences in length of pregnancy, Dolly became the first SCNT clone born alive.

Forsberg et al. (2002) also used cells from adult animals as donors for SCNT. Ear cells from a bull (age and breed not specified) were used to generate 32 embryo clones that were transferred into 17 surrogate dams, of which 10 became pregnant. Five pregnancies were lost prior to gestational day 60, and two more were terminated due to hydramnios or hydroallantois (these conditions are also referred to as hydrops). Three live animals were born, but one was euthanized at 11 days of age due to a heart defect. In a separate trial described in the same paper, cumulus cells from an in vivo matured oöcyte from a 17 year old cow were used to initiate 11 pregnancies, from which three calves were born. Although information on the health status of many of these animals is not available, 15 of these animals were bred, gave birth, and their milk studied by Walsh et al. (2003) (See Compositional Analysis Method - Section 3).

In addition, Pace et al. (2002) of the same group reported on the development of 117 cattle clones from the reconstructed embryo stage through to lactation. These animals were born between January 1998 and February of 2000. Some of the cell lines from which these animals were developed were transgenic (Forsberg et al. 2002), and 75 percent of the resulting clones were transgenic. Because this report does not distinguish individual animals by cell source, it is not possible to determine which of the animals are transgenic. Interpretation of adverse outcomes should therefore be considered within the context of the discussion of transgenic animals in Appendix D. Of the 117 clone births, 106 were born alive, and 82 remained alive at the time of publication. Birth weights of the surviving clones ranged from 11-72 kg, with an average birth weight of 51 ?± 14 kg. The distribution of birth weights was skewed in excess of birth weight ranges for conventional Holsteins.

Pace and his colleagues (2002) divided the calf clone deaths into preventable and non-preventable causes (summarized in Table VI-1). Of the 24 animals that did not survive, 12 died between post partum days 1-5, nine died between days 6-122, and three died at more than 123 days of age. Many of the animals appear to have experienced complications resulting from enlarged umbilici, and three of the deaths were directly related to this condition. For subsequent births, this condition was managed by prophylactically tying or clamping off the umbilical arteries. Difficulties with the umbilicus were also observed at levels apparently higher than in conventional animals by Kishi et al. (2000); Gibbons et al. (2002); Cyagra (Appendix E); Edwards et al. (2003); and Batchelder (2005). Nonetheless, 77 percent of the clones reported on by Pace et al. (2002) in this study were alive and apparently healthy at the time of the study publication (2004). 

In another example of the successful production of clones, Chavatte-Palmer et al. (2002) reported on clinical, hematological, and endocrine characteristics of 21 apparently normal cattle clones and 16 abnormal SCNT-produced fetuses compared with similar outcomes in animals derived by AI (and summarized in Table VI-2). Initial measurements such as pregnancy outcome (e.g., abnormal development, stillbirth, live birth) and birth weight were also compared with IVF-derived animals. (Data were presented as summaries, and individual animal data were not presented.) Detailed discussion of the health outcomes of these clones are in the section describing the next developmental node (Juvenile – Developmental Node 3), as they extend from the perinatal period to approximately 50 days after birth.

In a follow-up study by this same group, including animals from the 2002 study (Chavatte-Palmer et al. 2004), the authors noted a 76 percent survival rate (44/58) among clones following the first week after birth. Causes of death during the neonatal 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.

Reports from research groups noting no differences between clones and naturally bred animals provide very few details about the health status of the clones. For example, Kubota et al. 2000 reported that although 30 blood measurements were taken on four clone calves, and that they observed no differences between the clones and their age-matched peers, neither the nature nor the numerical values of the measurements were provided.

Matsuzaki and Shiga (2002) evaluated the potential link between endocrine status and perinatal difficulties in Japanese Black clone calves delivered via C-section (selected by the investigators on the basis of a comparison of fetal size and maternal pelvic diameter, or rapidly expanding hydroallantois) relative to clones delivered vaginally, or Japanese Black calves produced via AI, and IVF calves born via spontaneous vaginal delivery. Birth weight, plasma cortisol levels, Adreno Cortico Tropic Hormone (ACTH), and components of the insulin-like growth factor signal transduction pathway (IGF) were evaluated. Average birth weights of clones delivered by C-section were heavier than AI controls; average birth weights of vaginally delivered clones and IVF animals were intermediate compared with C-section clones and AI control animals. Clones delivered by C-section had lower cortisol and IGF-I levels than AI and in vitro produced controls, similar ACTH levels, and had more IGF binding protein-2 (IGFBP2) relative to controls. The authors concluded that in C-section delivered clones the expected prepartum rise in plasma cortisol did not occur, and that these animals failed to initiate the switch to extra-uterine IGF-I system during late gestation. Four of five C-section delivered clones died within the first week following birth; one of the eight vaginally delivered clones died in that same time period of unspecified causes.

In their first study, Kato et al. (1998) reported that eight of 10 blastocysts derived by SCNT from a Japanese Black beef cow completed gestation and were born. Seven were delivered vaginally, while one was delivered by emergency C-section due to dystocia. Two of the calves were born prematurely. Four of the eight calves died. No abnormalities were noted, and the authors attributed the deaths to “environmental factors” as described in Table VI-3.

In a second publication, Kato et al. (2000) reported the production of 13 surviving clones of 24 deliveries of Japanese Black and Holstein donor cells. Pregnancy duration was approximately equivalent to that of the donor cell breed, except that “a few” recipient cows had shorter gestations. Calves were either born vaginally or delivered via C-section; no criteria were given for the decision to perform C-section. Seven animals were either stillborn or died at delivery. Two clones died during C-section due to dystocia, but presented no gross abnormalities. One clone born appeared normal at birth but died 19 days later from septicemia. Six dead clones had significant morphological abnormalities of the kidney or outer extremities, including severe tendon contracture. One clone was born disemboweled, and another had a “warped” face. All of these abnormal births were attributed to infection with Akabane virus, a known teratogen (birth defect inducer), as antibodies to the virus were detected in the serum of afflicted animals. Mean body weights of clones were higher than those of controls,⁷ with nine clones exceeding the mean body weight of controls by >40 percent. Interestingly, Kato et al. report on the unusual appearance of some male clones derived from a bull that was 10 years of age when cells were taken for donors in the SCNT process. At birth, the bull calves were reported to exhibit “an adult appearance, displayed as many wrinkles in the skin, thick bone structure and rough hairs resembling those of adult males.” They speculate that these might result from mutations in the donor cells that increase with age or to telomere length.

In the Kubota et al. (2000) study of clones from the 17 year old Japanese Black bull described in the Cell Fusion/Fetal Developmental Node (Developmental Node 1), two calves died shortly after birth, one of which was diagnosed as having Akabane Virus. The other died due to complications following a difficult delivery (dystocia). Four others survived, and were reported to be healthy and normal. The average gestation periods for the clone pregnancies was 294 days (range of 291-299 days), which was nine days longer than the breed average of 285 days. Average birth weight of the clones was 36 kg (range of 30.7 to 42.5 kg), approximately 20 percent heavier than the breed average of 30 kg.     

Kishi et al. (2000) used fibroblast cells from ear punches of Holstein or Japanese Black cattle, and somatic cells isolated from the colostrum of mammary gland epithelial (MGE) cells from Holstein cows as SCNT donors. Of the 45 embryos implanted into 31 recipients, three pregnancies were confirmed on gestation day 60, and two calves were born from colostrum derived MGE cells. One clone was delivered at 279 days of pregnancy by C-section and weighed 44 kg; the other was vaginally delivered after induction of parturition at 280 days of gestation and weighed 45 kg. For the fibroblast-derived clones, 43 embryos were implanted into 37 recipients. Five pregnancies were confirmed on gestational day 60, and 2 calves were born (one Holstein and one Japanese Black). The clone derived from the Japanese Black fibroblast died six hours after birth due to internal hemorrhage of the umbilical artery. Two of the Holstein clones (the origin of the cells is unclear) received blood transfusions due to anemia at some unspecified time after birth. The three remaining Holsteins (presumably including the post-transfusion clones) were reported as “normal and healthy.”

A series of papers (Taneja et al. (2000); Tian et al. (2000); Xu and Yang (2001); Enright et al. (2002); Govoni et al. (2002); Xue et al. (2002); Savage et al. (2003)) has been published on a group of female Holsteins cloned from a 13 year old cow by the laboratory of X. Yang at the University of Connecticut. Most of these studies report on the birth and later development of these calves, and are discussed in the sections appropriate to those developmental nodes.

In a meeting abstract, Taneja et al. (2000) described the premature delivery of 10 Holstein clones and the supportive care that they required. Normal gestation length for a Holstein averages 282 days (range 280 to 285 days). All the calves born in this study were premature (average gestation length 266.6 ± 2.0 days), regardless of whether labor was induced or occurred naturally. Three cows initiated labor spontaneously at 263.0 ± 3.8 days gestation. Twin calves born to one surrogate dam were stillborn, with one requiring manual delivery. One of the calves in the spontaneous labor group was delivered by C-section, showing signs of stress and hypothermia (body temperature <100ºF). This calf was hospitalized after 36 hours, when it began running a fever. A chest x-ray revealed immature lung development, and blood gas measurements indicated low blood oxygen concentration. The calf also underwent surgery for an umbilical abscess and for patent urachus (the canal connecting the bladder with the umbilicus) on day 6, after which it recovered and survived. The last calf born in the spontaneous labor group was delivered vaginally with some assistance, was diagnosed with immature lung development and low blood oxygen concentration; it died within 12 hours of birth. Necropsy of this calf indicated bacterial infection and septicemia, as well as immature lung development. The remaining five surrogate dams were treated with dexamethasone 17 hours prior to planned C-sections. Four single calves and a pair of twins were born in the induced labor group. Two calves were delivered vaginally without assistance at 8 and 15 hours post induction treatment. The first calf (born after eight hours) was healthy and did not require supportive care. The second calf (born after 15 hours) died three hours after birth; necropsy revealed that it had died of hypoxia and immature lungs. A set of twin calves and another single calf were delivered by C-section. One of the twin calves and the singleton survived, while the other twin and another single calf died soon after birth. Necropsy revealed that they had inhaled mecomium (the first intestinal discharge that normally occurs after birth that can appear in the amniotic fluid if the fetus is distressed) and the lungs failed to inflate completely. All but one of the surviving calves required supportive care ranging from supplemental oxygen to surgery. The four surviving clones were the subject of additional studies by this lab, including Enright et al. (2002) and Govoni et al. (2002). In the study by Xue et al. (2002) comparing the relative effectiveness of different cell types as donors for SCNT, four of the six calves from the ovarian cumulus group survived the perinatal period; all four of the calves born from donor skin fibroblast cells died. All deaths occurred within 24 hours of birth due to respiratory distress.

Batchelder (2005) reported on the birth of eight clones (three Hereford and five Holstein) and nine comparators produced by AI (n=3) or ET (n=6). She noted an interaction between cloning and cattle breed, such that Hereford clones were heavier (range 50.0 to 71.0 kg; n=3) than their breed-matched ET comparators (range 31.5 to 48.0 kg; n=3), while Holstein clones had similar birth weights to their breed-matched ET comparators (37.1 vs. 39.4 kg). Neonatal clones had lower RBC and hematocrit at birth and for the first hour, but were similar to comparators thereafter. Clones also exhibited lower blood glucose and lactate levels than comparators during the first 24 hours, but were similar to comparators by 48 hours. No differences were noted between clones and comparators for WBC and differential patterns. Although Batchelder noted several clinical signs often associated with LOS in both Holstein and Hereford clones (delayed time to suckle and stand, hypoglycemia, forelimb flexor tendon contracture, enlarged umbilicus, patent urachus, and respiratory distress), many of the same signs were noted in the AI-derived comparator group in this study (see Chapter V for more details). In this study all clones survived the first 48 hours after birth, but two clones were lost between 72 hours and six days of age. All comparator calves survived.

Wells et al. (2004) reported that a total of 133 clone calves were delivered as a result of 988 embryo transfers of somatic cell nuclear transfers (SCNT) using adult and fetal donor cells. Embryonic cloning resulted in 27 delivered clone cattle from 210 embryos derived from embryonic blastomeres (ENT). Both techniques were reported to result in a live birth success rate of 13 percent. Approximately two thirds of these calves survived to weaning (3 months of age).

Yonai et al. (2005) reported on the growth, reproduction, and lactation of clones whose nuclear donors were a high milk performance 13 year old Holstein and a six year old Jersey that had previously been used for embryo transfer. These animals had previously been characterized as having shortened telomeres, but are otherwise indistinguishable from cattle of presumably normal telomere length (Miyashita et al. 2002). (Discussions of growth and reproductive and lactational performance of these clones are found in Developmental Nodes 3, 4, and Compositional Analysis, respectively). Table VI-4 summarizes the success rates for the two breeds of dairy clones. All embryos, regardless of the breed of the donor cows, were implanted into multiparous Holstein surrogate dams. One of the recipients of Holstein embryos had twin calves. The overall success rates, as measured by surviving calves as a function of embryos implanted were approximately 5 and 10 percent for the Holsteins and Jerseys, respectively.

The authors state that although there is an approximately two-fold difference in the production rates between breeds, this difference is not statistically significant due to the low numbers in the study. The abortion rate in the surrogate dams carrying Holstein clones was approximately two times higher than the Jersey group (68.4 percent v 31.8 percent). No dystocia was noted in surrogates carrying Jersey clones; incidence of dystocia in the surrogates carrying Holsteins was not reported. The authors attribute the differences in outcomes to the smaller size of the Jersey fetuses relative to the Holstein fetuses. Gestational periods and birth weights were reported as being within normal ranges for dairy cows of these breeds. Although there was more variability in birth weights of the Holstein clones than the Jerseys, no symptoms of LOS were noted in these two clone cohorts. The authors note that although cell culture conditions have been implicated as a potential source of large calves, the two cell lines used for nuclear transfer were cultured under identical conditions, implying that differences between the cell lines (i.e., heredity) was likely responsible.

In summary, the survival rate of clones appears to be in the range of 5-18 percent, depending on how it is calculated. Many of the perinatal clones die of complications or sequellae of LOS. Newborn cattle clones may be more physiologically fragile than their comparators, and differences between clones and comparators include body weight, body temperature, alterations in the amounts of circulating IGF-II, leptin, growth hormone, T4, and differences in mean erythrocyte volume either on the day of birth or shortly thereafter. None of the differences between clones and AI- or IVF-derived controls persisted through the longest observation period (up to three months) (Chavatte-Palmer et al. 2002; 2004), and most resolved within a week or two of birth (Hill et al. 1999 (for transgenic clones); Enright et al. (2002); Govoni et al. (2002); and Tian et al. (2001)) (See subsequent discussions in the sections on the appropriate developmental nodes).

(c)           Unpublished data

In response to requests by CVM, various groups involved in cloning submitted unpublished data. One such group, a commercial cloning company, submitted body temperature, pulse and respiration rates on 19 cattle clones (breed(s) and gender not identified) during the first 72 hours of life. These data has been discussed in greater detail in Chapter V. Body temperatures were elevated during the observation period (mean 103°F at birth; 102.7°F at 72 hours); heart rates appeared to increase (95.2 beats/min at birth; 138.6 beats/min at 72 hours); while respiration rates remained fairly constant (53.9 breaths/min at birth; 53.1 breaths/min at 72 hours). It is often difficult to evaluate data on heart rate and respiration in livestock, since the stress of handling tends to increase these rates. Body temperature in neonatal clones appears to be quite variable, with some studies reporting hyperthermia (Chavatte-Palmer et al 2002; Batchelder 2005) which may persist through the first 50 to 60 days of life and then appears to normalize.

Another cloning firm presented birth records on two Holstein heifer clones delivered by C-section. The calves weighed 45 and 47.7 kg at time of delivery, within the normal range for Holstein cattle; body temperatures were 100 and 102.6°F at birth, slightly below and above normal (101.5°F) for cattle. These two calves were otherwise normal, according to the veterinarian’s notes and limited blood chemistry (See Chapter V for details).

(d)           Summary for Perinatal Developmental Node in Bovine Clones (Developmental Node 2)

The combined information from the peer-reviewed literature and the Cyagra dataset indicates that newborn clones tend to be more fragile than their comparators, with a higher incidence of perinatal death. Abnormalities noted among both dead and surviving clones include respiratory distress, organ malformations, flexor tendon contracture, and umbilical difficulties. None of the adverse outcomes observed are qualitatively different from adverse outcomes that have been observed in natural breeding or other assisted reproductive technologies. Some animals succumbed to infection, but there does not appear to be a decrease in immune function in the population of clones at the perinatal stage. Despite the perinatal deaths and noted anomalies, most clones that survive parturition, either with or without assistance, appear to stabilize.

“Sentinel” markers were sought that might predict a successful outcome for perinatal clone calves. Based on the literature and the Cyagra data, it does not appear that any one analyte or analyte profile is predictive of whether a particular animal, or indeed, the entire cohort of animals will develop into normal, fully functioning, healthy animals. The laboratory data are consistent with the hypothesis that animals that look and behave normally are normal with respect to laboratory values, implying that consideration of the complete dataset on an individual animal is the best predictor of the health of that animal. Further, the seven surviving Cyagra clones that were sampled twice (# 71, 72, 73, 78, 79, 119, and 132) provide the baseline data for a small subcohort of animals for which there are laboratory measurements at two different time points, as described more fully in the following section.

iii.               Juvenile Development in Bovine Clones (Developmental Node 3)

Most of the information on this developmental node has been extracted from publications that primarily address the perinatal period.

(a)           Peer-reviewed Publications

For purposes of following the cohorts of animals, these reviews have been grouped by institution.

The Institut National de la Recherche Agronomique (INRA) Studies: Renard et al. 1999 and Chavatte-Palmer 2002

Renard et al. (1999) reported one case of lymphoid hypoplasia in a clone generated from cells in an ear biopsy of an animal that had herself been the product of blastomere (or embryo) nuclear transfer (BNT). An echocardiogram performed on the animal immediately after birth revealed an enlarged right ventricle of the heart. The animal was treated with an angiotensin converting enzyme (ACE) inhibitor and given diuretics for one month, at which time the condition was reported to be resolved. Blood samples taken every two days after birth revealed relatively high reticulocyte counts and immature blood cells in the blood during the first three weeks of life. Lymphocyte (white blood cell) counts were also reported as normal for about a month after birth, but counts fell rapidly after that time. Hemoglobin levels in the animal also decreased at about day 40. On day 51, the animal died from severe anemia. Histological examination of the calf revealed hypoplasia (lack of development) of the thymus, spleen, and lymph nodes or global lymphoid aplasia (absence of lymphoid cells in all organs in which they would likely be found) that likely began at birth. No evidence for the endogenous synthesis of immunoglobulin G was detected. Bovine Viral Diarrhea virus, which has been known to induce thymic atrophy, was ruled out. SCNT was implicated as the cause of the lymphoid aplasia, possibly due to the selection of a cell with a mutation responsible for the expression of the portion of the genome governing lymphoid development, or lack of appropriate reprogramming of the somatic cell nucleus. In a follow-up study by this group (Chavatte-Palmer et al. 2004) an additional four clones were diagnosed with thymic aplasia. Histological examination of the thymus glands of these calves indicated abnormal tissue organization, suggesting the aplasia was the result of epigenetic errors. It is not clear from the late report whether these four clones were also the result of serial cloning. To our knowledge, this is the only laboratory reporting thymic aplasia in clones. Three other calves in this cohort died suddenly with few or no clinical signs: two died of diarrhea, and one died without any apparent cause.

In a separate report of the larger cohort of clones produced by the same laboratory (see Perinatal Developmental Node), Chavatte-Palmer (2002) monitored the growth and development of 21 clones. For the first week after birth, the mean rectal body temperature was higher in clones than AI controls, and some temperature spikes (up to 41º C; normal temperature is considered to be approximately 38.5 ºC in dairy cows) were observed. Elevated temperatures in the clones persisted for 24-36 hours, and were not sensitive to pharmacological intervention. Animals were cooled by wrapping in wet towels and providing ventilation, although they did not appear to be distressed during the temperature spikes. No bacterial infection was detected, and no changes in hematology or clinical chemistry were observed. The authors state that the mean temperature remained elevated for 50 days, although data are only provided for the first week. Thyroxine (T4) levels were tested to determine if they could help explain the temperature difference between clones and controls. Plasma thyroxine levels were lower in clones than controls during the first two weeks of life, and then reverted to normal levels. Chavatte-Palmer et al. (2002) noted that lower plasma T4 levels coupled with elevated body temperatures in young calves was consistent with the findings of Carstens et al. (1997). (See discussion in Chapter V on metabolism and body temperature.)

Although lower circulating IGF-I levels may be partially responsible for the later onset at puberty observed in this group of clones (Enright et al. 2002) (as IGF-I is involved in development of ovarian follicles and uterine growth (Le Roith et al. 2001)), the concentration of IGF-I required for normal sexual development is not known. Despite the reported differences in these protein levels, the clones appeared otherwise healthy and grew normally. Appendix F: Comprehensive Veterinary Examination discusses the relative weight that individual clinical chemistry values should have in the overall evaluation of the health of cattle. Interpretation of these results should occur within the context of that discussion.

Savage et al. (2003) evaluated the behavior of the clones and age-matched controls described in the Govoni et al. (2002) study. Between 32 and 36 weeks of age, there were no differences in weight or height between the clones (205.5 ± 9.9 kg; 117.0 ± 1.8 cm) and controls (211.4 ± 7.4 kg; 119.5 ± 1.4 cm). All calves were raised together under the same management conditions. Based on a series of studies evaluating approach to other animals and novel objects, clones exhibited age-appropriate behaviors, but were reported to be more aggressive and inquisitive than controls, and spent more time grooming and socializing. Clones tended to spend less time in playful behavior than controls. Review of records on the cow that served as the donor for the clones indicated that she had displayed similarly aggressive and inquisitive behavior as a young animal, suggesting that at least some of these behavioral traits may be genetically controlled. Clones spent more time in proximity to adult animals in an adjacent pen (which also housed the nuclear donor), and in proximity to the feed bunk compared to control animals. In general, clones were reported to spend more time with each other rather than socializing with control animals, with the authors speculating as to whether clones exhibit some form of genetic kinship recognition. Nonetheless, the overall conclusion of this study was that the clones behaved normally.

Other Studies

Wells et al. (2004) and Wells (2005) followed the growth and maturity of cattle clones generated at their facility in New Zealand through approximately four years of age. Approximately 80 percent of the clones delivered alive at term survived the first 24 hours of live. They reported that two-thirds of the 20 percent that died was due to spinal fractures syndrome or to deaths from dystocia, associated with LOS (Wells 2005). Another 15 clones died in the time period before weaning, most commonly of musculoskeletal abnormalities, including tendon contracture and chronic lameness, and umbilical infections, attributable to complications of LOS. They also reported two clones dying as the result of bloat, and an unspecified number of clones dying due to endophyte toxicity after eating fungus-infected ryegrass. Bloat and other gastrointestinal disorders have been reported by others (Cyagra 2003; Batchelder 2005), but also may result from feeding or grazing management problems. Wells et al. use the phrase “clonal family” to refer to clones derived from a particular donor, and note that the bloat and susceptibility to endophyte toxicity was restricted to one clone family, and likely due to their genetics. Another clone family consisting of three clones (and five half-siblings produced by AI) survived with no health anomalies and at the time of reporting was 18 months old. Other health problems observed during the juvenile period included anemia, chronic heart failure, and degenerative nephrosis, problems that have also been noted by other researchers (Chavatte-Palmer et al. 2004). Additional deaths were categorized as being due to misadventure and accidental deaths due to clostridial disease, parasitism, and over feeding. Surviving animals from this group were characterized with respect to general health and physiological measurements; these are found in the discussion of Developmental Node 5 (Post-Pubertal Maturation).

Similar to Chavatte-Palmer et al. (2002), Batchelder (2005) also noted periodic moderate to severe hyperthermia in Holstein and Hereford clones up to 60 days of age. As with the Chavatte-Palmer clones, the Batchelder clones also showed no indication of infection, were unresponsive to anti-inflammatory drugs, and their behavior was unchanged; the hyperthermia also resolved spontaneously.

In their study of Japanese Black beef cattle clones described in the section on the Perinatal Developmental Node, Kato et al. (1998) reported that all of the clones that survived the perinatal period were alive and healthy at 85 and 120 days of age. In the subsequent study (Kato et al. 2000) of 13 clones that survived the perinatal period, 12 clones were alive and healthy at 117-350 days, and one clone died at three months “for no clear reason.”

Kubota et al. (2000), in their study of four surviving clones of a 17 year old Japanese Black bull, reported that the clones were 10-12 months of age at the time of publication. Based on veterinary examinations, growth curves, and 30 blood parameters no differences were found between the clones and their age-matched peers. No data were provided in the publication. Other groups have also reported normal growth rates for cattle clones (Wells et al. 2004; Heyman et al. 2004).

Yonai et al. (2005) (previously mentioned in the Perinatal Developmental Node) studied the growth of Holstein (n= 6) and Jersey (n=4) clones with shortened telomeres. Clones were given at least two liters of warmed colostrum immediately after birth, fed colostrum twice a day for the first five days of life, and monitored for physiological functions until they stabilized. Clones were fed according to the guidelines presented by the US National Research Council Nutrient Requirements of Dairy Cattle (1989). From Day 5 through Day 45, calves were given milk replacer twice daily, and offered calf starter pellets, hay and water during this time. After Day 45, all calves (clones and comparators) were weaned from milk replacer, and their feed gradually changed from calf starter pellet to formula feed over a two week period. Calves were fed 2-3 kg/day of formula feed, hay and water from Day 60 until one year of age. For the first 45 days after birth, the clones were reared in individual calf huts, after which they were reared together with other calves produced by AI or embryo transfer. Calves were held in a large pen in mixed groups of clones and age-matched comparators during the weaning period. After weaning, groups of 10-20 animals were moved into pens, and after one year of age, all animals were moved to a