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Safety & Health

Chapter VI: Food Consumption (continued, part 2)

Yonai et al. 2005

Study overview: In the most comprehensive study of reproductive function in cattle clones, Yonai et al. (2005) (previously mentioned in other Developmental Nodes) performed an extensive analysis of reproductive performance in Holstein and Jersey clones with shortened telomeres, including puberty onset, estrus behavior, hormone cycling, the appearance of follicular waves, fertility and birthing for three estrus cycles. Once puberty onset had been determined, ovulation and formation of corpora lutea were monitored thrice weekly, with plasma samples to monitor progesterone levels collected every three days. After puberty, the estrous behavior of the clones was monitored twice daily until the animals became pregnant, with the length of the estrous cycles and occurrence of standing estrus recorded. Plasma samples and ultrasonography were used to identify follicular waves and monitor progesterone and 17-ß estradiol concentrations between day 18 of estrus and the day of ovulation over 17 estrous cycles in the Jersey clones and 28 estrous cycles in the Holstein clones. All clones were bred by artificial insemination using semen from the same lot of one bull (breed unspecified). Pregnancies were diagnosed by ultrasonography at 40 days after AI. For the first and second postpartum cycles, all clones were artificially inseminated at first estrus, which usually occurred 90 days after parturition. The length of gestation and resulting calves’ birth weights were recorded. Table VI 7 summarizes the data collected in this very detailed study.

 

Table VI-7: Reproductive Parameters Evaluated for Jersey and Holstein Clones
(adapted from Yonai et al. 2005)
Parameter Mean ± Standard Deviation
Jerseys (n = 4)
Age at puberty -
Reproductive records from puberty to first parturition
Length of estrous cycle 1 (days) 20.2 ± 1.4
Follicle waves per cycle 1 (number) 2.3 ± 0.8
Plasma estradiol-17 concentration on estrous day 2
Detectable (17/17 cycles; pg/ml) 8.12 ± 2.40
Not detectable (0/17 cycles; pg/ml) -
Plasma progesterone under the curve 3 (ng/ml per cycle) 190.6 ± 59.4
Number of AI for first conception 2.3 ± 1.9
Age at first conception (days) 503 ± 54.9
Gestation period (days) 279 ± 2.5
Calf weight (first parturition) (kg) 22.0 ± 2.1
Reproductive records after first parturition
Interval from parturition to first ovulation (days) 51.3 ± 42.8
Interval from parturition to first estrus (days) 85.0 ± 52.7
Number of AI for second conception 1.3 ± 0.5
Interval from parturition to second conception (days) 115 ± 16.8
Age of second conception (days) 897 ± 44.8
Calf weight (second parturition) (kg) 26.4 ± 1.1
Reproductive records after second parturition
Interval from parturition to first ovulation (days) 32.5 ± 19.3
Interval from parturition to first estrus (days) 50.0 ± 27.8
Number of AI for third conception 1.5 ± 1.0
Interval from parturition to third conception (days) 129 ± 49.9
Age of third conception (days) 1,304 ± 46.6
Holsteins (n = 6)
Age at puberty 323 ± 0.6
Reproductive records from puberty to first parturition
Length of estrous cycle 4 (days) 20.3 ± 1.5
Follicle waves per cycle 4 (number) 2.3 ± 0.7
Plasma estradiol-17 concentration on estrous day 5
Detectable (19/28 cycles; pg/ml) 6.94 ± 2.64
Not detectable (9/28 cycles; pg/ml) 3.95 ± 1.74
Plasma progesterone under the curve 6 (ng/ml per cycle) 154.0 ± 58.0
Number of AI for first conception 2.0 ± 2.0
Age at first conception (days) 481 ± 35.0
Gestation period (days) 277 ± 5.8
Calf weight (first parturition) (kg) 37.8 ± 5.0
Reproductive records after first parturition
Interval from parturition to first ovulation (days) 56.0 ± 41.5
Interval from parturition to first estrus (days) 86.0 ± 33.0
Number of AI for second conception 1.2 ± 0.4
Interval from parturition to second conception (days) 126 ± 41.7
Age of second conception (days) 881 ± 61.7
Calf weight (second parturition) (kg) 44.2 ± 1.9
Reproductive records after second parturition
Interval from parturition to first ovulation (days) 79.3 ± 18.9
Interval from parturition to first estrus (days) 92.3 ± 19.2
Number of AI for third conception 1.3 ± 0.5
Interval from parturition to third conception (days) 138 ± 34.9
Age of third conception (days) 1,297 ± 75.0
1 Twenty-six estrous cycles in four cloned heifers were included.
2 Plasma samples were collected from 17 estrous cycles in four cloned heifers.
3 Plasma samples were collected every three days during the 26 estrous cycles.
4 Thirty-three estrous samples in five cloned heifers were included.
5 Plasma samples were collected from 28 estrous cycles in five cloned heifers.
6 Plasma samples were collected every three days during the 33 estrous cycles.

Reproductive function: First Estrus: Yonai et al. grouped their analysis of reproductive function into three stages: pubertal, post-pubertal conception and gestation, and post-parturition, including rebreeding. Although some of the clones entered puberty prior to the initiation of this stage of the study, Yonai et al. reported that changes in plasma progesterone were consistent with previous reports on puberty in conventional cows. They also reported that corpus luteum formation was consistent with that reported in conventionally bred cows, and that the clones exhibited appropriate estrous behavior at puberty. Overall, the observations at puberty indicated that these clones exhibited normal early reproductive development. With respect to post-pubertal maturation of the heifer clones, Yonai et al. noted that there was some difficulty detecting estrus by behavior in the Holstein heifer clones, and that there were differences in their estradiol levels, these were consistent with similar observations in conventionally bred Holstein heifers. There were no difficulties in observing estrus in the Jersey clones. Estrous cycle lengths in both clone lines were comparable to those observed in conventionally bred cattle. Additionally, the levels of progesterone secretion per cycle were reported as similar to those of conventionally bred heifers, which the authors interpreted as normal post-pubertal corpus luteum function. They conclude that the estrous cycles of the heifer clones were normal.

All of the heifers conceived upon artificial insemination, although one heifer clone and one comparator needed multiple cycles of insemination; the remaining clones and comparators all conceived after no more than two rounds of AI. All of the clones but one Holstein delivered healthy, live calves. The exception delivered a stillborn calf two weeks before expected parturition. No obvious abnormalities were observed in the stillborn. Two of the Holstein clones required limited assistance for delivery; the remaining Holsteins and all the Jersey clones did not require any assistance in delivery. The average gestational periods were normal for the clones and all of the resulting calves were within normal body weight ranges for their breeds. All the live-born calves were reported as being normal.

Second and Third Estrus. Yonai et al. noted a wide variation in the interval between parturition and first post-partum ovulation and estrus. The first postpartum ovulation in the Holstein clones occurred between 14 and 188 day (Table VI-7), and between 11 and 108 days in Jersey clones; the interval between parturition to first estrus was between 62 and 149 days for the Holstein clones, and 30 and 135 days for the Jersey clones. All clones had confirmed follicular waves, and pregnancy ensued in all of the clones following an average of 1.2 and 1.3 rounds AI for the Holstein and Jersey clones, respectively. The second parturition was largely uneventful for all of the clones, with one Holstein requiring minimal assistance calving. Gestation times for the all of the clones fell within normal ranges for the breeds; all of the calves had normal body weights, appeared to be normal at birth, and survived. Similar responses were noted for the third conception.

 

Table VI-8: Results of Milk Yield in First and Second Lactations of Jersey and Holstein Clones
(adapted from Yonai et al. 2005)
Animal Milk Yield
Jerseys (n = 4)
First Lactation
Clone 1 5,637.4
Clone 2 6,077.9
Clone 3 6,272.6
Clone 4 5,597.7
Mean ± Standard Deviation 5,896.4 ± 332.0
Donor Animal 5,064.0
Second Lactation
Clone 1 7,006.8
Clone 2 7,539.2
Clone 3 7,309.6
Clone 4 7,195.6
Mean ± Standard Deviation 7,262.8 ± 222.6
Donor Animal 6,087.0
Holsteins (n = 6)
First Lactation
Clone 1 8,591.2
Clone 2 9,219.5
Clone 3 9,586.5
Clone 4 9,836.0
Clone 5 9,029.1
Clone 6 9,735.6
Mean ± Standard Deviation 9,333.0 ± 476.4
Donor Animal 10,968.0
Second Lactation
Clone 1 10,678.6
Clone 2 12,402.6
Clone 3 11,341.4
Clone 4 10,376.0
Clone 5 10,110.2
Clone 6 12,719.4
Mean ± Standard Deviation 11,271.4 ± 1084.7
Donor Animal 11,442.0


Milk Production. Table VI-8 summarizes the yield of milk produced by the clones and their half-siblings and donor for the two lactation cycles following the first and second calvings. Data on the composition of this milk are addressed in the Food Composition portion of this chapter. Milk yield, although varying among the clones, was within the normal range for each breed for each lactation cycle. Interestingly, the Holstein clones produced less milk on average than their nuclear donor animal, while the Jersey clones produced more milk on average than their nuclear donor. The authors reported that mastitis was observed in the Holstein group of clones in two animals towards the end of the lactation cycle, and bloat was observed in two clones (not specified if the same animals) at approximately 130 days post-parturition. Neither was observed in the Jersey clones. Although not specified, the affected animals were most likely treated, and appear to have recovered as the number of animals did not change between cycles.

This study, which is the first to study multiple cycles of reproductive function in any species of clone provides detailed information on both the individual physiological parameters measuring growth and reproduction (including lactation), as well as integrated measures of those functions. The authors conclude that despite the observation that all of these clones had shortened telomeres, these Holstein and Jersey clones exhibited normal growth, reproductive and lactation characteristics.

Other Studies

Although Lanza et al. (2001) reported on transgenic clones, conception rates for female clones after AI were high, with 87.5 percent of the animals conceiving on the first insemination and the remainder conceiving on the second insemination attempt. The two transgenic clones that had given birth, as of the publication date, were reported to have delivered calves that appeared normal in all respects, although no specific data are provided.

Pace et al. (2002) reported that heifers began to display signs of reaching puberty at 10-11 months of age, within the normal age range of conventional Holstein heifers (9 to 12 months). They further report that all of the heifer clones that were inseminated (n=22) became pregnant, and calved at the age of 23-25 months, similar to non-clone cattle (approximately 75 percent of the cattle in Pace et al. (2002) were transgenic). No specific information on gestation length or health of the progeny was provided. Analysis of the milk from non-transgenic clones of this cohort (Walsh et al. 2003) is presented within Section 3 of this Chapter.

In an abstract, Aoki et al. (2003) present a preliminary report on the milk and milking behavior of two first-lactation Holstein clones derived from somatic cells isolated from the colostrum of mammary gland epithelial (MGE) cells described by Kishi et al. (2000), previously discussed in the Perinatal section. These two clones were housed near the same automatic milking system as eight second-lactation control cows produced by AI. Comparisons were made between first lactation clones and second lactation controls. These cow clones were apparently followed for at least two calvings, and results were reported for the first through third post-partum ovulation and follicular development per estrous cycle. First postpartum ovulation was delayed in both of the clones, as well as the interval between the first to second postpartum ovulation. Clones were reported to have had two waves of follicular development per cycle. Both clones and comparator cattle were reported to calve normally, and did not appear to have different body weights and body condition scores, although no data were provided. The authors did not report differences between gestation length and duration of estrous cycle. They concluded that the clones were “normal in regard to delivery, lactation, and growth, and were similar in regard to the functions of their reproductive physiology.” Differences were observed, however, in the milking behavior, including the number of times that they voluntarily entered the automatic milking system relative to controls. In general, first lactation animals lack experience with milking equipment, and produce less milk than second and later parity cows, which likely contributed to differences in milking behavior between the two groups (Vasconcelos et al. 2004; Flis and Wattiaux 2005). Given that this is an abstract, the number of animals is very small, and the difference in the total number of lactation cycles the cows had experienced, the significance of the observation is unclear. Presentation of these data in a complete publication would aid this risk assessment and other analyses of clones.

Heyman et al. (2004) reported that first lactation milk yields (9,341 ± 304 kg vs. 8,319 ± 1,800 kg for a 305 day lactation) and somatic cell counts (SCC), which are a measure of mammary gland health) for three female Holstein clones were similar to those of three age-matched non-clone comparators. Somatic cell counts for both clones and comparators (116 ± 103 x 103 vs. 113 ± 50 x 103) were well below the level indicative of subclinical mastitis (1,000 x 103), and the SCC limit cited by the Pasteurized Milk Ordinance for fluid milk entering commerce.

Reproductive Function of Male Clones

The reproductive function of male bovine clones has also been studied. Wells (2005) reported on the reproductive function of six bulls cloned from the same steer. The rates of in vitro embryo development following fertilization of abattoir-derived oöcytes using sperm from these sires varied among the sires, but the development of blastocysts to quality grades suitable for embryo transfer were similar to that for four comparator bulls (10-25 percent for the clones and 13-30 percent for the comparators). Likewise, Heyman et al. (2004) reported that three clones of an eight year old bull were enrolled in an AI center, and semen was collected when the clones were between 13 and 15 months of age. Percentages of normal sperm, cleavage rate and blastocyst rate following IVF were not different between the clones and their nuclear donor. Results of AI trials were only presented for one clone (no comparator). Forty-one cows became pregnant out of 63 animals inseminated, yielding a 65 percent pregnancy rate. Two pregnancies were lost by day 90 (5 percent loss). Only 26 pregnancies were allowed to go to term, yielding 25 live, healthy calves and one stillborn.

Shiga et al. (2005) reported on the semen quality of two clones of a 12 year old Japanese Black bull. Semen was collected over a four month period beginning when the clones were approximately 12 months old. Comparisons were made using frozen semen from the nuclear donor and using averages for the breed. Although ejaculate volumes of the two bulls were lower than the range for the breed (2.34 and 2.76 mL vs. 5-8 mL), sperm concentration, pH, and pre-freezing motility were within established ranges for Japanese Black bulls. Development of IVF embryos to the blastocyst stage was not different between clones and their nuclear donor (23.4 and 28.4 vs. 30.9 percent). Semen from one of the clones was used to inseminate 22 cows, compared to 102 cows inseminated by the nuclear donor. Pregnancy rates were similar between the clone semen and semen from the nuclear donor (54.5 vs. 62.7 percent). Two of the 12 (17 percent) resulting pregnancies from the clone aborted spontaneously in mid-pregnancy, compared to 5/64 (8 percent) abortions among the cows bred by the nuclear donor.

Semen and reproductive profiles of 3 cloned Holstein-Friesian bulls were reported by Tecirlioglu et al. (2006).  Development of the reproductive organs and scrotal circumference were reported to be normal in the clones. Semen was collected from these clones at 16-18 months of age.  Sperm morphology was similar between clones and their nuclear donors. One bull clone had lower motility of spermatozoa in fresh semen compared to its nuclear donor. Sperm velocity parameters in fresh semen were higher in clones compared to donors, but this difference was not observed in frozen-thawed semen. When used for in vitro fertilization, semen from clones resulted in higher cleavage rates (= 85 percent in clones vs. = 66 percent for donors).  The proportion of cleaved embryos that developed into blastocysts and the quality of blastocysts, however, were similar between clones and their donors. There were no differences between clones and donors in either pregnancy rate or offspring rate, but the authors point out that their animal numbers (a total of 26 calves sired by clones and donor bulls) were too small to conduct a valid statistical comparison for these parameters. No phenotypic abnormalities were observed in any of the calves sired by the clones.
Circulating testosterone concentrations in the three clone bulls (26.5 ± 2.6 nmol/L) were higher compared to age-matched controls (6.1 ± 1.4 nmol/L).  Reasons for this difference are unclear, but Tecirlioglu et al. (2006) speculate that testosterone concentrations in control bulls may have been suppressed by elevated glucocorticoids due to handling stress whereas clones were more accustomed to blood sampling and interaction with humans.  While the authors considered the results of this study to be preliminary due to the limited numbers of animals and traits investigated, they concluded that the reproductive potential of cloned bulls is not different from their genetic donors.

(b)  Unpublished data

Semen evaluations on four healthy post-pubertal clones derived from an Angus-Chianina nuclear donor cross were shared with CVM (Chapter 5, Table V-17). Semen was collected by a commercial reproduction service from May through June 2003, three times daily, the usual industry practice. The age of the bulls at the time of collection was not recorded. Semen evaluation showed that one clone had a low sperm concentration (average 169.5 x 106 cells/ml vs. the normal range 800-1,200 x 106 sperm/mL (Sorenson 1979; Beardon and Fuquay 1980; Hafez and Hafez 2000)) and low percentage of normal sperm (between 2 and 8 percent) during the observation period. This bull likely would have failed a breeding soundness exam, and if it had been a conventional animal, it would most likely have been sold to a feedlot for eventual slaughter. A second bull clone had marginal semen quality, and might have been retained depending on the perceived value of his genetics. The remaining two clones exhibited acceptable semen characteristics, and would likely have been retained for breeding.

Galli et al. (unpublished data) also presented data on breeding soundness and performance of three clones of a Holstein bull (Chapter V, Table V-10). Breeding soundness exams indicated that clones were acceptable for breeding. Artificial insemination trials using semen from one of the clones on four farms resulted in pregnancy rates ranging from 33 to 80 percent; however, few cows were actually bred (n=63 for all farms combined), there were no contemporary comparators used, and no details regarding farm management were provided, making these data difficult to interpret. Pregnancy rates to AI for this clone were within the range of the U.S. average for Holstein cattle.

(c) Summary Statement for Reproductive Development and Function in Bovine Clones (Developmental Node 4)

Although specific animals are rarely cited, all reports of reproductive function in bovine clones appear to indicate that the animals respond normally to developmental signals governing puberty onset and that they subsequently reproduce effectively. The results of the study by Yonai et al.

(2005) provide further confidence by reporting on detailed physiological parameters required for successful reproduction, and demonstrate that the clones continued to cycle and function normally after the first pregnancy. The studies of lactation and milk yield indicate a consistent response demonstrating that these animals function normally post-partum and during subsequent reproductive cycles. Reproductive failure is a common phenomenon in conventional cattle, and among one of the most frequent causes for culling. Although cases of reproductive failure have been reported among clones, they are not unusual among conventional cattle, and do not raise food safety concerns. Reproductive function is among the most tightly regulated functions that a mammal performs; the demonstration that clones can reproduce normally appears to indicate that those clones are functioning normally for this biological criterion.

v. Post-Pubertal Maturation in Bovine Clones (Developmental Node 5)

(a) Peer-reviewed Publications

Post-pubertal maturation includes the very long period of time between the development of reproductive capacity and the natural end of the animal’s life. Most cattle in US agriculture never reach the end of their “natural” life-spans for economic reasons. In commercial dairy establishments, dairy cows are sent to slaughter some time between the end of their third to fifth lactations, or sooner, depending on their health and productivity. Beef cattle that are not being used for breeding are generally sent to slaughter when they reach about 1,000 to 1,400 lbs, or at approximately 18 to 24 months of age (depending on breed, season, environmental conditions, etc.). Most of the possible food consumption risks arising from edible products of clones (e.g., milk or meat) would occur during this Developmental Node.

We have not conducted a survey of clone producers or the investigators who have published on the health status of clones earlier in the clones’ lives to determine their vital or health status. At this time, there are economic disadvantages to maintaining healthy clones without being able to realize financial investments, so many otherwise healthy clones have been euthanized. The following discussion therefore summaries reports that have been obtained from the literature, and tends to focus on anomalies that have been noted.

Kato et al. (2000) reported that as of September 1, 1999, all of the surviving clones from their Holstein and Japanese Black cumulus cell and fibroblast donors were healthy and aged 117-350 days. No further publications were found regarding the fate of these animals.

Because of the relatively short time that cloning has been practiced, (Gene, the first bovine SCNT clone was born in 1997 (Cibelli et al. 1998)), little information is available on animals during this developmental phase, and much of that information comes in the form of single sentences or short mentions in journal articles that address some other issue. Abnormalities that have been noted in mature cattle clones appear to be sequellae of anomalies or defects noted earlier in life, and may be related to LOS or other earlier diseases. For example, Batchelder (2005) reported that one clone died suddenly at 25 months of age. Necropsy results indicated severe trace mineral deficiency (selenium and copper) as the cause of death. None of the non-clone cattle grazing the same pasture developed signs of mineral deficiencies. Nonetheless, this particular clone was reported to have exhibited frequent but mild signs of bloat as a juvenile, and it is possible that its subsequent death may have been the result of gastro-intestinal tract problems resulting in reduced ability to absorb micro-nutrients. The two surviving clones were reported as healthy at 19 months of age.

Second Chance, the Brahman bull clone described by Hill et al. (2000a), has been outlined in detail in the preceding section. The researchers speculate that the early diabetes had resolved at eight months of age and the calf was clinically normal. At a conference in September of 2002, the bull was reported to be 3 years of age, with normal weight, growth, behavior, and normal semen production. The investigator presenting this information also reported that the bull’s glucose level was elevated, although they could not rule out the role of stress resulting from medical procedures as a cause (Westhusin in PIFB 200390 ). In a subsequent conversation, Dr. Westhusin indicated that the blood glucose has remained within normal limits since the previous report.

Lanza et al. (2000) reported on 24 sexually mature transgenic bovine clones. Physical examinations were reported as normal including temperature, pulse, respiratory rate, general appearance, lymph nodes, and abdominal palpation. Blood and urinalysis indicated that in general, those variables were within normal ranges although six animals had total urine protein levels slightly below the comparator average. Studies with adaptive T-cell responses indicated that these transgenic clones had functional immune systems, and that the animals responded to periodic infection in the same manner as conventional cattle.

Pace et al. (2002) measured weight gain in their transgenic clones until the age of 540 days. Although comparison of the overall cohort with any comparator group is difficult because the clones were raised at different facilities, 52 of the clones raised at the same facility had similar weight gain over the first 120 days of life (approximately 1.15 kg/day). Weight gain of 17 clones from the same genetic line declined to 1.09 ± 0.14 and 0.92 ± 0.10 kg/day at 365 and 540 days, respectively, entirely consistent with weight gain profiles of conventional animals.

Yonai et al. (2005) reported on the growth characteristics of six Holstein and four Jersey clones with shortened telomeres from birth through two years of age. Those data have been summarized in Table VI-6. Evaluation for clones aged 12-24 months indicates that animals had normal weight gain for their breeds, indicating their overall health. With the exception of brief mentions of bloat and mastitis, no other illnesses were reported in this study. All of the animals that entered the study were alive at the time the manuscript was submitted for publication.
Wells et al. (2004) have reported that clones produced at AgResearch have an overall annual mortality of eight percent over four years. Most of the mortality observed appears to be due to the sequellae of LOS or accidents or mishaps; no contemporaneous comparator exists. They also note that one clonal family and their half-siblings were all alive and healthy at 18 months of age, implying that there may be an association between the cell line used, susceptibility to LOS and its sequellae.

The immunologic competency of three Holstein bull clones was tested using skin allografts (Thoret et al. 2006). Skin grafts were chosen for this study because skin is the most immunogenic transplantable tissue and is easily obtained with little harm to the animal. The bulls were cloned using fibroblasts from 60-day fetuses as donor cells. At 18 months of age, the bull clones received a skin graft from an unrelated, 12-month old Holstein bull. Each clone also received a skin graft from the other two clones in the cohort. Grafts were placed along the back and left in place for 13 weeks. No immunosuppressive therapy was administered, and grafts were examined both macroscopically and microscopically via punch biopsies.  In every case, skin grafts among clones were accepted, but third-party grafts from the unrelated bull were rejected. 
These results indicate that genetically identical adult bovine clones are immunologically compatible and sufficiently immunocompetent to be able to recognize and reject allografts from genetically unrelated cattle.

Immune function in cow clones was also investigated by Tanaka et al. (2006). Numbers of granulocytes, monocytes, B cells and several T cell subsets were measured in blood obtained from six Holstein clones and five age-matched, sexually-derived Holstein cows during early lactation (58-62 days post-calving) and mid to late lactation (179-300 days post calving). Cows were 2-4 years old at the time of sampling.  Proportions of granulocytes, monocytes, B cells and most T cell subsets were similar in cloned and normal cows.  At calving, there is normally a decrease in the proportion of WCl+?d T cells.  In this study, the percentages of ?d ?and WCl+?d T cells were significantly lower during early lactation in clones compared to controls.  By mid to late lactation, both T cell subsets recovered and were no longer different from those in normal cows. As WCl+?d T cells produce interferon-??in response to several bovine pathogens, the authors suggest that decreased populations of these cells may cause cow clones to be more susceptible to infection during early lactation.  It is important to note, however, that the milk yield of these clones (11,731 ± 1,397 kg/300 days) was significantly higher than that of the comparators (9,577 ± 1,960 kg/300 days). T cells migrate from the blood into the mammary gland during lactation, and there is evidence that this migration may be selective for certain subsets of T cells.  Thus it is possible that the observed decrease in WCl+?d T cells found in the milk of the clones in clones in this study was related to their higher milk production, resulting in a greater influx of these cells from the blood into the mammary gland. 

Tecirlioglu et al. (2006) measured 13 biochemical parameters in blood collected from three post-pubertal, Holstein-Friesian bull clones. Blood samples were collected weekly for three weeks, and values were compared to age-matched controls housed under similar conditions at the same farm. Although serum concentrations of calcium, protein, albumin, aspartate aminotransferase, creatine and testosterone were higher in clones compared to controls, and glutamate dehydrogenase was lower in clones, because all these values were within the normal reference ranges for cattle at the laboratory where the analyses were done, the authors considered these differences to be minor.

As part of a three year study of clones at INRA, Heyman et al. (2007) reported on the health and development of cloned Holstein heifers from four to 36 months of age. Data describing the post-pubertal period will be discussed here; other findings from this study are described in the Juvenile and Reproductive Development sections. The authors reported that repeated clinical evaluations revealed no differences in cardiovascular, respiratory, and locomotive functions, clinical biochemistry, or immune parameters through 36 months of age (data not shown). Concentrations of plasma cortisol and urinary epinephrine and norepinephrine, measured every two months, and were similar between clones (n=5) and controls (n=5) during the postpubertal period.  Cortisol concentrations were more variable among the clones, however, than the comparators. These results do not support the hypothesis that clones are more sensitive to stress than conventional animals, and the authors conclude that the clones in their study were not suffering from chronic stress. 

Extensive blood chemistry and hematological analyses were conducted in 11 cloned cattle and 11 sexually produced comparators produced at the University of Connecticut (Yang X et al. 2007b).  Ages ranged from “>12 months” to 43 months. The six females and fives males in each group represented several breeds: Angus, Brangus, Holstein, and Red and White Holstein, and cross-breeds.

Blood samples were collected at the time of slaughter and analyzed at the Cornell University College of Veterinary Medicine Diagnostic Laboratory. No differences between clones and comparators were observed for the 24 blood chemistry parameters evaluated or the 16 hematological parameters. For “essentially all” parameters examined, standard deviations of the means were similar for clones and comparators, implying that the variability in blood chemistry and hematological measurements within the populations of clones and non-clones in this study were similar.

Cyagra Dataset: 6-18 Month Cohort

The oldest cohort of Cyagra animals spans 6-18 months of age, and actually overlaps the Juvenile and Post-pubertal Maturation developmental nodes. Clearly, the younger clones in this cohort have more in common with the older, but still juvenile, animals of the preceding cohort, while the older clones are more appropriately considered as nearing “adulthood.”

The 6-18 month Cyagra clones were virtually indistinguishable from the comparators. None of the animals had any visible anomalies on physical examination (See Appendix E for details). The laboratory values derived from blood samples drawn from the clones are virtually superimposable on those of the comparators. Only three of the 294 hematological values and six of the 592 clinical chemistry measurements were outside the clinically relevant range. In aggregate, 99 percent of the laboratory measurements were within the clinically relevant range established by the comparators.

Review of Chart E-301 indicates that only two analytes initially appeared to marginally exceed the range characterized by the comparators: estradiol-17ß (E2), and insulin-like growth factor-1 (IGF-I). Neither of these findings was judged to pose clinical significance for the animals or any food consumption risk. Although the E2 levels of five animals exceeded the comparator range, none exceeded the Cornell Reference range, which as previously discussed, is derived from adult cattle. By comparison, 14 of the 20 comparators had measurements that were lower than the Cornell Reference Range (Chart E-302). For a more complete discussion of the normal fluctuation of E2 levels in cattle, see Appendix E. IGF-I levels in the Cyagra cohort were slightly higher in males than in females, and in three of the bull calves (# 24, 33, and 35) were slightly increased (less than 10 percent) relative to the comparator Group. Review of the literature on IGF-I levels in cattle indicated that basal circulating levels of IGF-I vary with a range of factors and fluctuate dramatically among individual animals in herds (Vega et al. 1991). Plasma concentrations of IGF-I are strongly influenced by a number of factors including gender, age, and diet (Plouzek and Trenkle 1991 a,b). The primary nutritional determinants of basal IGF-I levels appear to be crude protein and the number of calories absorbed by the animal (Elsasser et al. 1989). Given that most non-transgenic clones are derived from animals of superior genetic merits for traits such as growth and development, 10 percent elevations in IGF-I levels are likely of no clinical significance for the animal, and pose no food consumption risk.

No remarkable dissimilarities were noted in the blood variables of clones and comparators. There were no indications of problems with respect to red or white blood cell measurements. One animal (Clone #98) exhibited higher basophil counts than the comparator range, but there appeared to be no clinical correlate to that value, and as a result it was judged insignificant to the health of the animal or food safety.

(b) Unpublished data

Hematology data for two Holstein heifer clones aged 14 months old were submitted to CVM by a private veterinary firm. They consisted of a Veterinary Certificate of Inspection, results of serological testing showing the animals were free of Bovine leucosis virus and Bovine viral diarrhea, and standard clinical chemistry and hematology panels. All hematology and clinical chemistry results were within the range of the laboratory’s reference values except red cell distribution width, which was slightly below the reference range used by the testing laboratory (see Chapter V). As discussed in Chapter V and Appendix E, RDW is a secondary indicator, and does not on its own suggest a health problem. Certificates of Veterinary Inspection accompanying the hematology data indicate that both heifers were healthy.

(c) Summary Statement for Post-Pubertal Maturation in Bovine Clones (Developmental Node 5)

Clones in this age group exhibited no remarkable differences from non-clones with respect to their overall health. The Cyagra clones were indistinguishable from the comparator group on the basis of clinical and laboratory tests. The study of Yonai et al. 2005 indicates that clones continued to grow well for the duration of the study (two years). No residual health problems were noted in any of the clones in this Developmental Node that had not been identified in earlier developmental nodes. Some clones died prematurely for different reasons, including the sequellae of earlier disease. Individual animal reviews indicated no health problems, or changes in physiological parameters that would indicate a food consumption risk that would not be detected in existing food safety regulations (e.g., mastitis in milking cows).

vi. Progeny of Bovine Clones

From a food safety perspective, information on the progeny of clones is probably more important than data on the clones themselves because it will be primarily the progeny, not the clones, that enter the food supply.

Starbuck II Progeny

Ortegon et al. (2007) studied physiological parameters and telomere length in a group of seven Holstein heifers sired by a bull clone, Starbuck II. Dams were conventionally bred normally cycling Holsteins. Starbuck II progeny were monitored monthly for 12 months, from weaning to puberty, and data were compared to those from a group of breed- and age-matched comparators. At 14-15 months of age, five of female progeny heifers were inseminated with semen from a conventionally bred bull.

The Starbuck II progeny heifers appeared phenotypically normal and were healthy throughout the observation period. There were no differences between the clone progeny heifers and comparators in growth parameters (body weights, height, length) or several reproductive endpoints (range in age of puberty, serum progesterone concentrations, ovarian follicular dynamics, estrous behavior). Starbuck II heifers also responded normally to drugs commonly used in cattle for estrous synchronization (prostaglandin F2a) and superovulation (follicle stimulating hormone). Clinical chemistry values in Starbuck II progeny were similar to values in controls and within normal ranges. Relative to their comparators, the clone progeny were described as “less excitable” during handling in the chute. This difference in behavior was reflected in the physiological measurements showing that the Starbuck II progeny heifers exhibited lower heart rate (81.4 ± 17.2 beats per min (bpm) vs. 99.4 ± 17.8 bpm), lower respiratory rate (35.2 ± 10.2 respirations per min (rpm) vs. 45.8 ± 14.0 rpm), and lower body temperature (38.9 ± 0.4°C vs. 39.2 ± 0.3°C) compared to controls. However, all of these values fell within the range of normal values for dairy cattle. The authors speculated that the lower heart rates, respiratory rate and temperature in Starbuck II progeny were due to a faster adaptation to handling and manipulation. It is also possible that these results reflect more frequent human contact (observation and handling) of the Starbuck II progeny relative to their comparators.  Pregnancy was confirmed in two of the five Starbuck II progeny that were inseminated, but these pregnancies were terminated at 45 days due to housing constraints.

Telomere lengths, measured in skin biopsies taken at 30 days of age, were similar in Starbuck II progeny (n = 32) and age-matched comparators (n = 20). Telomere length in Starbuck II was also similar to age-matched control bulls, but the lengths of Starbuck II’s telomeres and his progeny’s telomeres were not compared. Chromosomal analysis in peripheral blood leucocytes indicated similar frequencies of diploid karyotypes in progeny and comparators.  Therefore, it appears that telomere length homeostasis and chromosome stability were normal in the Starbuck II clones.

None of the abnormalities previously reported in cattle clones, such as cardiac and respiratory abnormalities, hyperthermia during the first two months of life, or altered hematological and endocrine parameters (Chavatte-Palmer et al. 2002), were observed in the progeny of Starbuck II.  Although no laboratory assays of immune function were performed, the authors concluded that the absence of illness and need for therapeutic treatments provides evidence of functional immune systems in Starbuck II progeny. The authors concluded that the clone progeny had normal phenotypic characteristics, normal behavior, and their growth, health, hematological and reproductive parameters were comparable to age-matched comparators.

Progeny of Clone x Clone Mating

In some cases, female clones may be mated to male clones to produce offspring for human consumption. This raises the question of whether progeny derived from mating pairs of clones will be different from those derived from conventional breeding, from matings in which only one of the parents is a clone. To begin to address this question, growth performance was described for a calf produced by a mating in which the dam and sire were both clones (Kasai et al. 2007).  Donor cells were cultured cumulus and ear cells, respectively, obtained from Japanese Black cattle. Comparators in this study were “full siblings” (n=7; heifer calves) produced by artificially inseminating the cow used as the nuclear donor with semen from the bull used as the nuclear donor. Comparator embryos were flushed from the nuclear donor and transferred to recipients. To produce the calf of the clones, the female cow clone was mated to the male bull clone by artificial insemination. 

Following a 292-day gestation (11 days longer than the average for comparator pregnancies), the dam gave birth to a heifer calf. No assistance was needed at the time of parturition. Birth weight, clinical examination, hematology, serum biochemistry and telomere length of the calf of the clones were within the ranges measured for the comparator calves. Growth of the calf from birth to 12 months, as measured by body weight and shoulder height, was similar to that of the comparators. No serious health problems were observed in the calf and at the time of publication, the heifer was four months pregnant following artificial insemination at 18 months of age. To the authors’ knowledge, this was the first report demonstrating normal growth performance in a calf derived from mating two clones. These results are consistent with those of Heyman et al. (2004), who did not observe LOS in offspring produced by mating either pairs of clones or clones to non-clones.

vii. Conclusions Regarding Food Consumption Risks from Bovine Clones and their Progeny

As the first prong of our strategy to address the food consumption risks associated with clones, we have used the Critical Biological Systems Approach (CBSA) as a framework to search for subtle differences between clones and comparators that may pose food consumption risks. In general, these differences cannot be detected macroscopically but may be evident as differences in physiological parameters during the five developmental nodes. For bovine clones, our health-based assessment of food consumption risks is facilitated by a significant body of evidence from the peer-reviewed literature together with a large data set from Cyagra. Many bovine clones do not survive the neonatal period, and several abnormalities (e.g., those related to LOS, prolonged recumbency, umbilical malformations) have been described during this developmental node.  None of these abnormalities is unique to clones, and all have been observed in calves produced by other ARTS such as in vitro fertilization or following natural mating.

In clones that survive the neonatal period, some studies have identified differences in physiological measures between clones and comparators during the first few weeks of life. 

These findings support the notion that bovine clones are more physiologically unstable during the early juvenile period. There is evidence that the physiological transition from neonatal period to the juvenile period may take longer in calf clones (e.g., elevated body temperature during the first two months of life). Once their physiological status is stabilized, however, there is ample evidence to indicate that growth and development proceed normally in bovine clones.  Similarly, several studies indicate that fertility in clones is normal, and there are no indications that the physiology or health of clones is compromised during the post-pubertal period. 

In summary, we have searched for subtle differences between clones and their comparators to identify differences that may pose food consumption hazards. We have not found any such subtle differences, and based on this review of the health and physiology of bovine clones using the CBSA approach, we conclude that there is no reason to expect that food from bovine clones would pose additional food safety risks compared with the same products derived from conventionally-bred cattle.

Clone progeny are not expected to pose any increased food consumption risks compared with other sexually reproduced animals (NAS 2002b). Although the amount of data describing the health of progeny of clones is more limited than the amount describing the health of clones themselves, the results are consistent with the biological assumption. In the two studies that characterized the physiology of heifers produced by clones, growth, reproductive function, and telomere length were normal in clone progeny, and the incidence of general health problems was not increased in clone progeny compared with progeny of other sexually reproduced animals. Based on the CBSA approach, we therefore conclude that sexually reproduced progeny of clones are indistinguishable from other sexually reproduced animals, and pose no additional food consumption risks.

b. Swine Clones

There are approximately 45 papers, including some reviews, within the peer-reviewed literature that address cloning of swine; many of these report on the production of transgenic swine by SCNT. Unlike cattle, where improvement of breeding stock has been a major driving force for advances in reproductive technologies, many of the earlier studies of SCNT in swine have focused on transgenic animals for use as xenotransplant organ sources (reviewed by Prather et al. 1999; Westhusin and Piedrahita 2000; Wheeler and Walters 2001; Carter et al. 2002; Machaty et al. 2002; and Prather et al. 2003). Nonetheless, cloning swine for agricultural purposes has become the focus of at least one large commercial venture (ViaGen, Inc.), and others (Archer et al. 2003a,b) have also reported extensively on the health and physiological status of non-transgenic swine clones.

The cloning of swine was first described in 2000 by Polejaeva and her colleagues at what was then PPL Therapeutics in Blacksburg, Virginia and Roslin, UK. Several laboratories followed that publication with their own reports of swine cloning using different approaches to cell fusion, oöcyte maturation, or other technical issues (Betthauser et al. 2000; Onishi et al. 2000; and Bondioli et al. 2001). In the subsequent years, additional studies have reported on the difficulties of overcoming the early stage failures (Boquest et al. 2002, and Yin et al. 2002a; Lee GS et al. 2005a; Zhu et al. 2004).
 
Another issue contributing to the difficulty of cloning swine is that unlike cattle, sheep, and goats, swine require a minimum number of viable embryos, thought to be approximately four, to initiate and sustain pregnancy (Polge et al. 1966; Dzuik 1985). This has posed a technical limitation for the development of cloning in this species because the high loss of embryo clones throughout the pregnancy necessitates the transfer of a very large number of clone embryos into the surrogate dam (between 150 and 500) to ensure that the minimum number of embryos is maintained. A recent paper by King et al. (2002) explored hormonal treatments to sustain limited numbers of viable embryos to term, and demonstrated that pregnancies can be established with a mixture of fertilized and parthenote embryos and that small numbers of fertilized embryos can develop to term successfully with hormonal support.

Because of these difficulties, most of the available reports describe only the implantation and early perinatal phase. Two publications by Archer et al. (2003a,b) describe the behavior and clinical chemistry of juvenile swine clones.

i. Cell Fusion, Nuclear Reprogramming, Embryonic and Fetal Development Through the Perinatal Developmental Period in Swine Clones (Developmental Nodes 1 and 2)

(a) Peer-reviewed Publications

In the first published report of swine clones by Polejaeva et al. (2000), two rounds of nuclear transfer were employed, with in vivo matured oöcytes as recipients and cultured granulosa cells as donors, to produce five live female piglet clones. Piglets were delivered by C-section on day 116 of the pregnancy. The only data on the health of these piglets indicated that the average birth weight of the clones of 2.72 pounds (range 2.28-3.08 pounds) was approximately 25 percent lower than in piglets produced using natural mating in the same population as the donor cells (average birth weight of 3.6 pounds, range 3.3-3.9 pounds in an average litter size of 10.9 piglets).

In the second report of swine cloning, after several unsuccessful attempts, Onishi et al. (2000) produced a single female piglet named “Xena” from cultured embryo fibroblast cells. The clone’s birth and placental weights were 1.2 kg and 0.3 kg, respectively, which the authors state were in the normal range for conventional offspring of that breed (Meishan). Xena was described as a “healthy female” but, with the exception of a photograph, no data were provided to confirm that observation.

Betthauser et al. (2000) also describe multiple attempts at establishing successful pregnancies in surrogate dams receiving swine embryos resulting from SCNT. Of the seven pregnancies that were established, three were with non-transgenic embryo clones. Four live births resulted from two pregnancies, out of 427 embryos implanted into surrogate dams. The first litter yielded two male piglets born alive by vaginal delivery, weighing 2.0 and 3.0 pounds each. The second litter also produced two live vaginally delivered male clone piglets and one mummified fetus. The live piglets in this litter weighed 2.2 and 3.5 pounds. The third pregnancy was aborted at 40 days of gestation. No further information was provided on the health status of the clones at birth. Subsequently, the senior author on this report wrote a Letter to the Editor of the publication (Bishop 2000) to inform that the piglets from the second litter had died one week after their birth due to the aggressive behavior of the first-time surrogate mother. This behavior limited the amount of time the piglets were able to nurse, and the consequent lack of adequate nutrition proved to be fatal to the piglets (Bishop 2000). CVM is unaware of any publications providing additional information on the health status of the first litter.

An Australian group (Boquest et al. 2002) described the birth of live piglets from cultured fetal fibroblast cells that were frozen for two years, employing a novel cell fusion method in which donor nuclei were exposed to inactivated oöplasm for a period of time prior to chemical activation (to begin the process of cell replication). They believe that the lag time between fusion and activation allows for the more efficient reprogramming of the donor cell nuclei. The investigators transferred between 40 and 107 embryos to 10 surrogate dams, resulting in five pregnancies. Three of those pregnancies were aborted, and each of the two remaining pregnancies yielded one live piglet. No information is provided about the health status of the clones.

Yin et al. (2002a) also developed a novel method for the production of pig clones by treating oöcytes to be used as recipients with demecolcine such that the condensed chromosomes produce a protrusion at the cell membrane that can easily be removed by micro-aspiration. Donor cells were obtained from an adult female four year old Landrace pig, and included cultured heart and kidney cells. Six surrogate dams were implanted with between 137 and 341 embryos. Three of the recipients never became pregnant, and one aborted the pregnancy on day 62. The remaining two pregnancies, both with embryos of heart tissue origin, resulted in live births. The first litter included four live female clones, and one dead fetus. The second resulted in another four live female clones, and two dead fetuses. None of the clones, live or dead, exhibited any morphological anomalies. The authors reported that the eight surviving clones were eight months old at the time of publication, and “appear quite healthy.” No further information is provided.

Lee GS et al. (2005a) found that supplementing culture media with epidermal growth factor (EGF) improved cleavage rate of NT embryos, but not the rate of blastocyst formation compared to unsupplemented media, although total cell numbers in surviving blastocysts were higher in EGF supplemented media. Adding EGF after morula formation did not affect blastocyst formation rate or cell numbers. Zhu et al. (2004) found embryos produced with stem cells isolated from fetal porcine skin cultures had higher preimplantation development rates than embryos produced using fetal fibroblast cells. Karyotypic analysis of the two donor cell cultures indicated that porcine stem cells accumulated fewer abnormalities and were more stable through multiple passages compared to fibroblast cells. Porcine stem cells also yielded more blastocysts than fibroblast cells. Because neither of these groups attempted to transfer embryos to recipients, there is no way to know whether these improvements in early embryo developmental efficiency would have resulted in a higher proportion of live clones.

Bondioli et al. (2001) reported on the generation of transgenic pig clones from cultured skin fibroblasts derived from an a-1,2-fucosyltransferase (H-transferase) transgenic boar. (H-transferase is involved in producing the sugars on the surface of a pig cell that are partially responsible for the acute phase of rejection observed when non-human tissues are transplanted into humans.) Of the 217 embryos transferred into five surrogate dams, two pregnancies resulted. One of the surrogate dams was euthanized at 90 days of gestation for health reasons that the authors state were unrelated to embryo transfer. One mummified fetus and one apparently viable fetus were recovered. The other pregnancy yielded two live piglets that were delivered by C-section at 116 days of gestation. The piglets were reported as “healthy,” and a photograph of two apparently normal piglets at two months of age is provided in the paper.

Walker et al. (2002) have reported on the largest litters of piglets produced by SCNT. Donor cells were derived from Duroc fetal fibroblasts, and fused with in vitro matured oöcytes. A total of 511 embryos were transferred into five surrogate dams, with between 59 and 128 embryos per recipient. All five recipients were confirmed pregnant by ultrasound between days 28 and 40 post-implantation. Four of the five pregnancies went to term, and litters containing between 5 and 9 piglet clones (total of 28) were delivered. Three of the four surrogate dams were induced and delivered on gestational day 115. The fourth was allowed to deliver naturally, and produced her litter on gestational day 117. One of the 28 clones was stillborn, but no abnormalities were noted on necropsy. One of the live born clones presented with anal atresia (no anus or tail), and was the smallest of all of the clones (birth weight of 0.72 kg, and crown rump length of 23.5 cm).

The authors noted that anal atresia is a developmental abnormality seen at a natural low frequency in conventional piglets. The question of whether this is a random event due to genetic or inappropriate reprogramming cannot be answered from this dataset.

 

Table VI-9: Summary of Birth Characteristics of Piglet Clones
(source: Walker et al. 2002)
Litter size Mean Birth Weight (kg)1 Mean Placental Weight (kg)1 Crown-Rump Length (cm)1
9 1.15 ± 0.17 0.29 ± 0.09 68.8 ± 2.1
5 1.06 ± 0.23 0.23 ±0.02 71.6 ± 7.6
7 1.35 ± 0.13 0.29 ± 0.07 74.9 ± 1.8
7 1.29 ± 0.26 NR NR
Control2 1.37 ± 0.12 NR NR
1 All values presented as means ± SD.
2 The control birth weight was derived from the average weight ± SD from 10 litters of piglets from naturally bred Duroc pigs.
NR = Not reported.

The remaining piglets had birth weights that appear to be a little lower than conventional piglets of the same breed. The authors noted with explicit surprise that there was little correlation between litter size, placental weights, and fetal weights (Table VI-9). They predicted a correlation of 0.639 between placental and fetal weight, but noted that the lowest mean birth weights occurred in the litters with the smallest number of piglets. The authors asserted that without the appropriate controls for litter size, in vitro oöcyte maturation and other manipulations, it is inappropriate to assign the SCNT process as the cause of the difference in birth weights. Two of these litters subsequently served as the source of the clinical and behavioral studies of Archer et al. (2003a,b).

Longer gestational length was reported for dams carrying two groups of clone pregnancies produced by Carroll et al. (2005).  These groups of piglets were derived using two different fibroblast cell lines developed from two porcine fetuses (day 35). Gestational length in clone pregnancies (n = 5 litters total) was 118.8 ± 0.97 days compared to 114 ± 0.41 days in natural pregnancies in the same herd from which the cells for the clones were obtained. The first group of clones (C1) consisted of just 2 piglets from a single litter (no information is provided about the littermates of these piglets), while the second group of clones (C2) consisted of 7 piglets from 4 litters. Birth weights are not reported in this study, and the authors did not report any abnormalities in the clone piglets. The acute phase immune response of these clones was subsequently evaluated at 27-30 days of age (for a discussion see the Juvenile Developmental Node).

Jiang et al. (2007) described body weights, organ weights, and gene expression in nine piglet clones that died at birth or shortly thereafter. Five of these were transgenic with one disrupted allele of the porcine a1,3-galactosyltransferase gene, and the other four were not transgenic. Controls (n=5) were age-matched piglets generated via sexual reproduction. The number of litters from which piglets were derived is not specified.  The results for controls were compared with results from all nine piglet clones (transgenic and non-transgenic). Three of the dead piglets “had minor phenotypic abnormalities, such as curled toes”.  The authors note that this anomaly has been observed occasionally in piglet clones, and that piglets with similar minor abnormalities can survive and develop normally. Consistent with other studies (Polejaeva et al. 2000; Lai et al. 2002; Yin et al. 2002a), the clones weighed less than controls at birth (1.2 kg. vs. 2.1 kg, respectively). Weights of heart, liver, kidney and spleen were similar between newborn clones and control piglets. Mean lung weight, expressed as a percentage of body weight, however, was significantly lower in the deceased clones (1.6 percent vs. 2.1 percent in controls).

To investigate whether lower body weight in piglet clones is correlated with expression of genes that regulate growth, Jiang et al. (2006) measured the expression of four imprinted genes in their cloned piglets: IGF-2, PEG3, IGF-2R, and GRB10.  These genes either promote (IGF-2, PEG3) or inhibit (IGF-2R, GRB10) growth. Using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR), the authors observed lower levels of expression of the IGF-2R gene in lung, brain and spleen tissue from the deceased newborn clones compared to controls, but these levels of gene expression were not related to body weight.

Shibata et al. (2006) used SCNT to produce eight female pigs of the Jin Hua breed. Compared with non-clone Jin Hua control piglets, the clones had similar mean birth weights (0.87 kg in controls vs. 0.91 kg in clones; no estimate of variance was provided) and were phenotypically normal. Reproductive and growth performance was subsequently characterized in these pigs, as described in the Juvenile and Reproductive Development nodes. 

To determine whether the cellular age of donor cells is altered by nuclear reprogramming during SCNT, Jeon et al. (2005) analyzed telomere length and telomerase activity in 12 newborn piglet clones. Donor cells for SCNT were fibroblasts isolated from day 30 fetuses. Clones had longer telomeres compared to donor fetal fibroblasts.  Although telomerase activity in these clones was similar to that in the donor cells, telomerase activity was higher in SCNT blastocysts. The authors suggest that in clones derived from porcine fetal fibroblasts, telomeres can be rebuilt/elongated at the blastocyst stage of development.

(b) Unpublished data

ViaGen Inc. provided birth weights of seven male swine clones as part of the data package presented to CVM. Clones were smaller at birth than AI comparators of similar genetic background (See Appendix F: ViaGen Dataset). No detailed health data were available on these clones for this developmental node. All clones survived the neonatal period.

Additional data submitted to CVM included birth weight, average daily weight gain (ADG), body temperature, and pulse rates on another cohort of neonatal swine clones (see Chapter V). Birth weights for three clones ranged from 1.1 to 1.4 kg, and ADG ranged from 0.46 to 0.55 kg; however, because the breed of swine was not identified, it is not possible to determine whether these data are within normal ranges. The report indicated that two of the five piglets, both from the same litter and weighing 1.0 kg at birth, died within the first 48 hours. The cause of death was not reported, and no other details were provided. Body temperatures of the piglets were low (range 98.8 to 101.8°F) during the first 48 hours compared to reference body temperature for adult swine (102-103°F). This finding is not unusual, however, as neonatal swine generally have difficulty regulating body temperature, and require supplemental heat after birth (see Chapter V).

(c) Summary Statement on the Embryo/Fetal to Perinatal Developmental in Swine Clones (Developmental Nodes 1 and 2)

The production of swine clones differs from the other livestock species discussed in this risk assessment because of the requirement for a minimum number of viable fetuses to maintain the pregnancy. The gestational losses observed are a function of the combined low “success rate” for embryonic and fetal development for the individual clone and the requirement for a minimum number of growing fetuses to implant. Clone piglets do not appear to exhibit the overgrowth phenomena observed in cattle, and if anything, newborn swine clones may be smaller than their non-clone counterparts. Most swine clones at this developmental stage appear to be healthy; there is only one report of a fairly commonly observed congenital malformation (anal atresia), and one that is less frequently observed (curled toes). 

Continued...